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Full text of "Laboratory manual in general microbiology"

AGRICULTUR 



BIOLOGY 
;W 

G 



LABORATORY MANUAL 

IN 

GENERAL MICROBIOLOGY 



PREPARED BY THE 

LABORATORY OF BACTERIOLOGY AND HYGIENE 

MICHIGAN AGRICULTURAL COLLEGE 



FIRST EDI T ION 
SECOND THOUSAND 



NEW YORK 

JOHN WILEY & SONS, INC. 

LONDON: CHAPMAN & HALL, LIMITED 

1916 



BiOLOGY 
R 
G 



COPYRIGHT, 1916 

BY 
WARD GILTNER 




LIBRAY 



PRESS OF 

BRAUNWOHTH & CO. 

BOOK MANUFACTURERS 

BROOKLYN, N. Y. 



PREFATORY NOTE 



LABORATORY instruction in bacteriology at the Michigan 
Agricultural College developed under the direction of Dr. 
C. E. Marshall. This laboratory guide represents the accu- 
mulated efforts of instructors working for a period in excess 
of a decade. To Assistant Professor L. Zae Northrup is 
due the credit for collecting and arranging the material pre- 
sented as well as for preparing de novo many of the experi- 
ments and much of the supplementary matter. She has 
been assisted by Mr. W. L. Kulp. Dr. E. T. Hallman and 
Dr. L. R. Himmelberger have taken the responsibility for 
arranging the exercises relating to immunity, serum therapy 
and pathologic bacteriology. Great praise is due Dr. F. H. 
H. Van Suchtelen for introducing many new features into 
the laboratory work during the academic year 1912-13, 
and also Dr. Otto Rahn for his several years of admirable 
effort immediately preceding. Others whose influence has 
been felt in creating this guide and to whom credit is due 
are Professors W. G. Sackett, S. F. Edwards, L. D. Bush- 
nell, C. W. Brown and W. H. Wright. 

While some claim to originality may be made for this 
laboratory guide, it is to be expected that much of the 
material herein has been presented in various other manuals 
and perhaps in better form in many instances. The greatest 
effort has been made to make this a laboratory guide to 
General Microbiology, leaving the particular fields of dairy, 
soil, water, medical and other phases of bacteriology to 
special guides already in print or at present projected. The 
presentation of this manual to the public is in no way an 

iii 



IV PREFATORY NOTE 

intimation that the special iields have not been admirably 
dealt with by others. 

The subject matter given under Part I of this manual is 
primarily for the purpose of giving a working knowledge of 
laboratory methods used in the study of microorganisms. 
Molds, yeasts and bacteria are taken up in the order of their 
comparative sizes and studied as to their identification by 
morphological and cultural methods. It is presupposed that 
the student has a knowledge of these microorganisms 
acquired from preceding lectures in microbiology. 

Part II consists of exercises demonstrating the various 
physiological activities of microorganisms. 

Part III deals with applied microbiology. After the 
student has familiarized himself with the ordinary tools and 
technic, etc., as dealt with in Parts I and II, it is not neces- 
sary that he be burdened with minute, detailed instructions. 
We have had this in mind in preparing Part III. 

No attempt has been made to compile an exhaustive 
list of exercises; the aim has been only to cover a wide 
range of activities under each different subject. In many 
cases, exercises have been taken directly, with few or no 
modifications, from laboratory manuals already in print. 
Credit has not been given directly; the list of references, 
however, includes all books from which material has been 
taken. 

The purpose of this laboratory manual is to make the 
student more independent. Practically all directions for 
work to be done are contained in it; for this reason the work 
as assigned from day to day should be read -over carefully 
before beginning an exercise and then followed step by step. 
Any desirable changes in directions may be indicated by 
the instructor. 

WARD GILTNER, 

Head of Department, 
EAST LANSING, MICH. 
Sept. 1, 1915. 



CONTENTS 



PART I 

GENERAL MORPHOLOGICAL AND CULTURAL 
METHODS 

PAGE 

Exercise 1. Cleaning Glassware 1 

Sterilization 5 

Exercise 2. Preparation of Glassware for Sterilization 15 

Nutrient Media 18 

Exercise 3. Titration of Media 20 

Milk 23 

Exercise 4. Preparation of Litmus Milk 25 

Exercise 5. Preparation of Glycerin Potato 26 

Exercise 6. Preparation of Meat Infusion 28 

Exercise 7. Preparation of Nutrient Broth 29 

Gelatin 31 

Exercise 8. Preparation of Nutrient Gelatin 36 

Agar : . . . 37 

Exercise 9. Preparation of Nutrient Agar 41 

Exercise 10. Preparation of Dunham s Peptone Solution 43 

Exercise 11. Nitrate Peptone Solution 44 

Cultures 44 

Exercise 12. Preparation of Plate Cultures, Loop or Straight 

Needle Dilution Method 49 

Exercise 13. Preparation of Plate Cultures, Quantitative Dilu- 
tion Method 52 

Exercise 14. Methods of Counting Colonies in Petri Dish Cul- 
tures 56 

Exercise 16. Isolation of Microorganisms from Plate Cultures 

and Method of Making Agar Streak Culture. ... 58 
Exercise 16. Method of Making Transfers of Pure Cultures into 

a Liquid Medium 60 

Exercise 17. Method of Making Stab Cultures 61 

Exercise 18. Preparation of a Giant Colony 61 

The Microscope , . . . 63 



VI 



CONTENTS 



PAGE 

Exercise 19. Method of Measuring Microorganisms 70 

Exercise 20. Determination of the Rate of Movement of Motile 

Organisms 74 

Exercise 21. Preparation of a Hanging Drop 74 

Exercise 22. Preparation of the Adhesion Culture 78 

Exercise 23. Preparation of the Moist-chamber Culture 80 

Exercise 24. Preparation of the Agar Hanging-block Culture .... 81 
Exercise 25. Lindner's Concave-slide Method of Demonstrating 

Fermentation 83 

Exercise 26. Lindner's Droplet Culture 84 

Exercise 27. Chinese Ink Preparation. . 85 

Exercise 28. The Staining of Microorganisms 87 

Exercise 29. Anjeszky's Method of Staining Spores 90 

Exercise 30. Method of Staining Tubercle and Other Acid-fast 

Bacteria 92 

Exercise 31. Method for Staining Flagella 93 

Exercise 32. Gram's Method of Staining 95 

Exercise 33. Method for Staining Capsules 97 

Exercise 34. Method of Making Impression Preparations 98 

Exercise 36. Method of Staining the Nuclei .of Yeast Cells 99 

General Characteristics of Mold Growth 100 

Exercise 36. Microscopical Examination of Molds 105 

Exercise 37. The Study of Molds 107 

Exercise 38. To Determine the Acidity Changes Produced by 

, Molds in Cider 112 

Exercise 39. To Demonstrate the Pathogenic Nature of Molds. . 113 

Yeasts - 114 

Exercise 40. To Isolate a Pure Culture of Saccharomyces Cere- 
visiaB and to Study the Flora of a Compressed 

Yeast Cake 114 

Exercise 41. Apparatus and Methods for the Study of Gaseous 

Fermentation 117 

Exercise 42. The Study of Yeasts 119 

Exercise 43. The Study of Bacteria 125 

Exercise 44. Ehrlich's Method of Testing Indol Production 132 

Exercise 45. Tests for the Reduction of Nitrates 133 

Descriptive Chart Society of American Bacteriologists 134 

Exercise 46. To Demonstrate the Efficiency of Intermittent 

Heating as a Method of Sterilizing Media 139 

Exercise 47. To Compare Morphologically Protozoa with Bac- 
teria 140 

Exercise 48. To Study the Natural Decomposition of Milk 141 



CONTENTS 



Vll 



Exercise 49. To Isolate Spore-forming Bacteria and to Study 

Spore Formation 144 

Exercise 50. To Demonstrate the Efficiency of Filtration as a 

Means of Removing Microorganisms from Liquids 145 
Exercise 61. To Demonstrate the Presence of Microorganisms in 

Air, on Desk, Floor, etc 146 

Exercise 62. Qualitative Study of the Microorganisms of the Skin 

and Hair , 148 

Exercise 53. Qualitative Study of the Microorganisms of the 

Mucous Membrane ... .... 150 



PART II 

PHYSIOLOGY OF MICROORGANISMS 

Exercise 1. To Demonstrate the Small Amount of Food Needed 

by Bacteria 152 

Some Physiological Classifications of Bacteria 153 

Anaerobic Culture Methods 155 

Exercise 2. The Effect of Anaerobic Conditions upon Micro- 
organisms from Manure 165 

Exercise 3. To Demonstrate that Acids Are Formed from Car- 
bohydrates by Bacteria 167 

Exercise 4. To Show that Organic Acids May Serve as a Food 

for Some Organisms 169 

Exercise 5. To Demonstrate the Variation in Food Requirements 

of Bacteria 170 

Exercise 6. To Demonstrate the Splitting of Carbohydrates into 

Alcohol and CO 2 171 

Exercise 7. To Demonstrate the Necessity of Nitrogen in Some 

Form for Microbial Growth 172 

ExerciseS. To Demonstrate the Production of H 2 S by Bacteria. 174 
Exercise 9. The Effect of Physical and Chemical Agencies on 

Microbial Pigment 175 

Exercise 10. To Illustrate One of the Physical Products of 

Metabolism 177 

Enzymes: Classifications and Reactions 178 

Exercise 11. A Comparison of Acid and Rennet Curds 185 

Exercise 12. To Show the Action of Proteolytic Enzymes upon 

Gelatin . . .187 



Vlll 



CONTENTS 



Exercise 13. - To show the Action of Proteolytic Enzymes upon 

Casein. 188 

Exercise 14. To Show the Action of Enzymes upon Starch 189 

Exercise 16. To Show the Action of Reducing Enzymes 190 

Exercise 16. To Show the Action of the Enzyme Catalase 191 

Exercise 17. To Demonstrate the Oxidizing Enzyme of Vinegar 

Bacteria 192 

Exercise 18. To Demonstrate the Necessity for an Activator 

for the Enzymic Action of Rennet 193 

Exercise 19. Effect of Concentrated Solutions upon Microor- 
ganisms 195 

Exercise 20. The Effect of Desiccation upon Bacteria 197 

Exercise 21. The Determination of the Optimum, Maximum 
and Minimum Temperature Requirements for 
Certain Organisms 198 

Exercise 22. The Effect of Freezing upon Spore-forming and 

Non-spore-forming Bacteria 199 

Exercise 23. The Determination of. the Thermal Death Point of 
a Spore-forming and a Non-spore-forming Or- 
ganism 200 

Exercise 24. To Determine the Relative Effect of Moist and 

Dry Heat on Bacteria 202 

Exercise 25. To Determine the Effect of Pasteurization upon the 

Growth of Microorganisms 203 

Exercise 26. To Illustrate the Effect of the Reaction of the 

Nutrient Medium upon Microorganisms . : 205 

Exercise 27. To Determine the Effect of Diffused Light upon 

Molds 206 

Exercise 28. To Show the Influence of Direct Sunlight upon the 

Growth of Microorganisms 208 

Exercise 29. Determination of the Phenol Coefficient of Some 

Common Disinfectants 210 

Exercise 30. To Determine the Action of Formaldehyde upon 

the Microflora of Milk 213 

Exercise 31. To Illustrate Symbiosis 214 

Exercise 32. To Illustrate One of the Phases of Mutual Rela- 
tionship of Microorganisms 215 

Exercise 33. To Demonstrate the Effect of the Metabolic Prod- 
ucts of Bact. Lactis Acidi on its Activities 217 



CONTENTS 



IX 



PART III 
APPLIED MICROBIOLOGY 

AIR MICROBIOLOGY 
Exercise 1. Quantitative Bacterial Analysis of Air . . . 



PAGE 

. 220 



WATER AND SEWAGE MICROBIOLOGY 

Exercise 1. Bacteriological Analysis of Water from a Source 

Suspected of Sewage Contamination 223 

Exercise 2. Bacteriological Analysis of Water Suspected of 

Sewage or Other Pollution 228 

Exercise 3. To Demonstrate the Efficiency of Chloride of Lime 

as an Agent in the Purification of Drinking Water . 236 
Exercise 4. To Demonstrate the Efficiency of the Berkefeld 

Filter Candle as a Means of Water Purification . . . 238 

SOIL MICROBIOLOGY 

Exercise 1. To Test the Calalytic Power of Soil 239 

Exercise 2. A Comparative Study of the Number and Types of 

Microorganisms in Soil 241 

Exercise 3. To Illustrate the Effect of Aeration of Soils on the 

Activities of Microorganisms Contained Therein.. 245 

Exercise 4. To Demonstrate the Cellulose-decomposing Power 

of Aerobic Organisms Found in the Soil 246 

Exercise 5. To Illustrate the Anaerobic Decomposition of Cel- 
lulose by Soil and Fecal Organisms 248 

Exercise 6. To Illustrate Nitrification in Solution 249 

Exercise 7. To Illustrate Denitrification in Solution 251 

Exercise 8. To Illustrate the Non-symbiotic Fixation of Nitrogen 
by Soil Organisms and Isolation of Azotobacter 
Through Its Mineral Food Requirements 253 

Exercise 9. A study of the Symbiotic Nitrogen-fixing Organisms 

of Legumes, Ps. Radicicola 256 

Exercise 10. To Demonstrate the Change of Insoluble Phosphates 
to a Soluble Form Through the Agency of Micro- 
organisms , 263 



X CONTENTS 

DAIRY MICROBIOLOGY 

PAGE 

Exercise 1. A Comparative Study of the Number and Types of 

Microorganisms and Other Cells in Milk 264 

Exercise 2. The Determination of the Bacterial Content of 

< Milk in the Udder 268 

Exercise 3. To Illustrate Extraneous Contamination 270 

Exercise 4. To Investigate the Amount and Kind of Dirt in 
Milk and its Relation to the Microbial Content of 
the Milk 273 

Exercise 5. To Determine the Influence of Temperature upon 
the Keeping Quality of Milk; Pure Milk Com- 
pared with Market Milk 278 

Exercise 6. A Study of the Pasteurization of Milk or Cream by 

Laboratory Methods 281 

Exercise 7. Determination of the Number and Types of Bacteria 

in Butter 284 

Exercise 8. To Determine the Number and Types of Micro- 
organisms in Cheese 286 

Exercise 9. A Comparison of the Bacterial Content of Sweetened 

and Unsweetened Condensed Milks 288 

Exercise 10. To Determine the Number and Types of , Micro- 
organisms in Cream 289 

PLANT MICROBIOLOGY 

Exercise 1. To Demonstrate that Plants are Subject to Microbial 

Diseases 291 

ANIMAL DISEASES AND IMMUNITY 

Exercise 1. Animal Inoculation in Bacteriology for the Deter- 
mination of the Identity of a Microorganism, Its 
Pathogenicity or Virulence, or for Production of 

Immunity 295 

Exercise 2. Isolation of Pathogenic Bacteria from Fluids and 

Tissues of Dead Animals 301 

Exercise 3. A Study of Bact. Anthracis 302 

Exercise 4. The Preparation of Tubuculin 303 

Tuberculin Test Chart 306 

Exercise 5. The Preparation of Black-leg Vaccine 307 

Exercise 6. The Preparation of Tetanus Toxin 308 

Exercise 7. The Preparation of Tetanus Antitoxin 309 



CONTENTS xi 

PAQE 

Exercise 8. A Demonstration of the Agglutination Test 310 

Exercise 9. A Study of Filterable Viruses 313 

Exercise 10. The Preparation of Bacterins or Bacterial Vaccines . 316 
Exercise 11. To Demonstrate Opsonins and to Determine the 

Opsonic Index 319 

Exercise 12. To Demonstrate the Precipitin Test 321 

Exercise 13. The Production of a Hemolytic Serum 323 

Exercise 14. To Demonstrate the Complement Fixation Test . . . 325 

APPENDIX 

Outline for the Study of Microbiology Special Media Table for 
Identification of Bacteria in Polluted Water Characters of 
B. Coli B. Typhosus Groups Common Disinfectants So- 
lutions for Cleaning Glassware Standard Solutions Indi- 
cators Salt Solutions Test Solutions Mounting Media 
Stains Solutions for Use in Staining Steam Table Tem- 
perature Conversion Formulae Metric System List of Text 
and Reference Books, . , .331 



LABORATORY RULES 



1. Do not bring coats, sweaters, hats, etc., into the 
laboratory and lay them on desks, etc.; hang them in the 
place provided for the purpose. 

2. Before beginning and after finishing work, the top 
of the desks must be washed off with a liberal supply of 
1-1000 mercuric chloride. This will destroy all micro- 
organisms and their spores and aid greatly in rendering 
aseptic technic possible. A large bottle of this disinfectant 
will be found near each desk. 

3. Do not put string, paper, pencils, pins, etc., in the 
mouth nor moisten labels with the tongue while in the 
laboratory. Follow this practice outside of the laboratory 
also. Food should not be eaten in the laboratory. 

4. Observe all possible cleanliness and neatness in the 
care of apparatus, desk, microscope, etc. 

5. Apparatus must be kept inside the desks, but not 
cultures. Cultures must be kept at a constant temperature 
in the place fitted for this purpose. 

The microscope and accessories must be returned to 
the case at the close of the work. 

6. Water, gas, steam and electricity are to be turned off 
when not in use. This applies to the individual desks, large 
sinks, steam (including autoclav), hot-air sterilizers, etc. 

7. Put all solid waste material, cotton, paper, matches, 
coagulated milk, etc., and waste liquid which will solidify 
when cold (agar, gelatin) into receptacles provided for that 
purpose, not into the sinks. 

8. Apparatus, media, etc., should be removed from steam 
heaters, immediately after steaming. 

xiii 



xiv LABORATORY RULES 

9. No cultures are to be taken out of the laboratory 
without the permission of the head of the department. 

10. All accidents, such as spilling infected material 
(pathogenic or non-pathogenic), cutting or pricking the 
fingers, must be reported at once to the instructor in charge. 

Additional rules will be given if necessary, in conjunction 
with special exercises or technic. 

11. 1-1000 mercuric chloride will not injure the skin if 
not used too often. Wash your hands in it thoroughly each 
time before you leave the laboratory to avoid carrying away 
undesirable organisms. Use every precaution against in- 
fection. 

12. At the beginning of each laboratory period read over 
carefully the directions for the next exercise in order to 
understand its purpose and to make any necessary pre- 
liminary preparations. 

13. Take careful notes on all observations made in the 
study of cultures and preparations made from them. 



FORM FOR WRITING UP EXERCISES IN THE 
NOTEBOOK 



I. Object. A concise statement of what the exercise is 
intended to prove or demonstrate is to be given. 

II. Apparatus. This includes everything with the excep- 
tion of the every-day tools such as burner, platinum needles, 
etc. 

III. Cultures. A brief morphological and cultural de- 
scription characteristic of each organism should be given, 
also its occurrence and importance. Certain organisms are 
used for a certain purpose. If this purpose is not evident, 
ascertain from the references given why these particular 
organisms were used. 

IV. Method. State briefly but clearly your method of 
procedure. 

V. Results. Give your results in full. Tabulate data so 
that they may be comprehended at a glance. Results often 
may be tabulated as + and . Plot curves whenever 
possible. 

VI. Conclusions. Draw the conclusion which your own 
results warrant. 

VII. Error. You may know that your results and the 
consequent conclusions are in error. If so, state what you 
consider to be the correct results and conclusions, noting 
any irregularities or abnormalities which may have occurred 
to change the results. 

VIII. Practical Application. Apply the principles in- 
volved in the exercise to some practical purpose. 

XV 



xvi FORM FOR WRITING UP EXERCISES 

IX. References. Give the substance of the references 

placed at the end of each exercise in your own words 

and apply to the exercise in question. Do not copy 
verbatim. 

Note. In writing up the notebook, details under II and IV should 
be omitted, only the headings are necessary. 



PART I 

GENERAL MORPHOLOGICAL AND CULTURAL 
METHODS 



EXERCISE 1. CLEANING GLASSWARE 

Glassware for use in microbiological laboratory work 
should be not merely clean, but chemically clean. Test 
tubes, Petri dishes, flasks, etc., are the receptacles used in 
the microbiological laboratory for containing the different 
nutrient substances upon which microorganisms are to 
subsist. Very frequently free alkali may be present on 
new glassware in sufficient quantity to prevent microbial 
growths in the nutrients contained therein. Prescott and 
Winslow in testing out different glassware say that, " The 
more soluble glassware yielded sufficient alkali to the me- 
dium to inhibit four-fifths of the bacteria present in certain 
cases." 

Glassware which looks clean may have been used previ- 
ously and should be given a thorough cleaning to rid it of 
possible traces of mercuric chloride, or other chemical having 
germicidal properties. 

Follow directions carefully and clean all new and appar- 
ently clean glassware in the order given. 

Cleaning New or Apparently Clean Glassware. All new 
glassware should first be treated with chromic acid cleaning 
solution (see appendix for all formula) before proceeding 
with the directions for cleaning glassware. 

Return used cleaning solution to the glass receptacle 
provided for the purpose. Do not throw it away. This 

1 



2 GENERAL 1 MICROBIOLOGY 

solution may 'be Used Wtii dxhUzed, i.e., until dark green 
in color. 

Heat will facilitate the action of the cleaning solu- 
tion. 

Small amounts of organic matter adhering to glass- 
ware are oxidized by this solution, but will not dis- 
appear until removed by a suitable brush and cleaning 
powder. * 

New Petri dishes and test tubes may conveniently be 



(a) 




(d) 



o 




FIG. 1. (a) Pipette, (6) Smith's Fermentation Tube, (c) Erlenmeyer 
Flask, (d) Test Tube, (e) Roux Tube, (f) Petri Dish, (g) Roux 
Flask. 

placed in a large glass jar, covered with cleaning solution 
and allowed to stand over night. Heavy glass jars will 
not stand heating in steam. New flasks may be partially 
filled with cleaning solution and placed in steam for fifteen 
minutes. 

Test Tubes. New test tubes should be filled with 
cleaning solution, placed in a wire basket and heated for 

* Any inexpensive fine-grained cleaning powder as powdered pum- 
ice stone, Bon Ami, etc., may be used. 



CLEANING GLASSWARE 3 

at least fifteen minutes in the steam. After removing test 
tubes from the cleaning solution: 

1. Wash them in water with a test-tube brush, using 
cleaning powder if necessary. 

2. Rinse with tap water till clean and free from cleaning 
powder. 

3. Rinse with distilled water. 

4. Drain. 

5. Test tubes and other glassware, flasks, pipettes, etc., 
may be rinsed with alcohol to facilitate drying, then drained. 

Flasks. After treating flasks with cleaning solution: 

1. Wash them as clean as possible with tap water 
and a flask brush ; use cleaning powder if necessary. (When 
using cleaning powder, empty all water out of the flask, 
wet the flask brush with tap water, dip it in the cleaning 
powder and then rub the soiled portions vigorously.) 

2. Rinse with tap water till clear and free from cleaning 
powder. 

3. Rinse with distilled water. 

4. Drain. 

Petri Dishes. After removing Petri dishes from the 
cleaning solution: 

1. Wash them in water, using cleaning powder if nec- 
essary. 

2. Rinse with tap water. (It is not necessary to use 
alcohol or distilled water.) 

3. Wipe immediately with a clean physician's cloth. 
Pipettes. 1. Place pipettes delivery end down, in a 

glass cylinder (graduate) in cleaning solution and allow 
them to stand over night. (Steam may break the glass 
cylinder) . 

2. Pipettes which have been used should be washed 
immediately. Grease which cannot be removed with 
water should be treated with 10% NaOH and then with 
cleaning solution. 

3. Rinse with tap water, followed by distilled water. 



4 GENERAL MICROBIOLOGY 

4. Rinse with alcohol. (Alcohol may be used repeatedly.) 

5. Drain. 

Fermentation Tubes. 1. Rinse with tap water. 

2. Fill with cleaning solution and heat fifteen minutes 
in steam or allow to stand over night if more convenient. 

3. Wash thoroughly in tap water, using a test-tube 
brush if necessary. 

4. Rinse in distilled water and drain. 
Cover-glasses and Slides. 1. Immerse the cover-glasses 

or slides, one by one in a 10% solution of sodium hydrate 
(NaOH) for thirty minutes only. This strength of NaOH 
will etch the glassware if left longer. 

2. Wash separately in tap water, handling with ordinary 
forceps.* 

3. Put, one at a time, in cleaning solution, and leave 
over night as convenient. 

4. Wash separately in water. 

5. Immerse in clean alcohol (95%). 

6. Wipe with a clean physician's cloth. 

7. Store in clean Esmarch and deep culture dishes 
respectively, to keep free from dust. 

Other Glassware. Some modification of these methods 
will be adaptable to nearly all glassware. 

Note 1. Glassware containing liquefiable solid media is best 
cleaned by heating and pouring out the material while in liquid 
condition, then treating as above. (Solid media when liquefied by 
heat should never be thrown in the sink, as it will solidify when 
cold and clog up the traps and drains.) 

Note 2. Flasks, test tubes, Petri dishes, etc., containing 
cultures, must be heated one hour in flowing steam before cleaning. 

Cultures containing spores should be autoclaved previous to 
cleaning. 

Note 3. If cultures or media have become dry, add water 
before heating. 

Especial care must be used in cleaning glassware in which mer- 
curic chloride or any other disinfectant has been used. 

* Always handle cover-glasses and slides with forceps. 



STERILIZATION 5 

STERILIZATION 

Sterilization consists in the destruction of all forms of 
life. It may be effected by various agents. As applied 
to the practical requirements of the bacteriological lab- 
oratory many of those agents such as electricity, sunlight, 
etc., are of little value and are limited in their applications; 
others are so well suited to particular purposes that their 
use is almost entirely restricted to such applications. 

The Two General Methods of Sterilization are: 

A. Physical. 

1. Plasmolysis or Plasmoptysis. 

2. Desiccation. 

3. Heat (a) dry heat; (6) moist heat. 

4. Light. 

5. Filtration. 

6. Dialysis. 

7. Comminution. 

B. Chemical. 

1. Disinfectants, etc. 

A. PHYSICAL AGENTS 

I. Concentrated solutions destroy microorganisms by 
withdrawing water from their cells (plasmolysis) , e.g., in the 
preservation of food by concentrated salt or sugar solu- 
tions. 

Microorganisms accustomed to a concentrated nutrient 
substrate may suffer plasmoplysis (bursting of the cell) 
if placed in a less concentrated medium. 

In either case, if they are subjected gradually to the 
changing conditions, death is delayed or prevented. 

II. Desiccation is destructive to many microbes, espe- 
cially those which do not form spores. For example, Ps. 
radicicola is very sensitive to desiccation on the ordinary 
cover-glass or on cotton. 



6 GENERAL MICROBIOLOGY 

III. Sterilization by Dry Heat. 

1. Sterilization in a naked flame. 

2. Sterilization in an ether flame. 

3. Sterilization in a muffle furnace. 

4. Sterilization by hot air. 

1. Sterilization in a Naked Flame, (a) The simplest 
means of sterilizing a metal instrument is to heat it to red- 
ness in a flame. This method is always adopted for ster- 
ilizing platinum, copper, etc., wires and iron and nickel 
spatulas, forceps, etc. 

A platinum needle should always be carefully dried 
before sterilization, by holding it near the flame. This 
avoids sputtering, which scatters microorganisms, especially 
if moist material, e.g., fat or protein, on the needle is 
immediately thrust into the flame. 

(6) An instrument may be sterilized by flaming it, 
i.e., by passing it rapidly through a hot flame. This method 
is useful for instruments, etc., having polished surfaces 
devoid of creases in which microorganisms might escape 
destruction, e.g., knives, glass rods, handles of platinum 
needles, mouths of test tubes, flasks, pipettes, etc. 

(c) Deep wounds are sterilized by cautery with an 
instrument heated to a dull red heat. 

2. Sterilization in an Ether Flame. In an emergency, 
small instruments, needles, etc., may be sterilized by dip- 
ping them in ether or absolute alcohol and after removal 
lighting the adherent fluid and allowing it to burn off the 
surface of the instruments. Repeat the process. It may 
then be safely assumed that the apparatus so treated is 
sterile. 

3. Sterilization in a Muffle Furnace. Porcelain filter 
candles are sterilized by heating them to white heat in 
the muffle furnace. This method of sterilization cannot 
be applied to porcelain filters with metal fittings, such as 
Berkefeld filters. 

The destruction of autopsied animals and accumulated 



STERILIZATION 7 

wastes of the laboratory is also best accomplished in this 
manner. 

4. Sterilization by Hot Air. Exposure to hot air is the 
usual method of sterilizing all glassware, instruments with 
metal handles, etc., but it is not suitable for organic sub- 
stances, with the exception of wool, cotton and paper. 

To insure efficient sterilization, the prepared glassware, 




FIG. 2. Hot Air Sterilizer. 

etc., must be placed in a gas or electrically heated oven 
(containing a thermometer registering over 200 C.) whose 
temperature is maintained at approximately 150 C. for 
one hour, or 180 C. for ten minutes. The oven must be 
allowed to cool down to 60 C. before opening the door 
to avoid the breaking of glassware by cold-air currents. 
Cotton, wool, and paper are slightly scorched at this tem- 
perature, 



8 GENERAL MICROBIOLOGY 

Apparatus must be absolutely clean and dry before 
being sterilized. 

IV. Sterilization by Moist Heat. Sterilization by moist 
heat may be effected in one of four ways: 

1. By continuous or discontinuous heating at low 
temperatures (56-80 C.). 

2. By continuous or discontinuous heating in water 
at 100 C. 

3. By continuous or discontinuous heating in flowing 
steam at 100 C. 

4. By one heating in superheated steam (steam under 
pressure) at temperatures above 100 C., generally 115 C. 
(about 10 Ibs. pressure) or 120 C. (about 15 Ibs.). 

1. Sterilization by Continuous or Discontinuous Heating at 
Low Temperatures. Some substances used as culture media, 
being rich in volatile or otherwise chemically unstable 
substances, cannot be heated to 100 C. without a marked 
alteration (e.g., coagulation) and to some extent a destruc- 
tion of their properties; blood serum, for example. 

Pasteur showed that such media can be better ster- 
ilized by heating them at a low temperature (55-60 C.) 
for a long time than at a high temperature (70 C. or 
even 100 C.) for a short time. In this process, heat is 
not applied 'directly, as a rule. Control of the temperature 
is ordinarily accomplished by means of water heated to 
the degree desired. 

Prolonged heating at a low temperature constitutes 
pasteurization. In practice, however, it is found that in 
order to kill all organisms pasteurization must be com- 
bined with the method of discontinuous heating devised 
by Tyndall. Albuminous media subjected to the Tyndall 
method must be incubated finally at 37 C. for forty-eight 
hours to eliminate all specimens showing contamination. 

2. Sterilization by Continuous or Discontinuous Heating 
in Water at 100 C. (a) Continuous Heating. Water at 
100 C. destroys the vegetative forms of bacteria almost 



STERILIZATION 



9 



instantaneously, and spores in from five to fifteen minutes 
ordinarily, although many spores of resistant species are not 
killed by several hours' heating at 100 C. Water suspected 
of sewage contamination may thus be rendered safe for 
drinking purposes simply by boiling for a few minutes. 

This method is applicable to metal instruments, syringes, 
rubber stoppers, rubber and glass tubing, and other small 
apparatus. 

(b) Discontinuous Heating. (Tyndall method.) Tyndall 
observed that certain resistant 
forms found in an infusion made 
from hay were not destroyed by 
heating the infusion at 100 C., 
once, even when the temperature 
was sustained for a prolonged 
period, yet by boiling it for a 
short time on three successive 
days all living organisms were 
destroyed. His theory was that 
by heating at 100 C., the vege- 
tative forms but not the spores 
were killed. The latter germinate 
as the fluid cools and are killed 
during the second heating. A 
few spores, however, escape de- 
struction at the second heating; 

these will have germinated by the time the third heating is 
due. After the third heating sterilization is accomplished. 

The explanation now given, however, is that the resist- 
ance of microorganisms is gradually lowered under the 
influence of repeated heatings. This principle of heating 
on three successive days, a medium to be sterilized is now 
known as the Tyndall method of sterilization. In general 
laboratory practice, steam is used instead of water at 100 
C., but this necessitates special apparatus, whereas water 
lends itself readily to the means at hand. 




FIG. 3. Arnold Steam 
Sterilizer. 



10 GENERAL MICROBIOLOGY 

The physical nature of the medium, the extraordinary 
resistance of the spores of certain species of bacteria or 
both in combination, may require that this intermittent 
heating be carried on over a longer period of time, i.e., 
four, five, six, etc., days in succession for the same or a 
longer period each time, or that the period between inter- 
mittent heatings be lengthened from twenty-four hours 
to forty-eight hours. 

Tyndall's method is valuable in that media of delicate 
composition may be sterilized without producing undesirable 
changes, such as are often produced by the high tempera- 
ture of the autoclav. 

3. Sterilization in Flowing Steam at 100 C. Continuous 
or Discontinuous, (a) Continuous Heating. Simple boiling 
or exposure to steam at 100 C., even though the exposure 
be prolonged, is not a reliable method of sterilization. When 
microorganisms have been dried, their resistance to the 
effects of heat is much enhanced, and especially is this the 
case when they are mixed with substances of a colloidal 
nature. Certain resistant forms of protoplasm known as 
spores may not be destroyed by one heating to 100 C., 
even when the temperature has been maintained for sev- 
eral minutes. 

(6) Discontinuous Heating. General use for the ster- 
ilization of media. 

This principle of sterilization advanced by Tyndall 
finds its widest application in bacteriological work with 
the use of flowing steam. High-pressure steam may be 
utilized to good advantage if a central heating station is 
available. The Arnold sterilizer makes use of steam for 
the sterilization process and lends itself readily to both the 
continuous and discontinuous method. 

4. Sterilization by Superheated Steam (under pressure 
and therefore above 100 C.). Water, syringes, surgical 
dressings, bedding, india-rubber apparatus, filters, old 
cultivations, culture media, etc., not injured by high tern- 



STERILIZATION 



11 



peratures, may be more quickly sterilized by heating in 
steam under pressure. 

Exposure to steam at a temperature of 115 C. for twenty 
minutes is in most cases sufficient to insure sterilization, 





FIG. 4. Autoclav, Horizontal, 
for Steam or Gas. 



FIG. 5. Autoclav, Vertical, 
for Gas Only. 



but some media, potato for instance, require a temperature 
of 120 C. for ten to fifteen minutes. It is now realized 
that media subjected to this high temperature undergo 
hydrolytic changes which render them unsuitable for the 
cultivation of more delicate microorganisms. Sterilization 



12 GENERAL MICROBIOLOGY 

in the superheated steam is carried on in a special apparatus 
called an autoclav, which may be so constructed as to run 
by direct or indirect steam. The latter is the more desir- 
able for the sterilization of media. 

V. Sterilization by Light. Light seems to act by pro- 
ducing powerful chemical germicides, probably organic per- 
oxides, in the medium surrounding the bacteria. Certain 
rays of light, the blue, violet and ultraviolet in particular 
are destructive to living cells. It is to these rays that sun- 
light owes its disinfecting action. Practical use has been 
made of the ultraviolet rays in water sterilization by 
employing the Cooper-Hewitt mercury vapor lamp having 
a quartz instead of a glass tube, as these rays do not pass 
through glass. 

VI. Sterilization by Filtration. Sterilization may be 
effected by the filtration of gases or liquids through 
materials which will retain microorganisms. 

The best example of the filtration of gases is the use 
of cotton plugs in flasks and tubes containing microorgan- 
isms. The cotton is porous enough to allow the necessary 
interchange of gases but will allow neither dust nor foreign 
microorganisms to enter. The sterilization of air or other 
gases if fore 3d through cotton would depend upon the 
thickness of the cotton layer and also upon the force which 
was exerted. 

Certain fluids used in bacteriological work cannot be 
subjected even to a moderate amount of heat without pro- 
foundly altering their nature. In order to make such a 
fluid sterile, it is passed through a cylindrical vessel, closed 
at one end like a test tube, and made either of porous 
" biscuit" porcelain, hard burnt and unglazed (Chamber- 
land filter) or of kieselguhr, a fine diatomaceous earth 
(Berkefeld filter) and termed a bougie or a candle. 

The pores of the finer filters are so small that while 
liquids, and solids in solution pass through, microorganisms 
are retained and the liquid passes through in a germ-free 



STERILIZATION 13 

condition. Pasteur in his early work utilized plaster plates 
as the filtering medium, but as a result of Chamberland's 
researches, porous porcelain now supersedes plaster. Finely 
shredded asbestos packed tightly in a Gooch crucible will 
serve as a bacterial filter provided the layer of asbestos 
is sufficiently thick. The rate of filtration is usually very 
slow because the pores of the filter are so very minute; 
therefore to overcome this disadvantage either aspiration 
or pressure is generally employed to hasten the process. 
This method may not exclude filterable organisms. 

VII. Sterilization by Dialysis. In one of the more 
recent methods devised for the preparation of antirabic 
vaccines the vaccine is prepared by placing the virus (spinal 
cord of a rabid rabbit) in a collodion sac and dialyzing it 
in running distilled water. The living virus is destroyed, 
yet its immunizing properties are retained unimpaired. 
Quite the opposite effect may be obtained under some- 
what different circumstances. If a collodion sac containing 
a suspension of a pathogenic organism be placed in the 
body cavity of a susceptible animal the organisms within 
the sac thrive, being nourished by the body fluids which 
diffuse through the semi-permeable membrane. 

GUMMING, J. G.: Rabies Hydrophobia. A study of fixed virus, 
determination of the M. L. D., vaccine treatment (Hogyes, 
Pasteur, and dialyzed vaccine), and immunity tests. Journal 
of Infectious Diseases, Vol. XIV (1914), pp. 33-52. 

VIII. Comminution or the actual crushing of the micro- 
bial cells is resorted to for demonstrating intracellular 
enzymes. 



14 GENERAL MICROBIOLOGY 



B. CHEMICAL AGENTS 

I. Sterilization by Disinfectants. Sterilization by dis- 
infectants has but limited use in bacteriological work. 
The amount of disinfectant necessary to destroy existing 
organisms in a nutrient medium is greater than the amount 
necessary to inhibit multiplication of an organism which 
may subsequently be used as an inoculum; the medium 
is therefore rendered useless. 

1. Disinfectants may be used for any apparatus which 
will not come in direct contact with culture media or with 
the organisms under investigation. Fixed non-volatile dis- 
infectants must be employed, since the vapors given off by 
volatile compounds hinder the growth of organisms on 
culture media. 

2. Disinfectants are in general use for sterilizing the 
hands, woodwork, for washing out vessels and sterilizing 
instruments during inoculation and other experiments. 

As an example, 1-1000 mercuric chloride, 1.5% formalin, 
5% phenol, 2% compound solution of cresol, etc., are cheap 
and adaptable in many cases. Tincture of iodin is valuable 
for painting wounds. 

The common soaps, and more particularly green soap, 
have a plight germicidal value, and this in conjunction 
with their solvent action upon fats and protein, and the 
mechanical cleansing which accompanies their use, justifies 
assigning them an important place among the chemical 
disinfectants. 

Disinfectants used for sterilizing the skin before col- 
lecting pus, blood, etc., from the living subject must be 
carefully removed by washing the part well with alcohol 
before collecting material, otherwise the presence of the 
disinfectant would materially interfere with the subsequent 
growth of organisms in the culture. 

3. Disinfectants are also added to sterile filtrates which 
are no longer required as culture media. For this pur- 



GLASSWARE FOR STERILIZATION 15 

pose a small quantity of some disinfectant (such as thy- 
mol or camphor) which is without chemical action on the 
constituents of the fluid is selected. 

An amount of carbolic acid (0.5%) or other chemical 
is frequently added to vaccines, bac terms, serums, etc., for 
preservative purposes. 

4. Disinfectants are sometimes used to sterilize a culture 
when the products of the microorgansims are under inves- 
tigation. Chloroform, ether, toluol, oil of garlic or mustard, 
etc., which may be driven off afterward by evaporation, 
are among the most useful in this connection. 

II. Sterilization by Antiseptics. Chemical reagents such 
as belong to the class known as antiseptics, i.e., substances 
which inhibit the growth of, but do not destroy bacterial 
life, are obviously useless. 

REFERENCES 

EYRE: Bacteriological Technic. Second Edition (1913), pp. 26-48. 
BESSON: Practical Bacteriology, Microbiology, and Serum Therapy 

(1913), pp. 3-27. 

MARSHALL: Microbiology (1911), pp. 64-67. 
EULER: General Chemistry of the Enzymes (1912), pp. 118-123.. 

EXERCISE 2. PREPARATION OF GLASSWARE FOR 
STERILIZATION 

The mouths of test tubes, fermentation tubes, pipettes, 
etc., are ordinarily plugged with cotton before sterilization. 
For this purpose cotton is ideal as it is cheap and adaptable, 
serves to filter out microorganisms from the air, while allow- 
ing the ready diffusion of gases, and after once used it may- 
be burned. 

Paper (ordinary newspaper) may be used to wrap glass- 
ware as Petri dishes, deep-culture dishes, pipettes, etc., which 
one wishes to store in a sterile condition and for which 
cotton is not adaptable. 

Glassware is sterilized for the purpose of destroying 



16 GENERAL MICROBIOLOGY 

microorganisms present on its surface and in or on the cotton 
or paper used respectively for plugging or wrapping. After 
sterilization the cotton and paper serve to prevent micro- 
organisms from entering and contaminating the sterile 
utensils. 

Dry heat, though not as effective a germ destroyer as 
moist heat, is more adaptable to the sterilization of empty 
culture flasks, pipettes and other glassware. Hot-air steril- 
ization not only accomplishes the sterilization of the glass- 
ware, cotton plugs, etc., but " sets " the plugs so that they 
may be handled with greater facility. 

All glassware must be absolutely clean and dry or contain 
traces of alcohol only before preparing for sterilization; 
otherwise sterilization cannot be accomplished. If consider- 
able moisture is present in test tubes, flasks, etc., it will not 
evaporate during the hot-air sterilization process, and it is 
very evident that the temperature of such moist portions 
of the glassware will not reach or at least- will not exceed 
100 C. 

Directions. Test tubes and flasks are plugged with 
cotton. The ordinary forceps are used for this purpose. 
(A 'glass rod may also be used.) A small piece of cotton is 
grasped on the edge with the forceps and inserted in the 
mouth of the test tube. Plugs should project into test 
tubes from 3 to 4 cms., and from 3 to 5 cms. into the 
neck of flasks, according to the size of the flask. Only an 
amount of cotton should project out of the mouth that is 
sufficient to protect the outward turned portion (lip) of the 
test tubes or flasks from dust. A " Christmas-tree " effect 
is to be avoided. Plugs should not be so tight as to be 
removed with difficulty, nor so loose as to offer no resistance 
to removal. A little experience will suffice to demonstrate 
the amount of cotton to use and the firmness with which 
the plug should fit. 

Cotton plugs for test tubes, flasks, etc., may be rolled. 
This kind of plug is more stable and may be used several 



GLASSWARE FOR STERILIZATION 



17 



times. Have the instructor demonstrate the method of 
rolling. 

For hot-air sterilization, test tubes plugged with cotton 
may be tied in large bundles or placed in wire baskets 




FIG. 6. (a) Proper Plug: (6) Plug 
too Shallow and too Loose, too 
Much Projecting; (c) Plug too 
Loose, too Little Projecting. 



FIG. 7. Copper Cyl- 
inder for Sterilizing 
Pipettes. 



(never in agate cups), cotton plugs up. A few test tubes 
should not be placed in a large wire-basket or in a wire 
test-tube rack, as it is necessary to economize space in the 
hot-air sterilizer. 

Petri dishes are wrapped separately in paper and tied 
together in sets of three, One sheet of newspaper makes 



18 GENERAL MICROBIOLOGY 

four papers of proper size for wrapping Petri dishes and is 
inexpensive. 

Three or more Petri dishes may be wrapped together 
if all are to be used at the same time. Mark each plainly 
with the desk number. 

Pipettes. Place a piece of cotton in the bottom of a test 
tube, plug the top only of the pipette with cotton (not too 
tightly}, leaving but little of the cotton projecting out. 
Wrap a small portion of cotton around the lower third of 
the pipette, insert the pipette into the test tube until the 
tip rests on the cotton, making the cotton wrapping serve 
as a plug for the tube. 

Wrap pipettes so prepared in paper (one layer) and tie 
and mark them plainly with the desk number. 

A covered metal case is often used for holding pipettes 
to be sterilized. The upper end of the pipettes are plugged 
with cotton, the pipette inserted in the case, the open end 
of the case plugged with cotton, and the cover replaced. 
(This latter method is not recommended for the new student, 
as the necessity of careful technic in removing a sterile 
pipette from the case without contaminating those remaining 
is difficult to impress upon him) . 

Fermentation tubes are plugged with cotton as directed 
for test tubes; the cotton plug should not project into the 
bulb. 

Deep culture dishes are wrapped singly in paper as 
directed for Petri dishes. 

Slides and cover-glasses are generally sterilized by 
flaming, but only as needed. 

NUTRIENT MEDIA 

" Chemically, like all other living cells, microorganisms 
consist of organic and inorganic nitrogen and mineral salts; 
it is therefore necessary in order to grow a microorganism, 
that these three classes of substances be made available, 
together with oxygen, which is an essential to the life of all 



NUTRIENT MEDIA 19 

living structures. Finally a certain amount of moisture 
is absolutely necessary." (Besson.) 

A food prepared for the growth of microorganisms is 
given the general term nutrient medium. A large number of 
microorganisms will grow readily in or upon easily available 
nutrient media, as milk, bouillon, etc. Some microorgan- 
isms have widely differing food requirements and need for 
growth nutrient media differing widely in their composition. 

However, there are a few general rules that must be 
applied in the preparation of all nutrient media for the use 
of microorganisms. These are briefly, that: Every culture 
medium must 1. Contain substances necessary for giowth. 
2. Be of suitable reaction. 3. Be contained in vessels 
which afford protection from contamination from without. 

Classification of Nutrient Media. Culture media may 
be classified as: 

I. Natural Media as occurring in nature, e.g., milk, 
potato and other vegetables, meat and meat products, blood 
and blood serum, egg, soil, etc. 

II. Prepared media, i.e., made in the laboratory. These 
are: 

(a) Of unknown chemical composition; e.g., nutrient 
agar, gelatin, etc. 

(6) Synthetic; i.e., chemical composition known, e.g., 
Giltay solution for denitrifying organisms. 

Or as: 

I. Liquid Media. These include: 

A. Media made from animal tissue and fluids, e.g., 
nutrient broth, serum broth, carbohydrate broths, milk, 
blood, nitrate peptone solution, Dunham's solution. 

B. Media made from vegetable tissue. Among these are: 
Malt extract (germinated barley), beer wort, yeast extract, 
hay infusion, natural fruit juices, wines (fermented fruit 
juices). 

C. Synthetic media. 

II. Solid Media. These mav be classified as: 



20 GENEKAL MICROBIOLOGY 

A. Liquefiable, e.g., nutrient agar, nutrient gelatin. 

B. Non-liquefiable, including: 1. Media liquid in a 
natural state but which, once solidified, cannot be liquefied 
by physical means, e.g., media prepared from albuminous 
fluids and tissues such as egg, blood serum, etc., or synthetic 
media solidified with sodium silicate. 

2. Media which are solid in the natural state, e.g., 
vegetable media such as potato, carrot, banana, etc. 

EXERCISE 3. TITRATION OF MEDIA 

The titration of bacteriological media made from meat 
is an important step in their preparation, as microorganisms 
are sensitive to the reaction of the nutrient substrate. 

Procedure. The following method is used for laboratory 
media, with the exception of milk, wort, cider, vinegar, fruit 
juices, etc. See p. 22. 

1. Put 5 c.c. of the medium to be tested and 45 c.c. of 
distilled water in an evaporating dish. 

2. Boil briskly one minute with constant stirring (to 
drive off all dissolved CO2 which registers as acidity). 

$. Add 1 c.c. phenolphthalein solution for indicator. 

4. Titrate while hot, preferably while boiling, with N/20 
sodium hydroxide, or N/20 hydrochloric acid as the case de- 
mands. ' A faint but distinct permanent rose color marks the 
end point. This color should remain permanent for five minutes. 

5. Compute and record the reaction of the medium in 
degrees of Fuller's scale, which is the number of cubic centi- 
meters of normal* acid or alkali present in 1000 cubic 

* A solution is said to be normal when it contains 1 gram equiv- 
alent of an acid or base in 1 liter. 

A gram equivalent of an acid or a base is that quantity which is 
equivalent to or will neutralize 1 gram molecule of a mono-basic acid 
or of a mon-acid base. 

The advantage of the system is that 1 c.c. of any normal solution 
will exactly neutralize or be exactly equivalent to 1 milligram equiva- 
lent of any acid or base. (Noyes, Wm. A., Textbook of Chemistry, 
1913, p. 184.) 



TITRATION OF MEDIA 21 

centimeters of the medium, using phenolphthalein as indi- 
cator. 

6. Alkaline media are denoted by placing a minus ( ) 
sign before the number of degrees of alkalinity; thus, 15 
would indicate that the medium was 15 alkaline, or that 
15 c.c. normal acid must be added per liter to neutral- 
ize it. 

Acid media are denoted by placing a plus. (+) sign 
before the number of degrees of acidity; thus, +15 
would indicate that the medium was 15 acid or that 
15 c.c. of normal alkali must be added per liter to neu- 
tralize it. 

Example. 

Burette reading after titrating 5.4 c.c. 

Burette reading before titrating 2.0 c.c. 

Number of c.c. N/20 NaOH required 

to neutralize the acid in 5 c.c. of 

the medium .4 c.c. 

If 5 c.c. of the medium (which is 1/20 of 100 c.c.) require 
3.4 c.c. of 1/20 normal NaOH to neutralize the acid pres- 
ent, 100 c.c. of the medium would require 20X3.4 c.c. or 
68 c.c. of 1/20 normal NaOH. 

As a normal solution is 20 times the strength of a 1/20 
normal solution, 100 c.c. of the medium would require 1/20 
of 68 c.c. or 3.4 c.c. of normal NaOH for neutralization; and 
one liter or 1000 c.c. of medium would require 10X3.4 
c.c. or 34 c.c. N/l NaOH for neutralization; i.e., the 
medium is 34 acid. Fuller's scale. This is the litre of the 
medium. 

When N/20 acid or alkali and a 5 c.c. portion of medium 
(in 45 c.c. of distilled water) are used, each 1/10 of 1 c.c. 
corresponds to 1 Fuller's scale. 

Adjustment of Reaction. If it is desired to leave the 
medium with a, e.g., +15 reaction, we have: 



22 GENERAL MICROBIOLOGY 

Acidity of the medium 

(+34) 3.4 c.c. per 100 c.c. of the medium 

Desired acidity (+15) . . 1.5 c.c. per 100 c.c. of the medium 
Amount of normal alkali - 

to be added 1.9 c.c. per 100 c.c. of the medium 

or 10X1.9 c.c. = 19 c.c. N/l NaOH per 1000 c.c. of medium 

Since normal solutions are of equal strength by volume, 
that is, 1 c.c. of N/l acid will just neutralize 1 c.c. of N/l 
alkali, it will readily be seen that if 15 c.c. N/l NaOH are 
required to neutralize the acid present in 1 liter of medium, 
then there must be present in that liter exactly 15 c.c. of 
N/l acid, or we should say the reaction is ( + 15) fifteen 
degrees acid. For any other degree of acidity add enough 
normal alkali to reduce the acidity to the point desired. 

The reaction of a medium changes somewhat after its 
neutralization, especially during sterilization, but also upon 
standing afterward at ordinary temperature. - This change 
is toward an increased acidity, and is most marked in 
media rich in dextrose. Consequently it is necessary to 
determine the titre of a medium at the time it is used rather 
than to quote figures obtained before sterilization. 

MILK, CIDER, VINEGAR, WORT, AND FRUIT JUICES 

Procedure. 1. Into an evaporating dish measure 5 c.c. 
of the medium to be tested, by means of suitable pipette. 
Make up to 50 c.c. with distilled water. 

Do not heat. The above media should not be heated 
before titration, as they contain volatile acids or other organic 
substances which may register as acid and which may be 
driven off by boiling, 

2. Add 1 c.c. phenolphthalein solution. 

3. Add, gradually, from an accurate burette, IN/20 
NaOH until the first permanent pink appears. 

4. Note the amount of NaOH required for the titration. 

5. Always run duplicates. 



MILK 23 

6. Record as degrees of acidity the number of c.c. of 
N/l NaOH which would be required to neutralize one liter 
of medium. 

MILK 

Milk is valuable as a nutrient medium for microorganisms 
because: It is a natural nutriment and almost ideal for a 
large number of microorganisms. Its composition, averag- 
ing 3.40% fat, 3.50% casein and albumen, 4.50% milk 
sugar, 0.75% ash, 87.75% water, is an evidence that it 
furnishes food in an excellent form for most microorganisms. 

The biochemical activities of many bacteria reveal them- 
selves definitely in the changes which milk, especially litmus 
milk, undergoes. Many of these changes are seen macro- 
scopically. Some of these are: 

(a) Acid Production. The lactose, C^IfeOn (milk 
sugar), is first inverted, forming two hexose molecules, 1 mol. 
dextrose and 1 mol. galactose. 



And each molecule of hexose yields two molecules of 
lactic acid: 

hexose = lactic acid. 

C 6 H 12 6 = 2CH 3 CH(OH)COOH. 

The blue litmus is turned red. 

(b) AlKali Production. Litmus becomes darker blue. 
This change very often accompanies peptonization. 

(c) Reduction (Decolorization of Litmus). This is due 
to the reduction of the coloring matter (litmus). Many 
microorganisms secrete enzymes which produce hydrogen. 
The hydrogen combines with the litmus, reducing it to its 
leuco-compound (colorless) . (Methylen blue becomes color- 
less under like conditions.) That this is a reduction and 
not a destruction may be demonstrated by shaking the 
decolorized culture with a few cubic centimeters of hydro- 
gen peroxid. The bacteria which decolorize the litmus also 



24 GENERAL MICROBIOLOGY 

reduce the hydrogen peroxid to E^O and nascent oxygen 
which reoxidizes the reduced litmus (showing by the reac- 
tion of the milk the type of microorganisms present). Re- 
oxidation takes place slowly under natural conditions. 
Reduction may take place when milk is acid, alkaline or 
neutral. 

(d) Curdling through Acid Production. The casein, like 
most proteins, is amphoteric, i.e., it is capable of reacting both 
as a weak acid and a weak base. The otherwise insoluble 
casein is found to be in the milk in a partially dissolved 
state (colloidal), due to its combination with the calcium 
salts: the calcium that was formerly combined with the 
casein, through the production of acid by certain micro- 
organisms, now combines with the lactic acid; as a result 
the casein precipitates, causing curdling (coagulation). Lit- 
mus is turned decidedly red. Milk having an acid curd 
will titrate above +50. 

(e) Rennet Curd. Coagulation may also take place when 
the medium is neutral or only slightly^ acid. This pro- 
duction of curd is due to a rennet-like enzyme produced by 
microorganisms, and is similar to the action of the rennet 
used to curdle milk in cheese factories. 

Many spore-forming species are found under the group 
of rennet-producing organisms. Rennet curd is usually 
followed by peptonization. 

(/) Peptonization. The curd produced by acid or ren- 
net-forming microorganisms may gradually disappear, leav- 
ing only a whey-like liquid. This is caused by certain 
bacteria which produce proteolytic enzymes that digest the 
curd and render it soluble. This liquefaction of solid pro- 
teins like gelatin, fibrin, boiled egg white, milk curd, etc., is 
due to two groups of enzymes, pepsin and trypsin. 

The pepsin of the animal body acts only in an acid 
medium (present in the stomach). 

The trypsin of the animal body acts only in alkaline 
medium (present in the intestine). 



PREPARATION OF LITMUS MILK 25 

The pepsin- and trypsin-like enzymes produced by micro- 
organisms cannot be thus separated by their activity in a 
medium of certain reaction; this varies with the species of 
microorganism and with environmental conditions. Pep- 
tonization of milk usually takes place in a neutral, slightly 
alkaline, or more infrequently slightly acid reaction. 

Some organisms peptonize milk without forming a 
rennet curd. 

(g) Gas Production. This is characterized by the for- 
mation of gas bubbles in the milk, and is generally accom- 
panied by the formation of acid curd. Very commonly the 
curd shrinks, causing extrusion of whey. 

EXERCISE 4. PREPARATION OF LITMUS MILK 

Apparatus. Fresh separated or skimmed milk; titra- 
tion apparatus; N/20 NaOH; phenolphthalein (indicator); 
5 c.c. pipette; azolitmin, 2% solution; filling funnel; 
pinch cock; sterile test tubes; apparatus for steam sterili- 
zation. 

Method. 1. Fresh separated or skimmed milk should 
be used. Whole milk is undesirable on account of its fat 
content. 

2. Titrate and record the reaction of the milk. If the 
milk titrates above 17 acid, the reaction must be adjusted 
to +15. Sour, curdled or uncurdled milk, after neutrali- 
zation, does not make a desirable nutrient medium for 
microorganisms, therefore, milk whose titre is above 20-25 
acid should be discarded. 

Fresh milk varies in acidity from 12 to 18. Milk 
with an acidity above 18 to phenolphthalein will not give 
a satisfactory blue color with azolitmin, as at 18 it begins 
to show the acid coloration. 

3. Add 2% of a standard solution of Kahlbaum's azo- 
litmin. Litmus or azolitmin is added merely as an indi- 
cator and should be of sufficient strength so as not to dilute 
the milk to any extent. 



26 GENERAL MICROBIOLOGY 

4. Mix the milk and the azolitmin thoroughly and tube, 
using approximately 8 c.c. of the litmus milk in each tube. 

Note. Care should be taken to prevent the milk from coming in 
contact with the top of the tubes, as it will cause the cotton fibers to 
adhere to the tube. This may be avoided by the use of a " filling 
funnel." 

5, Sterilize by heating in flowing steam for twenty 
minutes on four successive days. Milk is difficult to sterilize, 
owing to the resistant spores which are frequently present. 
If it is desired to sterilize a larger bulk than in tubes, the 
time of heating should be lengthened. 

Caution: Overheating tends to change (caramelize) the milk 
sugar. The color of the azolitmin may also be destroyed. These 
changes are not desirable. 

EXERCISE 5. PREPARATION OF GLYCERIN POTATO 

A number of chromogenic and pathogenic organisms 
thrive especially well on media containing glycerin. The 
manner in which glycerin favors the growth of these organ- 
isms is not known, but in some instances it seems to be 
directly utilized for the construction of fat (Bact. tubercu- 
losis) . 

Apparatus. Large healthy potatoes; cylindrical potato 
knife, or cork borer; ordinary knife; tumbler; sodium car- 
bonate, 1 : 1000 solution; glycerin, 5% solution; large 
sterile test tubes, or Roux potato tubes; absorbent cotton or 
short glass rod; 1 c.c. pipette; distilled water; apparatus 
for steam sterilization. 

Method. 1. Carefully clean one or two large potatoes. 

2. By means of a cylindrical potato knife or cork borer, 
cut cylinders of potato, 4 to 6 cm. long and 1.5 to 1.8 cm. 
in diameter. With an ordinary knife, halve each cylinder 
by a diagonal cut so that each piece resembles in shape an 
agar slant. Remove any portions of the skin on these pieces. 

3. Place in a tumbler and soak in a dilute (1 : 1000) 
solution of sodium carbonate* for twenty-four hours only. 

* Sodium carbonate is used to neutralize the natural acids of the potato. 



PREPARATION OF GLYCERIN POTATO 



27 



4. Transfer the pieces to a 5% solution of glycerin in 
water for a further twenty-four hours only. 

5. Place in sterile tubes 
prepared as follows: Select 
extra large test tubes 1.5 
to 2 cm. in diameter and 
clean and dry them. Place 
a small piece of absorbent 
cotton or glass rod 0.5 cm. 
X2.5 cm. in the bottom of 
each. Plug with cotton 
and sterilize in the usual 
way. (Roux tubes need 
only to be cleaned and 
sterilized.) 

Just before introducing 
the pieces of potato, add 
about 1 c.c. of distilled water 
to each tube, using a pi- 
pette. The potato should 
not touch the water. 

6. Sterilize by heating 
at 100 G. on four successive 



days for twenty minutes 
each day. 



FIG. 8. Potato Tubes. (Orig. 
Northrup.) 



Caution: The time stated in 3 and 4 must be strictly adhered to, 
else the potatoes will have to be discarded on account of contamina- 
tion with resistant spore-forming organisms. 



REFERENCE 

SMIRNOW, M. R.: The value of glycerinated potato as a culture 
medium. Cent. f. Bakt., II Abt., Bd. 41, p. 303. 



28 GENERAL MICROBIOLOGY 

EXERCISE 6. PREPARATION OF MEAT INFUSION 

Meat infusion is the foundation of the ordinary nutrient 
media, as broth, gelatin and agar, and also of a large number 
of special nutrient media, as sugar broths, etc. 

Under these directions sufficient meat infusion is pre- 
pared to make 1 liter each of nutrient broth, gelatin and 
agar. 

Apparatus. 1.5 kilograms (3 Ibs.) finely chopped fresh 
lean beef; 1500 c.c. tap water; 3.5 liter agateware pail; 
large funnel; ring stand; clean cloth; 1 liter measuring 
cup; three sterile 1 liter Erlenmeyer flasks; refrigerator; 
apparatus for steam sterilization (autoclav preferable). 

Method. 1. To 1.5 kilograms of finely chopped, fresh 
lean beef in a 3.5 liter agateware pail, add 1500 c.c. of tap 
water,* mix thoroughly and allow to stand in a cool place 
(refrigerator preferred) for sixteen to twenty-four hours only. 

2. Set up a large funnel in a ring stand and place a piece 
of clean cloth in the funnel. Place a measuring cup under 
the funnel. 

3. Strain the meat infusion through clean cheesecloth, 
thoroughly pressing out all the juice. 1.5 liters should be 
recovered. If any loss occurs make up to 1500 c.c., using 
tap water. 

This resulting sanguineous fluid contains the soluble 
albumins of the meat, the soluble salts, extractives and coloring 
matter, chiefly hemoglobin. 

4. Place 500 c.c. of meat infusion in each of three sterile 
1 liter Erlenmeyer flasks. Replace the plugs, and heat in 
the autoclav at 120 C. for thirty minutes. This is a safer 
procedure than heating for a longer time in flowing steam. 

During this heating the albumins coagulable by heat are 
precipitated. 

It has been found necessary and also more convenient 
to prepare and sterilize meat infusion before proceeding with 
the preparation of the different media in which it is used, 
* Approximately 500 c.c. of water to each pound of meat. 



PREPARATION OF NUTRIENT BROTH 29 

on account of the resistant spore-forming organisms which 
are almost universally present in the chopped meat; economy 
of time also is a consideration. Unless sterilized immedi- 
ately, meat infusion decomposes quickly owing to the 
abundance and diversity of the microflora acquired during 
the various processes of preparation for market. 

Infusion made from freshly chopped lean beef will vary 
in acidity between +15 and +25 Fuller's scale. If the 
reaction is markedly lower or higher, microbial action is 
taking place, which is, or may be, injurious to the food 
value of the medium in which the meat infusion is 
used. 

The infusion contains very little albuminous matter and 
consists chiefly of the soluble salts of the muscle, certain 
extractives, and altered coloring matters along with slight 
traces of protein not coagulated by heat. 

EXERCISE 7. PREPARATION OF NUTRIENT BROTH 

Nutrient broth is the standard liquid employed for cul- 
tivating microorganisms. It is practically a beef tea con- 
taining peptone. Peptone, a soluble protein not coagulable 
by heat, is added to replace the coagulated albuminous 
substances which precipitate when the meat infusion is 
sterilized. Salt is added to take the place of the phosphates 
and carbonates, some of which are precipitated on adjusting 
the acidity of the medium by sodium hydroxide. 

The reaction of ordinary nutrient media is adjusted to 
about +15 with phenolphthalein as indicator, as it is found 
that most microorganisms grow best on a medium neutral 
or slightly alkaline to litmus. 

When it is required that nutrient media be clear, egg 
albumen reduced to a smooth paste with water (or the well- 
beaten white of an egg) is added. By coagulation, the egg 
albumen removes mechanically all small particles in suspen- 
sion which otherwise would pass through the filter paper. 



30 GENERAL MICROBIOLOGY 

This process is most efficient when the egg albumen coagu- 
lates slowly. 

As egg albumen begins to coagulate at about 57 C. it is 
absolutely imperative for good results that the medium be 
cooled to 40-50 C. before the addition of egg albumen. 

Although egg albumen contains small amounts of sol- 
uble matter not coagulable by heat, as sugar, extractives 
and mineral matter, all of which will serve as microbial 
food, its purpose in nutrient media is primarily for its clari- 
fying action. 

Apparatus. 500 c.c. sterile meat infusion; 500 c.c. tap 
water; 10 gms. peptone, Witte's; 5 gms. salt; 10 gms. egg 
albumen (or one egg); 3.5 liter agate-ware pail; titration 
apparatus; N/20 NaOH; N/l NaOH; phenolphthalein 
(indicator); distilled water; 5 c.c. pipette; large stirring 
rod; coarse balances; large gas burner; large funnel; 
plaited filter paper; filling funnel; sterile test tubes; sterile 
1 liter flask; apparatus for steam sterilization-. 

Method. 1. Put the contents of a flask of meat infusion 
(500 c.c.) in an agate pail and add 500 c.c. of tap water. 

2. Add 1% of Witte's peptone and 0.5% of salt. 

3. Add 10 gms. of egg albumen which has been well 
mixed with 100 c.c. of tap water. (Put the egg albumen 
in a tumbler and add enough water to form a paste. Stir 
until smooth. Then add the remaining water. One egg* 
well beaten may be substituted.) Mix all thoroughly. 

4. Heat in flowing steam for forty-five minutes or in the 
autoclav at 120 C. for thirty minutes. 

5. Titrate with N/20 NaOH. 

6. Adjust the reaction of the medium to +15 with 
normal NaOH or normal HC1. Retitrate and adjust again 
if necessary. 

7. Counterpoise and note the weight. 

8. Boil fifteen minutes over a free flame, stirring con- 
stantly. 

* It is not necessary to add water to the egg. 



GELATIN 31 

9. Counterpoise and restore any loss by evaporation 
with distilled water. 

10. Filter while boiling hot through plaited filter paper 
just previously washed with 1/2 liter of boiling water. 

11. Pass the filtrate through the same paper till it is 
bright and clear. 

12. Fill thirty sterile test tubes, using approximately 
8 c.c. of this medium for each tube. Put the remaining 
broth in a large, sterile flask. 

13. Heat the test tubes and contents in flowing steam 
twenty minutes on three successive days. 

14. To sterilize a large flask of broth, heat for twenty 
minutes four days in succession. 

GELATIN 

Gelatin is one of the tools of the microbiologist. As 
such, it serves two purposes: as a solid culture medium, a 
technical device by which the isolation of a single species 
of microorganism is made possible, and, to those organisms 
which secrete proteolytic enzymes, it serves as a nitrogenous 
food material. 

Gelatin bears the distinction of being the first substance 
used for a solid culture medium. This medium was origi- 
nated in 1882 by Robert Koch and has since revolutionized 
the science of microbiology. Prior to the introduction of 
solid media, the isolation of a single species of microorganism 
involved much difficulty and almost always a certain measure 
of uncertainty. To quote from Jordan: " It cannot be a 
mere coincidence that the great discoveries in bacteriology 
followed fast on the heels of this important technical 
improvement, and it is perhaps not too much to claim that 
the rise of bacteriology from a congeries of incomplete 
although important observations into the position of a 
modern biologic science should be dated from about this 
period (1882)." 

Koch's first plates were made by pouring the liquefied 



32 GENERAL MICROBIOLOGY 

nutrient gelatin upon sterile, flat pieces of glass. The 
student on becoming familiar with the difficulties of pre- 
paring satisfactory plates with the use of the " Petri dish " 
will appreciate those met with in Koch's first gelatin plates. 

Gelatin is a protein, i.e., a nitrogenous food material. 
It contains as its essential elements carbon, hydrogen, 
oxygen, and nitrogen (other elements, however, such as 
sulphur, phosphorus, etc., may be present). Its empirical 
formula according to Schiitzenberger and Bourgeois is 
C7eHi24N24O29, but such a formula only gives information 
of the chief constituents and allows one to form some idea 
of the huge size of the molecule; no idea of the structure 
of the molecule is given. However, by digesting with 
dilute sulphuric acid, gelatin breaks down in the same way 
as the proteins, yielding glycin, leucin and other fatty 
amino-acids. 

Gelatin is an animal protein, but does hot occur as 
gelatin in the animal tissues. It exists there as the albu- 
minoid collagen which is the principal solid constituent of 
fibrous connective tissue, being found also, but in smaller 
percentage, in cartilage, bone and ligament. Collagen from 
these various sources is not identical in composition and 
gelatin varies correspondingly, e.g., gelatin from cartilage 
differs from that of other sources in that it contains a lower 
percentage of nitrogen. 

Gelatin, the body resulting from the hydrolysis of 
collagen, is also an albuminoid. (Hofmeister regards this 
hydrolysis as proceeding according to the equation : 



collagen + water = gelatin 

but in dealing with substances of such variable composition, 

empirical formulae of this kind have no great significance). 

Commercially, it is prepared from certain kinds of bones 

and parts of skin. These are selected, washed and extracted 



GELATIN 33 

by water and with a dilute acid (hydrochloric), with rela- 
tively little exposure to heat, so that as few as possible of 
the fluid disintegration products of the stock are formed and 
the jellying power of the resultant solution is not destroyed. 

The term gelatin is derived from the Latin verb gelare, 
to congeal, and calls to mind the principal attribute of this 
substance, that of its stiffening or jellying property. 

Gelatin belongs to that interesting class of substances 
called colloids. It is a typical example of the class, and 
exhibits the characteristic properties of the class. Colloids, 
in marked contrast to crystalloids, do not crystallize, do 
not readily diffuse and are impermeable to each other. 
The ultimate particles of colloids are much smaller than 
what we would ordinarily term a physical subdivision, but 
rather larger than chemical molecules; the diameter of the 
smallest particles in a colloidal solution, e.g., red colloidal 
gold, which have been counted by means of the ultra-micro- 
scope, is 6 millimicrons or 6 thousandths of a micron. A 
micron is one thousandth of a millimeter. (Bacteria are 
much larger, the smallest visible by means of the ordinary 
microscope being from 0.3 to 1.0 micron in diameter.) 
Consequently their reactions stand midway between the 
physical and the chemical changes of matter, as may be 
seen by considering the properties of gelatin. 

Gelatin will absorb a considerable quantity of warm 
water (it is almost insoluble in cold water) and swells up, 
yielding a jelly which, upon application of heat, melts to 
a viscous, sticky solution that gelatinizes again upon cooling. 
The name of hydrogel is applied to colloids showing this 
property. Ordinary gelatin media for microbiological work 
contain 12% to 15% gelatin. When dried at medium tem- 
peratures, gelatin can again be redissolved and redried in- 
definitely. From this property it is called a reversible 
colloid to distinguish it from other colloids which, when 
their physical state is once changed, are insoluble, e.g., 
casein and silicic acid. 



34 GENERAL MICROBIOLOGY 

If superdried at about 130 C., or superheated when in 
the gelatinous state either for a short time at a temperature 
above 100 C., or for a long time at 100 C., as in inter- 
mittent sterilization, the gelatin is so modified that its 
redissolving or resolidifying power respectively is lost. In 
superdrying, the loss of the redissolving property is laid to 
the too close contact of the constituent particles, a change 
in the physical state; in the superheated gelatin, the loss of 
the resolidifying power is probably due to the disintegra- 
tion of the gelatin molecule, a more purely chemical 
phenomenon. This loss of the gelatinizing property is also 
caused by the enzymic activities of many microorganisms 
and is also a disintegration process. 

Gelatin possesses a liquefaction point which, however, 
varies considerably under different conditions. Ordinarily, 
media containing 12% to 15% gelatin will liquefy or melt 
at a temperature in the vicinity of 24 to 26 C., solidify- 
ing again at 8 to 10 C. to a clear, transparent jelly. As a 
consequence, gelatin media may be employed only for 
organisms which do not require a higher temperature than 
22 to 24 C. for development. Overheating in the process 
of preparation or sterilization will cause a considerable 
lowering of the liquefaction point, perhaps ultimately so 
low that the medium will be liquid at room temperature 
(20 to 21 C.) It will readily be seen how the latter 
gelatin medium could not handily be used for the isola- 
tion of organisms. A few data will assist in fixing this in 
mind. 

The solidifying property of gelatin varies in inverse 
proportion with the time of heating during the process of 
sterilization; its liquefying point is lowered on an average of 
2 C. for each hour of heating at 100 C. This makes clear 
why such care must be taken in the preparation of a gelatin 
medium, in the fractional sterilization of this medium in 
streaming steam, and why immediate cooling is necessary 
aftei; each fractionation in the process of its preparation. 



GELATIN 35 

Although temperatures above 100 C. are much more 
destructive to the solidifying property than that of 100 C., 
it is possible to sterilize a medium containing 12% to 15% 
of gelatin in the autoclav (7 to 8 Ibs. pressure) at 112 to 
113 C. for twenty minutes or at 15 Ibs. pressure (120 C. 
for five minutes) without impairing its usefulness as a solid 
culture medium. 

This use of steam under pressure (dry steam) is almost 
necessary in the case of a gelatin medium to effect sterili- 
zation, since gelatin, from its source, method of preparation, 
and later liabilities to contamination, is almost certain to 
contain or bear upon its surface a large number of very 
resistant spores. Heating at 100 C. for thirty minutes 
on three or even four or five consecutive days is not always 
efficient, as these spores do not always germinate within 
twenty-four hours after heating and, referring to the data 
above, it is readily seen that the lowering of the lique- 
faction point is not to be considered as negligible in the 
process of intermittent sterilization. 

Gelatin possesses another property which renders it 
valuable for bacteriological work: i.e., in gelatin plate 
cultures no water of condensation ordinarily collects on the 
cover of the Petri dish (as with agar) later to drop on the 
surface of the gelatin and thus obliterate forms of colonies 
and cause isolated colonies to become contaminated with 
neighboring ones. The storing of this medium either 
in test tubes or in plates, sterile or inoculated, is thus 
rendered much more simple than with agar. 

REFERENCE 

VAN DERHEIDE, C.C.: Gelatinose Losungen und Verflussigungspunkt 
der Nahrgelatine, Arch. f. Hyg., Bd. 30, 1897, pp. 82-115. 



36 GENERAL MICROBIOLOGY 



EXERCISE 8. PREPARATION OF NUTRIENT GELATIN 

Apparatus. 500 c.c. sterile meat infusion; 500 c.c. tap 
water; 150 gms. gelatin; 10 gms. peptone, Witte's; 5 
gms. salt; 10 gms. egg albumen (or one egg); water bath; 
thermometer; 3.5 liter agate ware pail; long heavy stirring- 
rod; titration apparatus; N/20 NaOH; N/l NaOH; 
phenolphthalein (indicator); distilled water; coarse bal- 
ances; large gas burner; large funnel; plaited filter paper; 
filling funnel; sterile test tubes; sterile 500 c.c. Erlenmeyer 
flasks; apparatus for steam sterilization; running-water 
bath or refrigerator. 

Method. 1. Put the contents of a flask of meat infusion 
(500 c.c.) in an agate pail and add 500 c.c. of tap water. 

2. Add 15% gelatin, 1% Witte's peptone, and 0.5% 
salt to the mixture. 

3. Heat this mixture in a water bath to dissolve the 
gelatin, peptone and salt, stirring occasionally. 

4. Cool to 40-50 C. This is imperative. 

5. Then add 10 gms. of egg albumen which has been well 
mixed with 100 c.c. of tap water. (Put the egg albumen 
in a tumbler, add enough water to form a paste and stir 
until smooth; then add the remaining water. One egg 
well beaten may be substituted.) Mix all thoroughly. 

6. Heat in flowing steam for forty-five minutes or in the 
autoclav at 105 C. for thirty minutes. 

7. Titrate with N/20 NaOH. 

8. Adjust the reaction of the medium to +15 with 
normal NaOH or normal HC1. Re titrate and adjust again 
if necessary. 

9. Counterpoise and note the weight. 

10. Boil fifteen minutes over the free flame, stirring con- 
stantly. 

11. Counterpoise and restore any loss by evaporation 
with distilled water. 

12. Filter while boiling hot through plaited filter paper 



AGAR 37 

just previously washed with 1/2 liter boiling water. Pass 
the filtrate through the same paper until it is bright and 
clear. 

13. Fill thirty sterile test tubes, using approximately 
8 c.c. of medium for each tube. Divide the remainder into 
two equal portions and place in sterile 1/2 liter Erlenmeyer 
flasks. 

14. Heat in flowing steam twenty minutes on three 
successive days. 

15. Cool the gelatin in a running-water bath, immediately 
after each heating. Care must be taken to heat the gelatin 
as little as possible, since part of the solidifying power of 
gelatin is lost with each application of heat. 

16. To sterilize a large flask of nutrient gelatin, heat 
for twenty minutes on four days in succession. 

AGAR 

Agar or agar-agar (from a Malay word meaning " vege- 
table "), the substance which is used in preparing one kind 
of solid culture medium for bacteriological work, is a pro- 
duct prepared from various seaweeds found near the Indian 
Ocean and in Chinese and Japanese waters. This type of 
seaweed has several common names, as Ceylon or .Jaffna 
moss, Bengal isinglass, etc. Various species are used for 
food and the trade is considerable. 

Payen, a French chemist -(about 1859), obtained the 
agar jelly from the seaweed, Gelidium corneum, in the fol- 
lowing manner : The seaweed was allowed to stand for some 
time in a cold dilute solution of hydrochloric acid; the acid 
was removed by rinsing several times with water, then the 
seaweed was placed in a cold dilute solution of ammonia; 
next the ammonia was removed by repeated rinsing with 
cold water. During this process, the seaweed lost 53% 
of its weight in mineral salts, coloring matter, and organic 
constituents. The remaining portion was boiled in water, 



38 GENERAL MICROBIOLOGY 

during which process the vegetable jelly was extracted. 
The solution so obtained was poured off, leaving the useless 
sediment behind. This jelly is the same in composition 
as that existing in the vegetable tissues; it has not been 
changed chemically, as is collagen in the preparation of 
gelatin. The commercial agar is most probably prepared 
by evaporating this solution to dryness by different means. 

Agar usually comes into the hands of the bacteriologist 
as long, slender, grayish-white strips, or as blocks, or more 
especially in recent years, in the form of a gray-white pow- 
der of European manufacture. 

Agar, in contrast with gelatin, is a carbohydrate, i.e., 
it consists of a combination of carbon, hydrogen and oxygen 
only. Traces of nitrogen are present as impurities. The 
above qualitative determinations of its elementary constit- 
uents were made by Payen, by Parumbaru and by Hueppe, 
who made their determinations on agar from different 
sources. As far as can be ascertained, its empirical formula 
has not yet been investigated to any extent. 

Like gelatin, however, agar is a reversible colloid. It 
soaks up in cold water, dissolves in hot water after a long 
boiling to a tasteless and odorless clear solution, and solid- 
ifies upon cooling to a more or less opaque jelly. Its 
watery solution is neutral or nearly neutral to phenol- 
phthalein; still, a drop or two of twentieth normal sodium 
hydrate is sufficient to make the pink color perceptible. 

The colloidal properties of agar are not destroyed by a 
long-continued heating at a high temperature, nor by the 
action of ordinary microorganisms as are those of gelatin. 
The above properties, however, are influenced and may 
be wholly impaired by the reaction of the liquid in which 
the agar is dissolved. 

The reaction of the liquid, i.e., whether it is acid or 
alkaline, influences the agar as to its solubility, solidity, 
color, transparency, filterability and amount of condensa- 
tion water. If agar is dissolved in a liquid of an acidity 



AGAR 39 

equivalent to 0.1% HC1, the agar dissolves very readily, 
filters quickly, the resultant filtrate being a light yellow, 
transparent, slippery, watery solution which does not 
solidify upon cooling. If a smaller percentage of hydro- 
chloric acid is used, solidification occurs (below 40 C.) 
but the jelly will not " stand up " and is therefore useless 
for agar slant or plate cultures. A large amount of con- 
densation water is present also. 

If agar is dissolved in a weak alkaline or neutral broth, 
a thick, reddish-brown, viscous liquid is obtained which 
filters slowly and solidifies quickly at 40 C., to a very solid, 
opaque, dry jelly, having but little condensation water; 
it retains its shape well in slants and in plates. Thus 
the value of the agar as a solid culture medium is raised or 
lowered according to the cjegree of alkalinity or acidity. 

It must be noted in addition, however, that when once 
the solidifying property of agar is destroyed by the presence 
of an excess of acid in its solution, this property can never 
be regained by neutralization with alkali; the acid per- 
manently destroys the reversibility of the colloid. 

The melting-point of agar (of 1.5% in neutral solution) 
is 97 C. and although its solidifying point is at 40 C., 
when once it has solidified it will stand up in the thermostat 
at a temperature of 50 C. For bacteriological purposes, 
only that form of agar can be used which remains fluid at 
from 38 to 40 C. Agar which remains fluid only at a 
temperature above this point would be too hot when in 
a fluid state for use; the vitality of organisms introduced 
would be impaired or destroyed by the high temperature. 

Difficulties are encountered in the preparation of a solid 
culture medium from agar, due to its slow solubility, vis- 
cosity and consequent slow filterability. Its solution 
(digestion) is effected, as mentioned above, by a long 
heating in a water-bath, steam sterilizer, autoclav, or over 
a free flame. The length of time required for complete 
digestion depends upon three things: The reaction of the 



40 GENERAL MICROBIOLOGY 

liquid in which the agar is dissolved, the per cent content 
of agar, and the method of dissolving. The influence of the 
reaction of agar solutions has been treated above. For 
general culture use, however, ordinary agar is made +15 
Fuller's scale (agar solidifies with difficulty above +30 
Fuller's scale). 

