Library OF THE Mew Work State Veterinary College Cornell University Library willl A laboratory guide in bac | ii i a - UNITES ———<$<$—— Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000234629 A LABORATORY GUIDE IN BACTERIOLOGY THE UNIVERSITY OF CHICAGO PRESS CHICAGO, ILLINOIS Agents THE CAMBRIDGE UNIVERSITY PRESS LONDON AND EDINBURGH THE MARUZEN-KABUSHIKI-KAISHA TOKYO, OSAKA, KYOTO KARL W. HIERSEMANN LEIPZIG THE BAKER & TAYLOR COMPANY NEW YORK A LABORATORY GUIDE IN BACTERIOLOGY FOR THE USE OF STUDENTS, TEACHERS AND PRACTITIONERS BY PAUL G. HEINEMANN, Pu.D. Instructor in Hygiene and Bacteriology The University of Chicago THIRD REVISED EDITION THE UNIVERSITY OF CHICAGO PRESS CHICAGO, ILLINOIS | Lai \ \ Q PR Not Sell tes CopyRIGHT 1905 By 3 Tae UNIVERSITY OF CHICAGO bd, 4. 6 First Edition June 1905 Second Edition June 1911 / y ¢ = Second Impression October 1911 Third Impression October 1913 Third Edition September 1915 Composed and Printed By The University of Chicago Press z Chicago, Illinois, U.S.A. PREFACE TO FIRST EDITION The considerations which led the writer to add this laboratory guide of bacteriology to the number of such guides already in existence were of various nature and may be briefly set forth here. Probably no branch of biological science has ad- vanced so rapidly during the past few years as the science of bacteriology, and it is difficult even for an active laboratory worker to keep abreast of this ad- vance. A textbook or guide fixes the status of the science at the time of its writing, but almost before it leaves the press it becomes antiquated. Revisions, corrections, and additions are necessary at short intervals in order to keep a publication of this kind approximately up to date. There is, therefore, almost at any given moment room for a new publication to fill the want of a progressive instructor for a guide that gives the latest accepted rules and practices of the laboratory. The value of such a publication will Be enhanced by a plan and arrangement of sufficient flexibility and latitude to allow the instructor and the student to enter such additions and corrections as serve to bridge over the time between editions. Medical students entering on a course in bacteri- ology often have had too little previous laboratory training in methods of precision. It is a matter of importance for the instructor to put himself in the atti- tude of mind of the student and to try to appreciate his difficulties in understanding details. Many of v vi PREFACE TO FIRST EDITION the pieces of apparatus employed in a bacteriological laboratory are novel even to the student trained in chemistry and biology, and it has been thought best to exhibit these, to the smallest detail, by means of illustrations—a feature not sufficiently considered in other guides. The formulae for stains and the methods of staining have not been collected in one chapter, as is usually the practice, because this tends to confuse the student. They are described during the progress of the course, as occasion offers to put them to practical use. Culture-description charts have not been included in this volume. A beginner naturally makes incomplete descriptions and many alterations, and thus defaces the book and impairs its future utility. A sufficient num- ber of loose charts perforated for binding should be furnished to the student at a nominal figure. A point of inestimable importance is how best to stimulate the student to consult textbooks, special monographs, and other references, as often and as freely as possible. This guide has been written with the aim of not only not interfering in any manner with, the reading, through including such points and char- acteristics as might make a textbook superfluous in the judgment of the inexperienced, but also of making it necessary for the student to read the best textbooks with freedom and understanding. Cultural and mor- phological features are left entirely to the actual ob- servation of the student, supplemented by instruction and the reading of textbooks. The course, as outlined, is identical with the medical course given at the University of Chicago, with a few PREFACE TO FIRST EDITION vii additional chapters which may be used during courses for non-medical students. A chapter containing a fairly complete list of formulae for culture media employed in advanced work has been added with the object of making them easily accessible to those engaged in advanced work. The laboratory guides of Novy, Eyre, Frost, Gorham, Kanthack and Drysdale, and Connell, and the American edition of the Manual of Bacteriology of Muir and Ritchie have been freely consulted. I take this occasion of expressing my deep gratitude to Professor Edwin O. Jordan and Dr. Norman MacLeod Harris for their invaluable help and suggestions in the preparation of this guide. Paut G. HEINEMANN Catcaco, ILL. June, 1905 PREFACE TO SECOND EDITION The influence of applied bacteriology is extending rapidly, and while formerly the science of micro- organisms was chiefly confined to the medical field, it has become of vast industrial importance. The gigan- tic interests of fermentation industries, of the dairy, of agriculture, of municipal sanitation, and of water puri- fication are largely controlled by bacteriologists. A knowledge of the principles of bacteriology is becoming more and more desirable to the student of general science, and teachers of domestic science in some institutions are required to take an