PREPARED BY THE
LABORATORY OF BACTERIOLOGY AND HYGIENE
MICHIGAN AGRICULTURAL COLLEGE
FIRST EDI T ION
JOHN WILEY & SONS, INC.
LONDON: CHAPMAN & HALL, LIMITED
BRAUNWOHTH & CO.
BROOKLYN, N. Y.
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
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
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
Head of Department,
EAST LANSING, MICH.
Sept. 1, 1915.
GENERAL MORPHOLOGICAL AND CULTURAL
Exercise 1. Cleaning Glassware 1
Exercise 2. Preparation of Glassware for Sterilization 15
Nutrient Media 18
Exercise 3. Titration of Media 20
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
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
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-
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
Exercise 19. Method of Measuring Microorganisms 70
Exercise 20. Determination of the Rate of Movement of Motile
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
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
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
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-
Exercise 48. To Study the Natural Decomposition of Milk 141
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
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
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
Exercise 13. - To show the Action of Proteolytic Enzymes upon
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
Exercise 18. To Demonstrate the Necessity for an Activator
for the Enzymic Action of Rennet 193
Exercise 19. Effect of Concentrated Solutions upon Microor-
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-
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
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
Exercise 1. Quantitative Bacterial Analysis of Air . . .
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
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
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
Exercise 1. To Demonstrate that Plants are Subject to Microbial
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
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
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
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
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.
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-
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-
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
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,
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
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
VI. Conclusions. Draw the conclusion which your own
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.
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
Note. In writing up the notebook, details under II and IV should
be omitted, only the headings are necessary.
GENERAL MORPHOLOGICAL AND CULTURAL
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
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
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
2 GENERAL 1 MICROBIOLOGY
solution may 'be Used Wtii dxhUzed, i.e., until dark green
Heat will facilitate the action of the cleaning solu-
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
New Petri dishes and test tubes may conveniently be
FIG. 1. (a) Pipette, (6) Smith's Fermentation Tube, (c) Erlenmeyer
Flask, (d) Test Tube, (e) Roux Tube, (f) Petri Dish, (g) Roux
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
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
3. Rinse with distilled water.
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
3. Rinse with distilled water.
Petri Dishes. After removing Petri dishes from the
1. Wash them in water, using cleaning powder if nec-
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
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
3. Rinse with tap water, followed by distilled water.
4 GENERAL MICROBIOLOGY
4. Rinse with alcohol. (Alcohol may be used repeatedly.)
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
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
Note 3. If cultures or media have become dry, add water
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 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:
1. Plasmolysis or Plasmoptysis.
3. Heat (a) dry heat; (6) moist heat.
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-
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
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
The destruction of autopsied animals and accumulated
wastes of the laboratory is also best accomplished in this
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-
8 GENERAL MICROBIOLOGY
Apparatus must be absolutely clean and dry before
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
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
(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
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-
(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-
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
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
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
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
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
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 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
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.
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
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-
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
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
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
times. Have the instructor demonstrate the method of
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
(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
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
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
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.
" 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
(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.
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
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-
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-
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-
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
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.
6. Record as degrees of acidity the number of c.c. of
N/l NaOH which would be required to neutralize one liter
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
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
(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
(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-
Method. 1. Fresh separated or skimmed milk should
be used. Whole milk is undesirable on account of its fat
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
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-
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
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
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
FIG. 8. Potato Tubes. (Orig.
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.
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
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
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
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
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
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-
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-
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
7. Counterpoise and note the weight.
8. Boil fifteen minutes over a free flame, stirring con-
* It is not necessary to add water to the egg.
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 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
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
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
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
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
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.
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
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.
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
9. Counterpoise and note the weight.
10. Boil fifteen minutes over the free flame, stirring con-
11. Counterpoise and restore any loss by evaporation
with distilled water.
12. Filter while boiling hot through plaited filter paper
just previously washed with 1/2 liter boiling water. Pass
the filtrate through the same paper until it is bright and
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
14. Heat in flowing steam twenty minutes on three
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 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
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
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
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.
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
off; replace with the same amount of clean tap water.
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
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
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-
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-
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
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-
6. Tube, putting 8 c.c. in each tube.
7. Sterilize for fifteen minutes on three successive
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
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
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.
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
A pure culture is the growth of one species of micro-
organism only, in or on a nutrient medium, that was sterile
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-
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.
kept at a constant temperature. Organisms which natur-
ally grow at body temperature (37 C.) as Bacillus coli,
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
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
EXERCISE 12. PREPARATION OF PLATE CULTURES,
LOOP OR STRAIGHT-NEEDLE DILUTION METHOD
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-
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.
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
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
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
5. Number the tubes of agar 1, 2 and 3 and flame the
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,
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
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-
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
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
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
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
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
5. For plating, transfer 1 c.c. with a sterile 1 c.c. pipette
from the flask containing the desired dilution to a sterile
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
9. Place the plates on a level surface until the medium
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.
FIG. 17. Jeffer's Counting
FIG. 18. Wolfhugel'g
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
3. Using the tripod lens count the colonies in each sector
of the smallest circle, then in each division between the
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-
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
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
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
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
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
3. Distribute the medium evenly over the large side,
* Most valuable for molds, especially Rhizopus nigricans.
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
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
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
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,
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.
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.
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
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
2. Objectives made parfocal for one tube-length or eye-
piece are not parfocal for a different length or a different
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
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.
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
Light. 3. Never use direct sunlight. The best light is
obtained from white clouds. Northern or eastern light is
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
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.
7. With the eye at the ocular, focus up slowly with the
coarse adjustment until the specimen comes plainly into
// 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
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
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.
GAGE: The Microscope.
70 GENERAL MICROBIOLOGY
EXERCISE 19. METHOD OF MEASURING MICRO-
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
A micron is 0.001 mm., and is expressed by the Greek
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
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
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
Scale in Ocular of
the Leitz Ocular
VALUES FOR LEITZ ACHROMATIC OBJECTIVES
No. of Objective.
Mark on Draw-tube.
Micrometer Value in Microns of Each
Division Line of Step Micrometer.
1/12 oil imm.
5. Multiply the number of division lines of the ocular
micrometer covered by the organism in question by the value
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
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
or 0.1 micron.
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-
Method. 1. Place the object micrometer under object-
ive No. 3 and ocular No. 1, drawing out the draw-tube to
2. Bring the lines on the object micrometer into a sharp
3. Replace ocular No. 1 with the filar ocular microm-
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
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
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
PREPARATION OF A HANGING DROP
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-
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-
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
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
EXERCISE 22. PREPARATION OF THE ADHESION
The purpose of the exercise is to show the germination
of mold spores or the budding of yeast cells, i.e., colony
Apparatus. Clean cover-glasses; clean concave slides;
melted paraffin; small glass rod or camel's-hair brush;
28. Lindner's Adhesion Culture. (Adapted from Lafar's
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
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
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-
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;
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
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-
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-
*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-
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
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
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
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
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
11. Do your results coincide with those in the refer-
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.
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,
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-
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
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
EXERCISE 27. CHINESE INK PREPARATION
Chinese ink may be used to make bacteria more easily
visible microscopically and to aid in taking correct measure-
Apparatus. Sterile, dilute Chinese ink; * clean flamed
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
3. Distribute the organisms well with a platinum
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
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
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
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
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 "
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
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.
rial to the drop, and only sufficient to make it very slightly
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
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
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.
EXERCISE 29. ANJESZKY'S METHOD OF STAINING
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-
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;
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
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.
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
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
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-
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-
5. Place a number of absolutely clean cover-glasses in
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
7. Allow the preparation to dry for about five min-
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-
10. Wash off the mordant in a small stream of
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
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-
Culture. Agar slant cultures preferably.
Method. 1. Prepare a cover-slip film and fix in the
2. Stain in anilin-water gentian violet three to five
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-
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
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
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
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
5. Examine under the microscope.
6. Wash, dry and mount,
EXERCISE 34. METHOD OF MAKING IMPRESSION
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
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
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-
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
Rhizopus nigricans Black mold
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
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.
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.
Germination of Rhizopus Spore, Mycelium, Rhizoids and Development
of Sporangiophores and Sporangium.
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.
MICROSCOPICAL EXAMINATION OF MOLDS 105
EXERCISE 36. MICROSCOPICAL EXAMINATION OF
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,
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
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.
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
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
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
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.
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.
Name of student
Name of organism
Method of isolation
Stages of germination
Drawn from preparation
Method of reproduction
Drawn from preparation
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
Cider or wort
A gar streak
Age of colony
Size of colony
Gelatin or agar
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),
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.
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
3. Then with needle contaminated with the mold spores
inoculate the circle on the opposite side by puncturing as
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?
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.
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
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-
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
Torula rosea is an example of the pseudo-yeast. These
look like true yeasts, reproduce by budding, but seldom
produce alcoholic fermentation.
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
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-
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
7. Study this yeast according to directions in Exercise
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
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
10. Repeat every seven days until all the yeast cells
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.
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);
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
2. Sterilize. Carbohydrate broths are sterilized by the
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
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-
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
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
10. Record the relative proportions of C02 and H2
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
EXERCISE 42. THE STUDY OF YEASTS
The object of this exercise is to demonstrate how
to differentiate yeasts by microscopical and cultural
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
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
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
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-
12. Study the fourteen to twenty-day old wort cultures
in hanging drop for endospores. When do these form?
HANSEN, E.: Practical Studies in Fermentation, pp. 215-217.
LAFAR: Technical Mycology, Vol. II, Part II, pp. 394-406, 430-
EYRE: Bacteriological Technic. Second Ed., p. 7.
Name of student
Name of organism
Method of isolation
Total organism Drawn from preparation
Stages of budding Drawn from preparation
Method of reproduction Drawn from preparation
% Of gas in
Ratio of CO 2 :H 2
and other gases
THE STUDY OF YEASTS
Cider or wort
1 day .days days
Age of colony
Size of colony
Gelatin or agar
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
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
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.
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.)
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-
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
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 gelatin (stab culture).
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
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
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
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
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
CONN, ESTEN and STOCKING: Classification of Dairy Bacteria.