One per cent agar is much more easily soluble under 
equal conditions than a higher per cent. One and one- 
half per cent is the amount used in ordinary agar media, 
giving a somewhat stiffer and thus more desirable jelly. 

Agar is digested most rapidly over a free flame. If not 
heated sufficiently, after the filtration and sterilization of 
the agar by the intermittent method, a flocculent precip- 
itate frequently appears in the previously clear medium. 
This can be made to disappear in most cases by subjecting 
to the temperature of the autoclav (120 C. 15 Ibs.). 

Agar for culture media should be entirely clear when 
liquid, and homogeneously opaque-translucent when solid; 
it should have a translucence sufficient to allow deep colonies 
on plates or stab cultures to be observed readily; it should 
not contain flocculent material, sediment, or pieces of cotton 
or filter paper, as these hinder typical colony development 
of microorganisms and, to the inexperienced, may some- 
times be mistaken for colonies. 

In the first methods ever used for making agar culture 
media, instead of filtering the hot agar through filter paper, 
absorbent cotton, or asbestos, it was allowed to cool, dur- 
ing which process the sediment settled to the bottom; when 
solid the sediment was cut off. This method was not 
desirable, as the clearness of the resultant agar would depend 
upon the rate of cooling; the slower the cooling, the more 
completely would sedimentation take place. 

Agar is not a food for microorganisms in general, i.e., 
it is not affected by the digestive enzymes of most bacteria, 
as is gelatin. However, a few bacteria are known which 
have the power of liquefying agar, among which are B. 



PREPARATION OF NUTRIENT AGAR 41 

gelaticus n. sp. (gran) and Bad. nenckii, both of which are 
found, as would be expected, in sea water. This compara- 
tive inertness of agar renders it valuable for the preparation, 
of solid synthetic media, the value of which may be en- 
hanced by subjecting the commercial agar to natural 
fermentation during which process any traces of avail- 
able food substances are used up by the microorganisms 
present. (Beijerinck.) 

Agar is of special use in bacteriological work in which 
the cultivation of microorganisms must be conducted at a 
temperature above the melting-point of gelatin. This 
feature has made possible the great strides that have been 
taken in medical bacteriology, as many pathogenic bacteria 
can be isolated and grown only with difficulty at tempera- 
tures below that of the body. 

REFERENCES 

SMITH, ERWIN F.: Bacteria in Relation to Plant Diseases. Vol. I, 

pp. 31-36. Several illustrations. 
SCHULTZ, N. K.: Zur Frage von der Bereitung einiger Nahrsubstrate. 

Cent. f. Bakt. I. Orig., Bd. 10, 1891, p. 57. 

EXERCISE 9. PREPARATION OF NUTRIENT AGAR 

Apparatus. 3.5 liter agate-ware pail; 15 gms. agar; 
10 gms. peptone; 5 gms. salt; 10 gms. egg albumen (or one 
egg); 500 c.c. sterile meat infusion; 500 c.c. tap water; 
titration apparatus; N/20 NaOH; N/l NaOH; phenol- 
phthalein (indicator); distilled water; large funnel; 
plaited filter paper; filling funnel; sterile test tubes; 
sterile liter flask; coarse balances; large gas burner; 1 
liter measuring cup; apparatus for steam sterilization. 

Method. 1. In a 3 liter agate ware pail place 15 gms. 
of agar in 500 c.c. of tap water. 

2. Wash the agar well, separating the shreds and squeez- 
ing it through the hands. 

3. Decant the dirty water, measuring the amount poured 



42 



GENERAL MICROBIOLOGY 




off; replace with the same amount of clean tap water. 

Repeat. 

4. Dissolve over a free flame and boil for five minutes, 
stirring constantly. The solu- 
tion must be entirely free from 
lumps of agar. 

5. Add 1% Witte's peptone 
and 0.5% salt to the boiling 
agar. 

6. To 500 c.c. of meat in- 
fusion add 10 gms. of egg al- 
bumen which has been well 
mixed with 100 c.c. of tap water. 
(Put the egg albumen in a 
tumbler and add enough water 
to form a paste. Stir until 
smooth and then add the remain- 
ing water. One egg; well beaten, 

FIG. 9. Hot Water Funnel for may be substituted.) Mix all 
Filtering Agar or Gelatin. thoroughly 

7. Pour the melted agar mixture slowly into the meat 
infusion, stirring constantly. Heat in the autoclav at 
120 C. for forty-five minutes or for an hour in flowing 
steam. 

Note. The time for this heating may be lengthened to advantage, 
but never shortened. If agar has not been heated sufficiently before 
filtration, a flocculent precipitate will form in the tubes upon heating 
in flowing steam. In most cases this may be caused to disappear by 
heating for a short time in the autoclav at 15 Ibs. 

8. Titrate with N/20 NaOH. 

9. Adjust the reaction of the medium to +15 with 
normal NaOH or normal HC1. Retitrate and readjust 
the reaction if necessary. 

10. Counterpoise and note the weight. 

11. Boil fifteen minutes over a free flame, stirring con- 
stantly, 



DUNHAM'S PEPTONE SOLUTION 43 

12. Counterpoise and make up any loss in weight with 
boiling distilled water. 

13. Filter boiling hot through plaited filter paper just 
previously washed with boiling water. Pass the filtrate 
through the same paper until clear. 

14. Fill 60 to 70 sterile test tubes, using approximately 
8 c.c. of the medium for each tube. 

15. Heat in flowing steam twenty minutes on three suc- 
cessive days. 

16. At the end of the final heating, place the tubes of 
agar in an inclined position to solidify (do not allow the 
medium to touch the plug) so that a large surface is pre- 
sented for the cultivation of microorganisms. These are 
called agar slants. 

Note. If agar tubes are to be used only for agar slants, less of the 
medium is needed in the tube than when they are to be used for plating. 

17. To sterilize a large flask of agar, heat for thirty 
minutes on four successive days. 

EXERCISE 10. PREPARATION OF DUNHAM'S PEPTONE 
SOLUTION 

Dunham's solution is utilized for determining the 
power of microorganisms to produce indol, ammonia or 
nitrites from peptone, which properties are character- 
istic of certain species. 

Apparatus. 1000 c.c. of tap water; 10 gms. peptone, 
Witte's; 5 gms. salt; large burner; large funnel; plaited 
filter paper; filling funnel; sterile test tubes; apparatus 
for steam sterilization. 

Method. 1. Mix 1% peptone and 0.5% salt to a 
smooth paste with a measured (small) amount of water. 

2. Dilute to 1000 c.c. with tap water. 

3. Counterpoise and note the weight. 

4. Boil ten minutes over a free flame; counterpoise and 
make up any loss in weight with distilled water. 



44 GENERAL MICROBIOLOGY 

5. Filter while hot through a plaited filter previously 
washed with hot water. (Filtrate must be perfectly trans- 
parent.) 

6. Tube, putting 8 c.c. in each tube. 

7. Sterilize for fifteen minutes on three successive 
days. 

Microorganisms which will not produce ammonia or 
nitrites from peptone may show this power if nitrogen is 
added to this solution in the form of inorganic nitrogen as 
potassium nitrate (0.2%). 

EXERCISE 11. NITRATE PEPTONE SOLUTION 

This solution is used to determine the power some 
organisms have of reducing nitrates to nitrites, free ammonia 
or nitrogen. 

Apparatus. 1000 c.c. distilled water; 1 gm. peptone, 
Witte's; 0.2 gm. nitrite-free potassium nitrate; 'large agate- 
ware pail; filling funnel; sterile test tubes; apparatus for 
steam sterilization. 

Method. 1. Mix the following ingredients: 1000 c.c. 
distilled water; 1 gm. Witte's, or other peptone; 0.2 gm. 
nitrite-free potassium nitrate. Filter if necessary, 

2. Tube, placing 8 c.c. in each tube. 

3. Sterilize by heating for fifteen minutes on three suc- 
cessive days or for five minutes in the autoclav at 120 C. 

CULTURES 

Definitions. A culture consists of the active growth 
of microorganisms in or on a nutrient medium. 

A mixed culture is a culture composed of two or more 
species of microorganisms growing together in or on a 
nutrient medium. 

A pure culture is the growth of one species of micro- 
organism only, in or on a nutrient medium, that was sterile 
before inoculation. 



CULTUKES 45 

Pure cultures are used for studying the morphological 
and physiological characteristics of microorganisms. 

From mixed cultures, pure cultures may be obtained by 
the plating method. Mixed cultures of known micro- 
organisms may be employed in studies on symbiosis, meta- 
biosis, or antibiosis. 




FIG. 10. Mixed Culture in Petri Dish (Plate Culture) Showing 
Various Forms and Sizes of Colonies, (Orig, Northrup.) 

Plate cultures are cultures grown in Petri dishes contain- 
ing a nutrient medium. 

Slant culture is the term generally applied to cultures 
grown on the inclined surface of any medium, such as agar, 
potato, blood serum, etc., and are designated specifically 
as agar slant cultures, potato slant cultures, etc. They are 
generally prepared by drawing a contaminated needle in 
a straight line along the surface of the medium. Cul- 



46 



GENERAL MICROBIOLOGY 



tures prepared in this way are also frequently termed 
streak cultures. The term streak cultures may also be 
applied to cultures made similarly but grown on a horizon- 
tal flat surface as in a Petri dish. 

Slant or streak cultures are valuable in offering a large 
surface for growth, to aerobic organisms. 

Stab (or Stich) culture is the 
term applied to a culture, generally 
a pure culture, which is prepared 
by stabbing a translucent, liquefi- 
able solid medium to a considerable 
depth with a contaminated straight 
needle. Gelatin stab cultures are 
invaluable for studying gelatin 
liquefaction. Agar is frequently 
used for stab cultures. If sugar is 
added to the medium, gas produc- 
tion may be demonstrated. Aerobic 
and anaerobic bacteria may be 
easily differentiated by their be- 
havior in stab culture. 

Liquid cultures are cultures 
grown in a liquid medium such as 
milk, broth, cider, wort, etc. 

Shake cultures are made by 

FIG. 11. Liquefaction of inoculating with a pure or mixed 
culture, a liquefied nutrient me- 
dium (40-45 C.). The inoculum 
is distributed immediately through- 
out the medium by means of the needle used, or by rotat- 
ing or shaking. 

This type of cultivation is valuable for determining the 
oxygen relation of the organisms introduced and is espe- 
cially useful for demonstrating the presence of gas-producing 
organisms if a suitable medium is used. 

Care of Cultures. 1. Incubation: Cultures should b. 




Gelatin, Saccate becom- 
i n g Infundibuliform. 
(Orig. Northrup.) 



CULTURES 



47 



kept at a constant temperature. Organisms which natur- 
ally grow at body temperature (37 C.) as Bacillus coli, 




I 



a 
I 



1 



Streptococcus pyogenes, etc., may, w#Ji rffte exception of 
gelatin cultures, be kept in the 37 C. incubator. 

Always place cultures in tumblers with cotton in the 
bottom or in small wire baskets; never place them in a 



48 GENERAL MICROBIOLOGY 

horizontal position or incline them carelessly against a 
vertical surface without proper support. 

2. Care of Broken Cultures. If a culture of any organism 
is accidently broken pour 1 : 1000 mercuric chloride, 2% 
compound solution of cresol or 5% phenol over it and also 
over any articles which may have been infected; let stand 
ten minutes before wiping up. Always disinfect your hands 
after handling broken cultures. 

3. Disposal of Old Cultures. Heat glassware contain- 
ing cultures to be discarded one hour in flowing steam. 
Cultures of pathogenic spore-forming organisms should be 
autoclaved. Glassware so treated may safely be washed by 
the student. 

Never throw living cultures into waste crocks, sinks, 
or elsewhere. You safeguard yourself and others in the 
laboratory by destroying all living cultures. Carelessness 
in regard to this matter will not be tolerated. 

4. Care of Slides, Cover-glasses, etc. Slides and cover- 
glasses used for hanging drop mounts, etc., should be 
immersed in 1 : 1000 mercuric chloride or chromic acid 
cleaning solution for at least ten minutes before cleaning. 

5. Care of Cuts and Other Wounds. In case of cuts or 
wounds, consult the instructor at once. All wounds should 
be attended to immediately. Tincture of iodin is recom- 
mended for painting skin abrasions and deep wounds; in 
the latter case a bandage should be applied to keep extra- 
neous matter from entering and setting up infection. In 
case of serious injury, a physician should be consulted. 
Every laboratory should keep a stock of rolled bandages, 
etc., for emergencies, 



PREPARATION OF PLATE CULTURES 



49 



EXERCISE 12. PREPARATION OF PLATE CULTURES, 
LOOP OR STRAIGHT-NEEDLE DILUTION METHOD 
(QUALITATIVE) 

Plate cultures are a valuable asset to the microbiologist, 
as they offer a means by which pure cultures of micro- 
organisms may most easily be obtained; they also allow 
a quantitative and qualitative 
study of the micron 1 ora of differ- 
ent substances. 

Their preparation consists in, 
(1) inoculating a liquefied solid 
culture medium with micro- 
organisms, (2) mixing them well 
throughout the medium, (3) 
pouring the inoculated medium 
into a sterile Petri dish and, 
when it has solidified, (4) placing 
the Petri dish or plate culture 
at a constant temperature. 

The culture medium in so- 
lidifying fixes in situ the micro- 
organisms introduced, and well- 
separated organisms develop into 
more or less well-separated 
" colonies " which become visible 
to the naked eye after twenty- 
four to forty-eight hours. From FlG - 13. -Water-bath for Melt- 
these isolated colonies usually 
pure cultures may then be ob- 
tained, or a quantitative or quali- 
tative study may be made. 

Isolated surface colonies are most frequently round 
(concentric in growth) and generally are quite typical for 
each species, while isolated sub-surface colonies are lenticu- 




ing Agar or Gelatin for Plat- 
ing, containing a Removable 
Copper Test Tube Rack. 
(Orig. Northrup.) 



50 



GENERAL MICROBIOLOGY 



lar (double concave) or compoundly lenticular in shape as a 
rule, species differences not being as well denned. 

Apparatus. Tripod leveling stand; glass plate about 




I 



14 inches square; small spirit level; water-bath; thermom- 
eter; sterile Petri dishes; tubes of sterile media (gelatin 
or agar); culture; platinum needle and loop; Bunsen 
burner; wax pencil; mixed or pure culture. 



PREPARATION OF PLATE CULTURES 51 

I. Procedure for Agar Plates. The loop or straight- 
needle dilution method is valuable as a quick method of 
obtaining pure cultures when quantitative results are not 
desired. 

1. Place the glass plate on the leveling stand. 

2. Place the spirit level on the glass plate and make 
level by means of the leveling screws. 

Note. The plate-leveling stand facilitates the uniform distribution 
of the medium over the bottom of the Petri dish, but is not necessary 
for the accomplishment of favorable results. If the desk top is level 
this apparatus is unnecessary. 

3. Place three sterile Petri dishes, labeled 1, 2 and 3, 
in a row on the glass plate. 

4. Liquefy three tubes of agar at 100 C. in the water- 
bath or steam and keep at a temperature of 40 to 
45 C. 

5. Number the tubes of agar 1, 2 and 3 and flame the 
plugs. 

6. With the sterilized platinum needle, merely touch 
the culture and transfer to tube No. 1. 

Note. Hold cultures and plugs while transferring as in Fig. 20, 
p. 59. 

7. Distribute the microorganisms through the medium 
with the needle. 

8. Transfer one loopful from tube No. 1 to tube No. 2 
and mix with the needle, as in 7. 

9. Slightly raise the cover of Petri dish No. 1. Intro- 
duce the flamed mouth of tube No. 1 and pour the melted 
agar into the plate; remove the mouth of the tube, and 
replace the cover of the Petri dish. If the medium has 
not entirely covered the bottom of the plate, tilt slightly 
in different directions to distribute evenly. 

Note. Passing the Petri dish several times through the flame just 
previous to pouring the plate will aid greatly in preventing the forma- 
tion of condensation water on the cover. 



52 GENERAL MICROBIOLOGY 

10. Transfer two loopfuls from tube No. 2 to tube No. 
3 and mix. 

11. Plate tube No. 2 in Petri dish No. 2 (see 9). 

12. Plate tube No. 3. 

13. Label the plates with name of culture, number of 
dilution and date, and with your own name or desk number. 

14. When the agar has solidified firmly, invert the 
plates and place in the incubator at 37 C., or at room 
temperature. 

Note. If the plates are placed right side up, condensation water 
forms on the cover and drops down upon the surface of the agar, caus- 
ing the colonies to run together and thus destroying their character- 
istic appearance. 

II. Procedure for Gelatin Plates. 

1-3. Proceed as in I. " Procedure for Agar Plates." 

4. Liquefy three tubes of gelatin in the water-bath 

and keep at a constant temperature of 30 to 35 C. 
5-13. Proceed as in I. " Procedure for Agar Plates." 
14. Place at a constant temperature of 21 C. The 

gelatin may not harden until placed at this temperature. 

Note. Gelatin plates are kept right side up, as the organisms may 
liquefy the gelatin. The liquefied part would then fall from the medium 
upon the cover and ruin the plate for study. 

EXERCISE 13. PREPARATION OF PLATE CULTURES, 
QUANTITATIVE DILUTION METHOD 

In the method given below, " dilution flasks " are pre- 
pared containing measured amounts of water or salt solu- 
tion in which a measured amount of the substance under 
investigation is placed. 

As to whether water or salt solution is used depends upon 
the nature of the material to be dissolved or placed in 
suspension. If the substance whose microflora is to be 
studied contains a certain amount of various salts or other 
electrolytes in solution, an effort should be made to approx-* 



QUANTITATIVE DILUTION METHOD 



53 



imate this amount in the preparation of the diluting fluid, 
e.g., in obtaining a quantitative estimation of the micro- 




FIG. 15. Incubator. 



organisms from the blood, dilutions should be made in 0.85% 
salt solution; from tap water, in tap water, etc. 

Theoretically, dilutions made in a liquid of a markedly 
different electrolyte concentration from that of the sub 



54 



GENERAL MICROBIOLOGY 



stance to be studied, might cause either plasmolysis or 
plasmoptysis as the concentration was respectively too 
great or too weak. 

Microorganisms of different species differ markedly 
in their susceptibility to osmotic pressure. This cannot 
be determined, however, unless studies are made of pure 
cultures of each, therefore the percentage of salt in the 
diluting liquid should approximate 
that of the substance whose micro- 
flora is to be studied. 

The method below is applicable 
to substances in the liquid condi- 
tion only. Modifications of this 
method may be utilized to apply to 
nearly every class of substances. 

Plates are generally made from 
three different dilutions, so that 
well-separated colonies may be ob- 
tained on at least two plates. 

Apparatus. Sterile 1 c.c. pipettes 
(graduated to 0.1 c.c.); sterile 
10 c.c. pipettes (graduated) ; sterile 

FIG. "16. -Koch's Safety Erlenmeyer flasks of 200 c.c. ca- 
pacity containing 90 c.c. and 99 c.c. 
of sterile water or salt solution; 
three sterile Petri dishes; three tubes of sterile agar or gelatin. 




Burner for Incubator or 
Water Bath. 



Note. Use only freshly prepared dilution flasks, otherwise evapora- 
tion takes place so rapidly that accuracy is not possible. 

Culture. Substance under investigation. 

Method. 1. With a sterile 1 c.c. pipette, transfer 1 c.c. 
of the original sample or culture to a flask containing 99 
c.c. of sterile water or salt solution. The flask now con- 
tains 100 c.c. of liquid containing 1 c.c. of the original 
sample, giving a dilution of 1 in 100. 

Note. A sterile pipette must be used for each separate operation. 



QUANTITATIVE DILUTION METHOD 55 

2. Shake the flask to secure an even suspension of the 
microorganisms. 

Do not allow the liquid to touch the cotton plug. 

3. With a sterile pipette, transfer 1 c.c. of the first 
dilution into a flask containing 99 c.c. of sterile water 
and shake. The second flask now contains 100 c.c. of a 
liquid containing 1/100 of the original sample, a dilution 
of 1/100 in 100, or 1 in 10,000. 

4. If a higher dilution is required, 1 c.c. from the flask 
containing the 1/10,000 dilution placed in a flask con- 
taining 99 c.c. sterile water gives 100 c.c. of a liquid con- 
taining 1/10,000 of the original sample, or a dilution of 
1 in 1,000,000. 

If a lower dilution of the original sample than 1/100 
is desired, make use of the 90 c.c. dilution flasks as follows: 

With a sterile 10 c.c. pipette place 10 c.c. of the original 
sample into 90 c.c. of sterile water and shake. This flask 
now contains 100 c.c. of liquid containing 10 c.c. of the 
original sample, giving a dilution of 1 in 10. A dilution 
of 1 in 1000 may be made either by placing 1 c.c. of the 1/10 
dilution in 99 c.c. of sterile water, or by placing 10 c.c. of 
the 1/100 dilution in 90 c.c. of sterile water. 

Note. Almost any desired dilution can be made by the use of these 
flasks. 

5. For plating, transfer 1 c.c. with a sterile 1 c.c. pipette 
from the flask containing the desired dilution to a sterile 
Petri dish. 

Note. Never use less than 1 c.c. Run duplicates when absolute 
accuracy is necessary. 

6. Liquefy the desired number of agar or gelatin tubes 
in the water-bath or steam at 100 C. 

7. Cool to a temperature of 40 to 45 C. 

8. Pour the plates, tilting each carefully so that the 
1 c.c. of the diluted sample may be mixed well throughout 
Jhe medium. 



56 



GENERAL MICROBIOLOGY 



9. Place the plates on a level surface until the medium 
solidifies. 

10. Incubate at the desired temperature. 

EXERCISE 14. METHODS OF COUNTING COLONIES 
IN PETRI DISH CULTURES 

Apparatus. Jeffer's counting plate; black glass plate or 
cardboard; tripod counting lens, magnifying four diam- 
eters; plate cultures. 

Note. In Jeffer's counting plate (see illustration) each division 
has an area of 1 square centimeter. The figures denote the number 
of square centimeters in the respective circles. 





123156789 10 



FIG. 17. Jeffer's Counting 
Plate. 



FIG. 18. Wolfhugel'g 
Counting Plate. 



Method. 1. Invert the Petri dish culture to be counted 
upon the black glass plate or upon some black surface. 

Note. If liquefiers are present on the gelatin plate, place the Petri 
dish right side up upon the counting plate; this necessitates refocusing 
the lens. The cover may be removed to facilitate counting if the 
plate is to be discarded. 

2. Place the counting plate upon the Petri dish, making 
the circumference of the Petri dish coincide as nearly as 



COUNTING COLONIES IN PETRI DISH CULTURES 57 

possible with that of one of the circles on the counting 
plate. 

3. Using the tripod lens count the colonies in each sector 
of the smallest circle, then in each division between the 
concentric circles. 

Note 1. The tripod counting lens must be used if the colonies 
are very small, as they/ otherwise may be confused with air bubbles 
in the medium. If there are less than 500 colonies present, the 
entire plate should be counted. If the number is much greater, 
from ten to twenty divisions, in some definite order, should be 
counted, an average taken, and the results multiplied by the area 
of the plate in square centimeters. 

Note 2. Wolfhugel's counting plate is very desirable for 
counting a large number of colonies. It is ruled in square centi- 
meters and the squares on the diagonals of the plates are sub- 
divided into smaller squares. The colonies appearing in from ten 
to twenty of these smaller squares may be. counted, an average 
taken and the result multiplied by the number of small squares in 
1 sq. cm. times the area of the Petri dish in square centimeters. 
(The entire area of the plate may be obtained most quickly by 
placing a Jeffer plate upon the Petri dish in question.) 

4. Ascertain the number of colonies per cubic centi- 
meter in the original sample by multiplying the whole num- 
ber of colonies on the plate by the dilution; e.g., if there 
are 386 colonies on the plate and the original culture was 
diluted 1 in 1000, the number of colonies contained in each 
cubic centimeter of the original sample is 386,000. 

Note. When there is an excessive number of colonies on a plate 
the vigorous microorganisms will inhibit the growth of the less vigorous 
and thus the number of colonies counted is smaller than the number 
of microorganisms present. Moreover, the colonies may become 
confluent and the counts will again be in error. 



58 GENERAL MICROBIOLOGY 



EXERCISE 15. ISOLATION OF MICROORGANISMS 
FROM PLATE CULTURES AND METHOD OF 
MAKING AGAR STREAK CULTURE 

Apparatus. Straight platinum needle; several tubes 
of sterile agar slants; Bunsen burner; wax pencil; plate 
containing from 30 to 200 well-separated colonies. 

Method. 1. Examine the plate to determine the colo- 
nies which differ macroscopically and microscopically, (Use 



FIG. 19. Various Forms of Platinum Needles. (Orig. Northrup.) 

a counting lens or the lowest power of a compound micro- 
scope.) 

2. Note the most isolated of each kind, and mark them 
with the wax pencil upon the bottom of the plate to insure 
picking up the proper colonies later. Also note how the 
deep and surface colonies differ. 

3. Examine each marked colony under the lowest power 
of the microscope to make sure of its purity. If the colony 
does not appear to be wholly isolated, pick up a small 
portion of it with a sterile platinum needle and stain with 
one of the common stains (see p. 88) or examine it in the 



ISOLATION OF MICROORGANISMS 



59 



hanging drop (see p. 76) to determine if more than one 
kind of organism is present. 

4. If the colony is pure, pick up a portion with the 




o 

i 



sterile needle, or, in case of extremely small colonies, remove 
the cover from the plate, focus the low power of the micro- 
scope on the desired colony and while looking through the 
microscope, fish the colony. 



60 GENERAL MICROBIOLOGY 

5. Transfer to one of the agar slants, making a streak 
along the median line of the inclined surface of the agar, 
drawing the needle from the base to the top of the slant. 

Note. For investigational purposes, when dealing with unknown 
microorganisms, the following method is more accurate for obtaining 
them in pure culture: Transfer to a tube of broth; incubate for 
twenty-four hours and plate a second time. Isolate from the twenty- 
four-hour plate culture. 

6. Incubate at the optimum temperature. 

Note. If the agar slants haye become dried out to any extent, it 
is necessary that the agar be melted and re-slanted in order that 
optimum growth may take place. 

EXERCISE 16. METHOD OF MAKING TRANSFERS OF 
PURE CULTURES INTO A LIQUID MEDIUM 

Directions for making transfers of pure cultures from 
one medium to another must be followed very carefully, 
otherwise extraneous microorganisms may enter and hope- 
less confusion result. 

Apparatus. Test tubes containing a sterile liquid 
nutrient medium; platinum needle; Bunsen burner. 

Culture. Pure culture. 

Method. 1. Flame the cotton plugs of the test tubes 
containing the pure culture and the sterile liquid nutrient 
medium. 

2. Sterilize the platinum needle in the flame. 

3. Permit it to cool (about one minute is required). 

4. Hold it in the right hand and remove the cotton plug 
of the culture tube with the little finger of the same hand. 

5. Take up a very little of the culture with the needle. 

6. Replace the plug of the culture tube. 

7. Remove the plug of the tube of sterile liquid medium 
in the same manner. 

8. Insert the infected needle into the liquid. 

9. Replace the plug. 

10. Sterilize the needle before laying it down. 



METHOD OF MAKING STAB CULTURES 61 

EXERCISE 17. METHOD OF MAKING STAB CULTURES 

Apparatus. Tubes of sterile agar or gelatin; straight 
platinum. needle; Bunsen burner. 

Culture. Pure culture. 

Method. 1. Liquefy the gelatin or agar tube and re- 
solidify it in a vertical position in cold running water or 
in some cold place. 

2. With a sterilized straight platinum needle pick up a 
very little of the culture or colony. 

3. Insert the needle at the middle of the circle made by 
the surface of the medium and push the needle about 
5 cms. into the solid medium (within 1 cm. of the bottom 
of the tube), then withdraw carefully so that the path of 
the needle be as limited as possible. The microorganisms 
grow along the path of the needle. 

Avoid having the shoulder of the rod come in contact 
with the surface of the medium lest its heat disfigure the 
surface or even kill the microorganisms. The surface of 
the medium should remain intact during this process. 

4. Replace the plug in the new culture and sterilize 
the needle. 

EXERCISE 13. PREPARATION OF A GIANT COLONY 

Purpose. To show the development of a single colony 
of a microorganism. 

Apparatus. Sterile Roux culture flask* or Petri dish; 
tubes of agar or gelatin. 

Culture. Organism to be studied. 

Method. 1. Melt two tubes of dextrose agar or gelatin. 

Pour into the culture flask. 

Note. Allow the medium to touch and cover one large side only. 

2. Heat in this horizontal position in flowing steam 
fifteen minutes. 

3. Distribute the medium evenly over the large side, 
* Most valuable for molds, especially Rhizopus nigricans. 



62 



GENERAL MICROBIOLOGY 



and set on a level surface to cool. When the medium has 
solidified, place the flask with the medium-side up. 

4. Mark the center of the flask on the outside with a 




FIG. 21. Giant Colony of Mold in Roux Flask. (Orig.) 

wax pencil and inoculate with a bent platinum needle in 
one spot only. 

Note. When making mold inoculations moisten the sterile needle 
with sterile water or medium before touching the spores; this insures a 
positive inoculation. 

5. Keep at room temperature medium-side up. 

6. Examine and measure the diameter of the giant 
colony from day to day and describe the typical growth 



THE MICROSCOPE 63 

of the colony, using the terms on the descriptive chart 
of the Society of American Bacteriologists, p. 134, as far 
as possible. 

7. Compare the giant colonies of a Mucor, Pencillium, 
a yeast and Bacillus subtilis or Bacillus mycoides. Use 
agar for giant colonies of these bacteria, as they liquefy 
gelatin. 

8. Giant colonies of yeasts and bacteria and some molds 
may be grown in Petri dishes, or in flat-bottomed flasks. 

For illustrations of giant colonies of bacteria see: 

FUHRMAN: Vorlesungen iiber Technische Mykologie, pp. 41, 43. 
LOHNIS: Vorlesungen iiber Landwirtschaftliche Bakteriologie, pp. 38, 

170. 
LEHMANN AND NEUMANN: Bakteriologie und Bakteriologische Diag- 

nostik, Bd. I. (Atlas.) 

For illustrations of giant colonies of yeasts see: 

LAFAR: Technische Mykologie, Bd. 4, German Ed., pp. 24-25, 306, 
and above references. 

THE MICROSCOPE 

Care of the Microscope. For microbiological work a 
compound microscope is necessary. This should be fitted 
with a minimum of two oculars corresponding to the Leitz 
No. 1 (lowest power) (Spencer, 6X), and No. 3 or 4 (Spencer, 
10 X) and three objectives corresponding to the Leitz 
J in. (lowest power) (Spencer, 16 mm.), -f in. (Spencer, 4 
mm.) objectives (dry) and ^ in. oil immersion objective. 
A coarse and a fine adjustment permit the accurate focus- 
ing of any combination of lenses. The substage shoi Id be 
fitted with a good condenser and iris diaphragm for regulating 
the amount of light, and a plane-concave mirror. 

Great care should be exercised in the use and care of the 
microscope as it is a delicately adjusted instrument. 

The following rules should be heeded: 

The Stand. The stand is the body of the microscope 
carrying the optical parts. 



64 



GENERAL MICROBIOLOGY 




FIG. 22. Compound Microscope with Mechanical Stage Attached and 
Side Fine Adjustment. 



THE MICROSCOPE 65 

1. Leave the microscope in the case when not in use. Dust 
works into the bearings of the instrument, making them 
work hard and unnecessarily wearing them. 

2. When handling the microscope do not grasp it by the 
arm which contains the fine adjustment unless the micro- 
scope is designed to permit this. Grasp it by the pillar 
below the stage. 

3. Never use alcohol on the lacquered parts. Rubbing 
gently with a very little xylol and drying quickly will 
remove any oily material. 

The Stage. The stage is that portion of the microscope 
on which the mounted object is placed for examination. 

1. Should the stage become soiled with balsam, immer- 
sion oil or anything which water will not remove, it can be 
cleaned with xylol or chloroform. A little heavy oil will 
restore the stage to its original black color. 

The Fine Adjustment. The fine adjustment is used for 
bringing out details in very small objects and is necessarily 
of limited range and delicate in its mechanism. 

1. If, when looking into the eye-piece, no change of focus 
is noticed by turning the micrometer head, or if the microm- 
eter head ceases to turn, the adjustment has reached its 
limit. To adjust, focus down or up, respectively, with the 
coarse adjustment, and turn the micrometer head until 
the fine adjustment is midway within its range. 

2. When the fine adjustment screw stops, do not force it. 
The Draw Tube. The draw tube is the tube receiving 

the ocular. 

1. The draw tube should work easily and smoothly. 
On the draw tube will be found graduations in millimeters 
or inches, some fixed point at which certain combinations 
of objectives and oculars give the clearest image. This 
differs with different microscopes and should be known for 
the microscope used. 

The Nose-piece. The triple nose-piece on the compound 
microscope serves a double purpose; to obviate the neces- 



66 GENERAL MICROBIOLOGY 

sity of screwing the different objectives in as needed and 
to protect the back lens of the objective from dust. The 
later microscopes have a " collar " nose-piece which keeps 
the objectives free from dust at all times. 

1. Nose-pieces and objectives of the best makes are now 
made so that the objectives are parfocal, i.e., when one lens 
is in. focus the others on the nose-piece will be nearly in 
focus when they are swung into the optical axis. They 
are also approximately centered so that a point in the 
center of the field of one lens will be in the field of the 
others. 

2. Objectives made parfocal for one tube-length or eye- 
piece are not parfocal for a different length or a different 
eye-piece. 

3. Objectives of one microscope should not be inter- 
changed with those of another, even if of the same make. 

4. Always focus up, slightly, before turning from a lower 
to a higher power. Otherwise the front of the objective 
may be swung against the cover-glass and injure both the 
specimen and the objective. 

The Optical Parts. The optical parts are the lenses of 
the objectives, oculars and condenser and the mirror. 

1. Wipe dirty lenses gently with Japanese lens paper to 
remove dirt. 

2. Never rub a lens vigorously with anything. 

3. Avoid touching the surface of a lens with the fingers. 
Cutaneous secretions are hard to remove. 

4. Always clean the oil immersion objective with lens 
paper immediately after using. If the oil is allowed to dry, 
xylol must be used to clean the lens. 

5. Always leave an ocular in the tube to keep dust from 
settling on the back lens of the objective. Dust on the 
back lens may be removed with a earners hair brush. 

6. Never take an objective apart. 

7. Oculars, condenser and mirror should be kept clean 
by the use of lens paper. 



THE MICROSCOPE 



67 



Use of the Microscope. Position. 1. Always use the 
microscope with the tube in the perpendicular position. 
This is indispensable in examining fresh mounts or fluids. 

2. Work with both eyes open and if possible use both 
eyes interchangeably. 

Light. 3. Never use direct sunlight. The best light is 
obtained from white clouds. Northern or eastern light is 
preferable. 

The best artificial light is a Welsbach burner (gas). 
When employing artificial light use a blue glass between 




FIG. 23. Double Demonstration Ocular with Pointer Enabling Two 
Observers to View Simultaneously the Image Indicated by the 
Adjustable Pointer. 

the light source and the specimen. Often an eye-shade 
or some appliance with a similar purpose is desirable. 

4. Use the plane mirror in daylight, the concave mirror 
with artificial light. 

Focusing. 5. After putting in place a low-power ocular 
and objective, place the specimen on the stage, and while 
looking through the microscope, adjust the mirror so as to 
illuminate the field as evenly as possible, but not so brightly 
as to irritate the eyes. 

6. By means of the coarse adjustment, focus the body 
tube until the objective nearly touches the cover-glass, 
being careful not to touch it. 



68 



GENERAL MICROBIOLOGY 



7. With the eye at the ocular, focus up slowly with the 
coarse adjustment until the specimen comes plainly into 
view. 

// the light is too intense the focal point may be passed 
without noticing it. 

8. When the object is brought fairly well into focus by 




FIG. 24. Comparison Ocular Enabling One to Observe Two Different 
Microscopic Fields Side by Side. Any two microscopes may 
be used, (Healy.) 

means of the coarse adjustment, use the fine adjustment 
to obtain the sharpest focus to bring out details. 

9. Move the specimen when trying to obtain a focus, 
as a moving object is more apt to be noticed as the lens 
comes into focus. 

The microscope reverses the image. This will be noticed 
when the specimen is moved. The microscope magnifies 
the movement as well as the image; it therefore requires 



THE MICROSCOPE 69 

a certain delicacy of movement to put a specimen in a 
desired position. 

10. Beginners should always use the low-power objectives 
and oculars first. The low-power objectives have longer 
working distances and always show a larger portion of the 
specimen. After obtaining a general idea of the specimen, 
desired portions may be examined with the higher power 
objectives. 

11. In using high-power objectives for finding and exam- 
ining a specimen, it is always more desirable to use the low- 
est power ocular (corresponding to Leitz No. 1). If a higher 
ocular is used, there is a loss in the depth or sharpness and 
size of field, since they are both inversely proportional to 
the magnification. Illumination is also lost, which varies 
inversely as the square of the magnification. Remember 
that the largest field, the greatest penetration, and the best 
illumination are obtained by using the lowest magnification 
which makes all the detail in the image visible. 

Oil Immersion Objective. The highest power objective 
is the oil immersion lens. This is so termed because a drop 
of oil must be used between the front lens and the cover- 
glass. The oil used must have the same index of refrac- 
tion as glass to prevent the dispersion of the rays of light 
coming from the condenser. 

Working distance is the free distance between the cover- 
glass and the objective when the latter is focused. High- 
power objectives have short working distances. 

REFERENCE 

GAGE: The Microscope. 



70 GENERAL MICROBIOLOGY 



EXERCISE 19. METHOD OF MEASURING MICRO- 
ORGANISMS 

1. Using the Leitz Ocular " Step " Micrometer. In 
this ocular micrometer the intervals are arranged in groups 
of ten, each group being indicated by black steps rising 
from the first to the tenth interval. 

This arrangement possesses the great advantage that the 
divisions can always be seen distinctly whether the objects 
be light or comparatively dark. 

The intervals of the scale, instead of being 0.1 mm. or 
0.5 mm. wide, as in ordinary ocular micrometers, have a 
definite value of 0.06 mm. This gives for each objective 
and for a given tube length, convenient and in many cases 
integral micrometer values, which renders a greater facil- 
ity in the use of this instrument. The actual tube length 
differs in most cases but little from the standard length. 
The tube length and the micrometer value of each micro- 
scope, however, should be separately calibrated. 

It is of importance to be able to determine the size of 
microorganisms: (1) because it is of general interest to 
know the size of the microorganisms with which we are 
dealing; (2) because the difference in size is an important 
factor in' identifying and describing the organism; (3) 
because the size is necessary for purposes of comparison 
with other microorganisms. 

Apparatus. Microscope; Leitz ocular " step " microm- 
eter; object micrometer; specimen to be measured. 

Method. 1. With the aid of the Leitz ocular " step " 
micrometer the size of stained or unstained microorganisms 
on either a light or a dark field may be measured directly 
in microns. 

A micron is 0.001 mm., and is expressed by the Greek 
letter p.. 

2. One hundred divisions of the step micrometer cover 
100, 15 and 10 divisions of the object micrometer 



METHOD OF MEASURING MICROORGANISMS 71 



when Leitz objectives 3, 7 and 1/12 oil immersion are 
used. 

The object micrometer is simply a 
cover-glass (mounted on a slide in 
Canada balsam) upon whose surface 
has been ruled a scale 2 mm. in length, 
each millimeter being divided into 100 
equal parts, the space between each di- 
vision therefore being equal to 0.01 mm. 

3. If 100 division lines of the 
ocular step micrometer cover 0.01 mm. 
of the object micrometer, then each 
division line of the step micrometer has 
the value 0.0001 mm. or 0.1 micron. 

These values are only accurate when 
the draw-tube of the microscope is 
drawn out according to the following 
table. 

4. Using the ocular step micrometer 
and the object micrometer, find the tube 
length at which each objective gives a 
definite value in microns. This will vary 
some even with the Leitz oculars and 

objectives, so the tube length for each FIG. 25. Micrometer 
combination of lenses must be deter- 
mined separately for any make of 
microscope. 



-:JO 



40 



70 



80 



Scale in Ocular of 
the Leitz Ocular 
Step Micrometer. 



VALUES FOR LEITZ ACHROMATIC OBJECTIVES 



No. of Objective. 


Mark on Draw-tube. 


Micrometer Value in Microns of Each 
Division Line of Step Micrometer. 


3 

7 
1/12 oil imm. 


141 
174 
150 


10 
1.5 
1.0 



5. Multiply the number of division lines of the ocular 
micrometer covered by the organism in question by the value 



72 



GENERAL MICROBIOLOGY 



of the division line as determined in the above table. This 
gives the measurement directly in microns. 

Microorganisms may be measured more accurately by 
mounting them in Chinese ink, as they cannot move, are 
not shrunken or distorted as often occurs with stained speci- 
mens, and are clearly seen. Preparations stained with 




FIG. 26. Ocular Filar Micrometer for Very Exact Measurements. 
By means of a micrometer screw a line is moved across the 
field. The distance is measured by means of the divisions on 
the drum. 

aqueous-alcoholic dyes stand next in preference, never 
strong stains like carbol-fuchsin, anilin-water dyes, or 
saturated alcoholic solutions of dyes. 

II. Using a Filar Ocular Micrometer. The filar ocular 
micrometer is an instrument for the accurate measure- 
ment of microscopic objects. It consists of an ocular, 
between the eye and field lenses of which there is a scale 
ruled on glass in millimeters and half millimeters, below 



METHOD OF MEASURING MICROORGANISMS 73 

and across which a single-line index is made to travel by the 
use of the micrometer screw. 

The micrometer screw is fitted with a drum divided into 
100 parts, one revolution of which moves the index line 
one division or 5 microns. The drum is divided into fifty 
parts, so that each mark on the drum scale corresponds to 

5 microns 

or 0.1 micron. 

oU 

The micrometer value of each interval should be cali- 
brated for each objective with the aid of the object microm- 
eter. The eye-lens of the micrometer is adjustable to enable 
the observer to focus the scale accurately. 

The filar ocular micrometer slips into the draw-tube 
of the microscope like any ordinary ocular and may be 
fixed in position by the milled-head screw on the side. 

A. Calibration of the Filar Ocular Micrometer. 

Apparatus. Filar ocular micrometer; microscope; ob- 
ject micrometer. 

Method. 1. Place the object micrometer under object- 
ive No. 3 and ocular No. 1, drawing out the draw-tube to 
17 mm. 

2. Bring the lines on the object micrometer into a sharp 
focus. 

3. Replace ocular No. 1 with the filar ocular microm- 
eter. 

4. Focus again so that the division of the object microm- 
eter and the ocular micrometer are equally clear and turn 
the ocular micrometer so that the lines of both micrometers 
are parallel to each other. 

5. Determine how many microns one space of the ocular 
micrometer represents. 

Example. Six divisions of the ocular micrometer-scale cover the 
same length as three divisions of the object micrometer on which each 
division is 1/100 millimeter or 10 microns; therefore, six divisions of 
the ocular micrometer scale equals 30 microns and one division equals 
1/6 of 30 or 5 microns. 



74 GENERAL MICROBIOLOGY 

6. Determine how many revolutions of the drum (from 
to 0) are necessary to move the movable line one division 
and from this determination calculate the value in microns, 
of one division on the drum. One determination of this 
value is sufficient. 

B. Method of Using the Filar Ocular Micrometer. 

1. Replace the object micrometer by a slide containing 
organisms, focus, and measure an organism, counting the 
number of divisions the drum is turned in moving the 
movable line from end to end of the organism. 

Example. If the drum is turned two divisions the organism was 
two times 0.1 micron in length or 0.2 micron. 

2. To measure the microorganisms with a higher power 
objective, the value of each division of the scale has to be 
recalibrated. 

EXERCISE 20. DETERMINATION OF THE RATE OF 
MOVEMENT OF MOTILE ORGANISMS 

Apparatus. Microscope; Leitz "step" micrometer; stop- 
watch; hanging-drop preparation of motile organisms. 

Method. Using a hanging-drop preparation of the 
organism to be examined, determine the rate of movement 
per second, using the step micrometer and a stop-watch. 

EXERCISE 21. PREPARATION OF A HANGING DROP 

The purpose of the hanging-drop preparation is to study 
bacteria in the living condition; to demonstrate (a) their 
form, (6) arrangement, (c) motility (this is best ob- 
served from twenty-four-hour cultures), (d) appearance, 
(e) division of cells, (/) formation or presence of spores; 
(g) to determine the presence and types^of microorganisms in 
any material and to watch the changes in the predominat- 
ing types of the microbial flora in a medium from day to 
day; (h) and, in pathogenic bacteriology, to demonstrate 
agglutination. 



PREPARATION OF A HANGING DROP 



75 



Bacteria have two kinds of movement, the so-called 
Brownian or molecular movement, and true motility. The 
former may be demonstrated by examining the movement 
of powdered carmen rubrum in the hanging drop. A very 
little of the powder is sufficient. Brownian movement 
is shown more or less by all small particles of insoluble 
matter (including living non-motile or dead bacteria) in 
suspension. It is characterized by a vibratory movement 
affecting the entire field; the relative positions of the 
insoluble particles are never altered. This type of move- 




FIG. 27. Hanging Drop Slide. (Orig.) 



ment must be distinguished from that of true motility, 
which is characterized by the progressive movement, more 
or less rapid, of an organism across the field of the micro- 
scope, changing its position in the field independently of 
and in a direction contrary to other organisms present. 

There should be no currents of air entering under the 
cover-glass and passing through the concavity of the slide 
nor should there be currents in the liquid. The latter may 
occur if the organisms have not been well mixed through 
the drop in the process of preparation. If large numbers 
of the microorganisms in the drop are moving in one direc- 



76 GENERAL MICROBIOLOGY 

tion, this is an indication of currents in the liquid which 
have been induced by the liquid touching the side of the 
concavity, by the drop being too large, by improper mixing, 
or by air currents; this fault may be remedied by thoroughly 
mixing the bacteria in the drop with the straight needle 
or by resealing the cover-glass upon the slide. 

Apparatus. Clean cover-glasses; clean concave slides; 
platinum loop; straight platinum needle; Bunsen burner; 
distilled water; cover-glass forceps; melted paraffin or 
vaselin if preparation is to be sealed permanently. 

Method. 1. With a platinum loop place four small 
drops of water about the edge of the depression of the con- 
cave slide. 

2. For cultures: 

In liquid media. 

(a) With a sterile platinum loop transfer a portion 
of the culture to the center of a clean cover- 
glass. 

On solid media. 

(a) With a sterile platinum loop place a small drop 
of water or physiological salt solution in the 
center of a clean cover-glass. 

(6) With a sterile platinum needle transfer a minute 
portion of the culture to the drop of water so 
that only the faintest cloudiness appears. 

3. Quickly invert the cover-glass over the depression in 
the concave slide and gently depress the margin on the water 
until the chamber is sealed air tight. The hanging drop 
must not touch the bottom of the concavity. Note the 
illustration. If it is desired to keep the hanging drop 
longer than five to ten minutes, it may be sealed with 
paraffin as with the adhesion culture, or with vaselin. 

The drop must remain over the center of the concavity. 
If the drop touches the side of the concavity, the hanging 
drop as such is destroyed and it will be necessary to remake 
the preparation. If pathogenic organisms are used, both 



PKEPARATION OF A HANGING DROP 77 

slide and cover-glass must be placed in 1/1000 mercuric 
chloride or some equally efficient disinfectant for at least 
one hour before cleaning or reusing. 

4. Examine first with objective No. 3, then with object- 
ive No. 7 or the 1/12 oil immersion lens, using ocular No. 1 
in each case. After a perfect focus is obtained, ocular 
No. 4 may be used if desired. 

Manipulation of Microscope. Using the lowest power 
objective and ocular, focus the tube of the microscope 
down by means of the coarse adjustment until the objective 
nearly touches the cover-glass, being careful not to touch it. 
Then, with the eye at the ocular, focus up with the coarse 
adjustment and move the preparation until the edge of the 
drop comes plainly into view. This focal point may be passed 
without noticing it if the light is too intense or too dim. The 
edge of the drop is a curved line. The preparation 
should be so moved that this line cuts the center of the 
field. 

Focus up slightly, swing the No. 7 or 1/12 objective as 
desired, into place and after the field desired is obtained 
with the coarse adjustment, focus down until the objective 
nearly touches the cover-glass. Then with the eye at the 
ocular, focus up carefully with the coarse adjustment until 
the edge of the drop comes plainly into view. Use the fine 
adjustment to bring out details. 

In using the 1/12 oil immersion lens a small drop of im- 
mersion oil is placed in the center of the cover-glass, the 
1/12 objective swung into place as above. Greater care 
must be exercised in focusing, as this objective has a shorter 
working distance. 



78 



GENERAL MICROBIOLOGY 



EXERCISE 22. PREPARATION OF THE ADHESION 
CULTURE 

The purpose of the exercise is to show the germination 
of mold spores or the budding of yeast cells, i.e., colony 
formation. 

Apparatus. Clean cover-glasses; clean concave slides; 
melted paraffin; small glass rod or camel's-hair brush; 





FIG. 



28. Lindner's Adhesion Culture. (Adapted from Lafar's 
Technische Mykologie.) 



sterile cider in tubes. (Wort, milk and other media may be 
used as conditions demand.) 

Cultures. Pure culture of mold, or yeast. If mold 
spores are to be germinated, an old culture having spores is 
necessary. 

Method. 1. Inoculate a tube of sterile cider from the 
pure culture of the organisms to be studied. (Use spores 
in the case of mold and some of the cells for yeasts.) Dis- 
tribute well with the platinum needle. 

2. Transfer one loopful to a flamed cover-glass and 
spread in a thin film over the entire surface of the cover- 
glass, using the straight needle. If any of the cider adheres 
in droplets, shake them off. 



PREPARATION OF THE ADHESION CULTURE 79 

3. Breathe into the concavity of a concave slide until 
small droplets of moisture are visible on the glass. Before 
this moisture evaporates and while the cover-glass is 
still wet turn the cover-glass, culture side down, corner- 
wise, covering the concavity on the slide. 

4. Using the small glass rod or a camel's-hair brush 
dipped in hot paraffin, neatly seal the cover-glass on the 
slide so that the cavity will be air tight and the moisture 
will be retained. Success depends largely on quick work. 

5. Examine with objective No. 7 and ocular No. 1. 
There should be five to twenty spores or cells on a slide. 
If more are found, a new culture should be made. It 
may be necessary to inoculate a second tube of cider 
from the first to secure the proper dilution. 

6. If you are not familiar with the spores or cells of 
the organism to be studied, before making an adhesion 
culture, mount them in a drop of water heavily inoculated, 
cover with a cover-glass and examine microscopically. 

7. Keep the cultures at room temperature. Examine 
as often as possible for thirty-six hours and then every 
twenty-four hours till growth ceases. 

8. Draw as many stages as possible. The time required 
for spore germination is usually six to forty-eight hours. 

Note. Some molds grow quite extensively in the adhesion culture, 
even producing fruiting bodies. Very often both the mycelium and fruit- 
ing bodies show peculiar abnormalities and should never be drawn to repre- 
sent normal structures. These abnormalities are the result of the 
peculiar environment. 

9. Failure to obtain growth of the mold spores or yeast 
cells may be due to imperfect sealing, insufficient moisture 
at the start or too many cells on the cover-glass. If the 
adhesion culture fails to grow, a fresh tube of cider must 
be inoculated before making new adhesion cultures, as the 
food materials contained in the medium are partly or en- 
tirely used. In the case of mold spores it is reasonable 
to expect that any mold spores in the adhesion culture 



80 GENERAL MICROBIOLOGY 

will have germinated within forty-eight hours after prepar- 
ing the mount. 

Note. This method may be utilized to study the colony develop- 
ment of bacteria also. 

EXERCISE 23. PREPARATION OF THE MOIST- 
CHAMBER CULTURE 

The purpose of the exercise is to study colony formation 
in molds, yeasts and bacteria. 

Apparatus. Clean cover-glasses; small glass rings, 




FIG. 29. Moist Chamber Culture. (Orig.) 

clean; clean slides; sterile pipette; paraffin or vaselin; 
sterile distilled water in test tube; tube of a sterile liquid 
nutrient medium; platinum needle and loop; forceps; 
cover-glass. 

Culture. Pure culture of organism to be studied. 

Method. 1. With forceps, carefully sterilize in a 
flame a glass slide and a glass ring designed for this pur- 
pose. This should be done by a swinging motion to insure 
uniform distribution of the heat. 

2. Around the edges of the ring, after it has cooled suf- 
ficiently, place a little vaselin, while the ring is still held 
in sterile forceps. 



AGAR HANGING-BLOCK CULTURE 81 

3. Place the ring on the slide and press it down gently 
to make contact complete. The vaselin renders the cham- 
ber water tight. 

4. Seal the ring to the slide with melted paraffin as in 
the adhesion culture, to keep it from slipping around. 

5. With a sterile pipette convey into this chamber 
just enough (boiled) water to cover the bottom. 

6. Vaselin the upper edge of the ring. 

7. Inoculate lightly the tube of liquid medium with the 
organism to be studied. Distribute throughout the liquid 
with the needle. 

8. Transfer one loopful to a cover-glass. 

9. Using the straight needle, spread in a thin film over 
the entire surface of the cover-glass. If any of the liquid 
adheres in droplets, shake them off. 

10. Press the cover-glass, medium side down, upon the 
upper vaselined edge of the ring. 

11. Seal the edge of the cover-glass to the glass ring in 
several places with paraffin to prevent it from slipping 
around. 

12. Incubate at the desired temperature. 

This possesses some advantages over the adhesion cul- 
ture, as more air and moisture and consequently more favor- 
able conditions are furnished for growth. With a little 
more delicate manipulation agar or gelatin can be used 
in place of the liquid medium. 

EXERCISE 24. PREPARATION OF AGAR HANGING- 
BLOCK CULTURE 

This method was devised by Hill * for studying to 
better advantage the morphology and manner of multi- 
plication of bacteria. 

Carry out this procedure in a special plating room or 
chamber if possible, to avoid contamination from air cur- 
rents. 

*Hill, Journal of Medical Research, Vol. VII, March, 1902, p. 202. 



82 GENERAL MICROBIOLOGY 

Apparatus. Clean cover-glasses; clean concave slides; 
ordinary slides, clean; tube of sterile nutrient agar or 
gelatin; paraffin; two sterile Petri dishes: scalpel; plati- 
num loop. 

Culture. Pure culture of the organism to be studied. 

Method. 1. Liquefy a tube of nutrient agar or gelatin, 
pour it into a sterile Petri dish to the depth of about 4 mm. 
and allow it to harden. 

2. With the flame-sterilized scalpel, cut out a block of 
agar about 8 mm. square. 

3. Raise the agar block on the blade of the scalpel and 
transfer it, under side down, to the center of a sterile slide. 

4. With a sterile platinum loop, spread a drop of the 
liquid culture (or suspension of organisms from a solid 
culture medium) over the upper surface of the agar block 
as if making a cover-glass film. 

5. Place the slide and block in a sterile Petri dish and 
incubate for ten minutes at 37 C. to dry slightly. 

6. With sterile forceps, lower a clean, dry, sterile cover- 
glass carefully on the inoculated surface of the agar (avoid- 
ing air bubbles), so as to leave a clear margin of cover- 
glass overlapping the agar block. 

7. Invert the preparation and, with the blade of the 
scalpel, remove the slide from the agar block. 

8. With the platinum loop, run a drop or two of melted 
agar around the edges of the block. This solidifies at once 
and seals the block to the cover-glass. 

9. Sterilize a concave slide. 

10. Invert the cover-glass with the block attached on 
the concave slide and seal it in place, firmly, with paraffin. 

11. Observe immediately and later from time to time 
with ocular No. 1 and objective No. 7 or the oil immersion 
lens. 



LINDNER'S CONCAVE-SLIDE METHOD 83 



EXERCISE 25. LINDNER'S CONCAVE-SLIDE METHOD 
FOR DEMONSTRATING FERMENTATION 

The object of this exercise is to test the fermenting 
power of yeasts. 

Apparatus. Three clean concave slides; three clean 
cover-glasses; sterile filter paper (place several pieces 8 cm. 
square in a Petri dish and sterilize in the hot air); three 
tubes of sterile wort or cider; three sterile 1 c.c. pipettes; 
forceps; melted paraffin; platinum needles; Bunsen burner. 

Cultures. Saccharomyces cerevisice; Saccharomyces apicu- 
latus; Torula rosea. 

Method. The following procedure is to be used for each 
organism to be tested: 

1. Using the straight needle, inoculate a tube of wort 
with Saccharomyces cerevisioe and mix well through the 
medium. 

2. Sterilize a concave slide in the flame. 

3. Using a sterile pipette, fill the concavity of the 
slide until the liquid " rounds up " over the concavity. 

4. Holding a cover-glass in the forceps, sterilize it in 
the flame. 

5. Lay the cover-glass on the end of the slide and push 
it over with the forceps until the cover-glass covers the 
concavity, thus sealing in the inoculated liquid. There must 
be no air bubbles. The preparation must be made over again 
if this occurs. 

6. Remove the excess liquid with sterile filter paper, 
using forceps to hold the paper. 

7. Seal the cover-glass with paraffin as with the adhesion 
culture. 

8. Place the slides in a horizontal position in Petri 
dishes, or in a slide box as convenient. 

9. Incubate at 25 to 30 for twenty-four hours. Gas 
bubbles will be formed in twenty-four to forty-eight hours, 
if any fermentation occurs. 



84 GENERAL MICROBIOLOGY 

10. Record the time of fermentation and the relative 
fermentation of each yeast and draw conclusions from 
your results. 

11. Do your results coincide with those in the refer- 
ences given? 

12. State in detail your results with any conclusions 
which follow from them, and point out the practical applica- 
tions which may be made. 

By the use of sugar broth in place of wort, this method 
may be employed for bacteria as well. 

REFERENCES 

LAFAR: Technical Mycology, Vol. II, Part 1, pp. 113, 114, and Part 2, 
pp. 401-407, 430-436. (Index of three volumes is in Vol. II, 
Part 2.) 

GREEN: Soluble Ferments and Fermentation, pp. 333-362. 

CONN: Bacteria, Yeasts and Molds, pp. 56-99. 

EXERCISE 26. LINDNER'S DROPLET CULTURE 

The object of the exercise is to isolate a single yeast 
cell and watch its development. 

Apparatus. Sterile cover-glass (sterilize in flame); con- 




FIG. 30. Lindner's Droplet Culture. (Adapted from Lafar's Tech- 
nische Mykologie.) 

cave slide; forceps; sterile toothpick (sterilize in a test 
tube in hot air) ; paraffin; India ink. 

Culture. Pure culture of some yeast. 

Method. 1. Inoculate a tube of cider with yeast. 
Distribute the organisms well. 

2. Using the sterile toothpick, make five rows of small 



CHINESE INK PREPARATION 85 

2. Using the sterile toothpick, make five rows of small 
droplets (five droplets in a row) on a sterile cover-glass and 
place, culture side down, over the concavity of a sterile slide. 

3. Seal the cover-glass with paraffin as in the prepara- 
tion of the adhesion culture. Examine microscopically. 

4. Locate one droplet which contains only one cell. 
Using India ink, write the location of this droplet on the 
slide. 

5. Make a drawing of each stage of development until 
growth ceases. Why does the cell stop growing? 

6. State in detail your results with any conclusions 
to be drawn a*nd point out the practical applications which 
may be made. 

This method may be used to advantage with mold 
spores. 

EXERCISE 27. CHINESE INK PREPARATION 

Chinese ink may be used to make bacteria more easily 
visible microscopically and to aid in taking correct measure- 
ments. 

Apparatus. Sterile, dilute Chinese ink; * clean flamed 
glass slides. 

Cultures. Pure cultures (young agar streaks are best). 

Method. 1. Place one loopful of distilled water and 
three loopfuls of sterile Chinese ink in a row on a clean 
glass slide, about 2 cm. apart. 

2. Inoculate the loopful of water from the original 
culture. 

3. Distribute the organisms well with a platinum 
needle. 

4. Then inoculate the adjoining drop of ink from the loop- 
ful of water, the second drop of ink from the first, etc. 

6. Stir each loopful of ink well and then spread it so as 
to cover an area about 1 cm. square. 

* See appendix for method of preparation. 



86 GENERAL MICROBIOLOGY 

6. Let dry. If desired the specimen may be mounted 
in Canada balsam before examining. 

7. Examine with either the 1/7 or the oil immersion 
objective. 

8. Write the name of the organism, the date, and your 
name on the glass with India ink. 

By the use of the Chinese ink preparation, it is possible 
to examine any organism unstained. Organisms so treated 
neither shrink nor in any way change their form, making 




FIG. 31. Chinese Ink Preparation. (Orig. Northrup.) 

accurate measurement possible. Stains often cause organ- 
isms to appear swollen or shrunken. 

The motility of bacteria may be more easily demon- 
strated by adding a very slight amount of this ink to a 
hanging drop of the organism being studied. 

Caution. Chinese ink is very expensive. When making 
preparations, use every precaution to keep your supply 
sterile, as contaminating organisms may be confused with 
the culture under study. A control preparation to which 
no microorganisms have been added will serve to detect 
their presence, 



THE STAINING OF MICROORGANISMS 87 



EXERCISE 28. THE STAINING OF MICROORGANISMS 

Microorganisms are devoid of color as a rule and are 
stained for the purpose of observing their morphology to 
better advantage than in a hanging drop. Staining also 
often serves to bring out certain morphological character- 
istics which are otherwise not evident, such as the presence 
of metachromatic granules or a peculiar arrangement of 
the protoplasm, resulting in what are known as " beaded 
forms." 

The stains best suited to bacteria are the basic anilin 
dyes which are derived from the coal-tar product anilin 
(CeHsNH^). Many of them have the constitution of salts. 

Such compounds are divided into two groups, according 
as the staining action depends on the basic or the acid 
portion of the molecule. Fuchsin, gentian violet and methy- 
len blue are basic dyes, while eosin, picric acid and acid 
fuchsin are acid dyes. 

These groups have affinities for different parts of the liv- 
ing cells. The basic stains have a special affinity for the 
nuclei of tissues and for bacteria, the acid for the proto- 
plasm and not for bacteria. The violet and the red anilin 
dyes In order, are the most intense in action, easily over- 
staining the specimen. It is difficult to overstain with 
methylen blue. For this reason this stain is to be pre- 
ferred where the bacteria occur in thick or viscid substances, 
like pus, mucus or milk. In the presence of alkali, how- 
ever, the stain acts more energetically. 

Stock solutions of the ordinary dyes are commonly 
used. These are prepared by making a saturated solution 
of the dye in absolute alcohol; this is diluted with water 
as needed. 

Saturated alcoholic solutions of dyes will stain bacteria 
with difficulty. The best results are obtained with the 
diluted stain, spoken of here as an " aqueous-alcoholic " 
stain. 



88 . GENERAL MICROBIOLOGY 

Apparatus. Clean cover-glasses; clean slides; cover- 
glass forceps; platinum loop and needle; Bunsen burner; 
small pieces of filter paper; distilled water; aqueous-alco- 
holic solution of fuchsin, methylen blue, etc.; Canada 
balsam; microscope. 

Note. See appendix for formulae of stains. 

Method. 1. Flame a clean cover-glass, holding it by 
one corner with cover-glass forceps. 

2. Place one loopful of distilled water in its center. 

3. Touch the growth on slant agar lightly with a ster- 
ilized platinum needle and transfer a very little of the mate- 




FIG. 32. For use in mounting permanent cover-glass preparations. 

(Orig.) 

rial to the drop, and only sufficient to make it very slightly 
cloudy. - 

4. Flame the needle and allow it to cool. 

5. Spread the drop over the entire cover-glass with one 
or two strokes of a straight needle. In the case of path- 
ogenic microorganisms use a flat loop 2 mm. in diameter, 
and limit the spreading to the inner three-fourths of the 
cover-glass. 

6. Allow to dry in air. 

7. Fix the preparation on the cover-glass by passing 
the cover-glass, specimen side up, three times through the 
flame of a Bunsen burner. The speed is measured by 
moving the cover-glass and forceps in a circle of 1 ft. diam- 
eter in one second. 



THE STAINING OF MICROORGANISMS 89 

8. Flood the entire specimen-side of the cover-glass 
with stain, using a pipette. 

9. Allow the stain to act a short time. 

Note. The time required for staining varies so much with the 
different stains, different organisms and their physiological conditions, 
that no exact time can be given. In general, a good specimen is 
obtained by staining one-half to one minute with fuchsin or gentian 
violet, or one to five minutes with methylen blue. 

10. Wash the specimen in running water. 

11. Mount the cover-glass in water, specimen side 
down, on a clean slide. 

12. Dry the upper surface of the cover-glass and take 
up any excess of water by means of filter paper. 

13. Examine the slide under the microscope, using 
objective No. 7 and ocular No. 1. 

14. If satisfactory, remove the cover-glass carefully 
from the slide, floating it off if necessary. 

15. Allow it to dry in the air, specimen side up. 

16. Place a clean slide exactly on the figure (Fig. 32). 

17. Let a small drop of Canada balsam fall in the center 
of the slide, marked by the circle. 

Note. The consistency of the Canada balsam should be like thin 
cream. The diameter of the glass rod should not be more than 4 mm. 

18. Place the cover-glass, specimen side down, on this 
drop. 

19. Allow the balsam to spread over the entire under 
surface of the cover-glass (without pressing it down on the 
slide) and keep the cover-glass straight, coinciding with the 
lines of the figure. 

20. Label, stating in order, the name of the organism, 
the age and kind of culture, the stain used, the date, your 
own name and the purpose of the stain if otherwise than 
ordinary, e.g., spore stain. 

21. Allow the slide to stand in a horizontal position for 
a few days until the balsam becomes hard. 



90 



GENERAL MICROBIOLOGY 



EXERCISE 29. ANJESZKY'S METHOD OF STAINING 

SPORES 

Spores are not stained by the ordinary staining methods, 
as their physical nature differs from that of the vegetative 
rods within which they are formed. By proper treatment 
with strong anilin dyes, however, it is possible to force the 
stain into the spore. Once within the spore it is as dif- 
ficult to remove the dye as it was to cause it to enter. 




FIG. 33. Contrast Spore Stain, Carbol-fuchsin and Methylen Blue, 
X1500. (Orig. Northrup.) 

By a careful decolorization with a weak acid, it is pos- 
sible to remove the stain from everything on the cover- 
glass except from the spores. Then, on application of a 
dye of a contrasting color, the specimen will show, e.g., a 
bright red spore within a blue bacterium. 

The fundamental principles of this method are also 
used for staining " acid-fast " organisms, ^ Bad. tuber- 
culosis. 

Apparatus. Clean cover-glasses; clean slides; cover- 
glass forceps; platinum loop and needle; Bunsen burner; 



ANJESKY'S METHOD OF STAINING SPORES 91 

carbol-f uchsin ; methylen blue, aqueous^alcoholic ; hydro- 
chloric acid, 0.5%; sulphuric acid, 5%; Canada balsam; 
microscope. 

Culture. Culture of an organism just beginning to 
show spore formation. 

Method. 1. Prepare a cover-glass film of the spore- 
containing organism and allow it to dry. 

2. While it is drying, warm some 0.5% HC1 in the small 
evaporating dish over a Bunsen burner until it steams 
well and bubbles begin to form. 

3. When the solution is hot and the smear dry, drop 
the cover-glass upon the fluid and allow it to act upon the 
unfixed smear for three to four minutes. 

4. Wash and dry the cover-glass. 

5. Fix in the flame for the first time. 

6. Stain with carbol-fuchsin by flooding the cover- 
glass with the stain, warming twice until fumes arise. 

7. Allow to cool, and wash in water. 

8. Decolorize with 5% H 2 SO 4 . Spores are treated with 
a mild decolorizing agent, as they are much less resistant 
to acid than are acid-fast bacteria. (See p. 93, step 7.) 

9. Wash in water. 

10. Counterstain for one to two minutes with methylen 
blue. 

11. Wash, dry and examine the specimen in water. 
If satisfactory, dry it and mount in balsam. 

The whole procedure should not take longer than eight 
to ten minutes. 

REFERENCE 

MCFARLAND: Textbook of Pathogenic Bacteriology, p. 188. 



92 GENERAL MICROBIOLOGY 



EXERCISE 30. METHOD OF STAINING TUBERCLE 
AND OTHER ACID-FAST BACTERIA 

Acid-fast bacteria are so termed from their reaction to 
a special staining process. This process consists in staining 
the specimen containing, for example, tubercle bacteria, 
with hot carbol-f uchsin and decolorizing for a short time 
with acid; the acid takes the dye out of all other material, 
bacteria and blood or other body cells that may be present, 
leaving the tubercle bacteria stained red. This staining 
process is essentially the same as for spores, but the prin- 
ciple is different. 

The property which some bacteria possess of being 
acid-fast is attributed to the presence of fat and wax-like 
substances in their cells. This seems to be proved by the 
fact that when the bacterial cell substance of tubercle 
bacteria has been freed from these fats and waxes by 
extraction with absolute alcohol and ether, this property 
is lost. 

Apparatus. Clean slides; clean cover-glasses; plati- 
num loop; copper staining dish; Bunsen burner; forceps; 
carbol-f uchsin; sulphuric acid, 20%; methylen blue, aque- 
ous-alcoholic; immersion oil; Canada balsam; specimen 
to be examined. 

Method. 1. Using a sterile loop, smear some of the 
specimen in the center of one surface of a clean slide, 
taking care not to come within 0.5 cm. of the edge. 

Note. This may be applied to sputum, pus, etc. In case of 
tubercles or diseased organs or tissues these may be cut open with a 
scalpel, a portion incised, and grasping this portion with the forceps 
a smear made directly on the slide, following the precautions above. 
If pure cultures are to be examined, a cover-glass specimen may be 
made in the usual way. 

2. Dry the slide in air. 

3. Fix in the flame. 

4. Support the slide on the copper staining dish; flood 



METHOD FOR STAINING FLAGELLA 93 

the slide with carbol-fuchsin until the stain " rounds 
up." 

5. Heat the under side of the slide directly, with a flame 
until the carbol-fuchsin steams (but not boils). Keep the 
stain steaming for five minutes. 

6. Wash in water. 

7. Decolorize by dipping the slide preparation into 
20% H2SC4 for an instant and washing immediately. This 
process may have to be repeated two or three times. If 
not careful, however, the tubercle bacteria may be decolor- 
ized. If this happens, their acid-fast property will be 
destroyed to some extent. 

8. Counterstain with aqueous-alcoholic methylen blue. 

9. Wash in water, dry and examine directly with the 
oil immersion lens. The specimen, if a good one, may then 
be mounted in the usual way without removing the immer- 
sion oil. 

EXERCISE 31. METHOD FOR STAINING FLAGELLA 

Flagella, the exceedingly delicate organs of locomotion 
of bacteria, cannot be seen in an unstained or in an ordinary 
stained preparation. Special staining methods must be 
employed to make them visible. They are generally ren- 
dered visible by precipitating some chemical on them; 
this generally increases their width considerably. 

The staining of the flagella of bacteria is the most 
difficult of all bacteriological procedures and it generally 
requires considerable practice to insure good results. 

There are many methods for staining flagella. This 
one, however, has met with considerable success with stu- 
dents. Failure to make a good flagella stain with any 
method is no sign that the student is not a good work- 
man, nor is success the sign of a good bacteriologist. 

Apparatus. Clean glass slides; absolutely clean cover- 
glasses; small platinum loop; several cover-glass forceps; 



94 GENERAL MICROBIOLOGY 

distilled water; mordant for flagella staining; anilin-water 
fuchsin or gentian violet. 

Culture. Agar slant culture, twelve to eighteen hours old. 

Note. The best results are obtained if successive generations of 
this organism have been transplanted every eighteen to twenty-four 
hours for several generations. 

Method. 1. Place three drops of distilled water on a 
clean glass slide. 

2. Transfer a small amount of bacterial material from 




- FIG. 34. Flagella Stain. B. typhosus, X1500. 

the moist portion of the agar slant culture to the first drop 
by means of a small platinum loop, using only enough of 
the material to make this drop very slightly cloudy. 

3. Flame the needle and transfer a small portion from 
the first drop to the second. 

4. Proceed in like manner in preparing the third dilu- 
tion. 

5. Place a number of absolutely clean cover-glasses in 
cover-glass forceps. 

6. By means of a platinum needle bent at right angles 
near the end, make smears on the cover-glasses from the 



GRAM'S METHOD OF STAINING 95 

second and third drops on the slide by drawing the bent 
needle once, lightly across the cover-glass. // there is any 
tendency of the smear to roll or " gather in drops," the cover- 
glass should be discarded and a clean one substituted. This 
is imperative. 

7. Allow the preparation to dry for about five min- 
utes. 

8. Filter on each of the fixed smears enough of the mor- 
dant to cover the cover-glass. 

9. Allow to stand about five minutes at room tempera- 
ture. 

10. Wash off the mordant in a small stream of 
water. 

11. Draw off the excess water from the edge of the cover- 
glass by means of filter paper. 

12. Stain with anilin-water fuchsin or anilin-water gen- 
tian violet for about five minutes, either cold or by warm- 
ing somewhat over a low flame. 

13. Wash off the excess stain with clean water. 

14. Mount on a slide in water. 

15. Absorb the excess water with filter paper. 

16. Examine under the microscope. If the prepara- 
tion has been successful, it may be dried and mounted 
in balsam, 

EXERCISE 32. GRAM'S METHOD OF STAINING 

Certain organisms, when stained with anilin-water 
gentian violet and afterwards treated with a solution of 
iodin and washed in alcohol or anilin oil, give up the stain; 
others retain the color when subjected to this process. 
These latter organisms are said to be Gram-positive, those 
losing the stain are Gram-negative. 

This phenomenon is interpreted by Benians to be due 
to the possession of a definite cell-envelope which by the 
action of iogiin is rendered impermeable to alcohol. His 



96 GENERAL MICROBIOLOGY 

experiments show that so long as the Gram-stained cell 
is intact, the solvent is unable to remove the stain, but 
that as soon as the cell is crushed and injured, the stain 
is, in great part, dissolved out. The amorphous debris 
obtained from broken-up Gram-positive bacteria does not 
retain Gram's stain. 

Apparatus. Clean slides; clean cover-glasses; plati- 
num loop and needle; cover-glass forceps; distilled water; 
anilin- water gentian violet; Lugol's iodin solution; aceton- 
alcohol. 

Culture. Agar slant cultures preferably. 

Method. 1. Prepare a cover-slip film and fix in the 
usual way. 

2. Stain in anilin-water gentian violet three to five 
minutes. 

3. Wash in water. 

4. Treat with Lugol's iodin solution until the film is 
black or dark brown. 

5. Wash in water. 

6. Dry in air. 

7. Wash in aceton-alcohol until no more color is dis- 
charged. 

8. Wash in water. (Counterstain at this point if desired.) 

9. Dry in air. 

10. Mount in Canada balsam. 

Note. The Gram-Weigert method is more applicable in case of 
sections of tissues. The directions from 1-6 are the same. The speci- 
men is washed in anilin oil 1 part, xylol 2 parts, instead of alcohol, 
washed further in xylol and mounted at once in Canada balsam. Bad. 
bidgaricum in milk is very beautifully demonstrated by this modified 
method. 

A few of the more common Gram-positive and -negative 
organisms are appended. This is not as important a diag- 
nostic method as has been formerly supposed, because 
the reaction occurring often depends upon the age of the 
culture, the medium on which it is grown, etc. 



METHOD FOR STAINING CAPSULES 97 

GRAM-POSITIVE ORGANISMS. GRAM-NEGATIVE ORGANISMS. 

Staph. pyogenes aureus and albus Bact. mallei 

Strept. pyogenes Bact. aerogenes 

Bact. anthracis B. typhosus 

Bact. tuberculosis B. coli communis 

B. alvei B. cholera? suis 

B. tetani M. gonorrhea? 

Bact. acidi lactici Msp. deneke 

Bact. bulgaricum Msp. finkler-prior 

B. megaterium Spirocheta obermeieri 

B. subtilis Proteus tulgaris 

B. myeoides Ps. medicaginis 

B. mesentericus vulgatus B. amylovorus 

M. tetragenus Ps. campestris 

Streptothrix actinomyces B. phytophthorus 

Sacch. cerevisice and other yeasts B. caratovorus 

Molds 

REFERENCE 

BENIANS, T. H. C.: "Observations on the Gram-positive and Acid- 
fast Properties of Bacteria." Jour, of Path, and Bact., Vol. 
XVII, pp. 199-211 (1912). 

EXERCISE 33. METHOD FOR STAINING CAPSULES 

Some bacteria possess a gelatinous envelope or " cap- 
sule " which in some species surrounds each individual 
organism, and in others, groups of organisms. The pres- 
ence of this capsule may be demonstrated by various 
special staining methods. The capsule takes the stain 
much less quickly than does the organism, leaving a light- 
colored halo about it. The presence of a capsule does not 
always indicate that the organism forming it is a slime- 
forming organism, nor does the fact that an organism is a 
slime-former preclude the possession of a capsule. 

Apparatus. Clean cover-glasses; clean slides; platinum 
loop; cover-glass forceps; filter paper, pieces; glacial 
acetic acid; gentian violet, aqueous-alcoholic. 



98 GENERAL MICROBIOLOGY 

Cultures. Cultures in milk, serum, etc., media. 

Method. 1. Prepare the cover-glass specimen directly 
from the medium without the use of water. Spread and fix 
in the usual manner. 

2. Flood the specimen side of cover-glass with glacial 
acetic acid. 

3. Drain immediately without washing. A piece of 
filter paper may be touched to the edge of glass to take 
up surplus water and facilitate drainage. 

4. Stain with aqueous-alcoholic gentian violet for a 
few seconds. 

5. Examine under the microscope. 

6. Wash, dry and mount, 

EXERCISE 34. METHOD OF MAKING IMPRESSION 
PREPARATIONS 

Impression preparations (Klatschpraparat) are pre- 
pared from isolated colonies of bacteria in order that their 
characteristic formation may be examined by higher powers 
than can be used with the living cultivation in situ. They 
are prepared from plate cultivations. Young colonies of 
Bad. anthracis produce beautiful impression preparations. 

Apparatus. Clean cover-glasses; clean slides; Novy 
cover-glass forceps; dissecting needle; stain. 

Culture. Agar plate culture containing well-isolated 
colonies of organism to be studied. 

Method. 1. Taking a clean cover-glass in the Novy 
forceps, open the plate and rest one edge of the cover- 
glass on the surface of the medium a little to one side of 
the selected colony. 

2. Lower it carefully over the colony until horizontal. 
Avoid any lateral movement or the inclusion of air bubbles. 

3. Press gently on the center of the upper surface of 
the cover-glass with the points of the forceps to insure 
perfect contact with the colony. 



STAINING THE NUCLEI OF YEAST CELLS 99 

4. Steady one edge of the cover-glass with the forceps 
and pass the point of the dissecting needle just under the 
opposite edge and raise carefully; the colony will be adher- 
ent to it. 

When nearly vertical, grasp the cover-glass with the 
forceps and remove it from the plate. Re-cover the 
plate. 

5. Place the cover-glass specimen side up on desk and 
cover with half a Petri dish until dry. 

6. Fix in the flame. 

7. Stain and mount as with ordinary cover-glass speci- 
men, being careful to perform all washing operations with 
extreme gentleness. 

EXERCISE 35. METHOD OF STAINING THE NUCLEI 
OF YEAST CELLS 

The nuclei of yeast cells are not visible in unstained or 
in ordinary stained specimens. A special method of pro- 
cedure must be used. 

Apparatus. Clean cover-glasses; clean slides; forceps; 
ferric ammonium sulphate, 3% aqueous solution; Ehrlich's 
hematoxylin solution; two staining dishes for slides. 

Culture. Culture of Saccharomyces or Torula. 

Method. 1. Prepare and fix the film upon the slide in 
the usual way. 

2. Soak in 3% ferric ammonium sulphate "for two hours. 

3. Wash thoroughly in water. 

4. Stain in hematoxylin solution for thirty minutes. 

5. Wash in water. 

6. Differentiate in ferric ammonium sulphate solution 
for one and a half to two minutes, examining wet under 
the microscope during the process. 

7. Wash, dry and mount. 



100 GENERAL MICROBIOLOGY 



GENERAL CHARACTERISTICS OF MOLD GROWTH 
AND HINTS FOR STUDY 

A brief description of the molds to be studied in the 
laboratory is here given. The references cited will give 
more in detail of their structure, importance and occur- 
rence. 

In these descriptions, there have been noted the parts 
of the structure of each mold that are to be found micro- 
scopically and drawn, also the quickest method of obtain- 
ing the best results. All microscopic drawings and meas- 
urements can be secured from the adhesion culture or the 
moist-chamber culture. 

Rhizopus nigricans Black mold 
(Mucor stolonifer) 

The mycelium in the advanced stage consists of rhizoids 
(rootlets), bearing clusters of sporangiophores, joined by 
long hyphse (the stolons) to the mycelium proper. The 
hyphse are non-septate. 

The fruiting bodies consist of typical sporangia (spore 
cases containing spores) borne on the enlarged end (col- 
umella) of the sporangiophore. Spores are liberated by the 
bursting of the sporangium. 

The columella can be observed in fruiting bodies of a 
light brown color; white sporangia are too young and black 
too old to show this structure. If no fruiting bodies grow 
in the adhesion culture, they may be studied directly from 
a plate culture by preparing a glycerin slide. Take care 
not to burst the sporangium when transferring it to the 
slide. 

