Marine Biological Laboratory Library Voods Hole, Massachusetts Manual of Microbiological Methods fiCr>^ MANUAL OF Microbiological Methods BY THE SOCIETY OF AMERICAN BACTERIOLOGISTS Committee on Bacteriological Technic ]M. J. Pelczar, Jr., Chairman R. C. Bard G. W. Burnett H. J. Conn, Editor R. D. DeMoss E. E. Evans F. A. Weiss M. W. Jennison A. P. McKee A. J. Riker J. Warren O. B. Weeks McGRAW-HILL BOOK COMPANY, INC New York Toronto London 1957 MANUAL OF MICROBIOLOGICAL METHODS Copyright (c) 1957 by the Society of American Bacteriologists. Copyright, 1923, 1926, 1936, 1946, by the Society of American Bacteriologists. Printed in the United States of America. All rights reserved. This book, or parts thereof, may not be reproduced in any form without permission of the publishers. The methods given here have not been approved formally by the Society of Ameri- can Bacteriologists. The use of portions of the text of the United States Pharma- copeia, 15th Revision, official December 15, 1955, is by permission received from the Board of Trustees of the United States Pharmocopeial Convention. The said Board is not responsible for any inaccuracies of the text thus used. Library of Congress Catalog Card Number 57-8629 10 11 12 13 14 15 16 17 - MP - 1 9 8 7 69556 Committee Members and Other Contributors Allen, 0. N. Department of Bacteriology, University of Wisconsin, Madison, Wis. Ark, p. a. Division of Plant Pathology, University of California, Berkeley, Calif. *Bard, R. C. Smith Kline & French Laboratories, Philadelphia 1, Pa. Bartholomew, J. W. Department of Bacteriology, University of Southern California, Los Angeles 7, Calif. *BuRNETT, G. W. Dental Division, Walter Reed Army Institute of Research, Washington, D.C. tCoHEN, Barnett Formerly, Johns Hopkins Medical School, Baltimore, Md. *CoNN, H. J. Emeritus, Division of Food Science and Technology, N.Y. Agricultural Experiment Station, Geneva, N.Y. *DeMoss, R. D. Department of Bacteriology, University of Illinois, Urbana, 111. *EvANS, E. E. Department of Microbiology, Medical Center, University of Alabama, Birmingham 3, Ala. HiLDEBRAND, E. M. Horticultural Field Station, U. S. Department of Agri- culture, Beltsville, Md. HiLDEBRANDT, A. C. Department of Plant Pathology, University of Wisconsin, Madison, Wis. *Jennison, M. W. Department of Bacteriology, Syracuse University, Syracuse 10, N.Y. LiNDBERG, Robert B. Department of Bacteriology, Walter Reed Army Insti- tute of Research, Washington, D.C. McClung, L. S. Department of Bacteriology, Indiana Universitj^, Blooming- ton, Ind. *McKee, a. p. Department of Bacteriology, College of Medicine, State Uni- versity of Iowa, Iowa City, Iowa *Pelczar, M. J., Jr. Department of Microbiology, University of Maryland, College Park, Md. *RiKER, A. J. Department of Pknt Pathology, University of Wisconsin, Madison, Wis. *Warren, Joel Division of Biological Standards, National Institutes of Health, Bethesda 14, Md. *Weeks, 0. B. Department of Bacteriology, University of Idaho, Moscow^ Idaho *Weiss, F. a. American T3'pe Culture Collection, 2029 M Street, N.W., Washington, D.C. *Members of Committee on Bacteriological Technic, 1955-7 tDeceased Preface This manual is intended for use in those types of microbiological work which involve the study of microbial cultures or of viruses, either for identification or for learning the properties of the organisms investigated. This book takes the place of the loose-leaf publication issued during the period of 1923-1956 under the name of ''Manual of Methods for Pure Culture Study of Bacteria." The present manual covers a wider scope but still includes the subject of ''pure culture study, " the meaning of which is discussed. The methods given here are not to be regarded as official. The com- mittee has always taken the stand that official methods should not be adopted in the case of research work, because it is continually necessary to modify research methods in order to keep them up to date. The standardization of methods tends to hinder the development of new technics, while the chief function of this committee is to stimulate its development. The methods in this manual, therefore, are merely claimed to be those regarded as satisfactory by the committee at the time of publication. Whenever practical, the methods have been tested by the committee in comparison with other procedures. Of the chapters in this book, V, VIII, IX, X, and XI are almost entirely new. The others are revisions, more or less complete, of leaflets of the old manual. In the case of these revised chapters, the names of the contributors at the chapter heads are the latest revisers, not the original authors. In the case of the five entirely new chapters, however, the actual authors are cited at the chapter heads. H. J. Conn M. J. Pelczar, Jr. Vll Contents Preface Vll Chapter I. Introductory. Precautions. Practical Hints Chapter II. Staining Methods. General Principles. General Bacterial Stains. Negative Staining of Bacteria. The Gram Stain. Acid-fast Staining. Spore Staining. Staining the Diphtheria Organism. Fla- gella Staining. Capsule Stains. Stain for Fat Droplets. Robinow's Stains for Nuclear Apparatus. Stains for Spirochaetes. Dye Solubil- ities, by H. J. Conn, J. W. Bartholomew, and M. W. Jennison 10 Chapter III. Preparation of Media. Introductory. Cultivation and Storage Media. Enrichment Media. Differential Media. Media for Determination of Physiological Properties. Media for Special Bacteriological Procedures. Media for Special Purposes. Appendix: Specifications for Bacteriological Grade Agar, Gelatin, and Peptone. byG. W. Burnett, M. J. Pelczar, Jr. and H.J. Conn 37 Chapter IV. The Measurement of pH, Titratable Acidity, and Oxidation- Reduction Potentials. The Measurement of pH. Titratable Acidity. Buffer Action, and pH Adjustment of Culture Media. The Measure- ment of Oxidation-Reduction Potentials, by Barnett Cohen ... 72 Chapter V. Maintenance and Preservation of Cultures. Introduc- tion. Selection of Materials. Dormant Conservation. Maintenance Methods. Media Commercially Available. Media Recommended. by Freeman A. Weiss 99 Chapter VI. The Study of Obligately Anaerobic Bacteria. Anaerobic Culture Methods and Equipment. Technics for the Study of Anaer- obic Bacteria, by L. S. McClung and Robert B. Lindberg .... 120 Chapter VII. Routine Tests for the Identification of Bacteria. Intro- duction. The Descriptive Charts. Determining Optimum Condi- tions for Growth. Incubation. Variation. Micromethods. Cul- X CONTENTS tural Characteristics. Study of Cell Morphology. Relation to Free Oxygen. Action on Nitrates. Chromogenesis. Indole Production. The Production of Hydrogen Sulfide. Liquefaction of Gelatin. Cleavage of Sugars, Alcohols, and Glucosides. Hydrolysis of Starch. The Methyl Red and Voges-Proskauer Tests. Acid Production in Milk. Rennet Production, by H. J. Conn, M. W. Jennison, and 0. B. Weeks 140 Chapter VIII. Physiological and Biochemical Technics. Introduction. Growth Measurements. Preparations of Cells and Extracts. Bio- chemical Technics, by R. D. DeMoss and R. C. Bard 169 Chapter IX. Serological Methods. Introduction. Preparation of Anti- serum. Agglutination. Precipitation. Complement-fixation Test for Microbial Antigens, by E. Edward Evans 199 Chapter X. The Detection of Bacterial Pathogenicity. Introduction. General Factors Underlying Virulence. Preparation of Inocula. Selection and Preparation of Laboratory Animals for Pathogenicity Studies. Methods of Inoculation. Care of Experimental Animals. Clinical Signs in Animals. Recovery of Bacteria from Infected Ani- mals. Detection of Antigen in Tissues. Conclusion. Chart of Identification Procedures, by Joel Warren 224 Chapter XI. Virological Methods. Introduction. Precautions. Stains. Cultivation: Tissue Cultures. Hemagglutination. Serological Proce- dures. Complement Fixation. Concentrating Viruses. Storage. by Albert P. McKee 246 Chapter XII. Inoculations with Bacteria Causing Plant Disease. Intro- duction. Simple Representative Inoculation Methods. Treatment with Bacterial Products. Antibody Production. Cognate Considera- tions. Records. Interpretation of Results, by A. J. Riker . . 286 Chapter XIII. Glossary of Terms Used on the Charts and in the Manual 299 Index 307 CHAPTER I Introductory SCOPE OF THE MANUAL There has sometimes been misunderstanding as to the sense in which the Committee on Bacteriological Technic uses the expression "pure culture study of bacteria." It is occasionally thought that such an expression would cover nearly the whole field of bacteriological technic. On the other hand, the definition of pure culture study of bacteria which has been drawn up by the committee is the study of bacterial cul- tures with the object of learning their characteristics and behavior or determining their identity, or both. Such a study may be regarded as including isolation methods, methods for the cultivation and the storage of various kinds of bacteria, microscopic study of pure cultures either stained or unstained, determination of cultural characteristics of an organism, a study of its physiological characteristics, the chemical methods necessary in making the last-mentioned study, the determina- tion of pathogenicity and study of pathological effects, the serological characterisics of an organism when used as a means of description. It is clear from such a statement that pure culture study of bacteria is fairly comprehensive but that there are many fields of bacteriological technic not included within it, e.g., methods for the enumeration of bac- teria in their natural habitats, the diagnosis of disease and many other phases of medical bacteriology, methods employed in the study of food spoil- age and controlling the processes of fermentation, etc. Such a list might be extended almost indefinitely, for the field of pure culture study, although fairly broad, is actually a small part, though basic, of bacteriological technic. The scope of the present manual has been widened to include viro- logical methods and procedures for the maintenance and preservation of bacteria, and it may in the future be expanded to include methods for yeasts and molds. Nevertheless its subject matter still does not cover those topics listed in the preceding paragraph. RELATION TO TAXONOMY Clearly, one of the objects of pure culture study is to determine the identity of any bacterial culture under investigation. This brings the 2 MANUAL OF MICROBIOLOGICAL METHODS subject very close to the field of bacterial taxonomy, i.e., the naming and classifying of bacteria. Inasmuch as bacteria cannot be classified with- out studying their characteristics in pure culture, it is an obvious conclu- sion that pure culture study is a necessary prelude to bacterial taxonomy. It must be recognized, nevertheless, that one can consider pure culture study without regard to taxonomy and that one can study the taxonomy of bacteria without paying special attention to the methods of pure cul- ture study. Since this distinction can be made and the committee edit- ing this series of publications is a committee on technic, care has always been taken to maintain the distinction so as not to interfere with the functions of other committees that have been appointed and to deal with matters of nomenclature and classification. PUBLICATIONS OF THE COMMITTEE ON TECHNIC Descriptive charts. The first descriptive chart actually adopted by the Society of American Bacteriologists was in 1907. The chart has been revised from time to time and at present there are two forms : one known as the Standard Descriptive Chart and the other as the Descriptive Chart for Instruction. The latter is very much simpler than the former. The formxer is printed on both sides of an 83^- by 11-in. sheet of light cardboard; the latter on a sheet of heavy paper of the same size. The object of the descriptive chart is to provide a space for recording the most important characteristics of a single culture. The Standard Chart is the most complete and is intended especially for advanced work in bacteriology. Unfortunately, however, it does not meet modern research needs at all perfectly because each group of bacteria requires its own set of tests and no form can be drawn up sufficiently detailed to cover all of them. The Chart for Instruction, on the other hand, is so much simpler and contains so much blank space that it sometimes is found to be more satisfactory in research work than the Standard Chart. It is, however, intended primarily for students to use in characterizing cultures furnished them in connection with their classwork. Manual of Microbiological Methods. The origin of the present manual traces back to a committee report which was printed in the Journal of Bacteriology and was distributed in reprint form by the committee (1918). It was only 14 pages long and covered only the methods used in carrying out the determinations called for on the descriptive chart of those days. After one or two minor revisions it was converted in 1923 into the ''Man- ual of Methods for Pure Culture Study of Bacteria," which as remarked above was published in loose-leaf form until 1956. The first edition was only 48 pages in length and, like its predecessor, was confined wholly to the methods needed in using the chart. Gradually, hoAvever, it was INTRODUCTORY 3 expanded until it included 10 leaflets, and it has come to include a variety of methods other than those called for in the use of the descriptive chart. By 1953 several other subjects had been selected as desirable to include in future editions, and plans were made for converting the manual into a larger pubhcation. While the old manual was in loose-leaf form, it was kept up to date by periodic revision of its leaflets, one or two at a time, and by means of a continuation service owners were enabled to secure the latest editions to insert in their copies. This feature now, unfortunately, has to be given up because of the increased size of the book and of certain practical diffi- culties involved in loose-leaf publication. The committee regrets the necessity of abandoning the old system, but there seems to be no other course than to convert it into a regular book and to hope that revisions to bring the contents up to date can be accomphshed by means of periodic new editions. HISTORICAL The first efforts toward producing a descriptive chart for characterizing bacteria were made by two different individual investigators, H. W. Conn and S. de M. Gage. The work of these two investigators called the matter to the attention of bacteriolo- gists in general, and it was finally brought before the Society of American Bacteriolo- gists by F. D. Chester at the Philadelphia meeting in December, 1903, and then again at the 1904 meeting, when he explained his idea of a "group number" which would be descriptive of the salient characters of an organism. On his recommendation the society appointed a Committee on Methods for the Identification of Bacterial Species of which Professor Chester was made chairman. This committee drew up the first descriptive chart with which the Society of. American Bacteriologists had any connection. This chart was put before the society at its 1905 meeting. It was presented at this time as a preliminary effort, and no endorsement of it was given by the society, nor apparently was such endorsement requested. The committee was instructed to con- tinue its work, and a second chart was prepared during 1906 and presented at the society meeting in December of that year. At this meeting it was decided that the chart should call for more complete data concerning bacteria than provided for by either of the two charts already submitted, so the committee was instructed to do further work along this same line. The committee at this time was conrposed of F. D. Chester, F. P. Gorham, and E. F. Smith, but Professor Chester was largely responsible for the first two charts presented at society meetings. Before the committee undertook a further revision, however, he had left bacteriological work and hence was no longer active on the committee. During 1907, therefore. Dr. Smith acted as chairman of the committee, and under his supervision the committee drew up another chart which was presented to the society at its meeting in December of that year. This chart was oflScially endorsed by the society and was put on sale by the secretary of the society. For several years following no changes were made in the chart. The next step in its development was brought about by H. A. Harding (1910), who published a paper in which he outlined the complete history of the chart, with copies of the early charts. 4 MANUAL OF MICROBIOLOGICAL METHODS and discussed improvements that might be made. This paper is available for those desiring more detail concerning this early history than is given here. As the society felt that further modifications were now needed, a new committee was appointed in 1911 consisting of F. P. Gorham, C. E. A. Winslow, Simon Flexner, H. A. Harding, and E. O. Jordan. This committee gave a report at the 1913 meeting, presenting a chart which was put on sale by the society but was not officially endorsed. As this committee was unable to continue the work, an entirely new one was appointed at this time consisting of H. A. Harding, H. J. Conn, Otto Rahn, W. D. Frost, and L. J. Kligler. This committee soon lost Dr. Rahn, who left the country in 1914, and M. J. Prucha was added in his place. The committee was called the Committee on Revision of the Chart for the Identification of Bacterial Species. The new committee was instructed by the society to make a conservative revision of the chart and at the same time to draw up a manual of methods to be used in connec- tion with it. At the 1914 meeting of the society, therefore, a chart was presented for approval, much like the 1907 chart except for its more logical arrangement of data. This chart was given the society's endorsement and was issued during 1915. The 1914 chart was printed on a sheet with its back entirely blank, the glossary previously on the back having been omitted. The committee gave as the reason for this that the glossary would be included in the manual on methods shortly to be pub- lished. The publication of this manual was delayed, however, pending investigation of the methods to be included in it. This investigation of methods was to be under- taken not only for the sake of the manual but also as a preliminary step toward radical revision of the chart, which was felt to be badly needed. Early in 1917, however, and before this program could be carried out, the chairman of the committee was forced by pressure of other duties to drop the work. As he wished to remain on the commit- tee, however, no change in membership was made, but H. J. Conn was asked to become chairman. The committee then undertook the first step toward the preparation of a manual on methods. A report was presented at the 1917 meeting, giving the methods recom- mended at that time for use with the chart. The report was printed in the Journal of Bacteriology, March, 1918, and was subsequently sold by the society in the form of reprints. This report was considered a preliminary manual on methods. The committee proposed at the same time a much simplified chart in the form of a four-page folder, which it recommended for use in instruction until the official chart could be given the revision it needed. This chart was not endorsed by the society but was printed and sold by the society for two or three years. This same committee (but now called the Committee on the Descriptive Chart) issued another report on methods which appeared in the Journal of Bacteriology, March, 1919, dealing with the gram stain, production of acid, and the reduction of nitrates. At the 1919 meeting it issued a further report which appeared in the Journal of Bacteriology in two parts, March and May, 1920. The first part of the report was a revision of the one which had been published in March, 1918, and was sold as a revised manual of methods until the reprints were exhausted in 1922. At the 1920 meeting the Committee on the Descriptive Chart was discharged with the understanding that its functions would be taken over by a committee of broader scope then appointed and called the Committee on Bacteriological Technic. This committee was appointed with the understanding that its membership should fluctu- ate from year to year in order to keep on it men actively interested in the work. The new committee made a further revision of the chart, which was presented at the 1920 meeting and endorsed by the society. Later editions of this chart have been drawn up by the committee but have not been submitted to the society for oflJicial endorsement. In order to avoid committing the society in favor of any of the methods INTRODUCTORY 5 concerned, recent editions of the chart have merely been presented by the committee and permission asked to put them on sale. The committee issued four further reports in the Journal of Bacteriology (1921, 1922a, 6, and c) before the manual was prepared. One of these reports (19226) pro- posed certain revisions of methods in the case of the gram stain, fermentation, nitrate reduction, indole and hydrogen sulfide production. The committee presented this report at the 1922 meeting of the society with the recommendation that the revised material be published as part of a " Manual of Methods for Pure Culture Study of Bacteria." The committee was thereupon instructed by the society to publish this manual, using the loose-leaf form of binding, with the understanding that new folders be issued from time to time to keep it up to date. This was done, and the system con- tinued till 1956, when, as explained above, it proved necessary to convert the manual into book form and its name was changed to "Manual of Microbiological Methods." The Committee on Bacteriological Technic has seen the following changes in personnel : 1920 H. J. Conn,i K. N. Atkins, I. J. Kligler, J. F. Norton, G. E. Harmon. 1921 H. J. Conn,i K. N. Atkins, G. E. Harmon, Frederick Eberson, Alice Evans. 1922 H. J. Conn,i K. N. Atkins, G. E. Harmon, Frederick Eberson, F. W. Tan- ner, and S. A. Waksman. 1923 H. J. Conn,i K. N. Atkins, J. H. Brown, G. E. Harmon, G. J. Hucker, F. W. Tanner, and S. A. Waksman. 1924-5 H. J. Conn,i K. N. Atkins, J. H. Brown, Barnett Cohen, G. J. Hucker, F. W. Tanner. 1926-7 H. J. Conn,i Barnett Cohen, Elizabeth F. Genung, W. L. Kulp, W. H. Wright; with G. J. Hucker and S. Bayne-Jones as a subcommittee on serological methods. 1928 H. J. Conn,i Victor Burke, Barnett Cohen, Elizabeth F. Genung, W. L. Kulp, W. H. Wright. 1929-30 H. J. Conn,i Victor Burke, Barnett Cohen, Elizabeth F. Genung, I. C. Hall, W. L. Kulp, W. H. Wright (deceased. May, 1929). 1931-4 H. J. Conn,i Barnett Cohen, Elizabeth F. Genung, Victor Burke, I. C. Hall, J. A. Kennedy. 1935 H. J. Conn,i Victor Burke, Barnett Cohen, M. W. Jennison, J. A. Kennedy. 1936-42 H. J. Conn,i J. H. Brown, Victor Burke, Barnett Cohen, C. H. Werkman, M. W. Jennison, J. A. Kennedy, A. J. Riker. 1943-5 H. J. Conn,i Victor Burke, Barnett Cohen, C. H. Werkman, M. W. Jenni- son, J. A. Kennedy, L. S. McClung, A. J. Riker. 1946-7 H. J. Conn,^ G. H. Chapman, Barnett Cohen, I. C. Gunsalus, M. W. Jenni- son, L. S. McClung, A. J. Riker, C. E. ZoBell. 1948 M. W. Jennison,! G. H. Chapman, Barnett Cohen, H. J. Conn, I. C. Gunsalus, J. A. Kennedy, L.^ S. McClung, A. J. Riker, C. A. Stuart, C. E. ZoBell. 1949 M. W. Jennison,! G. H. Chapman, H. J. Conn, I. C. Gunsalus, L. S. Mc- Clung, C. A. Stuart, A. J. Riker, C. E. ZoBell. 1950 M. W. Jennison,! R. C. Bard, G. H. Chapman, H. J. Conn, I. C. Gunsalus, L. S. McClung, C. A. Stuart, A. J. Riker, C. E. ZoBell. 1951-2 M. W. Jennison,! R. C. Bard, G. W. Burnett, H. J. Conn, H. C. Lichstein, L. S. McClung, A. P. McKee, M. J. Pelczar, A. J. Riker, C. A. Stuart, C. E. ZoBell. ^ Chairman. 6 MANUAL OF MICROBIOLOGICAL METHODS 1953-4 M. J. Pelczar,! R. C. Bard, G. W. Burnett, H. J. Conn, E. E. Evans M. W. Jennison, H. C. Lichstein, L. S. McClung, A. P. McKee, A. J. Riker, J. Warren, O. B. Weeks, F. A. Weiss. 1955-7 M. J. Pelczar,! R. C. Bard, G. W. Burnett, H. J. Conn, R. D. DeMoss, E. E. Evans, M. W. Jennison, A. P. McKee, A. J. Riker, J. Warren, O. B. Weeks, F. A. Weiss. GENERAL CONSIDERATIONS Pitfalls to Be Avoided by the Student In studying microbial cultures with the object of identifying them or describing them, the student is apt to run into certain pitfalls. Some of these apply specifically to certain types of work and are therefore best taken up in the various chapters of this book where they seem properly to fit. Others are more general; some, in fact, are well known even to beginning students in bacteriology. However, as others are less fully appreciated, a few words concerning some of these pitfalls seem called for here — even at the risk of repeating cautions that may seem too ele- mentary. These pitfalls arise primarily from three sources: (1) the danger of impure cultures, (2) confusing results because of variation of bacterial species, (3) differences in methods of study. The danger in impure cultures is, of course, thoroughly understood. Unfortunately, however, the second consideration just mentioned makes it more important to emphasize the danger of impure cultures today than was the case before 1920. In those days bacteriologists quite generally accepted the idea of monomorphism, and whenever a culture was observed to be noticeably abnormal in either morphology or physiology, it was promptly discarded as a contaminant. When, however, it began to be learned that even the most strictly guarded pure cultures might show changes in morphology during their life history, and then later when it was realized that the same organism might occur in two or more phases showing distinctly different cultural and physiological characteristics, the old ideas of monomorphism were decidedly upset. As a result of the changing point of view, it is very easy for a careless student today to believe that he is observing two phases of the same pure culture when, actually, one of his ''phases" is a contaminant. This makes constant checking as to purity of cultures even more important than it was before dissociation into phase variants was generally accepted by bacteriologists. Accepting the idea of dissociation presents other difficulties to the student. Without exhaustive study, it is sometimes very easy to describe two phases of the same species as though they were different organisms. It is also easy to prepare a description of some culture w^hich is an illogical jumble of the characteristics of two or more phases, due to * Chairman,, INTRODUCTORY 7 the fact that it was first studied in an unstable form and dissociation was taking place during the course of the study. On the other hand, some of the methods employed in the hopes of inducing phase variation may actually cause contamination and be incorrectly interpreted. Some of these points are very adequately discussed by Frobisher (1933). The third source of error mentioned above (variation in methods) also needs emphasis. When a species is described in such terms as one fre- quently encounters in published descriptions, e.g., ''produces acid (with- out gas) from glucose and lactose but not from sucrose; does not reduce nitrates," one has to guess at the answers to such questions as these: What basal medium was used in each instance? What indicator of acid production was employed? How thorough a study was made to show the absence of any acid from sucrose or of any reduction of nitrate? Or, in the last instance, is it safe to assume that the author of the species merely failed to find nitrite in some nitrate medium? Unless such ques- tions are answered correctly, the description is meaningless; the attempt to identify an unknown culture with such a description may well give misleading results. With all these pitfalls to avoid, it is easy to see how the same set of data, no matter how carefully prepared, can be differently interpreted by two different bacteriologists. As a result extreme caution is urged, both in determining the identity of a culture and in deciding whether or not to pronounce it a new species. Practical Hints Determining the characteristic of a culture. One should always, if possible, make a complete study of a culture promptly after its first isola- tion while it is in a condition to display its true characteristics. When a culture has been carried in the laboratory for a long period of time, it may change in some respects from the original. When practical, such cultures should be exposed to conditions which might bring them back to the "normal." When this is done, however, the possibility should always be recognized that by such manipulation dissociation may be induced so that the phase subsequently studied may be quite different from the original isolation. Whenever distinct evidence of dissociation is observed, each phase should be studied and recorded separately, and efforts should be made to reverse ihe change or to obtain the same change with other strains until the possibility of impure cultures seems to be out of the question. No importance should ever be attached to a single determina- tion unless supported by repUcations giving the same results. In describ- ing morphology, one should not be contented with one or two observa- tions but should study several transfers and should follow up each of them day by day for about a week. When changes are observed, a careful 8 MANUAL OF MICROBIOLOGICAL METHODS study should be made to learn whether they indicate morphologic varia- tion, dissociation, or merely contamination. In making special staining tests, like the gram stain, several determinations should be made on separate transfers of the culture and at different ages, because there are species that vary in their staining reactions and such variation cannot be detected by single determinations. As a check on the technic, a known positive and a known negative culture should be included in the study. For example, when making a gram stain, it is good practice to place on the slide, beside the culture under study, a smear containing a mixture of a known gram-positive and a known gram-negative organism (which differ markedly in morphology). Then it is possible to observe if the expected results are obtained with the known cultures and thus to have some degree of control on the technic. Identification. After recording the characteristics of an organism, the next step is identification, if possible, with a previously described species. This should never be attempted until at least six representative strains of the unknown organism isolated from more than one source, if possible, have been studied. No rules can be given for identifying the culture. Descriptions of bacteria are scattered so widely through the literature and vary so greatly in their form that identification is often extremely different. Bergey's ''Manual of Determinative Bacteriology" is a great help, but it is usually necessary to go back to original descriptions and often to secure transfers of authentic strains before certain identification can be made. Difficult as this procedure is, no one is justified in naming a new species of bacteria until a comprehensive search through the litera- ture of species already described has been made. Frequently it is neces- sary to refer in some publication to a previously described species on the basis of such an identification as this. In this case it is important to state in the publication whether or not an authentic strain of the species has been obtained for comparison; if so, from where obtained; if not, what published description of the species was followed in making the identification. As to a name to use for such a species, one may follow the original author's nomenclature or may give it the name employed in some modern system (e.g., Bergey). Whatever name is chosen, no con- fusion will result if it is accompanied by the name of the original author of the specific name and by that of the one making the combination of generic and specific names. Thus, whether one says ^^ Bacillus coli Migula" or ^'Escherichia coli (Migula) Castellani and Chalmers," it is entirely clear what species is intended. Naming a new species. When it proves impossible to identify a cul- ture with any species described in the literature, it is often desirable to publish a description of it as a new species. When publishing such a description, there are five important points to be kept in mind : INTRODUCTORY 9 1. The description should be based on at least six representative isola- tions of the organism. 2. If variations are found to occur among these strains, a critical study must be made to be sure that they are not the result of contamination. 3. In naming any characteristic of the species, especially if it is a nega- tive character (e.g., ''nitrates not reduced"), the technic by which it is determined must be stated. 4. Before giving the results of any test as positive or negative, compari- sons must be made with a control culture known to be positive and one known to be negative. 5. Before actually assigning a name one should consult a specialist in bacterial taxonomy, both as to the necessity for a new name and as to the validity of the name selected. The Board of Editor-Trustees of Bergey's Manual, for example, are always very glad to offer such advice. If these hints were followed by all who are trying to identify species or to publish descriptions of them, much of the confusion in bacterial nomenclature would be eliminated. REFERENCES Committee on Bacteriological Technic. 1922a. An investigation of American stains. J. Bacteriol, 7, 127-248. . 19226. Methods of pure culture study. /. Bacteriol., 7, 519-528. . 1922c. An investigation of American gentian violets. /. Bacteriol., 7, 529-536. Committee on Descriptive Chart. 1918. Methods of pure culture study. /. Bacteriol, 3, 115-128. . 1919. Methods of pure culture study. Progress report for 1918. /. Bacteriol, 4, 107-132. . 1920a. Methods of pure culture study. Revised. /. Bacteriol, 6, 127-143. 19206. Progress report for 1919. /. Bacteriol, 5, 315-319. Frobisher, M. 1933. Some pitfalls in bacteriology. /. Bacteriol, 26, 565-571. Harding, H. A. 1910. The constancy of certain physiological characters in the classification of bacteria. N.Y. State Agr. Expt. Sta. Tech. Bull 13. CHAPTER II Staining Methods H. J. Conn in collaboration with J. W. Bartholomew and M. W. Jennison GENERAL PRINCIPLES The staining of bacteria depends in general upon the same properties of dyes as does the staining of animal or plant tissue for histological pur- poses. Short discussions of the nature of dyes, with special reference to staining are given elsewhere (Conn, 1953), and only the briefest summary of the subject need be given here. All bacterial dyes are synthetic products — anilin dyes, or coal-tar dyes, as they are generally called. Although the synthetic dyes vary greatly in their chemical nature and staining properties, they are for practical purposes often divided into two general groups, the acid dyes and the basic dyes. These terms do not mean that the dyes in question are free acids or free bases. The free color acids and bases, when obtainable, are colored, to be sure, but they are often insoluble in water and rarely have appreciable staining action; i.e., the colors do not ''stick." The salts of these compounds, on the other hand, are more soluble, penetrate better, and stain more permanently; they are the true dyes. An acid dye is the salt of a color acid; a basic dye the salt of a color base. In other words, acid dyes owe their colored properties to the anion, basic dyes to the cation. The actual reaction of an aqueous solu- tion of a dye, however, depends on several factors, and an acid dye may well be basic in reaction, while a basic dye may be acid. This is because the reaction of such a solution depends on the relative strengths of the dye ion and of the anion or cation with which it is combined in the dye salt. Basic dyes have greatest affinity for the nuclei of cells, probably because of the acid nature of the nuclear material. Acid dyes have a stronger tendency to combine with the cytoplasm. Bacteria do not show typical cell structure, and they tend to stain fairly uniformly with nuclear, i.e., 10 STAINING METHODS U the basic, dyes. Hence, the stains in common use by the bacteriologists are rarely acid dyes. Preparation of Smears Pure cultures of bacteria can ordinarily be prepared for staining by the simple process of making an aqueous suspension and drying a drop of it on a slide or cover glass, without any fixation other than gentle heat. The use of this simple procedure depends upon the fact that most bacteria, because of their small size or their stiff walls, can be dried without great distortion. For this reason it is not always necessary, as with higher organisms, to coagulate the tissues before microscopic preparations can be made, although for cytological studies and for accurate determinations of size and shape of the cells, some fixation other than heat is needed. The best bacterial smears are usually made by removing a small amount of surface growth from some solid medium and mixing it with distilled water. It is often possible to use a drop of a culture growing in a liquid medium, but such a smear is not always so satisfactory, since certain constituents of the medium may prevent the bacteria from adhering to the slide or may interfere with the staining. The suspension used should always be sufficiently dilute. Ordinarily, only a faint turbidity should be visible to the naked eye, for it is always best to avoid the occurrence on the slides of solid masses of bacteria, piled one on top of the other. If a smear after staining does not show any por- tions where the bacteria are well separated one from another, a new, more dilute smear should be made. This is particularly important in the case of the gram stain or flagella staining. The usual method of fixing the suspension to the slide or cover glass is to pass it rapidly after drying through a bunsen flame two or three times. Another very satisfactory method is to allow the drop of material to dry on a slide lying on a flat, moderately hot surface, such as a plate of some nonrusting metal resting on a boiling-water bath. With many bacteria an aqueous suspension of the surface grow^th from agar can be dried in the air at room temperature and stained without any fixing; this method is not universally successful, however. For some cytological procedures special methods of making bacterial preparations are necessary, sometimes calling for fixing solutions rather than heat. It must be seen that the technic described for staining dried smears is too crude for accurate measurements of cells or for studying cytological details. One cytological method is given on page 30. It is also beyond the scope of this pubHcation to give staining methods for other than pure culture work. In using any of the methods it must be remembered that blind adher- ence to a staining technic is no guarantee that the result will be satis- 12 MANUAL OF MICROBIOLOGICAL METHODS factory. Even experienced workers sometimes discover to their dismay that they took too much for granted as to the purity of their reagents, cleanUness of sUdes and covers, or proper compounding of the staining solutions. A technic should, therefore, be checked upon known organ- isms as controls. It is, furthermore, important to know that the solutions and water used for dilution are reasonably free from bacteria and spores. Staining Formulas There has always been a surprising amount of inaccuracy in the litera- ture concerning staining solutions. This is due to a variety of causes: indefiniteness in the original publication, mistakes of copying by later authors, modifications of the original which are not described as modifica- tions and come later to be ascribed to the original author, failure of authors to cite references when giving their methods. For such reasons it has proved necessary in this publication to give in many instances both the original (rather indefinite) formula and an emended formula as interpreted by the committee. The committee, however, assumes no responsibility for the identity of the two and offers the emendation merely to prevent the perpetuation of formulas which are clearly ambiguous or indefinite as to their ingredients. Recent cooperation among this committee, the Biological Stain Commission, and the National Formulary Committee of the American Pharmaceutical Association has resulted in the virtual adoption of these emended formulas. Staining schedule. Tap vs. distilled water. When washing slides after applying any stain, tap water is ordinarily more convenient to use than distilled water, and in the staining schedules that follow, tap water is specified in those instances where its use is considered to be ordinarily unobjectionable. It must be remembered, however, that the use of dis- tilled water is never contraindicated for such purposes, and many bacteri- ologists perfer it for all steps where washing is called for, because it is not subject to variation in composition, buffer content, etc. GENERAL BACTERIAL STAINS— RECOMMENDED PROCEDURES^ ZiehVs Carbol-fuchsin Old statement of formula Emended statement of formula Solution A Sat ale sol basic fuchsin 10 ml Basic fuchsin (90 % dye content) 0.3 g 5 % sol carbolic acid 100 ml Ethyl alcohol (95 %) 10 ml Solution B Phenol 5 g Distilled water 95 ml Mix solutions A and B 1 In these discussions, small-size type is used for all formulas and directions for pre- paring them, text-size type for all other directions concerning recommended pro- cedures, and small-size type for similar matters concerning alternate procedures. STAINING METHODS 13 Ammonium Oxalate Crystal Violet {Hucker*s) Solution A Solution B Crystal violet (90 % dye content) ^ . 2 g Ammonium oxalate 0.8 g Ethyl alcohol (95 %) 20 ml Distilled water 80 ml Mix solutions A and B Crystal Violet in Dilute Alcohol Crystal violet (90 % dye content) 2 g Ethyl alcohol (95 %) 20 ml Distilled water 80 ml Loeffler^s Alkaline Methylene Blue Original statement of formula Emended statement Solution A Cone sol methylene blue in al- Methylene blue (90 % dye con- cohol 30 ml tent) 0.3 g Sol KOH in distilled water Ethyl alcohol (95 %) 30 ml (1 :10,000) 100 ml Solution B Dilute KOH (0.01 % by weight) 100 ml Mix solutions A and B Methylene Blue in Dilute Alcohol Methylene blue (80 % dye content) 0.3 g Ethyl alcohol (95 %) 30 ml Distilled water 100 ml Carbol Rose Bengal Rose bengal (90 % dye content) " 1 g Phenol (5 % aqueous solution) 100 ml CaCh 0.01-0.03 g (The amount of CaCh added determines the intensity of staining.) Staining schedule: Follow the general procedure given under "Prepa- ration of Smears," page 11, allowing 5-60 sec for application of the stain. Overstaining rarely occurs except with carbol f uchsin ; understaining does not have to be feared except with rose bengal. Results: The results depend on which of the above staining fluids is selected. They are listed in the order of intensity of action; i.e., carbol fuchsin gives the most intense stain and is not indicated when selective staining is desired or when much debris is present on the shde. The crystal violet solutions are very good for routine purposes. The methyl- ^ It is not necessary that dry stains of ine exact dye content specified be used in this or in the preceding and following formulas. Samples of higher or lower dye content may be employed by making the proper adjustment in the quantity used. 14 MANUAL OF MICROBIOLOGICAL METHODS ene blue solutions are much more selective, vrith special affinity for metachromatic granules. The rose bengal solution is much less com- monly used; it is specially valuable when mucus or colloidal organic material is present, as such material is not ordinarily stained by it. GENERAL BACTERIAL STAINS—ALTERNATE PROCEDURES Kinyoun^s Carbol Fuchsin Basic fuchsin (dye content not specified; probably 90 %) 4 g Phenol crystals 8 g Ethyl alcohol (95 %) 20 ml Distilled water 100 ml This formula is preferred in some quarters to the Ziehl carbol fuchsin. It is attrib- uted to Kinyoun, but the reference to its original publication has not been located. Carbol Crystal Violet (Nicolle) Original statemeiit of formula Emended statement Solution A Sat ale gentian violet 10 ml Crystal violet (90 % dye content) 0.4 g 1 % aqu sol phenol 100 ml Ethyl alcohol (95 %) 10 ml Solution B Phenol 1 g Distilled water 100 ml Mix solutions A and B This formula is sometimes preferred either as a general stain or in the gram technic. If properly prepared it is permanent, but it has a tendency to gelatinize if the amount of dye is too great. To prevent this sort of deterioration the quantity of dye in the above amended formula has been reduced to 0.4 g from the 1.0 g recommended pre- viously. Even when the solution is so prepared as to be permanent, however, it seems to have no advantage over the ammonium oxalate crystal violet given above. Anilin ''Gentian Violet'' (Ehrlich) Original statement of formula Emended statement Solution A Sat ale sol gentian violet 5-20 ml Crystal violet (90 % dye content) 1.2 g Anilin water (2 ml anilin shaken Ethyl alcohol (95 %) 12 ml with 98 ml water and filtered) 100 ml Solution B Anilin 2 ml Distilled water 98 ml Shake and allow to stand for a few min- utes, then filter. Mix solutions A and B This formula is given largely for its historic interest. It is a quite unstable solution and has no special value today. It was, however, one of the first important bacterial staining fluids and was formerly regarded as the standard formula for the gram stain. It is not, however, certain what was the "anilin gentian violet" originally employed in the gram stain, even though ascribed to Ehrlich. As a matter of fact Ehrlich seems to be properly credited only with the idea of using anilin water in the formula, as he apparently did not recommend any one definite formula. STAINING METHODS 15 NEGATIVE STAINING OF BACTERIA—RECOMMENDED PROCEDURES Dorner's Nigrosin Solution Nigrosin, water soluble (nigrosin B Griibler recommended by Dorner; Amer- ican nigrosins certified by Commission on Standardization of Biological Stains ordinarily satisfactory) 10 g Distilled water 100 ml Immerse in boiling water bath for 30 min, then add as preservative Formalin 0.5 ml Filter twice through double filter paper and store in serological test tubes, about 5 ml to the tube. This staining solution is used for the negative demonstration of bac- teria, in place of the Burri india ink. For its use in Dorner's spore stain, see page 20. Staining schedule : 1. Mix a loopful of the bacterial suspension on the slide with an equal amount of the staining solution. (If prepared from growth on solid media, the suspension must not be too heavy.) 2. Allow the mixture to dry in the air, and examine under microscope. Results : Unstained cells in a background which is an even dark gray if the preparation is well made. Benians* Congo Red Congo red (80 % dye content) 2 g Distilled water 100 ml Staining schedule: 1. Place a drop of the above staining fluid on a slide. . 2. Mix culture with the drop and spread out into a rather thick film. 3. After film has dried, wash with 1 per cent HCl. 4. Dry, either in the air or by blotting. Results : Cells unstained in a blue background. Good results are not to be expected from broth cultures or from cultures in salt solutions unless the cells are first removed by centrifuging. THE GRAM STAIN— RECOMMENDED PROCEDURES There are numerous modifications of the gram stain, many of which have been listed by Hucker and Conn (1923, 1927). The two modifica- tions given below have proved especially useful to the committee. The Hucker modification is valuable for staining smears of pure cultures; that of Kopeloff and Beerman (1922) for preparations of body discharges such as gonorrhoeal pus, also for pure cultures of stronglj^ acid-forming organ- isms. The latter is itself a variation of the modification by Burke (1921). 16 MANUAL OF MICROBIOLOGICAL METHODS Hucker Modification Ammonium Oxalate Crystal Violet (See page 13) Gramas Modification of LugoVs Solution Iodine 1 g KI 2g Distilled water 300 ml Counterstain Safranin O (2.5 % solution in 95 % ethyl alcohol) 10 ml Distilled water 100 ml Staining schedule: 1. Stain smears 1 min with ammonium oxalate crystal violet. This formula has sometimes been found to give too intense staining, so that certain gram-negative organisms (e.g., the gonococcus) do not properly decolorize. If this trouble is encountered, it may be avoided by using less crystal violet. 2. Wash in tap water for not more than 2 sec. 3. Immerse 1 min in iodine solution. 4. Wash in tap water, and blot dry. 5. Decolorize 30 sec with gentle agitation, in 95 per cent ethyl alcohol. Blot dry. 6. Counterstain 10 sec in the above safranin solution. 7. Wash in tap water. 8. Dry, and examine. Results: Gram-positive organisms, blue; gram-negative organisms, red. Burke and Kopeloff-Beerman Modifications Alkaline Gentian Violet Solution A Solution B Gentian or crystal violet^ 1 g NaHCOs 1 g Distilled water 100 ml Distilled water 20 ml Burke's Iodine Solution Iodine, 1 g; KI, 2 g; distilled water, 100 ml Kopeloff and Beerman^s Iodine Solution Iodine 2 g Normal NaOH (40.01 g per liter) 10 ml After the iodine is dissolved, make up to 100 ml with distilled water. Burke^s Counterstain Safranin O (85% dye content), 2 g; distilled water, 100 ml ^ The authors specify either crystal violet or methyl violet 6B. Probably any of the gentian violets now sold under the commission certification are satisfactory, i.e., either crystal violet or one of the bluer grades of methyl violet (e.g., methyl violet 2B). STAINING METHODS 17 Kopeloff and Beerman^s Counter stain Basic fuchsin (90% dye content), 0.1 g; distilled water, 100 ml Staining schedule: 1. Dry thinly spread films in the air without heat. 2. Flood with solution A; mix on the slide with 2-3 drops (or more, depending on size of flooded area) of solution B, and allow to stand 2-3 min. Kopeloff and Beerman mix the two solutions in advance, 1.5 ml of solution A to 0.4 ml of solution B, and allow to stay on slide 5 min or more. 3. Rinse with either of the above iodine solutions. (The committee indicates no preference between the two; some workers prefer one, some the other.) 4. Cover with fresh iodine solution, and let stand 2 min or longer. 5. Rinse with tap water; then blot water from surface of smear, without drying. (Kopeloff and Beerman omit the washing.) The amount of drying is important in this step. One must get rid of all free water but not allow the cells to dry. 6. Follow the blotting very quickly with decolorization in ether and acetone (1 vol of ether to 1-3 vol of acetone), adding to the slide drop by drop until practically no color comes off in the drippings (usually less than 10 sec). In this step the speed of decolorization can be varied by varying the ratio of ether to acetone; the more acetone, the more rapid the process. It is sometimes desirable to slow down the process by using a ratio of 1:1. 7. Dry in the air. 8. Counterstain 5-10 sec in one of the above given counterstains. Burke's (i.e., safranin) is preferred. The Kopeloff and Beerman counterstain is too powerful to be used when the shorter staining time recommended by Burke is followed. 9. Wash in tap water. 10. Dry, and examine. Results: Gram-positive organisms, blue; gram-negative organisms, red This technic is claimed to have the advantage of not giving false positives due to vacuolar bodies that resist decolorization by other gram-staining procedures. Interpretation of the Gram Stain A word of caution is necessary as to the interpretation of the gram stain. The test is often regarded with unjustified finahty because organ- isms are generally described as being either gram-positive or gram-nega- tive. Many organisms, however, actually are gram-variable. Hence, 18 MANUAL OF MICROBIOLOGICAL METHODS one should never give the gram reaction of an unknown organism on the basis of a single test. He should repeat the procedure on cultures having different ages and should use more than one staining technique in order to determine the constancy of the organism toward the stain. Two phe- nomena deserve consideration. (1) Henry and Stacey (1943) and Bartholomew and Umbreit (1944) have shown that gram-positive organisms can be made gram-negative by treatment with ribonuclease and that their gram-positive reaction can be restored subsequently by treatment with magnesium ribonucleate. (2) Some organisms have granules which resist decolorization and which may cause misinterpreta- tion. Such observations show that the gram stain does not always give a clear-cut reaction and that the results must be interpreted with care. ACID-FAST STAINING— RECOMMENDED PROCEDURES Ziehl-N eelsen Method Ziehl (1882); Neelsen (1883) Staining schedule: 1. Stain dried smears 3-5 min with Ziehl's carbol fuchsin (page 12), applying enough heat for gentle steaming. 2. Rinse in tap water. 3. Decolorize in 95 per cent ethyl alcohol, containing 3 per cent by volume of cone HCl, until only a suggestion of pink remains. 4. Wash in tap water. 5. Counterstain with one of the methylene blue solutions given on page 13. 6. Wash in tap water. 7. Dry, and examine. Results: Acid-fast organisms, red; others, blue. Gross' ''Cold'' Method Of recent years an effort has been made (see Darrow, 1948; Gross, 1952) to eliminate the necessity of applying heat during the fuchsin stain- ing so as to simplify the technic and to avoid ''messy" preparations. Such procedures seem to have justified themselves and can be recom- mended for pure culture work ; whether or not they are reliable for diag- nostic purposes would require detailed comparison in actual use, and to the committee's knowledge no such comparison has been made. Gross' method is as follows : Preparation of basic fuchsin solution : Add 25 ml of a stock 4 per cent alcoholic basic fuchsin solution to 75 ml of 6 per cent aqueous phenol. STAINING METHODS 19 To this add 3-4 drops of Tergitol No. 7 (a Carbide & Chemical Corp. product), and stir thoroughly. Preparation of methylene blue solution: Add 30 ml of a stock 1.5 per cent alcoholic methylene blue solution to 100 ml of 0.01 per cent aqueous KOH. Staining schedule: 1. Stain 5-10 min, without heating, in the above basic fuchsin solution. 2. Rinse in warm water. 3. Agitate for 30-60 sec in acid alcohol (3 ml of cone HCl in 97 ml ethyl alcohol). 4. Rinse with cold water. 5. Counterstain 3-5 min in the above methylene blue solution. Note : The only essential difference between this method and Darrow's is that the latter author states that equally good results were obtained with a weaker (0.3 per cent) fuchsin solution in phenol. In the hands of one the committee's collaborators, however, Gross' 1 per cent solution has proved more satisfactory. ACID-FAST STAINING—ALTERNATE PROCEDURES Fluorescence Method Richards and Miller {1941) Although this method is not of special importance in pure culture work, special mention should be made of it because of the amount of attention now given to it in diagnostic work. Its real advantage is that it can be used with relatively low magnifi- cation, and the large fields that can be examined assure positive diagnoses in cases where the numbers of tubercle organisms are few. Solution A Solution B Auramine O (90 % dye content) . 