elementary course. The second edition of this laboratory guide has been revised and enlarged with the view of meeting modern requirements. The first edition was devoted chiefly to medical bacteriology. This has been partly rewritten, and in addition courses in general bacteriology, in the bacteriology of water, in the bacteriology of milk, in soil bacteriology, and a course on molds, yeasts, and acetic-acid bacteria have been outlined. This change of plan necessitated an entirely new arrangement of the material. The preparation of culture media, of stains, etc., has been incorporated in a separate part, and reference to this part is made at the beginning of each special part. The course in the bacteriology of water is calculated to be combined with the physical, chemical, and microscopic examination, the student being thus prepared for sanitary water analysis. Similarly, the course on the bacteriology of milk is to be given in ix x PREFACE TO SECOND EDITION connection with the physical and chemical examina- tion of milk. ; The author hopes that, by the radical changes indi- cated, the usefulness of the book may be extended, and that the study of applied bacteriology may be en- couraged. I am under special obligations to Professor Edwin O. Jordan for advice and suggestions as to the general plan of the book, to Dr. Mary Hefferan for critical reading of the manuscript and the proof, and to Dr. R. E. Buchanan for valuable advice in outlining the course in soil bacteriology. Paut G. HEINEMANN June, 1911 PREFACE TO THIRD EDITION ' In this third edition the general plan of the book has not been changed. The progress of science necessitated alterations and additions which have been incorporated in the text. A fermentation chart of cardboard has been added to the colony counter in the back cover. The author is under obligations to Professors Edwin O. Jordan and Norman MacLeod Harris for valuable suggestions. Pau G. HEINEMANN June, 1915 INTRODUCTION The advent of bacteriology into the realm of the biological sciences not only brought with it a new con- ception of the nature of many complicated phenomena, such as fermentation and disease, but also placed in the hands of experimental workers a new tool. The method of sterilization, of asepsis, made it possible for the first time to attack problems hitherto incapable of solution, or even of approach. This development of bacteriological technic, of rigid and undeviating adherence to definite rules and principles, is not likely to be passed over lightly by the historian of nineteenth- century science. The art of practical medicine and theoretical medical research alike owe much of their recent brilliant success to a ready adoption of the new method. At the present time an active campaign is being set on foot by public health authorities against several widespread and serious diseases of the human race. In various parts of the world malaria, tuberculosis, and typhoid fever are being fought energetically and with much success. In these systematic and organized movements the resources of bacteriology are being utilized as never before, and a full understanding of technical procedure and devices is deemed essential by all workers in this subject. The problems of water- supply and sewage disposal, of urban infantile mortal- ity, and of the control of contagious diseases are all bound up with the intelligent application of bacterio- logical methods. xi xii INTRODUCTION In the almost untilled field of industrial bacteri- ology there is need for a fuller appreciation of the value of bacteriological methods and principles. Many great industries are based wholly upon the proper selection and adaptation of micro-organisms, and a timely and discriminating utilization of their products. Loose and empirical methods have been in force in the past, but these must eventually give way to a more precise and truly bacteriological technic. Agricultural bacteriology is just now much in the public eye, and it would be gratuitous to prophesy the results that may reasonably be anticipated in this direction. Here again crude, rule-of-thumb, “prac- tical” ways of doing things are being supplanted by the scientific, the reasoning, and the precise. To the student, whether in medical, hygienic, or industrial bacteriology, proper technical methods of work must always have a peculiar value, since without their aid advance is impossible, and stumbling and disastrous missteps are certain. A comprehensive out- line of modern bacteriological methods, therefore, is a necessary adjunct to obtaining a true and full under- standing of the underlying principles and tendencies of the science. The technic of bacteriology is one of its greatest contributions to both science and art, and the use of so valuable and simple a tool should be mastered not only by the biological teacher and in- vestigator, but by practical workers in medicine, hygiene, and many other fields. Epwin O. JORDAN TABLE OF CONTENTS Part I. BACTERIOLOGICAL TECHNIC PAGE Section 1. Laboratory Rules . ... . ‘ 3 Section 2. General Directions . . . ws. 8 4 Section 3. Preparation and Cleaning of (assware ‘ 8 Section 4. Methods of Sterilization . . . . . Io Section 5. Preparationof CultureMedia . . . 18 Section 6. Preparation of Staining Solutions . . 43 Section 7. The Microscope . . . 45 Section 8. Scheme for Routine Study at anterla . 50 Section 9. Method of Describing Cultures . . 