NOVY: Laboratory Manual of Bacteriology.
JORDAN: General Bacteriology.
EXERCISE 44. EHRLICH'S METHOD OF TESTING
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:
UC/ C CH
I D II
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.
BOEHME, A.: Die Anwendung der Ehrlichschen Indol-reaktion fur
Bakteriologische Zwecke. Cent. f. Bakt. Orig. Bd. 40 (1906),
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
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;
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
(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
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
(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
A yellow color shows the presence of nitrates.
TESTS FOR THE REDUCTION OF NITRATES 135
Name of student
Name of organism Isolated from
Method of isolation
Shape of organism
Method of reproduction
Stages of germination
Aqueous-alcoholic stain Gram's stain Aci
1 day days days
1 day days days
1 day days days
1 day days days
Age of agar colony
Size of colony
Titre ..... .
TESTS FOR THE REDUCTION OF NITRATES 137
Age of gelatin colony
Size of colony
% Of gas in
Ratio of CO 2 :H 2
and other gases
NH 3 from peptone
H^S from peptone
Indol from peptone
Nitrites from peptone
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
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
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
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
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,
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
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
6. Roughly sketch the different species observed, giving
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
12. State your results in detail and point out any con-
clusions that may be drawn and any practical applications
that may be made.
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-
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-
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
142 GENERAL MICROBIOLOGY
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
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-
(26 hrs. old)
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
17. Compare the flora of fresh milk, 70 acid milk and
ten-day milk, both microscopically and from the plates.
18. State your results in detail and give any conclusions
to be drawn and any practical applications that may be
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-
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
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-
5. Make three loop-dilution plates from the heated broth
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
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-
10. Give in detail the results obtained, state any con-
clusions that may be drawn and point out any practical
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
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
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-
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.
MARSHALL: Microbiology, pp. 185-191.
BESSON: Practical Bacteriology, Microbiology and Serum Therapy,
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
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
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
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,
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
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.
MARSHALL: Microbiology, pp. 522-524, 545, 578, 591-593.
BESSON: Practical Bacteriology, Microbiology aud Serum Therapy,
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;
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
3. Allow to dry, fix and stain with aqueous-alcoholic
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.
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
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
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-
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.
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.
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
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'
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
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.
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.
SOME PHYSIOLOGICAL CLASSIFICATIONS OF
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
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
According to the necessity of one kind of food or environ-
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
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-
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
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
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
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
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.
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
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-
B. Cultivation in Vacuo. This requires special apparatus
for obtaining a vacuum and for cultivation in some cases.
FIG. 39a. Novy Jar for
FIG. 396. Novy Jar for
Apparatus. Special tubes:
1. Vacuum tubes (Fig. 129, p. 238, Eyre's Bacteriological
2. Pasteur, Joubert and Chamberland's tube (Fig. 80,
p. 93, Besson's Practical Bacteriology, Microbiology and
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,
6. Roux's tube for potato cultures (Fig. 92, p. 101,
7. Esmarch's tube (Fig. 95, p. 103, Besson, ibid.).
8. Vignal's tube (Fig. 96, p. 103, Besson, ibid.).
ANAEROBIC CULTURE METHODS
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,
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,
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
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
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
Apparatus. Tubes for use in oxygen absorption method.
1. Simple test-tube method.
2. Giltner's H tube.
ANAEROBIC CULTURE METHODS
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
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
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
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
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,
(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
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
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-
8. Compare your results with those of others and draw
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
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
12. What other methods may be used for obtaining
anaerobic conditions for microbial growth? Name the
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.
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 ,-
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
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.
LOHNIS, F.: Laboratory Methods in Agricultural Bacteriology, pp.
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
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
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
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
10. State your results in detail and point out any con-
clusions to be drawn. Point out any practical applications
that may be made.
LAFAR: Technical Mycology, English Ed., Vol. II, Part 2, pp. 417, 418,
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,
170 GENERAL MICROBIOLOGY
EXERCISE 5. TO DEMONSTRATE THE VARIATION IN
FOOD REQUIREMENTS OF BACTERIA AND THEIR
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
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
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
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
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-
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
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
8. State your results in full and draw any conclusions
warranted. What practical applications may be made of
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
Apparatus. Four tubes each of:
Ordinary broth (organic nitrogen, soluble albumins and
Dunham's solution (organic nitrogen, soluble peptone,
Uschinsky's asparagin medium (organic nitrogen, pro-
Cohn's solution (inorganic nitrogen combined with
* 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
Cultures. B. subtilis; Ps. radicicola; Aspergillus niger;
Method. 1. Inoculate heavily a tube of each medium
with Ps. radicicola. Proceed likewise with the three remain-
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,
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
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-
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 =
6. Is there any relation between the power of organisms
to liquefy gelatin and to produce " lead-blackening " sul-
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
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
CHEMICAL AGENCIES ON MICROBIAL PIGMENT 175
7. Give your results in full. Draw any conclusions
possible and point out any practical applications that may
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
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
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
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.
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-
F. Blue milk and " bloody " milk.
1. Inoculate milk tubes as follows:
+ Bact. lactia acidi.
and keep at 25
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-
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
LAFAR: Technical Mycology, Vol. I, pp. 105-122.
THRESH: Examination of Waters and Water Supplies, 2d Edition, pp.
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
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
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
7. State your results in full, and draw any conclusions.
What practical application of the above may be made?
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
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
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,
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
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
a. Saccharose: sucrase (invertase, invertin)
b. Lactose: lactase specific term.
c. Maltose: maltase specific term.
3. Glucosides: glucosidases general term.
a. Amygdalin: amygdalase (emulsin, synaptase)
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.
a. Proteins broken down to proteoses and
peptones : peptase (pepsin or aci'd-proteinase)
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
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
CLASSIFICATION OF ENZYMES 181
B. Acid amides (urea): amidases general term, to
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-
C. Sulphur to [28 : sulphur reductase specific. term.
D. Nitrates to nitrites, nitrates to NHs, etc. : nitrate-,
V. Coagulating enzymes.
A . Protein-coagulating.
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
1. Pectin : pectinase specific term.
182 GENERAL MICROBIOLOGY
ENZYMIC REACTIONS OF WELL KNOWN FERMEN-
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.
Ci 2 H 22 On + H 2 O + maltase =2C 6 H 12 6 .
III. dextrose + enzyme producing intra- = alcohol + carbon
molecular change dioxide.
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
B. Glucoside decomposition, by molds, bacteria and yeasts.
glucoside + water -f hydroly tic enzy me = sugar -f- aldehydes, acids, etc.
amygdalin-f- water -f emulsin = dextrose + benzaldehyde + hy-
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.
f at + water +hydroly tic enzyme = fatty acid + glycerin.
stearin+3 mols. water+steanrazse = 3 mols. stearic acid+glycerin."
H 2 O C 17 H 35 COOH CH 2 OH
C 17 H 36 CO-O-CH + H 2 O = Ci 7 H 35 COOH + CH 2 OH
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
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
EXERCISE 11. A COMPARISON OF ACID AND RENNET
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;
Cultures. B. prodigiosus; Bad. lactis addi; B. megater-
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
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
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.
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
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.
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
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
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;
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.
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)
EXERCISE 14. TO SHOW THE ACTION OF ENZYMES
Apparatus. Three sterile Petri dishes; three test tubes;
soluble starch; three tubes sterile agar; Lugol's iodin solu-
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
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
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.
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,
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
Apparatus. Petri dish; medium fine sand; sulphur;
cake of Fleischmann's compressed yeast, fresh (obtain
this yourself); small mortar and pestle; lead acetate
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
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
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
Apparatus. Four fermentation tubes of nutrient broth
(sterile) ; hydrogen peroxide (full strength) .
Cultures. B. coli; B. subtilis; B. mycoides; Bad.
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
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
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
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
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,
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
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?
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-
7. State the results of your experiment in detail and
draw conclusions. Point out any practical applications
that may be made.
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
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
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
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-
5. Mark the flasks as follows :
194 GENERAL MICROBIOLOGY
Flask No. l=unheated milk + rennet.
11 2 = unheated milk + calcium phosphate -f ren-
" ll 3 = heated (sterilized) milk + rennet.
" " 4 = heated (sterilized) milk + rennet + calcium
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
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?
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
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
8. State the results of your experiment in full and draw
conclusions. Point out any practical applications that may
MARSHALL: Microbiology, pp. 147-151.
FISCHER, A.: Structure and Functions of Bacteria, pp. 5, 8-9.
EXERCISE 20. THE EFFECT OF DESICCATION UPON
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
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.
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;
Method. 1. Inoculate four tubes of dextrose broth with
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
EFFECT OF FREEZING ON BACTERIA 199
What inter-relations have the optimum, minimum and
maximum temperature requirements of one species of
What influence will the reaction of the medium have
upon the extremes of temperature at which microorganisms
5. Discuss your results in detail and draw any conclu-
sions permitted. Point out any practical applications.
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
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
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-
10. Give all results and answers in full and draw any
conclusions permissible. Point out the practical applica-
tions that may be made.
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
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-
202 GENERAL MICROBIOLOGY
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
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
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?
9. Give all data and results in full. Draw any conclu-
sions possible and point out any practical applications.
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.,
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-
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
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-
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
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
* See note on page 203.
REACTION OF THE NUTRIENT MEDIUM 205
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
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
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
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
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
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
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.
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
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
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-
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-
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-
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
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
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
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-
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
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
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
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
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
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
Time in minutes during which culture is exposed to
action of disinfectant.
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
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
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) :
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
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
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?
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
Cultures. Bad. lactis acidi; Oospora lactis.
Method. 1. Place 150 c.c. milk in each flask and sterilize
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
4. Plot curves. How do you explain the direction these
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.
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,
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.
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
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
11. Give data and observations in full and draw conclu-
sions. Point out any practical applications.
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
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:
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.
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.
EXERCISE 1. QUANTITATIVE BACTERIAL ANALYSIS
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.
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
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
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
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
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.
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
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
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
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-
pec ted the sample must be diluted. Use distilled water
for dilution flasks.)
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
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.
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-
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
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
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-
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.