0.1 g Ethyl alcohol (70 %) 100 ml Liquefied phenol 3 ml Cone HCl 0.5 ml Distilled water 97 ml NaCl 0.5 g Staining schedule: 1. Stain dried smears 2-3 min in solution A. 2. Wash in tap water. 3. Destain 3-5 min in solution B, freshly prepared. 4. Dry, and examine under a monocular microscope, using 8 mm dry objective and a 20 X ocular; illumination should \)Q a low-voltage, high-amperage microscope lamp, supplied with a blue (ultraviolet-transmitting) filter, a complementary yel- low filter having been provided for the ocular. Results: Acid-fast bacteria, bright yellow, fluorescent; other organisms, not visible; background, nearly black. Much^s Method Much {1907) Much's method 2, which is now quite widely used, employs carbol gentian violet of essentially the formula given on page 13 for carbol fuchsin except that in the place of basic fuchsin the author calls for methyl violet BN, Preparations are stained cold 20 MANUAL OF MICROBIOLOGICAL METHODS for 24 hr or b}'- gentle application of heat until steaming. They are then washed in water and treated with Lugol's iodine (see page 16) from 1 to 5 min. After a second washing they are treated with 5 per cent nitric acid for 1 min followed by 3 per cent hydrochloric acid for 10 sec. They are then decolorized 1 min in equal parts of acetone and 95 per cent ethyl alcohol. Weiss (1909) has modified this procedure by staining with a mixture of 3 parts of carbol fuchsin to 1 part of carbol gentian violet and coun- terstaining with 1 per cent aqueous safranin (5 to 10 sec) or with Bismarck brown (1 min). The counterstain is applied immediately after the decolorization, the ace- tone-alcohol being removed merely by blotting. In some laboratories this method of counterstaining is employed following the Much technic with carbol gentian violet alone for the primary stain. Cooper^s Method Cooper (1926) The Cooper method calls for staining in Ziehl's carbol fuchsin to which 3 per cent of a 10 per cent aqueous sodium chloride solution is added just before use. Smears are stained either by steaming 3-4 min, then allowing them to cool until a precipitate forms, or else by standing overnight in a 37° incubator and cooling in an icebox for 20 min to allow precipitation to occur. After the precipitation, the smears are washed with tap water and decolorized 1-10 min in acid alcohol (5 ml of nitric acid, sp gr 1.42, to 95 ml of 95 per cent ethyl alcohol) ; washed again with water and finally for 1 min with 95 per cent ethyl alcohol. They are counterstained with 1 per cent brilliant green or, if the smear is heavy, with a greater dilution of this same stain; washed with water; dried; and examined. SPORE STAINING—RECOMMENDED PROCEDURES Dorner^s Method Dorner {1922, 1926) \ Staining schedule: 1. Make a heavy suspension of the organism in 2-3 drops of distilled Avater in a small test tube. 2. Add equal quantity of freshly filtered Ziehl's carbol fuchsin (page 13). 3. Allow the mixture to stand in a boiling water bath 10 min or more. 4. On a cover slip or slide mix one loopful of the stained preparation with one loopful of Dorner's nigrosin solution (page 15). 5. Smear as thinly as possible and do not dry too slowl3^ Note: If even backgrounds for exhibiting or photographing are required, especially in the case of slime-producing bacteria, the following procedure is recommended : 1. Make the suspension in 0.5 ml of nutrient broth or water. 2. Add 1 ml of 10 per cent gelatin solution. 3. Add 1 ml of carbol fuchsin, and stain as in steps 1 and 2 above. 4. Wash out the colloids with warm tap water, with the help of centrifuge or sedimentation. 5. Mix with nigrosin, and proceed as above. Results: Spores, red; vegetative cells, unstained; background, gray. STAINING METHODS 21 Dorner's Method — Snyder's Modification Snyder (1934) Staining schedule: 1. Prepare a dried smear on a slide, and cover with a small piece of blotting paper. 2. Saturate blotting paper with freshly filtered ZiehPs carbol fuchsin. 3. Allow to steam 5-10 min, keeping paper moist by adding more staining fluid. 4. For neat preparations, decolorize instantaneously with 95 per cent ethyl alcohol (but omit this step if the organisms do not hold color well) . 5. Wash with tap water. 6. Apply a drop of saturated aqueous nigrosin (or Dorner's fluid), and spread evenly. 7. Allow slide to dry quickly with gentle heat, without prior washing. Results: Same as with original method, but this modification proves applicable to some bacteria (e.g., Bacillus subtilis) that are difficult to stain by Dorner's technic. Conklin's Modification of Wirtz Method Wirtz {1908); Conklin {1934) Staining schedule: 1. Make smears as usual and fix by heat. 2. Flood slide with 5 per cent aqueous malachite green, and steam for 10 min, keeping slide flooded by addition of fresh staining fluid. 3. Wash 30 sec in running water. 4. Counterstain 1 min with 5 per cent aqueous mercurochrome. 5. Wash in running water. 6. Blot dry, and examine. Results: Spores, green; rest of cell, red. Trouble is sometimes experi- enced with the green fading after the slides have stood a few days. Apparently this is the result of an alkaline reaction and can be prevented by treating the slides in acid before making the smears. (The alkalinity may be due to an invisible film of soap or washing powder.) SPORE STAINING— ALTERNATE PROCEDURE Bartholomew and Mittwer's "Cold" Method Just as recent work is showing that heat is not necessary in making an acid-fast stain, it is proving that it may also be eliminated from spore staining, in which a very similar principle is involved. The following modification of the Wirtz method by Bartholomew and Mittwer (1950) is a good illustration: 22 MANUAL OF MICROBIOLOGICAL METHODS Staining schedule: 1. Fix the smear by passing through a flame 20 times. 2. Stain 10 min with saturated aqueous malachite green (i.e., about 7.6 per cent), with- out heat. 3. Rinse with tap water for about 10 sec. 4. Stain 15 sec in 0.25 per cent aqueous safranin. 5. Rinse, blot, and dry. Results are the same as with the Conklin modification. STAINING THE DIPHTHERIA ORGANISM— RECOMMENDED PROCEDURES Various special procedures have been devised for staining the diph- theria organism in such a manner as to render it distinctive in appearance by differentiation of its characteristic metachromatic granules. Staining with Methylene Blue Staining schedule: 1. Prepare smear as usual, and fix with gentle heat. 2. Stain for a feAV seconds with either of the methylene blue solutions (i.e., Loeffler's or dilute alcoholic) given on page 13. 3. Wash in tap water. 4. Dry, and examine. Results: Metachromatic granules, dark blue to violet; bacteria without such granules, evenly stained. The picture varies a little according to which of the two methylene blue solutions is employed. The Loeffler formula gives purplish shades of staining because of the oxidation of methylene blue caused by the alkali. Some users consider the poly- chrome effect thus obtained to give better differentiation; others think the metachromatic granules show more sharply with the clear blue of the unpolychromed dye. Albert's Diphtheria Stain Albert {1920) Toluidine blue 0.15 g ]\Iethyl green 0.20 g Acetic acid (glacial) 1 ml Ethyl alcohol (95 %) 2 ml Distilled water 100 ml Laybourn's Modification Lay bourn (1924) has modified the Albert stain by replacing the methyl green with an equal amount of malachite green. Staining schedule: 1. Make smears as usual, and fix with gentle heat. 2. Stain 5 min in either Albert's staining fluid or Laybourn's modification STAINING METHODS 23 of it. The latter is claimed to give deeper staining of both granules and body of the cells without lessening the contrast between them. 3. Drain without washing. 4. Treat 1 min in a modified Lugol's solution (iodine, 2 g; KI, 3 g; dis- tilled water, 300 ml). 5. Wash briefly in tap water. 6. Blot with filter paper, and examine. Results : INIetachromatic granules, black ; bars of diphtheria cells, dark green to black; body of cells, light green. Ljuhinsky Stain (from Blumenthal and Lipskerow, 1905) Original formula Emended form ida Solution A Solution A Pyoktanin (Merck) 0.25 g Methyl violet 2B or crystal vio- 5 % acetic acid 100 ml let (85 % dye content) 0.25 g Glacial acetic acid 5 ml Distilled water 95 ml Solution B Solutio7i B Vesuvin 0.1 g Bismarck brown Y 0.1 g Distilled water 100 ml Distilled water 100 ml Staining schedule: 1. Make smears as usual and fix with gentle heat. 2. Stain 30 sec to 2 min in solution A. 3. Wash in tap water. 4. Stain 30 sec mth solution B. 5. Wash in tap water. 6. Dry, and examine. Results: Metachromatic granules, dark blue or black; rest of cell> reddish or yellowish. STAINING THE DIPHTHERIA ORGANISM— ALTERNATE PROCEDURES Neisser^s Diphtheria Stain Neisser (1903) Solution 1 Solution 2 Methylene blue (dye content not Crystal violet (dye content not specified; probably 90 %) 1 g specified; probably 85 %) . . . . 1 g Alcohol (e.g., 95 %) 20 ml Alcohol (e.g., 95 %) 10 ml Acetic acid (glacial) 50 ml Distilled water 300 ml Distilled water 1000 ml Solution 3 Mix, and agitate until dye is dissolved Chrysoidin 1 or 2 g Hot water 300 ml Filter after dissolving 24 MANUAL OF MICROBIOLOGICAL METHODS Dried films are stained 10 sec in a mixture of 2 parts of solution 1 and 1 part of solution 2. Wash. Stain 10 sec in solution 3. Wash briefly in water, or not at all. Blot dry. Pondefs Diphtheria Stain Ponder {1912); Kinyoun (1915) Toluidine blue Azure I Methylene blue Glacial acetic acid Ethyl alcohol (see below) Distilled water Original As modified formula by Kinyoun 0.02 g 0.1 g 0.01 g 0.01 g 1 ml 1 ml 2 ml 5 ml 100 ml 120 ml Dissolve the dyes in the alcohol; add the water, then the acid; and let stand 24 hr before using. Do not filter. After prolonged standing, action may be intensified by adding 1 or 2 drops of glacial acetic acid. According to Kinyoun, smears are fixed with heat, allowed to cool, and stained 2-7 min. In the source of the original formula above cited, absolute alcohol is specified; Kin- youn calls for 95 per cent alcohol. On theoretical grounds, indeed, absolute alcohol is not indicated, and the 95 per cent strength may well be substituted even in the original formula. Although the committee has had no personal experience with either formula, information is at hand indicating the superiority of the Kinyoun modification. FLAGELLA STAINING— RECOMMENDED PROCEDURES Flagella staining is a difficult technic, and there have been numerous methods proposed for the purpose. It has long been realized that flagella are actually below the visual limit in size, but of recent years the electron microscope has given a definite idea how small they really are — around 0.02-0.03 fjL in diameter. Electron micrographs, in fact, often show many more flagella than do stained preparations. Until the electron micro- scope, however, has become a routine laboratory instrument, one must have resort to the principle introduced by Loeffler of mordanting the preparations before staining to increase the apparent size of the flagella. A second difficulty in staining flagella is the ease with which bacteria shed these delicate appendages unless the cultures are properly handled. To prevent this one ordinarily employs specially cleaned slides and specially prepared smears on the slides. Methods for preparing slides. Ordinary cleaning of glassware is not sufficient for the purpose. Various methods have been proposed, but the following directions seem to give as good results as any: STAINING METHODS 25 Use new slides if possible, preferably of Pyrex glass or similar heat- resistant properties. (This is because under the drastic method of clean- ing to remove grease, old slides have a greater tendency to break.) Clean first in a dichromate cleaning fluid, wash in water, and rinse in 95 per cent alcohol; then wipe with a clean piece of cheesecloth. (Wiping is not always necessary but is advisable unless fresh alcohol is used after every few slides.) Pass each shde back and forth through a flame for some time, ordinarily until the appearance of an orange color in the flame ; some experience is necessary before the proper amount of heating can be accurately judged. Unless heat-resistant slides are used, cool sHdes gradually in order to minimize breakage. An ordinarily satisfactory method of doing this is to place the flamed slides on a metal plate (flamed side up) standing on a vessel of boiling water and then to remove the flame under the water so as to allow gradual cooling. (Too rapid cooling may result in breakage, sometimes as long as 2 weeks after the heating.) Methods of handling cultures. Of various methods proposed, it is not possible to recommend any one as uniformly the best. As any laboratory worker becomes familiar with one particular method, he soon finds he can get better results with that than with any other. The following method, however, can be given as one of the most satisfactory, especially for students who have not had previous experience with some other method : Use young and actively growing cultures (e.g., 18-22 hr old) on agar slants. Before proceeding, check the culture for motility in hanging drop. If motile, wash off the growth by gentle agitation with 2-3 ml of sterile distilled water. Transfer to a sterile test tube, and incubate at optimum temperature for 10 min (30 min for those producing slime). At this point, again check motility under a microscope. Transfer a small drop from the top of the suspension (where motile organisms are most numerous) by means of a capillary pipet to one end of the sHde prepared as above described. Tilt the slide, and allow the drop to run slowly to the other end. (Two or three such streaks can be placed on a shde.) Place the shde in a tilted position, and allow it to dry in the air. Staining Procedure Good results can be obtained with any of the following methods, especially after famiharity has been obtained with it. Special recom- mendation must be given to the last of the four procedures (modified Bailey method). Although seeming a little more complicated on first reading, it has been found to give the most uniformly satisfactory results in inexperienced hands. 26 MANUAL OF MICROBIOLOGICAL METHODS Casares-Gil Flagella Stain^ As Published by Plimmer and Paine {1921) Mordant : Tannic acid 10 g AlCl3-6HoO 18 g ZnCh 10 g Basic fuchsin^ 1-5 g Alcohol (60 %) 40 ml The solids are dissolved in the alcohol by trituration in a mortar, adding 10 ml of the alcohol first, and the rest slowly. This alcoholic solution may be kept several years. For use, mix with an equal quantity of water (Thatcher, 1926) or dilute with 4 parts of water (Casares-Gil), filter off precipitate, and collect filtrate on the slide. Staining schedule: 1. Prepare smears of young cultures, on scrupulously cleaned slides as above directed. 2. Filter mordant onto slide as above directed (preferably using Thatch- er's 1 : 1 dilution) ; allow to act for 60 sec without heating. 3. Wash in tap water. 4. Flood slide with freshly filtered Ziehl's carbol fuchsin (page 13), and allow to stand 5 min without heating. 5. Wash with tap water. 6. Air-dry, and examine. Sometimes considerable search may be needed before finding a satisfactorily stained part of the smear. Results: Fagella well stained (red) in the case of those bacteria (e.g., colon-typhoid group, aerobic sporeformers) that do not have extremely delicate flagella. Gray\s Flagella Stain Gray {1926) Mordant: Solution A KA1(S04)2-12H20 (sat aqu solution) 5 ml Tannic acid (20 % aqu solution) 2 ml (A few drops of chloroform must be added to this if a large quantity is made up) HgCh (sat aqu solution) 2 ml Solution B Basic fuchsin (sat ale solution) 0.4 ml Mix solutions A and B less than 24 hr before using. Both solutions separately may be kept indefinitely, but deteriorate rapidly after mixing. Staining schedule: 1. Prepare smears from young cultures as above directed. 2. Flood slide with freshly filtered mordant, and allow to act 8-10 min. 1 See Galli-yalerio (1915). - The authors specify rosanilin hydrochloride. There are, however, other basic fuchsins more universally available which ought to prove equally satisfactory. STAINING METHODS 27 3. Wash with a gentle stream of distilled water, and follow steps 4-6 of above schedule (Casares-Gil method). Results: Same as with Casares-Gil method. Leif son's Stain^ Leifson (1930) KA1(S04)2-12H20, or NH4A1(S04)2-12H20 (sat aqu solution) 20 ml Tannic acid (20 % aqu solution) 10 ml Distilled water 10 ml Ethyl alcohol, 95 % 15 ml Basic fuchsin (sat solution in 95 % ethyl alcohol) 3 ml Mix ingredients in order named. Keep in tightly stoppered bottle, and the stain may be good for a week. Staining schedule: 1. Prepare slides as for the preceding methods. 2. Flood sHdes with the above solution, and allow to stand 10 min at room temperature in warm weather or in an incubator in cold weather. 3. Wash with tap water. (If a counterstain is desired, borax methylene blue may be applied, without heat, followed by another washing. See page 29.) 4. Dry and examine. Results: When no counterstain is used, same as with the two above procedures; with methylene blue counterstain, flagella red, cells blue. Bailey Method Bailey {1929) Modified by Fisher and Conn (1942) This method is specially recommended for bacteria on which flagella are difficult to stain (as is frequently the case with soil and Vv^ater non- sporeformers and with plant pathogens) because of slime production, unusually fine flagella or flagella that are readily lost. Mordant: Solution A Tannic acid (10 % aqu solution) 18 ml FeCls-eHsO (6 % aqu solution) 6 ml Solution B Solution A 3.5 ml Basic fuchsin; (0.5 % in ethyl alcohol) 0.5 ml HCl, concentrated 0.5 ml Formalin 2.0 ml Staining schedule: 1. Prepare smears of young cultures, following carefully the procedure recommended on page 25 under ''Methods of handling cultures." 2. Filter the above solution A onto the slide and allow it to remain 33^ min without heating. ^This stain, already mixed, is now available commercial!}' in powder form. 28 MANUAL OF MICROBIOLOGICAL METHODS 3. Pour off solution A, and without washing add solution B, also through a filter, and allow it to stand 7 min without heating. 4. Wash with distilled water. 5. Before the slide dries, cover with ZiehFs carbol fuchsin (page 13), allowing it to stand 1 min on a hot plate heated just enough for steam to be barely given off. 6. Wash in tap water. 7. Dry in the air, and examine. Results: Similar to the preceding methods, but the background pre- cipitate is usually finer and less conspicuous, thus interfering less with the demonstration of unusually fine, delicate flagella. Staining flag ella of anaerobes. O'Toole (1942) calls attention to cer- tain difficulties in staining the flagella of anaerobes and gives a modifica- tion of the above Bailey stain which is intended to overcome them. The method is not unlike that of Fisher and Conn, who had the O'Toole pro- cedure in mind when working out their modification. CAPSULE STAINS— RECOMMENDED PROCEDURES Bacterial capsules are more easily confused with artifacts than any other structure pertaining to the organisms. Inasmuch as capsules sometimes show merely as unstained areas around the cells, there is a temptation to call any such surrounding area a capsule; very often, how- ever, they merely represent the tendency of a lightly stained surrounding medium to retract from the cells on drying. For this reason the best way to demonstrate capsules is actually to stain them by some procedure which differentiates them from the cell itself. Several of the flagella stains accomplish this, notably those of Bailey and Leifson, given above. Much simpler is the procedure of Anthony described below. The Anthony method can be recommended because of both its simplicity and its dependability. Any of the other methods which follow give satis- factory results. The student is specially urged, however, not to pro- nounce any organism capsulated, as a result of any of these staining procedures, until he has carefully compared it with other organisms generally recognized as having capsules. Leifson Method Leifson {1930) This method is described in detail above (page 27) and does not need to be repeated here. The special methods of handling slides and cultures, outlined for flagella staining, do not need to be observed, but the following is essential: STAINING METHODS 29 After step 3 : 4. Stain 5-10 min, without heating, in borax methylene blue (methylene blue, 90 per cent dye content, 0.1 g; borax 1 g; distilled water 100 ml). 5. Wash in tap water. 6. Dry, and examine. Results: capsules, red; cells, blue. Anthony^s Method with Tyler^s Modification Anthony {1931) Original formula Tyler's modification^ Crystal violet (85 % dye content) 1 g Crystal violet (85 % dye con- Distilled water 100 ml tent) 0.1 g Glacial acetic acid 0.25 ml Distilled water 100 ml Staining schedule: 1. Prepare smears, and dry them in the air. 2. Stain 2 min in the above aqueous crystal violet or, according to Tyler, 4-7 min in the above acetic crystal violet. 3. Wash with 20 per cent aqueous CuS04-5H20. 4. Blot dry, and examine. Results: capsules, blue violet; cells, dark blue. Hisses Method Hiss (1905) Original statement of formula Emended formula Sat ale basic fuchsin or gentian Basic fuchsin (90% dye con- violet 5-10 ml tent) : 0.15-0.3 g Water to make 100 ml Distilled water 100 ml or Crystal violet (85 % dye con- tent) 0.05-0.1 g Distilled water 100 ml Staining schedule: 1. Grow organisms in ascitic fluid or serum medium, or mix with drop of serum and prepare smears from this mixture. 2. Dry smears in the air, and fix with heat. 3. Stain with one of the above solutions a few seconds by gently heating until steam rises. 4. Wash off with 20 per cent aqueous CuS04-5H«0. 5. Blot dry, and examine. Results: Capsules, faint blue; cells, dark purple. 1 See Park and Williams (1933), p. 84. 30 MANUAL OF MICROBIOLOGICAL METHODS STAIN FOR FAT DROPLETS Burdon's Method Bur don (194.6) Staining solution: 0.3 g of Sudan black B (commission certified) in 100 ml of 70 per cent ethyl alcohol. After the bulk is dissolved, shake at intervals and allow to stand over night. Staining schedule: 1. Prepare smears as usual from 18- to 24-hr cultures, and fix by heat. 2. Flood the entire slide with the above staining solution and allow it to stand undisturbed at room temperature for 5-15 min. (Exact time is unimportant, as good results are often obtained after only 1 or 2 min; on the other hand no harm results if the slides stain until the solution is completely dry.) 3. Drain, and blot slide completely dry. 4. Cover with xylene by pouring from a dropping bottle or dipping several times in a staining jar. Blot till dry. 5. Counterstain 5-10 sec with 0.5 per cent aqueous safranin, taking care not to overstain. Note: For acid-fast organisms, ZiehFs carbon fuchsin diluted 1:10 with distilled water may be applied for 1-3 min, instead of safranin. 6. Wash in tap water, blot, and dry. Results: Fat droplets blue-black or blue-gray; rest of cell pink. ROBINOW'S STAIN FOR NUCLEAR APPARATUS Slightly modified from Eohinow {1944) Among several methods given by Robinow for staining nuclear material the following seems as generally applicable as any : Staining solution: Add 1 drop of Giemsa stain to 1 ml of Sorensen's buffer of pH 6.9-7.0. Robinow specifies Gurr's R66. Giemsa stain as certified by the Biological Stain Commission, however, is equally good and requires less prolonged staining ; the staining time given below is that called for by the American-type Giemsa. Fixation and smearing: 1. Incubate petri-dish cultures 2-5 hr. 2. Remove a block of agar, and fix it from a few seconds to several hours in the vapor of 2 per cent osmic acid. 3. Make an impression smear on a cover slip or glass slide. 4. Store in 70 per cent alcohol till needed. Staining procedure: 1. Remove preparation from alcohol, and wash in water. 2. Place for 5-10 min in normal HCl at 60°C. STAINING METHODS 31 3. Remove, and wash three times in tap water. 4. Stain 1-15 minutes, at 37°C, in the above diluted Giemsa stain. 5. Mount in water for oil immersion examination. Note: If it is desired to mount in balsam, the staining time must be increased to several hours. Results: The deeper colors (blue and violet) tend to be localized in the chromatinic material comprising the nuclear structures. STAINS FOR SPIROCHAETES—RECOMMENDED PROCEDURE Fontana Stain Preparation of ammoniacal silver nitrate: Dissolve 5 g of AgNOs in 100 ml of distilled water. Remove a few milliliters, and to the rest of the solution add drop by drop a concentrated ammonia solution until the sepia precipitate which forms redissolves. Then add drop by drop enough more of the silver nitrate solution to pro- duce a slight cloud which persists after shaking. It should remain in good condition for several months. Staining schedule: 1. Prepare smear, and fix with heat. 2. Pour on a solution of 5 per cent tannic acid in 1 per cent phenol, and allow to steam 30 sec. 3. Wash 30 sec in running water. 4. Cover with a drop of the above ammoniacal silver nitrate, heat gently over a flame, and allow it to stand 20-30 sec after steaming begins. 5. Wash in tap water. 6. Blot dry, and examine. Results: Spirochaetes, dark brown or black, in a dark maroon field. STAINS FOR SPIROCHAETES— ALTERNATE PROCEDURE Tunnicliff's Stain Tunnicliff has employed carbol gentian violet (3 to 4 sec) followed by Lugol's iodine (see page 16) for the same period in staining bacterial smears. With a slight modifi- cation this proves a good spirochaete stain. The modification is: Carbol crystal violet (1 vol of 10 per cent ale crystal violet to 10 vol of 1 per cent aq phenol) 30 sec; wash with water; the Lugol-Gram iodine solution 30 sec; wash with water; safranin 30 sec; wash with water and dry. STAIN FOR RICKETTSIAE Macchiavello's Method Staining solution: 0.25 g of basic fuchsin (90 per cent dye content) dis- solved in 100 ml of distilled water, buffered to pH 7.2-7.4 with the proper phosphate buffer mixture. 32 MANUAL OF MICROBIOLOGICAL METHODS Table 1. Dye Solubilities at 26°C Per cent soluble in Color index Name of dye number Water 95 % alcohol 1027 Alizarin nil 0.125 1034 Alizarin red S 7.69 0.15 40 Alizarole orange G 0.40 0.57 36 Alizarole yellow GW 25.84 0.04 184 Amaranth 7.20 0.01 847 Amethyst violet 3.12 3.66 655 Auramin 0.74 4.49 12 Aurantia nil 0.33 146 Azo acid yellow 2.17 0.81 88 Azo Bordeaux 3.83 0.19 448 Benzopurpurin 4B 0.13 280 Biebrich scarlet 0.05 332 Bismarck brown R 1.10 0.98 331 Bismarck brown Y 1.36 1.08 252 Brilliant croceine 5.04 0.06 29 Chromotrope 2R 19.30 0.17 21 Chrysoidin R 0.23 0.99 20 Chrysoidin Y 0.86 2.21 370 Congo red 0.19 89 Crystal ponceau 0.80 0.06 681 Crystal violet (chloride) 1 gentian Crystal violet (iodide) /violets 1.68 13.87 0.035 1.78 Cresyl violet (National Aniline) 0.38 0.25 715 Cyanole extra 1.38 0.44 771 Eosin B (Na salt) 39.11 0.75 768 Eosin Yt (Na salt) 44.20 2.18 Eosin Yt (Mg salt) 1.43 0.28 Eosin Yt (Ca salt) 0.24 0.09 Eosin Yt (Ba salt) 0.18 0.06 130 Erika B 0.64 0.17 254 Erythrin X 6.41 0.06 773 Erythrosint (Na salt) 11.10 1.87 Erythrosint (Mg salt) 0.38 0.52 Erythrosint (Ca salt) 0.15 0.35 Erythrosint (Ba salt) 0.17 0.04 770 Ethyl eosin 0.03 1.13 Fast green FCF 16.04 0.35 176 Fast red A 1.67 0.42 16 Fast yellow 18.40 0.24 766 Fluorescein (color acid) 0.03 2.21 Fluorescein (Na salt) 50.20 7.19 Fluorescein (Mg salt) 4.51 0.35 Fluorescein (Ca salt) 1.13 0.41 Fluorescein (Ba salt) 6.54 0.56 STAINING METHODS Table 1. Dye Solubilities at 26*^0 {Continued) 33 Color index Name of dye Per cent soluble in number Water 95 % alcohol Fuchsin, basic: 676 Pararosanilin (chloride) 0.26 5.93 Pararosanilin (acetate) 4.15 13.63 Rosanilin (chloride) 0.39 8.16 678 New fuchsin (chloride) Gentian violet (see crystal or methyl violet) 1.13 3.20 666 Guinea green B 28.40* 7.30 1180 Indigo carmine 1.68 0.01 133 Janus green 5.18 1.12 670 Light green SF yellowish 20.35 0.82 657 Malachite green (oxalate) 7.60 7.52 9 Martins yellow, Na salt 4.57 0.16 Martins yellow, Ca salt 0.05 1.90 138 Metanil yellow 5.36 1.45 142 Methyl orange 0.52 0.08 Methyl orange (acid) 0.015 0.015 680 Methyl violet (gentian violet) 2.93 15.21* 922 Methylene blue (ZnCh double salt) 2.75 0.05 Methylene blue (chloride) 3.55 1.48 Methylene blue (iodide) 0.09 0.13 924 Methylene green 1.46 0.12 10 Naphthol yellow G 8.96 0.025 152 Narcein 10.02 0.06 825 Neutral red (chloride) 5.64 2.45 Neutral red (iodide) 0.15 0.16 826 Neutral violet 3.27 2.22 927 New methylene blue N 13.32* 1.65 728 New Victoria blue R 0.54 3.98 520 Niagara blue 4B 13.51 nil 914 Nile blue 2B 0.16 0.62 73 Oil red nil 0.39 150 Orange I 5.17 0.64 151 Orange II 11.37 0.15 27 Orange G 10.86 0.22 714 Patent blue A 8.40 5.23 774 Phloxinef (Na salt) 50.90* 9.02 Phloxinef (Mg salt) 20.84 29.10 Phloxinef (Ca salt) 3.57 0.45 Phloxine (Ba salt) 6.01 1.17 7 Picric acid 1.18 8.96 28 Ponceau 2G 1.75 0.21 186 Ponceau 6R 12.98 0.01 741 Pyronin B (iodide) 0.07 1.08 739 Pyronin Y 8.96 0.60 34 MANUAL OF MICROBIOLOGICAL METHODS Table 1. Dye Solubilities at 26°C {Continued) Color index Name of dye Per cent soluble in number Water 95 % alcohol 148 Resorcin yellow 0.37 0.19 749 Rhodamine B 0.78 1.47 750 Rhodamine G 1.34 6.31 779 Rose bengal t (Na salt) 36.25 7.53 Rose bengal! (Mg salt) 0.48 1.59 Rose bengal t (Ca salt) 0.20 0.07 Rose bengal t (Ba salt) 0.17 0.05 831 Safranin 5.45 3.41 689 Spirit blue nil 1.10 24 Sudan I nil 0.37 248 Sudan III nil 0.15 258 Sudan IV nil 0.09 920 Thionin 0.25 0.25 925 Toluidine blue 3.82 0.57 690 Victoria blue 4R 3.23 20.49 659 Victoria green 3B 0.04 2.24 8 Victoria yellow 1.66 1.18 * These figures are grams per hundred grams of saturated solution (the others being grams per hundred milliliters). t The color acids of these dyes (not listed here) are practically insoluble in water. Note: These figures are ordinarily for recrystallized dyes. Commercial samples are generally less soluble often by as much as 30 per cent. Source: Based on data obtained at the Color Laboratory of the U. S. Department of Agriculture. See Conn (1953), pp. 289-290. Staining schedule: 1. Smear a bit of tissue on a slide. 2. Dry in the air, and fix with gentle heat. 3. Pour the above staining fluid onto the slide through a coarse filter paper. Allow to stand 4 min. 4. Rinse very rapidly with 0.5 per cent aqueous citric acid. 5. Wash quickly and thoroughly with tap water. 6. Counterstain about 10 sec with 1 per cent aqueous methylene blue. 7. Rinse in tap water. 8. Dry, and examine. Results: Rickettsiae, red; cell nuclei, deep blue; cytoplasm, light blue. REFERENCES Albert, Henry. 1920. Diphtheria bacillus stains with a description of a "new" one. Am. J. Public Health, 10, 334-337. . 1921. Modification of stain for diphtheria bacilli. J. Am. Med. Assoc, 76, 240. STAINING METHODS 35 Anthony, E. E. 1931. A note on capsule staining. Science, 73, 319. Bailey, H. D. 1929. A flagella and capsule stain for bacteria. Proc. Soc. Exptl. Biol. Med., 27, 111-112. Bartholomew, J. W., and Tod Mitwer. 1950. A simplified bacterial spore stain. Stain TechnoL, 25, 153-156. Bartholomew, J. W., and W. W. Umbreit. 1944. Ribonucleic acid and the Gram stain. /. Bacteriol., 48, 567-578. Benians, T. H. C. 1916. Relief staining for bacteria and spirochaetes. Brit. Med. J. 1916 (2), 722. Blumenthal, J. M., and M. Lipskerow. 1905. Vergleichende Bewertung der differ- entiellen Methode zur Farbung des Diphtheriebacillus. Centr. BakterioL, I Abt., Orig., 38, 359-366. Burdon, Kenneth L. 1946. Fatty material in bacteria and fungi revealed by stain- ing dried, fixed slide preparations. /. Bacteriol., 52, 665-678. Burke, Victor. 1921, The Gram stain in the diagnosis of chronic gonorrhea. ./. Am. Med. Assoc, 77, 1020-1022. . 1922. Notes on the Gram stain with description of a new method, /. Bacterial., 7, 159-182. Conklin, Marie E. 1934. Mercurochrome as a bacteriological stain. /. Bacterial., 27, 30. Conn, H. J. 1953. "Biological Stains," 6th ed. Biotech Publications, Geneva, N. Y. , and Mary A. Darrow. 1943-1945. "Staining Procedures." Biotech Publi- cations, Geneva, N. Y. Cooper, F. B. 1926. A modification of the Ziehl-Neelsen staining method for tuber- cle bacilli. Arch. Pathol. Lab. Med., 2, 382-385. Darrow, Mary A. 1948. Staining of tubercle organism in sputum smears. Stain TechnoL, 24, 93-94. Dorner, W, C. 1922. Ein neues Verfahren fiir isolierte Sporenfarbung, Landwirtsh. Jahrh. Schweiz., 36, 595-597. . 1926, Un procede simple pour la coloration des spores. Lait, 6, 8-12. . 1930. The negative staining of bacteria. Stain TechnoL, 5, 25-27. Fisher, P. J., and Jean E. Conn. 1942. A flagella staining technic for soil bacteria. Stain TechnoL, 17, 117-121. Fontana, Artur. 1912. Verfahren zur intensiver und raschen Farbung des Tro^ ponema pallidum und anderer Spirochaten. Derm. Wochschr., 55, 1003-1004. Galli-Valerio, B. 1915. La methode de Casares-Gil pour la coloration des cils des bacteries. CenL BakterioL, I Abt. Orig., 76, 233-234. Gray, P. H. H. 1926, A method of staining bacterial flagella. /. Bacteriol., 12, 273-274. Gross, Milton. 1952. Rapid staining x>i acid fast bacteria. Ayn. J. Clin. Pathol., 22, 1034-1035. Henry, H., and M. Stacey. 1943. Histochemistry of the Gram-staining reaction for micro-organisms. Nature, 151, 671. Hiss, P, J., Jr. 1905. A contribution to the physiological differentiation of Pneumo- coccus and Streptococcus, and to methods of staining capsules. /. Exptl. Med., 6, 317-345, Hucker, G, J. 1922. Comparison of various methods of Gram staining. (Pre- liminary Report.) Abstr. Bacteriol., 6, 2. ■ , and H. J. Conn, 1923. Methods of Gram staining. N.Y. State Agr. Expt. Sta Tech. BulL 129. 36 MANUAL OF MICROBIOLOGICAL METHODS . 1927. Further studies on the methods of Gram staining. N.Y. State Agr. Expt. Sta. Tech. Bull. 128. Kinyoun, J. J. 1915. A modification of Ponder's stain for diphtheria. Am. J. Public Health, 5, 246-247. Kopeloff, N., and P. Beerman. 1922. Modified Gram stains. J. Infectious Dis- eases, 31, 480-482. Laybourn, R. L. 1924. A modification of Albert's stain for the diphtheria bacilli. J. Am. Med. Assoc, 83, 121. Leifson, Einar. 1930. A method of staining bacterial flagella and capsules together with a study of the origin of flagella. /. Bacteriol., 20, 203-211. Loeffler, F. 1884. Untersuchungen iiber Bedeutung der Mikroorganismen fiir die Entstehung der Diphtherie beim Menschen, bei der Taube und beim Kalbe. Mitt. Gesundheitsamte, 2, 421-499. (See p. 439.) Much, H. 1907. tJber die granulare, nach Teil nicht farbbare Form des Tuberku- losevirus. Beitr. Klin. Tuberk., 8, 85-99. Neelsen, F. 1883. Ein casuistischer Beitrag zur Lehre von der Tuberkulose. Centr. Med. Wisse., 21, 497-501. (See p. 500.) Neisser, M. 1903. Die Untersuchung auf Diphtheriebacillen in centralisierten Untersuchungsstationen. Hyg. Rundschau., 13, 705-717. Nicolle, Ch. 1895. Pratique des colorations microbiennes. Ann. inst. Pasteur., 9, 664-670. O'Toole, Elizabeth. 1942. Flagella staining of anaerobic bacilli. Stain Technol., 17, 33-40. Park, W. H., and Anna W. Williams. 1933. "Pathogenic Microorganisms." 10th ed. Lea & Febiger, Philadelphia. Plimmer, H. G., and S. G. Paine. 1921. A new method for the staining of bacterial flagella. J. Pathol. Bacterial., 24, 286-288. Ponder, C. 1912. The examination of diphtheria specimens. A new technique in staining with methylene blue. Lancet, 2, 22-23. Richards, O. W., and D. K. Miller. 1941. An efficient method for the identifica- tion of M. tuberculosis with a simple fluorescence microscope. Am. J. Clin. Pathol, 11, 1-7. Robinow, C. F. 1944. Cytological observations on Bad. coli, Proteus vulgaris and various aerobic spore-forming bacteria, with special reference to the nuclear structures. J. Hygiene, 43, 413-423. Snyder, Marion A. 1934. A modification of the Dorner spore stain. Stain Technol., 9,71. Thatcher, Lida M. 1926. A modification of the Casares-Gil flagella stain. Stain Technol., 1, 143. Tunnicliff, Ruth. 1922. A simple method of staining Gram-negative organisms. J. Am. Med. Assoc, 78, 191. Wirtz, R. 1908. Ein einfache Art der Sporenfarbung. Centr. BakterioL, I Abt. Orig., 46, 727-728. Ziehl, F. 1882. Zur Farbung des Tuberkelbacillus. Deut. med. Wochschr., 8, 451. CHAPTER III Preparation of Media G. W. Burnett, M. J. Pelgzar, Jr., and H. J. Conn INTRODUCTORY Scope The presentation of the data in this chapter is an attempt to describe growth media which have proved to be of general value to bacteriologists. The media to be discussed are usually employed for the isolation and maintenance of pure cultures and for the identification of species accord- ing to physiological properties. No group of bacteria will receive par- ticular attention except to the extent that data will naturally be more complete concerning media for groups rather thoroughly described. The information presented herein is intended primarily as an introduction to the description of growth media for those unfamiliar with the problems of bacterial cultivation. It is realized that the experienced specialist may employ media considerably different from those described below, but the fundamental ideas for a given medium would probably be similar to those presented here. For the purposes of this chapter, the media included are classified as follows: "Cultivation and Storage Media/' ''Enrichment Media," ''Differential Media,'' "Media for Determination of Physiological Prop- erties," "Media for Specific Bacteriological Procedures," "Media for Special Purposes." It is obvious that all previously described media will not be included; some time ago Levine and Schoenlein (1930) compiled a few thousand of the published formulas. Suggestions for media of par- ticular value in clinical diagnostic laboratory procedures may be found in Kitchens (1945), Gradwohl (1948), Marshall et al. (1947), Schaub and Foley (1952), Simmons and Gentzkow (1955), Stitt, Clough, and Branham (1948), Wadsworth (1947), the laboratory manuals of the U.S. Army, Navy, and other such sources. Very valuable information is available also in the manuals and catalogues of the companies which pre- pare dehydrated media; these catalogues are available free of charge directly from the companies. 37 38 MANUAL OF MICROBIOLOGICAL METHODS Bacterial Growth Requirements All living organisms require a utilizable source of energy in order to grow. Those using radiant energy are known as phototrophs, whereas those utilizing the chemical energy liberated from oxidation-reduction reactions are referred to as chemotrophs . In addition to an energy source, all living organisms require suitable carbon and nitro- gen sources, as well as inorganic salts. The autotrophic organisms can utilize CO2 as the sole carbon source, whereas heterotrophic organisms, although they may need CO2, also require carbon sources more complex than CO2, either for the carbon skeleton proper or for the hydrogen atoms linked to this skeleton, or both. The requirement for nitrogen may be satisfied in the form of NH4'*', NOs", or N2, although many organ- isms need complex organic nitrogenous compounds for this purpose. Such elements as Na, K, Ca, Mg, Mn, Fe, Zn, Cu, S, P, CI, etc., are required for growth and are utilizable gencrall}^ in the form of inorganic salts. In addition, many organisms require growth factors — organic substances which the organisms cannot synthesize at a significant rate and which are usually required in small amounts. Many bacteria require atmospheric oxygen for growth (obligate aerobes), whereas others fail to grow in the presence of oxygen (obligate anaerobes) ; some can grow under either set of conditions (facultative organisms), whereas still others can grow only under low oxygen tension (rnicroaerophiles) . The relationship of a given organism to oxygen is a manifestation of the oxidation-reduction potential range commensurate with the physiological activity of that organism (see Chap. IV). The requirement for oxygen by some bacteria can be satisfied by oxidizing agents such as NO3", S04~, etc. On the other hand, obligate anaerobes can be grown in the presence of oxygen pro- vided a sufficiently^ low 0/R potential is obtained by the addition of reducing agents or by permitting the formation of reduced products (see Chap. V). Thus, although all the physiological phenomena associated with the oxygen relationship have not been explained completely, this relationship must be considered in order to obtain vigorous bacterial growth. Most bacteria grow well only Vv^ithin a limited pH range. To maintain this range, at least during the initial growth buffers are added to the medium; these buffers may be of the types described in Chap. IV, or for the neutralization of acids, CaCOs may be included in the medium. Finally, to be available to the organism, all the components necessary for growth must be in aqueous solution. Bacteria apparently display all possible variations of the major nutritional require- ments. The autotrophs and many heterotrophs can be grown in chemically simple media of defined composition (synthetic media). By this means, qualitative and quantitative assay of each ingredient of such media can be made, leading to the micro- biological assay of vitamins, amino acids, carbohydrates, inorganic ions, etc. Because of our imperfect knowledge of the exact nutritional requirements of most bacteria, the comparatively few truly synthetic media have been devised chiefly for the cultivation of autotrophs or in connection with specific research problems involving the growth of certain heterotrophs. However, the large number of heterotrophic bacteria familiar to most bacteriologists continue to be grown in rather complex media. To a great extent this complexity is associated merely with the empirical manner in which bac- teriology has been practiced since the days of Pasteur and probably can be eliminated as exact nutritional information becomes available. A sort of tradition has developed in regard to the complex growth media made from natural plant and animal materials, which having been employed for many years remain as the familiar means of growing bacteria. The purpose of this brief review of bacterial nutrition is to stimulate reflection on the part of the laboratory investigator regarding the cultivation of bacteria. Growth PREPARATION OF MEDIA 39 media selected arbitrarily or according to tradition may be less adequate for many purposes than media whose composition has been determined according to the nutri- tional rationale; indeed, the latter approach will more certainly lead to a clearer understanding of the nature of the microorganism being cultivated. General Procedures in Media Preparation In selecting the components of a medium, it is desirable to employ sub- stances of defined composition, purity, or mode of preparation. In the preparation of synthetic media, known chemical compounds of chemically pure (C.P.) or reagent grade should be used. It is less easy to define the composition of some of the complex substances used in certain formulas. Peptones and gelatin should conform to the minimal standards given in the United States Pharmacopeia XV. Gelatin, in addition, should meet the additional specifications as set forth in the Military Medical Purchase Description, ASIMPA 1-212-000. Tentative specifications for bacteriological-grade agar have been proposed. The specifications referred to above for agar, gelatin, and some peptones are included in the appendix to this chapter. In addition to chemical analysis, the growth response of selected test organisms is used in designating a product as satisfactory for bacteriological use. Distilled water is used generall^^ to dissolve the components of the medium, although it is obviously unneces- sary when crude plant or animal extracts are employed. Many media, especially those prepared from natural products, are turbid or become turbid upon heating and require filtration through paper or other agents prior to sterilization. The reaction of most bacteriological media is usually adjusted to a hydrogen-ion concentration near neutrality. Since sterilization by wet heat usually causes a drop of pH of about 0.2-0.4 unit, it is necessary to adjust the pH of the medium before sterilization to a value higher by the amount indicated. Although solutions of natural extracts often do not require pH adjustment, each batch of medium should be tested to deter- mine if the desired pH is obtained. Detailed instructions for testing and adjusting the reaction of media are presented in Chap. IV. For ordinary purposes, satisfactory results will be obtained by adjusting the medium to slightly above (pH 7.2-7.4) the neutral point with bromthymol blue, the aqueous solution of the sodium salt or the alcoholic (95 per cent ethanol) solution of the unneutralized indicator (Table 2) being employed. A comparator block containing either standard solutions of known pH values and colors or a comparator consisting of colored glass standards for the corresponding pH and indicator color values should be used to deter- mine pH. For deeply colored media, a glass electrode pH meter should be used. 40 MANUAL OF MICROBIOLOGICAL METHODS Table 2. Acid-Base Indicators Indicator Bromphenol blue Bromcresol green Methyl red Bromphenol red Bromcresol purple Bromthymol blue Phenol red Cresol red Thymol blue (alk. range) . Phenolphthalein Concentration recommended, %* 0.04 0.04 0.02 0.04 0.04 0.04 0.02 0.02 0.04 0.10 Sensitive pH range 3.1-4.7 3.8-5.4 4.2-6.3 5.2-6.8 5.4-7.0 6.1-7.7 6.9-8.5 7.4-9.0 8.0-9.6 8.3-10.0 Full acid color Yellow Yellow Red Yellow Yellow Yellow Yellow Yellow Yellow Colorless Full alkaline color Blue Blue Yellow Red Purple Blue Red Red Blue Red * Stock solutions in 95 per cent ethanol for the indicator acids or in water for the indicator salts. See Chap. IV, Table 6, for details of preparation. Buffers are often required in medium to facilitate growth. This is par- ticularly true of media composed of simple compounds or in which acid- producing bacteria are cultivated. Mixtures of sodium and potassium phosphates are generally employed (see Chap. IV), although dibasic phosphate is also used singly. Buffer requirements will be indicated in the media described below. To determine pH changes during growth, indicators may be included in the medium. For this purpose, an indicator covering the desired range (Table 2) is selected and 1-2 ml of a 1-2 per cent alcoholic solution is added to each liter of medium before sterilization. Although litmus is an insensitive pH indicator, it has the advantage of also indicating major changes in oxidation-reduction potential and is thus useful to show these changes, particularly in milk media. General directions for media sterilization will not be presented here, since these are available in many bacteriological laboratory guides. A few critical points will, however, be emphasized. Most tubed media are safely sterilized in an autoclave, using 121°C (250°F) for 15 min, care being taken not to pack containers tightly; media in large flasks or bottles require a longer sterilization time (15-30 min). The autoclave should reach this temperature rapidly (within 10 min) and should cool down just as rapidly after interrupting the steam flow. It is to be noted that temperature is the important factor in heat sterilization, pressure being merely the means of obtaining water at elevated temperatures. Thus, during sterilization the temperature must be observed (a pressure gauge is not reliable as an indicator of temperature) and used as the criterion of an adequate level of heat. It should be realized that overheating a medium may lead to modifications of its composition. In general, main- PREPARATION OF MEDIA 41 taining most media at 121°C for 15 min will not cause important changes. The chemical composition of many substances, particularly carbo- hydrates, is changed, however, even by this limited heat treatment (Davis and Rogers, 1939 j. For critical study, such substances should be steril- ized separately by filtration through bacteriological filters (Seitz or sintered glass) and added aseptically to the remainder of the medium previously sterilized by heat. If filtration facilities are lacking, concen- trated stock solutions (20-25 per cent) of such substances can be sterilized by heat in the autoclave and then dispensed aseptically to the desired final concentration. In cases where conclusions based on comparative studies of the sterilization modifications described above are not avail- able, the effect of heat upon the medium must be considered in each new investigation. Dehydrated media refer to powdered, water-soluble commercial products which yield a growth medium. Usually all that is required in the preparation is to dissolve the proper amount of the powder (according to the directions accompanying the medium), to dispense as desired, and to sterilize. A wide variety of types, each suitable for a specific purpose, is available, and these have been found to be adequate for bacteriological use. Indeed, in most instances, the greater uniformity of these products over that attained by preparation of individual batches prepared in the laboratory from separate ingredients indicates their desirability for com- parative work. No attempt will be made here to describe each of these dehydrated media (in some instances formulas for preparation of media will be given even though satisfactory dehydrated products are avail- able), but those persons desiring to use these products should seek information from the manufacturing companies. Companies in this country which specialize in dehydrated media preparation include (1) Albimi Laboratories, Inc., 16 Clinton Street, Brooklyn 1, New York, (2) Baltimore Biological Laboratories, 1640 Gorsuch Avenue, Baltimore 18, Maryland, and (3) Difco Laboratories, Inc., 920 Henry Street, Detroit 1, Michigan. Products such as peptone, beef or yeast extract, agar, etc., are also available from other supply houses not specializing in dehydrated media preparation. CULTIVATION AND STORAGE MEDIA The media to be described in this section will include the formulas for various complex, nonsynthetic media which may be used for the general cultivation of bacteria, either from a natural sample or after a pure cul- ture has been obtained. No attempt will be made to designate any one medium as the standard for a particular purpose, but it may be noted that for certain purposes (for example, estimation of organisms present 42 MANUAL OF MICROBIOLOGICAL METHODS in water and milk) ''standard media" have been designated by other organizations (American Public Health Association, 1946, 1948). The peptone^ listed as an ingredient in some of the formulas is a product derived by digestion of proteinaceous materials of either plant or animal origin, by use of acid, alkali, or added or natural proteolytic enzymes (Asheshov, 1941; Brewer, 1943; Gladstone and Fildes, 1940; Leifson, 1943; Mueller and Johnson, 1941). Since the composition of peptone varies with the origin and the method of preparation, not all types may be suitable in all instances ; any type of bacteriological grade found to give best results for a particular purpose may be used. Data available at present indicate that peptone prepared by pancreatic digestion of casein (for example, B.B.L. "trypticase" and Difco "casitone") will often con- tain growth-promoting substances, required by many organisms, which are not found in some other peptones. In the formulas listed below in which peptone is specified, a particular type is indicated in a few instances; when this procedure is not followed, the worker may choose the type giving more satisfactory results. Agar,^ a complex carbohydrate refined from marine algae, is the usual agent for solidification of media. This material should be free from starch and debris and capable of producing a clear solution when hot ; the exact concentration to be used to give the desired degree of solidity may vary with the degree of purification, although usually 1.5 per cent is sufficient. Agar media should not be adjusted to a pH lower than 6.0 prior to sterilization, since the agar is hydrolyzed under these conditions. When such agar media are required, the pH is adjusted after sterilization by the aseptic addition of acid. All laboratory-prepared infusions, especially those from meat, should be checked microscopically to assure freedom from bacterial growth, especially if the infusion is held for even a few hours at temperatures which. will permit microbial reproduction. Beef -extract peptone broth (often called nutrient hroth) ordinarily has the following composition: beef extract, 3 g; peptone, 5.0 g; distilled water, 1,000 ml. The Avater is heated to 60°C to promote solution of ingredients; after cooling, the pH is adjusted to 7.0-7.2. After dispens- ing in tubes or other containers, it is autoclaved at 121°C for 15 min. This medium, still used quite widely for the general cultivation of aerobic organisms and as a basal medium for a variety of physiological tests, is now recognized to be nutritionally inadequate for many types of fastidious organisms. In many instances addition of 5 g of yeast extract will support growth of such types. For heef-extract agar (nutrient agar), 1.5 per cent of agar is added to the above medium before dispensing and autoclaving. ^ See appendix to this chapter for specifications of some peptones and agar. PREPARATION OF MEDIA 43 Thiogly collate broth, originally used for the growth of anaerobic bac- teria, is now being used also for other types with regard to oxygen rela- tionships. The liquid medium may be prepared as follows: Dissolve 15 g of peptone (preferably a pancreatic digest of casein), 5 g of glucose, 5 g of yeast extract, 0.75 g of L-cystine, 2.5 g of NaCl, 0.75 g of agar, and 0.1-0.5 g of sodium thioglycollate in 1 liter of water; adjust pH to 7.2. Sterilize for 15 min at 121°C. The small amount of agar which is neces- sary to maintain a low oxidation-reduction potential does not affect appreciably the fluidity of the medium. This medium is unsatisfactory for stock cultures unless CaCOs is added. If a dye to indicate the 0/R potential is desired, add 0.002 g of methylene blue or 0.001 g of resazurin per liter. If the medium to be used contains an indicator dye, for obligate anaerobes, the dye should show the medium to be reduced except in the upper layer. Therefore, unless the dye indicates that the medium has been reoxidized to a considerable extent, it is unnecessary to follow the usual practice of heating the medium for exhaustion of oxygen immediately prior to inoculation. This medium should not be stored in the refrigerator after preparation, as it absorbs more oxygen at lower temperatures. For special purposes, such as the sterility testing of bio- logical products in which inactivation of mercurials presents a