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BIOLOGY DEPT.

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DESMOIDS CGLLE *'ACY

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Bacteriological Methods

IN

Food and Drugs Laboratories

WITH AN

^action to Micro-analytical Methods

BY

ALBERT SCHNEIDER, M. D., PH. D.

' (Columbia University),

professor of pharmacognosy and bacteriology in the

college of pharmacy of the university of

CALIFORNIA, SAN FRANCISCO

LIBRARY

DES MOINES COLLEGE BF PHARfMCY

1019 High Street Des foir.es, Iowa
> i

87 ILLUSTRATIONS
AND 6 FULL PAGE PLATES

PHILADELPHIA
P. BLAKISTON'S SON & CO.

1012 WALNUT STREET

Copyright, 1915, by P. Blakiston's Son & Co.

THE MAPLE PRE S'S YORK PA

b I^.V LIBRARY

S des m:m c ^acy

1015 ; l»M ^

PREFACE

The administration of the Federal Pure Food and Drugs Act
and of the several State Pure Food and Drugs Laws has made the
introduction of bacteriological methods into food and drugs
laboratories a necessity. Because of the close relationship be-
tween the work of the bacteriologist and that of the micro-analyst,
it is advised that, wherever possible, these two laboratory branches
be combined in the most effectual cooperative manner. With
such cooperation in mind, a brief introduction to micro-analytical
methods is added. Fuller details on micro-technique will be
found in special works on the microscopy of fibers, foods, spices,
drugs, of water supplies, of sewage, etc.

As is more fully set forth in the text, the bacteriological as
well as micro-analytical methods in our food and drugs labora-
tories are not yet fully worked out, and the present volume is
submitted hoping that it will be instrumental in bringing about
a unification of methods and that it will perhaps also serve as a
guide to the working out of newer and inadequately tested older
methods.

The volume is primarily intended as a guide to students who
are interested in the bacteriological examination of foods and
drugs. Its use as a laboratory guide presupposes a thorough
2§nowledge of general bacteriology.

> Acknowledgments are made to the following authors for the
"se of illustrations: E. R. Stitt (Bacteriology, Blood Work and
nimal Parasitology), R. L. Pitfield (Compend on Bacteriology),
. J. MacNeal (Pathogenic Micro-organisms), C. E. Marshall
licrobiology), John F. Anderson and Thomas B. McClintic
lethod of Standardizing Disinfectants), and G. W. Hunter (Es-
ntials of Biology).

v

Vi PREFACE

Grateful acknowledgments are also made to A. E. Graham,
Inspector in Charge, San Francisco Laboratory, Bureau of Animal
Industry, for valuable suggestions regarding the examination of
meats and meat products with special reference to the isolation
and examination of animal fat crystals and the examination of
sausage meats for starch fillers; to Professor Karl Frederick
Meyer, of the Department of Bacteriology and Protozoology of the
University of California, for the article on "The Precipitin Test
for Meats," and to Merck's Report for permission to use those
parts of the text which had been published in that journal. It is
also desired to acknowledge the loan of several cuts by the Bausch
and Lomb Optical Company. Additional acknowledgments are
made throughout the text.
San Francisco, California.

CONTENTS

[. Outline of Micro- analytical Methods in Food and Drugs Laboratories

Page

i. Introduction i

2. Grouping of Substances to be Examined in Food and Drugs Laboratories . . i

3. The Work of the Micro-analyst in Relationship to that of the Chemist and

Bacteriologist 3

4. Equipment for Micro-analytical Work 4

5. Organoleptic Testing 8

6. Methods Useful in the Examination of Vegetable Drugs, Spices, Etc ... 9

7. Methods Useful in the Examination of Vegetable Food Products .... 11

8. Micro-chemical Color Reaction Tests 15

9. Making Analytical Reports 17

:o. The More Important Histological Elements of Plants 18

II. Bacteriological Methods in Food and Drugs Laboratories

1. Introduction 23

2. Direct Bacteriological Examinations 51

3. Numerical Limits of Micro-organisms in Foods and Drugs 53

4. Quantitative Estimations by the Cultural Methods 68

5. Preparation of Standard Culture Media. General Suggestions 71

6. Preparation of Required Standard Culture Media 75

7. Technique for Making Quantitative and Qualitative Estimations by the

Plating Methods 83

8. Practical Application of the Quantitative Estimations by the Plating

Methods 89

9. Qualitative Determinations 9°

to. Evidence of Sewage Contamination 97

[i. Possible Contamination of Foods with the Typhoid Bacillus 102

Possible Contamination of Food Substances with the Cholera Bacillus. . .112

Biological Water Analysis * 114

Bacteriological Examination of Mineral Waters . 118

The Microscopical and Bacteriological Examination of Milk 120

The Bacteriological Examination of Shellfish 144

The Bacteriological and Toxicological Examination of Meats and Meat

Products i52

vii

Vlll' CONTENTS

Page

18. The Bacteriological Examination of Eggs and Egg Products 187

19. The Bacteriological Examination of Pharmaceutical Preparations .... 197

20. The Microscopical and Bacteriological Examination of Syrups 202

21. The Microscopical and Bacteriological Examination of Fermented Foods

and Beverages 210

22. Standardization of Disinfectants 230

23. Determining the Purity and Quality of Sera, Bacterins and Related Products 265

24. Special Biological and Toxicological Tests 268

OUTLINE OF MICRO-ANALYTICAL METHODS IN
FOOD AND DRUGS LABORATORIES

i. Introduction

The value of the compound microscope in the examination of
foods and drugs is as yet not generally recognized. Efficiency in
this line of work depends very largely upon a long and wide range
of experience, in this regard differing very markedly from effi-
ciency in the field of chemical analyses. All that is required of
the chemist, as far as routine analytical work is concerned, is a
very close adherence to the methods laid down for him. He is
pronounced skilled in direct proportion to his adherence to
methods and skill shown in the manipulation of apparatus and
reagents. The micro-analyst in order to be efficient must be very
familiar with the appearance of the multitudinous forms of cells,
tissues, cell-contents and with the behavior of certain micro-
chemical reagents and this familiarity can be acquired only through
long and careful observation.

2. Grouping of Substances to be Examined in Food and Drugs

Laboratories

The analytical methods, as they apply to the critical examina-
tion of foods and drugs, are chemical, microscopical and bacteriolog-
ical. The substances to be analyzed may be grouped as follows:

2 MICRO- ANALYTICAL METHODS

i. Vegetable drugs, crude and powdered, pharmacopceial and other simple and
compound medicinal powders.

2. Spices and condiments, whole, ground and powdered. Prepared spices and
condiments.

3. Coffee, tea, cocoa, chocolate, confections, candies.

4. Tobacco and preparations made from tobacco, as snuff, smoking tobacco,
cigars, etc.

5. Chemicals, minerals, solutions of chemicals, etc.

6. Tablets, pills, powders.

7. Meats of all kinds, raw, cooked, canned, sausage meats, etc.

8. Dairying products, as milk, cream, cheese, butter, ice-cream, ice cream fillers,
etc.

9. Insect powders, dusting powders, cosmetics.

10. Cattle and poultry powders.

11. Unknown powders, wholly or partly of vegetable origin.

12. Starches, dextrins, sausage meat binders (starches).

13. Vegetable foods, as jams and jellies; fresh, pickled, cooked, canned and
preserved.

14. Flours and meals.

15. Breakfast foods, infant and invalid foods.

16. Breads and similar materials; biscuits, doughnuts, cakes, pies, pastries,
etc.

17. Macaroni, spaghetti and similar preparations, noodles, etc.

18. Nuts and nut-like fruits and seeds, etc.

19. Beverages of all kinds, liquids generally.

20. Pharmaceuticals of all kinds.

21. Patent and proprietary medicines.

22. Unknown foods and medicines.

In the examination of some of these substances the chemical
method is all important, as in chemicals generally; in the examina-
tion of others the microscopical method is all-important, as in
meals, flours, spices; and again the bacteriological testing is all-
important, as in sewage, contaminated water, contaminated milk,
infected foods and drinks generally, etc. A properly equipped
analytical laboratory, whether federal, state or private, should
be prepared to apply all three methods. The bacteriological in-
vestigations should be made by the micro-analyst rather than by
the chemist, because of the closer relationship between bacteriology
and microscopy.

INTRODUCTION 3

3. The Work of the Micro -analyst in Relationship to that of the
Chemist and Bacteriologist

Just what work should or should not be done by the micro-
analyst is as yet not definitely determined; at least, there is no
uniformity as to scope of action in the different analytical labora-
tories. It is suggested that the following work be assigned to the
micro-analyst:

1. Gross and net weight determination of all such samples as require it.

2. Moisture determination of substances which require it.

3. Ash and acid insoluble determinations of substances which are primarily
subject to microscopical analysis, as vegetable drugs, pills, powders, vegetable
compound powders, etc.

4. Use of certain special tests, as sublimation tests for benzoic acid, salicylic acid
and boric acid; Grahe's cinchona test, wheat gluten test, color reactions for boric
acid, capsicum, guaiac, salicylic acid, morphine, etc., tests for cholesterol and phy-
tosterol crystals, and others which may prove useful.

5. Bacteriological testing of foods and drugs generally, of sera, vaccines, galen-
icals, syrups, milk, water, jams, jellies, catsups, etc., as may be required, following
the method of the Society of the American Bacteriologists, and limiting the testing
to determining the presence or absence of the colon bacillus and other sewage organ-
isms, and the usual quantitative bacterial determinations for milk, water and other
substances, of which the quality is usually based upon the quantitative bacterial
content.

Substances subject to analysis in the laboratories mentioned
should be grouped or classified according to the special or pre-
ferred methods of examination to be applied. It is, of course,
evident that in the majority of cases chemical as well as micro-
scopical methods should be used. In some cases even all three
must be used in order that conclusive results may be obtained.
The following grouping is suggested:

1. Substances in which the chemical analysis is of first importance. Chemicals
generally, and chemicals in solution, alcohol, alcoholic drinks, flavoring extracts,
syrups, oils, fats, etc.

2. Substances in which the microscopical analysis is of first importance —
vegetable substances and preparations which are essentially of vegetable origin.
Meats of all kinds, variously prepared, cooked, spiced, etc.

3. Substances in which the chemical and microscopical examinations are of equal

4 MICRO-ANALYTICAL METHODS

importance — assayable vegetable drugs, all prepared food substances with chemicals
in solution, compound powders, pills, tablets.

4. Substances to which the microscopical examination is not generally applied
■ — chemicals, liquids in which the insoluble particles are slight in amount, as wines,
brandies, comparatively pure solutions, etc. Here the centrifuge plays an im-
portant part.

5. Substances in which the bacterial testing is of prime importance — milk,
sewage or otherwise organically contaminated water supplies, and other liquids,
beers, etc., contaminated foods generally. In this class of substances the micro-
scopical and chemical examinations become necessary in addition to the bacterio-
logical; in fact, a bacteriological test is incomplete without the use of a good com-
pound microscope.

The work of the micro-analyst is, so to speak, on trial. The
doubt in the minds of the critics is due, very largely, to the un-
satisfactory results traceable to the efforts of those who are not
sufficiently qualified. Even the most skillful analysts admit
numerous defects in methods and shortcomings in results. For
example, the quantitative estimates based upon optical judg-
ment are approximate only, and with most workers there is a
very marked tendency to make these estimates volumetric rather
than gravimetric. This can in a measure be corrected by bring-
ing into play the judgment of the relative weights of the several
substances under comparison. For example, the amount of
sand present in powdered belladonna root may be volumetrically
estimated at 20 per cent. In this case the acid insoluble ash
residue may show 35 to 40 per cent, of silica. An example like
this also indicates why the micro-analyst should make the sand
and ash determinations. The percentage estimates based upon
microscopical examination may vary within 25 to 50 per cent,
when the amounts of the admixtures are small or slight. For
example, the actual amount of arrow-root starch in the so-called
arrowroot biscuit is 2.5 per cent. The micro-analyst's estimates
may range from a trace or small amount to 5 per cent. When
the quantities of admixtures are large, from 30 to 90 per cent.,
the estimations may approximate within 10 or 15 per cent, of
the actual amount present. These estimates can no doubt be

INTRODUCTION 5

made much more accurate by uniform methods of technique,
aided by certain mechanical devices. For example, in the ex-
amination of vegetable powders, spices, meals, flours and similar
substances, the samples should be thoroughly mixed, and slide
mounts should be of standard and uniform thickness and the
relative amounts of the ingredients should be estimated by means
of microscope slides having uniform ruled squares of definite
measuring value in microns. These and other details in the
methods should be more fully worked out.

Several micro-analysts have declared themselves as opposed
to giving percentage estimates of the several ingredients of a
compound. However, not to give the approximate percentages
will cause great confusion and very materially lessen the value of
the work done. For example, to report a pancake flour as com-
posed of " buckwheat and wheat flour, the former predominating,'7
instead of "buckwheat approximately 75 per cent, and wheat
approximately 25 per cent.," would certainly be unsatisfactory.

The following examples will serve to explain the relative value
of the chemical and microscopical analyses. Suppose the sub-
stance to be examined is a baby food. The microscope may re-
veal approximate percentages of oil globules, steam dextrinized
wheat starch, unchanged wheat and arrowroot starch, wheat
tissue and milk sugar. The chemical analysis will show a definite
percentage of sugar, soluble starch, insoluble starch, fat, vege-
table fiber and ash. This is a good example of a case where the
two methods of analysis are of equal importance; one without
the other would be unsatisfactory, incomplete and inconclusive.
Again, the chemical assay may show that a sample of powdered
belladonna leaf contains 0.35 per cent, of mydriatic alkaloids, and
yet the microscopical examinations may prove the presence of
20 per cent, or more of some foreign leaf.

An adjunct in analytical work, much neglected by the chemist,
is the organoleptic testing. This is especially important in
the examination of unknown substances, fruit products, spices,

6 MICRO-ANALYTICAL METHODS

meats, etc., as it often gives a clue to the quality of the sub-
stances and to the means of getting quick results.

4. Equipment for Micro -analytical Work

The equipment and apparatus required by the micro-analyst
is comparatively inexpensive, and it is very earnestly advised to
secure only those appliances which are useful or essential for the
work in hand. The following list is submitted without entering
into detail, as it may be assumed that the microscopist does not
require explanations:

1. Simple lens.

2. Compound microscope.

a. Ocular with micrometer scale.

b. Oculars, Nos. 2 and 4.

c. Objectives, Nos. 3, 5 and 7.

d. 1/12 in. oil-immersion objective for bacteriological work.

3. Slides and covers.

4. Section knife or razor, and strop.

5. Polarizer, for the study of starches, crystals and other substances. Should
be convenient to use. The selenite plates are useful.

6. Thoma-Zeiss hemacytometer; for counting bacteria and yeast cells.

7. Stage mold and spore counter, as described in Part. II (Fig. 5).

8. Accurate metal or hard rubber millimeter ruler for measuring seeds (in fruit
products), etc.

9. The required glassware and adjunct apparatus.
10. The required reagents.

n. Equipment for making moisture determinations.

12. Equipment for making ash determinations.

13. Equipment for the required bacteriological tests and determinations.

The laboratory in which the work is done should be roomy, well-
lighted, provided with the necessary shelves, apparatus and supply
cases, reference books, etc. The details need not be given here.
The analyst must see to it that the necessary things are provided.
A skillful and experienced worker should have the tools of his
choice, not those selected for him by some one not qualified to
judge.

The skilled micro-analyst has little difficulty in determining

LABORATORY EQUIPMENT 7

the purity and comparative quality of the simple spices, as pepper,
allspice, cloves, cinnamon and ginger. However matters are

Fig. i. — Form of compound microscope suitable for bacteriological and general
microscopical work in food and drugs laboratories. Note the desirable and necessary
accessories as given in the text. The form of polarizing apparatus convenient to be
used with the compound microscope, sets into the substage diaphragm ring with the
iris diaphragm opened to the maximum. The analyzer takes the place of the ocular.
— {Bausch b° Lomb Co.)

quite different when it comes to the examination of powdered
vegetable drugs, compound vegetable powders and vegetable
products of unknown composition. A thorough knowledge of,

8 MICRO-ANALYTICAL METHODS

and a wide familiarity with, cell-forms, tissue elements and formed
cell contents is an absolute essential in order that accurately re-
liable and conclusive results may be obtained and serious con-
fusion may be avoided. Differences in the reports of findings by
micro-analysts are in part due to the personal equation, in part
due to variable methods and differences of judgment in estimat-
ing the quantity of tissue elements present and also in part due
to a lack of extensive and intensive experience.

5. Organoleptic Testing

The organoleptic tests are indeed valuable adjuncts to the micro-
scopical work. There are, however, some differences of opinion
regarding the interpretation and valuation which are to be placed
on comparisons of color, odor and taste, even among those having
had considerable experience and endowed with a fairly normal
special sense development. Our color terminology is in great
confusion, and so far as the olfactory sense is concerned, there
are only comparatively few odors or flavors which admit of ready
comparison such as tea flavor, coffee odor, vanilla odor, raspberry
flavor, loganberry flavor, and the odor of such drugs as valerian,
cubeb, fenugreek, asafetida, aloes, turpentine, camphor, the essen-
tial oils, calamus, etc., and the odor of the spices. Our compara-
tive judgment of tastes is more reliable. Much experience is
necessary to form fairly reliable estimates of flavors (associations of
tastes and odors), though pure fruit flavors are, as a rule, readily
distinguishable, as that of apples, dried apples, peach, dried peach,
quince and strawberry. Manufactured fruit preparations gener-
ally lose much of their flavor due to many causes, as cooking,
steaming, fermentative changes, presence of decayed (moldy)
fruits, mixing of several kinds of fruits or fruit juices, etc., to say
nothing of the wholly artificial or imitation fruit flavors and the
flavors of the imitation fruit products which have little or no fruit
in their composition.

SPECIAL TESTS 9

6. Methods Useful in the Examination of Vegetable Drugs,
Spices, Etc.

We shall give a few tests which have proven useful in the ex-
amination of drugs and food products. It will be found that
many of the test results are largely approximate, and some of
them are primarily intended to serve as aids or checks to the
chemical examination.

i. Mace Test. — To a pinch of the powdered mace add a few
drops of 10 per cent, sodium hydroxide solution. Banda or true
mace changes color only slightly, whereas wild or Bombay mace
turns a deep orange color.

2. Conium Test. — To the substance to be tested for the presence
of conium fruits (as anise, caraway or other umbelliferous fruits) ,
add 25 per cent, sodium or potassium hydroxide solution. In
the presence of i per cent, or more of conium fruits a distinct
mouse odor is developed in time (10 min. to }/2 hr.). This test
is not reliable with old umbelliferous fruits, as many of them de-
velop a more or less marked mouse odor with alkalies.

3. Lignin Test. — The classic phloroglucin-hydrochloric acid test
is useful in making estimates of the amount of lignified tissue
present, as in old belladonna root, aconite roots and stems,
lobelia herb, fruit products, spices, etc.

4. Grahe's Cinchona Test.— Drive the moisture from the inner
surface of a small test-tube by holding it over a Bunsen burner.
Into this dried test-tube place a pinch of finely powdered cinchona
bark (No. 80) and heat rather carefully over an alcohol lamp or
Bunsen burner. When the bark begins to char, red fumes begin
to fill the tube and condense on the side of the tube as a reddish
purplish liquid. The intensity of the reaction is approximately
proportional (direct proportion) to the percentage of alkaloids
present. Some skill and experience is necessary to perform this
test well. The tube must not be heated too quickly or too much,
and the powder should be uniformly fine.

IO MICRO-ANALYTICAL METHODS

5. Beaker Sand Test. — Pour a definite amount of the powdered
spice or vegetable drug into a beaker, add water, stir until the
sand is washed away from the vegetable particles and settles to
the bottom of the beaker. Let a stream of water run into beaker
so as to wash out the vegetable matter. The final washing and
decanting must be done carefully so as not to lose the sand. Salt
brine may be used instead of water, should the vegetable matter
have a comparatively high specific gravity. Dry the sand and
weigh to obtain the percentage of sand present.

6. Ash Determination.— According to the regulation method.
The percentage of the acid-insoluble residue should also be de-
termined. It should be borne in mind that the ash determination
gives only approximate results as far as the presence of clay and
dirt is concerned, since the organic matter of dirt is combustible.
The ash percentage varies greatly in vegetable drugs, especially
in herbs and leaves. The sand percentage is comparatively high
in those herbs and leaves having abundant trichomes, especially
if the drug plants (or herbaceous spices) bearing such trichomes are
grown in dry sandy soil. Dirt (and sand) percentage is apt to
be high in roots and rhizomes, particularly when rootlets are
abundant and when the gathering, garbling and cleaning is
carelessly done.

There are a number of chemical tests giving color reactions
which can be done conveniently by the micro-analyst, as the boric
acid reaction with curcuma, the H2SO4 color reaction with some
barks, capsicum, guaiac, resin, cubeb, etc.; the H2SO4 plus for-
maldehyde color reaction with morphine; the ferric chloride
color reaction with salicylic acid, etc. These tests should be
used when, in the judgment of the analyst, they may serve to
give better information regarding the identity, purity and quality
of the drug.

SPECIAL TESTS II

7. Methods Useful in the Examination of Vegetable Food

Products

i. Sublimation Test for Benzoic Acid. — Place a drop or two
of the suspected liquid or semi-liquid food substance into a
deep watch crystal of i in. diameter. Place over it a clean dry
slide. Now hold the watch crystal over a flame (alcohol lamp1)
until the substance (as wine, vinegar, catsup, jam, jelly, etc.),
comes to an active boil. The steam vapor, carrying with it
the benzoic acid, is condensed on the slide. Remove the slide
and set it aside until the condensed moisture has evaporated; very
moderate heat may be used to hasten evaporation. Examine
under the microscope, whereupon the benzoic acid crystals may
be seen, provided any were present. The test is delicate, very
reliable and very few substances interfere with it. It is very
pronounced in the presence of o.oi per cent, of benzoic acid.

2. Sublimation Test for Salicylic Acid.^Made like the benzoic
acid test. The crystal formation (plates) is very pronounced in
dilutions of i : iooo. After having examined the crystals under
the microscope, add a drop of weak solution of ferric chloride to
the crystals upon the slide, whereupon a blue coloration develops.
Boric acid is likewise deposited by sublimation, but the test is
not as satisfactory as those for benzoic acid and for salicylic acid.

The sublimation test may also be extended to other crystalline
substances which undergo sublimation on exposure to heat.

3. Curcuma Thread Test for Boric Acid. — Boil 5 grams of
powdered curcuma in 10 cc. of alcohol. To the evaporated alco-
holic extract add a little soda and several cc. of 50 per cent,
alcohol. In this place paper (bast fiber), cotton or linen threads
and bring to a brisk boil for a few moments. Remove threads
and dry between blotting paper, lay them in a very weak solu-
tion of sulphuric acid and rinse in water. When dry the threads
should be a golden yellow.

1 Alcohol lamp is preferable because the flame is small and yet the heating is more
quickly done.

12 MICRO-ANALYTICAL METHODS

The test for the presence of boric acid (also for borax) is made
as follows: Dip the end of a prepared thread in a 10 per cent,
solution of hydrochloric acid and allow to dry. Lay the thread on
a slide, cover with cover glass and examine. It should be of a
reddish-brown color. To the edge of cover glass apply a droplet
of a 10 to 13 per cent, solution of sodium carbonate, followed
by a droplet of the suspected solution. In the presence of boric
acid, the thread is colored blue, which coloration remains for a
longer or shorter period and then changes to gray and violet. The
test is a very delicate one and is not hindered by the presence of
sodium chloride, magnesium sulphate and aluminium sulphate.
Strong solutions of phosphoric acid, silicic acid, calcium chlorite
and magnesium chlorite, interfere with the reaction more or less.

4. Formaldehyde Test. — Concentrated hydrochloric acid
added to weak solutions of formaldehyde (1 15000) or substances
containing formaldehyde, forms stellate clusters having a some-
what crystalline appearance. The formaldehyde can be de-
posited on a slide by sublimation (as for benzoic acid) and the
acid added. The stellate clusters appear upon evaporation
of the hydrochloric acid. The test requires further verification
to determine its value.

5. Sulphurous Acid Test. — Moisten starch paper with a very
dilute solution of potassium-iodide iodine solution which colors
it blue. In the presence of the merest trace of sulphurous acid
the paper is decolorized. Do not use heat in this test.

6. Iodine Reaction. — The color reaction of starch with N/50
iodine solution is of great importance in the examination of fruit
products, such as jams, jellies, catsups, etc., as it shows whether
or not ripe or green fruits and juices of unripe fruit were used
and whether or not starch paste may have been added as a filler
or thickening agent. As is known, green fruits generally contain
more or less starch, whereas ripe fruits are quite generally free
from starch. The reaction may be observed only in the fruit
pulp cells, indicating the presence of unripe fruit, or it may be

SPECIAL TESTS 13

limited to the non-cellular portions of such substances as jams and
jellies, indicating the use of fruit juices obtained from unripe
fruits.

7. Microscopical Examination of Bacteria and Metals by
Direct Sunlight.1 — Very minute quantities of certain minerals
as iron, copper, mercury, and a few others, can be detected in
liquids and semiliquids (in the form of metallic hydroxides)
when examined (on slide mounts) by means of direct sunlight.
All transmissible light must be cut off.

Direct sunlight can also be used in making bacterial counts in
liquids, using the Thoma-Zeiss hemacytometer (Turck ruling).
The bacteria are readily recognizable on the dark background,
standing out far more clearly than in the usual examination by
transmitted light, because of the more pronounced color contrasts .

8. Micro-gluten Test. — Mount a bit of the flour in water on a
slide, being careful not to use too much water. Cover with
cover glass and move cover glass to and fro a few times on the
mounted material. The gluten separates into stringy fragments
which may readily be seen under the low power of the compound
microscope. The use of a weak solution of carbol-fuchsin,
sofranin, or other stain, will bring out the gluten particles more
clearly.

9. Hand Gluten Test. — Moisten wheat flour with water, making
it into a dough. Knead constantly and carefully under a slow

1 The optical principles of the ultra-microscope of Zsygmondy and Siedentopf
depend upon the use of direct sunlight (or other intense light) combined with an
absolutely dark field, with or without the use of a condenser, the rays of light being
directed upon the object to be examined approximately at right angles to the
optical axis of the compound microscope.

The limits of vision with the ultra-microscope are approximately 0.003^1, however,
solid particles (as of metallic colloids) of not more than 0.003^1 in diameter show no
structure, they appear rather as points of light.

The limits of vision with the ordinary microscope are, for air (white light) about
0.30/i, for homogeneous immersion (white light) about 0.25/z, and for homogeneous
immersion when rays of shorter wave length than white light (as the blue spectrum)
are used, are about 0.15/1.

14 MICRO -ANALYTICAL METHODS

stream of water, washing out all of the starch. The gluten sepa-
rates out as a tenacious gummy mass. With care fairly accurate
quantitative results may be obtained. Weigh the dried flour
and compare with weight of the dried gluten mass. With cereal
flours other than wheat, the entire dough mass is gradually washed
away, leaving no gluten.

10. Agar in Jams, Jellies and Similar Fruit Products. — The
method generally recommended is to ash a sample of the jam or
jelly at as low a temperature as possible, and to add weak hydro-
chloric acid for the purpose of decomposing the carbonates, etc.
If agar has been added to the substance the silicious skeletons
of diatoms will appear in the ash residue examined under a com-
pound microscope.

A far better method is to dissolve (with heat) about 10 grams
of the substance in 200 cc. of distilled water and centrifugalize
(while still hot) for half an hour. Decant off the supernatant
liquid and examine the residue microscopically. If agar has been
added, characteristic agar diatoms (mostly Arachnodiscus ehren-
bergii Baillon) will be found, also undissolved agar cell fragments
and remnants of undissolved parasitic algal forms, which are
quite universally found upon agar. The undissolved agar rem-
nants and the algal parasites, which are in fact almost as character-
istic as the diatoms, would be wholly destroyed by the ashing
process. Furthermore, the ashing-acid process, no matter how
carefully done, results in a comminution and destruction of some
of the diatom shells. Finding one or more diatoms and one or
more algal remnants in one slide mount (or in 5 to 20 fields of
view) is conclusive evidence that agar has been added, though
this does not indicate the exact amount that is present. If the
characteristic structures (diatoms and algal remnants) are com-
paratively abundant then it is safe to conclude that agar has been
added in considerable amount (2-4 per cent.) or that an impure
grade of agar was used. The purer the grade of agar the fewer
are the diatoms present, but no agar has yet been found on the

SPECIAL TESTS 1 5

market which is wholly free from diatoms, undissolved agar cells
and algal parasites.

The reason why distilled water should be used in making the
solution for centrifugalizing is because ordinary hydrant water
may contain diatoms, which might be confusing, especially to a
beginner, although the marine diatoms are mostly quite different
in form from the fresh water diatoms. With a high-speed centri-
fuge less material and less time need be consumed. Also, the
more complete the solution the better the results.

8. Micro-chemical Color Reaction Tests

There are certain micro-chemical color reactions, other than
those already mentioned, which are of great value in determining
the presence of impurities or adulterants in liquids and semi-
liquids. The methods as perfected by F. Emich depend upon the
use of cotton fibers treated with certain chemicals which convert
the metallic compounds into the sulphides. The prepared threads
can be readily transferred to the several solutions used and the
color and precipitation effects can be observed under the micro-
scope. The following are the more important reagents and
reactions:

1. Cotton Threads for Metal Tests. — Dip absorbent cotton threads alternately
into 15 per cent, solutions of sodium sulphide and zinc sulphate, pressing between
blotting paper, and air-dry each time.

The threads thus prepared should assume a deep black color with a 1 per cent,
solution of silver nitrate. They may be kept for a long time and are used to demon-
strate the presence of As, Sb, Au, Pt, Cu, Hg, Pb and Bi, in various chemical
compounds.

2. Ammonium Sulphide Vapor Test. — Place a few fibers of absorbent cotton into
a drop of the suspected solution and allow the moisture to evaporate. Suspending
the threads in the vapor of ammonium sulphide will indicate the presence of Cd,
Hg, Ag, Fe, Co and Ni (dark to black coloration)

The prepared threads are used in the following tests:

a. Arsenical Test. — Dip a sodium sulphide thread into the suspected solution
and allow to dry. In the presence of 0.008 per cent, arsenic there is a distinct yel-
lowish coloration, due to the sulphide of arsenic formed in and upon the threads.
The arsenical threads will also show the characteristic reactions with hydrochloric

1 6 MICRO-ANALYTICAL METHODS

acid, ammonia and ammonium sulphide by bringing a drop of the reagent in contact
with the thread upon the slide. (See also Biological Test for Arsenic in Part II.)

b. Zinc Test. — Dip cotton fibers into the suspected solution, allow the moisture
to evaporate, and then dip the threads into a solution of gold chloride. A violet
coloration develops which remains in the presence of acids but vanishes in the presence
of chlorine water, indicating the presence of zinc chlorite. The reaction is appreciable
in the presence of 0.003 ng of zinc chlorite, whereas in the form of the sulphite,
0.1 fig of zinc is required to show the reaction.

c. Antimony Test. — Dip a sulphide thread into the solution, allow solution to
evaporate and then expose the thread to the vapor of ammonium sulphide. If
the solution to be tested contains considerable hydrochloric acid, sulphide of anti-
mony is formed upon evaporation.

d. Gold Test. — Gives a brown coloration with the sulphide thread, which color
disappears upon prolonged exposure to ammonium sulphide, more quickly on ex-
posure to chlorine, bromine and sodium hypochlorite. The threads which have
been decolorized with chlorine are colored blue to black with iron chlorite and violet
to red with zinc chlorite.

e. Silver Test. — A neutral or faintly acid silver nitrate solution gives a brown to
black coloration with the sulphide thread, the depth of the reaction depending upon
the concentration of the solution. The fibers can be decolorized by placing in sodium
hypochlorite, and the color can be restored by means of zinc chlorite or an alkaline
solution of grape sugar. Sulphuric acid will again decolorize.

/. Mercuric Chloride. — Cotton threads dipped into a solution containing mer-
curic chloride and exposed to the vapors of ammonium sulphide or ammonia, are
colored black. The color is quite permanent in the presence of acids. A sulphide
thread is colored yellow in neutral solution of mercuric chloride, changing to black
in the ammonium sulphide vapor.

g. Lead Test. — Neutral lead solutions (lead nitrate) turn the sulphide threads
yellow and black on prolonged exposure to ammonium sulphide. In acid solutions
the color reaction with the sulphide thread is black. The yellow coloration is
promptly changed to black upon exposure to ammonium sulphide, or when placed
in weak sulphuric acid (1 : 15). The latter reaction distinguishes between lead and
mercury, as the yellow coloration of the mercury is changed very slowly with dilute
sulphuric acid.

h. Bismuth Test. — Solutions color the sulphide thread reddish-brown. Bromine
causes the color to disappear. Potassium dichromate causes a yellow coloration,
while alkaline solutions of zinc chlorite produce a black coloration. Lead solutions
are not reduced by alkaline solutions of zinc chlorite.

i. Iron Test. — Ammonium sulphide vapor gives a black precipitate which is
soluble in weak solutions of hydrochloric acid. Potassium ferrocyanide gives a
blue coloration.

j. Copper Test. — Solutions of copper sulphate give a brown coloration to the
sulphide thread, which color persists in 10 per cent, hydrochloric acid, but disappears
on exposure to bromine vapor. The threads which have been bleached with bro-

SPECIAL TESTS 1 7

mine give the copper ferrocyanide reaction when placed in an acidulated solution of
potassium ferrocyanide.

The following table from the work by Koenig gives the relative
sensitiveness of the tests above described:1

^W^S&i, WMn+i„., Limit Comparative

valency Reaction (mg_ x iq6) j sensitiveness

Bo'" Curcuma thread

As'" Sulphide thread

Sb'" Sulphide thread

Sn" Violet color with sulphide thread

Au'" Sulphide thread — brown, purple. .

Pt"" Sulphide thread

Cu" Sulphide thread + ferrocyanides.

Ag' Sulphide thread + Ag

Hg' NH3 vapor

Hg" Sulphide thread

Pb" Sulphide + PbCr04

Bi'" Sulphide + chromate + Bi

Cd" (NH3SH) vapor

Fe" (NH3SH) — blue

Co" NH3SH or nitroso — beta — naph-

thol

Xi" NH3SH

9. Making Analytical Reports

The methods of micro-analysts, whether in private, commercial
or government laboratories, should be uniform. Much could be

1 The comparative degree of sensitiveness of the different chemical compounds
concerned in the color reactions above described and tabulated is indicated by the
number of cubic centimeters in which 1 gram of the substance in solution is
still appreciable. The actual limit, determined experimentally, is indicated in
terms of milligrams, that is 0.00 1 mg., represented by jug. Expressing the com-
parative sensitiveness (CS) in a formula we have

_ jug limit molecular weights

amount limit combination valency
or to give the example for boron, we have

0.00001 59

CS = - , X = 33,ooo.

0.00000006 3

0. 1

1

in

33,000

10. 0

1

in

2,500

1 .0

1

in

40,000

3-o

1

in

20,000

3-o

1

in

22,000

8.0

1

in

6,000

8.0

1

in

4,000

5.o

1

m

22,000

8.0

1

in

25,000

5-o

1

in

20,000

8.0

1

in

13,000

8.0

1

in

9,000

6.0

1

in

9,000

8.0

1

in

3,5oo

0.3

1

in

100,000

0.3

1

in

100,000

1 8 MICRO-ANALYTICAL METHODS

done to bring this about if the analysts were to meet for the
purpose of comparing methods and results. Uniform blank re-
port forms should be adopted and used in the micro-analytical
laboratories, somewhat like those used by chemists. It cannot,
however, be denied that the efficiency in the work done depends
largely upon the ability, judgment and experience of the analyst.
The reports of the micro-analysts may be made according
to the following groups :

I. Drugs and foods of vegetable origin, including dry or solid products of both
animal and vegetable origin.

II. Liquid or moist products of animal and vegetable origin (canned and pre-
served products generally).

III. Bacterial examinations of liquids, foods and drugs.

There should be a special blank report card for each group of
substances, arranged as follows:

Form No. I

No (I. S., laboratory or other serial number).

Label

Sample received Sample examined.

Condition of wrappings and seals

Organoleptic tests

Consistency of feel

Color

Odor

Taste

Adjunct tests

Sand (beaker test) Per cent.

Ash Per cent.

Acid-insoluble ash Per cent.

Special tests

Microscopical findings.

Conclusions.

Analyst.

ANALYTICAL REPORTS 1 9

Form No. II

(No., label, dates, condition of seal and organoleptic tests, as for form I.)
Adjunct tests.

Sublimation tests for

Benzoic acid

Salicylic acid

Boric acid (curcuma thread)

Iodine reaction

Intracellular

Extracellular

Special tests

Microscopical findings.

General

Cytometric counts.

Dead yeast cells per cc.

Living yeast cells per cc.

Bacteria per cc.

Mold (hyphal fragments and hyphal clusters) . . . per cc.

Mold spores per cc.

Conclusions

Analyst.

20 MICEO-ANALYTICAL METHODS

Form No. Ill
Bacteriological Examination

(No., label, dates, condition of seals as for form I.)

i. Direct count. (Thoma-Zeiss hemacytometer with Turck ruling.)

i. Bacilli per cc

2. Cocci per cc

II. Plate and tube cultures. (Lactose-litmus-agar.)
i. Temperature differential test.

a. (200 C.) colonies per cc

b. (380 C.) colonies per cc

2. Color differential test.

a. Pink or yellow colonies per cc

b. Not pink or yellow colonies per cc

3. Gelatin liquefying colonies per cc

4 . Indol reaction ( ± )

5. Neutral red reduction ( + )

6. Gas (hydrogen) formula

7. Gram stain behavior ( + )

8. Presumptive colon bacillus test ( + ).

a. Amounts used

b. Number of tests

c. Rating

III. Special tests

IV. Conclusions.

Analyst.

ANALYTICAL REPORTS 21

We may give an example of a report as follows:

Form No. II

Lab. No. 462.

Label: Pure currant jelly. Madeby Smith, Jones & Co., Nan-
tucket, Wis.

Sample received August 5, 1914. Sample examined August 5,

1914.
Condition of seals: Good, unbroken sample.
Organoleptic tests: Not conclusive.

Consistency or feel: Poorly jellied.
Color: Normal for eur rant jelly.
Odor: Faint, somewhat disagreeable.
Taste: Not characteristic, bitterish, quite acid.
Adjunct tests.
Sublimation tests for

Benzoic acid: Negative.
Salicylic acid: Very marked.
Boric acid (curcuma thread): Negative.
Iodine reaction: Very marked.
Intracellular : Negative.
Extracellular: Positive, very marked.
Special tests: Salicylic acid color reaction, with ferric chloride,

very marked.
Microscopical examination.

General. Some apple tissue (window cells and pulp cells) and
currant tissue (selerenchyma) present. Added wheal
starch about 5 per cent.
Cytometric counts.

Dead yeast cells, 80,000,000 per cc.

Living yeast cells, none per cc.

Bacteria, 600,000,000 per cc.

Mold (hyphal fragments and clusters), 84,000 per cc.

Mold spores, 5,000,000 per cc.

Smut spores, none per cc.

Conclusions: Misbranded. Adulterated with apple and with wheat
starch and made from fermented and decomposed ma-
terial, preserved with salicylic acid. Not fit for human con-
sumption because of the quantity of yeast, mold and bacteria
present.

John Doe, Analyst.

2 2 MICRO-ANALYTICAL METHODS

The great advantage of the micro-analytical work as compared
with chemical work lies in the fact that small amounts of the
substances are used for analysis, the equipment is comparatively
inexpensive and the results are quickly attained. From twenty
to forty and even sixty samples of simple spices can be examined
in one day, from rive to twelve samples of powdered vegetable
drugs, cocoas, chocolates, flours, meals, etc., and perhaps an
equal number of jams, jellies, etc.

Because of the very close relationship between the micro-
scopical and bacteriological work, as already explained, certain
essentially micro-analytical methods will be given under bac-
teriological methods, more especially in Chapter 2 of Part II
which deals with the direct bacterial counts, and also under milk
analysis, water analysis and meat analysis.

DESCRIPTION OF PLATE I

Fig. i. — Types of Pollen Grains. — i. Saffron flower. 2. Flax. 3. Pink. 4.
Pumpkin and squash. 5. Cloves. Mature pollen grain. 6. Cloves. Immature
pollen grain. 7. Onagraceae. Circea lutitiana (Enchanter's Nightshade). 8.
Scutellaria. 9. Mallow. Distended by moisture. 10. Mallow. Normal form.
11. Albuco. 12. Lobelia inflata. 13. Compositae, showing one mature and two
immature pollen grains. 14. Hibiscus. 15. Pine pollen. 16. Santonica. 17.
Mentha species. 18. Hyoscyamus niger.

Fig. 2. — Potato Starch. — The granules are large and the markings (hili, lamel-
lations) are distinct. The cross bands under the polarizer are very distinct. Potato
starch, mounted in water, makes a good test object for judging the resolving power
of objectives. Dried and ground potatoes and potato parings are sometimes used
for adulterating purposes.

Fig. 3. — Starches. — 1. Sago starch from Cycas revoluta (Cycadaceae). The
commercial article known as sago is usually in the form of small granules (pearl
sago). There are many false sagos made from other than Cycad or Palm starch.
Much of this false sago is made from corn starch.

2. Canna starch from several species of Canna. The markings are very distinct,
the hili being at the larger end as a rule. Also called arrowroot (tous le mois arrow-
root).

3. Cassava or tapioca starch from the tuberous roots of Manihot utillissima and
other species of Manihot. Simple and compound granules; the granules are largely
separated in the processing, thus giving the appearance of simple granules. Their
compound origin is, however, recognizable by the contact facets.

4. Maranta starch (Arrowroot starch) from Maranta arundinacecE (Marantaceae).
The granules have many of the structural characteristics of potato starch.

5. Yam starch from several species of Dioscorea (Dioscoreaceae).

Fig. 4. — Dextrinized Starch. — The process of baking and cooking causes the
starch granules to undergo marked structural changes. They become much
enlarged, the outline becomes quite indistinct and the hili and lamellations are
distorted and correspondingly indistinct. 1. Normal wheat starch granules. 2.
Normal rye starch granules. 3. Dextrinized wheat and rye granules. 4. Normal
and dextrinized corn starch. 5. Normal and dextrinized bean starch. 6. Normal
and dextrinized ginger starch.

Plate I

Fig. i.

Fig. 2.

1 IP
O

o

o

go®

Ml &

£> 02

2 W %

(S

5,0 q

©O1

Ml3

OA W^f

vy

VS% I

Fig. 3.

Fig. 4.

DESCRIPTION OF PLATE II

Fig. 5. — Types of Crystals of Calcium Occurring in Different Plants. — 1. A
parenchyma cell containing a bundle of needle shaped (acicular) crystals of calcium
oxalate (raphide). 2, 3, 4, Acicular crystals differing in length, as they occur in
Scilla and in other representatives of the liliaceous groups of plants. 5. Much
elongated prismatic crystals as they occur in Quillaja and in Iris jlorentina. 6.
Prismatic crystals very widely distributed in the plant kingdom. 7. Elongated
prismatic crystals. 8. Twin crystals as they occur in Ulmus bark. 9. Very large
aggregate crystals as they occur in Rheum and Polygonum species. 10, 11. Smaller
aggregate crystals very widely distributed in the vegetable kingdom. 12, 13. Very
minute prismatic (pyramidal) crystals as they occur in Belladonna. 14. Prismatic
crystals as they occur in Hyoscyamus and in other plant groups.

Calcium oxalate crystals are among the highly diagnostic structural characteris-
tics of drug plants and should be studied not only as to form but also as to size.
They are not dissolved in the usual mounting media and are not destroyed by heat.
They dissolve slowly in the stronger acids (hydrochloric acid).

Fig. 6. — Types of Bast Cells as They Occur in Barks and in Other Plant
Parts. — 1. Shorter bast cell as they occur in the cinnamon barks. 2. Typical
bast cell (showing a portion of a cell only) as they occur in willow bark, in Ulmus, in
Mezereon, etc. 3. Branching bast cells as they occur in Quillaja and in Prunus
bark. 4. Greatly thickened sclerenchymatous bast cells as they occur in the
Cinchonas.

Fig. 7. — Types of Sclerenchyma (Stone) Cells. — 1. Typical sclerenchyma cells
as they occur in the endocarp of drupaceous fruits and nuts. 2. Elongated bast-
like sclerenchyma cells. 3. Thin- walled typical sclerenchyma cell. 4. Scleren-
chyma cell with unequally thickened walls as they occur in the cinnamons. 5.
Large thin-walled sclerenchyma cells as they occur in the seed coat of Amygdala.

6. Branching sclerenchyma cells as they occur in tea leaves and in peanut exocarp.

7, 8, 9. Forms of sclerenchyma cells.

Fig. 8. — Typical Sclerenchyma Cells (in groups) as they occur in the pulp of
the pear.

Plate II

Ftg. 5.

Fig. 6.

Fig. 7.

Fig. 8.