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),
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
1. Collect the sample in the sterile 500 c.c. flask, using
all precautions as with an unpolluted water sample. (See
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,-
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
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
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-
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.
3 Kf) K^"
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.
15. Examine gelatin plates after two days and then
every few days for seven days.
([ i| r\
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
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.
Standard Methods of Water Analysis, 1913 edition, pp. 79-82, 87-88,
PRESCOTT and WINSLOW: Water Bacteriology, 3d. ed., pp. 61-201,
SAVAGE: Bacteriological Examination of Water Supplies, pp. 27-69.
MARSHALL: Microbiology, pp. 97, 108, 162, 182, 204, 212, 221,
BACTERIOLOGICAL WATER ANALYSIS
Date . . . . 19
Sample No. .
Name of sender
Source of water
Per cent Per cent T> 4
C0 2 . H 2 . Reaction.
Plates made from ferm't'n tube or agar shake . .
Z OO : Z H
S -3ua-uo D
J13313 '^OBl ^I r [
q^oaq paa ^na N
JBSB paa '^na^
u^pa jo -b n
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
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
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
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-
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
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,
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
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.
MARSHALL: Microbiology, pp. 205-207.
DON and CHISHOLM: Modern Methods of Water Purification (1911),
EXERCISE 1. TO TEST THE CATALYTIC POWER OF
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.
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-
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
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-
Cubic centimeters of oxy-
Time for maximum oxy-
8. What part does each factor play in the liberation
of oxygen? Is this action in soil of any value? If so,
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-
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-
3. At the laboratory, carefully mix and pulverize the
composite sample with the spatula on the large piece of
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
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
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
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-
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
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?
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-
Method. 1. To 1 gm. of soil or excrement in a test
tube add 4 c.c. of sterile water and shake vigorously for
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
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
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
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp.
LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp. 7-9.
MARSHALL: Microbiology, pp. 238-244.
THE EFFECT OF AERATION OF SOILS
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
4. Place 50 c.c. of the 1% peptone solution in the remain-
5. Add sufficient peptone solution to the other flasks
to make 10%, 20% and 30% moisture content according
to the following table :
Coarse sand \
Medium sand -\
Fine sand !
1% peptone solution
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
10. Draw any conclusions warranted by your results
and point out any practical applications.
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
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
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
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-
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
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp.
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
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-
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
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
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
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,
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
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
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:
0.5 gm. manure
0.5 gm. soil
and keep at 25 to 30 C., to hasten the action of the micro-
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
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
(See appendix for media used for the isolation of these
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?
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp.
96, 97, 106, 109, 110.
LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp.
MARSHALL: Microbiology, pp. 259-263.
EXERCISE 7. TO ILLUSTRATE DENITRIFICATION IN
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
4. Using sterile pipettes, test 1 c.c. portions of each
after forty-eight hours, seven days, etc., for nitrates with
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
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?
How do denitrification and nitrate reduction differ?
How may colonies of nitrate-reducing bacteria be
14. Give all results in full and draw conclusions. Sug-
gest any possible practical applications of the above.
LIPMAN and BROWN: Laboratory Guide in Soil Bacteriology, pp.
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;
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
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
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
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-
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
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-
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.
MARSHALL: Microbiology, pp. 98-99, 230-231, 248, 250, 266, 270-273,
286, 288, 291.
LIPMAN and BROWN: Laboratory Guide in Soil Microbiology, pp.
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,
Further Studies with Some Azotobacter. Cent, f . Bakt. II Abt.,
Bd. 42, pp. 68-69.
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 40,
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-
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
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-
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
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
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
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.
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-
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-
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
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-
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-
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
B. TEST OF THE PHYSIOLOGICAL EFFICIENCY OF
PS. RADICICOLA AND OBSERVATION OF NOD-
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-
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.
(6) Wash off the disinfectant with sterile distilled water,
handling the seeds with sterile forceps.
FIG. 56. Alfalfa Plants from Inoculated and Uninoculated Seed.
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
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
12. In about a month examine all test tubes and look
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.
MARSHALL: Microbiology, pp. 273-283.
LAFAR: Technical Mycology, Vol. I, pp. 259-271.
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp.
LIPMAN and BROWN; Laboratory Guide in Soil Bacteriology, pp.
CHANGE OF INSOLUBLE PHOSPHATES 263
SMITH, E. F.: Bacteria in Relation to Plant Diseases. Vol. II,
Inoculation with Nodule-forming Bacteria, Cir. 5, Mich. Exp.
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.
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
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
10. Give results and any conclusions in detail. Point
out any possible practical applications.
MARSHALL: Microbiology, pp. 287-288.
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 115-
SACKETT, PATTEN and BROWN: The Solvent Action of Soil Bacteria
Upon Insoluble Phosphates, etc., Spec. Bui. 43, Mich. Exp. Sta.,
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
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
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
Culture: Fresh milk same as used
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
2. Place a clean glass slide on the
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
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
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-
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-
TYPES OF MICROORGANISMS IN MILK
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.
No. per c.c.
800,000 per c.c.
400,000 per c.o,
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
15. Give your results in detail and point out any prac-
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.
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),
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
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
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
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
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
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.
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-
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
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
FIG. 59. Bad. bulgaricum
colony, x75. (Orig. Nor-
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
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
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.
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.
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-
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-
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.
SAVAGE: The Bacteriological Examination of Food and Water (1914),
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
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
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
6. Fill the tubes with physiological salt solution and mix
the sediment well throughout the dilution fluid with a plat-
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
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
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-
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.
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.
EXERCISE 5. TO DETERMINE THE INFLUENCE OF
TEMPERATURE UPON THE KEEPING QUALITY OF
MILK; PITRE MILK COMPARED WITH MARKET
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
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
8. Plate in litmus lactose agar, using the following
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
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
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
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
15. Give your results in detail and point out any
practical applications or conclusions.
PASTEURIZATION OF MILK OR CREAM 281
WARD: Pure Milk and the Public Health (1909), pp. 15-16, 24-25,
RUSSELL and HASTINGS: Outlines of Dairy Bacteriology, pp. 54-56.
ERNST: Milk Hygiene, transl. by Mohler and Eichhorn (1914), pp.
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
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
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-
6. Pour water into the water-bath until the height of
the water corresponds to the height of the milk in the
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
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
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
17. Give your results in full and any conclusions that
may be drawn.
284 GENERAL MICROBIOLOGY
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-
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
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,
RUSSELL and HASTINGS: Practical Dairy Bacteriology, pp. 95, 97.
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp.
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
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
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
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.
LOHNIS: Laboratory Methods in Agricultural Bacteriology, pp. 83-88.
RUSSELL and HASTINGS: Experimental Dairy Bacteriology, pp.
MARSHALL: Microbiology, pp. 346-357.
CONN: Practical Dairy Bacteriology (1907), pp. 223-259.
SAVAGE: The Bacteriological Examination of Food and Water (1914),
DECKER: Cheese Making (1905), pp. 48-51, 63, 66, 69, 75, 88, 108-
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
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
4. With the sterile can-opener make an opening in the
can only large enough to admit the introduction of a 5 c.c.
Note. Only one can is to be opened at a time to avoid contam-
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,
MARSHALL: Microbiology (1911), 363-366.
SAVAGE: The Bacteriological Examination of Food and Water (1914),
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
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
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
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-
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
8. Make a microscopic count, using the method in Exer-
cise 1, Dairy Microbiology. How do microscopic and plate
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
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.
SAVAGE: The Bacteriological Examination of Food and Water (1914),
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.
WASHBURN, R. M.: Principles and Practice of Ice-cream Making.
Bui. 155. Vermont Agr. Expt. Sta., pp. 9-10, 34-46, 53-54,
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.
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.
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
Culture. B. carotovorus (culture of high physiological
Method. 1. The root of the carrot, turnip, rutabaga,
the cucumber or radish; the cotyledons of immature pea
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
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
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
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
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.
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.
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.
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.
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),
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
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.
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
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
(c) With fine-pointed sterile forceps insert the inoculum
298 GENERAL MICROBIOLOGY
into this pocket. Further treatment should not be neces-
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
(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.
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
V. Replace the periosteum and suture the skin.
8. Intrapulmonary. I. Pull the animal's front leg for-
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
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-
4. Give all results, observations and conclusions in
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
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
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-
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
7. With a sterile platinum loop, make transfers to agar
slants, shake cultures, and plates for isolation into pure
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.
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;
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
3. Make cover-glass preparations and stain with meth-
ylen blue, fuchsin and Gram's stain. Stain for spores
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
5. Make cultures on agar slants, and smear prepara-
tions from the blood, liver, spleen and kidney after the
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.
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-
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
Method, 1. Place about 200 c.c. of glycerinated veal
bouillon in each of two 500 c.c. Erlenmeyer flasks and
sterilize for twenty minutes each day for three consecu-
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
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.
THE PREPARATION OF TUBERCULIN
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,
MOORE: Bovine Tuberculosis.
KOLMER: Infection, Immunity and. Specific Therapy (1915), pp. 582-
ZINSSER: Infection and Resistance (1914) ,pp. 355-357, 438-442.
THE PREPARATION OF BLACK-LEG VACCINE 307
EXERCISE 5. THE PREPARATION OF BLACK-LEG VAC-
Apparatus. Sterile mortar and pestle; sterile cheese-
cloth; two sterile glass plates; sterile water; sterile homeo-
Culture. Diseased muscle of calf affected with black-
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.
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.
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-
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
For laboratory purposes, the rabbit may be used to furnish the
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-
1st day, 3 c.c. of toxin mixed with 1 c.c. of Gram's iodin
5th day, 5 c.c. of toxin mixed with 2 c.c. of Gram's iodin
9th day, 12 c.c. of toxin mixed with 3 c.c. of Gram's
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.
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,
ZINSSER: Infection and Resistance (1914), p. 463.
EXERCISE 8. A DEMONSTRATION OF THE AGGLU-
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.
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
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.
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),
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
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
(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
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
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
t " : ; , .,^nir]
A STUDY OF FILTERABLE VIRUSES
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
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
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
8. State your results and conclusions in full,
FIG. 71. Pasteur-Cham-
berland Filter Adjusted
for Filtration by Suc-
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.