problem, dehydrated media are available which meet the specifications of the National Institutes of Health and other agencies. Sodium caseinate agar. This medium is often used for the enumera- tion of bacteria, including the Actinomycetes, in soil. It is prepared as follows: sodium caseinate, 1.0 g; glucose, 1.0 g; MgS04, 0.2 g; K2HPO4, 0.2 g; FeS04, trace; distilled water, 1,000 ml; agar, 15 g. It is adjusted to pH 7.0. Before pouring plates or dispensing in other containers, it is shaken gently to disperse precipitate. Infusion broths may be prepared by extraction of either lean skeletal muscle or heart muscle in the cold or by heat. In the former case the following is typical : Add 1 ,000 ml of water to 400-600 g of lean veal or beef tissue which has been finely ground after removing as much fat as possible. Allow to infuse overnight at refrigerator temperature, remove scum of fat, squeeze infusion through mushn cloth, and restore volume to 1,000 ml. Add 5 g of peptone, ^nd heat for 20 min at 100°C; filter through paper; adjust pH to desired value. For infusions prepared by heat, use the same proportion of meat to water and boil over free flame for 15 min, with or without a previous overnight infusion period in a refrigerator. Add peptone, and if desired as the basal medium for blood agar, add 5 g NaCl to 1,000 ml, and continue as above. For aerobic organisms adjust the pH to 7.0-7.2, but for anaerobic types adjust to 7.6 and tube the liquid deep over a 1- to 2-cm column of desiccated tissue particles saved from the infusion. Autoclave for 20 min at 121°C. The M MANUAL OF MICROBIOLOGICAL METHODS use of infusions is diminishing owing to their replacement by media pre- pared from peptones or other materials of more uniform composition, better growth-promoting properties, and less complicated preparation details. Beef -liver infusion, used principally for anaerobic types, is prepared as follows : Remove fat from 500 g of fresh beef liver, grind, and heat, with occasional stirring, in 1,000 ml of tap water for 1 hr in flowing steam. Cool, and strain through cheesecloth. Restore filtrate to original volume, and add 1 per cent of peptone and 0.1 per cent of K2HPO4. Dry the tissue (at 55° C if possible) rapidly. Tube broth over several chunks of tissue. Use the broth (before addition of peptone and phosphate) in the original strength or diluted five times. Sterilize 20 min at 121°C. Avoid longer heating of medium, for this diminishes its value with respect to initiation of growth from small inocula. Brain medium is used to stimulate spore production by many Clostridia and hence is a valuable stock culture medium. The blackening reaction produced by certain species has some diagnostic value (Hall and Peterson, 1924). The medium is prepared as follows: Secure fresh sheep (or calf) brains which are as free as possible from injury. Using forceps, remove blood and membranous material from brain tissue. Add distilled water, in the ratio of 100 ml of water to 100 g of brain, and boil slowly for }4 hr. Put brains through potato ricer. Add 1.0 per cent of peptone and 0.1 per cent of glucose to the resulting mixture, and heat slightly to dissolve peptone. Tube in deep columns while the mixture is stirred in order to effect an even distribution of the brain tissue. It is sometimes recom- mended that reduced iron (''iron reduced by hydrogen," Merck & Co., Rah way. New Jersey), a thin strip of iron, or iron wire be added to the tube before tubing the liquid mixture. Sterilize in autoclave for 30 min at 121°C and check sterility by incubation at 37°C for a minimum of 24 hr. The finished medium should have approximately an equal amount of liquid broth above the brain particles. Proteolysis is indicated by putrefactive odors, a disintegration of the particles, and a blackening reaction. Yeast infusion may be prepared by several procedures. A satisfactory one follows: Obtain fresh yeast, preferably starch-free, and add 10 per cent by weight to several liters of tap water. Autoclave for 3 hr or more. Allow cells to settle by standing for several days at room temperature. Remove liquid infusion by siphon or with the Sharpies centrifuge. Steril- ize the liquid, after removal from the cells, in screw-capped bottle, and store indefinitely. If fresh yeast is not available, or if a simpler procedure is preferred, a similar medium may be prepared by adding 0.5 per cent of dehydrated yeast extract to distilled water. Care should be observed in selection of the yeast extract, since not all are equal in growth-promoting PREPARATION OF MEDIA 45 properties and some contain intact yeast cells which may be misleading in microscopic preparations. Semisolid agar. Some organisms, especially microaerophiles, are cultivated more successfully in semisolid media in which agar, in concen- trations varying from 0.2 per cent to 0.5 per cent according to the purpose for which the medium is intended, is added to a suitable base medium. Such a medium may be useful also for determination of motility and for fermentation reactions. To aid in reducing the degree of oxidation of the medium the lower concentration of agar is sufficient; for determina- tion of motility, concentrations approaching 0.5 per cent are necessary in order to stiffen the medium sufficiently to show distinctly the hazy zone characteristic of motile bacteria (Tittsler and Sandholzer, 1936). Since the concentration for this purpose is critical and the percentage to be used will vary with the purity of the agar, the exact concentration to be used must be tested for each batch of agar and each lot of medium checked with a known motile culture. Silica gel. It is sometimes desired to cultivate bacteria on inorganic gels to avoid unknown and possibly undesirable chemical contaminants in agar, to avoid liquefaction of agar by certain types, and to eliminate agar in the cultivation of certain autotrophic bacteria. Sterges (1942a and b) published full details (which cannot be condensed here) of prepara- tion of such media based on the reaction between sodium silicate and hydrochloric acid. A simplified technic recommended by Ingelman and Laurell (1947) follows: Mix 1 vol of ortho-silicic acid tetraethyl ester, Si(OC2H5)4, with 1 vol of ethanol; add 6 vol of boiled water a little at a time with stirring. Remove turbidity by centrifuging, and dispense in tubes or plates. Sterilize at 120°C for 30-40 min in autoclave, during which time gel formation occurs. Cool slowly, and remove ethanol by flooding with sterile water. Remove water, and replace with suitable nutrient solution. Reautoclave if necessary for sterility. Temple (1949) recommends a commercially available, highly purified colloidal silica preparation such as Ludox (duPont) ; for use a 30 per cent aqueous solu- tion is diluted to 10 per cent, nutrients are added, pH adjusted, it is dis- pensed in petri dishes and autoclaved. Storage media include those media in which bacteria are stored in "stock culture" condition for indefinite periods to provide a source of viable cultures as needed. The medium to be used will vary with the species to be maintained. A critical factor to be considered is whether or not the presence of a fermentable carbohydrate reduces viability; fre- quently 2 per cent CaCOa (sterilized separately by dry heat) is added to a medium to neutralize the acids produced by an organism which will not grow well in the absence of a fermentable carbohydrate. After incuba- tion in the chosen medium at the optimum temperature for a period 46 MANUAL OF MICROBIOLOGICAL METHODS allowing approximately half -maximal growth, stock cultures should be stored at refrigerator temperature between transfer periods, which may vary from two to several weeks according to the longevity of the types under study. Screw-capped tubes or small bottles may be used to minimize contamination and drying of medium during the storage period. Some workers (see Mc Clung, 1949) prefer to cover the growth with a layer of mineral oil (sterilized in shallow layers for 2 hr at 160°C with dry heat on three successive days). In some laboratories, stock cultures are prepared as agar slant cultures, but usually the stab technique is employed. Spore-producing types may be kept in stock condition in the form of spore suspensions ; often these are dried on sterile soil. McClung (1949) gives references on this technic and on details for lyophilization of all types. For those organisms which will grow on nutrient agar, peptone (1-2 per cent) agar, or yeast-infusion-glucose (0.5 per cent) agar, these media are generally used. For maintenance of Acetobacter species, Vaughn (1942) recommends agar containing yeast infusion, glucose, calcium carbonate, or liver-infusion broth prepared as above but diluted with an equal vol- ume of water before the addition of peptone and phosphate. For aerobic nitrogen-fixing types {Rhizohium, Azotohacter) , the y east-extra ct-man- nitol agar of Fred, Baldwin, and McCoy (1932) is prepared as follows: mannitol, 10.0 g; K2HPO4, 0.5 g; MgS04, 0.2 g; NaCl, 0.2 g; CaCls, 3.0 g; yeast infusion (pH 6.8), 100 ml; distilled water, 900 ml; agar, 15.0 g. The following medium is recommended for Neisseria by Vera (1948) and can be used for a variety of genera: peptone (pancreatic digest of casein), 20.0 g; cystine, 0.5 g; NasSOs, 0.5 g; NaCl, 3.0 g; agar, 3.5 g; distilled water, 1,000 ml; pH 7.3. In addition to the above, many formulas will be found in the literature which are of special value for particular types; dehydrated media are available for this purpose also. ENRICHMENT MEDIA The enrichment culture technic constitutes a means for the isolation of a wide variety of bacteria by adjusting the nutritional environment in such a manner as to enhance selectively the growth of a certain bacterial type within a given mixed inoculum. Use of this simple methodological approach, so ably exploited by Beijerinck (1921-1940), has assured the ready isolation of nearly all types of bacteria (van Niel, 1949) and con- stitutes a powerful tool for the bacteriologist in the isolation and identi- fication of pure cultures from an initially mixed population. Even fastidious pathogenic bacteria can be isolated in this manner, using for this purpose the animal body as a selective medium. In addition, selec- PREPARATION OF MEDIA 47 tion of the optimum temperature and optimum pH for growth aids in the cultivation of certain bacterial types, as does the proper adjustment with regard to oxygen relationship or 0/R potential. In this discussion, a few examples will be presented of the media used foi enrichment cultures; detailed information concerning many other media similarly employed will be found in the literature deaUng with specific groups. It is to be noted that certain of the formulas given in the section on ''Cultivation and Storage Media'' and on ''Differential Media" are of media which also may be used as enrichment media. In contrast, many of the media listed in the latter section contain com- pounds which inhibit the growth of certain types which might be expected in the initial sample in addition to the organism sought. In other instances, the composition of the medium allows the demonstration of the growth characteristics of the desired organism to make presumptive identification possible. The sulfur oxidizing bacteria. The chemoautotrophic growth of Thiohacillus thiooxidans is accomplished by inoculating a shallow layer of the following medium with about 1 g of mud or soil and incubating at 30°C: powdered sulfur, 10 g (or NasSsOa-SHsO, 5 g); (NH4)2S04, 0.4 g; KH2PO4, 4 g; CaCh, 0.25 g; MgS04-7H20, 0.5 g; FeS04, 0.01 g; water, 1 liter (Starkey, 1935). When good development has occurred, a transfer is made to fresh medium of the same composition. From the latter, iso- lations are made on the following solid medium: Na2S203-5H20, 5 g; K2HPO4, 0.1 g; NaHC03, 0.2 g; agar, 20 g; water, 1 liter. Thiohacillus colonies are recognized by the deposition of free sulfur: 2Na2S203 +O2 — > 2Na2S04 + 2S. The nonsulfur photosynthetic bacteria (Athiorhodaceae) utilize organic compounds as H-donors and require growth factors for their develop- ment. Some are strict anaerobes, growing only photosynthetically, but others are facultative aerobes and can grow well heterotrophically in the dark under aerobic conditions, obtaining energy by the oxidation of organic substrates. Here, general enrichment media for cultivation under anaerobic conditions will be described, the source of energy being light (van Niel, 1944). The basal medium consists of (NH4)2S04, 1 g; K2HPO4, 0.5 g; MgS04, 0.2 g; NaCl, 2 g; NaHCOs, 5 g; water, 1 liter. This medium is supplemented by the addition of a single organic sub- stance (ethanol, glycerol, mannitol, formate, acetate, succinate, malate, alanine, asparagine) in a final concentration of 0.15-0.2 per cent, after which the reaction is adjusted to pH 7.0 with H3PO4. The various media are dispensed into glass-stoppered bottles, inoculated with a small amount of surface water or mud, and the completely filled and stoppered bottles incubated in a light cabinet at 25-30°C under continuous illumi- nation with electric bulbs (25-40 watts). For more rapid and abundant 48 MANUAL OF MICROBIOLOGICAL METHODS growth, peptone and yeast extract may be substituted for the single organic compound. Isolations are made by preparing shake cultures of successive dilutions, using a medium of yeast extract, 5 g; NaHCOs, 2 g; Na2S, 0.1 g (to provide anaerobiosis) ; agar, 20 g; water, 1 liter; pH 7.0. Streaked plates of this medium (less the Na2S) may also be used for the isolation of facultatively aerobic strains, in which case growth will occur in the dark or in the light. Lactic acid bacteria. Enrichment cultures of the extensively fermenta- tive tj^pes such as the lactic acid bacteria depend upon the use of poorly buffered media rich in organic nitrogenous compounds and growth factors and containing fermentable carbohydrates which serve as energy sources. Lactic acid bacteria will predominate in such a nutritional environment oecause they withstand the high concentrations of acid produced by the breakdown of the carbohydrates, whereas most other bacteria are killed or inhibited. In general, lactic acid bacteria can be isolated from fer- menting plant juices, dairy products, buccal and vaginal cavities, raw sewage, etc. Here, a few common enrichment media and methods will be indicated which will permit growth of a wide variety of types, although additional study is required for their isolation and identification. Incu- bation temperatures are commonly 30, 37, or 45°C. Milk, raw or pasteurized, can be used both as a source of lactic acid bacteria and as an enrichment medium. Glass-stoppered bottles are filled with raw milk (with or without previous heating at 60°C for 10 min) and incubated. Similarly, glass-stoppered bottles completely filled with a medium con- sisting of 0.5 per cent yeast extract and 2 per cent of glucose or 10 per cent of sucrose are inoculated with raw sewage. Shredded cabbage or other plant tissue, pressed tightly and covered with water, or ground grain mash can serve as enrichment media. Further types can be isolated by streaking throat swabs on blood agar plates (described below) and incubating at 37°C. When growth has occurred in the enrichment media, plates of yeast-extract-glucose (or sucrose) agar containing 1 per cent CaCOs (sterilized separately by dry heat) are streaked, and portions of isolated colonies tested for catalase (lactic acid bacteria are catalase negative) with 5 per cent H2O2 before streaking again to obtain pure cultures. With certain types which show some sensitivity to atmospheric oxygen in primary cultures, poured, seeded plates may be more successful than streaked plates. Plates should be incubated at temperatures used for the corresponding enrichment. It is apparent that many variations of enrichment media may be employed and that the predominant occur- rence of given lactic acid bacterial types depends upon the source used. The coliform group. Owing to the vast amount of work performed with the Enter ohacteriaceae, highly specialized media have been developed for their rapid isolation and identification. Enrichment media for the PREPARATION OF MEDIA 49 noDpathogenic Escherichia and Aerobacter include nutrient broth plus 1 per cent of lactose. Gas production gives presumptive evidence of the group, and the necessary confirmation is accomplished by use of one of a variety of media listed under ''Differential Media." Similarly the pathogenic Salmonella may be enriched from feces, urine, water, sewage, contaminated foodstuffs, etc., by use of tetrathionate and selenite (Leifson, 1936) broths. These media are available in dehydrated form. For obligately anaerobic bacteria, a primary requirement is the initial attainment of a low 0/R potential, either by eliminating oxygen from the nutritional environment or by counteracting its effect by the addition of chemicals. The material in Chap. VI outlines methods for the cultiva- tion of members of the genus Clostridium. For the enrichment of the sporeforming anaerobic bacteria a variety of media are available, and choice among them will depend upon the species desired and the sample material. For the pathogenic species the thiogly collate medium^ or beef heart infusion listed under "Cultivation and Storage Media" is suitable. For certain soil types (organisms producing butyric acid, or butyl alcohol, and some others) the liver infusion medium (same section), corn liver medium, or potato infusion is useful. In the last two, the fermentation of starch may be observed, and some bacterial types give a characteristic ''head" (a slimy mass of unfermented cellulosic material). The starch-fermenting types usually sporulate readily on these natural infusions. The corn liver medium is prepared as follows: Add 50 g of ground (white or yellow) corn meal and 10 g of dried liver powder to 1 liter of tap water. Heat in flowing steam for 1 hr with occasional stirring. Remove from steam, cool almost to room temperature, and dispense in tubes, flasks, or bottles. Sterilize 45 min at 121°C. The resulting medium, on cooling, should be semisolid, with the coarser particles of corn settling to the bottom leaving a 2- to 3-cm layer of starchy material at the top. For determination of pigment production, omit the liver powder. The potato infusion is prepared as follows: White potatoes, 200 g; glucose, 5 g; (NH4)2S04, 1.0 g; CaCOs, 3 g; tap water to make 1 liter. Peel potatoes, and add water. Steam for 1 hr, or boil slowly until soft, and put through potato ricer. Add other ingredients, and bring up to original volume. Cool, and tube, with stirring, so as to obtain an even distribution of the potato particles. As an example of the enrichment of an anaerobic bacterium, which has been isolated (Baker and Taha, 1942; Bornstein and Barker, 1948) by use of a medium constituting a simple and chemically defined nutritional environment, Clostridium kluyveri may be isolated from black mud using a medium containing the following components: ethanol, 8 g; sodium acetate hydrate, 8 g; KH2P04-Na2HP04 buffer, 1 M, pH 7.0, 25 ml; (NH4)2S04, 0.5 g; NaaCOa, 0.1 g; MgS04-7H20, 0.2 g; CaS04-2H20, 50 MANUAL OF MICROBIOLOGICAL METHODS 10 mg; FeS04-7H20, 5 mg; MnS04-4H20, 2.5 mg; NaMo04-2H20, 2.5 mg; biotin, 3 jug; p-aminobenzoic acid, 50 /xg; sodium thiogly collate, 0.5 g; distilled water, 1 liter. The thiogly collate may be replaced by 0.2 g of Na2S-9H20 which is best added after steriUzation of the medium; the growth factors may be replaced by 0.5-1.0 g of yeast extract. The procedure is : Add heavy inoculum (5 per cent of black mud from fresh- water or marine sources) and incubate culture at 35° C in completely filled glass-stoppered bottles in order to exclude oxygen. To isolate pure cultures, use a solid medium of the same composition in an anaerobic jar. Use of bacteriostasis. A principle sometimes invoked in the enrich- ment culture of a particular type of organism from a mixed population is to utilize the inhibitory (bacteriostatic) property of a specific chemical without which the medium would be suitable for many species in the sample. For example, addition of crystal violet in a final concentration of 1 : 100,000 will inhibit most gram-positive types without affecting the gram-negative group. Similarly, the antibiotics penicillin and strepto- mycin may be used for selective inhibition. Sodium azide (0.03 per cent) has bacteriostatic action for gram-negative bacteria, the aerobic gram- positive sporeforming bacilli, and certain other aerobes (Lichstein and Soule, 1944). DIFFERENTIAL MEDIA The formulas presented in this section include media employed, com- monly for original isolation, to determine differential reactions which may permit presumptive identification of bacterial species. In some instances the media constitute selective growth environments, often as a result of the inclusion of specific compounds which inhibit the growth of those organisms not under investigation. Blood agar. This medium is used frequently in the study of strep- tococci and other groups exhibiting hemolytic properties. The medium is valuable also in the routine cultivation of fastidious pathogenic species which may or may not show hemolytic reactions. To prepare medium: Add aseptically 5 per cent of sterile rabbit, sheep, horse, or human blood to a satisfactory carbohydrate-free agar base medium containing 0.5- 0.85 per cent of NaCl (to maintain isotonicity). Blood agar seeded with a light inoculum yields more typical hemolytic reactions than do heavily streaked plates. Chocolate agar is prepared as above except that the blood is added to the base medium at 80°C; this causes a distinct darken- ing of the added blood (protein coagulation). Egg -yolk agar. This medium is used as a presumptive identification medium for certain pathogenic Clostridia and for sporeforming aerobes. Plates of an agar-base medium to which sterile egg yolk has been added PREPARATION OF MEDIA 51 are streaked, and the characteristic reactions obtained are due principally to production of lecithinase. To prepare the medium: After washing shell with disinfectant, asceptially withdraw first the white and then the yolk of fresh hen's egg. Dilute yolk with equal volume of 0.85 per cent NaCl, and add 1 ml of yolk suspension to each 9 ml of the following medium which is sterilized before addition of egg: peptone, 40 g; Na2- HPO4, 5.0 g; KH2PO4, 1.0 g; NaCl, 2.0 g; MgS04, 0.1 g; glucose, 2 g; agar, 25 g, 1 liter water, pH 7.6. Streak plates in such a manner as to yield well-isolated colonies. Consult McClung and Toabe (1947) for details of species characters displayed by Clostridia and Colmer (1948) and McGaughey and Chu (1948) for information on the aerobic baciUi. Media for gram-negative nonsporeforming bacteria. Many differ- ential media have been developed for the presumptive identification of the gram-negative rods occurring in the normal and diseased intestines and in samples contaminated with fecal material. Not all these media can be discussed, and although those which are mentioned may be pre- pared in the laboratory, readily available dehydrated products are gen- erally employed because of their greater uniformity. On Endo's agar other lactose nonfermenting types, such as Salmonella tijphosa, produce clear, colorless colonies which do not affect the faint pink color of the medium, whereas colonies of coliform organisms which ferment lactose are surrounded by a dark red zone. On eosin-methylene-blue agar (E.M.B.), colonies of lactose nonfermenting organisms are translucent; of the lactose fermenting types, Escherichia coli colonies are small, dark and have a greenish metallic sheen, while those of Aerohacter aerogenes are large, moist, and gray-brown in color with a pronounced tendency to coalesce. On the desoxycholate agar of Leifson (1935) growth of gram- positive organisms is inhibited. Colonies of the lactose nonfermenting Salmonella and Shigella are colorless, those of E. coli are red, and those of Aerohacter are pale with pink centers (Paulson, 1937). Medium for isolation of staphylococci. Chapman (1948) suggests the following medium for selective isolation of chromogenic staphylococci associated with food poisoning: D-mannitol, 10 g; peptone, 10 g; gelatin, 30 g; yeast extract 2.0 g; NaCl, 55 g; K2HPO4, 5 g; (NH4)2S04, 75 g; 10 per cent NaOH, 6 ml ; water, 1 ,000 ml ; agar, 15 g. Following steriHza- tion, shake medium to disperse precipitate. Media for Brucella. Although liver-infusion agar has been used widely for cultivation and differentiation of Brucella, it is being replaced, owing to batch variations, by one of the following media which are available in dehydrated form: (1) Difco tryptose agar (''tryptose," 20 g; glucose, 1 g; NaCl, 5 i; agar, 15 g). (2) B.B.L. trypticase soy agar (''casein peptone,'' 15 g; ''soy peptone," 5 g; NaCl, 5 g; agar, 15 g). Incubate cultures at 37°C in an atmosphere of 10 per cent CO2 and in original isolations, if 62 MANUAL OF MICROBIOLOGICAL METHODS gram-positive contaminants are suspected, add crystal violet in concen- tration of 1 : 700,000. For differentiation of Brucella species on the basis of bacteriostatic action of dyes, add 1 : 100,000 concentration of thionin and 1 : 100,000 concentration of basic fuchsin. Brucella melitensis and B. suis grow in the presence of thionin, but B. abortus in inhibited, whereas on agar containing basic fuchsin, B. suis is inhibited by B. melitensis and B. abortus is not inhibited. Media for cultivation of Corynebacterium diphtheriae. Perhaps the most widely used medium for the cultivation of C. diphtheriae, which yields cells of characteristic morphology and metachromatic granule staining reactions, is coagulated blood serum. Although it may be pre- pared from fresh serum (horse, cow, sheep, or pig) by adding 1 vol of 1 per cent dextrose broth to 3 vol of serum, users of small quantities of this medium will find the dehydrated product more satisfactory. Special precautions are necessary in the successful sterilization of this medium. It may be sterilized by inspissation or by the following method: Add desired quantity of dehydrated powder to warm (50°C) water, and main- tain this temperature for 45 min during which time stir medium gently to avoid bubbles. Tube in amounts such that a deep butt will not be pro- duced when tube is slanted. Place tubes in slanted position in autoclave equipped with manually operated air-escape valve. Cover tubes with several layers of newspaper, or pack in metal box between layers of non- absorbent cotton. Close door and air-escape valve of autoclave, and admit steam, raising pressure of air and steam mixture quickly to 15 lb. After 20 min open air-escape valve cautiously to permit escape of air while maintaining steam pressure. When all air is removed, close escape valve and continue heating at 121°C for 20 min. Reduce pressure slowly, and open door when pressure is at zero. Other media, often containing potassium tellurite (0.03-0.05 per cent), are valuable in the differentiation of this organism. The details of these cannot be condensed here, but those interested should consult the original papers, for example, Kellogg and Wende, 1946; Frobisher et al., 1948; Petran, 1948; Whitley and Damon, 1949; Buck, 1949. Media for the cultivation of Neisseria gonorrhoeae. The problems involved in the successful isolation of this organism from samples of clinical material will not be discussed here. For suggestions concerning the media to be used, consult Carpenter et al. (1949). Media for the cultivation of Mycobacterium tuberculosis. Many media have been devised for the cultivation of this organism from sputum, urine, pleural exudates, gastric contents, etc. The media of Dorset, Loewenstein, Petragnani, Petroff, etc., have been used widely; descrip- tions of such media are available in manuals for laboratory clinical pathology, particularly Gradwohl (1948). Recently, Dubos and his PREPARATION OF MEDIA 53 associates described growth of this organism in liquid semisynthetic medium, containing surface-active agents (esters of long-chain fatty acids and polyhydric alcohols), which supported comparatively rapid cell multiplication with a small inoculum and yielded cultures of uniform turbidity without granule or pellicle formation (see Frobisher, 1949). MEDIA FOR DETERMINATION OF PHYSIOLOGICAL PROPERTIES In the study of pure cultures, the so-called cultural characters and physiological reactions assume importance in the classification of many groups of heterotrophic bacteria. The latter reactions include the dis- similation of various carbohydrates and polyalcohols, the breakdown of simple and complex nitrogenous compounds, and the production of specific compounds as end products of the metabolism of the bacteria under study. The media for some of the more commonly used reactions will be included in this section. (See also Chap. VII, ''Routine Tests for the Descriptive Chart.") Carbohydrate indicator media. The common procedure is to add the carbohydrate or polyalcohol to be studied to a basal medium (either liquid or agar) to which an indicator has been added to detect changes in pH which develop during growth. Growth of some organisms, particu- larly the sporeforming anaerobes, may result in a marked reduction of the indicator, in which case the indicator must be added after rather than before growth ; use of a spot plate or other methods of pH determination is then made at the time of observation. Early observation of fermenta- tion results generally eliminates the difficulty due to reduction of the indi- cator, since acidity changes usually precede reduction. Production of gas is detected in liquid media by placing Durham tubes (small inverted vials which will fill with liquid during sterilization) in the tubes at the time the medium is dispensed. The tubes are unnecessary if a solid or semisolid medium is used. Semisolid agar is prepared by adding 0.3-0.5 per cent of agar to a satisfactory liquid medium and making stab inocula- tions in the column of medium with a straight inoculating needle. In such a medium (or in full-strength agar) gas production will be denoted by the appearance of gas bubbles and cracks in the medium; the same semisolid medium may also be used to determine motility. Full-strength (1.5 per cent) agar should be cooled m slanting position and inoculated on the surface of the slant. The basal medium to be employed for fermentation tests should pro- vide the necessary nutrients for the organism to be studied and must be free of fermentable carbohydrates. If good growth can be obtained on 2 per cent casein or gelatin-peptone solutions (or agar), these media are preferred. According to Vera (1949), some samples of beef and yeast 54 MANUAL OF MICROBIOLOGICAL METHODS extracts contain fermentable carbohydrate. In all instances, control tubes to which carbohydrate has not been added must be inoculated to check changes of pH due to the breakdown of carbohydrates or other substances. The synthetic medium of Ayers, Rupp, and Johnson (1919) may be used if a peptone-free medium is desired and if the organism being studied can utilize ammonium salts as a source of nitrogen. It is pre- pared as follows: NH4H2PO4, 1.0 g; KCl, 0.2 g; MgS04-7H20, 0.2 g; water, 1,000 ml; carbohydrate, 10.0 g; adjust the pH by addition of 1 N NaOH. The soluble carbohydrates or polyalcohols are added to the basal medium at a level of 0.5-1 .0 per cent. (For precautions taken in sterihza- tion see page 39.) The indicator (commonly 1 ml of a 1.6 per cent alco- holic solution per 1,000 ml of medium) is added before sterilization. Although litmus and Andrade's indicator (acid fuchsin decolorized with alkali) have been used widely, they do not give accurate results in terms of H-ion concentration; thus, except for special purposes, (see Chap. VII, page 165) it is recommended that sulfon-phthalein indicators be employed. Select the appropriate indicator from the list given in Table 2, governing choice by following considerations: phenol red, with pH range 6.9-8.5, is useful for indication of changes on the alkahne side of neutrality and slight changes to acid ; bromthymol blue has a sensitive range extending slightly in either direction from neutrality; bromcresol purple, with a pH range of 5.4-7.0, is useful in synthetic media and for pronounced pH changes in more highly buffered media. Bromthymol blue is frequently the most useful of these but must be used with caution in synthetic media, since it indicates even the minor pH changes due to CO2 absorption. Double and triple sugar agars are of particular value in the rapid identification of the gram-negative enteric bacteria. These media are tubed in columns sufficiently deep to allow a 1.5-in. butt in addition to a slant. They should be inoculated by smearing the slant and stabbing the butt with a straight inoculating needle. RusselVs double sugar agar is prepared as follows: to a liter of peptone broth or beef-extract peptone broth add glucose, 1.0 g; lactose, 10.0 g; NaCl, 5.0 g; phenol red, 0.025 g; and agar, 15 g. After incubation, those organisms {Salmonella typhosa) which attack glucose but not lactose will show an acid reaction (yellow) in the butt but not in the slant, whereas those types (Escherichia coli) which ferment lactose will give an acid reaction throughout the entire medium. Gas production is indicated by formation of gas bubbles or splitting of the medium. Krumwiede^s triple sugar agar is prepared by adding 10.0 g of sucrose to the above formula. Fermentation of either sucrose or lactose, or both, will give rise to an acid reaction throughout PREPARATION OF MEDIA 55 the medium, whereas fermentation of only glucose will produce acid in the butt but not in the slant. The addition of 0.2 g of ferrous sulfate and 0.3 g of sodium thiosulfate to the above formula allows the determina- tion of the production of hydrogen sulfide in the same medium {trij)le sugar iron agar); cultures producing H2S show an extensive blackening of the agar due to iron sulfide precipitation (Sulkin and Willett, 1940). Starch agar. Add 0.2 per cent of soluble starch to a suitable nutrient agar basal medium, sterilize, and pour plates. After inoculation and growth, test for starch hydrolysis by flooding the plate, which has been streaked across center line, with dilute (Lugol's) iodine. Absence of the bluish-purple color characteristic of the starch-iodine complex indicates hydrolysis. Media to demonstrate gelatin liquefaction. Plain gelatin may be made by adding 12 per cent of bacteriological grade gelatin to distilled water, adjusting the pH to 7.0 before sterilization, sterilizing 12-15 min at 120°C, and cooling the tubes immediately. Nutrient gelatin is prepared by adding the above amount of gelatin to a sugar-free nutrient broth. For most obhgate anaerobes, it is necessary to add 0.25 per cent of glucose and to tube the medium in deep columns. If growth does not occur, sodium thioglycollate (0.1 per cent final concentration) is added to serve as a reducing agent. Utilization of gelatin may be determined in an agar medium by adding gelatin (0.4 per cent final concentration) to a nutrient agar. A sufficient amount of the sterile medium is poured into a petri dish to avoid a thin spot if center of dish is sUghtly raised. A single streak of the culture is made across the surface of the hardened medium, and following incubation, the plate is flooded with a gelatin precipitant: acid HgCU (mercuric chloride 15.0 g; concentrated HCl, 20.0 ml; distilled water, 100 ml) ; or saturated ammonium sulfate solution. A white precipitate indicates presence of nonhydrolyzed gelatin ; absence of the precipitate in the region of growth indicates gelatin hydrolysis. Proteolysis. Gelatin hydrolysis (liquefaction) represents enzymatic action upon an incomplete protein, and positive action is not necessarily an indication that the organism can hydrolyze complex proteins; this is a characteristic of particular value in the study of certain obhgate anae- robes. The beef -heart infusion with particles of tissue represents one of the media in which muscle protein hydrolysis may be observed. Coagu- lated serum (as slants) represents another type of protein to be tested; organisms with abihty to hydrolyze serum proteins will cause partial or complete hquef action. For action on coagulated egg albumin one should include a small cube of hard-boiled egg white in a tube of a suitable base medium ; disintegration of coagulated egg white during growth is evidence for proteolytic activity. Another medium for the indication of proteo- lytic activity is alkaline egg medium which is prepared as follows: Mix the 56 MANUAL OF MICROBIOLOGICAL METHODS yolk of one and the whites of two fresh eggs (preferably in a Waring blender). Add 500 ml of distilled water, and adjust pH to 7.6. Stir well, or mix in blender. Add 1 part of above to 5 parts of nutrient broth, tube in deep columns, and sterilize for 20 min at 121°C. The final medium should be an opaque, whitish liquid. During growth proteolysis is indicated by progressive clearing of the medium. Nitrate broth. Add 0.1 per cent of KNO3 to a nutrient broth or agar. For obligate anaerobes, also add 0.1 per cent of glucose and 0.1 per cent of agar to basal medium and tube in deep columns. For organisms not reducing nitrates in a peptone medium the following synthetic medium of Dimmick (1947) is recommended: K2HPO4, 0.5 g; NaCl, 0.5 g; MgS04' 7H2O, 0.2 g; NaNOs, 2.0 g; glucose, 10.0 g; agar, 15 g; distilled water 1,000 ml. If the organism being studied requires more calcium, add 0.05 g of CaCl2 to the above; in this case it is important that the final pH be 7.2; to assure this after sterilization, adjustment before sterilization should be to about 7.8. Indole determination. Use 1 per cent concentration of a peptone high in tryptophane, such as those prepared by enzymatic digestion of casein or lactalbumin (see Chap. VII for methods of testing for indole). H2S production. If the lead acetate paper tests recommended in Chap. VII are not desired, use dehydrated media or consult the original papers of Vaughn and Levine (1936), Hunter and Crecelius (1938), and Untermohlen and Georgi (1940) for directions for preparation of media containing lead, bismuth, or iron salts which will precipitate as the sulfides in the presence of II2S. Methyl red and Voges-Proskauer reaction. Prepare basal medium for these tests as follows: peptone, 7.0 g; glucose, 5.0 g; K2HPO4, 5.0 g. Since all peptones are not suitable, use for peptone an enzymatic digest of casein. For details concerning these reactions consult Chap. VII. Medium for determination of utilization of citrate. Within the gram- negative nonsporef orming bacilli, differentiation of some types is based on utilization of citrate as the sole source of carbon. Koser's synthetic medium is suitable for this and is prepared as follows: KH2PO4, 1.0 g; MgS04, 0.2 g; NaNH4P04, 1.5 g; NaaCeHBOy, 3.0 g; water, distilled, 1 liter. Growth, as evidenced by turbidity, in the water-clear medium indicates utilization of the citrate radical as a carbon source. One should make certain that a small enough inoculum is used in this medium so that it is not noticeably turbid before incubation. This can be accompHshed by using a straight needle for inoculation or by using a loopful of a sus- pension in sterile water. Litmus milk. Prepare saturated aqueous solution of litmus. Add a sufficient quantity of the solution to give a light lavender color to fresh skimmed milk (some grades of dried milk powder may be substituted, but PREPARATION OF MEDIA 67 many are unsatisfactory); sterilize 12-15 min at 120°C; cool tubes immediately by immersing in cold water. For anaerobic organisms, Spray's system of classification is based upon use of this medium (tubed in a deep column) to which is added 0.05 g of reduced iron or a thin strip of iron. The reactions determined for aerobic bacteria on this medium are explained in Chap. VII; those for anaerobes in Spray's (1936) original paper. Pigment production. This character may be observed on a variety of media, and reports describing characteristics of new species should indicate the medium used. Starch agar (as described above) is often satisfactory, and if the organism will grow on potato slants, this medium may be used. Potato slants are prepared as follows: Peel white potatoes and cut plug from center, using cork borer of appropriate size. Slice plugs obliquely to make slants. Wash slants overnight in slowly running cold tap water. Place plugs in tube supporting them with small glass rod, stick of wood, or potato slice; add 1 ml of water to keep slants moist during incubation, and sterilize. Pigment production by Clostridia may be observed in corn-meal infusion medium (page 49) prepared without addition of liver or in potato infusion. Lipolysis. For media to be used to detect Hpolytic action and for methods of proper preparation of fat emulsions, consult the papers of Castell (1941), Castell and Bryant (1939), Collins and Hammer (1934), Eisenberg (1939), Knaysi (1941), and Starr (1941). MEDIA FOR SPECIFIC BACTERIOLOGICAL PROCEDURES In the preceding sections a variety of media have been presented which are suitable for the isolation, cultivation, characterization, and mainte- nance of bacteria. Many of these media can be employed for multiple purposes, and conversely, in some instances, several different formula- tions may prove satisfactory for the same purpose. The established per- formance of a medium and the personal preference of the laboratory worker result in the adoption of one formula in place of another. However, there are numerous bacteriological procedures which require that media of a designated composition be used. Listed in Table 3 are several such microbiological procedures, together with references which specify the composition of media required for performance of the tests. MEDIA FOR SPECIAL PURPOSES Voluminous experimental evidence attests to the fact that the composi- tion of the culture medium has a profound influence on the microbial cell with respect to formation of enzymes, toxins, antibiotics, and other 58 MANUAL OF MICROBIOLOGICAL METHODS products. Some aspects of this subject have been reviewed by Gale (1951). A few examples are presented below. Metabolically active cells. In many studies dealing with the physio- logical activity of cells harvested from culture media, comparatively little attention has been paid to the conditions of growth, a large cell crop being the usual criterion of the adequacy of the medium. Wood and Gunsalus (1942) have pointed out the limitation of this criterion and have studied the effects of the components of the growth medium upon the dehydro- genase activity of suspensions of Streptococcus mastitidis. A suitable and easily prepared medium was described consisting of the following ingredi- ents: 'Hryptone," 10 g; yeast extract, 10 g; K2HPO4, 5 g; glucose, 2 g; water, 1 liter. Metabolically active cells were obtained after incubation at 37°C for 12-15 hr. The final pH was about 6.8. Tsuchiya and Halvorson (1947) made similar studies to obtain suspensions of glyco- lytically-active Lactohacillus casei and L. arahinosus. The significance of this type of study rests upon the knowledge of the effects of each major component of the medium upon the particular physiological activity being studied, and such an analysis should precede biochemical investigation. Production of enzyme, toxin, or antibiotic. The report of Bellamy and Gunsalus (1945) on the composition of a pyridoxine-deficient growth medium for Streptococcus faecalis, which yielded cells containing large amounts of tyrosine decarboxylase apoenzyme, will serve as an example of a medium for the production of a bacterial enzyme available in a con- venient form to study coenzyme function. The composition of the medium used is described in the paper cited above; at this point, the major intention is the statement of the availability of a cultural method of great potential advantage in biochemical investigations. The production of bacterial toxins has engaged the attention of many bacteriologists. In at least one case {Clostridium perf ring ens) , one of the toxins produced by the organism is an enzyme (lecithinase) . It may be assumed, therefore, that the effect of the constituents of the medium upon toxin production is similar to the situation with respect to the pro- duction of other enzymes and may be studied effectively along parallel lines of investigation. The nutritive requirements for toxin production by the organisms of diphtheria (Mueller and Miller, 1941), botulinus (Lamanna, Eklund, and McElroy, 1946; Lamanna and Glassman, 1947; Lewis and Hill, 1947; Stevenson, Helson and Reed, 1947), tetanus (Mueller and Miller, 1943, 1948), and gas gangrene (Adams and Hendee, 1945; Logan et at., 1945) are available. The production of antibiotic compounds varies considerably with the nature of the culture medium and other factors. For a discussion of this topic consult Prescott and Dunn (1949). PREPARATION OF MEDIA Table 3. Literature References Concerning Special Media Microbiological procedure Reference for composition of media required Assay of antibiotics Grove and Randall, 1955 U.S.P. XV, 1955 59 Assay of vitamins Association of Vitamin Chemists, 1947 Barton- Wright, 1952 Johnson, 1949 U.S.P. XV, 1955 Assav of amino acids Barton- Wright, 1952 Dunn, 1947 Horn et al., 1950 Sterility testing of pharmaceutical prod- National Institutes of Health Specifica- ucts tions. U.S.P. XV, 1955 Disinfectant testing A.O.A.C, 1950 Enumeration of bacteria in milk and dairy Am. Assoc. Med. Milk Commissions, products Inc., 1954-1955 Standard Methods for the Examination of Dairy Products, 1953 Enumeration of bacteria in waters Standard Methods for the Examination of Water and Sewage, 1955 REFERENCES Adams, M. H., and E. D. Hendee. 1945. Methods for the production of the alpha and theta toxins of Clostridium welchii. J. Immunol., 51, 249-256. American Association of Medical Milk Commissions, Inc. 1954-1955. "Methods and Standards for the Production of Certified Milk." New York. American Public Health Association, American Water Works Association, and Federation of Sewage and Industrial Wastes Associations. 1955. "Standard Methods for the Examination of Water and Sewage," 10th ed., 522 pp. American Public Health Association, New York, American Public Health Association and Association of Official Agricultural Chemists. 1953. "Standard Methods for the Examination of Dairy Products," 10th ed., 315 pp. American Public Health Association, New York. Asheshov, I. N. 1941. Papain digest media and standardization of media in gen- eral. Can. J. Public Health, 32, 468-471. Association of Official Agricultural Chemists, sis of the A.O.A.C," 7th ed, 910 pp. Chemists, Washington. Association of Vitamin Chemists, Inc. 1947. Interscience Publishers, Inc., New York. Ayres, S. H., P. Rupp, and W. T. Johnson. 1919 bacteria in milk. U. S. Dept. Agr. Bull. 782. 1950. "Official Methods of Analy- Association of Official Agricultural Methods of Vitamin Assay," 189 pp. A study of the alkali-forming 60 MANUAL OF MICROBIOLOGICAL METHODS Barker, H. A., and S. M. Taha. 1942. Clostridium kluyverii, an organism con- cerned in the formation of caproic acid from ethyl alcohol. J. BacterioL, 43, 347-363. Barton- Wright, E. C. 1952. ''The Microbiological Assay of the Vitamin B Com- plex and Amino Acids," 79 pp. Pitman Publishing Corporation, New York. Beijerinck, M. W. 1921-1940. "Verzamelde Geschriften," 6 vols. M. Nijhoff, 's-Gravenhage. Bellamy, W. D., and I. C. Gunsalus. 1945. Tyrosine decarboxylase. II. Pyri- doxine-deficient medium for apoenzyme production. /. BacterioL, 50, 95-103. Bornstein, B. T., and H. A. Barker. 1948. The nutrition of Clostridium kluyveri. J. BacterioL, 55, 223-230. Brewer, J. H. 1943. Vegetable bacteriological media as substitutes for meat infu- sion media. /. BacterioL, 46, 395-396. Buck, T. C, Jr. 1949. A modified Loeffler's medium for cultivating Coryne- bacterium diphtheriae. J. Lab. Clin. Med., 34, 582-583. Carlquist, P. R. 1950. Culture Media in "Diagnostic Procedures and Reagents," 3d ed., pp. 1-48. American Public Health Association, New York. Carpenter, C. M., M. A. Bucca, T. C. Buck, E. P. Gasman, C. W. Christensen, E. Crowe, R. Drew, J. Hill, C. E. Lankford, H. E. Morton, L. R. Peizer, C. I. Shaw, and J. D. Thayer. 1949. Evaluation of twelve media for the isolation of the gonococcus. Am. J. Syphilis, Gonorrhea, Venereal Diseases, 33, 164-176. Castell, C. H. 1941. P-aminodimethylaniline monohydrochloride as an indicator of microbial action on fats. Stain TechnoL, 16, 33-36. , and L. R. Bryant. 1939. Action of microorganisms on fats. I. The signifi- cance of color changes in dyes used for the detection of microbial action on fat. Iowa State ColL J. Sci., 13, 313-328. Chapman, G. H. 1948. An improved Stone medium for the isolation and testing of food-poisoning staphylococci. Food Research, 13, 100-105. Collins, M. A., and B. W. Hammer. 1934. The action of certain bacteria on some simple tri-glycerides and natural fats, as shown by Nile-blue sulphate. /. BacterioL, 27, 473-485. Colmer, A. R. 1948. The action of Bacillus cereus and related species on the lecithin complex of egg yolk. /. BacterioL, 55, lll-l^b. Davis, J. G., and H. J. Rogers. 1939. The effect of sterilization upon sugars. Zentr. BakterioL, Abt. II, 101, 102-110. Dimmick, I. 1947. Phosphorous deficiency in relation to the nitrate reduction test. Can. J. Research, Sect. C, 25, 271-273. Dunn, M. S. 1947. Amino acids in food and analytical methods for their determi- nation. Food TechnoL, 1, 269-286. Eisenberg, G. M. 1939. A Nile blue culture medium for lipolytic micro-organisms. Stain TechnoL, 14, 63-67. Fred, E. B., I. L. Baldwin, and E. McCoy. 1932. Root nodule bacteria and legumi- nous plants. Univ. Wis. Studies in Sci., 5, 343 pp. Frobisher, M., Jr. 1953. "Fundamentals of Microbiology," 5th ed., 633 pp. W. B. Saunders Company, Philadelphia. , E. I. Parsons, E. L. Yeates, and K. L. Gay. 1948. A comparative study of tellurite plating media for Corynebacterium diphtheriae. Am. J. Hyg., 48, 1-5. Gale, E. F. 1951. "The Chemical Activities of Bacteria," 213 pp. Academic Press, Inc., New York. Gladstone, G. P., and P. Fildes. 1940. A simple culture medium for general use without meat extract or peptone. Brit. J. Exptl. Pathol., 21. 161-173. PREPARATION OF MEDIA 61 Gradwohl, R. B. H. 1948. "Clinical Laboratory Methods and Diagnosis," 4th ed., vol. 2, 1297-1721. The C. V. Mosby Company, St. Louis, Mo. Grove, D. C, and W. A. Randall. 1955. "Assay Methods of Antibiotics," 238 pp. Medical Encyclopedia, Inc., New York. Hall, L C, and E. C. Peterson. 1924. The discoloration of brain medium by anaerobic bacteria. /. BacterioL, 9, 211-224. Horn, M. J., D. B. Jones, and A. E. Blum, 1950. Methods for microbiological and chemical determinations of essential amino acids in proteins and foods. U.S. Dept. Agr. Misc. Publ. 696. Hunter, C. A., and H. G. Crecelius. 1938. Hydrogen sulfide studies. L Detection of hydrogen sulfide in cultures. /. Bacteriol, 35, 185-196. Ingelman, B., and H. Laurell. 1947. The preparation of silicic acid jellies for the cultivation of microorganisms. /. BacterioL, 63, 364-365. Johnson, B. C. 1948. "Methods of Vitamin Determination," 109 pp., Burgess Publishing Co., Minneapolis, Minn. Kellogg, D. K., and R. D. Wende. 1946. Use of a potassium tellurite medium in the detection of Corynebacterium diphtheriae. Am. J. Public Health, 36, 739-745. Knaysi, G. 1941. On the use of basic dyes for the demonstration of the hydrolysis of fat. J. Bacteriol, 42, 587-589. Lamanna, C, H. W. Eklund, and O. E. McElroy. 1946. Botulinum toxin (type A); including a study of shaking with chloroform as a step in the isolation procedure. J. BacterioL, 52, 1-13. , and H. N. Glassman. 1947. The isolation of type B botulinum toxin. /. BacterioL, 54, 575-584. Leifson, E. 1935. New culture media based on sodium desoxycholate for the isola- tion of intestinal pathogens and for the enumeration of colon bacilli in milk and water. J. Pathol. BacterioL, 40, 581-599. . 1936. New selenite enrichment media for the isolation of typhoid and paratyphoid {Salmonella) bacilli. Am. J. Hyg., 24, 423-432. 1943. Preparation and properties of bacteriological peptones. I; Enzy- matic hydrolysates of casein. Bull. Johns Hopkins Hosp., 72, 179-199. Levine, M., and H. W. Schoenlein. 1930. "A Compilation of Culture Media for the Cultivation of Microorganisms," 969 pp. The Williams & Wilkins Company, Baltimore. Lewis, K. H., and E. V. Hill. 1947. Practical media and control measures for pro- ducing highly toxic cultures of Clostridium botulinum. Type A. /. BacterioL, 63, 213-230. Lichstein, H. C, and M. H. Soule. 1944. Studies of the effect of sodium azide on microbic growth and respiration. I. The action of sodium azide on microbic growth. J. BacterioL, 47, 221-230. Logan, M. A., A. A. Tytell, I. S. Danielson, and A. M. Griner. 1945. Production of Clostridium perfringens alpha toxin. J. Immunol., 61, 317-328. McClung, L. S. 1949. Recent developments in microbiological techniques. Ann. Rev. Microbiol., 3, 395-422. , and R. Toabe. 1947. The egg yolk plate reaction for the presumptive diag- nosis of Clostridium sporogenes and certain species of the gangrene and botulinum groups. J. BacterioL, 53, 139-347. McGaughey, C. A., and H. P. Chu. 1948. The egg-yolk reaction of aerobic sporing bacilli. J. Gen. Microbial, 2, 334-340. Marshall, M. S., J. B. Gunnison, A. S. Lazarus, E. L. Morrison, and M. C. Shevky. 1947. "Applied Medical Bacteriology," 340 pp. Lea & Febiger, Philadelphia. 62 MANUAL OF MICROBIOLOGICAL METHODS Mueller, J. H., and E. R. Johnson. 1941. Acid hydrolysates of casein to replace peptone in the preparation of bacteriological media. /. Immunol., 40, 33-38. , and P. A. Miller. 1941. Production of diphtheric toxin of high potency (100 Lf) on a reproducible medium. /. Immunol., 40, 21-32. , and . 1943. Large scale production of tetanal toxin on a peptone- free medium. J. Immunol., 47, 15-22. and . 1948. Unidentified nutrients in tetanus toxin production. /. BacterioL, 56, 219-233. Paulson, M. 1937. The clinical use of desoxycholate and desoxycholate-citrate agars — new culture media — ^for the isolation of intestinal pathogens. Am. J. Med. Sci., 193, 688-690. Petran, E. 1948. Trypticase tellurite as a primary plating medium for the identi- fication of C. diphtheriae. Public Health Lab., 6, 39-40. Prescott, S. G., and C. G. Dunn. 1949. "Industrial Microbiology," 2d ed., 923 pp. McGraw-Hill Book Company Inc., New York. Schaub, I. G., and M. K. Foley. 1952. ''Diagnostic Bacteriology: A Textbook for the Isolation and Identification of Pathogenic Bacteria," 4th ed., 356 pp. The C. V. Mosby Company, St. Louis, Mo. Simmons, J. S., and C. J. Gentzkow. 1955. "Medical and Public Health Labora- tory Methods." Lea & Febiger, Philadelphia. Society for General Microbiology. 