53 Section 10. Directions for Filling out Culture Charts 58 Part JI. GENERAL BACTERIOLOGY Section 1. Preparation of CultureMedia . . . 69 Section 2. Collecting and Cultivating esas asl ismsfromthe Air . . . . 70 Section 3. Study of Molds, Yeasts, and Toile : 78 Section 4. Bacteriological Examination of Water, Air,andMilkk . . . . . 84 Section 5. Exercises on Infection and Sterilization - gt Section 6. Influence of Disinfectants, Light, and Heat on the Growth of Micro-organisms 94 Section 7. Study of Chromogenic Bacteria . . . 97 Section 8. Study of Micrococci . . . . . . 100 Section 9. Study of Intestinal Bacteria . . . . 101 Part III. Important PATHOGENIC BACTERIA Section 1. Preparation of Culture Media . . . 105 Section 2. The PyogenicGroup . . . 105 Section 3. The Group of Colon-Typhoid Bacilli 112 Section 4. The Proteus Group 3 sow oe EOE Section 5. The Capsulated Group . . . . . 123 Section 6. The DiphtheriaGroup. . . . . . 124 xiii xiv TABLE OF CONTENTS PAGE Section 7. The Hemorrhagic Septicemia Group. . 127 Section 8. The AnthraxGroup . . . . . . 127 Section 9. The Spirillum Group . . ae ik EZ) Section 10. The Group of Acid-proof Bacilli ¢ @ = 33r Section 11 Aand B. Miscellaneous Organisms . 132, 133 Section 12. Pathogenic Trichomycetes . . . . 134 Section 13. The Group of Anaérobic Bacilli . . . 134 Section 14. Isolation of Unknown Bacteria from a Mixture: 2. . 1. eee ee OQ PartIV. THE BACTERIOLOGICAL EXAMINATION OF WATER .AND SEWAGE Section xz. Preparation of Culture Media and of Dilution Flasks. . . . 144 Section 2. Bacteriological Examination of Water - 145 Section 3. Examination of Sewage . . . 148 Section 4. Determination of Anaérobes in Sewage . 149 Section 5. A Study of B. coliand Streptococci . . 149 Section 6. Isolation of B. typhosus from Water . 150 Section 7. Study of Reaction of Bacteria on Neutral- red Broth . . . . 1 . ee O50 Part V. THE BACTERIOLOGICAL EXAMINATION OF MILK Section 1. Preparation of Culture Media and of Dilution Flasks. . . . 154 Section 2. Quantitative Bactedoloeical: Fanti: tionof Milk. . . 154 Section 3. Examination of Market ‘Milk for Tuberde Bacilli . . . 155 Section 4. A Study of the Acid ermentatien ‘of Milk 155 Section 5. Determination of B. coli and Streptococci inMilkk . . 157 Section 6. Study of the Bifects ‘af Basteurtéation and So-called Sterilization of Milk . . 158 Section 7. A Qualitative and Quantitative Study of Anaérobesin Milk . .. 158 Section 8. A Study of Some Organisms Gaticing Ab- normal Fermentations in Milk . . . 159 TABLE OF CONTENTS XV PAGE Section 9. Examination of Milk for Molds and Yeasts . . «se 8 460 Section 10. Examination for Leukocptest in 1 Milk . 160 Section 11, A Study of Groups of Bacteriain Milk . 162 Part VI. THE BACTERIOLOGICAL EXAMINATION OF SOIL Section 1. Quantitative Determination of Bacteria and Sporesin Soil . . .. 165 Section 2. A Study of the Peptonization of Preteins by Soil Bacteria... 168 Section 3. The Formation of Amido Gonpounis and Ammonia... 170 Section 4. The Formation of Nitrites from Antenna and Isolation of Nitrite Bacteria. . . 171 Section 5. The Formation of Nitrates from Nitrites and Isolation of Nitrate Bacteria. . . 174 Section 6. The Assimilation of Free Atmospheric Nitrogen and Isolation of the Bacteria . 175 Section 7. The Reduction of Nitrates to Nitrites and Isolation of the Bacteria . . 177 Section 8. The Reduction of Nitrates to Free Nitro- gn .. 77 Section 9. Growing Laswsiee in Gand and § in Sand Inoculated with Legume Bacteria . . 178 Part VII. Motps, Yeasts, ToRULAE, AND AcETIc-ACID BACTERIA Section 1. Preparation of CultureMedia . . . 181 Section 2. AStudyofMolds . . . . . . . 182 Section 3. AStudyof Yeasts . . . sb hs Ge SS Section 4. Examination of Baker’s Wea, - . « Igo Section 5. Examination of Yeast of Salt-rising Bread 191 Section 6. AStudyofTorulae . . a 4 TOS Section 7. A Study of Acetic-Acid Bacteria on we 02 APPENDIX Dilution Tables . . . adie Gh Ma eecee SO? Table of Weights and Nicaeares' eee . 198 Table of Centigrade and Fahrenheit Thermonieters . 198 INDEX a ee we Ow a ae 203 PART I BACTERIOLOGICAL TECHNIC SECTION 1 LABORATORY RULES 1, Familiarize yourself with the laboratory rules. Upon their careful observance depend good work and your own safety. 2. Food must not be eaten in the laboratory; pencils, labels, or fingers must not be moistened with the tongue. 3. If any portion of a culture is spilt by accident upon the desk or floor, it should be covered immediately with a germicide (HgCl, 1:1000, or carbolic acid in 5 per cent solution). After this germicide has acted for 10 or 15 minutes, wipe it up and throw the cloth or paper into a waste jar. 4. In case the hands should come in contact with infectious material, they should be washed with one of the above mentioned germicides, and then scrubbed with soap and water. 5. The platinum needles used in making cultures should be sterilized in a flame before and after use, and before they are laid down. When the needles are covered with viscous material, as milk, for instance, they should be held at the side of the flame until dry before being sterilized. This will avoid the danger of scattering infectious material about the desk. 6. All possible care should be observed in handling apparatus, etc. Solid material should not be put into sinks. Burned matches, paper, cotton, broken glass, 3 4 LABORATORY GUIDE IN BACTERIOLOGY etc., should be put into crocks and not on the floor or into the sink. 7. Discarded cultures should be killed in the auto- clave (5 minutes at 120° C.) before being emptied into the crocks. 