DESCRIPTION OF PLATE III

Fig. 9. — Buckwheat. — 1. Proteid-bearing tissue. 2. Starch-bearing endosperm
tissue. Cell walls are very thin and the entire cell lumen is packed with starch
granules. 3. Starch granules. The granules resemble those of corn, being some-
what smaller. 4. Sclerenchymatous fibers.

Buckwheat is the predominating ingredient of the buckwheat pancake flours
and is occasionally used as an adulterant of spices.

Fig. 10. — Tissues of the Pine. — 1. The characteristic tracheids with bordered
pits. 2. Bast-like fibers of the bark. 3. Crystal-bearing bark parenchyma cells.
4. Tracheids in radial view. 5. Medullary ray cells in radial view. Pine wood
(pulp) is much used in making paper.

Fig. ii.- — Sclerenchyma Cells of Olive Pits. — Ground olive pits were, until
recently, extensively employed as an adulterant of spices and drugs.

Fig. 12. — Clove Stems. — A very common adulterant of cloves and of allspice.
1. Typical sclerenchyma cells. 2. Sclerenchyma cells with unequally thickened
walls. 3. Sclerenchymatous bast fibers.

Plate III

Fig. 9.

Fig. 10.

DESCRIPTION OF PLATE IV

Fig. 13. — Cassia Buds and Cassia Stems. — 1. Sclerenchymatous fibers of the
cassia stems. 2. Bast fibers of cassia stems. Parenchymatous cells of the buds. 4.
Trichomes of buds. 5. Thick- walled parenchyma cells. Cassia buds and cassia
stems are frequently used in adulterating cloves, allspice and cinnamon.

Fig. 14. — Coffee Adulterants. — 1. Sclerenchyma cells of date pits. 2. Scleren-
chyma cells of the walnut shell. 3, 4, 5. Tracheids and inulin-bearing parenchyma
cells of chicory. Figs and prunes are also much used as coffee adulterants, also
cereals, fleshy roots, acorns, etc.

Fig. 15. — Wheat Tissues. — 1. Wheat starch. 2. Trichomes from the bran.
3. Starch-bearing parenchyma. 4. Epicarp cells. 5. Proteid-bearing cells from
middlings. Rye histology is similar to that of wheat. Wheat flour is used in
macaroni, spaghetti, noodles, etc. Wheat flour, bran and middlings are much
used for adulterating purposes. Rye starch differs from that of wheat in the larger
size of the granules and the greater prominence of the hili.

Fig. 16. — Rice Tissues. — 1. Starch. Single granules and aggregates. These
aggregates are characteristic of rice and of oats. 2. Starch-bearing endosperm
cells. 3, 4, 5. Epicarp and pericarp cells. In form the starch granules of rice, oat,
corn, darnel, millet, fox-tail, buckwheat and chess resemble each other. The size
varies very much.

Plate IV

Fig. 13.

Fig. 14.

°°00

0°S „^

Fig. 16.

DESCRIPTION OF PLATE V

Fig. 17. — Bean Tissues. — 1. Epidermal palisade tissue with the crystal-bearing
hypoderm. 2. Starch-bearing endosperm tissue. 3. Starch granules with promi-
nent fissured hili. 4. Spongy tissue. 5. Epidermal palisade cells in vertical view.
6. Prismatic crystals of calcium from hypoderm.

Ground beans, peas and lentils are used for adulterating purposes.

Fig. 18. — Histology of Mallow Leaf. — 1. Transverse section of leaf showing
stellate trichome, epidermal, palisade and spongy tissue cells. Aggregate crystals
of calcium oxalate are present. 2. Stellate or aggregate trichomes. 3. Epidermal
cells (lower) showing stomata. Mallow leaves are extensively employed for adul-
terating leafy spices and drugs.

Fig. 19. — Histology of Corn. — 1. Corn starch. 2. Starch-bearing endosperm
of corn kernel. 3. Trichomes of the chaff of the corn cob. 4. Sclerenchymatous
cells of the corn cob. Ground corn cobs are used for adulterating purposes and
also in the manufacture of artificial maple syrup flavor.

Fig. 20. — A Few Types of Trichomes. — 1. Branching trichome of mullein. 2.
Many-celled simple trichome of henbane showing wart-like marking on outer surface.
3. Simple single-celled trichome as of rye and wheat. 4. Glandular trichome with
two secreting cells. 5. Glandular trichome with one secreting cell. 6. Many-
celled glandular trichome. 7. Simple, single-celled trichome of Indian hemp. 8.
Much elongated and twisted single-celled trichome, as of sage. 9. Sessile glandular
trichome (Eriodictyon). 10. Indian hemp. n. Pyrethrum. 12. Simple trichome.

Plate V

Fig. 19.

Fig. 20.

DESCRIPTION OF PLATE VI

Illustrating the Histology of a Typical Bark Showing all of the Tissues
Which May be Found in a Bark. — A, Longitudinal section in the radial direc-
tion but not showing the medullary rays. B, Transverse section, i. Outer
bark. The demarkation between outer and inner bark is not always distinct. 2.
Inner bark. 3. Beginning of wood tissue, a, Epidermis. Always wanting in tree
trunks and older branches, b, Cork tissue, c, Bark parenchyma. Cell-walls are
usually not suberized and the cells may contain various inclusions such as crystals
of calcium oxalate, tannin, starch granules and resin, d, Groups of sclerenchyma
cells. These, when present, normally predominate in the outer bark, e, Crystal-
bearing fibers which usually accompany the bast fibers. /, Bast fibers. These,
when present, normally predominate in the inner bark. The fibers may occur singly
or in groups, g. Cambium, h, Wood fibers, i, Ducts. Usually of the typically
porous type, k, Medullary rays.

An excellent typical bark having all of the histological elements indicated in
Plate VI is Rhamnus purshiana. The demarkation between outer bark and inner
bark is well defined in Ulmus and Quillaja.

Plate VI

SlIIIIlEerail''

^i '■^'•■:<i:'{tii&N:ti K

Fig. 21

BACTERIOLOGICAL METHODS IN FOOD AND
DRUGS LABORATORIES

i. Introduction

The study of the significance of bacteria in foods of all kinds is
one of the most important and interesting of scientific subjects
and one which has received much attention ever since the science
of bacteriology has become more highly developed as the result
of the perfection of the compound microscope. For a long
period of time the popular notion has prevailed that bacteria were
essentially harmful and to designate any substance as bacterially
contaminated was to pronounce it dangerous and to condemn it
without trial. We now know that many, in fact most bacteria,
are beneficent rather than harmful, and that many different
species of bacteria are concerned in the preparation of food
substances. It cannot be denied, however, that many kinds of
bacteria as well as other organisms are concerned in the pro-
duction of changes in food substances which we know to be highly
detrimental to the well being of the human race. It is the duty
of modern sanitary science to guard against disease and the
contamination of food substances through the invasion of patho-
genic and otherwise objectionable organisms. It is the work of
the food bacteriologist to detect objectionable contaminations in
foods and to aid in developing those processes and methods of
food preparation and manufacture which will prevent the re-
currence of such contamination. The food bacteriologist will
center his attention on the following:

23

24 BACTERIOLOGICAL METHODS

i. Chemical (decomposition) changes in foods and drugs induced by the various
organic infecting agents, as bacteria and other living organisms, which render such
substances unfit for human use or which render them dangerous for human use.

2. Foods and drugs as actual or possible carriers of infecting agents which are
or may be dangerous to life or which may or might be injurious to the physical
well-being of the human species.

It goes without saying that the food bacteriologist must pro-
ceed carefully in order that there may be no hasty decisions re-
sulting in the condemnation of food products which are not in-
jurious. There is, however, little excuse for hasty or unjustifi-
able passing of judgments as regards the quality of food. Bac-
teriological and toxicological methods have been sufficiently
perfected so that the careful analyst need not make unfair or
unwarranted decisions. The men entrusted with the critical
examination of foods and drugs as to their fitness for human
use should be investigators of authority and should have had
wide range of practical as well as laboratory experience, and they
should furthermore be possessed of good judgment. While
the condemnation of food materials should not be hasty it should
on the other hand not be too tardy or conservative. The prime
object of the work by the food bacteriologist is to protect the
consumer, not the dealer or manufacturer. This very important
point is most unfortunately not properly heeded with the result
that some of the work done in the administrative laboratories is,
or appears to be, in the interests of the dealer or manufacturer.

A goodly number of infections enter the human system by way
of the mouth with the ingested foods and drinks. Food substances
form excellent pabula for the bacteria and other parasitic agents
which enter the digestive tract or which may already have entered.
Foods and drinks are exposed to infection in a great variety of
ways. For purposes of illustration we may cite bread, the so-
called "staff of life," as one of the foods which is liable to infec-
tion. It may be assumed that the loaf of bread, when it is taken
from the oven, is entirely sterile and free from living organisms
of all kinds. Just as soon as the loaf is cool enough to permit it,

INTRODUCTION 25

the promiscuous manipulation begins and is continued until the
bread enters the digestive tract of the consumers. The loaves
are handled by the dirty, sweaty and oftentimes diseased hands
of the baker or his helper. Basketfuls of uncovered bread are
dragged over the dirty floors, over sidewalks, and through the
filthy alleys. The uncovered loaves are repeatedly handled by
the bakery drivers whose hands and clothing are generally very
filthy. The uncovered loaves are left on doorsteps and other
exposed places on the premises of the consumer. This much-
handled bread is finally eaten, crust and all, without any attempt
at sterilization. Such bread may be contaminated with a great
variety of disease germs. Infections from hands, disease-bearing
dust from the streets and alleys, excreta from disease-carrying
flies, excreta from the intestinal tract of man and of animals
are among the deposits which have been found on the exterior of
bread. Miss Katherine Howell has traced an epidemic of typhoid
fever to the consumption of contaminated bread and she has
demonstrated the presence of typhoid fever germs and of in-
testinal bacteria on numerous loaves of bread. Edward Bartow,
director of the Illinois State Water Survey, has also demonstrated
a bread-borne typhoid epidemic in Rockford, Illinois. Colon
bacilli are usually found in considerable numbers on every loaf
of unwrapped bread. Every loaf of bread from the public bakeries
should be wrapped in sterilized paper bags just as soon as it leaves
the oven and it should remain in these bags until ready to be
placed before the consumer.

Polluted water may carry the germs of dysentery, of cholera,
of typhoid fever and the larvae of intestinal and other parasites.
Clams and oysters have caused typhoid epidemics. Fruits and
vegetables are frequently polluted with fertilizer, especially where
human fertilizer is used, as is the custom with the Chinese truck
gardeners not only in China but also in other lands where the
Chinese are found. Using human excrement as a fertilizer of
soil should be prohibited by law. American army surgeons at

26 BACTERIOLOGICAL METHODS

the time of the American occupation of Cuba made the filthy
farming customs of the Chinese the object of a special report but
apparently nothing ever came of the recommendations made.
The Chinese also import dried human feces and dried human
urine for medicinal purposes and a recommendation was made
to Washington to prohibit such importations but apparently
nothing has ever been done about it.

Pollution of fruits and berries of all kinds may come from
the hands of pickers. Gathering of fruit is usually done by the
very ignorant, those who have no proper conception of personal
cleanliness and of sanitation. Entire families, men, women, and
children, migrate to the fields and work during the hottest part of
the season. They live in the open or in tents or perhaps in covered
wagons. The environment of these temporary abiding places
is anything but sanitary. Sickness often prevails in these camps,
such as typhoid fever, scarlet fever, measles and dysentery, to say
nothing of the more common body and intestinal parasites which
infest many of the laborers. These multitudinous infections are
brought in contact with the fruit, berries, peas, beans, lettuce,
cabbage, cucumbers, etc., etc. The products of the field are then
carried to the consumer by a driver who disseminates the contami-
nation by mixing and frequent handling. And in spite of all this
there are those who insist on eating berries unwashed because
they might lose some of the natural flavor.

Next to bread, milk is the most popular food substance. Most
unfortunately milk is also one of the best food substances for
all manner of germs, harmful and harmless. Sickness in those
employed about the dairying establishment has time and again
caused epidemics, such as diphtheria, typhoid fever, scarlet fever,
tuberculosis, dysentery, and streptococcic tonsillitis. Diseased
animals transmit infection to humans, as will be more fully ex-
plained in the chapters following.

It is generally believed that the usual processes of baking and
cooking as practised in the household are a sure guarantee that

INTRODUCTION 27

the foods so prepared are entirely free from living bacterial infection
of all kinds. This is true of some foods but not by any means of
all of them. Dr. W. A. Sawyer, Director of the California State
Hygienic Laboratory, in reporting upon an epidemic of 93 cases of
typhoid fever (at Hanford, California) due to a single carrier,
traced the source of the infection to cooked Spanish spaghetti,
prepared by the typhoid carrier. The following tests were made
in the California State Hygienic Laboratory to ascertain the effects
of baking on the presence of typhoid fever germs in the interior of a
mass of spaghetti.

"A large hot-air sterilizer was heated and kept between 1600
and 1700 C. (3200 and 3380 F.). The pan of spaghetti was in-
troduced and subjected to this heat for 30 min. When the dish
was removed the surface was of a golden brown color. The ap-
pearance and aroma suggested that the spaghetti was thoroughly
cooked and very hot. The temperature near the top was 54° C.
(129. 20 F.) and at the middle, 230 C. (73.4° F.). Ten minutes
later the temperature at the middle was 240 C. (75.20 F.) and the
dish was then returned to the oven. Cultures taken at various
levels showed that the typhoid bacilli were alive even close to the
surface.

"In the next baking the oven was kept at temperatures ranging
between 2070 and 2140 C. (4050 to 41 70 F.). After half an hour
the pan was removed. The surface was dark brown and the
points sticking up from it were charred. The liquid around the
margin was boiling vigorously and the whole dish was sizzling.
The temperature just under the surface was 830 C. (181. 40 F.).
At the middle it was 280 C. (82. 40 F.) and near the bottom it
was 480 C. (118.40 F.). An hour later the temperatures had be-
come nearly equalized and were 460 C. (114. 8° F.) near the top,
42. 50 C. (108. 50 F.) at the middle, and 430 C. (109.40 F.) near the
bottom. This showed that the interior of the dish did not reach
even a pasteurizing temperature.

"Cultures taken at the surface soon after the pan had been

28 BACTERIOLOGICAL METHODS

removed from the oven showed no typhoid colonies and very few
of other kinds. Cultures taken at a distance of half an inch from
the surface showed a few colonies of the typhoid bacillus, most of
the organisms having been killed. Cultures from a depth of
2% in. showed abundant colonies of typhoid bacilli. In these
cultures the typhoid colonies were identified by their appearance
on Endo medium and Russell medium and also by agglutination
by an ti- typhoid serum."

Dr. Sawyer sums up the experimental evidence as follows:

"The laboratory experiments completed the explanation of the
Hanford outbreak by showing that the sauce used in making the
Spanish spaghetti was a good culture medium and that the dish
had not been sterilized after leaving the house of the typhoid
carrier.

" Moreover, it was demonstrated that cooked dishes must
be considered as possible conveyors of infection unless it can be
shown that the method of cooking would produce complete
sterilization. The slowness with which heat penetrates dishes
like the Spanish spaghetti shows that very prolonged heating
would be necessary for sterilization of large dishes of such food.
Ordinary baking merely incubates the interior of these masses
of food."

This report by Dr. Sawyer is of special significance to the
food bacteriologist as it illustrates two very important factors
concerned in the study of food sanitation: First, the possible
contamination of food materials through carriers of disease,
and secondly, the necessity of studying more carefully our pres-
ent methods of sterilization (of food materials) through the
agency of heat. As will be more fully set forth in subsequent
chapters, the examination of canned foodstuffs shows that sterili-
zation is far from complete in the great majority of cases.

In addition to the more or less acute infections traceable to
the consumption of contaminated food products, there are the
multitudinous infections which are of slow development or

INTRODUCTION 29

chronic in character. In many of these cases it is not possible
to acertain definitely how the infection entered the system.
There are numerous so-called autointoxications which are said
to be due to autolytic changes in the ingested food substances
resulting in the formation of toxins which often give rise to very
serious and even fatal poisoning. As is generally known, certain
toxin-forming bacteria after once gaining access to the intestinal
tract may remain there for years feeding upon the contents of
the intestines and producing enough of the toxin to give rise to
symptoms of poisoning of a more or less chronic character. In
some instances the toxin-forming bacteria are not present in suf-
ficient numbers or do not multiply in sufficient numbers to give
rise to any marked symptoms, and in still other cases the originally
pathogenic or toxin-forming bacteria lose their virulency after
having lived in the intestinal tract for some time. As is known,
there is constant warfare in the intestinal tract between the harm-
ful and the really beneficent bacteria, and it is this discovery which
has led Metschnikoff and other bacteriologists to find germs
which upon being introduced into the intestinal tract would
overcome or crowd out the objectionable toxin formers.

Food poisoning has received considerable attention in re-
cent years. Vaughan and Novy have suggested a nomenclature
applicable to certain recognizable forms of poisonings traceable
to foods, as:

Bromatotoxismus or food poisoning.
Galactotoxismus or milk poisoning.
Tyrotoxismus or cheese poisoning.
Kreatoxismus or meat poisoning.
Ichthyotoxismus or fish poisoning.
Mytilotoxismus or mussel poisoning.
Sitotoxismus or cereal poisoning.

The poisonings mentioned are generally due to toxins or
related products elaborated by bacteria, but in some instances the
exact species responsible for such toxin formation have not yet
been determined. The identification of the species of bacteria

30 BACTERIOLOGICAL METHODS

responsible for the poisoning of foods and drinks is of minor im-
portance. What is of prime importance to the food bacteriologist
is to find the poison and if possible to ascertain the manner in
which the poison gained access to the food substance, in order that
methods may be devised to guard against the recurrence of such
contamination. It may also be stated that in the great majority
of cases of food poisoning the nature of the poison and its source
have already been determined and means are available to pro-
tect the consumer. If the manufacturers of foods and of food
products would give proper attention to the modern methods of
manufacture, then poisonings due to the eating of such prod-
ucts will be a rare occurrence indeed. It is regrettable that so
many of the smaller establishments engaged in the manufacture
of food products are not better informed regarding the available
modern methods of preparing and storing food substances in such
a manner as to guard against infection and contamination. It
is also regrettable that the various pure food and drugs laws and
regulations intended to protect the consumer are not more effi-
ciently and more strictly enforced.

We have already suggested a more efficient coordination of
the chemical, microscopical and bacteriological methods of analy-
sis in our food and drugs laboratories — federal, state, munici-
pal and private. The following are the bacteriological methods
applicable in the examination of foods and drugs as to quality
and purity. It is hoped that the suggestions offered may serve
as a basis for establishing more complete practical working
methods and at the same time indicate lines for further research.

Just what bacteriological analyses and tests should be made
in pure food and drugs laboratories has as yet not been decided
upon. However, based upon the present purpose and scope of
such laboratories, we submit the following outline as covering
the field fairly well and which outline will be followed quite closely
in the text, however not necessarily adhering to the same sequence
of the subject-matter.

introduction 3 1

Quantitative and Qualitative Determinations of Organisms
in Foods and Drugs

Substances to be analyzed.
Liquids of all kinds.

Semiliquids and semisolids miscible with water.
Solids of all kinds.
Numerical and quantitative limits of contamination in different
substances.
For molds — quantity of spores and hyphae.
For yeasts — number and kind.
For bacteria — number and kind.
For pus, dirt, sand, etc.
Methods.

Making concentrations.

Making dilutions.

Making the counts and estimates.

Bacteria.

Yeasts.

Mold spores and mold hyphae.

Algae, in drinking waters, etc.

Protozoa.

Pus cells, in milk, etc.

Dirt, sand, etc.
Plate counts — Petri dish cultures.

Culture media used.

Optimum temperature.

Time of incubation.
Qualitative determinations.
Apparatus.
Culture media.
Stains.
Special methods.

Colon group of bacilli.

Presumptive colon bacillus test.

Sewage streptococci.

Dysentery bacilli and amebae.

Bacillus typhosus.

Paratyphoid group.

Cholera vibrio.

Yeasts.

Molds.

Animal parasites.

Larvae, ovae, etc.

2,2 BACTERIOLOGICAL METHODS

Biological water analysis.

Bacteria, number and kind.

Diatoms.

Desmids.

Nostoc.

Other algas.

Molds; significance of.
Bacteriological milk analysis.

Quantitative.

Standards for different geographic areas.

Summer and winter standards — temperature standards.

Qualitative.

Pus and blood corpuscles; significance of.

Milk diseases.
Blue milk.
Ropy milk.
Bad odors, bad taste, etc.

Sour milk.

"Buttermilk" tablets.

Kefir, koumys, etc.
Bacteriological examination of shellfish.
The bacteriological and toxicological examination of meat

and meat products.
The bacteriological examination of eggs and of egg products.
Bacteriological examination of mineral waters.
Bacteriological examination of pharmaceuticals.
Bacteriological examination of sera, vaccines, bacterins, etc.
The microscopical and bacteriological examination of syrups.
Standardization of disinfectants.

Phenol coefficient.

Albumen coagulation coefficient.

Toxic coefficient.

The efficiency value of disinfectants.
Biological toxicity tests.

Upon first consideration it would appear that the bacteriolog-
ical methods in food and drugs laboratories might be closely simi-
lar to those in hygienic laboratories. Such is the case in a gen-
eral way, however, with certain well-defined differences. Whereas
the bacteriological work in hygienic laboratories pertains to the
prevention of disease and finding the primary causes of disease f
the work in the food and drugs laboratories has to do with the

INTRODUCTION 33

investigation of the biological factors influencing the quality of
food and drugs and the significance of pure food and drugs in
the maintenance of the public health and the physical well-being
of the human race, as against the pernicious effects of contami-
nated foods and drugs.

The question for first consideration is what bacteriological
methods are necessary and practicably applicable in testing
foods and drugs? This phase of the subject is comparatively
new and accordingly there are but few food and drugs bacteri-
ologists who have had any considerable general range of ex-
perience, and as a consequence there are comparatively few
methods fully worked out. Most of the bacteriological in-
vestigations and researches pertaining to foods have been along
special lines, and indeed much valuable information and useful
data have been brought together. Within the last 10 years the
work on the sanitary examination of milk and of water supplies
has become monumental in volume as well as in importance.
Numerous methods have been tried, some to be entirely abandoned
after being for a time heralded as the final word in determining
the potability of water supplies. The same may be said of the
development of the bacteriological examinations of milk supplies.

Quite recently bacteriologists have given considerable atten-
tion to the sanitary examination of shellfish, more especially
with reference to sewage contamination. In this investigation
American bacteriologists have taken the lead. European bac-
teriologists have done an enormous amount of work in the ex-
amination of sewage and of sewage disposal, to say nothing of
the classical researches on yeasts and on fermentation in general.
However, the general bacteriology of foods and of drugs is as yet
an unexploited field. It is true, the Bureau of Chemistry of
the Department of Agriculture has, within recent years (since
1906), done considerable work on the sewage contamination of
oysters and other shellfish (Bulletin No. 136, Bureau of Chemistry.
U. S. Department of Agriculture, by George W. Stiles) and in

34 BACTERIOLOGICAL METHODS

the quantitative estimation of the microbic contamination of
certain food supplies, and still more recently the laboratory
division of the U. S. Public Health Service has done much efficient
work on the standardization of disinfectants. We must also
mention the work on milk, meat inspection, etc., by the Bureau
of Animal Industry and the work on sanitation and related sub-
jects by the U. S. Public Health Service, not forgetting to men-
tion the vast amount of routine analyses in state and municipal
health laboratories and the sporadic research work in the bio-
logical 'and bacteriological laboratories of our colleges and uni-
versities and the individual investigations of food and drug
contamination on the part of a few of the more enterprising state
and municipal health officers. Very recently the sanitary study
of mineral waters has received a great deal of attention on the
part of individual workers. The Committee of the Laboratory
Section of the American Public Health Association has prepared
a report covering the general conclusions regarding some of the
methods of analysis.

The purely microscopical examination of food substances and
of drugs, with reference to contamination by mold, yeast and
bacteria, should be a part of the work of the bacteriologist rather
than that of the chemist. Therefore, for the sake of completeness,
this phase of the subject is included in the present report. We
shall now proceed with the discussion of the bacteriological method
applicable in food and drugs laboratories, giving only the essential
details, however adding certain suggestions intended as a guide
for further investigation with a view to the improvement of the
present working methods. Detailed description of apparatus and
of technique will be given only when thought necessary.

For all practical purposes, the examination of foods and drugs
for the presence of biologic contamination (inclusive of bacteria,
yeasts, molds, protozoa, ova and larvae of higher animal parasites,
etc., etc.) is either made directly or indirectly. That is, the sub-
stance is either placed on a slide or counting apparatus and the

INTRODUCTION 35

quantitative or qualitative determinations made directly under the
suitable power of the compound microscope; or, certain quantities
of the substances are placed in or upon certain culture media
(Petri dish cultures, tube cultures, etc.) in order to bring out the
biological and biochemical characteristics of the contaminating
organisms, whereupon the cultural products are examined micro-
scopically. In this latter instance the microscopical examination
may even be entirely omitted.

The direct microscopical method has some very marked ad-
vantages and should be carried out whenever feasible, particularly
when purely quantitative results or estimates is the main object
sought after. In other instances the direct method must be
combined with cultural tests and the two are often checks upon
each other.

2. Direct Bacteriological Examinations— Quantitative Tests

Substances to be examined include waters and liquids of all
kinds; sewage; milk1 and cream; ice cream, liquid pharmaceuti-
cals and medicamenta, oils, catsups, beverages of all kinds, all
semisolids such as pastes, jams, jellies, etc., other semiliquids and
semisolids which may be readily diluted with water if necessary;
solids as powders, pills, tablets, soils, clays, meats, starches,
dextrins, flours, meals, dried fruits, dried eggs, dried albumen,
sugar, etc. In fact all substances which are in any way liable to
contamination by micro-organisms.

The following is an outline of the methods of making determina-
tions of the number of organisms in food and drugs.

a. Substances Requiring Concentration. — Certain substances
which contain comparatively few micro-organisms, as drinking
waters, mineral waters, beverages generally, tinctures, fluid ex-

1 In the case of milk, the centrifuge is first used to separate out the fat as much
as may be necessary to make the ready counting of the organisms possible. (See
also Chapter on Milk Analysis.)

36 BACTERIOLOGICAL METHODS

tracts, aquas, etc., must be subjected to processes which will con-
centrate the organisms, as by passing the liquid through a filter
in which the pores are sufficiently small to leave the organisms
behind, as for example a Berkefeld or Chamberland clay tube.
In addition to the filter, the centrifuge will be found useful as will
be explained later.

Any liquid containing not more than from 100 to 1,000,000
organisms per cc. does not lend itself to direct examination quanti-
tatively without concentration. The amount or volume of sub-
stance (liquid) to be passed through the filter will depend upon the
degree of concentration required. Since the Thoma-Zeiss
hemacytometer (with Turck ruling) is to be used in making the
counts, the organisms should average at least 4 to 5 in the 3^5 0
c.mm. areas of the counting apparatus, or 1,000,000 to 1,250,000
organisms per cc. Let us suppose that a direct count is to be made
of a drinking water which is very pure, having not more than from
50 to 500 bacteria per cc. In order to make direct counting with
the hemacytometer possible, it would be necessary to pass from
20 to 30 liters of the water through the clay filter and thoroughly
mix the organisms left in the tube with 10 or even 1 cc. of
filtered sterile water. To filter that amount of water requires
too much time unless a large specially constructed apparatus is
installed. For practical purposes, 1 liter is the largest amount
of liquid that it will be necessary to filter and reduce to 1 cc,
making a concentration of 1000. For special purposes the 1 cc.
may be further concentrated in the centrifugal tube described in
Fig. 3. Weaker concentrates may answer the purpose in some
cases, as ten or one hundred, as in sewage, badly contaminated
milk and in other liquids in which the number of organisms
present may range from 100,000 to 1,000,000 per cc.

The special centrifugal tube described in Fig. 3 is used as
follows: After passing a liter of the liquid to be examined through
the clay filter tube and thoroughly washing out the organisms and
other particles left in the tube, pour the contents into the special

DIRECT EXAMINATION

37

/Occ.

B

Fig. 2. Fig 3.

Fig. 2. — Kitasato filtering outfit ready to be attached to the exhaust pump.
A two-opening flask or bottle is interpolated to receive the backflow water, should
there be any. Various types of clay bougies may be used with this filter. The
rubber tubing for the connection must be heavy so as to prevent collapse by the ex-
haust pressure. — (Pitfield.)

Fig. 3. — Special centrifugal tube (.4) for concentrating bacteria and other micro-
organisms in liquids and also used in isolating or separating motile bacteria from
those which are not motile, as is explained under water analysis. The tube has a
capacity of 15 cc. with 1 cc. and 10 cc. marks. The tube is in two parts. The lower
narrowed end, having a capacity of 1 cc, is attached to the larger part by means of
a rubber-band ring. Centrifugalization is done at high speed. After centrifugali-
zation, the 1 cc. tube is removed and the contents thoroughly mixed by means of a
platinum wire loop. To avoid loss of the contents of the tube during the mixing.
attach the rubber ring. After the mixing the material is ready for the microscopical
counting and other examination.

A suitable stopper attached to a brass or other metal rod (B) may be inserted
into the narrowed portion of the upper part of the tube in order to prevent mixing
of contents when removing the 1 cc. tube. These tubes will also be found useful
in measuring the amount of sediment in milk, water and other liquids. For this
purpose the 1 cc. portion should be graduated into tenths and hundredths.

$8 BACTERIOLOGICAL METHODS

centrifugal tube. For washing use about 10 cc. of filtered sterile
water, adding up to the 10 cc. mark if necessary. Centrifugalize
at high speed for 30 min., which will throw the bacteria and other
solids down into the narrow 1 cc. end of the tube.

The following is a brief outline of the method of procedure:
Use a Kitasato filter with the usual hydrant suction pump attach-
ment. Pass a liter of the liquid to be examined through the
filter, continuing suction until nearly all of the liquid has passed
through. Remove the clay bougie and wash down the organ-
isms clinging to the sides of the tube with not more than 10 cc.
of distilled water which has been filtered and boiled. Place thumb
over the opening of the tube and mix the contents thoroughly by
shaking for 20 sec, then pour the thoroughly mixed contents
into a sterile cylindrical graduate and add sterile distilled and
filtered water up to the 10 cc. mark, shake thoroughly and make
the counts at once by means of the hemacytometer. This pro-
cedure gives a concentration of 100. By means of the special
centrifugal tube the concentration may be increased to 1000,
as already explained. The method gives approximate results
only, the counts as a rule being less than the actual number of
organisms present in the liquid, a difference due to three chief
sources of error: First, a small number of bacteria (especially
the smaller motile forms) will pass through the clay filtering tube ;
second, some of the smaller bacteria are caught and held in the
pores of the clay tube; and third, some organisms will remain
clinging to the inner surface of the tube after the mixed contents
are poured out for counting purposes. These sources of error
are, however, not great, perhaps not exceeding 8 to 10 per cent.,
and are on the side of conservative estimates. The clay bougies
used should be of the finest quality and should be of uniform and
standard thickness. The sources of error by the direct method
are perhaps not as great, certainly not greater, than by the usual
plating methods and offer some very decided advantages. The
concentrates show, in addition to the bacteria, other organisms

DIRECT EXAMINATION

39

Fig. 4. — Apparatus for fractional filtration, designed for use with Pasteur-
Chamberland or Berkefeld filters, a, Glass mantle surrounding filter; b, Chamber-
land filter; c, paraffin joint; d and e, rubber stoppers; /, double side-arm suction
flask; g, pinchcock controlling outlet from suction flask; h, outlet tube surrounded
by glass shield and attached to lower end of suction flask by means of short rubber
tubing; /. glass shield fused to and surrounding outlet tube as a protection against
contamination when the filtrates are drawn off; j, glass inlet tube plugged with cotton,
for admitting air into suction flask; k, pinchcock governing the admission of air
into flask; I, vacuum gauge; m, stopcock connected with vacuum pump. — (U. S
Dept. of Agriculture, Bureau of Animal Industry, Bull. 113.)

40 BACTERIOLOGICAL METHODS

as mold hyphae, mold spores, protozoa, diatoms, etc., besides
dirt particles, sand particles, starch granules, body cells, pus
cells, etc., etc., which would be lost or rather which would not
appear in the plating method. Furthermore, the counts can be
made with a great saving of time — in a few hours as against 24
to 48 hr., and longer, by the plate cultural method. It is true
that in many instances the direct method must be supplemented
by the cultural methods when, in the judgment of the analyst,
this becomes desirable or necessary.

Concentrates may also be made by evaporation under reduced
pressure. With a little ingenuity a suitable equipment may be
constructed in the laboratory. The container of suitable ca-
pacity (1 liter and more) is connected with an exhaust pump
which lowers the pressure sufficiently to cause boiling at a tem-
perature not to exceed 370 C; 24 hr. is usually sufficient time
to evaporate the liquid to nearly dryness. After the evaporat-
ing process has continued for several hours, various enrichment
media may be added to the liquid to be evaporated, which will of
course aid the intended isolation and development of the de-
sired bacteria. If the enrichment medium is added from the
first, annoying bubbling and frothing may take place. This
method is especially useful in isolating the typhoid bacillus, the
paratyphoid group and the intestinal bacteria in general.

b. Substances Which do not Require Concentration. — Badly
contaminated substances as sewage, milk from badly managed
dairying establishments, badly contaminated liquids of all kinds,
soups, broths, beer, wines and such products as tomato catsups,
jams, jellies, canned oysters, etc., may be examined directly
without the necessity of making concentrations, or of centrifugali-
zation, in order to make quantitative and certain qualitative
estimates. The substances of this class may be divided as follows,
based upon the approximate number of organisms per cc. as
determined by means of the spore and yeast counter described
under Fig. 5, and the Thoma-Zeiss hemacytometer.

DIRECT EXAMINATION

41

COUNTING f

APPARATUS L
for

MOLDS OOSPORES

m

AREAS
'/zs sq.m.m.

I sq mm.
25 sq mm.

ISsq.mm.

CUBIC CONTENTS
'//25 cmm.
ysc.mm.
5 cmm.
15 cmm.

A B

/ 2 3 4 5 6 / 2 3 4 5 6

a r |~ 1 1 i 1

AZZZZZZZZZZZI

a _

A 1 I 1 1 1 11 1 1 _

C D

Yig. 5. — A, Counting apparatus for molds (hyphae and spores) and yeasts. The
rulings are 75 sq. mm., 25 sq. mm., 1 sq. mm. and ^5 sq. mm. On either side of the
ruled area are glass slips 0.2 mm. thick, so that the entire capacity of the space
within the ruled area is 15 cmm., subdivided into 5 cmm., >£ cmm. and >f 25
cmm. areas.

42 BACTERIOLOGICAL METHODS

i. Substances in which the organisms are not too numerous to
permit the use of the ^5 sq. mm. areas without making dilutions.
That is, substances in which the number of organisms does not
exceed 10,000,000 per cc, hence the number of yeast cells, spores,
bacteria, etc., may not exceed forty in one of the 3^50 c.mm. areas
of the hemacytometer. The limit for the spore and yeast counter
would be 5,000,000, before making the dilution is necessary.

2. Substances in which the number of organisms and spores
are too numerous to permit the use of the J^5 sq. mm. areas of the
hemacytometer, but permitting the use of the J^oo sq. mm. areas
without making dilutions. The total number of spores, bacteria
and other organisms may range from 10,000,000 to 100,000,000
per cc, numbers derived from finding on an average from 2.5 to
25 organisms in one of the Kooo c.mm. areas of the hemacytometer.

The counter is used as follows: A bit of the thoroughly mixed substance, as
jam, jelly, tomato paste, catsup, etc., is placed on the slide in the ruled areas and
covered with a rectangular cover glass (No. 2). Slight pressure may be necessary
to make the cover glass rest evenly on the two slips. The counting is done in areas
entirely filled (from slide to cover glass) by the substance mounted. The larger
areas may prove useful in estimating the amount of sand particles, dirt, etc., present.
The 3^25 c.mm. areas will be used in counting spores, yeast cells and mold hyphae
and similar contaminations. It is possible to make counts without dilutions as
long as the number of organisms in the areas does not exceed forty. If- more
organisms are present in one area dilution becomes necessary, as already ex-
plained. Making dilutions of 1-10, 1-100 and 1-1000 makes the counting limits
50,000,000, 500,000,000 and 5,000,000,000 per cc. The 3-1 2 5 c.mm. areas are also
used in estimating the quantity of mold hyphae present. Finding clusters of mold
hyphse in 25 per cent, of these smallest areas is presumptive proof that the substance
is unfit for human consumption. Naturally the more finely divided the substance
is the more numerous are the mold f ragmen ts. For making mold counts the material
to be examined should be reduced to uniform fineness. This could be accomplished
by rubbing a thoroughly mixed sample through a sieve of standard mesh, say y± mm.

B, a simplified modification of the counting apparatus just described, is made as
follows : The two slips 0.2 mm. thick are placed in position, but the ruling is omitted
and in place thereof an eye-piece scale C is used, the measuring value of which has
been carefully determined by means of the stage micrometer. The rulings on the
eye-piece must be delicate and the analyst must be careful not to move the eye or
change the direction of his vision while making counts.

The ruled slide (D) will be found useful for making quantitative estimates of
seeds, sand particles, dirt, larger parasites such as vinegar eels, ova of intestinal
parasites, etc., in catsups, crushed berries (strawberries, raspberries, loganberries,
etc.), jams and in other vegetable substances. Definite quantities of the substance
to be examined are placed upon the ruled area of the slide by means of a small
measuring spoon (0.25 gram, 0.5 gram, 1 gram), spread and covered with a suitable
cover glass and the counts made in the entire amount placed on the slide, using the
low power (80 diam.) of the compound microscope.

DIRECT EXAMINATION 43

3. Substances in which the organisms are too numerous to
permit ready counting by means of the 0.004 c.mm. areas of the
Thoma-Zeiss hemacytometer. It now becomes necessary to use
dilutions, which are made as follows.

Making the Dilutions. — The dilutions generally used are i-io.
Rarely will it be found necessary to use higher dilutions. Should
this, however, become desirable, a dilution of i-ioo is to be made.
The highest counts so far recorded were in the case of two tomato
pastes which showed 2,400,000,000 and 4,000,000,000 bacilli per
cc. In these instances dilutions of i-io were used and proved
quite satisfactory, though it was evident that a greater number of

.SC.H8rV.QMB OPT\<
• ROCHESTER,*:

Fig. 6. — Thoma-Zeiss hemacytometer. Complete equipment for blood count-
ing. This is very convenient for making bacterial counts in catsups, jams, jellies
and other vegetable foods and also in animal food substances.

bacilli per cc. would have necessitated the use of a dilution of
1-100. However, a dilution of i-io is all that is required for
practical purposes, as a bacterial count of 4,000,000,000 and more
per cc. would indicate the decomposed condition of the food
substance and its unfitness for human consumption.

In case of liquids and near liquids, 9 cc. of distilled water is
added to 1 cc. of the substance, and in the case of pastes and
similar products, 9 cc. of distilled water is added to 1 gram (or
1 cc. semiliquid) of the substance. The dilutions are made in
25 cc. graduated cylinders', which answer the purpose very well.
Or 100 cc. graduates may be used for making the dilutions, adding

44

BACTERIOLOGICAL METHODS

90 cc. of distilled water to 10 grams (or 10 cc.) of the substance.
Place the thumb firmly over the opening of the graduate; the con-
tents are thoroughly mixed by shaking vigorously for about 20 sec.
By means of a slender glass rod slipped well into the mixture, take
up a droplet of the mixed material and touch the end of the rod
lightly and quickly upon the middle of the ruled area of the hem-
acytometer. All this must be done rapidly, before the organisms

Fig. 7. Fig. 8.

Fig. 7. — Zappert ruling of the Thoma-Zeiss hemacytometer. This form of
ruling is especially convenient for making bacterial counts and counts of fat globules
in milk. — {Carl Zeiss.)

Fig. 8. — Turck ruling of the Thoma-Zeiss hemacytometer. This is especially
useful if it is desired to combine the bacterial count with the spore and yeast count.
The smaller areas (1-400 sq. mm.) may be used for making the bacterial counts, while
the larger areas (1-25 sq. mm.) may be used for making the spore and yeast counts. —
(Carl Zeiss.)

have had time to settle to the bottom of the graduate, and before
they have had time to accumulate at the end of the glass rod.

Making the Count. — After having cleaned the hemacytometer
(do not use alcohol), it is sometimes desirable to rub a very soft,
grit-free graphite pencil over the ruled area so as to render the
lines more readily visible. Usually, however, this is not necessary.
After placing the droplet of material as above described, cover
with a No. 2 cover glass and orientate the ruled area by

DIRECT EXAMINATION

45

means of the low power and make counts under the suitable high
powers. From ten to twenty of the ruled areas should be counted
and from these countings figure the average. It is desirable to
make two or three mounts of each sample, thus giving the average
of from twenty to thirty areas counted. The countings are to
be made in areas free from pulp
fragments and including all organ-
isms lying within the ruled bound-
ing lines and inclusive of half
averages of those organisms which
lie across the rulings. All count-
ings which present characters of
doubt are omitted from the final
estimates.

Those organisms which occur
within the cell-lumen of the vege-
table tissues are not to be counted.
To do so is practicably impossible
and such countings, even if pos-
sible, would add nothing to the
value of the findings. In case the

cells contain numerous bacteria this should be noted in the report,
as it certainly indicates decomposition of the material. The prin-
cipal decomposition changes due to the invasion of bacteria and
other organisms are, however, largely limited to the exterior of
cells, especially by those organisms which develop during or after
the factory processing. The numerical determinations are there-
fore limited to organisms which occur in the matrix and those which
have been washed from the exterior of cells by the thorough mixing.
The thorough mixing of the samples is a very important part of
the procedure. In the case of liquids and semiliquids, mixing is
done by thorough shaking, and in the case of pastes and similar
materials, by means of a spatula or a small spoon.

In making counts of very small or comparatively short bacilli,

Fig. 9. — Btirker ruling, useful in
making counts of milk fat globules,
spores, and yeast cells. The average
of many counts is taken. — (Carl Zeiss.)

46

BACTERIOLOGICAL METHODS

some difficulty is caused by those organisms which happen to be
vertically suspended in the counting chamber, thus presenting an
end view which gives the appearance of small granules or spherical
particles which the comparatively inexperienced observer may not
recognize, or which may be mistaken for inorganic particles or
organic particles other than microbic. In case of doubt, allow the

'mm.

N£

!/£>

9

si

i

Fig. io. — Tomato pulp cells in normal catsup. The cells are large, thin- walled,
containing granular particles. The coloring matter of the tomato frequently ap-
pears as deep scarlet-red crystalline particles usually arranged in groups within the
cell. — (Howard, Yearbook U. S. Dept. of Agriculture, 19 11.)

mount to remain at rest for 10 or 15 min., thus allowing the
bacilli to settle to the bottom of the cell where they will assume
the horizontal position, thus presenting the long axis to view
and making counting easier.

In order that all of the cells (individuals) of the bacilli may be
counted, it is necessary to use a high power (480 to 500 diam.).
Lower powers are not satisfactory for counting bacteria. For

DIRECT EXAMINATION

47

counting spores and yeast cells a magnification of 180 diam.
would prove very satisfactory, especially with a well -corrected
wide aperture objective. The counting of cocci is more confusing
than the counting of bacilli, but fortunately the microbic contami-

Fig. ii. — -Cluster of mold hyphse in granular (decomposed) tomato pulp. This
type of mold is traceable to field-rotted tomatoes. The finding of hyphae of this
type in tomato catsup indicates the use of rotted tomatoes, therefore, indicates
inadequate culling at the factory. — {Howard, Yearbook U. S. Dcpt. of Agriculture,
1911.)

nations of most vegetable substances are bacillar, though there are
some notable exceptions.

Mold Counting. — Thus far no satisfactory method for making
estimates of the amount of mold hyphae present in fruit and in
animal products has come into use. The method recommended
by B. J. Howard, Chief of the Micro-chemical Laboratory of the
U. S. Bureau of Chemistry, namely, determining the degree of

48 BACTERIOLOGICAL METHODS

mold contamination from the number of microscopic fields of the
compound microscope which show the presence of hyphal clusters,
is far from satisfactory. It indicates the amount of contamination
in a general way only. More reliable and more accurate estimates
could be obtained through the use of a counting apparatus in which
the number of hyphal clusters could be ascertained in definite
quantities of the material under examination. The hemacy-
tometer already mentioned does not serve the purpose because of
the smallness of the counting areas. The special counter described
in Fig. 5 would serve the purpose very well. It is furthermore
necessary to reduce the material to a uniform and standard fineness
by rubbing it through a sieve. A very small standard mesh sieve
would answer the purpose. Take i gram of the thoroughly mixed
material and by means of a small spatula rub all of it through the
sieve and make the estimations from the pulp which has been
passed through the meshes of the sieve.