1956. Constituents of bacteriological culture media. Special Report, edited by G. Sykes. Cambridge University Press, London. Spray, R. S. 1936. Semi-solid media for cultivation and identification of the sporulating anaerobes. J. BacterioL, 32, 135-155. Starkey, R. L. 1935. Isolation of some bacteria which oxidize thiosulfate. Soil Sci., 39, 197-219. Starr, M. P. 1941. Spirit blue agar: A medium for the detection of lipolytic micro- organisms. Science, 93, 333-334. Sterges, A. J. 1942a. Adaptability of silica gel as a culture medium. /. BacterioL, 43, 317-327. . 19426. Simplified method for the preparation of silica gels. /. BacterioL, 44, 138. Stevenson, J. W., V. A. Helson, and G. B. Reed. 1947. A casein digest medium for toxin production by Clostridium. Can. J. Research, Sect. E., 25, 9-13. Stitt, E. R., P. W. Clough, and S. E. Branhara. 1948. "Practical Bacteriology, Hematology and Parasitology," 10th ed., 991 pp. The Blakiston Division, McGraw-Hill Book Company, Inc., New York. Sulkin, S. E., and J. C. Willett. 1940. A triple sugar-ferrous sulfate medium for use in identification of enteric organisms. /. Lab. Clin. Med., 25, 649-653. Temple, K. L. 1949. A new method for the preparation of silica gel plates. /. BacterioL, 57, 383. Tittsler, R. P., and L. A. Sandholzer. 1936. The use of semi-solid agar for the detection of bacterial motility. /. Bacterial, 31, 575-580. Tsuchiya, H. M., and H. O. Halvorson. 1947. The preparation of glycolytically active washed cells of lactobacilli. ,/. BacterioL, 53, 719-727. United States Pharmacopeia XV. 1955. Untermohlen, W. P., Jr., and C. E. Georgi. 1940. A comparison of cobalt and nickel salts with other agents for the detection of hydrogen sulfide in bacterial cultures. /. BacterioL, 40, 449-459. PREPARATION OF MEDIA 63 Van Niel, C. B. 1944. The culture, general physiology, morphology, and classifi- cation of the non-sulfur purple and brown bacteria. Bacteriol. Revs., 8, 1-118. . 1949. The " Delft School " and the rise of general microbiology. Bacteriol. Revs., 13, 161-174. Vaughn, R. H. 1942. The acetic acid bacteria. Wallerstein Labs. Communs., 5, (14), 5-26. , and M. Levine. 1936. Hydrogen sulfide production as a differential test in the colon group. J. Bacteriol., 32, 65-73. Vera, H. D. 1948. A simple medium for identification and maintenance of the gonococcus and other bacteria. /. Bacteriol., 55, 531-536. . 1949. Accuracy and sensitivity of fermentation tests. Soc. Am. Bac- teriologists, Abstr. Papers, 49th gen. meeting, p. 6. Wadsworth, A. B. 1947. "Standard Methods of the Division of Laboratories and Research of the New York State Department of Health," 3d ed., 990 pp. The Williams & Wilkins Company, Baltimore. Whitley, O. R., and S. R. Damon. 1949. Raffinose serum tellurite agar slants as a replacement for LoeflEler's medium in diphtheria diagnosis. Public Health Rept., 64, 457-460. Wood, A. J., and I. C. Gunsalus. 1942. The production of active resting cells of streptococci. /. Bacteriol., 44, 333-341. APPENDIX TO CHAPTER III Specifications for Bacteriological Grade Agar, Gelatin, and Peptones BACTERIOLOGICAL GRADE AGAR^ Suggested Specifications Definition. Agar is any phycocoUoid derived from Rhodophyceae which meets the requirements given below for gelation temperature and gel melting temperature. Bacteriological grade agar meets all the stated requirements. Forms. Bacteriological grade agar shall be in the form of shreds, flakes, strips, sheets, or granules. Table 4. Requirements Determination Total solids Solubility, cold, % Solubility, hot, % Gelation temperature, °C Gel melting temperature, °C Rate of dissolution, min Sol turbidity, ppm Threshold gel concentration, % Protein nitrogen, % Reducing substances as galactose, % Chlorides as NaCl, % Viable spores, per g Debris count, per g Limits Max Min 78 2.0 99.8 39 33 70 15 10 0.25 0.32 10 1.5 3 30 Analytical Methods Total solids. Dry accurately weighed duplicate samples of 0.6-1.0 g for 5 hr at 105°C. Cover, cool to ambient temperature in desiccator, and weigh. Caution: ^ These specifications were prepared by Mr. H. H. Selby of the American Agar and Chemical Co., San Diego 12, California. They were originally presented to the Society of American Bacteriologists Committee on Bacteriological Technic at the 53d general meeting, San Francisco, California, 1953. 64 BACTERIOLOGICAL GRADE AGAR, GELATIN, AND PEPTONES 65 Agar itself is a good desiccant when dry. Weighings should be made promptly after cooling and done rapidly. An efficient desiccant is essential. Solubility, cold. Dissolve 1.5 g of solids (1.87 g of agar of 80 per cent of total solids, for example) in 100 ml of H2O by autoclaving at 120°C for 20 min in 250-ml tared flask. Remove flask at 99-100°C, swirl thoroughly to mix, and make up to 100 g net. Take 15.2-15.5 ml of solution into Luer-type syringe. Cap with pinched-off needle. Weigh to 0.1 g. Uncap syringe, and eject contents slowly into 50-mm aluminum-foil dish. Recap Luer, and reweigh. Difference is weight of aliquot (A). After 15 min cut gel freehand with knife into 3- to 4-mm squares and place dish in 3- to 5-mm layer of H2O in ice-cube tray or equivalent. Put tray on 1- to 2-mm cardboard in freezing compartment at —5 to — 10°C. Hold overnight. Transfer dish contents to petri plate, break into squares with single-edged razor blade, and retransfer to 30-ml medium-porosity fritted glass crucible. Wash with four 10-ml portions of H2O at 10-15''C, using suction at end of each wash. Stir and gently press flakes with flat- tened rod while washing. Evaporate combined washings in porcelain dish tared to 0.1 mg on steam bath. Dry residue 2 hr at 105°C, cool in desiccator, and weigh. Residue is B. Per cent cold solubles = -^— j — A Hot-water solubles. Dissolve 1.5 g of solids in 100 of ml H2O at 120°C for 20 min. Swirl at 99-100°C. Filter at 80-90°C through fine-porosity 30-ml fritted glass crucible tared to 0.1 mg. Wash flask and crucible three times with 25 ml of 80-90°C H2O. Dry crucible 2 hr at 105°C, and weigh. Net is C. Run a blank to obtain cor- rection for solubility of sintered glass membrane (D). Correction may approach 1 mg. T> .u . 1 u^ 100[1.5 - {C + D)] Per cent hot solubles = ^-= 1.0 Gelation temperature. Dissolve 1.5 g of solids in 100 ml of H2O at 120°C for 20 min. Swirl at 99-100°C. Cool to 60-70°C, and make to 100 g net. Place 15 ml in 15- by 150-mm test tube, and insert calibrated Weston-type dial thermometer. Insert 2- to 3-mm-OD glass tube with 90° bent tip and 0.5-mm orifice. Run air through tube at }i to 1 bubble per second. Note temperature at which course of rising bubbles becomes impeded. Gel melting temperature. Dissolve 2.0 g of solids in 100 ml of H2O at 120°C for 20 min. Swirl at 99-100°C. Cool to 60-70°C, and make to 100 g net. Place 10 ml in 15- by 150-mm test tube. Stopper, and support tube at 30° angle. Hold 1 hr at 15-25°C. Place tube vertically in 70°C water bath 1 hr. If slant does not collapse, sample passes test. Rate of dissolution. Arrange a 500-ml spherical three-necked flask with reflux condenser, sealed agitator with circular, segment paddle, and stopper. Place 200 ml of hot water in flask, start agitator, and so place and adjust a bunsen burner that the flame covers approximately half the wetted portion of the flask and maintains slow but definite ebullition (3 to 5 drops per second). Place 3 g of solids in length of glass tubing small enough to enter neck of flask. Provide a glass-rod-rubber-stopper plunger for tubing. Force sample from tube into flask with plunger in such a manner that particles do not become attached to flask. Restopper flask. Stop heating and agitation after 10 min. If no undissolved agar remains in liquid, sample passes test. Sol turbidity. Dissolve 2.0 g of solids in 100 ml of H2O at 120°C for 20 min. Swirl at 99-100°C. Cool to 60-70°C, and make to 100 g net. Stopper, and hold at 45- 47°C for 3 hr. Simultaneously hold the glassware of a Jackson Coleman or Hellige 66 MANUAL OF MICROBIOLOGICAL METHODS turbidimeter at 45-60 °C. Run turbidity estimation as with waters, using standard notation based on turbidities equivalent to Si02 content in parts per million. Threshold gel concentration. Use solution from gel-melting-temperature deter- mination. To duplicate 140-mm lengths of 14.5- to 15.5-mm-ID glass tubing, stoppered at one end, add 17.5-ml portions of 70-90°C H2O, using Luer-type syringe without needle. Immediately add 2.5-ml portions of sample solution with syringe, making 20 ml per tube. Stopper, and mix by inverting six times. Hold tubes 1 hr at 20-25°C, then 1 hr at 0-5°C, and, finally, 1 hr at 20-25°C. Remove top stoppers, and gently add 3-1 ml of H2O to tubes. Taking each tube individually, lay on hori- zontal wooden surface, remove bottom stopper, and induce cylinder of gel to slide from tube by elevating bottom end slightly and moving tube backward as cylinder moves forward relative to tube. Object is to remove tube without motion of gel with respect to wood. If neither cylinder of gel breaks under pull of gravity in 10 sec, sample passes test. Protein nitrogen. Use Hen wood & Garey (Hengar) modification of Kjeldahl method with 0.1 g of solids. Not more than 0.32 per cent of N (approximately 2 per cent protein) shall be found after correcting for reagents by running a blank on sucrose. Reducing substances as galactose after autoclaving. Dissolve 1 g of solids in 100 ml of H2O in 500-ml short-neck spherical flask containing one 3-mm glass bead. Use 120°C for 40 min. Swirl at 99-100°C. Transfer immediately to flame or heater preadjusted to maintain a steady boil. Add 0.2 g of NaOH and 4 mg of methylene blue chloride, dry or as freshly made solution. Close flask with two-hole stopper, one hole of which holds 90° vent tube. Titrate through open stopper hole to blue which is stable 0.5 min. Total titration time 2.5 ± 0.25 min. Use Soxhlet's solutions standardized according to National Bureau of Standards Circular 0440, page 187, 10 ml of solution A plus 10 ml of solution B diluted to 100 ml on day of use. No more titrating solution must be needed by the sample than the quantity necessary to react with 80 mg of Eastman galactose No. 141 dried to constant weight over boiled H2SO4 and carried through the same procedure. Chlorides as NaCl. Blend 1 g of solids in 200 ml of H2O in Waring blender or equivalent device provided with rheostat or auto transformer speed control. Start slowly to avoid splash. Blend, covered, at top speed until visually textureless. Cut speed to point of splash-free agitation; rinse top and sides into mixture. Add 2 ml of 10 per cent K2Cr04, and titrate in blending jar with 0.0171 A^ AgNOs to an end point permanent for 1 min. Subtract the value of a blank run on 200 ml of water. Each milliliter of AgNOs solution represents 0.1 per cent of NaCl. Viable spores. Transfer 2-g sample as received to 100 ml of sterile tryptone- glucose-beef extract broth in 6-oz screw-capped prescription bottle, using sterilized vegetable parchment for weighing and transferring. Close bottle tightly, and auto- clave at 115°C for 5 min. Agitate gently at 90-95°C, cool to 50-60°C, add 1 ml of sterile skim milk, and pour entire charge into three petri dishes, glass-covered. Invert when cool, and incubate at 35°C for 36 hr. Divide total colonies visible in Quebec- type counter by 2 to get spores per gram. Debris count. Dissolve 1.5 g of solids in 100 ml of H2O at 120°C for 20 min. Swirl at 99-100°C, and immediately pour entire charge into two 90-mm petri dishes. Cover with porous tops, and allow to congeal. Examine plates on Quebec-type colony counter in dim light at standard magnification of 1.5 diam, adjusting lens posi- tion for maximum resolution. Note all objects which a trained microbiologist might mistake for a microbial colony. To avoid the counting of bubbles, examine each object found with a 7.5 X magnifier without moving petri dish or 1.5 X lens. The smaller magnifier is to be used for examination only, not for counting. Count only true debris particles clearly visible at 1.5 X. Run a control with each series of BACTERIOLOGICAL GRADE AGAR, GELATIN, AND PEPTONES tj7 samples, omitting agar. Divide total number of counted particles by 1.5 to obtain count per gram. GELATIN^ ''Gelatin is a product obtained by the partial hydrolysis of collagen derived from the skin, white connective tissue, and bones of animals. Gelatin derived from an acid-treated precursor exhibits an isoelectric point between pH 7 and pH 9, known as Type A, while Gelatin derived from an alkali-treated precursor has an isoelectric point between pH 4.7 and pH 5, known as Type B. "Description. Gelatin occurs in sheets, flakes, or shreds, or as a coarse to fine powder. It is faintly yellow or amber in color, the color varying in depth according to the particle size. It has a slight, characteristic bouillon-like odor. It is stable in air when dry, but is subject to microbic decomposition when moist or in solution. ''Solubility. Gelatin is insoluble in cold water, but swells and softens when immersed in it, gradually absorbing from 5 to 10 times its own weight of water. It is soluble in hot water, in acetic acid, and in a hot mixture of glycerin and water. It is insoluble in alcohol, in chloroform, in ether, and in fixed and volatile oils. "Identification A. To a solution of Gelatin (1 in 100) add trinitrophenol T.S. or a solution of potassium dichromate (1 in 15) previously mixed with about one-fourth its volume of diluted hydrochloric acid: a yellow precipitate is formed. "Identification B. To a solution of Gelatin (1 in 5000) add tannic acid T.S.: turbidity is produced. "Residue on ignition, page 912 — Incinerate 5.0 Gm. of Gelatin without the use of sulfuric acid: the weight of the residue does not exceed 100 mg. (2 per cent). Save the residue. "Odor and water — insoluble substances — A hot solution of Gelatin (1 in 40) is free from any disagreeable odor, and when viewed in a layer 2 cm. thick is only slightly opalescent. "SuZ^fe— Dissolve 20 Gm. of Gelatin in 150 ml. of hot water in a flask having a round bottom and a long neck, add 5 ml. of phosphoric acid and 1 Gm. of sodium bicarbonate, and at once connect the flask with a condenser. Distil 50 ml., receiving the distillate under the surface of 50 ml. of 0.1 N iodine. Acidify the distillate with a few drops of hydrochloric acid, add 2 ml. of barium chloride T.S., and heat on a steam bath until the liquid is nearly colorless. The precipitate of barium sulfate, if any, when filtered, washed, and ignited, weighs not more than 3 mg., corresponding to not more than 40 parts per million of sulfur dioxide, correction being made for any sulfate which may be present in 50 ml. of the 0.1 iV iodine. "Arsenic — Heat 15 Gm. of Gelatin with 60 ml. of dilute, arsenic-free hydrochloric acid (1 in 4) in a covered flask until all insoluble matter is flocculated and the Gelatin dissolved. Add an excess of bromine T.S. (about 15 ml.), and heat until the excess bromine is expelled. Neutralize with ammonia T.S., add 1.5 Gm. of sodium phos- phate, and allow to cool. Add a slight excess (about 30 ml.) of magnesia mixture T.S., allow to stand for 1 hour, filter, and wash with five 10-ml. portions of ammonia T.S. diluted with 3 volumes of water. Drain the precipitate well, and dissolve it in dilute hydrochloric acid (1 in 4) to make exactly 50 ml. Subject 5 ml. of this solution to the test for Arsenic, page 803 : the stain, if any, does not exceed in length or intensity of color that produced in a test made with similar quantities of the same reagents and 1.5 ml. of the standard arsenic test solution (1 part per million). 1 Quoted (with permission) from U.S.P. XV, pp. 305-306. 68 MANUAL OP MICROBIOLOGICAL METHODS "Heavy metals, page 898 — To the residue obtained in the test for "Residue on igni- tion add 2 ml. of hydrochloric acid and 0.5 ml. of nitric acid, and evaporate to dryness on a steam bath. To the residue add 1 ml. of 1 A^ hydrochloric aci^ and 15 ml. of water, and warm for a few minutes. Filter, and wash with water to make the filtrate measure 50 ml. To 25 ml. of the filtrate add 10 ml. of hydrogen sulfide T.S.: the heavy metals limit for Gelatin is 50 parts per million. *'Gel strength — Place 1 Gm. of Gelatin, accurately weighed, and 99 ml. of water in a 200-ml. flask, allow to stand for 15 minutes, place the flask in a water bath at 60°, and swirl occasionally until solution is complete. Transfer 10 ml. of the solution to a test tube having an internal diameter of 12 mm., and place the tube in an ice bath, making certain that the top of the solution is below the level of the ice and water. Place the bath containing the tube in a refrigerator, and maintain it at about 0° for 6 hours. When the tube is removed from the bath and inverted, no movement of the gel is observed. "Bacterial content — ^Vhen Gelatin is examined as directed under Gelatin — Bacterio- logical Test, page 839, the total bacterial count does not exceed 10,000 per Gm., and coliform bacteria are not present in 10 mg. or less. Gelatin — Bacteriological Test ''Preparation of sample — Employ aseptic conditions throughout. If the Gelatin is in sheets, flakes, or shreds, reduce it to a powder under aseptic con- ditions in a sterile grinder or mortar. Mix thoroughly and weigh 1 Gm. of the powdered sample into a sterile dilution bottle containing 99 ml. of sterile water. After the Gelatin is thoroughly wetted, place in a water bath heated to between 40° and 45°. Shake well, and allow not more than 15 minutes for solution. "Total count — Using a sterile, 1-ml. pipet, place 1 ml. of the well-shaken Gelatin solution in each of two sterile Petri dishes 10 cm. in diameter and 15 mm. in depth. Promptly add 10 ml. of liquefied Tryptone Glucose Yeast Agar or Milk Protein Hydrolysate Glucose Agar warmed to 40°. Cover the dishes, and mix thoroughly the gelatin solution with the added medium by tilting and rotating the dishes. Allow the contents to solidify as promptly as possible, invert the dishes, and incubate them at 35° to 37° for 48 hours. Using a lens of 25 diameters magnification and of about 78-mm. focal length, count the colonies: the average of the two plates does not exceed 100 colonies (10,000 organisms per Gm.). "Coliform bacteria — Using a sterile, 1-ml. pipet, place 1 ml. of the well-shaken Gelatin solution in each of two fermentation tubes containing Lactose Broth. Incu- bate at 35° to 37°, and examine the tubes at the end of 24 and 48 hours: coliform bacteria are absent if no gas is produced. If gas is produced, transfer a culture there- from as soon as possible after gas formation is observed. Streak the culture upon Eosin-methylene-blue Agar and incubate at 35° to 37° for 24 hours. If typical coliform colonies have appeared, transfer a culture from at least two of them both to agar slants and to fermentation tubes containing Lactose Broth. If typical coliform colonies have not developed in 24 hours, continue incubation for another 24 hours, and select at least two of the colonies considered most likely to be species of the coli- form group, and transfer as directed above. Incubate the Lactose Broth at 35° to 37° for 24 to 48 hours. No gas is produced. Incubate the agar at 35° to 37° for 24 hours, and examine the growth microscopically following Gram-staining: no Gram-negative, non-sporulating bacilli are observed. "Formation of gas in the lactose broth and demonstration of Gram-negative, non- sporulating bacilli confirm the presence of coliform bacteria." In addition to the U.S. P. XV specifications as given above, gelatin should conloxT' BACTERIOLOGICAL GRADE AGAR, GELATIN, AND PEPTONES 69 to the following requirements as set forth in the Military Medical Purchase Descrip- tion, ASMPA 1-212-000, dated 14 September 1953: "A 12 per cent solution in distilled water, after autoclaving for 15 minutes at 121 C, shall be clear and free of gross suspended particles. After solidifying, it shall melt over a range of from 30 to 36 C. "It shall be free of fermentable carbohydrate when tested under the following conditions: "Prepare a medium of 4 per cent Gelatin and 0.3 per cent NaCl with sufficient phenolsulphonthalein added to give a readable color, tube in Durham fermentation tubes and autoclave 15 minutes at 121 C. Inoculate with a loop of a 24-hour culture of Escherichia coli. Neither acid nor visible gas shall be produced in 48 hours incuba- tion at 37 C." PAPAIC DIGEST OF SOYBEAN MEAL A soluble nutrient material prepared by the action of the enzyme papain on soybean meal followed by suitable purification and concentration. It meets the specifications under Pancreatic Digest of Casein, except that it shows substantial amounts of reduc- ing sugars. It contains fermentable carbohydrates and gives positive tests for indole, acetylmethylcarbinol, and sulfide upon inoculation and incubation with the specified organisms. (These specifications are essentially those contained in U.S. P. XV, page 1026, except for changes in wording.) PEPTIC DIGEST OF ANIMAL TISSUE (A BACTERIOLOGICAL PEPTONE)^ "A tan powder, having a characteristic, but not putrescent, odor. Soluble in water; insoluble in alcohol and in ether. An autoclaved solution (2 in 100) is clear and is neutral or nearly so in its reaction. "Degree of digestion. Dissolve 1 Gm. in 10 ml. of water, and use this solution for the following tests: " (a) Overlay 1 ml. of the digest solution with 0.5 ml. of a solution of 1 ml. of glacial acetic acid in 10 ml. of diluted alcohol: no ring or precipitate forms at the junction of the two liquids, and on shaking, no turbidity results, indicating the absence of undigested protein. "(6) Mix 1 ml. of the digest solution with 4 ml. of saturated zinc sulfate: a small amount of precipitate is formed, indicating the presence of proteoses. Retain the filtrate. " (c) To 1 ml. of the filtrate from the preceding test add 4 drops of bromine T.S.: the light yellow color changes to a red-brown, indicating the presence of tryptophane. "Nitrogen content, loss on drying, residue on ignition, and nitrite. Proceed as directed under Pancreatic Digest of Casein. "Microbial content. Dissolve 1 Gm. in 10 ml. of water. Spread 0.01 ml. on one square centimeter of a glass slide. Stain by the Gram method, and examine with an oil-immersion lens: not more than a total of 50 microorganisms, or clumps, are visible in 10 consecutive fields. "Bacteriologic test. It meets the following tests for bacteria-nutrient properties. Prepare media of the following compositions : " (a) 2 % of peptone and sufficient phenol red T.S. to give a perceptible color in water " (6) 0.1 % of peptone in water 1 Quoted from U.S. P. XV, pp. 1024-1027. 70 MANUAL OF MICROBIOLOGICAL METHODS " (c) 0.1 % of peptone and 0.5 % of dextrose in water '* (d) 1 % of peptone in water ''Adjust all media to a final pH of 7.2 to 7.4. Place 5 ml. of (a) in Durham fermen- tation tubes, and 5 ml. each of (6), (c), and (d) in ordinary test tubes. Autoclave the media at 121° for 15 minutes. After autoclaving, and after standing for 24 hours, all media are clear. ''Presence of fermentable carbohydrate. Inoculate medium (a) with Escherichia coli and with Streptococcus liquefaciens : acid is produced by E. coli but not by S. liquefaciens during incubation for 24 hours. ''Production of indole. Inoculate medium (6) with Escherichia coli and with Aero- bacter aerogenes, and incubate for 24 hours. Test by adding about 0.5 ml. of p-di~ methylaminobenzaldehyde T.S.: the appearance of a pink or red color (soluble in chloroform) indicates the production of indole by E. coli. The A. aerogenes culture gives a negative test. "Production of acetylmethylcarbinol. Inoculate medium (c) with Escherichia coli and with Aerohacier aerogenes, and incubate for 24 hours. Test by adding to the culture an equal volume of sodium hydroxide solution (1 in 10), shaking well, and allowing to stand at room temperature for several hours: the appearance of a pink color indicates the production of acetylmethylcarbinol by A. aerogenes. The E. coli culture gives a negative test. "Production of hydrogen sulfide. Inoculate medium {d) with Salmonella iyphosa. Hold a strip or loop of lead acetate test paper between the cotton plug and the mouth of the test tube so that it hangs about 5 cm. above the medium. Then incubate for 24 hours: the lower part of the lead acetate test paper shows an appreciable amount of brownish blackening (lead sidjide). PANCREATIC DIGEST OF CASEIN (A BACTERIOLOGICAL PEPTONE) "A grayish yellow powder, having a characteristic, but not putrescent, odor. Freely soluble in water; insoluble in alcohol and in ether. The casein used in prepara- tion of this digest meets the following specifications: Residue on ignition not more than 2.5 % Loss on drying not more than 8 % Free acid (as lactic acid) not more than 0.25% Fat not more than 0.5 % Reducing sugars not more than a trace Fineness All passes through a 20 mesh sieve "Degree of digestion. Dissolve 1 Gm. in 10 ml. of water. " (o) Overlay 1 ml. of the digest solution with 0.5 ml. of a solution of 1 ml. of glacial acetic acid in 10 ml. of diluted alcohol: no ring or precipitate forms at the junction of the two liquids, and when shaken no turbidity results (indicating the absence of undigested casein). " (6) Mix 1 ml. of the digest solution with 4 ml. of a saturated solution of zinc sulfate: a moderate amount of precipitate is formed (indicating the presence of proteoses). Filter, and retain the filtrate for the next test. " (c) To 1 ml. of the filtrate from (6) add 3 ml. of water, and follow with 1 drop of bromine T.S.: a violet-red color is produced, indicating the presence of tryptophane. "Nitrogen content. Determine the nitrogen content of the digest, previously dried at 105° to constant weight, by the Kjeldahl method (see page 909) : not less than 10 % of nitrogen (N) is found. BACTERIOLOGICAL GRADE AGAR, GELATIN, AND PEPTONES 71 "Loss on drying. Weigh accurately about 1 Gm., and dry at 100'^ to constant weight: it loses not more than 7% of its weight. "Residue on ignition. Weight accurately about 500 mg., and heat slowly until thoroughly charred. Cool, add 1 ml. of sulfuric acid, and ignite to constant weight: the weight of the residue corresponds to not more than 15%. "Nitrite. To 5 ml. of a solution of the digest (1 in 50) add 0.5 ml. of sulfanilic- a-naphthylamine T.S., mix, and allow to stand for 15 minutes: no pink or red color develops. "Bacteriological test. The digest meets the following tests for bacteria-nutrient properties. Prepare media of the following compositions: " (a) 2 % of peptone, in water; " (b) 0.1 % of peptone, in water; " (c) 1 % of peptone, 0.5 % of dextrose, in water; " (d) 1 % of peptone, in water; " (e) 2 % of peptone, 1.5 % of agar, in water. ''Adjust all media to a pH of 7.2 to 7.4. "Freedom from fermentable carbohydrate. To medium (a) add sufficient phenol- sulfonphthalein T.S. to give a readable color, tube in Durham fermentation tubes, and autoclave. Inoculate with a loop of 24-hour culture of Escherichia coli: no acid, or only a trace in the inner tube, and no gas are produced during incubation for 48 hours. "Production of indole. Inoculate 5 ml. of medium (h) with Escherichia coli, incu- bate for 24 hours, and test by addition of about 0.5 ml. of dimethylaminobenzaldehj-de T.S.: it shows a distinct pink or red color which is soluble in chloroform. "Production of acetylmethylcarbinol. Inoculate 5 ml. of medium (c) with Aero- bacter aerogenes, and incubate for 24 hours. Test by adding to the culture an equal volume of sodium hydroxide solution (1 in 10), shake, and allow to stand at room tem- perature for several hours: appearance of a pink color indicates the presence of acetylmethylcarbinol. "Production of hydrogen sulfide. Inoculate 5 ml. of medium (d) with Salmonella typhosa. Hold a strip or loop of lead acetate test paper between the cotton plug and the mouth of the test tube so that it hangs about 5 cm. above the medium. After incubation for 24 hours, the lower tip of the lead acetate test paper shows little if any darkening. After 48 hours, it shows an appreciable amount cf brownish blackening (lead sulfide) . "Growth-supporting properties. In the foregoing tests the media support good growth of Escherichia coli, Aerohacter aerogenes, and Salmonella typhosa. Medium (e) stab-inoculated with a stock culture of Brucella abortus shows good growth in the line of the stab after incubation for 48 hours. Slants of medium (e), inoculated with Escherichia coli, Aerobacter aerogenes. Salmonella typhosa, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus albus, show characteristic growth after incubation for 24 hours. Medium (e), to which about 5% of rabbit blood has been added and which has been inoculated and poured into Petri dishes, show^s character- istic alpha or beta zones about colonies of pneumococci and beta hemolytic streptococci (serological groups A and B), recognizable within 24 hours and fully developed after 48 hours' incubation. Medium (e), to which about 10 % of blood has been added and which then has been heated to 80 to 90° until the blood has turned chocolate-brown, permits the growth of gonococcus colonies within 48 hours when incubated in an atmosphere containing about 10 % of carbon dioxide." CHAPTER IV The Measurement of pH, Titratable Acidity, and Oxidation-reduction Potentials^ Barnett Cohen^ THE MEASUREMENT OF pH Originally, pH was defined as the logarithm of the reciprocal of the hydrogen-ion concentration. However, certain assumptions regarding indeterminate factors enter the theoretical treatment of any method of measuring this quantity. It is now recognized that the pH scale is standardized on a basis that is arbitrary with respect to a small and indeterminate uncertainty, although any pH number closely approxi- mates the logarithm of the reciprocal of the corresponding hydrogen-ion activity. The activity of any substance is virtually the product of that substance's molar concentration and a factor called the activity coeffi- cient. This factor expresses the departure from that behavior which would obtain were there no van der Waals and Coulomb (attraction and repulsion) forces operating. The common methods for the measurement of pH are of two types: (1) potentiometric and (2) colorimetric. The theoretical and practical aspects of the subject are treated extensively in the monograph by Clark (1928). Potentiometric Methods The several potentiometric methods to be cited depend upon the fact that the pH of a solution suitably incorporated in a so-called half-cell is proportional to the electric potential difference established between this half-cell and some reference half-cell used as a standard. ^ This presentation is confined to the brief description of general procedures that may be applied in the bacteriological laboratory. For theoretical discussions and the elaboration of detail, the reader should consult the texts, monographs, and original references cited. * Deceased. 72 THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 73 The hydrogen electrode method. This is regarded as the basic experi- mental method whereby the various other methods are standardized. It consists in the measurement of the potential difference (emf) estab- lished under conditions of maximum work between the ''hydrogen half- cell/' or ''hydrogen electrode," and a calomel or other half-cell which is employed as a working standard. The standard reference half-cell is usually a calomel electrode. The hydrogen half-cell consists of a suitable vessel provided with (1) a platinum foil electrode, coated with platinum-black, which is immersed or intermittently dipped in the solution to be measured, and (2) an inlet and outlet for oxygen-free hydrogen to saturate both solution and electrode at atmospheric pressure. A convenient reference half-cell is the ''saturated calomel electrode" which consists of a vessel containing a layer of purified mercury covered with a paste of calomel (HgoCh), mercury, and saturated KCl solution; the calomel paste is layered with crys- tals of KCl, and the rest of the vessel is filled with saturated KCl solution which has been saturated with calomel. A platinum wire provides the electrical lead to the mercury of the calomel cell, and a siphon containing saturated KCl solution provides liquid junction with the solution to be measured in the hydrogen half-cell. In the normal hydrogen half-cell, which provides the standard of potential for all measurements of potential in electrochemistry, the hydrogen partial pressure is one normal atmosphere and the hydrogen ions are at unit activity. The potential differ- ence between electrode and solution in the normal hydrogen half-cell is assumed to be zero at all temperatures. In standardizing the pH scale by means of measurements with a cell composed of a hydrogen half-cell and a saturated KCl calomel half-cell, it is customary to ignore the small and indeterminate liquid junction potential between the saturated solution of KCl and the solution in the hydrogen half-cell. The combination of the two half-cells to make an electric cell is indicated schema- tically as follows: (Pt)H2; H+ in solution X | sat. KCl | sat. KCl; HgsCh; Hg (Pt) Hydrogen KCl sat. calomel (reference) electrode bridge electrode For a pH determination, purified hydrogen is bubbled, through the test solution to saturate it and the platinized platinum electrode until equi- hbrium is attained as indicated by constancy of the emf determined potentiometrically between the metal terminals of the hydrogen and the calomel half-cells. The observed emf, in volts, ^ is converted to pH by the following equation, where T is the absolute temperature. TT _ Observed emf — emf of calomel cell _ Eh ,. ^ " 0.000,198,3227^ ~ 0.000, 198,322^ ^ ' ^ The electrical units employed herein are based on the "international" system in which, according to the National Bureau of Standards, 1 international volt (U.S.) equals 1.00033 absolute volts. The Bureau has announced that, as of January 1, 1948, absolute electrical units will supersede international units. However, the effect of this new convention for potentiometry is to introduce change? which may be regarded as negligibly small in ordinary measurements of pH and oxida- tion-reduction potentials. For example, in Eq. (2) — A^^/AkH equals 0.05912 inter- national volt and 0.05914 absolute volt, at 25°C (298.1° absolute). 74 MANUAL OF MICROBIOLOGICAL METHODS For this equation to be applicable, the temperature must be constant. For precise measurements, a correction must be made for any departure of the hydrogen partial pressure from one atmosphere. The correction seldom exceeds 0.001 volt (0.017 unit of pH) for the ordinary ranges of barometric pressure and vapor pressures of solutions. As indicated by Eq. (2), ApH = 0.000,198,322^ (2) the slope of the straight line relating potential to pH is a constant depend- ent on the absolute temperature. For example, at 25°, the potential of the hydrogen electrode becomes more negative by 0.0591 volt^ for each unit increase in pH. Values of this constant at certain temperatures are shown as constant A on page 75. Standardization of the saturated calomel half-cell. For ordinary meas- urements, the values at different temperatures of the saturated calomel half-cell, referred to the normal hydrogen half-cell, are as follows: °c Ecal, volts °C Ecal, volts 20 25 30 0.250 0.246 0.242 35 38 40 0.238 0.236 0.234 The potential of this half-cell after continued use may change as a result of dilution and contamination, and it is advisable to check its value regularly as a routine procedure. The precise standardization of the calomel half-cell is discussed in detail by Clark (1928). It consists in measuring the potential of this half-cell against the hydrogen electrode in a solution of known hydrogen-ion activity or against other carefully con- structed half-cells of reproducible, known potential. For measurements of ordinary precision, the quinhydrone electrode (see below) in O.IA^ HCl can serve for standardi- zation of the calomel half-cell. The quinhydrone electrode. Ignoring refinements and minor details, we may state that the potential of a noble-metal electrode in an acid or neutral solution saturated with quinhydrone varies linearly with the pH of the solution, and this so-called quinhydrone electrode may, therefore, be used to measure the pH of such solutions. The linear relationship of potential to pH holds only for acid and neutral solutions to about pH 8. In more alkaline solutions two effects disturb this regularity. One is the ionization of the reductant, and the other is deterioration of the components of the system. The quinhydrone electrode, within its range of usefulness, may often be employed in cases where the hydrogen electrode cannot be applied. ^ See footnote on page 73. THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 75 It comes to equilibrium rapidly, and its manipulation is simple and con- venient. Consult Clark (1928) for fuller details. Its utilization may be illustrated in the standardization of the satu- rated calomel half-cell. The potential Ecai of this half-cell is to be deter- mined relative to that of a standard solution of fixed pH and saturated with quinhydrone, e.g., 0.1 M HCl, the pH of which is 1.082 at 38°. This is done with purified quinhydrone and accurately prepared HCl solution as follows. Place about 5 ml of the standard HCl solution in a suitable electrode vessel. Add 50-100 mg of quinhydrone crystals to saturate the solution; some quinhydrone in the solid phase must be present. Insert a clean platinum or gold electrode preferably in contact with the solid phase at the bottom of the vessel. Then join this half-cell with the calomel half-cell by means of a siphon containing saturated KCl solution, bring the system to constant temperature, and measure the potential which should reach a constant value in a few minutes. The observed potential, Fobs, is related to the potential of the calomel cell, Ecai, as follows : Er.nl = E, Ec A-pTl (3) Eg and A are constants at any given temperature, and have the following values : °c E, A 20 0.7029 0.0581 25 . 6992 0.0591 30 0.6955 0.0601 35 0.6918 0.0611 38 . 6896 0.0617 For example, at 38°, with a quinhydrone electrode in 0.1 M HCl, E cal 0.6896 - £o6, - (0.0617 X 1.082) (4) from which the value of Ecai can b6 calculated after substitution of the experimentally determined value of Eobs- To determine the pH of an unknown solution, proceed as above except that the unknown solution is substituted for the standard HCl. The *'glass electrode/' Under suitable conditions, a properly pre- pared thin membrane of special glass separating two solutions of different pH exhibits an electric potential that is proportional to the difference in pH of the solutions. Based on this property, a device called the glass electrode is now widely used for the comparative determination of pH. 76 MANUAL OF MICROBIOLOGICAL METHODS The glass probably most generally employed is that known as Corning No, 015; Beckman-type E glass has been advocated for alkaline solutions (pH 9-14) because of its low sodium error as comparsf. with that of glass 015. One of the common forms of the glass electrode consists of a tube of the glass terminating in a thin-walled bulb which contains an electrode of definite potential in a solution of fixed pH. A combination of electrode and buffer solution frequently employed is a platinum wire, silver-plated and then coated with AgCl, in a half-cell containing 0.1 M HCl. For the construction, operation, and theory of the glass electrode, consult Dole (1941). The carefully rinsed bulb of the electrode, after seasoning in water or buffer solution, is immersed in the solution to be tested and coupled through a saturated KCl liquid junction with the saturated calomel half- cell as indicated schematically below, Ag; AgCl; HCl (0.1 M) \ glass membrane | solution X | KCl (sat.); HgsCla; Hg all parts of the cell being maintained at a uniform temperature. The potential difference between the terminals of this cell can be related to the pH of solution X if the glass electrode has been standardized in buffer solutions of known pH. Standardization of the glass electrode. The potential of a properly functioning glass electrode should vary linearly with pH, from about pH 1-9, in solutions of low salt content (up to 0.1 M). For this range, therefore, the electrode requires standardization in buffer solution at one point of pH, but preferably at two, within this linear range. Standard buffer solutions convenient for this purpose may be selected from Tables 5 and 7. Table 5. Some Standard Buffer Solutions Solution pH 25° 38° 0.1 M HCl 1.085 2.075 4.000 6.855 9.180 1.082 01 M HCl, 0.09 M KCl 2.075 0.05 M acid potassium phthalate 0.025 M KH0PO4, 0.025 M Na2HP04-2H20 4.015 6.835 0.05 M Na2B4O7-10H2O 9.070 Such standardization should be performed at least daily; preferably, it should be done immediately before a measurement. As occasion requires, a series of buffer solutions of known pH should be used to estab- lish more carefully the linearity of response of the electrode. In solutions more alkaline than about pH 9, the 015 glass electrode responds also to THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 77 cations other than H ions, the potential being influenced by the activity and kind of such cations. Sodium and Uthium ions produce the most marked effects; potassium and bivalent cations smaller effects. When working under these conditions, it is advisable to standardize the electrode with known buffer solutions of about the same composition and of pH closely above and below the pH of the sample being tested. The standardization for linearity of response from pH 1 to 9 is a neces- sary check on the operation of the glass electrode, since its results are comparative, not absolute. The slope, — A£';i/ApH, should be not merely constant at any temperature but also equal or closely equal to 0.000,198,3227". Obviously, a ''pH-meter" with its pH scale adjusted to the theoretical slope for a given temperature cannot give correct readings at all points from pH 1 to 9 if its glass electrode follows a significantly differ- ent slope at the same temperature. For a brief discussion of the effects of temperature, see Clark (1948). Cleaning of the glass surface, by immersion in a hot mixture of concentrated nitric and sulfuric acids followed by soaking in water, may restore a sluggish or erratic electrode to normal functioning. A somewhat drastic procedure that may be effective is to dip the glass electrode for a second or two in dilute HF or in a 20 per cent solution of ammonium bifluoride and then to wash it thoroughly in water. If the electrode still behaves erratically, it should be discarded. For such an emergency, it is highly advisable to have available a reserve electrode. This may obviate any mistaken tendency to carry on with an electrode of doubtful reliability. The instructions accompanying the various glass-electrode "pH-meters" now on the market are usually sufficient to aid the user in tracing out sources of trouble and error in operation. A major source of trouble is electrical leakage due to accumulation of films of moisture at critical parts of the circuit, and perhaps the most frequent sites of such accumulation are the electrode support and lead, both of which are apt to be spattered with water or salt solution during careless manipulation. The glass electrodes now available are fairly rugged and easilj^ adaptable to use under a variety of conditions and on different types of biological material (e.g., liquid and "solid" culture media). Measurements with an accuracy of 0.05 pH may be made rapidly in poorly buffered, colored, or turbid solutions and in blood or serum. The monograph by Dole (1941) discusses many of its uses. The Colorimetric Method The colorimetric method of measuring pH makes use of acid-base indi- cators, which, within certain limits, vary in color with the pH of the solution. Such indicators are compounds capable of existing in solution as conjugate proton (H-ion) donor and proton acceptor, with one of the conjugate pair differing in color from the other. The relation of these two forms to pH is defined by the equation TT r-/ . 1 [proton acceptor] ,.. pH = pK' + log ,p,^ton donor] ^^^ 78 MANUAL OF MICROBIOLOGICAL METHODS in which brackets represent concentrations, and pK' (= — log K') is called the apparent ionization exponent of the indicator's proton donor- acceptor system. Simple calculations, using, for example, 0.8, 0.5, and 0.3 as values for the ratio [proton acceptor]/[proton donor] at each of the pK' values 3, 6, and 9, will show that indicators with different pK' values cover different ranges of pH (see Fig. 1). For a full discussion of the properties and uses of pH indicators, see Clark (1928) and Kolthoff and Rosenblum (1937). Within a short range on the pH scale on each side of the pK' value, every dolor gradation of the indicator corresponds to a definite pH number; this zone may be called the sensitive range of the indicator. Throughout its sensitive range, an indicator can be used to determine the pH of a solution by comparing its color in the solution with that produced in standard solutions representing known pH numbers. The indicators. A selection of indicators is presented in Table 6. All but three of the compounds are sulfonphthaleins which are particularly useful in bacteriological work because of their high tinctorial power, low or moderate salt and protein errors, and relative resistance to bacterial action. Table 6 gives the pK' values of the indicators and their sensitive Table 6. Acid-base Indicators Name VK' pH range and colors Recom- mended cone %"■ Ml of 0.01 M NaOH per 0.1 g^ Thymol blue (acid range) . Methyl orange'^ Bromphenol blue Bromcresol green Methyl red Chlorophenol red Bromcresol purple Bromthymol blue 1.7 3.5 4.0 4.7 5.0 6.0 6.2 7.1 7.8 8.3 8.9 9.7 Red 1.2-2.8 yellow Red 3.1-4.4 yellow Yellow 3.1-4.7 blue Yellow 3.8-5.4 blue Red 4.2-6.3 yellow Yellow 5.1-6.7 red Yellow 5.4-7.0 purple Yellow 6.1-7.7 blue Yellow 6.9-8.5 red Yellow 7.4-9.0 red Yellow 8.0-9.6 blue Colorless 8.3-10.0 red 0.04 0.05 0.04 0.04 0.02 0.04 0.04 0.04 0.02 0.02 0.04 0.10 21.5 d 14.9 14.3 e 23.6 18.5 16 Phenol red Cresol red Thymol blue (alk range) . . Phenolphthalein 28.2 26.2 21.5 " Stock solutions in 95 per cent ethanol for the indicator acids, or in water for the indicator salts, unless otherwise specified. ^ Grind 100 mg of the pure indicator acid with the amount of NaOH specified, and when solution is complete, dilute with water to a volume that will yield the con- centration recommended in column 4. " Do not use with phthalate buffers. ^ Dissolve 50 mg in 100 ml of water. • Dissolve 20 mg in 60 ml of 95 per cent ethanol, and add 40 ml of water. ^ Dissolve 100 mg in 65 ml of 95 per cent ethanol, and add 35 ml of water. Source: See Clark (1948) and Kolthoff and Rosenblum (1937). THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 79 pH 6 PER CENT DISSOCIATION Fig. 1. Ionization curves of some sulfonphthalein indicators, illustrating the general relationships among the acid-base indicators and the applications of Eq. (5). Note: In some cases, the positions of the curves on the pH ordinate are approximate. Table 2 should be consulted for accurate values of pK'. 80 MANUAL OF MICROBIOLOGICAL METHODS ranges. The last column and footnote h of the table give specifications for the preparation of stock solutions of the monosodium salt of each oi the sulfonphthaleins. It will be noted from footnote a that ethanolic solutions are ordinarily satisfactory. For precise work, however, aqueous solutions of the indi- cator salts are preferable to the alcoholic solutions of the free acids. To obviate the labor of preparing the neutralized solutions, some makers now offer the soluble salts of the sulfonphthaleins. They are ammonium, sodium, or possibly other salts of these compounds. In ordinary use, the indicator salts contribute negligibly to the total ions present in a test solution, and the nature of the cation may be of no consequence. How- ever, in some studies of bacterial nutrition, the kind of cation and even the small amounts thus added may be of significance. In such cases, it is advisable to learn from the maker what cations (Na, NH4, etc.) are present in the indicator salt in order to make due allowance for their possible effects. The colorimetric method of pH determination depends on matching the color of a suitable indicator in the unknown solution with that of the same indicator in a standard. The standards can be set up in two differ- ent ways: by means of buffer standards or by means of ''drop ratios." These will be considered in detail presently. In brief outline, the colorimetric method includes these major steps: 1. Selection of the appropriate indicator 2. Preparation of color standards 3. Color comparison for pH determination Later paragraphs will outline essential specifications that must be observed in each of these steps in order to assure reliable results. Selection of the appropriate indicator. Test successive small portions (1 ml) of the unknown with a drop of bromthymol blue (BTB). If the color produced is orange or red, then the unknown is probably in the range of pH covered by thymol blue (acid range). If the BTB color is yellow, repeat the test with the indicators of successively lower pK^ (see Table 6) until that indicator is found which gives a color within its sensi- tive or useful range. If the BTB color is blue, proceed in like manner with indicators of higher pK' until the appropriate indicator is found. Of course, if the unknown is more acid than pH 1 or more alkaline than pH 10, none of the indicators listed in Table 6 will serve. If the unknown solution is unbuffered (e.g., water or saline) or very weakly buffered, the buffering effect of the added indicator may prevail and significantly change the pH of the unknown. In such cases, special methods are required (see Clark, 1928). It is plain that a rough idea can be obtained as to the pH value of a sufficiently buffered solution by simply finding which indicators give their THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 81 acid color in it and which give their alkahne color. Indeed, the intelligent employment of indicators with overlapping pH ranges can be made to define the upper and lower limits of a relatively narrow zone of pH within which lies the pH of the solution under study (Small, 1946). Accuracy, however, can be obtained only by actual comparison with the colors produced by the indicators in solutions (buffers) whose pH values are known, or produced by application of Eq. (5) (drop-ratio method, page 85). Buffer solutions and color standards. A considerable variety of buffer solutions have been proposed; and many of them are discussed and described by Clark (1928). The compositions of the series of buffer standards proposed by Clark and Lubs (1917) are given in Table 7. Preparation of the stock solutions is described by Clark (1928). After finding the appropriate indicator, prepare or select a series of properly graded standard buffer solutions sufficient in number to bracket the estimated pH of the unknown solution as determined in the prehmi- nary trials. If, for example, the indicator selected is bromcresol green and the estimated pH of the unknown is near 6.0, then not more than five standards, namely, buffers of pH 5.6, 5.8, 6.0, 6.2, and 6.4, should suffice to safely bracket the actual pH of the unknown. In preparing for the actual measurement, the unknown and the color standards should be contained in clear glass tubes selected for uniform bore, wall thickness, and inherent color. It is essential that the total concentration of indicator in the unknown be exactly the same as that in each of the color standards. This is best accomplished by accurately measuring, with a pipet equal amounts of indicator (e.g., 0.50 ml) into equal amounts (e.g., 10.0 ml) of each of the selected standard buffer solutions. The indicator may be satisfactorily measured in drops provided the dropper tip is properly shaped (not too blunt) and the dropper is held vertically during the measurement. The use of excessive amounts of indicator may introduce difficulties; the minimum quantity necessary to produce recognizable coloration is desirable from the theoretical standpoint. It is essential, of course, that the indicator be uniformly distributed throughout the solutions to which it is added. Prepared buffer standards can be obtained from supply houses, either as solutions or as powders or tablets to be dissolved as needed. They may also be obtained in sealed glass tubes containing the indicator. Such commercial color standards are convenient and satisfactory. They presuppose the use of comparable concentrations of indicator in the solution under test, and they must be used with the understanding that they are not permanent and may need to be checked or renewed at least once a year. All such indicator standards should be kept in the dark when not in use. Color comparison. This procedure, commonly miscalled colorimetry, requires intelhgent application to yield reliable results. The subject is adequately discussed by Clark (1928, 1948). Accurate color comparison of a standard solution with an unknown requires uniformity of the follow- ing conditions: the optical path (i.e., distance through the solutions traversed by the light), transparency, wall thickness and color of the 82 MANUAL OF MICROBIOLOGICAL METHODS Table 7. Composition of Mixtures Giving pH Values at 20°C at Intervals of 0.2 From Clark (1928), pp. 200-201. KCl, HCl mixtures pH 0.2 M KCl, in ml 0.2 M HCL, in ml Dilute to, in ml 1.2 50 64.5 200 1.4 50 41.5 200 1.6 50 26.3 200 1.8 50 16.6 200 2.0 50 10.6 200 2.2 50 6.7 200 Phthalate, HCl mixtures pH 0.2 M KH phthalate, in ml 0.2 M HCL, in ml Dilute to, in ml 2.2 50 46.70 200 2.4 50 39.50 200 2.6 50 32.95 200 2.8 50 26.42 200 3.0 50 20.32 200 3.2 50 14.70 200 3.4 50 9.90 200 3.6 50 5.97 200 3.8 50 2.63 200 Phthalate, NaOH mixtures pH 0.2 M KH phthalate, in ml 0.2 M NaOH, in ml Dilute to, in ml 4.0 50 0.40 200 4.2 50 3.70 200 4.4 50 7.50 200 4.6 50 12.15 200 4.8 50 17.70 200 5.0 50 23.85 200 5.2 50 29.95 200 5.4 50 35.45 200 5.6 50 39.85 200 5.8 50 43.00 200 6.0 50 45.54 200 6.2 50 47.00 200 THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 83 Table 7. Composition of Mixtures Giving pH Values at 20°C at Intervals of 0.2 {Continued) KH2PO4, NaOH mixtures pH 0.2 M KH2PO4, in ml 0.2 M NaOH, in ml Dilute to, in ml 5.8 50 3.72 200 6.0 50 5.70 200 6.2 50 8.60 200 6.4 50 12.60 200 6.6 50 17.80 200 6.8 50 23.65 200 7.0 50 29.63 200 7.2 50 35.00 200 7.4 50 39.50 200 7.6 50 42.80 200 7.8 50 45.20 200 8.0 50 46.80 200 Boric acid, KCl, NaOH mixtures pH 0.2 M H3BO.3 0.2 M KCl, in ml 0.2 M NaOH, in ml Dilute to, in ml 7.8 50 2.61 200 8.0 50 3.97 200 8.2 50 5.90 200 8.4 50 8.50 200 8.6 50 12.00 200 8.8 50 16.30 200 9.0 50 .21.30 200 9.2 50 26 . 