8. See that the air inlets of Bunsen burners are open before lighting, and relight if the flame strikes back. 9. Always return stockbottles to the proper places on the shelves. to. At the close of the day’s work the desks should be washed off with corrosive sublimate, and the hands . Cleaned by thorough washing. 11. Before leaving the laboratory, see that the gas is shut off under all apparatus, that water faucets are closed, and that all glassware, etc., is replaced in the lockers. Culture tubes containing media or cultures should be replaced in their proper places, in order to avoid settling of dust or other foreign material on the stoppers. Dust and air draughts are frequently the cause of contaminations, and in order to avoid these the utmost cleanliness should be observed. A bacteri- ological laboratory should present an orderly appear- ance at all times. SECTION 2 GENERAL DIRECTIONS The following directions should be followed in all the work outlined in this guide: 1. After obtaining the key to a locker, the student should examine the outfit, check all apparatus, and see that everything is in good condition. BACTERIOLOGICAL TECHNIC 5 2. The student should familiarize himself with the program before him for each day, as this will facilitate intelligent and systematic work. 3. The student should procure description charts and fill them out carefully according to directions. 4. Store cover slips in a stender dish and cover them with alcohol. A soft linen cloth should be used for cleaning. The following outfit will be needed for each course. Additions to this outfit will be designated as required in the respective courses. 200 culture tubes. 20 potato tubes. 12 fermentation tubes. 20 petri dishes. 3 Erlenmeyer flasks, one 1,000 c.c., two 500 c.c. each. 2 glass funnels, one 4 inches, one 6 inches. 4 bottles for staining fluids. * i balsam bottle. t stender dish. : 3 staining dishes. 1 saltcellar. I glass rod. 2 platinum needles. Turn the end of one of the needles around a sharp pencil point so as to form a closed loop. 8 tin cups or glass tumblers, the bottoms of which should be covered with a layer of cotton. 4 wire baskets. zr Bunsen burner. I saucepan and cover, or better, a double boiler. 3 graduates, one 500 c.c., one Ioo c.c., and one Io c.c. I pinchcock. I pipette and hose attached. I magnifier (hand lens). I tripod. 6 LABORATORY GUIDE IN BACTERIOLOGY Pinchcock Bottle for “ . Wire Basket Staining Fluid Fermentation Tube Bunsen Burner Culture Tube Fic. - Pipette BACTERIOLOGICAL TECHNIC 7 Magnifier een Balsam Bottle (Hand Lens) Stender Dish Fic. 1 8 LABORATORY GUIDE IN BACTERIOLOGY I retort stand with three rings. 1 thermometer in case. 1 box matches. 40 grams pepton. 120 grams gelatin. 50 grams agar. 6 sheets filter paper. 50 glass slides. i camel’s-hair brush. 50 cover glasses. 2 towels. 1 tube brush. 50 labels. 1 box for slides. 3 hollow-ground slides. 1 glass pencil. I pair forceps. 2 pairs cover-slip forceps. SECTION 3 PREPARATION AND CLEANING OF GLASSWARE , Culture tubes, flasks, fermentation tubes, and petri dishes must be free from organic matter, acids, and alkalis. They should be cleaned as follows: 1. Immerse them in a vessel containing soapsuds or soap powder, boil 10 to 15 minutes, then clean them with a tube brush. Or immerse them for an hour ina solution of potassium bichromate and sulphuric acid: Potassium bichromate................ 60 parts Water seca natu Seoceaanae sc uea es 300 parts Concentrated sulphuric acid........... 460 parts The sulphuric acid should be added slowly with con- stant stirring. 2. Rinse in tap water. BACTERIOLOGICAL TECHNIC 9 3. Again use tube brush, and soap and water if necessary. 4. Rinse again in water to remove every trace of acid or soap. 5. Place the tubes in a wire basket, mouth down, and heat in a hot-air sterilizer for 20 minutes or longer until dry. All other glassware should be treated in the same manner, excepting fermentation tubes, which: should not be heated in the hot-air oven, as this would be likely to cause breakage. The tubes should be plugged with cotton. Non- absorbent cotton is suitable for this purpose. The cotton plug allows free communication with the air, admitting oxygen, which is necessary for the growth of many bacteria; at the same time the admitted air is filtered germ-free and contamination of cultures is avoided. Various methods for plugging tubes are employed in different laboratories. The simplest method is as follows: Take a small amount of cotton and ph it gently into the tube with a glass rod. The cotton should reach into the tube for about 3 of an inch and be sufficiently firm to support the weight of the tube (Fig. 2). The cotton may also be rolled into a cylinder of thickness equal to that of the tube and then pushed into the mouth. The plugged culture tubes Fic. 2 should be placed in a hot-air oven _ Plugged Culture Tube 10 LABORATORY GUIDE IN BACTERIOLOGY at a temperature of 150° C. for about 30 minutes, or until the plugs are slightly browned. The tubes are not necessarily sterile, but the plugs have become set so as to fit the mouth of the tube, and may be removed and replaced readily. SECTION 4 METHODS OF STERILIZATION Sterilization is the process of removing all living organisms. This may be accomplished by heat, by ) t ' a Fic. 3 Berkefeld Filter a. Berkefeld filter d. Intercepting flask b,. Filtered liquid e. Connection with aspirator c. Side tube with