Aspergillus niger Black mold 

The mycelium of this mold consists of septate hyphse 
with frequent dichotomous branching. 



CHARACTERISTICS OF MOLD GROWTH 101 

The fruiting body (asexual) consists of an erefet- condio- 
phore usually ending more or less abruptly in a dilation or 
head which bears closely packed sterigmata each of which 
in turn bears a single chain of conidia, the newly formed 
conidium being pushed away by the formation of a new 
spore; thus the conidium at the end of the chain is the 
oldest. The conidia of this mold are black. 

Penicillium italicum Blue-green mold 

The mycelium consists of septate hyphae, having fre- 
quent dichotomous branching. 

The conidial fructification resembles a brush, the conidia 
(spores) being borne on the end of conidiiferous cells (ster- 
igmata) ; in this genus before the conidia appear, there is 
generally a primary and even a secondary branching of the 
condiophore in some species before the conidiiferous cells 
are formed. The species of Penicillium have more of a 
brush-like appearance than the species of Aspergillus. The 
spores of P. italicum are blue-green. 

Oospora (Oidium) lactis White mold 

The mycelium consists of septate hyphse, having di- 
chotomous branching; the hyphse are almost entirely sub- 
merged in the nutrient substrate. 

This differs from the other molds in that it does not have 
typical fruiting bodies. It reproduces by means of conidia, 
which are formed by a simple division of the hyphse. 
The conidia are colorless. 

REFERENCES 

MARSHALL: Microbiology, pp. 12-27. 

LAFAR: Technical Mycology, Vol. II, Part I, pp. 5, 15, 71-77; Part II, 

pp. 300-346, 451-455. 
KLOCKER: Fermentation Studies, pp, 184, 185, 274-282, 303, 304. 



PLATE I* 






Germination of Rhizopus Spore, Mycelium, Rhizoids and Development 
of Sporangiophores and Sporangium. 



Ripe Sporangium. 



PLATE II* 



103 





Sporangiophore with Columella Attached and" Ripe Sporangium 
Showing Spores Within. 




Various Stages in the Formation and Germination of a Zygospore. 

* Plates I-VI photographed from models manufactured by R. 
Brendel, Berlin Grunewald, Bismarck Allee 37, Germany. 



PLATE III 




MICROSCOPICAL EXAMINATION OF MOLDS 105 

EXERCISE 36. MICROSCOPICAL EXAMINATION OF 

MOLDS 

Apparatus. Clean cover-glasses; clean slides; hand 
lens or compound microscope; platinum needle and loop; 
dissecting needle; glycerin, 10%. 

Cultures. Plate culture of mold. 

Method. The gross structure of a mold colony upon 
a plate may be examined with a hand lens or by placing 
the inverted Petri dish culture on the stage of the compound 
microscope and examining with objective No. 3 and ocular 
No. 1. The structure may be examined in detail as follows: 

1. Select a young colony which shows colored fruiting 
bodies, if such are produced by the organism to be studied. 
(Growth from natural or artificial media may be treated 
in the same general way.) 

2. Using a sterile platinum needle, transfer a small 
portion of the mycelium and fruiting bodies to a drop of 
glycerin on a plain glass slide. If the mold growth is 
closely confined to the surface of the media (as with Peni- 
cillium or Aspergillus) , it is often desirable to cut out a 
small piece of the medium bearing the mold and lift to the 
slide by means of a sterile platinum loop. 

3. Tease out very gently, using dissecting needles or 
common pins. The mold structure is extremely delicate, 
so this operation must be performed with the utmost care. 

4. Place a cover-glass over the preparation. 

5. Examine with the microscope, using objective No. 3 
and ocular No. 1. When a portion of mycelium bearing 
fruiting bodies is found, examine with objective No. 7. 
Draw the young and the old fruiting organs. 



DESCRIPTION OF PLATE III 
Aspergillus, Showing Septate Mycelium, Conidiophore with 

Conidia, also Formation of As:ogonium. 

Penicillium, Germination of Spore, Formation of Mycelium, 

Septate Conidiophores with Conidia, Ripe Ascospore, 



PLATE IV 




THE STUDY OF MOLDS 107 



EXERCISE 37. THE STUDY OF MOLDS 

Apparatus. Ten sterile Petri dishes; four tubes of 
sterile slanted agar; ten tubes of sterile agar, for plates; 
four tubes of sterile cider or wort; four tubes of sterile 
gelatin; clean glass rings, slides and cover-glasses; hand 
lens; compound microscope; centimeter scale. 

Cultures. Pure or mixed cultures of the following four 
molds: Rhizopus nigricans; Aspergillus niger; Penicillium 
italicum; Oospora lactis. Mixed (or impure) cultures of 
two molds growing in their natural habitat will be found 
on each table. 

Method. 1. Plate out each mixed culture* making three 
straight needle dilution plates for each. Use agar as a 
medium. Place the plates in the constant-temperature room 
in the place assigned. Note the temperature. 

2. When the plates are twenty-four hours old, mark 
and draw a well-isolated typical colony of the mold from the 
most thinly populated plate. Measure and record the 
diameter of the colony in millimeters. 

3. When fruiting bodies begin to show, isolate a pure 
culture of each mold in cider (see Exercise 16). 

4. (a) As soon as growth begins to show in the tubes of 
cider (about twenty-four to thirty-six hours) make a 
macroscopic drawing of each. State the age of the culture. 

(b) When mycelium is well developed and fruiting 
bodies appear (as noted on plates) make a second drawing. 

* Two mixed and two pure cultures are furnished for study. These 
cultures owe their color to the presence of fruiting bodies or spores. 
Always endeavor to obtain spores when making inoculations from 
molds. 

DESCRIPTION OF PLATE IV 

Sexual Reproduction of Penicillium, Ascus Formation. 
Formation of Chains of Asci in Process of Ripening, Some Con- 
taining Ascospores, Section of a Ripe Ascus. 
Saccharomyces, Budding, Colony Formation, Produc- 
tion of Ascus and Germination of Ascospores. 



108 



GENERAL MICROBIOLOGY 




FIG. 35. Oospora (Oidium) lactis (after Hansen) see p. 104, Jorgensen. 

1. Hyphae with forked partitions; 2, two ends of hyphse one with 
forked partition, the other with commencement of development 
of a spherical link; 3-7, germinating conidia; 6-6'", germination of 
a conidium, sown in hopped beer-wort in Ranvier's chamber, 
and represented at several stages; at each end germ tubes have 
developed; after nine hours (6'") these have formed transverse 
septa and the first indications of branchings; 11-14, abnormal 
forms; 15, 16, hyphsn with interstitial cells, filled with plasma; 
17, chain of germinating conidia; 18, conidia which have lain 
for some time in a sugar-solution; the contents show globules 
of oil; 19, old conidia. 



THE STUD.Y OF MOLDS 109 

State the age of the culture. These cider tubes are then of 
the proper age from which to make inoculations. 

5. Pure cultures of the two remaining molds will be 
found in tumblers marked " Laboratory Cultures." 

Always leave such cultures in the place assigned, after 
using. 

Make cultures of each of the four molds as follows: 

(a) Agar slant (for method see Exercise 15). 

(b) Agar plate (giant colony) . Use Roux culture flask for 
Rhizopus nigricans (see pp. 2, 62). 

(c) Cider or wort (test-tube culture).* For method of 
inoculation of c and d see pp. 60, 61. 

(d) Gelatin stab (test-tube culture). Keep all gelatin test- 
tube cultures in cold-water bath or in a cool place (15 to 20 C). 

(e) Adhesion or moist-chamber culture. (See pp. 78-81). 
Prepare these cultures from the freshly inoculated cider tubes. 

6. Make drawing of spores of each mold from adhesion 
or moist-chamber culture as soon as preparation is made. 
Measure the spores and record the limits of size. 

7. Draw the twenty-four hour cultures each of a, b, c, d, 
and e and label in detail. Measure the spores which have 
germinated in e, and record the diameter and length of the 
mycelium. 

8. Make drawings of branched mycelium and several 
stages of development of fruiting bodies from a glycerin 
mount. This mount is most easily prepared from an agar 
plate colony. 

9. Make drawings of all cultures as soon as a marked 
development is seen over that of the preceding drawing. 
Three drawings of each culture should be sufficient. 

10. Measure giant colony of each mold every day 
and record the measurements. What is noted of the com- 
parative rate of growth? 

All drawings must be made directly on charts or in note- 

* Cider cultures have already been made of the two molds isolated 
from mixed culture. 



110 



GENERAL MICROBIOLOGY 



books as assigned. Describe the drawings at the time they 
are made. Descriptions are to be recorded in ink; use a 6H 
pencil for drawings. 

This outline or some modification of it may be employed 
for the various species of molds. 

MOLDS 



Name of student 


Desk No. 


Name of organism 


Isolated from 


Method of isolation 


Occurrence 


Importance 


Spore 



Stages of germination 



Drawn from preparation 



Mycelium 



Drawn from 



. preparation 



Method of reproduction 



Drawn from preparation 



Total organism 



Drawn from preparation 



Note: Mold, yeast and bacteria charts (pp. 110-111, 122-123, 135- 
138) may be procured from the College Printery, East Lansing, Mich. 



THE STUDY OF MOLDS 



111 



Cider or wort 
culture 
Titre 


Iday 




days 




days 




Incubated at 
C. 
















^J 









[^ 




Nutrient gelatin 
stab 
Titre 


1 day 




days 




.....days 




Incubated at 

c. 
















^J 




^J 




^J 




A gar streak 
Titre 


1 day 

r\ 




.days 

r\ 




days 

r\ 




Incubated at 

c. 
















^ 




^ 




^ 




Age of colony 














Size of colony 














Surface elevation 














Gelatin or agar 
colony 
Titre 














Incubated at 















112 GENERAL MICROBIOLOGY 



EXERCISE 38. TO DETERMINE THE ACIDITY CHANGES 
PRODUCED BY MOLDS IN CIDER (OR OTHER 
LIQUID MEDIA HAVING A LOW ACIDITY AND 
LOW SUGAR CONTENT) 

Apparatus. Four 100 c.c. Erlenmeyer flasks; 200 c.c. 
of cider; six 1-c.c. pipettes for titration (sterile); normal 
NaOH; N/20 NaOH. 

Cultures. Pure cultures of four molds. 

Method. 1. Determine the titre (reaction) of the cider 
and neutralize with normal sodium hydrate. 

2. Place 50 c.c. in each of the Erlenmeyer flasks and 
sterilize by the Tyndall method. 

3. Inoculate each, using a different mold for each flask. 

4. As soon as the mold mycelium shows in the flask 
(examine by looking through the flask toward the light), 
titrate. 

Note. One cubic centimeter of neutral cider is diluted to 50 c.c. 
with distilled water for titrating as the larger quantity, 5 c.c., when 
diluted, is of such a dark color that it is practically impossible to obtain 
a uniform or satisfactory end point. The burette reading must be 
multiplied by 5. 

Each pipette must be used only once. After using a pipette once, 
clean and resterilize it for future use. 

5. Titrate every three days thereafter, making eight 
titrations in all. 

6. Tabulate -your results. Plot a curve showing the rise 
in acidity, making all curves on one sheet, starting from the 
same zero point, and using different inks or different kinds 
of lines to represent the different acidity curves. Use 
acidity values as ordinates, days as abscissae. 

7. State fully any conclusions which may be based 
upon your data and point out the practical application 
which may be made. 

REFERENCE 
LAFAR: Technical Mycology, Vol. II, Part II, pp. 353-361. 



THE PATHOGENIC NATURE OF MOLDS 113 



EXERCISE 39. TO DEMONSTRATE THE PATHOGENIC 
NATURE OF MOLDS 

Apparatus. One deep culture dish; one perfect fruit 
the same as that from which the mold was isolated, or 
any fruit which is the natural habitat of the mold. 

Cultures. Pure culture of a mold isolated, from a fruit. 

Method. 1. Make small circles on opposite sides of 
the fruit with the wax pencil. 

2. Puncture the center of one circle with a sterile 
platinum needle. 

3. Then with needle contaminated with the mold spores 
inoculate the circle on the opposite side by puncturing as 
in 2. 

4. Place at about 25 C. and observe from day to day 
for two weeks. 

5. How do fruits usually become contaminated with 
molds? What preventive measures would you suggest? 

6. What is a perfect fruit from the bacteriological 
standpoint? From the horticultural standpoint? May 
these view-points differ? If so, how? What other fruits 
would be susceptible theoretically to the mold you used? 
Why? 

What other types of microorganisms may be path- 
ogenic to fruits? 

7. State in full the results obtained, with any con- 
clusions that may be drawn, and point out the practical 
application which may be made. 

REFERENCES 

MARSHALL: Microbiology, p. 513. 

SMITH, ERWIN F.: Bacteria in Relation to Plant Diseases, Vol. I, p. 202, 

Plates 29, 30 and 31. 
SMITH, ERWIN F.: Bacteria in Relation to Plant Diseases, Vol. II, 

Fig. 13, pp. 60 and 174-181. 



114 GENERAL MICROBIOLOGY 



YEASTS 

The so-called yeasts are divided into true yeasts " Sac- 
charomycetes " (wild and cultivated), and pseudo-yeasts 
or false yeasts, " Torulce " and " Mycodermata." 

By true yeasts are meant those which usually produce 
alcoholic fermentation (Sacch. membrancefaciens is an ex- 
ception), and which are able to form endospores. 

Pseudo-yeasts do not form endospores and produce 
little or no alcoholic fermentation. 

Sacch. cerevisice, the yeast used in the manufacture of 
beers and in bread-making, is a good example of the culti- 
vated yeast. 

Sacch. apiculatus and Sacch. ellipsoideus are examples 
of wild yeasts which are necessary in the making of wines. 
(These yeasts are cultivated and pure cultures used to some 
extent.) 

Torula rosea is an example of the pseudo-yeast. These 
look like true yeasts, reproduce by budding, but seldom 
produce alcoholic fermentation. 

REFERENCES 

MARSHALL: Microbiology, pp. 28-36, 420-423, 440, 460. 
KLOCKER: Fermentation Studies, pp. 205, 249, 289, 296. 

EXERCISE 40. TO ISOLATE A PURE CULTURE OF SAC- 
CHAROMYCES CEREVISICE AND TO STUDY THE 
FLORA OF A COMPRESSED YEAST CAKE 

Apparatus. Cover-glasses; concave slide; sterile 
Esmarch dishes; potato knife; platinum needles; Bunsen 
burner; sterile pipette; three tubes of sterile dextrose 
agar; iodin solution; methylen blue (0.0001% aqueous 
solution) . 

Culture. Fresh compressed yeast cake. 



ISOLATION OF SACCH. CEREVISLE 115 



A. Isolation of Saccharomyces Cerevisiae 

Method. 1. Sterilize the potato knife in the flame of 
the Bunsen burner. 

2. As soon as cool, cut a piece off the yeast cake. 

3. Make three dilution plates in dextrose agar imme- 
diately from this freshly cut surface. Use the straight 
needle and transfer only a very minute quantity of the 
yeast. Distribute well with the platinum needle. Use 
the straight needle for making dilutions in all cases. 

4. When the colony develops (three to six days) examine 
under objective No. 3, ocular No. 1, inverting the plate 
for this purpose. 

The individual cells of most yeast colonies may be 
seen under objective No. 3, while individual bacteria can 
seldom be distinguished in the colony at this low magni- 
fication. 

5. When you have located a yeast colony make a hang- 
ing drop from it in water and determine the shape of the 
individual yeast cells. 

6. If they have the shape and size of Sacch. cerevisice 
(see Marshall, p. 32), inoculate a tube of wort from this 
colony. 

7. Study this yeast according to directions in Exercise 
42. 

B. Study of Flora of Compressed Yeast Cake 

Method. 1. After preparing plates, place the yeast 
cake in a sterile Esmarch dish. 

2. Add 1 c.c. of boiled water, using a sterile pipette. 

3. From the freshly cut surface, prepare a hanging drop 
of the yeast in water, adding a loopful of iodin solution 
to it. Yeast cells will be unstained, while starch grains 
become blue. 

4. Repeat every seven days. 



116 GENERAL MICROBIOLOGY 

6. Is the cake made up mostly of starch grains or yeast 
cells? What is the purpose of the starch in the yeast 
cake? Do the starch grains remain intact or do they 
disappear? Explain. What kinds of starch are used? 

6. Draw and measure the starch grains. A drawing of 
the individual yeast cell may be made from this mount. 

7. Prepare a second hanging drop of yeast in water from 
the fresh cake. 

8. Stain by adding a loopful of 0.0001% aqueous 
methylen blue. Dead yeast cells are stained blue, while the 
living cells remain unstained. 

9. Count the number of living and dead cells in each of 
several fields. Estimate the per cent of living and dead 
yeast cells. 

10. Repeat every seven days until all the yeast cells 
are dead. 

11. How long does this take? What factors influence 
the death rate? Do other microorganisms enter? If so, 
what types? Why? From what source? Do they 
influence the value of the yeast cake? How? 

12. Each time you record the percentage of living and 
dead cells, note the macroscopical appearance of the cake. 
Also note ,the presence of new microorganisms, consistency 
of the cake, odor, and color. 

13. Record the results of this experiment in tabulated 
form, and state, any conclusions that may be drawn or 
practical application to be made. 

REFERENCES 

IAGO, WM. and IAGO, WM. C.: The Technology of Breadmaking. 

(1911), pp. 235-239. 

CONN: Yeasts, Molds and Bacteria, pp. 56-99. 
SCHNEIDER: Bacteriological Methods in Food and Drugs Laboratories. 

(1915). Plate I, Figs. 2, 3 and 4. 



STUDY OF GASEOUS FERMENTATION 117 



EXERCISE 41. APPARATUS AND METHODS FOR THE 
STUDY OF GASEOUS FERMENTATION 

Various forms of yeasts, bacteria and other micro- 
organisms have the ability to ferment carbohydrate, nitrog- 
enous, and other food substances with the liberation of gas. 

A. Smith's Fermentation Tube 

Theobald Smith (1893) introduced the use of a special 
tube for studying fermentation and gas production, and 
now Smith's fermentation tube is in general use in this 
and other countries. 

Its value lies in the fact that it is a simple apparatus, 
yet it allows not only of testing the relative fermentative 
powers of different species of microorganisms or of different 
strains of the same species, but of determining the gases 
produced qualitatively and their relative proportions 
quantitatively to some extent. 

Apparatus. Smith fermentation tubes; gasometer; 
nutrient carbohydrate broth (or any desired solution); 
platinum needles. 

Culture. Culture of the organism to be tested. 

Method. 1. The carbohydrate broth (or other liquid 
medium) is placed in the fermentation tubes, filling the long 
arm by carefully tilting. The bulb should be filled with 
the liquid only to the extent that air will not enter the long 
arm upon slightly tilting. The tube should not be filled 
so full that the bulb will not contain all of the liquid in the 
long arm. 

2. Sterilize. Carbohydrate broths are sterilized by the 
intermittent method. 

3. Inoculate fermentation tubes of the desired medium 
with the organism to be tested, using a loop or straight needle. 

4. Incubate at optimum temperature. 

5. Examine in twenty-four hours for gas production, 
and mark the level of the liquid in the long arm of the 



118 



GENERAL MICROBIOLOGY 



fermentation tube each day if gas is being formed. (If 
the level is higher than it was the previous day, the gas 
(CO2) is being absorbed. Do not allow this absorption 
to proceed further, but test the gas present for C02 
and H 2 ). 

6. Measure and record the amount of gas by means of a 
gasometer (see illustration). The total amount is not 




FIG. 36. Smith's Fermentation Tube Showing use of Frost's Gas- 
ometer. 



exact quantitatively, as some gas is given off from the 
open arm of the fermentation tube. 

7. When the maximum amount of gas is formed, test 
the gas for C02 and other gases as follows: 

Fill the short arm of the fermentation tube with 10% 
NaOH. Place the thumb over the mouth of the tube and 
shake vigorously, so that the gas contained in the long 
arm comes in contact with the NaOH. 

2NaOH+C0 2 = Na 2 CO 3 +H 2 O. 



THE STUDY OF YEASTS 119 

Sodium carbonate and water are formed, leaving the other 
gases free. 

Collect in the long arm of the tube all the gases remain- 
ing. Remove the thumb. The difference in the per cent 
of gas before and after treating with the NaOH equals the 
per cent of CCb which was present. 

8. Place the thumb over the mouth of the tube and 
collect all remaining gas in the short arm. Light a match, 
remove thumb and immediately touch off the remaining gas. 
If Eb is present the typical reaction occurs. Other gases 
are often present, but in too small amounts to allow of 
testing. 

10. Record the relative proportions of C02 and H2 
formed. 

B. Durham's Fermentation Tube 

Durham's fermentation tube is simply an ordinary test 
tube containing a sugar broth, in which a smaller test tube, 
inverted, has been placed before sterilization. 

This apparatus possesses some advantages over the 
Smith fermentation tube if only the presence of gas produc- 
tion is to be noted, as the tubes are more easily cleaned, 
sterilized and handled. 

The amount of gas may be roughly estimated, but the 
kind of gas may not be determined by the use of this 
apparatus, 

EXERCISE 42. THE STUDY OF YEASTS 

The object of this exercise is to demonstrate how 
to differentiate yeasts by microscopical and cultural 
methods. 

Apparatus. Clean cover-glasses; three clean concave 
slides; five clean fermentation tubes; one tube sterile 
2% dextrose broth;* one tube sterile 2% lactose broth; 

* The sugar and glycerin broths are furnished by the laboratory. 



120 GENERAL MICROBIOLOGY 

one tube sterile 2% saccharose broth; one tube sterile 
2% glycerin broth; one tube sterile nutrient broth; three 
tubes sterile wort; three tubes sterile gelatin; four tubes 
sterile dextrose agar; gasometer; 10% NaOH. 

Cultures. Saccharomyces cerevisice;* Saccharomyces 
apiculatus; Torula rosea. 

Method. 1. Fill one fermentation tube with each broth. 
Sterilize by heating one-half hour in the steam on three 
consecutive days. 

2. Make cultures of each yeast in 
(a) Beer wort. 

(6) Gelatin (stab culture). 

(c) Dextrose agar slant. 

(d) Dextrose agar plate (giant colony). f 

(e) Linder's concave slide culture (p. 83). 

3. Make cultures of Saccharomyces cerevisice only, in 
fermentation tubes of 

(a) Plain broth control (without carbohydrate). 

(b) 2% dextrose broth. 

(c) 2% lactose broth. 

(d) 2% saccharose broth. 

(e) 2% glycerin broth. 

4. Prepare an adhesion culture from freshly inoculated 
wort culture of the yeast (see p. 78). 

5. Examine microscopically immediately after prepara- 
tion and draw single cells and cells in various stages of 
budding (germination); show interior structure of cells. 

6. Examine all cultures after twenty-four hours, and make 
drawings of (a), (b), (c), (d), and (e) under 2 ; place as 
indicated on chart, and label correctly. 

7. Describe all gelatin and agar cultures according to 
the descriptive chart of the American Society of Bacteriol- 

* Saccharomyces cerevisioe has been previously isolated from a fresh 
cake of Fleischmann's compressed yeast. (See Exercise 40.) 

t Only one plate is necessary. All yeasts may be grown on one 
plate. Use dextrose agar. 



THE STUDY OF YEASTS 121 

ogists (p. 134). In describing the wort culture use the 
descriptive chart terms under the heading " Nutrient 
broth." 

8. If any gas has formed in the fermentation tubes 
mark the level of the liquid in the long arm with a 
wax pencil and record the percentage of gas, using the 
gasometer. 

9. Test quantitatively and qualitatively for gas in the 
fermentation tubes. (See Exercise 41, p. 117). 

10. What is the ratio of the CO2 to H2 and other gases? 
Is this ratio constant for all fermentations? For one 
organism? Why? Do all organisms cause fermentation? 
Why? What causes fermentation? 

11. Examine adhesion cultures after forty-eight hours 
and seventy-two hours and make drawing of colony forma- 
tion. 

12. Study the fourteen to twenty-day old wort cultures 
in hanging drop for endospores. When do these form? 
Why? 

REFERENCES 

HANSEN, E.: Practical Studies in Fermentation, pp. 215-217. 

LAFAR: Technical Mycology, Vol. II, Part II, pp. 394-406, 430- 

436. 

EYRE: Bacteriological Technic. Second Ed., p. 7. 



122 



GENERAL MICROBIOLOGY 



YEASTS 



Name of student 


Desk No. 


Name of organism 


Isolated from 


Method of isolation 


Occurrence 


Importance 


Total organism Drawn from preparation 


Stages of budding Drawn from preparation 


Method of reproduction Drawn from preparation 


Spore 



Fermentations 



% Of gas in 


Control 


Dextrose 


Lactose 


Saccharose 


Glycerin 


24 hours 












48 hours 












3 days 












5 days 












Total gas 
production 












Ratio of CO 2 :H 2 
and other gases 












Alcohol (odor) 












Acid 












Growth in 
closed arm 













THE STUDY OF YEASTS 



123 





Iday 




days 




days 




Cider or wort 














culture 














Reaction . 














Incubated at 














. C. 
















O 




[^ 




[^ 






Iday 




days 




days 




Nutrient gelatin 














stab 














Reaction 














Incubated at 














C. 
















^J 




^J 




U 





Agar streak 

Reaction 

Incubated at 

..C. 



1 day .days days 



A 




Age of colony 



Size of colony 



Surface elevation 



Gelatin or agar 

colony 
Reaction 
Incubated at 



124 



PLATE V 





THE STUDY OF BACTERIA 125 



EXERCISE 43. THE STUDY OF BACTERIA 

Studies will be made of ten bacteria representing the 
different morphological types. These are to be identified 
by morphological and cultural characteristics. 

Pure cultures of these organisms will be found on each 
desk in the tumblers marked " Laboratory cultures." 
Always return laboratory cultures to these tumblers imme- 
diately after using. 

DANGER. Some of these organisms are pathogenic. 
If you do not handle them with care and according to 
directions you endanger not only yourself, but all working 
in the laboratory. Do not be careless. Handle all organ- 
isms as if they were pathogenic. This is a good habit; get 
it immediately. (See " Care of Cultures," pp. 46-48) The 
instructor will designate which organisms are pathogenic. 

Apparatus. Clean cover-glasses; clean concave slides; 
clean plain slides; ten agar slants; ten tubes sterile agar 
for plates; ten tubes nutrient broth; ten tubes nutrient 
gelatin; ten tubes litmus milk; ten tubes glycerin potato; 
ten tubes Dunham's solution; ten tubes nitrate peptone 
solution; four fermentation tubes of plain broth; four 
fermentation tubes of dextrose broth; four fermentation 
tubes of lactose broth; four fermentation tubes of sac- 
charose broth; centimeter scale; gasometer; lead acetate 

DESCRIPTION OF PLATE V 

I. 1, Bad. tuberculosis; 2, B. typhosus; 3, Bad. leprce; 4, Bad. 
anthracis (strepto-bacterium, two with spores); 5, Bad. diph- 
therias (club-shaped); 6, anthrax spore, germinating (polar); 
7, B. amylobader (clostridium) ; 8, Streptococcus pneumonice 
(diplococcus with capsule). 

II. 1, B. subtilis (strepto-bacillus, peritrichous flagella, one with 
spore); 2, B. subtilis (peritrichous flagella); 3, formation of a 
new filament from a germinating spore; 4, spore of B. subtilis; 
5, germinating spore of B. subtilis (equatorial); 6, beginning 
germination. 



PLATE VI 



\ \A 




III. 1, Spirillum volutans (Cohn) with lophotrichous flagella 
(chain of three); 2, Sp. volutans, single cell; 3, Microspira 
comma, monotrichous flagellum; 4, Spirocheta obermeieri. 







IV. 1, Sardna lutea; 2, Micrococcus tetragenus with capsule; 3, 
streptococcus; 4, planococcus; 5, staphylococcus. 



THE STUDY OF BACTERIA 



127 



paper; aqueous-alcoholic fuchsin and methylen blue; 
mordant for flagella stain; Lugol's iodin solution; anilin- 




FIG. 37. Cycle of Development of Bacterial Cell. (Adapted from 
Fuhrmann's Technische Mykologie.) 

water gentian violet; carbol-fuchsin; acetic acid-alcohol 
for decolorizing spore stain; indol test solutions; nitrate 
test solutions; ammonia test solutions. 



128 



GENERAL MICROBIOLOGY 



Method. 1. Make an agar slant culture of each organism 
and incubate each at its optimum temperature. (Instructor 
will designate the optimum temperature of each.) 




11 



FIG. 38. Comparative Sizes of Bacteria. 

1, Micrococcus progrediens, 0.15^,' 2, Micrococcus urea;, 1-1.5^; 
3, Sarcina maxima, 4^; 4, Thiophysavolutans (sulphur bacteria), 
7-18^; 5, influenza bacillus, 4.2X0.4/x; 6, methane bacillus, 
5X0.4/*; 7, Urobacillus dudauxii (Miquel), 2-10X0.6-0.8^; 
8, Bacillus nitri (Ambroz), 3-8X2-3^; 9, Beggialoa alba 
(sulphur bacteria), 2. 9-5. 8 X 2. 8-2. 9/*; 10, Chromatium okenii, 
(sulphur bacteria), 10-15X5/*; 11, Beggiatoa mirabilis (sul- 
phur bacteria), 20-25 X40-50/X. (From Fuhrmann's Tech- 
nische Mykologie.) 

2. Draw and describe twenty-four-hour old agar slant 
cultures, then examine microscopically in hanging drop to 
determine the morphology, size, grouping or arrangement, 



THE STUDY OP BACTERIA 129 

motility, spores. Use ocular No. 1 and objective No. 7. 
The greatest motility will be observed in organisms growing 
in the condensation water at the base of the slant. 

3. Draw the total organism and record the presence or 
absence of motility. Describe all cultures at the time the 
drawings are made of each, following the terminology of 
the " Descriptive Chart of the American Society of Bac- 
teriologists," p. 134. 

4. Use drawing pencil for making drawings and ink for 
recording descriptions. 

Any descriptive terms may be added which will aid in 
identifying organisms, but descriptive chart terms must 
be followed as closely as possible, otherwise drawings will 
not be accepted. 

Always state the age of the culture, the temperature at 
which the organism is grown, the medium upon which it is 
cultivated and the litre of the medium. 

Use one chart for each organism. 

5. When the agar slant culture of each organism shows 
good growth, make inoculations from this culture into the 
following media : 

Agar plate (see below for method). 

Gelatin plate (see step 6, below). 

Nutrient broth. 

Nutrient gelatin (stab culture). 

Litmus milk. 

Glycerin potato. 

Dunham's solution. 

Nitrate peptone solution. 

Plain broth fermentation tube (control). 

Dextrose broth fermentation tube. 

Lactose broth fermentation tube. 

Saccharose broth fermentation tube. 

6. In preparing agar plate from bacterial cultures, proceed 
as follows: Inoculate a tube of nutrient broth lightly, using 



130 GENERAL MICROBIOLOGY 

the straight needle. Then, still using the straight needle, 
from the freshly prepared broth culture, inoculate lightly 
one tube of melted agar (at 40 to 50 C.) and pour into a 
sterile Petri dish. If the organism shows only a slight growth 
on the stock culture, transfer directly to melted agar. 

7. Moisten a strip of lead acetate paper and insert with 
cotton plug in tube of Dunham's solution. Blackening of 
this paper shows the formation of H2S. 

Between what substances does a chemical reaction take 
place? What are the resulting products? 

8. Draw and describe twenty-four-hour cultures of the 
first four bacteria in all media. If at any time presence 
of growth is doubtful, compare with a tube . of sterile 
medium. In the absence of growth, reinoculate. 

9. Record macroscopical changes only, in litmus milk; 
and in fermentation tubes note only, the place of growth, 
presence and percentage of gas; also the formation of H2S 
in Dunham's solution. 

10. Make a permanent stained preparation of each organ- 
ism (following directions under Exercise 28). Young 
(twenty-four to forty-eight hour) cultures must be used. 
Use either aqueous-alcoholic fuchsin or aqueous-alcoholic 
methylen blue. 

11. Make a flagella stain of the largest motile organism 
among your cultures. 

It is absolutely necessary that a young (eighteen to twenty- 
four hour, not older) culture be used for this purpose. Fol- 
low the directions under Exercise 31. 

12. Make further drawings and descriptions from day 
to day if any change in the growth from that of the preceding 
day is observed. Three drawings of a culture will be suf- 
ficient. Endeavor to illustrate typical growth by careful 
drawings. 

13. State whether the agar plate colony described is a 
surface or a subsurface colony. How do these two types of 
colonies differ? Why? 



THE STUDY OF BACTERIA 131 

14. Note the presence of condensation water, whether 
a small or large amount is present. How does this affect 
colony development? 

15. Draw and measure a typical surface and subsurface 
colony produced by each organism. 

The form and size often vary with the physical con- 
dition under which the colony grows or with physiological 
conditions, i.e., the proximity of colonies producing poison- 
ous metabolic products. 

16. Examine cultures three to six days old in hanging 
drop for presence of spores. Spores may be seen free or 
enclosed in the bacterial cells. They are easily distinguished 
by their refractivity. Ordinary anilin dyes will not stain 
them. 

17. Make a contrast spore stain of a spore-forming 
organism. (For method see Exercise 29.) 

Draw and describe only the mature cultures of the last 
six organisms (five to eight days old) . 

18. Make the indol, nitrate and ammonia tests also 
on the mature cultures. 

19. In fermentation tube cultures note and record the 
oxygen requirements of each organism; total per cent of 
gas; ratio of CO2 : H2 and other gases. 

20. Test each organism after seven days for indol, 
nitrate and ammonia production. The culture in Dun- 
ham's peptone solution is tested for indol (for method 
see Exercise 44). 

Divide the nitrate peptone solution culture into two parts; 
test one for nitrates, the other for ammonia (for method 
see Exercise 45). 

21. Prepare permanent stained preparations of one 
Gram-positive and one Gram-negative organism. 

22. Making use of morphological and cultural charac- 
teristics ascertained microscopically and by the various 
cultural tests, identify each organism, using Chester's 
Manual of Determinative Bacteriology for tracing out 



132 GENERAL MICROBIOLOGY 

the genus and species. Other valuable reference texts 
are: 

CONN, ESTEN and STOCKING: Classification of Dairy Bacteria. 
NOVY: Laboratory Manual of Bacteriology. 
JORDAN: General Bacteriology. 

EXERCISE 44. EHRLICH'S METHOD OF TESTING 
INDOL PRODUCTION 

The purpose of the exercise is to test the power of an 
organism to produce indol from peptone. 

Cultures for comparison should be of the same age and 
grown in the same kind of medium. Some peptones con- 
tain a trace of indol and, to avoid all possibility of mis- 
take when testing for indol, a control tube of sterile medium 
should be used at the same time. This reaction is char- 
acteristic for indol or for methyl indol (skatol). 

There are other tests for indol, but this one is by far 
the most delicate. The Salkowski-Kitasato test (cone. 
H2SO4 and NaNCb) will detect indol in a dilution of only 
1 : 100,000, while Ehrlich's test will give a reaction in a 
dilution ten times greater, or 1 : 1,000,000. 

Indol is one of the most important of protein decom- 
position - products. It is noted for its intense fecal odor. 
However, in highly dilute solutions it has the odor of orange- 
blossoms, hence is used extensively in perfumery. The 
jessamine blossom contains indol and has its odor. 

Indol has the following graphic formula: 

H 



UC/ C CH 

I D II 



H 



According to Emil Fischer, the reaction of Ehrlich's 
test, produces, by means of the oxidizing action of the potas- 



TESTS FOR THE REDUCTION OF NITRATES 133 

sium persulphate, a condensation of two molecules of indol 
with the aldehyde group of the para-dimethyl-amido- 
benzaldehyde, water splitting off. 

Apparatus. Solutions I and II for Ehrlich's test for 
indol;* two clean 5 c.c. pipettes. 

Culture. Dunham's peptone solution or broth culture 
of the organism to be tested. 

Method. 1. To about 10 c.c. of the liquid culture 
add 5 c.c. of solution I, then 5 c.c. of solution II. 

2. Shake the mixture. The reaction may be accelerated 
by heating. The presence of indol is indicated in a few 
minutes by a red color which increases in intensity with 
time. For standard compaiisons, five minutes is taken 
as the maximum time limit. 

REFERENCES 

BOEHME, A.: Die Anwendung der Ehrlichschen Indol-reaktion fur 

Bakteriologische Zwecke. Cent. f. Bakt. Orig. Bd. 40 (1906), 

pp. 129-133. 
BESSON: Practical Bacteriology, Microbiology and Serum Therapy 

(1913), p. 374. 

LOHNIS: Laboratory Methods in Agricultural Bacteriology (1913), p. 42. 
LEWIS, F. C.: On the detection and estimation of bacterial indol and 

observations on intercurrent phenomena. Jour. Path, and Bact., 

Vol. 19 (1915), pp. 429-443. 

EXERCISE 45. TESTS FOR THE REDUCTION OF 
NITRATES 

The purpose of the exercise is to test the power of an 
organism to reduce nitrates. 

Apparatus. Sulphanilic acid, nitrite test solution I; 
a-naphthylamin, nitrite test solution II; Nessler's solution; 
phenolsulphonic acid. 

Cultures. Seven-day old nitrate peptone solution cul- 
tures grown at 20 to 25 C., or four-day old nitrate pep- 
tone solution cultures (pathogenic) grown at 37 C. 

Method. (A) For nitrites: 1. Add 0.1 c.c. each of 
solutions I and II to each culture to be tested. 
* See Appendix. 



134 GENERAL MICROBIOLOGY 

2. Repeat with uninoculated control. 

3. The development of a red color in ten minutes indi- 
cates the presence of nitrites, the intensity of the color 
depending upon the amount of nitrites present. 

(B) For ammonia. 1. Add 0.2 c.c. of Nessler's solu- 
tion to each culture to be tested. 

2. Repeat with uninoculated control. 
The presence of ammonia is shown by a yellow color 
or precipitate. 

(C) For nitrates unchanged or free nitrogen liberated. 
1. When either or both of the preceding tests are positive, 
no further determination need be made, but if negative, 
then one of two conditions may prevail: (a) Either the 
nitrates have not been changed, or (6) they may have been 
reduced to free nitrogen. To ascertain which is true, it 
will be necessary to determine the presence or absence 
of nitrates. 

2. Test as follows: (a) Evaporate 10 c.c. 'of each cul- 
ture and the controls almost to dryness in an evaporat- 
ing dish and add to the residue 1 c.c. of phenolsulphonic 
acid. 

(6) Dilute with 10 c.c. distilled water, then add suf- 
ficient ammonium hydroxide, diluted 1 : 1 with distilled 
water, or concentrated potassium hydroxide solution, 
to make alkaline. 

(c) Transfer the liquid to a 50 c.c. Nessler tube or grad- 
uated cylinder and make up the volume to 50 c.c. with 
distilled, water. 

A yellow color shows the presence of nitrates. 



TESTS FOR THE REDUCTION OF NITRATES 135 
BACTERIA 



Name of student 


Desk No. 


Name of organism Isolated from 


Method of isolation 


Occurrence 


Importance 


Shape of organism 


Arrangement 


Size 


Motility 


Flagella 


Method of reproduction 


Involution forms 


Spore 


Stages of germination 


Aqueous-alcoholic stain Gram's stain Aci 


d-fast stain 



1 day days days 



Agar streak 

Titre 

Incubated at 





1 day days days 



Gelatin stab 

Titre 

Incubated at 



C. 



136 



GENERAL MICROBIOLOGY 

1 day days days 



Broth culture 
Titre 
Incubated at 



Potato culture 
Titre ........... 

Incubated at 
............ C. 



1 day days days 




v 



Age of agar colony 


days 


days 


days 


Size of colony 








Surface elevation 









Agar colony 
Titre ..... . 

Incubated at 



TESTS FOR THE REDUCTION OF NITRATES 137 



Age of gelatin colony 


days 


days 


days 


Size of colony 








Surface elevation 








Gelatin colony 
Titre 








Incubated at 
C. 









Litmus milk 


Acid 




Gas 




Acid curd 




Rennet curd 




Reduction 




Alkali 




Peptonization 





Fermentations 



% Of gas in 


Control 


Dextrose 


Lactose 


Saccharose 


Glycerin 


24 hours 












48 hours 












3 days 












7 days 












Total gas 
production 












Ratio of CO 2 :H 2 
and other gases 












Acid 












Growth in 
closed arm 













138 



GENERAL MICROBIOLOGY 



Chromogenesis 
on 


Nutrient broth 




Nutrient gelatin 




Nutrient agar 




Potato 




Production of 


NH 3 from peptone 




H^S from peptone 


Indol from peptone 




Nitrites from peptone 




Reduction of 
nitrates to 


NH 3 




Nitrites 





Remarks: 



EFFICIENCY OF INTERMITTENT HEATING 139 

EXERCISE 46. TO DEMONSTRATE THE EFFICIENCY 
OF INTERMITTENT HEATING AS A METHOD OF 
STERILIZING MEDIA. ALSO TO COMPARE THE 
EFFICIENCY OF CONTINUOUS AND INTERMITTENT 
HEATING 

Apparatus. 400 c.c. fresh skim milk; forty sterile 
test tubes; 2% azolitmin solution. 

Method. 1. Prepare litmus milk according to direc- 
tions on p. 25. 

2. Fill the tubes, using approximately 8 c.c. per tube. 

3. Set five away without heating. 

Heat five for fifteen minutes on the first day. 
Heat five for one hour on the first day. 
Heat five for fifteen minutes on two successive days. 
Heat five for fifteen minutes on three successive days. 
Heat five for fifteen minutes on four successive days. 
Heat ten for fifteen minutes on five successive days. 

4. Keep all tubes at room temperature. Examine 
every two or three days and describe the macroscopical 
changes of each set, as described under the discussion on 
litmus milk (pp. 23-25.) 

Why do not all the tubes of a set change alike? Why 
do not all sets present the same appearance? 

Save all tubes that do not show macroscopical changes. 
These are probably sterile. 

6. Tabulate your results after ten days to two weeks, 
recording the number and per cent of each lot that shows 
macroscopical changes. 

6. Is milk difficult to sterilize? Why? What other media 
present the same problem of sterilization as milk? Why? 

Would any other method for the sterilization of milk be 
preferable to the ones you used? Give reasons for your 
answer. 

7. State your results in detail and point out any con- 
clusions that may be drawn and any practical applications 
that may be made. 



140 GENERAL MICROBIOLOGY 



REFERENCES 

MARSHALL: Microbiology, pp. 153-161, 306-313, 363-365. 
CONN, H. W.: Bacteria, Yeasts and Molds, pp. 191-193. 
BESSON, A.: Practical Bacteriology, Microbiology and Serum Therapy, 
pp. 35-36. 



EXERCISE 47. TO COMPARE MORPHOLOGICALLY 
PROTOZOA WITH BACTERIA 

Apparatus. Deep culture dish; concave slide; clean 
cover-glasses; cover-glass forceps; platinum loop; tube 
of sterile broth; tube of sterile Chinese ink. 

Cultures. Rich soil or slimy leaves from a pond. 

Method. 1. Place the soil or leaves in the deep culture 
dish. 

2. Fill the dish two-thirds full with tap water and 
add the contents of a tube of broth. 

3. Keep the dish at room temperature for twenty- 
four to forty-eight hours. 

4. At the end of the incubation period, make a hanging 
drop from the supernatant liquid. Before inverting the 
drop on the slide, add to it that amount of Chinese ink 
that adheres to the end of a platinum needle. 

By the use of this ink, organisms are brought out by 
contrast, showing white on a dark field. The organisms 
are not killed or injured by the ink. 

5. Observe and measure any protozoa, using the lowest 
power objective with the step micrometer. Record the size 
in micra. 

6. Roughly sketch the different species observed, giving 
comparative measurements. 

7. Using the highest power dry objective, observe bac- 
teria, noting morphology and size. 

8. Draw lines to represent the ratio between the size 
of the predominant types of each. 

9. Are the protozoa present visible to the naked eye? 



THE DECOMPOSITION OF MILK 141 

How many of the largest protozoa present, placed end to 
end, would make an inch? 

10. How do the protozoa and bacteria in the drop 
compare in numbers? Do these organisms have any rela- 
tion to each other? If so, explain. 

11. Of what importance are protozoa? Name several 
well-known protozoa. 

12. State your results in detail and point out any con- 
clusions that may be drawn and any practical applications 
that may be made. 

REFERENCE 

MARSHALL: Microbiology, pp. 10-11, 68-80, 82-84. 

EXERCISE 48. TO STUDY THE NATURAL DECOMPO- 
SITION OF MILK 

Apparatus. 500 c.c. sterile Erlenmeyer flask; two 
5 c.c. sterile pipettes; ten 1 c.c. sterile pipettes; four 
10 c.c. sterile pipettes; six 200 c.c. sterile Erlenmeyer 
flasks; fifteen sterile Petri dishes; physiological salt solu- 
tion. 

Cultures. Fresh skim milk. 

Method. 1. Prepare " dilution flasks " as given in 
Exercise 13, p. 52, making two 90 c.c. and four 99 c.c. 
flasks. Sterilize by heating one hour in flowing steam or 
five minutes in the autoclav at 120 C. (15 Ibs. pressure). 
Dilution flasks and all glassware must be sterile before the 
experiment proper can be started. 

2. Place 200 c.c. of the fresh skim milk in the sterile 
500 c.c. flask and use this sample for the entire experi- 
ment. 

3. Plate the milk immediately on nutrient agar, using 
dilutions according to the age of the milk, as follows. 
(See Exercise 13, p. 52, for method of using dilution 
flasks.) 



142 GENERAL MICROBIOLOGY 

Age. Dilutions. 

Fresh milk. . 1 : 1,000, 1 : 10,000 and 1 : 100,000 

One day old 1 : 10,000, 1 : 100,000 and 1 : 1 M * 

Four days old 1 : 10 M, 1 : 100 M and 1 : 1,000 M 

Eight days old 1 : 10 M, 1 : 100 M and 1 : 1,000 M 

Ten days old 1 : 1 M, 1 : 10 M and 1 : 100 M 

Keep the plates at room temperature. 

Sterile pipettes are to be used always in making dilu- 
tions, plating and titrating. 

After the milk curdles it is advised to make the first 
dilution 1 : 10 to give a more uniform sample, from which 
further dilutions are made. Use a 10 c.c. pipette having 
a large opening in the delivery end to prevent clogging. 

4. Titrate the milk sample every day. After the milk 
curdles, shake well before titrating and choose a 5 c.c. 
pipette having a large aperture for delivery for obtaining 
the sample for titration. 

5. Record the reaction in degrees of Fuller's scale. 
After using pipettes, dilution flasks, etc., clean, refill 

and sterilize them at once for future use. 

6. Note the macroscopical changes in the milk sample 
(due to microbial growth), e.g., kind and consistency of 
curd, extrusion of whey, gas formation, peptonization; also 
note odor from time to time. 

7. Note the macroscopical evidences of microbial growth 
such as molds, etc., and the time of appearance. Identify 
the group to which these organisms belong, giving genus 
and species if possible. 

8. Determine the changes in the numbers of micro- 
organisms by counting the colonies of the different sets of 
plates after they have developed seven days at room tem- 
perature (see p. 56, Exercise 14, for method). 

9. Estimate the number of colonies of each type (see 
Exercise 14, p. 56). 

*M = Million. 



THE DECOMPOSITION OF MILK 



143 



10. Record your results, noting the date on which the 
plates were made, the age of the milk, the dilution, number 
of colonies on the plate and the average number of organisms 
per cubic centimeter. 

11. Examine in hanging drop and note the morphology 
of the microorganisms producing the most predominant 
types of colonies on each set of plates. Indicate after the 
drawing, the comparative numbers on each set of plates 
by the signs , + , +, + + , etc., to indicate absence, 
presence of few, or many of the type. 

12. Note whether molds or yeasts are present on any 
set of plates. Should either be found on fresh milk plates? 
Why? What types of microorganisms would you expect 
on fresh milk plates? 

13. Prepare your data according to the following dia- 
gram: 



Date 


Age 


Acidity 


Dilution 


Count 
per cc. 


Organisms 


Types 


Relative Nos. 


Feb. 2 


Fresh 
(26 hrs. old) 


+ 15 


1-1,000 
1-10,000 
1-100,000 


278,900 
325,500 
300,000 


Acid 
Yellow 


+_ 



Average count per cc 301,470 

14. Plot the curve showing the change in acidity and one 
illustrating the count per cubic centimeter on the same 
paper, using different colored inks or different types of 
lines. Use days for abscissae, acidity and count for ordinates. 
Start at the same origin. 

15. Is there any relation between the change in acidity 
and the change in flora? 

Should the acidity and count curves run parallel? If 
they do not, give a reason why. 

How could the bacterial count be made to increase 
after it goes down to a constant number? 



144 GENERAL MICROBIOLOGY 

16. What biochemical changes have occurred in the 
decomposition? 

17. Compare the flora of fresh milk, 70 acid milk and 
ten-day milk, both microscopically and from the plates. 
Explain. 

18. State your results in detail and give any conclusions 
to be drawn and any practical applications that may be 
made. 

REFERENCES 

CONN: Practical Dairy Bacteriology, pp. 21-57, 81-85. 
MARSHALL: Microbiology, pp. 298-299, 306-313, 321-326. 

EXERCISE 49. TO ISOLATE SPORE-FORMING BAC- 
TERIA AND TO STUDY SPORE FORMATION 

Apparatus. Two tubes of sterile broth; small piece 
of hay; three sterile Fetri dishes; clean test tube; three 
tubes sterile agar for plates; three sterile agar slants; 
carbol-fuchsin; acetic acid alcohol; aqueous-alcoholic 
methylen blue; platinum needle and loop; ordinary for- 
ceps. 

Culture. Hay. 

Method. 1. Place a piece of hay in the clean test 
tube, plug the tube, and sterilize in the hot-air oven. 

2. Using sterile forceps, place the sterile hay " aseptic- 
ally " in one tube of broth and an unsterilized piece in the 
other. 

3. Incubate both at room temperature for forty-eight 
hours. Do both tubes show growth? 

4. Heat in a water-bath at 80 C. for ten minutes the 
tube which shows marked growth. What does this accom- 
plish? 

5. Make three loop-dilution plates from the heated broth 
tube. 

6. Place the plates at room temperature and examine 
them daily for colony development. 



REMOVING MICROORGANISMS FROM LIQUIDS 145 

7. Make pure cultures on agar slants from three differ- 
ent well-isolated colonies of the predominant types and in- 
cubate at room temperature. 

8. Examine these in thirty-six to forty-eight hours 
in a hanging drop for morphology and spore formation. 

9. Make a spore stain as soon as spores are found. 
Where are the spores located in the bacterial cell? 

10. Have you studied any pure culture of bacteria which 
is similar to the types you have isolated? What organism 
is commonly found in hay? In what form does it exist on 
the hay? What do you know of the habitat of this organ- 
ism and related forms? Of the pathogenicity? 

11. State the results obtained in detail; draw the con- 
clusions which follow and point out any possible practical 
applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 5, 45, 154, 189, 242. 
JORDAN: General Bacteriology, 4th Ed., pp. 235-236. 
EYRE: Bacteriological Technic, 2d Ed., p. 140. 

EXERCISE 50. TO DEMONSTRATE THE EFFICIENCY 
OF FILTRATION AS A MEANS OF REMOVING 
MICROORGANISMS FROM LIQUIDS 

Apparatus. Six small funnels; two small filter papers; 
two small pieces of absorbent cotton; two small pieces of 
clean hospital gauze; eight tubes sterile agar; eight sterile 
Petri dishes; ten sterile 1 c.c. pipettes; sterile 10 c.c. 
pipette; three dilution flasks; six sterile test tubes; tube 
of sterile broth. 

Culture. B. coli. 

Method. 1. Inoculate broth with B. coli and incubate 
for twenty-four hours at 21 C. 

2. Sterilize filter paper in each of the two small funnels, 
a small piece of absorbent cotton in each of two more; 
fold two pieces of clean gauze several thicknesses and 



146 GENERAL MICROBIOLOGY 

sterilize in the remaining two funnels. Wrap all in paper 
and sterilize in the hot-air oven. 

3. Shake the broth culture of B. coli and plate, using 
dilutions 1 : 1,000 and 1 : 10,000. 

4. Filter each dilution (1 : 1,000 and 1 : 10,000) through 
each of the different substances, catching the filtrate in 
sterile test tubes. 

5. Plate 1 c.c. from each filtrate immediately and incu- 
bate the plates at 21 C. 

6. At the end of five days, count the plates. 

7. Which method of filtration is most efficient? Why? 
What factors could greatly influence the numbers of micro- 
organisms developing on the plates after filtration? 

8. What methods are most efficient in removing micro- 
organisms from liquids? Why? 

9. Suggest some natural methods of filtering micro- 
organisms. 

10. Give in detail the results obtained, state any con- 
clusions that may be drawn and point out any practical 
applications. 

REFERENCES 

EYRE: Bacteriological Technic, 2d Ed., pp. 42-48. 
MARSHALL: Microbiology, pp. 64-67. 

EXERCISE 61. TO DEMONSTRATE PRESENCE OF MI- 
CROORGANISMS IN AIR, ON DESK, FLOOR, ETC. 

Apparatus. Six sterile Petri dishes; six tubes of sterile 
agar. 

1. Air. Method. 1. Pour six plates with uninoculated 
sterile agar and set on a level surface until solid. 

2. Expose one plate for one minute to (a) laboratory 
air; (6) air of campus; (c) air of your room while sweep- 
ing or dusting. 

II. Floor. 1. Bend the straight platinum needle tilf 
it forms a right angle. 



PRESENCE OF MICROORGANISMS IN AIR, ETC. 147 

2. Sterilize it in the flame. 

3. Moisten the needle with sterile water. 

4. Rub it along the floor, and then, 

5. Draw it lightly across the surface of the agar in the 
fourth Petri dish. 

III. Desk. 1. Sterilize the needle and repeat operation 
(II, 5) obtaining the inoculum from the surface of a desk 
which has not just previously been washed with 1 : 1,000 
mercuric chloride. 

2. Then wash the surface of the desk well with this 
solution and when the desk top is dry, repeat the operation, 
using the sixth plate. 

3. Mark all plates with the date on which they were 
exposed or inoculated and place them at a constant tem- 
perature. 

4. Watch any developments from day to day. What 
organisms predominate on the plates? Why? 

5. Examine different colonies in a hanging drop. What 
types of bacteria are found? 

6. Upon what does the species and number of micro- 
organisms depend? What becomes of them when air cur- 
rents are present? When the floor is swept in the ordinary 
way? Mopped? When the desk is washed with water? 
With mercuric chloride? 

7. Are these types deleterious to health? Why should 
and how may they be avoided in the laboratory? Out- 
side of the laboratory? 

8. State your results for I, II and III in detail, draw any 
conclusion possible and point out any practical operations. 

REFERENCES 

MARSHALL: Microbiology, pp. 185-191. 

BESSON: Practical Bacteriology, Microbiology and Serum Therapy, 

pp. 862-863. 

CONN: Bacteria, Yeasts and Molds, pp. 114-123. 
CONN: Practical Dairy Bacteriology, pp. 65-67. 
TYNDALL: Floating Matter of the Air. 



148 GENERAL MICROBIOLOGY 



EXERCISE 52. QUALITATIVE STUDY OF THE MICRO- 
FLORA OF THE SKIN AND HAIR IN HEALTH AND 
IN DISEASE 

Apparatus. Ordinary forceps; two sterile, small white 
enamel basins (steam-sterilized); three sterile 1 c.c. pipettes; 
six sterile Petri dishes; six tubes of sterile agar; one liter 
flask containing about 700 c.c. of sterile water or salt 
solution; sterile cloth (J yd. hospital gauze wrapped in 
paper and sterilized at 180 C.); soap; clean slides and 
cover-glasses. 

Method. I. Skin, (a) Normal. 1. Place about half 
the sterile water in a sterile basin. 

2. Wash the hands thoroughly in the sterile water. 

3. Plate 1 c.c. of this water immediately in ordinary 
agar. 

4. Then wash the hands well with soap and tap water, 
rinse with tap water till free of soap and dry the hands on 
the sterile cloth. 

5. Place the remaining sterile water in the second 
sterile basin and wash the hands again. 

6. Plate 1 c.c. of this water. 

7. Incubate both plates (inverted) at 37 C. 

8. How long before starting this experiment did you 
wash your hands? How might this influence your results? 

9. What types of microorganisms would you expect 
to find on the skin? Why? 

(6) Diseased. 1. With a sterile needle obtain a small 
amount of purulent material from a pustule, boil, or abscess, 
etc. 

2. Make three loop-dilution plates in agar. Incubate 
at 37 C. 

3. Examine some of this material microscopically by 
preparing a stained smear. 

4. Draw and describe the latter. Do you find the 
same organisms on the plates as on the slide? 



MICROFLORA OF THE SKIN AND HAIR 149 

6. Isolate the most predominant organism on the plates 
and identify them. 

6. What is the source of all these microorganisms? 
What becomes of them when we wash our hands and wipe 
them in the ordinary way? Are they detrimental to health? 

7. What is pus? Of what does it consist? What care 
should be taken with discharges from suppurating sores? 

II. Hair, (a) Normal. 1. Using flame-sterilized forceps 
(ordinary type), obtain several hairs and place them in a 
sterile Petri dish. 

2. Using a sterile pipette, add 1 c.c. of sterile water 
or salt solution to the Petri dish and stir the hairs about 
in it with the pipette or sterile loop in order to dislodge the 
organisms adhering to them. 

3. Pour into the plate a tube of melted agar (at 40 
to 45 C.), and when hard, incubate at 37 C. 

4. After twenty-four to forty-eight hours, examine 
predominating colonies in a hanging drop. 

(b) Diseased. 1. With sterile forceps obtain a few hairs 
from the growing edge of the infected portion of the skin 
affected with ringworm or barber's itch. These .hairs will 
come out easily in comparison with healthy hairs. 

2. Mount and examine for the fungus, Trichophyton 
tonsurans. (See illustration on p. 578 in Marshall's 
Microbiology.) Draw. 

3. Continuous application of a glycerinated solution 
of 1 : 500 HgCl 2 (glycerin 1 part, HgCl 2 1 : 500, 9 parts) 
will kill this fungus. 

State in detail your results for I and II, draw any con- 
clusion permissible and point out any practical application. 

REFERENCES 

MARSHALL: Microbiology, pp. 522-524, 545, 578, 591-593. 
BESSON: Practical Bacteriology, Microbiology aud Serum Therapy, 
pp. 679-688. 



150 GENERAL MICROBIOLOGY 



EXERCISE 63. QUALITATIVE STUDY OF THE MICRO- 
FLORA OF THE MUCOUS MEMBRANE (MOUTH 
AND THROAT OR NOSE) 

Apparatus. Absorbent cotton, small piece; wire rod 
about 15 cms. long; clean slides and cover-glasses; Petri 
dish, sterile; tube of sterile agar; tube of sterile broth; 
aqueous-alcoholic fuchsin. 

Method. I. For Teeth. 1. Place a small drop of dis- 
tilled water on a clean cover-glass or slide. 

2. Introduce some material obtained by scraping along 
the base of and between the teeth with a sterile platinum 
needle. 

3. Allow to dry, fix and stain with aqueous-alcoholic 
fuchsin. 

4. Examine microscopically with the oil immersion lens. 

5. Draw all forms seen. Would all of these forms 
grow on an agar plate? Give reasons for your answer. 

II. For Throat or Nose. 1. Prepare a swab by winding 
a small piece of absorbent cotton snugly about one end of 
the wire rod. 

2. Place in a test tube, swab end down, and prepare 
for sterilization as with pipettes. 

3. Dry-sterilize. 

4. Pour an agar plate and allow it to harden. 

5. Moisten the sterile swab in sterile broth, using 
aseptic precautions, and then swab the throat or nose. 

6. Lightly brush the inoculated swab over the surface 
of the agar plate and place the plate inverted, at 37, to 
develop. 

7. Using the same swab, make a smear on a clean 
glass slide, dry, fix, stain and examine as with the prep- 
aration from the teeth. 

8. Return the swab to the tube of broth, incubate for 
twenty-four hours and examine the growth in a hanging 
drop. 



MICROFLORA OF THE MUCOUS MEMBRANE 151 

9. Draw and describe the predominating organisms. 

10. Find a streptococcus, if possible, on the agar plate 
from the swab. 

11. Make a stained slide and have the instructor in- 
spect the same, when you think that you have been suc- 
cessful. 

12. How does the microflora of the mucous membrane 
differ from that of the outer skin? 

CAUTION. Aseptic precaution must be taken in all instances, as 
some of the microorganisms may be pathogenic ! 

State in detail your results from I and II, draw any 
conclusions possible and point out any practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 522-524, 528, 546, 591-599, 609-613. 
BESSON: Practical Bacteriology, Microbiology and Serum Therapy, 
pp. 81, 191, 197, 270, 592-610, 617-626. 



PART II 
PHYSIOLOGY OF MICROORGANISMS 



EXERCISE 1. TO DEMONSTRATE THE SMALL AMOUNT 
OF FOOD NEEDED BY BACTERIA 

Apparatus. Distilled water; eleven sterile Petri dishes; 
sterile 1 c.c. pipette; sterile 10 c.c. pipettes; eight sterile 
200 c.c. flasks; eleven tubes of sterile agar (ordinary). 

Cultures. B. coli. 

Method. 1. Place 150 c.c. of distilled water in each of 
two sterile flasks. 

2. Sterilize one flask (flask B) in the autoclav for ten 
minutes at 15 Ibs. pressure (120 C.). 

3. Plate 1 c.c. from the remaining flask (flask A), imme- 
diately on agar. 

4. As soon as flask B is cold, plate 1 c.c. 

5. Then inoculate the water in flask B with B. coli, using 
the straight needle and transferring a very small amount, and 
plate 1 c.c. 

6. Place flasks A and B and the plates made from the 
flasks at room temperature. 

7. Prepare four 90 c.c. and two 99 c.c. dilution flasks and 
sterilize. 

8. At the end of five days, plate from flasks A and B, 
using 1 c.c. direct and dilutions 1 : 10, 1 : 100 and 1 : 1000. 
Incubate the plates at room temperature. 

9. Count each set of plates at the end of five days' 
incubation. 

152 



PHYSIOLOGICAL CLASSIFICATIONS 153 

10. Compute the weight of the bacteria in the flask of 
distilled water at its highest count. 

What is the smallest amount that may be weighed on the 
ordinary analytical balances? Conclusions? 

11. Plot curves to show whether bacteria are decreasing 
or increasing. Offer a logical explanation for the direction 
the curve takes in each instance. 

12. Note the conditions under which distilled water is 
obtained and dispensed in the laboratory. Why is the 
distilled water not sterile? 

13. By what process of distillation may distilled water be 
obtained free from microorganisms? What several factors 
outside of errors in technic may have influenced your 
results? 

14. What would be the comparative influence of a large 
and a small inoculation upon the number of B. coli surviv- 
ing the 5 days sojourn in the distilled water? 

16. State your results in detail, draw any possible con- 
clusions and point out any practical applications. 

REFERENCES 

MARSHALL, C. E.: Microbiology, pp. 88-89. 

FISCHER, ALFRED : Structure and Functions of Bacteria, pp. 52-54. 
PRESCOTT and WINSLOW: Elements of Water Bacteriology, 3d Ed. 
pp. 151-153. 

SOME PHYSIOLOGICAL CLASSIFICATIONS OF 
BACTERIA 

Bacteria are often classified, in general terms, according 
to their functions, into: 

Saprogenic, or putrefactive bacteria; 

Zymogenic, or fermentative bacteria; 

Pathogenic, or disease-producing bacteria. 
According to their food requirements, into : 

Prototrophic, requiring no organic food (e.g., nitrifying 
bacteria) j 



154 GENERAL MICROBIOLOGY 

Metatrophic, requiring organic food (e.g., zymogenic 

bacteria, saprophytes and facultative parasites) ; 
Paratrophic, requiring living food (e.g., obligate para- 
sites); (A. Fischer). 

According to special food preferred, into: 
Acidophile: acid loving; 
Halophile: salt loving; 
Saccharophile: sugar loving; 
Saprophile: loving dead organic matter; 
Coprophile: loving barnyard manure. 
According to their oxygen requirements, into : 

Aerobic: requiring atmospheric oxygen for growth; 
Anaerobic: requiring the absence of atmospheric oxygen; 
Partial anaerobic: requiring an intermediate oxygen 

tolerance. 

According to the necessity of one kind of food or environ- 
ment, into: 
Obligate: indicating absolute requirements, e.g., obligate 

parasite, obligate anaerobe; 

Facultative: indicating a variability in requirements; 
the word following indicates the condition under 
which the organism may live but does not prefer for 
growth, e.g., B. coli is a facultative anaerobe. 
According to their metabolic products, into : 
Chromogenic, or pigment-producing bacteria; 
Photogenic, or light-producing bacteria; 
Aerogenic, or gas-producing bacteria; 
Thermogenic, or heat-producing bacteria. 
Chromogenic bacteria are classified in accordance with 
the nature and location of the coloring matter which they 
elaborate, as 

Chromophorus bacteria, the pigment being stored in the 
cell protoplasm of the organism analogous to the chlorophyll 
of higher plants, e.g., green bacteria and red sulphur bac- 
teria, purple bacteria. 

Chromoparous bacteria, true pigment formers. The pig- 



ANAEROBIC CULTURE METHODS 155 

ment is set free as a useless excretion, may be excreted as a 
colored body or as a colorless substance which becomes 
oxidized upon exposure to the air. Individual cells are 
colorless and may cease to form pigment, e.g., B. prodigiosuSj 
B. ruber, B. indicus. 

Parachrome bacteria. The pigment is an excretory prod- 
uct but is retained within the cell, e.g., B. violaceus. (Bei- 
jerinck.) 
According to their temperature relations, into: 

Pecilothermic (poikilothermic) bacteria: adaptability to 

temperature of environment; 
Stenothermic bacteria: a very narrow temperature 

range (strict parasites); 

Eurythermic bacteria: a very wide temperature range 
(metatrophic bacteria), often 30 between maximum 
and minimum temperatures. 
According to their optimum temperature, into: 

Cryophilic (psychrophilic, term 

used chiefly for water organ- Min. Opt. Max. 

isms) bacteria C. 15 C. 30 C. 

Mesophilic bacteria (includes 

pathogenic bacteria) 15 C. 37 C. 45 C. 

Thermophilic bacteria 45 C. 55 C. 70 C. 

ANAEROBIC CULTURE METHODS 

The cultivation of strict anaerobes is accompanied by 
certain technical difficulties arising from the necessity of 
removing all traces of oxygen from the medium and from 
the atmosphere to which this medium is exposed. It is, 
therefore, necessary to employ special apparatus or special 
methods for their cultivation. 

The recent investigations of Tarrozzi, which have been 
confirmed by others, seem to show that oxygen does not exert 
any direct harmful effect on anaerobic organisms, but that 
the presence of free oxygen prevents the medium furnishing 



156 GENERAL MICROBIOLOGY 

the nutritive substances necessary for anaerobic life. Anae- 
robic organisms can, in fact, as Tarrozzi has shown, be grown 
in the presence of the oxygen of the atmosphere by simply 
adding pieces of animal tissue or some reducing agent to 
the culture medium. 

Several principles are employed as a basis for the different 
methods of anaerobic cultivations, as follows: 

I. Exclusion of air from the cultivation. 

II. Exhaustion of air from: 

1. The medium by boiling. This should always imme- 
diately precede the inoculation of the medium for anaerobic 
cultivations. 

2. The vessel containing the medium by means of an air 
pump, i.e., cultivation in vacuo. 

III. Absorption of oxygen from the air in contact with the 
cultivation, i.e., cultivation in an atmosphere of nitrogen, 
by means of: 

1. Chemical action upon a readily oxidizable substance 
in a sealed vessel containing the cultures, e.g., sodium 
hydroxide upon pyrogallic acid. 

2. Burning a filter paper saturated with alcohol in a 
sealed vessel. (Moore.) If the paper is well saturated no 
deleterious products of combustion are formed which would 
inhibit growth. 

3. Adding to the medium some easily oxidizable sub- 
stance as dextrose (2%), sodium formate (0.5%), sodium 
sulphindigotate (0.1%) or fragments of sterile tissue to 
absorb all the available oxygen held in solution by the 
medium. 

The chemicals are generally employed in the case of deep 
stab cultures, the fragments of sterile tissue in broth cul- 
tures (Tarrozzi's method). The tissue must be freshly 
removed from an animal (rabbit, mouse, guinea pig, etc.) 
and only pieces of liver, spleen, kidney or lymphatic glands 
may be used with success; blood, milk, or the connective 
tissues are useless for the purpose. Vegetable tissue (potato, 



ANAEROBIC CULTURE METHODS 157 

elder pith, mushrooms, etc.) have been used similarly with 
success (Wrzosek, Ori and others). Spongy platinum has 
also been used similarly with satisfactory results. 

The vitality of anaerobic organisms is exhausted much 
more quickly on media prepared on these principles than on 
media under anaerobic conditions (Jungano and Distaso). 

Perhaps if these methods were used in conjunction with 
anaerobic methods the vitality of the anaerobes would not 
be impaired. 

4. Growing the anaerobe in the presence of a vigorous 
aerobe by the use of special methods or apparatus. 

IV. Displacement of air by an indifferent gas such as 
hydrogen, carbon dioxid, etc. 

V. A combination of two or more of the above methods. 
The following methods are those best adapted for class 

use and can be utilized in a regular exercise as desired : 

I. EXCLUSION OF AIR 

Hesse's Method. This method may be used either with 
a pure culture or for determining the presence of anae- 
robes in any substance. 

Apparatus. Tubes of agar or gelatin for stab cultures; 
sterilized oil (olive oil, vaselin or paraffin oil) ; sterile 1 c.c. 
pipette. 

Culture. Pure culture of an anaerobe. 

Method. 1. Make a stab culture of the anaerobe, using 
a tube containing a deep column of the medium, and thrust- 
ing the inoculating needle to the bottom of the tube. The 
stab culture and a test tube shake culture also may be 
treated as follows: 

2. With the sterile pipette place a layer of sterile oil,* 
1 to 2 cm. deep, upon the surface of the medium. 

3. Incubate at the optimum temperature. 

* Sterile melted agar or gelatin may be substituted for the 
sterile oil. 



158 



GENERAL MICROBIOLOGY 



II. EXHAUSTION OF AIR 

A. By Boiling. It is well to expel all the air from a 
medium to be used for isolating or growing anaerobes by 
boiling twenty to thirty minutes, and cooling rapidly just 
previous to inoculating, and placing under anaerobic con- 
ditions. 

B. Cultivation in Vacuo. This requires special apparatus 
for obtaining a vacuum and for cultivation in some cases. 





FIG. 39a. Novy Jar for 
Tube Cultures. 



FIG. 396. Novy Jar for 
Plate Cultures. 



Apparatus. Special tubes: 

1. Vacuum tubes (Fig. 129, p. 238, Eyre's Bacteriological 
Technic) . 

2. Pasteur, Joubert and Chamberland's tube (Fig. 80, 
p. 93, Besson's Practical Bacteriology, Microbiology and 
Serum Therapy). 

3. Pasteur's tube (Fig. 81, Besson, ibid.). 

4. Lacomme's tube (Fig. 82, Besson, ibid.). 

5. Roux's tube for stroke cultures (Fig. 91, p. 101, 
Besson, ibid.). 

6. Roux's tube for potato cultures (Fig. 92, p. 101, 
Besson, ibid.). 

7. Esmarch's tube (Fig. 95, p. 103, Besson, ibid.). 

8. Vignal's tube (Fig. 96, p. 103, Besson, ibid.). 



ANAEROBIC CULTURE METHODS 



159 



Special flasks: 

1. Pasteur's flask (Fig. 79, p. 92, Besson, ibid.). 

2. Flasks with long necks (Fig. 83, p. 94, Besson, ibid.). 

3. Bottle (Fig. 84, p. 94, Besson, ibid.). 

4. Kitasato's dish (Fig. 93, p. 10, Besson, ibid.). 

5. Bombicci's dish (Fig. 94, p. 102, Besson, ibid.). 

6. Ruffer's or Woodhead's flask (Fig. 33, p. 41, Eyre, 
ibid.). 

Special jars in which test tube or plate cultures may be 
placed and a vacuum produced. 




FIG. 40. Bulloch's Anaerobic Jar. 

1. Novy's jar for plates (Fig. 135, p. 245, Eyre, ibid.). 

2. Novy's jar for tubes (Fig. 136, p. 245, Eyre, ibid.). 

3. Bullock's anaerobic apparatus (Fig. 137, p. 247, Eyre, 
ibid.). 

4. Tretrop's apparatus (Fig. 97, p. 105, Besson, ibid.). 

5. Botkin's apparatus (Fig. 134, p. 244, Eyre, ibid.). 
Apparatus for obtaining a vacuum: 

1. Electric pump adaptable to vacuum or pressure. 

2. Water vacuum pump. 

3. Mercury vacuum pump. 

Method. 1. The tube and flask cultivations are prepared 
by, (a) placing the desired medium in the vessel ; (6) inoculat- 
ing from the desired source; (c) attaching to the vacuum 



160 GENERAL MICROBIOLOGY 

pump and (d) while the pump is running, sealing the tube 
or flask in the flame, at the constriction provided for the 
purpose. 

2. The special jars have the advantage that tube and 
plate cultivations may be prepared in the usual way and 
then placed in the special jar which is then attached to the 
vacuum pump; when sufficient vacuum has been produced 
the stopcock is turned between the jar and the pump. 

Isolation of anaerobic organisms may be accomplished 
with much greater facility by the use of these jars. 

In practically every instance these same jars may also 
be employed in the methods given under the absorption of 
oxygen. 

III. ABSORPTION OF OXYGEN 

Different methods illustrating this general principle are 
much used because of its simplicity and general applicability. 
Any vessel with a tight cover as a Novy jar, an ordinary 
chemical desiccator, a Mason fruit jar, etc., may be used 
as a container for the tube or plate culture. 

A. Pyrogallic add method. 1. Dry pyrogallic acid is 
placed on top of some absorbent cotton in the bottom of 
the jar or tube. 

2. A solution of sodium hydroxide is poured in, but not 
directly upon it. 

3. The cultures are put in place. 

4. The jar or tube is immediately sealed and care is 
taken to mix the chemicals. The organisms thus grow in 
the presence of the inert gas nitrogen. 

The chemicals are used in the proportion of 1 gm. of 
pyrogallic acid to 10 c.c. of 10% aqueous solution of 
potassium or sodium hydroxide for each 100 c.c. of air 
space. 

Apparatus. Tubes for use in oxygen absorption method. 

1. Simple test-tube method. 

2. Giltner's H tube. 



ANAEROBIC CULTURE METHODS 



161 



3. Buchner's tube (Fig. 130, p. 239, Eyre, ibid.). 

4. Turro's tube (Fig. 86, p. 95, Eyre, ibid.). 

By the use of these tubes no sealed jar is necessary. 






FIG. 41. Giltner's Tube. (Orig.) FIG. 42. Buchner's Tube. 

1. The simple test-tube method is advantageous in that 
it requires no special apparatus. It has disadvantages, how- 
ever, which will be mentioned later. 

Apparatus. Test tube of sterile medium; rubber stopper 
to fit tube; pyrogallic acid and sodium hydroxide; paraffin. 



162 GENERAL MICROBIOLOGY 

Method. 1. Inoculate the medium with the material 
under investigation and replace the plug. 

2. Cut off the plug even with the mouth of the tube. 

3. Push the plug into the tube, 4 to 5 cm. 

4. Place on top of the plug the pyrogallic acid and only 
enough of the alkaline solution to saturate the plug. 

5. Insert the rubber stopper and seal with paraffin if 
necessary. If the cotton is more than saturated, the strong 
alkaline solution will run through the plug and kill the organ- 
isms in the culture. 

This preparation is valuable only for noting the presence 
of anaerobes in any substance or studying the growth of an 
anaerobe in pure culture, on account of the difficulties of 
technic. 

2. Giltner's H Tube. This is simply two test tubes 
connected near their mouths by a short piece of glass tubing. 
By this method the tube cultivation may be placed in one 
test tube, the chemicals in the other and both' tubes stop- 
pered. (Fig. 41, p. 161). 

The use of this apparatus presents a distinct advantage 
over any other tube cultivation method, as the culture is 
readily discernible at all times and may be handled without 
the disagreeable features of the other methods. 

The H tube lends itself also to the method depending 
upon the absorption of oxygen by an aerobic organism. 

3. Buchner's tube consists of a stout glass test tube 
having dimensions of about 23 cm. in length and 4 cm. in 
diameter, fitted with a rubber stopper. i \ 

a. A test-tube culture of the organism or mixed culture 
to be tested is prepared. 

6. A little cotton, the pyrogallic acid, and sodium 
hydroxide solution are placed in the Buchner tube, the cul- 
ture immediately introduced and the rubber stopper imme- 
diately fitted tightly in the mouth of the large tube. (Fig. 
42, p. 161). 

4. In Turro's tube, the medium is poured through the 



ANAEROBIC CULTURE METHODS 163 

small inner tube, sterilized and inoculated. The pyrogallic 
acid and sodium hydroxide are then placed in the bulb and 
the stopper immediately replaced. 

This method has advantages over Buchner's in that the 
oxygen is much more rapidly absorbed and the culture is 
visible during incubation. 

Plates. 1. Ordinary deep culture (Petri) dish. 

2. McLeod's plate base (used with the bottom of a deep 
Petri dish). (Muir and Ritchie, 6th Ed., Fig. 23, p. 66.) 

The principle of using these two plates is the same 
throughout and is illustrated in Exercise 2. 

Jars. As has been noted before, the jars designed for 
obtaining vacuum may be utilized in the pyrogallic acid 
method and in the method making use of burning alcohol 
to exhaust the oxygen. 

B. Liborius-Veillon Method and Roux's Biological 
Method depend upon the abstraction of oxygen from the 
medium by aerobic organisms. Liborius makes use of the 
aerobes already present in the mixed culture, while Roux 
uses a pure culture of an obligate aerobe. Nowak first grew 
Bad. abortus by this method. 

Liborius-Veillon Method. 1. Fill long test tubes (22 
cm. XI. 5 cm.) to a depth of 10-15 cm. with glucose agar or 
gelatin and sterilize (below 120 C.). 

2. Place the tubes in a water bath and boil twenty to 
thirty minutes to liquefy the agar and drive off the air dis- 
solved in the medium; then cool to 40-45 C. until sown. 

3. Make loop dilutions in the melted agar and, as soon as 
the tubes are sown, cool them rapidly in an upright position. 

Aerobic organisms grown in the upper part of the medium 
which contains a certain amount of air in solution, while 
the anaerobes multiply in the deeper layer. 

Roux's Method. 1. Make a deep agar or gelatin stab or 
shake culture of the organism or substance to be studied. 

2. Pour upon the surface of this medium a layer 1 to 2 cm. 
deep of a broth culture of a vigorous obligate aerobe as 



164 GENERAL MICROBIOLOGY 

B. subtilis, or an equal depth of liquefied agar or gelatin and 
inoculate this when solid with the aerobe. 

The growth of the aerobe will use up all the oxygen that 
reaches it and will not allow any to pass through to the 
medium below, which will consequently remain in an 
anaerobic condition. 

Giltner's H-tube Method. The use of Giltner's H-tube 
allows the anaerobe in a certain medium to be grown on one 
side of the H either as a stab culture or a streak, while the 
aerobe in the same or a different medium, liquid or solid, 
may be grown on the other side. Rubber stoppers, fitted 
to mouths of both tubes, are superimposed on cotton plugs. 
The aerobe soon exhausts the oxygen from the tube, allow- 
ing the anaerobes to develop. 

This is the method recommended for determining the 
presence of and isolating Bad. abortus from infected mucous 
membranes and tissues. This organism when first isolated 
from tissues is a partial anaerobe, i.e., when an agar shake 
culture is made in an ordinary test tube the colonies develop 
in a zone about 0.5 cm. in width about 1.5 to 2 cm. below the 
surface of the agar. 

By the use of the H tube, surface colonies of this organism 
may be readily obtained for study. 

Novy Jar Method. This same principle may be applied 
by the use of separate tube or plate cultivations of anaerobes 
and aerobes in a Novy jar or similar apparatus; the aerobic 
organisms should be offered a large surface for growth in 
each case, 

IV. DISPLACEMENT OF AIR BY INDIFFERENT GASES 

The special tubes, flasks and jars adapted to cultivation 
of anaerobes in a vacuum are equally applicable in this 
method. 

The gas generally employed is hydrogen. It is preferable 
to other gases not only because it is easily prepared, but that 
it has no injurious effects on the organisms. 



THE EFFECT OF ANAEROBIC CONDITIONS 165 

A Kipp generator is connected up with three wash bottles, 
containing: 

(a) 10% lead acetate solution to remove H^S ; 

(6) Silver nitrate solution to remove AsHs ; 

(c) 10% pyrogallic acid solution made alkaline to remove 
any trace of oxygen, may be used to furnish hydrogen. 

Hydrogen is most conveniently obtained by keeping a 
cylinder of the compressed gas in the laboratory. This 
gas generally contains about 99.6% hydrogen, the remain- 
ing 0.4% is mostly or entirely air, which represents 0.08% 
oxygen. The gas so kept requires no preliminary washing, 
but may be passed direct from the cylinder into the jar 
or flask. 

Carbon dioxide is harmful to a large number of organ- 
isms, as is also coal gas. Nitrogen is satisfactory, but its 
method of preparation is so difficult that its use should be 
abandoned in practical bacteriology unless it can be obtained 
compressed in cylinders. 