70 200 9.4 50 32.00 200 9.6 50 36.85 200 9.8 50 40.80 200 10.0 50 43.90 200 Notes. Overlapping members of the above series should be checked for consis- tency; i.e., phthalate "5.8" to "6.2" should match phosphates of the same pH num- bers when tested with bromcresol purple; likewise for phosphate and borate "7.8" and "8.0" when tested with cresol red. According to more recent assumptions used in standardization, the pH values given in the above table are too low about 0.03-0.04 unit of pH. containers, concentration of indicator in each of the solutions, and radiant power incident upon the systems under comparison. Also, any inherent color in the unknown must be compensated by an equivalent amount in the optical path through the standard. These conditions are met by selecting clear, unscratched tubes of uniform bore, glass thickness, and color, by having the same concentrations of indicator in the unknown and 84 MANUAL OF MICROBIOLOGICAL METHODS the standard, by dispersing the color uniformly in the solutions, and by employing proper illumination. The color comparison is conveniently made in a comparator block of the type described by Clark (1928, 1948). Various forms of this are obtainable from supply houses. Two pairs of tubes are arranged in the comparator as follows: (1) a tube containing buffer standard plus indicator behind which is placed a tube containing the unknown solution to compensate for inherent color and (2) a tube containing the unknown solution plus indicator backed by a tube containing distilled water. The two pairs of tubes are viewed against a uniform source of white light so placed that the beams incident upon the two systems are of the same radiant power. The color stand- ards are successively compared with the unknown until a match is obtained, thereby establishing the pH of the unknown. If the color of the unknown falls between those of two adjacent standards, an interpolated pH number may be estimated. Systems of fixed or "permanent" color standards are also available. These stand- ards consist of colored glasses or other transparent material. Since the spectral absorptions of such standards would hardly be expected to be exactly the same as those of the indicators that they are supposed to match, the applicability and accuracy of these fixed standards must be determined in each case before they are placed in service. Acceptable sets of such standards can be of great convenience in the bac- teriological laboratory, especially for approximate determinations. The drop-ratio standards of Gillespie. If commercial color standards are not available and there are no facilities for making standard buffer solu- tions, color standards may be prepared by the drop-ratio method as refined by Gillespie (1920). The method of preparing the standards con- sists in setting up pairs of tubes, containing stepwise proportions, of the full alkaline color and the full acid color of an indicator in such a manner that the resulting color of each pair, when properly viewed, represents a definite pH within the sensitive range of that indicator. A general notion of the arrangement and composition of the drop-ratio color standards may be obtained from inspection of Table 8. The preparation of the standards is explained in the next two paragraphs and in Table 9. Although the alcoholic solutions of the indicator acids mentioned in Table 6 may be used, Gillespie recommends for accurate work the use of aqueous solutions of the indicator salts (the preparation of which is speci- fied in Table 6), except in the case of methyl red. Table 9, lower half, gives specifications for the recommended concentrations of seven of the indicator stock solutions. The exact concentration of the indicator solu- tions is not very significant in much bacteriological work. Select 18 test tubes of approximately the same bore (between 12 and 15 mm). They can be selected by adding 10.0 ml of water to a large number of test tubes and choosing a lot in which the columns of water come to approximately the same height (i.e., ±1.5 mm). Place these 18 tubes in two rows in a rack, 9 tubes in each row. To the left-hand tube in the front row add 9 drops of the indicator solution, in the second tube THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 85 Table 8. Drop-ratio Color Standards for pH Determinations Quantity of indicator solution to be added to each tube later to receive Tube dilute alkali or acid and then brought to final volume of 5 ml pairs Acid tubes AJkpli tubes 1 9 drops* 1 drop 2 8 drops 2 drops 3 7 drops 3 drops 4 6 drops 4 drops 5 5 drops 5 drops 6 4 drops 6 drops 7 3 drops 7 drops 8 2 drops 8 drops 9 1 drop 9 drops * If a little more accuracy is desired one may use a 1-ml pipet graduated in tenths and use the specified number of tenths of a milliliter instead of drops in preparing these tubes. In that case each tube should be brought up to a total volume of 10 instead of 5 ml. place 8 drops, and so on to the last tube, which should contain 1 drop. In the back row of tubes place 1 drop in the left hand tube, 2 in the next, etc., up to 9 in the last. Make up approximately 20iV stock solutions of NaOH and HCl [i.e., 0.2 per cent NaOH and 1 ml of concentrated HCl (sp gr 1.19) diluted to 240 ml]. Then, except in the case of those indi- cators for which different directions are given in Table 9, add 1 drop of the stock acid solution to each tube in the front row and 1 drop of the stock alkali solution to each tube in the back row; add enough distilled water to each tube to bring its total contents to 5 ml, thoroughly mix the contents of each tube, and return to its place in the rack. It will be seen from Table 9 that two of the indicators, namely thymol blue and bromphenol blue, require more of the alkali or the acid, respectively, than the other standards in order to ensure the appearance of full alkaline or acid color. In the case of thymol blue (alkaline range) and cresol red, the production of the required acid color (yellow^) requires not a strong acid but a weaker one such as mono-potassium phosphate or, in the case of thymol blue, distilled water alone. The arrangement of tube pairs indicated in Table 8 produces progres- sively different colors corresponding to steps of 10 per cent in the trans- formation of the indicator from its acid to its alkaline color. That is, each pair of tubes, when aligned between the eye and a source of white light, will show a color mixture corresponding to a definite pH. This pH can be computed by means of Eq. (5), which can be rewritten as ^^, , , drops of alkalinized indicator .> x pH = pK' + log -^ ^ — . ,.^ ■■ . ,. — 7 — (5a) ^ drops of acidified indicator 86 MANUAL OF MICROBIOLOGICAL METHODS The fraction on the right side of the above equation is called the drop ratio. The values of the standards for seven of the indicators are given Table 9. Data FOR Determining pH Value by the Drop -ratio Method No. of drops pH value represented by each pair of tubes Pair Brom- phenol blue Methyl red Brom- cresol purple Brom- thymol blue Phenol red Cresol red Alkali tube Acid tube Thymol blue 1 1 9 3.0 4.05 5.2 6.15 6.85 7.35 7.95 2 2 8 3.4 4.4 5.6 6.5 7.2 7.7 8.3 3 3 7 3.6 4.6 5.8 6.7 7.4 7.9 8.5 4 4 6 3.8 4.8 6.0 6.9 7.6 8.1 8.7 5 5 o 4.0 5.0 6.2 7.1 7.8 8.3 8.9 6 6 4 4.2 5.2 6.4 7.3 8.0 8.5 9.1 7 7 3 4.4 5.4 6.6 7.5 8.2 8.7 9.3 8 8 2 4.6 5.6 6.8 7.7 8.4 8.9 9.5 9 9 1 4.9 5.95 7.0 8.05 8.75 9.25 9.85 Data as to stock solutions Per cent concentra- 0.008 0.008 0.012 0.008 0.004 0.008 0.008 tion of indicator salt salt in acid in salt in salt in salt in salt in salt in or acid water 95% alcohol water water water water water Quantity 0.05N NaOH to produce alkaline color* 1 drop 1 drop 1 drop 1 drop 1 drop 1 drop 2 drops Quantity of acidf to produce acid color . . 1 ml 1 drop 1 drop 1 drop 1 drop 1 dropt 1 dropt * If the standards are prepared by the method suggested in the footnote to Table 8 (i. e., measuring the indicator in tenths of 1 ml and diluting to 10 ml) it is well to use O.IA'^ instead of 0.05 AT NaOH to assure proper strength. The exact concentration or the exact number of drops used is of no great importance. t Use approximately 0.05iV HCl (or O.liV if the method is modified as indicated in the footnote to Table 8) except in the case of cresol red and thymol blue. In the case of these two indicators a weaker acid must be used. Gillespie recommends 2 per cent KH2PO1, or in the case of thymol blue no acid need be used, water alone having a suffi- ciently high pH value to bring out the full acid color. in Table 9. They may be computed for the other indicators by using the above equation and the pK' values in Table 6. For approximate work it is often possible to compare the Gillespie standards with the unknown by merely holding the two tubes of the standard in the hand between the eye and a source of light. For accurate work, however, a comparator block must be used, but one with six holes THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 87 instead of four, so that a tube of the unknown solution (without indicator) can stand behind the pair of tubes of the standard. The tube of the unknown for comparison with the standard should contain the same amount of indicator as the sum of those in the two standard tubes, i.e., 10 drops per 5 ml; and, of course, this tube must be backed by two tubes of water to equalize the optical path through the standard pair. Indicator papers. Passing mention may be made of these laboratory aids for the approximate measurement of pH. Red and bhie litmus papers for the detection of alkalinity and acidity are well known. Papers impregnated with other indicators, singly or in various combinations, can be made or obtained on the market. Those with a single indicator may be of use to detect roughly (about ±0.3 to 0.4 pH) values within a relatively narrow zone of pH; those with indicator combinations enable one to detect, more roughly, pH values over wider zones of pH. Such papers are more reliable in buffered solutions than in unbuffered ones. To be emphasized is the fact that the capillary action of the paper and of the sizing materials on the paper fibers may interfere, through selective sorption, with the normal interaction of solution and indicator. Generally speaking, a generous time of soaking of the paper for the establishment of equilibrium seems desirable. On the other hand, a standardization of the procedure may permit a short exposure (30 sec) to yield reproducible results, which are approximate in any case. See Kolthoff and Rosenblum (1937). Indicator papers are not recommended, except when the use of indicator solutions is precluded and a mere approximation is sufficient. TITRATABLE ACIDITY, BUFFER ACTION, and pH ADJUSTMENT OF CULTURE MEDIA In the titration of an acid with an alkali, or vice versa, a pH is reached at which the number of equivalents of acid equals the number of those of alkali. This pH is the equivalence point (''end point"). If both the acid and the alkali are completely ionized, e.g., HCl and NaOH, it is simple to calculate that this pH is about 7 and that in the case of O.IA^ reactants, the pH of the HCl solution will sweep precipi- tously from about pH 4 to 7 upon the addition of the last tenth per cent of NaOH ; further, the addition of the first tenth per cent excess of NaOH will cause a shift from pH 7 to about 10. In other words, the titration curve, constructed by plotting pH as ordinates and per cent neutraliza- tion as abscissas, is very steep at the equivalence point (pH 7) in this titration. The ideal indicator for the detection of this equivalence point would be one capable of giving a distinctive color at pH 7, e.g., bromthymol blue. In practice, however, the steepness of the titration curve of the HCl at the equivalence point in the above example will permit this indicator to pass sharply from yellow to blue upon the final addition of a negligibly small excess of NaOH. For this reason, phenolphthalein {pK' 9.7) is fre- 88 MANUAL OF MICROBIOLOGICAL METHODS quently used for this purpose because the first appearance of its pink color, at about pH 8.5, is a convenient and usually sufficiently accurate indication of the end point of such a titration. In fact, except for refinements that may be neglected for ordinary pur- poses, pH 8.5, detectable by means of phenolphthalein, is a fairly satis- factory end point for the titration of strong acids and of all weak acids with pK' values of less than 6.0. In the case of acids with pK' values greater than 6.0, it is necessary, by application of Eq. (5), to calculate the pH of the equivalence point, and to refine the method of end-point determination. For a discussion of the elementary theory of acid-base titration, see Clark (1928). Titratable acidity of a culture. The titration of an acid (or a base) to an equivalence point, as discussed above, is a rational application of simple acid-base theory. On the other hand, in the titration of complex mixtures such as milk, tissue extract, or culture media, an equivalence point has no precise meaning. In such a case, the selection of an end- point pH is arbitrary, and fixed by custom (e.g., pH 8.5 with phenol- phthalein) or by some special requirement. In bacteriology, there is frequent need for determining the so-called titratable acidity produced during the growth of a culture in a fluid medium. To do this, it is necessary first to select a base line — that is, a pH number which is to be used as an end point in the titration and for the selection of an appropriate acid-base indicator. In the absence of special criteria, it is reasonable to choose as a base line the pH of the uninocu- lated medium. The selection of pH 7 as a base line may be acceptable, because many bacteria grow optimally in this region, not necessarily because it represents the pH of theoretical ''neutrality." Other base lines may be chosen in accordance with the special requirements for which the titration is to be made. The titratable acidity of the culture can be measured by titration of a known volume of the fluid with O.XN NaOH to the predetermined end point as shown by a standardized glass electrode or by the color of a suit- able indicator. In the latter case, it is necessary to prepare for com- parison an appropriate color standard representing the pH of the chosen end point (see earlier discussion of the essential requirements for adequate color comparison) . If the end-point pH is other than that of the uninocu- lated control, a titration is made of the latter and its titration value is subtracted algebraically as a correction, or ''blank," from that of the culture. The result is usually recorded as millihters of 0.1 normal acid per 100 ml of the culture fluid. If the culture produces an alkaline reaction, the titration is performed with 0.1 A^HCl and recorded after correction, if any, in the same way but as a minus quantity of titratable acid. Special precautions are necessary if the titratable acidity is to THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 89 include all the volatile acids, including CO2 and bicarbonate, that may be present in the culture that is being titrated. It should be emphasized that in most cases, the titratable acidity is merely a measure of the buffering capacity (see below) of the medium within the pH range observed. It does not permit further interpretation without additional data on the components of the culture. The titratable acidity is of some importance, along with final pH, in the comparison of high-acid-producing organisms. For such comparisons to be valid, it is necessary that the different organisms be grown in the same medium. Different media which vary in buffering capacity may yield misleading results. Buffer action. The titration curve of a weak acid has a sigmoid shape, each end of the curve having a large (steep) slope and the main central portion having a small slope. This small slope expresses the buffer action of the system, that is, the ability of the system (comprising the weak acid and its salt) to resist large change in pH on the addition of acid or alkali. The sigmoid shape of the titration curve expresses, therefore, the fact that the buffer action of such a system is maximal at the mid-point and decreases on either side of this point, first gradually and then more extensively as either end of the curve is approached. The limits of the pH zone of effective buffer action may be* arbitrarily set at 1.5 pH units greater and less than the pK' of the acid of the buffer system. It is obvi- ous that increasing the concentration of the buffer system will increase its buffer action; therefore buffer action also depends upon the concentration of the buffer system. The buffer action of a culture medium is dependent on its composition and may vary considerably in different regions of pH. Significant results as to final pH and titratable acidity in cultures depend to a large extent on comparisons made in media having buffer action that is uniform and adjusted in amount to the purpose of the test. A method for estimating such buffer action is as follows : Assume, for example, that the initial pH of a culture medium is 6.8 and that it is desired to measure the buffering capacity of the medium between the pH limits 5.0 and 8.0. This can be done by titrating an aliquot, e.g., 5 ml, of the medium with 0.057V HCi to pH 5.0 and another aliquot with 0.05A^ NaOH to pH 8.0. The sum of these titers gives a simple and useful measure of the buffering capacity of the medium within the pH zone 5.0-8.0. Brown (1921) has described the procedure and some of its practical uses. The pH -adjustment of a culture medium. This is done with the medium at about 80-90 per cent of its final volume. Prepare approxi- mately normal NaOH and HCI stock solutions and also about 100 ml of each of these solutions diluted with distilled water exactly to one-tenth 90 MANUAL OF MICROBIOLOGICAL METHODS concentration. Assume, for example, that the adjustment of a colorless medium is to be made to pH 7.0 before sterilization. Test the pH of the medium to establish whether acid or alkali will be required for adjustment to pH 7. To determine the amount required, titrate 5 ml of the medium plus 5 drops of the appropriate indicator (e.g., bromthymol blue) with the diluted acid or alkali until the color almost matches that of 10 ml of standard buffer pH 7.0 plus 5 drops of the same indicator. Next, add water to the tube with the medium to bring the volume to 10 ml, mix well, and make a proper comparison with the standard. If the color difference is small, then small additions of either acid or alkali may be made to bring about a correct match without changing significantly the necessary volume relations. If the color difference is large, the titration should be tried again. (In the case of a medium with inherent color, this should be compensated as previously described.) From the titration value, a calculation can be made of the amount of the stronger acid or alkali to be added to bring the bulk of the medium to the desired pH. The pH of the medium is checked after the addition, and when the pH is correctly adjusted, the medium is diluted with dis- tilled water to the final volume. In making a colorimetric pH determination of a well-buffered medium that is already colored, it is permissible to dilute the test sample of the medium 1:5 or 1:10 with distilled water to thin out the inherent color before proceeding with the test. The change in pH due to such dilution of a well-buffered solution is usually negligible. On the other hand, cau- tion must be observed in employing the dilution procedure on poorly buffered solutions, because the results may be misleading should the distilled water, or even the indicator solution, be too far from the desired pH. THE MEASUREMENT OF OXIDATION-REDUCTION POTENTIALS Introduction. The oxidation-reduction reaction Clo + 21- -^ 2C1- + I2 represents an exchange of electrons between the chlorine : chloride system and the iodine : iodide system. These systems may be represented by the hypothetical ' ' half -reactions ' ' CI2 + 2, ;=i 2C1- I2 + 2, ;:± 21- to show the participation of electrons. In the interaction, chlorine is the electron-acceptor, and iodide the electron-donor. THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 91 The chlorine, the iodine, and a considerable number of other systems can be studied by means of electric cells in which such systems can display their relative oxidation-reduction tendencies in terms of electrode poten- tials. The latter permit evaluation of the change in Gibbs free energy (see later) in the interaction of any two such oxidation-reduction systems. Without going into details of derivation or refinements, we may state that the elec- trode equation for a reversible oxidation-reduction system has the general form: F — F — ^^] [r^ H fl fi^ K5 fH (1) < ^ c3 o '^ H m f§ 03 ^ c3 w > Pi % o O Q 1—1 Z o HH Xi 5^ Oj O H ^ C! U ^ ■n o O g .S2 ^ rn o -d s i o 3 H (J OJ H C/J 2 OJ 1-H 1—1 H H-1 H^ 3 H 3 S, CO eo ■* r,* r>< U3 d d d d d d d 1 1 1 1 1 1 1 r ooooooooo 1 1 1 1 1 1 1 1 1 « c5c5Sc5SMcoecco <6Sd><6(6ci<6<6d 1 1 1 1 1 1 1 1 1 - t-l>-»-lO"50«D'-lt-- ddaS<6<=i<6o<6 1 1 1 1 1 1 1 1 1 c ^ -^ (M C^ .ogc2gMg oooooooo 1 1 1 1 1 1 1 1 6 o E d 1 iii^^sSi oooooooo 1 1 1 1 1 1 1 1 ~ COOMlOCJOOC^CO^ ddddddddd + 1 1 1 1 1 1 1 1 -« d + cD>-ieo<— it>-coooco ssssssss oooooooo - o + ooooooo^ oooooooo + 1 1 1 1 1 1 - s o oooooooo oooooooo + 1 1 1 1 ^ 05 o 4- :=l2oooooo oooooooo + 1 1 c» 00 o §iSisisi oooooooo >-, o 4 c^_^^oooo oooooooo + 1 - o -f iilS^Sis oooooooo "O o -1- |SS^|S2| oooooooo " — ( CT3 CO >C UO t^ (M O «0 CO 03 lii T-c oo CO COCi5M(M o «o o «o o «o O ) «b «b t>; t^ 00 CO o> THE MEASUREMENT OF PH AND TITRATABLE ACIDITY 97 The relation of percentage reduction to potential as defined by the last term in Eq. (10) is given in Table 12. For example, if methylene blue is observed to be 80 per cent reduced at pH 7, Eh = 0.011-0.018 = -0.007 volt. Color standards. Since the compounds listed in Tables 10 and 1 1 are practically one-color oxidation-reduction indicators, color standards of sufficient approximation can be prepared simply by graded dilutions of the colored component, the oxidant. It should be borne in mind that some of the compounds are also acid-base indicators; therefore it may be necessary to set up the color standards in a buffer at the same pH as the solution or culture under test. Colorimetric measurement. The general principles of color com- parison as outlined for the indicator method of pH determination are applicable here. In addition, special precautions are required to make certain that the measurement is a valid one. An indicator may fade in a test solution for reasons other than simple reduction. The compound may precipitate or adsorb on suspended particles, or it may be decom- posed; in such cases judicious treatment with a suitable oxidizing agent (e.g., ferricyanide or air) will not immediately restore the initial color of the oxidant. Moreover, many reversible oxidation-reduction systems are so sensitive to oxygen as to require extreme precaution for its exclu- sion. This applies to the electrometric method as well as to the colorimetric. Table 12 Reduction, -0.03 log ratio, Reduction, -0.03 log ratio, % volts % ■ volts 1 -1-0.060 60 -0.005 10 0.029 70 -0.011 20 0.018 80 -0.018 30 0.011 90 -0.029 40 0.005 99 -0.060 50 0.000 100 (- ~) It is a fact that many biological systems act as if they contain, at any moment, only minute amounts of electromotively active oxidation-reduc- tion substances; therefore the addition to such a system of even a small amount of indicator oxidant may suffice to oxidize the system at once without appreciable reduction of the indicator. This drawback cannot be overcome except by allowing sufficient time for the biological system to overcome the poising^ effect of the added indicator. However, the 1 Poising action of an oxidation-reduction system is analogous to buffer action of an ^cid-base system. (Compare paragraph on buffer action, p. 89.) 98 MANUAL OF MICROBIOLOGICAL METHODS time required may be very long (especially in relation to the most active period of a growing bacterial culture) so that it may be difficult or impossible to determine successive Eh values colorimetrically at brief intervals. Furthermore, the indicator may not merely come into simple oxidation- reduction equilibrium with the components of the system under test. It may act catalytically to displace the oxidation-reduction equilibrium that it is supposed to measure, or it may be toxic toward living cells or combine chemically with components of the system under test. In summary, the indicator method, often applicable where it is impossi- ble to employ an electrode, may give results that require considerable caution in interpretation, especially the results obtained on unstable oxidation-reduction systems or on biological material containing them. REFERENCES Allyn, W. P., and I. L. Baldwin. 1932. Oxidation-reduction potentials in relation to the growth of an aerobic form of bacteria. /. BacterioL, 23, 369-398. Borsook, H., and H. F. Schott. 1931. The role of the coenzyme in the succinate- enzyme-fumarate equilibrium. /. Biol. Chem., 92, 535-557. Brown, J. H. 1921. Hydrogen ions, titration and the buffer index of bacteriological media. J. BacterioL, 6, 555-568. Clark, W. M. 1928. "The Determination of Hydrogen-Ions," 3rd ed. The Wil- liams & Wilkins Company, Baltimore. . 1948. "Topics in Physical Chemistry." The Williams & Wilkins Com- pany, Baltimore. Clark, W. M., Barnett Cohen, et al. 1928. Studies on Oxidation-Reduction, I-X. U. S. Public Health Service, Hyg. Lab. Bull. 151. Clark, W. M., and H. A. Lubs. 1917. The colorimetric determination of hydrogen- ion concentration. /. BacterioL, 2, 1-34, 109-136, 191-236. Cohen, Barnett. 1926. Indicator properties of some new sulfonphthaleins. Public Health Rpts., 41, 3051-3074. . 1933. Reversible oxidation-reduction potentials in dye systems; (also) Reactions of oxidation-reduction indicators in biological material, and their interpretation. Cold Spring Harbor Symposia Quant. BioL, 1, 195-204, 214-223. 1935. Oxidations and Reductions, chap. 19 in "A Textbook of Bio- chemistry," by B. Harrow and C. P. Sherwin. W. B. Saunders Company, Philadelphia. Dole, M. 1941. "The Glass Electrode." John Wiley & Sons, Inc., New York. Gillespie, L. J. 1920. Colorimetric determination of hydrogen-ion concentration without buffer mixtures, with especial reference to soils. Soil Sci., 9, 115-136. Glasstone, Samuel. 1942. "An Introduction to Electrochemistry." D. Van Nostrand Company, Inc., Princeton, N.J. (See Chap. 8.) Hewitt, L. F. 1936. "Oxidation-Reduction Potentials in Bacteriology and Bio- chemistry," 6th ed. The Williams & Wilkins Company, Baltimore. Kolthoff, I. M., and Charles Rosenblum. 1937. "Acid-base Indicators." The Macmillan Company, New York. Small, James. 1946. "pH and Plants." D. Van Nostrand Company, Inc., Princeton, N.J. CHAPTER V Maintenance and Preservation of Cultures Freeman A. Weiss INTRODUCTORY The title of this chapter suggests that there are two somewhat distinct aims in keeping cultures of microorganisms — maintenance and preserva- tion, or perhaps conservation would be a better term. Sometimes the preservation of cultures is only a continuation of the process of maintain- ing them, but often it implies something more. Maintenance means essentially supporting the culture and keeping it alive, pure, and in recognizably typical growth. Preservation has the connotation of long- term maintenance, but in addition, in view of the propensity of all organisms to vary — especially microorganisms because of the high fre- quency of generations of which they are capable — it also means main- taining them at essentially constant biological potentials. A preserved culture, for example one lyophilized (from lyo, loose, + philos, loving) or in dry soil, may appear macroscopically anything but typical, but it can be restored to its typical morphology and usually to its initial physio- logical and biochemical characteristics by suitable manipulation. On the other hand, cultures that are merely maintained may show typical form, may grow luxuriantly, but if they have lost some biological charac- teristic for which they were primarily selected, they have not been satis- factorily preserved. The basic function of a culture collection is to conserve cultures, not merely maintain them. This distinction does not always appear, but it exists, and it is with this in view that the methods and materials recom- mended in this chapter were selected. They are essentially the pro- cedures adopted by the American Type Culture Collection after long experience in both maintaining and preserving cultures. These methods also are subject to constant change as better techniques or materials are discovered, and no claim to universal application or general superiority is or can be made. In fact the procedures outlined here should always be supplemented, at least for routine maintenance of cultures, by technics 99 100 MANUAL OF MICROBIOLOGICAL METHODS recommended in the manuals published by the manufacturers of dehy- drated and other ready-prepared culture media (Difco Laboratories, Baltimore Biological Laboratory, and others). SELECTION OF MATERIALS As the maintenance of cultures is largely a mechanical task (for which, however, no suitable machine has been devised), it is desirable to reduce all mechanical factors to a minimum consistent with efficient manipula- tion of the cultures. The size and number of culture vessels, their con- venience in handling, the volume of culture media required, the storage space for cultures, especially refrigerated storage, all must be taken into consideration. For the conditions under which the American Type Culture Collection operates, the following specifications for materials have proved very satisfactory and are recommended to others, though re- cognizing that other laboratories may have varied preferences of their own. Culture tubes. These are 125 by 16 mm with straight sides (without flare) for cotton plugs or screw-cap tubes with rubber liners (plastic liners are prone to drop out, at times they leak, and they may give off toxic emanations that suppress growth of some cultures). These tubes hold 5-6 ml of culture media. For slow growers like Mycobacterium fMbercu- losis, and those requiring special media, as marble chips for Nitrobacter or a large surface of floating sulfur for Thiobacillus, larger culture tubes or flasks are needed. Shell vials for lyophilizing. Outer vial is 85 by 14 mm; inner vial 35 by 9.5-10.00 mm (outside dimensions). Culture tube racks. For the size of tube specified, a standard zinc- coated wire rack about 7^i by 3}i by 33^ in. will hold 40 tubes; two of these j oined end to end will fit the shelf depth of average-sized household or laboratory refrigerators. A storage space 40 by 16 by 16 in. will accommodate 800 cultures. Special racks or trays for holding smaller culture tubes or lyophiUzed specimens that are even more economical of space are commercially available. Milk solution for lyophilizing. Although various fluids, including serum (usually from beef blood), corn-steep liquor, and solutions of lactose or glucose, have been proposed for obtaining cell suspensions for lyophilizing, skim-milk powder, reconstituted at twice the original solid content of milk, possesses general advantages. It is readily obtainable and of uniform quality, can be stored without deterioration for many months (refrigerated), and has no adverse chemical or physical effects on most organisms. It is not suitable for all bacteria, however; for example. Vibrio comma must be lyophilized from serum. Milk must be carefully prepared for use in lyophilizing, as an excess of heat will cause clotting or partial caramelization , making it difl&cult to obtain a uniform suspension MAINTENANCE AND PRESERVATION OF CULTURES 101 of cells and perhaps interfering with dehydration. The following speci- fications have given consistent satisfaction. Warm 200 ml of distilled water, and add 40 g of powdered skim milk. Stir thoroughly, preferably with an electric mixer. Filter through cheesecloth and tube, 5-6 ml per tube. Autoclave exactly 13 min at 115°C (approximately 11 lb). Ample space for the circulation of heat around the tubes must be provided to ensure sterilization at this exposure. Vaspar, a mixture of equal parts of 45° mp paraffin and white petro- latum, makes an excellent seal for cultures of anaerobes and for liquid cultures that are transported or mailed. Sometimes a layer of melted agar is added above the culture, for example of Lactobacillus in milk, to accomplish the same purpose. This effects a tighter seal, since wax may shrink away from the glass and permit leakage of fluid, but a wax seal has the advantage, on the other hand, that a pipet can be pushed through it without disturbing the seal or clogging, thus permitting easy transfer of the culture. White mineral oil or lipuid petrolatum, of the medicinal grade, is com- monly used to seal cultures of various kinds of microorganisms, on agar slants or stabs and also broth cultures, against desiccation and oxidation, thus preserving them for months or sometimes several years beyond the survival of unsealed cultures. Both vaspar and mineral oil can be steril- ized by autoclaving at 15 lb pressure for 60 min and then driving off any entrapped moisture by heating in a drying oven at 110°C for about 1 hr. DORMANT CONSERVATION Lyophilization. The essentials of the freeze-drying method of pre- serving cultures may be stated as follows: (1) Obtain as dense as possible a suspension of cells of the organism to be preserved by washing down a culture that is grown to the proper stage of maturity with a suitable fluid, commonly serum, milk, or 3 per cent lactose; (2) transfer 0.1-0.2 ml of the suspension to vials; (3) quick-freeze these in a mixture of dry ice and 95 per cent alcohol; (4) evacuate while frozen until completely desiccated; (5) hermetically seal the vials while evacuated. There are many varia- tions of the method, and some elaborate types of equipment have been devised to accommodate a large number of vials at one time and to con- trol environmental factors. In commercial establishments and some institutions where lyophilized preparations are produced in great quantities, it is customary to use glass tubing, cut in short lengths and sealed at one end, or very small culture tubes, which are handled en bloc through the freezing, dehydrating, and sealing processes, by attaching them to a multioutlet manifold. The tubes or vials may be as small as 3-4 mm in outside diameter, 1.5-2 mm 102 MANUAL OF MICROBIOLOGICAL METHODS inside. To fill such small vials or reconstitute the culture requires a capillary pipet or hypodermic syringe, and the vials, especially if hard glass is used, are difficult to open and are subject to considerable risk of contamination by inrush of air when the vacuum is broken. The procedure which has long been in use (15 years) at the American Type Culture Collection eliminates most of these difficulties, and espe- cially the hazard of contamination when the culture is opened, by using a double-vial arrangement, one within the other, the inner one plugged with cotton like an ordinary culture tube. Specifications for the vials have been given. The small vials are cleaned, cotton-plugged, and autoclaved in advance. Prior to use they are labeled with glass-marking ink. Filling is done with an ordinary measuring pipet, 1 ml size, graduated in 0.1 ml to a basal line (not to the tip, as it is undesirable to blow the last drop of fluid into the small vial). For most cultures, the use of slightly over 1 ml of fluid to wash down the slope will proA^ide a cell suspension of sufficient density so that 0.15- 0.2 ml dispensed in each vial yields satisfactory lyophilized preparations. Thus from one growing culture five preserved ones can be obtained. The last drop or two from the pipet is customarily placed on an agar slant and spread evenly over the surface or transferred to other suitable media as a check on the purity of the culture during these manipulations. After filling, the cotton plugs are trimmed just above the rim of the vials, and the vials are quickly transferred to a freezing mixture of roughly equal parts of finety crushed dry ice and alcohol. A convenient receptacle for this is a large petri dish, covered with a coarse wire screen through Avhich the vials will pass and which holds them upright. The freezing mixture need not be more than about 1 cm deep. As soon as the culture has solidified, the vials are transferred by means of long forceps to a stout bottle having a ground-glass top, to which is connected a U tube terminating in a moisture trap and having a side outlet near the top which is connected to a vacuum pump. The vessel holding the vials is enclosed in a suitable container which is packed with coarsely crushed dry ice, serving as a cold jacket to keep the material frozen through the initial stage of dehydration. This will ordinarily be within an hour or two, and this time should not be greatly exceeded, as prolonged freezing at this temperature is inimical to survival. Throughout the process of dehydration the distal end of the U tube, with the moisture trap, is kept in a thermos vessel surrounded with dry ice and alcohol, hence serving as the cold element of the distilling apparatus. The moisture trap serves to protect the oil in the vacuum pump from absorption of water. The pump must run until desiccation is complete, which will ordinarily require about 6 hr, but overnight operation is a convenient way to ensure enough time, and the sealing is completed the next morning. In the meantime a set of the large vials is prepared to receive the small MAINTENANCE AND PRESERVATION OF CULTURES 103 ones by placing a pad of cotton in the base; then the small vial is inserted and in turn is covered with a wad of shredded asbestos which is pressed rather firmly against its top. The asbestos plug protects the cotton from scorching and the inner vial from excessive heat while the upper part of the large vial is drawn out to a thin neck. This step is carried out by heating the vial, about 4 cm from its open end, in a gas-air torch set to give a narrow pointed flame, rotating it so as to soften and constrict uniformly, until an isthmus about 5 cm long and 2-3 mm in outside diameter can be drawn out. Great care must be taken that the bore of the isthmus is not closed in this process. The open end of the vial, which still has its original diameter, serves for attachment to a manifold for the final exhaustion and sealing. A six-outlet manifold is convenient for this purpose, and for expeditious progress if many vials are to be sealed, two such manifolds, each provided with a shutoff valve, are connected by a Y tube to a manometer and then to a vacuum pump. The connection between vial and manifold outlet is made through a snug-fitting rubber cork, which must be kept well lubri- cated with stopcock grease. The vials remain on the manifold under as high a vacuum as the apparatus will pull (it should be at least 100 to 150 /x, and 50 is preferable) for about 10 min; then each is separated in succession by burning off the neck in a micro bunsen burner. Care must be taken not to overheat the rounded end of the vial back of the tip; otherwise it may collapse because of the vacuum within and thus form a precarious seal. Subsequently, usually within a day or two and again for about 10 days, before the preparations are filed away as reliable, the vials are tested with a high-frequenc}^ vacuum tester. A glow within the vial when the tip of the apparatus is brought near it is indicative of a suitable vacuum, but only momentary exposure to the discharge is advisable, as microscopic perforations of the glass may result, with loss of vacuum. If the survival capacity of the organism under lyophilization is not known, a check for viability should be made after one month and again after six. If viability is demonstrated at these intervals, it may be assumed that the preparation will remain viable for the average survival period of the species. This may vary from 2 to 10 or more years. The over-all viability gradually declines, the rate depending largely on the number of viable cells in the original preparation. Most lyophilized preparations survive well if stored at room tempera- ture, but some species of Hemophilus^ Lactobacillus, Neisseria, Pasteurella, and doubtless others are short lived unless refrigerated. For reconstituting the culture the tip of the vial above the asbestos wad is heated moderately in a gas flame, then a drop of water is placed on it. The resultant cracking breaks the vacuum, and the inner vial, protected by its cotton plug from contamination, is withdrawn. Reviving the 104 MANUAL OF MICROBIOLOGICAL METHODS culture is the reverse of the filUng process. Enough nutrient broth to hquefy the pellet is added by means of a pipet, then withdrawn and transferred to a tube of broth or an agar slant. It is often desirable to inoculate both a broth culture and a slant. The latter permits spreading the inoculum out so that often discrete colonies develop from which sub- sequent transfers can be made. The broth, on the other hand, permits enfeebled cultures to start development when direct inoculation of a slant might fail. Oil Sealing Cultures of many kinds of bacteria can be preserved for periods greatly exceeding the viability of ordinary agar or broth cultures by sealing them with sterile paraffin oil. This is essentially a process of preventing mois- ture loss from the medium, although suppression of growth by excluding oxygen may also contribute to longevity. Among the bacteria, as a rule only species that produce a copious surface growth and can therefore be easily transferred are well adapted to this technique. Some kinds of Rhizobium (but not all species) have survived for 4 or 5 years by this method. Other genera that survive well in oil-sealed cultures are Achromohacter, Bacillus, most of the Enter ohacteriaceae, Flavohacterium, Micrococcus (some species), Proteus, Pseudomonas, Sarcina, Serratia, Streptococcus, Vibrio (saprophytic forms). On the other hand, cultures of the following have not been successfully preserved under oil seals in our experience: Azotohacter, Lactobacillus, Leuconostoc, Mycobacterium, Rhodo spirillum. Salmonella. The procedure of oil sealing is simplicity itself. Screw-cap tubes are preferable to cotton-plugged ones, as there is always some risk of getting oil on cotton plugs. The preparation of the oil has been described. For treating only a few cultures the oil is simply poured over the culture until all the agar is covered. The use of a separatory funnel as a reservoir for the oil and of a glass sleeve enclosing the tip of the funnel, into which the culture tube is inserted as a shield against atmospheric contamination, may be advisable when a considerable number of cultures are to be oiled. It is important not to let the oil get too warm from repeated flaming of the reservoir tube or from contact with the flamed lip of the culture tube. No portion of the agar must remain uncovered; otherwise loss of water will continue through any exposed surface. It is advisable to store oiled cultures at the same temperature at which unsealed ones are kept for preservation. Soil Cultures Soil is sometimes used directly as a growing medium (though more often for molds and Streptomycetes than for bacteria), and it is also used MAINTENANCE AND PRESERVATION OF CULTURES 105 as an absorbent or desiccating medium for preserving cultures. In the first case soil that is suitably pulverized and screened is placed in culture tubes, and enough water or very dilute culture medium is added to raise it to about 60 per cent of its maximum water-holding capacity. It must be autoclaved at least 1 hour^ and tested for sterility before use. After the culture has sufficiently grown, it is simply allow^ed to become air dry and is then suitably sealed and stored at room temperature or refrigerated. If soil is to be used merely as an absorbent, it is first air dried, then tubed and autoclaved, and finally heated in a drying oven until moisture- free, after which the tubes are stored in a desiccator until needed. They are then inoculated with a broth culture or with a cell suspension washed from an agar slope, as described under ''LyophiHzation." For preparing a cell suspension a 2 per cent solution of peptone is useful. A 0.05- to 0.1-ml portion of this suspension is placed with a pipet on 1 ml of soil, and the tubes are returned to a desiccator until dry or may be tightly stop- pered if the soil already appears dry. A variation using silica gel granules as an absorbent has been proposed by Dr. J. Lederberg, of the University of Wisconsin. Silica gel of the grade known as 40, 6-16 mesh is used, and 1-1.5 g is placed in culture tubes about 75 by 8 mm, which are then plugged and baked in an oven at 170°C for 2 hr. They are cooled and stored in a desiccator until needed. A cell suspension in 2 per cent peptone is prepared and pipetted on to the granules as described above. The tubes may then be hermetically sealed, without evacuating, or stoppered and stored in a desiccator. The method is still in the trial stage but has given promise of being successfully adaptable. Other Desiccants Pieces of thread and strips of filter paper have long been used as absorb- ents of spore suspensions of both aerobes and anaerobes. After immer- sion in a spore suspension, followed by air drying, these materials can be stored in glassine envelopes or culture tubes for many months with satis- factory retention of viability. Deep Freezing The storage temperatures indicated in the follomng section for holding cultures of various bacteria between routine transfers are only low enough to retard or suppress normal growth; they are not necessarily the mini- mum temperatures at which the cultures will survive. Temperatures 1 Some technicians advise much more drastic sterilization, up to 2 hr at a time and repeated three times at 1-day intervals. Excessive heating of soil should be avoided, however, because it breaks down the organic components and may release toxic products. We consider 1 hx at 15 lb as generally enough. 10(3 MANUAL OF MICROBIOLOGICAL METHODS Table 13. Maintenance Methods (American Type Culture Collection) Genus Acetobacter Acetobacter Achromobacter . . . . Achromobacter . . . . Actinomyces Aerobacter Agrobacterium . . . . Alcaligenes Arthrobacter Azotobacter Azotobacter Bacillus . . . Bacillus Bacillus Bacter aides Brucella Cellulomonas Cellvibrio Chro7nobacterium. . Clostridium Corynebacterium . . Corynebacterium . . Corynebacterium . . Corynebacterium . . Desulfovibrio Diplococcus Erwinia Erwinia Erysipelothrix . . . . Escherichia Flavobacterium . . . . Gaffkya Hemophilus Hemophilus Klebsiella Lactobacillus Leuconostoc Listeria Malleomyces Micrococcus (including Staphylococcus) Mycobacterium. . . Mycobacterium. . , Moraxella Neisseria Neisseria Neisseria Nitrobacter Nitrosomonas. . . . Species Glucose positive Mannitol positive Various Fischeri Bovis Various Various Various Various Glucose positive Mannitol positive Various Starch-hydrolyz- ing Urea-decomposing Various Various Various Vulgaris Various Various Acnes Diphtheriae Animal sources Plant pathogens Pneumoniae Various Tracheiphila Insidiosa Various Various § Tetragena Influenzae Pertussis Various Various Various Various Mallei Various Saprophytic Tuberculosis Various Gonorrhoeae Meningitidis Saprophytic Agile Europeae Medium formula No. 2,3 4 23t 23a 10 (stab cul- ture under seal) 23t 23 1 23 1 23t 5 6 23 1 27 36 8,10 26 23t 12 23 1 7.17 10 10,20 8,10 23,25 30 8 23 1 20 8 23t 1 20 8,24 9 with blood 20 17,33,34 17,33 10 16 23t 16 14,18 8 1 1 with CO2 8,20 10,13,20 21 22 Transfer time 1 mo 1 mo 1 to 2 mo 1 mo 1 mo 2 mo 2 mo 3 mo 3 mo 4 mo 4 mo 12 mo-indefi- nite 12 mo-indefi- nite 12 mo-indefi- nite 1 mo 6 wk 3 mo 1 mo 6 wk 6 mo-indefi- nite 1 mo 1 mo 1 mo 1-2 mo 6 mo 3 mo 1 mo 1 mo 3 mo 3 mo 3 mo 1 mo 2 mo 2-4 wk 6 wk 4 mo 1 mo 3 mo 4 mo 3 mo 10 days 10 days 1 mo 1 mo 1 mo Incubation temp, °C 28 28 Room Room 34-37 30 Room 34-37 Room Room Room 28J 28 28 37 34-37 30 Room Room 28 34-37 35-37 30 Room— 28 34-37 35-37 Room Room 34-37 34-37 30 35-37 35-37 35-37 34-37 34-37 t Room 34-37 34-37 30t 30 1 34-37 34-37 35-37 35-37 34-37 Room Room Storage temp, °C 10 10 10 Room Room 10 10 10 10 10 10 10 10 10 28 10 10 10 18 Room 18 18 18 18 10 10 Room 10 10 10 10 10 10 10 10 10 Room 10 10 10 35-37 35-37 Room Room Room MAINfENANCE AND PRESERVATION OF CULTURES 107 Table 13. Maintenance Methods (American Type Culture Collection) {Continued) Genus Species Medium formula No. Incubation time Storage temp, °C Storage temp, °C Lyophil- ization* Pasteurdla Propionibacterium. . Proteus Various Various Various Aeruginosa Plant pathogens Various Rubrum Various Various Various Various Various A and B groups D group Various 10,13,20 with blood 33 23 1 23t 23,25.37 2811 23 23t 23t 23t 23 1 23,23a 19 with blood under oil 33,34 15 31,32 20 25,38 1 mo 2 mo 3 mo 3 mo 3 mo 1 mo 6 wk 3 mo 3 mo 3 mo 34-37 30 34-37 34-37 Room— 28 Room Room 34-37 30 30 34-37 Room 34-37 30J Roomt 26 34-37 Room— 28 18 10 10 10 10 18 18 10 10 10 Room 10 10 10 26 10 10 + + + + + + ? + + + + + + Pseudomonas Pseudomonas Rhizohium Rhodo spirillum .... Salmonella Sarcina Shigella 1 mo 1 mo 1 mo 3 mo 7-10 days 2 mo 2 mo Streptococcus Streptococcus Streptomyces Thiobacillus 4- + + Vibrio Xanthomonas Comma Various + * Plus sign indicates successful preservation by this method, + + that the organism in question should preferably be kept lyophilized; minus sign that lyophilization is unsuccessful or not tried at ATCC. t Tryptone-glucose-yeast medium (No. 35) as used by the Northern Regional Research Laboratory, U.S. Department of Agriculture, may be substituted. X The optimum temperature for some species is different; consult Bergey's Manual. § A special medium is required by certain species. il Some strains are favored by the addition of CaCOs to this medium. below 0°C are sometimes employed for long-term preservation of bac- terial cultures, including some that are not amenable to dry methods of storage. The principle involved is similar to that applied in quick freez- ing of food products, red blood cells, spermatozoa, and vital tissues. Relatively little is known as yet about the effect of subfreezing tempera- tures on the viability of bacterial cultures, including such factors as minimum temperature that is tolerated and duration and rate of freezing. The available information is reviewed in references cited at the end of this chapter. Since essentially aqueous solutions or emulsions such as broth and agar become solid at these temperatures, often with rupture of glass containers even if the organisms survive, some means of counteracting the physical effect of ice formation is desirable. Hollander and Nell (1954) have sug- gested the use of 15 per cent glycerol to accomplish this purpose. In general, it appears that moderately low temperatures, as —10 or — 20°C, have been more favorably regarded for the preservation of bacterial cultures than lower or higher subfreezing temperatures. There are few conclusive data, however. 108 MANUAL OF MICROBIOLOGICAL METHODS MEDIA Formulas employed by the American Type Culture Collection: 1. Ayers & Johnson Agar Stock culture agar (Difco) 50 g Distilled water 1,000 ml pH 7.4 2. Acetobacter Agar (Glucose) Autolyzed yeast 10 g CaCOs 10 g Agar 15 g Distilled water 1,000 ml Heat to 100°C, and add Glucose 3 g In tubing, the CaCOs should be distributed evenly between tubes. After auto- claving, the tubes should be shaken, then cooled quickly and slanted so as to keep the CaCOs in suspension. 3. Acetobacter Agar with Liver Extract Liver extract 100 ml Tryptone 5 g Agar 20 g Distilled water 900 ml Heat to 100°C, and add Glucose 20 g CaCOa 10 g Observe precautions as in formula 2 to keep CaCOs evenly suspended. 4. Acetobacter Agar (Mannitol) Yeast extract 5 g Peptone 3 g Agar 15 g Distilled water 1,000 ml Heat to 100°C, adjust pH to 7.4, and add Mannitol 25 g 6. Azotobacter Agar (Glucose) K2HPO, 1.0 g MgS04 0.2 g NaCl 0.2 g FeS04 Trace Agar 15 g Soil extract 100 ml Tap water 900 ml Dissolve, adjust pH to 7.6, and autoclave. Add 1 ml of sterile 10 per cent glucose to each tube for use. To prepare soil extract place 500 g of air-dry field soil in 1,300 ml of 0.1 per cent Na2C0a, autoclave for 1 hr, filter through paper, and make up vol- ume to 1,000 ml. MAINTENANCE AND PRESERVATION OF CULTURES 109 6. Azotobacter Agar {Mannitol) K2HPO4 1.0 g MgSO* 0.2 g NaCl 0.2 g FeSO* Trace Agar 15 g Soil extract 100 ml Tap water 900 ml Dissolve, add mannitol 20 g Adjust pH to 8.3, autoclave. 7. Beef Liver for Anaerobes Ground beef liver 500 g Tap water 1,000 ml Peptone 10 g K2HPO4 Ig Soak liver in 1 liter of water overnight in refrigerator; skim off fat. Autoclave 10 min at 15 lb. Strain through cheesecloth; save the meat. Add peptone and K2HPO4 to broth, and heat to 100°C. Adjust pH to 9.0, filter through paper, and make up to 1 liter with tap water. Place a small amount of CaCOs in each tube; add meat to depth of 3^^ in.; cover with broth to total depth of 2 in. Autoclave. 8. Blood Agar Heart infusion agar (Difco) 40 g Distilled water 1,000 ml Heat to 100°C; adjust pH to 7.4. Tube, and autoclave, but do not slant. Melt blood base agar, cool to 45°C, add 0.5 ml of sterile blood aseptically, mix, and slant, 80. Blood Glucose Agar Same as formula 8 with addition of 0.5-0.75 ml of sterile 10 per cent glucose solution per tube, besides blood. 9. Bordet-Gengou Medium Potato infusion 100 ml Prepare by dicing 12.5 g of potato in 100 ml of distilled water, let stand overnight at 60°C, filter through cheesecloth, and restore volume. Agar 1.5 g NaCl 0.55 g Proteose peptone 1.0 g Heat to 100 C, adjust pH to 7.4, and add Glycerol 1.0 ml 110 MANUAL OF MICROBIOLOGICAL METHODS 10. Brain- Heart Infusion Agar Brain-heart infusion agar (Difco) 37 g Agar 15 g (for semisolid) 2 g Distilled water 1,000 ml pH7.4 11. Chocolate Agar Heat blood agar (formula 8) to 80°C; reslant. 12. Cellvibrio Medium NaNOs 2.0 g MgS04 0.5 g KCl 0.5 g Fe2(S04)3 0.01 g KH2PO4 0.14 g K2HPO4 1.2 g Yeast extract 0.02 g Agar 7.5 g Distilled water 1,000 ml Place a strip of filter paper 3^^- by 4-5 cm on the agar slope, or suspend in liquid medium. 13. Cystine-trypticase Medium Cystine-trypticase agar (BBL) 29.5 g Carbohydrate (optional) 5-10 g Distilled water 1,000 ml Mix well; heat gently with agitation; boil for 1 min. Tube, and autoclave 15 min at 12 lb. Store at room temperature. 14. Dorsett Egg Medium Commercial preparations are advised. 15. Emerson Medium NaCI 2.5 g Peptone 4.0 g Yeast extract 1.0 g Beef extract 4.0 g Distilled water 1,000 ml Adjust to pH 7.0 with KOH; add Glucose 10 g Agar 30 g 16. Glycerol Agar Blood base agar (Difco) 20 g Nutrient agar 15.5 g Distilled water 1,000 ml Heat to 100°C; adjust pH to 7.3; add Glycerol 60 ml MAINTENANCE AND PRESERVATION OF CULTURES 111 17. Liver Medium (Northern Regional Research Laboratory) Liver extract (commercial) 10 g Yeast extract 5 g Tryptone 10 g K2HPO4 2g Glucose 5 g Distilled water 1,000 ml pH7.4 If stabs are desired, add 10 g of agar. Liver extract can be prepared by placing 1 lb of ground beef liver in 2 liters of water and heating in flowing steam for about 3 hr until the liquid assumes a yellow fluorescence. Filter through cheesecloth, place in flasks, and autoclave. Keep aseptically, and use 100 ml in above formula; reduce water to 900 ml. Save the solid meat, and place a few particles in each tube. 18. Low enstein- Jensen Medium Commercial preparations are advised. 