cotton filter f. Rubber hose BACTERIOLOGICAL TECHNIC II certain chemicals, or by filtration. Chemicals are used chiefly for sterilizing the skin, surgical instru- ments, and cultures which have been accidentally spilt. Filters for sterilization are made of some porous material, either infusorial earth or unglazed porcelain. Substances which may be injured by heat are sterilized in this manner. Positive or negative pressure is necessary for this kind of sterilization (Fig. 3). Sterilization by dry heat. —Sterilization by dry heat is applicable to the steriliza- tion of most glassware. This method of sterilization is car- ried out by means of hot-air sterilizers (Figs. 4 and 5). Fic.q4 0 These hot-air sterilizers are ay ane boxes with double walls of sheet iron. The bottom shelf should always be covered with a piece of asbes- tos, to prevent ‘heating the apparatus too rapidly. The temperature is maintained at 160° or more for one hour. The flame enters a hole provided at the bottom of the box. Care should be taken to avoid the possi- bility of the flame becoming luminous, otherwise the glassware will be covered with soot. Culture media and all substances liable to be in- jured by heat of 160° C. or over must be sterilized by the application of moist heat. Experience has taught that hot steam has greater germicidal powers than air of the same temperature. Hot steam, therefore, is the most common means of sterilizing culture media. 12 LABORATORY GUIDE IN BACTERIOLOGY Steam is applied in two ways. The first method is that of exposing media to steam of 100° C. for 20 min- utes. This is done in an apparatus generally known as the Arnold steam sterilizer. The usual form is illus- trated in Figs. 6 and 7. Fig. 6 shows the appearance Fic. 5 Lautenschlager Hot-Air Sterilizer of the ordinary form with the hood off. Fig. 7 shows the inside arrangement. The two compartments, @ and 8, are connected by small holes, and a certain amount of water has to be kept here. The water is brought to a boil and the steam rises through a number of holes in the bottom (c) into the chamber (d). The steam condenses at the top and returns between two BACTERIOLOGICAL TECHNIC 13 sheet-copper walls (e, ¢) to the large compartment (b). Larger forms on the same principle are in use (Fig. 8). The media to be sterilized are placed in the large chamber (d, Fig. 7). The water is then heated until steam is generated, and the action of the steam on the media is continued for 20 minutes from the time steam Fic. 6 Fic. 7 Arnold Steam Sterilizer Arnold Steam Sterilizer The hood is taken off and a. Inner water compartment the door opened, showing 6. Outer water compartment inside arrangement c. Perforated bottom d. Sterilizing chamber e. Sheet-copper walls begins to rise. This process is repeated on two succeed- ing days, so that the media have been exposed to the steam for three days. On the first day all vegetative forms are killed. The media are then kept at room or incubator temperature, so that spores which may be present and are not killed by the first exposure may develop into vegetative forms and be killed by the second exposure. If after this any spores should 14 LABORATORY GUIDE IN BACTERIOLOGY survive, they will develop in the next 24 hours, and the third exposure to steam will complete sterilization. Sterilization is accomplished in a shorter time by the use of steam under pressure. The autoclave is the usual apparatus used for this purpose. Certain bac- teria, some of which are widely distributed in nature, Fic. 8 Arnold Sterilizer have the faculty of forming spores. These spores are highly resistant to heat and do not lose their vitality either by boiling or by application of steam under ordinary atmospheric pressure. By adding the pres- sure of one atmosphere to ordinary pressure, the boiling point is raised to121.4° C. 120° C. is sufficient to kill all spores during an exposure of 5 minutes, if the media are in tubes. Larger amounts of media require a proportionately longer exposure. The autoclave consists of a strong cylinder made of BACTERIOLOGICAL TECHNIC 15 iron. Some forms have a cover, others a door on one side. A basket, or a set of shelves, is on the inside. A gauge indicates the pressure and temperature. A safety valve opens automatically when the desired pressure is reached. Two forms are illustrated in Figs. 9 and 10. Before using the auto- clave the inside should be examined. It must be clean and contain a suff- cient quantity of clean water. Water containing impurities is liable to foam up when boiling, wet the plugs, and ruin the media. If the lid is on top, it should be fastened care- fully by tightening the thumbscrews. In order to distribute the pressure of the lid uniformly the diametrically opposite screws should be tight- ened simultaneously. The valve should be open and left open until the steam has escaped for about one minute. Then the valve Fic. 9 Autoclave a. Steam valve d,d. Thumbscrews b. Safety valve e. Bunsen burner c. Gauge and opening is closed and when the desired pressure has been reached, the gas should be turned down so as to maintain pressure for the requisite length of time. 16 LABORATORY GUIDE IN BACTERIOLOGY At the end of this period the gas is shut off and the pressure allowed to decrease gradually. The valve should not be opened, nor the lid removed, until atmospheric pressure has been restored, otherwise the sudden release of pressure would cause the media to boil suddenly and push the