EXERCISE 2. THE EFFECT OF ANAEROBIC CONDI- 
TIONS UPON MICROORGANISMS FROM MANURE 

Apparatus. Modeling clay; tubes of sterile gelatin; 
three sterile Petri dishes; three sterile deep-culture dishes 
(use top of Petri dish for cover); sterile 1 c.c. pipettes; 
sterile dilution flasks; six tubes of sterile agar; pyrogallic 
acid; 10% solution of sodium hydrate; absorbent cotton. 

Culture. Horse manure. 

Method. 1. Plate the manure (1 gm. in 99 c.c. dilu- 
tion flask) in duplicate in the Petri dishes and in the deep 
culture dishes, using dilutions 1 : 100, 1 : 10,000 and 
1 : 1,000,000. 

2. As soon as the agar is solid, invert the deep culture 
dishes containing the dilutions. 

3. Place a small piece of absorbent cotton in the center 
of the cover. This must not touch the agar. 

4. On the absorbent cotton, place 1 gm. of pyrogallic 



166 GENERAL MICROBIOLOGY 

acid crystals; then place 10 c.c. of 10% NaOH in the cover 
of the dish. (The cotton prevents a too rapid reaction be- 
tween the chemicals.) 

5. Seal at once by packing the space between the cover 
and bottom air tight with modeling clay. Then mix the 
chemicals. 

6. Place all six plates at room temperature. 

Note. In the reaction which takes place between pyrogallic acid 
and NaOH, oxygen is used and an anaerobic condition is established 
within the culture dish (exact reaction not known.) 

7. Count the organisms after seven days. Estimate the 
number of different types of colonies developing under the 
varying conditions of air supply and note growth. Con- 
clusions? 

8. Compare your results with those of others and draw 
conclusions. 

9. Make gelatin stabs of three or four of the predomi- 
nant types of colonies and cultivate anaefobically by 
Hesse's method. What types of organisms are these 
morphologically and culturally? 

10. Are any types found on aerobic plates which are 
lacking on the anaerobic plates and vice versa? 

What type of anaerobe is frequently found in horse 
manure? 

11. When do anaerobic conditions exist in milk? In 
soil? Is this beneficial or otherwise in each case? What 
relation may there be between age of milk and type of colo- 
nies? Can this same relationship apply in the case of 
soil? 

12. What other methods may be used for obtaining 
anaerobic conditions for microbial growth? Name the 
obligate anaerobes. 

13. How is an organism isolated which is tolerant of an 
amount of oxygen less than that of the atmosphere, but will 
not grow under strictly anaerobic conditions? 

14. State your results for the experiment in detail and 



ACIDS FORMED FROM CARBOHYDRATES 167 

point out any conclusions that may be drawn. Mention 
any practical applications to be made. 

REFERENCES 

EYRE: Bacteriological Technic, 2d Ed., pp. 236-247. 
MARSHALL: Microbiology, pp. 93-98, 228-232, 306-331, 634-640. 
BESSON: Practical Bacteriology, Microbiology and Serum Therapy ,- 

pp. 87-105. 
GILTNER: Suggestions for partial anaerobic cultures. Science, n. s., 

Vol. XLI, No. 1061, p. 663. 

EXERCISE 3. TO DEMONSTRATE THAT ACIDS ARE 
FORMED FROM CARBOHYDRATES BY BACTERIA 

Apparatus. Three tubes of sterile litmus lactose agar; 
three tubes of sterile dextrose agar containing CaCO 3 ; 
sterile dilution flask (containing about 150 c.c. sterile salt 
solution) ; six sterile Petri dishes-; sterile 1 c.c. pipettes. 

Culture. Fresh milk culture of Bad. lactis addi. 

Method. 1. Place a very small loopful of the Bad. lactis 
addi culture in the dilution flask. (Transfer from the white 
portion of the litmus milk culture.) Shake well. . 

2. Make three plates from each medium, using widely 
varying amounts, for example, 0.5 c.c., 0.1 c.c. and 1 drop. 
Just before plating, mix the CaCOs well with the agar (avoid 
air bubbles). 

3. Place the plates (inverted) at room temperature. 

4. Examine each daily after forty-eight hours. Note 
how each medium is changed by the growth of the colonies. 
Explain what has happened. 

How is the object of the experiment demonstrated in 
the case of each medium? 

6. Compare the size of colonies on the different media; 
also on each dilution of one medium; explain. Why are 
the colonies smaller on the thickly seeded plates? 

6. Write the chemical equation with a specific enzyme 
for each change in the case of each medium. 



168 GENERAL MICROBIOLOGY 

7. Write the reaction involving the CaCOs. 

8. State your results for the experiment in detail, draw 
any conclusions and point out any practical applications 
that may be made. 




FIG. 43. Solution of Calcium Carbonate by Bact. lactis acidi. 
(Orig. Northrup.) 

REFERENCES 

LOHNIS, F.: Laboratory Methods in Agricultural Bacteriology, pp. 

71-73. 

VERNON: Intracellular Enzymes, p. 98. 

EULER-POPE: General Chemistry of the Enzymes, pp. 31 and 58. 
MARSHALL: Microbiology, pp. 96, 102, 107, 108, 306-312. 
RAHN, O. : Tech. Bui. No. 10, Mich. Experiment Station, Fermenting 

Capacity of a Single Cell of Bact. lactis acidi, pp. 22-23. 



ORGANIC ACIDS SERVE AS A FOOD 169 

EXERCISE 4. TO SHOW THAT ORGANIC ACIDS MAY 
SERVE AS A FOOD FOR SOME ORGANISMS 

Apparatus. Two sterile 200 c.c. Erlenmeyer flasks; 
sterile 5 c.c. pipettes; 200 c.c. whey, soured by Bact. lactis 
acidi. 

Cultures. Oospora lactis. 

Mycoderma (pickle scum yeast). 

Method. 1. Titrate the acid liquid and record the titre. 

2. Place 100 c.c. in each flask. 

3. Heat for one hour, cool and inoculate each flask with 
one organism. 

4. Titrate every two days from the time growth shows 
until the reaction becomes constant. Always use sterile 
pipettes for obtaining a sample for titration. 

5. Plot curves on the same paper, using the same zero 
point. 

6. Did you place the organism in its natural habitat? 
Will either of these organisms use another acid except 

that common to its habitat? 

7. What is the chemical nature of this organic acid? 

8. What has happened to the organic acid in question? 
Write the chemical equation showing this action. 

9. What type of enzyme is concerned in the change which 
takes place? 

10. State your results in detail and point out any con- 
clusions to be drawn. Point out any practical applications 
that may be made. 

REFERENCES 

LAFAR: Technical Mycology, English Ed., Vol. II, Part 2, pp. 417, 418, 

452-455. 
LASER: Biological Test for Butter. Zeitschrift fur Hygiene (1891), 

Vol. X, p. 513. 

MARSHALL: Microbiology, p. 111. 
NORTHRUP: Tech. Bui. No. 15, Michigan Experiment Station. The 

Influence of Certain Acid-destroying Yeasts upon Lactic Bacteria, 

pp. 5-7. 



170 GENERAL MICROBIOLOGY 



EXERCISE 5. TO DEMONSTRATE THE VARIATION IN 
FOOD REQUIREMENTS OF BACTERIA AND THEIR 
SELECTIVE POWER 

Apparatus. Two tubes of sterile fermented agar; two 
sterile Petri dishes; potassium phosphate, di-basic; aspara- 
gin; peptone; ammonium sulphate; sodium nitrate; dex- 
trose; lactose; saccharose. 

Culture. B. prodigiosus. 

Method. 1. Melt the tubes of agar in the steam and, 
when cool but still liquid (about 40 C.), inoculate each 
heavily with B. prodigiosus and pour the plates. Allow to 
stand twenty-four hours at room temperature before pro- 
ceeding. Is there any visible growth on the plate? 

2. Mark on the bottom of each plate with drawing ink, 
dividing it into three equal sectors. 

3. Use ink to indicate the places of chemicals, which 
should be deposited at the center of each plate and of each 
sector. 

4. Use very small quantities of the chemicals and be 
very careful not to scatter them over the plate while convey- 
ing them to their proper places, otherwise the purpose of the 
experiment will be defeated. 

5. Incubate at room temperature and examine the plate 
from day to day for growth. 

Does the fermented agar support growth of itself? 
What explanation can you give for the action which 
occurs? 

6. How is the variation in food requirements of B. pro- 
digiosus shown? The selective action? Give another 
example of the demonstration of the selective action of 
bacteria. Which source of nitrogen is seemingly least 
available? Which most available? Why? Which carbo- 
hydrate is most easily digested? Which least? Why? 

Beijerinck, knowing that agar in and of itself is a food 
for but very few microorganisms, reasoned that this sub- 



SPLITTING OF CARBOHYDRATES INTO ALCOHOL 171 

stance might be used for making solid synthetic media 
if it could be freed in some way from all traces of food 
materials. This he hoped to accomplish by allowing 
the agar to ferment spontaneously upon the addition of 
water. 

In order that agar may not support microbial growth 
it must be allowed to ferment over a long period of time to 
exhaust every possible trace of food. 

7. State your results in full, draw any conclusions that 
follow and point out the practical applications that may 
be made. 

REFERENCES 

MARSHALL: Microbiology, pp. 89-93, 98-100. 

FISCHER, A.: Structure and Functions of Bacteria, p. 115. 

EXERCISE 6. TO DEMONSTRATE THE SPLITTING OF 
CARBOHYDRATES INTO ALCOHOL AND CO 2 

Apparatus. Clean 375 c.c. Erlenmeyer flask fitted with 
one-hole rubber stopper containing a bent glass tube plugged 
at the end with cotton; two calcium chloride tubes; potash 
bulb ; calcium chloride (small granules) ; potassium hydrox- 
ide solution (1 part KOH, 2 parts H 2 0); rubber tubing for 
connecting up apparatus; 400 c.c. fractional distillation 
flask; thermometer; 250 c.c. 5% saccharose bouillon. 

Culture. Sacch. cerevisice. 

Method. 1. Place the saccharose broth in the 375 c.c. 
flask, insert the rubber stopper and sterilize by the discon- 
tinuous method. 

2. When sterile, inoculate with the yeast and connect 
the flask in " train " with a CaCk tube (to remove moisture) 
a tared potash bulb (to take up 62) and a second CaCl2 
tube. 

3. Place at 25 to 30 C. and allow to stand until no more 
gas evolves (about two weeks) . 

4. Test quantitatively for alcohol (distill off over 10 c.c. 



172 GENERAL MICROBIOLOGY 

of liquid, measure the distillate and determine its specific 
gravity*) and estimate percentage of yield. 

6. Weigh the potash bulb to find the amount of CO2 
given off. Does it correspond to the yield of alcohol? 
Explain. Are your results according to theory? 

6. Write the chemical equation for each change, giving 
the specific enzymes concerned in each reaction. What 
types of enzymes are concerned? 

7. Would alcohol be formed in bouillon containing no 
sugar? In a 5% aqueous solution of sugar? Why? 

What fermentable substances are present in ordinary 
meat bouillon? 

8. State your results in full and draw any conclusions 
warranted. What practical applications may be made of 
the above? 

REFERENCES 

MARSHALL: Microbiology, pp. 135, 140-141. 

LAFAR: Technical Mycology, Vol. II, Part II, pp. 473-481, 511-515. 

HAWK: Physiological Chemistry, 4th Ed., pp. 255, 357. 



EXERCISE 7. TO DEMONSTRATE THE NECESSITY 
OF NITROGEN IN SOME FORM FOR MICROBIAL 
GROWTH 

Apparatus. Four tubes each of: 

Ordinary broth (organic nitrogen, soluble albumins and 
proteins) . 

Dunham's solution (organic nitrogen, soluble peptone, 
no albumen). 

Uschinsky's asparagin medium (organic nitrogen, pro- 
tein-free) . 

Cohn's solution (inorganic nitrogen combined with 
organic acid). 

* Table for determining per cent of alcohol from specific gravity 
in Sadtler's Industrial Organic Chemistry (1912), pp. 579-584. 



NITROGEN FOR MICROBIAL GROWTH 173 

Winogradski's medium for nitrate formation (inorganic 
nitrogen combined with an inorganic acid) . 

Winogradski's medium for symbiotic nitrogen-fixation 
(nitrogen-free) . 

Cultures. B. subtilis; Ps. radicicola; Aspergillus niger; 
Sacch. ellipsoideus. 

Method. 1. Inoculate heavily a tube of each medium 
with Ps. radicicola. Proceed likewise with the three remain- 
ing organisms. 

2. Record the growth at the end of five days. What 
conclusions may be drawn? 

3. Compare the formulae of the different media given 
above. Organic nitrogen is present in the radical NH2, 
inorganic, NH4. 

4. What is the explanation for the growth of one organ- 
ism and not another on a certain medium? 

Do the organisms obtain their carbon from an organic 
or an inorganic compound in each case? Is organic or in- 
organic nitrogen the most available in each case? What 
is the value of such a medium? Why are other chemicals 
added besides the main nutrient? 

Why is distilled water used in all these media? To 
what does the term " auxanography " refer? 

5. Give your results in detail and draw any conclusions 
warranted. What practical applications may be made of 
the above? 

REFERENCES 

MARSHALL: Microbiology, pp. 91-93, 99, 242, 362. 
LAFAR: Technical Mycology, Vol. I, pp. 361, 468. 
LOHNIS: Laboratory Methods in Agricultural Bacteriology, p. 58. 



174 GENERAL MICROBIOLOGY 



EXERCISE 8. TO DEMONSTRATE THE PRODUCTION 
OF H 2 S BY BACTERIA 

Apparatus. Three tubes ordinary gelatin; tube ordinary 
agar; sterile Petri dish; lead carbonate, 0.1 gm. 

Cultures. B. coli communis; B. mycoides; B. mesen- 
tericus vulgatus. 

Method. 1. Make stabs of all organisms in gelatin and 
place these at a temperature not exceeding 20 C. 

2. Melt a tube of agar and while hot add 0.1 gm. of 
lead carbonate to the tube and mix well by rolling it vigor- 
ously between the hands (avoid air bubbles) . 

3. Pour into the sterile Petri dish and when cold make a 
streak (2.5 cm. long and 3 cm. apart) of each organism on 
the plate in the order named. Invert and place at 25 C. 

4. Examine the gelatin stabs from day to day for lique- 
faction; examine the plate culture at the same time. Note 
the action on lead carbonate (Beijerinck's test). 

5. Write chemical equations for the action of sulphur- 
eted hydrogen on lead carbonate. 

/\ 

Lead carbonate = Pb<Q >C = 

x cr 

6. Is there any relation between the power of organisms 
to liquefy gelatin and to produce " lead-blackening " sul- 
phur? Explain. 

From what compounds is the H^S produced in this 
experiment? What type of organisms can be detected by 
this test? Where do they occur in the largest numbers in 
nature? 

Which of the ordinary laboratory media offer the greatest 
source from which this gas may be produced? Explain. 

By what other means may H 2 S production by bacteria 
be demonstrated? 



CHEMICAL AGENCIES ON MICROBIAL PIGMENT 175 

7. Give your results in full. Draw any conclusions 
possible and point out any practical applications that may 
be made. 

REFERENCES 

LAFAB: Technical Mycology, Vol. II., Part 2, pp. 558-560. 
MARSHALL: Microbiology, pp. 113-117. 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 42, 116. 
EYRE; Bacteriological Technic, 2d Ed., 1913, pp. 290-291. 

EXERCISE 9. THE EFFECT OF PHYSICAL AND CHEM- 
ICAL AGENCIES ON MICROBIAL PIGMENT AND 
THEIR FORMATION 

Apparatus. Six tubes of gelatin; six dextrose agar slants 
+ 15; eight tubes of plain milk, sterile; hydrochloric acid; 
sodium hydroxide; chloroform; ether; benzol; carbon 
bisulphide; litmus paper; clean slide; small funnel. 

Cultures. Ps. pyocyanea; R. violaceus; B. prodigiosus; 
Sarcina lutea; Torula rosea; B. cyanogenus; Bad. lactis acidi. 

Method. A . Effect of temperature on pigment formation. 

1. Make two dextrose agar streak cultures of each 
organism. 

2. Place the cultures in duplicate at 25 C. and 37 C. 

3. Examine every day or so and at the end of a week 
record the degree of pigment formation by + +, +, H , . 
Brightness of pigment formation should be considered in all 
cases, not the amount of growth. 

4. Where is the pigment seen macroscopically in each 
case? Explain. 

Does temperature have any influence on pigment forma- 
tion? Does this correspond in each case with that of the 
natural habitat of the organism? 

How do these several pigments differ? Of what impor- 
tance is pigment production? 

B. Relation of air to pigment formation. 

Make gelatin stabs of all organisms and keep at or below 
20 C. Note the place of pigment formation. Explain. 



176 



GENERAL MICROBIOLOGY 



C. Relation of light to pigment formation. 

Make two streaks of B. prodigiosus. Place one in bright 
sunlight, keep the other in the dark. Explain the results. 

D. Effect of chemicals on pigment. 

1. To one of the brightest pigmented cultures of B. 
prodigiosus, add 10 c.c. of 95% alcohol and shake vigorously. 
Alcohol dissolves the pigment. 

2. Pour off into a flask and allow to settle. Filter. 

3. Divide the clear filtrate into four parts. 

To one, add a drop or two of HC1; note the result and 
explain. To the second add a drop or two of NaOH; note 
the result and explain. 

Place the third in bright sunlight and note what happens. 

Place a few drops of the fourth portion on a clean slide 
and allow to evaporate slowly. Examine crystals under 
microscope and draw. What are these crystals? Explain. 

E. Solubility of pigment. 

1. Make five dextrose agar streak cultures of B. pro- 
digiosus and, when well pigmented, try the solubility of the 
pigment in (a) water, (b) chloroform, (c) ether, (d) benzol, 
(e) carbon bisulphide. Results? 

2. Are any of the different bacterial pigments formed, 
water-soluble? What is the simplest method for determin- 
ing whether the pigment produced by an organism is water- 
soluble? 

F. Blue milk and " bloody " milk. 
1. Inoculate milk tubes as follows: 



Organism. 


Alone. 


+ Bact. lactia acidi. 


ToTuld rosea 






B cyanogcnus 






B prodigiosus. 






Control 







and keep at 25 
Observe daily. 



C. along with uninoculated control. 



PHYSICAL PRODUCTS OF METABOLISM 177 

2. At the end of seven days test the reaction of each. Is 
there any relation between the reaction and pigment pro- 
duction? 

3. What conditions are conducive to the formation of 
red milk? of blue milk? 

How would you describe and explain " bloody " milk as 
produced by microorganisms to anyone unfamiliar with the 
phenomenon? How differentiated from true bloody milk? 

4. State all results in full. Draw any conclusions war- 
ranted and point out the practical applications that may be 
made. 

REFERENCES 

LAFAR: Technical Mycology, Vol. I, pp. 105-122. 

THRESH: Examination of Waters and Water Supplies, 2d Edition, pp. 

43-44. 

STERNBERG: Textbook of Bacteriology, pp. 130-132. 
CONN: Agricultural Bacteriology, pp. 156-157. 

EXERCISE 10. TO ILLUSTRATE ONE OF THE PHYSICAL 
PRODUCTS OF METABOLISM 

Apparatus. Three gelatin slants (20 alkaline, 3% salt) ; 
rubber stopper to fit one of the gelatin tubes. 

Culture. Ps. lucifera or some actively phosphorescing 
organism. 

Method. 1. Make a streak culture of the above organ- 
ism upon each of the gelatin slants. 

2. With one of the cultures, boil a rubber stopper and 
insert in place of the cotton plug. 

3. Place the stoppered culture and a second one (cotton- 
plugged) at 20 C., the third cotton-plugged culture at 
5-10 C. 

4. Examine in the light and in the dark after twenty-four 
and forty-eight hours. Compare (in the dark) the two 
cultures at 20 C.; if there is a marked difference, loosen the 
rubber stopper and note what happens. 

5. If there is no immediate result from loosening the 



178 GENERAL MICROBIOLOGY 

stopper, replace the stopper with a sterile cotton plug and 
note both cultures after twenty-four hours. What occurs 
in either case? 

6. Which is the better temperature for the growth of 
this organism? Can you suggest a reason why? What 
is the natural habitat of this type of organism? Of what 
importance are they? 

What would you conclude regarding the respiration of 
phosphorescent bacteria? What term is applied to bacteria 
exhibiting this phenomenon? Of what importance is this 
phenomenon? 

7. State your results in full, and draw any conclusions. 
What practical application of the above may be made? 

REFERENCES 

LAFAR: Technical Mycology, Vol. I, pp. 123-126. 

MARSHALL: Microbiology, (1911), p. 129 

FISCHER, A.: Structure and Functions of Bacteria (1900), pp. 63-64. 

ENZYMES: CLASSIFICATIONS AND REACTIONS 

Enzymes can be classified in several different ways : 

I. According to their place of activity as endo-enzymes 
(intracellular) or exo-enzymes (extracellular) ; 

II. According to the type of food substance acted upon, 
as proteolytic (protein-digesting), lipolytic (fat-digesting), 
enzymes attacking carbohydrates, etc.; 

III. The most satisfactory and inclusive classification 
is that denoting the chemical reactions produced by 
the enzyme during its activity. Enzymes may thus be 
called : 

1. Hydrolytic, the addition of one of more molecules of 
water to the molecule of the substance acted upon. 

2. Enzymes producing intramolecular changes, i.e., caus- 
ing a rearrangement of the atoms within the molecule. In a 
few cases these changes may be hydrolytic (urease) but a 



CLASSIFICATION OF ENZYMES 179 

number of the enzymes of this class causes this rearrange- 
ment, splitting the molecule without the addition to or sub- 
traction from any elements therein. 

3. Oxidizing, the addition of oxygen to (or the subtrac- 
tion of hydrogen from) the molecule of the substance acted 
upon. 

4. Reducing, the subtraction of oxygen from (or the 
addition of hydrogen to) the molecule of the substance 
acted upon. The reducing enzymes are the only class of 
enzymes in the above classification acting upon inorganic 
compounds; some organic compounds are also acted upon, 
viz., litmus, methylen blue, etc. 

5. Coagulating, unknown processes accompanied by 
coagulation. Enzymes whose actions are not so well known 
are those producing syntheses, isomers, acting anaerobically, 
etc. 

Note. Euler's suggestion that the names of enzymes be formed from 
the compound acted upon, by suffixing "-ase," will be adhered to in all 
subsequent study of enzymes, the suffix u -lytic " for the adjective, and the 
suffix "-ese " for synthesizing enzymes. 

Bayliss has suggested the ending "-clastic" for the adjective, criticiz- 
ing the ending '-lytic" because the definition of electrolytic," which 
must be granted priority, implies action by the agent rather than upon 
the substance indicated by the term. He also questions the existence of 
Euler's "synthesizing enzymes," 

CLASSIFICATION OF ENZYMES 

I. Hydrolytic Enzymes of: 

A. Carbohydrates, including Glucosides, carbohydrases 

general term. 
1. Polysaccharides. (CeHioOs)^. 

a. Celluloses: cellulases general term. 
b' Hemicelluloses : cytases general term. 

c. Starches, insoluble and soluble: amylases, 

(ptyalin, diastase) general term. 

d. Glycogens: glycogenases general term. 

e. Dextrins: dextrinases general term. 



180 GENERAL MICROBIOLOGY 



2. Disaccharides. 

a. Saccharose: sucrase (invertase, invertin) 

specific term. 

b. Lactose: lactase specific term. 

c. Maltose: maltase specific term. 

3. Glucosides: glucosidases general term. 

a. Amygdalin: amygdalase (emulsin, synaptase) 

specific term. 

b. Tannin (digallic acid) : tannase specific term. 

4. Pentoses: (C 5 Hio0 5 ):r. 

a. Pectoses: pectases general term. 

B. Esters: ester ases general term. 

1. Fats: Upases (steapsin) general term. 
a. Stearin: stearinase specific term. 

C. Proteins: proteinases or carbamases general term. 
1. Protein-digesting. 

a. Proteins broken down to proteoses and 
peptones : peptase (pepsin or aci'd-proteinase) 

general term. 

6. Proteins broken down further to polypeptids 
and occasionally to a-amino acids with a 
trace of ammonia: tryptase (trypsin or 
alkali-pro teinase) general term. 

c. Proteoses, peptones, polypeptids and pro- 

tamins broken down completely to a-arnino 
acids with a trace of ammonia: ereptase 
(erepsin, protease) general term. 

II. Enzymes producing intramolecular changes,acting on : 
A. Carbohydrates (d-hexoses) CeH^Oe to form: 

1. Alcohol, ethyl and carbon dioxide: zymase 

general term. 

a. Dextrose: dextro-zymase specific term. 
6. Levulose: levulo-zymase specific term. 
c. Galactose: galacto-zymase specific term. 

2. Lactic acid: lactic acid bacteria zymase specific 

term. 



CLASSIFICATION OF ENZYMES 181 

B. Acid amides (urea): amidases general term, to 

form : 
1. Ammonium carbonate: urease specific term. 

III. Oxidizing enzymes: oxidases general term, of: 

A. Ethyl alcohol: alcoholase (alcoholoxidase, vinegar- 

oxidase) specific term. 

B. Organic acids: 

1. Lactic acid: lactacidase specific term. 

2. Acetic acid: acetaddase specific term. 

C. Tyrosin: tyrosinase specific term. 

IV. Reducing enzymes: reductases general term, of: 

A. Hydrogen peroxide: 

1. Catalase specific term, free oxygen liberated. 

2. Peroxidase specific term, transference of oxygen. 

B. Organic dyes to leuco-compounds. 

1. Methylen blue, litmus, azolitmin, indigo, etc.: 

methylen-blue reductase, etc. specific term. 

2. Methylen blue in the presence of formaldehyde 

(Schardinger's reaction) : perhydridase spe- 
cific term. 

C. Sulphur to [28 : sulphur reductase specific. term. 

D. Nitrates to nitrites, nitrates to NHs, etc. : nitrate-, 

nitrite-reductase, etc. 

V. Coagulating enzymes. 

A . Protein-coagulating. 

1. Casein: 

a. Of cow's milk: caseinase (rennin, rennet, 

chymosin) specific term. 
6. Of human milk: parachymosin specific term. 

2. Fibrin of blood: thrombase (thrombin) specific 

term. 

B. Carbohydrate-coagulating. 

1. Pectin : pectinase specific term. 



182 GENERAL MICROBIOLOGY 

ENZYMIC REACTIONS OF WELL KNOWN FERMEN- 
TATION PROCESSES 

A. Beer or bread fermentation by Saach. cerevisice. 

I. starch + water +hydroly tic enzyme = maltose. 

2(C 6 Hi O 6 ) + xH 2 O + amylase = zCi 2 H 22 O u . 

II. (a) maltose + water +hydrolytic enzyme = 2 mols. dextrose. 

(from yeast) 
Ci 2 H 22 On + H 2 O + maltase =2C 6 H 12 6 . 

III. dextrose + enzyme producing intra- = alcohol + carbon 

molecular change dioxide. 

(from yeast) 

C 6 H 12 O 6 + yeast zymase = 2CH 3 CH 2 OH+2CO 2 . 

This same yeast, and other yeasts also, can ferment saccharose 
(.cane sugar) corresponding to II above, as follows: 
II. (6) saccharose -f- water -j-hydroly tic enzyme = d-dextrose + 
fj (from yeast) d-levulose. 

Ci 2 H 22 On + H 2 O + sucrose =C 6 H ;2 O 6 +C 6 Hi 2 06. 

Both of these simple sugars can be fermented to alcohol 
and CO2 according to III above. 

Comparatively few yeasts can attack lactose (milk sugar) , 
e.g., Sacch. kefir (p. 369, Marshall), Sacch. fragilis, Sacch. 
tyricola, etc. (See p. 106, Guilliermond's Les Levures.) 
The reactions are similar to II (a) above, as follows : 

(c) lactose + water+hydrolytic enzyme = d-dextrose +d-ga- 

(from yeast) lactose. 

Ci 2 H 22 On + H 2 + lactose = C 6 H 12 O 6 +C 6 Hi 2 O 

Both simple sugars are changed to alcohol and CO 2 according to 
III above. 

B. Glucoside decomposition, by molds, bacteria and yeasts. 

General reaction, 

glucoside + water -f hydroly tic enzy me = sugar -f- aldehydes, acids, etc. 
Specific reaction, 

amygdalin-f- water -f emulsin = dextrose + benzaldehyde + hy- 
drocyanic acid. 

C 20 H 27 OnN + 2H 2 O + emulsin* = 2C 6 H 12 O 6 + C 6 H 6 CHO+HCN. 
* Emulsin is a mixture of four different enzymes, 



ENZYMIC REACTIONS 183 

C. Fat decomposition, by a few molds, yeasts and bacteria. 
Only microbial method of fat decomposition. 

General reaction, 

f at + water +hydroly tic enzyme = fatty acid + glycerin. 
Specific reaction, 
stearin+3 mols. water+steanrazse = 3 mols. stearic acid+glycerin." 



H 2 O C 17 H 35 COOH CH 2 OH 
I I 

C 17 H 36 CO-O-CH + H 2 O = Ci 7 H 35 COOH + CH 2 OH 
I I 

Ci 7 H 35 CO.O-CH 2 H 2 C n H 35 COOH CH 2 OH 

Lipases decompose more especially the natural fats, 
i.e., the glycerin esters of palmitic, stearic and oleic acids. 
Lipases from different sources differ markedly in reactions. 

D. Vinegar fermentation. 

In order that this fermentation may take place, alcohol 
must be present in the nutrient solution, either added arti- 
ficially or as a product of fermentation. In the latter case, 
reactions I, Ha and III, 116 and III, or He and III under A 
above must precede those of the vinegar fermentation. 

Assuming that alcohol is present in the liquid in which 
the vinegar bacteria are growing, the reactions take place in 
two stages, as follows: (See p. 448, Marshall.) 

I. ethyl alcohol -f oxygen + oxidizing enzyme = acetaldehyde+ water 
CH 3 CH 2 OH + O + alcoholase = CH 3 CHO + H 2 O. 

II. acetaldehy de+oxy gen + oxidizing enzyme = acetic acid 
CHaCHO + O + acetaldehydase = CH 3 COOH 

If there is not plenty of air present the oxidation may not 
become complete and small amounts of acetaldehyde may 
form, i.e., the reaction stops at the first stage. 

If the initial percentage of alcohol is below 1 to 2% the 
vinegar bacteria will soon attack the acetic acid, oxidizing 
it completely to carbon dioxide and water, as follows : 

III. acetic acid +oxy gen + oxidizing enzyme = carbon dioxide+water 
CHsCOOH + 4O + acetacidase = 2CO 2 +2H 2 O. 



184 GENERAL MICROBIOLOGY 

This cannot take place, however, if above 10 to 12% 
acetic acid is present, as this amount is antiseptic to the 
vinegar bacteria. (See pp. 450-451, Marshall.) 

E. Organic acid decomposition, by acidophile organisms 
(organisms of the Oidium and Mycoderma type). 

lactic acid +oxygen +oxidizing enzyme = carbon dioxide + water 
CH 3 CHOHCOOH + 6O + lactacidase = 3CO 2 +3H 2 O. 

The destruction by oxidation of acetic acid by the acetic 
bacteria is given under D above. 

Nearly all organic acids are decomposed in a similar 
manner, by total combustion. 

F. Reactions of reductases. 

hydrogen peroxide oxygen + reducing enzyme = water +oxygen. 
H 2 O 2 O + catalase = H 2 O + O. 

methylen blue +hydrogen + methylen blue = leuco-basa of 

reductase methylen blue. 

C 6 H 3 N=(CH 3 ) 2 C 6 H 3 N=(CH 3 ) 2 

N As 



reductase J 

C 6 H 3 =N=(CH 3 ) 2 C 6 H 3 N=(CH 3 ) 2 

G. Lactic acid fermentation, produced in milk by 
Bact. lactis acidi. 

lactose + water -fhydroly tic enzyme = d-dextrose-fd-galactose. 
Ci 2 H 22 Ou + H 2 + ' lactose = C 6 H 12 O 6 +C 6 H 12 O 6 . 

dextrose 1 +enzyme producing intra- = lactic acid. 
galactose J molecular change 

c acid bacteria zyraa*e = 4CH 3 CHOHCOOH. 



Bact. lactis acidi will ferment a nutrient solution con- 
taining only a simple sugar, e.g., dextrose, the reaction then 
being according to the second equation. 



A COMPARISON OF ACID AND RENNET CURDS 185 

H. Urea fermentation, by urea bacteria. 

Urea + water + hydrolytic endo-enzyme = ammonium carbonate. 
(producing intramolecular change) 



H 2 N 



\ 



C=0 



EXERCISE 11. A COMPARISON OF ACID AND RENNET 

CURDS 

Apparatus. Three 200 c.c. flasks containing 100 c.c. each 
of sterile skim milk; 200 c.c. fresh skim milk; small funnel; 
eighteen large test tubes; absorbent cotton; 10% lactic acid; 
5% phenol. 

Cultures. B. prodigiosus; Bad. lactis addi; B. megater- 
ium. 

Method. 1. Inoculate flasks containing 100 c.c. sterile 
milk with B. prodigiosus, Bad. ladis addi and B. megaterium. 

2. Place about 30 c.c. fresh skim milk in a 200 c.c. flask. 

3. Add 10% lactic acid drop by drop, shaking constantly. 

4. When the first finely divided curd appears, titrate. 
What degree and percent of acid were necessary to curdle the 
milk? 

5. Titrate fresh skim milk. 

6. Prepare 150 c.c. of 0.5% phenol milk (by adding 
15 c.c. of 5% phenol to 135 c.c. of milk and sterilize). 
Mix well and titrate again. Is the acidity of the milk 
increased perceptibly by the addition of phenol? 

7. As soon as curd appears in inoculated flasks, titrate. 
Determine the degree and percent of acidity present. 

8. Allow the cultures to develop several days until 
decided proteolysis is evident. Then titrate again and 



186 GENERAL MICROBIOLOGY 

filter each culture through absorbent cotton (a small piece 
in small funnel); 15 c.c. of each filtrate is necessary. 

9. Mix the filtrate from each culture with phenol milk 
in the following proportions : 

1 0.5 c.c. filtrate +9. 5 c.c. phenol milk. 

2. . 1.0 c.c. filtrate+9.0 c.c. phenol milk. 

3 2.0 c.c. filtrate+8.0 c.c. phenol milk. 

4 3.0 c.c. filtrate+7.0 c.c. phenol milk. 

5 4.0 c.c. filtrate+6.0 c.c. phenol milk. 

6 Heat 4 c.c. of filtrate only, in steam 

for fifteen minutes. After cooling, add 6 c.c. phenol milk. 
Shake these mixtures well and incubate at 37 C. 

10. Record the time necessary for coagulation in each 
case. Why do not all tubes change alike? Explain. 

11. Can corrosive sublimate be used to replace phenol 
in this experiment? Explain. 

What types of enzymes are concerned in these changes? 

Are these intra- or extra-cellular in each case? Will the 
place of occurrence of the enzymes explain the action taking 
place in the different sets of tubes? 

What enzymes produce each type of curd? 

What are the differences between an acid and a rennet 
curd? , 

Which type is produced by each of the organisms used? 

What effect has heat upon enzymes? 

12. Give results in full and draw any conclusions per- 
mitted. Point out any practical applications of the above. 

REFERENCES 

LAFAR: Technical Mycology, Vol. I, pp. 184-187. 

BAYLISS: Nature of Enzymic Action, p. 37. 

EULER: General Chemistry of the Enzymes, pp. 45-48, 58. 

MARSHALL: Microbiology, pp. 139-141. 

VERNON: Intracellular Enzymes, pp. 220-221. 

COHNHEIM: Enzymes, pp. 29, 87-89, 



PROTEOLYTIC ENZYMES UPON GELATIN 187 

EXERCISE 12. TO SHOW THE ACTION OF PRO- 
TEOLYTIC ENZYMES UPON GELATIN 

Apparatus. Phenol, 0.5% solution (10 c.c. of 5.0% 
phenol +90 c.c. distilled H2O) ; water bath and thermometer; 
gelatin, 7 gms.; 15 tubes sterile gelatin; formalin; xylol; 
5 sterile 1 c.c. pipettes; centimeter scale. 

Cultures. B. ramosus; B. fluorescens; B. subtilis; 
B. mycoides; B. prodigiosus. 

Method. 1. Make two gelatin stab cultures of each 
organism and when nearly all liquefied (2-5 days old) pro- 
ceed with the experiment. 

2. Dilute the 5% phenol to 0.5% as above, with distilled 
water. 

3. Add 7 gms. gelatin, dissolving by heating not over 
70 C. Neutralize carefully. 

What is the source of acid in phenol gelatin? Why is 
the gelatin neutralized? 

4. Select five test tubes having the same diameter. Fill 
each half full. Solidify in an upright position. 

5. Shake each of the liquefied cultures with 3 to 4 c.c. 
of xylol. 

6. After one hour, add 1 c.c. of the clear supernatant 
xylol solution of each culture to a tube each of solid phenol 
gelatin and of ordinary gelatin. With a blue pencil, mark 
the surface of the solid gelatin. 

7. Examine the tubes daily. Is there any evidence of 
growth? Of liquefaction? If liquefaction is noted, measure 
its progress in millimeters. 

8. Save the original cultures with which the xylol 
has been shaken. Is there any evidence of further growth? 

9. If 1 c.c. of a liquefied gelatin culture of a liquefying 
organism were added to a tube of solid ordinary gelatin, 
what would happen? What would result if it were added 
to a tube of solid phenol gelatin? Explain. 

10. What action does the xylol have? How can you 



188 GENERAL MICROBIOLOGY 

prove that xylol has this action? What other chemicals 
could be used in place of xylol? 

11. What is the object of adding phenol to the gelatin? 
Would 5% phenol serve the same purpose? Give reason 
for answer. What other chemicals could be used in place of 
phenol? Why? What chemicals could not be used in 
place of phenol? Why? How else may pure enzyme action 
be demonstrated? 

12. Add 5 drops of 40% formaldehyde (formalin) to 
each tube of the duplicate liquefied gelatin cultures and 
note whether they become solid again in a. few days. Ex- 
plain the action. 

13. Give your results in full and draw any conclusions 
possible. What practical applications of the above may be 
made? 

REFERENCES 

BAYLISS: Nature of Enzymic Action, p. 37. 

MARSHALL, C. E.: Microbiology (1911), pp. 134, 138, 141. 

EULER, HANS: General Chemistry of the Enzymes (1912), pp. 115-123. 

VERNON, H. M.: Intracellular Enzymes (1909), pp. 215-220. 

EXERCISE 13. TO SHOW THE ACTION OF PRO- 
TEOLYTIC ENZYMES UPON CASEIN 

Apparatus. Five tubes of sterile milk; two tubes 
nutrient agar; sterile 5 c.c. pipettes; two sterile Petri dishes. 

Cultures. Bad. lactis acidi; B. ramosus; B. coli; 
B. violaceus. 

Method. 1. Warm the milk (40-45 C.). 

2. Place 2 c.c. in each sterile Petri dish and pour one tube 
of melted agar upon it, mix thoroughly by carefully tilting. 

3. When solid, make parallel streaks with Bact. lactis 
acidi and B. ramosus upon one and of B. coli and B. viola- 
ceus upon the other. Transfer cultures -to litmus milk also. 

4. Examine streak cultures every day for evidences of 
proteolysis. Make drawings and compare the rate of action 
of the different bacteria. Compare streak with milk cultures. 



ACTION OF ENZYMES UPON STARCH 189 

5. Is there any relation between the power of enzymes 
to liquefy gelatin and their ability to dissolve casein? What 
type of proteolytic enzyme dissolves casein? 

6. Give your results in detail. Draw any conclusions 
which follow and point out any practical applications that 
may be made. 

REFERENCES 

MARSHALL, C. E. : Microbiology, pp. 138-139, 354. 

HASTINGS : The action of various classes of bacteria on casein as shown 

by milk agar plates. Cent. f. Bakt., II Abt., Bd. 12 (1904) 

p. 590, 

EXERCISE 14. TO SHOW THE ACTION OF ENZYMES 
UPON STARCH 

Apparatus. Three sterile Petri dishes; three test tubes; 
soluble starch; three tubes sterile agar; Lugol's iodin solu- 
tion. 

Cultures. Soil for inoculation. 

Method. 1. Place 0.1 gm. of soluble starch in each 
test tube, plug and sterilize in the hot air sterilizer. 

2. To each tube of starch add one tube of melted agar. 

3. When at the correct temperature (40-45 C.) inoculate 
one tube with one loopful of soil. (State type used.) 
Inoculate the second from the first, etc., then plate all three 
dilutions. 

4. When the colonies are well developed, pour iodin solu- 
tion on the plate and note any clearing around the colonies. 
What does this indicate? 

5. Examine microscopically different types of colonies 
attacking starch. Are they molds, yeasts, or bacteria? 
Which type predominates on your plates? 

6. What enzymes are concerned? Give specific action. 
How is pure enzymic action demonstrated? 

7. Write the theoretical chemical equation. What is 
soluble starch? 



190 GENERAL MICROBIOLOGY 

8. What is the value of such microbial action in soil? 
Where are starch-digesting microorganisms present in 
nature? Of what importance? 

9. State results in full and draw any conclusions. 
Point out any practical applications of the above. 

REFERENCES 

BAYLISS: Nature of Enzymic Action, pp. 25, 113. 
MARSHALL, C. E.: Microbiology (1911), pp. 90, 106, 248, 463, etc. 
EULER, HANS: General Chemistry of the Enzymes, pp. 13-15, et al. 
HAWK, PHILIP B.: Physiological Chemistry (1914), pp. 10, 48, 50, 

61, 65. 

SADTLER, S. P.: Industrial Organic Chemistry, p. 186. 
LAFAR: Technical Mycology, Vol. II, Part 2, pp. 351-353. 

EXERCISE 15. TO SHOW THE ACTION OF REDUCING 
ENZYMES 

Apparatus. Petri dish; medium fine sand; sulphur; 
cake of Fleischmann's compressed yeast, fresh (obtain 
this yourself); small mortar and pestle; lead acetate 
paper. 

Method. 1. Thoroughly grind the sulphur, sand and 
yeast cake in a small mortar. 

2. Place the contents of the mortar in a covered dish 
with a piece of moistened lead acetate paper. What odors 
are noted? 

3. What reaction is demonstrated by the lead acetate 
paper? What reactions are taking place? Give a chemical 
equation which will cover the final changes. May other 
enzymes be released from the yeast cells during the process 
of maceration? If so, what enzymes? 

What names are applied to the specific enzyme acting on 
sulphur and to the class to which it belongs? Where does 
this action occur in nature? 

This enzymic action was first observed in 1888 by a Frenchman, 
J. de Reypailhade, who found that the alcoholic extract of yeast would 
convert elementary sulphur into sulphuretted hydrogen. 



ACTION OF THE ENZYME CATALASE 191 

4. Give all results in full and draw any conclusions 
permissible. What practical applications may be made of 
the above? 

REFERENCES 

MARSHALL, C. E.: Microbiology, pp. 135, 142-143. 

LAFAR, F.: Technical Mycology, Vol. II, Part II, pp. 558-560. 

KRUSE, W. : Allegemeine Mikrobiologie, pp. 652-655. 

EXERCISE 16. TO SHOW THE ACTION OF THE 
ENZYME CATALASE 

Apparatus. Four fermentation tubes of nutrient broth 
(sterile) ; hydrogen peroxide (full strength) . 

Cultures. B. coli; B. subtilis; B. mycoides; Bad. 
lactis acidi. 

Method. 1. Inoculate the fermentation tubes. 

2. After growth is well started, add 1 c.c. of hydrogen 
peroxide to each tube and mix well. 

3. After the tubes have stood for half an hour measure 
the gas formed. Compare your results with those of other 
students. 

Note. If the bottle of H 2 O 2 stands uncorked or in a warm place 
it decomposes very rapidly and the gas formed in the fermentation 
tubes will be much less than from a full strength solution. 

4. What is the strength of commercial hydrogen 
peroxide? 

Where else is catalase found? What is the type of action 
supposedly taking place? Write chemical equation showing 
the general reaction. 

Have you ever observed the action of catalase produced 
in animal tissues? What is the difference between catalase 
and peroxidase? 

5. State the results of your experiment in full and draw 
any conclusions permissible. Point out any practical 
applications that may be made. 



192 GENERAL MICROBIOLOGY 

REFERENCES 

BAYLISS: Nature of Enzymic Action, pp. 140-141. 

EULER-POPE: General Chemistry of the Enzymes, pp. 65, 67-68. 

VERNON: Intracellular Enzymes, pp. 127-132. 

LOHNIS, F.: Laboratory Methods in Agricultural Bacteriology, pp, 

66-67, 79. 
MARSHALL; Microbiology, pp. 135, 142-143. 

EXERCISE 17. TO DEMONSTRATE THE OXIDIZING 
ENZYME OF VINEGAR BACTERIA 

Apparatus. 200 c.c. fermented cider (or other fruit 
juice); sterile 375 c.c. Erlenmeyer flask; sterile 10 c.c. 
pipette; water bath; specific gravity bottle. 

Culture. Bad. aceti. 

Method. 1. Place the cider in a sterile flask and heat 
in a water bath at 60 C. for one hour. Cool quickly. What 
is this process called? 

2. Determine the specific gravity of the fermented 
cider. 

3. Inoculate with a pure culture of Bact. aceti and titrate 
every three days until the titre is constant. 

4. Plot the curve showing and explain the direction 
which the curve takes. What is taking place? Enzyme? 
Chemical equation? 

5. Determine the specific gravity of the solution at the 
last titration. ..How does this compare with specific gravity 
of cider vinegar of legal standard? What is the legal stand- 
ard for vinegar in this state? Can you explain why all 
vinegar does not come up to the legal standard? 

6. Is it practicable to use pure cultures for preparing 
vinegar? How do various species of vinegar bacteria differ 
from one another? 

Under what conditions will acetic fermentation set in 
" spontaneously? " 

What raw materials will give rise to a vinegar by a 
normal acetic fermentation? 



ACTIVATOR FOR ENZYMIC ACTION OF RENNET 193 

How does a scarcity of alcohol influence the amount of 
acid produced? An excess of alcohol? 

How may vinegar be prepared artificially? How adul- 
terated? 

7. State the results of your experiment in detail and 
draw conclusions. Point out any practical applications 
that may be made. 

REFERENCES 

EULER-POPE: General Chemistry of the Enzymes, 60-61. 
MARSHALL: Microbiology, pp. 135, 142, 448-451. 
SADTLER: Industrial Organic Chemistry, pp. 266-272. 
LAFAR: Technical Mycology, Vol. I, pp. 295-307. 
Circular on " Vinegar " prepared by Bacteriological Laboratory, East 
Lansing, Mich. 



EXERCISE 18. TO DEMONSTRATE THE NECESSITY 
OF AN ACTIVATOR FOR THE ENZYMIC ACTION OF 
RENNET (FROM CALF'S STOMACH) 

Apparatus. Four clean 200 c.c. Erlenmeyer flasks; 
sweet skim milk (not over -f!5); rennet, fresh com- 
mercial; two 1 c.c. pipettes; sterile saturated solution of 
monobasic calcium phosphate (Ca(H2PO 4 )2+H 2 O); water 
bath; thermometer. 

Method. 1. Place about 150 c.c. of skim milk in each 
of two 200 c.c. flasks, plug these and sterilize them by the 
Tyndall method. 

2. When ready to start the experiment, obtain 300 c.c. 
of fresh skim milk and place 150 c.c. of milk in each of the 
two remaining flasks. 

3. Mark the fresh milk flasks Nos. 1 and 2; the sterilized 
milk flasks Nos. 3 and 4. 

4. Place all four flasks in a water bath and heat 
the water to 35 C., not higher. (Steam cannot be sub- 
stituted.) 

5. Mark the flasks as follows : 



194 GENERAL MICROBIOLOGY 

Flask No. l=unheated milk + rennet. 

11 2 = unheated milk + calcium phosphate -f ren- 
net. 

" ll 3 = heated (sterilized) milk + rennet. 
" " 4 = heated (sterilized) milk + rennet + calcium 

phosphate. 

6.. Add 1 c.c. of the calcium phosphate solution to one 
flask of fresh milk and mix. (Flask No. 2). 

7. To each flask of milk add a drop of rennet, shake 
quickly, replace the flask in the water bath and leave for ten 
to twenty minutes without disturbing. 

8. Add 1 c.c. of calcium phosphate solution to flask 
No. 4. Shake quickly, return the flask to the water bath 
and leave for ten to twenty minutes without disturbing. 
Observe. If no curd appears, set the flasks at 37 C. and 
observe after about twenty-four hours. What is the expla- 
nation for the phenomena occurring in this flask? 

9. Observe the milk in all flasks for curdling. Which 
flasks of milk curdled? Why? 

10. What are the various synonyms of "rennet?" 
What is the specific action of this enzyme? 

What is the source of the enzyme used? How prepared? 

What living organisms produce coagulating enzymes? 

Does the rennet produced by various bacteria require 
soluble calcium salts for an activator? How would you 
determine this? 

What is an activator? To what property of an activator 
is its action attributed? What are the different classes of 
activators? Do all enzymes require activators? 

11. Give all results in full and draw any conclusions pos- 
sible. What practical applications of the above may be made? 

REFERENCES 

LAFAR: Technical Mycology, Vol. I, pp. 185, 186. 

BAYLISS: Nature of Enzymic Action, pp. 120-121, 132-133. 

RICHMOND: Dairy Chemistry, p. 301. 

EULER-POPE: General Chemistry of the Enzymes, pp. 94, 106-109. 



EFFECT OF CONCENTRATED SOLUTIONS 195 



EXERCISE 19. EFFECT OF CONCENTRATED 
SOLUTIONS UPON MICROORGANISMS 

Apparatus. 750 c.c. nutrient broth; gelatin; salt; 
dextrose; saccharose; five 10 c.c. pipettes; 100 c.c. graduate. 

Cultures. Mycoderma; B. coli; M. varians; Sacch. 
cerevisice; Penicillium; B. prodigiosus. 

Method. 1. Make up four tubes each of the following 
concentrations : 

Electrolytes: sodium chloride, 5%, 10%, 15%, 20%, 25%. 

Non-electrolytes: dextrose and saccharose, 30%, 45%, 
60%, 75%, respectively. 

Colloids: gelatin, 5%, 10%, 30%, 50%. 

2. With the exception of the gelatin the separate weigh- 
ing out for each concentration can be avoided by using the 
following method of mixing, with the stock solution con- 
taining 50% or 75% of the substance under study: 

(a) Weigh out the correct quantity of material and place 
it in a 100 cc. graduate. 

(6) Fill the graduate to the 100 c.c. mark with nutrient 
broth. Place the hand over the mouth of the graduate and 
shake until solution is complete. If necessary, fill to the 
mark again with broth. For example: Dissolve 25 g. of 
salt in about 90 c.c. of broth, fill the graduate to 100 c.c., 
to obtain a broth of which 100 c.c. contain 25 g. of salt. 
Mix this salt broth with common broth in the following 
proportions, by means of pipettes: 

Salt Plain Salt content 

broth. broth. of mixture. 

2 c.c. + 8 c.c. 5% 

4 c.c. + 6 c.c. 10% 

6 c.c. + 4 c.c. 15% 

8 c.c. + 2 c.c. 20% 

10 c.c. + c.c. 25% 

Broth will give a precipitate after heating with salt, 
consequently each salt broth mixture has to be filtered 
separately after heating. What is this precipitated material? 



196 GENERAL MICROBIOLOGY 

Note 1. The stock solution of dextrose is best prepared by adding 
to 75 g. dextrose, 50 c.c. of broth, heating the mixture in the steam 
until dissolved and then making up to 100 c.c. with broth. The sugar 
solutions may have to be filtered also. 

Note 2. As it is a difficult procedure to make up as high concen- 
trations of gelatin as 30% and 50% with any degree of ease and 
accuracy, gelatin prepared according to the following procedure will 
serve to illustrate the point of the exercise. 

With a blue pencil, mark the 10 c.c. level on each of sixteen tubes. 
To make up 5% gelatin, place 0.5 gm. gelatin in each of four tubes 
and make up to the 10 c.c. mark with broth. Proceed similarly with 
the remaining concentrations. After heating once, mix well with a 
sterile platinum loop. 

Gelatin is practically the only colloid that can be obtained in solu- 
tions concentrated enough for this experiment (up to 70%). Great 
care must be taken to avoid the condensation of moisture on the sides 
of the test tubes or flask, because this moisture will reduce the con- 
centration of the surface gelatin and thus cause incorrect data. 

3. Sterilize the tubes by the intermittent method. 

4. Inoculate heavily one tube of each concentration of 
the salt with Mycoderma, one with B. coli and one with M. 
varians. Inoculate tubes of each concentration of dextrose, 
saccharose and gelatin with Penicilliwn, Sacch. cerevisice, 
and B. prodigiosus, leaving a tube of each concentration 
uninoculated for control. 

Note. The inoculation must be heavy, because experience teaches 
that a small inoculum is sometimes not sufficient to secure growth. 

5. Note and tabulate the growth after seven days. 

6. Reinoculate the lowest concentration of each set 
that does not show growth from the highest of the same 
set that does grow, e.g., if Penicillium grows at 45% 
dextrose but not at 60%, inoculate 60% from 45%. If 
it now grows, what is indicated? Is there not plenty of 
water and food material present? Explain your results. 

Does the natural habitat or food requirements of each 
organism explain in any. way the action occurring? 

7. What is meant by osmotic pressure? Electrolyte? 
Colloid? What is known of the relative osmotic pressure 



EFFECT OF DESICCATION UPON BACTERIA 197 

of electrolytes, non- electrolytes and colloids? How are 
these differences explained? Is the relative preserving 
power of these different substances according to the molec- 
ular weight theory? Explain. 

Why is a large inoculum more apt to insure growth than 
a small inoculum? 

Note. Salt-resisting organisms can be secured by plating in agar 
containing 10 to l7>% of salt, from butter, brine pickles, salt pork, 
salt fish, and other salted food. Sugar-resisting organisms can be 
obtained similarly. 

8. State the results of your experiment in full and draw 
conclusions. Point out any practical applications that may 
be made. 

REFERENCES 

MARSHALL: Microbiology, pp. 147-151. 

FISCHER, A.: Structure and Functions of Bacteria, pp. 5, 8-9. 

EXERCISE 20. THE EFFECT OF DESICCATION UPON 
BACTERIA 

Apparatus. Four sterile cover-giasses ; four sterile 
Esmarch dishes; potato knife; eighteen tubes of sterile broth. 

Cultures. B. violaceus (non-spore-producing, non-slime- 
forming) ; slimy milk bacillus (non-spore-producing, slime- 
forming); B. subtilis (spore-producing, non-slime-forming); 
meat bacillus (spore-producing, slime-forming). 

Method. 1. Using a platinum needle, smear one cover- 
glass thickly with a culture of B. violaceus, the second with 
the slimy milk bacillus, the third with the spore-former 
(B. subtilis) and the fourth with the spore and slime produc- 
ing meat bacillus. 

2. Place each of these cover-glasses in separate sterile 
Esmarch dishes and break each into five or six small pieces 
with a sterile potato knife. 

3. Transfer a piece of each cover-glass to a tube of 
nutrient broth after 1, 3, 7, 14, etc., days. 



198 GENERAL MICROBIOLOGY 

Stop transferring when you find that there is no growth 
in the test tube last inoculated. 

4. What influence has the physical condition of the sub- 
strate upon which the microorganisms are dried upon their 
longevity? Illustrate. 

What dried cultures of microorganisms have been used 
with success commercially? Without success? What other 
methods may be employed to demonstrate the effect of 
desiccation on microorganisms? 

5. State all results in full and draw any conclusions. 
Point out any possible practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 151-152, 280-283, 338-343, 367-369, 

374-380, 428-429, 453-460. 

SMITH, E. F.: Bacteria in Relation to Plant Diseases, Vol. I, pp. 70-71. 
EYRE: Bacteriological Technic, 2d Ed., pp. 306-308. 

EXERCISE 21. THE DETERMINATION OF THE OPTI- 
MUM, MAXIMUM AND MINIMUM TEMPERATURE 
REQUIREMENTS FOR CERTAIN ORGANISMS 

Apparatus. Sixteen tubes of dextrose broth. 

Cultures. Sacch. cerevisice; B. subtilis; Oospora lactis; 
Bad. aerogenes. 

Method. 1. Inoculate four tubes of dextrose broth with 
each organism. 

2. Place one culture of each organism at each of the 
following temperatures: 5, 25, 37, and 45. 

3. Note the growth as to vigor after twenty-four, forty- 
eight, seventy-two hours and seven days. Tabulate the data. 

4. What is the natural habitat of each organism? Does 
this explain your results in any way? 

What is the biological significance of the cardinal points 
of temperature? In what industries making use of micro- 
organisms is the regulation of temperature especially 
important? 



EFFECT OF FREEZING ON BACTERIA 199 

What inter-relations have the optimum, minimum and 
maximum temperature requirements of one species of 
microorganism? 

What influence will the reaction of the medium have 
upon the extremes of temperature at which microorganisms 
will grow? 

5. Discuss your results in detail and draw any conclu- 
sions permitted. Point out any practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 153-157. 
LAFAR: Technical Mycology, Vol. I, pp. 58-60. 
JORDAN: General Bacteriology, 4th Ed., pp. 70-72. 

EXERCISE 22. THE EFFECT OF FREEZING UPON 
SPORE - FORMING AND NON - SPORE - FORMING 
BACTERIA 

Apparatus. Small ice dish; thermometer; three tubes 
each of sterile cider, sterile milk, sterile broth and sterile 
wort; coarse salt; ice. 

Cultures. Sacch. cerevisice; Bad. lactis atidi; B. 
megaterium; Aspergillus niger. 

Method. 1. Heavily inoculate the cider tubes with the 
yeast, the milk tubes with Bad. ladis acidi, the broth tubes 
with B. megaterium and the wort tubes with Aspergillu niger. 

2. Incubate one set of cultures at 25 C. 

3. Make sufficient freezing mixture of ice and salt 
to nearly fill the ice dish. 

4. Carefully insert the two remaining sets of cultures 
in the freezing mixture and keep the freezing mixture at or 
below C. for two hours. 

5. Remove one set of tubes and incubate at 25 C. 

6. Then place the ice dish containing the third set of 
cultures in the refrigerator (note the temperature) . 

7. Examine both sets of cultures at the end of twenty- 
four hours and forty-eight hours for growth. 



200 GENEKAL MICROBIOLOGY 

8. Compare the three sets of cultures and note the 
variations from the normal type of growth. Tabulate your 
data. 

9. Are all of these organisms pecilothermic? What 
are termed the cardinal points of temperature for micro- 
organisms? What is the lowest temperature at which 
growth, even of the feeblest kind, is possible? What term 
is applied to organisms which grow best at low tempera- 
tures? 

10. Give all results and answers in full and draw any 
conclusions permissible. Point out the practical applica- 
tions that may be made. 

REFERENCES 

FISCHER: Structure and Functions of Bacteria, pp. 73-75. 
MARSHALL: Microbiology, pp. 154-155, 158-159, 199, 318, 395-401. 
LAFAR: Technical Mycology, Vol. I, pp. 58-60. 

EXERCISE 23. THE DETERMINATION OF THE THER- 
MAL DEATH POINT OF A SPORE-FORMING AND A 
NON-SPORE-FORMING ORGANISM 

Apparatus. Water bath; test-tube rack to fit bath; 
ring tripod; Bunsen burner; thermometer; thirteen test 
tubes of uniform diameter containing exactly 10 c.c. of 
broth; platinum loop 4 mm. in diameter; dish containing 
cold water (20 G. or below). 

Cultures. Twenty-four to thirty-six hour broth cultures 
of B. typhosus and B. mycoides. 

Method. 1. Examine the cultures for the presence or 
absence of spores. 

2. Set up the water bath on the ring tripod, place only 
sufficient water in it to cover the medium in the test tubes 
and insert the test-tube rack. 

3. Insert the thermometer into one of the test tubes 
of broth, passing it through the cotton plug. 

4. After flaming the plugs, place all the remaining tubes 



THERMAL DEATH POINT 201 

of broth in the rack in the water bath and heat slowly until 
the thermometer in the tube of broth registers 45 C. 

5. Hold at this temperature for fifteen minutes. 

Note. Slow heating is necessary in order that the respective 
temperatures may be held for the desired period of time. 

6. Without removing the tubes from the bath, inoculate 
one tube of broth witfy a loopful of the broth culture of 
B. typhosus, a second with B. mycoides. Carefully mix the 
inoculum with the broth without removing the tubes. Mark 
each tube carefully. 

7. Allow these inoculated tubes to remain in the water 
bath at 45 C. for ten minutes. 

8. Remove and place immediately in cold water. 

9. Incubate each organism at its optimum temperature 
after each trial. 

10. Next, raise the temperature of the bath five degrees, 
i.e., to 50 C. and inoculate the tubes as before with B. 
typhosus and B. mycoides. 

11. Keep the tubes at 50 C. for ten minutes, remove 
them from the bath, cool and incubate. 

12. In the same manner expose the organism to the 
following temperatures, 55, 60, 65 and 70, for a period 
of ten minutes each. 

13. In all cases incubate seven days and record as the 
thermal death point (t. d. p.) the lowest temperature at 
which growth fails to appear. 

14. What are the standard methods for the determina- 
tion of the t. d. p.? What are the flaws in the above 
method? What different factors may influence the thermal 
death point of an organism? 

Do all organisms possess the same t. d. p.? Explain. 

15. Give data and results in full. Draw any conclusions 
that properly follow and point out any practical applica- 
tions. 



202 GENERAL MICROBIOLOGY 

REFERENCES 

ROSENAU: Preventive Medicine and Hygiene, pp. 780-781. 
JORDAN: General Bacteriology, 4th Ed., pp. 36-37, 72. 
MARSHALL: Microbiology, pp. 159-161. 
NOVY: Laboratory Work in Bacteriology, pp. 513-518. 

EXERCISE 24. TO DETERMINE THE RELATIVE EFFECT 
OF MOIST AND DRY HEAT ON BACTERIA 

Apparatus. Ten tubes of nutrient broth (large tubes); 
ten sterile Esmarch dishes; ten sterile (flamed) cover-glasses; 
autoclav; steam sterilizer; hot-air sterilizer. 

Cultures. Agar culture of a spore-forming organism 
(having spores at the time). 

Milk culture of slimy milk organism, non-spore-forming. 

Method. 1. Make thick smears of each organism on five 
cover-glasses. 

2. Place each cover-glass of the separate cultures in a 
sterile Esmarch dish and mark. 

3. Place two Esmarch dishes of each culture in the hot- 
air sterilizer; heat to 120 C. and remove one dish of each 
culture after ten minutes at 120 C., the other two after 
thirty minutes. 

4. Place two smears of each organism in the steam 
sterilizer;, remove one of each after ten minutes, the two 
remaining after thirty minutes. 

6. Place the two remaining Esmarch dishes in the auto- 
clav and heat for ten minutes at 120 C. 

6. When cool, transfer each of the cover-glasses to a 
tube of sterile broth; mark carefully. 

7. Note in which tubes growth appears. 

8. What is one of the most necessary factors for the 
prompt destruction of microorganisms by heat? Why? 

Not considering moisture, what various conditions in- 
fluence the destruction of microorganisms by heat? How 
are molds and yeasts influenced by moist and dry heat? 

To what factors are the greater destructive powers of 
the autoclav due? 



PASTEURIZATION 203 

9. Give all data and results in full. Draw any conclu- 
sions possible and point out any practical applications. 

REFERENCES 

HITE, B. H., GIDDINGS, N. J., and WEAKLEY, CHAS. E.: The effect of 
pressure on certain microorganisms encountered in the preservation 
of fruits and vegetables. Bui. 146, W. Va. Univ. Agr'l Expt. Sta., 
1914. 

MARSHALL: Microbiology, pp. 159-161. 

LAFAR: Technical Mycology, Vol. I, pp. 79-84. 

FISCHER: Structure and Functions of Bacteria, pp. 75-77. 

EXERCISE 25. TO DETERMINE THE EFFECT OF PAS- 
TEURIZATION UPON THE GROWTH OF MICRO- 
ORGANISMS 

Apparatus. 300 c.c. each of milk (not sterile) and of 
some fermenting fruit juice; water bath; two thermometers; 
four sterile 200 c.c. Erlenmeyer flasks; twenty-four tubes 
of dextrose agar; dilution flasks; twenty-four sterile Petri 
dishes; sterile 1 c.c. and 10 c.c. pipettes. 

Method. 1. Place 150 c.c. of milk in each of two 200 c.c. 
sterile Erlenmeyer flasks; do the same with the fruit juice. 

2. Make three dilution plates each (1-100, 1-10,000, 
1-1,000,000) from the milk and from the fruit juice in agar 
and incubate (inverted) at room temperature. 

3. Place a flask of each nutrient liquid in the water bath 
(cold water) and heat rapidly to 75-80 (thermometer in 
each flask), shaking the flasks frequently to obtain an even 
temperature throughout their contents. 

4. Remove the flasks when the temperature reaches 80 
C. and cool* them quickly. 

5. Make dilution plates (1-10, 1-1,000, 1-10,000) in 
agar; mark each carefully. Place the flasks and plates at 
room temperature. 

6. Place the other two flasks in the water bath (in cold 
* It has been found by experiment that the quick cooling must take 

place through the temperatures 40-36 C. in order to be most efficient 
in preventing further bacterial growth. 



204 GENERAL MICROBIOLOGY 

water) and heat slowly up to 60 (thermometer in each flask). 
Keep at 60-65 for twenty minutes. 

7. Remove the flasks from water bath and cool* quickly. 

8. Make dilution plates, using the same dilutions as 
before, and place the flask and plates at room temperature, 
marking each carefully. 

9. .Watch daily for signs of growth in each medium. 

10. Make plates from each flask after six days, deter- 
mining the range of dilutions by consulting your former 
plates. Will the organisms have increased or decreased in 
this time? W^hy? 

11. Compare the types of organisms on the plates before 
and just after pasteurizing and six days after pasteurizing. 
Examine each type microscopically. Of what does the 
predominant flora of each nutrient fluid consist before 
pasteurization? After pasteurization? 

Note. The fruit juice may be saved for the experiment on meta- 
biosis. 

12. Count each set of plates and record the average 
number of microorganisms per c.c. 

13. Plot the curve to show the destruction of micro- 
organisms by pasteurization. 

Compare the milk data with milk data and also the cider 
with cider. 

14. Keep the original flasks for one or two weeks. If 
any marked changes occur, plate qualitatively and ascer- 
tain the type of organism causing the change. 

15. How does the physical nature of the two nutrient 
substances influence their response to pasteurization? 
Give reasons for explanations offered. 

What changes are brought about in milk by pasteuriza- 
tion? In cider or other fermenting fruit juice? 

16. Give all data and results in full and draw any conclu- 
sions. Point out any practical applications that may be 

made. 

* See note on page 203. 



REACTION OF THE NUTRIENT MEDIUM 205 



REFERENCES 

MARSHALL: Microbiology, pp. 319-320, 386-388. 

RUSSELL and HASTINGS: Experimental Dairy Bacteriology, pp. 89-91. 

LAFAR: Technical Mycology, Vol. II, Part I, pp. 142-144. 



EXERCISE 26. TO ILLUSTRATE THE EFFECT OF THE 
REACTION OF THE NUTRIENT MEDIUM UPON 
MICROORGANISMS 

Apparatus. One liter of ordinary broth (should be 
enough for three students); normal NaOH; normal acid; 
four sterile 1 c.c. pipettes; 10 c.c. pipettes; sterile test 
tubes. 

Cultures. B. prodigiosus (broth culture); B. subtilis 
(broth culture); Oospora lactis (broth culture); Torula 
rosea (broth culture). 

Method. 1. By adding normal acid or alkali produce in 
100 c.c. portions of ordinary broth the following reactions: 
-40, -30, -20, -10, 0, +10, +20, +30, +40, +50 
degrees Fuller's scale, and titrate after readjusting the 
reaction, as a check. 

2. Tube, using 9.9 c.c. in each tube (mark the tubes 
plainly), and sterilize (refiltration may be necessary before 
tubing in some cases). 

3. Using a sterile 1 c.c. pipette, inoculate one set (ten 
tubes) with 0.1 c.c. of the broth culture of each of the above 
organisms (four sets) and incubate the tubes at room 
temperature. 

4. Examine the tubes as often as possible for the first 
twenty-four to thirty-six hours, and record the tube or 
tubes in which macroscopic growth is first visible. What 
do you conclude as to the effect of the reaction of the medium 
in these instances? 

5. Examine the tubes every day for seven days. Tabu- 
late your observation. Note the range of reaction in 
which each organism is capable of growing. Does this 



206 GENERAL MICROBIOLOGY 

range differ with different organisms? Explain the action 
occurring. 

6. In each case inoculate heavily the first tube at either 
or both extremes, in which the organism fails to grow, 
from the tube just next it in series which shows growth. 
Does this freshly inoculated tube show signs of growth after 
twenty-four to forty-eight hours? Explain the action which 
occurs. 

7. Which organisms are acidophiles? 

What is the optimum, the minimum and the maximum 
reaction for each organism according to this experiment? 

What factors not considered in this experiment might 
influence results? 

How would you determine the exact optimum reaction 
of an organism? 

8. Give all data and results in full and draw conclusions. 
Point out any practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 173-180, 235-237. 
EYRE: Bacteriological Technic, 2d Ed., pp. 305-306. 

EXERCISE 27. TO DETERMINE THE INFLUENCE OF 
DIFFUSED LIGHT ON MOLDS 

Apparatus. Sterile deep culture dish; tube of dextrose 
agar or gelatin; black paper. 
Culture. Rhizopus nigricans. 
Method. 1. Pour a tube of agar into a deep culture dish. 

2. When solid, inoculate with Rhizopus nigricans. 

3. Wrap the dish closely in black paper so that no light 
can penetrate. 

4. Cut a hole 2 to 3 cm. in diameter in the top edge of 
the paper and place the dish in a north window so that 
only diffused light will enter the aperture. 

5. Allow the dish to stand ten days, then examine it. 
How does diffused light influence this mold? 



DIFFUSED LIGHT ON MOLDS 



207 



6. What is the term applied to this type of action? 
How are mold spores influenced by light? What influence 
does diffused light have on other microorganisms? Is it 
to be expected that other common molds, Penicillium, 
Oospora, Aspergillus, etc., would exhibit this same phenome- 
non? 

7. Give all data and observations in detail. Draw any 




FIG. 44. Phototropism Exhibited by Rhizopus nigricans. The 
mold was grown on gelatin with diffused light coming from the 
right side. (From Marshall.) 

conclusions that follow and point out any practical applica- 
tions. 

REFERENCES 

MARSHALL: Microbiology, pp. 162-165. 
JORDAN: General Bacteriology, 4th Ed., p. 73. 



208 GENERAL MICROBIOLOGY 



EXERCISE 28. TO SHOW THE INFLUENCE OF DIRECT 
SUNLIGHT UPON THE GROWTH OF MICRO- 
ORGANISMS 

Apparatus. Two tubes of sterile agar; two sterile Petri 
dishes; two sterile 1 c.c. pipettes; two tubes of sterile 




FIG. 45. Action of Direct Sunlight on Bacteria. These plates were 
heavily inoculated with R. coli and B. prodigiosus, respectively, 
and then were exposed bottom side up to the direct rays of the 
January sun for four hours. At the moment of exposure the 
figure 0, cut from black paper, was pasted to the plate, shading 
the bacteria underneath. After one, two and three hours the 
corresponding figures were pasted to the plates. The above 
picture was taken twenty-four hours after exposure, proving that 
three or four hours' exposure to direct sunlight weakens and may 
even kill bacteria. B. prodigiosus proved more sensitive than 
B. coli. (From Marshall.) 

distilled water, salt solution or broth for dilution purposes; 
black paper; glue. 

Cultures. Ps. campestris; B. typhosus. 

Method. 1. Inoculate a tube of sterile liquid heavily 
with Ps. campestris. Mix the contents well. 

2. Place 1 c.c. of this suspension in a sterile Petri dish 
and pour the plate. 



SUNLIGHT UPON MICROORGANISMS 209 

3. Duplicate with the B. typhosus culture, placing the 
pipettes immediately after using in 1-1,000 HgCb. 

4. Cut any design out of black paper and paste on the 
bottom of the Petri dish. 

5. Place the dish bottom side up in direct sunlight for 
two hours. 

6. Set the dish away in the dark at room tempera- 
ture. Observe the growth and explain. Which organism 
is the more sensitive to sunlight? Conclusions? 

Note. Heat B. typhosus plate 1 hour in steam before cleaning the 
Petri dish! 

7. What theories have been advanced as to the mechan- 
ism of destruction by direct sunlight? Does sunlight have 
any effect on bacterial spores? 

How are other forms of organisms affected by light? 

What is phototaxis? Do bacteria ever exhibit this 
phenomenon? 

Which portion of the spectrum is most active? 

What relation does the wave-length of light rays bear 
to the activity of the rays? 

How do diffuse light, electric or other forms of artificial 
light, X-rays, radium rays, etc. compare with direct sun- 
light as to their action on bacteria in general? 

8. Give all data and state results in full. Draw any 
conclusions that follow and point out any practical applica- 
tions. 

REFERENCES 

MARSHALL, C. E.: Microbiology, pp. 162-163. 

SMITH, ERWIN F. : Bacteria in Relation to Plant Diseases, Vol. I, 

p. 71; Vol. II, p. 324. 
LAFAR, F,; Technical Mycology, Vol. I, pp, 60-63, 



210 GENERAL MICROBIOLOGY 



EXERCISE 29. DETERMINATION OF THE PHENOL 
COEFFICIENT OF SOME COMMON DISINFECTANTS 

(Two students working together are required in this exercise.) 

Apparatus. Copper water bath; test-tube rack for 
above bath, thirty-two test tubes of uniform size containing 
exactly 5 c.c. of sterile nutrient broth (use a graduated 
burette or a similar apparatus for filling tubes) ; eight clean 
dry test tubes of uniform size; several (4 or 5) platinum 
loops, of 4 mm. inner diameter; sterile 1 c.c. pipettes with 
fine point; three clean 5 c.c. pipettes; phenol, 5%; mer- 
curic chloride, 1 : 500; small funnel; filter paper to fit 
funnel; sterije test tube; watch with second hand. 

Culture. B. typhosus, twenty-four hour broth culture 
grown at 37 C. 

Method. 1. Place the filter paper in the funnel, wrap in 
paper and sterilize in the hot air. 

2. Filter the twenty-four-hour broth culture of B. 
typhosus into the sterile test tube. This is for the purpose 
of removing clumps of bacteria and any foreign matter. 
Funnel and filter paper are to be treated immediately after 
use with 1 : 1000 HgCl 2 . 

3. Regulate the water bath at 20 C. and keep at this 
temperature. 

4. Mark the thirty-two test tubes, each containing 
exactly 5 c.c. of nutrient broth, with the name of the disin- 
fectant, the dilution, and the time exposed, according to the 
following table. Then place the tubes, in order, in the rack 
in the water bath. 

6. Mark each set of clean, dry test tubes carefully with 
the name of the disinfectant and the dilution to be added 
(see table on p. 212), and place in each, 5 c.c. of the dilu- 
tion of the disinfectant as indicated on the labels. Keep in 
a test-tube rack at 20 C. Work with one disinfectant at a 
time. 



PHENOL COEFFICIENT OF DISINFECTANTS 211 

N. B. Have the assistant carefully keep track of the exact 
time of all operations, to the second. 

In actual practice determinations are made oftener than 
every five minutes, two and one-half minutes being the 
standard interval. This requires the most careful attention 
of both operator and assistant. 

6. Using the 1 c.c. pipette, add 0.1 c.c. of the culture to 
one tube of each dilution of the disinfectant and mix quickly 
with a sharp rotary motion of the tube. 

7. At the end of one minute from the time of each 
separate operation, make a loop transfer from the tube of 
each dilution of the disinfectant inoculated with the cul- 
ture into the corresponding tube of broth in the water 
bath. 

Note. The assistant takes the tubes from the water bath and 
hands them to the operator, then, after the operation of transferring, 
returns the inoculated broth tube to the water bath, sterilizes the 
needle and places it in the most handy position for the operator. j 

8. This operation is then repeated; working as quickly 
as possible, add 0.1 c.c.- of the culture to the remaining 
tubes of the different dilutions of the disinfectant. 

9. When, in each case, the culture has been exposed for 
exactly five minutes, ten minutes and fifteen minutes respec- 
tively to the action of the disinfectant, a loop transfer is to 
be made to the corresponding tube of broth. 

10. When all transfers are made, place the broth cul- 
tures at 37 C. Examine after forty-eight hours for growth 
and record growth as + or . 

Note. The phenol coefficient of a disinfectant is the ratio of the 
strength of the unknown disinfectant which will kill a filtered 24- hr. 
broth culture of B. typhosus in a certain length of time, to the strength of 
phenol which will accomplish the destruction in the same length of time, 
the dilution of phenol taken as 1 . 

For example: The dilution of an unknown disinfectant required to 
kill B. typhosus in 7| minutes was 1 : 550, and the dilution of phenol 
necessary to kill B. typhosus in the same time was 1 : 100. 550-;- 100 
= 5.5, the phenol coefficient of the unknown disinfectant. This means 



212 



GENERAL MICROBIOLOGY 



that the unknown disinfectant undiluted is 5 times the strength of 
the undiluted phenol.* 

11. Determine the approximate phenol coefficient of 
mercuric chloride according to the results of your experi- 
ment. How does this compare with results in literature? 

12. What are some of the principal factors involved in 
the examination of disinfectants (pp. 12-20, Hyg. Lab. Bui. 
No. 82). How would each of these come into consideration 
in actual practice? 

METHOD OF MAKING DILUTIONS OF DISINFECTANT FOR 

TEST 

1 part of 5% phenol +1 part distilled water =2.5% phenol. 
1 part of 5% phenol +4 parts distilled water = 1.0% phenol. 
1 part of 5% phenol+9 parts distilled water = 0.5% phenol. 



1 part of 1 : 500 HgCl 2 + l part distilled water =1 
1 part of 1 : 500 HgCl 2 +3 parts distilled water = 1 
1 part of 1 : 500 HgCl 2 +9 parts distilled water = 1 



1000 HgCl 2 . 
2000 HgCl 2 . 
5000 HgCl 2 . 



METHOD OF RECORDING RESULTS 



Disinfectant. 



Time in minutes during which culture is exposed to 
action of disinfectant. 





1 min. 


5 min. 


10 min. 


15 min. 


Phenol 5.0% 
Phenol 2.5% 










Phenol 1.0%. 










Phenol 0.5% 










HgCl 2 1 500 
HgCl 2 1 1000 










HgCl 2 1 2000 










HgCl 2 1 5000 











13. Give data and results in full. Draw any conclusions 
that properly follow and point out any practical applications. 

* In a large class it would be interesting to have determined the 
phenol coefficient of the chromic acid cleaning solution and of the 10% 
sodium hydroxide used for cleaning glassware, as each is recommended 
for immersing slides, cover-glasses, etc. contaminated with bacteria. 



FORMALDEHYDE UPON MICROFLORA OF MILK 213 



. REFERENCES 

JOHN F. ANDERSON and THOMAS B. McCLiNTic : I. Method of Standard- 

izing Disinfectants with and without Organic Matter. Hygienic 

Laboratory Bui. No, 82 (1912), pp. 1-20, 34, 73. 
S. RIDEAL and E. K. RIDEAL: Some Remarks on the Rideal- Walker 

Test and the Rideal-Walker Method. Jour, of Infectious Diseases, 

Vol. X (1912), pp. 251-257. 
H. C. HAMILTON and T. OHNO: Standardization of Disinfectants. 

Reprint No. 45 (1913), from Research Laboratory of Parke, 

Davis and Co., pp. 451-458. 
- The Bacteriological Standardization of Disinfectants. Reprint 

No. 65 (1914), from Research Laboratory of Parke, Davis and Co. 
M. L. HOLM and E. A. GARDNER: Formaldehyde Disinfection with 

Special Reference to the Comparative Value of Some of the 

Proprietary Products. Journal of Infectious Diseases, Vol. VII 

(1910), pp. 642, 643, 650, 658-659. 
MARSHALL: Microbiology, pp. 173-180. 

EXERCISE 30. TO DETERMINE THE ACTION OF 
FORMALDEHYDE UPON THE MICROFLORA OF 
MILK 

Apparatus. Fresh milk, skim or whole; 1% solution of 
formaldehyde; azolitmin solution; twelve sterile test tubes; 
HoS04, concentrated commercial. 

Method. 1. Make the following mixtures in sterile 
test tubes in plain milk, and duplicate in litmus milk (adding 
2% azolitmin solution to the milk) : 

Milk. Formaldehyde 



9.0 c.c. + 1 c.c. ofl% =0.1% 

9.3 c.c. +0.7 c.c. of 1% =0.07% 

9.7 c.c. + 0.3 c.c. of 1% = 0.03% 

9.0 c.c. + 1 c.c. of 0.1% = 0.01% 

9.0 c.c. + 1 c.c. of 0.07% = 0.007% 

9.0 c.c. + 1 c.c. of 0.03% = 0.003% 

Place at room temperature. 

2. Record the action in each tube, the time required 
for spoilage and the amount of formaldehyde necessary to 
preserve the milk. 



214 GENERAL MICROBIOLOGY 

3. What is the lowest per cent of formaldehyde that has 
inhibitive action? That has preservative action? What 
terms are applied to these different percentages in each 
case? 

4. Make a "ring" test for formaldehyde as follows: 
Add several drops of concentrated commercial H2SO<i to 
each tube of plain milk, allowing it to run down the side of 
the tube as in making an ordinary " ring " test. A violet 
coloration at the junction of the H 2 S04 with the milk 
demonstrates the presence of formaldehyde in the milk. 
The presence of ferric chloride, an impurity in commercial 
sulphuric acid, is essential to this test. 