Precautions. — The following are some of the factors which
necessitate caution in making counts of microbes, yeast cells,
spores and mold fragments.

a. Badly decomposed factory pulp which compels prolonged
heating in order to render it suitable for canning, often presents
such a granular appearance as to make accurate counting of the
microbes rather difficult. In such materials many of the more
or less disintegrated pulp cells are filled with bacteria and these
cannot be included in the count. Commonly in such substances
many of the mold fragments are also very much disintegrated
through decomposition changes, probably initiated by enzymes
formed by the bacteria and other organisms.

b. While it is quite easy to distinguish between living yeast
cells, dead yeast cells and spores, it is not thought advisable to
attempt such differentiation in routine laboratory practice, ex-
cepting in cases where identification is simple and where there is
very little room for doubt. One of the first important problems
for the food and drugs bacteriologist to solve is the identification

DIRECT EXAMINATION 49

of those micro-organisms which commonly attack foods and
drugs, more especially the molds and yeasts.

c. It is neither practicable nor necessary to differentiate
between the different kinds of spores which may be present in a
product, excepting as suggested under (b). ■

d. In many instances it would be desirable to resort to plat-
ing methods in order to determine the number of viable organ-
isms present. This would be simple for bacteria and mold spores,
but more difficult for yeasts.

Differentiating between Living and Dead Bacteria and other
Low Forms of Organisms. — It would be most desirable to deter-
mine some practical working method for distinguishing between
living and dead bacteria in foods and drugs. Often the question
arises as to the time and place source of the contamination. Did
the organisms present develop in the fruit, in the pulped material
during the processing or in the containers after manufacture?
Again, are the organisms estimated by the direct count dead or
alive?

Several investigators have stated that dead and living bacteria
react differently with certain stains. For example G. Broca, an
Italian bacteriologist, declares that the use of the following mixed
stain will serve this purpose. To 8 cc. of concentrated carbol-
fuchsin add ioo cc. of Loeffler's methylene blue. Let the mixture
stand for 24 hr. before using. Exposed to this stain, dead bacteria
(killed by heat or by disinfectants) are colored red while living
bacteria are colored blue. It is declared that other stains, as
Giemsa's, will react in a similar manner.

More recent experiments would indicate that selenium and
tellurium compounds will serve to differentiate living bacterial
contaminations. It would appear that these substances are
decomposed into metallic tellurium and selenium when brought
in contact with living organisms. Much experimental work
along this line has been done by Hansen, Gmelin, Gosio and
others, and still more recently (1913) by King and Davis of the

5<0 BACTERIOLOGICAL METHODS

Research Laboratory of Parke, Davis and Company. Potassium
tellurite is said to be the most satisfactory reagent. In dilutions
of i : 50,000 this substance forms characteristic black compounds
with all of the more common micro-organisms when in the living
state. The reaction does not take place in the presence of dead
micro-organisms and the different organisms do not all react in
the same degree or manner. Some are much more susceptible
than others. The Bacillus coli appears to be the most sensitive
to the reagent. With most species of bacteria the time re-
quired to produce the characteristic color and precipitation reac-
tion ranges from 12 to 96 hr. at a temperature of 370 C, but with
the colon bacillus a distinct coloration or color ring becomes visible
several minutes after the reagent is added. King and Davis
summarize the experimental results as follows:

1. Nearly all of the more common micro-organisms (bacteria and yeasts) react
with potassium tellurite, forming characteristic, black compounds.

2. This capacity depends on an active stage of metabolism of the reacting
organism, and the action is, in all probability, a reduction of the tellurite.

3. The "tellurite reaction" can be used as an indicator of microbial life, and is
especially suitable for revealing microbic contamination.

4. A dilution of 1 : 50,000 of the salt seems to be most suitable for its action as a
general microbic indicator. In this concentration, it produces no irritative action
when introduced into test animals.

5. The bacteria of the "colon-typhoid group" show differences in resistance
to the antiseptic action of potassium tellurite and in the appearance of their reaction
with this salt. These variations are sufficient to suggest the experimental use of
potassium tellurite for differential diagnosis in the group.

6. The intensity of bacterial action on potassium tellurite depends upon the
individual resistance of the bacterium and the concentration of the salt present.
The velocity of reduction of the tellurite is apparently a specific function of an organ-
ism, apart from its resistance to antiseptic action. With the colon bacillus, the
"tellurite reaction" is almost instantaneous.

7. Treatment with potassium tellurite has practically no influence on the bio-
logical characteristics of an organism.

3. Numerical Limits of Micro-organisms in Foods and Drugs

What should be the maximum limit of the number of bacteria
and other micro-organisms in food and drugs within the intent

DIRECT EXAMINATION

51

of the U. S. Pure Food and Drugs Act? This is as yet an un-
settled question and one that requires further careful considera-
tion, even calling for some extensive investigation in order that
certain disputed points may be finally settled. There are,
however, certain results based upon extensive observation which

*

•.+

.

Fig. 12. — Type of mold development in the tomato pulp during and after the
processing. According to tests made by B. J. Howard of the Bureau of Chemistry,
mold will develop in tomato catsup containing 0.1 per cent, sodium benzoate. Com-
pare the hyphce with those shown in Fig. n. They are much larger in transverse
diameter and the walls of the cells are much thinner. — (Bitting, Bull. 119, Bureau
of Chemistry, U. S. Dept. of Agriculture.)

may be set down as conclusive. The organisms of all kinds which
may occur in and upon clean and uncontaminated ripe fruit,
for example, are negligible quantitatively as well as qualitatively.
Such organisms as do occur are limited to the exterior. Only
under abnormal conditions do micro-organisms find their way
5

52 BACTERIOLOGICAL METHODS

into the tissues beneath the epidermis and into the parenchyma-
tous cells of whole fruits. It would be interesting to determine
the average number of bacteria on the exterior of such fruits as
the apple, the peach, the pear, the apricot, the tomato, the
cucumber, etc., and from these figures to estimate the number of
organisms per cc. of the fruit substance. The practical value of
such information would, however, not be great, as may be understood
from the statements already made. It must be admitted without
question or doubt that fruit products of any kind, which contain
only such organisms as normally occur on clean uncontaminated
ripe fruit, will never come under the ban of the pure food and
drugs act. This also applies to foods and drugs in general. The
organisms which concern the analyst are those which occur in and
upon contaminated and diseased fruits and those which are in-
troduced or added or allowed to develop and multiply during
the processing, and afterward. We may therefore make the
following postulate: All fruit products from clean uncontami-
nated fruit (ripe or green), prepared under modern sanitary con-
ditions, contain micro-organisms in negligible quantities only.
It is true that the ideal conditions implied in this postulate may
not always be attained in practice, yet we are warranted in
making a second postulate, namely: that the number of organ-
isms present in fruit products, over and above the negligible
quantities mentioned, are in direct proportion to the careless-
ness in the various steps of the processing. Stating it conversely,
as the manufacturers of food products attain the practically ideal
conditions, the number of organisms in their products will become
gradually negligible. That such conditions are attainable is
clearly shown by the canned products of the careful housewife and
of the careful manufacturer. What may be done by the careful
housewife may be done even better by the careful manufacturer,
because the latter can employ the most approved modern methods,
aided by special machinery, which are not at the disposal of the
housewife or even of the small manufacturer.

DIRECT EXAMINATION

53

In a general way, the number of micro-organisms in food prod-
ucts and in liquids intended for internal use, not including the fer-
mented products, is negligible when they do not exceed 250,000
per cc. (ranging from 5000 per cc. to the maximum). In fer-

Fig. 13. — Various stages in the germination of spores in catsups. Note trans-
verse septation and branching of the hyphae. Germinating spores may be traceable
to the tomato from the field or they may be from spoiling factory pulp. — (Bitting,
Bull. 119, Bureau of Chemistry, U. S. Dept. oi Agriculture.)

mented products, as cider, vinegar, wines, beer, etc., the number
of organisms present may be much greater, but even here the
quantitative estimates generally become negligible if the modern
methods of purifying or clarifying (through sedimentation, the
use of albumen, gelatin, casein, etc.), filtration, centrifugalization,
and sterilization are carried out. Of course, in such products as

54

BACTERIOLOGICAL METHODS

sour milk, ripened cream, ripened cheese, sauerkraut, pickles,
etc., the processes of clarification are not applicable, and hence we
always find a large number of certain predominating types or
species of organisms present.

The microscopical examination of products which have under-
gone normal fermentation shows that the number of organisms
present is quite variable, depending upon a variety of causes and
conditions. This can readily be ascertained from the examination

Fig. 14. — Substances frequently found in tomato catsup, a, Heat dextrinized
corn starch. Starch is frequently used as a filler or stiffening agent, b, bacteria
which frequently appear in great numbers, c, Vinegar eels derived from cider or
wine vinegar. Soil nematodes may also be found, indicating gross soil contamina-
tion and inadequate washing at the cannery, d, Nematode larvae derived from the
soil, e, f, g, h, Spore types frequently met with in catsups, i, Yeast cells.

of such common household products as vinegar, sour cream, cider,
apple butter, sour milk, etc. It would be most desirable to de-
termine the exact identity of the organisms which produce the
most favorable fermentation changes in fermented food products.
This has been done in some cases and pure cultures of the specific
organisms are used for manufacturing purposes, resulting in the
production of superior food articles. When the fermentation

DIRECT EXAMINATION

55

processes are left to nature the result is not by any means uniform
and we have products which are often so vitiated by the develop-
ment of undesirable associated organisms as to make the food unfit
for use. There is a definite biological relationship between those
organisms which initiate desirable fermentations and those which
are objectionable; both kinds are generally present, but fortunately

Fig. 15. — Vinegar eels from decomposed blackberry pulp. The small particles
scattered through the field are yeast cells. Bacteria were also present but they do
not show in the illustration. — (Howard, Yearbook U. S. Dept. of Agriculture, 1911.)

the desirable or beneficent forms overgrow the objectionable
forms very rapidly, but not always. It should be one of the prin-
cipal efforts of the food and drugs bacteriologist to isolate and
identify the organisms which are desirable in the production of
fermented food products and those which are unquestionably
undesirable and objectionable, for in these products it is not a
question so much of quantity as of quality of the organisms present.

56 BACTERIOLOGICAL METHODS

This is by no means a simple problem. Much of this field of work
is as yet untouched, and it is not likely that definite conclusions
will be reached in the very near future. It means an investigation
of those conditions which are recognized as diseases in industrial
or manufactured products, characterized by unaccountable de-

Fig. 16. — Mold from decomposing plum. — {Howard, Yearbook U. S. Dept. of
Agriculture, 191 1.)

teriorations in flavor, in taste, in color, in nutritive value, etc. It
means a very careful study of organisms which are similar in
morphology and yet quite different in specific functional activities,
giving rise to objectionable fermentation products.

The following tables will give some idea of the number of organ-
isms which occur in certain canned food products. Animal food

DIRECT EXAMINATION

57

products are not included in Tables II and III because there are not
sufficient data available on which to base suggestions. There
appears to be no plausible reason why canned animal products
should not be subjected to the same method of examination as
vegetable substances, particularly sausage meats, canned meats,
canned oysters and shellfish generally, canned eggs and canned
soup stocks. Pickled herring which shows 8,000,000,000 bacteria
per cc. in the liquor is certainly a questionable food article. In

Fig. 17. — Spores and hyphal fragments from decaying sweet pepper. "Dry rot"
fungus. — {Howard, Yearbook U. S. Dept. of Agriculture, 191 1.)

this particular instance there was no objectionable odor noticeable,
but the meat of the herring was somewhat soft. Smoked meats
and fish should be examined for mold in addition to bacteria.
This subject should receive immediate careful consideration on the
part of food bacteriologists.

Table I shows the number of organisms which may occur in
some of the more common household food substances, fermented
and unfermented. The figures are based upon direct counts.
Table II is based upon the examination of factory products ob-
tained in the open market. The numerical extremes in the
micro-organisms given in Table II, are in direct ratio to the relative

58

BACTERIOLOGICAL METHODS
Table I1]

Number of Organisms per Cc.

Name of Substance

Bacteria

Yeasts

Spores

Hyphae

Blackberry jam . .
Blackberry jelly. .
Cheese, California

500,000
Few

Few.

Few.

80,000,000

50,000 to
500,000
1,000,000
Few

Few

Entirely per-
meated.

Cider

50,000 to
30,000,000
Few. . .

Cider vinegar. . . .
Currant jelly

Fruits, canned. . .

Few

Few. . .

Herring, pickled..
Jams

8,000,000,000
Few. . .

Few

Few

Few.

Jellies

Few

Few. . .

Meat, sausage

Milk, ordinary. . .
Milk, certified

1,000,000 to
150,000,000
25,000 to
2,000,000
1,000 to
15,000
2,000,000,000 to
7,000,000,000
Few

Milk, sour

Plum preserve.. . .

Few

Few

Few.

Plum relish

100,000,000

800 to
32,000,000

Few

2,000,000 Some.

Water, drinking
(San F.).

' '

unsanitary conditions in the factories. It is quite evident that
the products of the manufacturers who employ modern methods are
fully up to the quality of those prepared by the careful housewife.

1 The counts recorded in Tables I, II, and III were made by the direct method
using the hemacytometer. In the case of the sausage meat some of the counts were
checked by the plating method and it was found that the count by the plating
method was invariably higher than by the direct method. Other investigators have
noted similar discrepancies. The direct examination of meats for bacteria is occasion-
ally unsatisfactory because of the confusion due to granular fragments traceable
to broken up blood corpuscles, fragments of coagulated albumen, etc.

DIRECT EXAMINATION
Table II

59

Number of Organisms per Cc.

Name of Substance

Hyphae

Bacteria

Yeasts

Spores

Apple jam

Apple jam

Apple jam

Apricot jam

Blackberry with
apple.

Catsup

Catsup 440,000,000

Catsup 560,000,000

Catsup 800,000,000

Few.

500,000

400,000

25,000

40,000

5,000,000

3,750,000
494,000

9,250,000 Some.

None I None.

1,728,000 Numerous. . Very abundant.

Some.
Some.

Catsup

Catsup

Catsup

Catsup

Cherry jam with
apple.

Currant jam
Currant jam

Fig jam

Loganberry jam..
Loganberry jam..
Orange marma-
lade

Plum jam

Peach jam

Strawberry jam.
Strawberry jam.

Tomatoes

Tomatoes

Tomato paste. . .
Tomato paste. . .
Tomato paste. . .
Tomato paste. . .
Tomato paste. . .
Tomato paste. . .

200,000,000

5,000,000

80,000,000

400,000,000
5,000,000

1,000,000

Few

12,000,000

1,000,000

40,000,000

45,000

1,250,000

6,250,000

8,750,000

27,500,000
20,000,000

1,500,000
Few

5,000,000

7,500,000

200,000

500,000
1,250,000

12,000,000
2,000,000,000
2,000,000,000
2,000,000,000
1,400,000,000
4,000,000,000
1,000,000,000
2,000,000,000

500,000

750,000

4,500,000

500,000

10,000

80,000,000

Few

750,000
1,400,000
4,000,000
1,000,000
1,200,000
5,000,000
6,000,000
1,000,000
100,000,000

Abundant.
Very abundant.
Very abundant.
Entirely per-
meated.
Very abundant .
Trace.

Very abundant.
Very abundant.
Very abundant.

Some.

Quite abundant.

Some.

Some.
Abundant.
Very abundant.
Very abundant.
Very abundant.
Very abundant.
Very abundant.
Very abundant.
Quite abundant.
Entirely per-
meated.

6o

BACTERIOLOGICAL METHODS
TABLE II.— {Continued)

Name of Substance

Numt

er of Organisms per Cc.

Hyphae

Bacteria

Yeasts

Spores

Tomato pulp1. . . .

Less than

5,000,000

1,900,000,000
Few

Less than
500,000

37,000,000
Few

Practically none
(1-3 per cent,
of fields).

Entirely per-
meated (100
per cent.).

Few.

Tomato pulp1. . . .

Imitation jam.. . .

30,000,000

Table III

Maximum

No. of Organisms per Cc.

Hyphae2

Bacteria

Yeasts

Spores

Apple butter

Berries

Catsup

Cider

5,000 to

1,000,000

Few

10,000,000 to
50,000,000
500,000 to
2,000,000
Few

1,000,000

Few

1,000,000 to
10,000,000
500,000
Few

500,000 to
5,000,000
50,000 to
500,000
1,000,000 to
10,000,000
1,000,000

500,000
500,000

15 per cent.
18 per cent.

Fruits

Jams

500,000 to

1,000,000
500,000

Few

10 to 1 2 per cent.
10 per cent.
1 to 5 per cent.

Jellies

Marmalade3

Tomato pastes. . .

500,000,000
5,000,000

Few

2,000,000

20 to 25 per
cent.

Vinegars (fruit) . .

Few. . . .

1 Both samples were from large factories and represent the extremes in the factory
conditions. The first sample is from a factory where the conditions are what they
should be, the second from a factory where the conditions are just the reverse.

2 Percentages given this column refer to the number of the 1/125 c.mm. areas of
the mold counter described in Fig. 5 which contain hyphal clusters. As a rule abun-
dant spores indicate the presence of abundant hyphal tissue, and vice versa.

3 The organism in orange marmalade, under ordinary conditions of manufacture,
are negligible in amount.

DIRECT EXAMINATION 6 1

Other manufacturers, either through greed, ignorance or careless-
ness, or through all three causes combined, refuse to employ
modern methods and as a result their products are very often in
an undescribably filthy condition, wholly unfit for consumption.
In addition to the bacteria, yeast cells, mold, sand and dirt
particles present in the inferior grades of catsup, jams, jellies,
etc., there are found insect remnants (flies, aphides, beetles),
vinegar eels, larvae of various nematodes (from soil), etc. The
presence of numerous fly remnants is certainly an indication of
highly unsanitary factory conditions. The presence of vinegar
eels indicates the use of bad vinegar and the presence of soil
nematodes and of sand and dirt particles indicates insufficient or
no washing. Laboratory experience has demonstrated that
there is a definite relationship between the number of bacteria
and other organisms and the amount of dirt and other impurities
present in factory products. Unsanitary factory conditions en-
courage a certain recklessness in such factories, inducing the
laborers about the place to even go out of the way to add more
filth. Thus shovelfuls of refuse are taken up from the filth-coated
floors and thrown into the mixing vats, the idea evidently being
that it will add to the bulk and that no one will know the difference.
Vats are often not cleaned until the conditions are almost unde-
scribable. Refuse is added, often of such a character as to be un-
fit as food even for animals. This criminal negligence, care-
lessness and indifference is too frequently engendered by ignor-
ance which, gives heed to nothing else than a strict enforcement
of the law.

The filthy condition of some of these products is very generally
not apparent to the layman because of certain methods employed
primarily intended to hide or mask such defects. The odors of
decomposition are quite effectually dissipated by the steaming
and cooking process. The vitiated taste is quite effectually
masked by the heavy spicing. Any appreciable change in color is

62 BACTERIOLOGICAL METHODS

restored by means of added coloring substances. Any change
in consistency is corrected by adding fillers, such as starch, gelatin
and agar. The unscrupulous manufacturer will work up a supply
of spoilt canning tomatoes, including rejected " swells" and
" leaks," making them into catsup or paste. Overripe and par-
tially decomposed fruits (culls and rejects) are worked up into
jams preserves and into combinations in which the objectionable
character and appearance are hidden or lost sight of.

We are justified in the conclusion that the number of micro-
organisms in food products is a reliable guide to the wholesomeness
and sanitary quality of such products and the very natural ques-
tion arises, what are the maximum numbers of bacteria, yeast cells
and mold spores (including mold hyphae) permissible under
reasonable and practicable sanitary conditions. While ideal
factory conditions may not always be practicably attainable,
yet it is wholly reasonable to expect the operation or methods which
will bring the maximum quantitative counts per cc. within the
numerical limits given in Table III. These proposed maximum
numerical limits are tentative only. As the sanitary conditions
in the canneries are improved, as they undoubtedly will be, the
limits can be correspondingly decreased, finally reaching the
negligible quantities as already explained. Where numbers are
omitted in the tables it indicates that the quantity of organisms
is negligible. "Few," indicates that the number of organisms is
somewhat more than in negligible amounts, yet not sufficient to
make counting necessary or to question the suitableness of the
article for food purposes.

It is quite evident that different numerical limits must be
adopted for different classes or kinds of food products. This can
be seen from a study of the tables. Some fruits and fruit products
are more susceptible to the attacks by bacteria, yeasts and molds,
than others. Acid fruits, as the cherry, the plum, tomatoes,
loganberries, blackberries, etc., are much more likely to be attacked

DIRECT EXAMINATION 6$

by molds than are apples, peaches, pears and apricots. Yeasts
very rarely appear in the whole fruit, but they develop very
rapidly in fruit pulps which contain sugar (natural or added).
Yeasts require in addition to sugar, a high percentage of moisture
for their active growth, including an ample supply of oxygen (air) .
The presence in canned fruit products of numerous yeast cells
indicates fermentation during the processing. The presence of
numerous bacteria in fruit products indicates the use of rotted
(bacterially) fruit or bacterial contamination and development
during the processing, or both.

It would appear that most of the bacteria which develop in fruit
pulps, especially those from fruits which are quite acid, as for ex-
ample tomato pulps, belong to the lactic acid group. Numerous
tests in the laboratories of the Bureau of Chemistry show a paral-
lelism between the number of bacteria and the amount or per-
centage of lactic acid present in tomato catsups. The usual
rotting bacteria require more air (oxygen) then is present in the
pulp mass and as a result these are soon overgrown by the lactic
acid bacilli, if the pulp is allowed to stand for a time without steril-
ization. It is, however, very evident that the contamination of
such products as catsups, tomato pastes and tomato purees is
never wholly limited to lactic acid bacilli. The inclusion of field
rotted tomatoes and the rotted pulp material from filthy mixing
vats and other parts of the machinery of the unsanitary factories,
adds a sufficient number of rotting bacteria to render the article
dangerous to health, if consumed. Ravenel and other investiga-
tors have shown that when certain food products, as cream and
milk, are kept in cold storage, particularly after pasteurization
or incomplete sterilization, the development of lactic acid bacilli
is checked and the growth of toxin forming bacteria is encouraged,
resulting in occasional poisoning to the consumer. It is very
likely that similar conditions may exist in some of the incompletely
sterilized canned food products (vegetable as well as animal) which
have been stored for some time at a comparatively low temperature.

64 BACTERIOLOGICAL METHODS

The question is frequently asked, what percentage of rotten or
moldy fruit must be present to render the product unfit for human
consumption? This question cannot be answered definitely. In
a general way, it may be stated that where there is not over 5 per
cent, of rotted or moldy fruit used, the number of organisms in the
finished products will not reach the maximum limits given in Table

Fig. 18. — A type of mold, Spicaria sp., very frequently found on decaying
tomatoes. Some of the filaments and numbers of spores are shown. — {Howard,
Yearbook U. S. Dept. of Agriculture, 191 1.)

Ill, in fact the counts will in all probability be considerably less.
A careful culling of spoilt fruit in the field and at the factory,
coupled with reasonably sanitary factory methods and modern
methods of sterilization, will furnish products which will meet all
of the requirements of any pure food law.

The statement is frequently made by manufacturers that even

DIRECT EXAMINATION

65

though bacteria, yeasts and mold are present in considerable
numbers, they are harmless and do not produce toxic effects
when introduced into the digestive tract. This statement is
wholly without foundation in fact. On the contrary it is known
that certain bacteria, yeasts and molds do cause disease and
more or less severe intoxications and intestinal disturbances.
The objectionable character of mold is universally recognized

Fig. 19. — Mold colonies in gelatin seen under the low power of the microscope
(X 80). This mold developed in the gelatin after it was spread on the screen
to dry. This gelatin also contained numerous bacteria. Gelatin thus infected is
not suitable for bacteriological purposes neither is it suitable for use as food.

and nearly all animals refuse to eat moldy and mold contaminated
food materials. Various ulcerative diseases of the skin and of
the digestive tract are caused by mold organisms. While many
of the yeasts are entirely harmless and cause very important
fermentative changes, some of them are pathogenic to man while
others initiate objectionable fermentation changes in the food
substances.

66

BACTERIOLOGICAL METHODS

As already indicated the number of organisms in food sub-
stances is in direct ratio to the following conditions:

i. Insufficient culling of partially and wholly decomposed fruits.
2. Unsanitary factory conditions and unsuitable methods.

Ova of the Parasitic Worms or Man
TREMATODA

O SCALE X ItOO

Heterophyes
heterophyes

(cvftcrLooss.igoa) i

Dicro- \fr%

coelium

l&nceatum

Fasciola Fasciolopsis

Opistorchis
felineus (*r<crLoo»s.K>o&)

i i

Clonorcliis Clonorchis
sinensis endemicus

(Modified from Loon. IW)

nmnsomw«$

cVJT/f at ■&/ t/rc/zr/i/Scfioc/.

Fig. 20. — Intestinal ova. Trematodes. Ova of intestinal parasites may possibly
occur in foods of vegetable origin contaminated by soil, sewage and fecal matter.
Note comparative size and the actual measurements according to the scale. It may
be mentioned that the extremely small seeds of Vanilla planifolia have been mistaken
for ova of intestinal parasites. — (Stitt.)

We are warranted in establishing a maximum limit as to the
number of organisms permissible in food substances. The
method of estimating the quality of foods based upon the number
of micro-organisms present has been tested out in different coun-
tries and has proven very reliable and satisfactory; and those who

DIRECT EXAMINATION

67

are entrusted with the enforcement of the laws governing the
physical well-being of the people are most emphatically in the
right when they insist that the sanitation in and about our
factories should be of a high order.

In addition to the purely quantitative estimates of micro-
organisms based upon direct examination, the analyst is enabled

Ova or the Parasitic Worms of Man
CESTODA

DRAWN TO SCALE X IOOO

1
W J

2

0|

EM
0_

Teenia v* >v>b>

solium DiDlo^=£^ Dibothrio

■ oonoporus cephalus nymenolepis nana

® drandis lalus(^rLoo„,w) ****""—■•-»
[ riyrnenolepis

>-*. ^ dimmuta

saginata

\ * Dipylidium

caninuniinr «»*■*.«»)

Cestode segments

ORAWN TO SCALE X IO W

IO MILUMITIRS ___,

1 : 1 I jf5fc

a£

Davainea
madag^cajiensis j^no.

, * lepis #i /^

ag diminuta, LS v—

Tbenia Dipylidium f lMlg| -r m%<\ H.nanaIfeQja .

solium caninum Dibothriocephalus latus saginata

d/X » '<s/ aJ.lfrd/eaJSehoaJ.

Fig. 21. — Intestinal ova. Cestodes. — (Stilt.)

to form certain opinions and conclusions regarding the source of
the contamination. For example, the hyphal development in
mold infested fruit is in marked contrast to the hyphal develop-
ment in the fruit pulp, due to unsanitary factory conditions.
This difference in hyphal structure is due to a difference in the
amount of oxygen (air) supply, of moisture, of light and the
6

68 BACTERIOLOGICAL METHODS

added ingredients (spices, sugar, vinegar, etc.), of the canning
product. The analyst can thus determine approximately how
much of the hyphal tissue present is derived from the use of
moldy fruit and how much is traceable to unsanitary factory
conditions. Again, the presence of one or more ova or larvae of
intestinal parasites, as the tape worm, would indicate sewage
contamination or contamination with fecal matter. Sand and
dirt particles indicate insufficient washing, etc. It is self evident
that the value of the report by the analyst depends upon his
knowledge of the subject and the range of his experience. Until
the work is well under way and the methods are perfected, there
is no place for inexperienced analysts in our food and drugs
laboratories.

4. Quantitative Estimations by the Cultural Methods

Estimating the number of bacteria per cc. in foods and drugs,
etc., by planting or plating definite amounts of the substances
into plate (Petri dish) culture media, is a well-known and standard
procedure. The general and special technique of the plating
method is described in the various text-books and manuals on
bacteriology. Some of the details of the method are standard,
in so far as they are generally adopted by investigators, such
as the preparation of certain culture media, making the dilutions,
counting, etc. ; in other regards there is anything but uniformity .
It is generally admitted that the results of different investigators
differ widely but there appears little unanimity of opinion as to
the factors which are responsible for these variations in quanti-
tative results.

Micro-organisms are sensitive to a degree and they respond
readily to the slightest variations in moisture, temperature and
food supply. A failure to recognize this fully in laboratory
practice leads to confusion and erroneous results. The following
are some of the more important factors which are responsible for
errors and variations in results.

CULTURE MEDIA

69

1

I. Culture Media. — Differences in the quality of the meat
used in making the meat infusions has given some marked varia-
tions in the quantitative results. Meat extracts from younger
animals give higher counts than do extracts from the meat of
older animals. Again, the prepared extracts of the different
packing houses give different results and the results obtained

Ova of the Pamsitic Worms of Man
NEMATODA

D R AW

Median focus BrSurfo.ce focus

/Modijied from StilesJyQ2\ ( lifter Stiles VMi\
Loossl«K)5 ) \ I

Q A

ArShowing Jk
sides syrfH -, \
metrically 1 •
convex

J (after Loom,

V/r^B.- Turned
m-j-n over to
r^-'ii show one
H&f!sldeflatten-

:i|^ed(Original)

Oxyuris

vermicularisA.B

IS

D.~Atypicai. unfertilized

egg ( <\ fur Loess, IQW)

Strongylus

subtilis

(after Looss. IQ05^

C. Without outer
envelope (Modified

from Stiles. r»o2. and
Looss.l^OS.]

NeccYtor
americrmus

Asccxris
lumbricoides ^ii
a B CD. Trichuris
tnchiura

(Original)

Embryo
in stool
after 12 to 4ti
. «*»>_ houra

Agchylostoma
duodenale

Atfchylostonia

. duodenale EmbrvJ

Loossiy06) 111 frt.'Sh StOOl Loos* mo.-,

Strongyloides
stercoral is

US,Vm a/. yfcd/ca/ScfiooJ.

Fig. 22. — Intestinal ova. Nematodes. — (Stitt.)

from the use of media made with manufactured meat extracts
differ from those obtained from the use of the laboratory made
meat infusions. In fact most workers are opposed to the use of
the manufactured meat extracts because of the fact that they are
mixed products and also because of the uncertainty of the amount
and number of the added ingredients any or all of which may

70 BACTERIOLOGICAL METHODS

interfere with the growth and development of certain bacteria.
Investigators have also noted great variations in results with
different brands or makes of peptones used, the quantitative
differences amounting to 50 per cent, in some cases. Equally
remarkable are the differences due to the kinds of water used in
the preparation of the culture media. For instance it is known
that agar made up with sewage encourages the development of
sewage organisms while the same medium made up with tap
water encourages the growth of bacteria predominating in such
tap water.

The gelatine used is yet another important factor in cultural
results, depending upon the age of the gelatine, its purity, the de-
gree of heating to which it has been exposed, its origin, possible
contamination with arsenic, with bacteria and mold. Other
ingredients used in the preparations of culture media cause more
or less marked variation in comparable results. The above
statements make it evident that it is absolutely necessary to
adopt and to adhere to uniform methods in order that the com-
parable results may be approximately uniform.

2. Glassware.- — Different investigators have found that the
number of bacteria in and upon culture media varied with the
composition of the glass containers used. The comparatively
soluble glass, for example, yielded enough free alkali to inhibit the
development of the more sensitive bacteria. The size of the
containers and the thickness of the glass yielded differences in
the results. It is therefore very desirable to adopt Petri dishes
and test-tubes of standard form and thickness of standard cubic
contents.

3. Other Factors. — The form and size of the incubating
chamber, the degree of ventilation, degree of darkness, amount of
oxygen present, etc., cause variations in the results.

Of even greater importance than any of the factors so far
mentioned, is the personal equation in the laboratory technique.
No two workers follow out the same details in the different steps

CULTURE MEDIA 7 1

of the laboratory procedure and very frequently proper judgment
is lacking in the application of certain details of the methods.
For example, there is lack of uniformity in the degree of heat
to be used in melting gelatin media preparatory to planting,
in the amount of material to be planted in each Petri dish, manner
of planting, time of incubating, etc.

We hereby submit the following technique in the preparation
of culture media and in the methods of making cultures in plates
as well as in test-tubes, following very generally the suggestions
as given in the report of the Committee of the American Health
Association.

5. Preparation of Standard Cultural Media, General Suggestions

1. Ingredients. — Distilled water is to be used in the prepara-
tion of all of the standard media. The distilled water must be
comparatively free from bacteria and must be kept in clean
sterilized containers and as free as possible from mineral and
organic impurities. If other than distilled water is used, this is
to be stated and the special reasons for using it indicated.

For making meat infusions, fresh lean meat is to be used,
from comparatively young animals, free from disease. Meat
extracts may be used in place of the meat infusion.

Unless otherwise specified, the peptone used should be made
from fresh beef by pancreatic digestion. It should be dry and
recently made. Workers should be sure to specify the kind of
peptone desired. Egg albumen or fibrin peptone is not to be
used in any of the standard media. The article should be secured
from some reliable house.

The gelatin to be used in the preparation of the standard media
should be of the best obtainable, the so-called French brand be-
ing, as a rule preferred. A 10 per cent, solution should not
soften when kept at a temperature of 250 C. It should be en-
tirely free from arsenic and as free as possible from acids, micro-
organisms, molds, and other impurities. A good grade of gelatin

72 BACTERIOLOGICAL METHODS

should respond to the following test: Place 0.30 gram of the
gelatin in a medium sized test-tube and add 15 cc. of distilled
water, let stand for half an hour, warm gently until all of the
gelatin has dissolved, then place the tube in water at a tempera-
ture of 1 5. 5° C. and leave undisturbed for half an hour. The
solution should remain in place when the tube is inverted.

The commercial gelatin is a variable product, being made from
varying proportions of animal tissues as hides, ligaments, bone and
bone cartilage. The purest and best gelatin is made from liga-
ments and this kind would no doubt give the most uniform re-
sults in bacteriological work, but it is apparently not possible
to obtain such gelatin in the market. The next best grade (prac-
tically obtainable) would be that made from hides of compara-
tively young domestic cows free from all foreign additions as salt,
arsenic and other hide preservatives.

Each lot of gelatin should be examined microscopically before
making it into culture media. Old yellowed and brittle material
should not be used. Examine from five to six sheets from each
pound package, using the low power of the compound microscope.
The examinations are made directly without mounting. If numer-
ous mold colonies are found as shown in Fig. 19, or numerous
mold filaments more or less scattered through the mass, it is
unfit for use as a culture medium. Numerous formed mold
colonies in the matrix indicate growth during the drying process
after the gelatin was spread on the drying screens. More or
less torn and disintegrated hyphal fragments unequally distributed
through the mass indicate infection and growth before the
gelatin was spread for drying. To examine for bacteria, mount
small bits of the sheet on a slide in water covered with cover
glass. If bacteria are numerous, approximating 10,000,000 per
cc. and more, it should not be used. In order to make more
accurate counts, take 1 gram of the gelatin and rub up in 9 cc.
of boiled distilled water and make the counts of the thoroughly
mixed sample by means of the hemacytometer. As a rule it is

STERILIZATION 73

not necessary to make plate or tube cultures to determine the
fitness of the gelatin for bacteriological work. Incidentally it
may be remarked that a gelatin which is unsuitable for bacterio-
logical work is also unfit for use as human food.

The agar should be the highest grade obtainable, and if the
shredded form is used it should always be washed in sterilized
distilled water before making into culture media.

With regard to the other ingredients required in making cul-
ture media, such as dextrose, lactose, maltose, saccharose, glycerin,
salt litmus, etc., etc., special efforts should be made to get these
as pure as possible. The degree of purity should be determined
by actual tests.

2. Sterilization.- — Thorough sterilization of all culture media
is absolutely necessary. It is, however, known that heating pro-
duces some marked changes in the molecular composition of the
media, even inducing actual chemical decomposition. It is
therefore desirable to make the time of heat exposure as brief as
possible. Ordinarily it is therefore preferable to use the auto-
clave, bringing the temperature up to 1200 C. (15 lb. pressure)
for a period of 15 min. This temperature will sterilize all
media. A shorter period does not insure complete sterilization
and a longer exposure is apt to produce inversion of the sugars
used and also permanently lower the melting point of the gelatin.
Solid media as gelatin and agar should be liquefied before placing
in the autoclave.

The following rules should be strictly observed in using the
autoclave :

a. The sterilizer should be hot when the media are introduced.
About ioo° C. Let all air escape from chamber.

b. At the end of the period of sterilization (15 min.), remove
the media and cool them as rapidly as possible.

Compliance with these rules will reduce to a minimum the
tendency toward liquefaction of the gelatin and a tendency to
decompose the various chemicals used, due to prolonged heating.

74 BACTERIOLOGICAL METHODS

If streaming or live steam is to be used in place of the steam
under pressure in the autoclave, intermittent sterilization is to
be practised. Place the media in the steam sterilizer for 30
min. on each of 3 successive days. Wait until the tem-
perature in the sterilizer has risen to approximately ioo° C.
before placing the media therein. Agar media should first be
liquefied. At the end of each period, remove the media and cool
as rapidly as possible for reasons already given.

When media are prepared under the proper laboratory condi-
tions and sterilized as above suggested, they are as a rule free from
all living germs. However, if practicable, the media should be
watched for a period of 2 days, stored in a room at ordinary
temperature, in order to note possible bacterial developments.

3. Adjustment of Reaction of the Mediae — As a rule bacteria
develop most actively in media which are slightly alkaline to
litmus and since certain media are quite acid in reaction (gelatin
in particular) it becomes necessary to reduce them to a standard
reaction. The standard indicator to be used is phenolphthalein.
When phenolphthalein is not obtainable, litmus paper (or a 1
per cent, aqueous solution of Kahlbaum's azolitmin) may be
used. The reaction adjustments are to be made as follows:

Place 5 cc. of the medium to be tested in 45 cc. of distilled
water (making a dilution of 1-10). Boil briskly for 1 min.,
with stirring or rotary shaking. Add 1 cc. of the phenolphthalein
solution (made by dissolving 5 grams of the salt in 1 liter of
50 per cent, alcohol). Titrate while hot with N/20 caustic
soda solution (in distilled water). A distinct pink coloration
marks the proper reaction. To be more precise, the pink should
correspond to a mixture or combination of 25 per cent, red and

75 per cent, white of the color top recommended by the Com-
mittee on Standard Methods of the American Health Associa-
tion. The reactions of the media are stated in terms of the
percentages of normal acid or alkaline solutions required to
neutralize them. Alkalinity is indicated by the minus ( — )

STANDARD MEDIA 75

sign and acidity by the plus ( + ) sign. Thus, if the reaction
of a medium is given as + 1.00 it indicates that it would be
necessary to add 1 per cent, of normal sodic hydrate solution to
the medium in order to bring it to the neutral point (to phenol-
phthalein). It will be observed that while the titrating is done
with the N/20 caustic soda solution, the normal solution is added
to bring the medium to the desired reaction, the stronger solution
being preferred because it reduces the amount of liquid intro-
duced. The Committee on Standard Methods specifies that the
reaction of all standard culture media shall be + 1.0 per cent,
and if it differs in reaction by more than 0.20 per cent, the medium
shall be readjusted and when a reaction other than the standard
is used it shall be indicated and the reasons for using a different
reaction shall be fully stated.

Media are preferably made in large quantities as this will
reduce to a minimum the discrepancies due to variation in the
composition of the ingredients used. As soon as made and
titrated, the media should be put into tubes and in other culture
containers, after which media containers and all are to be sterilized
according to the methods already described. To guard against
the evaporation of moisture from the media, the tubes, flasks, etc.,
should be sealed by dipping the plugged ends into melted paraffin,
or they may be capped with rubber coverings especially made
for that purpose. In case media are to be used within a few
days, sealing is not necessary but they should be kept in a moist
place, preferably in the ice-box.

6. Preparation of Required Standard Culture Media

Culture media used in bacteriological work may be divided
into those which are required for general purposes and those
which have special uses. The former should by all means be
prepared according to the standards suggested by the Committee
of the A. H. A. If special media are used, their exact composi-
tion and mode of preparation should be fully and explicitly given.

76 BACTERIOLOGICAL METHODS

Furthermore, the reasons why the special media are used should
be clearly set forth, so that co-workers may judge of their special
value and may try them out intelligently, should they care to
do so.

As special media are adopted into general use by the majority
of bacteriologists they are to be relegated into the group of general
media. For example, a few years ago, lactose-litmus-agar, Endo
medium, Hess' medium, lactose-bile medium, etc., were special
media. They are now in general use and they should be pre-
pared according to a standard method. We hereby give the
methods of preparing some of the more important media used in
general bacteriological work, following the directions of the
Committee of the A. H. A.

1. Nutrient Broth. — Infuse 500 grams of chopped lean meat
for 24 hr. in distilled water. Shake occasionally and keep in
the refrigerator. Any loss by evaporation is to be restored.
Strain the infusion through cotton or through cotton flannel.
Add 1 per cent, of peptone and warm over water bath or steam
until the peptone is entirely dissolved. Heat for 30 min. in
rice cooker or in steam sterilizer and restore any loss by evapora-
tion. Titrate with normal sodic hydrate (or normal hydro-
chloric acid) to a reaction of + 1.0 per cent. Boil for 2 min.
over open flame, stirring constantly. Restore loss by evapora-
tion. Filter through cotton (placing the cotton on cotton
flannel or on perforated filter paper). Pass the medium through
this filter until it comes out perfectly clear. Again titrate and
record the final reaction. Pour into tubes (10 cc. in each tube)
and sterilize in the manner as already directed.

This medium is much used for general cultural purposes.
It is used in making the cultures of typhoid fever germs, for
determining the phenol coefficient of disinfectants by the Ander-
son-McClintic method of rating the germ destroying power of
disinfectants. It is also used in culturing motile bacteria, etc.
Variousjndicators may be added.

STANDARD MEDIA 77

2. Sugar Broths. — Broths to which sugars are to be added
are prepared in the same manner as nutrient broth, adding i
per cent, of dextrose, lactose, saccharose or other sugar. The
sugar is to be added before sterilizing. Sterilizing in the auto-
clave is to be preferred because the longer steam sterilization is
apt to cause inversion of the sugar. The reaction of the sugar
broths shall be neutral to phenolphthalein.

These media are much used in testing for the presence of
Bacillus coli (dextrose broth). The committee states that the
removal of muscle sugar by inoculating with B. coli is not nec-
essary if small amounts of gas formation are to be disregarded.
In the routine work of testing water for the presence of the B.
coli a sufficient volume of the water to be tested is added so that
the resulting mixture will be one of normal strength. The com-
mittee also advises against the use of beef extracts in place of the
laboratory made beef infusions.

3. Nutrient Gelatin. — Make the beef infusion in the manner al-
ready described. After the first filtering through cotton or cotton
flannel, add 10 per cent, of gelatin (the per cent, being based on the
weight of the beef infusion instead of volume and the weight of
the gelatin to be on a basis of dry condition, and i per cent, of
peptone) and warm over water bath with constant stirring until
the peptone and gelatin are entirely dissolved. While dissolving
the peptone and gelatin the temperature should not rise above
6o° C. Boil for 2 min. and adjust the reaction to +1.00 per
cent. Heat for 40 min. over water bath or in steam sterilizer
and restore any loss by evaporation. Again adjust the reaction
if necessary and boil over open name for 5 min. with constant
stirring. Restore loss, filter until clear, titrate and record this
final reaction. Tube and sterilize as for beef broth and at once
store in ice chest. Protect against evaporation as already
explained.

4. Nutrient Agar. — Boil 15 grams (dry weight) of washed
thread agar in 500 cc. of distilled water for half an hour and make

78 BACTERIOLOGICAL METHODS

up weight to 500 grams. Infuse 500 grams of lean meat in 500
cc. of distilled water for 24 hr. in ice chest. Make up loss by
evaporation, strain, weigh filtered infusion and add 2 per cent,
of peptone. Warm on water bath with constant stirring until all
of the peptone is dissolved. To 500 grams of the meat infusion add
500 cc. of the 3 per cent, agar solution, keeping the temperature
below 6o° C. Boil for 1 min. and titrate to +1.0. Sterilize
in steam for 40 min. and restore any loss by evaporation. Re-
adjust if necessary and then boil for 5 min. with constant stirring.
Restore any loss due to evaporation and filter by passing it through
the filtering material (cotton and cotton flannel or perforated
filter paper) at least three times. Titrate and record the final
reaction. Tube, sterilize and store as for gelatin media. It
must be borne in mind that agar media are never as clear as broth
or gelatin media.

5. Lactose Litmus Agar. — To make this medium add 1 per cent,
of lactose to nutrient agar just before sterilizing and make the
reaction neutral to phenolphthalein.

If this medium is to be used in tubes the sterilized azolitmin
(1 per cent, aqueous solution) is added just before the final mass
sterilization, that is, the sterilization before pouring into the
tubes.

If the medium is to be used in Petri dishes, the azolitmin is
not added until ready to pour into the dishes.

The azolitmin and the lactose should be sterilized separately
before adding to the agar medium, though it is permissible to mix
the lactose with the agar and sterilize together, preferably in the
autoclave (1200 C. for 15 min.).

It would appear that the azolitmin of the market varies con-
siderably and many bacteriologists prefer the pure litmus. A 1
per cent, aqueous suspension of azolitmin should dissolve readily
when boiled for 5 min.

This medium is much used in bacteriological work on pre-
sumptive sewage contaminations, as estimating the temperature

STANDARD MEDIA 79

differential colonies (200 C. and 370 C), red colonies and total
colonies, etc.