19. Meat-infusion Broth Lean ground beef 500 g Distilled water 1,000 ml Mix thoroughly; store in refrigerator overnight. Skim off fat, strain the infusion through cheesecloth, and add Peptone 10 g NaCl 5 g Dissolve by heating, strain through cheesecloth, and make up to volume. Adjust pH to 7.4. Heat in autoclave 10 min at 15 lb, filter through paper, and adjust pH. Dispense in tubes, and autoclave 20 min at 15 lb. 20. NIH Semisolid Medium (National Institutes of Health) Meat infusion l^roth . 675 ml Nutrient agar 1.8 g Distilled water 75 ml KCl (1.5 g in 10 ml of water) 1 ml CaCh (1.5 g in 10 ml of water) 0.5 ml Heat to 100°C; adjust pH to 7.0, tube for stabs. 21. Nitrobacter Medium (a)NaN02 10 g K2HPO4 r 0.5 g NaCl 0.3 g MgS04 0.5 g MnSO* Trace Fe2(S04)3 Trace Distilled wcder 1,000 ml pH7.5 Put 100-ml portions in flasks. Autoclave. (6) Wash marble chips thoroughly in distilled water; place in large test tubes, about one-third full; autoclave 1 hr at 15 lb. Add (a) to (b) aseptically. 112 MANUAJj OF MICROBIOLOGICAL METHODS 22. Nitrosomonas Medium (NH4)2S04 2.0 g K2HPO4 1-0 g NaCl 0.5 g MgS04 0-5 g MnS04 Trace Fe2(S04)3 Trace Distilled water 1,000 ml pH8.5 Dispense 100-ml portions in flasks, autoclave, and add to tubes of marble chips as in formula 21. 23. Nutrient Agar Blood base agar (Difco) 20 g Peptone 2.5 g Beef extract 1.5 g Agar 15.5 g Distilled water 1,000 ml pH7.4 23a. Alternate Formula with Salt Peptone 5 g Beef extract 3 g NaCl 30 g Agar 14 g Distilled water 1,000 mi 24. Peptic Digest of Blood Autoclave 150 ml of 0.9 per cent NaCl in a bottle having a ground-glass stopper but with the stopper separately wrapped in paper and the bottle cotton-plugged. Place in 55°C water bath, and add HCl (cone) 6 ml Sterile sheep blood, defibrinated 50 ml Pepsin 1 ml Keep in water bath at 55°C overnight, shaking occasionally during first 2 hr. Add 12 ml of 20 per cent NaOH, then slowly with intermittent shaking enough more to raise the pH to 7.6. Then restore to pH 7.0-7.2 with HCl. Add 0.25 per cent chloroform; store glass-stoppered in refrigerator. For use add 0.1 ml to brain-heart infusion agar or semisolid medium. 25. Potato Dextrose Agar Potato, peeled and diced 300 g Distilled water 1,000 ml The potatoes should be handled with minimum exposure to air. Boil in 500 ml of water until thoroughly cooked. Filter through cheesecloth, make up the volume to 1,000 ml, and add Agar 15 g Glucose 20 g Dissolve by heating. Autoclave. MAINTENANCE AND PRESERVATION OF CULTURES 113 26. Potato Agar Potato infusion 500 ml Water 500 ml Peptone 10 g Beef extract 5 g NaCl 5g Agar 30 g Heat to 100°C, adjust pH to 7,3, autoclave 5 min at 15 lb, filter through cotton, and add Glucose 10 g Tube, autoclave, and slant. The potato infusion is made by slicing 250 g of peeled potatoes into 500 ml of dis- tilled water. Cover, and hold in 60°C incubator overnight. Filter through cheese- cloth, and make up to 1,000 ml. 27. Potato-starch Agar (a)Nutrient agar 3.1 g Distilled water 80 ml pH7.4 (6)Potato starch 1 g Distilled water (cold) 20 ml Mix thoroughly, then heat with constant stirring until a smooth paste is formed. Combine with (a), autoclave, and slant. 28. Rhizobium Medium Yeast extract 1 g Agar 15 g Soil extract (see formula 5 for preparation) 200 ml Tap water 800 ml Heat to 100°C, adjust pH to 7.4 and add Mannitol 10 g 29. spirillum Medium a. Fresh-water species Peptone 5 g Beef extract 3 g Yeast autolysate 3 g Calcium lactate 1 g Agar 2 g Tap water 1,000 ml b. Marine species Use sea water or sea-salt solution of equivalent concentration. An artificial sea salt can be compounded of NaCl 2.75 g MgCh 0.50 g MgS04 0.20 g CaCh 0.05 g KCl 0.10 g FeS04 Trace Distilled water 100 ml 114 MANUAL OF MICROBIOLOGICAL METHODS 30. Sporovibrio Medium (Starkey) Peptone 5.0 g Beef extract 3.0 g Yeast extract 0.2 g MgS04 1.5 g NazSO* 1.5 g Ferrous ammonium sulfate 0.1 g (increase to 0.2 g if medium does not blacken after growth) Agar 15 g Tap water 1,000 ml Heat to 100°C, adjust pH to 7.4, add Glucose 5 g Tube for stabs, using long (150-mm) tubes. Boil tubes to be inoculated for 10 min; cool quickly. After inoculating, push cotton plug down about half way, place two pieces (0.5 mm) of pyrogallic acid on it, add 8 to 10 drops of strong NaOH. Cork tightly with rubber stopper, invert, and incubate at 37°C. 31. Thiobacillus Thioparus Medium (a)K2HP04 2.0 g CaCh 0.1 g MgS04 0.1 g MnS04 Trace FeS04 Trace Tap water 900 ml pH7.8 Dispense 90 ml in 250 ml-flasks; autoclave. (6)Na2S203 10 g Tap water 50 ml Autoclave in flasks. (c)(NH4)2S04 0.1 g Tap water 50 ml Autoclave in flasks. At time of inoculating add 5 ml aseptically of each of (6) and (c) to (a) 32. Thiobacillus Thiooxidans Medium (NH4)2S04 0.2 g MgS04 0.5 g CaCh 0.25 g FeS04 Trace KH2PO4 3.0 g Precipitated sulfur 10 g Tap water 1,000 ml One-gram portions of sulfur are placed in 10 dry 250-ml flasks, and 100 ml of a solu- tion of the other components is carefully poured into each flask so that the sulfur floats. Sterilize in flowing steam for % hr on 3 consecutive days. MAINTENANCE AND PRESERVATION OF CULTURES 115 33. Tomato-juice Agar Tryptone 10 g Yeast extract 10 g Agar 12 g Distilled water 800 ml Heat to 100°C; adjust pH to 7.2; add Tomato juice 250 ml Tube for stabs; autoclave. The tomato juice must be taken from canned unsea- soned tomatoes, not from commercial juice 'preparations. It is prepared by filtering the juice from a No. 2 can, keeping refrigerated overnight, then adjusting pH to 7.0. 34. Tomato, Yeast, Milk Medium Dehydrated skim milk 100 g Yeast extract 5 g Distilled water 1,000 ml Mix well and add Tomato juice, pH 7.0, as in formula 33 100 ml Methylene blue 1.5% solution in alcoholic KOH 2 ml Filter through cheesecloth, tube, and autoclave. 35. Tryptone Glucose Yeast Agar (Northern Regional Research Laboratory) Tryptone 5 g Yeast extract 5 g Glucose 1 g K2HPO4 1 g- Agar 20 g Tap water 1,000 ml pH 7.0 36. Urea Agar Urea 10 g Distilled water 100 ml Sterilize by filtration. Add ig ml per tube aseptically to nutrient agar (No. 23) after melting and cooling to 45-50°C. Mix well; cool for slants. 37. V-8 Juice Agar V-8 juice (Campbell Soup Co.) 200 ml CaC03 3g Agar 15 g Tap water 800 ml pH7.2 The V-8 juice can be filtered or not depending on the clarity desired, and the amount of CaCOs may be varied to give a different pH. 116 MANUAL OF MICROBIOLOGICAL METHODS Table 14. Commercially Available Media Recommended for Culture Maintenance Recommen iations of Baltimore Biological Laboratory, Inc. Difco Laboratories, Inc.* Organism Media Storage temp, °C Suggested transfer interval Media Storage temp, °C Suggested transfer interval CTA medium Room 3-6 mo Brain-heart infu- Room 1-12 mo / (cystine- sion, or same (less \ trypticaset with 0.2 or 1.5% than Actimomyces .... < agar) / Thioglycollate I medium 135C with CaCOi agar Bacto-tryptose agar 25) Algaet Euglena Trypticase agar slants Above 6 Chlorella Trypticase salts medium Brucella CTA medium Room 3-6 mo Tryptose agar Brain-heart infu- Room 3 mo sion agar /Cooked-meat Room Yearly Cooked-meat Room 10 years 1 phy tone § medium plus \ medium Egg meat Clostridia J Thioglycollate j medium 135C / with CaCOi 1 Trypticase agar \ base medium Coliform group: Trypticase agar Room or 6-12 mo Cooked-meat Room 1 year Salmonella and base refriger- medium plus other enteric Trypticase soy ator Nutrient agar bacilli agar with oil [CTA medium Room 3-6 mo LoeflSer blood Room 1 mo Corynebacteria. . . j Trypticase soy [ agar under oil serum Tryptose agar Heart-infusion agar Hemophilus: [ CTA medium Room 3 mo Chocolate agar 37 Weekly blood-requiring. I Trypticase soy [ agar with blood prepared with proteose No. 3 agar or GC Medium base en- riched with hemoglobin and bacto-supple- ment B or C /Lactobacillus Room Monthly to Micro assay cul- Room 1-12 mo Lactobacilli and 1 agar with oil 6-8 wk ture agar Leuconostoc I Thioglycollate 1 medium 135C V with CaCOi Tomato-juice agar Litmus milk * Trade names of all products of this company include the prefix "Bacto-." It is omitted in this table to economize on space. t Media and storage times and temperatures vary with organisms. Periods of transfer may vary with types of closure of container and concentration of media. Dehydration occurring in cotton-plugged tubes may impair longevity of cultures. t U.S. P. pancreatic digest of casein. § U.S. P. papaic digest of soybean meal. MAINTENANCE AND PRESERVATION OF CULTURES 117 Table 14. Commercially Available Media Recommended for Culture Maintenance {Continued) I Recommendations of Organism Micrococci. Mycobacteria . Nonpathogens. Neisseria. Gonococcua. Pasteurella- Listena P. multocida. P. tiUarensis . Pleuropneumonia Pneumococci. Baltimore Biological Laboratory, Inc. Media CTA medium Trypticase soy agar with oil Dorset medium ATS medium Lowenstein- Jensen medium Same, or CTA medium or trypticase soy agar with oil CTA medium CTA medium CTA medium Cystine heart agar with blood CTA medium with inacti- vated sheep serum PPLO agar with serum CTA medium Storage temp, C Room Room or refriger- ator Room 35 Room Room 35 Room Suggested transfer interval 6-12 mo 3 mo 10 days 3-6 wk 1 mo. 10-21 days Difco Laboratories, Inc.* Media Cooked-meat medium Brain-heart infu- sion, or same with 0.2 or 1.5 per cent bacto agar Blood agar base Dorset medium Petragnani medium Lowenstein medium Glycerol agar Dextrose starch agar 37 3-2 strength + sup- plement C GC medium base with hemoglobin -|- supplement C Cooked-meat medium Tryptose agar Cystine heart agar -f bacto- hemoglobin PPLO agar with ascitic fluid or PPLO serum fraction Cooked-meat medium Brain-heart infu- sion agar Tryptose blood agar base -|- blood Stock culture agar Storage temp, °C Room Room Suggested transfer interval 3 mo Room 37 Room 1 year plus 4 wk 1 year 1 wk * Trade names of all products of this company include the prefix "Bacto-." It is omitted in this table to economize on space. t Media and storage times and temperatures vary with organisms. Periods of transfer may vary with type of closure of container and concentration of media. Dehydration occurring in cotton-plugged lubes may impair longevity of cultures. 118 MANUAL OF MICROBIOLOGICAL METHODS Table 14. Commercially Available Media Recommended for Culture Maintenance (Continued) Recommendations of Baltimore Biological Laboratory, Inc. Difco Laboratories, Inc.* Organism 1 Media Storage temp, °C Suggested transfer interval Media Storage temp, °C Suggested transfer interval Protozoat Trichomonas Simplified trypticase Serum medium Room 35 2 weeks 3 days Endamoeba medium over- layed with horse 37 14 days overlaid vaginalis serum saline with rice flour Lash serum medium Salmonella- Shigella (see also Coliform group) Trypticase soy agar under oil /OTA medium Room or refriger- ator Room 6-12 mo 3-6 mo Cooked-meat medium Nutrient broth Nutrient agar Cooked-meat Room Room 2 years plus 1 mo Streptococci ThioglycoUate < medium 135C 1 Trypticase soy V agar under oil medium Brain-heart infu- sion agar Tryptose blood agar base + blood Stock culture CTA medium Room or 6 mo or agar Brain-heart infu- Room 3 mot / Trypticase agar \ base refriger- ator more sion agar Potato dextrose Streptomyces / Trypticase soy 1 agar or my- agar Yeasts 1 cophil agar un- der oil Mycophil agar under oil Room or refriger- ator 6 mo or more Dextrose starch agar Potato dextrose Room 3 mo Molds Mycophil agar under oil Room or refriger- ator 6 mo or more agar Mycological agar Sabouraud mal- tose agar Mycological agar Littman oxgall Room 12 mo 1 agar 1 * Trade names of all products of this company include the prefix " Bacto-." It is omitted in this table to economize on space. t Media and storage times and temperatures vary with organisms. Periods of transfer may vary with type of closure of container and concentration of media. Dehydration occurring in cotton-plugged tubes may impair longevity of cultures. MAINTENANCE AND PRESERVATION OF CULTURES 119 REFERENCES British Commonwealth Collections of Microorganisms. 1954. "A Discussion on the Maintenance of Cultures by Freeze Drying," 48 pp. Her Majesty's Stationery Office, London. Fennell, Dorothy I., K. B. Raper, and May H. Flickinger. 1950. Further investi- gations on the preservation of mold cultures. Mycologia, 42, 135-147. Flosdorf, Earl W. 1949. "Freeze-Drying. Drying by Sublimation," 280 pp. Reinhold Publishing Corporation, New York. Gordon, Ruth E., and N. R. Smith. 1947. Preservation of certain microorganisms under paraffin oil. J. BacterioL, 63, 669. Harris, R. J. C. 1954. "Biological Applications of Freezing and Drying," 415 pp. Academic Press, Inc., New York. Hartsell, S. E. 1947. The longevity of bacterial cultures under paraffin oil. J. BacterioL, 53, 801. . 1953. The preservation of bacterial cultures under paraffin oil. Appl. Microbiol., 1, 36. Hauduroy, Paul. 1951. "Techniques bacteriologiques," 167 pp. Masson et Cie, Paris. Haynes, W. C, L. J. Wickerham, and C. W. Hesseltine. 1955. Experience in main- taining industrially important microorganisms. Appl. Microbiol., 3, 361-368. Hollander, David H., and E. Ellen Nell. 1954. Improved preservation of Tre- ponema pallidum and other bacteria by freezing with glycerol. Appl. Microbiol., 2, 164-170. Proom, H., and Louis M. Hemmons. 1949. The drying and preservation of bac- terial cultures. /. Gen. Microbiol., 3, 7-18. Raper, K. B., and D. F. Alexander. 1945. Preservation of molds by the lyophil process. Mycologia, 37, 499-525. Weiser, R. S., and C. M. Osterud. 1945. Studies on the death of bacteria at low temperatures. I. The influence of the intensity of the freezing temperature, repeated fluctuations of temperature, and the period of exposure to freezing temperatures on the mortality of Escherichia coli. J. BacterioL, 50, 413-439. CHAPTER VI The Study of Obligately Anaerobic Bacteria^ L. S. McClung and Robert B. Lindberg It is impossible to list here all the methods which have been proposed for the study of anaerobic bacteria; an attempt is made, however, to out- line a number of technics which have been used widely and which should ordinarily be suitable for routine studies of anaerobic species. Those interested in other technics are advised to consult Sec. B of the sub- ject index bibliography relating to the anaerobic bacteria (McCoy and McClung, 1939; McClung and McCoy, 1941). The worker who has had no experience with anaerobic bacteria should study some of the articles which deal with principles of anaerobic culture or which record the results of a study of a considerable number of strains: Fildes, 1931; Hall, 1922, 1928, 1929; Heller, 1921; Knorr, 1923, 1924; McCoy et al, 1926, 1930; Mcintosh, 1917; Meyer, 1928; Reed and Orr, 1941; Spray, 1936; Zeissler, 1930; Zeissler and Rassfeld, 1928. Valuable suggestions in English will be found in Smith (1955), and in French in Lehert and Tardieux (1952). The recent literature has been reviewed by McClung (1956). The organisms which we call obligate anaerobes are those that require a low (reduced) oxidation-reduction potential, which can be brought about by exclusion or removal of atmospheric oxygen, alone or in com- bination with chemical reducing agents added to the medium. It is not easy to answer the question of the best method of determining whether or not a given organism is an obligate anaerobe. The catalase reaction, when applied to pure culture, gives presumptive evidence, for obligate 1 The methods and technics suggested herein are those recommended for use with the more common sporeforming anaerobic species. Many of these methods are suit- able also for the study of the nonsporeforming types, but for the present no attempt will be made to outline particular methods of study for these. If the technics herein outlined do not prove satisfactory, the worker interested in the pathogenic nonspore- formers should consult the review of Dack (1940) and the books by Pr6vot (1948, 1955) and Smith (1955). Nonpathogenic types exist, as for example, the methane organisms discussed by Barker (1936). For the complete literature on all types refer to Sec. Id (nonsporeformers) in the bibliography of McCoy and McClung (1939) and McClung and McCoy (1941). 120 THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 121 anaerobes usually are catalase-negative. For this reaction a plate culture of the organism in question is flooded with a 10 per cent solution of H2O2. The evolution of gas bubbles from the colonies denotes the presence of catalase. If the proper material for the catalase reaction is not available or in doubt, the following technic will usually suffice to characterize an anaer- obic strain and to differentiate it from the aerobes: Inoculate, while the agar is molten, several deep tubes (8- to 9-cm columns of medium) of a suitable nutrient agar medium containing 1.0 per cent glucose; allow these to solidify in an upright position and incubate the tubes at several tem- peratures or at the optimum temperature for the organism in question; adjust the seeding so that relatively few (e.g., 25-50) colonies per tube will result. With an obligate anaerobe, all the colonies should be local- ized in the bottom of the tube and none should appear on the surface or in the upper 1-cm layer. Likewise, with pathogenic organisms cultured in fluid thioglycollate medium, the growth should be confined to the lower section of the medium and no growth should result in the upper layer wherein the methylene blue is recolorized. If growth does occur in the upper layer of either medium, the culture is not an obligate anaerobe or is contaminated with an aerobic or a facultative species. In the case of clinical cultures, in which speed is important, two blood agar or egg- yolk plates (McClung and Toabe, 1947) may be inoculated and incubated in parallel, one anaerobically, the other aerobically. The appearance of growth in only the anaerobic environment is evidence of presence of an obligate anaerobe. ANAEROBIC CULTURE METHODS AND EQUIPMENT All the procedures which have been devised for the cultivation of anaerobic bacteria have the single purpose of excluding atmospheric oxygen from the environment in which the growth is to take place. With certain tubed media the oxygen potential may be reduced sufficiently by constituents of the medium to permit anaerobic growth (Brewer, 1940; Hewitt, 1950; Knight, 1931; Reed and Orr, 1943; and Holland, 1944). Since this is rarely possible for -surface cultures on a solid medium, usually plate and slant cultures are incubated within a closed container from which the oxygen is removed by one or another means. A study of the various methods shows that no single procedure may be proposed as the best technic but the method of choice will depend upon the pre- vailing circumstances. A procedure which is ideal for one situation may be impractical or impossible to apply with other conditions. Each of the technics outlined below is recommended within the limits proposed in the discussions. 122 MANUAL OF MICROBIOLOGICAL METHODS Use of methylene blue as indicator of anaerobiosis. For all types of anaerobic jars and containers, except individual-plating or tube-culture systems, it is convenient to include an indicator tube which will serve as a check on the development of anaero- biosis. The most commonly used system utilizes the change of methylene blue from the colored (oxidized state) to the leuco form (reduced state). Using the solution prepared as given below, any system which gives sufficient degree of removal of oxy- gen from the atmosphere for anaerobic growth to develop will cause the blue color of the solution to disappear or will maintain the colorless condition if the solution is boiled (heat reduction) immediately prior to its being placed in the container. A somewhat less sensitive system can, in an emergency, be prepared by adding a tinge of color from Loeffler's alkaline methylene blue to a tube of glucose broth. The procedure recommended (Fildes, 1931) is: Prepare three stock solutions, (1) 6.0 ml of O.IN NaOH diluted to 100 ml with distilled water, (2) 3.0 ml of 0.5 per cent aqueous methylene blue diluted to 100 ml with distilled water, (3) 6.0 g of glucose in 100 ml of distilled water to which has been added a small crystal of thymol. Each time the indicator solution is needed, mix equal parts of the three solutions in a test tube and boil in a cup of water until the color disappears. Place tube in anaerobic container immediately and begin process of securing anaerobic conditions. If the container is satisfactorily deoxygenated, the color in the solution should not reappear. If the blue color does return, it is a sign that the container leaks or has not been satis- factorily exhausted of oxygen. (In the vegetable tissue jar, to be described, the color may appear but will disappear with the development of anaerobiosis during the incu- bation period.) Oxygen Removal by Combustion Using Laidlaw Principle For laboratories which are engaged in problems where anaerobic plating is to be done frequently, it is advisable to plan for this and to purchase equipment accordingly. Although the systems discussed above may be adequate for this purpose, it is well to consider one of the jars which utilize, on the Laidlaw (1915) principle, combustion as a means of securing the anaerobic environment. These methods were designed especially for incubation of plates, but other culture vessels (flasks, tubes, bottles, etc.) may be used. Jars using this principle are those of Brewer (Brown and Brewer, 1938) and Mcintosh and Fildes (Fildes and Mcintosh, 1921). Brewer Anaerobic Jar^ Materials for method of Evans, Carlquist, and Brewer (1948): (1) Brewer jar complete with electric cord, (2) source of illuminating gas or hydrogen, (3) plasticene (see footnote 2 on page 123), (4) vacuum pump for evacuation. Method, Place plates in jar. Add tube of methylene blue solution. Include a tube of soda lime in the jar to absorb excess CO2. Place roll of (warmed) plasticene around rim of jar. Put on lid and press down on plasticene to form seal. Add the lid clamp but tighten only slightly. If used with illuminating gas, attach the jar by the rubber tubing to the vacuum pump. Evacuate until the manometer or gauge reads approxi- 1 Brewer jar. Baltimore Biological Laboratory, Baltimore, Maryland, and Fisher Scientific Company, Pittsburgh, Pennsylvania. THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 123 n lately 20 cm or 8 in. After this degree of evacuation is reached, con- nect the rubber tube to the gas supply (a three-way stopcock facilitates this change without loss of vacuum). Attach the electric plug (110- volt alternating or direct current) and allow the gas and electric cur- rent to remain attached for 30 to 45 min. At the end of this time clamp the rubber tube tightly, remove the electric cord, and place the jar in the incubator. (Formation of water droplets on the inside walls of the jar indicates the proper functioning of the apparatus.) To open the jar, remove the clamp. Insert a knife blade between lid and rim of jar, using caution to avoid scoring the soft metal rim of the top and making subsequent leakage more likely. If used with hydrogen, attach the jar, without evacuation, to the hydrogen tank and admit the gas at a pressure of 1-2 psi. Attach the electric connection, and allow the current and gas both to remain on for 30 min. Then treat the jar as above. The jar may be used as a gas-replacement system by evacuating the jar on a water aspirator pump to 700 mm Hg negative pressure; fill with illumi- nating gas, repeating this process three times or more. Avoid dropping of agar from inverted plates due to excessive evacuation. This method may fail to produce anaerobiosis as adequate as that achieved by catalytic combustion as described above. Advantages. Convenient system for incubation of a number of plates in experi- ments where speed of obtaining anaerobiosis is essential. Recommended for clinical laboratories. Inexpensive system after the initial outlay for apparatus. Dis- advantages. Some possibility of explosion or cracking of jar. Initial expense of equipment is more than for other methods, but this may be a good investment if routine work is to be done over a period of time. Requires source of hydrogen or illuminating gas and electricity; while these are available in most laboratories, they are not available in others such as some mobile laboratory units, temporary labora- tories in field surveys, etc. Biological Methods for Oxygen Removal Vegetable-tissue Jar Materials for method of McClung, McCoy, and Fred (1935): (1) jar or other container which may be^ sealed airtight (recommended: 6- by 18-in. or 6- by 12-in. Pyrex cylinder^) ; (2) square (7 by 7 in.) of plate glass or a glazed plate; (3) plasticene,^ 3^ lb; (4) glass tumbler; (5) supply of 1 Pyrex cylinder. Pyrex Catalogue No. 850. Corning Glass Works, New York, or supply house. 2 Sealing materials. Plasticene of a suitable grade is sold by Baltimore Biological Laboratory, Baltimore, Maryland, and by J. L. Hammet Co., Cambridge, Massa- chusetts. Care should be taken in choosing products for sealing, since some dry to a hard cake upon incubation. A silicone grease is preferred by many workers, since it gives a seal less likely to leak, and jar tops are more easily removed than with plasticine. A suitable product is marketed as Dow-Corning High Vacuum Grease by Do\y Corning Corp., Midland, Michigan. 124 MANUAL OF MICROBIOLOGICAL METHODS oats or other grain (other tissues, particularly chopped Irish potatoes, may be used but are less conveniently stored for occasional use, and in some cases produce objectionable odors which are evident when the jar is opened) ; (6) tap water. Method. Place inverted tumbler (if plates are to be used) or other support in bottom of cylinder. Add oats to fill at least one-tenth of the capacity of the cylinder. Add sufficient tap water to moisten the oats. Stack plates or other cultures on support. Add tube of methylene blue solution (see above). Place layer of plasticene, previously softened by placing in incubator, on rim of cylinder. Push plate-glass square firmly against plasticene; using fingers, press the clay against both the square and the cylinder until a satisfactory seal is obtained. Place jar in incubator immediately. (A 40- to 48-hr incubation period is recom- mended.) If plate cultures are employed, replace the ordinary petri dish cover with unglazed porcelain (''clay") tops^ to absorb the moisture which collects within the cylinder. If porcelain tops are unavailable, add a petri dish lid containing CaCl2 to absorb the moisture. Advantages. The method is inexpensive and employs easily available materials. No special apparatus is required, an advantage in laboratories where anaerobic cul- tures are not usually prepared. It may be used at any incubation temperature with- out danger of explosion. It is particularly suitable in problems requiring large numbers of plate cultures. It is recommended especially for cultural and physiologi- cal studies of strains which have been purified by other methods. Disadvantages. Several hours may be needed for anaerobic conditions to become established, and therefore the method is not suitable when the results are required quickly. It is not recommended for routine clinical use where speed of isolation of pure culture is an important factor. With certain enrichments it is not suitable for purification of species contaminated with aerobic sporeforming bacteria because of the quick growth of these forms. In plate-culture experiments, as in the isolation of new strains, no one plate may be removed from the cylinder for observation until the end of the incu- bation period, for to do so would destroy the anaerobic conditions within the cylinder. Use of Aerobe to Absorb Oxygen Another biological method for oxygen removal utilizes the growth of an aerobic organism (usually Staphylococcus aureus, Serratia marcesceus, or Saccharomyces cerevisiae). A wide variety of applications of this system have appeared in the literature. The technics suggested^ involve the growth of the aerobic organism in pure culture on a medium separate from that on which the anaerobe is to be cultured. ^Unglazed porcelain (''clay") tops for petri dishes. The Coors porcelain dish, sold by Arthur H. Thomas Company, has been found to be more uniform in size and quality than others tested, 2 These are similar to the Fortner method and are recommended in place of it. In the Fortner method the aerobe is streaked on one half of the plate and the anaerobe on the other half of the same dish. THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 125 Materials for method of Snieszko (1930): (1) two petri dishes of ordinary size; (2) paper tape, Scotch tape, adhesive plaster, or plasticene; (3) culture of Serratia marcescens or other fast-growing aerobic organism; (4) tube of nutrient agar. Method. Select two petri dishes which have bottoms of exactly the same size, and sterilize these in position in their usual top sections. Pour nutrient agar into the bottom half of plate A, and after solidification streak the medium heavily (or flood across surface with 0.5 ml of broth culture) with the aerobic organism. (As an alternate method, seed the agar before pouring.) Pour into plate B, a medium suitable for the anaerobe (see Chap. Ill) ; when hard, streak with the sample or culture of the anaerobe (or seed with the latter prior to pouring). Remove the two bottoms from their respective tops, and fit together at their rims. Use tape or other sealing device around the juncture to provide an airtight seal. Place plate in the incubator immediately. If thermophilic anaerobic cultures are to be made, replace the S. marcescens by a thermophilic aerobe, or before placing plates in thermophilic incu- bator, incubate for 18 hr at 32°C to allow S. marcescens to grow and to use the oxygen. Advantages. No elaborate equipment is needed, since the method uses ordinary petri plates and other common materials. Thus it is available as an emergency method in almost any laboratory at any time. The technic is extremely simple and can be set up by inexperienced individuals. Since each set of plates is an individual unit, observation of the growth of each anaerobe may be carried out without destroy- ing anaerobic conditions for other cultures. Disadvantages. The method is somewhat time-consuming when large numbers of cultures are to be made and is therefore not suitable in laboratories where routine platings of a number of cultures is not an unusual event. Anaerobiosis may not be attained promptly enough to prevent death of the inoculum of nonsporeforming species of vegetative cells of anaerobic sporeformers. Negative results on isolation attempts with this procedure are hence not reliable. Chemical Methods for Oxygen Removal Many of the methods proposed for removal of oxygen from the environ- ment for anaerobic culture involve the initiation of a chemical reaction in which oxygen is consumed. Of the various systems which have been sug- gested, those which are recommenxled have been tested and used suffi- ciently to show their utility, and they do not require elaborate apparatus. Phosphorus Jar Materials. (1) Sticks of yellow (or white) phosphorus (which must he kept under water in tightly stoppered, wide-mouth bottle; the small sticks, %6-in.-diameter, are the most useful); (2) Pyrex cylinder, or any con- venient jar or container w^hich may be sealed airtight; (3) pair of long forceps or chemical tongs; (4) plasticene; (5) small amount of tap water. 126 MANUAL OF MICROBIOLOGICAL METHODS Method. Place small amount of tap water in bottom of cylinder to remove the P2O5 which forms. Stack inoculated plates or tubes on sup- port. Add tube of methylene blue solution (see page 122). Place smal) (50-ml) beaker on top of cultures. Remove two or three short (l}i- to 2-in.) pieces of phosphorus from water with forceps or tongs, and place in beaker. Immediately put lid on jar and seal with plasticene. (Upon drying for a few minutes the phosphorus should ignite spontaneously and remain burning as long as there is oxygen present.) If experience shows that the phosphorus used does not ignite spontaneously but merely gives off a gray smoke, ignite it before the jar is sealed by a match held with the forceps. Since considerable heat is developed, place beaker, unless resist- ant glass is used, 3 in. from the top of the container and put a '^ blank'' plate under the beaker rather than an inoculated plate. After the phos- phorus ignites and the jar is tightly sealed, place it directly in the incu- bator. At the time the container is opened have available a crock or pan filled with water. As soon as the lid is taken from the jar, remove the beaker containing the phosphorus with the tongs and submerge under the water in the pan and save for later use. After this remove the cultures from the jar. Advantages. Quick method of obtaining anaerobiosis. It is relatively inexpensive since the only materials are phosphorus and a container which may be sealed. Dis- advantages. Care must be exercised to prevent accidental burns which are very pain- ful. Inexperienced technicians should be cautioned concerning the dangers. Alkaline Pyrogallol Methods Another chemical method for removing oxygen in order to promote anaerobic growth is to utilize the oxygen absorptive capacity of the reaction between alkali and pyrogallic acid. Of the technics and devices reported which make use of this reaction two may be recommended as being especially useful. One of these concerns a technic applied to indi- vidual plate culture, and the other relates to a system for individual tube cultures. Spray (or Bray) Plate Cultures Materials. (1) Spray (1930) anaerobic dish;^ (2) plasticene, or tape for sealing; (3) 20 per cent aqueous NaOH; (4) 40 per cent aqueous pyrogallic acid. Note: The Spray dish consists of an ordinary glass petri-dish top and a special bottom which is deep and has a raised ridge across the center. The top of the bottom dish has a lip into which the top section of the dish fits. Although constructed of heat-resistant glass, in practice considerable breakage may be encountered during ^ Spray anaerobic dish. Fisher Scientific Company, Pittsburgh, Pennsylvania, or E. H. Sargent Company, Chicago, Illinois, THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 127 sterilization and handling of the Spray dish. This is eliminated in the Bray^ dish, which is Pyrex and essentially the same design as the Spray dish. In the Bray dish, however, the need for the lip is eliminated, since the top of the bottom section is slightly smaller in diameter than the remainder of the bottom section. This allows the top to fit down over the rim of the bottom section. Method, Pour anaerobic medium in the top half of the dish, and after soHdification, streak from sample or culture, or pour seeded plate. After inverting dish, place 10 ml of 20 per cent aqueous NaOH solution in one section of the bottom dish and 4 ml of 40 per cent aqueous pyrogallic acid in the other. Seal dish with plasticene or tape. Tilt dish to mix solu- tions and place in incubator. Advantages. Anaerobiosis is attained quickly. It is a useful method for single plate culture. Since each plate is a single unit, observations may be made at any time and any particular plate of a series may be opened when visual inspection reveals growth to be at the desired stage. Recommended for clinical laboratory technicians seeking a quick method of purification of possible pathogenic types. Disadvantages. Preparation of individual dishes is time-consuming, and in work on any scale, anaerobic jars may be preferred. Loss of anaerobiosis due to leaks in seal is not uncommon. The excess of alkali present results in absorption of CO 2, which may be disadvantage- ous with some strains. In addition, a small amount of CO is given off during oxida- tion of the pyrogallate. Some strains are inhibited by this substance. Preliminary replacement of air by an inert gas before introduction of the pyrogallate will minimize this effect. Anaerobic Jar with Alkaline Pyrogallol for Plate Culture For each 100 ml of jar capacity 1 g of pyrogallic acid and 10 ml oi 2.5N NaOH are used. Plates are stacked in the jar, together with anaerobic indicator tube, and pyrogallic acid is added to alkali in a large- diameter test tube. The jar is quickly sealed, using plasticine or silicone grease. Advantages. General availability of materials and suitability for a variety of jars, which need be only sealable. Disadvantages. As noted above, absence of CO2 and presence of CO may inhibit growth of some strains. Tube Culture Materials for method of Griffin (1932) : (1) Two test tubes with approxi- mately 5^ in. diameter (one empty and the other containing a liquid or slant culture of the anaerobe), (2) two one-holed rubber stoppers to fit tubes, (3) short piece of small-diameter rubber tubing, (4) two short pieces of glass tubing of diameter to fit tightly in holes of rubber stoppers, (5) small glass vial, (6) dry pyrogallic acid, (7) strong aqueous NaOH. 1 Bray anaerobic dish. Corning Glass Works, Corning, New York, Pyrex No. 3155, or dealer. 128 MANUAL OF MICROBIOLOGICAL METHODS Method. Put a column of pyrogallic acid, approximately IJ^ in. high, in the bottom of the empty tube. Stand empty vial in this acid. With pipet, fill vial two-thirds full of NaOH solution. Fashion a connecting unit from the rubber stoppers and rubber and glass tubing. Insert one of the stoppers in the tube with the chemicals. Push down cotton plug in culture tube to a level 1 in. above the medium. Insert second stopper in this tube. Tilt tube containing chemicals sufficiently to allow NaOH solution to spill over the acid. Advantages. If a supply of chemicals is at hand, it is useful as an emergency system, when the special equipment required by other systems is not available. Disadvantages. Not suitable for large numbers of cultures, or at least, such use would be more time consuming than other methods. Plating System Using Strongly Reducing Medium The single plating device introduced by Brewer (1942) is an ingenious method which offers a means of plating cultures without added equip- ment. The dish is used with agar containing strong reducing agents and is designed so that at the periphery the top rests on the medium, forming a seal. The remainder of the top is slightly raised, so that a small amount of air is trapped over the surface. The oxygen entrapped is removed by the reducing agents in the medium. Brewer Culture Dish^ Materials. (1) Brewer anaerobic culture dish; (2) regular petri dish with bottom either 15 or 10 mm deep; (3) infusion agar suitable for anaerobes which contains suitable reducing agents, such as the following: 0.2 per cent sodium thioglycollate, 0.1 per cent sodium formaldehyde sulfoxylate, and 0.0002 per cent methylene blue. Method. Pour sterilized medium in bottom of regular petri dish (25 ml minimum in 10-mm dish and 40 ml minimum in 15-mm dish). Streak center area from sample or culture. Replace the lid of the regular dish with the Brewer anaerobic lid. (The lid at its periphery should touch the agar at all points in order that a perfect seal be obtained. In the success- fully prepared dish, the agar in the center of the dish remains colorless while the blue color returns to the agar at the end of the dish because of oxygenation of the dye which serves as an oxidation-reduction-potential indicator.) Place plates in the incubator immediately after they are pre- pared, and examine as needed during the incubation period. When trans- fers are to be made from the plate, break the seal by a slight turn of the lid. Advantages. A useful, quick method of single-plate culture. An extremely simple method which is easy to learn and use. The only trick in the technic is to have 1 Brewer anaerobic dish. Baltimore Biological Laboratory, Baltimore, Maryland, and Kimble Glass Company, Vineland, New Jersey. THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 129 sufficient agar in the original dish so that a perfect seal is formed when the special lid is added. Recommended for routine use in hospital laboratories, and particularly for mobile laboratories, where anaerobic cultures for pathogens may be encountered. Disadvantages. Surface moisture may result in film formation in some instances; this may be reduced by using a porcelain top (see footnote 1 on page 124) on the regular dish prior to the Brewer anaerobic lid or by drying the plates in incubator before streaking. Some organisms apparently are inhibited by the reducing agents. This is not serious, since the reports indicate that all pathogenic types are easily cultured by this method. The Brewer anaerobic lids are, at the present time, relatively expensive. TECHNICS FOR STUDY OF ANAEROBIC BACTERIA^ In the above section the various pieces of apparatus and methods for their use with anaerobic bacteria have been considered. Formulas for the particular media which are recommended may be found in Chap. III. The remainder of this chapter will be devoted to a discussion of the details of certain technics which should aid the worker who has not had previous experience with anaerobes. It may not be amiss to insert here a precautionary note concerning the necessity of very careful inspection of the purity of cultures. There are instances on record, in the older literature, where two species grew sym- biotically on plate culture with such constancy that recorded observations were made of the colony type of mixture, the investigator being unaware of the existence of more than one type. In all studies concerning obligate anaerobes, a check on the purity of the culture should be made with regard to aerobic contaminants. The following test is suggested: For most cultures, streak a glucose nutrient agar slant and incubate it at 37°C; but for anaerobic species having a lower or higher optimum tem- perature, incubate a second agar slant at the temperature which is optimum for the anaerobe. If the culture appears free of aerobic types, investigate the purity with respect to anaerobic contaminants. Make repeated platings, and scrutinize intensely the colonies which develop. For cultures which will grow on the egg-yolk agar (with 0.3 to 0.5 per cent glucose added for the butyric group) of McClung and Toabe (1947), contamination in cultures is more readily revealed on this medium than on media not containing egg yolk. Preliminary Microscopic Examination If the sample is suitable, one should make preliminary examination using the gram stain. The conventional method of staining a smear, heat ^ In this chapter reference will be made to the "pathogenic group " and the "butyric- butyl group." The former term is used to designate such organisms as Clostridium tetani, C. septicum, C. histolyticum, C. chauvoei, C. perfringens, C. parahotulinum, C. hotulinum, and C. sporogenes. In the butyric-butyl group are included C. butyricum, C, beijerinckii, C. butylicum, C. pasteurianum, C. acetobutylicum, C. felsineum, C. roseum, and C. thermosaccharolyticum. 130 MANUAL OF MICROBIOLOGICAL METHODS fixed on a glass slide, should be used, except that the decolorizer should be either 95 per cent ethyl alcohol {'preferred) or 25 parts of acetone and 75 parts of ethyl alcohol. The use of greater amounts of acetone must be avoided because of the ease with which anaerobes are decolorized. Decolorization of young cultures of Clostridia is relatively common even when alcohol is used with caution. The presence of numerous gram- negative cells does not rule out Clostridia. The usefulness of the gram method is limited in smears prepared from blood, fibrin, or albumin. In samples of pathologic material, large, gram-positive rods are likely to prove to be anaerobic bacilli, but a final diagnosis must not be based on microscopic observations unsupported by cultural tests. Of the strictly aerobic gram-positive species, Bacillus anthracis Koch is the only usual pathogen. The characteristic morphology of Clostridium perfringens (syn. C. welchii) and the regularity of its appearance in certain clinical conditions frequently combine to give presumptive evidence of value; similarly, the typical microscopic picture presented by a spore-bearing C. tetani culture should be remembered when such forms are encountered in pathologic material. All anaerobic species are non-acid-fast; there- fore, this stain has no diagnostic importance. Microscopic Examination of Pure Cultures Gram stain. If the organism in question will grow within this period, apply the gram stain to a 16- to 18-hr culture and observe the same cau- tion with reference to the decolorizer as noted above. Ordinarily the stain is satisfactory when prepared from any enrichment medium in which the organism will grow. In recording the gram reaction of a new species, state the medium from which the smear was made and the age of the culture. Examination for motility. The majority of the sporeforming anaerobic bacilli are motile ; the most important exception is Clostridium perfringens (C. welchii) . The technic by which the motility examination is made is often of utmost importance in securing the correct results. Unless the culture is known to he nonpathogenic, discard all cover slips and slides into a disinfectant solution or sterilize by steam before washing. Use young cul- tures (12-18 hr) except as noted. Accept the results of hanging drop or wet-mount preparations under cover slips only if observation reveals posi- tive motility. If motility is doubtful or appears to be negative, initiate other procedures. For example, use a flattened capillary tube sealed at each end. Heat glass tubing, of small diameter, and flatten a small area. Prepare a capillary tube from the flattened section. Draw a small amount of culture into this tube, and seal the tube in the flame on both sides of the drop of culture. Examine this preparation with the high- power objective. If the motility is still recorded as negative, make THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 131 further observations on younger (4-6 hr) cultures. For these, examine the third or fourth tube of a serial passage series, using the medium which appears to give the best growth of the culture. Semisolid agar (0.5 per cent) may be inoculated by stab and observed for clouding due to motility. In some cases this procedure is complicated by breaking up of the agar as a result of gas production. Because of the relatively small number of species which are nonmotile, considerable caution should be exercised in reporting cultures which appear to be nonmotile. Naturally occurring nonmotile variants of motile species, however, have been encountered. Flagella stain. For material for preparation of flagella stains use young cultures growing in the medium which is most favorable to the organism being studied. A temperature lower than that generally considered optimum is recommended (Leifson, 1951). If difficulty is encountered in securing positive slides from cultures known or thought to be motile, use the technic of Leifson, and consult the directions given by O'Toole (1942) for suggestions in technic which refer particularly to anaerobic bacteria. Capsule stain. For the capsule stain one may use any of the conven- tional methods. The most important capsulated species is Clostridium perfringens (C welchii). Material taken from artificially infected labora- tory animals generally serves as the origin of smear preparations. If stains from in vitro cultures are desired, the medium of Svec and McCoy (Chap. Ill) is useful if other media prove unsuccessful. Demonstration of spores. Cultures surviving 20 min heating at 80°C may be presumed to be sporeformers. It is, however, useful to demon- strate the spores microscopically. The exact method of making the spore stain is of little importance in comparison with other factors, as each of the common methods (Dorner, Moeller, and malachite green) appears satisfactory. One must, however, pay some attention to the medium in which one expects to induce sporulation. Media containing fermentable carbohydrates are not satisfactory, in general, for the pathogenic group. The media naturally containing carbohydrate (e.g., corn mash or potato infusion), on the other hand, appear ideal for most of the butyric-butyl group. For the pathogens one should use the deep brain, beef heart, or alkaline egg medium. In some instances spores may be demonstrated within 24-28 hr after inoculation, but if the culture is negative at this time, older cultures should be examined. Protection from evaporation must be given cultures which are to be incubated longer than one week. Clostridium perfringens (C. welchii) appears to be one of the most difficult species in which to demonstrate spores microscopically with regularity. If success is not attained using the above-mentioned media in cultures having the characteristics of this organism, one may use the medium recommended by Ellner (1956). 