5. Did all percentages of formaldehyde used give this 
test? Did all percentages which preserved give the test? 

Is formaldehyde a desirable preservative for milk? 
Why? Are any chemicals more desirable for this purpose 
than formaldehyde? 

What are the main uses of formaldehyde? What is 
paraformaldehyde? Its use? 

6. State your results in full and draw any conclusions 
that follow. What practical applications may be made? 

REFERENCES 

MARSHALL: Microbiology, p. 179. 

SAVAGE: Milk and Public Health, pp. 383-391. 

HAWK: Practical Physiological Chemistry, 4th Ed., p. 239. 

EXERCISE 31. TO ILLUSTRATE SYMBIOSIS 

Apparatus. Sterile 5 c.c. pipettes; three sterile 200 c.c. 
Erlenmeyer flasks; 450 c.c. skim milk; three tubes of litmus 
milk (sterile). 

Cultures. Bad. lactis acidi; Oospora lactis. 

Method. 1. Place 150 c.c. milk in each flask and sterilize 
(Tyndall method). 

2. Mark the flasks, A, B and C. Inoculate flask A 



MUTUAL RELATIONSHIP OF MICROORGANISMS 215 

with Bad. ladis acidi, flask B with Bad. ladis acidi and 
Oospora ladis, and flask C with Oospora ladis alone. 

3. Make ten titrations, titrating every two or three days 
(not oftener) and record the titrations. Tabulate the 
data. 

4. Plot curves. How do you explain the direction these 
curves take? 

5. At the end of the titrations, make loop transfers 
from each flask into litmus milk tubes and watch these 
carefully in the next twenty-four to forty-eight hours. 
Record the results. 

6. Does the action in the flasks appear to be symbiotic? 
If so, how is it shown? 

Is this symbiosis desirable or not? Explain. 
What other well-known examples of symbiosis occur in 
nature? Give a reason for your statement. 

7. Give all data and results in full. Draw any conclu- 
sions that follow and point out any practical operations. 

REFERENCES 

MARSHALL: Microbiology, pp. 181-182, 273-282, 323. 

NORTHRUP: The Influence of Certain Acid-destroying Yeasts upon 

Lactic Bacteria. Tech. Bui. No. 15, Mich. Expt. Sta., pp. 8-16, 

32-34. 

EXERCISE 32. TO ILLUSTRATE ONE OF THE PHASES 
OF MUTUAL RELATIONSHIP OF MICROORGANISMS 

Apparatus. Sterile 5 c.c. pipette; 3 sterile 200 c.c. 
Erlenmeyer flasks; 450 c.c. sweet cider (that from pas- 
teurization experiment may be used). 

Cultures. Sacch. ellipsoideus, Bad. aceti. 

Method. 1. Place 150 c.c. of sweet cider in each flask. 

2. Determine and record the reaction of the cider, 
then heat the flasks thirty minutes in the steam. 

3. Cool the flasks and inoculate flask A with Sacch. 
ellipsoideus. 



216 GENERAL MICROBIOLOGY 

4. Determine the weight at once and then every day 
until the weight becomes constant. 

5. Inoculate flask B with Bad. aceti and flask C with both 
Bad. aceti and Sacch. ellipsoideus. 

6. Titrate B and C every two days. Titrate flask A 
only at the end of the experiment. 

7. Determine the amount of alcohol formed in flask A 




FIG. 46. Antibiosis. This peculiarity of growth is the result of the 
inhibitive action of metabolic products diffused through the 
medium. (Orig. Northrup.) 

by distilling the contents of the flask and determining the 
specific gravity of the distillate. 

How much CO2 was given off? Calculating from this 
amount, how many grams of sugar (CeH^Oe) were present 
in the flask? What percent sugar was this solution? 

What was the theoretical amount of alcohol present? 

8. Plot curves showing the acid formation in each 
case. Explain the direction which these curves take. 



EFFECT OF THE METABOLIC PRODUCTS 217 

9. Explain the mutual action and the changes which 
occur. 

10. What enzymes are responsible for each change? 
Write out the chemical equations for each change, giving 
enzyme concerned in each case. 

Was the theoretical amount of alcohol changed into 
acetic acid? Give a reason for what really does happen. 

What phase of mutual relationship is illustrated? 

What is the classical example of this type of mutual 
relationship? 

11. Give data and observations in full and draw conclu- 
sions. Point out any practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 182-183, 448-458. 
LAFAR: Technical Mycology, Vol. I, pp. 297-307; Vol. II, Part 2, 
pp. 473-481, 511-515. 

EXERCISE 33. TO DEMONSTRATE THE EFFECT OF 
THE METABOLIC PRODUCTS OF BACT. LACTIS 
ACIDI ON ITS ACTIVITIES 

Apparatus. Two sterile 200 c.c. flasks. 200 c.c. sweet 
skim milk; azolitmin solution; apparatus for titration; 
sterile dilution flasks; sterile Petri dishes; sterile 1 c.c. 
pipettes; sterile 5 c.c. pipettes; sterile 10 c.c. pipettes; 
ten to fifteen tubes of sterile litmus milk. 

Culture. Bad. lactis addi (twenty-four-hour culture). 

(At least two weeks should be allowed for the completion of 
this experiment.) 

Method. 1. Place 100 c.c. skim milk, +10 to +15 
(record acidity before adding azolitmin), in each flask and 
sterilize by the Tyndall method. 

2. Mark the flasks A and B. 

3. Inoculate each flask with a loopful of a twenty-four- 
hour milk culture of Bact. lactis acidi. Mix well with a 
needle and plate dilutions 1-10, 1-100, 1 10,000 from flask 



218 GENERAL MICROBIOLOGY 

A for obtaining the initial number of Bad. lactis acidi 
introduced per c.c. 

4. Continue as follows: 

Flask A; 

2d day, titrate and use dilutions 1-10,000, 

1-100,000 and 1-1,000,000. 
3d day, titrate and use dilutions 1-1,000,000, 

1-10,000,000 and 1-100,000,000. 
6th day, titrate and use dilutions 1-100,000, 

1-1,000,000 and 1-10,000,000. 
7th day, titrate and use dilutions 1-1,000, 

1-10,000 and 1-100,000. 

N. B. Shake the flask of milk well each time before titrat- 
ing and making dilutions. 

5. Titrate and plate every third day thereafter, until the 
acidity remains constant. 

6. In flask B from day to day note in millimeters the 
extent of the re-oxidation of the azolitmin. 

7. Flask B. Without disturbing the milk any more than 
necessary, make a loop transfer every day or so for 10 to 
14 days from this flask into a tube of sterile litmus milk. 

What occurs in each case? In what respects does 
flask B check up with flask A? Give explanations for sim- 
ilarity or dissimilarity of actions occurring. 

8. Milk contains on an average about 4.5% lactose. 
Has this sugar been fermented entirely to lactic acid? 
Explain what really occurs. 

9. What titre would milk containing 5% lactose have 
if this sugar were entirely changed to lactic acid? 

Does any lactic-acid-producing organism approximate 
this reaction (in milk) at the height of its activity? 

10. Give reasons for what occurs in each flask. What 
practical applications may this experiment have? 

11. Tabulate your results and plot number and acidity 
curves. Explain these curves. 



EFFECT OF THE METABOLIC PRODUCTS 219 

12. Draw any conclusions that follow from the above 
and point out any practical applications. 

REFERENCES 

RAHN, O.: The Fermenting Capacity of a Single Cell of Bad. lactis 

atidi. Tech. Bui. No. 10, Mich. Exp. Sta., p. 25, et al 
MARSHALL: Microbiology, pp. 157, 308-312. 



PART III 
APPLIED MICROBIOLOGY 



AIR MICROBIOLOGY 

EXERCISE 1. QUANTITATIVE BACTERIAL ANALYSIS 

OF AIR 

Apparatus. One carbon tube, dia. 15 mm.; cork stopper, 
perforated, to fit carbon tube; short piece of glass tubing 
bent at right angles; sand which has passed through a 150 
mesh sieve; 8-liter aspirator bottle complete with rubber 
stoppers and glass tubing; sterile test tubes * containing 
10 c.c. of sterile physiological salt solution; sterile 1 c.c. 
pipettes; four sterile Petri dishes; four tubes of sterile 
agar for plating; sterile agar slants; tubes of sterile broth; 
tubes of sterile litmus milk. 

Method. 1. Prepare a sand filter aeroscope by placing 
a layer of cotton in the bottom of the carbon tube. 

2. Upon this place 1 cm. of sand which has been run 
through a 150 mesh sieve. 

3. Insert a cork stopper through which is passed a bent 
glass tube plugged at the outer end with cotton. 

4. Sterilize the apparatus in hot air oven. 

5. Place 8 liters of water in the aspirator bottle and 
mark the level of this amount of liquid. 

6. Adjust the delivery tube so that it aspirates one 
liter of air per minute. 

* For convenience in shaking the sample, it is recommended to use 
test tubes with aluminum screw caps, having cork packing. 

220 



QUANTITATIVE BACTERIAL ANALYSIS OF AIR 221 




7. Attach the aeroscope (lower end of carbon tube), to 
the aspirator so that the aspirated air will be filtered through 
the sand. 

8. Remove the cotton plug from the upper end of the 
aeroscope and filter 8 liters of air in 
approximately eight minutes. 

9. Using " aseptic " precautions, trans- 
fer as much sand as possible to one of the 
tubes of sterile salt solution. 

10. Mix well by bumping the tube 
against the hand at least fifty times (do 
not wet the cotton plug). 

11. Then, with a sterile 1 c.c. pipette, 
transfer 1 c.c. of the suspension to each 
of four Petri dishes and pour plates. 

12. Incubate two plates at 37 C. for 
two days, and the remaining two plates 
at room temperature for five days. 

13. Count at the end of these respec- 
tive periods and determine the number 
of bacteria per liter. How do your counts 
compare with air counts obtained by 
other students? from other data? (See 
Marshall's Microbiology, p. 789.) 

Make separate counts of molds and 
identify them as far as possible. 

14. Make sub-cultures of different 
types on agar and study their cultural 
characteristics on this medium. 

15. Transfer these cultures to tubes of 
broth and litmus milk and note their 

action on these media. Draw conclusions from these 
results. 

16. What morphological types are found? Are any of 
the types of bacteria present constantly found in air? What 
are the sources of microorganisms in the air? 



FIG. 47. Modified 
Standard Aero- 
scope. (Ruehle 
and Kulp.) 



222 GENERAL MICROBIOLOGY 

Are any of the types isolated related to pathogenic forms? 
May pathogenic bacteria be isolated from air? If so, 
under what circumstances? 

How do microorganisms enter the air? What types of 
microorganisms are most apt to be present in air? What 
is the explanation for this? 

What other methods may be employed for obtaining 
quantitatively the bacteria in the air? 

Of what importance is the quantitative or qualitative 
determination of microorganisms in air? 

17. Give data and results in full and draw any conclu- 
sions permitted. Point out any practical applications of the 
above. 

REFERENCES 

RUEHLE, G. L. A. and KULP, W. L.: Germ content of stable air and 

its effect upon the germ content of milk, Bui. 409, N. Y. Expt. 

Sta., 1915. 

MARSHALL: Microbiology (1911), pp. 185-191. 
EYRE: Bacteriological Technic: 2d Ed. (1913), pp. 468-470. 
BESSON: Practical Bacteriplogy, Microbiology and Serum Therapy, 

transl. by Hutchens (1913), pp. 862-867. 
CHAPIN, C. V.: The air as a vehicle of infection. Jour. Amer. Med. 

Ass'n., Vol. LXII, pp. 423-430 (1914). 
WINSLOW,- C. E. A.: Bacteriology of air and its sanitary significance. 

Cent. f. Bakt. Abt. II. Bd., 42, p. 71 (1914). 
WINSLOW, C. E. A. and BROWN, W. W.: The microbic content of 

indoor and outdoor air. Mo. Weather Rev., Vol. XLII (1914), 

pp. 452-453. Abst. in Exp't. Sta. Record, Vol. XXXII, No. 3 

(1915), p. 211. 



BACTERIOLOGICAL ANALYSIS OF WATER 223 



WATER AND SEWAGE MICROBIOLOGY 

EXERCISE 1. BACTERIOLOGICAL ANALYSIS OF WATER 
FROM A SOURCE NOT SUSPECTED OF SEWAGE 
CONTAMINATION 

Apparatus. Sterile 500 c.c. flask for collecting water 
sample; litmus lactose agar shake; twelve tubes of litmus 
lactose agar; twelve salt-free gelatin tubes; two litmus lac- 
tose bile fermentation tubes; six agar slants; six tubes of 
sterile broth; six tubes of Dunham's solution; six tubes of 
nitrate peptone solution; six dextrose fermentation tubes; 
six tubes of litmus milk; twelve sterile Petri dishes; sterile 
100 c.c. volumetric pipette; sterile 1 c.c. and 5 c.c. pipettes; 
record sheet for recording data obtained; record sheet for 
recording pure cultures isolated; water sample. 

Cultures. B. coli. 

Water from the local water system should be used for 
the experiment. This method can be used also for water 
from deep wells, springs, etc. 

Method. 1. Flush the water pipes thoroughly by 
allowing the water to run, or by pumping, at least thirty 
minutes. 

2. Hold the collection flask, mouth downwards, remove 
the plug and still holding in this inverted position, wash 
the mouth off with the running water, then fill quickly 
and replace the plug. The plug must not be laid down 
during this process. 

3. The sample must be analyzed at once. In routine 
work, if this is not practicable, place the sample on ice 
and analyze as soon as possible. Samples kept at 10 C. 
or less should never be left over a maximum of six hours 
before analysis. 

4. Plate immediately in duplicate, 1 c.c., 0.5 c.c. and 
0.1 c.c. of the sample direct in litmus lactose agar and in 
gelatin (6 plates each). (If sewage contamination is sus- 



224 



GENERAL MICROBIOLOGY 



pec ted the sample must be diluted. Use distilled water 
for dilution flasks.) 




FIG. 48. 



FIG. 49. 



FIG. 48.^-A Model Dug Well Constructed to Avoid Microbial Con- 
tamination of Water. (From Gerhard's Sanitation, Water 
Supply and Sewage Disposal of Country Houses.) 

FIG. 49. A Shallow Driven or Tube Well. (From Gerhard.) 



5. To the melted agar shake (at 45 C.), add 100 c.c. 
of the sample, using the volumetric pipette, and shake well 



BACTERIOLOGICAL ANALYSIS OF WATER 225 

to mix the sample thoroughly with the medium. Avoid 
shaking so violently as to produce gas bubbles. 

6. Using a sterile 5 c.c. pipette, add 5 c.c. of the water 
sample to each litmus lactose bile fermentation tube. 

7. Incubate the gelatin plates (cover-side up) in a cool 
place. 

8. Incubate the agar plates (cover-side down), the agar 
shake and the fermentation tubes at 37 C. 

9. Make note of the time of day these are placed in the 
incubator. All cultures placed at 37 C. must be examined 
within twenty-four hours. Types of colon-like organisms if 
present, may be quite easily recognized within twenty- 
four hours by the type and reaction of the colony on the 
agar plate, by the fermentation test, and by acid and gas 
formation in the agar shake. 

10. Examine the agar plates after eighteen to twenty- 
four hours incubation. 

11. If acid colonies are present, make morphological 
determinations (hanging drop) for their similarity to B. 
coli. 

12. If these characteristics are positive, transfer five 
different colonies to agar slants. 

13. Agar shake and fermentation tubes must be examined 
in eighteen to twenty-four hours also for gas and acid produc- 
tion. 

14. If gas is present in either, and no acid colonies have 
appeared on the original plates, make dilution plates in litmus 
lactose agar in order to isolate the acid and gas producing 
organisms. 

15. Transfer five different acid colonies which show a 
morphology similar to that of B. coli to agar slants. 

16. At the end of forty-eight hours, remove the agar 
plates from the incubator, make counts, record the types 
of colonies present and the number of each type. 

17. Transfer each type of colony not previously isolated, 
to an agar slant. 



226 GENERAL MICROBIOLOGY 

18. After all agar slant cultures have grown sufficiently 
(twenty-four hours at least), make sub-cultures from each 
pure culture into litmus milk, gelatin stab, nutrient broth, 
dextrose fermentation tube, Dunham's peptone solution 
and nitrate peptone solution for corroborative tests, and 
record the characteristics of growth according to the descrip- 
tive chart of the Society of American Bacteriologists, on 
the form on p. 235. 

19. Determine to which group of water organisms each 
pure culture belongs. (Consult the table in Savage's Bac- 
teriological Examination of Water Supplies, pp. 192-1S3.) 

20. Compare pure cultures isolated from acid colonies 
with a pure culture of B. coli in each case. 

21. Keep the plates at room temperature after forty- 
eight hours of incubation and count at the end of seven 
days. Record the counts as above. 

22. Keep the agar shake and fermentation tubes for 
seven days and record any changes that take place. 

23. Examine gelatin plates after forty-eight hours and 
then every day or so for seven days. 

24. Count before the liquefying colonies get so numerous 
or large as to render counting difficult. 

25. Record the total number of organisms per cubic 
centimeter; also the proportion of the liquefying to the 
non-liquefying organisms. Deep-well water should contain 
none or but very few liquefying organisms. Why? 

26. If liquefying organisms are present in large propor- 
tion or in great numbers on the plates from deep-well 
water, re-plate from the same source (not from the original 
sample) to determine whether the liquefying colonies 
came from the original sample or from some fault of 
technic. 

27. Fish each type of colony and determine to which 
group of water organisms it belongs. Are the same organ- 
isms found on gelatin as on agar plates? 

28. Record and compare the number and types of organ- 



BACTERIOLOGICAL ANALYSIS OF WATER 227 

isms developing on the agar and gelatin plates. Explain 
why your results vary on different media. 

29. Does one medium seem more favorable to the 
development of a larger number of organisms? If so, 
which? Give reasons for answers. 

30. If the gelatin count is less than 100 organisms per 
cubic centimeter the water is good, 200 per cubic centimeter 
will pass but if many more, i.e., 500, 1000 or over, the water 
is suspicious and effort should be made to determine the 
presence of B. coli. 

31. Read the statements on pp. 41-43 and 62-64, et al, 
in Prescott and Winslow's Elements of Water Bacteriol- 
ogy and compare with those on p. 77, Standard Methods 
of Water Analysis. After experiments 1 and 2 are com- 
pleted, draw your own conclusions from the above and give 
reasons for statements you make. 

32. Each student must know the morphological and cul- 
tural characteristics of B. coli, Bad. aerogenes and B. typho- 
sus. 

Read about and comment on the methods used in other 
countries, giving references to the literature read. 

33. What other methods are employed to determine 
the potability of water? Discuss these. 

REFERENCES 

Standard Methods of Water Analysis, American Public Health Asso- 
ciation (1913), pp. 77-80, 88-96, el al. 

PRESCOTT and WINSLOW: Elements of Water Bacteriology, 3d Ed., 
pp. 1-51, 215-228. 

MARSHALL: Microbiology, pp. 192-204. 

SAVAGE: Bacteriological Examination of Water Supplies (1906), 
pp. 192-193, 194-264. 

THRESH: Examination of Waters and Water Supplies, 2d Ed. (1913), 
pp. 184-271, 488-524. 

DON and CHISHOLM: Modern Methods of Water Purification (1911), 
pp. 260-271, 



228 GENERAL MICROBIOLOGY 



EXERCISE 2. BACTERIOLOGICAL ANALYSIS OF WATER 
SUSPECTED OF SEWAGE OR OTHER POLLUTION 

Apparatus. Sterile 500 c.c. flask for collecting sample; 
litmus lactose agar shake; six litmus lactose agar tubes for 
making plates; ten litmus lactose (or ordinary) agar slants 
for pure cultures isolated; six salt-free gelatin tubes for 
plates; ten tubes of gelatin for pure cultures isolated; two 
litmus lactose bile fermentation tubes (p. 122, Prescott and 
Winslow); ten tubes of litmus milk; ten tubes of Dunham's 
peptone solution; ten tubes sterile esculin bile for B. coli 
test (p. 129, P. and W.); ten tubes of nitrate peptone 
solution; ten each fermentation tubes of dextrose, lactose 
and saccharose broth; 99 c.c. dilution flasks; twelve sterile 
Petri dishes; sterile 100 c.c. volumetric pipette; sterile 
1 c.c. and 5 c.c. pipettes; sample of water or sewage from 
source indicated by instructor; record sheet for recording 
data; record sheet for pure cultures isolated. 

Cultures. B. coli, Bad. aerogenes, B. typhosus. 

Method. Water for this experiment may be obtained 
from a lake, river, etc., just below a sewer outlet, or 
from a surface well, etc., the source will be designated by 
instructor. 

1. Collect the sample in the sterile 500 c.c. flask, using 
all precautions as with an unpolluted water sample. (See 
Exercise 1.) 

2. This sample must be analyzed at once. . 

3. Using a wide range of dilutions, plate immediately 
in litmus lactose agar and in salt-free gelatin, making six 
dilution plates in each medium. 

4. If the water is not suspected of great pollution, 0.1 
c.c. of the sample may be plated directly, using low dilu- 
tions for the remaining five plates. 

5. If pure sewage is to be plated, use dilutions 1 : 100, 
1 : 1000, 1 : 10,000, 1 : 100,000, 1 : 1,000,000 and 1 : 10,- 
000,000. 



BACTERIOLOGICAL ANALYSIS OF SEWAGE 229 




6. Make an agar shake as in Exercise 1 and inoculate 
lactose bile fermentation tubes with 0.1 c.c. direct and 1 
c.c. of the 1 : 100 dilution of the sample respectively (or 

greater quantities if only 
a small amount of pollu- 
tion is suspected). 

7. Incubate the agar 
shake, fermentation 
tubes and agar plates at 
37 C. Place gelatin 
plates at 15 to 20 C. 

8. Make note of the 
hour at which the cul- 
tures are placed at 37 C. 
Examine these within 
twenty-four hours for in- 
dications of the presence 
of the colon-type of 
organisms. 

9. Examine and count 




FIG. 50. General Ground Plan of Individual House Sewer System 
Showing Exit from House, Anaerobic Tank, (A) Switch, and 
(B) Subsurface Irrigation Tile. 

the agar plates after eighteen to twenty-four hours incuba- 
tion at 37 C. (Keep at room temperature after forty- 
eight hours.) 



230 



GENERAL MICROBIOLOGY 



10. Fish acid colonies, placing them on agar slants, and 
incubate at 37 C. for twenty-four hours. 




11. Examine each colony microscopically in a hanging 
drop. If characteristic of B. coli, make sub-cultures in 
litmus milk, gelatin (stab), Dunham's solution, nitrate 



BACTERIOLOGICAL ANALYSIS OF SEWAGE 231 

peptone solution, dextrose, lactose and saccharose fermenta- 
tion tubes, and esculin bile, for identification. 

12. Test the gas for H and CO2 and record the ratio. 




















O=: 






<N 

5 






1 




1 


-, ,,8,8 


1 




1 





3 Kf) K^" 

O rj 

*! 



^ I 
O I. 



13. Transfer B. coli, Bad. aerogenes and B. typhosus 
to the same media and compare their cultural character- 
istics with those of organisms isolated. 

14. Determine whether the organisms are Gram-posi- 
tive or -negative. 



232 



GENERAL MICROBIOLOGY 



15. Examine gelatin plates after two days and then 
every few days for seven days. 



([ i| r\ 



y 

J9M9S f 




16. Count before the liquefying organisms get so numer- 
ous or large as to render counting difficult. 

17. Estimate the ratio of liquefying to non-liquefying 



BACTERIOLOGICAL ANALYSIS OF SEWAGE 233 

organisms. What is the significance in sanitary water anal- 
ysis of a large number of liquefying organisms? 

18. If acid colonies are not present in twenty-four to 
forty-eight hours on litmus lactose agar plates, and acid 
and gas are evident within this time either in the agar 
shake or lactose bile fermentation tubes, make dilution 
plates from either of these in litmus lactose agar, incubate 
at 37 C. for twenty-four hours and fish acid colonies, 
placing them on the different media for differentiating 
B. coli. 

19. Compare the cultures isolated with the pure culture 
of B. coli in every case. Also make comparisons with 
Bad. aerogenes and B. typhosus. 

20. Isolate several different colonies and record the cultural, 
etc., characteristics of each organism on the record sheet 
furnished. Were any of the types of organisms in this 
sample present in the first sample? Would you expect to 
find this the case? Give reason. 

21. The data and conclusions in the above should be 
given in detail. Point out also any practical applications. 

REFERENCES 

Standard Methods of Water Analysis, 1913 edition, pp. 79-82, 87-88, 

92, 95-102. 
PRESCOTT and WINSLOW: Water Bacteriology, 3d. ed., pp. 61-201, 

228-265. 

SAVAGE: Bacteriological Examination of Water Supplies, pp. 27-69. 
MARSHALL: Microbiology, pp. 97, 108, 162, 182, 204, 212, 221, 

323-324. 



234 



GENERAL MICROBIOLOGY 



BACTERIOLOGICAL WATER ANALYSIS 

Date . . . . 19 



Sample No. . 

Name of sender 

Address 

Source of water 

Surroundings 

Temperature 

Appearance 

Odor 

Remarks. . 



Age. 


Agar Shake. 


Lactose Bile. 


Per cent Per cent T> 4 
C0 2 . H 2 . Reaction. 


24 hours 






48 hours 










24 hours 


48 hours 


Remarks 


acid 


non- 
acid 


acid 


non- 
acid 


Litmus 
lactose 
agar 
plates 


1.0 c.c. 












0.5 c.c. 












0.1 c.c. 










Average count 






3 days 


7 days 




liquef. 


non- 
liquef. 


liquef. 


non- 
liquef. 


Gelatin 
plates 


1 c.c. 












0.5 c.c. 












0.1 c.c. 










Average count 



Plates made from ferm't'n tube or agar shake . . 
Organisms isolated 



] 

Ii 
pSl 


3ACTERIC 


LC 


>GI 


CA 


L 


AS 

L 


ALYS 

1 1 


5lS 





F 


\V^ 


LTJ 


SB 




235 


s3id BauuiQ 


























80IJM 






























Fermentati'on in 


qiq ^OBT 










i 




















q^oaq An 
































OSOUS^H 
































^uu^ 
































uipaj 
































^raopy 


__ 


_ 


_ 



























^lo^nQ 
























asoanqooBg 










1 


















aso^OBT - 
































aso^xaa 





























i 




Z OO : Z H 




1 




















eiiq uiinosa 
































Vai ^soaj-aBOA 












_ 




















S -3ua-uo D 






























omoj 

































3HIUI -n 































J13313 '^OBl ^I r [ 






























q^oaq paa ^na N 
































JBSB paa '^na^ 
































u^pa jo -b n 
































ppuj 
































SnN 





.._ 







_ 






















^uauiSijj 




















UlB^S UIBJQ 


_ 































ASopqdao^ 


























II 

ii 

|3 



























236 GENERAL MICROBIOLOGY 



EXERCISE 3. TO DEMONSTRATE THE EFFICIENCY OF 
CHLORIDE OF LIME AS AN AGENT IN THE PURI- 
FICATION OF DRINKING WATER 

Apparatus. Small can of commercial chloride of lime 
(one can is sufficient for the class); 6 liter precipitating jar; 
sterile 1.5 liter flask; 20 sterile Petri dishes; sterile 1 c.c. 
pipettes; two sterile 100 c.c. volumetric pipettes; ten tubes 
of litmus lactose agar; ten tubes gelatin; two litmus lactose 
agar shakes; six dilution flasks, four 99 c.c. and two 90 c.c, 
(distilled water) ; one liter of sewage. 

Method. 1. Using the large precipitating jar, prepare 
a 6% solution of chloride of lime (by weight), using the 
entire contents of a newly opened can. This stock solution 
will contain about 2% available chlorine calculated on the 
basis of 35% available chlorine in the commercial chloride 
of lime. 

2. Mix well and allow to settle over night before pro- 
ceeding with the experiment. 

3. Obtain the sewage in the large sterile flask. Save 
a small quantity of the sewage in a sterile test tube for 
microscopic examination. 

4. Make gelatin and litmus lactose agar plates of the 
fresh sample, plating 1 c.c. and 0.1 c.c. direct, and dilutions, 
1-100, 1-10,000 and 1-1,000,000 (five plates in each 
medium) . 

6. Add 1 c.c. of the 6% solution of chloride of lime 
(2% available chlorine) to exactly 1,000 c.c. of the sewage 
in the sterile flask; shake vigorously for one minute and 
allow it to stand one hour. This amount is over ten 
times the amount necessary for sterilization, calculating 
on the basis of 16 Ibs. of chloride of lime (containing 
35% available chlorine) per million gallons of water. (See 
table on page 11, Hooker's Chloride of Lime in Sanita- 
tion.) 

6. Plate in gelatin and litmus lactose agar, using 1 c.c. 



EFFICIENCY OF CHLORIDE OF LIME 237 

and 0.1 c.c. direct, and dilutions of 1-100, 1-10,000 and 
1-1,000,000 (five plates for each medium). 

7. Make duplicate agar shakes also, with the raw river 
water and with the same after the chloride of lime has been 
added. 

8. Incubate all plates and shakes at 37 C. Observe 
each at the end of twenty-four and forty-eight hours. 

9. Count the agar plates at forty-eight hours and record 
the results. Count the gelatin plates before the colonies 
are obscured by liquefiers. 

10. What types of organisms have been destroyed? 
What types remain? Is this according to the results of 
other investigators (see Hooker, p. 23). Would you feel 
safe in drinking this water? 

11. Examine the sewage in a hanging drop and draw 
the types of organisms present. Note which types pre- 
dominate, also note their comparative size and motility. 

12. Make records of these to compare with the data on 
cultural determinations. Are all of these types found by 
the cultural methods? 

13. Chloride of lime is used for purifying drinking water 
in the proportions of 5 to 25 pounds per million gallons of 
water. What effect does chloride of lime have on organic 
matter, discoloration, turbidity and swampy or other 
smells in raw water? 

14. 35% available chlorine is necessary for efficient 
sterilization. The available chlorine is merely an index of 
the efficiency of the chloride of lime. Chloride of lime in 
its industrial applications of bleaching, deodorizing or 
disinfecting does not act by its chlorine but by its oxygen. 
Its action is not chlorination but oxidation. (Hooker, 
P- 7.) 

15. What is the maximum limit for the amount of 
chloride of lime used in dosing drinking waters? What is 
the amount used for treating water on shipboard? Why? 

What is the minimum length of contact allowed between 



238 GENERAL MICROBIOLOGY 

the stock solution of chloride of lime and the water to be 
purified? Why is a minimum time limit set? 

What other means are used for the chemical steriliza- 
tion of water? Are these efficient? 

What is the Hazen theorem? Its explanation? 

What other uses has chloride of lime in sanitation? 

16. Give all data and observations in full. State any 
conclusions to be based on the above and point out any 
practical applications. 

REFERENCES 

HOOKER: Chloride of Lime in Sanitation (1913), pp. 7, 12-34, 34-77. 

WESBROOK, WHITTAKER and MOHLER: The Resistance of Certain 
Bacteria to Calcium Hypochlorite. Reprint from Jour, of Amer- 
can Public Health Association, Vol. I (1911). 

WHITTAKER: Field Equipment for Laboratory Work on Water 
Supplies. Reprint from Journal of American Public Health Asso- 
ciation, Vol. II (1912), pp. 948-954. 



EXERCISE 4. TO DEMONSTRATE THE EFFICIENCY 
OF THE BERKEFELD FILTER CANDLE AS A MEANS 
OF WATER PURIFICATION 

Apparatus. Berkefeld filter candle complete with cylin- 
der; water-power vacuum pump; filter flask with rubber 
stopper into which the filter candle fits; one liter sterile flask; 
eight tubes of salt-free gelatin for water analysis; eight 
Petri dishes; sterile 1 c.c. and 10 c.c. pipettes; dilution 
flasks; distilled water 

Method. 1. Set up the filtering apparatus, connect 
with the vacuum pump and wash the filter by running 
through it 500 c.c. of boiling, distilled water. 

2. Place a cotton plug in the cylinder and in the side 
arm of the filter flask; sterilize the filtering apparatus as set 
up, in the autoclav. 

3. Collect a liter of polluted river water from a point 
near the opening of a sewer. 



TO TEST THE CATALYTIC POWER OF SOIL 239 

4. Make dilution plates in gelatin immediately, using 
dilutions 1 : 10, 1 : 100, 1 : 10,000 and 1 : 1,000,000. 

6. Filter the remainder of the sample through the 
Berkefeld filter candle and plate immediately from the 
filtrate, making gelatin plates, using 1 c.c. and 0.1 c.c. 
direct, dilutions of 1 : 100 and 1 : 10,000. 

6. Incubate all plates at the same temperature. Ex- 
amine the plates daily and record the counts. 

7. Was the bacterial count reduced by the use of this 
filter candle? What type of organisms passed the filter? 
Is this filtered water fit for drinking purposes? 

How does this filter compare with other types as to 
its action? What other types of filters are used for water 
purification? Sewage purification? Upon what does the 
value of each depend? 

8. Give all data in full and draw any conclusions that 
are warranted. Point out any practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 205-207. 

DON and CHISHOLM: Modern Methods of Water Purification (1911), 
pp. 231-236. 

SOIL MICROBIOLOGY 

EXERCISE 1. TO TEST THE CATALYTIC POWER OF 

SOIL 

Apparatus. 3% hydrogen peroxide; three 375 c.c. 
Erlenmeyer flasks; three one-hole stoppers to fit flasks; 
three pieces of bent glass tubing; three pieces rubber tub- 
ing; three 100 c.c. glass graduates. 

Culture. Soil rich in humus. 

Method. 1. Fit the Erlenmeyer flasks with one-hole 
rubber stoppers through which a short piece of bent glass 
tubing is inserted. Fit short pieces of rubber tubing 
(about 40 cm. in length) to the glass tubing. 



240 



GENERAL MICROBIOLOGY 



2. Place 5 gms. of the soil in one of the flasks and mix 
it with 50 c.c. of water. 

3. Arrange the 100 c.c. graduate in a water bath for 
collecting gas, by filling with water and inverting mouth 
down, under water. Clamp it in place. 

4. Then add 20 c.c. of 3% hydrogen peroxide to the flask 
containing the soil, stopper the flask and, keeping the 
mixture moving, collect the oxygen in the graduated tube 
over water. Record the time for maximum oxygen libera- 
tion. 

5. At the same time determine the part played by bac- 
teria and enzymes, by repeating the catalase test on soil 
that has been heated in the autoclav at 15 Ibs. pressure 
(120 C.). 

6. Also determine the part played by humus by repeat- 
ing this test with the same earth, having burned the humus 
by heating with the flame. 

7. Record your results according to the diagram follow- 
ing: 





Fresh 
soil. 


Autoclaved 
soil. 


Burned 
soil. 


Cubic centimeters of oxy- 
een . 








Time for maximum oxy- 
gen liberation 

















8. What part does each factor play in the liberation 
of oxygen? Is this action in soil of any value? If so, 
what? 

9. Compare your results with those of the catalase 
test in milk. By which substance is the most oxygen 
liberated? Is there a logical explanation for this? 

10. Give all results in full, draw any conclusions that 



TYPES OF MICROORGANISMS IN SOIL 241 

are suggested and point out any possible practical applica- 
tions. 

REFERENCE 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, p. 103. 



EXERCISE 2. A COMPARATIVE STUDY OF THE NUM- 
BER AND TYPES OF MICROORGANISMS IN SOIL: 
THE PLATE VS. THE MICROSCOPIC METHOD 

A. THE PLATE METHOD 

Apparatus. Sterile soil borer; sterile paper bag; sterile 
large spatula; sterile large piece of paper; sterile paper 
15 cm. square; 500 c.c. wide-mouthed flask containing 200 




$690,750,000 $1,342,453,982 $2,225,700,000 

Total Value of Total Value of Total Value of 

Annual Timber Cut. Mineral Output, 1911. Natural Manure. 

FIG. 54. Showing the Comparatively Enormous Value of Organic 
Microbial Food which Should Enter the Soil. (Carver, Year- 
book of U, S. Dept. of Agr., 1914.) 

c.c. sterile water; 90 and 99 c.c. dilution flasks (sterile 
water); ordinary agar; sterile Petri dishes; soil (different 
types), manure, etc. 

Each student should use one type of soil and one type of 
manure for this experiment. Assignments will be made by 
the instructor. The results obtained by each student 
are to be compared with those of others. 



242 GENERAL MICROBIOLOGY 

Method. 1. Remove the coarser surface debris from 
the soil. Sink the soil borer to the depth of 30 cm., remove 
the borer and place the soil in the bag. 

2. Take six borings so that your composite sample 
will be representative of the entire plot under considera- 
tion. 

3. At the laboratory, carefully mix and pulverize the 
composite sample with the spatula on the large piece of 
paper. 

4. Weigh out 20 gms. on sterile paper and transfer 
immediately to the flask containing 200 c.c. of sterile water. 
A large amount of soil is used to reduce the error as much 
as possible. The manure should be treated in a similar 
manner. 

5. Shake thoroughly for one minute, allow the coarser 
particles to settle and transfer 10 c.c. (equivalent to 1 gm. 
of soil) of the supernatant liquid to 90 c.c. of .sterile water. 
Each cubic centimeter of this dilution then contains 0.01 
gm. soil. 

6. Make and plate from the following dilutions: 1-10, 
1-100, 1-1000, 1-10,000, 1-100,000, 1-1,000,000. 

7. Incubate at room temperature for four to eight 
days. 

8. Count and record the results as number of bacteria 
per gram of soil or manure, in tabular form. 

9. Record the number of the various types of micro- 
organisms. Note the numbers of chromogenic bacteria 
and the streptothrix forms. 

10. Examine some of the manure in the hanging drop. 
What forms are seen? Make drawings. Are all of these 
forms found on the plates? Give reasons for what does 
occur. Add sterile water to the manure in a sterile deep 
culture dish or flask and examine every three or four days 
during the course of the experiment. Record your obser- 
vations. 

11. When the peat sample is obtained, at the same time 



TYPES ^OF MICROORGANISMS IN SOIL 243 

partially fill a small sterile flask with swamp or marsh water. 
Examine immediately in the hanging drop and draw the 
forms seen. 

12. Place this swamp water in the sunlight (more or 
less direct) for two or three days and examine again in the 
hanging drop for any forms of life present. 

13. Compare the flora of these two microscopical prep- 
arations. Suggest why each type of organism is present. 

14. Compare all types of soil examined both quanti- 
tatively and qualitatively as to their microflora. Which 
soils are most alike in their flora? Suggest a reason why. 
Why do various soils vary in the number of bacteria found? 

REFERENCES 

MARSHALL: Microbiology, pp. 226-245. 

CONN: Agricultural Bacteriology, pp. 34, 70, 120. 

EYRE: Bacteriological Technic. Second Ed., pp. 470-478. 

B. MICROSCOPICAL METHOD 

Apparatus. Soil or manure of same type as used for 
plating; sterile water; sterile Chinese ink; platinum loop 
of known capacity; sterile watch glass; cover-glasses, 
absolutely clean] ocular micrometer; stage (object) microm- 
eter. 

Method. 1. To 1 gm. of soil or excrement in a test 
tube add 4 c.c. of sterile water and shake vigorously for 
five minutes. 

2. Place 0.5 c.c. in a clean, sterile watch glass. Add 
0.5 c.c. of Chinese ink. 

3. Mix with a platinum loop of known capacity. 

Note. To determine the capacity of the platinum loop, weigh 
two watch glasses. Into one put exactly 1 gm. (1 c.c.) of water. 
Transfer five loopfuls from the glass containing water to the empty 
watch glass. 

Weigh each. Then determine the weight and also the volume 
of one loopful. 



244 GENERAL MICROBIOLOGY 

4. Transfer one loopful of the " ink manure " solution 
to a clean, sterile cover-glass and spread in an even film 
over the entire surface. 

5. Let this dry in air and fix by passing three times 
through the flame. Mount at once in balsam. 

6. Measure the surface area of the cover-glass. Also 
the diameter of one field of the oil immersion lens (using 
the stage micrometer) and from that the area of the field. 

7. Count fifty fields and determine the average. 

8. From the data which you now have, determine the 
number of organisms on the cover-glass, which is the number 
in one loopful. 

9. Then from this calculate the number in 1 gm. of soil 
or excrement. 

10. Also calculate the weight of bacteria in 1 gm. (See 
p. 88, Marshall's Microbiology.) 

11. Compare the count thus obtained with the count 
obtained by the plate method. What is shown? How 
do you explain this result? 

12. Compare the manure and soil counts. Draw con- 
clusions and explain. 

13. What other methods are used for obtaining numbers, 
etc., of organisms in soil and like materials? 

14. State your data and observations in full. Draw 
any conclusions warranted and point out any practical 
applications. 

REFERENCES 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 

89-91. 

LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp. 7-9. 
MARSHALL: Microbiology, pp. 238-244. 



THE EFFECT OF AERATION OF SOILS 



245 



EXERCISE 3. TO ILLUSTRATE THE EFFECT OF AERA- 
TION OF SOILS ON THE ACTIVITIES OF THE MICRO- 
ORGANISMS CONTAINED THEREIN 

Apparatus. Coarse, medium, and fine sand; magnesium 
oxide; dilute HC1; N/10 NH 4 OH; N/10 HC1; indicator; 
sterile 1 c.c. pipette; ten sterile 250 c.c. Erlenmeyer flasks; 
condenser; sterile 1% peptone solution. 

Culture. B. mycoides, broth culture. 

Method. 1. Prepare the sand for use by first heating 
it with dilute HC1; then wash it several times, first with 
tap water then with distilled water and dry at 110 C. 

2. Place 50 gms. of coarse sand in each of three flasks; 
do the same with the medium and the fine sand. 

3. Sterilize the flasks in the autoclav at 120 C. for 
thirty minutes. 

4. Place 50 c.c. of the 1% peptone solution in the remain- 
ing flask. 

5. Add sufficient peptone solution to the other flasks 
to make 10%, 20% and 30% moisture content according 
to the following table : 



Soil. 


Moisture. 


After twenty 
days. 


Coarse sand \ 

( 


10% 
20% 
30% 




Medium sand -\ 


10% 

20% 






30% 




Fine sand ! 


10% 

20% 




1% peptone solution 


30% 
99% 





6. Inoculate each flask with 1 c.c. from a broth culture 
of B. mycoides and shake to distribute the organisms evenly. 



246 GENERAL MICROBIOLOGY 

7. Make ammonia determinations after twenty days. 

Note. Ammonia determinations from the above are made by the 
following procedure: Remove the cotton plugs, add 200 c.c. of water, 
10 gms. MgO and a small piece of paraffin. Distill off the ammonia 
present, collecting it in N/10 HC1 and titrating against N/10 NH 4 OH, 
us-ing methyl orange as indicator. 

1 8. Record your results in the form given above. 

9. How does aeration affect bacterial activity? Size 
of sand grains? 

What interrelation have grain-size and moisture con- 
tent of soil? 

How do microorganisms obtain their food in soil that 
is not wet? 

What influence does humus have on aeration? On 
bacterial activity? 

10. Draw any conclusions warranted by your results 
and point out any practical applications. 

REFERENCE 
RAHN, O.: Tech. Bui. No. 6, Mich. Agr'l Expt. Sta. 

EXERCISE 4. TO DEMONSTRATE THE CELLULOSE- 
DECOMPOSING POWER OF AEROBIC ORGAN- 
ISMS FOUND IN THE SOIL 

Apparatus. Two Petri dishes; four pieces of round filter 
paper to fit Petri dishes; 0.05% K 2 HPO 4 ; MgNH 4 P0 4 ; 
0.05% NH 4 NC>3; 1 c.c. pipette; soil rich in humus, or 
well-rotted manure. 

Method. 1. Put a thin layer of MgNH 4 P0 4 between 
two filter papers in a Petri dish. 

2. Moisten this with the solution of K2HP0 4 and in- 
oculate with a few drops from a water solution of the soil 
or manure. Keep at 25 to 30 C. 

3. In three to six days brown spots will occur and later 
holes will be formed by bacteria. Thin places in the filter 



CELLULOSE DECOMPOSING POWER 247 

paper can be detected by holding the Petri dish towards the 
light. 

4. With a sterile platinum needle, test the consistency 
of the paper in the spots which have been most probably 
attacked and compare with that of the undecomposed 
spots. Describe the results. 

5. Add more of the K2HPO4 solution when necessary 
to keep the filter paper moist. 

6. Start a second Petri dish in the same way but keep 
it moist with 0.05% NH 4 NO 3 and 0.05% K 2 HPO 4 . Here 
we find brown spots caused usually by fungi. 

7. Macerate some of the brown spots from each Petri 
dish with water and make a Chinese ink preparation. 

8. What types of organisms are seen in each prepara- 
tion? Make drawings. 

9. What organisms are especially active in the anaerobic 
decomposition of cellulose? Which type, aerobic or anae- 
robic, is responsible for the greater amount of cellulose 
decomposition in nature? When may the other types take 
precedence? 

What steps would you take to isolate the organisms 
which are growing on your plates? 

Are the chemicals used above present in the soil? In 
what form? In what forms does cellulose exist in culti- 
vated soils? 

Are cellulose-decomposing bacteria ubiquitous? Are 
they always found where cellulose in some form is depos- 
ited? Are cellulose decomposing bacteria limited to soil? 

10. Data and results are to be given in full, also draw any 
conclusions warranted and point out any possible practical 
applications. 

REFERENCES 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 

104-105. 
LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp. 

62, 63, 65. 



248 GENERAL MICROBIOLOGY 

MARSHALL: Microbiology, pp. 246-249. 

McBETH and SCALES: The destruction of cellulose by bacteria and 
filamentous fungi. Bui. 266, B. P. I., U. S. Dept. Agr. (1913). 

EXERCISE 5. TO ILLUSTRATE THE ANAEROBIC DE- 
COMPOSITION OF CELLULOSE BY SOIL AND 
FECAL ORGANISMS 

Apparatus. Six (large) tubes of Omelianski's synthetic 
medium for anaerobic cellulose fermentation; tall Novy 
jar; vacuum pump. 

Culture. Fresh and decayed manure. 

Method. 1. Inoculate one tube with small amounts 
of fresh horse or cow manure, a second with partially de- 
cayed manure. 

2. Place some cotton in the bottom of the Novy jar, 
insert the inoculated tubes in it, replace the stopper and 
exhaust the air by means of the vacuum pump. (Pyro- 
gallic acid and sodium hydroxide may be substituted.) 

3. Incubate the tubes in the Novy jar at 34 to 35 C. 
for four to six weeks. 

4. From time to time note any changes occurring in the 
filter paper. 

5. After the latter has been wholly or partially diges- 
ted, make transfers to new tubes of the medium and incu- 
bate anaerobically as before. 

6. Repeat this procedure from the cultures made just 
previously. (The jar is evacuated each time after obser- 
vations are made.) 

7. Examine the organisms causing the disintegration of 
the filter paper both in the hanging drop and with some 
ordinary stain (not the ink preparation). Make permanent 
stained preparations. 

8. Starch, cotton, straw, etc., digestion may be com- 
pared if these substances are substituted for filter paper 
in Omelianski's medium. 

Not taking soil into consideration, where do anaerobic 



NITRIFICATION IN SOLUTION 



249 



cellulose-decomposing organisms probably play a most 
important part? How is this determined? 

9. What types of anaerobic cellulose-decomposing bac- 
teria are favored by this synthetic medium? These bac- 
teria have only in exceptional cases been grown on solid 
media. How can these types be separated? 

10. Data and observations should be given in full. 
Draw any conclusions warranted and indicate any practical 
applications that may be made, 

REFERENCES 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, p. 93. 
LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, p. 64. 
MARSHALL: Microbiology, pp. 246-248. 
McBETH and SCALES: I.e., Exercise IV. 



EXERCISE 6. TO ILLUSTRATE NITRIFICATION IN 
SOLUTION 

Apparatus. Nine 50 c.c. Erlenmeyer flasks; 75 c.c. 
each of Solutions I, II and III for nitrification (see appen- 
dix); Nessler's solution; a-naphthylamin; sulphanilic acid; 
2% diphenylamin in sulphuric acid; aqueous alcoholic 
stains. 

Cultures. Rich soil from cultivated land; manure 
from surface layers of manure heap. 

Method. 1. Place 25 c.c. of each solution in a 50 c.c. 
Erlenmeyer flask and sterilize in the autoclav. 

2. Inoculate as follows: 





Solution I. 


Solution II. 


Solution III. 


0.5 gm. manure 
0.5 gm. soil 
Nothing control 

















and keep at 25 to 30 C., to hasten the action of the micro- 
organisms. 



250 GENERAL MICROBIOLOGY 

Owing to the presence of some carbon-monoxide in the 
air of the laboratory from the burning gas, the carbon- 
monoxide-assimilating B. oligocarbophilus often appears as 
a dry white skin on the surface of the solution in these flasks. 

Note. Solution I is adapted for relatively increasing the nitrite 
bacteria, Solution II the nitrate producers and Solution III the sim- 
ultaneous growth of both organisms as in nature. 

3. After eight to fourteen days, test all solutions and 
control flasks every second or third day by transferring 
0.1 c.c. with a sterile pipette to a white glazed surface 
(e.g., plate) using 

(a) Nessler's solution, for ammonia; 

(6) nitrite test solutions, for nitrites; 

(c) nitrate test solution, for nitrates. 

Tabulate your results. Discuss and explain the decom- 
position which is taking place in each inoculated flask, 
giving the successive steps in the disintegration of the 
crude nitrogenous organic matter. 

4. Examine a loopful of each solution in the hanging 
drop each time chemical tests are made. Morphologically 
what types predominate in each solution? In the sample 
of soil? Of manure? Are any of these spore-formers? 
If so, which type? 

5. Make permanent stained preparations from each 
flask. 

6. Nitrifying bacteria do not grow on the ordinary 
solid media. Why? Many different methods have been 
tried for the isolation of nitrifying organisms but the obtain- 
ing of pure cultures is still a most difficult bacteriological 
task. 

(See appendix for media used for the isolation of these 
organisms.) 

What methods are employed for their isolation besides 
the use of solid synthetic media? What is the principle 
of each method? 

What are the different types of nitrifying organisms? 



DENITRIFICATION IN SOLUTION 251 

Their respective functions? What interrelationships exist 
between these organisms? 

Where are nitrifying organisms found in nature? What 
is their significance? 

W^hat conditions in soil are necessary for their pro- 
liferation? What methods does the agriculturist use which 
serves to conserve these organisms? 

REFERENCES 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 

96, 97, 106, 109, 110. 
LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp. 

25-34. 
MARSHALL: Microbiology, pp. 259-263. 

EXERCISE 7. TO ILLUSTRATE DENITRIFICATION IN 
SOLUTION 

Apparatus. Eight tubes of sterile Giltay's solution; 
eight tubes of nitrate broth; eight to ten tubes of Giltay's 
agar; eight to ten tubes of nitrate agar; eight fermenta- 
tion tubes of nitrate bouillon, four with and four .without 
sugar (use 1% dextrose); peptone solution in tubes; sterile 
1% solution of KNOs; nitrate test solution; Nessler's 
solution, test for NHs; a-naphthylamin and sulphanilic acid 
(nitrite test solutions); sterile 1 c.c. pipettes; sterile Petri 
dishes; sterile dilution flasks. 

Cultures. Soil (record type used); straw from manure 
pile; horse manure. 

Method. 1. Inoculate test tubes of each solution in 
series as follows: 

1 and 2 

3 and 4 0.1 gm. soil. 

5 and 6 0.1 gm. straw; 

7 and 8 0.1 gm. horse manure. 

2. Incubate these at 37 C. for forty-eight hours. 

3. Note any changes occurring. Determine whether 



252 GENERAL MICROBIOLOGY 

any nitrites or ammonia have developed. To what are 
the gas bubbles due? 

The crystals deposited in Giltay's solution are magnesium 
phosphate. 

4. Using sterile pipettes, test 1 c.c. portions of each 
after forty-eight hours, seven days, etc., for nitrates with 
phenolsulphonic acid. 

5. From two tubes showing abundant gas formation, 
make nitrate agar plates, using a wide range of dilutions 
that one or two plates may show well-isolated colonies. 
Incubate at 37 C. 

6. From various colonies appearing on the plates, make 
stab cultures in nitrate agar. Incubate these at 37 C. 

Save one showing the most abundant gas formation under 
these conditions, for further study. 

7. Inoculate a nitrate bouillon fermentation tube with 
the pure culture just isolated, also add some of the crude 
material to a fermentation tube. 

8. Duplicate with a nitrate bouillon fermentation tube 
containing sugar. 

9. Determine the amount and nature of the gas formed 
in each tube and compare results. (Determine by elimi- 
nation; test for CO2 and H2.) 

What influence does dextrose have upon the rate and 
amount of gas formation? 

10. Inoculate in duplicate, tubes of peptone solution 
with the pure culture. Record the growth and gas forma- 
tion, if any, qualitatively. 

11. To an old culture (not necessarily pure) in which the 
nitrates have disappeared, add 1 c.c. of a sterile 1% solu- 
tion of KNOs. Does gas formation re-occur? 

12. Continue to add a small amount of KNOs as rapidly 
as the culture ceases to give a reaction for nitrates (an 
indication that the latter have been used up.) Note how 
much KNOs your culture can reduce. 

13. Theoretically, what would be the difference in 



NON-SYMBIOTIC FIXATION OF NITROGEN 253 

action of denitrifying organisms in soil and in solution? 
Why? 

How do denitrification and nitrate reduction differ? 

How may colonies of nitrate-reducing bacteria be 
detected? 

14. Give all results in full and draw conclusions. Sug- 
gest any possible practical applications of the above. 

REFERENCES 

LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp. 

35-40. 

MARSHALL: Microbiology, pp. 263-267. 
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 98-99. 

EXERCISE 8. TO ILLUSTRATE THE NON-SYMBIOTIC 
FIXATION OF NITROGEN BY SOIL ORGANISMS, 
AND ISOLATION OF AZOTOBACTER THROUGH 
ITS MINERAL FOOD REQUIREMENTS 

Apparatus: Mannit solution for nitrogen-fixing organ- 
isms; mannit agar; eight sterile 100 c.c. Erlenmeyer 
flasks; concave slides; cover-glasses; concentrated H2S04; 
filter paper. 

Culture. From clay loam, sandy loam, manure. 

Method. 1. Place 50 c.c. of the mannit solution in 
each flask and sterilize (in autoclav). 

2. Inoculate in series as follows: 
Flasks 1 and 2 

Flasks 3 and 4 0.1 gm. clay loam. 
Flasks 5 and 6 0.1 gm. sandy loam. 
Flasks 7 and 8 0.1 gm. manure. 

3. Incubate at room temperature and note the changes 
taking place. A wrkikled skin, wnite at first, brownish 
later, is gradually formed. This is composed of the aerobic 
Azotobacter. 

4. From time to time examine the cultures in the hang- 
ing drop and note the type of organisms predominating. 
Single, small, thin bacilli are visible between the large 



254 



GENERAL MICROBIOLOGY 



cells of Azotobacter. The former are almost always a type 
resembling the nodule bacteria, B. radiobacter, and which 
can also fix nitrogen to a slight extent. Besides these, 
especially when Azotobacter is less in evidence, many other 
sporing and non-sporing bacteria participate in the process. 
Azotobacter, however, is the most vigorous free nitrogen- 
fixing organism yet discovered. 
(See reference, E. B. Fred, 
Exercise 9, Soil Microbiology.) 

5. What characteristic odor 
is produced in these cultures? 
Add a drop of concentrated 
H2SO4 to a small portion of 
the culture liquid. This in- 
tensifies the odor. 

6. When a brownish surface 
film develops, make plates from 
this culture, using relatively 
high dilutions. 

7. After a rather long period 
of incubation (six to seven 
days) examine the organisms in 
the various colonies and isolate 
Azotobacter chroococcum if possi- 
ble upon a mannit agar slant. 
On account of the slimy prop- 
erty of its cell wall, its separa- 
tion from B. radiobacter is often very difficult. The quickest 
way is to reinoculate first into the mannit solution. 

8. Save several of the plates having well-isolated colo- 
nies and note any changes which may occur. 

9. If any brown colonies develop, examine them in 
stained preparations. Measure the bacteria stained. 

10. Are these pure cultures? If not, plate from several 
such colonies in mannit agar to isolate the different organ- 
isms present. 




FIG. 55. Azotobacter. xlOOO; 
Smear from Six-day Old Cul- 
ture on Ashby's Agar at 
25 C. Showing organisms 
and capsules in various 
stages of development. (Dan 
H. Jones.) 



NON-SYMBIOTIC FIXATION OF NITROGEN 255 

11. Is B. radiobacter present? What part does it play 
in the fixation of nitrogen? 

12. Make several agar slant pure cultures of B. radio- 
bacter. 

13. Study the morphology and the cultural character- 
istics of this organism. 

14. Inoculate a small flask of the mannit solution with 
a pure culture of the newly isolated organism. 

15. How does this organism compare, morphologically, 
culturally, etc., with Ps. radicicolaf With the Azotobacter? 

What part in the nitrogen cycle does the Azotobacter play? 

What practices of the farmer favor the development of 
the Azotobacterf In what way? What soil conditions are 
favorable to the Azotobacter species? Are these condi- 
tions favorable to other bacteria? To plants? 

Diseased spots in soil are said to be caused by an 
excessive nitrogen fixation and nitrification, e.g., the niter 
spots in Colorado soils. 

16. State your results in full and draw conclusions. 
Point out the practical applications of the above. 

REFERENCES 

MARSHALL: Microbiology, pp. 98-99, 230-231, 248, 250, 266, 270-273, 

286, 288, 291. 
LIPMAN and BROWN: Laboratory Guide in Soil Microbiology, pp. 

43-45. 
HOFFMAN, C., and HAMMER, B. W.: Some Factors Concerned in the 

Fixation of Nitrogen by Azotobacter. Research Bui. No. 12, 

Univ. of Wis. (1910). 
JONES, DAN II.: A Morphological and Cultural Study of Some 

Azotobacter. Cent. f. Bakt. II Abt., Bd. 38 (1913), pp. 13-25, 

5 plates. 
Further Studies with Some Azotobacter. Cent, f . Bakt. II Abt., 

Bd. 42, pp. 68-69. 
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 40, 

113-114, 127. 
HEADDEN, W. P. : The Fixation of Nitrogen in Colorado Soils. Buls. 

178, 186, Colorado Agr. Expt. Sta. 



256 GENERAL MICROBIOLOGY 

EXERCISE 9. A STUDY OF THE SYMBIOTIC NITROGEN- 
FIXING ORGANISMS OF LEGUMES, PS. RADICI- 
COLA 

A. ISOLATION OF PS. RADICICOLA FROM 
NODULES OF LEGU MINOS JE 

Apparatus. Spade or trowel; sterile Petri dishes; 
tubes of nitrogen-free ash agar; tumbler for mercuric 
chloride solution; small piece of filter paper; small pair 
of forceps; scalpel or chisel-edged platinum needle; platinum 
loop; clean slides; mercuric chloride, 1-500; alcohol, 95%; 
tubes of sterile water; aqueous-alcoholic gentian violet 
orfuchsin; eosin; LugoPs iodin solution; saturated alcoholic 
solution of gentian violet. 

Culture. From nodules of roots of leguminous plants. 

Method. 1. Using a spade or trowel, obtain the roots 
of some legumes which show nodule formation in, abundance. 
If the soil is firm, as with clay, do not forcibly pull up the 
legume to obtain the roots as this procedure strips off the 
nodules which develope almost without exception on the 
young rootlets. 

Note. Heavily inoculated legumes may be stored for winter use in a 
cool, dry, dark place. Living organisms have been found after more 
than two years in some of the larger nodules. 

2. Thoroughly wash the roots under the tap and place 
the plant in clean, cool water. 

3. Keep parts of the plant for identification if the 
species is unknown. Not all plants belonging to the family 
Leguminosce are attacked by symbiotic nitrogen-fixing 
bacteria, only those belonging to the sub-family, Papilio- 
nacece. 

4. Compare the size, numbers, and location of the nod- 
ules on the roots of the different legumes. 

5. Remove a good-sized nodule from the roots, wash 
in clean water and immerse for three minutes in mercuric 
chloride solution, 1 : 500. 



NITROGEN-FIXING ORGANISMS OF LEGUMES 257 

6. Remove the nodule with flamed forceps and take up 
the excess of the solution between folds of sterile filter 
paper, then dip into alcohol, the last traces of alcohol 
being removed by passing the nodule quickly through the 
flame. 

7. Place the nodule on a flamed and cooled slide. 

8. Holding the nodule in flamed and cooled forceps, 
cut into it, and break it open by means of a sterile scalpel 
or a chisel-edged platinum needle. 

9. Thrust a sterile platinum needle into the nodule in 
the middle of the newly exposed surface and gently rotate the 
needle so that some of the crushed tissue adheres to it. 

10. Touch the needle in a drop of sterile water in a sterile 
Petri dish. 

11. Transfer a loopful of this suspension to a second 
drop of sterile water in a second Petri dish and a loopful 
from this to a third drop in a third Petri dish. 

12. Pour the relates, using tubes of nitrogen-free ash 
agar and incubate at room temperature for two or three 
days. 

13. Make a smear on a clean slide from the freshly cut 
surface of the nodule, stain and examine microscopically. 
What is the morphology of Ps. radidcola found in the 
nodule? What are bacteroids? 

14. Make a smear directly from this same nodule on 
a clean slide, fix and stain with eosin followed by Lugol's 
iodin solution. The iodin demonstrates the starch which 
is usually present in nocjules. Is starch present? 

15. After a few days of incubation the colonies of Ps. 
radidcola will be noted on the plates as round, grayish- 
white, translucent, slime-like drops, finely granulated and 
often with compact white centers. 

16. Examine these colonies in the hanging drop. They 
contain the normal, short, rod forms which during the 
first days are very motile. 

17. Isolate several pure cultures of Ps. radidcola on 



258 GENERAL MICROBIOLOGY 

slanted nitrogen-free agar and note characteristic growth. 
Why is nitrogen-free agar used for the cultivation of Ps. 
radicicolaf 

18. Make permanent stains of pure culture and compare 
with organisms on stained smear from nodule as to size, 
shape, etc. Are involution forms present in either prepara- 
tion? 

Do all species produce organisms of the same general 
morphology in the respective nodules? In pure culture? 

19. Make a flagella stain from the pure culture as fol- 
lows: 

a. Take a loopful of the mucilaginous growth from a 
colony or an agar culture and spread it on a clean slide, 
lashing it out in slender tongues. 

6. Let the film dry in air without killing or fixing. 

c. Flood the film a moment with saturated alcoholic 
solution of gentian violet. 

d. Wash under the tap, dry and examine with the oil 
immersion lens. 

20. The mucilage in which the cells lie will be found 
to be deeply and evenly stained and the bacteria stained 
scarcely at all, so that the preparation presents somewhat 
the appearance of a photographic negative. 

The single polar flagellum may be demonstrated by 
this stain, since it, like the protoplasm of the cells, refuses 
the stain, and so it appears as a clear, uncolored streak in 
the surrounding deeply stained mucilage. The flagella 
are best seen at the margins and in thin places, inasmuch 
as the mucilage in the denser areas masks the slender fla- 
gella. 

21. Sometimes the roots of leguminous plants show, 
instead of the normal nodules, lesions of crown-gall caused 
by Bad. tumefaciens which somewhat resemble Ps. radici- 
cola. 

22. For a rapid diagnosis, plate in the synthetic Congo 
red medium which differentiates these two organisms; 



NITROGEN-FIXING ORGANISMS OF LEGUMES 259 

Ps. radicicola forms white colonies, while Bad. tumefadens 
absorbs the Congo red and therefore produces red or reddish 
colonies. 

B. TEST OF THE PHYSIOLOGICAL EFFICIENCY OF 
PS. RADICICOLA AND OBSERVATION OF NOD- 
ULE FORMATION 

To observe nodule formation and nitrogen fixation, it 
is necessary to have seeds germinating free from bacteria. 

Apparatus. 500 c.c. of nitrogen-free agar; six sterile 
large est tubes with foot; sterile ordinary test tubes 
(the agar should be distributed in all the test tubes to a 
depth of about 5 cm.); sterile Petri dishes; clean slides; 
mercuric chloride, 1 : 500; flask of sterile distilled water; 
sterile pipette; seeds of some leguminous plant. (The> 
smaller seeds are better for this experiment.) 

Culture. Ps. radidcola (specific strain). 

Method. 1. Obtain sound, mature pods of some legume 
as pea, bean, vetch, etc. 

For testing the physiological efficiency of the pure 
culture of Ps. radicicola just previously isolated, use seeds 
from the same legume as that from which this particular 
culture was isolated. 

2. Soak the pods for five minutes in mercuric chloride 
1 : 500 and remove the excess of solution with sterile cheese- 
cloth. 

3. Tear open the pods with flamed forceps, place the 
seeds between folds of sterile cotton, and put the cotton in 
a dry, warm place until the seeds are dry. 

4. Select the best of these seeds and store them in dry 
sterile test tubes until they are to be used. 

6. Whether seeds are procured as described above, or 
otherwise, proceed as follows: 

(a) Soak the leguminous seed in 1 : 500 mercuric chloride 
solution for five minutes. 



260 



GENERAL MICROBIOLOGY 



(6) Wash off the disinfectant with sterile distilled water, 
handling the seeds with sterile forceps. 




FIG. 56. Alfalfa Plants from Inoculated and Uninoculated Seed. 
(Orig. Northrup.) 



6. Seeds parepared as above should then be treated 
according to the following procedure : 

Using the sterile forceps, transfer several of these ster- 



NITROGEN-FIXING ORGANISMS OF LEGUMES 261 

ilized seeds to each of the large test tubes. Or, place the 
seeds between layers of moist sterile filter paper in a Petri 
dish until they have germinated and then transfer the seeds 
to the large test tubes. When using the larger seeds use 
only three to six per tube, and six to ten only of the smaller 
seeds as alfalfa, clover, etc. 

7. Put the tubes containing the ungerminated seed in a 
warm place (30 to 35 C.) until the seeds germinate. 
Keep the germinated seed in a well-lighted room for a few 
days. 

8. Examine the tubes and reject all that are contaminated 
with molds or bacteria. 

9. After a few days, inoculate four of these tubes contain- 
ing growing leguminous plants with a pure culture of Ps. 
radicicola, by dropping upon the seeds and surface of the 
agar a heavy suspension of the bacteria in sterile water, by 
means of a sterile pipette. 

10. Keep two tubes uninoculated as controls. 

Note. To imitate infection under more natural conditions, just 
before the seeds are placed upon the agar, the agar may be melted, 
cooled to 40 to 45 C. and inoculated with a loopful of Ps. radicicola 
culture, mixing the organisms well through the agar with the needle. 
After the agar has solidified the sterile seeds may be then placed on the 
surface of the agar as before. 

11. Label the test tubes and place in some location 
where they will be sufficiently protected from the sun, 
heat, or cold, etc. This is very important. A piece of 
cheese-cloth thrown over the tubes will protect them from 
the sun. 

12. In about a month examine all test tubes and look 
for nodules. 

13. Record the presence, number, size, and shape of 
nodules, place of formation, etc. Show nodule-bearing 
seedlings to the instructor. 

14. Isolate Ps. radicicola from one of these nodules. 
This completes the cycle. 



262 GENERAL MICROBIOLOGY 

If all of these operations are successful Koch's postu- 
lates have been fulfilled. (See reference, W. J. MacNeal, 
Exercise 1, Animal Diseases and Immunity.) 

15. What do you conclude as to the physiological 
efficiency of the culture of Ps. radicicola used? 

16. What several factors might be responsible for a 
failure of infection? Explain. 

Why are inoculated seeds kept from direct sunlight? 
What may be the advantage of seed inoculation? How 




FIG. 57. Ps. radicicola, Polar flagella, x!500. Twenty-five day old 
culture from sweet pea. (B. Barlow). 

do different methods of seed inoculation compare as to 
advantages and disadvantages? 

17. Give your results in detail and draw conclusions. 
Point out any possible practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 273-283. 

LAFAR: Technical Mycology, Vol. I, pp. 259-271. 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 

111-113. 
LIPMAN and BROWN; Laboratory Guide in Soil Bacteriology, pp. 

56-59, 76-77, 



CHANGE OF INSOLUBLE PHOSPHATES 263 

SMITH, E. F.: Bacteria in Relation to Plant Diseases. Vol. II, 

pp. 96-138. 
Inoculation with Nodule-forming Bacteria, Cir. 5, Mich. Exp. 

Sta., 1915. 
FRED, E. B.: A Physiological Study of the Legume Bacteria, Ann. 

Kept. Va. Polytechnic Institute, 1911, 1912, pp. 174-201. 

EXERCISE 10. TO DEMONSTRATE THE CHANGE OF 
INSOLUBLE PHOSPHATES TO A SOLUBLE FORM 
THROUGH THE AGENCY OF MICROORGANISMS 

Apparatus. Dextrose; di- or tri-calcium phosphate; 
tubes of soil-extract agar containing 2% dextrose; four 
100 c.c. Erlenmeyer flasks. 

Culture. Soil. 

Method. 1. Place 0.1 gm. of di- or tri-calcium phos- 
phate, and 60 c.c. of a 2% solution of dextrose in tap water 
in each flask. Sterilize. 

2. To two flasks add 0.1 gm. soil each, leaving two for 
controls. 

3. Incubate at 37 C., and after the fermentation has 
continued for some days, make plates from the inoculated 
flasks as follows: 

4. Sterilize about 0.1 gram of di- or tri-calcium phos- 
phate in each of three test tubes. 

5. Place the contents of each tube in a sterile Petri dish; 
make loop-dilution plates from flasks in soil extract agar 
containing 2% dextrose, being careful to mix the phos- 
phate well with the agar in the dish by carefully tilting. 

6. Incubate at 37 C. 

7. Note frequently the appearance of the plates. The 
colonies of acid-producing bacteria developing at this 
temperature dissolve the phosphate and thus become sur- 
rounded by a clear area similar to that produced by lactic 
acid-producing bacteria on dextrose calcium carbonate agar. 

8. Examine the colonies in a hanging drop for mor- 
phology, motility, etc. 

9. How is the action noted in 7 made use of practically? 



264 GENERAL MICROBIOLOGY 

In what compounds is phosphorus found in soil? Are 
these available as plant food? What are the functions of 
bacteria in this connection? 

What relation has phosphorus to decay and nitrogen 
fixation? 

10. Give results and any conclusions in detail. Point 
out any possible practical applications. 

REFERENCES 

MARSHALL: Microbiology, pp. 287-288. 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 115- 

116. 
SACKETT, PATTEN and BROWN: The Solvent Action of Soil Bacteria 

Upon Insoluble Phosphates, etc., Spec. Bui. 43, Mich. Exp. Sta., 

1908. 

DAIRY MICROBIOLOGY 

EXERCISE 1. A COMPARATIVE STUDY~OF THE NUM- 
BER AND TYPES OF MICROORGANISMS AND 
OTHER CELLS IN MILK 

A. PLATING METHOD 

Apparatus. Sterile Petri dishes; sterile 1 c.c. and 10 
c.c. pipettes; tubes of sterile litmus lactose agar; 90 c.c. 
and 99 c.c. dilution flasks; tubes of sterile litmus milk. 

Culture. Fresh milk from any source desired. 

Method. 1. Shake the milk vigorously one hundred 
times to obtain a homogeneous sample, and plate in dilu- 
tions, 1-100, 1-10;000 and 1-1,000,000 in litmus lactose 
agar. 

2. Incubate the plates inverted at room temperature 
for five days. 

3. Count at the end of this time, estimate the average 
number of bacteria per cubic centimeter and approximate 
the numbers of the different types of colonies. 

4. Are acid colonies present? Chromogenic colonies? 
B. subtilis or B. mycoides? 



TYPES OF MICROORGANISMS IN MILK 



265 



What do the types signify? 

5. Isolate the different types in litmus milk and note 
their action. To which group of microorganisms does 
each type belong? (See Marshall's Microbiology, pp. 
306-313.) Suggest from what source each type may come. 

B. MICROSCOPIC METHOD 

Apparatus. Special capillary pipettes graduated to 
deliver exactly 0.01 c.c.; clean glass slides; 
three staining jars; xylol; alcohol, 95%; 
Loeffler's alkaline methylen blue; stage 
micrometer; eyepiece micrometer for 
counting objects in microscopic field; stiff 
straight needle. 

Culture: Fresh milk same as used 
in A. 

Method. 1. Draw with ink a figure 
the size and shape of an ordinary micro- 
scopic slide and on either side and equi- 
distant from the center draw a square 
whose area is one square centimeter, 
making the homologous sides of all figures 
parallel. 

2. Place a clean glass slide on the 
figure. 

3. With the capillary pipette, drop 
over the center of one of the smaller 

figures exactly 0.01 c.c. of milk directly FIG. 58. Capillary 
from the well-shaped sample and with a pl P ette usea in 
stiff straight needle spread this drop of 
milk exactly over the area (one square 
centimeter) covered by this figure. 

4. Make a duplicate smear, placing 
the drop of milk containing 0.01 c.c. on 
the slide over the remaining small square. 

5. These smears may be dried by the use of gentle 



the Microscopic 
Method for 
Counting B a c - 
teria in Milk. 
Note the straight 
narrow bore and 
the square tip. 



266 GENERAL MICROBIOLOGY 

heat (e.g., level wooden surface over a steam radiator). 
Do not allow the smears to become too hot, as this causes 
them to check, making satisfactory staining impossible. 

6. As soon as dry, place the slides in a staining jar 
containing xylol for a short time to remove the fat. 

7. Remove the slide from the xylol, absorb the surplus 
xylol about the edges with filter paper and allow it to 
dry. 

8. Fix the film to the slide by immersing in 95% alcohol. 

9. Stain immediately by flooding the smears with Loef- 
fler's methylen blue for two or three minutes. 

10. Decolorize to a light blue in 95% alcohol. 

11. In counting, use the oil immersion objective. Place 
the draw tube at some convenient mark so that an even 
number of fields of the microscope covers one square centi- 
meter. 

To do this, determine the radius of the -microscopic 
field in millimeters with the stage micrometer and calculate 
its area by the formula irR 2 . (7r = 3.1416.) 

Then if z = the area of the smear in square millimeters 
and if 0.01 c.c. of milk is used, 



y the factor necessary to transform the number of bacteria 
found in one field of the microscope into terms of bacteria 
per cubic centimeter. 

TO simplify the calculation, place the draw tube so that 
y consists of as many ciphers as possible. Convenient 
factors will be obtained if the length of R be 0.101 mm. 
or 0.08 mm. 

Let z thousand equal the number of fields of the micro- 
scope in one square centimeter. Since 0.01 c.c. of milk was 
taken then each bacterium seen in one field represents lOOXz 
thousand or z hundred thousand bacteria per cubic centi- 
meter. 



TYPES OF MICROORGANISMS IN MILK 



267 



12. For careful quantitative work it is necessary to count 
one hundred fields for each sample, i.e., fifty fields per square. 
If n = the number of fields counted and m = the total num- 
ber of bacteria found, the number of bacteria per cubic 
centimeter is calculated by the following formula: 



z hundred thousand 



centimeter of milk. 



= number of bacteria per cubic 



In comparatively fresh milk where the bacteria are few, 
count the whole microscopic field. 

An eye-piece micrometer having a large square ruled 
into smaller squares is recommended where large numbers 
of bacteria are present. The area of the large square is 
different from that of the whole microscope field and con- 
sequently the factor necessary for computation is different. 
This factor can be determined by modification of the formula 
given in 11. 

13. Draw a typical smear from different samples of milk. 
Indicate the kinds of cells and the number found, also the 
quality of the milk. 



Quality of milk. 


Bacteria 
per field. 


No. per c.c. 


Tissue 
cells. 


Cell count. 


Good 
Fair 


None 
5 


2,000,000 


2 

1 


800,000 per c.c. 
400,000 per c.o, 


Souring normally 
Poor 


200 
250 


80,000,000 
100,000,000 


1 

7 


400,000 per c.c. 
2,800,000 per c.c. 













14. What types of bacteria are found microscopically? 
How do these compare with those found on plates as to 
types and numbers? 

What are the advantages and disadvantages of the plat- 
ing method? Of the microscopic method? For what type 
of work is each best adapted? What other microscopic 



268 GENERAL MICROBIOLOGY 

methods have been employed as a rapid means of setting 
bacteriological milk standards? 

Of what value are bacteriological milk standards and 
analyses? 

15. Give your results in detail and point out any prac- 
tical applications. 

REFERENCES 

BREW, JAMES D.: A Comparison of the Microscopical Method and 

Plate Method of Counting Bacteria in Milk.' Bui. 373, N. Y. 

Agr. Expt. Sta., Geneva, Feb., 1914. 
MARSHALL: Microbiology, pp. 293-296, 331-333. 
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 

62-65. 
SAVAGE: The Bacteriological Examination of Food and Water (1914), 

p. 85-89, 92, 95-99. 

WARD: Pure Milk and the Public Health (1909), pp. 126-128. 
ERNST: Milk Hygiene; translated by Mohler and Eichhorn (1914), 

pp. 24-31. 
FROST, W. D.: A Microscopic Test for Pasteurized Milk. Jour. Am. 

Med. Assn. Vol. LXIV, No. 10, p. 821 (1915). 

EXERCISE 2. THE DETERMINATION OF THE BACTE- 
RIAL CONTENT OF MILK IN THE UDDER 

Apparatus. Several large sterile test tubes; four 
sterile Petri dishes; 99 c.c. dilution flasks; sterile 1 c.c. 
pipettes; tubes of sterile litmus milk; four tubes litmus 
lactose agar. 

Method. 1. Wash off the end of the teat very care- 
fully with a solution of mercuric chloride, 1 : 1000; allow 
it to dry till the surplus solution has disappeared and 
only sufficient moisture remains to make the cells and any 
dirt adherent. 

2. Secure a sterile cotton-plugged test tube, remove 
the cotton pliig with the little finger and, while holding the 
mouth of the tube as near the end of the sterilized teat as 
possible and inclining the tube toward the horizontal posi- 
tion as far as feasible, milk the tube half-full. 



BACTERIAL CONTENT OF MILK IN THE UDDER 269 

By this method obtain from the same teat: 

a. One sample of the fore milk, 

b. One sample of the middle milk; 

c. One sample of the strippings; 

Note. For investigational purposes it may be better to employ 
a sterile milking tube adjusted to a sterile flask. This may easily be 
prepared. This method, however, is not recommended for student 
work. 

3. Secure one sample from the pail, gathered from the 
same cow at the same milking. 

4. Plate each sample on litmus lactose agar, using the 
following dilutions and amounts: 

1 c.c. of 2a diluted 1 : 100 for the plate. 
1 c.c. of 26 diluted 1 : 10 for the plate. 
1 c.c. of 2c diluted 1 : 10 for the plate. 
1 c.c. of 3 diluted 1 : 100 for the plate. 

5. Place the plates at a temperature of 21 C. for seven 
days. 

6. Count the number of colonies in each plate and 
record the average number in 1 c.c. of milk in each case. 
Explain any variation in counts. 

7. Compare the colonies of plates 2a, 26, and 2c with 
3. What types predominate? 

8. Estimate, so far as possible, the number of colonies 
of each type, and compare the relative numbers of each 
species in the different plates. 

9. Isolate the species in milk tubes to study their action 
upon milk. To what group of microorganisms found in 
milk does each of the species isolated belong? How do you 
account for the presence of these particular species? 

10. In which sample would you expect to find the greatest 
number of microorganisms? Why? 

Why should all samples be taken from the same quarter 
of the udder? 

How do bacteria ordinarily gain entrance to the udder? 



270 GENERAL MICROBIOLOGY 

By what means may bacteria cause infection of the 
udder? _ 

What is the significance of bacteria in the udder? As 
to numbers and types? 

11. Give your results in full and point out any conclu- 
sions and any practical applications possible. 

REFERENCES 

MARSHALL: Microbiology, pp. 297-299, 306-313. 
WARD: Pure Milk and the Public Health (1909), pp. 1-7, 69, 86-88. 
ROSENAU: The Milk Question (1912), pp. 71-74. 
RUSSELL and HASTINGS: Outlines of Dairy Bacteriology (1910), 
pp. 30-35, 75. 

EXERCISE 3. TO ILLUSTRATE EXTRANEOUS CON- 
TAMINATION 

Apparatus. Forceps; scalpel or spatula; 1 seventeen 
sterile Petri dishes; three 10 c.c. dilution flasks (for A and B) ; 
fifteen sterile 1 c.c. pipettes; fifteen tubes of sterile litmus 
lactose agar; soap; ordinary towel; two 1 qt. sterile basins; 
one milk pail; two 1 liter flasks each containing 500 c.c. 
sterile salt solution; one deep Petri dish; sterile glass rod. 

A. SCALES FROM COW'S SKIN (DEAD EPITHELIAL 

CELLS) 

Method. 1. With a sterile spatula scrape from the 
skin of a cow's udder some scales, such as usually fall into 
the milk, into a sterile Petri dish. 

2. Transfer by means of the sterile spatula some of these 
to a 75 c.c. Erlenmeyer flask containing 10 c.c. of sterile 
physiological salt solution. 

3. Shake thoroughly, then plate 1 c.c. of this suspension 
in litmus lactose agar, 



TO ILLUSTRATE EXTRANEOUS CONTAMINATION 271 



B. HAIRS FROM COW 

Method. 1. Select two hairs from the back of the cow 
where the usual or natural clean- 
liness exists and two from the 
hip stained with manure. By 
means of sterile forceps place 
them in sterile Petri dishes. 

2. One of each kind, that is, 
one from the back and one from 
the hip, place in 10 c.c. of a 
sterile salt solution, as under A. 