plugs out of place. When large autoclaves are used (Fig. 10) pro- vision must be made for proper circulation of air, or “air cushions” form and media will not be sterilized. If an aperture remains in the outlet the discharge of air is facili- tated. Attachment of a vacuum pump is often desirable to remove all air. Blood serum or egg media are the most diffi- cult to sterilize. The tem- perature of coagulation of these media is relatively low, and sudden heating causes the mass to break up, form bubbles, and be- come useless for cultural purposes. The Koch inspis- sator may be used, or, with certain precautions, the autoclave. The Koch inspissator (Fig. 11) allows the tubes to rest in an inclined position and to be heated gradually to 75° C. This temperature is maintained Fic. 10 Autoclave BACTERIOLOGICAL TECHNIC 17 for one hour. This process has to be repeated for five or six successive days, before sterilization is complete. If the autoclave is to be used, the tubes are placed in the autoclave in an inclined position. Good results are obtained by this method: Fic. 11 Koch Inspissator 1. Close the lid and the steam outlet. 2, Admit steam. After reaching 3 pounds pressure, keep this pressure for 5 minutes. 3. Increase the pressure slowly to 5 pounds and keep there for 5 minutes. 18 LABORATORY GUIDE IN BACTERIOLOGY 4. Increase to 10 pounds and hold for 5 minutes. 5. Increase to 15 pounds and hold for 5 minutes. 6. Open the steam outlet and keep at 15 pounds pressure for 20 minutes. It is advisable to sterilize the tubes again on the following day by slowly bringing the pressure to 15 pounds with the steam outlet slightly open and keep- ing at this pressure for 20 minutes. SECTION 5 PREPARATION OF CULTURE MEDIA EXERCISE I. PREPARATION OF DUNHAM’S PEPTON SOLUTION AND OF PEPTON BROTH (BOUILLON) 1. Weigh the saucepan, measure into it 1,000 c.c. of tap water, and heat over a flame or a water bath. 2. Dissolve in this, when hot, but not boiling, 10 grams Witte’s pepton. : 3. When dissolved, replace the evaporated amount of water and divide into two equal parts of 500 c.c. each. 4. One-half is then filtered until perfectly clear, tubed, and sterilized in the autoclave for 5 minutes at 120°C. -This is Dunham’s Pepton Solution. 5. Dissolve 1.5 grams extract of beef in the remain- ing 500 C.c. 6. Adjust the reaction with phenolphthalein paper or by titration against n. NaOH. Note.—The reaction of culture media is a matter of vital importance. Bacteria, especially pathogenic bacteria, grow preferably in a medium which is neutral or slightly acid to phenolphthalein. The neutral point of litmus is about 2 per BACTERIOLOGICAL TECHNIC 19 cent more alkaline than the neutral point of phenolphthalein, so that a medium which is neutral to phenolphthalein is about 2 per cent alkaline to litmus. It has been found that about 1 per cent acid to phenolphthalein is the most favorable reaction for the growth of pathogenic bacteria. A medium of this reac- tion is still alkaline to litmus. 7. After neutralization fill into an Erlenmeyer flask and autoclave for 10 minutes at 120° C. 8. Keep the sterilized broth for 24 hours and then filter until clear and distribute into culture tubes, -which have to be autoclaved again. Note.—The reason for exposing broth to a heat of 120°C. twice is this: The solution contains substances which are pre- cipitated by heat and appear as a sediment after cooling. As it is important to have a perfectly clear broth in tubes, these sub- stances are precipitated by the first heating, and, if tubed later, the second sterilization will not affect the appearance of the medium. For ordinary purposes it is sufficient to neutralize media by means of phenolphthalein paper. This is prepared by soaking filter paper in a 1 per cent solution of phenolphthalein in 50 per cent alcohol and then allowing the paper to become dry. A 2 per cent or 4 per cent solution of sodium hydrate is added to the medium to be neutralized until a faint, but decided, pink appears on phenolphthalein paper. A more precise method is as follows: Measure by means of a pipette 5 c.c. of the medium into a white porcelain evaporating dish, add 45 c.c. of distilled water and 1 c.c. of a 1 per cent solu- tion of phenolphathalein in 50 per cent alcohol. Heat the mix- ture to boiling and slowly add from a graduated burette 1-20th normal NaOH until a faint but decided and stable pink appears in the liquid. The amount of NaOH is read from the burette and the amount for neutralization of the whole volume calculated. It is desirable to make another titration after the NaOH has been added. The amount to be added to the medium has to be varied according to the reaction desired. If it is to be neutral, 20 LABORATORY GUIDE IN BACTERIOLOGY the above proceeding will accomplish the object. If it is desired to have a medium which has a reaction of 1 per cent acid, a pro- portionate amount should be deducted from the total amount of NaOH calculated. : Example—By reading the burette we find that it takes 3 c.c. 1-20th normal NaOH to neutralize 5 c.c. of the medium. This means that 60 c.c. 1-20th n.NaOH will neutralize roo c.c. medium, or 600 c.c. 1-20th n.NaOH will neutralize 1,000 c.c. medium. To find the requisite amount of n.NaOH divide the above figure by 20. Then 3 c.c. n.NaOH will neutralize 100 c.c. and 30 c.c. n.NaOH will neutralize 1,000 c.c. If the reaction is to be i per cent acid, deduct 10 c.c. from 30=20 c.c. If 20 c.c. normal NaOH are