6. Lactose Bile.- — This medium is to be made in two ways:
Add 1 per cent, of peptone and 1 per cent, of lactose to sterilized
undiluted fresh ox gall; or add the peptone and lactose to a 10
per cent, aqueous solution of freshly made dry ox gall. It is used
without titrating. Old dried ox gall should not be used. Obtain
it from a reliable dealer. If possible, make arrangements to get
the fresh undiluted ox gall from some abattoir.

This is the standard medium for making the quantitative as
well as qualitative tests for the colon group of bacilli.

7. Liver Broth. — Chop 500 grams of fresh beef liver into small
pieces and place in 1000 cc. of distilled water. Weigh infusion
and container. Boil for 2 hr. in rice cooker, starting cold
and stirring occasionally. Make up loss in weight and pass
through wire or cloth strainer. Add 10 grams of peptone, ■ 10
grams of dextrose and 1 gram of di-potassium phosphate (K2-
HPO4) . Dissolve the added ingredients by warming in rice cooker
with stirring and then titrate to the neutral point (to phenol-
phthalein). Boil for 30 min. in the rice cooker and for 5 min.
over open flame with constant stirring to prevent the carameliza-
tion of the dextrose. Make up loss due to evaporation and filter.
Tube, sterilize and store as for other media.

This is a much used enriching medium which gives asg forma-
tion with all of the species which ferment dextrose. It is also
much used to rejuvenate pure cultures of bacteria and encourages
the development of attenuated forms of bacteria.

8. HissTyphoidBacillus Medium— Two media are used. One
for the isolation of the typhoid bacillus by the plating method, and
the other for the differentiation of the typhoid germ from other
forms in tube cultures. The former is designated as the plate
medium and the second as the tube medium. They are prepared
as follows:

80 BACTERIOLOGICAL METHODS

a. Plate Medium

Agar 10 grams

Gelatin 25 grams

Salt 5 grams

Liebig's meat extract 10 grams

Dextrose 10 grams

Water (distilled) 1000 cc.

Add gelatin when the agar is melted, dissolve the gelatin, add
the other ingredients, titrate to +2.0 per cent., filter, etc., as for
other media. The medium is to be clarified by adding the whites
of one or two eggs, well beaten in 25 cc. of distilled water, boil for
45 min. and filter through absorbent cotton. Do not add the
dextrose until after clearing.

b. Tube Medium

Agar 5 grams

Gelatin 80 grams

Salt 5 grams

Liebig's meat extract 5 grams

Dextrose 10 grams

Water (distilled) 1000 cc.

The manner of preparation is the same as for the plate medium.
However, the reaction is to be +1.5 instead of +2.0 per cent.
Without the dextrose and less salt and titrated to +1.0 per
cent., the plate medium constitutes the ordinary nutrient agar-
gelatin medium which was formerly very much used because it
possessed the solidifying properties of agar combined with the
nourishing properties of gelatin.

9. Endo Medium. — This medium is much used in testing for
the colon bacillus. It is variously modified by different workers
and it is highly important that some standard method of pre-
paring the medium should be adopted and adhered to. The
following is the method of preparation and use recommended by
the committee.

Add 30 grams of powdered agar to 1 liter of cold water by
sifting slowly upon the surface of the water and allowing it to

STANDARD MEDIA 8 1

settle. Add 10 grams of peptone and 5 grams of Liebig's meat
extract. Heat in rice cooker until the ingredients are entirely
dissolved. Neutralize with sodium carbonate, using litmus as
an indicator, and then add 10 cc. of a 10 per cent, solution of
sodium carbonate.

Store the medium in lots of 100 cc. using flasks large enough
to permit the addition of the other ingredients. Sterilize for 2
hr. in streaming steam.

To use the Endo medium proceed as follows: Make a 10
per cent, aqueous solution of sodium sulphite and add 2 cc. of
fuchsin solution (10 per cent, of basic fuchsin in 96 per cent,
alcohol) to 10 cc. of the sulphite solution and steam this mixture
for a few minutes in the steam sterilizer. Add 1 gram of chemically
pure sterilized lactose to each 100 cc. of the Endo medium after
the medium has been liquefied and while the temperature is not
above 6o° C. While the medium is still liquid, add 0.5 cc. of
the fuchsin-sulphite solution and then pour into the Petri plates
and allow to harden in the incubator. The sulphite solution
must be prepared fresh as needed.

10. Milk. — The milk to be used for cultural purposes must be
pure and recently drawn. In all cases the milk of the grade or
quality known as " certified milk" is to be preferred. The
recently drawn milk is to be placed in the refrigerator for 12
hr., so as to permit the cream to rise to the top and any sus-
pended matter to sink to the bottom. Skim the milk and siphon
off all but the bottom sedimentary portion. Adjust to +1.0 per
cent. Tube and sterilize.

Litmus milk is made by adding 1 per cent, of sterilized azo-
litmin to the above. In using litmus milk always set aside a
control tube with the inoculated tubes for purposes of color
comparison.

Because of the difficulty of always getting a uniformly high
quality of cow's milk, it has been suggested that an artificial
substitute be employed. Hill and his pupils recommend a

82 BACTERIOLOGICAL METHODS

medium in which prepared casein (nutrose) is the principle in-
gredient. Chemically, nutrose is a caseinate of sodium and is
prepared as follows: Moist casein precipitated from skimmed
milk is washed with water in a solution of sodium hydroxide,
evaporating the solution to dryness in vacuo, powdering the residue
and washing successively with alcohol and ether and then dry-
ing. It is a coarse, white, odorless and tasteless powder, forming
a turbid adhesive solution with water, having an alkaline reac-
tion toward litmus and an acid reaction toward phenolphthalein.
It is a food product intended for the sick because of its easy di-
gestion. It is made in Germany but may be secured through any
of the larger American pharmaceutical houses (Victor Koechl &
Co., New York City).

The formula for making the artificial milk is as follows:

Nutrose 24 grams

Lactose 10 grams

Distilled water 1000 cc.

Dissolve the nutrose and lactose in the water (cold) for
12 hr. with occasional thorough shaking and then filter
through cotton. Tube and sterilize at no° C. for 20 min., or
in the steam sterilizer in the usual manner. No adjustment is
required.

This medium contains all of the nutritive ingredients of cow's
milk with the exception of fat which is not desired for the ordinary
cultural work. It is of uniform quality and is said to give far
more uniform results than cow's milk. It is furthermore more
translucent than cow's milk and shows the reactions with in-
dicators much better. It would be advisable to make the artificial
milk the standard substitute for cow's milk.

11. Peptone Medium. — This is simply a 1 per cent, peptone
solution in distilled water and is intended to be used for making
the indol test. Beef broth from which muscle sugar has been
removed by inoculating with B. coli is believed to be objection-

STANDARD MEDIA 8$

able because of the toxins present and which interfere with the
growth of many species of bacteria.

Other media of a more or less special character will be described
or referred to under the discussion of methods. Those described
above are the more important ones required in the bacteriological
examination of foods and drugs.

7. Technique for Making Quantitative and Qualitative Estimations
by the Plating Methods

As has been explained, the plating method is intended to de-
termine the number of living bacteria present in foods and drugs
and the results supplement the results of the method of making
the direct counts already described. From this statement it is
evident that the quantitative results by the two methods are not
the same. For example, the bacterial count of a catsup by the
direct method may be very high while the plating method may
give negative results, due to the fact that the heat sterilization
employed at the cannery killed all of the bacteria present. This
also shows why it is absolutely necessary to employ both methods
in order to form a correct estimate of the total contamination of
the substance.

The following suggestions on laboratory technique are given
with a view to the unification of methods, thereby leading to
greater uniformity in comparative results.

1. Apparatus. — Test-tubes to be used for the usual cultural
purposes shall be of medium weight and thickness, 15 cm. long
by 1.6 cm. diam. Petri dishes shall be 10 cm. in diam. Petri
dishes with porous covers are preferred. All glass ware must
be scrupulously clean and may be sterilized by exposing to a dry
heat of about 1500 C. for a period of 1 hr., after being cleaned,
wiped dry and plugged with a good grade of commercial cotton.
A browning of the free ends of the cotton plugs indicates that
the right degree of heating has been attained. A standard wire

7

84 BACTERIOLOGICAL METHODS

loop is made as follows: Bend the end of a No. 27 platinum wire,
10 cm. long, around a piece of No. 10 wire. The free portion of
the straight platinum wire inoculating needle, shall be 10 cm.
long (No. 27 wire). The standard fermentation tube shall be
of the following proportions. The length of the closed end of the
fermentation tube (diameter about 1.5 cm.) shall be about 14 cm.,
and the open end shall be of bulbous form (diameter of bulb about
3.8 cm.) large enough to hold all of the liquid in the closed end.
Larger and smaller fermentation tubes than the standard just
described may be used for special purposes. Standard and other
fermentation tubes may or may not be graduated as the special
purposes may require.

2. Amounts of Media to be Tubed. — The standard amount of
culture medium to be placed in each test-tube of standard size is
5 and 10 cc, the media to be introduced by means of a suitable
burette. Greater or lesser quantities may be used as occasion
may require. Tubes containing just 10 cc. of culture media are
required for the plating purposes. 5 cc. quantities (of gelatin,
agar and other solid media) are required for making slants.

3. Amounts of Culture Media to be Plated.— For the usual
quantitative determinations by the plating method, 10 cc. of the
culture medium shall be poured in each standard Petri dish.

The required number of tubes each containing 10 cc. of agar or
gelatin culture medium are placed in the steam sterilizer until
the medium is entirely liquefied and then placed in a beaker or other
suitable container with lukewarm water, with thermometer.
Plate the gelatin medium when the thermometer registers be-
tween 2 50 and 300 C. The temperature of the medium must not
be more than 300 C. If the temperature is less than 250 C. the
gelatin will begin to coagulate and will not pour and spread
properly. Agar media must be plated at a higher temperature
than gelatin media, usually 400 to 420 C. The Petri dishes should
be warm when the media are poured, the temperature being ap-
proximately the same as that of the medium when it is poured.

TECHNIQUE 85

This will insure a more uniform spreading of the medium over the
bottom of the dish.

To pour the liquefied agar or gelatin from the tubes, remove the
cotton plug and flame the mouth of the tube so as to kill any
bacteria or spores that may be present; raise one side of the cover
just high enough to permit bringing the tube to the middle of the
dish and pour contents into the dish over the material planted into
the middle of the dish. Let cover of the dish sink into place and
by very slight tilting of the Petri dish induce the culture medium
to spread evenly over the bottom of the dish before the medium
has had time to coagulate. As the medium spreads it also causes
the spreading of the planted material.

Many workers use 5 cc. of the medium for plating, instead of
10 cc. as above recommended. The smaller amount is satisfactory
when 1 cc. quantities are to be planted or inoculated. However,
in order to make sure that the entire area of the bottom of the
dish is well covered, 10 cc. quantities should be used. The larger
amount also minimizes the influences which the changes in
evaporation in the media may have upon the quantitative results.

4. Method of Making the Plate Cultures.— Absolutely clean
sterilized (dry heat of 1500 C. for 1 hr.) Petri dishes of the
standard size (10 cm. diam.) are used. 0.1 cc. quantities of
the substance to be cultured, or dilutions thereof, are planted or
delivered into the middle of the dish, an absolutely clean and
sterile 1 cc, pipette accurately divided into tenths. The cover of
the dish is to be lifted just high enough to permit placing the pipette
in position, and is to be replaced just as soon as possible.

In the usual water analysis work, 1 cc. quantities are generally
planted, instead of 0.1 cc. quantities as above recommended.
For purely quantitative results, the smaller amounts should be
planted because the larger amounts may include enough of the
inoculating liquid to interfere with the uniformity of results.

Formerly it was customary to mix the material to be planted
with the medium in the tube before plating. This method has

86 BACTERIOLOGICAL METHODS

some very objectionable features, chief of which is that the residue
remaining in the tube after pouring retained a certain percentage
of the organisms, thus interfering with the accuracy of the results.
It must, however, be admitted that the method has some ad-
vantages, chief of which is the more uniform mixing of the bacteria
with the medium and their more uniform distribution in the
plate, making accurate counting of the colonies easier.

5. Making the Dilutions. — Whether or not making dilutions
'is necessary depends upon the number of organisms present in
the substance to be analyzed. The number of colonies in a
Petri dish must not exceed 200 in order to make counting fairly
easy and accurate. In fact with the method of direct planting,
as usually recommended, which generally results in a some-
what irregular distribution of the bacteria (hence also the colonies
to be counted) it would be desirable to make the dilutions such
that the number of colonies in each plate shall not exceed 100.
If 0.1 cc. quantites are to be plated or planted, as above recom-
mended, it would follow that dilution would not be necessary
as long as the number of bacteria per cc. does not exceed
1000.

However, since most food and drugs contain more chan that
number of bacteria per cc, it becomes necessary to make dilu-
tions. The standard dilutions are made by tens, as 1-10, 1-100,
1-1000, and 1-10,000. The dilutions are made by adding 1 cc.
of the substance to be analyzed to 9, 99, 999 and 9999 cc. of
sterile distilled water, or other desirable sterile diluent, and
shaking thoroughly. In practice it is desirable to plate three
of the graded dilutions, so that the second higher dilution will in
all probability yield about 100 bacteria in the 0.1 cc. of the
material plated. Thus with fairly pure drinking water, the plant-
ings would be made from the undiluted water, the 1-10 and the
1-100 dilutions, presuming that there are about 10,000 bacteria
per cc. present. In case of unusually pure drinking water, that is
water in which the number of bacteria is probably not more

TECHNIQUE

87

than 50 per cc, it would be desirable to use 1 cc. quantities for
plating which would give about 50 colonies in the plate.

The thorough mixing of the sample before making the dilutions
is of the greatest importance, likewise the thorough mixing of
each dilution before taking out the quantity to be plated. Each

1

z

8

••

s

•"•

••
■i
••
••

M

•
•

I

\ • /

X^!^^/

/a

Fig. 23. — Types of growth in stab cultures. A, Non-liquefying. 1, Filiform
(Bacillus coli); 2, beaded (Streptococcus pyogenes); 3, echinate (Bacterium acidi
lactici); 4, villous (Bacterium murisepticum); 5, arborescent (Bacillus mycoides).

B, Gelatin liquefying. 6, Crateriform (Bacillus vulgar et 24 hr.; 7, napiform
(Bacillus subtilis, 48 hr.); 8, infundibuliform (Bacillus prodigiosus); 9, saccate
(Microspira finkleri); 10, stratiform (Pseudomonas^fluorescens). — (McFarland,
after Frost.)

test should be made in triplicate, taking up the 1 cc. amounts
for making the dilutions with three different clean sterile pipettes.
It is preferable to use a new pipette for each dilution. If it is
not convenient to have on hand a sufficient number of clean
sterilized pipettes, the pipette in use must be thoroughly

88 BACTERIOLOGICAL METHODS

rinsed in sterilized distilled water, using a fresh supply of dis-
tilled water for each rinsing.

Draw the liquid to be plated into the pipette, place thumb
over the upper end of pipette, let liquid run out until one of the
o.i cc. marks is reached, then bring the lower end of the pipette
close to the surface of the liquid in the diluting tube and let
just o.i cc. run out, the finer degrees of accuracy are attained
by using or not using the meniscus of the liquid projecting from
the lower end of the pipette after the last drop has fallen. This
correcting droplet is secured by touching the lower end of the
pipette lightly against the inside of the diluting tube at a point
near the surface of the liquid, without, however, actually touch-
ing the liquid. A similar adjustment may be made when taking
up the i cc. amount to be diluted, only in this case the pipette is
of course to be touched against the side of the tube or vessel
containing the liquid of which the dilution is to be made.

Thorough mixing of the contents of the diluting tube is at-
tained by vigorous shaking. Place the thumb over the open-
ing of the tube, interposing a piece of sterilized rubber sheeting
such as is used by dentists. Some workers mix the contents of
the tube by rapidly rotating between the two hands and by tap-
ping against the palm of one hand.

6. Incubation.— The regulation incubators are to be used.
It is highly important that there should be ample ventilation, a
matter to which amateurs and even experienced bacteriologists
as a rule give little or no attention. All modern incubators are
supplied with ventilating openings at the top which should be
kept open most of the time. The air in the incubating chamber
should be practically saturated with moisture, which may be
accomplished by placing a flat dish containing water in the lower
chamber.

Two standard incubating temperatures are employed, namely,
20° C. and 370 C, the first corresponding to the ordinary room
temperature and the second to the body (human) temperature

TECHNIQUE

89

or blood heat. The devices to regulate the temperature should
be such that the variation from the two standards given shall not
be more than 20, that is, not more than i° in either direction.

There is no standard time of incubation. For work in the
study of water sanitation as carried out in Germany, England
and also in the United States, gelatin plates are incubated for 2
days at a temperature of 200 C. It is suggested that the period
be extended to 3 days in order to get more accurate results.

fc\

•i >.

i

/A \

Fig. 24. — Types of streak culture. 1, Filiform (B. coli); 2, echinulate (B. acidi
lactici); 3, beaded (Str. pyogenes); 4, effuse (B. vulgaris); 5, arborescent (Bacillus
mycoides). — (McFarland after Frost.)

From 1 to 2 days is the usual time of exposure for the higher
temperature (370 C).

8. Practical Application of the Quantitative Estimations by the

Plating Methods

The relative importance of the quantitative bacteriological de-
terminations by the method of direct counting and by the plating
method has been explained. Both methods must be made
standard in every food and drug laboratory. Quantitative esti-
mations by the plating method should take precedence with all
substances containing largely living organisms such as water
supplies of all kinds, milk, raw meats, and shellfish, etc., and all
substances in which infection is suspected, even though such sub-

90 BACTERIOLOGICAL METHODS

stances may have been subjected to processes of sterilization during
some phase of the processing or of manufacture.

In a general way the quantitative results by the plating method
are to be interpreted in a manner similar to the results by the
direct count. In some cases the question at issue may be relative
to the presence or absence of viable bacteria in substances which
presumably do not contain living organisms, such as canned foods
generally. Manufacturers of canned products are of the opinion
that the methods of heat sterilization employed will kill all of the
bacteria which may have been present at the time of canning.
This is undoubtedly true in many cases, but in other instances it is
only too evident that retarded fermentation processes continue
after the cans are sealed, which accounts for the high counts in
canned food products which contained only small numbers of bac-
teria at the time the cans were sealed. These subdued fermenta-
tion processes as a rule do not result in the formation of sufficient
gas to produce " swells" and hence the article is not suspected
until the container is opened when a more or less disagreeable or
peculiar odor is noticeable, which is, as a rule, not sufficiently
pronounced to prevent the use of the article as food.

In addition to the purely quantitative results, the plating
method indicates the general qualitative character of the organ-
isms present and conveys some idea as to the course of the infection
or contamination as shown by the characters of the colonies de-
veloped in the Petri dish or in the tubes.

9. Qualitative Determinations

The chief qualitative determinations in food and drugs labora-
tories pertain to sewage contamination. The recognition and
determination of pathogenic bacteria as the typhoid bacillus,
the cholera bacillus, diphtheria bacillus, etc., is an incidental
possibility in the food laboratory routine and not a regular part
of it. Of far greater significance is the recognition of the evidence

QUALITATIVE DETERMINATIONS 9 1

of the presence of intestinal parasites, such as the segments of tape
worm, the larvae and ova of such parasites, etc., as already stated
under the discussion of the direct method of making counts. The
bacteriologists in food and drugs laboratories should be qualified
to recognize all of the possible disease germs and the smaller carriers
of disease which may be associated with food substances and they
should be able to demonstrate the presence of such contamination,
if necessary. The thus far recognized routine in food and drugs
laboratories and in public health laboratories is limited to making
the so-called presumptive colon bacillus test, as indicative of sewage
contamination or contamination with fecal matter. Sewage con-
tamination means primary contamination with fecal matter. The
reason why the colon bacillus test has been selected as giving satis-
factory evidence of sewage contamination is because this bacillus
is most abundant and is constantly present in fecal matter. Any
considerable number of colon bacilli in water supplies or in food
substances is evidence of gross negligence and defects as to sanitary
requirements.

As far as the practical work in finding evidence of the sewage
contamination of food substances is concerned, there is no effort
made to isolate and identify a definite bacillus recognizable as
Bacillus coli. It is rather the recognition of certain cultural
characteristics which have come to be recognized as being peculiar
to the bacilli, known as the B. coli group, all of which are traceable
to intestinal origin. Furthermore this group of bacteria is very
widely distributed in the animal kingdom, being in no wise limited
to the intestinal tract of man. The B. coli group of the lower
animals is in all probability different from that which inhabits
man and certain workers have made attempts to differentiate
them by means of special cultural methods, but thus far these
methods are not sufficiently perfected to be used practically in
food and drugs laboratories. These statements also apply to the
Streptococci group of intestinal origin. However, some of the
laboratory results thus far attained would indicate that in the

92 BACTERIOLOGICAL METHODS

near future it will be possible to differentiate between pollutions
traceable to human origin and such as are traceable to cow, horse
or hog manure, for example. It cannot be denied that food
materials intended for human consumption which are contamin-
ated to any distinctly appreciable amount with the contents of
the intestinal tract of any animal, are unsanitary and hence unfit
for human consumption.

In-as-much as the intestinal bacteria (bacilli and streptococci)
are very abundant and very widely distributed, it is quite evident
that it would be impracticable to pronounce all foods unfit for
use if only one or a few intestinal organisms were found to be present
in a comparatively large quantity. Human feces contains about
one-third bacteria (dry weight), the majority of which belong to the
colon group, and the exterior of the human body carries bacteria
derived from the intestinal tract, especially the hands and the
deposits under the finger nails. Flies are carriers and distributors,
of intestinal bacteria. The dust of the streets and street sweepings
contain large numbers of bacteria derived from the intestinal tract
of the horse, etc. It would be impracticable to enter into a
fuller discussion of the distribution of bacteria traceable to in-
testinal origin. Suffice it to state that it is the work of the food
bacteriologist to determine the presence, in articles intended as
food, of those bacteria which indicate contamination with fecal
matter, no matter what the source of such objectionable matter
may be. The basis for the condemnation of contaminated foods is
quantitative in the comparative sense. For example, the finding
of a few colon bacilli in large quantities of water or their occasional
presence in small quantities of water, does not indicate that the
water is unsuitable for drinking purposes. If, however, the colon
bacilli appear in a large proportion of many small samples (i cc.
or less) of water it is safe to conclude that there is considerable
recent sewage contamination and that such water is dangerous
to health. The intestinal bacteria are in themselves not seriously
pathogenic to man even when taken in considerable numbers.

QUALITATIVE DETERMINATIONS 93

The real source of danger lies in the fact that the intestinal bacteria
normal to man and the lower animals, may be and frequently are
associated with pathogenic bacteria, such as the typhoid bacillus
and the dysentery bacillus. Our long experience with the con-
sumption of sewage contaminated water supplies, has shown that,
as a rule, the first danger sign of the excessive contamination was
usually a marked increase in the number of cases of dysentery,
generally followed by sporadic cases and epidemics of typhoid
fever.

The practical results of the quantitative bacterial determi-
nations of food substances combined with the qualitative tests
for the colon group, has proven of the highest value and it is
considered entirely feasible to continue the application of the
tests and to suggest ways and means of improving the laboratory
technique covering such methods. The qualitative methods
thus far worked out are based upon a knowledge of the life history
of the bacteria concerned and may be briefly stated as follows:
Normal intestinal bacteria and such other bacteria as may develop
in the intestinal tract, such as the typhoid bacillus, the cholera
bacillus, the dysentery bacilli, etc., are adapted to a temperature of
about 37° C. and they feed upon the food materials found in the
intestinal tract and have a somewhat reduced oxygen supply.
Among the substances peculiar to the intestinal tract we find bile,
pancreatin, and other enzymes and a certain water percentage and
the various ingredients of food materials more or less digested
and the various products elaborated by the different species and
varieties of bacteria present. It is a study of the peculiarities of
the intestinal bacteria which has suggested the technique for
their isolation and their quantitative as well as qualitative esti-
mation in food supplies, as can be seen readily from a study of
the culture media and cultural methods recommended. To
enter into any fuller discussion of the work done by American and
European investigators on the bacteria which are normal to the
intestinal tract or which may occur in the intestinal tract in

94

BACTERIOLOGICAL METHODS

disease, is not practicable but we desire to give the following
tabulation showing the relationship between the different species
of the colon-typhoid group of intestinal bacteria in their behavior
with dextrose and lactose media.

Bacteria of the Colon-typhoid

Group

Species

Dextrose

Lactose

Gas Formation

Acid Formation

Gas Formation

Acid Formation

B. alcaligenes

B. typhi

B. dysenteries

none
none
none

active
active
active

active

none
slight
distinct

strong
strong
strong

strong

none
none
none

none
none
none

active

none
none
none

B. enter itidis

Paratyphoid group

Hog cholera bacillus

B. coli

slight
slight
slight

strong

There are numerous other distinguishing characteristics be-
side those indicated in the above tabulation, as agglutinating phe-
nomena and behavior with other special culture media. It is
simply desired to indicate somewhat more specifically the lines
of research which were necessary to determine the identity of the
related species and varieties of intestinal bacteria.

The Bacillus coli was isolated as early as 1884 from the feces
of a cholera patient, at which time this organism was supposed
to have some causal relationship to cholera. Later it was proven
that this bacillus was a normal inhabitant of the intestinal tract
of man and of other animals, being regularly present in their
excreta, and this discovery proved of the highest importance to
sanitarians as the presence of this germ in water supplies, in milk,
in mineral waters, etc., is generally regarded as evidence of
sewage contamination. The colon bacillus has been found in
sewage contamination, river water, in spring water, ice, milk,

QUALITATIVE DETERMINATIONS 95

cream, butter, buttermilk, sour milk tablets, mineral waters,
oysters, clams, flour, oatmeal, cornmeal, cereals, frozen eggs, dried
nuts and fruit, etc. B. coli is not normally present in sea water
and its occurrence in salt water shellfish is evidence of sewage
contamination. The occurrence and general distribution of
the colon bacilli is almost in direct proportion to the density of
the population. Animals of all kinds are disseminators of colon
bacilli, particularly the larger animals as the horse, cattle, the
dog and domestic fowls. Within and about the home, the house-fly
and the stable-fly are the chief distributors of colon bacilli. We
may repeat that colon bacilli are found on the skin of persons,
particularly on the hands and under the finger nails. The under-
clothing worn carries these bacilli and is the agent instrumental
in distributing them over the exterior of the body, especially in
those of uncleanly habits. Water in which the hands have been
rinsed will generally yield positive colon bacillus tests. It is
also apparent that the colon bacillus does not survive for a great
length of time outside of its natural environment; thus sewage-
contaminated waters purify themselves of colon bacilli after a
time, the period varying with the temperature and the amount
of organic matter present. Thus it sometimes happens that a
water supply may show a high bacterial count and yet be quite
free of colon bacilli. As a rule, however, water supplies and
substances brought in contact with such water supplies which
show a high general bacterial count, will also show a comparatively
high count in colon bacilli. There may be notable exceptions to
this rule. A water supply, or other liquid substance, may show
a comparatively low bacterial count and yet yield numerous
colon bacilli. Such an occurrence would indicate an unusual
source of extensive sewage contamination.

From the foregoing it is evident that the sanitary examination
of foods and drugs resolves itself into the making of quantitative
bacterial counts, as already fully explained, and the presumptive
colon bacillus test, with an occasional test for other specific

96 BACTERIOLOGICAL METHODS

organisms, as will be explained later. It is also evident that
the isolation or the identification of the colon bacillus in a mixed
contamination as all sewage-contaminated substances are, is
not as simple a matter as might appear on first consideration.
However, the presumptive tests for the presence of the colon
bacillus in definite quantites of the food materials or liquids used
with, or associated with, certain food materials, is almost uni-
versally accepted as evidence of the dangerous contamination
with sewage. It is, however, quite clear that health officers
should not adopt hard and fast rules or standards for the con-
demnation of foods because of such evidence of sewage con-
tamination. Very naturally, the standard for water supplies
will not apply to oysters and shellfish generally and the standard
for shellfish will not be practically applicable to mineral waters,
etc. With substances of which the standard or quality is quite
generally based upon a numerical count, as for example milk,
the presumptive colon bacillus test need not be applied, unless
it is to be carried out as giving corroborative evidence of the
sewage contamination.

One of the first important duties of the food and drugs bac-
teriologists will be for them to get together and agree upon uni-
form methods and to decide upon the kinds of bacteriological
examination under the pure food and drugs act to which the
quantitative as well as the qualitative (presumptive colon bacillus
test) determinations are applicable, in harmony with our present
knowledge of food bacteriology. The working laboratory methods
adopted must be practicable and must be carried out primarily
as a better protection of the physical well-being of the consumer,
incidentally also safeguarding the business interests of the con-
scientious manufacturers. The following suggestions are in-
tended to indicate along what lines the practical qualitative work
may be done and also to outline certain research work which should
be carried on in order to develop the working methods to greater
perfection and to add such new methods as may prove useful.

SEWAGE CONTAMINATION

97

io. Evidence of Sewage Contamination. General Methods.

It may be assumed that the presence of any or all of the
large group of colon bacilli in water or in food substances is indi-
cative of sewage contamination or contamination with fecal
matter. The colon bacilli are aerobic, nonsporeforming, motile,
short and produce acid and gas in dex-
trose and lactose media and develop
best at a comparatively high tempera-
ture (3 70 C). A practical presumptive
colon bacillus test depends upon the
characteristics thus indicated and is car-
ried out as follows:

1. Presumptive Colon Bacillus Test.
— Add the substances to be tested
(water, sewage, mineral water, shellfish
liquor, washings from vegetables, etc.)
in 0.01 cc, 0.10 cc, 1 cc, 5 cc. and 10
cc. quantities (or these equivalents in
dilutions) into fermentation tubes hold-
ing at least 40 cc. of lactose bile, incu-
bate at 370 C. and look for the forma-
tion of gas. If gas formation is observed
the presence of colon bacilli may be sus-
pected. If, in the case of water supplies
for example, the 0.10 cc. tubes show gas
formation then it may be reasonably as-
sumed that colon bacilli are present. If

two out of five of such tubes give positive gas reactions, the
test may be considered conclusive. To test the gas formed,
fill the tubes showing gas formation with a 2-per cent, solution
of sodic hydrate, hold thumb firmly over the opening of the fer-
mentation tube and mix contents by tilting back and forth
carefully. The volume of gas absorbed is C02 whereas the un-

Fig. 25. — Fermentation
tube. This type of fermenta-
tion tube is especially conven-
ient for making the gas deter-
mination with the colon bacil-
lus. Other forms of fermenta-
tion tube may be used. — {Pitt-
field.)

98 BACTERIOLOGICAL METHODS

absorbed portion is supposedly hydrogen. The colon bacillus
shows a gas formation of H hydrogen. The standard time of
incubation is 48 hr., but if colon bacilli are abundant, gas forma-
tion will be observed in the tubes carrying the larger amounts of
the inoculated material at a much shorter time, occasionally within
a few hours. Small numbers of attenuated colon bacilli may
require 2 and 3 days before there is any gas formation
noticeable. In this connection it may be mentioned that the
attenuated colon bacilli indicate remote contamination, as all
B. coli of recent contamination develop readily in lactose bile.

Fig. 26. — Bacillus coli. Superficial colony on a gelatin plate 2 days old (X 21).
— (McFarland after Heim.)

This constitutes the usual presumptive test for the presence of
sewage contamination. Some investigators, however, recommend
that the test be supplemented as follows: Plant the suitable
quantities or dilutions into liver broth (in test-tubes) and in-
cubate at 370 C. for about 12 hr. and then transplant these cultures
into the lactose bile as above explained. The liver broth en-
richment medium is said to bring out the attenuated forms of
colon bacilli. In routine procedures the liver broth culturing is
usually omitted as the important point at issue is the determina-
tion of fairly recent contamination with sewage, or of sewage

COLON BACILLUS TEST 99

contamination in large amount, and the lactose bile medium
gives conclusive results regarding this.

The presumptive colon bacillus test is to be supplemented
further as follows: Plate suitable dilutions of the substances to be
tested for sewage contamination (0.001 cc, 0.01 cc, 0.10 cc,
1. 00 cc.) into lactose litmus agar Petri dishes, making two sets.
Incubate one set of these plate cultures at 200 C. and the other at
370 C. and note the following:

1. The relative number of colonies which develop at the two temperatures.

2. The number of acid-forming colonies.

The time of incubation at the lower temperature (200 C.)
should be 3 days, although fairly conclusive results may be
noted at the end of the second day. The standard time of incuba-
tion at the higher temperature (370 C.) is 48 hr., although certain
results may be noted at the end of 24 and 36 hr. If the propor-
tion of high temperature colonies is high, it is indicative of the
presence of numerous bacteria derived from the intestinal tract.
If the high temperature colonies approximate (numerically) the
low temperature colonies, sewage contamination may be suspected.
If in addition many of the high temperature colonies show pink
or vermilion (on lactose-litmus agar), the sewage contamination
is practically proven. Both the colon bacilli and the sewage
streptococci show pink colonies on this medium, the latter being
the brighter, more vermilion in coloration. This coloration is
due to the formation of acid by the organisms named which reacts
with the litmus. Examine the pink colonies under the micro-
scope in order to determine which are the colon bacilli and which
the streptococci. As a rule, high temperature colonies should not
exceed i : 100 as compared with the low temperature colonies. It
must be kept in mind that the pink colonies may turn blue within
24 hr. due to the liberation of ammonia and amines. Red
colonies indicate lactose fermentation with formation of acid, but
since bacteria other than the colon bacillus form acid (notably

IOO

BACTERIOLOGICAL METHODS

the streptococci), it is desirable to examine such colonies micro-
scopically and to inoculate into other media and perhaps to
test for indol formation, in order to obtain satisfactory proof as
to whether or not they are colon bacilli.

Neutral red (a safranine dye) reduction was at one time con-
sidered a very important check test for the colon group. Stokes,
as early as 1904, recommended that neutral red be added to lactose
j bratk in the fermentation tubes which contain the required dilu-
>-

69

fr: J2

as eg

=3 3

— : CD TZ

CO JET

c* <rr>

CO

<

CO

-J

Fig. 27. — B. coli showing flagellar stained by the van Ermengen method (X
1000). — (MacNeal, from McFarland after Migula.)

tions of the liquids to be examined. 30 to 50 per cent, gas forma-
tion in the closed arms of the tubes and the change of the neutral
red to canary yellow, is said to be characteristic for the colon
group. It would appear that the majority of bacteriologists are
inclined to omit the neutral red test as being of little value.

The production of indol in peptone broth or solutions is another
colon bacillus test much used in the United States. Boehmes'
modification of the Ehrlich method is now generally employed,
made as follows: Two solutions are required.

COLON BACILLUS TEST

IOI

Solution No. I

Para-dimethyl-amido-benzaldehyde 4 parts

Absolute alcohol 380 parts

Concentrated HC1 80 parts

Solution No. II
Sat. sol. of potassium persulphate.

The indol test is performed as follows: Add 5 cc. of solution I
to 10 cc. of a broth culture and then add 5 cc. of solution II, the

s

\ ■

iBmmm

Fig. 28. — Bacillus of typhoid fever, stained by Loeffler's method to show flagella

(X 1000). — (Williams.)

whole being then shaken. A red color indicates indol. Some of
the leading bacteriologists consider this a very valuable test.

The so-called hog cholera group or the Gaertner group of bacilli
is important from the standpoint of the food bacteriologist. The
Gaertner group occupy a position intermediate between the
chemically active coli group and the chemically inert typhoid
group and includes the following important species or rather strains
— the Bacillus enteritidis strain which includes many of the bacteria
isolated in cases of food poisoning and some of the B. typhi murium

102 BACTERIOLOGICAL METHODS

varieties, as B. psittacosis, and B. suipestifer and B. paratyphosus B.
They differ from the typhoid group by gas formation in dextrose,
and from the colon group by the production of an alkaline reaction
in milk. They are concerned in the development of intestinal
disturbances such as dysentery and diarrhea. No practical
routine working method for the isolation of the Gaertner group
has as yet been recommended. Some of the more important
cultural characteristics are indicated in the table of Bacteria of
the Colon- typhoid Group.

Another important group of bacteria from the standpoint of
the food bacteriologist is the large group of sewage streptococci.
They occur in the intestinal tracts of many animals. There
are numerous strains of this group and they are somewhat less
widely distributed than the colon group. The determination of
sewage streptococci adds but little more than may be learned from
the colon test and for this reason we shall not enter into any fuller
discussion. This statement also applies to the host of other bac-
teria and related organisms which are more or less constantly
associated with sewage and sewage contaminations.

For all practical purposes, the presumptive colon bacillus test
supplemented, as the special cases may require, with certain
special tests, combined with the quantitative counts by the
plating method (gelatin media) will give all the information which
is necessary to judge of the quality of certain foods, drinks and
medicamenta, as far as the contamination with sewage is concerned.
These points will be more fully discussed under special heads.

ii. Possible Contamination of Foods with the Typhoid Bacillus

Testing food substances and medicamenta for the presence of
the typhoid bacillus will never become a regular routine in the
food laboratory. On occasion it will become an incidental pro-
cedure and must therefore receive some consideration. To under-
stand the special significance and importance of this organism
as a possible contaminator of foods, it is necessary to enter into

TYPHOID BACILLUS CONTAMINATION 103

a brief statement of the typhoid fever and the organism which
causes this disease. The primary cause of typhoid fever is the
Bacillus typhosus, which in its general morphological characteris-
tics resembles the colon bacillus, differing in that it is somewhat
longer and more actively motile. When introduced into the
intestinal tract of man it multiplies very actively and produces
the symptoms of the disease known as typhoid fever. In disease,
therefore, this organism grows in the same environment as the
colon bacillus, excepting that the temperature (fever temperature)
is higher. After recovery from the disease, the germs may remain

Fig. 29. — Bacillus typhosus, 72-hr. gelatin culture. — (Stilt, after Kolle and

Wassermann.)

in the intestinal tract for long periods of time, for months and years.
Furthermore, those who have never had the disease may become
infected with the germs and carry them for long periods of time
without developing the disease. Persons infected with the germs
of typhoid fever without suffering from the disease are known as
typhoid carriers, and it is self-evident that they may cause typhoid
fever in those with whom they may come in contact. Numerous
such carriers have been found and many sporadic case- oi typhoid
have been traced to such source. However, the majority of
typhoid epidemics are traceable to foods and drinks contaminated

io4

BACTERIOLOGICAL METHODS

with the intestinal secretions of typhoid patients. The subject
of typhoid contamination is therefore intimately associated with
the general subject of sewage contamination or contamination
with human fecal matter. Very naturally, the typhoid bacillus
is far less common than the colon bacillus. In a general way it
may be stated that the distribution of the typhoid bacillus is as
wide as the distribution of typhoid contaminated sewage. As
long as we adhere to the antiquated and highly unsanitary method

V*

Fig. 30. — B. typhosus from gelatin smear preparation stained with fuchsin (X

1000). — (MacNeal.)

of emptying our sewage into the drinking-water supplies just so
long will we continue to have epidemics of typhoid fever. Numer-
ous statistical records show that the mortality rate from typhoid
fever in our larger cities is directly proportional to the filthiness
of the drinking-water supply. House-flies are known to be carriers
of typhoid and the germs have been isolated from vegetable food
materials, from oysters and other shellfish, from milk, etc.

The laboratory procedure in the examination of foods and
liquids for the typhoid bacillus includes the isolation and identi-

TEST FOR TYPHOID BACILLUS

I05

fication of the germ. The proceedings are similar to those out-
lined for the colon bacillus, excepting that in this case the quan-
titative factor is not considered. The finding of a single typhoid
fever germ in a mass of food materials is sufficient to condemn it.
It may be assumed that where there is one typhoid bacillus there
are more in the same vicinity and these may initiate an epidemic
of typhoid fever.

The food bacteriologist may be called upon to examine food
substances for the presence of typhoid contamination (from the
feces of typhoid patients or of carriers) in instances where it is
known that food has been exposed to typhoid infection or where
such infection is merely suspected. The isolation from foods and
the positive identification of the typhoid bacillus is by no means a
simple matter. It is necessary to make use of special cultural
methods, supplemented by the agglutination test, etc. The
methods tried out by various bacteriologists are too numerous to
even review and most of them have after a time been abandoned
as unsatisfactory. The following tabulation from the work of
Prescott and Wilson indicates some of the more practical laboratory
procedures which have been tried with more or less success.

Examination of

water for typhoid (

bacilli

Physical concen-
tration

2. Enrichment

3. Isolation.

4. Identification.

a. By filtration.

b. By agglutination

(Schuder's 1
Fischer's I Proc-
Wilson's f ess.
[ Muller's J

a. Hoffman and Ficker's caffein process.

b. Jackson's lactose bile.

c. Parietti's carbol broth.

a. Eisner's gelatin medium.

b. Endo's medium.

c. Loeffler's malachite green medium.

d. Drigalski-Conradi agar.

e. Hiss's medium.
/. Hesse's medium.

a. Morphological and cultural charac-

teristics.

b. Agglutination.

io6 BACTERIOLOGICAL METHODS

Space will not permit discussing the methods thus outlined
nor is this essential for the present purpose. Those interested
arc referred to the work by Prescott and Winslow, Elements of
Water Bacteriology (19 13), which contains a fairly complete digest
of the methods. Furthermore, the methods adopted must be
suited to the special cases in hand. The most suitable procedure
for isolating the Bacillus typhosus from drinking water would
not be practicably applicable in the examination of typhoid con-

Fig. 31. — B. typhosus from an agar culture 6 hr. old. Highly magnified
(X iooo), showing the flagellar stained by the Loeffler method. — (McFarland after
MacNeal.)

taminated sewage or milk, for example. For the time being there
is no routine laboratory method for the isolation of the typhoid
bacillus and we must content ourselves with a brief consideration
of those methods which will in all probability give the best
results.

It is of the highest importance that the food bacteriologist
should search out typhoid contaminated foods before the occur-
rence of an epidemic. In fact, if such work is not undertaken
until cases of typhoid have developed, the bacteriological find-

TEST FOR TYPHOID BACILLUS 107

ings are often wholly negative, because of the long incubation
period (14 days), so that the bacilli may all have disappeared
from the sewage or water between the time of the infection and
the manifestation of the symptoms of the disease. Under con-
ditions favorable to the typhoid germs, as food supply, temperature,
absence of sunlight, etc., they may survive for several months.
It is generally conceded that the Bacillus typhosus is quite re-
sistent and persistent. According to Ravenel, the germ survives
for several months and longer in fecal matter deposited in snow
which when carried into the stream supplying a
city with drinking water by the early spring
rains caused an outbreak of typhoid.

The highly objectionable method of using
human excrement for fertilizing the soils of
truck gardens, as practised by the Chinese and
others, may lead to the typhoid contamination F _IU

of the vegetables grown in such gardens. Wash- trating the Widat
ings of the soil and of the vegetables should be nomenon.^Uppe^r

examined for typhoid germs. h.alf bef°re the pac-

tion. Lower half

The following general method for the isola- shows clumping of

the motionless
m—(Pittfield. I

tion of the typhoid bacillus is suggested, subject

to modification to suit special cases.

1. Concentration.- — Run from i to 10, and more, liters of
water (as from well, cistern, stream, water tank, etc.) through a
clay filter. Just before all of the water has passed through the
filter, shake it up and pour into a suitable centrifugal tube (the
special tube already described will answer the purpose very well)
and place in incubator for 30 min. at a temperature of 370 C.
The incubating is done for the purpose of increasing the motility
of the typhoid bacilli.

2. Separation by Centrifugalization. — Take tube from the
incubator and centrifugalize for from 5 to 30 min. at a high speed.
The non-motile bacteria will be thrown down first, while the

108 BACTERIOLOGICAL METHODS

highly motile Bacillus typhosus will tend to remain near the middle
and upper parts of the tube.

3. Cultural Separation on Basis of Motility. — By means of a
sterile pipette take up the upper half or third of the contents
of the centrifugalized tube (2) and place in the special loop tube
with phenol-broth and incubate at 370 C. for 24 hr., or longer if
necessary.

4. Plate Cultures. — Take up several platinum loopfuls from
the loop tube (the opening opposite the inoculated end) and
plant in lactose-litmus-agar (at 370 C.) and note the character
of the colonies which form. Compare with the colon bacillus
colonies. Examine colonies microscopically.