3. Shake thoroughly, then 
plate 1 c.c. of this suspension in 
litmus lactose agar. 

4. Embed each of the two 
remaining hairs in the litmus 
lactose agar after pouring the 

liquefied agar into a sterile Petri dish. These hairs should 
be placed in the agar just before solidifying by means of 
sterile forceps. 




FIG. 59. Bad. bulgaricum 
colony, x75. (Orig. Nor- 
thrup.) 



C. OTHER SUBSTANCES 

Method. Study straw, hay, dung, etc., in a similar 
manner. Instead of using dilution flasks containing 10 c.c. 
it will be more desirable to use 100 c.c. 

In the case of dung, a particle smaller than the head 
of a pin should be added to 100 c.c. for suspension, and 
in case of straw and hay very small segments unless they 
are very clean. 

Note. A sufficient number of such substances should be studied 
to familiarize the student with the amount of contamination which 
may take place from these sources. 



272 GENERAL MICROBIOLOGY 



D. HANDS 

Method. 1. Wash the hands in the ordinary manner, 
rinse them thoroughly, then wipe with an ordinary towel. 

2. After this has been done, put 500 c.c. of sterile water 
in a sterile dish, and rub the hands thoroughly with this 
water. 

3. Plate 1 c.c. of this water in litmus lactose agar. 

4. Again rub the hands, before they have been washed 
and after working for some time, in 500 c.c. of sterile water 
placed in a sterile dish. 

6. Plate 1 c.c. of this water in litmus lactose agar. 
6. Compare the numbers (using 1 c.c. as the unit) and 
kinds of bacteria in the two plates. 

E. PAILS 

Method. 1. Add to a milk pail washed in the usual 
manner, 500. c.c. of sterile salt solution, and plate in litmus 
lactose agar, 1 c.c. of this suspension after it has been 
moved over the inner surface of the pail. 

2. Repeat by using a milk pail heated in steam for ten 
minutes, or cleansed with boiling water. 

3. This same process may be repeated using milk bottles, 
milk cans, etc. 

F. AIR 

Method. 1. Determine qualitatively the microflora 
of the air of the stable before feeding or bedding or before 
any disturbing, and after feeding or bedding or after any 
disturbing, by the following methods : 

2. Pour the liquefied litmus lactose agar into several 
Petri dishes, and expose the poured plates for different 
lengths of time. 

3. Expose 10 c.c. of sterile 0.6% salt solution in a deep 
Petri dish 5 c.c. deep and 9 c.c. in diameter. Try to disin- 



AMOUNT AND KIND OF DIRT IN MILK 273 

tegrate the dust particles by stirring with a sterile glass 
rod and agitating. Plate 1 c.c. in litmus lactose agar. 

4. Quantitative studies of barn air under various con- 
ditions may be made according to Exercise 1, Air Micro- 
biology. 

5. What advantage has litmus lactose agar over ordi- 
nary agar in this exercise? 

What types of organisms are met most frequently under 
A, B, C, D, E and F? How may this occurrence be ac- 
counted for? 

Which sources furnish the greatest number of organisms? 
From which sources are the greatest number of micro- 
organisms most likely to enter milk? The most undesirable 
types? Explain in each case. 

What sources of milk contamination have not been dis- 
cussed under this exercise? Of what importance is each? 
What is the simplest method in each case of preventing 
contamination from the various sources mentioned above? 

6. Give your results in full and draw any conclusions 
and make any practical applications possible. 

REFERENCES 

SAVAGE: The Bacteriological Examination of Food and Water (1914), 

pp. 90-91. 
ERNST: Milk Hygiene, transl. by Mohler and Eichhorn (1913), pp. 

67-102, 125-131, 234-235. 

JENSEN: Milk Hygiene, transl. by Pearson (1907), pp. 70-82, 86-127. 
MARSHALL: Microbiology, pp. 300-306. 

EXERCISE 4. TO INVESTIGATE THE AMOUNT AND 
KIND OF DIRT IN MILK AND ITS RELATION TO THE 
MICROBIAL CONTENT OF THE MILK 

Apparatus. Six sterile 1 c.c. pipettes; 99 c.c. dilution 
flasks; six tubes sterile litmus lactose agar; six sterile 
Petri dishes; sedimentation tubes, 10 c.c. capacity; balance; 
centrifuge; clean slides; methylen blue, aqueous-alcoholic; 
physiological salt solution; pneumatic or other type of 



274 GENERAL MICROBIOLOGY 

sediment tester; cotton disks for sediment tester; clean 
empty milk bottle; one pint bottled milk from each of 
several miscellaneous sources. 

Note. The same sample of milk must be used for A, B and C. 
Proceed with tests in the order given. 

A. DETERMINATION OF MICROBIAL CONTENT 
OF MILK 

Method. 1. Shake the sample in the bottle vigorously. 

2. Plate the dilutions 1 : 100,1 : 10,000 and 1 : 1,000,000 
in litmus lactose agar. 

3. Place the plates at 25 C., and proceed with the 
microscopic sediment test. 

4. Count the plates at the end of five days and estimate 
the number of bacteria per cubic centimeter and the relative 
proportion of acid to other types of colonies. 

5. Determine the morphology of the organisms making 
up the colonies of each type and compare with the findings 
in the microscopical sediment test. 

6. Are all organisms present microscopically? Explain 
your results and draw conclusions. 

B. MICROSCOPIC SEDIMENT TEST 

Method. 1. Mix the milk well and warm about 30 c.c. 
to 60 to 70 C. 

2. Place 10 c.c. of this well-mixed, warmed milk into 
each of two sedimentation tubes. 

3. Place one tube on each of the scale pans and balance 
by adding more milk to the lighter tube. The tubes must 
be equal in weight or they will throw the centrifuge " off 
center." 

4. Centrifuge in a machine designed for this purpose for 
five minutes, till a more or less considerable compact sedi- 
ment separates out. 

6. Pour or pipette off the milk above the sediment. 



AMOUNT AND KIND OF DIRT IN MILK 



275 



6. Fill the tubes with physiological salt solution and mix 
the sediment well throughout the dilution fluid with a plat- 
inum needle. 

7. Balance the tubes and centrifuge again. 

8. Pour or pipette off the physiological salt solution. 

9. With a small platinum loop, obtain a small amount 
of the sediment and make a smear on a clean slide. 

10. Stain with aqueous-alcoholic methylen blue. 




FIG. 60. Wizard Sediment Tester for Milk. 

11. Determine the proportions of bacteria and leuco- 
cytes in ten fields. Also note the presence of bacteria in 
clumps and foreign matter. 

The presence of many leucocytes and streptococci asso- 
ciated together is generally indicative of an inflamed con- 
dition of the udder, as in mastitis (garget). On the other 
hand, sometimes the milk from normal udders may show a 
considerable quantity of leucocytes in the sediment. 



276 GENERAL MICROBIOLOGY 



C. MACROSCOPIC SEDIMENT TEST 

Method. 1. Put a cotton disk in place in the pneu- 
matic sediment tester, heat the sediment tester and clean 
empty milk bottle in steam thirty minutes, and allow to 
cool. 

2. Attach the sediment tester to the top of the milk 
bottle containing the sample of milk, using " aseptic " 
precautions, and invert the whole apparatus over the mouth 
of the sterile empty milk bottle. 

3. Pump the contents of the upper bottle into the lower 
bottle by means of the rubber bulb. The milk is forced 
through the cotton disk and leaves its larger particles of 
insoluble dirt on the cotton. 

4. Note the quality of the milk tested by this method. 
Is there any interrelationship between microscopic sedi- 
ment test, and the macroscopic sediment test? , , 

5. What does the presence of visible dirt on the cotton 
indicate? Is this sediment te^t an argument for straining 
milk before it goes to the consumer? Is it an argument 
for running milk through a milk clarifier before putting it 
on the market? 

6. Immediately after straining, plate the milk in lit- 
mus lactose agar, using dilutions 1 : 100, 1 : 10,000 and 
1 : 1,000,000 as before. 

7. Incubate, the plates for five days at 25 C. and count, 
estimating total average number and proportions of types 
as in A. 

8. Compare the counts with those of A, also the propor- 
tions of the various types. 

Note. This method was formerly used for obtaining an estimate 
microscopically of the numbers of bacteria in milk. It presents 
difficulties, however, which lead to many technical errors and there- 
fore it cannot be relied upon to give uniform results. The method is 
valuable, however, for determining something of the sanitary quality 
of the milk, 



AMOUNT AND KIND OF DIRT IN MILK 277 




FIG. 61. Cotton Disks Prepared by the Use of the Wizard Sediment 
Tester, (Circ. 41, Wise. Expt. Sta.) 



278 GENERAL MICROBIOLOGY 

Did straining have any effect on the numbers of organisms 
present in the milk? What effect may it have? Is this 
beneficial to the milk as a commercial product? 

9. In what way may the microscopic sediment test 
explain the results obtained by plating milk before and after 
straining? 

10. Make, stain and examine smears from the upper 
surface of the cotton disk. What is the nature microscopic- 
ally of the material retained by the cotton? How does this 
smear compare qualitatively with that from the centri- 
fuged sample? 

11. What is the nature of the dirt ordinarily found in 
milk? How may its presence be eliminated? 

12. Give all results in full and draw any conclusions 
permissible, Point out any practical applications. 

REFERENCES 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 63-65. 
JENSEN: Milk Hygiene, transl. by Pearson (1907), pp. 126-127. 
MARSHALL: Microbiology, pp. 326-327. 
ROSENAU: The Milk Question (1912), pp. 55-88. 
ERNST: Milk Hygiene, transl. by Mohler and Eichhorn (1914), p. 
182. 



EXERCISE 5. TO DETERMINE THE INFLUENCE OF 
TEMPERATURE UPON THE KEEPING QUALITY OF 
MILK; PITRE MILK COMPARED WITH MARKET 
MILK 

One of the most important considerations in the pro- 
duction of milk, either for factory use or for town or city 
supply, is the temperature at which the milk is maintained. 
The beneficial effects of scrupulous cleanliness in the pro- 
duction of milk will be largely counteracted unless the milk 
is cooled immediately after drawn and maintained at a 
temperature too low for development of the bacteria 
present. 



PUKE MILK COMPARED WITH MARKET MILK 279 

Apparatus. Three sterile 1 liter Erlenmeyer flasks; 
one sterile 2 liter Erlenmeyer flask; twenty-four sterile 
Petri dishes; sterile 10 c.c. pipettes; sterile 1 c.c. pipette 
graduated to 0.1 c.c.; twenty-four tubes of litmus lactose 
agar; 90 c.c. and 99 c.c. dilution flasks; ice and salt for pre- 
paring freezing mixture; fresh milk and bottled milk. 

Method. 1. In a sterile 2 liter Erlenmeyer flask place 
about 1 J liters of milk from a can of milk immediately after 
it has been filled by the milkers. 

Note. This exercise is to be repeated, for purposes of comparison, 
using three pint bottles of milk all obtained at one time from the 
same milkman. In this case the first plating is to be made from 
each separate bottle. 

2. Record the temperature. Plate from the sample 
immediately in litmus lactose agar, making dilutions of 
1 : 100 and 1 : 500. Determine the acidity of the sample, 
using a sterile 5 c.c. pipette to obtain the sample. 

Note. Portions for acidity determination and plating should be 
removed with sterile pipettes in all instances. 

3. Transfer the sample " aseptically " into the three 
1 liter flasks, placing an equal portion as nearly as possible 
in each flask. Label the flasks A, B, C. 

4. Cool flask A in a freezing mixture to 10 C., and set 
away in refrigerator to maintain the low temperature. 

5. Cool flask B in a freezing mixture to 10 C., then place 
it at a constant temperature of 21 C. 

6. Place flask C at a constant temperature of 21 C. 

7. At the end of twenty-four hours determine and record 
the acidity of each of the three portions of the original 
sample. 

8. Plate in litmus lactose agar, using the following 
dilutions : 

Flask A, 1 : 100 and 1 : 1,000. 

Flasks B and C, 1 ; 10,000 and 1 ; 1,000,000. 



280 GENERAL MICROBIOLOGY 

9. At the end of another twenty-four hours repeat the 
titrations and platings with all flasks, using the following 
dilutions : 

Flask A, 1 : 10,000 and 1 : 1,000,000. 

Flasks B and C, 1 : 1,000,000 and 1 : 100,000,000. 

10. At the end of five days determine and record the 
acidity of the milk in all flasks. Plate from flask A only, 
using dilutions of 1 : 1,000,000 and 1 : 100,000,000. 

11. All plates should be held at 21 C. for a period of 
five days before counting. 

12. Compare the relative kinds and numbers of colonies 
in plates from the three flasks. Note also the time of curd- 
ing and the nature of the curd formed in each case. 

13. Compile the results of the investigation in tabulated 
form. Plot bacterial count and acidity curves. 

14. From the results obtained, what conclusions would 
you draw as to the influence of cooling upon the keeping 
quality of milk? 

How does the age and original quality of the milk effect 
its keeping qualities when subjected to different temperature 
conditions? 

How dpes cooling milk and keeping it cool compare with 
merely cooling and then allowing the milk to acquire the 
temperature of the room? What is the explanation of the 
action occurring? 

What is the purpose of cooling the milk as soon as it 
comes from the cow? What different methods are used? 
What are some of the disadvantages of the different methods 
used for cooling? 

What bacterial action takes place in the refrigerator 
milk? Is the germicidal action of milk sufficiently important 
to recommend a change in the general practice of cooling 
milk? 

15. Give your results in detail and point out any 
practical applications or conclusions. 



PASTEURIZATION OF MILK OR CREAM 281 

REFERENCES 

WARD: Pure Milk and the Public Health (1909), pp. 15-16, 24-25, 

37, 121. 

RUSSELL and HASTINGS: Outlines of Dairy Bacteriology, pp. 54-56. 
ERNST: Milk Hygiene, transl. by Mohler and Eichhorn (1914), pp. 

148-149, 156. 
MARSHALL: Microbiology, pp. 318-319. 



EXERCISE 6.x A STUDY OF THE PASTEURIZATION OF 
MILK OR CREAM BY LABORATORY METHODS 

Apparatus. Water bath; test-tube rack of metal to 
fit water bath; sterile, large tubes selected for uniformity 
in diameter (2 cm.); sterile Petri dishes; sterile 1 c.c. pi- 
pettes, graduated to 0.1 c.c.; sterile litmus lactose agar 
tubes. 

Method. 1. Secure milk or cream, about 125 c.c. to 
be used for tubing and pasteurizing. 

Note. If time permits, it is desirable to test pasteurization upon: 

a. Fresh milk or cream. 

b. Milk or cream which has stood for twenty-four hours but is 
still sweet. 

c. Milk or cream which has reached an acidity of about 22. 

d. Milk or cream from different sources, supposedly having dif- 
ferent bacterial contents. 

2. Tube the sample or samples of milk or cream, pour- 
ing 10 c.c. into each tube, filling fifteen tubes for each sample. 

Note. Only one sample should be pasteurized at a time. 

3. Prepare one tube from each sample of milk or cream 
for the introduction of the thermometer. By so doing, 
the conditions practically identical, the temperature will 
be easily read and controlled. 

4. After the tubes are prepared mark tubes in duplicate 
as follows: 50, 60, 70, 80, 90 and 100, leaving two 
unmarked as controls. 

5. Place them in the rack so that the marks on the tubes 



282 GENERAL MICROBIOLOGY 

may be easily recognized, and insert the rack in the water- 
bath. 

6. Pour water into the water-bath until the height of 
the water corresponds to the height of the milk in the 
tubes. 

7. Put aside two tubes of milk or cream from each 
sample, one to be employed for comparative check-obser- 
vation, and the other for check-plating against those which 
will be subjected to pasteurization. 

8. Apply heat to the water-bath. 

9. At 50, 60, 70, 80, 90 and 100 C., remove two 
tubes of each sample of milk or cream undergoing pasteuri- 
zation and place in cold water. 

10. Employ one of the tubes thus removed for plating 
and the other place at a temperature of 25 to 28 C. along 
with the previous check-observation tube (7). 

11. Make two plates in litmus lactose agar from the tube 
held for check-plating (7) and from one of the two tubes 
removed at each of the temperatures designated above. 
The remaining tube is to be left undisturbed and placed 
at 25 C., to observe macroscopical changes. 

Dilutions for plating: 

Fresh milk, unpasteurized, 1 : 10 and 1 : 100. 

Milk twenty-four hours old, but sweet, unpasteurized, 
1 : 10,000 and I : 1,000,000. 

Milk with an acidity of 22, unpasteurized, 1 : 100,000 
and 1 : 10,000,000. 

Milk pasteurized at 50 C. (fresh) 1 : 10 and 1 : 100. 

Milk pasteurized at 50 C. (old) 1 : 10,000 and 
1 : 1,000,000. 

Milk pasteurized at 60 C., 1 : 10 and 1 : 100. 

Milk pasteurized above 60 C., 1 : 10. 

12. Keep the plates at 25 C. for seven days, counting 
colonies at the end of this time. 

13. Determine the character of the microorganisms 
left after pasteurization with those before pasteurization 



PASTEURIZATION OF MILK OK CREAM 283 

as to the relative number of each kind, to the fermentation 
of milk or cream, to spore formation, and to resistance. 
Which microorganisms have succumbed to pasteurization 
at different temperatures and which were able to withstand 
it? 

14. Record the results obtained from the study of plates 
and cultures made from colonies. 

15. Record your observations from day to day of macro- 
scopical changes in the pasteurized and unpasteurized con- 
trol tubes. Does pasteurization destroy organisms that 
are favorable, or detrimental to the milk? What influence 
does pasteurization have upon the keeping quality of 
milk? . 

16. What influence do the following factors have upon 
the efficiency of pasteurization: the age of milk? acidity? 
degree of temperature to which milk is subjected? dura- 
tion of pasteurization temperatures? presence or absence 
of air? pressure, whether atmospheric or greater? viscos- 
ity or other changes in milk or cream? 

What changes are accomplished by pasteurization? 
Why is milk pasteurized? Is this end always accomplished 
in commercial plants? 

What different methods are used commercially for the 
pasteurization of milk? What are the advantages and 
disadvantages of each method? Why? 

At what stage in the process of production should 
milk be pasteurized to accomplish the desired results? 
Must the after-treatment of pasteurized milk be any differ- 
ent from that of unpasteurized milk? 

Do you think that milk should be pasteurized before 
it reaches the consumer? 

Does pasteurization affect the digestibility of milk? 
What are the limitations of pasteurization as applied to 
milk? 

17. Give your results in full and any conclusions that 
may be drawn. 



284 GENERAL MICROBIOLOGY 

REFERENCES 

MARSHALL: Microbiology (1911), pp. 319-321. 

ROSENAU: The Milk Question (1912), pp. 16, 37, 76, 105, 112, 120, 

128, 132, 138, 161, 185-230, 294. 
WARD: Pure Milk and the Public Health (1909), pp. 71, 73, 74, 114- 

125, 



EXERCISE 7. DETERMINATION OF THE NUMBER AND 
TYPES OF BACTERIA IN BUTTER 

Apparatus. Three tubes litmus lactose agar; litmus 
milk tubes; fresh butter; sterile dilution flasks; three 
sterile Petri dishes; sterile 1 c.c. volumetric (bulb) pipettes. 

Method. 1. Melt a small quantity of butter in a 
test tube at the lowest possible temperature (not higher than 
40 to 45 C.). Mix well. 

2. Using a warm pipette, transfer 1 c.c. of the well- 
mixed melted butter to 99 c.c. of sterile (warm) salt solu- 
tion. Free the pipette from fat by filling it with the dilu- 
tion water several times. Use warm (50 C.) pipettes 
and dilution flasks throughout so that the butter will not 
stick to the pipettes and may be readily emulsified. 

3. Plate in litmus lactose agar, using dilutions 1 : 1,000, 
1 : 100,000 and 1 : 1,000,000. 

Note. These dilutions may have to be changed. Look up the 
average number of bacteria in the type of butter you are using and 
make dilutions accordingly. 

4. Incubate the plates at 25 C. 

5. Weigh 1 c.c. of well-mixed melted butter and record 
the weight in grams. 

6. Examine the plates after three to five days for acid 
and other types of colonies. 

7. Count and record the number of bacteria per cubic 
centimeter, also the types. Note the action of each type 
on litmus milk. 

8. Estimate the number of bacteria per gram. 



NUMBER AND TYPES OF BACTERIA IN BUTTER 285 

9. What is the melting-point of butter? Are bacteria 
ordinarily killed at this temperature? 

What kinds of microorganisms are found in fresh butter 
from ripened cream? In old butter? In fresh oleomar- 
garine? In renovated butter? In canned butter? 

Do bacteria increase or decrease in butter kept in stor- 
age? What other methods of making a bacteriological 
examination of butter may be employed? 

Are microorganisms in any way responsible for the 
flavors of butter? Explain. 

What pathogenic organisms may gain entrance to 
butter? 

What is the avenue of entrance? How long can bacteria 
exist in butter? How do bacterial numbers and types 
compare with those of fresh milk? of ripened cream? 

10. Give your data and conclusions in full and point out 
any practical applications, 

REFERENCES 

RUSSELL and HASTINGS: Practical Dairy Bacteriology, pp. 95, 97. 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 
81-82. 

MARSHALL: Microbiology, pp. 335-345. 

SAYER, RAHN and FARRAND: Keeping Qualities of Butter, I. Gen- 
eral Studies, Tech. Bui. I (1908), Mich. Agrl. Expt. Sta. 

RAHN, BROWN and SMITH: Keeping Qualities of Butter, II and III. 
Tech. Bui. 2 (1909), Mich. Expt. Sta. 

WELLS, LEVI: Renovated Butter: Its Origin and History. Year- 
book of Dept. of Agr. for 1905, pp. 393-398. 

ROGERS, L. A.: Studies Upon the Keeping Quality of Butter, I. 
Canned Butter. Bui. 57, B. A. I., U. S. Dept. of Agr. (1904), 
pp. 6-8, 22-23. 



286 GENERAL MICROBIOLOGY 



EXERCISE 8. TO DETERMINE THE NUMBER AND 
TYPES OF MICROORGANISMS IN CHEESE 

Apparatus. Cheddar cheese; cheese trier (sterile) if 
an uncut cheese is to be sampled; mortar, with pestle; 
two knives, sterile; quartz sand; sterile filter papers about 
6 and 8 cm. square; one dilution flask containing 95 c.c. 
of 0.85% salt solution; dilution flasks, containing 90 and 
99 c.c. sterile 0.85% salt solution; sterile 1 c.c. and 10 c.c. 
pipettes; three tubes litmus lactose agar; three sterile 
Petri dishes; sterile litmus milk tubes. 

Method. 1. Sterilize in the hot-air oven, 10 gms. of 
sand in a mortar with a pestle. 

2. Using a red-hot knife blade, sear a portion of the sur- 
face of the cheese. 

3. To weigh the cheese " aseptically," place the smaller 
sterile filter paper upon the larger on the balance pan. 

h 4, With a sterile knife remove the inner portion of the 
seared surface and obtain and weigh out 5 gms. of cheese, 
using aseptic precautions. 

5. Transfer the cheese to the sterile mortar and grind 
up well. 

8. Transfer the cheese and sand mixture with sterile 
knife or spatula to the 95 c.c. dilution flask. Shake well to 
free the sand from the cheese. 

Directions for making dilutions. In transferring with 
a pipette a portion of the first suspension to other dilution 
flasks the sample should be taken immediately after shaking 
before the sand has settled. Settling may be avoided by 
holding the pipette in a horizontal position until ready to 
deliver the contents. The grinding material should be fine 
enough to avoid clogging the pipette. 

7. Make and plate the following dilutions in litmus 
lactose agar: 1 : 100,000; 1 : 10,000,000 and 1 : 1,000,000,- 
000, if the cheese has been made recently. 

If the cheese is not perfectly fresh, use dilutions 1 : 100,- 



TYPES OF MICROORGANISMS IN CHEESE 287 

000; 1:1,000,000 and 1:10,000,000. Lower dilutions 
may be necessary if the cheese has been stored for some 
time. 

8. Incubate the plates at 25 C. for three to five days. 

9. Count and estimate the number of microorganisms 
per gram of cheese. What types predominate? Why? 

10. Transfer the different types to litmus milk and note 
the action after several days. Which of these types, as 
determined from the action on litmus milk, may have a 
prominent part to play in the ripening of the cheese? Why? 

12. Do microorganisms play any part in the formation 
of the flavor of cheddar cheese? Other cheeses? 

How does the cheese analyzed compare with the butter 
analyzed as to numbers and types of microorganisms? 

What qualitative tests are made for milk used for cheese 
making? What is the principle and application of these 
tests? 

What cheese " abnormalities " may be caused by 
microorganisms? During what stages' in the process of 
making cheese may these occur? 

What pathogenic organisms have been found in cheese? 
What is known of their longevity in this medium? 

13. State your results and conclusions in full and point 
out any practical applications. 

REFERENCES 

LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 83-88. 
RUSSELL and HASTINGS: Experimental Dairy Bacteriology, pp. 

103-109. 

MARSHALL: Microbiology, pp. 346-357. 
CONN: Practical Dairy Bacteriology (1907), pp. 223-259. 
SAVAGE: The Bacteriological Examination of Food and Water (1914), 

pp. 118-119. 
DECKER: Cheese Making (1905), pp. 48-51, 63, 66, 69, 75, 88, 108- 

110. 
VANSLYKE and PUBLOW: The Science and Practice of Cheese-making 

(1912), pp, 115-135, 285-312, 371-378, 



288 GENERAL MICROBIOLOGY 



EXERCISE 9. A COMPARISON OF THE BACTERIAL 
CONTENT OF SWEETENED AND UNSWEETENED 
CONDENSED MILKS 

Apparatus. Six sterile Petri dishes; six tubes of litmus 
lactose agar; 99 c.c. dilution flasks; two 95 c.c. dilution 
flasks; tubes of sterile litmus milk; two sterile 5 c.c. pipettes 
with large aperture for delivery; can-opener. 

Culture. Unopened can of sweetened condensed milk; 
unopened can of unsweetened condensed milk (contents 
guaranteed to be sterile) . 

Method. 1. Sterilize the can-opener in the flame. 

2. Thoroughly cleanse the outside of the unopened cans 
of condensed milk and then submerge in boiling water for 
five or ten minutes. 

3. Remove the cans from the water, being careful in 
handling them not to contaminate the upper surface of the 
cans. 

4. With the sterile can-opener make an opening in the 
can only large enough to admit the introduction of a 5 c.c. 
pipette. 

Note. Only one can is to be opened at a time to avoid contam- 
ination. ' 

5. With a sterile pipette obtain a 5 c.c. sample from the 
can just opened and transfer to a 95 c.c. dilution flask. 

Note. As the condensed milk is very viscous and adheres to tho 
sides of the pipette, after delivering the 5 c.c. into the dilution flask 
blow out the remainder into the sink or other suitable place, then 
replace in the dilution flask and wash out the adhering fluid by draw- 
ing the diluting fluid up into the pipette several times. The use of a 
5 c.c. volumetric pipette having a large aperture for delivery would 
lessen the possibilities of contamination. 

6. This resulting dilution is a 1 : 20 dilution of the con- 
densed milk, or a 1 : 40 dilution of the original milk (if 
the directions on the can give a dilution of 1 : 1 for pro- 
ducing a milk of original composition). 



TYPES OF MICROORGANISMS IN ICE CREAM 289 

7. Plate the following dilutions of the condensed milk 
in litmus lactose agar: 1 : 20, 1 : 2000 and 1 : 20,000. 
Place plates at 25 C. 

8. Examine and count at the end of five days. 

9. Record, the numbers and types of organisms develop- 
ing on the plates. Are any acid colonies present? Deter- 
mine the morphology of the acid colonies. 

10. Transfer each type of colony to a tube of sterile 
litmus milk and observe action from day to day. Are the 
types which are present desirable? Is Bad. lactis acidi 
present? Any organisms of the B. coli type? Are patho- 
genic bacteria apt to be present? 

11. To what factors are due the keeping qualities of 
each type of condensed milk? 

What care should be taken of opened cans of milk of 
either type? Of the milk after it has been diluted accord- 
ing to directions? 

In what other forms is concentrated milk sold? What 
factors are responsible for the keeping quality of each of 
these latter types? 

12. Give your results and conclusions in detail, 

REFERENCES 

MARSHALL: Microbiology (1911), 363-366. 

SAVAGE: The Bacteriological Examination of Food and Water (1914), 

pp. 111-113. 
SADTLER: Industrial Organic Chemistry (1912), pp. 281, 288. 

EXERCISE 10. TO DETERMINE THE NUMBER AND 
TYPES OF MICROORGANISMS IN ICE CREAM 

Apparatus. Litmus lactose agar shake; three tubes 
sterile litmus lactose agar; three sterile Petri dishes; ster- 
ile 1 c.c. pipettes; sterile dilution flasks; sterile wide- 
mouthed glass-stoppered bottle; sterile butter trier; sterile 
knife. 

Culture. From ice cream. 



290 GENERAL MICROBIOLOGY 

Method. 1. Remove the (frozen) ice cream sample 
from the container by means of the sterile butter trier. 

2. With the sterile knife discard the upper portion of the 
sample and place in the sterile wide-mouthed bottle. 

Note. Pack the sample in ice if it cannot be examined at once. 

3. To examine, allow the ice cream to melt quickly by 
placing it at about 37 C. and then treat as a milk 
sample. 

4. Plate on litmus lactose agar, using the following dilu- 
tions: 1 : 10,000, 1 : 1,000,000 and 1 : 100,000,000 and 
incubate plates at 37 C. 

5. Add a large quantity (25 c.c. to 50 c.c.) to the melted 
agar shake and incubate at 37 C. Examine in twenty- 
four to forty-eight hours for acid and gas. Is B. coli 
present? 

6. Count plates at the end of three days and estimate 
the total number of bacteria present per cubic centimeter, 
also the number of acid colonies and of any other predom- 
inant type. 

7. Transfer predominant types to litmus milk tubes and 
note action, also note rapidity with which each type pro- 
duces changes in the litmus milk. What may these results 
signify? 

8. Make a microscopic count, using the method in Exer- 
cise 1, Dairy Microbiology. How do microscopic and plate 
counts compare? 

9. Look up references for ascertaining bacteriological 
standards for ice creams. What is the quality of the 
ice cream you analyzed as compared with the maximum 
bacterial limit? What do you think this limit should be? 

10. From what diverse sources do bacteria enter ice 
cream? 

What is their significance in this product? 
What relation may some of the common practices of 
ice-cream makers have to the bacterial content of milk? 



PLANTS SUBJECT TO MICROBIAL DISEASES 291 

What effect does storage have upon the number of bac- 
teria in properly hardened ice cream? 

What significance has a pure ice-cream supply in relation 
to public health? 

11. Give results and conclusions in detail. 

REFERENCES 

SAVAGE: The Bacteriological Examination of Food and Water (1914), 

pp. 119-121. 

MARSHALL: Microbiology (1911), pp. 372-373. 
WILEY, H. W.: Ice cream, Hygienic Lab. Bui. 56.' Milk and its 

Relation to the Public Health (1909), pp. 251-311. 
HAMMER, B. W.: Bacteria and Ice Cream, Bui. 134 (1912), Iowa 

Agr. Expt. Sta. 
MORTENSEN, M. and GORDON, J.: Lacto: a New and Healthful 

Frozen Dairy Product. Bui. 119 (1911), Iowa Agr. Expt. 

Sta. 
WASHBURN, R. M.: Principles and Practice of Ice-cream Making. 

Bui. 155. Vermont Agr. Expt. Sta., pp. 9-10, 34-46, 53-54, 

64-66. 

BOLDUAN: Food Poisoning (1909), pp. 84-90. 
AYERS, S. H. and JOHNSON, Jr., W. T.: A Bacteriological Study of 

Retail Ice Cream, Bui, 303 U. S. Dept. Agr., 1915. 

PLANT MICROBIOLOGY 

EXERCISE 1. TO DEMONSTRATE THAT PLANTS ARE 
SUBJECT TO MICROBIAL DISEASES: INFECTION 
OF CERTAIN SPECIES OF VEGETABLES HAVING 
JUICY ROOTS, LEAVES, FRUITS, ETC., WITH B. 
CAROTOVORUS 

Apparatus. Tubes of sterile 2% saccharose broth; 
tubes of sterile agar; sterile water; sterile Petri dishes; 
three sterile deep culture dishes; sterile filter paper; sterile 
knife; sterile forceps; mercuric chloride, 1 : 500; juicy 
vegetables. 

Culture. B. carotovorus (culture of high physiological 
efficiency). 

Method. 1. The root of the carrot, turnip, rutabaga, 
the cucumber or radish; the cotyledons of immature pea 



292 



GENERAL MICROBIOLOGY 



seedlings, petioles of cabbage seedlings, potatoes, etc., 
may be used for this exercise. For what other plants is 
B. carotovorus pathogenic? 

2, Thoroughly wash the root, or vegetable to be in- 




FIG. 62. Crown Gall Produced by Bact. tumefaciens. (Orig.) 

oculated. Two or three vegetables of one kind should be 
employed. 

3. Disinfect a spot about 2 cm. in diameter with 1 : 500 
mercuric chloride and rinse with sterile water to get rid 
of disinfectant. Drain off excess moisture on sterile filter 
paper, handling vegetable with sterile forceps. 

4. Puncture the disinfected area on one vegetable with 



PLANTS SUBJECT TO MICROBIAL DISEASES 293 

the sterile stiff needle for control and place in a sterile 
deep culture dish. 

5. Obtaining some of the culture of B. carotovorus on 
the sterile needle, puncture the remaining vegetables in the 
center of the disinfected area and place vegetables in a 
sterile deep culture dish at 20 to 25 C. 

B. carotovorus is a wound parasite which invades the 
intercellular spaces, dissolving the middle lamellae and 
portions of the inner lamellae, thereby establishing a con- 
dition which is known as soft rot. 

6. Examine in twenty-four hours for evidence of action 
of B. carotovorus. This should be easily distinguishable 
in three days. 

7. Isolate the causal organism and determine its mor- 
phology and cultural characteristics. Compare with pure 
culture and with description given in Marshall's Micro- 
biology, p. 512. 

Is the organism newly isolated, capable of producing 
infection? Make inoculations from one, two, three and 
four-day old newly isolated cultures to sterile living vege- 
table tissue to determine this. Is there any difference in 
the infectivity of a one-day old and a three- or four-day old 
culture? 

8. What is known of methods of control of this disease? 

9. Grow four giant colonies of B. carotovorus on ordinary 
agar, one in each Petri dish and allow them to develop 
until nearly 1 cm. in diameter. 

10. Under sterile conditions, remove slices of fresh 
carrot, beet and rutabaga or turnip roots and potato and 
place in sterile Petri dishes. (Slices should be at least 
3 to 4 cm. in diameter.) 

11. With a sterile scalpel make a circular incision 0.5 
cm. from the edge of the colony through the layer of agar 
in the Petri dish. 

12. Remove this colony intact to the surface of one of 
the slices of vegetable and replace cover of Petri dish. 



294 GENERAL MICROBIOLOGY 



13. Repeat, removing a colony to the slice of each of the 
different vegetables. 

14. Examine in twenty-four hours for evidences of soft 
rot, and note progress of softening from day to day. What 
is demonstrated by this phenomenon? Are all vegetables 
attacked? 

15. What parts of the plant does B. carotovorus attack? 
What chemical constituents of these parts are decomposed 
through the agency of their action? 

What are the main features of difference in the mechanism 
of action of the various types of bacterial plant diseases? 

Give an example of a disease illustrating each. 

How is the progress of infection effected in these various 
types? What organisms produce a disease of similar type 
in other vegetables and plants? 

What is known of immunity in the plant kingdom? 

What methods of control are employed with different 
types of plant diseases? How are methods of control 
influenced by the type of disease? 

Note. This exercise may be made more interesting and instructive 
if combined with histological methods. 

Plates illustrating the invasion of root tissues by B. carotovorus 
are found in Smith's Bacteria in Relation to Plant Diseases, Vol. I, 
pp. 56, 103. 

16. State in full your results and conclusions. 

REFERENCES 

SMITH, ERWIN F.: Bacteria in Relation to Plant Diseases, Vol. I, 
pp. 5, 6, 65, 86, 103. Vol. II, pp. 51-52, 65, 81-88, 96, 292. 
Vol. III. 

MARSHALL: Microbiology, pp. 490-519. 

JONES, L. R.: A soft rot of carrot and other vegetables, pp. 299, 
13th Rept. of Vt. Expt. Sta. (1901). Also in Cent. f. Bakt. II, 
Bd. 14, pp. 369-377. 

JONES, L. R.: Pectinase, the cytolytic enzyme produced by B. caroto- 
vorus and certain other soft-rot organisms. Tech. Bui. 11, N. Y. 
Agr. Expt. Sta, (1909), 



ANIMAL INOCULATION IN BACTERIOLOGY 295 



ANIMAL DISEASES AND IMMUNITY 

EXERCISE 1. ANIMAL INOCULATION IN BACTERIOL- 
OGY FOR DETERMINATION OF THE IDENTITY 
OF A MICROORGANISM, ITS PATHOGENICITY OR 
VIRULENCE, OR FOR PRODUCTION OF IMMUNITY 

Apparatus. Experimental animals: rabbits; guinea 
pigs; white rats; white mice, etc.; scalpels; scissors; 
forceps; razor; syringe; trephine; sterile dishes; anes- 
thetic; disinfectant; cotton. 

Culture. Pure culture or infected material. 

I. INTRODUCTION 

1. Avoid the use of animals where the employment of 
other means answers the purpose equally well. 

2. Unless other factors prevent, always use the most 
susceptible and least expensive animals. 

II. PREPARATION OF ANIMAL * 

Method. 1. (a) Examine carefully each animal before 
subjecting it to experimentation. 

(6) Use no animal already showing symptoms of illness 
or general lack of vigor. 

(c) Record the weight and temperature of each animal. 

2. (a) Administer an anesthetic (general or local as 
indicated) whenever the operation is very painful or tedious 
or where perfect immobility of the parts is required. 

Note. For local anesthesia a 2% solution of cocaine hydrochloride 
may be made by dissolving 0.1 gm. of cocaine hydrochloride in 5 c.c. 
of sterile water. Instill a few drops into the conjunctiva! sac or inject 
1 to 5 c.c. into the subcutaneous tissues near the seat of operation. 
For general anesthesia 10 to 30 c.c. of a 5% solution of chloral hydrate 
may be injected per rectum, or ether or chloroform may be inhaled. 

*The instructor must arrange for experiments that must be started 
early in order to be completed before the term closes. 



296 



GENERAL MICROBIOLOGY 



Ether is probably safer in the hands of a novice. It may be adminis- 
tered by saturating cotton placed in a paper cone which is kept over 
the animal's nose. Care should be exercised to replenish the supply 
of the anesthetic on the cotton as fast as it volatilizes and not to force 
the anesthetizing too fast. Injury to the integument about the 
nose may be avoided by rubbing on vaseline before beginning the 
operation. The tissues should not be cut until anesthesia is complete. 

(6) Choose a site for operation where the results will not 
interfere with the animal's locomotion or normal functions, 
(c) Use sharp, sterile instruments. 




FIG. 63. Tray for Sterilizing Surgical Instruments. 



Note. Methods for holding different animals for different forms 
of operations vary. An assistant is usually required to hold the animal, 
where an anesthetic is not administered, and where an anesthetic is 
used it is usually better to have an assistant administer it, although 
this is not necessary. (For various devices for holding experimental 
animals see text-book: Eyre, Bacteriological Technic, 2d Ed. (1913), 
pp. 349-352.) 

3. Remove the hair with scissors or clippers from the 
field of operation and shave the surface. Wash the skin 
and disinfect it with 2% liquor cresolis compositus (U. S. P.). 
Wash off the disinfectant with alcohol and allow the 



ANIMAL INOCULATION IN BACTERIOLOGY 297 

alcohol to evaporate. The animal is now ready for the 
operation. 

Note. It is understood that a 2% solution of liquor cresolis com- 
positus (U. S. P.) shall be used wherever a disinfectant solution is indi- 
cated unless otherwise stated. 

III. METHODS 

Where the exact nature of the inoculum is unknown, 
the experimenter will be guided, as to what method to select, 
by his judgment, influenced by experience with other inocula 
in animal experimentation. The method most adaptable 
in the case of each specific microorganism will be indicated 
in the treatment of that organism. 

1. Cutaneous. Rub the inoculum on the shaved and 
disinfected skin or make several parallel, superficial inci- 
sions and rub the inoculum into the scarifications with a 
sterile scalpel. See that no disinfectant remains on the 
skin before operating. 

2. Subcutaneous. I. (a) Pick up the skin with the 
thumb and forefinger of the left hand and insert the needle 
through one side of the fold of skin thus made. 

Note. The point of the needle should not enter the skin on the 
other side of the fold, but should lie in the subcutaneous tissue. 

(6) Release the skin and inject the material. 

(c) Place the finger moistened with the disinfectant 
over the point where the needle enters the skin and remove 
the needle. 

II. (a) For solid material that will not pass through a 
hypodermic needle, make a short incision through the skin 
parallel to the horizontal plane of the body. 

(b) With a sterile probe separate the skin from the 
underlying tissues on the lower side of the cutaneous 
incision, making a small pocket in the subcutaneous 
tissue. 

(c) With fine-pointed sterile forceps insert the inoculum 



298 GENERAL MICROBIOLOGY 

into this pocket. Further treatment should not be neces- 
sary. 

3. Intramuscular. I. Plunge the needle deeply into the 
muscles, preferably on the inside of the thigh. 

II. Inject the material slowly with steady pressure if 
the volume is great. 

4. Intravenous. I. Inject the liquid into the ear vein 




FIG. 64. One Method of Injecting Hog-cholera Serum. (Orig.) 

of the rabbit (and other animals if possible) or jugular vein 
where accessible. The injection should be in the direction 
of the circulation. 

II. The femoral vein may be used where other veins 
are not readily entered with the needle. Use general 
anesthesia. Make an incision on the inside of the thigh 
over the femoral space. Separate the iliacus, pectineus 
and sartorius muscles. The femoral vein and artery are 
laid bare. After inoculation disinfect and suture the skin. 



ANIMAL INOCULATION IN BACTERIOLOGY 299 

Note. Solid substances, larger than leucocytes, and air bubbles 
should not be injected into the vascular system. Fatal emboli may 
result. Return of blood through the needle indicates that the vein 
has been entered. If swelling occurs at point of inoculation the inocu- 
lum is entering the subcutaneous tissue. Try again. 

6. Intraabdominal or intraperitoneal. I. The site for 
the operation is the center of the angle formed by the last 
rib, transverse processes of the lumbar vertebrae, and the 
external angle of the ilium. 

(a) Plunge the needle or trocar and canula through the 
abdominal wall with one thrust. 

Note. When the parietal peritoneum is punctured the sudden 
disappearance of resistance to the entrance of the needle is noticed. 
The intestines will not be entered if pressure on the needle stops at 
this point. 

(6) Inject the material and remove the needle, placing 
the thumb and finger on each side of the needle and press- 
ing gently on the skin during the removal so as to prevent 
separating the skin and underlying layers of tissue. 

II. Infectious material or cultures in a sterile collodium 
capsule may be introduced into the abdominal cavity by 
performing laparotomy. (General anesthesia is desired.) 

6. Intraorbital. Always perform under local anesthesia. 
(2% cocaine hydrochloride.) 

I. Steady the eye with fixation forceps. 

II. Pierce the cornea near to its periphery with a fine 
needle. The needle should incline with the point outward 
so that, upon entering the anterior chamber of the eye, 
the iris will not be damaged. 

III. Inject the material. 

7. Subdural. Operate under general anesthesia. 

I. Make a longitudinal incision through the skin at one 
side of the sagittal suture. Hold back the skin and sub- 
cutaneous tissue with tenacula. 

II. Make a crucial incision through the periosteum and 
push back the four corners. 



300 



GENERAL MICROBIOLOGY 



III. Expose the dura mater by removing a small button 
of the parietal bone (0.5 cm. in diameter) with a trephine. 

IV. Inject the inoculum immediately beneath the dura 
mater. 

V. Replace the periosteum and suture the skin. 

VI. Disinfect. 

8. Intrapulmonary. I. Pull the animal's front leg for- 
ward. 




FIG. 65. Another Method of Injecting Hog- cholera Serum, (Orig.) 

II. Plunge the needle through the fifth or sixth inter- 
costal space into the lung tissue. 

III. Slowly inject the contents of the syringe. 

Note. In large animals material may be injected into the trachea 
between the tracheal rings. 

9. Ingestion. If possible mix the infectious material 
with the animal's food. 

Note. See that the animal eats all that is intended to be eaten. 

Introduce the infectious material into a gelatin capsule 
and force the animal to swallow it; or give the material as 
a drench where advisable. 



ISOLATION OF PATHOGENIC BACTERIA 301 

Note. Fasting the animal before introducing unpalatable material 
into the food may be helpful in increasing the amount eaten. The 
chemical reaction of the stomach contents as governed by physio- 
logical activity will influence results. 

IV. CARE OF INOCULATED ANIMALS 

1. Watch each animal closely and take temperatures as 
the case demands. 

2. Treat each animal as a case of infectious disease in 
quarantine. 

Note. Whatever clinical, diagnostic or sanitary measures neces- 
sary in that given disease may be employed as seen fit. 

3. When the animal is removed from the cage for the 
last time, carefully destroy all refuse in the cage and dis- 
infect thoroughly. 

4. Give all results, observations and conclusions in 
detail. 

REFERENCES 

STITT: Practical Bacteriology, Blood Work and Parasitology, 3d 

Ed., 1914, pp. 48, 143, 152. 
MOORE and FITCH: Bacteriology and Diagnosis, pp. 114,' 118-120, 

124, 125, 130, 133. 

EYRE: Bacteriological Technic. pp. 332-369. 
KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 53-64. 

EXERCISE 2. THE ISOLATION OF PATHOGENIC BAC- 
TERIA FROM FLUIDS AND TISSUES OF DEAD 
ANIMALS 

Apparatus. Disinfectant; scalpel; scissors; forceps; 
bone forceps; ten sterile pipettes; 10 c.c. sterile pipettes; 
250 c.c. flask containing glass beads, sterile; sterile Esmarch 
dishes; spatula; platinum loop; special media. 

Method. 1. Disinfect the skin. 

2. Remove the spleen, kidney, lymph glands, and any 
other diseased tissue, to sterile Esmarch dishes, using sterile 
instruments. 



302 GENERAL MICROBIOLOGY 

3. Collect samples of pericardial and pleuritic fluids* 
blood, urine and bile with sterile pipettes and place 
these in small sterile flasks. Collect at least 25 c.c. of 
blood in a sterile flask containing glass beads for defibrinat- 
ing. 

4. Remove the organs collected to the laboratory and 
make cultures as follows: 

5. Sear the surface of the organ with a spatula heated to 
a white heat. 

6. Tear the seared surface with forceps, sterilized in 
flame. 

7. With a sterile platinum loop, make transfers to agar 
slants, shake cultures, and plates for isolation into pure 
cultures. 

8. Repeat 7, using any body fluids collected. 

Note. The different diseases require special procedures and 
media for successful results. Attention will be called to these varia- 
tions at the proper places. 

REFERENCES 

MOORE and FITCH: Bacteriology and Diagnosis, pp. 95-96. 
MOORE: Principles of Microbiology, pp. 156-162, 237-258. 
EYRE: Bacteriological Technic, pp. 248-258. 



EXERCISE 3. A STUDY OF BACT. ANTHRACIS 

Note. Bact. anthracis is the cause of anthrax, a disease very fatal 
to man and certain domestic animals. Great care should be taken 
while working with it. 

Apparatus. Six tubes of agar; three tubes of potato; 
three tubes of milk; tube of gelatin; slides and stains; 
autopsy instruments. 

Culture. Bact. anthracis. 

Method. 1. Inoculate three tubes each of agar, potato^ 
and milk and one tube of gelatin with Bact. anthracis. 



THE PREPARATION OF TUBERCULIN 303 

2. Incubate one tube of each at 20 C., one at 37 C., 
and one at 42 C. (Study and record the effect of these 
temperatures upon the growth and spore formation of the 
organism.) 

3. Make cover-glass preparations and stain with meth- 
ylen blue, fuchsin and Gram's stain. Stain for spores 
(Anjeszky's method). 

4. Transfer a small quantity of the agar culture to 
4 or 5 c.c. of sterile physiological salt solution and 
inject 0.25 c.c. subcutaneously into a guinea pig. Make 
daily observations and an autopsy of the animal at 
death. 

5. Make cultures on agar slants, and smear prepara- 
tions from the blood, liver, spleen and kidney after the 
autopsy. 

6. Fix the smears in the flame, stain with methylen blue 
or fuchsin. After twenty-four and forty-eight hours ex- 
amine the cultures microscopically. 

7. State your results and conclusions in full. 

REFERENCES 

MARSHALL: Microbiology, pp. 469, 476, 559, 561, 599-604. 
JORDAN: General Bacteriology, 4th Ed. (1914), pp. 223-236. 
BESSON: Practical Bacteriology, Microbiology and Serum Therapy, 

transl. by Hutchens (1913), pp. 517-535. 
KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 653- 

654. 
ZINSSER: Infection and Resistance (1914), pp. 15, 18, 53, 64, 296. 

EXERCISE 4. THE PREPARATION OF TUBERCULIN 

Apparatus. Two 500 c.c. Erlenmeyer flasks; glycerin- 
ated veal broth; evaporating dish; 0.5% phenol salt solu- 
tion; Berkefeld filter; heavy filter paper; 20 c.c. homeo- 
pathic vials; sealing wax. 

Culture. Bact. tuberculosis (specially adapted for 
tuberculin) . 

Method, 1. Place about 200 c.c. of glycerinated veal 



304 



GENEKAL MICROBIOLOGY 



bouillon in each of two 500 c.c. Erlenmeyer flasks and 
sterilize for twenty minutes each day for three consecu- 
tive days. 

R2. From a culture of the tuber- 
cle bacterium furnished, inoculate 
each flask of veal broth. In mak- 
ing the inoculations care should 
be taken to place the inoculum on 
the surface and to avoid agitation 
after inoculation. Seal the flasks 
with paraffin and place in the incu- 
bator at a temperature of 37 C. 

3. Allow the cultures to grow 
four weeks after the surface is 
covered, then shake well, place in 
a steam sterilizer and subject to 
steam for two and a half hours. 

4. Filter through a filter paper 
to remove most of the bacterial 
growth. 

5. Evaporate to one-tenth its 
original volume over a water bath 
at a temperature of 60 C. 

6. To one volume of the con- 
centrated tuberculin add seven 
volumes of sterile physiological 
salt solution containing 0.5% 
phenol or tricresol and then filter 
through a Berkefeld filter. 

7. Place the product in 20 c.c. 
homeopathic vials and seal with wax. Label the vials and 
place in a cool dark room. 




FIG. 66. Bact. tuberculosis 
(avian), on Banana. 
(Orig. Himmelberger.) 



THE PREPARATION OF TUBERCULIN 



305 



REFERENCES 

MARSHALL: Microbiology, pp. 485, 487. 

MOORE: Principles of Microbiology, pp. 251-253. 




FIG. 67. Glycerin Veal-broth Cultures of Bad. tuberculosis (Human), 
for Tuberculin, about Eight Weeks Old, (Orig. Keck,) 

BESSON: Practical Bacteriology, Microbiology and/ Serum Therapy, 

pp. 289-345. 

MOORE: Bovine Tuberculosis. 
KOLMER: Infection, Immunity and. Specific Therapy (1915), pp. 582- 

601, 661-680. 
ZINSSER: Infection and Resistance (1914) ,pp. 355-357, 438-442. 



306 



GENERAL MICROBIOLOGY 




THE PREPARATION OF BLACK-LEG VACCINE 307 



EXERCISE 5. THE PREPARATION OF BLACK-LEG VAC- 
CINE 

Apparatus. Sterile mortar and pestle; sterile cheese- 
cloth; two sterile glass plates; sterile water; sterile homeo- 
pathic vials. 

Culture. Diseased muscle of calf affected with black- 
leg. 

Method. 1. Place a piece of diseased muscle from a 
calf affected with black leg (this will be furnished by the 
instructor) in a sterile mortar, add a small quantity of 
water and triturate completely with a sterile pestle. 

2. Squeeze the pulp through a piece of sterile cheese 
cloth, spread the filtrate in a thin layer over a sterile glass 
plate or saucer and dry at a temperature of 35 to 37 C. 
in an atmosphere free from contamination. 

3. Mix approximately one part by volume of the dried 
virus with two parts of water, triturate until the mixture 
is converted into a semi-solid homogeneous mass and spread 
in a thin layer over a glass plate or saucer. 

4. Heat in an oven to a temperature of 100 to 104 C. 
for a period of seven hours. 

5. One centigram of the attenuated virus mixed with a 
small quantity of water is a dose for a calf. Place the 
product in sterile vials, ten doses to the vial, place a cork 
stopper in each vial and seal. 

REFERENCES 

NORGAARD, V. A., and MOHLER, J. R.: Black leg, its Nature, Cause 

and Prevention, B. A. I. Circular No. 31, revised (1911). 
MARSHALL: Microbiology, pp. 472-473. 
KOLMER: Infection, Immunity and Specific Therapy (1915) p. 655. 



308 GENERAL MICROBIOLOGY 



EXERCISE 6. THE PREPARATION OF TETANUS TOXIN 

Apparatus. Dextrose broth; two 100 c.c. Erlenmeyer 
flasks; paraffin oil; 5% phenol; Berkefeld filter. 

Culture. B. tetani. 

Method. 1. Place 50 c.c. of dextrose bouillon in each 
of two 100 c.c. Erlenmeyer flasks, plug and boil gently 
two or three minutes over a flame. 

2. Cover the bouillon with a layer of paraffin oil about 
5 mm. deep and heat in the autoclav. 

3. After cooling, inoculate the bouillon with B. tetani 
and incubate about two weeks. 

4. Examine the culture microscopically to determine 
the absence of contamination, add sufficient 5% phenol 
to make a 0.5% solution and filter through a Berkefeld filter. 

5. Incubate about 1 c.c. of the filtrate in 10 c.c. of 
dextrose broth under anaerobic condition, for forty-eight 
hours to make sure that the filtrate is sterile. 

6. The filtrate, if sterile, is to be used in immunizing a 
rabbit for the production of antitoxin. 

Note. A fairly potent toxin will kill a guinea pig in a 0.001 c.c. 
dose. The toxin in solution is very unstable and should be kept in a 
tightly stoppered bottle in a cool dark place. It may be kept for 
several months by precipitating with a saturated solution of ammo- 
nium sulphate and drying in vacuo over sulphuric acid. 

REFERENCES 

MARSHALL: Microbiology (1911), pp. 480-484. 

BESSON: Practical Bacteriology, Microbiology and Serum Therapy 

(1913), pp. 536-548. 
KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 115, 

234, 720, 819. 
ZINSSER: Infection and Resistance (1914), pp. 41, 107, 131-133. 



PREPARATION OF TETANUS ANTITOXIN 309 



EXERCISE 7. THE PREPARATION OF TETANUS ANTI- 
TOXIN 

Note. In the preparation of tetanus antitoxin for therapeutic 
purposes, healthy horses are used. For the first injection of toxin 
a small fraction of a cubic centimeter is given subcutaneously. The 
increase in the size of the dose and the frequency of injection depend 
upon the condition of the animal, but the quantity injected is gradually 
increased until the animal is able to stand 300 to 400 c.c. of toxin at 
one injection. 

For laboratory purposes, the rabbit may be used to furnish the 
antitoxin. 

The following method suggested by Roux and Vaillard 
produces a satisfactory antitoxin for laboratory study. 

Apparatus. Tetanus toxin; Gram's iodin solution; 
rabbits; 20 c.c. syringe; disinfectant; anesthetic; oper- 
ating tray; 50 c.c. sterile glass cylinder. 

Method. 1. Give the first five or six injections sub- 
cutaneously, subsequent ones may be given intraperi- 
toneally. 

1st day, 3 c.c. of toxin mixed with 1 c.c. of Gram's iodin 
solution. 

5th day, 5 c.c. of toxin mixed with 2 c.c. of Gram's iodin 
solution. 

9th day, 12 c.c. of toxin mixed with 3 c.c. of Gram's 
iodin solution. 

16th day, 5 c.c. of undiluted toxin. 

23d day, 10 c.c. of undiluted toxin. 

30th day, 15 c.c. of undiluted toxin. 

The quantity may be gradually increased until the 
rabbit is getting 100 c.c. of undiluted toxin. 

2. After the 6th injection has been given, wait a period 
of ten days and bleed the rabbit aseptically. This is accom- 
plished as follows: 

(a) Secure the rabbit in a dorsal position on an operating 
tray and anesthetize with ether. 

(b) Expose an area about 3 cm. square over the inferior 



310 GENERAL MICROBIOLOGY 

thoracic wall, in the region of the apex of the heart, shave 
and clean with alcohol. 

(c) Insert a sterile needle attached to a sterile 20 c.c. 
syringe, through the thoracic wall into the heart and slowly 
draw the plunger out. 

If only a small quantity of serum is desired for testing, 
the animal may be saved for subsequent bleedings. 

(d) Place the blood in a sterile container, allow to 
clot and draw off the serum for standardization. 

REFERENCES 

MARSHALL: Microbiology (1911), pp. 480-484. 

BESSON: Practical Bacteriology, Microbiology and Serum Therapy 

(1913), pp. 544-548. 
KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 234, 

242, 719-729. 
ZINSSER: Infection and Resistance (1914), p. 463. 

EXERCISE 8. A DEMONSTRATION OF THE AGGLU- 
TINATION TEST 

Note. There are two methods of applying the agglutination 
test: First, by combining the suspect's serum in varying amounts 
with a suspension of the specific organism and incubating eighteen to 
thirty-six hours; the results are then read with the unaided eye. 
Second, the serum may be combined in varying dilutions with a sus- 
pension of the specific organism, and hanging drop preparations made 
and examined microscopically. If agglutinins are present, clumping 
of the organisms will occur in a few minutes. With either method, 
controls, containing the organism but normal serum, should be pre- 
pared for comparative purposes. 

Apparatus. Four agar slants; test-tube rack for small 
test tubes; twelve small test tubes; antiserum; physio- 
logical salt solution; 1 c.c. pipettes, graduated to 0.01 c.c.; 
5 c.c. pipettes; cover-glasses; concave slide. 

Culture. B. typhosus or B. cholerce suis. 

Method. Macroscopic Test. 1. Antigen. This is a 
suspension of the specific organism obtained from a twenty- 
four to forty-eight hour agar culture in physiological salt 



DEMONSTRATION OF AGGLUTINATION TEST 311 

solution. Only a sufficient quantity of the growth to give 
a slight cloudiness to the salt solution in a small test tube 
should be used. 

Note on Antigen. Where a series of agglutination tests are to 
be made at intervals, the antigen should be standardized so that 
the same concentration will be used for each test. Great care should 
be used in preparing the antigen to avoid clumps in suspension. 
In some cases thoroughly shaking in a shaking machine will afford a 
satisfactory antigen, in others it must be filtered through a filter paper, 




FIG. 69. Macroscopic Agglutination of B. cholerce suis by Dorset- 
McBryde-Niles Serum. From left to right tubes show, first, 
complete agglutination, heavy sediment, clear supernatant 
liquid; in each succeeding tube the sediment becomes less, the 
turbidity greater, the tube at the right showing uniform cloudi- 
ness and no sediment, no agglutination. (Orig. Giltner.) 

2. The antiserum may consist of immune serum a 
rabbit immunized to the typhoid bacillus may be used to 
furnish the serum , or hog cholera serum or virus may be 
used with B. typhosus and B. cholerce suis respectively. 

3. The following table shows the various combinations 
of serum, antigen and salt solution to give definite dilu- 
tions. Physiological salt solution should be used in dilut- 
ing the serum. 



312 



GENERAL MICROBIOLOGY 



Tube. 


Antigen. 


Serum 
Diluted 1-10. 


Salt Solution. 


Dilution. 




c.c. 


c.c. 


c.c. 




1 


4 


1.0 


0.0 


1-50 


2 


4 


0.5 


0.5 


1-100 


3 


4 


0.2 


0.8 


1-250 


4 


4 


0.1 


0.9 


1-500 






Serum 










diluted 1-100. 






5 


4 


0.5 


0.5 


1-1000 


6 


4 


0.2 


0.8 


1-2500 


7 


4 


0.1 


0.9 


1-5000 



4. Shake all tubes well and incubate at 37 C. for twenty- 
four hours and record the results. 

Microscopic Test. 1. If this test is carried out during 
the same period with the macroscopic test, a small loopful 
of the dilution from any tube may be transferred to a clean 
cover-glass placed on a hanging drop slide and the edges 
sealed with vaselin or oil. It may then be examined with 
a microscope. 

2. If done independently of the macroscopic test, prepare 
the suspension of organisms in one test tube and the dilu- 
tions of serum in others. 

Mix a loopful of the diluted serum with a loopful of the 
antigen on a clean cover-glass, mount on a concave slide 
and observe with a microscope for a period of thirty minutes 
to one hour. 

3. Give results and any conclusions in detail. 

REFERENCES 

KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 68, 

69, 79, 152, 266-291. 

MARSHALL: Microbiology (1911), pp. 488, 567-570. 
MCFARLAND: Pathogenic Bacteria and Protozoa, 7th Ed. (1912), 

pp. 149-152. 
GELTNER: Studies of Agglutination Reactions in Hog Cholera during 

the Process of Serum Production (Preliminary) Tech. Bui. 3 

(1909), Mich. Agr. Expt. Sta. 

GILTNER: Same title Tech. Bui. 8 (1911), Mich. Agr. Expt. Sta. 
ZINSSER: Infection and Resistance (1914), pp. 218-247. 



A STUDY OF FILTERABLE VIRUSES 313 



EXERCISE 9. A STUDY OF FILTERABLE VIRUSES 

Apparatus. Physiological salt solution; Chamberland 
filter with water-suction or air pump and pressure gage; 
sterile flasks; clinical thermometer; syringe; flasks of bouil- 
lon, 50 c.c. in each; autopsy set. 

Culture. Hog cholera virus (blood of hog sick with 
cholera) . 

Method. 1. Preparation of the Filter. If the filter has 
been used once clean it by: 

(a) First rinsing with cold water under the tap. 

(6) Force about 1 liter of cold distilled water through 
it. 

(c) Then a solution consisting of 1 gm. KMn(>4 and 6.5 
gms. HC1 in 1000 gms. water. 

(d) Next, 1000 c.c. of a solution of 1% oxalic acid. 

(e) Boiling water is then forced through the filter until 
the liquid which runs through is free from acid. 

(/) Lastly, cold distilled water must be forced through 
the filter. 

Thus treated, any organic residue is destroyed and the 
filter is as good as new. 

This method of purification must always be used imme- 
diately after using a filter. Filter candles must not be left 
twenty-four hours without cleaning. 

A new filter may be prepared for use by forcing through 
it a large quantity of boiling distilled water and finally 
cold distilled water. 

The amount of liquid necessary to force through the 
filter for cleaning varies with the size of the filter. The 
ordinary 8 inch filter should receive the full amount 
(1000 c.c.) of each solution and distilled water for efficient 
purification. 

Filters are best sterilized by being set up ready to use 
and autoclaved. (See Fig. 71 for one method.) 

2. Procure some hog cholera virus and after diluting it 



314 



GENERAL MICROBIOLOGY 



t " : ; , .,^nir] 




s 

I 
I 

s 

'I 
fr 



A STUDY OF FILTERABLE VIRUSES 



315 



with equal parts of physiological salt solution, pass it 
through a clean, sterile, Chamberland filter at a pressure 
not to exceed one atmosphere and 
during a time not to exceed one 
hour. 

3. Make sub-cultures of the fil- 
trate by introducing 1 c.c. into each 
of several flasks of bouillon contain- 
ing 50 c.c. each. Take every pre- 
caution against contamination. Also 
make microscopical preparations. 

4. If no growth results under 2 
inject 2 c.c. into the muscles of a 
50 Ib. pig. Make daily observations 
of the pig and record the temperature 
each day, 

6. When undoubted symptoms 
of hog cholera have developed, kill 
the pig and make a careful autopsy. 
Save the blood in a sterile jar. 

6. Repeat the experiment, using 
blood procured in 4 as virus. 

7. By repeated nitrations and in- 
jecting into susceptible hogs, it 
may be proven that a living micro- 
organism, incapable of producing visible growth in vitro, 
passes through the filter and develops in the body of the 

pig- 

8. State your results and conclusions in full, 




FIG. 71. Pasteur-Cham- 
berland Filter Adjusted 
for Filtration by Suc- 
tion. 



REFERENCES 

DORSET, MCBRYDE and NILES: Further Experiments Concerning 
the Production of Immunity from Hog Cholera, Bui. 102, B. A. I., 
U. S. Dept. Agr. 

MCBRYDE: Filtration Experiments with B. choleras swis, Bui. 113, 
B. A. I., U. S. Dept. Agr. 



316 GENERAL MICROBIOLOGY 

GILTNER: What is the Antigen Responsible for the Production of 
Antibodies in Hog Cholera Serum? Tech. Bui. 13, Mich. Agr. 
Expt. Sta. 

KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 77, 78. 



EXERCISE 10. THE PREPARATION OF BACTERINS OR 
BACTERIAL VACCINES 

Apparatus. Scalpel; scissors; forceps; sterile tubes 
and Esmarch dishes; sterile swabs; sterile physiological 
salt solution; 50% alcohol; disinfectant; four agar slants; 
six agar tubes for plating; six sterile Petri dishes. 

Culture. Infected material or specific cultures to be 
furnished by the instructor. 

A. AUTOGENOUS BACTERINS 

Method. In the preparation of an autogenous bac- 
terin, it is first necessary to isolate the microorganism 
causing the disease. This is accomplished as follows: 

1. If there are any unopened abscesses, open one with 
a sterile scalpel after first disinfecting the field with 2% 
compound solution of cresol an4 washing with 50% alcohol. 
Collect some of the pus on a sterile swab and suspend 
in sterile 'physiological salt solution. 

2. If the abscess is already opened, using a sterile 
curette, obtain some of the diseased tissue at the bottom 
of the abscess and macerate this in sterile physiological 
salt solution. 

3. Pour agar plates from this salt solution suspension, 
using at least six, plated in series of two. 

4. Incubate the plates and after twenty-four hours 
make observations on the number and type of colonies. 
After forty-eight hours make transfers to agar slants of the 
most numerous type of colony. Colonies should be studied 
under low power of the microscope. 

5. Grow three or four cultures of the organism on slanted 



BACTERINS OR BACTERIAL VACCINES 317 

agar. Make a morphological study of the organism. After 
twenty-four hours wash off the growth from each tube 
with 3 c.c. of sterile saline solution. 

6. Put the suspension all in one container, reserving 1 
c.c. to be used in standardization. 

Note. In the hemocytometer method for standardizing bacterins 
it is desirable to use a special hemocytometer with a counting chamber 
0.02 mm. deep provided with a special cover-glass for counting bac- 
teria, but if this is not accessible, an ordinary hemocytometer and 
cover-glass as used for blood counting may be used. If the latter, 
a 4 mm. objective must be used for counting. 

Using the diluting pipette of the blood counting apparatus the 
suspension of bacteria is diluted to the desired dilution with Collison's 
fluid made as follows: 

Hydrochloric acid, 2 cc. 

Mercuric chloride 1-500, 100 cc. 

Acid fuchsin, 1% aqueous solution enough to color to a deep 
cherry red. 

Filter before using. 

The bacterial suspension is allowed to remain in the pipette eight 
to ten minutes to stain, then thoroughly agitated by rotating the 
pipette and the first few drops from the arm of the pipette discarded. 
The mount is then prepared and the slide placed on the stage of 
microscope which has been previously leveled, and the count made. 
The count and calculations are made as for blood counting. 

7. Heat in a water bath at 60 C. for one hour. This 
is usually sufficient to kill the bacteria, unless they are 
spore producers. 

8. To test the sterility of the suspension after heating, 
with a sterile loop make an agar streak and incubate for 
twenty-four hours. If growth is obtained the culture 
must be heated again. 

B. STOCK BACTERINS 

The procedure in the preparation of a stock bacterin 
is the same as in the preparation of an autogenous bacterin, 
except that the organisms used are from cultures kept in 
stock for that purpose. 



318 



GENERAL MICROBIOLOGY 



C. POLYVALENT BACTERINS 

Polyvalent bacterins are those which are prepared from 
several species of bacteria, e.g., M. (Staph.) albus, M. 
(Staph.) aureus, Strep, pyogenes, etc. 

The suspension of each must be prepared and standard- 




FIG. 72. Blood-counting Apparatus for Use in Standardizing Bacterial 

Vaccines. 



ized separately, and then the emulsions of all mixed. In 
this way, it is possible to have a known number of each 
species in the resulting product. 

REFERENCES 

FITCH C. P.: A Review of the Principal Methods Used to Standardize 

Bacterins (Bacterial Vaccines). Report of the N. Y. State Vet. 

College for the year 1913-1914, pp. 207-219. 
MCCAMPBELL: Laboratory Methods for the Experimental Study of 

Immunity, pp. 186, 188. 
STITT: Practical Bacteriology, Blood Work and Parasitology, 2d 

Ed. (1911), pp. 143-145. 
KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 611- 

661. 
ZINSSER: Infection and Resistance (1914), pp, 328-357. 



TO DEMONSTRATE OPSONINS 319 



EXERCISE 11. TO DEMONSTRATE OPSONINS AND TO 
DETERMINE THE OPSONIC INDEX 

Apparatus. Several .small test tubes; forty-five small 
mixing pipettes; sterile citrated salt solution; Wright's 
stain; suspension of leucocytes; normal serum; patient's 
serum. 

Culture. Organism producing the disease. 

Method. 1. The small mixing pipettes are made by 
drawing out 4 to 5 mm. glass tubing to a long, fine capil- 
lary tube; and providing with a small rubber bulb. (Con- 
sult the instructor for the method.) 

2. Prepare the suspension of leucocytes by collecting 
a few cubic centimeters of blood from any animal and 
immediately place in three or four volumes of citrated salt 
solution. 

Centrifuge and wash at least three times, being careful 
not to pipette off any of the cells during the washing 
process. 

After the last washing, pipette off the supernatant 
liquid and lay the tube in as nearly a horizontal position 
as possible for about twenty-five to thirty minutes. At 
this time there will appear an upper whitish layer of cells 
composed almost exclusively of leucocytes. 

Pipette off the leucocytes. They should be used within 
five to six hours from the time the blood is collected. 

3. Bacterial Suspension. Transfer a loopful from an 
eighteen to twenty-four hour agar culture to 2 or 3 c.c. of 
physiological salt solution and mix well. The suspension 
must be carefully made to avoid clumps and some method 
of standardization used so that successive tests will be 
comparable. (The nephelometer may be used for this 
purpose see an instructor.) 

4. Collect the patient's serum and normal serum at the 
same time and under the same conditions in order that the 
results may be comparable. The blood is collected in a 



320 GENERAL MICROBIOLOGY 

small test tube and either allowed to clot and the serum 
removed, or it is defibrinated and centrifuged. In either 
case the serum should be used within three to four hours 
from the time the blood is drawn. 

5. Make the test as follows : 

a. With a diamond point or wax pencil make a mark on 
the drawn out arm of the mixing pipette about 2 cm. from 
the end. 

b. With the aid of a rubber bulb on the opposite end, 
draw a column of the bacterial suspension up to the mark, 
admit a bubble of air, then draw a column of leucocytes 
up to the mark and another bubble of air, then a column 
of the serum to be tested. 

c. Mix these by forcing out on a glass slide or into a 
small test tube and drawing up again, repeating once or 
twice, being careful to avoid introducing air bubbles. 

d. Finally draw up the mixture into the pipette and seal 
the end of pipette in the flame, using care not to heat the 
mixture. 

e. Incubate fifteen minutes with frequent shaking. 

/. Then place a drop on a slide and make a thin film 
made as in the preparation of a blood film, dry and stain 
with Wright's or Jenner's stain. 

6. Repeat the experiment, using the normal serum. 

7. With an oil immersion lens count the number of 
organisms taken up by fifty leucocytes on each slide and 
calculate the average number taken up by each. The 
result is the opsonic power of the serum. 

8. The opsonic index is the ratio of the opsonic power 
of the suspected serum to that of the normal serum. 

Example. If the average number of bacteria taken up 
by the leucocytes in the presence of the suspect serum is 
5.6 and the average number taken up by the leucocytes in 
the presence of the normal serum is 4.8, the opsonic index 
of the suspect serum is determined as follows: 5.6^-4.8 = 
1.16+ opsonic index. 



TO DEMONSTRATE THE PRECIPITIN TEST 321 

McCampbell's Modification of the Opsonic Test. 1. 

Prepare the bacterial suspension as above and add 0.8% 
sodium citrate. 

2. (a) With a blood diluting pipette, draw the bacterial 
suspension up to the mark 0.5. 

(6) With the same pipette draw up the same amount 
of blood collected from the patient, then draw both into the 
bulb and mix quickly. 

(c) Place a flat rubber band around the ends of the 
pipette and incubate fifteen minutes. Prepare film, and 
stain. 

3. Repeat the experiment, using normal blood. The 
opsonic index is determined as above. 

Note. The sodium citrate is slightly antiopsonic but this factor 
is constant in both preparations, consequently the results are com- 
parable. 

4. Give results and any conclusions in detail. 

REFERENCES 

McCAMPBELL: Laboratory Methods for the Experimental Study 

of Immunity (1909), pp. 44-70. 

MCFARLAND: Pathogenic Bacteria and Protozoa, 7th Ed., p. 307. 
KOLMER: Infection, Immunity and Specific Therapy, pp. 187-205. 
ZINSSER I.e. Exercise 10, p. 318. 

EXERCISE 12. TO DEMONSTRATE THE PRECIPITIN 

TEST 

This test is of importance in identifying the source of 
blood in legal cases and may also be used in the examina- 
tion of various meat products for the presence of foreign 
meat substances. 

It is based upon the fact that if an animal is injected 
at intervals of six to eight days for four or five times with 
any foreign protein its serum acquires the property of pre- 
cipitating that specific protein even when in a very high 
dilution. 



322 GENERAL MICROBIOLOGY 

An antiserum for each specific protein to be tested for 
must be prepared by animal inoculation. Thus, if a test 
for human blood is to be made, an anti-human-blood serum 
must be used. 

Apparatus. Syringe and needles; sterile cow's blood; 
sterile 500 c.c. Erlenmeyer flask containing glass beads; 
rabbit; disinfectant; sterile flasks, tubes and pipettes; 
sterile physiological salt solution. 

Method. 1. Inject a rabbit intra-abdominally with 
6 c.c. sterile, defibrinated cow blood. On the sixth or seventh 
day, repeat the injection, using 10 c.c. On the twelfth or 
fourteenth day give 12 c.c. and again on the eighteenth or 
twenty-first day give another 12 c.c. 

2. On the twenty-fourth or twenty-eighth day draw 
a little blood from the rabbit and test it to determine its 
property of precipitating cow blood. If it has a high litre, 
the rabbit should be anesthetized and the bloo.d drawn from 
the heart as explained in Exercises 1 and 7, pp. 295 and 309. 

3. Place the blood in a sterile container and allow to 
clot. Draw the serum into small, sterile, glass bulbs hold- 
ing 0.5 c.c. and seal the bulbs by heating the arm in a small 
flame, using care to avoid heating the serum. Serum col- 
lected in this way and placed in a cool, dark place will retain 
its precipitating properties for several months. 

4. Dilute the blood to be tested with physiological salt- 
solution. Several dilutions should be made, e.g., 1-200, 
1-500, 1-1000, 1-10,000 and 2 c.c. of each dilution placed 
in small test tubes. Place six to eight drops of the anti- 
serum in each tube. If the suspect serum is from the same 
species of animal as that used in immunizing the rabbit 
(in this case, cow serum), immediate precipitation will 
occur. After a few minutes' observation the tubes should 
be incubated at 37 C. for twenty to thirty minutes and the 
results noted. 

5. If the blood is dried, as a blood stain on cloth, the 
quantity should be estimated and placed in a definite 



THE PRODUCTION OF A HEMOLYTIC SERUM 323 

quantity of salt solution so that the dilution may be approx- 
imated. 

6. Give results and conclusions in full. 

REFERENCES 

MCFARLAND: Pathogenic Bacteria and Protozoa, 7th Ed., p. 146. 

MARSHALL: Microbiology (1911), pp. 570-574. 

NUTTALL: Blood Immunity and Relationship (1904). 

KOLMER: Infection, Immunity and Specific Therepy (1915), pp. 70, 71, 

292-315, 517-519, 844-846. 
ZINSSER: Infection and Resistance (1914), pp. 248-271, 

EXERCISE 13. THE PRODUCTION OF A HEMOLYTIC 

SERUM 

For this work a rabbit will be immunized to washed 
sheep blood cells. 

Apparatus. Sterile physiological salt solution; glass 
beads; small glass funnel; two 200 c.c. Erlenmeyer flasks; 
five or six sterile centrifuge tubes; sterile 5 c.c. pipette, 
with rubber bulb attached for draining off serum and salt 
solution in centrifuge tubes; sterile 14-gage 2j-inch hypo- 
dermic needle ; sheep; rabbit. 

Note. Chemically pure salt and distilled water should be used in 
the preparation of salt solution for this work and for the complement 
fixation test. 

Method. 1. In one Erlenmeyer flask place eight or 
ten glass beads for defibrinating blood, plug and sterilize 
in hot air. 

Provide the other one with a small, sterile, glass funnel 
and two layers of sterile cheese-cloth for filtering and de- 
fibrinating blood. 

2. With an attendant holding the sheep, clip the wool 
over the area of the jugular vein and wash with 50% alcohol. 

3. Place the thumb over the jugular furrow about half 
way between the head and shoulder and press on the vein 
so that it will become distended. 



324 GENERAL MICROBIOLOGY 

4. Thrust the needle through the skin anterior to the 
thumb, into the vein and draw about 20 to 30 c.c. of blood 
into the flask containing the glass beads. 

5. Defibrinate by agitating three or four minutes and 
filter through two layers of cheese-cloth. 

6. Mix the blood with approximately an equal amount 
of sterile physiological salt solution and centrifuge in sterile 
centrifuge tubes. 

7. Draw off the supernatant fluid down to the corpuscles, 
using the sterile pipette with bulb attached. 

8. Fill the tube with sterile salt solution and mix 
thoroughly by pouring from one tube to another several 
times. 

9. Centrifuge again and repeat 6 and 7, at least five times. 

10. Mix the washed corpuscles with an equal volume of 
sterile physiological s,alt solution, warm to body tempera- 
ture and inject 14 c.c. into the peritoneal cavity of a rabbit. 

11. Six or seven days later inject the rabbit intra-peri- 
toneally with 20 c.c. of a 50% suspension of washed sheep 
blood cells and again on the fourteenth day with 24 c.c. of 
a 50% suspension. 

12. About a week or ten days from the last injection 
draw a 'small quantity of blood from the rabbit, allow to 
clot, pipette off the serum and inactivate at a temperature 
of 56 C., for thirty minutes and titrate. (For method of 
titration, see Exercise No. 14.) 

Note. Care must be exercised in the above operations to avoid 
contaminating the blood cells and they must be thoroughly washed 
and injected on the same day they are drawn. 

13. State in full your results and any conclusions that 
may be drawn. 

REFERENCES 
See Exercise 14. 



THE COMPLEMENT FIXATION TEST 325 

EXERCISE 14. TO DEMONSTRATE THE COMPLEMENT 
FIXATION TEST 

The complement fixation test is one of the most com- 
plicated biological reactions used as a means of diagnosis 
in infectious diseases. 

Apparatus. Guinea pig; rabbit; sheep; suspected 
serum (from aborting cow or other animal to be tested); 
small test tubes; test-tube rack; flasks; physiological 
salt solution; centrifuge tubes; disinfectant; syringe and 
needles. 

Culture. Bad. abortus (or other organism depending 
on disease for which test is made). 

I. TITRATION OF REAGENTS 

Method. Four reagents other than the serum to be 
tested are required: 1, complement; 2, hemolysin; 3, red 
blood cells from a sheep; 4, antigen. Above components 1, 
2 and 4 must be titrated before using in order to deter- 
mine the amounts to be used in the tests. 

1. Complement. This is contained in and obtained from 
fresh serum from a guinea pig. The complement is titrated 
for the purpose of determining the least amount which in 
the presence of a sufficient amount of hemolysin will produce 
complete hemolysis of a definite quantity of washed red 
blood cells from the sheep. This amount is spoken of as 
the litre. 

2. Hemolysin (see Exercise 13). The source of hemo- 
lysin is inactivated serum from a rabbit that has been 
previously immunized to washed red blood cells from a 
sheep. 

The selection of a rabbit and sheep is merely a matter 
of convenience. Any two animals of a different genus 
may be used. In the test for syphilis in man, human 
blood cells are usually used because more convenient to 
obtain in a number of laboratories. 



326 



GENERAL MICROBIOLOGY 



TITRATION OF COMPLEMENT 



Tubes. 




i 


2 


3 


4 


5 


6 


7 


Salt solution 


(A) 


c.c. 

1.5 


c.c. 

1.5 


c.c. 

1.5 


c.c. 

1.5 


c.c. 

1 5 


c.c. 
1 5 


c.c. 

1 5 


Hemolysin 
Suspension of blood 
corpuscles 


() 
(C) 


0.1 
5 


0.1 
5 


0.1 
5 


0.1 
5 


0.1 
5 


0.1 
5 


0.0 
5 


Complement 


(D) 


0.02 


0.04 


0.06 


0.08 


0.1 


0.0 


0.1 


Result after one-half 
hour 


(K) 


- 


* 


+ 


+ 


_!_ 


- 


- _ 



Incubate all tubes in water bath at 37 C. for one-half hour and read. 