132 MANUAL OF MICROBIOLOGICAL METHODS Since some taxonomic systems give considerable attention to the size and position of the spore, these characteristics should be recorded when the original laboratory examination is made. The characteristic appear- ance of Clostridium tetani spores has been noted above; these are round in shape and borne at the end of a slender vegetative rod. This is almost the only instance in which the picture of the spore and sporangium assumes importance in species diagnosis, and this observation must be supported by cultural or pathologic information as nontoxic organisms of similar microscopic characters occur. Granulose reaction. The cells of certain species, particularly during the early stages of spore formation, store granulose. To test for this, add a drop of LugoFs iodine to a wet mount preparation. Cells containing granulose will stain blue or violet, while others will appear yellow. Cultivation Technics^ Preliminary Enrichment Methods Ordinarily the best method to be followed in initiating growth of an anaerobe from a sample is to inoculate one of the tubed media rather than to proceed directly to plate culture. Certainly this should be done if there is question concerning the possible success of the preliminary cul- ture, and it is advised that parallel tube cultures be inoculated to serve as reserve cultures at the same time the plating is done, if the plating technic is favored. The medium to be used will be a matter of choice, as dis- cussed in Chap. Ill, depending upon the nature of the sample. If aerobic contamination is suspected and the anaerobe is thought to be in the spore state, a duplicate primary culture should be heated briefly (boil for 1 or 2 min, or hold at 80°C for 20 min). This should be a duplicate culture, however, in case the anaerobic form is a nonsporeformer or is a spore- former in the vegetative state. Almost all types of tubed media should have the dissolved oxygen driven off by boiling or heating in flowing steam. The specimen of choice for bacteriological analysis of deep wounds consists of bits of tissue taken at the time of debridement during the initial surgery, or at the time of change of dressing. Alternate, though less desirable, samples include swab samples or tissue exudate obtained by aspiration. Such samples should be planted in heart-infusion broth with heart particles (often called chopped-meat medium) and in addition, streaked on blood or egg-yolk agar. (See Chap. Ill for details of prepar- ation of these media.) Following incubation the heart-infusion cultures 1 The use of petrolatum, mineral oil, or other materials as a seal at the surface of liquid media is not recommended. THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 133 should be plated for isolation of pure cultures. It should be remembered that infections from such wounds are commonly polymicrobic in origin. Preliminary Purification Procedures It is often difficult to isolate anaerobic bacteria from enrichments which also contain aerobic bacteria. It would be presumed that aerobic bac- teria could ordinarily be eliminated merely by the anaerobic environment when this is introduced. Often in practice this is not the case, and other procedures must be instituted. It is of value frequently to attempt partial or complete elimination of the contaminants in tube culture using a liquid medium before plating is done. Materials derived from human or animal sources, other than feces, are usually contaminated with non- sporulating aerobic rods and cocci. Cultures derived from milk, soil, water, grains, feces, etc., contain, in addition, sporeforming aerobes. In fecal and perhaps other samples the contamination may include non- sporeforming anaerobes. If the nonsporeforming anaerobe is wanted, then anaerobic plating and picking of isolated colonies should be com- bined with optimum temperature and selective medium to secure the culture. In all cases the original enrichment tube should be preserved in the refrigerator, after growth is evident, until the purification routine is successfully completed. This will ensure a supply of starting material should something go wrong with the purification. Generally one of the easiest practices to be followed to get rid of non- sporeforming types is as follows : Heat subcultures from the contaminated enrichment, retaining the original tube, of course, unheated. Heat the newly inoculated tubes 20 min at 80°C or a shorter time a# higher tem- peratures. Take care to ensure the presence of the spores of the anaerobe. Use old cultures in a sugar-free medium as the best source of material to be heated, although other cultures may be satisfactory in special situations. For enrichments contaminated with sporeforming aerobes the above procedure may not be satisfactory, owing to the heat resistance of the aerobic spores. In this case, one may employ dyes as bacteriostatic agents. Nearly all, if not all, aerobic sporeformers are inhibited by crystal violet, and most of the anaerobic types are relatively resistant. Two or three serial transfers may therefore be made in a medium contain- ing this dye (approximately 1 : 100,000 final concentration) to eliminate the aerobe. The exact concentration of the dye to be used may vary with the medium and the conditions at hand. If the dye is used in some of the complex media, its effectiveness may be reduced during sterilization ; therefore, the dye should be added to such media after sterilization. Either liquid or solid media may be used. 134 MANUAL OF MICROBIOLOGICAL METHODS Sodium azide (0.02 per cent final concentration) together with chloral hydrate (0.01 per cent final concentration) may be used, singly or together, to inhibit gram-negative aerobic spreading organisms. Sorbic acid (York and Vaughn, 1944) in a final concentration of 0.12-0.15 per cent in thioglycollate broth will greatly aid in eliminating aerobic spore- forming bacilli, staphylococci, and many gram-negative contaminants. Initial enrichment broths are subcultured to sorbic acid thioglycollate broth, incubated 24 hr, then plated to an isolation plate. In the event that cultures are contaminated by Pseudomonas strains, polymyxin B may be added to the sorbic acid thioglycollate broth; a concentration of 10 /ig per ml will usually inhibit these organisms and permit recovery of Clostridia present (Lindberg, Mason, and Cutchins, 1954). Another method for elimination of aerobic sporeformers utilizes the fact that while growth of the aerobe may take place in an anaerobic environment, the conditions for sporulation are unfavorable. Under such conditions the anaerobe will be expected to sporulate freely. Thus liquid cultures in tubes or plate cultures taken from an anaerobic jar are chosen for material for heating as in the case of the nonsporeforming contaminants. Isolation Procedures From a purely theoretical viewpoint, microscopic single-cell methods of isolation are ideal, but the low percentage of successes with these pro- cedures excludes them from any uses except research. Several reports are in the literature indicating success with anaerobes using the Chambers micromanipulator or similar instruments, and wherever there is great need for strains of single-cell origin, the technic should be attempted. Because of the sensitivity of the vegetative cells toward oxygen, it is recommended that spores be picked rather than vegetative cells. One should use freshly exhausted media showing highly reducing activity for the subcultures, and naturally the medium should be suited to the organism being purified. If growth is not evident within the first 48 hr, the tubes may be protected from evaporation and incubated indefinitely. Reputable workers have reported dormancy of spores for six months' or longer duration. In routine problems either plating or deep agar tube methods are avail- able for purification of cultures from the original enrichment tubes. As stated above, the usual procedure in the isolation of anaerobes from samples in which contamination is excessive is best done by attempting partial purification in tube culture. This, however, need not be the case if the population of the sample is dominated by one species. In these the plating routine may be started without the preliminary enrichment procedure. Perhaps a few words should be included concerning details THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 135 of technic. Since some of the anaerobes tend to spread rapidly over the surface of the agar, in many instances it will be found that ''poured" agar plates are to be preferred to plates inoculated by streaking the surface. Two common methods are available for preparing these: (1) Melt tubes of the plating medium, cool, and inoculate before pouring; (2) place a small amount of sterile tap water in the culture dish, inoculate, and pour the agar into the dish immediately. If conditions warrant, use crystal violet in the agar. Place the plates in the anaerobic environment as soon as possible. (The size of inoculum to be used will vary so that some prac- tice may be necessary to give a dilution sufficient so that well-isolated colonies will appear.) If difficulty is encountered in obtaining discrete colonies, decrease the agar concentration in the plating medium to 0.75- 1.0 per cent. Another method is available for colony isolation which may be pre- ferred, particularly if the special apparatus needed for some of the plating methods is not at hand. This method involves the inoculation of a column of medium as mentioned in the opening pages of this chapter in the discussion of methods useful to determine whether or not a particular strain is an obligate anaerobe. For isolation purposes the fewer the number of colonies appearing in the medium the better. The percentage of fermentable sugar should be reduced to the lowest amount which gives good growth of the organism in order to prevent the production of gas, which may crack the medium. Assuming that we have available a deep tube of agar in which there appear several isolated colonies, two methods of isolation are available: (1) If soft glass tubes are used, cut the glass and break the tube at a short distance below the desired colony. Deposit the agar quickly in a sterile petri dish. Using a hot needle or small blade, cut across the plug of agar near the colony and transfer it to a suitable liquid medium. (This method is preferred if the tube shows aerobic contamina- tion in the upper layers.) (2) If Pyrex tubes are used, eject the plug of agar into the sterile dish by applying a bunsen flame to the bottom end. Before this, heat the sides of the tube and sterilize the mouth of the tube in the flame. During the ejection step of the technic, hold the mouth of the tube so that it points directly into the sterile dish. After the column of agar is deposited in the dish, proceed as discussed above. Inoculation Technics The following points of culture transfer and other routine technics are sufficiently different from the procedures used with aerobes so that some note is needed : Steam or boil most liquid media for a few minutes immediately prior to inoculation in order to drive off oxygen which may have been absorbed following sterilization. Attempt to deliver the inoculum to the bottom of 136 MANUAL OF MICROBIOLOGICAL METHODS the new tube of medium, for it is this portion of the medium which will stay reduced the longest. Although it is possible to initiate growth from a small number of cells, in routine studies use a more adequate inoculum. To facilitate the placing of the inoculum in the bottom of the tube with liquid and semisolid media, substitute a Wright or Pasteur pipet (used with small rubber bulbs) for the inoculation needle. By this means trans- fer a small drop (0.1 or 0.2 ml) of the culture to the new tube. Use the pipet also in the isolation of subsurface colonies particularly from media in which the concentration of agar is reduced. Prepare these pipets from 6- to 8-in. lengths of sterile 8- to 9-mm soft glass tubing (with cotton plug in each end) by applying heat to the center of the glass and pulling to form two capillary pipets. In general, use a culture from 16-20 hr old. With the pathogenic types this time may be extended a few hours with no harm. With the butyl- butyric types, however, which sporulate readily in many media, there is a critical period in which the culture is not very satisfactory for transfer purposes. As the culture goes into the spore stage, it is less and less suit- able until sufficient time elapses for the spores to mature. When spores are present in the inoculum, with these cultures and perhaps others as well, the new tube should be given a heat treatment (80°C for 20 min) after inoculation. Generally, if an anaerobic sporeforming culture is desired in an experi- ment, inoculate a tube of a favorable medium from a stock culture which contains spores, heat-shock it, and use the resulting culture for the experi- ment rather than the inoculation of the latter tube or flask directly from the spore-containing culture. Maintain the stock culture in the spore state, follow the above transfer routine rather than carry the anaerobe in a serial passage, and use such cultures for sources of inoculum for experi- mental flasks or tubes. This is particularly true with the actively fermentative types, where serial passage may yield a culture of undesir- able characters — even though it is descended in pure state from a culture that was satisfactory. Other Methods of Value Stock Culture Methods The anaerobes are susceptible to freezing-drying technic as a means of preservation of cultures over a long period of time as shown by Roe (1940). This technic is unnecessary, however, as species of Clostridium are usually viable in spore state over a long period of time. For the pathogenic group, one should use beef-heart infusion, alkaline egg medium, and brain mash, with the last perhaps being the best. With the butyric-butyl group, use plain corn mash or potato infusion. Prepare the plain corn THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 137 mash in a manner similar to the method given for corn-Hver medium with the exception that the Uver powder is omitted. Brain medium may be suitable also (see also Chap. III). In any medium, after all gassing has subsided and spores have been demonstrated microscopically, the tube should be sealed in the flame or the stopper covered to protect the medium from evaporation and the tube placed in a cool room or refrigerator. Viable subcultures may be obtained from such tubes for months or even years in some instances. Another method which has been used with success is worthy of mention. This involves the storage of cultures on sterile soil : Dry fresh garden soil and sift through a fine-mesh screen; add 5 per cent of CaCOs to neutralize any acidity of the culture. Place soil in tubes in 2-in. columns, and auto- clave overnight. Test each tube for sterility, using both aerobic and anaerobic media. If sterile, add 2 or 3 ml of a well-sporulated culture with a sterile pipet and dry the tube (preferably in a vacuum desiccator) . To obtain an active culture from this stock (which may be stored at room temperature) transfer a small amount of the soil to an enrichment medium and heat-shock. By the soil stock method a relatively permanent source is available from which cultures may be revived as needed without destroying the stock culture. Serological Reactions The serological relationships of the sporeforming anaerobes have been reviewed (McCoy and McClung, 1938; Smith, 1955). The toxin-anti- toxin reaction is of value as a taxonomic aid with certain species. In such an instance one takes advantage of the fact that relationships may be established by the success or failure of the reaction of antitoxin, pre- pared against the toxin of a kno\vn organism, with the toxin from the unidentified strain. In some instances the anaerobic species are mono- typic with respect to toxin formation. In other species this is not true, and subgroups have been established within these species or species groups on the basis of non-cross-neutralization tests. The problem of the complex relationships of the toxins produced by the Clostridia is beyond the scope of this chapter, but those interested in the details beyond those presented by Smith (1955) should consult Oakley (1954) and Van Heyningen (1950, 1955). REFERENCES Barker, H, A. 1936. Studies upon the methane-producing bacteria. Arch. Mikrob., 7, 420-438. Brewer, J. H. 1940. Clear liquid mediums for the "aerobic" cultivation of anae- robes. /. Am. Med. Assoc, 115, 598-600. . 1942. 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A modification of the Buchner method of cultivating anaerobic bacteria. Science, 75, 416-417. Hall, I. C. 1922. Differentiation and identification of the sporulating anaerobes. /. Infectious Diseases, 30, 445-504. . 1928. Anaerobiosis. Chap. XIII in "The Newer Knowledge of Bac- teriology and Immunology," edited by E. O. Jordon and I. S. Falk, University of Chicago Press, Chicago. 1929. A review of the development and application of physical and chemi- cal principles in the cultivation of obligately anaerobic bacteria. /. Bacterial. 17, 255-301. Heller, H. H. 1921. Principles concerning the isolation of anaerobes. Studies in pathogenic anaerobes. II. /. Baderiol. , 6, 445-470. Hewitt, L. F. 1950. "Oxidation-reduction Potentials in Bacteriology and Biochem- istry," 6th ed. E. S. Livingstone, Ltd., Edinburgh. Knight, B. C. J. G. 1931. Oxidation-reduction potential measurement in cultures and culture media. Chap. XIII in "System of Bacteriology," vol. 9. (Great Britain) Medical Research Council. Knorr, M. 1923. Ergebnisse neurer Arbeiten iiber krankheitserregende Anaerobien. I. Teil. Kjanksheitserregende anaerobe Sporenbildner, ausschliesslich Tetanus und Botulinus. Zentr. Gesam. Hyg., 4, 81-100, 161-180. . 1924. Ergebnisse neuerer Arbeiten iiber krankheitserregende Anaerobien. II. Teil, 1: Botulismus. Zentr. Gesam. Hyg., 7, 161-171, 241-253. Laidlaw, P. P. 1915. Some simple anaerobic methods. Brit. Med. J., 1, 497-498. Lebert, F., and P. Tardieux. 1952. "Technique d'isolement et de determination des bacteries ana6robies," 2d ed., 55 pp. Pacomhy, Paris. Leifson, E. 1951. Staining, shape, and arrangement of bacterial flagella. J. Baderiol., 61, 377-389. Lindberg, R. B., R. P. Mason, and E. Cutchins. 1954. "Selective Inhibitors in the Rapid Isolation of Clostridia from Wounds." Baderiol. Proc, pp. 53-54. McClung, L. S., E. McCoy, and E. B. Fred. 1935. Studies on anaerobic bacteria. II. Further extensive uses of the vegetable tissue anaerobic system. Zentr. BakterioL, II Abt., 91, 225-227. , and E. McCoy. 1941. "The Anaerobic Bacteria and Their Activities in Nature and Disease: A Subject Bibliography," Suppl. 1: Literature for 1938 and 1939. xxii and 244 pp. University of California Press, Berkeley, Calif. , and R. Toabe. 1947. The egg yolk plate reaction for the presumptive diagnosis of Clostridium sporogenes and certain species of the gangrene and botulinum groups. J. Baderiol., 53, 139-147. THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 139 . 1956. The anaerobic bacteria with special reference to the genus Clos- tridium. Ann. Rev. Microbiol., 10, 173-192. McCoy, E., E. B. Fred, W. H. Peterson, and E. G. Hastings. 1926. A cultural study of the acetone butyl alcohol organism. /. Infectious Diseases, 39, 457-483. , , , and . 1930. A cultural study of certain anaerobic butyric acid-forming bacteria. /. Infectious Diseases, 46, 118-137. , and L. S. McClung. 1938. Serological relations among the spore-form- ing anaerobic bacteria. Bacteriol. Revs., 2, 47-97. , and . 1939. "The Anaerobic Bacteria and Their Activities in Nature and Disease: A Subject Bibliography" (in two volumes) . rearm and 295 pp.; xi and 602 pp. University of California Press, Berkeley, Calif. Mcintosh, J. 1917. The classification and study of the anaerobic bacteria of war wounds. {Gt. Brit.) Med. Research Council, Spec. Rpt. Ser., 12, 1-58. Meyer, K. F. 1928. Botulismus. In W. Kolle, R. Krause, und P. Uhlenhuth, "Handbuch der pathogenen Mikroorganismen," 3 Aufl., 4, 1269-2364. Molland, J. 1944. Oxidation-reduction potentials in cultures of anaerobic bacteria. Acta Pathol. Microbiol. Scand., 21, 673-712 . Oakley, C. L. 1954. Bacterial toxins. Demonstration of antigenic components in bacterial filtrates. Ann. Rev. Microbiol., 8, 411-428. O'Toole, E. 1942. Flagella staining of anaerobic bacilli. Stain TechnoL, 17, 33-40. Prevot, A.-R. 1950. "Manual de classification et de determination des bacteries anaerobies," 2d ed., 290 pp. Masson et Cie, Paris. . 1955. "Biologic des maladies due aux anaerobies," 572 pp. Editions M^dicales Flammarion, Paris. Reed, G. B., and J. H. Orr. 1941. Rapid identification of gas gangrene anaerobes. War Med., 1, 493-510. , and . 1943. Cultivation of anaerobes and oxidation-reduction potentials. /. Bacteriol, 45, 309-320. Roe, A. F. 1940. Report on viability of 200 cultures of anaerobes desiccated for six years. /. Bacteriol, 39, 11-12. Smith, L. DS. 1955. "Introduction to the Pathogenic Anaerobes," 253 pp. Uni- versity of Chicago Press, Chicago. Snieszko, S. 1930. The growth of anaerobic bacteria in petri dish cultures. Centr. Bakteriol, II Abt., 82, 109-110. Spray, R. S. 1930. An improved anaerobic culture dish. J. Lab. Clin. Med., 16, 203-206. . 1936. Semisolid media for cultivation and identification of the sporulat- ing anaerobes. J. Bacteriol, 32, 135-155. Van Heyningen, W. E. 1950. "Bacterial Toxins," 133 pp. Charles C Thomas, Pubhsher, Springfield, lU. . 1955. Recent developments in the field of bacterial toxins. Schweiz. z. allgem. Pathol, u. Bakteriol, 18, 1018-1035. York, K., and R. H. Vaughn. 1944. Use of sorbic acid enrichment media fof species of Clostridium. J. Bacteriol, 68, 739-744. Zeissler, J. 1930. Anaerobenzuchtung. In W. Kolle, R. Krause, und P. Uhlenhuth, "Handbuch der pathogenen Mikroorganismen," 3 Aufl., 10, 35-144. Zeissler, J., and L. Rassfeld. 1928. Die anaerobe Sporenflora der europaischen Kriegsschauplatze 1917. Veroffentl Kriegs-Konstitutionspathol, 5, Heft 2. 99 pp. CHAPTER VII Routine Tests for the Identification of Bacteria H. J. Conn, M. W. Jennison, and O. B. Weeks INTRODUCTORY The Society of American Bacteriologists issues descriptive charts for use in characterizing bacterial species. The charts are blank forms to be recorded, at least one chart to be used for each culture studied. The ''Manual of Methods for Pure Culture Study of Bacteria" was originally published to secure uniformity in the methods used for determining these characteristics. The present ''Manual of Microbiological Methods" has become much broader than this, and practically all the methods covered in the first editions of the original manual are now included in this chapter. The methods described in this chapter are intended primarily for aerobic saprophytes and cannot therefore be considered applicable in general to strict anaerobes, nutritionally "fastidious" organisms, and bacteria having other "special" cultural characteristics. Chapter VI must be consulted in studying the latter group, while Chap. IX gives methods specially applicable to animal pathogens. Special methods for plant pathogens are given in Chap. XII. In the case of other special groups, the investigator will therefore be forced to modify the methods or to use others more suited to the group in question. THE DESCRIPTIVE CHARTS There are two descriptive charts, each printed on 83-^- by 11-in. sheets of heavy paper: the Standard Descriptive Chart and the Descriptive Chart for Instruction. The general plan of each is to have the body of it consist, under various headings, of a series of blanks to be completed and descriptive terms to be underlined as the various characteristics of the cultures are determined. In addition to this, there is a place on the mar- 140 ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 141 Is? & fi Ml 6 n t) 2 o s Z Q ^ 1 gills gill pi I "111 1 Hi! J I ■?^ii. Nliili ly-naiiao S|;i nVJl'JOlOlSAHd 1 21^1 it In If! fill II III' 3 ill *Ji M 5 I I illHli im 5 O ^< II 3 ! Si! K m ; '-11 iilli alii mi til •g 3 . 'V, "^ o o o a> ^ > >> 13 -a > . o t>» 1—1 rO \IM 'd r-l\ c3 00 CI C3 02 142 MANUAL OF MICROBIOLOGICAL METHODS I & Pimi \i 111 I illil lllllll 111 ill |ii liill 111 I jjiiyi liltlill ni It 5 IJ& iii I! m J ! II 1 J- .Mil I i i I i S ill! I iinl ill iiil III! •E S J g 5 5l« IV^i^V 111? m Jijii [yellow With indicator media it is difficult to learn the exact reaction by refer- ence to color standards, but a good estimate as to hydrogen-ion concen- tration can be obtained by inspection, particularly when three tubes are used, one with each of the three indicators recommended above. For this purpose Table 15, showing the relation of the ranges of these three indicators to one another, will be found useful. After some experience a bacteriologist can usually devise some method for recording on the chart, by a system of numerals or + signs, the strength of reaction observed with each indicator employed; such a system often proves practical for comparative purposes but gives no very definite information as to final H-ion concentration. Gas production in liquid media can be measured in percentage of gas in the closed arm of the Smith or the Durham fermentation tube. The Durham tube, which is most commonly used, consists of a small test tube (e.g., 75 by 10 mm) inverted in a large tube (e.g., 150 by 18 mm). In the case of solid media it is recorded as present or absent according to whether or not bubbles or cracks are present in the agar. This test is especially valuable if the organism is tested in a shake culture, but the presence of gas can usually be detected in an ordinary agar slant. These tests for gas production are chiefly useful if the organism produces pri- marily hydrogen; if the gas is all carbon dioxide, little or none will accumulate in the fermentation tube because of the great solubility and rapid diffusion into the air. , Interpretation of results. In case an organism produces gas or con- siderable increase in acidity in either broth or beef-extract peptone agar in the presence of some fermentable substance and this does not occur in the basal medium without the addition of the fermentable substance, it may safely be concluded that cleavage of this substance has occurred. Very often for routine diagnostic purposes such information is enough. To understand the true action of the organism on any carbon compound, however, much more investigation must be made as explained elsewhere (see Chap. VIII). This is particularly necessary in the case of organisms 162 MANUAL OF MICROBIOLOGICAL METHODS that produce a small amount of acid in some tubes but not in others con- taining the same carbon source and in cases where the addition of some carbon source results in a distinctly improved growth without the appear- ance of demonstrable acid or gas. In routine work, accordingly, one should record as positive only those organisms that produce considerable acid or gas from a given compound and as negative only those that con- sistently fail to show any acid or gas or any increase of growth when supplied with the carbon compound under investigation. All others should be regarded as border-line cultures, calling for further investigation. It is especially important to recognize that the question whether or not cleavage of a carbohydrate occurs depends greatly on the cultural charac- teristics. Clarke and Cowan (1952) remark that tests of fermentative ability are often tests of the ease with which the buffer capacity of a medium is overcome. Accordingly some bacteria fail to produce an acid reaction in a beef-extract-peptone medium but will do so from the same carbohydrate in a synthetic or semisynthetic medium. It must accord- ingly be recognized that although tests for fermentative ability in some medium may have diagnostic usefulness, they do not necessarily indicate actual ability to metabolize the carbohydrate therein. HYDROLYSIS OF STARCH The breaking down of starch is rather more complicated than that of sugars because of the extensive hydrolysis that is necessary before it can be utilized by the bacteria. The first stage of this process is generally known as diastatic action because of the similarity to that brought about by the enzyme diastase. The final end result is usually an increase in acid, so one may obtain good evidence as to the utilization of starch by substituting it for sugar in the above methods (pages 159ff.) and deter- mining acid produced or increase in H-ion concentration. It is often desirable, however, to secure evidence as to the intermediate products and as to whether the starch has been entirely consumed or not, and various methods have been proposed for this purpose. This test may be made on raw starch, dissolved by boiling, or on the so-called "soluble starch." The latter is a partly hydrolyzed product, but it is often used as "starch" in this test because its iodine reaction is like that of true starch and different from that given by typical dextrins. If soluble starch is used, its true nature must be taken into account, but at the same time it must be remembered that true starch is partly hydrolyzed when sterilized in culture media, and even cultures growing in such a substratum are not furnished with raw starch as the sole carbo- hydrate. When such media are filtered, possibly "soluble starch" is all that remains. ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 163 A satisfactory method has been proposed by Eckford (1927) for learn- ing the type of action on starch brought about by organisms capable of making good growth in broth. The same method may be adapted to organisms which prefer some other liquid medium by substituting it for broth in Eckford's method. The procedure, however, is not well adapted to those bacteria that fail to grow well in liquid medium. The technic is as follows: Add 0.2 per cent soluble starch to broth and incubate cultures a week to 10 days. Examine on second, fourth, seventh and tenth days for hydrolysis of starch, produc- tion of acid, and reduction of Fehling's solution. For this test a drop is placed in a depression on a porcelain plate and a larger quantity in a serological test tube. The latter is tested for acid production with an indicator of the proper pH range. To the drop on the plate add a drop of dilute iodine solution and read reaction as follows: if blue, no hydrolysis; if reddish brown, partial hydrolysis with production of erythro- dextrin; if clear, hydrolysis complete, with production of dextrin or perhaps glucose. The tubes showing complete hydrolysis may be tested for reducing sugar with Fehling's solution. For bacteria that do not grow well in liquid media, no better method has yet been proposed than the plate technic given in all previous editions of the manual with little modification. This method has its disadvan- tages but is often useful; it is as follows: Use beef-extract agar containing 0.2 per cent of soluble starch. Pour it into a petri dish, and after hardening make a streak inoculation on its surface. Incubate at optimum temperature for the organism under investigation. Observations are to be made on the second day for rapidly growing organisms but not until the seventh day for the more slowly growing ones. To make the test, flood the surface of the petri dishes with Lugol's iodine or with a saturated solution of iodine in 50 per cent alcohol. The breadth of the clear zone outside the area of growth indicates the extent of starch destruction. By means of a simultaneous inoculation on another plate containing the same medium with bromcresol purple as an indicator one may at the same time learn whether or not acid is produced as an end product. THE METHYL RED AND VOGES-PROSKAUER TESTS Special tests as to cleavage of glucose are commonly made in the differentiation of the organisms of the colon-aerogenes group. The medium ordinarily employed is as follows: 5 g of proteose peptone (Difco, Witte's, or some brand recognized as equivalent), 5 g of cp glucose, 5 g of K2HPO4 in 1,000 ml of distilled water. The dry potassium phosphate should be tested before using in dilute solution to see that it gives a distinct pink color with phenolphthalein. According to Smith (1940), the K2HPO4 in this medium should be replaced with the same amount of NaCl, if the tests are to be carried out on aerobic sporeformers. 164 MANUAL OF MICROBIOLOGICAL METHODS Tubes should be filled with 5 ml each, and each culture should be inocu- lated into duplicate (or triplicate) tubes for each of the two tests. Incu- bation should be at optimum temperature of the organism under investi- gation, and tubes should be incubated 2-7 days, according to the rate of growth of the organism in question. Although the same medium is used for both the methyl red and the Voges-Proskauer tests, they must be per- formed in separate tubes. The latter test depends upon the production of acetyl-methyl-carbinol from the glucose. Fabrizio and Weaver (1947) show the possibility of a rapid test for the production of this compound ; Cowan (1953) agrees as to its practicability. A similar micro- test for the methjd red reaction proves more difficult. A positive methyl red reaction is regarded as being present when the culture is sufficiently acid to turn the methyl red (0.1 g dissolved in 300 ml of 95 per cent ethyl alcohol and diluted to 500 ml with distilled water) a distinct red; a yellow color with the methyl red indicator is regarded as a negative reaction, while intermediate shades should be considered doubtful. For the Voges-Proskauer reaction, according to the ''Standard Meth- ods" of the APHA (1946), to 1 ml of culture add 0.6 ml of 5 per cent a-naphthol in absolute alcohol and 0.2 ml of 40 per cent KOH. It is important to shake for about 5 sec after addition of each reagent. The development of a crimson to ruby color in the mixture from 2 to 4 hr after adding the reagents constitutes a positive test for acetyl-methyl- carbinol. Results should be read not later than 4 hr after addition of the reagents. Various other tests have been suggested for this reaction, both to obtain results more quickly and because some organisms apparently give different results with dif- ferent tests. In any case, weakly positive reactions may be obscured by the color of the reagent. A procedure which has given excellent results with many thousand cul- tures run by a member of the committee (CAS) is the creatine test of O'Meara, as modified by Levine, Epstein, and Vaughn (1934). In this procedure the test reagent added to the culture is 0.3 per cent creatine in 40 per cent KOH. This reagent deterio- rates rapidly at temperatures over 50°C but may be kept 2 weeks at room temperature (22-25°C) or for 4 to 6 weeks in a refrigerator. A recent modification of Coblentz (1943) is similar to the APHA method but uses a massive inoculum in broth from an infusion-agar slant culture, followed by incuba- tion of the broth for 6 hr. Also, the 40 per cent KOH has 0.3 per cent of creatine added to it to intensify the reaction. After addition of the reagents (a-naphthol and KOH-creatine) the culture is shaken vigorously for 1 min; a positive reaction is charac- terized by an intense rose-pink color developing in a few seconds to 10 min. The microtest of Fabrizio and Weaver (1947) calls for inoculation with a loopful of growth from a 6- to 12-hr infusion agar slant into 0.5 ml of infusion medium with 1 per cent trypticase and 0.5 per cent NaCl, placed in small tubes and preheated in a 30° water bath. It is then incubated at 30°C in a water bath for 90 min. It is then tested for acetyl methyl carbinol by the same method as given above, except that smaller quantities of the reagent (0.15 and 0,05 ml, respectively) are added. ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 165 ACID PRODUCTION IN MILK Acid production in milk maj^ be determined verj^ simply, but the opacity of the milk must be taken into account if accurate determinations are desired. The milk must be considerably diluted before adding indi- cator for comparison with a buffer standard. Indicator milk is often useful. Litmus has been used most frequently, as it indicates reduction as well as pH changes (although roughly) . Neu- tral litmus milk (about pH 6.8) has a lavender color, which becomes red with acid production or blue with production of alkalinity. Reduction is indicated by a partial or complete fading of the color. The use of litmus milk has been seriously criticized because of the inaccurate nature of litmus as a pH indicator; nevertheless the differences it brings out have enough practical value so that it has not yet been superseded by any other indicator in milk. The use of bromcresol purple, as was recommended by Clark and Lubs (1917), does not show changes in 0/R potential. Table 16 Degrees of Acidity Easily Recognized in Milk Acidity Indicator, reaction, etc. Approximate pH value "Neutral" "Weak" Same color with bromcresol purple as sterile milk, i.e., blue to gray-green Color with bromcresol purple lighter than in sterile milk, i.e., gray-green to greenish yellow Yellow with bromcresol purple. Not curdled Curdled. Blue or green to bromphenol blue Yellow to bromphenol blue 6.2-6.8 "Moderate" "Strong" "Very strong" 5.2-6.0 4.7-6.0 3.4-4.6 Under 3 . 4 It is possible to recognize the five degrees of acidity listed in Table 16 by the use of bromcresol purple (either in the milk before inoculation or added after incubation), the subsequent addition of bromphenol blue, and observation as to the presence of curdling. This is only a rough method of measurement, but in the routine study of milk cultures it will often be found valuable. H. C. Brown (1922) proposed condensed milk diluted with 4 parts of water contain- ing phenol red. The reaction is adjusted by addition of alkali until first appearance of a brick red. Subsequent changes of reaction in either direction can be observed. RENNET PRODUCTION The production of the enzyme rennet (lab) can sometimes be recognized in litmus milk by noticing the occurrence of coagulation without the 166 MANUAL OF MICROBIOLOGICAL METHODS appearance of acid. It is often obscured by simultaneous digestion, how- ever, and two other methods have been proposed which often show rennet production with cultures that fail to show it when inoculated directly into milk. Conn (1922) grows bacteria in milk sterilized in the usual manner; after the appear- ance of whey or peptonized milk, 0.5 ml is transferred to 10 ml of unsterilized milk and placed in a 37° incubator. Examinations are made every 5 min for the first half hour and at less frequent periods thereafter for a few hours longer. First appearance of coagulation is noted. Undoubtedly this test could be standardized, if desired, so as to be performed like the other microtests given here. Gorini (1932) obtains vigorous growth on an agar slant, then covers the growth with milk, fractionally sterilized at temperatures not over 100° so as not to alter the color of the milk. The growth is mixed with the milk by use of a platinum needle, and the tube is incubated at 37° until coagulation occurs. Although the committee is not prepared to recommend either method, it is felt that by a combination of the two a good indication of rennet production can be obtained. REFERENCES American Public Health Association. 1946. "Standard Methods for the Examina- tion of Water and Sewage," 9th ed., published by the Association, New York. Arnold, W. M., Jr., and R. H. Weaver. 1948. Quick microtechniques for the identifi- cation of cultures. I. Indole production. J. Lab. Clin. Med., 33, 1334-1337. Bachmann, Barbara, and R. H. Weaver. 1947. A quick microtechnique for the detection of the reduction of nitrates to nitrites by bacteria. (Abstract) /. BacterioL, 54, 28. Bartholomew, J. W., and W. W. Umbreit. 1944. Ribonucleic acid and the Gram stain. J. BacterioL, 48, 567-578. Bisset, K. A. 1950. "The Cytology and Life-history of Bacteria." The Williams & Wilkins Company, Baltimore. Bohme, A. 1905. Die Anwendung der Ehrlichschen Indolreaktion fiir bacterio- logische Zwecke. Centr. Bakterioh, I Abt. Orig., 40, 129-133. Brough, F. K. 1950. A rapid microtechnique for the determination of nitrate reduc- tion by microorganisms. /. BacterioL, 60, 365-366. Brown, H. C. 1922. Use of phenol red as an indicator for milk and sugar media. Lancet, 202, 842. Clark, W. M., and H. A. Lubs. 1917. A substitute for litmus for use in milk cul- tures. J. Agr. Hesearch, 10, 105-111. , and S. T. Cowan. 1952. Biochemical methods for bacteriology. /. Gen. MicrohioL, 6, 187-197. Clarke, P. H., and Cowan, S. T. 1953. Hydrogen siilphfde production by bacteria /. Gen. MicrohioL, 8, 397-407. Coblentz, J. M. 1943. A rapid test for acetyl methyl carbinol production. Am. /. Public Health, 33, 815. Conn, H. J., and G. J. Hucker. 1920. The use of agar slants in detecting fermen- tation. /. BacterioL, 5, 433-435. ' . 1922. A method of detecting rennet production by bacteria. /. Bac- terioL, 7, 447-448. ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 167 , and Gladys E. Wolfe. 1938. Flagella staining as a routine test for bacteria. /. Bacteriol, 36, 517-520. Cowan, S. T. 1953a. Fermentations: Biochemical micro-methods for bacteriology. J. Gen. Microbiol, 8, 391-396. . 19536. A micromethod for the methyl red test. J. Gen. Microbiol., 9, 101-109. Eckford, Martha O. 1927. Thermophilic bacteria in milk. Am. J. Hyg., 7, 201- 221. (See p. 208.) Fabrizio, Angelina, and R. H. Weaver. 1947. A quick microtechnique for the detection of acetjdmethylcarbinol by bacteria. (Abstract) /. Bacteriol., 54, 69. Fellers, C. R., and R. W. Clough. 1925. Indol and skatol determination in bacterial cultures. /. Bacteriol., 10, 105-133. Fisher, P. J., and Jean E. Conn. 1942. A flagella staining technic for soil bacteria. Stain Technol, 17, 117-121. Foth, 1892. Zur Frage der Sporenfarbung. Centr. Bakteriol., 11, 272-278. Frazier, W. C. 1926. A method for the detection of changes in gelatin due to bacteria. /. Infectious Diseases, 39, 302-309. Frieber, W. 1921. Beitrage zur Frage der Indolbildung und der Indolreacktionen sowie zur Kenntnis des Verhaltens indolnegative Bacterien. Centr. Bakteriol., I Abt. Orig., 87, 254-277. Gnezda, J. 1899. Sur les reactions nouvelles des bases indoliques et des corps albuminoides. Comyt. rend. acad. sci., 128, 1584. Gore, S. N. 1921. The cotton-wool plug test for indole. Indian J. Med. Research, 8, 505-507. Gorini, C. 1932. The coagulation of milk by B. typhosus and other bacteria con- sidered inactive on milk. /. Pathol. Bacteriol., 35, 637. Hannan, John, and R. H. Weaver. 1948. Quick microtechniques for tha identifica- tion of cultures. /. Lab. Clin. Med., 33, 1338-1341. Harrison, F. C. 1929. The discoloration of halibut. Can. J. Research, 1, 214-239. Henry, H., and M. Stacey. 1943. Histochemistry of the Gram-staining reaction for microorganisms. Nature, 151, 671. Holman, W. L., and F. L. Gonzales. 1923. A test for indol based on the oxalic acid reaction of Gnezda. /. Bacteriol., 8, 577-583. Hunter, C. A., and H. G. Crecelius. 1938. Hydrogen sulphide studies. I. Detec- tion of hydrogen sulphide in cultures. /. Bacteriol., 35, 185-196. Knaysi, G. 1951. ''Elements of Bacterial Cytology," 2 ed. Comstock PubHshing Associates, Inc., Ithaca, N.Y. Koser, S. A., and R. H. Gait. 1926. The oxalic acid test for indol. /. Bacteriol., 11, 293-303. Kovacs, N. 1928. Eine vereinfachte Method zum Nachweis der Indolbildung durch Bakterien. Z. Immunitdtsforsch., 55, 311-315. Leifson, Einar. 1951. Staining, shape, and arrangement of bacterial flagella. J . Bacteriol, 62, 377-389. Levine, Max, S. S. Epstein, and R. H. Vaughn. 1934. Differential reactions in the colon group of bacteria. Am. J. Public Health, 24, 505-510. Magoon, C. A. 1926. Studies upon bacterial spores. J. Bacteriol, 11, 253-283. (See pp. 261-264.) Moeller, H. 1891. tJber eine neue Methode der Sporenfarbung. Centr. Bakteriol, 10, 273-277. Morse, M. L., and R. H. Weaver. 1947. A quick microtechnique for the detection of hydrogen sulfide production. (Abstract) /. Bacteriol, 54, 28-29. 168 MANUAL OP MICROBIOLOGICAL METHODS Shaw, C, J. M. Stitt, and S. T. Cowan. 1951. Staphylococci and their classifica- tion. /. Gen. Microbiol, 5, 1010-1023. Smith, N. R. 1940. Factors influencing the production of acetyl-methyl-carbinol by the aerobic spore-formers. J. BacterioL, 39, 757. — ■ , Gordan, R. E., and Clark, F. E. 1946. Aerobic mesophilic sporeforming bacteria. U.S. Dept. Agr. Misc. Publ. 5^9. Tittsler, R. P. 1930. The reduction of nitrates to nitrites by Salmonella pullorum and Salmonella gallinarum, J. Bacterial., 19, 261-267. — , and L. A. Sandholzer. 1936. The use of semi-solid agar for the detection of bacterial motility. /. Bacterial., 31, 575-580. Untermohlen, W. P., and C. E. Georgi. 1940. A comparison of cobalt and nickel salts with other agents for the detection of hydrogen sulfide in bacterial cultures. J. Bacterial, 40, 449-459. Vera, H. D. 1949. Accuracy and sensitivity of fermentation tests. Sac. Am. Bacteriologists, Ahs., 49th, p. 6. Wallace, G. I., and S. L. Neave. 1927. The nitrite test as applied to bacterial cul- tures. /. Bacterial, 14, 377-384. Zipfel, H. 1912. Zur Kenntnis der Indolreaktion. Centr. Bakteriol, I Abt. Orig., 64, 65-80. Zobell, Claude E., and Catherine B. Feltham. 1934. A comparison of lead, bismuth and iron as detectors of hydrogen sulphide produced by bacteria. J. Bacterial, 28, 169-176. CHAPTER VIII Physiological and Biochemical Technics R. D. DeMoss and R. C. Bard INTRODUCTORY Even the bacteriologist not experiencing repeated contacts with micro- bial biochemistry is aware of the vast accumulation of knowledge in this field during recent years. Indeed, the methods and interpretations of microbial biochemistry have attained leading roles in modern micro- biology, and as a result of this position, the importance of these phases of study in the training of new workers and in current research can hardly be overemphasized. The main guiding principle during the preparation of this chapter has been the concept of environmental adaptation. The extreme adaptabil- ity and response of bacteria allow the choice of specific growth conditions in order to understand more easily and more completely a given bio- chemical situation. Thus, the premise has been adopted which demands a thorough study of physiological conditions relating to a specific bio- chemical process prior to a detailed investigation of that biochemical process. Accordingly, the following technics apply to physiological studies, often requiring a measurement of growth, and to both general and specific biochemical reactions. The material presented will deal primarily with bacteria, although investigations with algae, molds, protozoa, and even viruses along similar lines are being pursued vigor- ously and a unity of concept is being formulated. The methods described in this chapter are not in general applicable to routine work in the sense of taxonomic investigations (see Chap. VII). In some cases these technics represent an extension of the routine tests, while the majority of the methods, because of the requirement for more precise and detailed measurements, are based upon different principles and usually depend upon instrumentation in addition to visual observation. 169 170 MANUAL OF MICROBIOLOGICAL METHODS GROWTH MEASUREMENTS In routine tests for taxonomic purposes, semiquantitative measure- ments of growth are usually deemed satisfactory. However, for research problems in the physiological and biochemical aspects of bacteriology, quantitative methods are generally necessary. Either the rate or extent of growth is used for quantitative purposes, and the extent of growth generally represents the most accurate expression, particularly w^hen related to small experimental differences. Microbiological assay methods for the determination of amino acids, vitamins, and other growth factors, as well as antibiotics, are usually dependent upon growth measurements either directly or indirectly. Investigations into the nutritional require- ments of bacteria, although often satisfied by qualitative methods, are more definitively answered by quantitative growth determinations. Within the area of nutritional studies is included the elucidation of bio- chemical pathways by means of biochemical mutants. Various mutants must be defined on the basis of nutritional requirements before becoming useful in such a study. Several methods for the quantitative estimation of growth are described in detail below. Since most investigators publish results in terms of the milligrams of N or milligrams of cells (dry weight) used, the method employed for measuring is generally related by means of a standard curve to one of these unitages. For example, using a given suspension of bac- terial cells, one may determine turbidity, dry weight, and total Kjeldahl nitrogen and then construct curves of turbidity as a function of dry weight and of nitrogen. With subsequent suspensions, only the turbidity needs to be determined, and the dry weight may then be read from the previously constructed curve. However, it should be pointed out that the margin of error will vary with cells harvested at different stages of growth. In addition, the turbidity-dry-weight relationships determined for one species do not necessarily apply to another species or under differ- ent conditions of growth. Quantitative Methods Cell counts. Indirect method. Dispense sterile saline solution (0.85 per cent; w/v) in 9.0-ml amounts in sterile, cotton-plugged test tubes. Serially dilute 1.0 ml of a culture or suspension of the organism in the saline to give 1:10, 1:100, 1:1,000, etc., dilutions, respectively, of the culture. Dispense 10- to 15-ml quantities of melted nutrient agar in sterile plugged tubes, and hold at 40-45°C. To each agar tube, add 1.0 ml of a suitable dilution of bacterial suspension, mix well, pour into a sterile petri dish; allow to harden, and incubate. The incubated agar plate should contain 30-300 colonies for accuracy in counting, and all dilutions should PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 171 be plated at least in triplicate. After the colonies have been counted, the number of cells in the original suspension is calculated. Alternately, the inocula may be spread over the surface of agar plates in the case of obligate aerobes. In the latter procedure, plates are first poured and allowed to solidify. To remove excess water from the surface of the agar, the plates are dried overnight at 37°C or for 2 hr at 50-55°C. The sample to be counted is deposited (0.1 ml in 3-4 separate drops) on the agar surface and spread uniformly by means of a sterile bent glass rod. Since excess surface water has been removed, difficulties due to colony spreading are obviated. A culture in the maximum stationary phase of the growth curve will often contain about 10^ cells per ml; thus, the suitable dilution for plating will be found in the seventh serial dilution tube for a fully grown culture. The sixth and eighth tubes should also be plated in order to allow for errors in estimation of the original culture population. It should be obvious that not all bacterial species mil grow on nutrient agar and will therefore require a different agar medium in order to develop colonies. Anaerobic species must, of course, be incubated under anaerobic conditions. Furthermore, some species will require different diluent fluids, since saline may allow lysis of some cells, thereby resulting in low counts. The method is based upon the assumptions that each cell develops into one colony and that each colony is derived from only one cell. These assumptions are not necessarily valid for those species which grow in chains or clumps. Since not all cells are capable of reproduction, each cell mil not, as assumed, yield a colony. For these reasons, the estimate of number of cells will nearly always be lower than the true value. In addition, the proportion of nonreproducing cells may vary at different stages of growth; thus the error will not be constant. Inconstancy of error also will obtain when the size of the clump or the length of the chain varies with age of the culture. For a discussion of the application of statistical method to bacterial enumeration procedures, see the review by Stearman (1955) and the pre- vious reports cited therein. Direct microscopic method. A counting chamber of the hemocytometer type is employed in this method. The chamber consists of a ruled slide and a cover slip constructed in a manner such that a definite known volume is delimited by the cover slip, slide, and ruled lines. Detailed description and instructions for use are furnished with the commercially available apparatus. The Petroff-Hauser or Helber bacterial counting chamber gives results superior to those obtained with the ordinary hemo- cytometer. The Petroff-Hauser slide is ruled in 0.05-mm squares, with the distance between the cover slip and slide equal to 0.02 mm. 172 MANUAL OF MICROBIOLOGICAL METHODS According to Wilson and Knight (1949) the bacterial suspension should contain about 10^-10^ cells per ml for accurate counting. This cell con- centration results in 3-12 cells per square of the counting chamber. A total count of about 400 cells per slide results in a final count correct to within 10 per cent. For best results, the direct count is made with dark- field illumination. Results obtained with the counting chamber will always be higher than those from the colony-count method, since those cells which are not capable of reproduction are also counted. The direct-count method is, however, more rapid. Cell volume. This technic involves the hematocrit principle of blood- cell determination. Unlike blood cells, however, bacterial cells occupy a smaller proportion of the volume of the suspending medium, and larger samples must be employed. A tube similar to the Hopkins vaccine tube, which holds 5- to 10-ml samples containing 0-0.05 ml of cell volume, is satisfactory. Using the Hopkins tube, the cell volume may be estimated directly, and if desired, the number of cells may be estimated by calcula- tion from the known cell size. Schmidt and Fischer (1930) centrifuged cell suspensions in capillary tubes and then measured the height of the columns of sedimented bacteria. A number of disadvantages accompany this method. Unless standard conditions are rigidly followed, the final cell volume observed will be variable, since the cells can be packed loosely or tightly, depending upon the rate and time of centrifugation. The average volume per cell also changes during the course of growth. Thus, the problem of relating total cell volume to other growth measurements becomes complex. Any variation in suspending medium will probably affect the water content of the cells, thus causing variable results in the total volume observed. In this method the differences observed are often small, thus allowing much greater opportunity for error. Dry weight. Using distilled water, wash and resuspend the bacterial cells. Add aliquots of varying volume to small tared watch glasses or weighing bottles, and dry in an oven overnight at 85°C. After accurate weighing on an analytical balance, the dry weight of the bacterial mass in the original suspension is calculated. If salt or other solutions are used in place of distilled water, it is neces- sary to determine the dry weight of the solution constituents. The determination, as calculated from such results, will contain an error intro- duced by the wet volume of the bacterial cells. On the other hand, the use of water for washing and resuspending the cells tends to extract salts or other soluble compounds from the cells, resulting in a final weight which is lower than the true weight. Since the turbidity of a cell suspension is more easily and rapidly PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 173 determined, a standard curve is often constructed, relating dry weight to optical density (see below). Turbidity. Any of the available photometers are suitable for turbidity determination, although the type (Evelyn) which utilizes the sample tube as a lens for concentrating the transmitted light on a single phototube is probably the more accurate. Turbidity is measured most accurately at 420 m/x, providing the suspending fluid or medium is colorless. For yellow or brownish media, a wavelength of 660 uifi is employed. Turbid- ity changes during growth are often expressed simply as changes in optical density. However, at the higher values of optical density (0.4- 2.0) turbidity is not a linear function of other expressions such as dry weight, total nitrogen, or cell numbers. Therefore, the culture or suspen- sion should be suitably diluted before measurement, in order to fall within the range 0.0-0.4 optical density unit. If turbidity is selected as the method of determination of growth, number of cells, dry weight, or cell N, a standard curve relating optical density to the desired unitage is constructed. Determinations of both optical density (optical density = 2 — logio per cent transmission) and, for example, dry weight are performed on aliquots of a given cell suspen- sion. The standard curve then consists of a plot of optical density vs. dry weight. Since the optical density of a given cell suspension varies with the wavelength employed, separate standard curves must be con- structed for each wavelength which is to be used. The measurement of turbidity during growth may be accomplished by growing the culture directly in colorimeter tubes or in specially fabricated flasks fitted with side arms which may be inserted in a colorimeter (Wiame and Storck, 1953). Production of acid or alkali. The growth of several acid-forming bac- teria may be followed by simple titration of the culture, using standard alkali. For example, nutritional studies or microbiological assays with certain lactic acid bacteria are commonly recorded in terms of milliliters of O.OIA^ NaOH required to titrate the culture to the bromthymol blue end point. Conversely, during grow^th of some Mycobacterium species, acid disappears from the medium and growth may be recorded as milli- liters of O.OIA'' HCl required to reach the indicator end point. One limitation of this method is obvious. After maximum growth is attained, metabolism (e.g., acid formation) continues; thus, the proper incubation time must be selected in order to determine true growth rather than true acid production. In addition, some species of bacteria accumu- late acid in the early stages of growth but subsequently metabolize the acid. Total nitrogen. Add a 2.0-ml ahquot (10-100 jug of total N) of the washed bacterial suspension to a 30-ml micro-Kjeldahl flask. Add 2.0 ml 174 MANUAL OF MICROBIOLOGICAL METHODS of a digestion mixture (500 ml of H2SO4, sp gr 1.84; 75 g of Na2S04; 2.0 g of CuSeOs; and 500 ml of H2O), and boil gently in a hood or on a Kjeldahl rack for 1 hr longer than is necessary to clarify the solution. Allow the mixture to cool; add a small amount of antifoam agent (caprylic or oleic acid, or silicone) and 5.0 ml of ION NaOH. Immediately connect the flask to a small distillation apparatus fitted with a graduated receiver tube containing 2.0 ml of 0.Q5N H2SO4. The delivery tube should extend well belov/ the surface of the acid in the receiver. Gently boil the sample for 5 min ; during the last minute, raise the delivery tube slightly above the surface of the acid. This procedure allows all the distilled ammonia to drip free of the delivery tube and prevents drawing the receiving solution back into the distilling flask when the flame is removed from the latter. Dilute the receiving solution to contain 10-15 ng per ml of ammonia-nitrogen. In a colorimeter tube, mix 2.0 ml of diluted dis- tillate, 2.0 ml of Nessler's reagent (contains per liter: 4.0 g of Hgl, 4.0 g oi KI, and 1.75 g of gum ghatti), and 3.0 ml of 2N NaOH. Allow to stand for 15 min at room temperature, and read in the colorimeter at 540 m/z. The amount of ammonia-nitrogen is determined from a curve previously constructed using known samples of ammonia-nitrogen. It should be remembered that this method cannot be used with aliquots of a culture taken directly from the medium, since the sample will include medium constituents and will yield falsely high results. The nessleriza- tion procedure can also be replaced by direct titration of the distillate when the receiver acid solution is measured quantitatively. Discussion and Recommendations It is obvious that application of any of the methods described above depends upon the experimental approach and the type of apparatus and materials available. The technic selected also will depend to a certain extent upon the investigator's definition of growth. If growth is viewed as a simple increase in size, then a cell count will not reveal the extent of growth. On the other hand, if growth is recognized as increase in both the amount of protein and the number of cells, then expressions of total N, turbidity, and cell count will each represent growth, although