added to each liter the reaction should be 1 per cent acid. This should be ascertained by a second titration. A normal solution is the equivalent weight in grams (Gram- Molecule) dissolved in distilled water and made up to 1,000 c.c. In the case of monovalent chemicals the molecular weight is taken, if bivalent the molecular weight is divided by two, etc. All media should be prepared with the utmost care and should be perfectly clear. EXERCISE 2, PREPARATION OF NUTRIENT AGAR-AGAR Agar-agar (or called simply “agar’’) is a watery extract of certain seaweeds found on the Pacific coast of Asia. A solution of agar containing about 1.5 per cent forms a firm jelly, which melts near the boiling point of water, and on cooling solidifies at about 39°. Gelatin solidifies at much lower temperature, and can- not be kept solid at body temperature. The use of agar is, therefore, of great importance in the study of pathogenic bacteria, a large number of which prefer body temperature for growth. 1. Weigh a saucepan, or, if available, a double boiler. Note the weight. 2. Measure 1,000 c.c. of tap water into the sauce- BACTERIOLOGICAL TECHNIC at pan. It is advisable to add about 200 c.c. of water to this to allow for evaporation. 3. Cut and shred 15 grams of agar, add this to the water, bring to the boiling point, and keep at this temperature until the agar is completely dissolved. Violent boiling should be avoided and the mixture should be stirred, so as to prevent overheating. NotEe.—The agar may be soaked in cold water over night. This removes some of the impurities and renders the agar more readily soluble. 4. Add 3 grams extract of beef and 10 grams Witte’s pepton. 5. Adjust the reaction. 6. Adjust the weight. Place 1,000 grams weight and the weight of the saucepan on one side of the scales and then add enough water to make the saucepan with the agar balance. If the weight is too high, it should be boiled gently until the weight has been brought down to the proper amount. 4. Make a paper filter as described below and arrange a retort stand as illustrated in Fig. 12. 8. When at the boiling point filter the agar and dis- tribute into culture tubes. g. The tubed agar should be sterilized in the auto- clave for 5 minutes. For agar slants each tube should contain about 7 c.c. or be filled one-third of the length of the tube. For plating, the tube should be half filled and contain ro c.c. Slants are prepared by allowing the agar after sterilization to cool in a slanting position. If it is de- sired to slant a large number a whole basket may be put in a slanting position. When a few tubes only 22 LABORATORY GUIDE IN BACTERIOLOGY are required they may rest with the plugged end on a glass rod or rubber hose until the agar has solidified. For the purpose of filtering media heavy filter paper (Schleicher and Schiill No. 598) of the best quality only should be used. This is especially im- portant when filtering agar or gelatin. After the filter Q ad anil aN Fic. 12 4 Apparatus for Filtering Media Filter . Large funnel - Small funnel . Rubber hose 4 . Pinchcock . Pipette . Culture tube 7. RAMS p ROSA has been folded and inserted into the funnel hot water should be run through the filter, until it is soaked and warm. Method of folding filters (Fig. 13).—@ and 5) Take a square piece of filter paper twice as wide as the depth of the funnel and fold to half the size so as to make No. 1 cover No. 2. (Compare with Fig. 13.) BACTERIOLOGICAL TECHNIC 23 c) Fold this to make 1 cover 2 and 3 cover 4 (result Fig. 13¢). It consists of four layers and forms a square. d) Fold the upper part, consisting of two layers, from 1 to 2 (Fig. 13d). The shaded triangle, 2-3-4, now has six layers; the other, 1-3-4, two layers. A 2 2 1 2 4 é 3 t £0 4 2 a Z 3 2 3 2 5 Z (2 Lt # I 0 4& o 4 3} A 2 Zi i “ Fic. 13 Method of Folding Paper Filter (For reference letters see text) ~ e) Fold the upper double layer so as to make 2 cover a point in the diagonal at 5, taking care to make a sharp point’at 4 (result Fig. 13e). The shaded part is now eight layers deep. f) Turn the folded part face down and repeat 24 LABORATORY GUIDE IN BACTERIOLOGY operations exactly on the other side as in d and e (re- sult Fig. 13f). g) Take up and open the large middle fold (result Fig. 13g). The two halves must now be symmetrical. h) Fold so as to make the lines 1-3 and 1-4-meet at the center line 1-2 (result Fig. 137). 4) Now pick up and fold backward so as to have 1 cover 2 in the back (Fig. 137). j) Cut through the line 1-2 and open up. The extreme ends will be found without a fold and may be folded so as to make g sharp edges. This filter is inserted evenly into the funnel, spread- ing the folds at a distance from each other as nearly alike as possible. Care should be taken to make the folds and the point sharp, as this insures rapid filtration and prevents the filter from tearing. If a vacuum pump is available, the medium may be filtered rapidly by the use of suitable apparatus, as illustrated in Fig. 14. At the connection with the vacuum pump a valve should be inserted or a flask arranged as in the illustration, to prevent the water from entering the flask if the water pressure should be reduced suddenly. If some precautions are properly observed, chiefly the making of a good filter with sharp edges and a sharp point, and the soaking of this in hot water, there is no difficulty in filtering agar or gelatin successfully in a short time. There is some danger of the point