KOLMER: Infection, Immunity and Specific Therapy (1915), pp. 77, 78.
EXERCISE 10. THE PREPARATION OF BACTERINS OR
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
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
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
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.
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
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.
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-
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
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
Centrifuge and wash at least three times, being careful
not to pipette off any of the cells during the washing
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
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
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%
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
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-
4. Give results and any conclusions in detail.
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
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
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
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
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-
6. Give results and conclusions in full.
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
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
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-
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
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
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.
See Exercise 14.
THE COMPLEMENT FIXATION TEST 325
EXERCISE 14. TO DEMONSTRATE THE COMPLEMENT
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
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
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
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.
TITRATION OF COMPLEMENT
Suspension of blood
Result after one-half
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-
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.
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
THE COMPLEMENT FIXATION TEST
TITRATION OF HEMOLYSIN
Salt solution . . .
Complement. . . .
Result after one-
Incubate all tubes in water bath at 37 C. for one-half hour and
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 smallest quantity causing complete hemolysis is called the
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
Positive serum . . .
Incubate for half an hour in a water bath at 37 C., then add the hemolytic sys-
tem as follows:
Suspension of blood
Results after in-
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
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-
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.
Suspect serum. . .
Incubate one-half hour at 37 C, in water bath.
THE COMPLEMENT FIXATION TEST
3. Then add the hemolytic system.
Hemolysin . . .
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.
MOHLER, J. R. and EICHHORN, A.: The diagnosis of glanders by
complement fixation test. Bui. 136, B. A. I., U. S. Dept. Agr.
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.
OUTLINE FOR THE STUDY OF MICROBIOLOGY *
I. MORPHOLOGICAL AND CULTURAL MICRO-
A. Morphology and Development.
1. Gross anatomy.
c. Arrangement or grouping.
e. Involution, variability and mutation.
2. Histology of cell.
a. Wall or outer membrane.
c. Protoplasm, beaded forms, granules.
d. Nuclear material.
e. Flagella and motion.
3. Classifications and their basic features,
B. Cultural Significance.
a. For morphologic and developmental studies.
b. For cultural effects.
3. Cultural features.
4. Biochemical features.
* Adapted from Marshall, vide 43d Annual Report of Michigan
State Board of Agriculture.
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.
4. Products of physiological significance of which little is
c. Aromatic compounds.
5. Absorption or assimilation of foreign bodies.
B. Studies in Metabolism.
1. Elements required in growth of microorganisms.
5. Temperature of cultivation.
6. Conditions of media; reaction, composition, etc.
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.
C. Studies in Association.
D. Common Fermentative Changes Produced by Micro-
1. Studies in enzymes.
c. Actions (specificity) and materials fermented.
d. Conditions under which enzymes act;
Radiation, light rays (solar, electric, etc.),
Rontgen rays, radium rays and emana-
(2) Chemical and physico-chemical.
Activators; kinases, organic acids, bases,
Paralysors and poisons.
Concentration of solutions.
Reaction of substrate.
Extent of accumulated products.
2. Products manufactured by fermentation.
a. Necessary and limiting conditions of production.
b. Most favorable conditions of production.
c. Methods of determination.
d. Constancy and variability of products.
e. Gradation in fermentation changes.
E. Products Significant through the Intermediation of a
b. Cell products.
Toxins, diffusible and endotoxin. '
c. Precipitins. ^
F. Influencing Agents and Their Effects.
*See p. 164, Kolmer's Infection, Immunity and Specific Therapy
Steam under pressure.
5. Mechanical pressure.
6. Mechanical agitation.
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-
e. Distribution of microorganism in body.
/. Management of disease.
g. Prevention of disease.
h. Care of dead from communicable diseases.
B. Surgical Significance.
3. Septicemia and pyemia.
4. Malignant growths.
C. Susceptibility and Immunity.
d. Individual idiosyncrasies.
e. Body components.
2. Acquired, active or passive.
a. Devitalization. ,
b. Hereditary predisposition.
c. One attack of disease.
g. Other bacterial products.
D. Serum Therapy Microbial Therapeutics
1. Diagnostic agents.
c. Bacterial suspensions.
d. Diphtheria toxin (Schick).
2. Remedial agents.
E. Disinfection and Antisepsis.
1. Agents employed.
a. Mode of action.
2. Determination of values, phenol coefficient.
F. Sanitary Studies.
1. Water analysis.
b. Interpretation of results.
2. Water contamination and filtration.
3. Sewage analysis.
b. Interpretation of results.
4. Sewage' destruction.
a. Aerobic filtration.
b. Anaerobic septic tank.
c. End products.
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.
A, Milk Supply.
1. Communicable diseases conveyed through milk.
a. Kinds of microorganisms.
b. Avenues of transmission.
2. Environment of animals and conditions of milk-
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.
b. Butter fat.
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.
3. Condition of formation.
C. Fermentations in Milk, Butter and Cheese.
Bitter flavor, barn-yard, tallowy.
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.
6. Conditions influencing it.
7. Controlled or fostered.
D. Pasteurization and Sterilization.
1. Determination of significance of each.
2. Methods employed.
3. Practical utilization.
a. Sour milk.
6. Sour cream.
a. Pure cultures.
b. Mixed cultures.
3. Value determined.
7. Influencing conditions.
8. Facts governing amounts to employ.
1. Microorganisms present.
2. Microorganisms compared with those of milk.
3. Environmental condition for bacterial life changed.
a. Influenced by pasteurization of the cream.
b. Influenced by growth of microorganisms.
c. Factors influencing stability.
d. Methods of preservation.
b. Factors influencing.
c. Correlation between the presence of certain
groups of organisms and specific flavors.
6. Significance of casein and buttermilk in butter.
1. Kinds of microorganisms employed in different
2. The study of microorganisms in the ripening process.
3. Influence of microorganisms on aroma and flavor.
4. Keeping values.
I. Disinfectants utilized.
A. The Making of Soil.
1. Microorganisms in soil.
a. Number at different depths and in different
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.
6. Starches and sugars.
c. Proteins, etc.
4. Action of iron and sulphur bacteria.
C. Nitrification The nitroso- and nitro-processes.
1. Conditions influencing.
d. Supply of oxygen.
e. Amount of organic matter present.
1. Factors influencing the loss of nitrogen.
E. Nitrogen Fixation.
2. Nonsymbiotic (aerobic and anaerobic).
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.
2. Microorganisms found as causal agents.
3. Cultural characteristics.
4. Resistance of microorganisms.
6. Methods of treatment.
C. Microbial Decomposition of Fruits, Vegetables and
Other Plant Substances.
2. Microorganism studies.
3. Conditions favoring.
5. Structural changes. .
A. Factors Controlling Fermentations.
1. Presence of microorganism.
2. Purity of culture.
3. Vigor of cell.
4. Character of fermentable material.
5. Air supply.
6. Reaction of medium.
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.
1. The Enzymes.
a. Hydrolytic of
Carbohydrates = Carbohydrases.
Fats = Esterases.
Lipases of natural fats.
Proteins = Proteinases.
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.
c. Oxidizing = oxidases.
d. Reducing = Reductases.
Methylen blue, indigo and azolitmin reduc-
Nitrate and nitrite reductase, etc.
2. Materials Acted Upon.
/. Organic acids, etc.
3. Products resulting.
a. Beer and distilled liquors.
6. Wine, cider and other fermented fruit juices.
c. Ginger beer.
d. Koumiss, etc.
2. Acetic acid.
3. Lactic acid.
c. Foods, sauer kraut, brine pickles, etc.
4. Butyric acid.
a. Urea, uric and hippuric acid.
b. Proteins and their nitrogenous fractions.
a. Proteins, albumins.
b. Proteoses, albumoses.
/. Amins and other ammonia derivatives.
i. Non-nitrogenous organic acids.
k. Ammonia, H 2 S, and other gases.
VIII. FOOD AND DRINK PRESERVATION
A. Preservation of Foods.
2. Cold storage.
4. Drying, evaporating or concentrating.
8. Chemical preservatives or antiseptics.
B. Preservation of Drinks.
1. Pasteurizing and sealing.
2. Cold storage.
3. Chemical preservatives.
CLASSIFICATION OF MIGULA (MODIFIED)
Streptococcus. . . 1
division in 1 1
plane, no fla- 1
division in 2 1
planes, no fla-
\ u u/r t/ wo
Py9 e s (albus
division in 3 1
planes, no fla- ,
Planococcus .-..:. 1
division in 2
planes, flagella J
Planosarcina . . .
division in 3
planes, flagella >
(straight rods) <
Bci ctarLcL CBCB
Pseudomonas. . . '
(straight or ir-
comma to spi-
ral forms, stiff,
ly polar fla-
CLASSIFICATION OF MIGULA (MODIFIED) Continued
Order. Family. Genus. Species.
cork screw, sev-
eral turns, non- rubrum
flexible spirals, 1 O 5 ema - er
motile, no fla- )
See pp. 10 and 56-62, Marshall's Microbiology.
unbranched threads, uniform in
tion by mo-
tile and non-
unbranched threads, filaments en-
larged at free end.
branched and unbranched filaments.
Cell division in 3 planes.
dichotomous branching, uniform di-
threads, non-motile and attached;
no sheath, flat cells, motile with un-
dulating membrane; no gonidia.
5 \ Rhabdochromatium
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
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
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
or several layers of clean cheese-cloth and allow to ferment
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
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.
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.
Sodium carbonate, Na2COs . 60 gm.
Calcium chloride, CaCk Trace
Distilled water 1000 . c.c.
Winogradski's medium for symbiotic nitrogen-fixation:
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
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-
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.
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!
Albuminoid-free culture solutions for studying urea
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
H. Distilled water 1000.0 c.c.
NaN0 2 1.0 gm.
K 2 HPO 4 0.5gm.
MgSO 4 0.3gm.
Na 2 CO 3 0.3gm.
This solution causes a greater relative increase in the
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.
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
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.
CaSO 4 0.1 gm.
CaC0 3 , 5.0 gms.