5. Other Cultural Tests. — Test for absence or presence of gas
formation. Enrichment in liver broth may be tried, etc.

6. Agglutination Tests. — Two methods may be used. The
microscopical and the macroscopical. The usual routine mi-
croscopical method is carried out as follows: By means of a
clean sterile pipette place 0.1 cc. of the typhoid serum and 0.9
cc. of physiological salt solution (salt is necessary to bring about
agglutination) in a clean sterile Syracuse watch crystal and
mix thoroughly by means of a clean sterile glass rod. This
gives a serum dilution of 1-10. Place one platinum loopful of a
24-hr. bouillon culture of the typhoid bacillus on a clean cover
glass and add one loopful of the mixture from the Syracuse watch
glass. This gives a dilution of 1-20. Two loopfuls of the
culture and one of the serum mixture gives a dilution of 1-40.
Three loopfuls of culture and one of serum mixture gives a dilu-
tion of 1-80. Make the dilutions one at a time and place the
cover glass holding them (inverted) on a vaselined hollow or
concave slide and examine at once under the high power, con-
tinuing the observation for 30 min. if necessary. The first
change noticeable will be a gradual loss of motility, followed
by a clumping of the now non-motile germs. This constitutes
a positive agglutination reaction. Clumping with the lower

TYPHOID AGGLUTINATION TEST IO9

dilutions (1-20, 1-40) is not considered characteristic for the
typhoid organism, since other bacteria may also produce agglutina-
tion with the typhoid serum. It is, however, not likely that
sera will agglutinate other than the specific one in dilutions as
high as 1-80. Higher dilutions should be tried on the principle
that the positiveness of the test is in proportion to the serum
dilution which will produce clumping. It should also be borne
in mind that the agglutination phenomena are more marked at
the body temperature (370 C.) and that in the case of the typhoid
serum, the paratyphoid group will also give positive results.
In reporting on the agglutinating phenomena always give the
dilution and the time factors. The novice must frequently be
reminded that all manner of solutions of salts, acids, etc., will
produce agglutination with most bacteria. We would not
recommend the use of the blood-counting pipette (which accom-
panies the hemacytometer) for making the dilutions and mixtures
of the serum and the bacterial cultures, as is advised by some
investigators, largely because of the danger of possible infection
in sucking up the quantities of bacteria, and also because this
method adds nothing to the value of the results.

For the so-called macroscopical method or precipitation method,
as it is also called, small test-tubes are used in which the suitable
dilutions of the serum (with normal salt solution) and the bacterial
cultures are mixed. A positive reaction is indicated by rlocculency
and the deposition of a slight precipitate. Dead (formalized)
typhoid cultures may be used. The method in general use in
Germany is preferred, a description of which may be found in
most text-books on bacteriology. Some of the American pharma-
ceutical houses (Parke, Davis & Co.) market a full equipment
for making the macroscopic agglutination test with the typhoid
germ. It contains full directions for using and according to re-
ports is as reliable as this test can be made for practical purposes.
It need hardly be stated that in all cases it is desirable to make a
control test with normal salt solution.

HO BACTERIOLOGICAL METHODS

The following is offered by way of fuller explanation of some
of the details of the method above outlined for the isolation and
identification of the Bacillus typhosus. The unusually active
motility of the typhoid germ has been utilized by several in-
vestigators (Drigalski and Starkey) as a means for separating
it from less highly motile forms. Drigalski allowed from 5 to
10 liters of the suspected water to stand in tall milk cans for 1 or
2 days at the room temperature, after which he plated definite
amounts taken from the surface of the container into litmus -
lactose-agar. By this method he was enabled to isolate typhoid
bacilli from several contaminated springs. Starkey used glass tubes
bent into four loops which after being filled with phenol broth were
inoculated at one end and incubated anaerobically at 370 C. for
24 hr. The more actively motile typhoid bacilli found their way
to the fourth loop from which they were isolated by plating. The
centrifugal method above recommended is merely an adjunct to
the methods employed by Drigalski and Starkey. The non-
motile bacteria are thrown down first and in a very short period
of time thus being an advantage over the Drigalski method in
which gravity is the separating force. It is true that in time the
motile forms would also be thrown down. It is therefore im-
portant not to prolong the centrifugalizing more than is necessary.
In place of the four-loop Starkey tube we would suggest the use
of four separate tubes; one a simple U-tube or single-loop, a W- or
double-loop, a three-loop and a four-loop tube. These tubes, after
being cleaned and sterilized are filled with phenol broth and in-
oculated at one end at the same time. Incubate at 370 C. or even
at 400 C. and examine loopfuls taken from the ends opposite the
ends inoculated as follows: The U-tube at the end of 6 hr.,
the double-loop tube and the three-loop tube at the end of 12 hr.,
the three-loop tube (reexamination) and the four-loop tube at the
end of 24 hr., and the four-loop tube again at the end of 36 hr.
if necessary. The phenol broth and the higher temperature hinders
the growth of most bacteria without checking the growth of the

TEST FOR TYPHOID BACILLUS

III

typhoid germs. These conditions will enable the highly motile
Bacillus typhosus to reach the more remote loops first where they
may be taken out by means of -the platinum loop or the pipette.
In place of the loop tubes above described and which can be

Fig. 33. — Loop tubes for culturing and isolating typhoid bacilli and other
motile bacteria as explained in the text. 1, Single-loop or U-tube; 2, double-loop or
W-tube; 3, three-loop tube; 4, four-loop tube. The tubes are filled with phenol
broth or other desirable media and inoculated at the ends marked (a). Material
for subculturing and for microscopical examination is taken from the opposite end
(b), at varying intervals of time.

made in the laboratory, it would be preferable to use a single tube
of four or five loops provided with openings at each of the upper
turns of the loops, thus making five or six openings in all, from
which the quantities to be examined and plated may be taken.

112 BACTERIOLOGICAL METHODS

The tubes must be fastened to suitable stands or supports to pre-
vent, as much as possible, the mechanical mixing of the contents
after the inoculations are made. It is perhaps self-evident that
concentrates or high contaminations are to be inoculated into the
tubes. The tubes should be large enough to hold at least 50 to
100 cc. of medium and suspected water in equal parts.

12. Possible Contamination of Food Substances with the Cholera

Bacillus

In the United States the contamination of foods with the
cholera vibrio is far less likely than the contamination with the
typhoid fever germ, yet it is a possibility to be reckoned with.

Fig. 34. — Spirillum cholera, from broth culture, stained with fuchsin (X 1000). —
(Slitt, after Kolle and Wassermann.)

The cholera germ' is found in the feces^(but not in the urine) of
patients and in the feces of carriers, in which regards it resembles
the typhoid bacillus. It is less resistent than the typhoid organ-
ism, disappearing rapidly from the stools, usually in 5 to 10
days. Under certain conditions (as in fresh water supplies)
the infection may endure for longer periods, for several months
and more. Like the typhoid germ, it shows some marked tend-

THE TEST FOR THE BACILLUS OF CHOLERA 113

encies to locate in the bile duct or gall-gladder, where it may re-
main dormant for a long period of time. This observation has led
to the use of bile as an enriching medium for both organisms.

The cholera vibrio work in the food and drug laboratory may
resolve itself into the isolation of the germ from water supplies,
from vegetables and possibly from feces and from sewage, and
consists in the use of special culture media, special cultural methods,
inoculation methods and agglutination tests. It is interesting to
note that the method now in use for isolating the cholera vibrio
from water supplies is the original
Koch method, done as follows.
Add 1 per cent, each of peptone
and salt to 100 cc. of the suspected
water and incubate at 380 C.
Examine microscopically at inter-
vals of 8, 12 and 18 hr. As soon
as curved and comma -shaped
organisms appear, plate on agar
and make such additional tests
as may be necessary to prove the .Fig. 35.— S. cholera showing invo-

c ,, , , , lution forms (X iooo). — (MacNeal,

presence of the cholera germ, such after VanEmengen.)
as the nitroso reaction, agglutina-
tion test, Pfeiffer's phenomenon, etc. It is not practical to enter
into a fuller discussion of the subject. More complete details will
be found in the works on bacteriology and in bulletins and reports
on bacteriology and on hygiene. For example, the U. S. Public
Health Service has worked out a quick routine method for isolat-
ing the cholera germ from feces, used in the U. S. Quarantine Ser-
vice and at the quarantine station of New York, as reported in
the Journ. of the Am. Pub. Health Association (Dec, 191 1) and
a condensed summary of the general methods may be found
in the admirable little work by Stitt (Practical Bacteriology,
Blood Work and Parasitology, 1913). Numerous special reports
will be found in American and foreign bacteriological literature.

114 BACTERIOLOGICAL METHODS

13. Biological Water Analysis

The complete biological analysis of water supplies is, as a rule,
not a regular routine of the food and drugs bacteriologist, yet he
should be prepared to make such analysis when occasion makes
it necessary. The food bacteriologist will have to do more with
the analysis of sewage contaminated water supplies and with
foods and other substances which have come in contact with such
contamination.

The complete biological analysis of water supplies may be out-
lined as follows the fuller details of which may be found in special
text-books, bacteriological journals and reports on water analysis.

Securing the sample.
Bacteriological examination.

Quantitative.

Qualitative; the presumptive colon bacillus test.
Algae; significance of.

Diatoms.

Desmids.

Nostoc and oscillaria.

Other algae.
Molds and spores; significance of.
Ova and larvae of higher parasites; significance of.
Sand, dirt, etc.

The water supply of a city or community should be watched
at all times, but perhaps more particularly in the early spring when
the melting snows and the heavy rains bring in materials accumu-
lated and held back during the winter months. Furthermore, the
rise in temperature encourages the rapid multiplication of various
organisms, such as algae and bacteria. In late summer and early
fall the drinking water often becomes vitiated, through a reduction
in supply, perhaps as the result of lack of rainfall. In the early
spring, after the first days of warm weather, the water supply often
becomes murky due to dirt washed in, green in tint due to the
enormous development of algae and generally accompanied by a
decidedly disagreeable odor which is traceable to the presence of

BIOLOGICAL WATER ANALYSIS 115

blue-green algae of the Nostoc and Oscillaria groups. Various
more or less futile attempts are made by the water companies to
correct these conditions. In order to reduce the growth of algae the
reservoirs are roofed over (the algae requiring sunlight for their
development), forgetting that while one evil is thus in a measure
corrected, another and perhaps greater, is encouraged by such pro-
cedure, namely, the growth and development of bacteria which
thrive best in the absence of sunlight. Numerous desmids, di-

Fig. 36. — S. cholera very highly magnified, showing flagellar. — (MacNeal, from
Kolle and Schiirmann.)

atoms and blue-green algae in drinking water, indicate the presence of
dead and decaying organic matter in comparatively large amount.
Diatoms are especially abundant in water supplies from old
wooden tanks and wood-lined reservoirs. Nostoc and Oscillaria
are especially abundant in water supplies fed from soil drainage.
Bacteria are present in all soil and sewage contaminated waters.
The well water of the farms may be contaminated with all manner
of organisms, such as sewage organisms and disease germs, includ-
9

Il6 BACTERIOLOGICAL METHODS

ing the larvae of Nematodes and the spores of fungi, to say nothing
of dead and decayed animals as mice, rats, rabbits, frogs, etc.

In many instances the contamination (by bacteria and algae) of
the water supplies of cities and towns is so extensive as to make di-
rect counting easy. We hereby give the report of the microscopical
examination of a sample of water from a Berkely (California)
reservoir, analyzed in March, 191 2.

Diatoms 1,500,000 per cc.

Desmids 860,000 per cc.

Oscillaria filaments 125,000 per cc.

Bacteria 16,500,000 per cc.

Paramecia 60,000 per cc.

Spores 5,000 per cc.

Hyphae of fungi 460 per cc.

The water was at the time decidedly greenish in tint with a dis-
agreeable odor, due to the numerous algae present. Water show-
ing such a high and varied biological count shows surlace seepage
and indicates sewage contamination and is not fit for drinking
purposes, yet the biologist for the water company declared it good
and harmless. The only interpretation that can be put upon a
count such as the above is that the water supply is dangerously
contaminated. Diatoms and desmids feed upon dead and decay-
ing vegetable matter. Oscillarias occur in wet soils rich in humus.
Paramecia feed upon decaying organic matter. The molds like-
wise are proof of the decay of organic matter, animal and vegetable.
In all cases of evidence of surface seepage, sewage contamina-
tion may be suspected and all sewage contaminated drinking
waters are a menace to the public health.

In no case should the examination of concentrated (1000 cc.
reduced to 10 cc.) and centrifugalized sediment be omitted, as this
will perhaps reveal contaminations which might be overlooked in
the direct examination. Nor must the presumptive colon bacillus
test be omitted when there is the least indication that sewage con-
tamination exists. In case of slight but suspicious contaminations

BIOLOGICAL WATER ANALYSIS 117

the colon bacillus test should be supplemented by the plate count
and the examination of the centrifugalized sediment.

Although the bacteriological examination of water supplies is
the work of the sanitarians, the food bacteriologists are frequently
called upon to pass judgment on the potability of water supplies.
There is no definite numerical standard for drinking waters. In
the United States the presence of the colon bacillus is almost wholly
the basis for condemnation, it being assumed that if bacteria are
present in great numbers the colon bacillus is also generally present.
This is, however, very frequently not the case. Distilled water may
contain numerous bacteria without any colon organisms. Stag-
nant waters may contain bacteria in great numbers without colon
bacilli. It is not practicable to adopt an arbitrary numerical
limit as has been suggested by various investigators. Miquel
(1891) suggested the following standards:

10 bacteria per cc. or less Excessively pure

10-100 bacteria per cc Very pure

100-1000 bacteria per cc Pure

1000-10,000 bacteria per cc Mediocre

10,000-100,000 bacteria per cc Impure

100,000 and more bacteria per cc Very impure

German sanitarians generally recognize a limit of 50 to 300 for
drinking water. Dr. Sternberg of the Public Health Service (1892)
suggested that less than 100 bacteria per cc. indicated a deep source
of the water supply and uncontaminated by surface drainage and
that a water supply with 500 bacteria per cc. was open to sus-
picion and that 1000 and over is presumptive indication of sewage
contamination or of surface drainage. It is quite evident that
there is very little excuse for the use of city and other communal
drinking water supplies with a count higher than 5-10,000 per cc,
and it is suggested that this be made the numerical limit for drink-
ing water in the absence of or irrespective of the presence of the
colon bacillus.

The general routine for making the tests for the presence of the

Il8 BACTERIOLOGICAL METHODS

colon bacillus has already been explained. It is suggested that
i cc, o.io cc. and o.oi cc. quantities of the water be run into
fermentation tubes with lactose-bile medium, making five sets of
these tube cultures, and incubate at 370 C. for 48 hr., noting pos-
sible gas formation. Gas formation indicates sewage contamina-
tion. If the gas is formed quickly, in 6 to 1 2 hr., the contamination
is probably recent, if more slowly, 24 to 36 hr., the contamination
is probably older. Gas in the 0.0 1 cc. quantities or less, indicates
very high sewage contamination, gas in the 0.0 1 to 0.10 cc. quan-
tities indicates serious contamination, and condemnation of the
water supply for drinking purposes may be based on the presence
of gas formation in two out of three tubes containing 0.10 cc.
quantities, or three out of five of the 1 cc. quantities, also tak-
ing into consideration the rate of gas formation and the numerical
plate count as well as the findings based on the direct microscop-
ical examination. In brief, condemnation of water supplies in-
tended for drinking purposes must be based upon the judgment of
a competent sanitarian, one who comprehends the significance of the
findings in relation to the source of the water supply and the sources
of the contaminations. It is not practicable to lay down hard and
fast rules. Each case must be considered by itself. In one in-
stance the gas formation may develop in 0.3 cc. quantities (three
out of five tubes containing 0.10 cc. quantities) or even in 0.10 cc.
quantities and yet the water may be considered potable, as might
be the case in deep well water into which street and road dust is
carried, or which might contain surface drainage from field or
garden. Again the water may be quite unfit for drinking pur-
poses with colon bacilli in 10 cc. or in 100 cc. quantities, as might
be the case in wells or springs highly contaminated with old or
much weathered sewage contamination.

14. Bacteriological Examination of Mineral Waters

The bacteriological analysis of bottled waters is very important
because it is an efficient means of ascertaining the conditions at the

MINERAL WATERS 119

bottling establishments. A general opinion prevails that mineral
waters are free from germs, due to the germ-destroying properties
of the mineral salts present. This is not the case. Many mineral
waters from contaminated sources or from unsanitary bottling
establishments contain bacteria in large numbers, 300,000,000 per
cc. and more. Even a medicinal water composed of concentrated
ocean water (Magpotine) gave a count of 10,000 bacteria per cc.
The Bureau of Chemistry has found mineral waters contaminated
with sewage. Often the contamination is traceable to the inade-
quate cleansing and sterilizing of used bottles and to the dirty
hands of those employed in the factory.

The bacteriological examination of mineral waters consists in
making the presumptive colon bacillus test and in making bacte-
rial counts by the plating method. It is, however, also desirable
to make direct microscopical examinations, including quantitative
cytometric counts of concentrates (1 liter quantities reduced to
10 cc.) and of centrifugalized samples, as already explained. This
will give information regarding factory conditions which could not
be ascertained by the usual plating methods.

In the case of bottled mineral waters, the securing, handling and
shipping of samples is a very simple matter as no extra precautions
and care are necessary. In the case of water from mineral springs
or artificial waters in bulk, the securing of samples for examination
must be done carefully to guard against outside contamination.
Containers for samples must be clean and sterile and as soon as the
sample is taken the container must be closed with a sterilized cork
or other suitable stopper, sealed and taken to the laboratory by the
shortest route for immediate examination. If the samples are to
be transported long distances or if for any other reason, the
examinations must be postponed for from 6 hr. to several days, the
sample must be kept on ice during the entire period.

Mineral waters are or should be quite free from bacteria and
other contaminating organisms. As yet no standards have been
adopted as to the maximum number of bacteria and other organ-

120 BACTERIOLOGICAL METHODS

isms permissible. The only quality test made by the Bureau of
Chemistry is for the colon bacillus.

15. The Microscopical and Bacteriological Examination of Milk

It is not practicable to enter into a discussion of the dairying
industry or the multitudinous factors which cause modification of
the quality of cow's milk. These are matters which concern the
food bacteriologist but little. Bovine diseases, inclusive of tuber-
culosis, must be left to the veterinarian and the making of dairy
products concern the manufacturer primarily. By this it is, how-
ever, not intended to imply that the food bacteriologist need not
have intimate knowledge of cattle diseases and of dairying meth-
ods. Not only should he be well informed regarding these things
but he should be qualified to examine cattle for diseases, tubercu-
losis in particular, and should be prepared to examine and report
upon the sanitary conditions, equipment and the moderness of dai-
rying establishments. However, the chief efforts of the food bac-
teriologist are devoted to the examination of the milk and dairying
products as they appear upon the market.

For the present purpose it will suffice to give a mere outline of
the methods of examining and testing milk microscopically and
bacteriologically. The report of the analysis should comprise the
following.

Securing the sample.
Sealing the sample.

Keeping sample on ice until ready for examination.
Examining the sample.
Direct examination.

Determining the fat content by the microscopical method.
Quantitative determination of
Bacteria.
Epithelial cells.
Blood corpuscles.
Pus cells and leucocytes.
Plate cultures.

Presumptive colon bacillus test.
Numerical count.

MILK

121

Milk may be described as a uniform suspension of fat globules
in an aqueous solution of milk-sugar and casein. The fat globules
represent the so-called butter fat of the milk. They are fairly uni-
form in size, very uniformly distributed and under ordinary con-
ditions do not tend to coalesce or clump. Pasteurization and
boiling the milk does cause some of the globules to unite or rather
to form aggregates but even in such cases it is possible to recognize
the individual globules.

On mounting a droplet of diluted milk (1-150 to 1-200) on the
hemacytometer it will be found that the fat globules soon rise to

Fig. 37. — Milk fat globules. Larger field as they appear under the low power of
the compound microscope (X 80), globules in the corner circle as they appear under
the high power (X 500). — {Hunter, after S. M. Babcock.)

the top while the heavier particles, such as bacteria and body cells,
sink to the bottom of the cell, thus separating these elements auto-
matically, and making the counting of globules and bacteria pos-
sible in the same mount by simply focusing sharply upon the fat
globules or upon the bacteria as may be desired. Some difficulty
in making the counts is caused by the fact that the oil globules are
out of focus when the rulings are in focus, making a constant shift-
ing of focus from fat globule to lines and vice versa from lines to fat
globule, necessary. Not only is this annoying but it makes accu-
rate counting difficult. This difficulty can be overcome by com-

122 BACTERIOLOGICAL METHODS

bining the use of an eye-piece micrometer scale with that of the
hemacytometer, and it is suggested that such a combination be
used, not only for milk examination, but also for making many of
the cytometric counts of food products.

A practical method for determining the fat content of milk by
means of the compound microscope was worked out in the bacterio-
logical laboratory of the California College of Pharmacy. The pro-
cedure is as follows: Make dilutions of the milk from 1-150 to
1-200, using distilled water or normal salt solution (0.6 per cent.)
and count the fat globules by means of the hemacytometer or the
special counter above suggested. Numerous counts made have
shown that 578,100,000 fat globules in 1 cc. of milk corresponds to
1 per cent, of butter fat. This number was obtained by comparing
the fat globule count with the fat determination by the standard
chemical method (combined with the use of the centrifugal
machine). The following are a few comparisons as they were ob-
tained in the laboratories of the California College of Pharmacy.

1,383,000,000 fat globules per cc. corresponded to 2.30 per cent, of butter fat.
933,000,000 fat globules per cc. corresponded to 1.60 per cent, of butter fat.
566,000,000 fat globules per cc. corresponded to 1 . 10 per cent, of butter fat.
470,000,000 fat globules per cc. corresponded to 0.80 per cent, of butter fat.

Dividing the sum total of the several counts of fat globules
made by the sum total of the corresponding percentages of butter
fat, gives 578,100,000 the average number of globules in 1 cc. of
milk corresponding to 1 per cent, of butter fat. From this it will
be seen that in round numbers, 2,000,000,000 fat globules per cc.
represent a fair quality of milk, that is, milk having somewhat over
3.50 per cent, of butter fat. According to comparative tests made,
the microscopical method is fully as accurate and reliable as the
chemical methods. The microscopical method is not recom-
mended for routine procedure in dairying establishments but it
is certainly a most valuable adjunct to the food laboratory
methods. It could at all times be employed as a substitute for
the chemical fat determination if for any reason the latter method

BACTERIOLOGICAL STANDARDS FOR MILK

123

is not applicable. Thus, it can be ascertained microscopically
whether or not water has been added to the milk or if it is full
milk, half milk or skimmed milk.

The bacteriological standardization of milk has received much
attention within recent years and all civilized countries have
adopted certain numerical limits of bacteria permissible in whole-
some milk. Unfortunately, however, there is very little uniformity
regarding these numerical limits in different countries or in differ-
ent parts of the same country. In some cities and communities

I ' tf&~-

■

■

'-•-.

s

'.

0

oi

1

0

infest' iSDte

O

V

Fig. 38. — Milk fat globules very highly magnified (X 1000).
acid bacteria at the left. — (Hunter.)

A group of lactic

there are two standards, a summer or low (numerical limit higher)
standard and a winter or high (numerical limit lower) standard.
The terms summer and winter are, however, misleading in certain
areas of the United States and, for regulatory purposes, it would be
better to base the standards on a temperature differential, irre-
spective of season, combining this with a sliding scale of bacterial
count. Under such a plan the Southern States, including the
immediate Pacific Coast region, would be under a single standard,
namely, the lower or so-called summer standard. The rest of the
United States would have both standards.

124

BACTERIOLOGICAL METHODS

The following is a tentative standard based upon the tempera-
ture differential as above suggested.

Number of Bacteria per Cc.

Standards

Ordinary
Milk

Certified
Milk

Inspected
Milk

Cream (Fresh
or Unripened)i

Temp, from lowest to 6o° F.
Winter standard

30,000 to

50,000

3,000 to
8,000

12,000 to

15,000

30,000 to

5,000,000

Temp, from 6o°F. to highest.
Summer standard

50,000 to
100,000

8,000 to

15,000

15,000 to

30,000

5,000,000 to

150,000,000

It is not practicable to fix a numerical bacterial limit for creams.
Tests made show that the count varies within wide limits, even in
cream from milk which has been kept under the most favorable
sanitary conditions and surroundings. Fresh creams, that is, the
cream removed from the milk as soon as formed, usually within
24 hr. after the milk is drawn, contains comparatively fewer bac-
teria than the cream which has been set aside to ripen. The
ripening process is far from objectionable, in fact it is encouraged
and regulated in the well-conducted dairying establishments in
order to develop the desirable butter flavor. Most of these
flavoring lactic acid bacteria are removed in the process of butter
making, being drawn away and worked out with the buttermilk,
only comparatively few remaining in the butter itself.

Taking milk samples is not unlike water sampling. Milk
should be examined not later than 6 hr. after being drawn. If it
cannot be examined within that time it must be kept on ice but
in no case should the examination be made later than 1 2 hr. after
the milk was drawn.

Body cell counts should not be omitted and proper judgment
should be exercised in interpreting the findings. Body cell counts

1 Ripened cream contains numerous lactic acid bacilli, 300,000,000 per cc, and
even more.

BODY CELLS IN MILK 1 25

give most valuable information regarding the health condition
of the cows and will serve to indicate the danger point as to the
usability of the milk. It is not practicable to give exact numerical
limits at the present time. Further investigation is necessary to
this end. However, the following suggestions will be of great
value to the analyst in arriving at a better estimate of the quality
of the milk under examination.

Epithelial cells few (1000 per cc), of no significance.

Epithelial cells many (5,000,000 per cc. and more), indicates some irritation or seri-
ous inflammatory condition of udder or in milk ducts.

Epithelial cells many with some pus cells, danger. The diseased animal should
be found and removed from the herd.

Pus cells few, indicates some slight abscess formation which should be treated if
possible.

Pus cells many (5,000,000 per cc. or more) indicates danger. The diseased
animal should be removed from the herd.

Blood corpuscles few, no special significance. Probably due to some slight injurv
resulting in capillary hemorrhage.

Blood corpuscles many. Indicates some mechanical injury which requires
attention.

For practical purposes it is not advisable to attempt to dis-
tinguish between leucocytes and pus corpuscles. Numerous leuco-
cytes indicate some serious inflammatory condition while numer-
ous pus cells indicates abscess formation perhaps following a
more severe inflammation.

Various methods have been submitted for making the body
cell counts. That by Prescott and Breed is perhaps the simplest
and also the most practical. It is carried out as follows. Spread
0.01 cc. of the milk on a glass slide1 over an area of 1 sq. cm.,
evaporating the milk to dryness using moderate heat. Next
dissolve out the butter fat by means of xylol, fix with alcohol,
again dry, and stain with methylene blue. Decolorize partially
with alcohol and examine under the compound microscope. The
body cells in the entire area of the mount are counted and the

1 The ruled slide elsewhere described (Z>, Fig. 5) will be found very useful for
counting body cells in definite quantities of the milk.

126

BACTERIOLOGICAL METHODS

entire number found multiplied by ioo gives the number of body
cells per cc. of the milk. Prescott and Breed have examined
numerous milk samples and declare that the average number of
body cells is 1,500,000 per cc. and that a count as low as 100,000
per cc. is uncommon.

Little can be said regarding the microscopical and bacteriolog-
ical examination of butter, cheese, cream and other factory products.

Fig. 39. — Unglazed porcelain filters. Chamberland system; A, without pressure:
B, fitted to main water supply; C, section of a porous porcelain filter.

There are no bacterial standards and the laboratory work is very
largely limited to the detection of adulterants such as excess of
salt, of water and the presence of lard and oleomargarine in but-
ter, fillers in cream and in ice cream, etc.

The following simple tests will be found useful in the labora-
tory:

1. Spoon Test for Oleomargarine and Renovated Butter. — Melt a small piece
of the suspected butter in a tablespoon or small dish, using a small flame. Stir the
melting substance with a small piece of wood such as a tooth-pick or match. At a

MILK BACTERIA 1 27

brisk boil, oleomargarine and renovated butter will sputter very briskly and noisily
without foaming. Genuine butter boils less noisily and with abundant foam
formation.

2. Fat Cohesion Test. — Fill a medium beaker about half full of sweet milk (pref-
erably skimmed) and heat to within near the boiling point. Add about 5 grams of
the sample and stir until completely melted. Remove from the fire and place beaker
in ice water. When the fat begins to congeal, stir with a small piece of stick. Fat or
oleomargarine will collect in one mass or lump at the end of the stick, whereas pure
butter granulates and will not adhere to the stick. This test is rot applicable to
renovated butter which behaves like unrenovated or fresh butter.

As is generally known, milk is an excellent culture medium for
a great variety of bacteria. For a time after the milk is drawn,
bacterial development is checked by the bacterolytic properties
which all fresh milk is said to possess. These lysins, however,
gradually grow less and less until there is no longer any evidence
of their existence.

Milk bacteria may be grouped into the acid formers, digesting
bacteria and those which appear to have but little effect on the
appearance of the milk. The acid-forming group is a large one and
includes the true lactic acid bacteria which are carried into the
milk from stable dust and other dirt in and about the stables and
elsewhere. The initial bacterial changes in the milk are, however,
not produced by the acid formers, but rather by those bacteria
which decompose proteids, to which belong the B. subtilis and its
aerobic allies. Streptococcus acidi lactici ferments both proteids
and lactose as does also B. coli communis and some of its allies.
In a short time, however, the true lactic acid bacteria multiply in
such large numbers as to crowd out or almost completely check the
development of the other species. They transform the lactose
into lactic acid. On longer exposure, Oidium lactis enters from the
atmosphere which fungus begins to decompose the lactic acid
and some of the remaining proteids, having the effect of lowering
the acidity which again encourages the renewed multiplication
of the lactic acid group. This alternating preponderance of
lactic acid bacteria and higher fungi continues until the proteids
and the milk sugar are almost completely used up. Butyric acid

128 BACTERIOLOGICAL METHODS

bacteria may enter the milk causing the very characteristic fer-
mentation changes resulting in the formation and liberation of bu-
tyric and propionic acids from the splitting of lactose. Butyric acid
milk has a very disagreeable odor. Various bacteria cause dis-
eases of milk as blue milk and ropy milk.

In some American cities the routine examination of milk for
B. coli is regularly adopted. The results in Baltimore have shown
the presence of this bacillus in 25 per cent, of 0.001 cc. quantities
of the milk in the winter time and 75 per cent, during the summer.
It would appear that three positive tests out of a total of five
from 0.001 cc. quantities of milk, would indicate the danger point
as to quality. For making plate counts of milk bacteria, lactose-
litmus-agar should be used in order to differentiate between
acid formers and non-acid formers.

In most communities the milk streptococci are considered
objectionable, as they belong to the group of pus-forming organ-
isms. It is frequently found that a high streptococcus count goes
with a high leucocyte count and the two are corroborative of the
existence of some severe inflammatory condition of the udder or
milk ducts. There is fairly conclusive evidence that the hemo-
lytic milk streptococci are frequently causative of more or less
severe and even fatal intestinal diseases among children, especially
during the hot summer weather. It is also fairly well proven that
some of the throat and mouth infections of children are traceable
to the staphylococci and streptococci of milk. The problem of
tuberculous milk is of lesser importance to the food bacteriologist
because the health authorities of the land have this matter under
jurisdiction. It is criminally unlawful to market milk from tuber-
cular cows. Ravenel states that approximately 20 per cent, of
the clinical cases of tuberculosis are of the bovine type and milk
from tuberculous cows continues to be a very serious menace to
the public health. It would be of the greatest value if some simple
and practical micro-chemical laboratory test for tuberculous milk
could be worked out. We would suggest this as one of the very

HYDROGEN DIOXIDE MILK TEST 1 29

important problems to be undertaken. It is evident that the con-
trol exercised by the health authorities, while it has accomplished
much, is not sufficiently stringent or far-reaching to stamp out
tuberculosis in cows.

A milk test much used in Holland and other European countries
is to ascertain the amount of gas formation in a unit of time, in a
fermentation tube containing a mixture of definite quantities of
milk and hydrogen dioxide. The amount of gas liberated is di-
rectly proportional to the amount of organic matter (bacteria,
body cells and other organic impurities) present. Tests made in
the laboratories of the California College of Pharmacy and in the
laboratories of the San Francisco Board of Health would indicate
that the method gives uniform results and that such a method
would prove a most valuable addition to the routine milk examina-
tion, serving as a check and confirmation of the bacterial and body
cell counts. In order that the test may yield uniform results in all
laboratories, a uniform method of procedure must be adopted.
The following tentative method is suggested.

A standard 10 percent, volume (of available oxygen) solution of
hydrogen dioxide should be used. The peroxide should be standard-
ized to the specified quality . For determining the valuation of the
peroxide we would recommend the Planes colorimetric test, made
as follows. Dilute the dioxide to be tested with nine parts distilled,
water. To 5 cc. of this solution (1-10) add 3 cc. of a 10 per cent
solution of potassium iodide and 1 cc. of 8 per cent, sulphuric acid,
in a standard test-tube. The color produced is matched against a
n/10 iodine solution in a second standard test-tube. 1.8 cc. of
the standard solution is equivalent to 1 cc. of oxygen.

Into graduated fermentation tubes with slender arms, having a
capacity of 25 cc, run 10 cc. of milk and 10 cc. of the standard
hydrogen dioxide, mix well in the bulb and at once run into the
arm, excluding all air from the upper end of tube. Set aside in the
incubator for 1 hr. at a temperature of 200 C. and record the
amount of gas formed at the end of this period.

13°

BACTERIOLOGICAL METHODS

Fig. 40. — Streptococcus (Staphylococcus) pyogenes and S. aureus. There are
three principal species of Streptococci (S. pyogenes albus, S. pyogenes aureus and S.
pyogenes citreus), similar in form and appearance, concerned in pus formation, as in
wound infection. These organisms are very widely distributed in soil and air.

Note the chain form arrangement of the cocci in A . B is a smear preparation. —
{Stitt (A) and Pitt field (£).)

TEST FOR WATERED MILK 131

The following quick and simple test is recommended to dis-
tinguish between raw and boiled milk!

Reagent

Methylene blue (alcoholic) 5 cc.

Formaldehyde (40 per cent.) 5 cc.

Water (distilled) 190 cc.

Add 1 cc. of this reagent to 20 cc. of the milk and heat for 10
min. at 40°-45° C. Raw milk is decolorized
while boiled milk retains the blue coloration.
This test should in all cases be checked by the
microscopical examination. Boiling the milk
causes the fat globules to unite and adhere more
or less, a characteristic which is also noticeable
in pasteurized milk. The flavor and odor of
boiled milk is in itself quite characteristic.

Knapp recommends the following test for
determining the addition of water to milk. 10
cc. of the suspected milk are run into a test-tube
and curdled by adding one drop of rennet and
placing the tube in the water bath for about 2
min. at a temperature of 35°-4o° C. The
whole is then poured upon a very fine wire sieve
and the liquid allowed to drain off into a tube
graduate, pressing the curd with a glass rod so
as to remove the liquid as completely as possible.
The amount of liquid remaining in the curd
is fairly constant in the tests and therefore
practically negligible for comparative purposes. cuiture of Staphylo-

If the amount of liquid drained off exceeds 8 coccus aun^ 1 week

n . old. — {MacNeal.)

cc, water has been added. This test should be

checked by the chemical butter fat tests and also by the microscop-
ical method for determining the fat content, as already explained.
Among the micro-organisms which cause the coagulation of
milk and which are often found in sour milk, particularly in old sour
10

132 BACTERIOLOGICAL METHODS

milk, is the Streptococcus lacticus of Kruse. The Bacillus (lactis)
aerogenes which is very closely similar to Bacillus coli, also sours
milk and is likely to be present at the beginning of the fermenta-
tion. The common pus streptococci and staphylococci are often
found in milk in large numbers, traceable to dirt and filth and to
diseased udders and less commonly to the hands of the milkers.
The colon bacillus when present is traceable to stable dust and
manure and to the unclean hands of the milkers.

The following are some of the organisms which cause diseases
of milk:

1. Bacillus cyanogenes — Blue milk.

2. Bacillus prodigiosus — Red milk.

3. Bacillus erythrogenes — Red milk.

4. Bacillus synxanthus — Yellow milk.

5. Torula amara — Bitter milk.

6. Streptococcus hollandicus — Ropy milk.

Naturally the bacilli normally present in the milk which is
stored for cream formation are also present in the cream after the
skimming and cause the so-called ripening of the cream. In
order that the ripening process may proceed in a desirable manner,
the objectionable butyric acid formers must be excluded. The
butyric acid formers are more generally associated with filth, hence,
a careful compliance with sanitary rules and regulations in the
dairying establishment will generally encourage the invasion and
development of the desirable lactic acid organisms to the exclusion
of the undesirable microbes, though this is by no means always
the case. Occasionally, even with the most scrupulous adherence
to sanitation, cream will not ripen properly and these occasional
failures have prompted the more progressive dairymen to in-
oculate the milk and cream with pure cultures of the desirable cream
ripening bacilli. Others use natural cream starters, that is, small
quantities of old cream which has ripened with a desirable flavor.

Cream should not show colon bacilli in less than 0.10 cc. quanti-
ties and fresh unripened cream should not contain more than 5,000,-

TUBERCLE BACILLI IN MILK 1 33

000 bacteria per cc. Ripened cream should not contain more than
150,000,000 bacteria per cc. and most of which bacteria should be
of the lactic acid group. Pathogenic bacteria which may be pres-
ent in milk may also be present in the cream. Tubercle bacilli,
diphtheria bacilli and typhoid bacilli are the most likely to occur.
In the case of doubtful cream, the colon bacillus test should not be
omitted and in the case of suspected contamination with patho-
genic organisms, the cream, as well as the milk from the same
source, should be examined, resorting to the
usual animal inoculation tests.

The tests for the presence of tubercle bac-
illi in milk, cream, meats, etc., comprises the
microscopic examination of stained (Ziehl-
Neelsen method of staining) sediments or con-
centrates-as may be required, and animal in-
oculations. For the animal inoculation test, , Q?: 4?~ T^bf^e

' bacilli in sputum.

guinea-pigs are . used. Centrifugalize (in a Stained with carbol-

j. , i • \ i c xi «n fuchsin and methy-

powerful machine) about 250 cc. of the milk ieneblue.— (Pittfield.)
in order to throw down the tubercle bacilli (with
the other inclusions), and from this make the desired cover-slip
preparations and inoculate (in the region of the left knee-joint
of hind leg) the remainder of the sediment into three healthy
guinea-pigs. Place the inoculated guinea-pigs in individual cages
and keep them under observation for from 2 to 4 weeks. The
reasons why several pigs should be inoculated are as follows.
Some of the pigs may be killed by bacteria other than the tu-
bercle bacilli and it is always desirable to duplicate the tests.
At the end of the second week, one of the guinea-pigs should be
dissected and the glands of the sublumbar region as well as the
glands of the superficial tissues and of the popliteal region exam-
ined. If tubercular infection has taken place, these glands will
be found much enlarged containing foci of tubercle bacilli. The
enlarged glands are dissected and suitable cover-glass prepara-
tions made therefrom. If the evidence of tubercular infection

134

BACTERIOLOGICAL METHODS

Fig. 43. — Tubercle ba-
cillus slant culture on glyc-
erin-agar, several months
old. — (Stitt, after Curtis.)

is not conclusive, the other two inocu-
lated guinea-pigs should be kept 2 weeks
longer then dissected and examined like
the first. Occasionally there is abscess
formation at the point of inoculation but
this need not necessarily interfere with the
tubercular development in the glands and
in the deeper tissues.

It is frequently possible to isolate the
bacillus of tuberculosis (from sputum,
glandular tissues, meat pulp, centrifugal-
ized sediments of milk, cream, etc.) by
special manipulation and the use of special
culture media. The following method
is suggested. Spread two or three drops
of the material (concentrate, sediment,
crushed, suspected tuberculous meat ex-
tract, etc.) evenly over the surface of two
or three glass slips and place the smear
preparations in the drying oven at ioo° C.
for 15 min., however, not before the ma-
terial on the slips is well dried at the room
temperature. Tubercle bacilli are quite
resistant to dry heat and will withstand
the temperature of ioo° C. for from 30
min. to 1 hr. The exposure to that tem-
perature for 15 min. will kill most of the
bacteria associated with the tubercle germs
and will in fact kill some of these. At
the end of 15 min. take the glass slips
from the drying oven and by means of a
small sterile spatula or scalpel, scrape the
dried suspected material over the surface

CULTURING THE TUBERCLE BACILLI OF MILK

135

of the special medium in Petri dishes. The medium used (Hesse's
agar) is made as follows:

Nutrose (sodium caseinate) 5 grams.

Sodium chloride 30 grams.

Glycerin 30 grams.

Agar 10 grams.

Na2C02 (crystalline) solution (28.6 per cent.) 5 cc.

Distilled water 1000 cc.

Mix ingredients. Heat until agar is dissolved. Filter through
cotton. Pour into Petri dishes. Sterilize fractionally.

After inoculating two or three Petri dishes in the manner
indicated, incubate at 37.50 C. in a moisture-saturated atmosphere
for several days. If tubercle bacilli are present young colonies
will appear which may be identified with a low power by the resem-
blance to broken wavy lines.

Instead of making the glass-slip smears as above suggested,
good results may be obtained through the use of the cotton throat
swabs such as are used by physicians for taking throat cultures in
diphtheria cases. Dip or roll the cotton ends of three swabs in
the suspected tuberculous material, suspend in air until perfectly
dry and then place in drying over (ioo° C.) for 15 min., then rub
the cotton over the surface of the special culture medium in Petri
dishes. Make some six or seven parallel streaks over the surface of
the medium. Incubate and examine as before. Should the glass-
slip or cotton-swab preparations be placed in the drying oven
before air drying them, all or nearly all of the tubercle bacilli would
be killed in the drying oven.

Several investigators have recommended a direct method of
examination for ascertaining the presence of tubercle bacilli in
milk, and in other materials, through the use of agents which will
completely dissolve all bacterial bodies excepting the acid-fast
group of organisms to which the tubercle bacillus belongs. For
this purpose antiformin (really a mixture of chlorinated sodium
hypochlorite and Labarraque's solution) has been highly recom-

136 BACTERIOLOGICAL METHODS

mended. This proprietary article is a strongly alkaline solution of
sodium hypochlorite. In each cc. it contains approximately 5.68
grams of sodium hypochlorite, sodium hydroxide 7.8 grams and
sodium carbonate 0.32 grams. The available chlorine amounts to
about 5 .68 grams. It dissolves all organic matter, such as that con-

Fig. 44. — Bacillus tuberculosis in the sputum of a consumptive; stained by Ziehl
method (X 2100). — (After Kossel.)

tained in sputum and feces, excepting the tubercle bacilli. It is in
itself an active antiseptic having a phenol coefficient of 3.

For bacteriological work, a 50 per cent, solution of the anti-
formin will be found satisfactory. Mix equal parts of the anti-
formin solution (50 per cent.) and milk or sputum or other mate-

ANTIFORMIN TUBERCLE CULTURES 137

rial supposed to contain the tubercle bacilli, in a suitable glass
container and bring to a boil over the Bunsen burner. When the
material is cool, add 1.5 cc. of a mixture of chloroform and alcohol
(chloroform one part and alcohol nine parts) to each 10 cc. of the
material and shake vigorously. The tubercle bacilli absorb some
of the chloroform and become heavier than the rest of the organic
matter. Next centrifugalize at a high speed for 15 min. which
separates the material into three layers; the antiformin at the top,
the sediment in the middle, and the chloroform with the tubercle
bacilli at the bottom. Pipette off the layer of chloroform and ex-
amine for tubercle bacilli by resorting to the usual staining methods.
The smear preparations can be made to stick to the cover or slide
by mixing with serum or egg albumen solution. This method may
also be tried in the examination of creams, cheese, buttermilk and
butter. The strength of the antiformin solution should be graded
according to the amount or percentage of organic matter to be dis-
solved, taking the strength required for sputum work as the high-
est. For milk work the 15 per cent, solution will be satisfactory.
For cheese a 50 per cent, solution should be used, likewise for
feces.

Stitt recommends the following antiformin method for cultur-
ing the tubercle bacilli. Mix 20 cc. of sputum, 65 cc. of sterile
water and 15 cc. of antiformin. Stir with a glass rod. After a
period ranging from 30 min. to 2 hr., the mixture should be
homogeneous. Centrifugalize for 15 min. or longer, decant, and
wash the sediment twice in sterile normal salt solution and smear
out the well-washed sediment over serum or glycerin egg albumen
or nutrose slants. It must be remembered that the tubercle bacillus
will not grow in sunlight and that the colonies form on the surface
of the culture media only.

Stitt also states that it is not wise to use the antiformin in
solutions stronger than is necessary to dissolve the organic matter
and bacteria other than the tubercle bacillus. For example for
sputum, it is suggested that 20 or 25 per cent, of antiformin be

i38

BACTERIOLOGICAL METHODS

used. If stronger solutions are used, many of the tubercle bacilli
are also disintegrated or considerably changed in form and in the
behavior with the acid-fast stains.

In addition to the routine examination of ice creams for the
presence of fillers and ingredients which do not properly belong
to ice creams, the food bacteriologist will have occasion to make
bacteriological and toxicological tests. According to Vaughan,
the toxic changes in ice cream are due to the presence of a poison
designated tyrotoxicon, presumably identical with the toxin occa-
sionally found in milk and cheese. Ice-cream poisoning depends
upon the development of the toxin-forming bacteria in the milk
and cream before it is frozen. It is not at all likely that ice cream
made from clean wholesome cream and milk will contain toxins,
provided it is kept well frozen and is not stored too long. There
is good evidence that slightly infected ice cream which is kept for
several days and longer, may show sufficient toxic bacterial de-
velopment to produce symptoms of poisoning. The virulency of
the toxins produced by the bacteria appears to increasewith the
lowering of the temperature.