of the filter breaking when the hot medium is poured on, This may be avoided by folding’a second filter of about two inches diameter and fitting this small filter on the bottom and outside of the larger one. BACTERIOLOGICAL TECHNIC 25 The basket which is to receive the tubes after filling should be placed in an inclined position, as this facili- tates the proper arrangement of the tubes. In filling the tubes the pipette at the end of the rubber hose Fic. 14 Filtering Media by Means of Vacuum Pump a. Liquid medium e. Reflux flask b. Absorbent cotton Ff. Rubber stopper with two holes c. Rubber stopper g. Connection with aspirator d. Filtered medium should be inserted to a depth of at least two inches and when the proper amount has been discharged should be removed carefully so as to avoid wetting the mouth of the tube. A wet tube-mouth will cause 26 LABORATORY GUIDE IN BACTERIOLOGY the cotton plug to stick to the glass, and later not only occasion much annoyance to the person using the tube but expose the medium in it to danger of contamina- tion. EXERCISE 3. PREPARATION OF DEXTROSE AGAR Dextrose is added to agar for the demonstration of. gas-forming organisms. Dextrose is decomposed by these bacteria with gas formation, the gas appearing as bubbles in the medium. Dextrose agar is prepared by adding a definite amount of dextrose, usually 1 per cent, to filtered agar. Note.—Dextrose agar cannot be distinguished from plain agar by appearance. It is therefore necessary either to label the tubes or to separate dextrose agar tubes from plain agar tubes in a basket by tying a piece of string or inserting a piece of paper between. EXERCISE 4. PREPARATION OF PEPTON GELATIN 1. Weigh the saucepan and measure 1,000 c.c. of tap water into it. To this 200 to 300 c.c. of water should be added to allow for evaporation. 2. Dissolve 3 grams extract of meat and 10 grams pepton. 3. When boiling dissolve ro per cent gelatin in cold weather and 12 per cent in hot weather. The gelatin must be of the best quality (gold label) and should be dissolved slowly, taking a few leaves at a time, and with constant stirring. 4. When completely dissolved, adjust the reaction as directed in the preparation of agar. Gelatin con- tains an appreciable amount of acid and it will require more NaOH solution for neutralization than agar. BACTERIOLOGICAL TECHNIC 27 5. Cool to about 60°C. Dissolve the whites of two or three eggs, or about 10 grams of pure powdered egg albumin in about 100 c.c. of tap water. Mix this solution with the gelatin and heat slowly to the boiling point, placing a piece of asbestos under the pan unless a double boiler is used. Boil gently until the egg white or egg albumin has coagulated and a solid film has formed which mechanically incloses the impurities. 6. Adjust the weight to 1,000 grams, allowing for the weight of the pan. Filter and tube, as in the preparation of agar. 7. Sterilize in the autoclave for 5-8 minutes at 120° C., or in the arnold for three successive days. If sterilized in the autoclave, care should be taken not to allow the temperature to go beyond 120°C., and gelatin should not be permitted to remain at this temperature beyond the prescribed time. Gelatin is readily decomposed by heat and then does not solidify after cooling. _ EXERCISE 5. PREPARATION OF LITMUS MILK Milk is one of the most important culture media. Only the cleanest milk obtainable should be used. “Certified milk” is most suitable. In many cases com- mercial milk powder may be used. If certified skim- med milk or fat-free milk is available step 1 is omitted. 1. Separate five-sixths of the cream from the milk. 2. Add a sufficient quantity of tincture of litmus to impart a decided blue color to the milk. If a solution of Merck’s pure extract of litmus 1:100 is at hand about 5 per cent of this will be sufficient. 3. Distribute in culture tubes and sterilize in the 28 LABORATORY GUIDE IN BACTERIOLOGY autoclave for 5 minutes at 120°C. After sterilization the blue is usually more or less lost, but returns upon standing. EXERCISE 6. PREPARATION OF POTATO 1. Select several large potatoes, and cleanse. by brushing the dirt off, cutting out the eyes and other blemishes, and washing in water. Fic. 15 Fic. 16 Fic. 17 Potato Cylinder Ordinary Style of Potato Tube Potato Tube (showing diagonal for u. Potato b. Cotton cutting) 2. Punch out cylinders with a borer of suitable size, trim them, and cut each cylinder into two equal parts on a diagonal line (Fig. 15). BACTERIOLOGICAL TECHNIC 29 3. Immerse the pieces in running water for 24 hours. 4. Trim the pieces of potato so they slide down the tube and insert one half-cylinder into each potato tube. The wide end rests on the constriction of the tube. Pour a small amount of water into it, and fill about one-half of the part of the tube below the constriction. Large culture tubes without constric- tions may be used. A small amount of cotton should be pushed to the bottom of these and the cotton soaked with water. The potato then rests on the cotton (Figs. 16, 17). 5. Sterilize in the autoclave for 8 to 10 minutes at 120° C., or in the arnold for three consecutive days. Note.—Potatoes usually harbor a spore-bearing bacillus, the spores of which are highly resistant. Therefore a longer expo- sure in the autoclave is necessary to insure sterilization. SUBSTITUTE FOR POTATO 1. Dissolve 15 grams agar in 600 c.c. water and filter. 2. Dissolve the following salts in 200 c.c. water: ASPATaP iia. cies