10% FeCl 6 solution 1.0 drop
Method. 1. Add -the above chemicals to 1000 c.c.
2. Titrate using phenolphthalein and neutralize using
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
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.
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.
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.
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-
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,
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,
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-
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
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
Media. Litmus lactose agar shake.
1. 2% washed agar.
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,
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-
1 . 0% peptone.
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
500 c.c. meat infusion.
500 c.c. distilled water.
Method. Prepare, tube and sterilize as for ordinary
Salt is omitted. Reaction +10.
Each student will need forty or fifty tubes of salt-free gela-
IV. Sugar-free broths and sugar broths. (Neutral red
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
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
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
Each student needs four litmus lactose bile salt fermentation
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.
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.
T? I < <0 I *ti
ni^qs s.urejo I
$+. I ^ +
i i i
+U I +U
^|+ ^1+ +LI+L
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,
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
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
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
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
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
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.
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,
Caution. This solution should not be left in contact
with any glassware longer than thirty minutes as it etches
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.
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-
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.
HC1 (sp.gr. 1.12) and make up to one liter with distilled
water. This makes a solution just a little stronger than
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
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
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
5. Proceed from this point as in the preparation of
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.
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
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.
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-
Sodium chloride, c.p 8.5 gms.
Sodium citrate 15.0 gms.
Distilled water 1000.0 c.c.
Ehrlich's test solution for indol production:
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.
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-
tilled water, allow the solution to cool and add it to the
5. Dilute to one liter with distilled water.
6. Allow to stand for one week and decant for use.
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
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.
Methylen blue for differentiating living from dead
Methylen blue 0.1 gm.
Distilled water. . . 1000.0 c.c.
Aqueous-alcoholic stains, fuchsin, methylen blue and
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
2. Allow the undissolved stain to settle over night.
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.
Basic fuchsin 1 gm.
Absolute alcohol 10 c.c.
Carbolic acid 5 gms.
Distilled water 100 c.c.
1. Dissolve the fuchsin in the absolute alcohol. (Solu-
2. Dissolve the carbolic acid in the distilled water
Note. Solutions A and B will keep indefinitely if kept sepa-
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.
Loeffler's alkaline methylen blue.
Saturated alcoholic solution of methylen blue 30 c.c.
Potassium hydrate, 0.1% aqueous solution 100 c.c.
SOLUTIONS FOR USE IN STAINING
Aceton-alcohol for decolorizing in Gram's method of
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.
STEAM TEMPERATURE PRESSURE TABLE
Mm. of Hg.
Pounds per sq.in.
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.
Degrees C X 1 .8 +32 = Degrees F.
= Degrees C.
+32 = Degrees F.
= Degrees R.
ALCOHOL BY VOLUME
(From the Chemlker Kalender.'publlshed by Julius Springer, Berlin.)
DEGREE f~\ DEGREES
FIG. 74. Comparison Fahrenheit-Centigrade Scale.
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.
. . 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,
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
METRIC SYSTEM Continued
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.
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
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
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.
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
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
EFFRONT, JEAN. Enzymes and Their Applications. 1st Ed.
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
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
FUHRMANN, FKANZ, Vorlesungen uber bakterien Enzyme, 1907.
FUHRMANN, FRANZ. Vorlesungen uber technische Mykologie.
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.
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.
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.
384 LIST OF TEXT AND REFERENCE BOOKS
JENSEN, C. 0. Essentials of Milk Hygiene. Transl. by Leonard
JESS, PAUL. Kompendium der Bakteriologie und Blutserum-
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
KISSKALT, K., und HARTMANN, M. Praktikum der Bakteriologie
und Protozoologie. 1907.
KITT, TH. Bakterienkunde und pathologische Mikroskopie.
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.
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,
LIPMAN, J. G. Bacteria in Relation to Country Life. 1908.
LIPMAN, J. G., and BROWN, P. E. Laboratory Guide in Soil Bacter-
LOEB, JACQUES. The Mechanistic Conception of Life. 1912.
LOEB, JACQUES. Studies in General Physiology, Part I. 1905.
LOHNIS, F. Handbuch der landwirtschaftlichen Bakteriologie.
LOHNIS, F. Laboratory Methods in Agricultural Bacteriology.
Translated by Wm. Stevenson and J. Hunter Smith. 1913.
LOHNIS, F. Vorlesungen iiber landwirtschaftliche Bakteriologie.
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
MCFARLAND, JOSEPH. Biology, General and Medical. 2d Ed.
McKAY, G. L., and LARSEN, C. Principles and Practice of Butter-
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.
Mum, ROBERT, and RITCHIE, JAMES. Manual of Bacteriology.
6th Ed. 1913.
MULLER, PAUL TH. Vorlesungen iiber Infektion und Immunitat.
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.
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
RIDEAL, SAMUEL. Sewage and the Bacterial Purification of
ROECHLING, H. H. Sewer Gas and its Influence upon Health.
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-
RUSSELL, H. L., and HASTINGS, E. G. Outlines of Dairy Bacter-
iology. 9th Ed. 1910.
RUSSELL, H. L., and HASTINGS, E. G. Agricultural Bacteriology.
SADTLER, S. P. Industrial Organic Chemistry. 1912.
SANTEE, E. M. Farm Sewage. 1912.
SAVAGE, WM. G. The Bacteriological Examination of Water
SAVAGE, WM. G. The Bacteriological Examination of Food and
SAVAGE, WM. G. Milk and the Public Health. 1912.
SCHNEIDER, ALBERT. Bacteriological Methods in Food and Drug
SHINKLE, C. A. American Commercial Methods of Manufactur-
ing Preserves, Pickles, Canned Foods, etc. Revised Ed.
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.
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.
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.
VERNON, H. M. Intracellular Enzymes. 1908.
WALKER, E. W. AINLEY. Inflammation, Infection and Fever.
WARBASSE, J. P. The Conquest of Diseases Through Animal
WARD, A. R. Pure Milk and the Public Health. 1909.
WARD, H. MARSHALL. Diseases in Plants. 1901.
WASSERMAN, A. Immune Sera. Translated byChas. Baldwin.
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.
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.
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.
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.
Jahresbericht liber die Forsch. d. path. Mikroorganismen, Baum-
Jahresbericht tiber die Fortschritte der Lehre von den Garungs
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.
Proceedings of the Royal Society of London.
Revue ge'ne'rale du Lait.
Zeitschrift fur Hygiene und Infektionskrankheiten.
Zeitschrift fur Immunitatsforschung.
STRtrCTtTKE OF COLONIES.
EDGES OF COLONIES.
* For illustration see next page.
ELEVATION OF COLONIES.
9. Rectangular depression.
Abortion, contagious, diagnosis
Acetacidase, 181, 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
digallic, enzyme of, 180
hydrochloric, 39, 245, 313
hydrochloric, effect on pigment,
hydrochloric, N/l, 20, 367, 368
hydrochloric, N/20, 20, 245,
lactic, 23, 181, 184, 218
lactic, enzyme of, 181
normal, 20-22, 367, 368
Acidophile, 154, 184
Acid, oxalic, 313
phenolsulfonic, 133, 371
pyrogallic, 156, 160, 165, 248
Acid, silicic, colloidal, state of, 33
sulfanilic, 133, 251, 371
sulfuric, concentrated, 213, 249,
308, 366, 371
sulfuric, for decolorization, 92
Acid-fast bacteria, 92, 97, 324
Acid production, 23, 112, 137, 169
of fresh beef, 29
organic, decomposition, 169,
organic, enzymes of, 180, 181
Action, selective, 170
Activator, 193, 194
Adhesion culture, 78, 79, 100,
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
Aeroscope, sand filter, 221
action of acid on, 38, 39
as a microbial food, 40, 41
commercial form of, 38
Congo red, 355
Conradi-Drigalski's, 361, 363
Agar, dextrose, 114, 175
dextrose calcium carbonate,
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
washing of, 41
plate culture, preparation of,
shake, litmus lactose, 223, 228,
slant, 26, 43
Agents, chemical, of sterilization,
physical, of sterilization, 5
Agglutination, macroscopic test,
microscopic test, 312
observation of, 74
Air, bacterial analysis of, 220-
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,
Albumin, egg, use of, 29
in milk, 23
Albumins, coagulation by heat, 28
soluble, as food, 172
effect of, on sterilization, 8
Alcohol, by volume, table of, 377
ethyl, 181-183, 215-217, 364,
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,
Aldehydases, 181, 183
Alkali, action of on methylen blue,
Alkali, free, 1
normal, 20-22, 368
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
sulfate, 170, 308, 350, 352, 353
Amphoteric reaction of casein, 24
Amygdalin, enzyme of, 180, 182
Amylase, 179, 182
Anaerobic bacteria, 46, 154, 248
culture methods, 155-167
ANDERSON, JOHN F. and McCLiN-
TIC, THOS. B., 213 .