A. 0.9% salt solution. 

B. 1% dilution of inactivated immune rabbit serum in salt solu- 
tion. 

C. 1% suspension of washed sheep-blood cells in salt solution. 

D. 20% solution of fresh guinea pig serum in salt solution (0.4 
c.c. complement made up to 2 c.c. with salt solution). 

E. * A variation of reaction according to strength of complement, 
-f = complete hemolysis. 

=no hemolysis. 

The inactivation is accomplished by heating to a tem- 
perature of 56 C. for one-half hour to destroy the com- 
plement. If not used for several days it is not necessary 
to heat, > as complement is destroyed on standing. If it 
is to be kept for some time, preserve by adding 5% 
phenol sufficient to make a 0.5% solution. It is then 
titrated to determine the smallest quantity which will bring 
about a complete solution of the same quantity of washed 
sheep blood cells used in the titration of the complement, 
when in the presence of a proper quantity of complement. 

3. Antigen. Antigen is an extract of the specific bacteria 
made by growing the bacteria on agar and washing off with a 
few cubic centimeters of salt solution, and is preserved with 
phenol sufficient to make 0.5% and glycerin sufficient to 
make 1%. The suspension is placed in a shaking machine 
for three hours a day for three consecutive days to obtain 
homogeneity. 



THE COMPLEMENT FIXATION TEST 
TITRATION OF HEMOLYSIN 



327 



Tubes. 




l 


2 


3 


4 


5 


6 


7 


8 






c.c. 


c.c. 


c.c. 


c.c. 


c.c. 


c.c. 


c.c. 


c.c. 


Salt solution . . . 


(A] 


1.5 


1.5 


1.5 


1.5 


1.5 


1.5 


1.5 


1.5 


Hemolysin 


(B) 


0.01 


0.02 


0.04 


0.06 


0.1 


0.15 


0.15 


0.0 


Suspension blood 




















cells 


(O 


0.5 


0.5 


0.5 


0.5 


0.5 


0.5 


0.5 


0.5 


Complement. . . . 


(D) 


0.1 


0.1 


0.1 


0.1 


0.1 


0.1 


0.0 


0.1 


Result after one- 




















half hour 


(E) 


* 


* 


* 


+ 


+ 


+ 









Incubate all tubes in water bath at 37 C. for one-half hour and 
read. 

A. 0.9% salt solution. 

B. 1% dilution of inactivated immune rabbit serum in salt solution. 

C. 1% suspension of washed sheep-blood cells. 

D. Titrated guinea pig serum diluted so that 0.1 c.c. contains 
1.5 times the titre. 

E. + indicates complete hemolysis. 
indicates no hemolysis. 

* indicates a variation in the reaction according to the strength of 
the hemolysin. 

The smallest quantity causing complete hemolysis is called the 
titre. 

A titration of this reagent is made to determine the 
smallest quantity that will prevent hemolysis in the presence 
of 1.5 times the titre of complement and the hemolysin, 
sheep cells and immune serjim. In other words, we must 
determine the smallest quantity of antigen that will fix 
the amount of complement used in the test. 

TIRATION OF THE ANTIGEN 



Tubes. 




1 


2 


3 


4 


5 


6 


7 


8 

c.c. 
1.5 
0.00 
0.15 
0.1 


9 


10 


11 


Salt solution 
Positive serum . . . 


(A) 
(B) 
(C) 
(D) 


c.c. 
1.5 
0.02 
0.01 
0.1 


c.c. 
1.5 
0.02 
0.02 
0.1 


c.c. 
1.5 
0.02 
0.05 
0.1 


c.c. 
1.5 
0.02 
0.1 
0.1 


c.c. 
1.5 
0.02 
0.15 
0.1 


c.c. 

1.5 
0.02 
0.2 
0.1 


c.c. 
1.5 
0.02 
0.25 
0.1 


c.c. 
1.5 
0.00 
0.2 
0.1 


c.c. 
1.5 
0.00 
0.25 
0.1 


c.c. 
1.5 
0.00 
0.3 
0.1 


Complement 



Incubate for half an hour in a water bath at 37 C., then add the hemolytic sys- 
tem as follows: 



328 



GENERAL MICROBIOLOGY 



HEMOLYTIC SYSTEM 



Tubes. 




1 


2 


3 


4 


5 


6 


7 


8 


9 


10 


11 


Hemolysin 


(E) 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


c.c. 
1 


Suspension of blood 
cells 


(F) 


5 


5 


5 


5 


5 


5 


5 


5 


5 


5 


5 




























Results after in- 


























cubation 


(G) 


* 


* 

















+ 


+ 


+ 


+ 



Incubate half an hour in a water bath at 37 C. and then keep in the ice box for 
twelve hours and read. 

A. 0.9% salt solution. 

B. Inactivated serum known to contain antibodies. 

C. Suspension of a culture of suspected bacteria, carbolized. 

D. Titrated guinea pig serum diluted so that 0.1 c.c. contains 1.5 times the 
titre. 

E. Immune rabbit serum of known titre diluted so that 0.1 c.c. contains three 
times the titre. 

F. 1% suspension of washed sheep blood cells in salt solution. 

G. * signifies a variable reaction according to the activity of t 
+ signifies a complete hemolysis. 

signifies no hemolysis. 

The smallest quantity of antigen (in combination with antibody) that com- 
pletely fixes the complement is known as the litre. 



ity of the antigen. 



II. COMPLEMENT FIXATION TEST. (Test proper) 

1. Suspected Serum. 'This is drawn from' the animal 
that is suspected of being infected with the infectious 
disease in question. 

The blood is drawn from the jugular vein. It is allowed 
to clot and the serum collected. It must be inactivated 
before testing unless it is to be held for a week or more 
before applying the test, in which case inactivation is not 
necessary, but 1% phenol should be added as a preserva- 
tive. 

Note. The test proper and controls must be run at the same time. 
If several tests are run at the same time one set of controls is sufficient. 

2. Test of Suspect Serum. 



Tubes. 






Test Proper. 


Controls. 


1 


2 


3 


4 


1 


2 


3 


4 


Salt solution 
Suspect serum. . . 


(A) 

(B) 
(C) 
(D) 


::;:: 


c.c. 
1.5 
0.1 
0.1 
0.1 


c.c. 
1.5 
0.02 
0.1 
0.1 


c.c. 
1.5 
0.04 
0.1 
0.1 


c.c. 
1.5 
0.04 
0.1 
0.0 


c.c. 
1.5 
0.0 
0.0 
0.0 


c.c. 
1.5 
0.0 
0.0 
0.1 


c.c. 
1.5 
0.0 
0.1 
0.1 


c.c 
1.5 
0.0 
0.1 
0.0 


Complement 



Incubate one-half hour at 37 C, in water bath. 



THE COMPLEMENT FIXATION TEST 



329 



3. Then add the hemolytic system. 



Tubes. 






Test Proper. 


Controls. 


1 


2 


3 


4 


1 


2 


3 


4 


Blood cells 
Hemolysin . . . 


(E) 

(F) 




c.c. 
0.5 
0.1 


c.c. 


c.c. 


c.c. 


c.c. 


c c 


c c 


c.c. 
0.5 
0.1 


0.5 
0.1 


0.5 
0.1 


0.5 
0.1 


0.5 
0.0 


0.5 
0.1 


0.5 
0.1 







Incubate one-half hour at 37 C. in water bath and then place in ice box for 
twelve hours and read. 

A. Salt solution, 0.9%., 

B. Suspect ser\im inactivated for one-half hour. 

C. Antigen, two times titre. 

D. Complement, 1.5 times titre. 

E. 1% washed sheep blood corpuscles in salt solution. 

P. Immune rabbit serum (hemolysin) diluted so that 0.1 c.c. contains three 
times the titre. 

Control tubes 2 and 3 should show complete hemolysis. 
Control tubes 1 and 4 should show complete absence of hemo- 
lysis. Control tube 4 is control on the inactivation of the 
suspect's serum and should show absence of hemolysis. 

Hemolysis in the other tubes indicates the absence of 
antibodies in sufficient quantity in the amount of serum 
used to fix complement. 

4. Give all results in detail and draw conclusions. 

REFERENCES 

MOHLER, J. R. and EICHHORN, A.: The diagnosis of glanders by 
complement fixation test. Bui. 136, B. A. I., U. S. Dept. Agr. 
(1911). 

SURFACE, F. M.: The diagnosis of infectious abortion in cattle. 
Bui. 166, Kentucky Agr. Expt. Sta. (1912). 

HADLEY, F. B. and BEACH, B. A.: The diagnosis of contagious abor- 
tion in cattle by means of the complement fixation test. Res. 
Bui. 24. Wis. Agr. Expt. Sta. (1912). 

MOHLER, JOHN R., EICHHORN, A. and BUCK, J. M.: The diagnosis 
of dourine by complement fixation. Jour. Agr. Res. Bui., Vol. I 
(1913), pp. 99-109. 

KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 146, 
164, 185, 316-501, 847-863. 

ZINSSER: Infection and Resistance (1914), pp. 134-217. 



APPENDIX 
OUTLINE FOR THE STUDY OF MICROBIOLOGY * 



I. MORPHOLOGICAL AND CULTURAL MICRO- 
BIOLOGY 

A. Morphology and Development. 

1. Gross anatomy. 

a. Form. 

b. Size. 

c. Arrangement or grouping. 

d. Multiplication. 

e. Involution, variability and mutation. 

2. Histology of cell. 

a. Wall or outer membrane. 

b. Capsule. 

c. Protoplasm, beaded forms, granules. 

d. Nuclear material. 

e. Flagella and motion. 
/. Spores. 

3. Classifications and their basic features, 

B. Cultural Significance. 

1. Media. 

a. For morphologic and developmental studies. 

b. For cultural effects. 

2. Colonies. 

3. Cultural features. 

4. Biochemical features. 

* Adapted from Marshall, vide 43d Annual Report of Michigan 
State Board of Agriculture. 

331 



332 APPENDIX 

C. Staining Values. 

1. Demonstrations of parts of cell. 

2. Identification of species. 

3. Differentiation of species. 

D. Determination of Microorganisms. 

1. Methods employed. 

2. Differential characteristics. 

II. PHYSIOLOGIC MICROBIOLOGY 

A. Cell Studies. 

1. Composition of cell contents. 

2. Composition of cell wall. 

3. Physical products of physiological significance. 

a. Heat. 
6. Light. 

4. Products of physiological significance of which little is 

known, 
a. Pigment. 
6. Enzymes. 

c. Aromatic compounds. 

d. Toxins. 

5. Absorption or assimilation of foreign bodies. 

6. Chemotaxis. 
5. Phototaxis. 

8. Aerotaxis, 

9. Plasmolysis. 
10. Plasmoptysis. 

B. Studies in Metabolism. 

1. Elements required in growth of microorganisms. 

2. Respiration. 

3. Nutrition. 

4. Moisture. 

5. Temperature of cultivation. 

6. Conditions of media; reaction, composition, etc. 



APPENDIX 333 

7. Physiologic test media. 

8. Identification and determination of species of micro- 

organisms by means of 

a. Cultural physiologic methods. 

b. Chemical tests. 

c. Physical tests. 

d. Biological tests. 

9. Enzymes. 

C. Studies in Association. 

1. Symbiosis. 

2. Metabiosis. 

3. Antibiosis. 

D. Common Fermentative Changes Produced by Micro- 

organisms. 

1. Studies in enzymes. 
a. Formation. 
Zymogen. 
Activator. 
Kinases. 
6. Kinds. 

c. Actions (specificity) and materials fermented. 

d. Conditions under which enzymes act; 

(1) Physical. 
Temperature. 

Radiation, light rays (solar, electric, etc.), 
Rontgen rays, radium rays and emana- 
tions. 

(2) Chemical and physico-chemical. 
Activators; kinases, organic acids, bases, 

neutral salts. 
Protective agents. 
Paralysors and poisons. 
Concentration of solutions. 
Reaction of substrate. 
Extent of accumulated products. 



334 APPENDIX 

2. Products manufactured by fermentation. 

a. Necessary and limiting conditions of production. 

b. Most favorable conditions of production. 

c. Methods of determination. 

Qualitative. 
Quantitative. 

d. Constancy and variability of products. 

e. Gradation in fermentation changes. 

Intermediate products. 
Ultimate products. 

E. Products Significant through the Intermediation of a 

Host. 

1. Antigens.* 

a. Cells. 

b. Cell products. 

Toxins, diffusible and endotoxin. ' 

Bacterial proteins. 

Enzymes. 

2. Antibodies.* 

a. Antitoxin. 

b. Agglutinins. 

c. Precipitins. ^ 

d. Opsonins. 

e. Aggressins. 
/. Cytolysins. 

g. Anaphylactins. 

F. Influencing Agents and Their Effects. 

1. Light. 

a. Direct. 
6. Diffuse. 
c. Special. 

*See p. 164, Kolmer's Infection, Immunity and Specific Therapy 
(1915). 



APPENDIX 335 

d. Phototropism. 

e. Phototaxis. 

2. Temperatures. 

a. Heat. 

Direct flame. 

Dry. 

Moist. 

Steam under pressure. 
6. Cold. 

c. Thermotaxis. 

d. Thermotropism. 

3. Electricity. 

4. Desiccation. 

5. Mechanical pressure. 

6. Mechanical agitation. 

7. Gravity. 

8. Chemicals. 

a. Chemotropism. 

b. Chemotaxis. 

c. Concentrated solutions. 

d. Antiseptics, disinfectants, 

III. HYGIENIC MICROBIOLOGY 

A. Communicable Diseases of 

1. Man and animals. 

a. Causal agent or microorganism. 
6. History of microorganism. 

c. Vitality or persistency of microorganism. 

d. Means of dissemination and avenues of infec- 

tion. 

e. Distribution of microorganism in body. 
/. Management of disease. 

g. Prevention of disease. 

h. Care of dead from communicable diseases. 



336 APPENDIX 

B. Surgical Significance. 

1. Wounds. 

2. Abscesses. 

3. Septicemia and pyemia. 

4. Malignant growths. 

5. Operations. 

C. Susceptibility and Immunity. 

1. Natural. 

a. Race. 

b. Species. 

c. Age. 

d. Individual idiosyncrasies. 

e. Body components. 

2. Acquired, active or passive. 

a. Devitalization. , 

b. Hereditary predisposition. 

c. One attack of disease. 

d. Vaccines. 

e. Bacterins. 
/. Toxins. 

g. Other bacterial products. 

D. Serum Therapy Microbial Therapeutics 

1. Diagnostic agents. 

a. Tuberculin. 

b. Mallein. 

c. Bacterial suspensions. 

d. Diphtheria toxin (Schick). 

e. Luetin. 

2. Remedial agents. 

a. Antitoxins. 

b. Serums. 

c. Vaccines. 

d. Bacterins. 



APPENDIX 337 

E. Disinfection and Antisepsis. 

1. Agents employed. 

a. Mode of action. 

2. Determination of values, phenol coefficient. 

3. Methods. 

F. Sanitary Studies. 

1. Water analysis. 

a. Methods. 

b. Interpretation of results. 

2. Water contamination and filtration. 

3. Sewage analysis. 

a. Methods. 

b. Interpretation of results. 

4. Sewage' destruction. 

a. Aerobic filtration. 

b. Anaerobic septic tank. 

c. End products. 

5. Ventilation. 

a. Currents as means of dissemination. 

b. Filtration and washing of air. 

c. Germ content of air. 

d. Methods of analysis. 

e. Interpretation of results of analysis. 

6. Foods. 

a. Poisonous. 

b. Infected. 

/ 
IV. DAIRY 

A, Milk Supply. 

1. Communicable diseases conveyed through milk. 

a. Kinds of microorganisms. 

b. Avenues of transmission. 

c. Prevention. 



338 APPENDIX 

2. Environment of animals and conditions of milk- 

ing. 

a. Stabling. 

b. Feeding. 

c. Milker. 

d. Utensils. 

3. Bacterial content of milk in udder. 

a. Non-pathogenic microorganisms. 

6. Pathogenic microorganisms and antibodies. 

c. Conditions of growth in udder. 

d. Abnormal udders. 

4. Bacterial action on constituents of milk. 

a. Proteins. 

b. Butter fat. 

c. Lactose. 

d. Mineral constituents. 

5. Analysis Of air of stables. 

a. Before cleaning. 

b. Immediately after cleaning. 

c. Before feeding. 

d. Immediately after feeding. 

e. Analysis of out-door air. 

6. Determination of value of staining. 

7. Determination of value of aeration. 

8. Determination of value of cooling. 

a. Simple cooling. 

b. Cooling and keeping cool. 

c. Cooling and warming, then cooling. 

9. Cleansing of utensils. 

a. Methods and their values. 

b. Water analysis. 
10. Milk control. 

B. Pigment in Milk and Cheese. 

1. Kinds. 

2. Character. 



APPENDIX 339 

3. Condition of formation. 

4. Control. 

C. Fermentations in Milk, Butter and Cheese. 

1. Kinds. 

a. Lactic. 

b. Butyric. 

c. Alcoholic. 

d. Gaseous. 

e. Peptic. 
/. Rennet. 
g. Ropy. 
h. Soapy. 
i. Taints. 

Bitter flavor, barn-yard, tallowy. 
j. Special. 

Kephir, koumiss, matzoon, leben, yoghurt, etc. 
k. Natural enzymes (galactase). 
I. Antibody formation (agglutinins, etc.). 

2. Microorganism involved. 

a. Its life history. 

3. Nature of fermentation. 

4. Constituents acted upon. 

5. Products. 

6. Conditions influencing it. 

7. Controlled or fostered. 

D. Pasteurization and Sterilization. 

1. Determination of significance of each. 

2. Methods employed. 

3. Practical utilization. 

E. Starters. 

1. Natural. 

a. Sour milk. 
6. Sour cream. 
c. Buttermilk. 
(J, Others, 



340 APPENDIX 

2. Artificial. 

a. Pure cultures. 

b. Mixed cultures. 

3. Value determined. 

4. Preparation. 

5. Employment. 

6. Constancy. 

7. Influencing conditions. 

8. Facts governing amounts to employ. 

F. Butter. 

1. Microorganisms present. 

2. Microorganisms compared with those of milk. 

3. Environmental condition for bacterial life changed. 

4. Quality. 

a. Influenced by pasteurization of the cream. 

b. Influenced by growth of microorganisms. 

c. Factors influencing stability. 

d. Methods of preservation. 

5. Decomposition. 

a. Products. 

b. Factors influencing. 

c. Correlation between the presence of certain 

groups of organisms and specific flavors. 

6. Significance of casein and buttermilk in butter. 

G. Cheese. 

1. Kinds of microorganisms employed in different 

cheeses. 

2. The study of microorganisms in the ripening process. 

3. Influence of microorganisms on aroma and flavor. 

4. Keeping values. 

H. Preservatives. 

I. Disinfectants utilized. 



APPENDIX 341 

V. SOIL 

A. The Making of Soil. 

1. Microorganisms in soil. 

a. Number at different depths and in different 

soils. 

b. Kinds at different depths and in different soils. 

c. Character of microorganisms found. 

d. Rate of growth. 

2. Disintegration of inorganic material. 

3. Decomposition of organic material. 

a. Celluloses. 

6. Starches and sugars. 

c. Proteins, etc. 

4. Action of iron and sulphur bacteria. 

B. Ammonification. 

C. Nitrification The nitroso- and nitro-processes. 

1. Conditions influencing. 

a. Physical. 

b. Reaction. 

c. Temperature. 

d. Supply of oxygen. 

e. Amount of organic matter present. 
/. Moisture. 

D. Denitrification. 

1. Factors influencing the loss of nitrogen. 

E. Nitrogen Fixation. 

1. Symbiotic. 

2. Nonsymbiotic (aerobic and anaerobic). 



342 APPENDIX 

VI. PLANT 

A. Nitrogen Accumulators. 

1. Microorganism involved. 

2. Cultural characteristics. 

3. Formulation of nodules. 

4. Character of nodules. 

5. Conditions under which they form. 

6. Determination of nitrogen accumulations. 

7. Significance of nodules. 

B. Microbial Diseases. 

1. Kinds. 

2. Microorganisms found as causal agents. 

3. Cultural characteristics. 

4. Resistance of microorganisms. 

5. Persistency. 

6. Methods of treatment. 

7. Pathology. 

C. Microbial Decomposition of Fruits, Vegetables and 

Other Plant Substances. 

1. Nature. 

2. Microorganism studies. 

3. Conditions favoring. 

4. Control. 

5. Structural changes. . 

VII. FERMENTATION 
A. Factors Controlling Fermentations. 

1. Presence of microorganism. 

2. Purity of culture. 

3. Vigor of cell. 

4. Character of fermentable material. 

5. Air supply. 



APPENDIX 343 

6. Reaction of medium. 

7. Temperature. 

8. Concentration of fermentation solutions. 

9. Concentration of products of fermentation. 

B. The Production of Enzymes by Microorganisms. 

1. Formation of enzyme in cell. 

2. Its secretion by the cell. 

3. Determinative methods for study. 

4. Environmental influences. 

C. The Fermentations. 

General. 

1. The Enzymes. 

a. Hydrolytic of 

Carbohydrates = Carbohydrases. 

Cellulases. 

Hemicellulases. 

Glycogenases. 

Dextrinases. 

Inulinase. 

Saccharase. 

Lactase. 

Maltase. 

Trehalase. 

Raffinase. 

Amygdalase. 

Tannase. 

Pectase, etc. 
Fats = Esterases. 

Lipases of natural fats. 

Stearinases, etc. 
Proteins = Proteinases. 

Peptases. 

Tryptases. 

Ereptases, etc. 



344 APPENDIX 

b. Producing intramolecular changes, acting on 

Carbohydrates, to form alcohol and C02. 

Zymases of d-dextrose, d-levulose, etc. 
Carbohydrates to form lactic acid. 

Lactic acid-bacteria zymase. 
Acid amides = amidases. 

Urease. 

c. Oxidizing = oxidases. 

Alcoholase. 

Lactacidase. 

Acetacidase. 

Tyrosinase. 

Laccase. 

d. Reducing = Reductases. 

Catalase. 

Peroxidase. 

Methylen blue, indigo and azolitmin reduc- 

tase. 

Perhydridase. 
Sulphur reductase. 
Nitrate and nitrite reductase, etc. 

e. Coagulating. 

Caseinase. 
Parachymosin. 
Thrombase. 
Pectinase. 

2. Materials Acted Upon. 

a. Celluloses. 

6. Starches. 

c. Sugars. 

d. Fats. 

e. Proteins. 

/. Organic acids, etc. 
g. Alcohols. 

3. Products resulting. 



APPENDIX 345 

Special. 

1. Alcoholic. 

a. Beer and distilled liquors. 

6. Wine, cider and other fermented fruit juices. 

c. Ginger beer. 

d. Koumiss, etc. 

2. Acetic acid. 

a. Vinegar. 

b. Mashes. 

c. Foods. 

I 

3. Lactic acid. 

a. Milk. 

b. Mashes. 

c. Foods, sauer kraut, brine pickles, etc. 

d. Ensilage. 

4. Butyric acid. 

a. Milk. 

b. Mashes. 

c. Foods. 

5. Ammoniacal. 

a. Urea, uric and hippuric acid. 

b. Proteins and their nitrogenous fractions. 

6. Proteolytic. 

a. Proteins, albumins. 

b. Proteoses, albumoses. 

c. Peptones. 

d. Peptids. 

e. Amino-acids. 

/. Amins and other ammonia derivatives. 

g. Ptomains. 

h. Leucomains. 

i. Non-nitrogenous organic acids. 

7. Alcohols. 

k. Ammonia, H 2 S, and other gases. 



346 APPENDIX 

7. Nitrification. 

8. Denitrification. 

9. Ammonification. 

VIII. FOOD AND DRINK PRESERVATION 

A. Preservation of Foods. 

1. Freezing. 

2. Cold storage. 

3. Salting. 

4. Drying, evaporating or concentrating. 

5. Smoking. 

6. Corning. 

7. Canning. 

8. Chemical preservatives or antiseptics. 

9. Preserving. 

10. Pressure. 

11. Fermentations. 

B. Preservation of Drinks. 

1. Pasteurizing and sealing. 

2. Cold storage. 

3. Chemical preservatives. 

4. Carbonating. 

5. Filtration. 

6. Fermentation. 



APPENDIX 



347 



CLASSIFICATION OF MIGULA (MODIFIED) 



Order. 


Family. 


Genus. 


Species. Variety- 






Streptococcus. . . 1 








division in 1 1 


pyogenes 






plane, no fla- 1 


erysipelatus 






geUa 








Micrococcus. 1 


t>'i/TCLQ^71AJLS 






division in 2 1 


( niivoitQ 






planes, no fla- 


\ u u/r t/ wo 

Py9 e s (albus 






geUa 






CoccacecB 


Sarcina ] 






round 
forms 


division in 3 1 
planes, no fla- , 


lutea 






gella J 


1 






Planococcus .-..:. 1 








division in 2 


agilis 






planes, flagella J 




I. 




Planosarcina . . . 




EUBACTERIA 

(true 




division in 3 
planes, flagella > 


' mobiiis 


bacteria) 




t 


lactis acidi 






Bacterium 


bulgaricum 






(straight rods) < 


aerogenes 


Suborder. 




(non-flagellate) 


abortus 








. tuberculosis 


A. 

Haplobacte- 


Bci ctarLcL CBCB 


Bacillus 


fluorescens lique- 
faciens 


TITiOB. 

(lower bac- 


rod forms. 


(straight rods) 
(flagellate) 


mycoides 
prodigiosus 
typhosus 


teria.) 






coli 






Pseudomonas. . . ' 








(straight or ir- 


radicicola 






regular rods, 


> campestris 






polar flagella) 








Spirosoma ' 








comma to spi- 


> nasals 




SpirillaceoB 
curved 


ral forms, stiff, 
no flagella. 






(comma) or 


Microspira 






spiral 
forms. 


comma- 
shaped, simple 


comma 
deneke 






curve, general- 
ly polar fla- 


finkleri 






gella. 





348 



APPENDIX 



CLASSIFICATION OF MIGULA (MODIFIED) Continued 



Order. Family. Genus. Species. 


I. 




Spirillum 


EUBACTERIA 




cork screw, sev- 


(true bact.) 
Suborder. 


Spirillaceoe 
curved 


eral turns, non- rubrum 
flexible spiral, 


A. 


(comma) or 


polar flagella. 


Haplobacte- 


spiral 


Spirocheta 1 


rincK 


forms. 


flexible spirals, 1 O 5 ema - er 


(lower bac- 




motile, no fla- ) 


teria). 




gella. J 


See pp. 10 and 56-62, Marshall's Microbiology. 






Chlamydothrix 


T 


Chlamydo- 


unbranched threads, uniform in 


JL 

EUBACTERIA 
(true bacte- 


bacteriacece 
cylindrical 


diameter. 

Crenothrix 


ria). 
Suborder 

B. 

Trichobacte- 
rinoB (higher 
bacteria). 


cells in 
threads, en-- 
sheathed; 
reproduc- 
tion by mo- 
tile and non- 
motile go- 
nidia. 


unbranched threads, filaments en- 
larged at free end. 
Phragmidiothrix 
branched and unbranched filaments. 
Cell division in 3 planes. 
Cladothrix 
dichotomous branching, uniform di- 






ameter. 




Beggiatoacece 


' Thiothrix. 
threads, non-motile and attached; 




cells con- 
tain sul- 


sheath; gonidia. 




phur gran- 
ules. 


Beggiatoa 
no sheath, flat cells, motile with un- 






dulating membrane; no gonidia. 


II. 




{Thiocystis 


THIOBACTE- 


Rhodobacteri- 


Thiocapsa 


RIA (sul- 


acece 


Thiosarcina 


phur bac- 
teria). 


cells con- 
tain bacte- 
riopurpurin, 


2 Lamprocystis 
3 Thiopedia 




sometimes 


(Amcebobacter 




sulphur 


Thiothece 




granules. 


Thiodictyon 




5 sub-fami- 


f Chromatiwn 




lies. 


5 \ Rhabdochromatium 






( Thiospirillum 



APPENDIX 349 

SPECIAL MEDIA 

Litmus lactose agar for demonstrating acid production 
of microorganisms: Prepared the same as ordinary nutrient 
agar (see Exercise 9, Part I), with the exception that 1% 
lactose and 2% of the standard azolitmin solution is added 
just after filtration, while the agar is still hot, and well 
mixed through the agar before tubing. Sterilize by Tyndall 
method. 

Dextrose calcium-carbonate agar for showing acid for- 
mation by microorganisms: Prepared the same as ordinary 
nutrient agar, with the exception that 1% dextrose and 1% 
CaCOs are added to the hot agar just after filtration. The 
added chemicals must be mixed well through the agar and 
care must be taken during tubing that the CaCOs remains 
in homogeneous suspension throughout the medium. Ster- 
ilize by discontinuous method. 

Sour whey for determining the acid-destroying power 
of microorganisms: Inoculate sweet milk with a pure active 
culture of Bad. lactis acidi or Bact. bulgaricum as desired, 
and place at about 30 C. Allow the maximum acidity to 
form, cut the curd and heat in flowing steam for twenty 
or thirty minutes. Strain through clean cheese-cloth and 
allow to drain. Filter through filter paper. If clear whey 
is desired, it will be necessary to clear the medium with 
egg albumin. 

Butter fat for demonstrating fat decomposition: Melt 
butter at about 100 C. and allow the casein to settle. 
Decant the clear fat, place about 8 c.c. in sterile test tubes 
and sterilize by the intermittent method. 

Other kinds of fat may be prepared similarly. 

Fermented agar for making solid synthetic media and 
for testing food requirements and selective powers of bac- 
teria: 1. Place a weighed amount (three parts) of agar in 
a large bottle and to this add 200 parts of distilled water. 

2. Cover the mouth of the bottle with parchment paper 



350 APPENDIX 

or several layers of clean cheese-cloth and allow to ferment 
spontaneously. 

3. Change the water in the bottle occasionally, replacing 
the amount of water removed, with the same amount of 
clean, distilled water. 

4. When the active fermentation (as noted by the evolu- 
tion of gas) has ceased entirely, this agar should be placed 
in an agateware pail, counterpoised, boiled over a free 
flame to dissolve the agar, counterpoised and any loss made 
up with distilled water. 

5. Place in tubes or flasks as desired and autoclav. 

Uschinsky's asparagin medium : protein-free. 

Asparagin, COOH - CH(NH 2 ) CH 2 CO - NH 2 . . 3.4 gms. 

Sodium chloride, NaCl 5.0 gms. 

Magnesium sulphate, MgS04 0.2 gm. 

Calcium chloride, CaCl 2 0.1 gm. 

Monobasic acid potassium phosphate, KH 2 PO4. 1 . gm. 

Iron sulphate, FeSO4 Trace 

Distilled water , , , 1000.0 c.c. 

Cohn's solution: inorganic nitrogen combined with an 
organic acid. 

Monobasic acid potassium phosphate, KH 2 P04. 5 . gms. 

Calcium phosphate, CasPCU 0.5 gm. 

Magnesium sulphate, MgSCU *. 5.0 gms. 

Ammonium tartrate, CH(OH) COO NH 4 .... 10 . gms. 

CH(OH)-COO-NH 4 
Distilled water 1000.0 c.c. 

Winogradski's medium for nitrate formation: inorganic 
nitrogen combined with inorganic acid. 

Ammonium sulphate, (NH4) 2 S04 0.40 gm. 

Magnesium sulphate, MgSO4 . 05 gm. 

Dibasic acid potassium phosphate, K 2 HP04 . . 0.10 gm. 



APPENDIX 351 

Sodium carbonate, Na2COs . 60 gm. 

Calcium chloride, CaCk Trace 

Distilled water 1000 . c.c. 

Winogradski's medium for symbiotic nitrogen-fixation: 

nitrogen-free. 

Dibasic acid potassium phosphate, K2HP04. . 1.00 gm. 

Magnesium sulphate, MgS04 . 50 gm. 

Sodium chloride, NaCl 0.01 gm. 

Ferric sulphate,Fe 2 (864)3 - 0.01 gm. 

Manganese sulphate, MnS(>4 ,. . . 0.01 gm. 

Dextrose, CH 2 OH(CHOH) 4 CHO 20 . 00 gms. 

Distilled water 1000.00 c.c. 

Gelatin for cultivating phosphorescent halophilic or- 
ganisms: Prepared as ordinary gelatin with the addition 
of 3% salt. The reaction is made -20. 

Fermented cider for the cultivation of acetic bacteria: 
Inoculate unfermented cider with Sacch. ellipsoideus and 
allow to proceed until the evolution of gas ceases. Filter, 
place in tubes and flasks as desired. Pasteurize. 

MEDIA FOR SOIL MICROBIOLOGY. 

Soil extract: 1. Boil 1 kg. of good rich garden soil 
with 2 liters of tap water for two hours over the free flame. 

2. Pour off the turbid liquid, mix some talc- and filter 
through a double filter paper. If the first filtrate is turbid 
refilter through the same paper. 

3. Make up to 800 c.c. with tap water. 

4. Place in tubes or flasks as desired and autoclav. 
Soil extract agar is prepared by adding 1.5% washed 

agar to the soil extract prepared as above. 

Soil may be plated either in soil extract agar (or other 
special agar) or in ordinary agar, gelatin, etc. On account of 
the diversity of the requirements of the various species of 



352 APPENDIX 

microorganisms in soil, no one medium will suffice for the 
cultivation of all species. Emphasis is therefore not laid 
on any particular medium for plating soils. 

Omeliansky's medium for anaerobic cellulose fer- 
mentation : 

Filter paper (in strips). Cotton, straw, or 

starch may be substituted for filter paper . 2.0 gms. 

CaC0 3 .. 20.0 gms. 

K 2 HPO 4 . . 1.0 gm. 

MgS0 4 0.5 gm. 

(NH 4 ) 2 SO 4 1 .0 gm. 

NaCl Trace 

Distilled water 1000.0 c.c. 

Method. 1. Introduce substances in order named 
into 1000 c.c. distilled water. 

2. Stir to dissolve all soluble substances and tube while 
insoluble substances are in homogeneous suspension, plac- 
ing about 10 c.c. in each tube. 

3. Sterilize in autoclav. 

Media for studying urea decomposition: Urea broth, 
gelatin and agar are generally prepared by adding 1% to 
2% urea to the ordinary media. This medium favors the 
growth of B. coli, B. proteus, B. erythrogenes, etc. 

Ordinary media to which 10% urea has been added favors 
the growth of B. pasteurii, a spore-producing bacterium. 

Urea gelatin and agar may be prepared by adding 1 
c.c. of a 15% aqueous solution of urea to each tube of the 
ordinary media after sterilization, and then heating the 
tubes again. This is the method preferred because the 
addition of urea reduces the solidifying power of the gelatin. 
A small amount of urea is converted into ammonia by 
heating in the steam, but this has little influence on the 
results obtained in the experiment. Heating in the auto- 
clav is to be avoided! 



APPENDIX 353 

Albuminoid-free culture solutions for studying urea 
decomposition : 

I. Soil extract 100 c.c. 

K 2 HP0 4 O.OSgm. 

Urea 5 . 00 gms. 

II. Sohngen's solution. 

Tap water 100.00 c.c. 

Urea 5 . 00 gms. 

K 2 HPO 4 . 0.05gm. 

Ammonium or calcium malate, 

or, calcium citrate or tartrate. 0. 50 to 1.00 gm. 

B. pasteurii will not grow in these solutions as it requires 
the presence of albuminoids in the medium. 

Solutions for cultivating nitrifying bacteria: 

I. Distilled water 1000.0 c.c. 

(NH 4 ) 2 SO 4 1.0 gm. 

K 2 HP0 4 1.0 gm. 

MgS0 4 .... 0.5gm. 

NaCl 2.0 gms. 

FeS0 4 0.4gm. 

Add basic MgCO 3 after sterilizing. 
This solution is adapted for relatively increasing the 
nitrite bacteria. 

H. Distilled water 1000.0 c.c. 

NaN0 2 1.0 gm. 

K 2 HPO 4 0.5gm. 

MgSO 4 0.3gm. 

NaCl 0.5gm. 

Na 2 CO 3 0.3gm. 

This solution causes a greater relative increase in the 
nitrate producers. 



354 APPENDIX 

III. The same as solution I, but instead of MgC0 3 
CaCOs is added after sterilizing. This solution stimulates 
the simultaneous growth of both organisms, as in nature. 

Culture solutions for denitrification studies. Nitrate 
broth or agar. Add 1 c.c. of a 1% solution of sodium or 
potassium nitrate to tubes of ordinary broth or agar (melted), 
mix well and re-sterilize. 

Giltay's solution. 

KH 2 PO 4 . 2.0 gms. 

MgS0 4 . . 2.0 gms. 

KNO 3 -.... 1.0 gm. 

CaCl 2 0.2 gm. 

Fe2Cle solution 2.0 drops 

Citric acid 5.0 gms. 

Method. 1. Dissolve the above substances in 800 c.c. 
of distilled water (solution I). 

2. Add a few drops of phenolphthalein -and, using a 
pipette, drop in just enough 10% NaOH to turn the solution 
a faint pink. 

3. Dissolve 10 gms. dextrose in 200 c.c. of distilled water 
(solution II). 

4. Mix solutions I and II very thoroughly. 

5. Sterilize in the autoclav at 15 Ibs. pressure for ten 
minutes. (Lipman and Brown.) 

Giltay's agar is prepared by adding 1.5% washed agar 
to the above solution. Boil until dissolved. Filter through 
absorbent cotton. Sterilize in autoclav. 

Mannit solution for nitrogen-fixing organisms. 

Mannit 15.0 gms. 

K 2 HP0 4 0.2gm. 

MgSO 4 0.2gm. 

NaCl 0.2gm. 

CaSO 4 0.1 gm. 

CaC0 3 , 5.0 gms. 

10% FeCl 6 solution 1.0 drop 



APPENDIX 355 

Method. 1. Add -the above chemicals to 1000 c.c. 
distilled water. 

2. Titrate using phenolphthalein and neutralize using 
normal NaOH. 

Do not filter. The presence of CaCOs offers an additional 
means of isolating Azotobacter, as these organisms are found 
in soil in much greater numbers around the particles of 
calcium carbonate. 

3. Sterilize at 120 C. (autoclav), for ten minutes. 
Mannit agar is prepared by adding 1.5% washed agar 

to the above solution, boiling until the agar is wholly dis- 
solved and sterilizing as above. Do not filter. 

Nitrogen-free ash agar for cultivation of Ps. radicicola. 

1. Stir 5 gins, of wood ashes (beech, elm, maple) into 
1000 c.c. distilled water for two to three minutes only. 
Filter. 

2. Add 1% washed agar. 

3. Heat in steam for thirty minutes. 

4. Then add 1% commercial saccharose. 

5. Boil five minutes over a free flame. 

6. Strain while hot through several thicknesses of clean 
cheese-cloth. This may be filtered if desired. 

7. For Exercise 9, Soil Microbiology, tube, placing 
about 6 cm. of agar in the large test tubes with foot, the 
rest in ordinary test tubes. Sterilize. (Tyndall method.) 

Nitrogen-free solution may be prepared as above, 
omitting the agar. 

Congo-red agar for differentiating Ps. radicicola from Bact. 
tumefaciens: 

Distilled water 1000.00 c.c. 

Saccharose 10.0 gms. ' 

K 2 HP0 4 1.0 gm. 

MgSO 4 0.2gm. 

Washed agar 15.0 gms. 

Congo-red 0.1 gm. 



356 APPENDIX 

Solution for sulphate reduction: 

Tap water 1000.0 c.c. 

K 2 HPO 4 7. 0.5gm. 

Sodium lactate 5.0 gms. 

Asparagin 1.0 gm. 

MgSO 4 1.0 gm. 

A few drops of FeSCU solution. Sterilize in the auto- 
clav. 

WATER ANALYSIS MEDIA 

Culture media for standard bacteriological water anal- 
ysis must contain ingredients of a special nature. 

Ingredients. 1. Distilled water in place of tap water. 

2. Infusion of fresh lean meat instead of meat extract. 

3. Witte's peptone (dry, from meat) . 

4. No salt. 

5. Gelatin of the best French brand and as free as pos- 
sible from acids and other impurities. 

6. Commercial agar of as high a grade of purity as 
possible. Agar may be purified by washing. 

7. Dextrose, lactose, saccharose, etc., of sugar media, 
chemically pure. 

8. A 1% aqueous solution of Kahlbaum's azolitmin 
may be used in place of litmus. 

Sterilization. Sterilize media in the autoclav at 
120 C. (15 Ibs. pressure) for fifteen minutes. A shorter 
period than this may result in incomplete sterilization, a 
longer period will probably result in inversion and cara- 
melization of the sugars and in lowering the melting-point 
of the gelatin. Have the sterilizer hot when the medium is 
inserted so that heating to the point of sterilization will be 
accomplished as quickly as possible; cool rapidly upon re- 
moving from the autoclav. 

The Tyndall (intermittent) method may be employed, 
heating for thirty minutes on three successive days, 



APPENDIX 357 

Reaction. Phenolphthalein is used as indicator. 

Titrate media while hot with N/20 NaOH and adjust 
the reaction if necessary. All media should have a +10 
reaction Fuller's scale unless otherwise stated in the direc- 
tions. 

Distribution of Work. It may be desirable to have 
students work in groups in preparing media. The fol- 
lowing plan has worked satisfactorily: 

Students may work in groups of five, one of the groups 
preparing a sufficient quantity of medium for himself and_the 
other four members of the group, dividing the work up as 
follows : 

One student prepare agar shakes and litmus milk. 

One student prepare gelatin. 

One- student prepare litmus lactose agar. 

One student prepare litmus lactose bile. 

One student prepare Dunham's solution and nitrate 
peptone solution. , 

In this arrangement each student must furnish the re- 
spective sterile glassware sufficient for containing the various 
necessary media, to the student preparing each medium. 

Each student of the group must so plan his work that the 
medium he prepares will be finished, sterilized and ready for 
use at the same time as those of the remaining members of 
his group. 

Media. Litmus lactose agar shake. 

1. 2% washed agar. 
2% peptone. 
2% lactose. 

2% azolitmin. 

500 c.c. meat infusion. 

500 c.c. distilled water. 

Method. 1. Strain the meat infusion through a piece 
of clean cheese-cloth. 

2. Place the washed agar in the distilled water, weigh, 



358 APPENDIX 

digest over a free flame, weigh again and make up any loss 
with distilled water. 

3. To the hot agar add the peptone and lactose and mix 
until dissolved; then add the strained meat infusion. 

4. Titrate and adjust the reaction to 0. 

5. Add the azolitmin, boil up over the free flame and 
place about 100 c.c. in sterile 250 c.c. Florence flasks. 

Each student will need four litmus lactose agar shakes. 

II. Litmus lactose agar. (To be used in tubes for plat- 
ing only.) 

1.5% agar. 

1 . 0% peptone. 

1.0% lactose. 

2 . 0% azolitmin solution. 

500 c.c. meat infusion. 

500 c.c. distilled water. 

Method. 1. This agar is prepared as ordinary nutrient 
agar making the reaction +10, adding the lactose and azo- 
litmin just before tubing. 

2. Tube and sterilize by the Tyndall method. 

Each student will need at least forty tubes of litmus lactose 
agar. 

III. Gelatin. 

15% gelatin. 

1% peptone. 
500 c.c. meat infusion. 
500 c.c. distilled water. 

Method. Prepare, tube and sterilize as for ordinary 
gelatin. 

Salt is omitted. Reaction +10. 

Each student will need forty or fifty tubes of salt-free gela- 
tin. 

IV. Sugar-free broths and sugar broths. (Neutral red 
dextrose broth.) 



APPENDIX 359 

Method. 1. Heavily inoculate a tube of sterile broth 
with B. coli and incubate at 37 C. 

2. Soak 1 Ib. finely chopped lean beef in 1000 c.c. dis- 
tilled water over night (twenty-four hours). 

3. Strain out the meat juice and make up to 1000 c.c. 
with distilled water. 

4. Pour the entire contents of the twenty-four-hour 
broth culture of B. coli into the meat juice and 

5. Incubate at 37 C. for twelve to sixteen hours, not 
longer. B. coli uses the fermentable substances, inosite 
(muscle sugar), dextrose, etc., as food, leaving the meat 
juice free from fermentable substances. // this action is 
allowed to proceed too long, poisonous decomposition products 
of the^ proteins are formed which will inhibit the growth of 
other microorganisms. 

6. Mix the peptone (1%) into a thin paste with as little 
water ' as possible and add to the twelve or sixteen-hour 
culture of B. coli in the meat juice. 

7. Heat in the autoclav for twenty minutes or in the 
steam for one hour. 

8. Titrate and make neutral to phenolphthalein. 

9. Boil over a free flame for three to five minutes. 

10. Add 1% dextrose and 10 c.c. of 0.5% solution of 
neutral red and stir until sugar is dissolved. 

11. Filter until clear. 

12. Fill ten fermentation tubes for each student. 

13. Sterilize in autoclav or in flowing steam. 

14. Other sugar broths are prepared by adding instead 
of dextrose, 1% of the sugar desired. 

Practically all sugar-fermenting organisms will ferment 
monosaccharides such as dextrose; comparatively few will 
ferment the disaccharides lactose, saccharose, etc. B. 
coli will ferment all three sugars to a greater or less extent. 
Bacteria of the typhoid group ferment none of the three and 
those belonging to the paratyphoid group ferment dex- 
trose but not lactose, therefore the use of lactose in culture 



360 APPENDIX 

media will inhibit to a great extent the growth of the last 
two groups and favor the development of the organisms 
of the B. coli group. This group is by far the largest, 
occurs most often and in greatest numbers in sewage and 
like 'material, therefore tests for this group are used as indi- 
cation of the presence of intestinal organisms in the material 
(water in this case) to be examined. 

V. Litmus lactose bile salt medium. 

Bile salts are invaluable for certain media used for 
water analysis as they inhibit organisms of practically all 
but the intestinal type. 

20 gms. peptone. 
5 gms. sodium taurocholate. 
10 gms. lactose. 
20 c.c. 2% azolitmin solution. 
1000 c.c. distilled water. 

Method. 1. Dissolve the bile salt and peptone in the 
water and boil. 

2. Add the lactose and sufficient azolitmin to give a 
distinct purple tint. 

3. Filter, fill into fermentation tubes and sterilize by 
intermittent method. 

Each student needs four litmus lactose bile salt fermentation 
tubes. 

VI. Esculin bile solution for B. coli test. 

10 . gms. peptone. 
5 . gms. sodium taurocholate. 
0.1 gm. esculin. 
. 5 gms. soluble iron citrate. 
1000.0 c.c. distilled water. 

Method. 1. Dissolve the ingredients in the order given, 
clear with egg albumen, tube and sterilize (see Prescott and 
Winslow's Elements of Water Bacteriology, 3d Ed., p. 279). 
This solution has a blue fluorescence. 



APPENDIX 361 

VII. Dunham's solution; twenty-five tubes for each stu- 
dent. (See p. 43.) 

VIII. Nitrate peptone solution; twenty-five tubes for 
each student. (See p. 44.) 

IX. Litmus milk; twenty-five tubes for each student. 
(See p. 25.) 

EXPLANATION OF TABLE ON PAGES 362-363 

Method. B. coli- and B. cholerce suis-like organisms: 
Place 5 c.c. of suspected water in each dextrose and liver 
broth fermentation tube and 50 to 100 c.c. in a litmus lactose 
agar flask. Incubate at 37 C. If gas appears in time of 
three days, make plating on Conrad-Drigalski's agar * from 
one showing most of gas production. Isolate different 
colonies on agar slants. From the growth on the agar 
slants inoculate different media to subgroup the organisms 
and consequently to identify them. 

B. typhosus: Hoffman and Fiske enrichment medium. 
Add to the suspected water 1.0% of nutrose; 0.5% of 
caffein; 0.001% of crystal violet. Incubate at 37 C. 
for not more than twelve to thirteen hours. Make Endo 
or Conradi-Drigalski agar plates. Isolate bluish colonies, 
transferring to agar slant, and identify. The Widal reac- 
tion should be used for the confirmatory test. 

(Data on pages 362-363 collected by 0. M. Gruzit.) 

* Other media for bacteriological water analyses will be found in 
the 1915 edition of the " Standard Methods for the Examination of 
Water and Sewage " published by the American Public Health Associ- 
tion, pp. 124-137. 

This publication is the standard work; references to special phases 
will be found in the bibliography following each chapter. 



362 



APPENDIX 




APPENDIX 



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364 APPENDIX 

COMMON DISINFECTANTS 

Mercuric chloride : (HgCl 2 ) White crystals. 

Synonyms: mercury bichloride, corrosive sublimate, 
bichloride of mercury. 

The stock solution (40% HgCk in HC1) is prepared by 
mixing 1 part mercuric chloride with 2.5 parts commercial 
hydrochloric acid. This dissolves readily and aqueous 
solutions of any desired dilution may be made from it much 
more quickly than by the use of the salt. 

The pure salt is soluble in 16 parts of cold water and 3 
parts of boiling water. 

Mercuric chloride 1 : 1000, the solution commonly used 
in the laboratory for disinfecting purposes, is prepared by 
adding to 2.5 c.c. of the stock solution, sufficient dis- 
tilled water to make 1000 c.c. of solution. 

As a germicide, mercuric chloride acts in solution by 
combining chemically with the protein of the microorganisms. 
Therefore its efficiency varies in inverse proportion to the 
amount of dead organic matter present. 

Mercuric chloride is exceedingly corrosive as is also 
the acid in which it is originally dissolved; therefore it 
should not be placed in metal containers or agateware 
pails, cups, etc., if the enamel is chipped sufficiently to 
expose the metal. 

Remember that mercuric chloride is a DEADLY POISON! 
Great care must be exercised in properly labelling all bottles, 
etc., containing it. 

Phenol: (CeHsOH) long colorless crystals that become 
pink upon exposure to light and air. 

Synonyms: Carbolic acid, phenic or phenylic acid, 
phenyl hydrate, hydroxybenzene (or -ol). 

The stock solution (95% phenol) is prepared by adding 
1 part of water to 19 parts (by weight) of crystalline phenol. 
Solution may be hastened by placing the vessel containing 
the crystals in a dish of warm water, 



APPENDIX 365 

Note. When making up the stock solution or dilutions from the 
stock solution always have a bottle of ethyl alcohol at hand as a remedy 
for burns caused by phenol. 5% phenol is prepared by adding one 
part of 95% phenol to nineteen parts of distilled water. 

Its value as a disinfectant is increased by the fact that 
it acts in the presence of albuminous substances. It does 
not corrode metals or destroy fabrics in a 5% solution. 

Liquor cresolis compositus, U. S. P. 

Cresol 500 gms. 

Linseed oil 350 gms. 

Potassium hydroxide 80 gms. 

Water, a sufficient quantity to make 1000 gms. 

Dissolve the potassium hydroxide in 50 gms. of water 
in a tared dish, add the linseed oil, and mix thoroughly. 
Then add the cresol and stir until a clear solution is pro- 
duced. Finally add sufficient water to make the finished 
product weigh 1000 gms., or more briefly: mix equal parts 
by weight of cresol and linseed oil-potash soap (Sapo mollis, 
U. S. P.). 

This mixture is a thick, dark, amber-colored fluid which 
mixes readily with water in all proportions to form a clear, 
soapy solution. A 3% or 4% solution will accomplish the 
same results as 5% phenol. It is not interfered with by 
albuminous substances and does not destroy metals or 
fabrics. 

Tincture of iodin, U. S. P. 

lodin '. . . 70 gms. 

Potassium iodid 50 gms. 

Alcohol, sufficient to make 1000 c.c. 

Triturate the iodin and potassium iodid in a mortar 
to a coarse powder and transfer at once to a graduated 
flask. Rinse the mortar with several successive portions 



366 APPENDIX 

of alcohol and pour the rinsings in the bottle; then add 
alcohol, shaking occasionally until the iodin and potassium 
iodid are all dissolved and the finished tincture measures 
1000 c.c. 

SOLUTIONS FOR CLEANING GLASSWARE 
Chromic acid cleaning solution for cleaning glassware: 

Potassium or sodium dichromate 60 gms. 

Commercial sulphuric acid 60 c.c. 

Water 1000 c.c. 

Prepare in a flask resistant to heat, never in a heavy 
glass jar. 

Add the potassium dichromate to about 500 c.c. water; 
shake well and add the sulphuric acid gradually, continually 
shaking with a rotary motion. The remaining water may 
then be added. The potassium dichromate should be all 
dissolved before using the solution. 

This solution may be used repeatedly until oxidized 
to a dark green color. Heat will hasten its action. 

Chromic acid cleaning solution is especially valuable 
for removing traces of oxidizable organic matter and neu- 
tralizing ' any free alkali adhering to glassware. However, 
effort should be made to previously remove as much extrane- 
ous matter as possible with water and a suitable brush 
before treating with this solution. This will economize 
time. 

Caution. This solution contains sufficient sulphuric acid 
to destroy fabrics, bristles of brushes, and corrodes metal 
quickly. For this reason neither cloth nor brushes should 
be used as an immediate aid to this cleaning agent, nor 
should this solution be placed in agateware utensils if the 
enamel is chipped, exposing the metal. 

If this solution is used cold, leave the glassware contain-^ 
ing it, over night on top of desk, never inside of desk. 



APPENDIX 367 

Sodium hydroxide solution for cleaning glassware and 

absorbing C02 : 

Sodium hydroxide, sticks 100 gms. 

Water 1000 c.c. 

Use only once if the glassware is very dirty. 

This solution is invaluable for cleaning greasy flasks, 
pipettes, etc. 

Caution. This solution should not be left in contact 
with any glassware longer than thirty minutes as it etches 
the glass. 

A sodium hydroxide solution of this strength is very 
corrosive, attacking cloth, laboratory desk tops, etc., and, 
therefore, should be wiped up immediately if spilled. 

This strength may also be employed to absorb C0 2 
in fermentation tube cultures of gas-producing organisms. 

STANDARD SOLUTIONS 

A. Preparation of N/10 Na 2 CO3 from titration against 
which normal acid is prepared. 

1. Dry finely powdered chemically pure Na 2 CO,3 in a 
drying oven at 105 C. for two hours. 

2. Weigh out carefully and as accurately as possible 
5.3 gms. of the dried salt. 

3. Dissolve in distilled water which has been boiled 
previously to expel C0 2 and then cooled. 

4. Make up solution to one liter, using a calibrated 
volumetric flask and observing temperature for which the 
flask was calibrated. 

5. Keep this N/10 solution of Na 2 COs in a stoppered 
bottle. It should be used as soon as possible after prepara- 
tion, as the Na 2 COs acts upon the glass and thus deterio- 
rates. 

B. Preparation of N/l, N/10 and N/20 HC1. 

1. Measure out 77.5 c.c. HC1 (sp.gr. 1.20) or 138 c.c. 



368 APPENDIX 

HC1 (sp.gr. 1.12) and make up to one liter with distilled 
water. This makes a solution just a little stronger than 
normal. 

2. To determine its exact strength, titrate 5 c.c. with 
N/10 Na2COs, using phenolphthalein as the indicator. 

3. Rim check determinations, which should check within 
one- or two-tenths of a cubic centimeter. 

4. From results, calculate by proportion how much a 
liter of the solution should be diluted to make it N/l. e.g. : 

5 c.c. HC1 was neutralized by 55 c.c. N/10 Na 2 C0 3 

.'. HC1 is N/l.l 
By proportion: 

N/l : N/l.l:: 1000 :x 

a: = 1100 

Hence each liter of the HC1 solution should be diluted to 
1100 c.c. with distilled water to make a N/l solution of 
HC1. 

5. N/10 and N/20 solutions of HC1 can be made by 
making the proper dilutions. Always use calibrated flasks 
and burettes when making these dilutions. 

C. Preparation of N/l and N/20 NaOH. 

1. Weigh out roughly 41 gms. of chemically pure NaOH. 

2. Dissolve in distilled water, which has been boiled 
to expel CO2 and then cooled. 

3. Make up to one liter, using a calibrated volumetric 
flask and observing the temperature for which it was cali- 
brated. This makes a solution a little stronger than normal. 

4. Determine its exact strength by titration with N/10 
HC1. 

5. Proceed from this point as in the preparation of 
N/l HC1. 

6. N/10 and N/20 solutions can be made from the N/l 
solution as in the preparation of N/10 and N/20 HC1. 



APPENDIX 369 

INDICATORS 
Phenolphthalein, indicator for titration: 

Phenolphthalein 0.5 gm. 

50% alcohol (neutral) 100.0 c.c. 

A drop of a weak solution of alkali should produce per- 
manent pink color when added to a small amount of this 
solution. Phenolphthalein is colorless in the presence of 
acid. 

Kahlbaum's azolitmin solution: Dissolve 2.5 gms. of 
Kahlbaum's azolitmin in 100 c.c. distilled water by heating 
in steam for half an hour. Filter (this will filter much 
more readily if allowed to settle for some time; decant 
upon the filter). Sterilize by heating fifteen minutes each 
day on three successive days. Sterilization is necessary, 
otherwise molds and other microorganisms will grow on the 
organic material present, often changing the reaction. 

A solution of litmus or azolitmin is often added to sugar 
and other media before sterilization for the purpose of 
detecting microorganisms which produce a change in the 
reaction of the media. 

Litmus is a mixture of dyes obtained from the lichens 
Roccella and Lecanom by allowing them to ferment after 
the addition of ammonia and potassium carbonate. When 
the mass has assumed a deep blue color, the liquid is pressed 
out, absorbed by chalk or gypsum, and dried. 

Merck's purified litmus, often used in bacteriological 
work, is made from commercial litmus solution by freeing 
it from the, red pigment orcin, and drying without absorb- 
ing it by means of chalk or gypsum. 

Azolitmin is a purified pigment from litmus. 



370 APPENDIX 



SALT SOLUTIONS 

Physiological salt solutions for immunity work, dilu- 
tion flasks, etc.: 

Sodium chloride, c.p 8.5 gms. 

Distilled water 1000.0 c.c. 

Chemically pure sodium chloride must be used for 
immunity work, especially for animal injection. For 
dilution flasks the best grade of cooking salt serves the 
purpose. Salt prepared for table use cannot be used on account 
of its starch content. 

Normal salt solution for dilution purposes, etc., 
not for immunity work: 

Sodium chloride, best commercial grade . 60 gms. 
Distilled water 1000 c.c. 

Citrated salt solution for used in demonstrating opso- 
nins: 

Sodium chloride, c.p 8.5 gms. 

Sodium citrate 15.0 gms. 

Distilled water 1000.0 c.c. 

TEST SOLUTIONS 

Ehrlich's test solution for indol production: 
Solution I. 

Para-dimethyl-amido-benzaldehyde 4 gms. 

96% alcohol 380 c.c. 

HC1, cone.. . . ....' 80 c.c. 

Solution II. Saturated watery solution of potassium 
persulphate (oxidizing agent). 

See Exercise 44, Part I, for the method of the test. 



APPENDIX 371 

Nitrate test solutions: 
I. Phenolsulphonic acid. 

1. Mix 3 gms. of pure crystallized phenol with 37 gms. 
of c.p. concentrated sulphuric acid (20.1 c.c., sp.gr. 1.84) 
in a round-bottom flask. 

2. Heat for six hours in a water bath at 100 C., keeping 
the flask submerged the whole time. 

This may crystallize on cooling, but it can be brought 
into solution easily by heat. 

Directions for making this test will be noted in Exercise 
45, Part I. 

II. Diphenylamin. A solution of 2% diphenylamin in 
sulphuric acid when added to a liquid containing nitrates 
or nitrites gives a blue color. 

Diphenylamin 2 gms. 

Sulphuric acid, c.p. cone 100 c.c. 

Nitrite test solutions: 

Solution I. 8.0 gms. sulphanilic acid dissolved in 1000 
c.c. of 5N acetic acid (sp.gr. 1.041;. 

Solution II. 5.0 gms. a-naphthylamin dissolved in 
1000 c.c. of 5N acetic acid. These solutions should be kept 
separate and mixed in equal parts just before use. 

Nessler's solution, for free ammonia: 

1. Dissolve 62.5 gms. of potassium iodid in 250 c.c. of 
distilled water. Reserve about 10 c.c. of this solution. 

2. Add gradually to the main portion a cold saturated 
solution of mercuric chloride, stirring constantly and in- 
creasing the quantity of mercuric chloride until a bright, 
permanent precipitate is formed. 

3. Now add the reserved potassium iodid solution and 
again add the saturated mercuric chloride solution, cautiously 
and with constant stirring until a distinct though slight 
red precipitate remains. 

4. Dissolve 150 gms. of caustic potash in 150 c.c. dis- 



372 APPENDIX 

tilled water, allow the solution to cool and add it to the 
above solution. 

5. Dilute to one liter with distilled water. 

6. Allow to stand for one week and decant for use. 

MOUNTING MEDIA 

Canada balsam for making permanent mounts of mi- 
croscopic preparations : 

Canada balsam, dry, hard, for microscopic use 4 parts 

Xylol 3 parts 

This gives a mounting medium of about the right con- 
sistency. It should not " thread " when a drop is taken 
out with the glass rod. Balsam should be kept in a bottle 
stoppered with a glass bell-stopper, and having a rim 
arranged so that the excess of balsam taken upon the 
glass rod can be drained off. 

Immersion oil for oil immersion objectives. 

It is necessary that the immersion oil have practically 
the same index of refraction as glass in order to avoid 
dispersion of any of the light rays. Cedar wood oil having 
a refractive index of 1.515 to 1.520 is the usual medium 
interposed between the specimen and the oil immersion 
objective as it has approximately the same index of refrac- 
tion as crown glass, 1.518. The refractive index of air is 
1.000. 

Chinese ink: 

Bum's " Pelikan " Chinese ink 1 part. 

Distilled water 7 parts. 

Tube, using 8 to 10 c.c. per tube, sterilize in the autoclav 
and allow to stand two or three weeks without disturbing, 
for sedimentation to take place. It is to be used without 
shaking or disturbing any more than necessary. 



APPENDIX 373 



STAINS 

Methylen blue for differentiating living from dead 
yeast cells: 

Methylen blue 0.1 gm. 

Distilled water. . . 1000.0 c.c. 

Aqueous-alcoholic stains, fuchsin, methylen blue and 
gentian violet: 

1. A saturated alcoholic solution of a stain is prepared 
by shaking frequently about 10 gms. of the stain with 
100 c.c. of absolute alcohol. If the stain dissolves quickly, 
add more dry stain. The alcoholic solution should be 
slightly supersaturated. 

2. Allow the undissolved stain to settle over night. 

3. Decant. 

4. Dilute 1 part of the alcoholic solution with 9 parts 
of distilled water. 

Note 1. If 95% alcohol is used instead of absolute alcohol to 
dissolve the stain, the dilution should be made 1 : 7. 

Note 2. These aqueous solutions may not keep longer than about 
a month, while the saturated alcoholic solutions keep indefinitely. 

Note 3. The vegetative forms of bacteria stain more or less 
readily with all aqueous-alcoholic stains but not with saturated alcoholic 
stains. Acid-fast bacteria, e.g., Bact. tuberculosis, are the exception 
to the former. 

Anilin- water gentian violet: 

1. Shake 5 c.c. of anilin oil vigorously with 100 c.c. of 
distilled water in a stoppered bottle for several minutes. 

2. Filter through a wet filter immediately before use. 

3. Add 1 part of saturated alcoholic solution of gentian 
violet to 9 parts of the freshly prepared anilin-water and 
filter immediately before use. 

Note. Anilin-water stains do not keep longer than about a week. 
The stock solutions will keep indefinitely if kept separate. 



374 APPENDIX 

Ziehl-Nielson's carbol-fuchsin. 

Solution A. 

Basic fuchsin 1 gm. 

Absolute alcohol 10 c.c. 

Solution B. 

Carbolic acid 5 gms. 

Distilled water 100 c.c. 

1. Dissolve the fuchsin in the absolute alcohol. (Solu- 
tion A.) 

2. Dissolve the carbolic acid in the distilled water 
(Solution B). 

Note. Solutions A and B will keep indefinitely if kept sepa- 
rate. 

3. Mix in the proportion of 10 c.c. of solution A to 100 
c.c. of solution B. 

Note. If A and B do not mix readily, warm slightly and add a 
few drops of absolute alcohol. 

4. Filter. 

Loeffler's alkaline methylen blue. 

Saturated alcoholic solution of methylen blue 30 c.c. 

Potassium hydrate, 0.1% aqueous solution 100 c.c. 



APPENDIX 375 



SOLUTIONS FOR USE IN STAINING 

Aceton-alcohol for decolorizing in Gram's method of 
staining: 

Aceton 10 c.c. 

Absolute alcohol 100 c.c. 

Acetic acid-alcohol for clearing in making impression 
preparations (also used for decolorizing in ordinary method 
of spore-staining) : 

Alcohol, 90% 2 parts 

Acetic acid, 1% 1 part. 

Mordant for staining flagella: 

Tannin, 20% 10 c.c. 

Ferrous sulphate, cold saturated solution . . 8 c.c. 
Fuchsin, cold saturated solution in absolute 

alcohol 1 c.c. 

Lugol's iodin solution, for use in Gram's staining method: 

lodin 1 gm. 

Potassium iodid 3 gms. 

Distilled water . . 300 c.c. 



376 



APPENDIX 



STEAM TEMPERATURE PRESSURE TABLE 



Temperature 
Centigrade. 


Mm. of Hg. 


Pounds per sq.in. 
Absolute Pressure. 


Atmospheres. 


Degrees, 








98 


707.1 


13.7 


0.93 


99 


733.1 


14.2 


0.96 


100 


760.0 


14.7 


1.00 


101 


787.8 


15.2 


1.03 


102 


816.0 


15.8 


1.07 


103 


845.2 


16.3 


1.11 


104 


875.4 


16.9 


1.15 


105 


906.4 


17.5 


1.19 


106 


938.3 


18.1 


.23 


107 


971.1 


18.8 


.27 


108 


1004.9 


19.4 


.32 


109 


1039.6 


20.1 


.36 


110 


1075.3 


20.8 


.41 


111 


1112.0 


21.5 


.46 


112 


1149.8 


22.2 


.51 


113 


1188.6 


22.9 


.56 


114 


1228.4 


23.7 


.61 


115 


1269.4 


24.5 


.67 


116 


1311.4 


25.3 


.72 


117 , 


1354.6 


26.2 


.78 


118 


1399.0 


27.0 


.84 


119 


1444.5 


27.9 


.90 


120 


1491.2 


28.8 


.96 


121 


1539.2 


29.7 


2.02 


122 


1588.4 


30.7 


2.09 


123 


1638.9 


31.7 


2.15 


124 


1690.7 


32.7 


2.22 


125 


1743.8 


33.7 


2.29 



APPENDIX 



377 



FORMULAE FOR CONVERSION OF DEGREES OF TEMPER- 
ATURE ON ONE SCALE INTO DEGREES ON ANOTHER 

Centigrade (Celsius) scale: Freezing-point = 0; boiling-point = 100. 
Fahrenheit scale : Freezing-point = 32 ; boiling-point = 212. 

Reaumur: Freezing-point = 0; boiling-point = 80. 

(F-32)4 



Degrees C X 1 .8 +32 = Degrees F. 
F-32 



Degrees 



1.8 
RX9 



= Degrees C. 



+32 = Degrees F. 



Degrees 



9 
RX5 

4 
CX4 

5 



= Degrees R. 



Degrees C. 



Degrees R. 



ALCOHOL BY VOLUME 
TRALLES 

(From the Chemlker Kalender.'publlshed by Julius Springer, Berlin.) 



Per 
Cent 
by Vol. 


Specific 
Gravity. 


Per 
Cent 
by Vol. 


Specific 
Gravity. 


Per 
Cent 
by Vol. 


Specific 
Gravity. 


Per 

Cent 
by Vol. 


Specific 
Gravity. 


1 


0.9976 


26 


0.9689 


51 


0.9315 


76 


0.8739 


2 


0.9961 


27 


0.9679 


52 


0.9295 


77 


0.8712 


3 


0.9947 


28 


0.9668 


53 


0.9255 


78 


0.8685 


4 


0.9933 


29 


0.9657 


54 


0.9254 


79 


0.8658 


5 


0.9919 


30 


0.9646 


55 


0.9234 


80 


0.8631 


6 


0.9906 


31 


0.9634 


56 


0.9213 


81 


0.8603 


7 


0.9893 


32 


0.9622 


57 


0.9192 


82 


0.8575 


8 


0.9881 


33 


0.9609 


58 


0.9170 


83 


0.8547 


9 


0.9869 


34 


0.9596 


59 


0.9148 


84 


0.8518 


10 


0.9857 


35 


0.9583 


60 


0.9126 


85 


0.8488 


11 


0.9845 


36 


0.9570 


61 


0.9104 


86 


0.8458 


12 


0.9834 


37 


0.9559 


62 


0.9082 


87 


0.8428 


13 


0.9823 


38 


0.9541 


63 


0.9059 


88 


0.8397 


14 


0.9812 


39 


0.9526 


64 


0.9036 


89 


0.8365 


15 


0.9802 


40 


0.9510 


65 


0.9013 


90 


0.8332 


16 


9791 


41 


0.9494 


66 


0.8989 


91 


0.8299 


17 


0.9781 


42 


0.9478 


67 


0.8965 


92 


0.8265 


18 


0.9771 


43 


0.9461 


68 


0.8941 


93 


0.8230 


19 


0,9761 


44 


0.9444 


69 


0.8917 


94 


0.8194 


20 


0.9751 


45 


0.9427 


70 


0.8892 


95 


0.8157 


21 


0.9741 


46 


0.9409 


71 


0.8867 


96 


0.8118 


22 


0.9731 


47 


0.9391 


72 


0.8842 


97 


0.8077 


23 


0.9720 


48 


0.9373 


73 


0.8817 


98 


0.8034 


24 


0.9710 


49 


0.9354 


74 


0.8791 


99 


0.7988 


25 


0.9700 


50 


0.9335 


75 


0.8765 


100 


0.7939 



378 



APPENDIX 



DEGREE f~\ DEGREES 
-110 



220 - 



210 
200 
190 
180 

iro 

160 
150 
140 
13JO 
120 
110^ 



90- 
80 -g 
70 -= 
60 -g 
50 j 
40 - 



30-= 
20 -J 

10 -| 

F 



100 



70 



50 



30 



20 



10 



10 



C 



FIG. 74. Comparison Fahrenheit-Centigrade Scale. 



APPENDIX 379 

METRIC SYSTEM 
Linear Measure 

1000 millimicrons = 1 micron (micromillimeter). 
1000 microns = 1 millimeter. 

10 millimeters = 1 centimeter. 

10 centimeters = 1 decimeter. 

10 decimeters = 1 meter. 

10 meters = 1 decameter. 

10 decameters = 1 hectometer. 

10 hectometers = 1 kilometer. 

10 kilometers = 1 myriameter. 

The unit of length, one meter, is equal to QQQQQQ P art of the 

distance measured on a meridian of the earth from the equator to the 
pole and equals about 39.37 inches. 



1,000,000 sq. millimicrons = 1 sq. . 

1,000,000 sq. microns = 1 sq. millimeter. 
= 



Square Measure 

micron. 

. . millime. 

100 sq. millimeters = 1 sq. centimeter. 
100 sq. centimeters = 1 sq. decimeter. 
100 sq. decimeters = 1 sq. meter = 1 centare 
100 sq. meters = 1 sq. decameter = 1 are. 

100 sq. decameters = 1 sq. hectometer = 1 hectare. 
100 sq. hectometers 1 sq. kilometer. 
100 sq. kilometers = 1 sq. myriameter, 

Cubic Measure 

1000 cubic millimeters = 1 cubic centimeter. 
1000 cubic centimeters = 1 liter. 

10 liters = 1 decaliter. 

100 liters = 1 hectoliter. 

1000 liters = 1 kiloliter = 1 cu. meter = 1,000,000 c.c. 

The unit of capacity is the liter and represents the volume of a 
kilogram of water at its maximum density, 4 C. and 760 mm. mercury 
pressure. 



380 APPENDIX 

METRIC SYSTEM Continued 
Weight 

The unit of weight is the gram and represents the weight of one 
cubic centimeter of water at its maximum density, 4 C. and 760 mm. 
mercury pressure. 

10 milligrams = 1 centigram. 

10 centigrams = 1 decigram. 

10 decigrams =1 gram. 

10 grams = 1 decagram. 

10 decagrams =1 hectogram. 

10 hectograms = 1 kilogram = 1000 grams. 

10 kilograms = 1 myriagram. 

10 myriagrams = 1 quintal. 

10 quintals = 1 millier or tonneau. 



LIST OF TEXT AND REFERENCE BOOKS 

ABBOT, A. C. The Principles of Bacteriology. 9th Ed. 1915. 
AIRMAN, C. M. Milk, its Nature and Composition. 1909. 
American Public Health Association. Standard Methods for the 
Examination of Water and Sewage. 1915. 

BASTIAN, H. C. The Beginnings of Life. 1872. 

BAYLISS, W. M. The Nature of Enzyme Action. 3d Ed. 1914. 

BAYLISS, W. M. Principles of General Physiology. 1915. 

BELCHER, S. D. Clean Milk. 1912. 

BESSON, A. Practical Bacteriology, Microbiology and Serum 
Therapy. Translated by H. J. Hutchens. 1913. 

BORDET, J. and GAY, F. P. Studies in Immunity. 1st Ed. 1909. 

BOSTON, L. NAPOLEON. Clinical Diagnosis. 1905. 

BOWHILL, T. Manual of Bacteriological Technique. 2d Ed. 1902. 

BRACHVOGEL, JOHN K. Industrial Alcohol: Its Manufacture and 
Uses. 1907. 

BRANNT, WM. T. Vinegar, Acetates, Cider, Fruit Wines, Pres- 
ervation of Fruits. 2d Ed. 1900. 

BUCHANAN, ESTELLE D. and R. E. Household Bacteriology. 1913. 

BUCHANAN, R. E. Veterinary Bacteriology. 1916. 

BURGESS, P. S. Soil Bacteriology Laboratory Manual. 1914. 

CALKINS, GARY N. Protozoology. 1909. 

CHAPIN, CHARLES V. The Sources and Modes of Infection. 2d 

Ed. 1912. 
Chemical Rubber Co., Cleveland, Ohio. Handbook of Chemistry 

and Physics. 1914. 

CHESTER, F. D. A Manual of Determinative Bacteriology. 1901. 
CITRON, JULIUS. Immunity. Translated by A. L. Garbat. 1912. 
COHNHEIM, OTTO. Enzymes. 1st Ed. 1912. 
CONN, H. W. Agricultural Bacteriology. 2d Ed. 1909. 
CONN, H. W. Bacteria in Milk and its Products. 1903. 
CONN, H. W. Bacteria, Yeasts and Molds. 1903. 

381 



382 LIST OF TEXT AND REFERENCE BOOKS 

CONN, H. W. Bacteria, Yeasts, and Molds. 1913. 

CONN, H. W. Practical Dairy Bacteriology. 1910. 

CONN, H. W., ESTEN, W. M., and STOCKING, W. A. Classification 
of Dairy Bacteria. 1906. Reprint from the Report of the 
Storrs (Connecticut) Agr'l Exp't, Sta. for 1906, 

DE BARY, H. Comoarative Morphology and Biology of the 

Fungi, Mycetozoa and Bacteria. 1887. 
DE BARY, H. Lectures on Bacteria. 2d Ed. 1887. 
DECKER, JOHN W. Cheese-making. 1905. 
DIEUDONNE, H. Bacterial Food Poisoning. 1909. 
DOANE, R. W. Insects and Disease. 1910. 
DON, JOHN, and CHISHOLM, JOHN. Modern Methods of Water 

Purification. 1911. 
DUCKWALL, E. W. Canning and Preserving of Food Products 

with Bacteriological Technique. 1905. 
DUGGAR, B. M. Fungous Diseases of Plants. 1909. 
DUGGAR, B M. Plant Physiology. 1912. 
DtiRCK, HERMANN. Allgemeine Pathologische Histologie. Teil 

III. 1903. 

EFFRONT, JEAN. Enzymes and Their Applications. 1st Ed. 
1904. 

EHRLICH, P. and BOLDUAN, CHARLES. Collected Studies in Im- 
munity. 2d Ed. 1910. 

ELLIOTT, S. MARIA. Household Bacteriology. 1914. From 
Handbook of Health and Nursing. Edited by the American 
School of Home Economics. 1912. 

ERNST, WILLIAM. Milk Hygiene. Transl. by John R. Mohler 
and Adolph Eichhorn. 1914. 

EULER, HANS. General Chemistry of the Enzymes. Translated 
by Thomas H. Pope. 1st Ed. 1912. 

EYRE, J, W, H. Bacteriological Technique. 2d Ed. 1913. 

FARRINGTON, E. H., and WOLL, F. W. Testing Milk and its 

Products. 1904. 
FISCHER, ALFRED. Structure and Functions of Bacteria. Transl. 

by A. Coppen Jones. 1900. 
FLEISCHMANN, W. The Book of the Dairy, 1896. Transl. by C. 

M. Aikmann and R. Patrick Wright. 



LIST OF TEXT AND REFERENCE BOOKS 383 

FLUGGE, C. Die Mikroorganismen, 1896. 
FOWLER, G. J. Bacteriological and Enzyme Chemistry. 1911. 
FRED, E. B. Laboratory Manual of Soil Bacteriology. 1916. 
FROST, W. D. A Laboratory Guide in Elementary Bacteriology. 

3d Ed. 1904. 
FROST, W. D. and MCCAMPBELL, E. F. A Text-book of General 

Bacteriology. 1910. 

FUHRMANN, FKANZ, Vorlesungen uber bakterien Enzyme, 1907. 
FUHRMANN, FRANZ. Vorlesungen uber technische Mykologie. 

1913. 
FULLER, G. W. Sewage Disposal. 1912. 

GAGE, S. H. The Microscope 1 . 1904. 

GERHARD, WM. P. The Sanitation, Water Supply and Sewage 

Disposal of Country Houses. 1909. 

GORHAM, F. P. Laboratory Course in Bacteriology. 1915. 
GREEN, J. REYNOLDS. Soluble Ferments and Fermentation. 

2d Ed. 1901. 
GUEGUEN, F. Les Champignons: Parasites de 1'Homme and 

des Animaux. 1904. 
GUILLIERMOND, A. Les Levures. 1912. 

HANSEN, EMIL CHR. Practical Studies in Fermentation. 1896. 
HARDEN, ARTHUR. Alcoholic Fermentation. 2d Ed. 1914. 
HASTINGS, E. G. and WRIGHT, W. H. A Laboratory Manual of 

General Agricultural Bacteriology. 1913. 
HAWK, P. B. Physiological Chemistry. 4th Ed. 1913. 
HEINEMANN, P. G. A Laboratory Guide in Bacteriology. 2d Ed. 

1911. 

HERTWIG, OSCAR. Allgememe Biologic. 1912. 
HERZOG, M. Disease-Producing Microorganisms. 1910. 
HEWLETT, R. T. Manual of Bacteriology, 5th Ed. 1915. 
Hiss, P. H. and ZINSSER, H. Text-book of Bacteriology. 2d Ed. 

1914. 

HOOKER, A. H. Chloride of Lime in Sanitation. 1913. 
HUEPPE, F. Methods of Bacteriological Investigation. Transl. 

by H. M. Biggs. 1886. 

IAGO, WM. and IAGO, WM. C. The Technology of Bread-making. 
1911. 



384 LIST OF TEXT AND REFERENCE BOOKS 

JENSEN, C. 0. Essentials of Milk Hygiene. Transl. by Leonard 

Pearson. 1907. 
JESS, PAUL. Kompendium der Bakteriologie und Blutserum- 

therapie. 1903. 

JORDAN, EDWIN 0. General Bacteriology. 3d Ed. 1914. 
JORGENSEN, A. Microorganisms and Fermentation. Transl. by 

A. K. Miller and E. A. Lennholm. 1893. 

KERSHAW, G. B. Modern Methods of Sewage Purification. 1911. 
KINNICUTT, L. P., WINSLOW, C. E. A., and PRATT, R. W. Sewage 

Disposal. 1910. 
KISSKALT, K., und HARTMANN, M. Praktikum der Bakteriologie 

und Protozoologie. 1907. 
KITT, TH. Bakterienkunde und pathologische Mikroskopie. 

1903. 

KITT, TH. Text-book of Comparative General Pathology. Trans- 
lated by Wm. W. Cadbury and Allen J. Smith. 1906. 
KLOCKER, ALB. Fermentation Organisms. Translated by G. 

E. Allen and J. H. Millar. 1904. 
KOLLE, W., und WASSERMANN, A. Handbuch der pathogenen 

Microorganismen I, II und III und Atlas. 1903. 
KOLMER, J. A. Infection, Immunity and Specific Therapy. 1915. 
KRAUS, R., und LEVADITI, C. Handbuch der Technik und Metho- 

dik der Immunitatsforschung. Bd. I, Antigene. 1908; Bd. 

II, Antikorper. 1909; Erster Erganzungsband. 1911. 
KRUSE, W. Allgemeine Mikrobiologie. 1910. 

LAFAR, FRANZ, Die Essigsaure-Garung. 1913. 

LAFAR, FRANZ. Technische Mykologie. German Ed. Bd. I, 

1904-1907; Bd. II, 1905-1908; Bd. Ill, 1904-1906; Bd. 

IV, 1905-1907; Bd. V, 1905-1914. English Ed. Vol. I, 

1898; Vol. II, Part 1, 1903; Vol. II, Part II, 1910. Translated 

by Charles T. C. Salter. 
LAW, JAMES. Veterinary Medicine. 3d Ed. Vol. I. 1910; 

Vol. II, 1911. 
LEE, H. BOLLES. The Microtomist's Vade Mecum. 6th Ed. 