The danger from ice cream is directly proportional to the un-
sanitary conditions of the milk and cream used and ice-cream
poisoning is far more likely to manifest itself during the hot summer
weather. All suspicious ice creams should be examined bacteri-
ally, making numerical plate cultures and also the presumptive co-
lon bacillus test and tests for streptococci and staphylococci. The
toxicological test as recommended for meats is, however, far more
important and should not be omitted. Ice cream should not con-
tain more than 1,000,000 bacteria per cc. and should not develop
colon bacilli in less than 0.10 cc. quantities by the standard pre-
sumptive colon bacillus test.

Of the more common ice-cream fillers we may mention starch
and tragacanth. Vegetable mucilages other than tragacanth may
be suspected. Gelatin is also used. Eggs are frequently added.
A filler to which a small amount of rennet had been added has

BUTTER AND CHEESE 1 39

been extensively advertised as an ice-cream producer which did
not require the use of cream or of ice.

Occasionally it may become necessary to examine sour milk
and buttermilk for the presence of toxins and objectionable bac-
teria and other undesirable organisms. Because of the careless
and more or less promiscuous handling of buttermilk before it
reaches the consumer, it is especially liable to the invasion of
foreign organisms. The routine examination of buttermilk is
largely limited to a direct microscopical inspection. Mold spores
and yeast cells should be sparingly present and the predominating
bacilli should be small1 (lactic acid formers) and of irregular and
rather indefinite outline. Mold and cocci should be very sparingly
present.

To examine butter for the presence of bacteria (direct micro-
scopical method) and other contaminations, place i gram of the
butter in 10 cc. of ether and shake until all of the butter fat is
dissolved. Pour the solution into the special centrifugal tube
and centrifugalize for 5 min. Wash the contents of the i cc.
end tube into 10 cc. of ether and again shake and centrifugalize.
Pour off the ether and add 2 cc. of a 2 per cent, sodic hydrate
solution and shake until the casein is dissolved. The sodic hydrate
solution emulsifies the small amount of fat present. Examine
the emulsion for bacteria, counting the bacteria and body cells
by means of the hemacytometer.

Butter and cheese made from the milk of animals suffering
from foot-and-mouth disease have transmitted this disease to
humans. The bovine type of tuberculosis has resulted from the
consumption of milk, cream and butter. Tubercle bacilli have
been found in the more quickly ripened cheeses. Tubercle bacilli
do, however, not survive long in soured cream or milk, perhaps
not over 2 or 3 days.

The following are some of the more important organisms con-
cerned in the ripening of cheese.

1 The Bacillus bulgarius is comparatively large (i X 6/jl).

140 BACTERIOLOGICAL METHODS

i. Lactic acid bacteria. — These are the chief agents concerned in the ripening of
Cheddar, American and Edam cheese. Pure cultures of the Bacillus acidi lactici are
often used as a starter. In the manufacture of the Edam cheese, slimy whey is
used as a starter {Streptococcus hollandicus) .

2. Penicillium glaucum the common green mold is the principle organism con-
cerned in the ripening of Roquefort, Gorganzola and Brie cheeses. In some coun-
tries the green mold is scraped from molded bread and added to the curd.

3. A great variety of other bacteria, yeasts and mold are concerned in the devel-
opment of the more specific flavors and aromas. Further investigation is necessary
to ascertain the special function performed by each and the mutualistic relationship
that may exist between them.

4. Gas generating bacteria are concerned in the formation of holes in the interior
of the ripening cheese. These gas formers also modify the aroma or flavor of the
cheese and in some instances constitute the chief ripening agents.

Spoiling of cheese is not uncommon, due to the invasion of a
variety of undesirable organisms. The cheese " hopper" or
" skipper" found in and upon old and overripened cheese and in
cheeses which have not been properly screened, is the larva of the
black two-winged fly Piophila casei. The insect deposits its
eggs in the surface cracks and crevices of the cheese upon which the
developing larva feeds. The name skipper or hopper is derived
from the fact that the larvae are capable of projecting themselves
some distance by coiling and suddenly uncoiling.

This fly is a common pest in the dairying establishments. A
less common but even more annoying pest is the larva of the
" bacon beetle." Cheeses which are comparatively hard and
smooth externally are not so likely to be infested by the skipper
or bacon beetle larva as are the cheeses which are rough externally.
It is customary to wipe the cheese in order to remove the para-
sites. If the cheeses which are stored for ripening are properly
screened, the fly and beetle cannot get access to them to deposit
the eggs. A small mite (Trioglyphis siro) also occurs on cheese
upon which it feeds.

Inadequately screened cheeses also permit flies and other pests
to deposit possible infections, thus typhoid contamination and
also pus streptococci and staphylococci may be found upon this
food substance.

DISEASES OE CHEESE

141

Bitter cheese is due to a variety of bacteria, as Tyrothrix
geniculatus (the bitter soft cheese bacillus), Micrococcus easel amari
(bitter cheese coccus), Weigmann's bitter milk bacillus, Conn's
bitter milk coccus, and others. Red coloration of cheese may be
caused by yeasts (Saccharomyces ruber) or by cocci. Black cheese

Fig. 45. — Oidium lactis. a, b, Dichotomous branching of growing hyphas; c, d, g,
simple chains of oidia breaking through substratum at dotted line x—y, dotted por-
tions submerged; e, /, chains of oidia from a branching outgrowth of a submerged
cell; h, branching chain of oidia; k, I, m, n, 0, p, s, types of germination of oidia under
varying conditions; /, diagram of a portion of a colony showing habit of Oidium
lactis as seen in culture media. — (From Bull. 82, Bur. Animal Industry, U. S. Dept.
Agr.)

may be due to the presence of iron in milk, perhaps traceable to the
action of slightly soured milk in rusty buckets. Some yeasts and
molds may produce dark to black decomposition changes. Blue
cheese is the result of the action of a bacillus. Putrid cheese is the
result of the invasion of saprophytic bacteria and other micro-

142

BACTERIOLOGICAL METHODS

organisms. Cheese poisoning is not uncommon, due to the pres-
ence of bacteria which give rise to toxins (tyro-toxicon) . In a
general way it may be stated that cheese diseases are due to
filthy and unsanitary conditions in the dairying establishment re-

Fig. 46. — Penicillium glaucum showing the characteristic spore formation. This
fungus is a true saprophyte, the common green mold, occurring on a great variety
of organic substances.

suiting in infected milk, or to filthy and unsanitary conditions in
the cheese factory, or the infection may be traceable to the im-
proper and careless storing and handling of the cheese. Ripened
cheese being in itself a decomposition product resulting from the
invasion of certain desirable micro-organisms usually entering

CANNED AND CONDENSED MILK

143

from the air, it is but reasonable to expect irregularities in the final
result unless the invasion of undesirable micro-organisms, which
are also present in the air, is carefully guarded against.

Condensed milk is prepared by concentrating full or skimmed
milk. It may be sweetened by adding cane sugar (40 per cent.).
While condensed milk contains relatively fewer bacteria than does
ordinary milk, due to the process of manufacture, yet none is
entirely sterile. The number of bacteria usually present ranges
from about 500 or even less to as high as
250,000 per cc. Colon bacilli, dysentery
bacilli and streptococci are generally
absent. Tubercle bacilli have been found.
The method for examining condensed milk
is much as for ordinary milk, with suitable
modifications in making the dilutions.

Canned condensed milk occasionally
spoils, due to the development of bacteria
and yeast organisms. Yeast organisms are
not likely to appear unless the milk is
sweetened with sugar. Spoiling may be-
come apparent through the "swelling" of
the can. Organoleptic testing is occasion-
ally a guide to the condition or quality of
the milk. A numerical bacterial limit
should be adopted for condensed milk. If
more than 1,000,000 bacteria per cc. are
present it is not suitable for human consumption. Tubercle bacilli
should be absent. According to the limited reports on the subject
we may assume that the process of condensing the milk kills all
pathogenic bacteria which may be present, including even the more
resistant tubercle bacilli. The contaminating bacteria may pro-
duce toxins and in marked bacterial invasion it would be well to
make inoculation tests with white mice or guinea-pigs, as for toxins
in meat and in ice cream. An examination of the centrifugalized

Fig. 47. — Penicillium
of Camembert and Roque-
fart cheese. This mold
grows at a very low tem-
perature. It is closely
similar to, if not identical,
with P. glaucum. — {Jor-
dan after Thorn.)

144 BACTERIOLOGICAL METHODS

sediment must not be omitted as this will convey information re-
garding the sanitary conditions of the factory as well as of the
dairying establishments which supplied the milk to the factory.

Dried or powdered milk is prepared by spraying milk (usually
skimmed) into a partial vacuum or by spraying it on a revolving
drum or on a moving belt in a partial vacuum. The dried material
is then placed in suitable containers. The dried milk contains
all of the ingredients of the milk excepting the water, the lysins
and certain enzymes. The fat globules are altered physically
but not chemically. Mixing dried milk with the required amounts
of water makes a liquid resembling ordinary milk. The micro-
scopical and bacteriological examination of dried milk is as for
condensed milk. Like the condensed milk it is quite free from
disease germs of all kinds but bacterial invasion is not excluded
from material which has been carelessly prepared or canned.
Toxins and ptomaines should be absent. The absence of moisture
in powdered milk prevents the ready growth of micro-organisms
and it may be kept in good condition for a period of 5 or 6 months
and even longer, in dry sterile containers stored in a cool dry
place.

Attempts have been made to commercialize frozen milk but
so far without success. It is rather difficult to handle frozen
milk and the article furthermore loses the milk flavor on thawing.

16. The Bacteriological Examination of Shellfish

The term shellfish includes oysters, mussels and clams. Only
those species and varieties which serve as food for man are of
interest to the food bacteriologist. Since it has been conclusively
proven that shellfish, oysters in particular, have been responsible
for typhoid epidemics, much attention has been given to the
bacteriology of this class of food. In tracing such epidemics it was
discovered that the causative oysters had been floated or grown in
heavily polluted waters. In several instances contamination of

SHELLFISH 145

the water supply washing the oyster beds, was traceable to the
discharges from typhoid fever patients.

All shellfish are easily adaptable to filthy habits and surround-
ings. They appear to thrive in proportion to the amount of or-
ganic contamination of the water supply constituting the food
beds. It must, however, not be supposed that sewage and other
highly objectionable (to man) contamination is normal to the life
of the shellfish. We know that the domestic hog is fond of the
highly contaminated refuse materials from the kitchen known as
swill but we also know that hogs thrive better on sanitary food.
Thus the filth feeding oyster grows equally well, if not better, in
clean sea water, that is, water free from sewage contamination and
decayed animal matter.

The danger from shellfish (to man) is due to the fact that these
animals are often from highly contaminated water supplies and
that they are generally eaten raw or only partially cooked. The
possible diseases traceable to the eating of shellfish are Asiatic
cholera (in countries where this disease prevails), typhoid fever
and a variety of less severe intestinal diseases such as dysentery,
colitis and intestinal ulcerations. The work of the food bacteri-
ologist is, however, not the rinding of the specific germs causing an
epidemic, but rather an endeavor to ascertain the danger point in
the quality of the food as represented by the positive colon bacillus
tests. The prime object of the pure food laws is the maintenance
of health rather than finding the cause of disease. This most
important fact is sometimes not understood as is clearly indicated
by a supreme court decision permitting the bleaching of flour.
It is the intent of the pure food law to clearly mark the danger
points in our food supplies so that the consumer may maintain
his physical well-being through the avoidance of such dangers.
He who advises against the heeding of the proper and timely warn-
ings set up by those entrusted with this duty, either through
ignorance or indifference, is a menace to the public welfare. The
danger sign, "avoid bleached flour" and not the actual physical

146 BACTERIOLOGICAL METHODS

disturbances which results from the eating of such flour, is the
proper warning. The presence of a limited number of colon bacilli
in foods and drinks is the danger mark and not the actual occur-
rence of cholera, of typhoid, of dysentery, due to the eating of
more highly contaminated foods. The danger signal must be
within the zone of safety and not beyond it.

The methods for the bacteriological examination of shellfish
are but modifications of the methods used in the examination of
water supplies. As in the case of drinking water, the chief index
to the pollution of shellfish is the colon bacillus test. The examina-
tion of the water source above the oyster beds very frequently
gives inferential information as to the possibility of the contami-
nation of the shellfish which obtain their food supply from beds
flooded by such waters. The following is the method for the
bacteriological examination of shellfish adopted by the American
Health Association at the 191 2 meeting.

1. Selection of Sample.- — Twelve oysters of average size of the
lot to be examined, having deep bowls, short lips and shell tightly
closed, are picked out by hand or by means of a sterilized long-
handled spoon and prepared for immediate transportation to the
laboratory.

2. Making a Record of the Sample. — This record should cover
the following points. The exact location of the bed from which the
sample was taken. The depth of the water at the time the oysters
were gathered. Weather conditions, direction and velocity of
wind, state of tide, day and hour when the stock was taken from
the water, the conditions under which the stock had been kept
since removal from the water and up to the time when the sample
was taken, presence of abnormal odors, temperature of stock, and
the day and hour of taking the sample.

3. Transportation of the Sample. — The sample oysters are to
be packed in a suitable metal or pasteboard container of the size
and shape convenient for shipping. The important points to bear
in mind are: the prevention of the mixing of the oyster liquors of

SHELLFISH 147

the different samples and avoiding the mixing of the oysters with
the ice water of the packing ice. The samples must in all cases be
placed on ice or packed in ice if they cannot be examined inside of
36 hr. or if the outside temperature is above 500 F. It is, however,
not necessary to place the oysters in absolutely tight containers
provided the above conditions are maintained.

4. Laboratory Procedure. — Record the date of receiving the
sample, condition of seals, of the sample oysters and the tem-
perature of the interior of the container at the time of opening.
The bacteriological examination should in all cases be started as
soon as possible after the receipt of the sample.

Before beginning operations the hands must be thoroughly
scrubbed and all vessels to be used must be sterilized. The shell
of the oyster may be opened by means of a sterilized oyster knife
or by drilling a hole through the shell near the hinge. The drill
must be sterilized and the area of the shell to be operated upon must
be cleaned, flamed before drilling and flamed at least once more
during the drilling process.

The simplest and quickest method for opening the oyster shells
is that employed by Stiles of the Bureau of Chemistry. By means
of a pair of sterilized wire nippers crush and break off enough of
the two valves so as to make the use of the oyster knife easy.

Before opening the oysters see that they are thoroughly
scrubbed and then rinsed in boiled (sterile) water, and each
oyster is wiped quite dry and flamed before it is opened.

5. Bacterial Counts.- — Bacterial counts are made of the compos-
ite sample of each lot obtained by mixing the shell liquor of five
oysters. Agar shall be used for the culture medium and in general
the procedure shall be in accordance with the method recommended
for the examination of water. The water used for making the di-
lutions shall contain 1 per cent, of sodium chloride, in order to
approximate the natural salinity of the oyster liquor. The agar
plate cultures shall be incubated at 200 C. for 3 days and the col-
onies counted in the usual manner.

148 BACTERIOLOGICAL METHODS

6. Determining Bacteria of the Colon Bacillus Group.— Meas-
ured quantities of the shell liquor of each of five oysters selected
from the dozen shall be placed in fermentation tubes containing
lactose-peptone-bile. The measured quantities shall be 1 cc,
0.10 cc, and 0.01 cc, or such other quantities or corresponding
dilutions as may be desired. The fermentation tube inoculations
thus prepared shall be incubated for 3 days at a temperature of 370
C, and the presence of gas noted daily. From 10 to 85 per cent,
of gas during this period shall be considered a positive test indi-
cating a presumption of the presence of at least one bacterium of
the colon bacillus group in the quantity of the water used in the
test. But no final colon bacillus rating shall be made unless con-
firmatory tests for the presence of organisms of the colon bacillus
group shall have been obtained from the tube of highest or next
highest dilution from each oyster showing the presence of gas.
These confirmatory tests shall be begun immediately upon noting
the formation of gas and shall be carried out in conformity with
the procedure recommended by the Committee on Standard
Methods of Water Analysis.

7. Statement of Results. — The results of the bacterial counts
shall be expressed as the number of bacteria per cc. The results
of the colon bacillus test shall be expressed either in the form of an
arbitrary numerical system or in estimated number of colon bacilli
per cc. of the sample.

It is suggested that the arbitrary numerical method proposed
by the American Health Association be given the preference. The
following are the rating valuations according to this method.

Colon bacillus in i .00 cc. but not in o. 10 cc, a value of 1
Colon bacillus in o. 10 cc. but not in 0.01 cc, a value of 10
Colon bacillus in 0.01 cc. but not in 0.001 cc, a value of 100, etc.

The sum of these values for five oysters gives the total value of
the sample examined and this figure indicates the rating for
Bacillus coli. According to this system the highest (best) rating

THE RATING OF SHELLFISH

149

is indicated by o and the lowest (worst) by 500, represented in
tabular form by the following possible results of two analyses:

Example A

Oysters

I. 00 cc.

O.IO CC.

0.01 cc.

Numerical
Value

I

0

0

0

O

2

0

0

0

O

3

0

0

0

O

4

0

0

0

O

5

0

0

0

0

Total rating for B. coli =

O

Example B

Oysters

1. 00 cc.

O.IO cc.

O.OI cc.

Numerical
Value

I

+

+

+

100

2

+

+

+

IOO

3

+

+

+

100

4

+

+

+

IOO

5

+

+

+

100

Total rating for B. coli =

500

The (+) mark means that gas formation in the lactose bile tubes took place,
indicating contamination with the colon bacillus.

The (o) mark indicates that no gas formation took place in the lactose bile tubes.

The results above indicated are, however, not generally ob-
tained in practice. The important question is at what rating
shall the shellfish be pronounced unfit for human use, or rather
what rating shall be the danger signal as to the quality of this food?
There seems to be no uniformity of opinion as regards this point.
Thus far, the Bureau of Chemistry has barred oysters from inter-
state shipment which gave three positive tests out of five in 0.10
cc. quantities of oyster liquor, which standard is also adopted by

ISO

BACTERIOLOGICAL METHODS

the Rhode Island Shellfish Commission. This standard may be
graphically represented as follows (Example C) :

Example C

Oysters

i.oocc. o.io cc.

O.OI cc.

Numerical
Value

I

+

+

o

IO

2

+

+

o

IO

3

+

+

o

IO

4

+

o

o

I

5

' +

o

o

I

Total rating for B. coll =

32

It sometimes happens in laboratory practice that the smaller
quantities of shell water from a number of oysters show positive
results, whereas larger amounts of liquor from an equal number of
oysters show negative results. In such cases it is customary to
give the next lower numerical value to the positive results in the
high dilutions, and such positive results shall be considered as
being transferred to a lower dilution giving negative results in
another oyster. This recession of assigned values shall, however,
not be carried beyond the point where the number of such reces-
sions is greater than the number of instances where other oysters
in the series of five failed to give positive results. This may be
illustrated as follows (Examples D and E) :

Example D

Oysters

i. oo cc.

O.IO cc.

O.OI cc.

Numerical
Value

I

+

+

o

IO

2

+

+

o

IO

3

+

+

o

IO

4

+

o

o

io (not i)

5

+

+

-f

io (not ioo)

Total rating for B. coli =

So

THE RATING OF SHELLFISH
Example E

151

Oysters

1. 00 cc.

0.10 CC.

O.OI cc.

Numerical
Value

I

+

+

+

IO (not 100)

2

+

+

+

10 (not 100)

3

+

0

0

I

4

0

0

0

1 (not 0)

5

0

0

0

1 (not 0)

Total rating for B. coli =

23

The bacteriological examination of oysters from opened or
shucked stock very naturally must be somewhat modified from the
method as outlined for oysters in the shell. The stock in the con-
tainer from which the sample is to be taken must be thoroughly
mixed. The containers (wide-mouthed glass jars) must be steril-
ized and should have a capacity of 1 quart. By means of a suit-
able sterilized ladle (may be flamed with alcohol on the spot),
half fill the containers with the oysters and seal containers in such
manner as to exclude all outside contamination. Unless the exami-
nation can be made within 3 hr. after taking the sample, said
sample must be placed on ice. It is very desirable to make the
bacteriological examination shortly after the sample is taken.
The laboratory technique is much as for oysters in the shell, though
it must be borne in mind that dilutions higher than 0.01 cc. are
usually required. The results of the bacteriological examination
of the opened or shucked stock shall be expressed in- the same way
as that specified for oysters in the shell, except that in the calcula-
tion for B. coli rating the values for the results of the positive
fermentation tests, after confirmation, shall be recorded for each
of the inoculations of each and every dilution. All tests are to
be made in triplicate, that is, three fermentation tubes are to be
inoculated for each dilution used.

Clams, mussels and other shellfish are to be examined in the
same manner as oysters, in so far as this is possible. In opening

152 BACTERIOLOGICAL METHODS

soft-shelled clams it will be found that if two incisions are made
through the mantle the shell water may be poured out without
opening the shell. It is stated that hard-shell clams may be
opened by striking the shell over the dorsal muscle with a hammer.
An opening is formed which will permit the insertion of a knife
with which to cut the muscle. In case any one shellfish does not
contain enough shell water to make a test, the water from several
individuals may be mixed.

The examination of shellfish for sewage pollution is of the utmost
importance, as dangerously contaminated oysters are very com-
mon. In fact it would be advisable to discontinue the oyster as
an article of diet. At its very best* it is a filthy article. It is
unquestionably a dangerous article of food, in this regard compar-
able to the mushrooms in the vegetable kingdom. However, there
is not the least likelihood that the oyster will be left from our
dining tables as long as there are any available. It is therefore
most desirable that the supervising of this food on the part of
those who are entrusted with the safeguarding of the health of the
people should be carefully and consistently done.

Some authorities (English) recommend that the liquor and
oysters be mixed, the latter finely chopped, for the purpose of
making the colon bacillus test. There appears to be no gain from
this procedure and the method cannot be recommended.

17. The Bacteriological and Toxicological Examination of Meat
and Meat Products

Remarkable as it may seem, food bacteriologists have given but
little attention to the examination of meats and meat products,
despite the fact that fatal poisoning from eating infected meats is
very common. Intoxications ranging from mild to very severe,
resulting from the ingestion of more or less highly contaminated
meats are of daily occurrence in every community. At each in-
stance of a death or deaths resulting from the eating of bad meat,

MEAT BACTERIA 1 53

the health authorities get busy and almost invariably find the true
source of the trouble, and there the matter usually rests. No ra-
tional attempt is made to prevent a repetition of the occurrence.
Meats of all kinds when left exposed to the air soon show signs
of decomposition. The aerobic forms of bacteria are first to de-
velop, causing the decomposition of proteids and sugars. Inas-
much as sugar is usually present in small amounts only, the sugar
decomposers are soon crowded out by the proteid-splitting forms.
The small amount of acid formed by the sugar decomposers is
neutralized by the ammonia which is formed during proteid de-
composition. The aerobes very naturally act on the outside of the
meat particles, using up the oxygen in the air on and within the
immediate surface tissues of the meat. This reduction in oxygen
gradually permits the anaerobes to get a start, especially B. per-
fringens and B. bifermentens sporogenes. These use up proteids
as well as sugar, and the complete removal of sugar encourages
the more active development of pure aerobes which act upon
proteids only. The following tabulation from the work by Ellis

Organisms Action on Products Formed

Proteus vulgaris (Gluten and fibrin Phenol, indol, amines, fatty acids.

Proteus vulgaris Casein Albumoses, peptones and amino-

acids.

Streptococcus longus 'Fibrin Tyrosin, leucin, amines and fatty

acids.

B. coli communis Casein Albumoses.

B. coli communis Peptone Ammonia and indol.

B. coli communis Mixture of eggs and Skatol, phenol, leucin and oxy-

meat acids.

Gluten Phenol, indol, amines and fatty

acids.

Casein Leucin, tyrosin, fatty acids, aro-

acid bacteria matic fatty acids and trypto-

phan.

B. subtilis and B. prodigiosus Albumoses Leucin, tyrosin and tryptophan.

Cholera vibrio Albumen Leucin, tyrosin, indol, amines and

fatty acids.

Micrococcus pyogenes

Aerobic peptonizing lactic

154 BACTERIOLOGICAL METHODS

indicates the activities of putrefactive bacteria in culture, which
correspond with the putrefactive changes produced in nature :

The rotting bacteria produce the familiar changes in meat
usually designated as spoiling, rotting and tainting, and such meat
is universally recognized as unfit for food because of the deleterious
effects following the ingestion of such meats. Tainted meats
may appear entirely normal to the naked eye and slight decay of
the inner tissues may not be appreciable to the sense of smell.
The decomposition changes resulting in the liberation of indol,
skatol and related substances having disagreeable odors usually
begin near the bones and joints, and such decomposition may not
become apparent until the bony structure is exposed by cutting
or until the odors are dissipated more actively by boiling. This
odor is very persistent; boiling for several hours will not cause it to
disappear entirely. It must not be supposed that meats free from
bad odors are necessarily free from ptomaines and toxins. For
example, perfectly fresh meat may absorb these poisonous sub-
stances when placed in contact with badly tainted meats and, again,
some toxin-forming bacteria do not produce odoriferous gases.

The bacteriology and toxicology of canned meats and soup
stocks containing meat has not received the attention that it should.
It is these substances which are so largely responsible for the mul-
titudinous lesser intestinal disturbances following their use as food.
The present methods of canning meats should be thoroughly in-
vestigated and ways and means devised to improve them in
accord with modern advance in the manufacture of food products.
It is generally believed that the eating of canned meats and soups
is fraught with danger to life and health, and this is not far from
the truth. The proper canning of meats requires infinitely more
care than the canning of vegetable substances. The careful super-
vision of the marketing of meats and meat products is vastly more
important than the supervision of vegetable foods. It is compara-
tively rare for toxins and ptomaines to be formed in vegetable
substances, whereas this is the rule in the decomposition of meats.

MEAT BACTERIA

155

Furthermore, meats decompose much more readily than vegetable
substances, which makes it necessary to observe greater care in the
preparation of this class of food for the market. Some meats
decompose much more readily than others. Meats of higher
animals resist decomposition longer than do meats of lower ani-
mals. Fish meats decompose quickly when exposed to the air.
The story is current among fishermen that certain kinds of fish

HHi^

: V

Fig. 48. — Bacillus welchii, also known as B. aerogenes caps ulat us and B. phlcg-
mones emphysematosa in smear preparation. This is the common "gas bacillus, " be-
cause of the abundant gas formation in the tissues invaded and in culture media. _ It
is a plump large nonmotile, anaerobic, capsulated, Gram positive, spore-bearing
bacillus and is very widely distributed in nature. — {Williams.)

begin to decompose before they can be removed from the hook.
This is of course exaggeration, but the statements made indicate
in a way the comparative resisting power of different kinds of
meat to rotting bacteria. It is highly probable that the difference
in the resisting power to decomposition is due in part at least to
the presence of bacteriolysins. Little is known regarding the
changes which take place in cold storage meats. Cold storage

156

BACTERIOLOGICAL METHODS

Fig. 49 — Typical cultural
characteristics of Bacillus aero-
genes capsulatus (B. welchii) in
agar. Culture 48 hr. old. The
agar mass is separated by the gas
which is formed. — (MacNeal.)

does check the growth of all kinds of
bacteria and of higher fungi, but not
in the same ratio. For example, the
freezing temperature inhibits the de-
velopment of the usual rotting bac-
teria very effectually, whereas many
of the toxin formers multiply slowly,
in time forming enough of the poison
to produce marked symptoms of
poisoning when meat thus affected is
eaten. Little is known of the changes
which take place in incompletely ster-
ilized canned meats, and no attempt
has so far been made to ascertain the
degree of decomposition which usually
takes place in the meats before they
are placed in the cans and sterilized.
This is a matter of the utmost impor-
tance and should receive the immediate
attention of the food bacteriologists.

What shall be the routine method
in the examination of meats? It is
quite evident that the methods which
are applicable in the examination of
vegetable substances are not suitable
in the examination of meats. We
hereby suggest the following outline of
methods applicable in the food labora-
tory:

1. Direct microscopical examination of
meats.

a. Bacteria on surface of meats.

b. Mold and spores present, as in moldy
bacon, pork, etc.

c. Presence of bladder worms, larvae of
parasites, etc.

EXAMINATION OF MEATS 1 57

d. Trichinae in pork.

e. Cereal fillers and starches in sausage meats. Tragacanth fillers.
/. Coloring substances and preservatives in sausage meats.

2. Plate cultures. (Lactose-litmus-agar and gelatin media.)

a. Numerical counts of bacteria.

b. Number of gas formers and acid formers.

c. B. botulinus in pork meats.

3. Toxicological tests.

a. Inoculation tests (guinea-pigs) to prove the absence or presence of ptomaines
or toxins.

b. Tests for tuberculous and other diseased meats.
4. Determining the source of the meat.

a. By the precsipitin test.

b. Sugar test for horse meat.

c. Microscopical identification based on differences in the size and structure of
the muscular fibers and the differences in the size and form of the fat crystals
derived from different animals.

Of the above tests the numerical bacterial count and the toxico-
logical tests are of the greatest importance and should be carried
out in the examination of suspected raw meats, sausage meats and
of canned meats and soup stocks. There certainly should be a
limit to the number of bacteria in all raw meats, whether ground
into sausage or not, as this would be the means of regulating the
sanitary requirements in the proper handling of meats. The only
practical method for determining the quality of canned meats is to
make inoculation tests on guinea-pigs or white mice, using filtered
aqueous extracts of the suspected meat products. If ptomaines
or toxins are present the tests will show it. It would be very
desirable to work out a micro-chemical test for determining the
presence of toxins and ptomaines in meats. As above indicated,
there are some very important differences between toxins and
ptomaines. The former are destroyed by the boiling temperature,
whereas the latter are not. For example, the thorough cooking
of sausage meats prevents botulism but it does not prevent the
ill effects resulting from the eating of meats with ptomaine poison.

Dried and smoked meats should be examined for the presence
of bacteria and molds. Dried fish in particular is very fre-

158 BACTERIOLOGICAL METHODS

quently highly contaminated with molds. It is very evident that
the present method of pickling fish of all kinds must be changed.
The method of pickling herring, for example, in wooden vats or
casks must be abandoned, as the containers are wholly unsuitable
from a sanitary standpoint. The liquor from canned fish (in tin
cans) is frequently very highly contaminated with bacteria in
spite of the high salt content. The gelatin of the market requires
careful examination, as much of the sheet variety is not infrequently

Fig. 50. , Fig. 51.

Fig. 50. — Bacillus botulinus from a sugar-gelatin culture. — (Pittfield, after Kolle
and Wassermann.)

Fig. 51.— Bacillus enter itidis. Under this name is included a number of organ-
isms of the Gaertner group which play a very important part in meat decomposition
and meat poisoning. It is also known as the dysentery group. The organisms are
actively motile, non-sporogenous, aerobic, non-liquefying and Gram negative. —
{Jordan after Kolle and Wassermann.)

entirely permeated by mold and bacteria, rendering it not only
unfit for food for man but also unsuitable for bacteriological work.
The entire subject of meat poisoning is as yet not very well
understood. Dr. Savage states that the bacteria concerned in
meat poisoning may be classed under three groups: (a) the Gaertner
group of bacilli, (b) aerobic bacilli not belonging to the Gaertner
group, such as B. proteus and B. coli, and (c) Bacillus botulinus.
In the majority of cases of outbreaks of fatal food poisoning, some

EXAMINATION OF MEATS 1 59

form of the Gaertner group of bacilli has been the infecting organ-
ism. The Gaertner bacilli are large coli-typhoid types which
occupy a position intermediate between the chemically active
colon group and the chemically inert typhoid group, and includes
B. enkritidis, B. typhi murium, B. suipestijer and B. paratyphosus
B. Sausages and ham are the commonest sources of botulism.
From the anaerobic character of the bacillus it follows that poison-
ing is rarely due to the eating of fresh sausage and pork. Invasion
of meats by B. botulinus can take place only when the necessary
anaerobic conditions exist, as for example when a ham is stored at
the bottom of the pickling vat and entirely covered by the pickling
solution, and in the interior of insufficiently cooked sausages and in
stored masses of sausage meats.

The use of the compound microscope in the. examination of
meats and meat preparations is still in its infancy. The work done
shows very clearly that with more experience very valuable in-
formation can be obtained from the microscopical examination
regarding the quality of meats of all kinds. It is highly probable
that the microscope will show diagnostic differences in the muscu-
lature of different animals, thus making it possible to determine the
source of the meat.

Considerable attention has already been given to the micro-
scopical study of fat crystals derived from the fats of different
species of animals. So-called rancid fats or fats which have aged
considerably, even though they may not yet give evidence of ran-
cidity to the unaided senses, will show more or less abundant
crystalline structure, arranged in clusters, which may be readily
seen under the low power of the compound microscope. These
crystals are not apparent in fresh fats, but are generally more or less
abundantly present in canned meats and soup stocks containing
animal products, in meat extracts and in other meat derivatives
having fat admixtures. The presence of crystal clusters indicates
fat decomposition and these are therefore an indication of the
quality of the meat product containing them. The degree of ran-

l6o BACTERIOLOGICAL METHODS

cidity or, to state it more accurately, the quality of the product
dependent upon age is in direct proportion to the quantity of crys-
talline clusters present. It would appear that the quantity of
crystals present is not proportional to the amount of bacterial
contamination and decomposition. The indications are that it is
possible to determine the source of the fat from the color, size and
arrangement of the fat crystal aggregates. For example, the
crystal clusters of lard are smaller than those of the fat of the do-
mestic fowl. The fat crystal aggregates of the hen are compara-
tively large and the individual crystals are long and slender. The
inexperienced analyst is apt to mistake the crystal clusters for
mold colonies (Leptothrix). This mistake can very readily be
avoided by applying heat which causes the prompt melting of the
fat crystals whereas the mold hyphae are not greatly disturbed or
changed. The differential characteristics which would be con-
cerned in the microscopical examination of fat crystals may be
given as follows:

i. Differences in the size of the aggregates.

2. Differences in the length of the individual crystals.

3. Differences in the diameter of the individual crystals.

4. Differences in the form of the ends of the individual crystal. Ends may be
rounded or pointed.

5. Differences in color. These will in all probability pertain to different races or
families of the animal kingdom. For example, lard crystals are colorless whereas
those of the domestic fowl are yellowish.

The use of certain chemicals will aid in the microscopical find-
ings. For example, sulphuric acid produces characteristic color
reactions with certain fats. If two drops of concentrated sul-
phuric acid are added to twenty drops of goose fat, a greenish-yel-
low color is produced which changes to reddish brown on stirring.
Under the same conditions cod-liver oil turns a violet color whereas
turtle oil turns brown. Castor oil turns yellowish to yellowish
brown and finally wine red with a very distinct zone. A similar
reaction is observed with neats foot oil. Raw linseed oil turns a
deep reddish brown to very dark brown. Lard oil shows a distinct

FAT CRYSTALS l6l

brown zone which deepens to wine red. The reaction for sperm
oil is much as for cod-liver oil. These color reactions with sul-
phuric acid are perhaps of little value in the detection of fat adul-
terations and admixtures but they will prove helpful aids in the
examination of these substances as to identity. Pure concen-
trated acid should be used. It must also be kept in mind that the
fat impurities which may be present modify the color reactions.
Pure samples of fats should be kept on hand for purposes of making
check and comparative tests. The Bureau of Animal Industry
has suggested a method for distinguishing between fats and oils
derived from the animal and the vegetable kingdoms based upon
differences in the appearance of the crystals (phytosterol and
cholesterol).

In addition to the study of fat crystals which are formed spon-
taneously in more or less decomposed and aged meat products as
above set forth, certain methods for testing fat crystals isolated
in the pure state by chemical methods are now generally carried
out in meat inspection and food laboratories. These tests com-
bine the use of the compound microscope and should therefore
be carried out by the micro-analyst or bacteriologist and for that
reason are hereby included. R. H. Kerr of the Biochemic Divi-
sion of the Bureau of Animal Industry has worked out a method
for detecting vegetable fats in mixtures of animal and vegetable
fats and vice versa, the method will also serve to demonstrate the
presence of animal fats in supposedly pure vegetable fats. The
method is a slight modification of several methods which have been
in use for some time and which are described in various text-books,
and it is hereby given in full as it appears in Circular No. 212 (May
10, 1913) of the Bureau of Animal Industry.

The Detection of Phytosterol in Mixtures of Animal and

Vegetable Fats

Sample. — The amount of sample used depends on the amount of material avail-
able. From 200 to 300 grams is the amount usually taken. The test is seldom

1 62 BACTERIOLOGICAL METHODS

attempted if less than ioo grams are available, and an amount greater than 500 grams
is never taken.

Extraction with Alcohol. — The sample is melted and poured into a flat-bottomed
flask of i-liter capacity which is closed with a rubber stopper perforated with three
holes. This flask is set on the top of the steam bath and connected to a reflux
condenser and to a 700 cc. round-bottomed flask containing 500 cc. of 95 per cent,
alcohol. A glass tube which is adjusted so that its lower end is about one-fourth of
an inch above the surface of the fat and whose upper end is bent at a right angle and
closed by means of a short piece of rubber tubing and a pinchcock fills the third hole in
the stopper. The distilling flask is set down in the steam so that the alcohol boils
briskly. The outlet tube reaches down to the bottom of the flask containing the
sample so that the alcohol vapor as it distills over bubbles up through the fat and
keeps it in a state of vigorous agitation. The alcohol vapor is condensed in the
reflux condenser and returned to the flask containing the fat. The distillation is con-
tinued until all of the alcohol has collected in the flask containing the fat. The dis-
tilling flask is now disconnected. The alcohol in the flask immediately ceases to boil
and soon separates from the fat. The empty distilling flask is next connected to the
bent tube by a piece of glass tubing of sufficient length, the pinchcock opened, and the
alcohol layer siphoned off into the distilling flask. This is then connected as before
and the distillation continued until the alcohol has again collected in the first flask.
It is then siphoned into the distilling flask as before, and a third extraction made.
After the third extraction the alcohol layer is again siphoned off into the distilling
flask and the fat is discarded. The alcohol now contains practically all of the choles-
terol and phytosterol originally present in the fat.

Saponification and Extraction with Ether. — The alcohol in the distilling flask is
next concentrated by boiling to about 250 cc, and 20 cc. of a concentrated potassium-
hydrate solution (100 grams KOH dissolved in 100 cc. water) added to the boiling
liquid. It is boiled for 10 min. to insure complete saponification of all the fat' and
is then removed from the steam bath and allowed to cool almost to room temperature.
After it has cooled sufficiently it is poured into a large separatory funnel containing
500 cc. of warm ether and shaken to insure thorough mixing. The mixture may be
clear, but is more often opalescent. There is now poured in 500 cc. of distilled water,
and the funnel is rotated gently. Shaking must be avoided, as it leads to the forma-
tion of extremely stubborn emulsions, but the water should be mixed with the alcohol-
ether-soap solution. Separation takes place at once and is clear and sharp. The
soap solution is drawn off and the ether layer washed with 300 cc. of distilled water,
shaking being still avoided. After this washing it is washed repeatedly with small
quantities of water until all soap is removed. The ether layer is then transferred to
a flask and the ether distilled off. Distillation is stopped when the contents of the
flask have been reduced to about 25 cc, and the concentrated ether solution contain-
ing the cholesterol, phytosterol, and all other unsaponifiable matter is transferred to a
tall 50 cc beaker. The evaporation is continued until all ether is driven off and the
residue is perfectly dry. If desired, a tared beaker may be used and the weight of the
unsaponifiable matter determined at this point.

FAT CRYSTALS

163

Preparation of the Acetates. — A small amount (3 to 5 cc.) of acetic anhydrid is
added to the dry residue in the beaker and heated to boiling over a free flame, the
beaker being covered with a watch glass during the process. After a brief boiling — a
few seconds is sufficient — the flame is removed and the beaker transferred to the
steam bath and left there until the acetic anhydrid is driven off.

Purification of the Acetates. — Thirty-five cc. of hot 80 per cent, alcohol are
added to the acetylated residue in the beaker and heated to boiling with vigorous
stirring. The liquid is then filtered quickly through a folded filter and the in-
soluble residue washed well with boiling 80 per cent, alcohol. The acetates of chol-
esterol and phytosterol are dissolved, while the greater portion of the impurities
present are not dissolved by the alcohol and remain on the filter. Paraffin and paraf-

Fig. 52. — Phytosterol crystals.

Fig. 53. — Cholesterol crystals.

fin oil, if present, are likewise separated by this treatment. The combined filtrate
and washings are next cooled to a temperature of io° to 120 C. and allowed to stand
at that temperature for 2 to 3 hr. During this time the acetates of cholesterol
and phytosterol crystallize from the solution. They are removed by filtra-
tion, washed with cold 80 per cent, alcohol, and then dissolved on the filter with a
stream of hot absolute alcohol from a wash bottle, as little alcohol as possible being
used. The alcoholic solution of the acetates is caught in a small glass evaporating
dish, two or three drops of distilled water being added to the solution and heat applied
if it is not perfectly clear. The dish is then set out on a desk in the laboratory and the
alcohol allowed to evaporate spontaneously. The contents are stirred occasionally
and the deposit of crystals which forms around the edges of the liquid and on the sides
of the dish rubbed down into the solution with the stirring rod. As soon as a good
deposit of crystals has formed they are removed by filtering through a hardened

1 64 BACTERIOLOGICAL METHODS

filter, washed twice with cold 90 per cent, alcohol, and dried by suction. After
drying by suction they are dried at ioo° C. for half an hour and the melting point
determined.

Determination of the Melting Point. — A tube of about 1 mm. diam., sealed at
one end and having a slight flare at the other, is filled to a depth of about 5 mm. with
the dried crystals, which are packed somewhat firmly in the lower end by tapping on
a hard surface. This is attached to the bulb of a suitable thermometer and the melt-
ing point determined. A thermometer graduated from 950 to 2000 C. in one-fifth
degrees is used in this laboratory. The determination is made in an Anschutz
apparatus, the outer bulb being filled with concentrated sulphuric acid and the inner
tube with glycerin. The apparatus is so adjusted that no correction of the observed
temperature is required. The melting point of the first crop of crystals usually gives
definite information as to the presence or absence of phytosterol, but the conclusion
indicated is confirmed by recrystallizing from absolute alcohol and again determining
the melting point. If the crystals are pure cholesterol acetate, the melting point of
the second crop should agree closely with that of the first. If phytosterol acetate is
present, however, a higher melting point should be noted, as phytosterol acetate is
less soluble than cholesterol acetate.

The Emery Method eor the Detection of Beef Fat in Lard

James A. Emery of the Biochemic Division of the Bureau of
Animal Industry recommends the following method1 for detect-
ing beef fat in lard. It is given here because of its value in isolat-
ing the crystals of fats for microscopical examination.

Technique of Method. — Five grams of the warm filtered fat is weighed (on a bal-
ance sensitive to o. 1 gram) in a glass-stoppered graduated cylinder of 25 cc. capacity,
150 to 1 75 mm. in height, with an internal diameter of about 18 mm., and warm ether
is added until the 25 cc. graduation is reached. The glass stopper is securely re-
placed and the cylinder is shaken vigorously until complete solution of the fat takes
place. The cylinder with its contents is then allowed to stand in a suitable place
where a constant temperature, at which it is desired to have the crystallization pro-
ceed, may be maintained. (An apparatus described by Rogers proved efficient for
the maintenance of this constant temperature.)2 After 18 hr. the cylinder is re-

1 Circular 132, May 23, 1908. Bureau of Animal Industry, U. S. Dept. of
Agriculture.

2 It is necessary to observe great caution in the use of this form of apparatus, as
the sparking of the thermo-regulator is a source of danger if the solutions are care-
lessly handled. A better form for this work would be one in which the temperature
is controlled by a circulating hot- water system heated by a small lamp outside of the
box, the regulation of which could be adjusted by using one of the many forms of gas
regulators on the market.

FAT CRYSTALS

165

moved and the supernatant ether solution carefully decanted from the crystallized
glycerids, which are usually found in a firm mass at the bottom of the vessel. Cold
ether is then added in three portions of 5 cc. each from a small wash bottle, care being
taken not to break up the deposit while washing and decanting the first two portions.
The third portion is, however, actively agitated in the cylinder with a sharp rotary
motion and by a quick movement transferred, with the crystals, to a small filter paper.
The crystals are then washed with successive small portions of the cold ether, with the
use of the wash bottle, until 10 to 15 cc. has been used, dependent on the amount of
crystals. Then by means of a slight exhaust the
small amount of remaining ether is rapidly re-
moved. The paper with its contents is then
transferred to a suitable place, where it should
be spread out and any large lumps of the glyc-
erids broken up by gentle pressure. When
dry the mass is thoroughly comminuted and the
melting point of the crystals determined.

As the difference between the melting points
of the glycerids obtained in this manner from
beef fat and lard is not very great, being only
about 3.5 degrees, and as the writer has men-
tioned a standard melting-point temperature for
the glycerids of pure lard obtained under certain
conditions, a description of the apparatus used
in determining the melting points, together with
its manipulation, is essential and may be of
some assistance.