Anesthesia, general, 295, 296,
local, 295, 299
Anesthetic, 295, 296, 309
administration of, 295, 296
Anilin, basis of dyes, 87
Anilin-water, gentian violet, 373
preparation of, 373
stains, 72, 94, 373
Animal, fluids as media, 19
method of holding for inocula-
preparation for inoculation.
tissues, as media, 19, 20
Animals, autopsied, destruction
inoculated, care of, 301
Anjesky's spore-staining method,
Anthrax, 302, 303
Antibiosis, 45, 216
Antibodies, 313-315, 328
Antigen, for agglutination test,
for complement fixation test,
preservation of, 326
titration of, 327
Antiseptics, sterilization by, 15
Anti-serum, 310, 322
tetanus, 309, 310
Apparatus, sterilization of, 6, 7
Aqueous-alcoholic stains, 72, 87,
Arnold sterilizer, 9, 10
Arrangement of microorganisms,
Artery, femoral, 298
Asbestos, shredded, as filter, 13
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
Azolitmin, source of, 369
solution, Kahlbaum's, -25, 181,
solution, purpose of, 369
Azotobacter, 253-255, 354, 355
Bacillus, meat, 197
slimy milk, 197
Bacillus acidi lactiti, 347, 362,
aerogenes, 347, 362, 363
aerogenes 2, 362, 363
Bacillus alvei, 97
caratovorus, 97, 291-294
cholerce suis, 97, 310, 311, 362,
cloaca, 362, 363
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
fluorescens liquefaciens, 187, 347
gelaticus, n. sp. (gran), 41
lactis aerogenes, 362, 363
megaterium, 97, 185, 199
mesentericus vulgatus, 97, 174
mycoides, 63, 97, 174, 187, 191,
200, 201, 245, 264, 347
paralyphosus A, 362, 363
paratyphosus B, 362, 363
pasteurii, 352, 353
prodigiosus, 155, 170, 175, 176,
185, 187, 195, 196, 205
proteus, 97, 352
radiobacter, 254, 255
ramosus, 187, 188
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
Bacteria, acetic, 183
biochemical activities of, 23-25,
compared with protozoa, 140,
Bacteria, effect of desiccation on,
effect of moist and dry heat on,
flagella of, 93
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,
phosphorescent, 177, 178
spores of, destruction by heat
study of, 125-132, 135-138
sulfur, 154, 348
true, 347, 348
vegetative forms, destruction
of, by heat, 5-11
vinegar, 183, 184, 192, 193
water, identification of, 361-363
weight of, 152, 153, 243, 244
Bacterins, autogenous, 316, 317
preparation of, 316-318
preservation of, 15
Bacterium abortus, 163, 325, 347
aceti, 192, 215, 216
acidi lactici, 97
aerogenes, 97, 198, 227, 228, 231,
233, 362, 363
Bacterium anthrads, 97, 98, 302,
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
tuberculosis, 90, 92, 97, 303-305,
tumefaciens, 258, 259, 355
Balsam, Canada, 65, 71, 372
Banana, as a medium, 20
Bath, running water, 36
Bedding, sterilization of, 10
Beef, chopped lean, 28
Beer fermentation, 182
manufacture, yeasts used, 114
BEIJERINCK, 41, 155, 170, 174
Bengal isinglass, 37
BENIANS, T. H. C., 97
Benzol, effect on pigment, 175,
Berkefeld filter, 238, 303, 304
filters, sterilization of, 6
BESSON, A., 15, 19, 133, 140, 147,
149, 151, 167, 222, 303, 305,
Bile, esculin, 360, 362, 363
isolation of pathogens from,
litmus lactose, 223, 360
salts, value in media, 360
Biochemical activities, 23
Black-leg vaccine, preparation of,
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,
cells, red, use of, 323-329
serum, as a medium, 19, 20,
serum, sterilization of, 8
BOHME, A., 133
Boiling, sterilization by, 10
Bombicci's anaerobic dish, 159
Books, text and reference, list of,
Botkin's anaerobic apparatus, 159
Bouillon, nutrient, 19
Bread fermentation, 182
Bread-making, yeasts used, 114
BREW, J. D., 268
Broth, adonit, 362, 363
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
sterilization of, 31
sugar, 19, 84, 358
Brownian movement, 75
Buchner's anaerobic tube, 161,
Budding of yeasts, 78, 114
Bullock's anaerobic apparatus,
fat, preparation of, 349
trier, use of, 289, 290
Calcium carbonate, 167, 168, 349,
352, 354, 355 ,
chloride, 351, 351
phosphate, di-basic, 263
phosphate, mono-basic, 193
phosphate, tri-basic, 263, 350
salts, action of, in milk, 24
Calculations for microscopic
counts, 266, 267
titration, 21, 22
Calibration of filar micrometer,
Candles, filter, sterilization of, 6, 7
Capsules, presence of, 97, 98'
Caramelization of sugar, 26
Carbohydrate, agar, 37
Carbohydrates as food, 167, 168
enzymes of, 179-182
Carbohydrates, fermentation of,
Carbol-fuchsin, 72, 90, 92, 374
Carbolic acid. See Acid
Carbon bisulfid, effect on pigment,
Carbon dioxid, 20, 117-119, 157,
165, 171, 172, 216, 231, 374
dioxid. absorption of, 117-119
dioxid, atmosphere of, 157
Carmen rubrum, 75
Carrot, as a medium, 20
Case, pipette, 17, 18
Casein, coagulating enzyme of,
colloidal state of,]24, 33
in milk, 24
proteolytic enzymes of, 188,
Catalase, 181, 184, 191, 239, 240
Cell division, 74
Cellulose, decomposition of, 246-
enzyme of, 179
Centrifuge, 273-275, 323, 324
Ceylon moss, 37
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,
Cheese, cheddar, 286
microfiora of, 286, 287
Chemical agents of sterilization,
5, 14, 15
CHESTER, F. D., 131
Chinese ink preparation, 85, 86
preparation of, 372
use of, 72, 85, 86
Chloral hydrate, use of, 295
Chloride of lime in water purifica-
Chlorine, available, 236, 237
Chloroform, 15, 65, 175, 176, 295
Chlorophyll, pigment analogous
Chromic acid cleaning solution,
1, 48, 212
Chromogenic organisms, 26, 154,
Chromoparous bacteria, 154
Chromophorus bacteria, 154
Cider, 20, 22, 46, 204
fermented, 192, 351
titration of, 20, 22
Classification of nutrient media,
Classifications, physiological, 153-
Clay, modeling, 165
Clean, chemically, 1
Cleaning glassware, 1, 2, 3, 4
solution, 1, 2, 48, 366
Cloth, physicians, 3
Coagulation, 8, 29, 179
Cocaine hydrochloride, 295, 299
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
formation, 49, 79, 80, 84
Combustion, total, 184
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,
Condenser, microscope, 66, 69
Congo red medium, 259
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
Cork borer, 26
Cornea, inoculation into, 299
Corpuscles, red, method of wash-
Corrosive sublimate, 186
Cotton, absorbent, 26
decomposition of, 248
Cotton, effect of sterilization on,
purpose of, 15
waste, disposal of, ix
plugs, preparation of, 16, 17
plugs, rolled, 16, 17
Counter-stain, 92, 93
Counting colonies, 56, 57
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
Cresol, compound solution of,
14, 48, 296, 297, 365
Crucible, Gooch, as filter, 13
Cryophilic bacteria, 155, 200
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
care of, 46-48, 125
description of, 129
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
stab, 46, 61
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
Data, tabulation of, xi
Death-point, thermal, 200-202
DECKER, J. W., 287
Decolorization in staining, 90-
of litmus, 23, 24
Defibrination of blood, 302, 320,
Degrees, Fuller's scale, 20, 21
Denitrification in solution, 251,
studies, culture media for, 354
Denitrifying organisms, 19, 251,
Deodorizer, chloride of lime as,
Desiccator, as anaerobic dish, 160
Desk, microorganisms on, 146, 147
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,
Diatomaceous earth, 12
Dichotomous branching, 100, 101,
Dilution flasks, 52, 54
method, loop, 49-52
straight needle, 51, 107, 115
Diphenylamin, sulfuric acid solu-
tion of, 249, 371
Directions, laboratory, x
Disaccharides, enzymes of, 180,
Discoloration in water, 237
Discontinuous heating, 8-10
Diseases, animal, 295-329
producing bacteria, 153
Dish, evaporating, use of, 20-22
Dishes, deep culture, wrapping of,
Petri, wrapping of, 17, 18
Disinfectant, phenol coefficient of,
Disinfectants, ix, 5, 14, 15, 236-
Distilled water, 20
micron 1 ora of, 152, 153
DON, J. and CHISHOLM, J., 227,
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
organic, reduction of, 181, 184
saturated alcoholic solution of,
stock solutions of, 87
Earth, diatomaceous, 12
Egg, albumin, liquefaction of, 24
as a medium, 19, 20
use of, in preparation of media
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
action, pure, 188
acting anaerobically, 179
classification of, 179-181
coagulating, 179, 181, 185, 186,
effect of heat on, 186
formation of name of, 179
hydrolytic, 178, 179, 180, 182-
Enzymes, intracellular, 178
lipolytic, 178, 180, 183
oxidizing, 179, 181, 183, 184
change, 178, 180-185
proteolytic, 24, 25, 178, 180,
reducing, 179, 181, 184, 190
specific, 167, 169, 172, 190, 192,
ERNST, WM., 268, 273, 278, 281
Esmarch's tube, 158
ESTEN, W. M., 132
Esters, enzymes of, 180
Ether, 6, 92, 175, 176, 295
flame, sterilization in, 6
Eubacteria, 347, 348
EULER, HANS, 15, 168, 179, 190,
Eurythermic bacteria, 155
Evaporating dish, use of, 20-22
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
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
lactic acid, 167, 184, 217, 218
Lindner's method of demon-
strating, 83, 84
tube, Durham's, 119
tubes, cleaning of, 4
tubes, plugging of, 18
Ferric chloride, 214, 354
Ferrous sulfate, 350, 353, 375
Fibrin, coagulating enzyme of,
liquefaction of, 24
Filar ocular micrometer, 72-74
Filter, Berkefeld, 12, 238, 239
candles, sterilization of, 6, 10
-paper, decomposition of, 246-
-paper in media, 352
pores of, 12
Filters, bacterial, 12
Berkefeld, sterilization of, 6
cleaning of, 313
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
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,
Flowing steam, 10, 11
Focal point, 68, 77
Focusing, 67-69, 77
Food, requirements, variation in,
small amount needed, 152, 153
Foods, microbial, 18-20
Forceps, sterilization of, 6
use of, 16
Form, for writing up exercises,
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,
juices, natural, 20, 22
juices, titration of, 22
Fruiting bodies, 79, 100-109
Fuchsin, 87, 88, 373
FUHRMAN, F., 63
Fuller's scale, 20, 21,40, 205
Funnel, filling, 25