not pre- cisely, and will also be in some measure of disagreement, since each is based upon different physical and chemical attributes of the cells. Thus, where possible, the method selected should be used throughout a com- plete study, since comparisons among experiments will then be valid. PREPARATION OF CELLS AND EXTRACTS Growth and Harvest of Metabolically Active Cells It is impossible to suggest specific growth media and conditions of incubation suitable for the production of microbial cells demonstrating PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 175 all types of high metabolic activity. In practice it is knoA^^i that the metabolism of a given organism reflects many contributing factors: the nature and age of the organism, its nutritional requirements, and reac- tions to physical and chemical conditions, etc. (see Werkman and Wilson, 1951). A medium yielding a large crop of cells is not necessarily one which will produce cells of high metabolic activity. The reader is referred to Chap. Ill for some details of media composition, to Snell (1951) for a discussion of bacterial nutrition, and to Gale (1943) and Stanier (1951) for consideration of the many factors affecting the enzy- matic activities of cells. Prior to a biochemical study of a given organ- ism, it is desirable and often necessary to investigate closely the relation- ship between the conditions of growth and of metabolic activity. Culti- vation of the organism under conditions which enhance the quantitative presence of the biochemical reaction of interest (e.g., adaptive enzyme and apoenzyme formation) is a povrerful tool available to the microbial biochemist, and once these conditions are recognized, their control is relatively simple. Harvesting of cells from a liquid medium is achieved by centrifugation, the type of machine employed depending primarily on the speed desired and the volume of medium involved. For large volumes the Sharpies type of ultracentrifuge is emploj^ed, while for smaller volumes many types of angle head centrifuges are available. Although refrigeration during centrifugation may be desirable and even obligatory in some cases, in most instances room-temperature centrifugation is adequate. Harvest- ing of cells from a solid medium is done simply by washing the surface of the medium with water or saline, using a spatula or glass rod and collec- tion of the resultant suspension. Preparation of Resting Cell Suspensions After centrifugation the medium is discarded and the packed cells resuspended in a suitable menstruum and washed once or twice to remove nutrients absorbed to the cell surface. The menstruum employed varies, although distilled water, saline, or buffer is most com.monly used. The washed cell paste is resuspended, and since it is often necessary in bio- chemical work to relate a given metabolic activity to cell number or cell mass, the cell suspension density is usually controlled and measured. Determination of cell number is performed in the usual manner : dilution and plate count or total microscopic count. How^ever, metabolic activity is not necessarily related to viability or total cell number, a measure of cell mass or protoplasm, being more directly related to biochemical activ- ity. If dry weight of cells is related to the turbidity of a cell suspension, a quick and accurate relationship of cell mass is obtained (see the previous section) . Si^ch a suspension is referred to as a resting cell suspension because the 1/6 MANUAL OF MICROBIOLOGICAL METHODS organisms are washed free of nutrients and are suspended in a nutrient- free liquid, thus rendering them nonproliferative. In actual use, the endogenous activity of the cell suspension must be determined as well as the activity upon the addition of a specific substrate. When the endog- enous activity is low, the experimental values can be readily corrected by subtraction of the endogenous rate. However, if the endogenous rate is high, certain complications may arise which may best be avoided by attempts to lower this rate. Bubbling air at room temperature into the suspension for an hour or more may result in lowered endogenous values by the oxidation of nutrients still present within the cells. Some of the advantages of the resting cell technic include (1) elimina- tion of growth in nonprolif crating cells, (2) ease of preparation with good reproducibility of data, (3) uniformity of suspensions for quantitative pipetting, (4) excellent spectrum of enzymes stable under these condi- tions, and (5) maintenance of activity at refrigerator storage tempera- ture, the time depending on the lability of the particular enzyme system. The major difficulties are (1) selective permeability of the living cell which prevents entrance of many types of compounds into the cell and (2) the number of reactions which may be competing for the substrate or the products or both. Dried Cells The use of dried preparations of microbial cells has certain advantages over the resting cell technic. The permeability barrier is markedly reduced and in some cases eliminated; the number of reactions occurring is reduced; the preparations are often stable for months or even years, thus allowing excellent reproducibility of results. Although some enzyme systems appear quite labile to drying, persistent efforts with some modification in technic have resulted in stable preparations of many enzymes. There are two methods of drying in general use : vacuum and acetone drying. Vacuum drying consists of drying the cell suspension in vacuo over a suitable desiccant, such as Drierite or phosphorus pentoxide, in a desiccator. A minimum volume of water is used to prepare the cell paste, since thin layers of cell paste present the maximum surface to the desiccant. A good high-vacuum pump and an amount of desiccant capable of absorbing the volume of water present are required. Acetone drying consists of the dropwise addition of a heavy cell paste to about 10 vol of ice-cold acetone with constant stirring. The cells precipitate and are removed immediately by vacuum filtration, the acetone being removed by suction. The dried cell preparations and, in most cases, the cell-free enzyme extracts below should be stored at to — 20°C to prevent destruction of enzyme activity. PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 177 Cell-free Enzyme Extracts For many types of studies, for example those involving reaction mecha- nisms, cell-free or soluble enzyme extracts are desirable. There are numerous technics available, none of which works in all cases. In prin- ciple, all methods attempt to disrupt the cell structure, thus allowing the liberation of intracellular material. The bacterial debris and any whole cells left are removed by high-speed centrifugation, usually in the cold, leaving the soluble elements in the supernatant liquid. If the desired enzymatic activity is not located in the supernatant liquid, it is possible that such activity may be found in the sedimented debris, and the latter should be examined before the particular procedure employed to obtain the cell-free extract is discarded as unworkable. Brief descriptions of these methods follow. Autolysis: cell suspension made to pH 7.5-8.5 with phosphate buffer and kept at 37°C under toluene for 12-24 hr. The degree of autolysis depends on the type of organisms. Although autolysis increases with time, so does inactivation of enzymes. Lysis : most commonly employed with Micrococcus lysodeikticus using the enzyme lysozyme to disrupt the cells (see, for example, McManus, 1951). Freezing and thawing: enzymes are often released by rupture of the cell membrane with alternate freezing and thawing (see, for example, Koepsell and Johnson, 1942). Wet grinding: (1) hand grinding of frozen cell paste in a mortar with powdered glass, alumina, carborundum, or other suitable abrasives (see, for example, Mcllwain, 1948) ; (2) semimechanical method of Kalnitsky et at. (1945) which involves the passage of a paste of bacteria and powdered glass between concentric cones of heavy glass, the inner cone revolving by connection to a motor and the outer cone held firmly in place; (3) a wet-crushing mill described by Booth and Green (1938) but not generally available; (4) shearing action of minute glass beads in a high-speed blender (Lamanna and Mallette, 1954). Dry grinding: con- sists of slow rotation in vacuo for several hours of dried powders of bac- teria mixed with glass beads. The shearing action of the beads appears to be the important factor. Pressure: A large instantaneous pressure forces the frozen cell material between two machined surfaces, with resultant cell disruption. The apparatus has been referred to as the ''Hughes press '^ (see Hughes, 1951, and a modification by Gest and Nordstrom, 1956). Sonic vibration: this technic is becoming more popular because of the ease of manipulations. The bacterial cells are disrupted by the sonic energy (see, for example, Shropshire, 1947, and Stumpf et at., 1946). The cells of various bacterial species differ in their susceptibility to sonic disruption. Gram-positive cells are more resistant than gram-negative cells; cocci are more resistant than rod forms; large cells are more resistant than small ceUs. Thus, the time of exposure in 178 MANUAL OF MICROBIOLOGICAL METHODS the sonic oscillator required for cell disruption will vary for different species. In addition, some enzymes are more labile than others under the conditions of sonic disruption. Although maximum protein concen- tration in the extract may be obtained after 50 min exposure, for example, maximum enzyme yield may be achieved after only 20 min and the total activity of the particular enzyme may decrease after that period (see Table 17). The nature of the suspending fluid markedly influences the Table 17. Effect of Disintegration Time on Enzyme Yield Time, min Enzyme, units per ml Protein, mg per ml Specific activity, units per mg protein 10 2.0 61.7 0.03 20 3.2 96.3 0.03 30 2.0 111.6 0.02 40 1.8 145.8 0.01 50 1.0 154.8 0.003 composition of the extract obtained by sonic disruption; alkaline buffers (pH 8-9) often give better results. Thus, the conditions used for the preparation of cell-free extracts must be carefully investigated in order to obtain adequate results, reflecting the particular biochemical reaction of interest to the investigator. Combination of methods may be employed: extraction of dried cells at low temperatures, oscillation of suspensions prepared from dried cells or frozen ceil pastes, etc. Protein Fractionation The cell-free extracts of bacteria, prepared according to the foregoing methods, contain many of the bacterial enzymes. It is often the case that a given biochemical reaction cannot be easily measured in such an extract owing to the presence of interfering reactions catalyzed by other enzyme systems which are also present. Therefore, it often becomes necessary to separate the desired enzyme system from some or all of the other enzyme systems present. This result may be achieved either by isolation of enzyme protein or by isolation of enzyme activity, using enzymatic activity as a measure of the process. Since bacterial enzymes are protein in nature, most of the problems encountered in enzyme protein isolation are similar to those of animal protein fractionation. The isola- tion of enzyme activity is often accomplished by utilizing fortuitous differences in physical properties (e.g., sensitivity to heat or pH) of the desired active protein. A useful example is the purification of the enzyme myokinase from rabbit-muscle extract (Colowick and Kalckar, 1943). In contrast to other enzymes, the enzymatic activity of myo- kinase is relatively stable to heat and acids. The protein is thus easily separated from interfering reactions by heating the extract at 90°C for PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 179 2 min in the presence of 0.5A^ HCL. The majority of the other proteins are denatured and precipitated by this treatment and may be removed by centrifugation. The myokinase remains in the supernatant fluid in a nearly pure condition. It should be noted that not all denatured pro- teins are precipitated by this and other methods. Thus, it would be as useful in isolating an enzj^me activity to denature all other activities yet allow the spurious proteins to remain in solution with the desired enzyme. As pointed out above, the preparation of bacterial protein extracts often requires somewhat more extensive methods than those needed for animal protein solutions. In addition, certain special problems arise which are peculiar to the fractionation of bacterial protein mixtures. The cell-free extracts obtained by most of the methods cited above contain relatively large amounts of nucleic acid. Because nucleic acid extends the range of precipitation of a given protein from a mixture, it is highly desirable to remove as much nucleic acid as possible from the mixture before proceed- ing with the common fractionation procedures. Either one of two methods is commonly used for precipitating nucleic acid. After the crude cell-free extract is prepared, the solution is dialyzed for at least 4 hr at 0-5° C against a solution (distilled water or a suitable buffer at about 0.01 M and pH 8 is often used) in order to remove as many small molecules as possible. After dialysis, the protein solution is adjusted to pH 6.0 by the dropwise addition of M CH3COOH. Nucleic acid is pre- cipitated from the adjusted mixture by the dropwise addition of either (1) 0.05 vol of M MnClo or (2) protamine sulfate. One milligram of protamine sulfate (17 mg per ml, pH 5.0) is added for each 100 mg of protein in the original cell-free extract. Protamine sulfate has a negative temperature solubility coefficient, and the above concentration repre- sents an approximately saturated solution at room temperature. The additions are made at 0°C with gentle stirring. After 20 min, the mixture is centrifuged and the supernatant solution, with a greatly decreased concentration of nucleic acid, may be fractionated with conventional procedures. For these and many other specific protein fractionation procedures, the treatise of Colo wick and Kaplan (1955) is recommended. Protein Estimation The optical method of Warburg and Christian (1941) for the estima- tion of protein is simple and rapid but is unfortunately less precise when protein solutions contain high concentrations of nucleic acid. Thus, this technic cannot be used reliably with crude cell-free bacterial extracts. The method depends upon the relative optical densities of the protein solution at 260 mju (due to the purine and pyrimidine components of nucleic acid) and at 280 m/x (due to the aromatic amino acids of the 180 MANUAL OF MICROBIOLOGICAL METHODS protein). Table 18 gives the factors necessary for calculation. For clarity, a sample calculation is given below. Table 18. Protein Estimation by U.V. Absorption Factor for 1.0-cm cell at Ratio 280/260 % nucleic acid 280 (factor X 280 reading = mg protein/ml)* 1.75 1.105 1.60 0.25 1.075 1.50 0.50 1.05 1.40 0.75 1.025 1.30 1.00 0.995 1.25 1.25 0.975 1.20 1.50 0.95 1.15 2.00 0.91 1.10 2.50 0.87 1.05 3.00 0.835 1.00 3.50 0.8 0.96 3.75 0.785 0.92 4.25 0.755 0.88 5.00 0.715 0.86 5.25 0.705 0.84 5.50 0.69 0.82 6.00 0.67 0.80 6.50 0.645 0.78 7.25 0.615 0.76 8.00 0.59 0.74 8.75 0.565 0.72 9.50 0.54 0.70 10.75 0.51 0.68 12.0 0.48 0.66 13.5 0.45 0.65 14.5 0.43 0.64 15.25 0.415 0.62 17.5 0.38 0.60 20.0 0.35 0.49 100 * For 0.5-cm cells, multiply values in last column by 2. Example. 0.2 ml of a protein solution (after protamine sulfate treatment) and 2.8 ml of distilled water are pipetted into a cuvette with a 1-cm light path. The reference cuvette contains distilled water. Readings obtained : 280 m/x 260 m/x 280 260 ratio = O.D. = 0.273 O.D. = 0.248 0.273 0.248 1.10 In the first column find 1.10. From the third column, find the factor 0.87. PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 181 Calculation of protein concentration: 0.87 X 0.273 = 0.237 mg of protein per milliliter in the cuvette 0.237 X 15 (dilution factor) = 3.585 mg of protein per milliliter of original protein solution. The sample may be recombined with the stock solution after estimation, since no protein is denatured during the procedure. This feature of the optical method is particularly advantageous at high degrees of enzyme purification when the enzyme solution often contains a very small amount of protein. The protein concentration of extracts containing large quantities of nucleic acid (e.g., before protamine treatment) may be estimated by any of several colorimetric procedures. The method of Lowry et al. (1951) utilizing the Folin-Ciocalteau phenol reagent is recommended because of its high sensitivity and is described here. Reagents: 1. 40 g of Na2C03 in 500 ml of distilled water 2. 0.3 g of CuS04 and 0.6 g of sodium tartrate in 500 ml of distilled water 3. 1 part of Folin-Ciocalteau phenol reagent (available commercially) and 2 parts of distilled water Procedure. To a protein sample containing 7-70 jug of protein, add sufficient water to make a total volume of 0.5 ml. Add 5.0 ml of carbon- ate-copper-reagent (equal volumes of reagents 1 and 2 mixed just before use), mix, and incubate at 37°C for 30 min. After cooling to room tem- perature, add 0.5 ml of the diluted phenol reagent, mix, let stand 20 min at room temperature, and read at 660 m/z. For preparation of the standard curve, a satisfactory standard protein solution may be prepared using crystalline bovine albumin (Armour and Co., Chicago). The dilute albumin solution is subject to surface denaturation and should be freshly prepared for each determination. BIOCHEMICAL TECHNICS Manomeiric Technics Manometric methods are employed widely because of accuracy and speed in the quantitative analysis and rate measurements of reactions involving either the evolution or uptake of certain gases. In addition, acid production may be measured by following CO 2 evolution in a bicar- bonate buffer in equilibrium with a CO2 gas mixture. The reader is referred to two excellent books for detailed discussions of the methods and types of apparatus available (Dixon, 1943; Umbreit et al., 194V>;. lliere are two major types of manometers in use, i.e., the constant-pres- 182 MANUAL OF MICROBIOLOGICAL METHODS sure type commonly referred to as the Barcroft respirometer and the constant-volume type or Warburg instrument. Inasmuch as the War- burg type is by far the most widely employed in this country, further comments will be concerned with this apparatus. The fundamental principle involved is that quantitative changes in the amount of a gas can be measured by determining pressure changes as long as the temperature and volume of the gas are kept constant. The apparatus consists of a flask or vessel having one or more side arms equipped with stoppers and commonly containing a small center well sealed to the bottom. The flask is attached to a manometer having one closed and one open arm and usually containing Krebs' solution (Krebs, 1951) or Brodie's solution (density 1.03) rather than mercury (density 13.6), thus increasing the sensitivity of the pressure changes. The vessel is placed in a water bath at constant temperature, and the system shaken in order to increase the rapidity of gas exchange between the liquid and gaseous phases. The level of the liquid in the closed arm of the manom- eter is brought to a previously calibrated point (usually 150 or 250 mm), and the level in the open arm recorded. In this manner the system is always read at constant volume, and the recorded values represent pres- sure changes. Inasmuch as the manometers are influenced by barometric changes in pressure by virtue of having one end open to the atmosphere, a thermobarometer (flask containing water attached to a manometer) is included in all experiments. The pressure changes (positive or negative) recorded for the thermobarometer are used to correct all readings of the manometers attached to an experimental A^essel. The prime purpose of the side arm of the flask is to allow separation of the components of the system under investigation so that one may con- trol the initial reaction time. For example, a bacterial suspension in a suitable buffer may be placed in the main compartment of the flask and the substrate in the side arm. After temperature equilibration, the sub- strate is added by removing the manometer from the water bath, closing the open end of the manometer with one's finger, and tipping the system at an angle sufficient to allow the material in the side arm to pour into the main compartment. The manometer is then returned to the water bath, and readings taken at suitable intervals, depending primarily on the speed of the particular reaction under study. The stopper of the side arm may be a venting plug, thus allowing introduction of gases of known composition other than air. The center well is used primarily to hold alkali to absorb CO2 as, for example, in the measurement of O2 uptake or H2 output. In actual operation certain information is required in order to make possible the quantitative calculation of the gas evolved or taken up. PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 183 One must know at a given temperature the gas volume of the flask and manometer (to the liquid reference level in the closed arm) , the volume of fluid in the flask, the gas being exchanged, the solubility of that gas, and the density of the manometric fluid. The Warburg technic is commonly used to measure rates of O2 or H2 uptake, CO2 or H2 production, and respiratory quotients with a variety of substrates. When properly used this method provides one of the most versatile tools for the microbial physiologist. It is pertinent here to point out that although the manometric apparatus itself allows only the measurement of gas exchange, it is often desirable to subject the contents of the vessel to chemical analysis at the conclusion of the manometric experiment. After deproteinization and centrifugation, the supernatant liquid may be analyzed for a variet}^ of common intermediates and end products of metabolism, for many of which excellent micromethods are available. It is quite possible with some bacteria and with certain sub- strates to obtain an accurate dissimilation balance with the usual volume of contents (about 3 ml) employed in the Warburg flask. Analytical Procedures The material in this section is usually presented under the heading of "Fermentation Analysis." The less restricted title used above, however, is considered more appropriate because fermentation represents only one of the dissiiTiilatory mechanisms found in microorganisms. The term ''fermentation," or intramolecular oxidation, is properly reserved for those anaerobic processes in which the hydrogen acceptor originates from the substrate. Other mechanisms include anaerobic oxidation or inter- molecular oxidation w^herein carbonate, nitrate, sulfate, or an organic compound acts as the hydrogen acceptor and respiration or aerobic oxida- tion wherein oxygen serves as the hydrogen acceptor. Coupled reactions between two substrates have also been described as, for example, the Stickland reaction between pairs of amino acids (Stephenson, 1949). These distinctions are important if a valid mechanistic interpretation of the results is to be obtained and^ used to describe the probable inter- mediary pathways of dissimilation. The majority of the studies pub- lished to date deal with the products of carbohydrate, polyalcohol, and organic acid dissimilation. The principles in all cases are the same. Measurement of dissimilatory products must include both their quali- tative identification and quantitative determination. The quantity of substrate dissimilated, as well as any substances reacting T\dth the sub- strate during its breakdown (e.g., oxygen), must be accounted for 184 MANUAL OF MICROBIOLOGICAL METHODS quantitatively by the products formed. In some instances, particularly during aerobic processes, part of the substrate disappearing is assimilated by the organisms while at the same time the rest of the substrate is broken down to various products (Clifton, 1946, 1951). Use of a growth medium during substrate dissimilation permits maximum assimilation and the elaboration of adaptive metabolic mechanisms, each with resultant effects upon the nature and quantity of end products. Thus, use of washed cell suspensions is desirable, employing a medium containing only the sub- strate to be dissimilated plus those other substances required for the process: phosphate, buffer, metallic ions, etc. Care must be exercised in sterilizing the reaction medium; heat-labile compounds are sterilized by filtration, usually of a concentrated solution, and added aseptically to the rest of the reaction medium. Sterilization of the reaction mixture is unnecessary if resting cell suspensions are employed, since short-term dis- similation (0.5-3 hr) is generally adequate. In order to obtain a quantitative accounting of the dissimilation proc- ess or a ''balance," it is necessary to determine the gases formed, used, or both, as well as the dissolved products. An atmosphere of defined com- position must therefore be employed in contact with the reacting solution. Both these objectives may be realized by shaking the solution in a closed system (e.g., Warburg apparatus) or by bubbling nitrogen continuously through the solution (provided nitrogen fixation does not occur), the emitted gases being passed through a suitable absorption train. At the end of the chosen period of time, the reaction is stopped by the addition of enough nonvolatile mineral acid to bring the pH to 2-3; this also releases bound CO 2 which is included in the gas measurements. The solution is then freed of cells and proteinaceous material by treatment with a suitable agent (barium or zinc hydroxide or trichloracetic acid) and centrifugation, and the clarified solution analyzed. A small sample of the reaction mixture may be removed just before the reaction is stopped to check for bacteriological purity ; this is necessary for reactions conducted in culture media and which proceed for long periods of incubation. Owing to space limitations, it is not possible to describe here in detail the methods employed in dissimilatory products analysis. Reference is made to the following books: Johnson et al. (1949), McNair (1947), Neish (1952), and Umbreit et al. (1949), which contain detailed descrip- tions of individual methods and apparatus. Sensitive micromethods have been described and continue to appear which are particularly useful for analysis of Warburg flask contents, eliminating the need in many cases for more cumbersome equipment (Black, 1949; Bueding and Yale, 1951; Kennedy and Barker, 1951; Neish, 1952). The methods described by Neish (1952) are most generally applicable to the problems discussed PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 185 herein, although slight modifications may be advisable for the particular problems which are under investigation. In any case, the appropriate analytical method should be thoroughly tested and proved under the particular conditions of application. In general terms, portions of the clarified solution referred to above are subjected to several treatments. Disappearance of the substrate is measured, as are the types of products listed in Table 19, according to the procedure described below. The pH of the cleared solution containing the dissolved dissimilatory products is adjusted to pH 7-8 and distilled Table 19 Neutral volatile products, pH7-8 Volatile acids, pH2-3 Nonvolatile products Acetone Formic 2,3-Butanediol* Diacetyl Acetic Glycerol * Acetaldehyde Propionic Ethanol Butyric Lactic acidf Isopropanol n-Valeric Pyruvic acidf n-Butanol Isovaleric Fumaric acidf Acetylmethylcarbinol n-Caproic Succinic acidf * Ether-extractable at pH 7-8. t Ether-extractable at pH 2-3. to obtain a fraction containing the neutral volatile products. The residue is adjusted to pH 2-3 and steam-distilled to obtain the volatile acid frac- tion. The nonvolatile products remain in the residue. Each of the products thus obtained is measured, taking into considera- tion the possible effects of other products upon the particular analysis. Partition chromatography may be used to separate and measure many of the components of the mixtures; the volatile acids may also be meas- ured by Duclaux distillation (Neish, 1952). After identification and quantitative determination of the dissimilatory products, two types of balances are drawn up to check the accuracy of the analysis. These are the carbon and oxidation-reduction (0/R) balances. An example of such balances, taken from Johnson et at. (1949), is given in Table 20. As a result of dissimilation, substrate carbon appears in the products formed, and complete carbon recovery is to be expected if all products have been identified and if the quantitative determinations are not in error. Usually the analytical data are calculated on the basis of milli- moles of product per 100 millimoles of substrate dissimilated ; micro- moles are used in microanalysis. To calculate the carbon balance, millimoles of any product are multiplied by the number of the carbon 186 MANUAL OF MICROBIOLOGICAL METHODS atoms in the molecule and indicated as millimoles of C. The total C millimoles of the products must equal the millimoles of the substrate similarly calculated or must be within the range of the experimental error of the analytical methods employed. Thus, the carbon-recovery figure equals 100 per cent theoretically; the value of 97.4 per cent in the balance presented below represents, however, very good carbon recovery. To obtain the figures for the calculated amounts of CO2 expected on a theoretical basis, a knowledge of the dissimilatory mechanism in opera- tion must be available or certain assumptions concerning its operation Table 20. Carbon and Oxidation-reduction Balances Com- pound Carbon Calc CO2 Oxid value Oxid prod Red prod Glucose utilized Lactic acid mM 100.0 96.2 6.8 85.9 7.3 89.3 mM 600.0 288.6 20.4 171.8 14.6 89.3 mM 0.0 0.0 85.9 7.3 -1 -2 +2 mM 178.6 mM Glycerol 6.8 Ethyl alcohol 171.8 Acetic acid . Carbon dioxide Totals 584.7 93.2 178.6 178.6 Carbon recovery = 0/R = 584.7 = 97.4% obs COj 600.0 ^'"^^"calc CO2 oxidation value _ 178.6 reduction value 178.6 ^ 89^ 93.2 = 1.00 = 0.958 are made. In the balance presented above, the data suggest the opera- tion of a mechanism of glucose dissimilation similar to the Embden- Meyerhof-Parnas scheme of glycolysis (Baldwin, 1947). The hexose glucose is split into two trioses which, by oxidation and reduction, are converted to the end products lactic acid and glycerol. Thus, on this basis, no CO2 is expected to be liberated during the formation of these end products. However, a number of the triose molecules originating from glucose are decarboxylated, at the pyruvic acid level, to a C2 com- pound and CO2. By reduction, some of the C2 compound is converted to ethanol while the rest of the C2 compound is oxidized to acetic acid. Thus, for each mole of ethanol and of acetic acid formed, a mole of CO 2 is also liberated. On this basis, the theoretical yield of CO2 is calculated. When this figure is compared with the actual experimental yield of carbon dioxide, a ratio of 1 is obtained if the reasoning and analyses are correct. In the balance presented above, the ratio actually obtained is 0.958, which is another indication of a very good balance. PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 187 A fiirther check of analytical accuracy is the 0/R balance. The O/R balance, or redox index, is the ratio of the number of equivalents of oxida- tion and reduction occurring during dissimilation. Since in any chemical reaction these must be equal, the O/R ratio should be 1. Oxidation- reduction balances are calculated by multiplying the millimoles of product by a value expressing its degree of oxidation or reduction com- pared with the general carbohydrate formula CH2O. The values are positive if the compound is more highly oxidized than CH2O and negative if more reduced. Compared with one oxygen atom with a value of -f-l, two hydrogen atoms have a value of —1. Thus, CO2 has an oxidation value of +2; pyruvic (C3H4O3) and formic (CH2O2) acids, +1; glucose (C6H12O6) and lactic acid (CsHeOs), 0; ethanol (C2H6O) and acetyl- methylcarbinol (C4H8O2), —2 (also known as reduction value if negative) ; etc. It is obvious that the addition or removal of water during the fermentation will not affect the O/R balance, since the O/R value of water is 0. However, the introduction of oxygen into the system w^il] alter the O/R balance, because of the positive O/R value involved. The balance presented above indicates an O/R index of 1.00, which is in excellent agreement with the remaining analytical data. The methods of calculation discussed above also may be applied to oxidations involving oxygen or to intermolecular fermentations (e.g., amino acid fermentation such as the Stickland reaction), always provid- ing that the quantities of the participating substrates and products are known. The theoretical calculations presented above are based on the assump- tion that the dissimilatory mechanism is kno^vn. The existing knowledge concerning the pathways of microbial dissimilation is, indeed, limited, although data are rapidly accumulating (Elsden, 1952; Gunsalus et at., 1955). However, since the results of analyses are often employed to postulate the probable dissimilatory pathway, caution must be exercised in such interpretation. More mechanistic studies of substrate dissimilation may be made. Individual stepwise enzymatic reactions may be observed, using dried cells, cell-free enzyme extracts, or purified enzyme preparations. Such reactions involve the breakdown of a given substrate, usually a phos- phorylated compound, to specific end products and may be examined by a variety of technics (Lardy, 1949; Sumner and Somers, 1947; Sumner and Myrback, 1950-1952). Such studies represent some of the final steps in the description of the mechanism of substrate dissimilation; recent advances are discussed contemporaneously in the annual review series for biochemistry and for microbiology (published by Annual Reviews, Inc., Stanford, California). 188 MANUAL OF MICROBIOLOGICAL METHODS Technics for Isolated Reactions Reactions Involving Oxidation Reduction Thunberg technic. The Thunberg technic for estimation of dehydro- genase activity is especially applicable to cell suspensions and to prepara- tions which are too dense for spectrophotometric measurement (see page 190). In some instances, the Thunberg technic, observed instru- mentally in a colorimeter, provides an economical substitute for a spectrophotometer. In general, a tube fitted with an outlet for evacuation and a side arm is used. Buffer, substrate, and methylene blue are placed in the tube, and the bacterial suspension introduced into the side arm. The type and concentration of buffer and substrate depend on the nature of the system studied, while the methylene blue is commonly employed in a final concentration of 1/20,000 to 1/60,000 (1.3 X 10-^ M to 4.4 X 10-^ M). Methylene blue in the oxidized state is blue-colored; in the fully reduced state, the dye is white (leuco) . The system is made anaerobic to prevent the autooxidation of the methylene blue by molecular oxygen; this is generally achieved by evacuation using a water aspirator or vacuum pump and then filling with nitrogen. The tube is placed in a constant- temperature bath, the bacterial suspension tipped in after temperature equilibration, and the rate, time, or both, required for dye reduction measured by visual inspection or by the aid of a photoelectric colorimeter. When methylene blue reduction is measured with the unaided eye, a standard (90 per cent reduction) is usually employed. This is prepared as the experimental tubes with two exceptions: adding one-tenth the amount of methylene blue and using a heat-inactivated cell suspension. As a control, a tube without substrate is included so that the rate of endogenous dye reduction may be ascertained; this endogenous rate is often significant, since the organism may contain a considerable quantity of hydrogen donors. A dehydrogenase is considered present when the methylene blue reduction time in the presence of added substrate is less than the endogenous time. A more complete discussion of this method is presented in Umbreit et al. (1944). Many other dyes have been substituted for methylene blue in the Thunberg method, including indophenol derivatives and, more recently, triphenyltetrazolium chloride (Kun and Abood, 1949; Gunz, 1949; Smith, 1951). In each case, however, the general principle is the same; i.e., electrons are transferred from the substrate to the acceptor. Acid formation. The availability of pH meters presents a quick, simple method for following acid production during the course of a reaction. When weakly buffered or unbuffered media are used, the time course of PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 189 pH change is a relative measure of the progress of a reaction in which hydrogen ions are produced. The method has limited application, since the activity of a given system is usually dependent on pH and the linear relationship of time and pH is of short duration. In addition, pH change is not a simple function of progress of the reaction. This method has been applied by Sable and Guarino (1952) to the purification of glucono- kinase from yeast. The formation of acid during a reaction or during a sequence of reac- tions may be followed manometrically, using essentially the same technics mentioned above in the section on manometric technics. This method takes advantage of the buffering capacity of the HCOa" + H+ :;:± CO 2 + H2O system. The formation of each acid is accompanied by release of a hydrogen ion into solution. As a result, CO 2 is evolved from the reaction mixture and may be quantitatively measured. A complete account of the buffer theory involved in this method, as well as a chart describing the relationships of pH, temperature, and bicarbonate concentration, may be found in Umbreit et al. (1949). The method is applicable to the fermentation or oxidation of substrates which yield lactic or other acids as end products. If CO2 is also an end product, a control flask is used containing a buffer other than bicarbonate (e.g., phosphate) at the same pH, allowing the determination of the true CO2 value. By difference, the CO2 evolved in the bicarbonate flask as a result of acid formation is calculated. In an isolated reaction involving the reduction of di- or tri-phospho- pyridine nucleotide, a hydrogen ion is produced; thus, with opaque sys- tems which cannot be measured in a spectrophotometer (see below), the reaction may be followed manometrically as acid production. In this case, a stoichiometric quantity of the pyridine nucleotide must be added or a second system must be included which is capable of oxidizing the reduced pyridine nucleotide. For the second system, several reagents are available which accomplish the oxidation of reduced pyridine nucleo- tide, the best probably being K3Fe(CN)6. When coupled with the ethanol-acetaldehyde system, for example, the reactions are written as follows : CH3CH2OH -h DPN+ ^ CH3CHO -f- DPNH -F H+ 2Fe(CN)6 + DPNH ^ 2Fe(CN)6 + H+ + DPN+ 2H+ + 2HCO3- ^ 2H2O + 2CO2 Sum: CH3CH2OH -f- 2Fe(CN)6— + 2HCO3 ;=± CH3CHO + 2Fe(CN)6 + 2H2O + 2CO2 Thus, under these conditions two molecules of CO2 are evolved for each molecule of ethanol oxidized and two molecules of ferri cyanide are 190 MANUAL OF MICROBIOLOGICAL METHODS required to oxidize one molecule of diphosphopyridine nucleotide. Since the oxidation of reduced diphosphopyridine nucleotide by ferricyanide is a relatively slow process in low concentrations of the latter, ferricyanide is added to the reaction mixture in an amount at least twice that required for oxidation of the substrate present. A slight difference in stoichiometry exists for the case of simultaneous acid production and diphosphopyridine nucleotide reduction. CH3CHO + DPN+ + H2O ^ CH3COO- + DPNH + 2H+ Here, three molecules of CO2 are produced per molecule of substrate oxi- dized when measured by the ferricyanide manometric method. Spectrophotometric measurements. The course of a given reaction is often followed spectrophotometrically, advantage being taken of the fact that the change in concentration of one of the products or reactants may be observed as a change in light absorption at a given wavelength. The classic examples of this technic are reactions which involve di- or tri- phosphopyridine nucleotide. These nucleotides exhibit an absorption peak at 340 m/i when in the reduced state, while the oxidized forms do not absorb an appreciable amount of light at that wavelength. Therefore, during the course of a reaction which results in reduction of pj^ridine nucleotide, light absorption at 340 mju increases. The measurement may consist of readings taken at definite intervals after initiation of the reac- tion, in which case the time course of the reaction is plotted, or readings may be taken at the beginning and end of the reaction, in which case only the extent of the reaction is determined. Other applications also involve changes in optical density (absorption) at a particular wavelength of light, such as the decrease in absorption at 265 mjjL during the deamination of adenosine-5-phosphate or the increase in absorption at 220 mju during formation of certain keto compounds. The method obviously requires previous knowledge of the spectrum of the compound to be measured. In addition, the extinction coefficient for the compound at the particular wavelength employed must be known if the measurement is to be completely quantitative. Although several types are available, the most commonly used spectro- photometer is the Beckman model DU. A complete instruction manual is supplied with this instrument. Nitrogen Metabolism Proteins. Proteins are estimated quantitatively by the methods pre- viously described (see pages 179-180). The results of total nitrogen determination (pages 173-174) are converted to protein values when PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 191 multiplied by 6.25. This conversion factor is merely an average value based on proteins containing 16 per cent of nitrogen and thus leads to some error. Although considerable attention has been given to proteolysis in ani- mals, there are comparatively few studies involving microorganisms. It is believed that native proteins probably do not enter the bacterial cell and that protein utilization reflects the ability of the cell to produce extracellular enzymes capable of hydrolyzing the protein to amino acids, the latter entering the cell during metabolism. Comparatively few bac- terial species produce proteolytic enzymes. The simplest test for proteo- lytic activity is to ascertain the ability of a given bacterial species to liquefy certain proteins such as solidified gelatin, coagulated casein, or coagulated serum. This may be done either by incorporating the protein into the growth medium or by testing the cell-free filtrate of a culture on the protein itself. One may also determine the degree of proteolysis by measuring the amino nitrogen changes either by the Sorensen formol titration (Brown, 1925), which depends on the increase in acidity w^hen neutralized formaldehyde is added to a solution containing ammonia, primary amines, amino acids, or polypeptides, or by the Van Slyke manometric technic (Peters and Van Slyke, 1932), which depends upon the production of gaseous nitrogen when nitrous acid acts on an aliphatic amine. Amino acids. The three methods most commonly employed for the quantitative estimation of amino acids are chromatography and ion exchange, microbiological assay, and enzymatic technic. All have their merits and disadvantages. Chromatography and microbiological assay allow estimation of essentially all the amino acids, while the enzymatic methods are limited to a few. The technics of chromatography depend upon the differential distribu- tion of amino acids between two phases using some form of supporting column. The paper-partition method is perhaps the simplest and most commonly employed, for it accomplishes both separation and estimation of the various amino acids. A two-dimensional system is usually employed with phenol water and n-butanol-acetic water as the developing solvents. Most amino acid spots are made visible with the ninhydrin spray or dipping technic. The amino acid spots are estimated quanti- tatively by (1) measuring the spot area (Berry et at., 1951) or (2) colori- metric measurement after elution of the spot (Housewright and Thorne, 1950). Complete discussion of paper chromatographic theory and methods, including solvents, spray reagents, and Rf values, particularly as applied to amino acids, has been pubhshed (Block et aL, 1955; Berry et al., 1951; Williams and Synge, 1950). 192 MANUAL OF MICROBIOLOGICAL METHODS Ion-exchange columns are useful for batch and analytical scale separa- tions of amino acid (Hirs et at., 1952; Moore and Stein, 1954) but, although quite precise, require more time and attention than the paper method. Microbiological assay is employed widely and depends upon strains of microorganisms (commonly lactic acid bacteria) of exacting but known nutrition, capable of growing in chemically defined media provided the amino acid to be assayed is supplied, and responding to graded amounts of this amino acid in a regular fashion. The limits and specificity of the growth response must be checked before unknown samples are assayed. Total growth is determined usually by acidity or turbidity measurements. These methods do not allow separation of amino acids but do permit the quantitative measurement of one amino acid in a mixture. Technics and organisms are available for the microbiological assay of all the amino acids (Snell, 1946; Dunn, 1949; Barton-Wright, 1955). Although several enzymatic methods are available, the specific decar- boxylase method is the one most widely used because of simplicity, specificity, accuracy, stability of dried bacterial preparations, and ease of manipulations. The method depends upon the manometric measurement of CO2 production from the amino acid by enzymes from selected strains of microorganisms. Six amino acids may be determined quantitatively in this manner: arginine, lysine, tyrosine, histidine, glutamic acid, and ornithine (Gale, 1948; Umbreit and Gunsalus, 1945; Archibald, 1946). The methods employed in the study of three of the major reactions of amino acids will be considered briefly. The enzymatic removal of the carboxyl group of an amino acid with the formation of the corresponding amine and CO 2 is known as decarboxyla- tion. Decarboxylase activity may be determined by measuring either the disappearance of the amino acid acted upon by one of the methods listed above or the resulting products. In most instances, CO2 evolution is measured manometrically. In the case of aspartic acid decarboxylase, CO 2 measurements cannot be used because of the low activity. Billen and Lichstein (1949) have therefore employed a microbiological assay method for the /^-alanine produced. The enzymatic removal of the amino group of an amino acid with the formation of ammonia and most commonly but not exclusively the cor- responding keto acid is known as deamination. Although the activity may be measured by determining the disappearance of the amino acid acted upon, the most common method is to measure the production of ammonia by nesslerization. The enzymatic transfer of the amino group of an a-amino acid to an a-keto acid resulting in the formation of another a-keto acid, the former corresponding in structure to the original a-keto acid and the latter to the PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 193 original a-amino acid, is known as transamination. The technics employed to study the transaminases are varied, but all measure the appearance or disappearance of the keto or amino acid. Green et al. (1945) discussed keto acid measurements, Cohen (1940) aspartate meas- urement by the chloramine-T reaction, Lichstein et al. (1945) amino acid measurement by decarboxylase method, and Feldman and Gunsalus (1950) amino acid measurement by paper-partition chromatography. The symposium on amino acid metabolism edited by McElroy and Glass (1954) is recommended as a source of reference to specific analytical procedures. Nucleic acids, purines, and pyrimidines. Studies of nucleic acid metabolism necessitate the measurement of these complex substances themselves as well as the constituent purines, pyrimidines, carbohydrates, and phosphorus. A preliminary step involves the removal of nucleic acids from the rest of the cell material, followed by separation of the two types of nucleic acids, desoxypentose and pentose, and their quantitative analysis. The nucleic acids are then hydrolyzed, and the quantities of the constituent parts measured. The treatise of Chargaff and Davidson (1955) contains a complete con- sideration of methods and results of nucleic acid research. Several useful general reference sources also are cited: Cold Spring Harbor Symposia (1947); Symposia of the Society for Experimental Biology (1947); Symposia on Biochemistry of Nucleic Acids (1951); as well as ion- exchange methods (Cohn, 1951) and paper chromatography methods (Hotchkiss, 1948; Carter, 1950) for purines and pyrimidines. Phosphorus Metabolism Although many phosphorylated compounds of biochemical importance have been described, isolated, and characterized, most of the work has dealt with animal tissues, yeast being the only microorganism that has received any appreciable attention in the past. However, it is probable that similar studies with microorganisms will yield analogous results, and indeed, work with the latter is accumulating. Thus, it appears valid to include here a brief description of phosphorylated compounds, regardless of their origin. The manual of Umbreit et al. (1949) presents methods for the analysis of phosphorylated intermediates as well as technics for their isolation and synthesis. The isolation and purification of phosphorylated compounds derived from cellular material are usually accomplished by fractional pre- cipitation of their metallic salts, but more recently, technics have been described employing the principle of solvent distribution (Plant et al., 194 MANUAL OF MICROBIOLOGICAL METHODS 1950) and two-dimensional chromatography (Bandurski and Axelrod, 1951). Most of the procedures described to date, however, leave room for improvement, and no single, universally satisfactory technic is known. Studies of phosphorus metabolism are now being pursued in which only a few biochemical steps are involved, employing dried cells or enzyme preparations, so that complexity of analysis is greatly reduced. The uptake or appearance of inorganic phosphorus is readily measured by the method of Fiske and Subbarow (1925). The usual investigation leads from this type of observation to the finding that a large number of phosphorylated compounds, largely carbohydrate derivatives, are formed or broken down during cellular metabolism. Thus, the amount of inorganic phosphorus released by hydrolysis in A^ HCl at 100°C in 7 min (A7 P) from such compounds as adenosine di- and tri-phosphate, glucose- 1-phosphate, etc., is measured and reflects the presence and quantity of such phosphorylated compounds. Similarly, hydrolysis under the same conditions for 180 min helps to characterize other phosphorylated com- pounds: phosphopyruvate, triose phosphate, etc., but care must be exer- cised in drawing conclusions from these results, since partial phosphorus liberation occurs even during shorter periods of hydrolysis and other com- pounds may be involved. Therefore, analysis is also made for the non- phosphorus moiety of the compound: pentose, glucose, fructose, etc. Many of the AT P compounds are referred to as ''high-energy phosphate'^ compounds, since, on hydrolysis, 10,000-15,000 cal are liberated com- pared with the 2,000-4,000 cal released upon hydrolysis of the more stable phosphate esters. Lipmann and Tuttle (1945) have described a specific method for acyl phosphate to form the corresponding hydroxamic acid; addition of ferric ions results in the formation of a purple complex which is a measure of the acyl phosphate present. Another type of phosphorylated intermediate which is readily measurable includes the ''alkali-labile" triose phosphates, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Inor- ganic phosphorus is released from these compounds when placed in N NaOH or KOH for 20 min at room temperature (Meyerhof and Lohmann, 1934) and is measured as increase in inorganic phosphorus. Fresh alkali must be used, since silicates, originating from the walls of the glass container, analyze as inorganic phosphorus. Mention should be made of the commercial availability of many phos- phorylated compounds of biochemical importance. Such preparations, albeit of stated limited purity in many cases, are useful in metabolic studies as substrates as well as standards for analytical procedures. A useful recent compilation of the role of phosphorus in the metabolism of organisms has appeared (McElroy and Glass, 1951, 1952), including references to many analytical procedures. PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 196 Other Methods Microbiological assay has become a powerful analytical tool and is widely used. Methods for amino acid assay have been mentioned above (Nitrogen Metabolism) ; for methods used in vitamin analysis, the com- pilation of Gyorgy (1951) is useful. Isotopes are being used at an ever-increasing pace in the study of microbial biochemistry. Several commercial organizations offer ready assistance with reference to the type of equipment best suited for specific purposes. Numerous centers exist for the training of new workers in this field, such as the laboratories of the Atomic Energy Commission. The literature in this field is rapidly accumulating, and several compilations of methods exist (Kamen, 1951; Wilson, 1948), with descriptions of new and improved procedures often appearing. Bacterial Genetics The use of biochemical mutants and of organisms adapted to metabo- lize specific substrates has played an important role in advancing bio- chemistry in the past decade. Such tools are being employed with great ingenuity, and methods for their use have appeared (Catcheside, 1951; Lederberg, 1951; Luria, 1950; Spiegelman and Landman, 1954). BIBLIOGRAPHY Archibald, R. M. 1946. In Amino acid analysis of proteins. Ann. N, Y. Acad. Sci.f 47, 181-186. Bandurski, R. S., and B. Axelrod. 1951. The chromatographic identification of some biologically important phosphate esters. J. Biol. Ckem., 193, 405-410. Barton-Wright, E. C. 1952. "The Microbiological Assay of the Vitamin B-complex and Amino Acids," 179 pp. Pitman Publishing Corporation, New York. Berry, H. K., H. E. Sutton, L. Cain, and J. S. Berry. 1951. Development of paper