Determination of the Melting Point. — A
large test tube approximately 150 by 25 mm.,
containing water (free from air) into which the
bulb of a thermometer1 with the melting-point
tube attached is immersed, is placed in a beaker

of water and so adjusted that the surface of the liquid contained in the two vessels
is at the same level. The water in the beaker should be heated rapidly to about
550 C. and that temperature maintained until the thermometer carrying the melt-
ing-point tube registers between 500 and 550 C, then heat is again applied and the
temperature of the outer bath carried somewhat rapidly to 670 C, when the lamp is
removed. The melting point of the crystals is regarded as that point when the
fused substance becomes perfectly clear and transparent. The use of a dark
background placed about 4 in. from the apparatus will prove of advantage.

The melting-point tube should be of about 1 mm. internal diam., sealed at one
end and with a slight flare at the other extremity, in order that the loading may be
expedited. The amount of the substance taken for each determination should be

1 The thermometer used was one graduated in one-fifth degrees and extending
from o° to ioo° C.

Fig. 54. — Beef fat crystals.
a, Clusters of crystals as seen
under the low power of the
compound microscope; b, crys-
tals highly magnified.

i66

BACTERIOLOGICAL METHODS

approximately the same and should occupy a space about 9 mm. in length, being
somewhat firmly packed in the lower end of the tube by tapping it sharply on a
hard surface. The water in the outer bath should be agitated frequently during the
determination.

Possible Sources of Error. — In applying the foregoing method too great care
cannot be exercised with the preparation of the sample. The presence of water, the
incomplete solution of the fat in the ether, or the presence of small particles of
extraneous matter may interfere with the process of crystallization, frequently caus-

a

Fig. 55. — Lard crystals, a, Clus-
ters of crystals as seen under the low-
power of the compound microscope; b,
crystals highly magnified.

Fig. 56. — Duck fat crystals, a,
Clusters of crystals as seen under the
low power of the compound microscope;
b, crystals highly magnified.

ing it to proceed too rapidly and resulting in the formation of a large mass of small
fluffy crystals instead of the compact mass of larger crystals desired. These fine
crystals render the preliminary washing by decantation with ether difficult, and they
also persistently hold the unsaturated glycerids in larger amount than is desirable.
The temperature at which the crystallization should be allowed to proceed should not
be less than 150 C. nor more than 200 C, with the best results obtainable in the neigh-
borhood of an average between the two. Although larger crystals are formed at the
higher temperature (200 C), only lards of high grade afford crystalline deposits in
working quantity, and in many cases where lards of inferior grades are tested the
amount of solid glycerids entering into their composition is so reduced as not to yield
any deposit at all.

HORSE MEAT 1 67

Pure fresh butter shows no crystalline structure. Salted butter
will of course show the characteristic salt crystals. Melted
butter which is allowed to cool slowly shows a marked crystalline
structure under polarized light, even under the low powers of the
compound microscope, but this is not a diagnostic character
inasmuch as other fats show a similar behavior with polarized
light.

Horse meat has been used as food for man for many ages and
is at the present time a regularly marketed food article in many
countries. During the siege of Paris (1870) when food became very
scarce, experiments with the meats of various animals were made,
as that of rats, mice, cats, dogs, mules and horses. Horse meat
especially met with general favor and since that time has become
quite common in the French meat markets. It is stated that it is
a frequent substitute for beef in our restaurants (the cheaper
eating places in our larger cities). Horse meat differs from beef
in that it is somwhat coarser grained, darker in color and that
it contains a higher percentage of glycogen. As a rule the meats
from cattle contain little or no glycogen, although it is stated that
fresh meat from well-nourished cattle may contain as much gly-
cogen as does the meat of the horse. It must also be borne in
mind that the meat from dogs, cats, starved calves and fetuses
contains considerable glycogen. Should such meats be added to
sausages the admixture might be recognized by the color, the meat
of fetuses and starved calves being much lighter than that of the
horse or of mature cattle.

In time the glycogen of horse meat is changed into grape sugar
and will respond to the Fehling's solution reaction for sugar.
For this purpose use a cold aqueous extract of the suspected meat.
In the case of fresh horse meat the following tests are recommended.
The Brautigam and Edelmann test for the presence of horse
meat is made as follows: Grind or chop (finely) 50 grams of the
meat and boil for 1 hr. in 200 cc. of water. Add 1.5 grams (3
per cent, by weight of the meat) of caustic potash and heat over

1 68 BACTERIOLOGICAL METHODS

water bath until the muscle fibers are disintegrated. Boil down to
50 grams and filter. When cool add an equal part of dilute nitric
acid (10 per cent.) to precipitate the albuminoids, and again filter.
Pour the filtrate into a test-tube and carefully pour iodine water
down the inside of the tube. If horse meat is present a burgundy
red zone appears at the point of contact of the two solutions. The
widt!h and intensity of the colored zone is in direct proportion to the
amount of horse meat present.

If starch is present (as in sausages and sausage meats seasoned
with starch-bearing spices or mixed with starch fillers), this must be
precipitated from the boiled meat extract and removed by filtra-
tion. To the extract add two or three times the volume of con-
centrated acetic acid and let stand for 2 or 3 hr., and then
filter through two or three thicknesses of filter paper. Test
the filtrate with the iodine water as above suggested. However,
before making the glycogen test the test for starch should be
applied, for if it responds to this test the precipitation of starch
must be repeated. Because of the dilution with the three or more
volumes of acetic acid (to precipitate the starch) negative re-
sults may be obtained in cases where horse meat is present. It
is therefore advisable to precipitate the glycogen by means of
alcohol, using from ten to twelve times the volume of the acidu-
lated meat filtrate. The cloudy alcoholic suspension is run
through a small filter and the precipitated glycogen on and in
the filter paper is washed out by means of hot acidulated (acetic
acid) water, and this filtrate is then tested with the iodine water.
This test is positive in the presence of 5 per cent, quantities of
horse meat. The wine-red color reaction is temporary only and
it must be kept in mind that dextrin interferes with the reaction.

Because of the fact that meats other than that derived from
the horse may contain glycogen, it is sometimes necessary to
supplement the above color reaction with the biological test or
the precipitin test which has come into use within recent years.
The general routine for making the test is as follows: Inject

PRECIPITATION TEST 1 69

(subcutaneously or intravenously) rabbits with 10 cc. of filtered
defibrinated horse blood (or serum) every other day five or six
times. At the end of this time draw blood from the rabbit,
allow it to clot kept on ice, remove the serum and filter, where-
upon the reagent is ready for use. Express and extract (in saline
solution) the juice from the meat suspected to contain horse meat,
filter and keep on ice until wanted for use. To the filtrate thus
prepared add a few drops of the equinized rabbit serum. If
cloudiness and slight whitish precipitate forms it constitutes a
positive test, proving conclusively that the suspected meat is
horse meat or contains horse meat. Only raw fresh meat re-
sponds to this test. Heating destroys the action of the reagent.
Inoculating rabbits with the defibrinated and filtered blood
serum of various animals, as of hog, domestic fowl, deer, dog,
bear, etc., and testing in the manner outlined in the following
method by Dr. Karl F. Meyer of the State University of Cali-
fornia, the meat of the responding animal may be identified.

The Precipitin Test for the Detection of Horse and Deer
Meat and for Meat Adulterations in General

The method can be used for fresh, dried, frozen, pickled, raw
and smoked, but not for boiled, meat. The meat may not be
heated above 6o°-7o° C. for the biologic test.

For the tests are needed:

a. Specific antisera (anti-horse or deer precipitin serum; pre-
cipitin) .

b. Aqueous extract of the meat to be identified (precipitinogen).
1. Antisera. — The sera must be specific and highly active

against the meat protein to be determined. Rabbits are in-
jected subcutaneously, intravenously or intraperitoneally with
serum, defibrinated blood or extract of the fat free meat. The
best results are obtained by inoculating fresh serum intravenously.
The sera for injection can readily be obtained from abattoirs

170 BACTERIOLOGICAL METHODS

or from serum institutes or laboratories. Horse serum is not as
toxic to rabbits as are some other sera. Meat extracts should
always be filtered to avoid infection of the animals to be im-
munized, but extensive sloughing is likely to occur with any
method of immunization and the mortality rate is high. The
blood or serum used as antigen can be preserved by the addition
of chloroform (1-2 per cent.), or by drying.

On account of the individual differences existing in rabbits
in regard to the development of precipitins, it is advisable to
treat at least six animals at the same time. The injections of
2-3 cc. of horse or deer serum are made at intervals of 5 days.
Ten days after the last injection the blood is tested for pre-
cipitins. The further treatment of the animals differs individu-
ally, depending on the precipitin contents of the rabbits. Ani-
mals which show a high precipitin reaction are given subsequent
inoculations subcutaneously or intraperitoneally, to avoid ana-
phylactic death which frequently results from intravenous in-
oculations. Some rabbits fail to produce precipitins, whatever
the method used.

Fornet and Muller1 recommend the intraperitoneal injection
of 5, 10 and 15 cc, respectively, of protein material on the 1st,
2d and 3d day, respectively. The test for antibodies is carried out
on the 1 2th day. Gay and Fitzgerald1 inject on three consecutive
days 1 cc. of the antigen, bleed, and test the serum on the 10th
day. Both methods frequently give very good results. The
precipitin content of an immune serum is occasionally titrated
during the process of immunization by withdrawing a few cubic
centimeters of blood from an ear vein. The hair over the marginal
vein is removed and the skin rubbed with alcohol. A fine pipette is
introduced into the vein and the blood collected by capillary attrac-
tion or by suction. It is, however, advisable for the beginner to
cut the vein transversely and to collect the blood in a centrifugal
tube. The hemorrhage is stopped by covering the wound with

1 University of California publications, Pathology, Vol. II, 75, 191 2.

PRECIPITATION TEST 171

cotton soaked in liq. ferri sesquichloridi (ferric chloride) or by
placing a small hemostat for H to i hr. on the incision. The
serum which has separated from the clot is centrifugalized and
the titer is determined as follows:

Preliminary Titration. — Into each of a series of six test-tubes
place 2.0 cc. of the following dilutions of serum (horse or deer)
antigen, mixed with 0.85 per cent, saline 1:100, 1:500, 1:1000,
1 : 5000, 1 : 10,000 and 1 : 20,000. To each cubic centimeters of the
dilution 0.1 cc. of antiserum is added. The solution of 1:1000
should become turbid instantaneously or within 1 to 2 min., the
other dilutions in from 3 to 5 min. The serum should have a
titer of 1:20,000; that means the serum should cause a turbidity
in a dilution (of horse serum or extract of meat) of 1 : 20,000 in less
than 5 min. The antiserum is either introduced by allowing
it to run down the side of the tube (no shaking is permissible),
or it is stratified on the diluted horse serum. In the first case the
turbidity appears from the bottom, in the second case in form of a
grayish ring; both reactions are positive. The coloration is
best seen against a dark background. The pipettes and test-
tubes must be perfectly clean and sterile. The equipment de-
signed by Uhlenhuth is very satisfactory. The test-tubes are
long and narrow, 10 cm. by 0.8 cm., and are suspended in beveled
holes of the test-tube rack. Pipettes of 1 cc. capacity graduated
into Koo cc, and 5 and 10 cc. pipettes graduated into Jfo cc.
will be found satisfactory.

Preservation of Serum. — In case the titer of the serum is
satisfactory, the rabbit is bled to death (aseptically) from the
carotids. For full details on technique, consult the text-books on
Immunity. The centrifuged serum should be perfectly clear
and sterile and should not be opalescent. Kept cool and in the
dark (ice chest) it will remain potent for months, even years.
To avoid opalescence the animal should be bled only after a period
of fasting. On account of autoprecipitation, it will lose some of
its potency. The precipitate formed can be removed by cen-

172 BACTERIOLOGICAL METHODS

trifugalizing or by filtration, but the titer must again be tested.
Preservatives such as carbolic acid, etc., should not be added to
the sera. Sterile sera are obtained by filtration through Berke-
feld filters. Drying of the sera on filter paper is the best method
known for preserving them {Jacobsthal und v. Elsler).

2. The Preparation of the Meat Extract. — To make the bio-
logic test for horse or deer meat, remove from the deeper parts
of the specimen, by means of a flamed or boiled knife and through
a fresh opening, a piece of muscle of about 30 grams weight.
It should contain as little fat as possible. On a sterilized tile
(best covered with unused writing paper) chop the meat carefully.
The finely minced meat is placed in a sterilized 100 cc. Erlenmeyer
flask and spread out with a sterile glass rod and covered with
50 cc. sterile saline solution. Salted meat is washed for 10
min. in a large flask with distilled water, renewing the water
several times, without shaking the flask.

The mixture of saline and meat is kept for about 6 hr.
at room temperature, or over night in the refrigerator. To
obtain a clear solution the flask should not be shaken.

Since the presence of fat interferes with the reaction, it is
advisable to remove it by means of ether and chloroform. To
make the extraction, take 75-100 grams of the minced meat,
place in a large Erlenmeyer flask and cover with equal parts of
ether and chloroform. After 24 hr. the ether and chloroform are
poured off, the meat is washed once or twice with saline solution
and then extracted, as stated above.

To determine whether a sufficient quantity of protein sub-
stances has passed into solution, place 2 cc. of the extract in a
test-tube and shake vigorously. If a fine foam develops and
persists for some time, the extraction may be said to be sufficiently
complete. The protein solution must be perfectly clear and must
therefore be filtered. With extracts from fresh meat this is
usually accomplished by filtering through a firm filter paper
previously moistened with saline solution. If it is not crystal

PRECIPITATION TEST 1 73

clear, and especially if the meat to be examined was fat or salt,
it is filtered through a sterile Berkefeld or through a layer of
infusorial earth stratified in a Buchner funnel.

The filtrate is suitable for the test when a foam is developed
by shaking and when it contains about i part of protein in 300
parts of salt solution. To determine this, 2 cc. of the clear filtrate
are placed in a test-tube and heated, and a drop of dilute nitric
acid (sp. gr. 1.153) is added; if a marked cloudiness and a
flocculent precipitate forms, the extract is too highly concentrated
and must be diluted with normal salt solution until the heat and
acid test causes only a diffuse, opalescent cloudiness which settles
to the bottom of the tube after 5 min. as a slight precipitate.

Before proceeding with the test, the reaction of the meat ex-
tract should be tested with litmus paper and if it is found to be
acid it should be neutralized very carefully with 0.1 per cent,
sodium hydroxide or magnesium oxide solution. Only slightly
acid or alkaline solutions should be used. For the extraction of
the meat, spigot, tap or distilled water should not be used. Fresh
meat frequently produces a sufficiently strong protein solution
in 1 hr. In boiled, preserved and decomposed meat, the ex-
traction proceeds very slowly (24 hr.) and the solutions are
difficult to clarify.

Technique of the Test. — If, for example, the object is to
determine whether a piece of meat is horse flesh or, if sausage,
contains the meat of this animal, the test is conducted as follows:

Tube 1. — 2 cc. of unknown extract (1 .-300) + 0.1 cc. of anti-horse serum.
Tube 2. — 2 cc. of unknown extract (1 : 30c) -|- 0.1 cc. of normal rabbit serum.
Tube 3. — 2 cc. of horse flesh extract (1 : 300) -\- 0.1 cc. of anti-horse serum.
Tube 4. — 2 cc. of pork extract (1 : 300) +0.1 cc. of anti-horse serum.
Tube 5. — 2 cc. of beef extract (1 : 300) +0.1 cc. of anti-horse serum.
Tube 6. — 2 cc. of saline solution + 0.1 cc. of anti-horse serum.

The immune serum is added to each tube very carefully and
run down the sides of the tube, or stratified. The tubes must
not be shaken. The tubes are kept at room temperature. The

174

BACTERIOLOGICAL METHODS

test must not be made with a mixture of the sera of different
rabbits.

Interpretation of the Results. — If in tubes i and 3 a misty cloudi-
ness should appear within 5 min., and if a definite precipitate forms
within 30 min., the other tubes remaining perfectly clear, the
extract is very probably one of horse flesh or the flesh of some
other single-toed animal. Precipitates which develop more slowly
cannot be considered as positive. The protein of horses and
donkeys cannot be differentiated by this test. In a similar man-
ner, tests may be made for the meat of deer, dogs or any other
animals, if the respective immune sera are used with the extract.

Fig. 57. — Types of syringes: 1, Roux's bacteriologic syringe; 2, Koch syringe;
3, Meyer's bacteriologic syringe. The Meyer syringe is the simplest and best for
general purposes. — (McFarland.)

Heterologous precipitates, which occur when antisera are
added to concentrated foreign protein solutions, rarely are
disturbing factors of the tests when the above technique is used.
The elective absorption (according to Kister and Weichardt)
with the foreign protein is occasionally necessary for scientific
tests.

The organoleptic tests are not always conclusive as to the
quality of the meat. It is a well-known fact that the stinking or
putrefactive odors are generally wholly absent in even highly
decayed salted and brine-pickled fish and meats and in heavily
seasoned sausage meats and in smoked meats. On the other

MEAT BACTERIA 175

hand, it is advisable to reject or condemn all meats which emit
offensive odors, provided such odors are not normal to the meat.
Under normal offensive odors may be mentioned the fishy odor
of meats from animals which feed upon fish, mussels and other
aquatic animals; the sex odor wrhich is often marked in the meats
from older males; the various vegetable odors due to feeding,
such as the turnip odor and taste in beef, fenugreek odor, etc.,
etc. Distinctively putrefactive odors in meats are a very reliable
indication of their unfitness for consumption. Marked changes
in consistency (sloppy, smeary and porous meats) and in color
(grayish, yellowish, greenish) usually indicate advanced stages of
decomposition. Some authorities have recommended that the
presence of free ammonia should be the test for putrefactive
changes in meats and should serve as the basis for condemnation
procedures, but others point out the fact that toxins are formed
even before there is any appreciable formation of ammonia. The
safest guide to the quality of meats is undoubtedly the bacterio-
logical test. As to the question on what bacteriological findings
shall the quality estimates of meat be based, it is suggested that
judgment be based upon the number of bacteria present and
generally irrespective of kind. If exposed and comminuted meats
do not contain more than 1,000,000 bacteria per gram, they may be
presumed to be reasonably wholesome. The exceptions to this
numerical limit are the finding of pathogenic and toxin-forming
bacteria. The conclusive proof of the mere presence in meats
of bacteria which are pathogenic to man is sufficient to condemn
such meats. It is reasonable to assume that most bacterial in-
vasions of meats are of the putrefactive kind and hence objection-
able, and it is therefore fair and just to all concerned to fix a nu-
merical limit at which such foods are still reasonably wholesome,
as suggested. There are, however, those notable exceptions where
meat contains toxins and ptomaines in quantities sufficient to
produce serious and even fatal poisoning without bacteria being
present, as when fresh meat has been in contact with decomposed

176

BACTERIOLOGICAL METHODS

and toxin-bearing meats from which it has taken up the poisons
by absorption. It is therefore desirable and often necessary
to supplement the bacterial count by the toxicity test.

The numerical limit above suggested (1,000,000 per gram
of the meat substance) pertains to bacteria found upon the ex-
terior of the meat bulk or in the outside cells and tissues of the
meat bulk or meat particles. Proper care must therefore be

observed in taking samples
and in preparing the sample
for plating. In the case of
bulk meats such as whole
slaughtered animals, hams,
bacon, etc., pieces as nearly
cubical as possible (about 1
gram each) are removed with
a sharp sterilized scalpel,
the outer surface of the meat
forming one face of the cube.
This is to be weighed and
pulped in a sterile mortar
with an equal amount of
sterile normal salt solution
and this pulped material is
then made up to the desired
dilutions for plating, using
normal salt solution. Gela-
tin media should be used for
culturing and incubation should be done at 200 C. for a period of
3 days and the counts made. In the case of sausage meats and
comminuted meats generally, take 1 gram quantities, pulp
thoroughly and mix thoroughly with the required amount of nor-
mal saline and plate. In the case of soups and soup stocks hav-
ing a meat or meat derivative base, take 1 cc. quantities, from
the thoroughly mixed sample, dilute and plate.

Fig. 58. — Illustrating the method of
making I an intravenous injection into a
rabbit. The ear is manipulated to induce
hyperaemia and the surface vein is com-
pressed near the base of the ear, to facilitate
the inserting of the syringe needle. —
{McFarland.)

TOXINS IN MEAT 1 77

Weinzirl and Newton describe a method of determining the
bacterial content of meat, in which the meat is ground in a mortar
with sterile sand and normal salt solution to obtain an emul-
sion for inoculation into the culture media, and report the appli-
cation of this method to the determination of the bacterial con-
tent of a number of samples of market Hamburger steak. The
result showed that the standard of 1,000,000 bacteria per gram
advocated as a maximum limit for the salable product is much
too low, as nearly all the samples examined would be condemned
on this basis, though showing no taint or other evidences of
putrefaction. The authors propose a limit of 10,000,000 bacteria
per gram.

For making toxicity tests of meats, broths, sausage meats,
soup stocks and other meat products, the following general
method is recommended. In case of solids such as meats (raw,
smoked, cooked, canned or pickled), sausages, sausage meats, etc.,
10 grams of a well-mixed average sample are well pulped in 10
cc. of boiled distilled water. Let stand for 20 min. with frequent
stirring. Express and filter the extract through a clay bougie.
The toxins being soluble will be found in the filtrate. Inject
2 cc. of the clear filtrate into the subdermal connective tissue
or intraperitoneally into guinea-pigs or white mice, using three
animals for each test. If one or more of the animals thus
inoculated die within 48 hr., or if they show marked symptoms
of intoxication without dying, the meat is unfit for consumption.
In the case of soups, broths, soup stocks, chop suey and other
meat products which contain liquid, the procedure is much simpler.
Take suitable quantities of the thoroughly mixed sample and
filter, first through filter paper and finally through the clay
bougie, as for the meat extract already described, and inject
2 cc. quantities as already explained. The toxicity tests should
in all cases be supplemented by the plate count.

Botulism or sausage poisoning is due to a toxin (botulin)
formed by the Bacillus botulinus (Lat., botulus, a sausage), a

178 BACTERIOLOGICAL METHODS

large anaerobic sporogenous saprophyte especially common in
sausages and sausage meats, particularly in liver sausages, blood
sausages, jelly sausages, in hams, in liver pate, canned meats,
etc., etc. The bacillus, inclusive of the spores and the highly
virulent toxins which it forms, are destroyed by boiling and
thorough cooking. The digestive ferments do not destroy the
toxin. The usual smoking of hams and sausages does not de-
stroy the toxin or the bacillus. The bacillus is killed by strong
brines, but this does not also destroy the toxin. The oval spores
are quite readily killed by heat and chemicals. Heating to 8o°
C. for 1 hr. kills them. Ichthyotoxism (fish poisoning) and
mytilotoxism (shellfish poisoning) are closely akin to botulism
and are in all probability caused by the same bacillus or perhaps
a varietal form of B. botulinus. The occurrence of the Bacillus
botulinus is, however, not limited to pork and sausage meats.
Well-authenticated cases are on record of the occurrence of this
bacillus in canned vegetables and in domestically prepared string
beans served without previous heating. There is no doubt
that the heat employed in the canning process destroys the toxin
formed, but the temperature may not always be high enough to
kill all of the bacilli and their spores even though the spores are
not very resistant to heat (8o° C). Bacillus botulinus does not
multiply in the living organism. It grows readily in slightly
alkaline media at a temperature of 180 to 250 C. At higher tem-
peratures (3 50 to 370 C.) it grows only sparingly and without
the formation of toxin. Cultures give out an odor of butyric
acid.

In pickled, canned and otherwise prepared and preserved
meats, and mixtures of meat and vegetables (chop suey, pork and
beans, etc.), the processes of bacterial development are greatly
modified. The use of deodorants, of preservatives and color-
ing agents mask or obscure many of the decomposition changes
in meats. " Very frequently the only cause for suspicion is an
unusually heightened color or a lack of the normal meat flavor.

MEAT BACTERIA

I79

Sausage meats are found on the market so highly colored as to
produce a red ink with the water in which they are boiled. The
meat dealer tries to deceive the housewife by stating that the
red color is derived from the rich red blood of the meat itself,
whereas the red coloring matter of the blood is decomposed by
the boiling and the boiled meat extract is only slightly colored.
Very frequently pickled pigs' feet appear on the market which look
quite normal, the only suspicious character being an unusual
pallor of the surface with a smeary consistency and a lack in the
flavor. On microscopical examination it will be found that the
surface of the meat is covered or coated with yeast cells, mold
hyphae and mold spores and bacteria. The American method
of making sausage and sausage meats from carelessly and pro-
miscuously handled meat trimmings which accumulate during
the day's work in the retail meat markets, is accountable for the
high contamination with bacteria and other organisms (10,000,000
to 100,000,000 per gram). Such sausage meats are also very
frequently colored to reduce the pallor due to the use of ex-
cessive amounts of fatty tissue trimmings, thus leading the cus-
tomer to believe that there is a considerable amount of muscular
(red meat) tissue present. The coloring also serves to hide the
beginnings of decomposition changes in the meat. Preservatives
are added to check and mask the decomposition changes which
have begun to manifest themselves. It is unlawful to add
coloring substances to sausage meats, but it is permissible to color
sausage casings.

Numerous chemical tests for ascertaining the existence of
putrefactive changes in meats have been recommended. The
Ebers test appears to have met with considerable favor and is
made as follows: Into a test-tube pour about 3 cc. of a mixture
composed of 1 part, of pure hydrochloric acid, 1 part ether and
3 parts alcohol. This tube may be closed with a perforated
rubber stopper carrying a glass rod which is pushed through
the opening of the stopper so that the end almost touches the
13

i8o

BACTERIOLOGICAL METHODS

liquid in the tube. Dip the free end of the tube into the meat
pulp, meat extract or meat broth and, after shaking the tube in
order to fill it with the acid vapors, insert the rod, closing the tube
with the rubber stopper. If the juice or the meat particle is
from decayed meat, a grayish smoky vapor appears at the end
of the glass rod, which settles to the surface of the liquid. There
must be no free ammonia in the room while making the test.

The test is not applicable to
pickled meats. This test
should be made supplementary
to the microscopical, bacterio-
logical and toxicological exami-
nations already explained. In

place of the test-tube or
reagent glass above recom-
mended, the small perfume
sample bottles with glass rod
stoppers may be used in mak-
ing the test.

Fig. sq.— Bacillus tetani as seen in a Sausage meat binders or

scraping from a wound. Some of the fiii p p v rParJilv detected

organisms show spore formation while nilers are very reaanY aetectea

others do not. The pale globules are by means of the compound

blood corpuscles. (X iooo). — (Kolleand . „ , ,

Wasserman.) microscope. Corn starch and

wheat starch fillers are most

commonly employed, the object in adding them being to increase

the water content of the sausage meat. Some brands of sausage

contain corn meal and other cereal products. Egg albumen and

tragacanth fillers are used occasionally, and it is said that it is

possible to increase the water content of the meats by 30 per

cent, with only 3 per cent, of the tragacanth filler. The increase

in water content through the use of the starch fillers is about 5

to 10 per cent. In examining meats for starch fillers or added

cereal it must not be forgotten that some of the spices used

contain starch (pepper, allspice).

CEREAL IN SAUSAGE MEAT l8l

Graham, of the laboratory division of the Bureau of Animal
Industry, has recommended a method for determining the per-
centage of starch added to sausages and sausage meats. A
small pellet of a thoroughly mixed sample of the meat preparation
is well pulped and teased j[out. Make* the usual slide mount,
using just enough of the prepared material to fill the space be-
tween slide and cover, using some pressure. Count the number
of starch granules in the areas (squares) of the ocular scale and
compare with the known number of similar starch granules in
i, 2, 3 and 4 per cent, mixtures of the same starch. Rarely does
the amount of starch filler added exceed 3 or 4 per cent. Mr.
Graham states that the method gives results accurate within 10
per cent., which is sufficiently accurate for all practical purposes.

It is suggested that the special spore and mold counter de-
scribed elsewhere (A or B, Fig. 5) be used with the ocular counting
scale (Whipple's) for making the starch determinations in sausage
meats. The exact number of starch granules in mixtures con-
taining 1 per cent, of starch should be carefully ascertained, follow-
ing the general method recommended for rinding the number of
oil globules representing 1 per cent, of butter fat in milk. For
determining the number of granules in 1 per cent, suspensions of
the starch, it is suggested that weak solutions of gum arabic (1 per
cent.) be used. The gum solution keeps the meat particles as well
as the starch granules in suspension until the counting is com-
pleted. Having once determined the exact number of granules
in 1 per cent, of the starch suspension, it is a simple matter to
make comparative determinations of homologous starch in
sausage meats, or in other substances, as may be required.

Add 1 gram of a well-mixed sample of the sausage or sausage
meat to about 2 cc. of water in a suitable dish and mix thor-
oughly, in order to wash the starch from the meat particles. Next
add enough of the gum arabic solution to make a total of 9 cc.
of thejiquid, thus making a dilution of 1-10. Mix thoroughly
in order that the starch present in the meat may be uniformly

1 82 BACTERIOLOGICAL METHODS

distributed and make the counts as for spores or yeast cells, and
from the findings determine the percentage of starch which has
been added. This quantitative method for determining added
starch is applicable even if the starch has been dextrinized through
the cooking of the sausages, provided the individual granules
are still recognizable and provided also the identity of the starch
is still ascertainable. Corn meal and corn starch are the more
common sausage fillers used in the United States.

The above method for determining the percentages of starch
in mixtures could also be employed, modified to suit special cases,
in the examination of compounds of flour, of meals, for ascertain-
ing the percentage of starch in baking powders, in almond meal,
in adulterated mustard and in other products where starch or flour
is used for purposes of adulteration, and to ascertain the pro-
portions in flour or meal compounds, etc.

In frozen meats the red blood corpuscles are almost com-
pletely decolorized and disintegrated (hemolyzed), changes
which are readily observed under the compound microscope.
The microscope will also prove useful in the detection of added
coloring substances. The micro-sublimation test will readily
demonstrate the presence of benzoic and salicylic acids in meats
and meat products.

The microphytic examinations of meat include the following
groups of the plant kingdom :

1. Penicillium Species. — Especially common on hams, bacon
and smoked meats generally. These molds are essentially aerobic
saprophytes and are therefore found on the exterior of meats.

2. Aspergillus Species. — These molds are apt to occur on
and in fish meats, in gelatin, in canned meats and in pickled
meats.

3. Mucor Species. — These small molds are less common
than the above. They may occur on pickled meats and on
meats that are kept in damp places.

4. Yeasts. — Yeast cells may occur on pickled meats and,

MEAT BACTERIA

l83

more especially, in meats and meat products which contain starch
and sugar.

5. Bacteria. — It is not necessary to enter into any extensive
discussion of the different species and varieties of bacteria which
may occur in and upon meats. The more important bacterial
invasions of meats have already been mentioned. The following
is a partial list of the more important species which the food bac-
teriologist may be called upon to look for in meats:

a. Bacillus botulinus. — Most common in sausages, as already

Fig. 60. — B. tetani, showing flagellae.

stated elsewhere. Forms highly virulent toxins and produces
rancid changes.

b. Bacillus tuberculosis. — Will be found in meats of tuber-
culous animals.

c. Bacillus tetani. — May occur in meat products, more es-
pecially in gelatin. It is essentially anaerobic but thrives better
in association with aerobes, and it produces one of the most
virulent toxins known wdiich is, however, very unstable in its
chemical composition and easily destroyed. A temperature of
6o° to 650 C. destroys it and it is also very quickly destroyed on
exposure to air and light. The danger from the tetanus bacillus
pertains to possible inoculation with the bacillus rather than the

1 84

BACTERIOLOGICAL METHODS

ingestion of the toxins, which might be formed outside of the
body and absorbed by the meat.

d. Cadaver bacilli. — Under this head are included a variety

of bacteria which cause putrefactive

U changes in dead animals and in meats,

with toxin and ptomaine formation,
and to which reference has already
been made.
e. Bacillus anthracis . — The anthrax
bacillus may occur in all food-produc-
ing animals, and its isolation from
beef and other meats may become an
occasional necessity in the food labo-
ratory.

/. Staphylococcus group. — These
may occur in great abundance in liv-
ing animals, causing septic decomposi-
tion changes in tissues and organs.

g. Streptococcus group. — Like the
Staphylococci, these organisms pro-
duce pyemic or septic changes in liv-
ing animals.

h. Numerous other bacteria may
on occasion come to the attention of
the food bacteriologist, as the bacillus
of hog cholera, of swine plague, of
swine erysipelas and others. In this
connection we must not forget the
possible presence in beef, and less fre-
quently also in pork, sheep and horses, of the ray fungus (Actin-
omyces bovis) which is the primary cause of "lumpy jaw" in
cattle and which disease is transmissible to man.

Examination of meats for the presence of encysted trichinae
(Trichinella spiralis) is incidental rather than a routine in the

Fig. 6i. — Tetanus bacillus
stab culture in glucose-gelatin 6
days old. — (McFarland, after
Fraenkel and Peijfer.)

TRICHINA

l85

food laboratory. Even if the meat is found to contain trichinae
it does not warrant condemnation procedures, because these organ-
isms are harmless provided the meat is properly cooked be-
fore eating; however, it cannot be denied that no consumer could
be persuaded to use meat thus infected. The examination of
pork for the presence of encysted trichinae was at one time a
regular routine in the larger slaughtering houses of America be-
cause of the European (largely German) boycott against Ameri-

• m

Fig. 62. — Actinomyces bovis from broth culture (X 1000). — (Williams.)

can pork. In recent years the routine examination for trichinae
has been very generally abandoned.

Trichinae are not uniformly distributed in the muscular
tissue of the animal. They are most abundant in the diaphragm,
next in the base of the tongue, in the laryngeal, lumbar, mas-
ticatory, and abdominal muscles and nearest the tendinous
insertions of the bones. They are never found in adipose tis-
sue. They may occur in wild hogs, in dogs and in bears and of
course also in man. To examine meat for trichinae, cut bits

1 86 BACTERIOLOGICAL METHODS

from the organs of chief distribution of the parasite. From
these samples cut small flat pieces and compress between two

Fig. 63. — Colony of Actinomyces bovis from cow. — (Williams.)

glass slips and examine under the low power of the compound
microscope. As a clearing agent a solution of acetic acid (1-30)

Fig. 64. — Encysted Trichina spiralis (Trichinclla spiralis) in muscle tissue. —

{Stitt, after Ziegler.)

may be used. To clear sections of salted hams or other meat,
use diluted potassium or sodium hydrate. Examining minced

EGGS 187

meats and sausages for trichinae requires greater care and
persistency.

Encysted trichinae retain their vitality for a long period
of time when kept at a low temperature, and persist even after
the meat has undergone decomposition through bacterial infec-
tion. The wandering embryos are harmless and the muscle
trichinae continue their development only in another host, as
man, dog or bear. In the intestinal tract of this second host
they become sexually matured, growing to a length of 0.5 to 0.75
mm., and produce young in large numbers. Trichinella does not
produce ova.

The inexperienced analyst might mistake vinegar eels (in
pickled meats), Miescher's bodies (Sarcocystis), lime concretions,
muscle degenerations and trichinae-like worms {Pseudo-trichina) ,
found in the muscles of the rat, mouse, rabbit, fowl, fish, mole
and other animals, for trichinae.

18. The Bacteriological Examination of Eggs and Egg
Products

Among the foods which require the attention of the bac-
teriologist are eggs and egg products such as evaporated eggs,
frozen eggs and dried egg albumen. Many fresh eggs are quite
free from bacteria, or if bacteria are present they do not exceed
negligible quantities, usually not over 500,000 per cc. Ex-
tensive investigations made by Stiles (Bureau of Chemistry)
show that the contamination of eggs is in proportion to age and
favorable temperature. Thus during warm weather the bacterial
development is quite rapid, whereas cold retards such develop-
ment. Placing contaminated eggs in cold storage clunks bacterial
development temporarily and even causes a reduction in the
number of organisms present at the time the eggs were phi cod
in storage, but within a short time the temporary numerical
reduction in bacteria is not only regained but there is a steady

1 88 BACTERIOLOGICAL METHODS

increase in proportion to the time of storage, until a maximum
development is reached. The bacterial flora of the white and
of the yolk of the egg differs quantitatively as well as qualita-
tively. It may happen that the yolk is badly infected while
the white is in comparatively good condition. As a rule, how-
ever, if the yolk is highly contaminated the white is similarly
affected. In fact the first decomposition changes generally take
place in the periphery of the egg albumen, the infection taking
place via the exterior of the shell.

Commercially, eggs are designated as fresh, stale, storage;
firsts, seconds and thirds (when sorted as to size); watery and
weak when the white is thin; heat eggs; leakers, checks and
mashed when the shell is more or less broken; eggy, strong,
musty, sour and stale as to odor; blood ring, sour rot, white rot,
light rot, spot rots, moldy, black rots, etc., when more or less
rotted and decomposed; green or grass eggs when the white is
more or less green colored through the invasion of bacteria.
These terms have no scientific importance and are of no signifi-
cance to the food bacteriologist, beyond that of indicating the
probable or likely condition and contamination and probable
cause of the change or deterioration of the eggs so designated.

The old-time popular methods of testing eggs by candling,
by shaking to determine "looseness," floating on brine, noting
discoloration of the shell, and by the odor, have their value in
practice but are far from reliable. An egg which gives off the
odor of sulphuretted hydrogen is universally recognized as bad,
rotten or spoiled. In Germany eggs are pronounced spoiled if the
white is gelatinous in consistency (as in old eggs from which
moisture has escaped) or yellowish in color (also due to age),
or if the yolk is more or less adherent to the shell or is more or
less mixed with the white. A fresh egg broken in the manner
customary in the kitchen allows the entire contents, yolk and
all, to fall out into a receptacle without rupturing the yolk.
The white should be of uniform consistency, uniformly trans-

EGGS

189

lucent and without marked yellowish or amber coloration. The
yolk should be uniformly soft and entirely free from all lumpiness
and should not be adherent to the shell.

Eggs are preferably used in the comparatively fresh state,
that is, within a few days or at the longest 8 days after they are
laid. It is, however, not always possible or practicable to use
the eggs while still fresh, and egg preservation has become a very
important industry. Eggs may be preserved in brine, in liquid
glass and in various chemical preservatives. They may also
be preserved in oil, in lard, or coated with tallow, wax or paraffin,

W

Fig.

65. — Egg membrane as seen under the high power of the compound
microscope (X 450).

in order to keep out air bacteria and molds and also to check the
evaporation of moisture from the interior. There is an opinion
among some poultrymen that eggs will keep much longer if
placed in a definite position (vertically with narrower end down).
The now generally employed and preferred method for preserv-
ing eggs is to keep them in storage at a temperature as low as it
is possible to make it. This is perhaps the simplest and
cheapest method for keeping eggs in the natural state. However,
as already stated, cold storage eggs gradually deteriorate, through
bacterial invasion and through loss of moisture, in direct ratio to

IQO BACTERIOLOGICAL METHODS

time, until they finally become unsuitable for consumption. If
fresh-laid eggs were thoroughly cleansed and sterilized externally,
coated with sterilized wax, tallow, paraffin or placed in liquid
glass and then put in cold storage, they would no doubt remain
wholesome for a period of 5 months to 1 year. Under the
usual conditions, cold storage eggs show marked deterioration
in the course of 2 or 3 months as indicated by loss of
moisture, yellowing of the albumen, softening of the yolk, loosen-
ing, increase in size of the air chamber and by the increase in the
bacterial count. The increase in the size of the air chamber is
due to the shrinkage of the egg mass, resulting from the loss of
moisture. According to Greenlee,1 the loss in weight of eggs
is due to evaporation of moisture to the external atmosphere but
the decrease in moisture of the white is not wholly due to external
evaporation, as the yolk takes up a part of the moisture, thus
increasing the moisture and weight of the yolk and also account-
ing for the increased liquidity and explaining the tendency on the
part of the yolk to rupture and the white to gelatinize. Fresh
eggs break well, whereas old eggs, including those kept in storage,
break badly as a rule. The egg mass does not leave the shell
readily, the yolk may be adherent to the shell, likewise the white,
and the yolk membrane ruptures easily and the result is generally
a mess.

Evaporated, dried and frozen eggs have come into extensive
use in recent years. For these purposes, the cheapest and hence
the poorest market eggs are generally employed. There is indeed
an attempt made to cull the bad eggs at the factory, but this is,
as a rule, not done in an efficient manner. The eggs are usually
broken by women and the egg mass is thoroughly mixed and
dried by spraying into a drying chamber or by spreading on a
drying belt, or the drying may be done in very shallow pans.
Of course the temperature must be kept below the coagulation

1 Deterioration of Eggs as shown by Changes in the Moisture Content. Circular
83, Food Research Laboratory, Bureau of Chemistry, Aug. 20, 191 1.

EGGS 191

point of the albumen. Instead of drying, the egg mass may be
preserved by freezing and keeping it frozen until wanted for
use. The important factors are the use of fresh wholesome eggs
and cleanliness. Pennington1 summarizes the importance of
cleanliness as follows: "The preparation of frozen and dried
eggs parallels the milk problem. In dairying it is first necessary
to obtain a cow giving good milk. Then her products must be so
handled that it is maintained in good condition until it reaches
the consumer, a question that has engaged the attention of
sanitarians for many years and is still

the subject of study. The hen seems C CP CO Q

to be more reliable as a producer of ^oJ^oO^

good eggs than is the cow of good £ Cb<0 8<£o

milk. In either case the ignorance g n Ooo ^ChQ 0^
or carelessness of man results in the o § ~Q> 9^3 c?A
addition of multitudes of bacteria a 00Q °^^Q Q r>
which will, and frequently do, spoil £> o o ° ^ r> o ^
the product for food purposes. The 0 <? 8 ^°cp£ °° <*>

fundamental in the handling of whole

6>

•11 • 7 7- t^u £ Fig. 66. — Diplococcus common in

some milk is cleanliness. I he tun- the white of storage eggs,

damental in the handling of good eggs

is also cleanliness, a cleanliness based upon and adapted to the
work to be accomplished."

Recently (19 13-19 14), American poultrymen of the Pacific
Coast have raised a hue and cry against the importation of stor-
age eggs from China. Analysts in food laboratories have been
called upon to examine these as to their suitableness for eating
and cooking purposes. Barring differences incidental to acci-
dents in shipping, the Chinese storage eggs compare favorably in
quality with those cornered by the American egg trust or combine.

What is needed in food laboratories is a method for determining
when an egg is or is not suitable for human consumption. Ac-

1 Practical Suggestions for the Preparation of Frozen and Dried Eggs. Circular
No. 98, Bureau of Chemistry, July 31, 1912.

192 BACTERIOLOGICAL METHODS

cording to observations made, it would appear that the direct
microscopical examination of the white of the egg will give this
information. The yolk of the egg does not lend itself to direct
examination because of the fat globules (cholesterin) and proteid
granules present which interfere with the observation of the
bacteria. The procedure as carried out in the laboratories of
the California College of Pharmacy is to break the egg into a
suitable sterilized dish, after having washed, dried and flamed the
egg thoroughly. The egg mass is carefully tilted and poured
from one portion of the shell into the other until most of the
white is separated from the yolk. Mix the white thoroughly
by means of a sterilized egg beater and examine under the com-
pound microscope, making the counts with the hemacytometer.
Fresh eggs contain bacteria in such small numbers as to make
counting difficult. The principal organism found in the white of
the egg is a coccus form, of fairly large size, having some of the
characters of a diplococcus combined with those of yeasts. Mul-
tiplication appears to be by a modified budding process. After
the cell has developed to maturity it sends out a second cell
which at first appears as a scarcely perceptible speck or protuber-
ance elevated above the surface. This protuberance grows
larger and larger until it has the dimensions of the mother cell,
whereupon the two cells separate. A chain of three cells is
not uncommon and chains of fours and even fives may be found.
At first these structures were believed to be proteid or perhaps
lecithin particles, and in fact attempts to cultivate them in arti-
ficial media resulted in failure. There is, however, little doubt
that they are micro-organisms which develop preferably in the
white of the egg. They apparently do not increase in large
numbers. The highest number recorded in cold storage eggs was
about 180,000,000 per cc. They appear to increase in direct
ratio to the age of the egg. They do not stain very readily. The
most satisfactory stain appears to be carbol-fuchsin, though
the organisms are not in the least acid fast. They do not stain

EGGS 193

with methylene blue. This egg albumen organism requires
further careful study.

The following general methods for making bacteriological
examinations of eggs and of egg products are recommended.

To examine fresh eggs for bacteria proceed as follows: Scrub
the egg well in clean sterilized water by means of a sterile hand
brush. Soak in corrosive sublimate solution (1-1000) for 3
min., rinse in boiled water and wipe dry with a. sterilized
cotton cloth. Flame the end to be opened and set into a suitable
holder (the ordinary breakfast table egg holder will answer the
purpose after being sterilized), and with a sterile instrument
crack open the flamed end and by means of a sterile forceps
pick away small pieces of the shell without rupturing the egg
membrane, making a hole large enough to introduce a sterile
pipette. Rupture the egg membrane with a sterile forceps and
take out 2 cc. of the white of the egg, place in a tared flask with
broken glass and reweigh. Add 10 cc. of physiological salt solu-
tion and shake for 10 min., and then plate definite volumes.