1905. 
LEFAS, E. La Technique histo-bacteriologique moderne. 1906. 



LIST OF TEXT AND REFERENCE BOOKS 385 

LEHMANN, K. B., u. NEUMANN, R. O. Bakteriologie und bak- 
teriologische Diagnostik. Teil I, Atlas, 1910; Teil II, Text, 
1912. 

LIPMAN, J. G. Bacteria in Relation to Country Life. 1908. 

LIPMAN, J. G., and BROWN, P. E. Laboratory Guide in Soil Bacter- 
iology. 1911. 

LOEB, JACQUES. The Mechanistic Conception of Life. 1912. 

LOEB, JACQUES. Studies in General Physiology, Part I. 1905. 

LOHNIS, F. Handbuch der landwirtschaftlichen Bakteriologie. 
1910. 

LOHNIS, F. Laboratory Methods in Agricultural Bacteriology. 
Translated by Wm. Stevenson and J. Hunter Smith. 1913. 

LOHNIS, F. Vorlesungen iiber landwirtschaftliche Bakteriologie. 
1913. 

MACFADYEAN, ALLAN. The Cell as the Unit of Life. 1908. 

MARSHALL, C. E. Microbiology. 1911. 

MASSEE, G. Diseases of Cultivated Plants and Trees. 1914. 

MAST, S. 0. Light and the Behavior of Organisms. 1911. 

MATTHEWS, C. G. Manual of Alcoholic Fermentation. 1901. 

McCAMPBELL, E. F. Laboratory Methods for the Experimental 
Study of Immunity. 1909. 

McFARLAND, JOSEPH. Pathogenic Bacteria and Protozoa. 7th 
Ed. 1912. 

MCFARLAND, JOSEPH. Biology, General and Medical. 2d Ed. 
1914. 

McKAY, G. L., and LARSEN, C. Principles and Practice of Butter- 
making, 1913. 

MERCK'S INDEX. 1907. 

METCHNIKOFF, ELIE. Comparative Pathology of Inflammation. 
Translated by F. A. Starling and E. H. Starling. 1893. 

METCHNIKOFF, ELIE. Immunity in Infective Diseases. 1905. 

MIGULA, W. System der Bakterien. Bd. I, 1897; Bd. II, 1900. 

MOLISCH, HANS. Die Eisenbakterien. 1910. 

MOLISCH, HANS. Leuchtende Pflanzen. 1904. 

MOLISCH, HANS. Die Purpurbakterien. 1907. 

MOORE, V. A. The Pathology and Differential Diagnosis of 
Infectious Diseases of Animals. 1916. 

MOORE, V. A. Principles of Microbiology. 1912. 



386 LIST OF TEXT AND REFERENCE BOOKS 

MOORE, V. A. Bovine Tuberculosis and its Control. 1913. 
MOORE, V. A. and Fitch, C. P. Bacteriology and Diagnosis. 

1914. 
Mum, ROBERT, and RITCHIE, JAMES. Manual of Bacteriology. 

6th Ed. 1913. 
MULLER, PAUL TH. Vorlesungen iiber Infektion und Immunitat. 

1904. 

NOCARD, Ep., and LECLAINGHE, E. Les Maladies imcrobiennes 

des Animaux. T. I, 1903; T. II, 1903. 
NOVY, F. G. Laboratory Work in Bacteriology. 1899. 
NUTTALL, G. H. F. Blood Immunity and Blood Relationship: 

Precipitin Tests. 1904. 

OGDEN, HENRY N. and CLEVELAND, H. BURDETT. Practical 

Methods of Sewage Disposal. 1912. 
OPPENHEIMER, C. Die Fermente und ihre Wirkungen. Bd. I, 

Vierte Auflage. 1913; Bd. II, 1913. 

PARK, WM. H. Pathogenic Bacteria and Protozoa. 4th Ed. 
1910. 

PASTEUR, L. Studies on Fermentation. Translated by F. Faulk- 
ner and D. C. Robb. 1879. 

PFEIFFER, L. Die Protozoen als Krankheitserreger. 1890. 

PORCHER, CH. Le Lait Desseche. 1912. 

PRESCOTT, S. C. and WINSLOW, C.-E. A. Elements of Water 
Bacteriology. 3d Ed. 1913. 

RICHMOND, H, D, Dairy Chemistry, 1899. 

RICKETTS, H. T. and DICK, G. F. Infection, Immunity and Serum 

Therapy. 1913. 
RIDEAL, SAMUEL. Sewage and the Bacterial Purification of 

Sewage. 1901. 
ROECHLING, H. H. Sewer Gas and its Influence upon Health. 

1898. 

ROGERS, A., and AUBERT, A. B. Industrial Chemistry. 1912. 
ROSENAU, M. J. Disinfection and Disinfectants. 1902. 
ROSENAU, M. J. The Milk Question. 1912. , 



LIST OF TEXT AND REFERENCE BOOKS 387 

ROSENAU, M. J. Preventive Medicine and Hygiene. 1913. 

RUSSELL, H. L., and HASTINGS, E, G. Experimental Dairy Bac- 
teriology. 1909. 

RUSSELL, H. L., and HASTINGS, E. G. Outlines of Dairy Bacter- 
iology. 9th Ed. 1910. 

RUSSELL, H. L., and HASTINGS, E. G. Agricultural Bacteriology. 
1915. 

SADTLER, S. P. Industrial Organic Chemistry. 1912. 

SANTEE, E. M. Farm Sewage. 1912. 

SAVAGE, WM. G. The Bacteriological Examination of Water 

Supplies. 1906. 
SAVAGE, WM. G. The Bacteriological Examination of Food and 

Water. 1914. 

SAVAGE, WM. G. Milk and the Public Health. 1912. 
SCHNEIDER, ALBERT. Bacteriological Methods in Food and Drug 

Laboratories. 1915. 

SHINKLE, C. A. American Commercial Methods of Manufactur- 
ing Preserves, Pickles, Canned Foods, etc. Revised Ed. 

1912. 
SMITH, ERWIN F. Bacteria in Relation to Plant Diseases. Vols. 

I, 1905; II, 1911; III, 1914. 
SNYDER, HARRY. Dairy Chemistry. 1911. 
STERNBERG, G. M. Textbook of Bacteriology. 1896. 
STEVENS, F. L., and HALL, J. G. Diseases of Economic Plants. 

1913. 
STITT, E. R. Practical Bacteriology, Blood Work and Animal 

Parasitology. 3d Ed. 1914. 
SYKES, W. J. The Principles and Practice of Brewing. 1897. 

THORP, F. H. Outlines of Industrial Chemistry. 1905. 
THRESH, JOHN C. Examination of Waters and Water Supplies. 

2d Ed. 1913. 
TYNDALL, JOHN. Floating Matter of the Air in Relation to 

Putrefaction and Infection. 1881. 

VAN SLYKE, Lucius L., and PUBLOW, CHARLES A. The Science 

and Practice of Cheese-making. 1912. 

.VAUGHAN, V. C., and NOVY, F. G. Cellular Toxins. 4th Ed. 
1902. 



388 LIST OF TEXT AND REFERENCE BOOKS 

VAUGHAN, V. C., VAUGHAN, V. C., Jr., and VAUGHAN, J. W. Pro- 
tein Split Products in Relation to Immunity and Disease. 
1913. 

VERNON, H. M. Intracellular Enzymes. 1908. 

WALKER, E. W. AINLEY. Inflammation, Infection and Fever. 

1904. 
WARBASSE, J. P. The Conquest of Diseases Through Animal 

Experimentation. 1910. 

WARD, A. R. Pure Milk and the Public Health. 1909. 
WARD, H. MARSHALL. Diseases in Plants. 1901. 
WASSERMAN, A. Immune Sera. Translated byChas. Baldwin. 

1904. 

WHIPPLE, G. C. Typhoid Fever. 1st Ed. 1908. 
WILLIAMS, H. U. Manual of Bacteriology. Revised by B. 

Meade Bolton. 5th Ed. 1908. 

WILLOUGHBY, EDWARD F. Milk: Its Production and Uses. 1903. 
WILSON, EDMUND B. The Cell in Development and Inheritance. 

1911. 

WING, HENRY H. Milk and its Products. 1904. 
WINSLOW, C. E. A., and WINSLOW, ANNE R. The Systematic 

Relationships of the Coccacese. 1908. 

ZIEGLER, ERNST. Pathologic und Anatomie. Bd. I, 1901; 

Bd. II, 1902. 

ZINSSER/HANS. Infection and Resistance. 1915. 
ZOPF, WILHELM, Die Pilze. 1890. 

PERIODICALS 

Annales de 1'Institut Pasteur. 

Arbeiten aus dem kaiserlichen Gesundheitsamte. 

Archiv fur experimentale Pathologie und Pharmakologie. 

Archiv fur Hygiene. 

Archiv fiir Schiffs und Tropenhygiene. 

Berliner klinische Wochenschrift. 
Biochemische Zeitschrift. 
British Medical Journal. 
Bulletin de 1'Institut Pasteur. 



LIST OF TEXT AND REFERENCE BOOKS 389 

Centralblatt fur Bakteriologie, I Abteilung, Originate. Medizin- 
isch-hygienische Bakteriologie und tierische Parasitenkunde. 

Centralblatt fur Bakteriologie, I Abteilung, Referate. 

Centralblatt fur Bakteriologie, II Abteilung. Allgemeine land- 
wirtschaftlich-technologische Bakteriologie, Garungs-phy- 
siologie und Pflanzenpathologie. 

Comptes Rendus Academic des Sciences. 

Comptes Rendus Societie de Biologic. 

Comptes Rendus des Travaux du Laboratoire de Carlsberg. 

Deutsche medizinische Wochenschrift. 
Experiment Station Record. 
Hygienische Rundschau. 

Jahresbericht liber die Forsch. d. path. Mikroorganismen, Baum- 

garten's. 
Jahresbericht tiber die Fortschritte der Lehre von den Garungs 

Organismen, Koch's. 
Journal of Agricultural Research. 
Journal of the American Medical Association. 
Journal of the American Veterinary Medical Association. 
Journal of Experimental Medicine. 
Journal of Hygiene. 
Journal of Infectious Diseases. 
Journal of Medical Research. 
Journal of Pathology and Bacteriology. 
Journal of the Royal Army Medical Corps. 
Journal of Tropical Medicine. 

Lancet. 

Proceedings of the Royal Society of London. 

Revue ge'ne'rale du Lait. 

Zeitschrift fur Hygiene und Infektionskrankheiten. 
Zeitschrift fur Immunitatsforschung. 








STRtrCTtTKE OF COLONIES. 

1. Areolate. 

2. Grumose. 

3. Moruloid. 

4. Clouded. 

5. Gyrose. 

6. Marmorate. 

7. Reticulate. 



EDGES OF COLONIES. 

8. Repand. 

9. Lobate. 

10. Efose. 

11. Auriculate. 

12. Lacerate. 

13. Fimbriate.* 

14. Ciliate.* 



* For illustration see next page. 



APPENDIX 



391 



13 




14 




B 




ELEVATION OF COLONIES. 

1. Flat. 

2. Raised. 

3. Convex. 

4. Pulvinate. 

5. Hemispherical. 

6. Umbilicate. 

7. UmbonatCk 

8. Concave. 

9. Rectangular depression. 



392 



APPENDIX 





APPENDIX 



393 




INDEX 



Abortion, contagious, diagnosis 

of, 329 
Accidents, X 
Acetacidase, 181, 183 
Acetaldehydase, 183 
Acetaldehyde, 183 
Acetic acid-alcohol, 375 
Aceton-alcohol, 96, 375 
Acid, acetic, 183, 215, 371 

acetic, enzyme of, 181 

acetic, glacial, 97 

carbolic, 48, 185-188, 210-212, 
303, 308, 364, 365, 371, 374 

citric, 354 

digallic, enzyme of, 180 

fatty, 183 

hydrochloric, 39, 245, 313 

hydrochloric, effect on pigment, 
175, 176 

hydrochloric, N/l, 20, 367, 368 

hydrochloric, N/20, 20, 245, 
367, 368 

hydrocyanic, 183 

lactic, 23, 181, 184, 218 

lactic, enzyme of, 181 

normal, 20-22, 367, 368 

oleic, 183 

Acidophile, 154, 184 
Acid, oxalic, 313 

palmitic, 183 

phenolsulfonic, 133, 371 

picric, 87 

pyrogallic, 156, 160, 165, 248 



Acid, silicic, colloidal, state of, 33 

stearic, 183 

sulfanilic, 133, 251, 371 

sulfuric, concentrated, 213, 249, 
308, 366, 371 

sulfuric, for decolorization, 92 
Acid-fast bacteria, 92, 97, 324 

stain, 135 

Acid production, 23, 112, 137, 169 
Acid-proteinase, 180 
Acidity, 20-22 

of fresh beef, 29 
Acids, 184 

a-amino, 180 

organic, decomposition, 169, 
184 

organic, enzymes of, 180, 181 
Action, selective, 170 
Activator, 193, 194 
Adhesion culture, 78, 79, 100, 

107, 120 
Adjustment, coarse, 65-69, 77 

fine, 65-69, 77 

of reaction, 21, 22, 30 
Aeration of soils, 245, 246 
Aerobic bacteria, 46, 154, 246, 247 
Aerogenic, 154 
Aeroscope, sand filter, 221 
Agar, 37-41 

action of acid on, 38, 39 

as a microbial food, 40, 41 

commercial form of, 38 

Congo red, 355 

Conradi-Drigalski's, 361, 363 
395 



396 



INDEX 



Agar, dextrose, 114, 175 

dextrose calcium carbonate, 

167, 349 

digestion of, 39, 40 
Endo, 361, 363 
fermented, 41, 349 
filterability of, 39, 40 
litmus lactose, 349, 357, 358 
mannit, 253, 254 
melting point of, 39 
nitrate, 251, 354 
nitrogen-free ash, 256, 355 
nutrient, 19, 20, 41-43 
nutrient, preparation of, 41-43 
percentage used in media, 40 
soil extract, 263, 351 
solidification of, 39, 40 
solubility of, 39, 40 
source of, 37 
sterilization of, 40 
urea, 352 
washing of, 41 
waste, IX 
plate culture, preparation of, 

129 
shake, litmus lactose, 223, 228, 

236, 357 
slant, 26, 43 
Agents, chemical, of sterilization, 

5, 14 

physical, of sterilization, 5 
sterilizing, 5 
Agglutination, macroscopic test, 

310-312 

microscopic test, 312 
observation of, 74 
Air, bacterial analysis of, 220- 

222 

displacement of, 157, 164, 165 
exclusion of, 156, 157 
exhaustion of, 156 
microorganisms in, 146, 147, 

220-222, 272, 273 



Air, relation to pigment formation, 

175 
Albumin, egg, use of, 29 

in milk, 23 
Albumins, coagulation by heat, 28 

soluble, as food, 172 
Albuminoids, 32 

effect of, on sterilization, 8 
Alcohol, by volume, table of, 377 

ethyl, 181-183, 215-217, 364, 
369 

ethyl, enzyme of, 181, 183 

ethyl, fermentation of, 183 

for cleaning microscope, 65 

for decolorization, 265, 266 

for drying, 3, 4, 16 

for extraction of fat, 92 

for producing vacuum, 156 

sterilization in, 6, 14, 257 
Alcoholase, 181, 183 
Alcoholic fermentation, 114, 122, 

171 

Alcoholoxidase, 181 
Aldehydases, 181, 183 
Aldehydes, 182 
Alkali, action of on methylen blue, 

87 
Alkali, free, 1 

normal, 20-22, 368 

production, 23 
Alkali-proteinase, 180 
Amidases, 181 
Amides, acid, 181 
Amino acids, enzymes of, 180 
Ammonia, 37, 44, 131, 134 

formation in soils, 245, 246, 252 
Ammonium carbonate, 181, 185 

hydroxide, 134, 245, 246 

malate, 353 

nitrate, 246 

sulfate, 170, 308, 350, 352, 353 

tartrate, 350 
Amoebobacter, 348 



INDEX 



397 



Amphoteric reaction of casein, 24 

Amygdalase, 180 

Amygdalin, enzyme of, 180, 182 

Amylase, 179, 182 

Anaerobe, 154 

obligate, 154 

partial, 154 
Anaerobic bacteria, 46, 154, 248 

culture methods, 155-167 
ANDERSON, JOHN F. and McCLiN- 

TIC, THOS. B., 213 . 
Anesthesia, general, 295, 296, 
299 

local, 295, 299 
Anesthetic, 295, 296, 309 

administration of, 295, 296 
Anilin, basis of dyes, 87 

dyes, 87 

oil, 373 
Anilin-water, gentian violet, 373 

preparation of, 373 

stains, 72, 94, 373 
Animal, fluids as media, 19 

inoculation, 295-301 

method of holding for inocula- 
tion, 296 

preparation for inoculation. 
295-301 

tissues, as media, 19, 20 
Animals, autopsied, destruction 
of, 6 

inoculated, care of, 301 
Anjesky's spore-staining method, 

90,303 

Anthrax, 302, 303 
Antibiosis, 45, 216 
Antibodies, 313-315, 328 
Antigen, for agglutination test, 
311 

for complement fixation test, 
325-327 

preservation of, 326 

titration of, 327 



Antiopsonic, 321 

Antiseptic, 184 

Antiseptics, sterilization by, 15 

Anti-serum, 310, 322 

Antitoxin, 309 

tetanus, 309, 310 
Apparatus, sterilization of, 6, 7 
Aqueous-alcoholic stains, 72, 87, 

88 

Are, 379 

Arnold sterilizer, 9, 10 
Arrangement of microorganisms, 

74 

Artery, femoral, 298 
Asbestos, shredded, as filter, 13 
Aseptic, IX 
Ash, in milk, 23 
Asparagin, 173, 350, 356 
Aspergillus niger, -100, 101, 104, 

105, 107, 173, 199, 207 
Aspiration, for filtration, 13 
Autoclav, IX, 11, 28 
Auxanography, 173 
Azolitmin, source of, 369 

solution, Kahlbaum's, -25, 181, 
369 

solution, purpose of, 369 
Azotobacter, 253-255, 354, 355 

chroococcum, 254 

B 

Bacillus, meat, 197 

slimy milk, 197 

Bacillus acidi lactiti, 347, 362, 
363 

aerogenes, 347, 362, 363 

aerogenes 2, 362, 363 
Bacillus alvei, 97 

amylovorus, 97 

caratovorus, 97, 291-294 

cholerce suis, 97, 310, 311, 362, 
363 

cloaca, 362, 363 



398 



INDEX 



Bacillus coli communior, 362, 363 
coli communis, 47, 97, 145, 146, 

152, 153, 154, 174, 188, 191, 

195, 196, 223-233, 289, 290, 

347, 352, 359-363 
cyanogenus, 175 
erythrogenes, 352 
fluorescens liquefaciens, 187, 347 
gelaticus, n. sp. (gran), 41 
indicus, 155 

lactis aerogenes, 362, 363 
megaterium, 97, 185, 199 
mesentericus vulgatus, 97, 174 
mycoides, 63, 97, 174, 187, 191, 

200, 201, 245, 264, 347 
oligocarbophilus, 250 
paralyphosus A, 362, 363 
paratyphosus B, 362, 363 
pasteurii, 352, 353 
phytophthorus, 97 
prodigiosus, 155, 170, 175, 176, 

185, 187, 195, 196, 205 
proteus, 97, 352 
radiobacter, 254, 255 
ramosus, 187, 188 
ruber, 155 
subtilis, 63, 97, 164, 173, 187, 

197, 198, 205, 264 
tetani, 97, 308 
typhosus, 97, 200, 201, 208, 209, 

210, 211, 227, 228,231, 233, 

310, 311, 347, 359-363 
violaceus, 155, 175, 188, 197 
vulgaris, 97 
Bacteria, acetic, 183 
acid-fast, 92 
aerobic, 46 
anaerobic, 46 
biochemical activities of, 23-25, 

182-185 

chromogenic, 242 
compared with protozoa, 140, 

141 



Bacteria, effect of desiccation on, 
197, 198 

effect of moist and dry heat on, 
202, 203 

flagella of, 93 

Gram-negative, 95-97 

Gram-positive, 95-97 

green, 154 

identification of, 125-138 

in air, 146, 147, 220-222 

longevity of, 197, 198 

nitrate, 249-251, 353 

nitrite, 249-251, 353 

pathogenic, 125, 221, 301 

pathogenic, isolation of, 301, 
302 

phosphorescent, 177, 178 

purple, 154 

slime-forming, 97 

soil, 241-244 

spores of, destruction by heat 
5-11 

study of, 125-132, 135-138 

sulfur, 154, 348 

true, 347, 348 

urea, 185 

vegetative forms, destruction 
of, by heat, 5-11 

vinegar, 183, 184, 192, 193 

water, identification of, 361-363 

weight of, 152, 153, 243, 244 
Bacleriacece, 347 
Bacterins, autogenous, 316, 317 

polyvalent, 318 

preparation of, 316-318 

preservation of, 15 

stock, 317 

Bacteriopurpurin, 348 
Bacterium abortus, 163, 325, 347 

aceti, 192, 215, 216 

acidi lactici, 97 

aerogenes, 97, 198, 227, 228, 231, 
233, 362, 363 



INDEX 



399 



Bacterium anthrads, 97, 98, 302, 

303 

bulgaricum, 96, 97, 347, 349 
lactis acidi, 167-169, 184, 185, 
188, 191, 199, 214, 215, 217- 
219, 289, 347, 349 
mallei, 97, 327, 328 
nenckii, 41 
tuberculosis, 90, 92, 97, 303-305, 

347, 373 

tumefaciens, 258, 259, 355 
Balsam, Canada, 65, 71, 372 
Banana, as a medium, 20 
Base, 20 

Bath, running water, 36 
Beaded, 87 

Bedding, sterilization of, 10 
Beef, chopped lean, 28 
Beer fermentation, 182 
manufacture, yeasts used, 114 
wort, 22 
Beggiatoa, 348 
BeggiatoacecB, 348 
BEIJERINCK, 41, 155, 170, 174 
Bengal isinglass, 37 
BENIANS, T. H. C., 97 
Benzaldehyde, 182 
Benzol, effect on pigment, 175, 

176 
Berkefeld filter, 238, 303, 304 

filters, sterilization of, 6 
BESSON, A., 15, 19, 133, 140, 147, 
149, 151, 167, 222, 303, 305, 
308, 310 

Bile, esculin, 360, 362, 363 
isolation of pathogens from, 

302 

litmus lactose, 223, 360 
salts, value in media, 360 
Biochemical activities, 23 
Black-leg vaccine, preparation of, 

307 
Bleaching, 237 



Blood, as a medium, 19, 20 

defibrination of, 302, 323 

dried, test of, 322 

enzyme of, 181 

isolation of pathogens from, 301 
Blood cells, red, injection of, 324 

cells, red, method of washing, 
324 

cells, red, use of, 323-329 

serum, as a medium, 19, 20, 
45 

serum, sterilization of, 8 
BOHME, A., 133 
Boiling, sterilization by, 10 
BOLDUAN, 291 

Bombicci's anaerobic dish, 159 
Books, text and reference, list of, 

381-388 

Botkin's anaerobic apparatus, 159 
BOURGEOIS, 32 
Bougie, 12 

Bouillon, nutrient, 19 
Bread fermentation, 182 
Bread-making, yeasts used, 114 
BREW, J. D., 268 
Broth, adonit, 362, 363 

carbohydrate, 19 

dextrose, 362, 363 

dulcit, 362, 363 

glycerinated veal, 303, 304 

lactose, 362, 363 

liver, 362, 363 

neutral red, 363 

nitrate, 251, 354 

nutrient, 19, 29, 46 

nutrient, preparation of, 29-31 

saccharose, 362, 363 

serum, 19 

sterilization of, 31 

sugar, 19, 84, 358 

sugar-free, 358 

urea, 352 
Brownian movement, 75 



400 



INDEX 



Buchner's anaerobic tube, 161, 

162 

Budding of yeasts, 78, 114 
Bullock's anaerobic apparatus, 

159 
Butter, 197 

canned, 285 

fat, preparation of, 349 

microflora, 284-285 

old, 285 

renovated, 285 

storage, 285 

trier, use of, 289, 290 



Caffein, 361 

Calcium carbonate, 167, 168, 349, 
352, 354, 355 , 

chloride, 351, 351 

citrate, 353 

malate, 353 

phosphate, di-basic, 263 

phosphate, mono-basic, 193 

phosphate, tri-basic, 263, 350 

salts, action of, in milk, 24 

sulfate, 354 

tartrate, 353 

Calculations for microscopic 
counts, 266, 267 

titration, 21, 22 

Calibration of filar micrometer, 
72-74 

lenses, 71-74 
Camphor, 15 

Candles, filter, sterilization of, 6, 7 
Canula, 299 

Capsules, presence of, 97, 98' 
Caramelization of sugar, 26 
Carbamases, 180 
Carbohydrases, 179 
Carbohydrate, agar, 37 
Carbohydrates as food, 167, 168 

enzymes of, 179-182 



Carbohydrates, fermentation of, 

117, 167 

Carbol-fuchsin, 72, 90, 92, 374 
Carbolic acid. See Acid 
Carbon bisulfid, effect on pigment, 

175, 176 
Carbon dioxid, 20, 117-119, 157, 

165, 171, 172, 216, 231, 374 
dioxid. absorption of, 117-119 
dioxid, atmosphere of, 157 
monoxid, 250 
Carmen rubrum, 75 
Carrot, as a medium, 20 
Case, pipette, 17, 18 
Casein, coagulating enzyme of, 

181 

colloidal state of,]24, 33 
in milk, 24 
proteolytic enzymes of, 188, 

189 

Caseinase, 181 

Catalase, 181, 184, 191, 239, 240 
Cautery, 6 
Cell division, 74 

-envelop, 97 
Cellulose, 179 
Cellulose, decomposition of, 246- 

249 

enzyme of, 179 
Centare, 379 
Centigram, 379 
Centimeter, 379 
Centrifuge, 273-275, 323, 324 

tubes, 323 
Ceylon moss, 37 
Chalk, 369 
CHAMBERLAND, 12, 158, 313, 315 

'filter, 12, 313, 315 
Changes in milk, 23-25 
CHAPIN, C. V. 222 
Chart, descriptive. Soc. of Am. 
Bacteriologists, 63, 121, 129, 
132, 226 



INDEX 



401 



Cheese, cheddar, 286 

microfiora of, 286, 287 

trier, 286 
Chemical agents of sterilization, 

5, 14, 15 

CHESTER, F. D., 131 
Chinese ink preparation, 85, 86 

preparation of, 372 

use of, 72, 85, 86 
Chlamydobacteriacece, 348 
Chlamydothrix, 348 
Chloral hydrate, use of, 295 
Chloride of lime in water purifica- 
tion, 236-238 
Chlorination, 237 
Chlorine, available, 236, 237 
Chloroform, 15, 65, 175, 176, 295 
Chlorophyll, pigment analogous 

to. 154 

Chromatium, 348 
Chromic acid cleaning solution, 

1, 48, 212 
Chromogenic organisms, 26, 154, 

264 

Chromoparous bacteria, 154 
Chromophorus bacteria, 154 
Chymosin, 181 
Cider, 20, 22, 46, 204 

fermented, 192, 351 

titration of, 20, 22 
Cladothrix, 348 
Classification of nutrient media, 

18-20 
Classifications, physiological, 153- 

155 

Clay, modeling, 165 
Clean, chemically, 1 
Cleaning glassware, 1, 2, 3, 4 

powder, 2 

solution, 1, 2, 48, 366 
Cloth, physicians, 3 
Coagulation, 8, 29, 179 
Cocaine hydrochloride, 295, 299 



Coccacece, 347 
Cohn's solution, 172 
Collagen.. 32, 38 
Collodion sac, 13, 299 
Colloid, reversible, 33, 38, 39 
Colloidal state of casein. 24, 33 
Colloids, 33, 195, 196 
Colonies, acid, 167, 225, 230 

counting, 56, 57 

zone development of, 164 
Colony, giant, 61-63, 109, 293 

isolated, 58 

mold, 105 

yeast. 120 

formation, 49, 79, 80, 84 
Columella, 100 
Combustion, total, 184 
Complement, 325-329 

destruction of, by heat, 326 

fixation test, 323, 325-329 

source of, 325 

titration of, 326 
Concentration of solutions, effect 

of, 53, 54 
Condensation water, 35, 38, 51, 

129, 131 

Condenser, microscope, 66, 69 
Congo red medium, 259 
Conidia, 101 
CONN, H. W., 84, 116, 132, 140, 

144, 147, 177, 243, 287 
Conradi-Drigalski's agar, 361-363 
Continuous heating, 8-11 
Contrast stain, 92, 93 
Cooling, value of, 203, 280 
Coprophyl, 154 
Cork borer, 26 

Cornea, inoculation into, 299 
Corpuscles, red, method of wash- 
ing, 323 

Corrosive sublimate, 186 
Cotton, absorbent, 26 

decomposition of, 248 



402 



INDEX 



Cotton, effect of sterilization on, 
7 

purpose of, 15 

waste, disposal of, ix 

plugs, preparation of, 16, 17 

plugs, rolled, 16, 17 
Counterpoise, 30 
Counter-stain, 92, 93 
Counting colonies, 56, 57 

lens, 56 

plate, Jeffer's, 56 

plate, Wolfhiigel's, 56 
Cover-glasses, cleaning of, 4 

sterilization of, 18, 48 
Cow, aborting, serum from, 325 
Cream, ripened, microflora of, 285 

pasteurization of, 281-284 
Crenothrix, 348 
Cresol, compound solution of, 

14, 48, 296, 297, 365 
Crucible, Gooch, as filter, 13 
Cryophilic bacteria, 155, 200 
Crystalloids, 33 

Crystals, formation of, 176, 252 
Culture, adhesion, 76, 78-80, 81 

hanging-block, 81, 82 

Lindner's droplet, 84, 85 

media, sterilization of, 8-13 

medium, 31, 37, 49 

methods, anaerobic, 155-165 
Cultures, ix, x, xi, 44-48 

broken, 48 

care of, 46-48, 125 

description of, 129 

dried, 198 

gelatin, 46 

impure, 44, 45, 107 

incubation of, 46, 47 

liquid, 46, 60 

mixed, 44, 45, 107 

old, disposal of, 10, 14, 48 

plate, 45, 49-56 

pure, 44, 45, 49, 107, 114 



Cultures, shake, 163 

slant, 45 

stab, 46, 61 

stick, 46 

streak, 46, 58, 60 

transferring, 51, 52, 60 
CUMMING, J. G., 13 
Cup, measuring, 28 
Curd, acid, 24, 185, 186 

liquefaction of, 24 

rennet, 24, 185, 186 
Curdling, cause of, 24 
Curette, use of, 316 
Curves, plotting, xi, 112 
Cutaneous inoculation, 297 
Cuts, care of, 48 
Cytase, 179 



Data, tabulation of, xi 
Death-point, thermal, 200-202 
Decagram, 379 
Decaliter, 379 
Decameter, 379 
Decigram, 379 
Decimeter, 379 
DECKER, J. W., 287 
Decolorization in staining, 90- 

93,96 

of litmus, 23, 24 
Defibrination of blood, 302, 320, 

323 

Degrees, Fuller's scale, 20, 21 
Denitrification in solution, 251, 

252 

studies, culture media for, 354 
Denitrifying organisms, 19, 251, 

252 
Deodorizer, chloride of lime as, 

237 

Desiccation, 197 

Desiccator, as anaerobic dish, 160 
Desk, microorganisms on, 146, 147 



INDEX 



403 



Dextrin, 179 
Dextrinase, 179 

Dextrose, 23, 156, 170, 180, 182, 
184, 195, 196, 351, 358, 359 
fermentation of, 180, 182 
influence on denitrification, 252 
Dextro-zymase, 180, 182 
Dialysis, as a sterilizing agent, 

5, 13 

Diastase, 179 
Diatomaceous earth, 12 
Dichotomous branching, 100, 101, 

348 

Dilution flasks, 52, 54 
method, loop, 49-52 
qualitative, 49-52 
quantitative, 52-56 
straight needle, 51, 107, 115 
Diphenylamin, sulfuric acid solu- 
tion of, 249, 371 
Directions, laboratory, x 
Disaccharides, enzymes of, 180, 

182 

Discoloration in water, 237 
Discontinuous heating, 8-10 
Diseases, animal, 295-329 
plant, 291-294 
producing bacteria, 153 
Dish, evaporating, use of, 20-22 
Dishes, deep culture, wrapping of, 

18 

Petri, wrapping of, 17, 18 
Disinfectant, phenol coefficient of, 

210-213 
Disinfectants, ix, 5, 14, 15, 236- 

238, 309 
DISTASO, 157 
Distilled water, 20 

micron 1 ora of, 152, 153 
DON, J. and CHISHOLM, J., 227, 

239 

DORSET, M., MCBRYDE, C. N. 
and NILES, W. B., 315 



Dourine, diagnosis of, 329 
Draw-tube of microscope, 65 
Drench, use of, 300 
Dunham's solution, 19, 43, 44, 

125, 129, 172 

Durham's fermentation tube, 119 
Dust, microorganisms in, 146 
Dyes, acid, 87 

basic, 87 

organic, reduction of, 181, 184 

saturated alcoholic solution of, 
87 

stock solutions of, 87 

E 

Earth, diatomaceous, 12 

Egg, albumin, liquefaction of, 24 

as a medium, 19, 20 

use of, in preparation of media 

30 
Ehrlich's method of testing indol 

production, 132, 133 
Electrolytes, 53, 195, 196 
Emboli, from injection, 299 
Emulsin, 180, 182 
Endo agar, 361-363 

-enzymes, 178, 190 
Endospores, 114, 121 
End-point, 20 

Enzyme, carbohydrate-coagulat- 
ing, 181 

action, pure, 188 
Enzymes, 179-194 

acting anaerobically, 179 

classification of, 179-181 

coagulating, 179, 181, 185, 186, 
193, 194 

effect of heat on, 186 

extracellular, 178 

formation of name of, 179 

hydrogen-producing, 23 

hydrolytic, 178, 179, 180, 182- 
185 



404 



INDEX 



Enzymes, intracellular, 178 

isomer-producing, 179 

lipolytic, 178, 180, 183 

oxidizing, 179, 181, 183, 184 

producing intramolecular 
change, 178, 180-185 

protein-coagulating, 181 

protein-digesting, 180 

proteolytic, 24, 25, 178, 180, 
187-189 

reducing, 179, 181, 184, 190 

rennet-like, 24 

specific, 167, 169, 172, 190, 192, 
217 

syntheses-producing, 179 
Eosin, 87 
Erepsin, 180 
Ereptase, 180 

ERNST, WM., 268, 273, 278, 281 
Esmarch's tube, 158 
ESTEN, W. M., 132 
Esterases, 180 
Esters, enzymes of, 180 

glycerin, 183 
Ether, 6, 92, 175, 176, 295 

flame, sterilization in, 6 
Eubacteria, 347, 348 
EULER, HANS, 15, 168, 179, 190, 

192-194 

Eurythermic bacteria, 155 
Evaporating dish, use of, 20-22 
Exo-enzymes, 178 
Extracellular enzymes, 178 
Extractives, from meat, 28, 29 
EYRE, J. W. H., 15, 121, 145, 146, 
158, 159, 161, 167, 175, 198, 
206, 222, 243, 296, 301, 302 



Facultative, 154 

Fat, butter, preparation of, 349 
construction of, 26, 183 
decomposition of, 183 



Fat, enzymes of, 180, 183 

in bacteria, 92 

in milk, 23 
Fats, natural, 183 
Fermentation, acetic, 192, 193 

alcoholic, 114 

gaseous, 117-119 

lactic acid, 167, 184, 217, 218 

Lindner's method of demon- 
strating, 83, 84 

spontaneous, 171 

tube, Durham's, 119 
Smith's, 117-119 

tubes, cleaning of, 4 

tubes, plugging of, 18 

urea, 185 

vinegar, 183 
Fermentative, 153 
Ferric chloride, 214, 354 

sulfate, 351 

Ferrous sulfate, 350, 353, 375 
Fibrin, coagulating enzyme of, 
181 

liquefaction of, 24 
Filar ocular micrometer, 72-74 
Filter, Berkefeld, 12, 238, 239 

candles, sterilization of, 6, 10 

-paper, decomposition of, 246- 
249 

-paper in media, 352 

pores of, 12 
Filters, bacterial, 12 

Berkefeld, sterilization of, 6 

Chamberland, 12 

cleaning of, 313 

porcelain, 12 

purification of, 313 
Filterable organisms, 12, 13 
Filtrate, germ-free, 12-14 
Filtration, as a sterilizing agent, 
5, 12, 13, 145, 146 

of liquid culture, 210 

rate of, 13 



INDEX 



405 



FISCHER, ALFRED, 153, 154, 171, 

178, 197, 200, 203 
FISCHER, EMIL, 132 
Fish, salt, 197 
Fishing a colony, 59 
Flagella, 93, 94 

staining method, 93-95, 258 
Flame, naked, sterilization in, 6, 

18, 82, 302 
Flasks, cleaning of, 3 
plugging of, 16, 17 
Roux, 2, 61, 62 
Floor, microorganisms on, 146, 

147 

Flowing steam, 10, 11 
Fluorescent, 360 
Focal point, 68, 77 
Focusing, 67-69, 77 
Food, requirements, variation in, 

170, 171 

small amount needed, 152, 153 
Foods, microbial, 18-20 
Forceps, sterilization of, 6 

use of, 16 
Form, for writing up exercises, 

xi, xii 
Formaldehyde, 188, 213, 214 

ring test, 214 
Formalin, 14, 188 
Formulae for conversion of de- 
grees of temperature, 377 
Freezing, effect of, 199, 200 
FROST, W. D., 268 
Fructification of molds, 100, 101 
Fruit juices, fermented, 20, 22, 

203, 204 

juices, natural, 20, 22 
juices, titration of, 22 
Fruiting bodies, 79, 100-109 
Fuchsin, 87, 88, 373 
acid, 87 
basic, 373 
FUHRMAN, F., 63 



Fuller's scale, 20, 21,40, 205 
Funnel, filling, 25 
Furnace, muffle, 6 

G 

GAGE, S. H., 69 

Galactose, 23, 180, 182 
fermentation of, 184 

Galacto-zymase, 180 

Garget, 275 

Gas, absorption of, 118, 119 

Gasometer, 117, 118, 120 

Gas-producing bacteria, 154 

Gas production, 25, 46, 83 
production in milk, 25 
production, qualitative, 117- 
119 

Gauze, hospital, 3 

Gelatin as a food material, 32 
constitution of, 32 
decomposition products of, 32 
discussion of, 31-35 
effect of enzymes on, 180 
effect of superdrying on, 34 
effect of superheating on, 34, 35 
liquefaction of, 24, 46, 174, 187, 

188, 195-197 
liquefaction, point of, 34 
lowering of liquefaction point, 

34, 352, 356 
nutrient, 19, 20, 36, 37 
nutrient, cooling of, 36, 52 
nutrient loss of solidifying 

power, 34, 35 

nutrient preparation of, 36, 37 
nutrient sterilization of, 35-37 
phenol, 187, 188 
physical properties of, 32, 33 
salt-free, 356-358 
size of molecule, 32 
solidifying point, 34, 352 
source of, 32 
acid in, 34, 187 



:406 



INDEX 



Gelatin, urea, 352 

waste, ix 
Gelatinizing property, loss of, 

34, 35 

Gelideum corneum, 37 
Gemmation, 120 
Gentian violet, 87 
Germicidal, 1, 365 
Germination of mold spores, 78, 79 

stages of, 79 
Giant colony, preparation of, 

61-63 
Giltay's agar, 354 

solution, 19, 251, 252, 354 
GILTNER, W., 160-162, 164, 312, 

316 

Giltner's H tube, 160-162, 164 
Glanders, diagnosis of, 329 
Glands, lymph, isolation of 

pathogens from, 301 
Glass rods, sterilization of, 6 
Glassware, cleaning, 1-4 

new, 1 

preparation of, for sterilization, 
15-18 

purpose of sterilization, 15, 16 

sterilization of, 6-15 
Glucosidases, 180, 182 
Glucosides, enzymes of, 180, 182 
Glycerin, 26, 183 
Glycogen, enzyme of, 179 
Glycogenase, 179 
Gonidia, 348 

Gooch crucible as filter, 13 
Gram, unit of weight, 380 

equivalent, 20 

molecule, 20 

negative, 95-97, 235 

negative bacteria, 97 

positive, 97, 235 

positive bacteria, 97 
Gram's stain, 95-97, 373 
Gram-Weigert staining method, 96 



Granules, metachromatic, 87 
GREEN, REYNOLDS, 84 
Growth, rate -of, 109 
GRUZIT, O. M., 361 

GUILLIERMOND, A., 182 

Guinea pigs, 295, 303, 308, 325 
Gypsum, 369 

H 

Hair, microflora of, 148, 149, 271 
Halophile, 154, 351 
HAMILTON, H. G. and OHNO, T., 

213 

HAMMER, B. W., 291 
Hands, bacteria on, 148, 272 

sterilizing of, 14 
Hanging block, agar, 81, 82 
Hanging drop, 59, 74-77 

purpose of, 74 
HANSEN, E., 121 
Haplobacterince,' 347 , 348 
HASTINGS, E. G., 189 
HAWK, P. B., 172, 190, 214 
Hay, bacteria on, 271 

infusion, 9, 19 
Hazen theorem, 238 
HEADDEN, W. P., 255 
Heat, as sterilizing agent, 5-12 
Heat, dry, 202 

for sterilizing, 5-7, 16 

moist, 5, 8-12, 202 
Heating, continuous, 8-11 

discontinuous, 8-10 

intermittent, 8-10 
Heat-producing bacteria, 154 
Hectare, 379 
Hectogram, 379 
Hectoliter, 379 
Hectometer, 379 
Hemicelluloses, enzymes of, 179 
Hemoglobin, 28 
Hemolysin, 325-329 

preservation of, 326 



INDEX 



407 



Hemolysin, source of, 325 

titration of, 327 
Hemolysis, 325-329 
Hemolytic serum, preparation of, 

323, 324 
system, 328, 329 

Hesse's method for anaerobes, 157 
Hexoses, 23, 180 
HILL, 81 
Hoffman and Fiske's enrichment 

medium, 361, 362 
HOFFMAN C. and HAMMER, B. W., 

255 

HOFMEISTER, 32 

HOLM, M. L. and GARDNER, E. A., 

213 

HOOKER, A. H., 236-238 
Hospital gauze, 3 
Hot air sterilization, 7 
HUEPPE, 38 
Humus, 239, 240 

Hydrochloric acid, N/l, prepara- 
tion ot, 367, 368 
acid N/20, 20 
acid N/20, preparation of, 367, 

368 

Hydrogel, 33 
Hydrogen, atmosphere of, 157, 

164, 165 
peroxide, enzyme of, 181, 184, 

191, 239, 240 

sulfid, Beijerinck's test for, 174 
sulfid, production of, 174, 175, 

190, 191 

sulfid, test for, 130 
Hydrophobia, 13 
Hyphse, 100, 101, 108 



IAGO, WM. and IAGO, WM. C., 116 
Ice cream, microflora of, 289-291 
Illumination for microscope, 67, 69 
Image, reversed, in microscope, 68 



Immunity, 295, 301, 303-329, 370 
in plants, 294 
production of, 295 
tests, 13 

Impression preparation, 98, 99, 
375 

Inactivated serum, 325-329 

Inactivation of serum, 326, 328 

Incision, crucial, 299 

Incisions, 297 

Incubator, 37, 53 

India ink, 85 

Indicators, 20, 369 

Indigo, 181 

Indol, 43, 131-133, 370 
Ehrlich's test for, 132, 133 
graphic formula of, 132 
in peptone, 132 
solutions, 370 
test for, 132, 133, 370 

Infection, x 

Infusion, hay, 9, 19 

meat, preparation of, 28, 29 

Inhibit, 1 

Inhibitive action, 216 

Ink, Chinese, 85, 86, 243, 247 
preparation of, 372 
preparation, Chinese, 85, 86 

Inoculation, animal, 295-300 
cutaneous, method of, 297 
ingestion, method of, 300 
intraabdominal, method of, 299 
intramuscular, method of, 298 
intraorbital, method of, 299 
intraperitoneal, method of, 299 
intrapulmonary, method of, 300 
intravenous, method of, 298 
of media, 45, 46, 59-61 
subcutaneous, method of, 297 
subdural, 299 

Inoculations, mold, 62 

Inoculum, 14, 197, 297 

Inosit, 359 



408 



INDEX 



Instruments, metal, sterilization 

of, 6, 14, 15 
Intermittent heating, 8-10, 37 

139 
Intraabdominal inoculation, 29 

322 

Intracellular enzymes, 178 
Intramolecular change, 178, 180 

182, 184, 185 

Intramuscular inoculation, 298 
Intraorbital inoculation, 299 
Intraperitoneal inoculation, 299 
Intrapulmonary inoculation, 300 
Intravenous inoculation, 298 
Inulin, 363 

Inversion of sugars, 23 
Invertase, 180 
Invertin, 180 
lodin solution, Lugol's, 95, 96, 

114, 115, 189, 309, 375 
tincture of, 14, 365 
Iris diaphragm, 63-69 
Iron, citrate, soluble, 360 

sulfate, 350 
Isinglass, Bengal, 37 
Isolation of pure cultures, 31, 

52-56 
Itch, barber's, 149 



Jaffna moss, 37 
Jenner's stain, 320 
JENSEN, C. O., 273, 278 
JONES, DAN H., 255 
JONES, L. R., 294 
JORDAN, E. O., 31, 132, 145, 199, 
202, 207, 303 

JOUBERT, 158 
JUNGANO, 157 

K 

Kidney, isolation of pathogens 
from, 301, 302 



Kieselguhr, 12 

Kilogram, 379 

Kiloliter, 379 

Kilometer, 379 

Kipp generator for hydrogen, 

165 

Kitasato's dish, 159 
Klatschpraparat, 98 
KLOCKER, A., 101, 114 
Knife, potato, 26 
Knives, sterilization of, 6 
KOCH, ROBERT, 31, 32 
Koch's first plates, 31, 32 
KRUSE, W., 191 



Labeling plates, 52 

Lacomme's tube, 158 

Lactacidase, 181, 184 

Lactase, 180, 182 

Lactic acid, 23, 184 

Lactose, 23, 167, 170, 223, 356 

enzyme of, 180, 182 

fermentation of, 182, 184 
LAFAR, F., 63, 78, 84, 101, 112, 
121, 167, 169, 172, 173, 177, 
191, 193, 199, 200, 203, 205, 
209, 217, 262 
Lamprocystis, 348 
Laparotomy, 299 
LASER, 169 
Lead acetate, 165 

acetate paper, use of, 125, 
190 

carbonate, 174 

Leguminosce, nodules of, 256-263 
LEHMANN, K. B. und NEUMANN, 

R. O., 63 

Leuco-compound of litmus, 23 
Leucocytes, 299, 319, 320 

in milk, 275 
Level, spirit, 50 
Leveling stand, 50 



INDEX 



409 



Levulose, 180 

fermentation of, 180 
Levulozymase, 180 
Liborius-Veillon anaerobic meth- 
od, 163 

Lichens, as source of litmus, 369 
Light, artificial, 209 

artificial for microscopical work, 

67 

diffused, 206, 207 
producing bacteria, 154, 177, 

178 
relation to pigment formation, 

176 
Lindner's concave slide culture, 

120 

droplet culture, 84 
fermentation method, 83, 84 
Lipase, 180, 183 
Lipases, action of, 183 
LIPMAN, J. G. and BROWN, P. E., 
244, 247, 249, 251, 253, 255, 
263, 354 
Liquefaction of boiled egg white, 

24 

of fibrin, 24 

of gelatin, 24, 46, 187, 188 
of milk curd, 24 
Liquor cresolis compositus, 48, 

296, 297, 365 
Liter, 379 
Litmus, decolorization of, 23, 25, 

181 

Merck's purified, 369 
milk, 23-26 

reduction of, 23, 24, 181 
solution, 23, 26 
solution, purpose of 369 
source of, 369 
Loam, clay, Azotobacter in, 253 

sandy, Azotobacter in, 253 
Loeffler's alkaline methylen blue, 
265 



LOHNIS, F., 63, 133, 168, 173, 175, 
192, 241, 244, 247, 249, 251, 
253, 255, 262, 264, 268, 278, 
285, 287 

Longevity, 198 

Loop dilution method, 51 

Lugol's iodin solution, 95, 96, 114, 
115, 189, 309, 375 

M 

Macroscopical changes, 23, 142 
Magnesium ammonium phos- 
phate, 246 

carbonate, basic, 353 
oxid, 245 
phosphate, 252 
sulfate, 350-355 
Magnification, 70-74 
Maltase, 180, 182 
Malt extract, 19 
Maltose, enzyme of, 180, 182 
fermentation of, 182 
from starch, 182 
Manganese sulfate, 351 
Mannit, fermentation of, 253, 254 

solution, 253-255, 354 
Manure, anaerobic bacteria in, 

165 

Azotobacter in, 253 
bacteria in, 165, 241, 243, 248, 

253 

cellulose-decomposing organ- 
isms in, 246, 247 
Measuring microorganisms, 70- 

74, 109 

MARSHALL, C. E., 15, 101, 113, 
114, 140, 141, 144-147, 149, 
151, 153, 167, 169, 171-173, 
175, 178, 186, 187-193, 197- 
200, 202, 203, 205-207, 209, 
213-215, 217, 219, 222, 227, 
233, 239, 243, 244, 248, 249, 
251, 253, 255, 262, 264, 268, 



410 



INDEX 



270, 273, 278, 281, 285, 287, 
289, 291, 294, 303, 305, 307, 
308, 310, 312, 323, 331 
Mason jar, 160 
McBETH, I. B. and SCALES, F. M., 

249 

McBRYDE, C. N., 315 
MCCAMPBELL, E. F., 318, 321 
MCFARLAND, J., 91, 312, 321, 323 
McLeod's plate base, 163 
Mastitis, 275 
Meat, as"a medium, 19 
infusion, preparation of, 28, 29 
products, as media, 19 
Media, acid, 22 
albuminous, 8 
alkaline, 20 

culture, sterilization of, 8-13 
liquid, 19, 20, 22-26 
mounting, 372 
natural, 19, 20, 22-26 
nutrient, 19, 20, 22-26, 44 
nutrient, classification of, 19, 20 
over-heating of, 26 
prepared, 19, 20 
solid, 19, 20 
solid liquefiable, 20 
solid liquefiable, disposal of, ix 
solid, nbn-liquefiable, 20 
synthetic, 19, 20, 40, 170, 171 
water analysis, 356-361 
Medium, culture, solid, 31, 37, 

45,46 
enrichment, Hoffman and 

Fiske's, 361, 362 
glycerin potato, 26, 27, 45 
liquefiable solid, 46 
physical condition of, 131 
standard liquid, 29 
Membrane, semi-permeable, 13 
Mercuric chloride, 364 
corrosive character of, 364 
germicidal action of, 364 



Mercuric chloride, poisonous na- 
ture of, 364 
solubility of, 364 
stock solution, 364 
synonyms of, 364 
1-500, 210, 212, 259 
1-1000, x, 1, 14, 48, 77, 147, 

210, 212, 268 
Mesophilic bacteria, 155 
Metabiosis, 45, 204, 215-217 
Metatrophic, 154, 155 
Meter, 379 
Methylen blue, 23, 87-93, 179, 

181, 184 

enzyme of, 179, 181, 184 
for yeasts, 373 
Loeffler's alkaline, 265, 266, 

374 

leuco-base of, 184 
reductase, 181, 184 
Methyl indol, 132 
Metric system, 379, 380 
Mice, white, 295 
Micrococcm gonorrhea, 97 
tetragenus, 97, 347 
varians, 195, 196 
Microflora of hair, 148, 149 
of the mucous membrane, 150, 

151 

of skin, 148 
of soil, 241-244 
Micrometer, head, 65 
object, 70-74 
ocular, 70-74, 265 
ocular, filar, 72-74 
stage, 70-74, 265 
step, 70, 71, 74 
Micromillimeter, 379 
Micron, 33, 70 ; 71, 73, 74, 379 
Microorganisms, arrangement of, 

74 

chromogenic, 26 
determination of size, 70-74 



INDEX 



411 



Microorganisms, effect of sunlight 
on, 208, 209 

filterable, 12, 13 

food requirements of, 18, 19 

in soil, 241-244 

measuring of, 70-74 

pathogenic, 26, 301, 303, 308, 
310, 313, 315 

products of, 15 

rennet-producing, 24 

resistance of, 10 

salt-resisting, 197 

starch-digesting, 189, 190 

sugar-resisting, 197 
Microscope, ix, 58, 63-69 

carrying the, 65 

cleaning the, 65, 66 

focusing the, 65-69 

how to use, 63-69 

lighting for, 67 
Microspira comma, 347 

deneke, 97, 347 

finkler prior, 97, 347 
MIGULA, W., 347, 348 
Migula's classification, modified, 

347, 348 
Milk, acid production in, 23 

action of calcium salts in, 24 

as a medium, 19, 20, 22-26, 
46, 129, 141-144 

" bloody," 177 

blue, 177 

bottles, bacteria in, 272 

cans, bacteria in, 272 

cells in) 267 

changes in, 23-25 

clarifier, value of, 276 

composition of, 23 

condensed, microflora of, 288, 
289 

cow's, coagulating enzyme of, 
181 

curd formation in 24, 280 



Milk, dilutions for plating, 142, 
264, 268, 269, 274, 276, 279, 
280, 282 

dirt in, 273-278 

effect of straining, 276-278 

extraneous contamination of, 
270-273 

fore, bacteria in, 268, 269 

gas production in, 25 

germicidal action of, 280 

human, coagulating enzyme of, 
181 

keeping quality of, 278-281 

litmus, 23, 25, 26, 361, 363 

litmus, preparation of, 25, 26 

market, 278-281 

microflora of, 264-268, 274 

middle, bacteria in, 268, 269 

pails, bacteria in, 272 

pasteurization of, 203-205, 281- 
284 

peptonization of, 24, 25 

phenol, 185,186 

pure, 278-281 

reaction of, 25, 177 

red, 177 

separated, 25 

skimmed, 25 

sour, 25 

staining of, 96 

standards, bacteriological, 268 

sterilization of, 26, 139 

strippings, bacteria in, 268, 
269 

titration of, 20-23 

viscosity of, 283, 288 

whole, 25 
Milking tube, 269 
Millier, 380 
Milligram, 380 

equivalent, 20 
Millimeter, 33, 63, 70, 379 
Millimicron, 33, 379 



412 



INDEX 



Mirror, concave, 67 

plane, 67 
MOHLER, J. R. and EICHHORN, A., 

329 
MOHLER, J. R., EICHHORN, A. and 

BUCK, J. M., 329 
Moist chamber, preparation of, 

80,81 

culture, 100, 109 
Moisture, 

relation to microbial destruc- 
tion by heat, 202, 203 
Mold colony, examination of, 105, 

107, 109 
growth, characteristics of, 100- 

104, 106, 108 

spores, germination of, 78-80 
Molds, 100-113, 182 
in air, 221 

influence of light on, 206, 207 
microscopical examination of, 

105 

pathogenic nature of, 113 
resistance to heat, 202 
study of, 107-111 
MOORE, V. A., 156, 302, 305 
MOORE, V. A. and FITCH, C. P., 

301, 302 
Mordant for flagella staining, 94, 

375 
MORTENSEN, M. and GORDON, J., 

291 
Moss, Ceylon, 37 

Jaffna, 37 

Motility, 74, 75, 86, 129 
Mounts, permanent, 372 
Mouth, microflora of, 150, 151 
Mouths of culture tubes, flasks, 

sterilization of, 6 
Movement, Brownian, 75 
molecular, 75 
rate of, 74 
Mucilage of Ps. radicicola, 258 



Mucor, 63, 100 

stolonifer, 100 
Mucous membrane, microflora of, 

150, 151 

Mucus, staining of, 87 
Muffle furnace, 6, 7 
MUIR, R. and RITCHIE, J., 163 
Mutual relationship, 215-217 
Mycelium, 79, 100-102, 105, 107- 

110, 112, 113 

Mycoderma, 169, 184, 195, 196 
Mycodermata, 114 
Myriagram, 380 
Myriameter, 379 

N 
a-naphthylamin, 133, 249, 251, 

371 
Needles, platinum, sterilization 

of, 6 

Nephelometer, 319 
Nessler's solution, 133, 249, 251, 

371, 372 

Neutralization, 20, 22, 25 
Newspaper, uses of, 17, 18 
Nitrate peptone solution, 19, 44 

reductase, 181 

Nitrates, reduction of, 44, 131, 
133, 134, 138, 181, 251-253, 
363 

test solutions for, 371 
Nitrification, 255 
in solution, 249-251 
studies, media for, 353, 354 
Nitrifying bacteria, 153, 353 
Nitrite reductase, 181 
Nitrites, 43, 44, 133, 134, 138, 250, 

252 

reduction of, 181 
test solutions for, 371 
Nitrogen as food, 172, 173 
atmosphere of, 157, 165 
cycle, 255 



INDEX 



413 



Nitrogen-fixation studies, media 

for, 354, 355 
-fixation, non-symbiotic, 253- 

255, 264 
-fixation, symbiotic, 256-263, 

264 

from nitrates, 44, 134 
inorganic as food, 172, 173 
organic, 172, 173 
Nodule formation, observation of, 

259-262 
Nodules, on leguminous plants, 

256-263 

Non-electrolytes, 195, 196 
NORGAARD, V. A. and MOHLER, 

J. R., 307 

Normal acid, 20-22, 367, 368 
alkali,>0-22, 368 
solutions, 20-22, 367, 368 
NORTHRUP, Z., 169, 215 
Nose, microflora of, 150 
Nosepiece, collar, 66 

triple, 65 
Notebook, xi 
NOVY, F. G., 132, 158, 160, 164, 

202 
Novy's jars, anaerobic, 158, 160, 

164 

NOYES, WM. A., 20 
Nuclei, stain for, 99 
Nutrient media, 18-20, 23-44 
Nutrose, 361 
NUTTALL, G. H. F., 323 

O 

Objective, oil immersion, 69, 71 
Objectives, 63, 66, 67, 69, 71, 77 

achromatic, 71 

cleaning of, 66 
Obligate, 154 
Oculars, 66-72 

cleaning of, 66 
Odor, fecal, cause of, 132 



Oidiam, 101 
Oil, cedar wood, 372 
immersion, 66, 69, 372 
linseed, 365 
of garlic, 15 
of mustard, 15 
olive, 157 

Oleomargarine, 285 
Omelianski's medium, 248, 352 
Oospora lactis, 101, 107, 108, 169, 

198, 205, 207, 214, 215 
Operation, site of, 297-300 
Opsonic index, determination of, 

319-321 

test, McCampbelPs modifica- 
tion, 321 
Opsonins, demonstration of, 319- 

321,370 
Optical axis, 66 
Orcin, 369 
Organic matter, effect of chloride 

of lime on, 236-238 
OBI, 157 

Osmotic pressure, 52-54, 196 
Outline, for study of microbiology, 

331-346 

Oven, for hot-air sterilization, 7 
Over-heating media, 26 
Oxidases, 181, 183, 184 
Oxidation, 183, 184, 237 
Oxygen, absorption of, 156, 160- 

164 

free, liberation of, 181, 184, 240 
requirements, 131 
tolerance, 154, 164 
transference of, 181 



Paper, effect of sterilization on, 

7 

filter, in media, 352 
lens, 66 
uses of, 15, 16 



414 



INDEX 



Papilionacece, attacked by Ps. 

radicicola, 256 
Parachrome bacteria, 155 
Parachymosin, 181 
Para - dimethyl - amido - ben- 

zaldehyde, 133, 370 
Paraffin, 76, 78, 80 

oil, 157, 308 
Paraformaldehyde, 214 
Parasite, wound, 293 
Parasites, facultative, 154 

obligate, 154 
Paratrophic, 154 
Paratyphoid group, 362 
Parfocal, 66 
PARUMBARU, 38 
PASTEUR, L., 8, 13, 158, 159 
Pasteurization, 8, 203-205, 215, 

281-284 
effect on digestibility of milk, 

283 
factors influencing efficiency, 

283 

Pathogenicity, 295 
Pathogenic material, care of, x 
nature of molds, 113 
organisms, 26, 88, 129, 151, 153 
organisms, care in staining, 88 
PAYEN, 38 

Peat, bacteria in, 242, 243 
Pecilothermic bacteria, 155, 200 
N Pectase, 180 
"Pectin, 181 
Pectinase, 181, 294 
Pectose, enzyme of, 180 
Pencillium, 63 
italicum, 101, 104, 105, 107, 195, 

196, 207 

Pentoses, enzymes of, 180 
Pepsase, 180 
Pepsin, 24, 180 
Peptase, 180 
Peptone, 29, 43, 44, 170, 172 



Peptones, enzymes of, 180 
Peptonization, 24 

of milk, 24 
Perhydridase, 181 
Pericardial fluid, isolation of 

pathogens from, 302 
Periodicals, list of, 388, 389 
Peroxidase, 181, 191 
Petri dishes, cleaning of, 3 

wrapping of, 15, 17, 18 
Phenol (see carbolic acid) 
coefficient, 210-213 
remedy for burns caused by, 365 
stock solution of, 364 
synonyms of, 364 
value as a disinfectant, 365 
Phenolphthalein as indicator, 20- 

22, 369 
Phosphates, insoluble to soluble, 

263, 264 

Phosphorescence, 177, 178 
Phosphorus, relation to decay, 

263, 264 

Photogenic, 154, 177, 178, 351 
Phototaxis, 206, 207 
Phototropism, 207 
Phragmidiothrix, 348 
Physical agents of sterilization, 5 

condition of medium, 198 
Physician's cloth, 3 
Physiological efficiency, 262, 293 
Physiology of microorganisms, 

152-219 

Pickles, brine, 197 
Pigment, crystals of, 176 
formation, effect of temperature 

on, 175 
formation, relation of air to, 

175 

formation, relation of light, 176 
-producing bacteria, 154, 155. 

175-177 
solubility of, 176 



INDEX 



415 



Pigments, microbial, effect of 
physical and chemical agen- 
cies on, 175-177 
Pipette case, 17, 18 
Pipettes, cleaning of, 3 

preparation of, for steriliza- 
tion, 18 

use of, 22, 54, 55, 112 
Planococcus agilis, 347 
Planosarcina mobilis, 347 
Plants, microbial diseases of, 

291-294 
Plasmolysis, 54 
Plasmoptysis, 54 
Plates, agar, 51, 52 
gelatin, 52 
inverting of, 52 
Plating, 45, 49-56 

room, 81 
Platinum, spongy for absorbing 

oxygen, 157 

needles, sterilization of, 6 
Pleuritic fluid, isolation of patho- 
gens from 302 
Plugs, cotton, 15-17 

rolled, 16 

Poikilothermic bacteria, 155 
Polypeptids, enzymes of, 180 
Folysaccharides, enzymes of, 179, 

180, 182, 184 
Porcelain niters, 12, 13 
Pores of filter, 12 
Pork, salt, type of rrJcroflora, 197 
Potability of water, 227 
Potassium dichromate, 366 
hydroxide, 134, 365, 374 
iodid, 365, 375 
nitrate as food, 44, 252, 354 
permanganate solution, use of, 

313 

persulfate, 133, 370 
phosphate, di-basic, 170, 246, 
247, 350-356 



Potassium phosphate, mono-basic, 

350, 354 
Potato, as a medium, 19, 20 

glycerin, 26, 27, 45 

knife, 26 

tubes, preparation for steriliza- 
tion, 26 

tubes, Roux, 26 
Precipitin test, 321-323 
Precipitate, flocculent, in media, 40 
PRERCOTT, S. C. and WINSLOW, C. 

E. A., 153,227,228,233,360 
Pressure, for filtration, 13 

osmotic, 53, 54 

steam under, 10, 12 

temperature table, 376 
Probe, use of, 297 
Products, metabolic, 217-219 

microbial, study of, 15, 217-219 
Protamins, 180 
Protease, 180 
Protein decomposition products, 

132 

Proteinases, 180 
Proteins, enzymes of, 180 

soluble, as food, 172, 173 
Proteolytic enzymes, 24, 180, 185- 

189 

Proteoses, enzymes of, 180 
Proteus vulgaris, 97, 352 
Protoplasm, stain for, 87 
Prototrophic, 154 
Protozoa, 140, 141 
Pseudomonas campestris, 97, 208, 
347 

lucifera, 177 

medicaginis, 97 

pyocyanea, 175 

radidcola, 173, 256-263, 355 

radicicola, isolation from no- 
dule, 256-263 

radicicola, physiological effi- 
ciency of, 256-263 



416 



INDEX 



Psychrophilic bacteria, 155 
Ptyalin, 179 
Pumice stone, 2 

Pump, electric vacuum or 
pressure, 159 

mercury vacuum, 159 

water vacuum, 159 
Purification of filter candles, 313 
Pus, collecting, 14 

isolation of bacteria from, 148, 
149 

staining of, 87, 92 
Putrefactive, 153 



Quintol, 380 



R 



Rabbits, 295, 309, 322-325 

Rabies, 13 

RAHN, O., 168, 219, 246 

Rats, white, 295 

Rays, light, 209 

radium, 209 

X, 209 

Reaction, 20-22, 29, 30, 36, 112, 
199, 206, 215 

adjustment of 21, 30, 36 

amphoteric, of casein, 24 

maximum, 206 

minimum, 206 

of water analysis media, 357 

optimum, 29, 206 
Reactions, enzymic, 182-185 
Reductase, methylen blue, 181, 
184 

nitrate, 181 

nitrite, 181 

sulfur, 181 
Reductases, 181, 184 
Reduction, of litmus, 23, 24 

organic dyes, 181, 184 
References, xii, 381-389 



Refraction, counteracted, 372 

index of, 69, 372 
Refractivity of spores, 131 
Refrigerator, use of, 28 
Rennet, 24, 181, 193, 194 

curd, 24 
Rennin, 181 

Resistance of microorganisms, 10, 
27 

spores, 10 

Respiration of bacteria, 165, 177 
DE REYPAILHADE, J., 190 
Rhabdochromalium, 348 
Rhizopus nigricans, 100, 102, 103, 

207 

Rhodobacteriacece, 348 
RICHMOND, H. D., 194 
RIDEAL, S. and RIDEAL, E. K., 

213 

Ringworm, 149 
ROGERS, L. A.', 285 
Root tissues, invasion by bacteria, 

291-294 

ROSENAU, M. J., 270, 278, 284 
Rot, soft, bacterial, 291-294 
Roux, 309 
Roux's anaerobic tubes, 158 

biological method for anaerobes, 
163, 164 

flask, 62, 109 

tubes for potatoes, 26 
Rubber apparatus, sterilization of 

9 

RTJEHLE, G. L. A., 221, 222 
Ruffer's flask, 159 
Rules for culture media, 19 

laboratory, ix, x 

RUSSELL, H. L. and HASTINGS, 
E. G., 205, 270, 281, 285, 287 

S 

Sac, collodion, 13 
conjunctival, 295 



INDEX 



417 



Saccharomyces, apiculatus, 83, 114, 

120 
cerevisice, 83, 97, 114, 115, 120, 

171, 182, 195, 198, 199 
ellipsoideus, 114, 173, 215, 216, 

351 

fragilis, 182 
kefir, 182 

membrancefaciens, 114 
tyricola, 182 
Saccharomycetes, 114 
Saccharophile, 154 
Saccharose, 170, 171, 195, 355, 

356, 359 

enzyme of, 180, 182 
fermentation of, 182 
SADTLER, S. P., 172, 190, 193, 289 
Salkowski- Kitasato test for indol, 

132 
Salt, citrated, 370 

effect on microorganisms, 196, 

197 

normal, 370 
phenol, 303, 304 
physiological, 370 
purpose of, in media, 29 
solution, 52, 370 
solutions, 370 
Salts, soluble, of meat, 28 
Sand grains, size of, 245, 246 

quartz, 245 

Sapo mollis U. S. P., 365 
Saprogenic, 153 
Saprophile, 154 
Saprophyte, 154 
Rartina lutea, 175, 347 
SAVAGE, W. G., 214, 227, 233, 268, 

273, 287, 289, 291 
SAYER, W. S., RAHN, O. and 

FARRAND, BELL, 285 
Scale, Fuller's, 20, 21 
Scarification, for inoculation, 297 
Schardinger's reaction, 181 



SCHULTZ, N. K, 41 

SCHUTZENBERGER, 32 

Sealing anaerobic cultivations, 156 

microscopical preparations, 76, 

79, 80, 82, 83 ; 85 
Seaweeds, agar from, 37 
Sediment test, macroscopic, 276- 
278 

microscopic, 274-276 

tester for milk, 274-278 
Sedimentation tubes, 274 
Seed inoculation, 259-263 
Seeds, sterilization of, 259-261 
Selective action, 170 
Septate, 100, 101 
Serum, blood, 310 

blood sterilization of, 8 

hemolytic, production of, 323, 
324 

hemolytic, titration of, 326, 327 

immune, 311 

inactivation of, 325, 328 

suspect, 325, 328 
Serums, preservation of, 15, 328 
Sewage, bacteriological analysis 
of, 223, 228-233 

purification of, 236-239 
Sheaths of bacterial cells, 348 
Sheep, use of, 325-329 
Silicic acid, colloidal state of, 33 
Silver nitrate solution, 165 
Size of microorganisms, deter- 
mination of, 70-74 
Skatol, 132 
Skin, microflora of, 148, 149, 272 

sterilization of, 14 
Slides, cleaning of, 4 

labelling of, 89 

permanent, 89 

sterilization of, 18, 48 
Slime-forming organisms, 97, 197, 

202 
SMIRNOW, M. R., 27 



418 



INDEX 



SMITH, ERWIN F., 41, 113, 198, 

209, 263, 294 
SMITH, THEOBALD, 117 
Smith's fermentation tube, 117 
Soap, linseed oil potash, 305 
Sodium carbonate, 26, 351, 353, 

367 

chloride, 350-354, 370^ 
citrate, 319, 37& 
formate, 156 

hydroxide, effect of, on pig- 
ment, 175, 176 
hydroxide for anaerobic culture, 

156, 367 
hydroxide for cleaning, 3, 4, 

212, 367 
hydroxide for CO 2 absorption, 

367 

hydroxide N/l, 21, 22, 29, 368 
hydroxide N/20, 20, 21, 368 
lactate, 356 
nitrate, 170, 354 
nitrite, 353 
sulfindigotate, 156 
taurocholate, 360 
Sohngen's solution, 353 
Soil, as a medium, 19 
borer, 241 

catalytic power of, 239-241 
cellulose-decomposing bacteria 

in, 246-249 
denitrifying bacteria in, 251- 

253 

diseased spots in, 255 
extract, preparation of, 351 
microscopical enumeration of 

bacteria in, 243, 244 
nitrifying bacteria in, 249-251 
Solution, albuminoid-free, 353 
citrated salt, 319 
Cohn's, 350 
culture for nitrifying bacteria, 

353, 354 



Solution, Giltay's, 354 

mannit, 253, 254, 354, 355 

Nessler's, 133, 134, 371, 372 

Sohngen's, 353 

Uschinsky's, 350 

Solutions, concentration of, 52-54, 
195-197 

normal, 20-22, 367, 368 

standard, 367, 368 
Spatulas, iron and nickel steril- 
ization, 6 

Specific gravity, alcohol by vol- 
ume, 377 
Spectrum, 209 
Spirillacece, 347 
Spirillum rubrum, 348 
Spirocheta obermeieri, 87, 348 
Spirosoma nasale, 347 
Spleen, isolation of pathogens 

from, 301 

Sporangiophore, 100, 102, 103 
Sporangium, 100, 102, 103 
Spore formation, study of, 144, 

145 

Spores, ix, 5, 74, 100-104, 107, 
109, 110, 144-145 

bacterial destruction of, by 
heat, 9, 10, 26, 27 

effect of sunlight on, 209 

mold, germination of, 78, 85 

resistance of, 10, 35, 144, 145, 

197-202 

Spore stain, 90, 91 
Sputum, staining of, 92, 93 
Staining, 58, 87-89 

capsules, 97, 98 

flagella, 93-95 

Gram's method, 95, 97 

Gram-Weigert method, 96 

in mvo, 116 

of tissues, 96 

purpose of, 87 

time necessary' for, 88 



INDEX 



419 



Stains, anilin-water, 72, 95, 96, 

373 

aqueous-alcoholic, 72, 373 
preparation of, 373, 374 
saturated alcoholic, 72, 373 
Stand, microscope, 63, 65 
Staphylococcus pyogenes albus, 97, 

318, 347 

aureus. 97, 318, 347 
Starch, decomposition of, 182, 

189, 248 

enzymes of, 179, 182, 189 
grains, in yeast cake, 116 
soluble, 179, 189 
Steam, flowing, 10 
high pressure, 10, 11 
sterilization, 10, 12 
superheated, 10, 11 
Steapsin, 180 

Stearin, enzyme of, 180, 183 
Stearinase, 180, 183 
Stenothermic bacteria, 155 
Sterigmata, 101 
Sterilization, 5-15 
by antiseptics, 15 
by continuous or discontinuous 
heating in water at 100 C., 
8,9 

by dialysis, 13 
by disinfectants, 14, 15 
by dry heat, 6, 7 
by dry heat in ether flame, 6 
by dry heat in hot air, 6, 7 
by dry heat in muffle furnace, 6 
by dry heat in naked flame, 6 
by filtration, 12, 13 
by flowing steam at 100 C.; 
continuous or discontinuous, 
10, 139 

by moist heat, continuous or 
discontinuous heating at low 
temperatures, 8 
fractional, 34, 139 



Sterilization of. a large bulk of 
medium, 26 

of agar, 43 

of litmus milk, 26 

of meat infusion, 28 

of nutrient broth, 31 

of nutrient gelatin, 37 

of potato medium, 27 

of water analysis media, 356 

superheated steam, 11 

Tyndall method, 8, 9, 139 
Sterilizer, Arnold, 9, 10 

hot air, 7, 17 

steam, ix 

STERNBERG, G. M., 177 
STITT, E. R., 301, 318 
STOCKING, W. A., 132 
Stolon, 100 
Straw, decomposition of, 248 

microorganisms on, 271 
Streptococci in milk, 275 
Streptococcus erysipelatus, 347 

pyogenes, 47, 97, 318, 347 
Slreptothrix, 242 

actinomyces, 97 
Subcutaneous inoculation, 297, 

309 

Subdural inoculation, 299, 300 
Substrate, nutrient, 101 
Sucrase, 180, 182 
Sugar, caramelization of, 26 

for demonstrating gas pro- 
duction, 46 

milk, 184 

muscle, 359 
Sugars as food, 167, 170 

complex as food, 167 

fermentation of, 171, 172, 182, 
184 

inversion of, 23 

simple, as food, 167 
Sulfur, lead-blackening, 174 

reduction of, 181, 190, 191 



420 



INDEX 



Sulfur, reductase, 181 

Sunlight, effect of, 176, 208, 209, 

243 

SURFACE, F. M., 329 
Surfaces, polished, sterilization 

of, 6 
Surgical dressings, sterilization 

of, 10 

Suture, 299, 300 
Swab, preparation of, 150 

use of, 316 

Symbiosis, 45, 214, 215, 256 
Synaptase, 180 

Synthetic media, 171, 248, 350 
Syphilis, hemolysin in, 325 
Syringe, 295, 309 
Syringes, sterilization of, 10 



Tannase, 180 

Tannin, 375 
enzyme of, 180 

TARROZZI, 155, 156 

Teeth, microflora of, 150 

Temperature, 16, 198-203 
body, 47 

cardinal points of, 198, 200 
constant, ix, 47, 49, 52 
degrees, conversion of, 377 
effect on microorganism and 

spores, 199-203 
effect on pigment formation, 

175 

for sterilization, 8-11 
influence on keeping quality of 

milk, 278-281 
low, for sterilization, 8 
maximum, 155, 198, 199 
minimum, 155, 198, 199 
optimum, 155, 198, 199 
pressure table, 376 
relations, 155 

Tenacula, 299 



Test tubes, cleaning of, 2 

plugging of, 16 
Tests, immunity, 13 

Tetanus, antitoxin preparation 
of, 309, 310 

toxin, preparation of, 308 
Thermal deathpoint, 200, 201 
Thermogenic, 154 
Thermometer, clinical, 313 
Thermophilic bacteria, 155 
Thiobacteria, 348 
Thiocapsa, 348 
Thiocystis, 348 
Thiodictyon, 348 
Thiopedia, 348 \ 
Thiosarcina, 348 
Thiospirillum, 348 
Thiothece, 348 
Thiothrix, 348 
THRESH, J. C., 177, 2271 
Throat, microflora of, 150 
Thrombase, 181 
Thrombin, 181 
Thymol, 15 

Time for sterilization, 8-11 
Tissue, diseased, isolation of 
pathogens from, 301, 302 

sterile for oxygen absorption, 
156 

vegetable, sterile, 156 
Tissues, connective, 156 

staining of, 96 

subcutaneous, 297, 299 
Titration, 20-22 

calculations, 21, 22 
Titre, 21, 25, 112 
Toluol, 15 
Tonneau, 380 
Torula rosea, 83, 114, 120, 175, 

176, 205 
Torulce, 114 

Toxin, tetanus, preparation of, 308 
Toxins, 308-310 



INDEX 



421 



TRALLES, 377 

Transferring cultures, 51, 59-61 

Tray, operating, 309 

Trephine, 295, 300 

Trjetrop's anaerobic apparatus, 159 

Trichobacterince, 348 

Trichophyton tonsurans, 149 

Tricresol, 304 

Trocar, 299 

Trypsase, 180 

Trypsin, 24, 180 

Tryptase, 180 

Tube length of microscope, 65, 66, 

71 
Tubercle bacteria, method of 

staining, 92, 93 
Tubercles, staining of, 92, 93 
Tuberculin, preparation of, 303, 

306 
Tubes, fermentation, cleaning of, 4 

plugging of, 18 

vacuum, 158 
Tumblers, use of, 47 
Tumors, crown-gall, 258 
Turbidity in water, 237 
Turro's anaerobic tube, 161 
TYNDALL, J., 8-10, 147 

method for sterilization, 8-10, 

193, 217, 356, 358 
Typhoid group, 362, 363 
Tyrosin, 181 
Tyrosinase, 181 

U 

Udder, bacteria in, 268-270 

infection of, 270 
Ultramicioscope, 33 
Urea, 181, 185, 352, 353 

decomposition, media for, 352, 

353 

Urease, 178, 181, 185 
Urine, isolation of pathogens 
from, 302 



Uschinsky's solution, 172, 350 
Utensils, sterilization of, 6-11 



Vaccine, antirabic, preparation of, 
13 

black-leg, preparation of, 307 
Vaccines, bacterial, pieparatiort 
of, 316-318 

preservation of, 15 
Vacuum, for anaerobic cultures, 

156, 158-160 
VAILLARD, 309 
VAN DER HEIDE, C. C., 35 
VAN SLYKE, L. L. and PUBLOW, 

C. K., 287 
Vaselin, 76, 157, 296 
Vegetables, as a medium, 19, 20 

diseases of, 291, 294 
Vein, femoral, 298 

jugular, 298 

VERNON, H. M., 168, 186, 188, 192 
Vignal's tube, 158 
Vinegar, bacteria, 192 

circular on, 192 

fermentation, 183 

legal standard of, 192 

oxidase, 181 

pure cultures for, 192 

titration of, 22 
Violet, crystal, 361 

gentian, 87, 373 
Virulence, 295 
Virus, attenuated, 307 

dialyzation of, 13 

filterable, 313-315 

hog cholera, 313-315 
Vitality, 157 
Vosges-Proskauer's reaction, 363 

W 

WARD, A. R., 268, 270, 281, 284 
WASHBURN, R. M., 291 



422 



INDEX 



Wastes, laboratory, destruction 

of, 6, 7 

Watch, stop, 74 
Water, as an end product, 183 
bacteria isolated from, 223-227 
bacteriological analysis of, 223- 

235 

' distilled, 20 
filtered, 238-239 
in milk, 23 

of condensation, 35, 38, 39, 52 
on shipboard, treatment of, 237 
purification of,. 236-239 
sterilization of, by heat, 8-12 
Wax-like substances in bacteria, 

92 
Weight of bacteria, calculation 

of, 153, 244 
WELLS, LEVI, 285 
Welsbach burner, 67 
WESBROOK, F. F., WHITTAKER, 
H. A. and MOHLER, B. M. 
237 

Whey, sour, 169, 349 
WHITTAKER, H. A., 238 
Widal test, 361 
WILEY, H. W., 291 
Wine-making, yeasts used, 114 
Wines, 19 
Winogradski's medium for nitrate 

formation, 173, 350, 351 
medium for symbiotic nitrogen- 
fixation, 173, 350, 351 
WINSLOW, C. E. A., 153, 222, 227 
WINSLOW, C. E. A. and BROWN, 
W, W., 222 



Wood ashes, 355 
Woodhead's flask, 159 
Woodwork, sterilizing of, 14 
Wool, effect of sterilization on, 7 
Working distance, 69, 77 
Wort, beer, 20, 46, 78 

titration of, 22 

Wounds, sterilization of, 6, 14, 48 
Wright's stain, 319 
Wrzosek, 157 



Xylol, effect on enzymes, 187, 188 
use of, 65, 66, 187, 188, 265, 
266, 372 



Yeast, 63, 78-80, 84, 97 

cake, flora of, 115, 116 

Fleischmann's Qompressed, 114, 

190 
Yeasts, budding of, 79 

cultivated, 114 

enzymes of, 182, 190, 191 

fermenting power of, 83, 84 

pseudo, 114 

resistance to heat, 202, 203 

study of, 114-123 

wild, 114 



Ziehl-Nielson's carbol-fuchsin, 374 
Zone development of colonies, 164 
Zymase, 180 

lactic acid bacteria, 180, 184 
Zymogenic, 153 




JBT?'-^ 




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