Furnace, muffle, 6
GAGE, S. H., 69
Galactose, 23, 180, 182
fermentation of, 184
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-
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,
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
size of molecule, 32
solidifying point, 34, 352
source of, 32
acid in, 34, 187
Gelatin, urea, 352
Gelatinizing property, loss of,
Gelideum corneum, 37
Gentian violet, 87
Germicidal, 1, 365
Germination of mold spores, 78, 79
stages of, 79
Giant colony, preparation of,
Giltay's agar, 354
solution, 19, 251, 252, 354
GILTNER, W., 160-162, 164, 312,
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
preparation of, for sterilization,
purpose of sterilization, 15, 16
sterilization of, 6-15
Glucosidases, 180, 182
Glucosides, enzymes of, 180, 182
Glycerin, 26, 183
Glycogen, enzyme of, 179
Gooch crucible as filter, 13
Gram, unit of weight, 380
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
Hair, microflora of, 148, 149, 271
Halophile, 154, 351
HAMILTON, H. G. and OHNO, T.,
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
Heat-producing bacteria, 154
Hemicelluloses, enzymes of, 179
preservation of, 326
Hemolysin, source of, 325
titration of, 327
Hemolytic serum, preparation of,
system, 328, 329
Hesse's method for anaerobes, 157
Hexoses, 23, 180
Hoffman and Fiske's enrichment
medium, 361, 362
HOFFMAN C. and HAMMER, B. W.,
HOLM, M. L. and GARDNER, E. A.,
HOOKER, A. H., 236-238
Hospital gauze, 3
Hot air sterilization, 7
Humus, 239, 240
Hydrochloric acid, N/l, prepara-
tion ot, 367, 368
acid N/20, 20
acid N/20, preparation of, 367,
Hydrogen, atmosphere of, 157,
peroxide, enzyme of, 181, 184,
191, 239, 240
sulfid, Beijerinck's test for, 174
sulfid, production of, 174, 175,
sulfid, test for, 130
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
Impression preparation, 98, 99,
Inactivated serum, 325-329
Inactivation of serum, 326, 328
Incision, crucial, 299
Incubator, 37, 53
India ink, 85
Indicators, 20, 369
Indol, 43, 131-133, 370
Ehrlich's test for, 132, 133
graphic formula of, 132
in peptone, 132
test for, 132, 133, 370
Infusion, hay, 9, 19
meat, preparation of, 28, 29
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
Inoculations, mold, 62
Inoculum, 14, 197, 297
Instruments, metal, sterilization
of, 6, 14, 15
Intermittent heating, 8-10, 37
Intraabdominal inoculation, 29
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
Inversion of sugars, 23
lodin solution, Lugol's, 95, 96,
114, 115, 189, 309, 375
tincture of, 14, 365
Iris diaphragm, 63-69
Iron, citrate, soluble, 360
Isinglass, Bengal, 37
Isolation of pure cultures, 31,
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
Kidney, isolation of pathogens
from, 301, 302
Kipp generator for hydrogen,
Kitasato's dish, 159
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
Lead acetate, 165
acetate paper, use of, 125,
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
fermentation of, 180
Liborius-Veillon anaerobic meth-
Lichens, as source of litmus, 369
Light, artificial, 209
artificial for microscopical work,
diffused, 206, 207
producing bacteria, 154, 177,
relation to pigment formation,
Lindner's concave slide culture,
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,
Liquefaction of boiled egg white,
of fibrin, 24
of gelatin, 24, 46, 187, 188
of milk curd, 24
Liquor cresolis compositus, 48,
296, 297, 365
Litmus, decolorization of, 23, 25,
Merck's purified, 369
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,
LOHNIS, F., 63, 133, 168, 173, 175,
192, 241, 244, 247, 249, 251,
253, 255, 262, 264, 268, 278,
Loop dilution method, 51
Lugol's iodin solution, 95, 96, 114,
115, 189, 309, 375
Macroscopical changes, 23, 142
Magnesium ammonium phos-
carbonate, basic, 353
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,
Azotobacter in, 253
bacteria in, 165, 241, 243, 248,
isms in, 246, 247
Measuring microorganisms, 70-
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,
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.,
McBRYDE, C. N., 315
MCCAMPBELL, E. F., 318, 321
MCFARLAND, J., 91, 312, 321, 323
McLeod's plate base, 163
Meat, as"a medium, 19
infusion, preparation of, 28, 29
products, as media, 19
Media, acid, 22
culture, sterilization of, 8-13
liquid, 19, 20, 22-26
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,
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
Methylen blue, 23, 87-93, 179,
enzyme of, 179, 181, 184
for yeasts, 373
Loeffler's alkaline, 265, 266,
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,
of skin, 148
of soil, 241-244
Micrometer, head, 65
ocular, 70-74, 265
ocular, filar, 72-74
stage, 70-74, 265
step, 70, 71, 74
Micron, 33, 70 ; 71, 73, 74, 379
Microorganisms, arrangement of,
determination of size, 70-74
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
resistance of, 10
starch-digesting, 189, 190
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,
Milk, acid production in, 23
action of calcium salts in, 24
as a medium, 19, 20, 22-26,
46, 129, 141-144
" bloody," 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,
cow's, coagulating enzyme of,
curd formation in 24, 280
Milk, dilutions for plating, 142,
264, 268, 269, 274, 276, 279,
dirt in, 273-278
effect of straining, 276-278
extraneous contamination of,
fore, bacteria in, 268, 269
gas production in, 25
germicidal action of, 280
human, coagulating enzyme of,
keeping quality of, 278-281
litmus, 23, 25, 26, 361, 363
litmus, preparation of, 25, 26
microflora of, 264-268, 274
middle, bacteria in, 268, 269
pails, bacteria in, 272
pasteurization of, 203-205, 281-
peptonization of, 24, 25
reaction of, 25, 177
staining of, 96
standards, bacteriological, 268
sterilization of, 26, 139
strippings, bacteria in, 268,
titration of, 20-23
viscosity of, 283, 288
Milking tube, 269
Millimeter, 33, 63, 70, 379
Millimicron, 33, 379
Mirror, concave, 67
MOHLER, J. R. and EICHHORN, A.,
MOHLER, J. R., EICHHORN, A. and
BUCK, J. M., 329
Moist chamber, preparation of,
culture, 100, 109
relation to microbial destruc-
tion by heat, 202, 203
Mold colony, examination of, 105,
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,
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.,
Mordant for flagella staining, 94,
MORTENSEN, M. and GORDON, J.,
Moss, Ceylon, 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
rate of, 74
Mucilage of Ps. radicicola, 258
Mucor, 63, 100
Mucous membrane, microflora of,
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
a-naphthylamin, 133, 249, 251,
Needles, platinum, sterilization
Nessler's solution, 133, 249, 251,
Neutralization, 20, 22, 25
Newspaper, uses of, 17, 18
Nitrate peptone solution, 19, 44
Nitrates, reduction of, 44, 131,
133, 134, 138, 181, 251-253,
test solutions for, 371
in solution, 249-251
studies, media for, 353, 354
Nitrifying bacteria, 153, 353
Nitrite reductase, 181
Nitrites, 43, 44, 133, 134, 138, 250,
reduction of, 181
test solutions for, 371
Nitrogen as food, 172, 173
atmosphere of, 157, 165
Nitrogen-fixation studies, media
for, 354, 355
-fixation, non-symbiotic, 253-
-fixation, symbiotic, 256-263,
from nitrates, 44, 134
inorganic as food, 172, 173
organic, 172, 173
Nodule formation, observation of,
Nodules, on leguminous plants,
Non-electrolytes, 195, 196
NORGAARD, V. A. and MOHLER,
J. R., 307
Normal acid, 20-22, 367, 368
solutions, 20-22, 367, 368
NORTHRUP, Z., 169, 215
Nose, microflora of, 150
Nosepiece, collar, 66
NOVY, F. G., 132, 158, 160, 164,
Novy's jars, anaerobic, 158, 160,
NOYES, WM. A., 20
Nuclei, stain for, 99
Nutrient media, 18-20, 23-44
NUTTALL, G. H. F., 323
Objective, oil immersion, 69, 71
Objectives, 63, 66, 67, 69, 71, 77
cleaning of, 66
cleaning of, 66
Odor, fecal, cause of, 132
Oil, cedar wood, 372
immersion, 66, 69, 372
of garlic, 15
of mustard, 15
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,
test, McCampbelPs modifica-
Opsonins, demonstration of, 319-
Optical axis, 66
Organic matter, effect of chloride
of lime on, 236-238
Osmotic pressure, 52-54, 196
Outline, for study of microbiology,
Oven, for hot-air sterilization, 7
Over-heating media, 26
Oxidases, 181, 183, 184
Oxidation, 183, 184, 237
Oxygen, absorption of, 156, 160-
free, liberation of, 181, 184, 240
tolerance, 154, 164
transference of, 181
Paper, effect of sterilization on,
filter, in media, 352
uses of, 15, 16
Papilionacece, attacked by Ps.
Parachrome bacteria, 155
Para - dimethyl - amido - ben-
zaldehyde, 133, 370
Paraffin, 76, 78, 80
oil, 157, 308
Parasite, wound, 293
Parasites, facultative, 154
Paratyphoid group, 362
PASTEUR, L., 8, 13, 158, 159
Pasteurization, 8, 203-205, 215,
effect on digestibility of milk,
factors influencing efficiency,
Pathogenic material, care of, x
nature of molds, 113
organisms, 26, 88, 129, 151, 153
organisms, care in staining, 88
Peat, bacteria in, 242, 243
Pecilothermic bacteria, 155, 200
N Pectase, 180
Pectinase, 181, 294
Pectose, enzyme of, 180
italicum, 101, 104, 105, 107, 195,
Pentoses, enzymes of, 180
Pepsin, 24, 180
Peptone, 29, 43, 44, 170, 172
Peptones, enzymes of, 180
of milk, 24
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)
remedy for burns caused by, 365
stock solution of, 364
synonyms of, 364
value as a disinfectant, 365
Phenolphthalein as indicator, 20-
Phosphates, insoluble to soluble,
Phosphorescence, 177, 178
Phosphorus, relation to decay,
Photogenic, 154, 177, 178, 351
Phototaxis, 206, 207
Physical agents of sterilization, 5
condition of medium, 198
Physician's cloth, 3
Physiological efficiency, 262, 293
Physiology of microorganisms,
Pickles, brine, 197
Pigment, crystals of, 176
formation, effect of temperature
formation, relation of air to,
formation, relation of light, 176
-producing bacteria, 154, 155.
solubility of, 176
Pigments, microbial, effect of
physical and chemical agen-
cies on, 175-177
Pipette case, 17, 18
Pipettes, cleaning of, 3
preparation of, for steriliza-
use of, 22, 54, 55, 112
Planococcus agilis, 347
Planosarcina mobilis, 347
Plants, microbial diseases of,
Plates, agar, 51, 52
inverting of, 52
Plating, 45, 49-56
Platinum, spongy for absorbing
needles, sterilization of, 6
Pleuritic fluid, isolation of patho-
gens from 302
Plugs, cotton, 15-17
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,
persulfate, 133, 370
phosphate, di-basic, 170, 246,
Potassium phosphate, mono-basic,
Potato, as a medium, 19, 20
glycerin, 26, 27, 45
tubes, preparation for steriliza-
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
Protein decomposition products,
Proteins, enzymes of, 180
soluble, as food, 172, 173
Proteolytic enzymes, 24, 180, 185-
Proteoses, enzymes of, 180
Proteus vulgaris, 97, 352
Protoplasm, stain for, 87
Protozoa, 140, 141
Pseudomonas campestris, 97, 208,
radidcola, 173, 256-263, 355
radicicola, isolation from no-
radicicola, physiological effi-
ciency of, 256-263
Psychrophilic bacteria, 155
Pumice stone, 2
Pump, electric vacuum or
mercury vacuum, 159
water vacuum, 159
Purification of filter candles, 313
Pus, collecting, 14
isolation of bacteria from, 148,
staining of, 87, 92
Rabbits, 295, 309, 322-325
RAHN, O., 168, 219, 246
Rats, white, 295
Rays, light, 209
Reaction, 20-22, 29, 30, 36, 112,
199, 206, 215
adjustment of 21, 30, 36
amphoteric, of casein, 24
of water analysis media, 357
optimum, 29, 206
Reactions, enzymic, 182-185
Reductase, methylen blue, 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
Resistance of microorganisms, 10,
Respiration of bacteria, 165, 177
DE REYPAILHADE, J., 190
Rhizopus nigricans, 100, 102, 103,
RICHMOND, H. D., 194
RIDEAL, S. and RIDEAL, E. K.,
ROGERS, L. A.', 285
Root tissues, invasion by bacteria,
ROSENAU, M. J., 270, 278, 284
Rot, soft, bacterial, 291-294
Roux's anaerobic tubes, 158
biological method for anaerobes,
flask, 62, 109
tubes for potatoes, 26
Rubber apparatus, sterilization of
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
Sac, collodion, 13
Saccharomyces, apiculatus, 83, 114,
cerevisice, 83, 97, 114, 115, 120,
171, 182, 195, 198, 199
ellipsoideus, 114, 173, 215, 216,
Saccharose, 170, 171, 195, 355,
enzyme of, 180, 182
fermentation of, 182
SADTLER, S. P., 172, 190, 193, 289
Salkowski- Kitasato test for indol,
Salt, citrated, 370
effect on microorganisms, 196,
phenol, 303, 304
purpose of, in media, 29
solution, 52, 370
Salts, soluble, of meat, 28
Sand grains, size of, 245, 246
Sapo mollis U. S. P., 365
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
Sealing anaerobic cultivations, 156
microscopical preparations, 76,
79, 80, 82, 83 ; 85
Seaweeds, agar from, 37
Sediment test, macroscopic, 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,
hemolytic, titration of, 326, 327
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
Skin, microflora of, 148, 149, 272
sterilization of, 14
Slides, cleaning of, 4
labelling of, 89
sterilization of, 18, 48
Slime-forming organisms, 97, 197,
SMIRNOW, M. R., 27
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,
chloride, 350-354, 370^
citrate, 319, 37&
hydroxide, effect of, on pig-
ment, 175, 176
hydroxide for anaerobic culture,
hydroxide for cleaning, 3, 4,
hydroxide for CO 2 absorption,
hydroxide N/l, 21, 22, 29, 368
hydroxide N/20, 20, 21, 368
nitrate, 170, 354
Sohngen's solution, 353
Soil, as a medium, 19
catalytic power of, 239-241
denitrifying bacteria in, 251-
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
culture for nitrifying bacteria,
Solution, Giltay's, 354
mannit, 253, 254, 354, 355
Nessler's, 133, 134, 371, 372
Solutions, concentration of, 52-54,
normal, 20-22, 367, 368
standard, 367, 368
Spatulas, iron and nickel steril-
Specific gravity, alcohol by vol-
Spirillum rubrum, 348
Spirocheta obermeieri, 87, 348
Spirosoma nasale, 347
Spleen, isolation of pathogens
Sporangiophore, 100, 102, 103
Sporangium, 100, 102, 103
Spore formation, study of, 144,
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,
Spore stain, 90, 91
Sputum, staining of, 92, 93
Staining, 58, 87-89
capsules, 97, 98
Gram's method, 95, 97
Gram-Weigert method, 96
in mvo, 116
of tissues, 96
purpose of, 87
time necessary' for, 88
Stains, anilin-water, 72, 95, 96,
aqueous-alcoholic, 72, 373
preparation of, 373, 374
saturated alcoholic, 72, 373
Stand, microscope, 63, 65
Staphylococcus pyogenes albus, 97,
aureus. 97, 318, 347
Starch, decomposition of, 182,
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
Stearin, enzyme of, 180, 183
Stearinase, 180, 183
Stenothermic bacteria, 155
by antiseptics, 15
by continuous or discontinuous
heating in water at 100 C.,
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,
by moist heat, continuous or
discontinuous heating at low
fractional, 34, 139
Sterilization of. a large bulk of
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
STERNBERG, G. M., 177
STITT, E. R., 301, 318
STOCKING, W. A., 132
Straw, decomposition of, 248
microorganisms on, 271
Streptococci in milk, 275
Streptococcus erysipelatus, 347
pyogenes, 47, 97, 318, 347
Subcutaneous inoculation, 297,
Subdural inoculation, 299, 300
Substrate, nutrient, 101
Sucrase, 180, 182
Sugar, caramelization of, 26
for demonstrating gas pro-
Sugars as food, 167, 170
complex as food, 167
fermentation of, 171, 172, 182,
inversion of, 23
simple, as food, 167
Sulfur, lead-blackening, 174
reduction of, 181, 190, 191
Sulfur, reductase, 181
Sunlight, effect of, 176, 208, 209,
SURFACE, F. M., 329
Surfaces, polished, sterilization
Surgical dressings, sterilization
Suture, 299, 300
Swab, preparation of, 150
use of, 316
Symbiosis, 45, 214, 215, 256
Synthetic media, 171, 248, 350
Syphilis, hemolysin in, 325
Syringe, 295, 309
Syringes, sterilization of, 10
enzyme of, 180
TARROZZI, 155, 156
Teeth, microflora of, 150
Temperature, 16, 198-203
cardinal points of, 198, 200
constant, ix, 47, 49, 52
degrees, conversion of, 377
effect on microorganism and
effect on pigment formation,
for sterilization, 8-11
influence on keeping quality of
low, for sterilization, 8
maximum, 155, 198, 199
minimum, 155, 198, 199
optimum, 155, 198, 199
pressure table, 376
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
Thermometer, clinical, 313
Thermophilic bacteria, 155
Thiopedia, 348 \
THRESH, J. C., 177, 2271
Throat, microflora of, 150
Time for sterilization, 8-11
Tissue, diseased, isolation of
pathogens from, 301, 302
sterile for oxygen absorption,
vegetable, sterile, 156
Tissues, connective, 156
staining of, 96
subcutaneous, 297, 299
calculations, 21, 22
Titre, 21, 25, 112
Torula rosea, 83, 114, 120, 175,
Toxin, tetanus, preparation of, 308
Transferring cultures, 51, 59-61
Tray, operating, 309
Trephine, 295, 300
Trjetrop's anaerobic apparatus, 159
Trichophyton tonsurans, 149
Trypsin, 24, 180
Tube length of microscope, 65, 66,
Tubercle bacteria, method of
staining, 92, 93
Tubercles, staining of, 92, 93
Tuberculin, preparation of, 303,
Tubes, fermentation, cleaning of, 4
plugging of, 18
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
Udder, bacteria in, 268-270
infection of, 270
Urea, 181, 185, 352, 353
decomposition, media for, 352,
Urease, 178, 181, 185
Urine, isolation of pathogens
Uschinsky's solution, 172, 350
Utensils, sterilization of, 6-11
Vaccine, antirabic, preparation of,
black-leg, preparation of, 307
Vaccines, bacterial, pieparatiort
preservation of, 15
Vacuum, for anaerobic cultures,
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
VERNON, H. M., 168, 186, 188, 192
Vignal's tube, 158
Vinegar, bacteria, 192
circular on, 192
legal standard of, 192
pure cultures for, 192
titration of, 22
Violet, crystal, 361
gentian, 87, 373
Virus, attenuated, 307
dialyzation of, 13
hog cholera, 313-315
Vosges-Proskauer's reaction, 363
WARD, A. R., 268, 270, 281, 284
WASHBURN, R. M., 291
Wastes, laboratory, destruction
of, 6, 7
Watch, stop, 74
Water, as an end product, 183
bacteria isolated from, 223-227
bacteriological analysis of, 223-
' distilled, 20
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,
Weight of bacteria, calculation
of, 153, 244
WELLS, LEVI, 285
Welsbach burner, 67
WESBROOK, F. F., WHITTAKER,
H. A. and MOHLER, B. M.
Whey, sour, 169, 349
WHITTAKER, H. A., 238
Widal test, 361
WILEY, H. W., 291
Wine-making, yeasts used, 114
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
Xylol, effect on enzymes, 187, 188
use of, 65, 66, 187, 188, 265,
Yeast, 63, 78-80, 84, 97
cake, flora of, 115, 116
Fleischmann's Qompressed, 114,
Yeasts, budding of, 79
enzymes of, 182, 190, 191
fermenting power of, 83, 84
resistance to heat, 202, 203
study of, 114-123
Ziehl-Nielson's carbol-fuchsin, 374
Zone development of colonies, 164
lactic acid bacteria, 180, 184
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