To plate the yolk, break the egg in the usual manner merely
being careful that the yolk membrane is not ruptured; remove
the white of the egg by pouring back and forth in the two parts of
the shell. Let the yolk rest in the larger part of the shell and
puncture the vitelline membrane by means of a sterile forceps.
Withdraw 2 cc. of the yolk and proceed as for the white of the

egg-
To make bacterial counts of eggs which are quite badly
spoiled (rotten eggs), simply break the thoroughly cleansed egg
in the usual manner into a suitable sterilized dish and mix thor-
oughly (white and yolk) by means of a sterile egg beater. Let
stand for 5 min., suitably covered to keep out air bacteria.
Skim off the foam caused by the stirring and take up 1 cc. of the
mixed egg mass and add to 9 cc. of boiled distilled water, and
shake for 10 min. as for the examination of the white of fresh
eggs. Make direct counts from suitable dilutions and also plate

194 BACTERIOLOGICAL METHODS

definite quantities (dilutions as i-io and i-ioo). Certain rotting
bacteria attack eggs very readily. As all housewives know,
eggs which are broken become unfit for use in a very short period
of time, because of decomposition changes. The dried eggs of
the market are very likely to show high bacterial counts and the
manufacture of evaporated eggs, dried egg albumen and other
egg products intended for use as food should be carried on under
suitable methods, keeping in mind the speedy decomposition of
the egg material.

Dried and evaporated eggs and dried egg albumen are ex-
amined for bacteria by the direct method and also by the plating
method.

It is certainly evident that no complicated bacteriological
testing is necessary to determine the unfitness of a rotten egg or
an egg which is highly musty or discolored as shown by the
candle test. The important problem in estimating the significance
of rotten eggs is what percentage of rotten eggs may be present
in an acceptable lot or shipment? It is evident that, under ordi-
nary conditions, the bacterial count of eggs not sufficiently
spoiled to be noticeable to the unaided senses (eggs taken from a
lot in which there are numerous rotten eggs) will exceed many
millions per cc. Condemnation of eggs for human consumption
should not be based upon the percentage of rotten eggs present,
but rather upon the finding of a given number of bacteria in a
mixed sample of the whites of one dozen average eggs taken from
the lot, exclusive of completely rotted eggs. Eighteen eggs
are taken from the lot, cleaned as already suggested, the eggs
broken one by one, pouring the white of each egg into a suitable
container and rejecting all eggs in which the white cannot be
separated from the yolk without mixing. If six or more out of
the eighteen eggs are decidedly bad, the lot is to be condemned
without further examination.

If twelve out of the eighteen eggs break in such a manner
as to make it possible to separate the whites from the yolks,

EGGS 195

then the whites are to be thoroughly mixed and the bacterial
count made in the manner already explained. It is suggested
that if the bacterial count (inclusive of the coccus form and motile
forms or any other recognizable forms) exceeds 200,000,000
per cc. the eggs are not suitable for human consumption. In
certain instances the limiting count should no doubt be lower.
The analyst should take into consideration some of the other
factors indicative of the quality of the eggs, as gelatinous con-
dition of the whites, yellowing of the whites, tendency to adhere
to the shell, etc. There is the possible occurrence of highly con-
taminated yolks with the white in passable condition. This is,
however, a condition not likely to occur in the entire dozen
selected for the count and may be ignored as a factor having any
practical value in the rating of eggs as to quality.

In case the direct examination gives doubtful results, it is
recommended that the plating method be resorted to for check
purposes. For culturing it is advised that egg albumen be used
for the bacteria in the white of the egg and yolk media for the
yolk bacteria. In all media for egg bacteria egg peptone should
be used instead of the ordinary meat peptone. The following
media will be found useful :

Whole Egg Medium
Contents of one egg

Egg albumen peptone (Merck's) 1 gram

Distilled water 100 cc.

Mix ingredients thoroughly in a sterile container by means
of a sterilized egg beater. Titrate to + 1.00. Filter through
cotton. Tube and plate as may be desired. Coagulate care-
fully and sterilize as for culture media in general.

This medium is recommended for making plate counts of
egg bacteria, of the yolk as well as those of the white of the egg.
It will also be found an excellent medium for culturing the tubercle
bacillus. For plating the egg albumen the following medium is
recommended :
14

196 BACTERIOLOGICAL METHODS

Egg Albumen Medium
Whites of two eggs

Egg albumen peptone (Merck's) 1 gram

Distilled water 100 cc.

Prepare as for whole egg medium. If egg yolk bacteria are
to be cultured, the following medium may be used:

Egg Yolk Medium
Yolk of two eggs

Egg albumen peptone (Merck's) 1 gram

Distilled water 100 cc.

Several investigators have reported toxins in eggs. It is
also known that some persons are peculiarly susceptible to
eggs, being more or less injuriously affected on eating even
perfectly fresh eggs. This phenomenon is by some ascribed to
personal idiosyncrasy and others suggest that this is due to
toxins present to which certain persons are perhaps peculiarly
susceptible. The poisonous principles present in eggs should be
more carefully investigated. The possibility of toxin forma-
tion in cold storage eggs also requires further careful study.

Eggs decompose very rapidly when the shell and membrane
are broken and soon become unfit for use, due to bacterial de-
velopment. The shell of the egg serves to prevent, or at least to
check, for a time the development of the egg-rotting bacteria
through exclusion of oxygen (of the air). It is, however, highly
probable that the egg membrane keeps out bacteria even more
effectually than does the shell. Shell-less eggs like those of the
oviparous snakes are well protected against bacterial infection
by the thick membrane, even though the eggs are deposited
in the soil and in decaying rubbish. It has also been suggested
that the egg membrane contains some bacteriolytic or perhaps
bactericidal properties. It is declared, on fairly reliable authority,
that fresh egg membrane applied to buccal inflammations and
threatened abscesses will effect a prompt cure. The Chinese
have used egg membranes as a medicine for many centuries.

MEDICINAL SUBSTANCES 1 97

In spite of shell and of egg membrane, the egg is gradually
contaminated more and more, until finally complete decomposi-
tion has taken place. Tests made by European investigators
show that fresh eggs inoculated with various molds resist penetra-
tion completely for about i month. After 8 weeks species
of Cladosporum had entered. In 12 weeks Phytopthora in-
festans developed and still later Rhizopus nigricans. Other fungi
which finally developed in the interior of the eggs were Clado-
sporum herbarum, Aspergillus niger, Penicillium glancum and
some yeasts. Fresh egg albumen is said to have marked bac-
teriolytic properties, but such properties are certainly quickly
lost upon exposure to the air and light. Some eight or more
species and varieties of bacteria are concerned in the decomposi-
tion of eggs, principally aerobes. Some of these liberate sul-
phuretted hydrogen {Bacillus oogenes hydrosulphnrciis group) ;
others belong to the B. coli group and still others cause decomposi-
tion of the white without any pronounced color development.
Liquefaction of the white as well as of the yolk is the most marked
physical change in eggs undergoing bacterial decomposition.
Mold infection is very generally indicated by an odor of mus-
tiness. Pronounced mold infection is further indicated by spots
shown in candling.

19. The Bacteriological Examination of Pharmaceutical Preparations

Thus far practically nothing has been done as to the bac-
teriological examination and standardization of medicinal sub-
stances. There is a popular belief that the ordinary pharmaceu-
ticals, particularly the tinctures and the fluidextracts, are quite
free from bacteria, the supposition being that these substances
are in themselves highly antiseptic. This is only partially in
accord with facts. Stronger alcoholic solutions of potent drug
constituents no doubt inhibit the more rapid multiplication
of most bacteria and higher fungi, but it is known that weak solu-

198 BACTERIOLOGICAL METHODS

tions (1 per cent.) of pilocarpine, atropine, cocaine, morphine
and of ergot, on standing for a time, show many millions of
bacteria per cc, often also molds, mold spores and some yeasts.
The variation in the resisting power of different bacteria to
different medicinal substances is noteworthy. The pus staphy-
lococci die at once in ether and in a saturated solution of quinine,
but will remain active in a 10 per cent, solution of cocaine, while a
2 per cent, solution of morphine kills them in 24 hr. The same
organisms will resist the action of pure glycerin for 6 to 8 days.
Ten per cent, iodoform, glycerin, camphorated oil (1-10),
solutions of apomorphine (0.2-20), quinine (1-10), antipyrin
(1-2), and cocaine (1-10) are usually quite free from bacteria.
The coal-tar derivatives are generally considered antiseptic in
property. Aquae are frequently found to contain bacteria in
enormous numbers and the syrups are generally more or less
contaminated with yeasts, bacteria and also with molds. It
is known that weak solutions of substances intended for hy-
podermic and intravenous use, when left exposed to the air for a
time, show numerous bacteria. Tinctures and nuidextracts
are always more or less contaminated, showing organisms in direct
proportion to age and the degree in unsanitary factory conditions.
Certain medicinal substances, as those intended for hypodermic
and intravenous use, are presumably free from living organisms.
Undoubtedly the extensive bacteriological examination of medi-
camenta would reveal some of the causes which are responsible
for irregularities in drug action, and would explain some of the
hitherto perplexing phenomena of poisoning resulting from the
administration of certain medicamenta in ordinary medicinal
doses.

The bacteriological examination of medicamenta may be
outlined as follows:

Direct microscopical examination.

1. Bacteria.

2. Molds.

MEDICINAL SUBSTANCES 1 99

3. Mold spores.

4. Yeasts.
Plating methods.
Colon bacillus test.
Tetanus bacillus test.

Tests for the staphylococcus and streptococcus groups.

In securing samples for examination, the precautions necessary
to guard against outside contamination must be observed.
Only rarely will it be necessary to pack the samples in ice. In
the preliminary routine of the laboratory, many of the more ex-
tensive contaminations will be apparent to the unaided senses.
Thus a change in color, in odor, in taste, and opacities in sub-
stances that should be clear, sediments in substances that should
be free from deposits, etc., generally indicate decomposition
changes due to bacteria and other organisms. More or less
cloudy deposits with a clear supernatant liquid indicate possible
spore sedimentation. Extensive contamination by bacteria,
yeasts and molds may be estimated quantitatively by means
of the hemacytometer and other suitable counting devices.
All are agreed that the counts for medicinal substances intended
for administration per mouth should not be very high. How-
ever, no numerical standards have as yet been adopted. It is
suggested that such remedial agents are unfit for use when they
contain bacteria in excess of 5,000,000 per cc. and yeasts and spores
in excess of 500,000 per cc. Plating methods should be resorted
to in order to ascertain the number of living bacteria present.
The potassium tellurite may also be tried in order to ascertain
microbic invasion.

All substances containing gelatin, intended for hypodermic
use or for application to mucous membranes or to abraded skin
surfaces, should be tested for the anaerobic tetanus bacillus
{Bacillus tetani). Comparatively large quantities should be
plated in large Petri dishes or in Erlenmeyer flasks (using agar
media), and incubated at 370 C. in the absence of oxygen. Oxygen
may be excluded by pouring a layer of sterile olive oil over the

200 BACTERIOLOGICAL METHODS

medium or by displacing the air by means of the hydrogen ap-
paratus. The colonies which appear should be examined micro-
scopically to ascertain whether or not the characteristic spore-
forming tetanus bacillus (drum stick bacillus, the Trommelschlager
Bacillus of the Germans) is present. As a confirmatory test, a
suspension of the suspected colony should be injected hypo-
dermically into guinea-pigs or white rats and symptoms noted.
Should other than the spore-forming bacteria be present in the
anaerobic culture, these may be killed by pasteurizing for i hr. at
8o° C, at which temperature all organisms excepting the spores
of the tetanus bacilli are killed. Again incubate at 370 C. for
several days and examine microscopically, and make inocula-
tion tests as already suggested. The finding of a single tetanus
bacillus (as represented by a single colony in the anaerobic culture)
in the gelatin renders it unfit for use.

To test medicinal substances of all kinds in powdered form,
the plating method must be relied on very largely, as the bacteria
which might be present would be hidden or obscured by the
granular particles present. However, extensive yeast and mold
contamination could be detected readily and estimated quantita-
tively by the direct microscopical method. The qualitative
test for this class of substances is very largely limited to the
determination of the absence or presence of the colon group,
the staphylococcus group and the streptococcus group. Face
powders and dusting powders should be free from any consid-
erable contamination with the pus-forming organisms. There
should be uniformly standard methods governing the manu-
facture of all medicamenta, intended for internal or external use,
which must be touched by the hands of the manufacturer. There
must be absence of all skin diseases and of transmissible conta-
gions of all kinds. There should be specific requirements as to
personal cleanliness and the sanitation of the laboratory. Just
as typhoid carriers, cholera carriers and diphtheria carriers em-
ployed as servants in the household and as laborers in the factory

MEDICINAL SUBSTANCES 201

may spread infections among those with whom they are brought
in daily contact, so may the manufacturing pharmacist convey
disease to his customers, through the articles which he offers
for the cure of disease. This subject should receive more attention
on the part of health officials.

Skin and scalp infections (acne, boils, abscesses, carbuncles)
are traceable to the use of powders and ointments. The more
common infections which may be carried by the usual hand pre-
pared face powders and face and scalp lotions and ointments are
pus streptococci and staphylococci, the colon bacillus and the
tubercle bacillus. The most common of these infections are the
staphylococcus group of pus germs and the germ of tuberculosis.
Very few women who use face powders persistently for a long time
escape without more or less severe facial infections. Particularly
is this true of women who use the more or less irritating chemical
skin renewers, that is, so-called cosmetics which act by removing
the superficial epithelial layers of the skin. The use of these
highly irritating agents is generally followed by the application
of the germ-carrying dusting powders and ointments, the more or
less raw skin favoring the infection.

The finger-nail deposits carry many different kinds of germs
accumulated by the skin and scalp scratching process and through
the manifold manipulations of all manner of articles during the
daily work. These various contaminations may be transmitted
to the hand manufactured toilet and face preparations offered
for sale in the retail drug stores. Among the bacteria most
commonly found with the finger-nail deposits are streptococci,
staphylococci and the colon bacilli. Less commonly the itch
mite and the larvas of intestinal parasites and molds are found
among the finger-nail deposits.

To examine finger-nail deposits, scrape the nail of the thumb
and second and third fingers of the right hand (in the case of
right-handed persons) and make the ordinary smear mounts,
using such stains and reagents as may be required to bring out

202 BACTERIOLOGICAL METHODS

the staining properties and the morphological characteristics of
the different kinds of bacteria and of other organisms. Cultural
methods may be desirable for purposes of identification.

The bacteriological testing of ampuls and all medicamenta in-
tended for hypodermic, intravenous and intramuscular use is
reduced to great simplicity. Since these substances must be
absolutely sterile, the finding of living bacteria (by the plating
method) would be proof that they are unfit for use. It must be
borne in mind, however, that cloudiness in ampuls (containing
substances which should be clear) does not necessarily indicate
bacterial contamination, as this condition is frequently the re-
sult of chemical change, possibly occasioned by the alkalinity
of the glass used in making the ampuls. However, all ampuls
which show cloudiness when they should be entirely clear are
to be rejected, even though no living organisms are present.
Oils, salves and plasters may be examined directly (microscopic-
ally), noting in addition to bacteria and other living and dead
organisms, possible decomposition changes in oils and fats, as
indicated by the presence of the characteristic fat crystals. For
plating, oils may be emulsified with measured quantities of the
liquefied gelatin or agar media and measured quantities poured
into Petri dishes; or definite quantities (o.i cc, o.oi cc, o.ooi cc,
etc.) may be planted into the Petri dishes in the regulation man-
ner and the liquefied gelatin or agar poured over it and spread.
Incubate for from 3 to 4 days at a temperature of 200 C. and
count the colonies formed. Plasters and salves may be plated
by liquefying them at a temperature not to exceed 400 C,
making the desired dilutions with sterilized olive oil.

20. The Microscopical and Bacteriological Examination of Syrups

Syrups are very important products extensively used in
medical and pharmaceutical practice and at the soda fountain,
and for practical purposes may be grouped as follows:

SYRUPS 203

1. Medicinal syrups.

a. Officinal, simple and medicated.

b. Patent and proprietary medicated syrups.

c. Medicinal preparations containing syrup or saccharine substances.

2. Soda fountain syrups.

3. Fruit juices containing sugar. Fruit juice concentrates.

4. Syrups, molasses, treacle.

Syrups contain cane sugar in variable amount. Many of
them also contain variable amounts of invert sugars. The
pharmaceutical syrups are numerous and may be prepared with
sugar or simple syrup. The chemicals and therapeutic agents
which are added undoubtedly have some influence on the keeping
qualities of the preparations, but thus far no one has made any
extensive report on the contaminations of medicinal preparations
containing sugar or syrup. The soda fountain syrups are essen-
tially sweetening and flavoring agents used in the preparation
of the familiar soda fountain beverages, ice creams and sundaes.
They may also contain physiologically active ingredients such as
caffeine, cocaine, cocoa, ginger, etc.

Manufacturers have more or less difficulty in preparing syrups
which will endure without spoiling. Simple syrup and the fruit
juices in particular are likely to undergo yeast fermentation, and
in many instances there is also mold development, more com-
monly the Penicillium glaucum. The contaminations of medicinal
syrups and medicines containing syrup are very variable. Some
of these preparations keep for a long time, while others appear
to be quite susceptible to the invasion of yeasts. In many in-
stances the initial yeast invasion is soon followed by the develop-
ment of bacteria and also mold.

Among the organisms which are most likely to attack syrups
and solutions containing sugar are the so-called potato group of
bacilli. The most common and most destructive members of
this group are Bacillus vulgatus,B. Uodermos,B. rnesentericusfuscus,
B. mesentericus ruber and B. levaniformans, which latter species
is really the group type. These bacteria are widely distributed in

204

BACTERIOLOGICAL METHODS

nature, occurring in the soil and in surface waters, and because
they very frequently comtaminate potato cultures, are known as
the potato group. They are spore forming, which spores are

Fig. 67. — Wild and pseudo-yeasts. A, S. pombe. {After Lindner); B, Torulce.
{After Pasteur); C, Mucor, (1) spores; (2) germinating spores and mycelium; D, S.
apicidatus; E, Mycoderma vini. — {After Bioletti.)

remarkably resistant to heat, being able to withstand boiling for
2 hr. Lafar maintains that they will resist the temperature of
streaming steam for 6 to 7 hr., thus making them the most re-

SYRUPS 205

sistant of all bacterial spores. They are further characterized by
their ability to form gummy products on potato, on bread and in
sugars.

Bacillus mesentericus niger and B. granulatus mesentericus
are also members of the potato group and may be found in sugars
and syrups. Leuconostoc mesenteroides and Bacterium gelatinosum
betce, which are frequently found in sugar beet juice, are evidently
related to the potato group. Bacillus gummosus, also a gum
former, is frequently found in digitalis infusions. This latter
bacillus is comparatively large, feebly motile, forms spores and
produces lactic and butyric acids. Another organism which is
probably closely related to the potato group is the Bacterium
mesentericus panis viscosum, the cause of stringy or slimy bread.
All of the organisms named are Gram positive, are spore formers,
liquefy gelatin and are motile, having flagellar The principal
biologic characteristics of the group may be given as follows:

1. They form gums (levan) from sugars.

2. The spores are very resistant to heat.

3. They have a very low nutrient requirement.

Very minute quantities of sugar are sufficient to induce them
to grow and multiply, resulting in the transformation of some of
the sugar into a gum known as levan.

From the above general characteristics of the sugar bacteria
it is almost self-evident how they may cause very serious harm to
solutions of all kinds containing sugar. The highly resistant
spores make thorough sterilization difficult and, since high tem-
peratures cause inversion of sugars, the use of the autoclave and
repeated and prolonged heating at the boiling temperature are
frequently not permissible. In cases where the use of the autoclave
is permissible, a single exposure at a temperature of 1200 C.
for a period of 30 min. is sufficient to kill the spores. More
generally the fractional method of heat sterilization must be
employed.

It is a noteworthy fact that after the gum formers have

2o6

BACTERIOLOGICAL METHODS

once gained access to the syrup they are not easily exterminated.
This emphasizes the importance of great care and cleanliness in
the preparation of all syrups. It is also a fact that high con-
centrates of sugar are not so liable to be attacked as are the
weaker solutions. The most favorable strength of sugar solution

Fig. 68. — Bacillus californiensis isolated from the roots of the sugar beet. A
typical gum former found in the soil, on the roots and in the surface tissues of the
sugar beet and in the juice of the sugar beet, a, Beet root cells showing a mixture
of cell plasm and B. californiensis (mycoplasm); b, epidermal cells of the root show-
ing bacteria within the cell and also on the exterior; c, a bit of mycoplasm removed
from the cell by pressure; d, B. californiensis removed from the cells by pressure;
e, B. californiensis from a pure culture in beet root gelatin; /, zoogloea form of
B. col.; g, gelatinized form of B. cal.; i, a single chain highly magnified (X 450
to 1000).

is about 20 per cent. Development may, however, take place in
60 per cent, solutions. The organisms are facultatively anaerobic
and their growth is greatly accentuated by free aeration. Com-
pletely rilling the containers will materially check and even
completely prevent the growth of the bacteria.

SYRUPS 207

The bacterial diseases of syrups and substances containing
sugar are by far the most important and the most difficult to
combat. We must, however, not forget the far more common
fermentation and decomposition changes induced by the yeasts
and molds. Yeasts are very likely to attack the openly exposed
fruit juices and fruit syrups at the soda fountain. Crushed
fruits of the soda fountain may contain yeasts and also bacteria
and molds. Fruit products invaded by yeasts are no longer
suitable for use at the soda fountain. The attempt to render
fermenting fruit juices usable by heating is not practicable as the
natural flavor is lost, and the attempt to use such fruits or fruit
juices would prove disastrous to the soda fountain business.

Little is known regarding the influence of the therapeutically
active ingredients of the medicinal syrups on the development of
the sugar-destroying bacteria but it would appear from the reports
of some observers that they do not materially check them.
It is quite evident that the carbonated sugar-bearing soda foun-
tain drinks are just as liable to bacterial invasion as are the
noncarbonated soft drinks. The sugar-destroying gum formers
frequently do great damage to the soda bottling business, at times
ruining the entire output. The trouble may be slow in making
itself evident. For a long time all appears to be well at the
factory but gradually complaints come in from the dealers (dis-
tributors) and the consumers. It is claimed that " gelatinous
lumps" appear in the drink and that the taste is insipid and not
sufficiently sweet. Very naturally these complaints are detri-
mental to the business. With proper attention to details at the
factory the trouble could have been avoided. In the manu-
facture of soda fountain syrups and medicinal syrups, one of the
most important essentials is thorough sterilization of the syrup
to be used. The other ingredients which enter into their com-
position should also be thoroughly sterilized. All containers
should be thoroughly sterilized and they should be completely

208

BACTERIOLOGICAL METHODS

filled while the containers and the syrup are still hot and then her-
metically sealed by means of sterilized stoppers.

In the case of medicinal syrups and sugar-bearing medicines
which are contaminated by bacteria, it must be borne in mind
that the active constituents present are also more or less com-
pletely decomposed. Such substances should be quite free from
contamination and the presence of marked contamination should

h

C -

^9?

M

-a

a

-b

I:

V:

a

- -b

m

I

-b

Fig. 69. — Stab culture appearance of B. californiensis in beef extract gelatin
tubes. 1, Appearance of growth on third day after inoculation; 2, deep stab cul-
ture 24 hr. old, from tube (1); 3, same as (2) 36 hr. old. Liquefaction of gela-
tin is noticeable; 4, same as (2) 3 days old. In the course of 2 weeks the entire
contents of tube became liquefied.

form the basis for the condemnation of such products. The
adoption of a numerical bacterial, yeast and mold standard would
appear highly desirable.

Among the products which are classed with the syrups are
syrup or molasses and treacle from the sugar cane, and the sorghum
molasses of the central and northern states, maple syrup and
other syrups of commerce including the so-called corn syrup which

SYRUPS

209

is largely starch glucose, the glucose syrups, honey and other
syrupy substances used as a food and condiment. As a rule
these do not come to the notice of the bacteriologist. A micro-
scopical examination may be made occasionally. For example,
the finding of pollen grains may be the means of distinguishing
between true and imitation honey. Molds occasionally attack
syrups and less frequently yeast cells may be found in some of the
improperly stored syrups.

ft

W W

Fig. 70. — Streak culture appearance of B. calif omiensis on beef extract gelatin. 1,
24 hr. old; 2, 48 hr. old; 3, 72 hr. old; 4, 4 days old.

The bacteriological examination of sugars, candies and condi-
ments is wholly incidental and need not be discussed. Analysis
of this class of substances is left almost entirely to the chemist
and the micro-analyst.

The bacteriological and microscopical examination of crushed
fruits, fruit juices and fruit juice concentrates is much the same
as for syrups. The crushed fruits at the soda fountain are very
prone to yeasty fermentation during the hot summer months.

2IO BACTERIOLOGICAL METHODS

Grape juice is apt to become moldy. The nature of the con-
tamination will depend upon the relative amounts of sugar and
acids present.

21. The Microscopical and Bacteriological Examination of Fer-
mented Foods and Drinks

The examination of alcoholic drinks and other fermented
liquids and fermented food substances including certain fermented
products used in the preparation of foods, on the part of the
bacteriologist, is of minor importance and is largely supplementary
to the analyses of the chemist and the organoleptic testings of
the expert taster. The methods of procedure will be largely
limited to the microscopical examination of concentrates, of natural
and centrifugalized sediments, of sedimentary suspensions and
of surface formations or deposits, with a view to the detection of
added or other impurities and the recognition of abnormal fer-
mentative changes, and invasions by objectionable bacteria, yeasts
and molds. " Diseased" or "sick" wines, beers, porters, ales,
vinegars, pickles, sauerkraut, etc., should be carefully examined
as to the quantity and identity of the objectionable organisms
present. In order that the report of the bacteriologist may
supplement the report of the expert taster, it is absolutely essential
that the bacteriologist have a thorough knowledge of the micro-
scopical appearance of normally fermented products. This
knowledge may be gained only through experience. The yeasts
and other organisms concerned in normal wine fermentation are
well known to the specialists who have made a long study of wine
ferments.

There appears to be no recognized standard as to the number
or kind of organisms which may be permissible in properly fer-
mented and properly clarified wines and in other fermented
drinks, nor does the present status of the subject warrant the
adoption of numerical limits as to the organisms present. There

FERMENTED FOODS AND DRINKS

211

are, however, many instances in which the findings of the bacteri-
ologist may be final and conclusive as to the quality and purity
of the wine or other fermented alcoholic beverages or of fermented
food products. If, for example, there is abundant mold forma-

Fig. 71. — Development of Mucor mucedo. a, b, c, d, stages in the formation of
the zygospore; d, mature zygospore; e, f, endospore formation; g, endospores; h,
germinating -spore, the beginning of the new zygospore forming cycle. The appear-
ance of the stalks and the spore bearing capsules explains why this is called the
"pin cushion fungus." Related molds occur on stale bread, on fruits, on damp
gloves and leather generally. It is the cause of a fatal infectious disease in house-
flies.

tion in a product which normally should be free from such organ-
isms, then the product should be pronounced unfit for human use.
Again it may be possible to recognize abnormal bacterial or
perhaps abnormal yeast development as the causes of the deterio-

212 BACTERIOLOGICAL METHODS

ration and undoubtedly the microscope alone will in most in-
stances reveal the presence of numerous abnormal and objec-
tionable organisms, even before the expert taster has been able
to appreciate any abnormal alteration in flavor or in bouquet.

The following is a very brief outline of the principal fermen-
tations concerned in the manufacture of alcoholic beverages.

A. Alcoholic Fermentation. — The alcohol forming ferments or
zymases, or yeast ferments proper, are by far the most common
and most widely distributed in nature and the most important
from a commercial and economic standpoint. The zymases act
upon sugars splitting these into alcohol and carbonic acid gas,
thus acting upon the end products formed by the diastases and
preparing them for the action of the acid forming ferments.

. Zymases are formed by a great variety of plants and animals,
more generally by the so-called yeast plants (the Saccharomyces
and Torula groups). The alcohol-generating enzymes formed
by these plants are capable of being isolated or separated from
the living cells which form them and may continue the fermenta-
tive activities indefinitely. Alcoholic fermentation is by no
means a simple process. The degree of alcohol production
and by-product formation varies greatly, depending upon a great
variety of factors and influences. To enter into a fuller dis-
cussion of the details of the fermentative processes and a de-
scription of the organisms involved, is not practicable or essential
for the present purpose. The number of saccharine substances
capable of undergoing alcoholic fermentation is legion, and it
has thus far not been possible to ascertain the number and variety
of yeast organisms and associated organisms which are involved
in the multitudinous fermentations (natural and artificial)
resulting in the formation of alcohol. In commercial practice
(in the manufacture of wine, beer, brandy, etc.), a distinction is
made between upper yeasts, lower yeasts, wild yeasts, etc. In
some breweries lower yeasts are the chief fermenters used and in
others the upper yeasts are preferred. For example, it is claimed

FERMENTED POODS AND DRINKS 213

that the use of bottom yeast (Unterhefe) makes it easier to guard
against the entrance of wild yeasts and other objectionable
organisms. On the other hand it is claimed that the use of the
upper yeast (Oberhefe) yields a better quality of beverage. These
are factors of the greatest importance to the bacteriologists and
zymologists employed by the breweries but concern the food
bacteriologist but little.

The following are some of the more important yeast organisms
concerned in alcoholic fermentation, giving the principal fermenta-
tive activities of each. Hansen's differentiation between the
genera Saccharomyces and Torula is based upon sporulation.
Saccharomyces forms spores (Ascospores; usually four spores in
each ascus or spore sac, rarely eight) whereas Torula does not
form spores. According to some authorities this is not a practical
basis of differentiation.

Saccharomyces

cereviseos Hansen. A typical top yeast.

pastorianus, Hansen. A bottom yeast.

intermedins, Hansen. A rather feebly acting top yeast.

validus, Hansen. A top yeast.

ellipsoideus, Hansen. A typical bottom yeast.

tnrbidans, Hansen. A bottom yeast the cause of turbidity.

willianus, Saccardo. A flavor-producing yeast.

boyanus, Saccardo. Causes turpidity in beer and wine.

logos, van Laer. A bottom yeast developing a flavor.

thermanitonum, Johnson. A rapidly acting ferment.

ilicis, Gronlund. A bottom yeast; isolated from Ilex.

aquifolii, Gronlund. Also isolated from Ilex species.

pyriformis, Ward. Found in ginger beer.

vordennanni, \V. and P. Isolated from Arrak.

sake, Yabe. Active in the fermentation of sake.

balatce, Saito. In )'am brandy.

cartilaginosus, Lindner. Isolated from Kephir.

multisporus, Hansen. A top yeast.

mali, Kayser. A cider ferment.

marxianus, Hansen. A Wine ferment.

exiguus, Hansen. In beer wort.

jorgcnscnii, Lasche. Causes turbidity.

zopfii, Artari. Found in syrup.

214 BACTERIOLOGICAL METHODS

bailii, Lindner. In beerwort.

hyalosporus, Lindner. In beerwort.

rouxi, lUitroux. Found in fruit juices.

soya, Saito. In soya sauce.

unisporus, Hansen. In dutch cream.

flava lactis, Krueger. Found in cheesy butter.

hanseni, Zopf. In cotton seed meal.

minor, Engelman. Found in bread.

membranaefaciens, Hansen.

anomalans, Hansen. Causing a fruity flavor.

saturnus, Klocker. Isolated from soil.

acidi lactici, Grotenfeldt. A milk-curdling yeast.

fragilis, Jorgensen. Found in Kephir.

barkeri, Saccardo. In ginger beer.

hidwigii, Hansen. From oak bark extract.

comesii, Covara. From millet seed.

octosporus, Bevjerinck. On dried currants.

mellacei, Jorgensen. A top yeast developing a pleasant odor.

guttulatiis, Robin. Found in a rabbit.

capsidaris, Schionning. From soil.

According to Hansen, Torulas also occur in great variety.
The Levure de sel is a yeast capable of developing in a 10 to 15 per
cent, sodium chloride solution. Those desiring to obtain detailed
information regarding the complete fermentation processes in-
volved in the brewing of beer and other fermented drinks, must
consult the special technical treatises of which there are many
available.

B. Acid-forming Ferments.— Dilute alcohol upon standing
exposed to the air, gradually becomes sour, losing its alcohol
more and more. This loss of alcohol and gain in acidity is due
to the action of ferments which split the alcohol into acetic acid
and water. The organisms which produce the acid-forming fer-
ments or enzymes mostly belong to the group bacteria (bacilli).
The more common and important species are Mycoderma {Bacillus)
aceti, B. Pasteurianum, B. kiitzingianum, B. oxydans and B.
acetosum. The yeast Saccharomyces mycoderma is also capable of
forming acetic acid. The vinegar organisms are most active
at a temperature of 250 C. to 300 C. They are very slowly active

FERMENTED FOODS AND DRINKS

215

at io° C. and are killed at a temperature above 350 C. The
so-called mother of vinegar consists of an agglutinated mass of
Mycoderma aceti and is used as a starter in the manufacture of
vinegar. Thus far it has not been possible to isolate the vinegar
ferment or enzyme from the living cells which form it.

There are also acids of nonalcoholic origin formed by living
ferments, such as oxalic acid, malic acid, citric acid and others,

Fig. 72. — Types of yeast organisms and yeast sporulation. A, Saccharomyces
pasteurianus showing spore formation in fours and eights {after Bioletti); B,
Schizosaccharomyces odosporus, showing simple septation instead of budding, and
spore formation {after Schionning); C, Saccharomyces anomalus, vegetative cells
and spore sacs. — {Marshall, after Kayser.)

which appear to be derived from the direct fermentation of
sugars. Citric acid is formed from sugars through the activity
of two fungi, Citromyccs pfefferianus and C. glaber. Saccharo-
myces hansenii forms oxalic acid from mannit and galactose, with-
out the intermediary alcohol formation.

The following are the more important products in which
there is alcohol formation through the action of yeast organisms.

2l6

BACTERIOLOGICAL METHODS

i. Whiskey and Brandy. — Whiskey and brandy are alcoholic
beverages with an alcohol content ranging from about 44 per
cent, to 55 per cent, (by volume). Whiskey (Spiritus frumenti
of the U. S. P. and Schnapps of the Germans) is usually made
from grain as rye, wheat, barley and corn. Brandy (Branntwein)

^ # • 1 ^'Sb

©

j)

Fig. 73. — Wine and beer yeasts. A, Saccharomyces ellipsoides showing the
young and vigorous cells; B, the same cells old (1) and dead (2); C, S. cerevisea as
top yeast and D, S. cerevisece as bottom yeast. — (Marshall.)

is usually made from grapes. In the manufacture of both whiskey
and brandy there is alcoholic fermentation followed by distilla-
tion, with or without the addition of coloring substances, as
caramel. In both whiskey and brandy, certain collateral prod-
ucts of distillation known as congeners, such as flavor (bouquet),
aldehydes, ethers, trace of fusel oil, trace of fruit or grain color, of

WHISKEY AND BRANDY 217

acids, etc., are present. These congeners are normally present
and vary somewhat, dependent upon variations in the method
of distillation, slight differences in the quality of the grain or
fruit used, the process of fermentation, temperature, etc. With
ageing whiskey as well as brandy undergo complex changes (chem-
ical as well as fermentative) indicated by changes in color, odor
and taste. These slow changes appear to be largely zymotic in
nature but they are not well understood.

Whiskey and brandy may be made from all substances ca-
pable of undergoing alcoholic fermentation, such as rice, wheat,
barley, rye, oats, potatoes, apples, pears, berries of all kinds, etc.
Any apparatus so constructed and equipped as to vaporize and
carry over and condense the alcohol existing in the fermented
product, may be used in distillation. In the process of distilla-
tion certain congeners are always carried over with the alcohol
and these constitute normal inclusions of the brandy or whiskey.
If the congeners are poisonous or otherwise objectionable, then
the distillate containing them is also poisonous or otherwise
objectionable and may render the product unsuitable for human
use. These poisonous congeners evidently exist in certain
products of alcoholic distillation and should be more carefully
investigated.

Alcohol per se (less all congeners) is a protoplasmic poison.
Small quantities, when taken into the system are oxidized and
in so far as it is oxidized, alcohol is a food, but because of its
toxic character, alcohol can never be used as a food having
practical value as such.

Rectified whiskey or brandy is redistilled or double distilled
whiskey or brandy. As a result of this redistillation there is an
increase in the alcoholic strength, with a decrease in the amount
of fusel oil, a change or decrease in the congeners, a change in the
color and bouquet "or flavor, etc. As generally comprehended
rectification implies purification and increase in alcoholic strength,
without foreign additions of any kind. Adding coloring sub-

2l8 BACTERIOLOGICAL METHODS

stances or flavoring agents to raw (unaged) whiskey or brandy so
as to imitate the product which has been allowed to age natu-
rally, constitutes adulteration under the Federal Pure Food and
Drugs Act. Adding whiskey or brandy to alcohol (ethyl) com-
monly known as rectified spirits, does not make rectified whiskey
or brandy.

Various medicamenta may be added to whiskey and brandy,
such as caraway, aloes, juniper berries, absinthium, etc. Many
alcoholic beverages are sold to the unsuspecting public under the
guise of tonics and blood purifiers.

2. Beer. — Beer is a fermented drink generally made from
barley. The carefully selected grain is washed in running water
and then macerated in water to induce germination. This
process liberates the ferment diastase which occurs in the
grain and this enzyme acts upon the starch present converting it
into saccharine compounds. The saccharine compounds are
next acted on by the yeasts (Saccharomyces cerevisecB and other
species) which convert the sugars into alcohol. Hops are added
to give the beer a bitter taste and also for the purpose of in-
fluencing the fermentation process favorably. After the al-
coholic fermentation is completed, the product is filtered, clarified,
pasteurized and occasionally preserved by adding salicylic acid
or other preservative. The alcoholic content of beer varies from
about 1.50 to 6 per cent. Some beers are fortified by adding
alcohol. There are many kinds or brands of beer, differing in
color, flavor, taste and consistency.

Brewers must observe great caution to guard against the inva-
sion of objectionable organisms as bacteria, yeasts and mold,
which might vitiate the normal or desirable process of fermenta-
tion. In spite of all precautions, things often go wrong. The
wort may undergo sour or other objectionable fermentation and
as a result the entire lot may have to be rejected. Wild yeasts
may gain the upper hand and ruin the beer. Even after the
product is finished and placed in the containers, abnormal fer-

BEER

219

mentations may be set up by various bacteria, yeasts and mold,
causing more or less serious spoiling and even complete deteriora-
tion. The following are the more common beer diseases which
may be brought to the attention of the food bacteriologist.

a. Ropiness. — This is quite common. The beer becomes thick
and mucilaginous capable of being drawn out into threads. Two
species of bacteria cause ropy beer; Bacillus viscosus I and B.

Fig. 74. — Saccharomyces cerevisece. The variety known as brewers' top

yeast. — (Oberhefe.)

viscosus II. These bacteria are rod-shaped and measure 0.8 by
1.6-2.4 microns. Bacillus I gives rise to yellowish-white viscous
patches on the surface of the beer whereas bacillus II does not
develop such patches. B. viscosus III has been isolated from
British ropy beer. The ropiness results from a change in the cell-
wall of the bacterium and not from any chemical change in the
beer itself. Ropiness may also be caused by a mold, Dcmatium

220

BACTERIOLOGICAL METHODS

pullulans, which shows septate branching hyphal filaments and
yeast-like sporulation, which might be mistaken for yeast cells.
b. Turning or Souring of Beer. — Soured, turned or spoiled
beers have a disagreeable taste and odor and are no longer clear or
brilliant and sedimentary deposits are usually found. Beers

Fig. 75. — Saccharomyces cerevisece. The variety known as brewers' bottom
yeast (Unterhefe). a, Spore formation; b, elongated cells (rudimentary filaments
or hyphae).

containing only a small amount of hops or of hop extract and
which are low in alcohol and inadequately filtered, pasteurized
and improperly bottled, are likely to spoil. The most common
cause of this kind of spoiling is due to the aerobic acetic acid
bacteria which are particularly apt to do great damage in the
top fermented beers where the conditions for their development

BEER

221

(aeration) is more favorable than in the bottom fermented beers.
The three most common and best known beer acidifiers are Bac-
terium aceti, B. pastorianus and B. kutzingianum.

Lactic acid and butyric acid bacteria may gain access to the
fermenting vats and render the beer wholly unfit for use. Of
these two kinds of bacteria, the butyric acid formers are by far the
most objectionable because of the very disagreeable odors which
they form.

Fig.

-Saccharomyces ellipsoides. The common wine ferment,
jams, jellies^and canned fruits.

Also common in

c. Bitterness. — Bitterness of beer may be caused by several
species of so-called wild yeasts, principally Saccharomyces pastori-
anus I, II and III, and of these, variety I is the most common and
most injurious. It is stated that small amounts of varieties
II and III are not objectionable as they transmit to the beer a
stronger taste and smell.

d. Turbidity. — Turbidity of beers may be the result of a
variety of factors which may be outlined as follows.

222 BACTERIOLOGICAL METHODS

1. Gluten turbidity, due to the precipitation of protein
substances.

2. Starch turbidity, due to the presence of unchanged starch.

3. Yeast turbidity, due to a high content of yeast cells.
If wild yeasts are the cause of the turbidity then there may be
noticeable a bad taste and bad odor.

4. Bacterial turbidity, due to the development of bacteria.
In this case there may be noticeable bad odor, bad taste and
ropiness.

5. Sarcina turbidity, caused by the members of the sarcina
group. Unless certain species are present in large numbers the
beer may not be appreciably affected in quality. It must be
remembered that some of the sarcinas cause disturbances in
gastric digestion.

3. Wines. — Wine is grape juice which has undergone alcoholic
fermentation through the action of yeast organisms. To enter
into the details of wine production is not necessary. Wines vary
in the amount of alcohol (8 to 16 per cent.) which they contain,
in color, in taste, in the amount of unchanged or added sugar,
in the amount of acid, etc. Saccharomyces ellipsoides is the most
common yeast concerned in the alcoholic fermentation of grape
juice. Wine diseases are frequently met with and are not unlike
those of beer. Ropiness, turning and lactic acidification are
perhaps the most common, induced by bacilli and cocci. It
may be stated that the greater natural acidity of wines in general
tends to retard or check bacterial invasion. Bacterial invasion is
also in a measure checked by the greater alcohol content of wines
over that of beers. Souring of wine is the most common malady,
induced by acetic acid bacteria which reduce much of the alcohol
into acetic acid. A tough membranous scum forms on the
surface of the wine, composed of a nearly pure culture of the
acidifying bacteria. Bacterium (Mycoderma) aceti, B. pastorianum
and B. kutzingianum are the most common of the acid formers
and the three species appear to be very closely related. Souring of

WINE AND SAKE

223

wine is in reality a normal process and which must be expected
to develop under ordinary condition. It is, however, most desirable
to retard or check this process as much as possible.

Fig. 77. — Sake. Steamed rice cells (c) attacked by the fungus (Aspergillus
oryza). The fungus changes the starch into saccharine substances. Yeasts and
bacteria are usually associated with the hyphal fungus, feeding upon the saccharine
substances formed.

4. Sake or Japanese Rice Wine. — Sake is a fermented drink
quite popular in Japan, China and in Corea. It is made from rice
which has been steamed to soften the grain and starch so that

224

BACTERIOLOGICAL METHODS

the fungus aspergillus oryzce may convert the starch into saccharine
compounds. The fungus is kept on hand in pure culture and

r-^

Fig. 78. — Sake. Aspergillus oryza, showing vegetative hyphae (a) and the spore-
forming hyphae (b, c, d).

mixed with the steamed rice and the sugar fermentation takes
place in a warm room. The alcoholic fermentation, which follows,
is much like that in beer making, likewise the final processes of

SAKE AND ARRAK

225

clarifying and pasteurizing. This drink contains from 14 to
18 per cent, alcohol and is essentially a wine. It may be taken
cold or hot. The Japanese usually drink it hot. There are several
brands of sake differing in quality. There is a sweet variety

Fig. 79. — Sake. A, Dead or dying yeast cells (Saccharomyces sake). Vacuoles
are wanting, the cell walls are generally more thickened and the cells are somewhat
shrunken in appearance; B, living yeast cells showing distinct vacuoles; C, D,
actively budding yeast cells {S. sake) and hyphas of aspergillus from the fermenting
vats.

(Mirin) and a white variety (Shiro). Sake has a peculiar aroma
or flavor which may be likened to that of bad champagne.

5. Arrak. — This Javanese alcoholic drink is made from rice
which is acted upon by a fungus (Ragi) similar to Aspergillus

226 BACTERIOLOGICAL METHODS

oryzce, and subsequently the alcoholic fermentation is carried on
by the saccharomyces. The method of preparing arrak is
therefore similar to that of making sake. More generally,
however, arrak is made from fermented molasses.

6. Yoghurt. — This is Bulgarian sour, thick or klabbered
sheep's or cow's milk. The milk is boiled and evaporated to about
half its volume, then cooled to about 450 C. and the ferment known
as may a or podkoassa is added. The maya is simply the dry
residue from a previous fermentation. The fermented product
has a sour aromatic taste. The most important organism in this

Fig. 80. — Showing a Kephir granule or mass natural size and three types of bacteria
found in Kephir. — {Marshall.)

fermentation is the Bacillus bulgaricus. Other bacilli, cocci
and yeasts are also present. The Yoghurt tablets of the market
are presumably pure cultures of the Bacillus bulgaricus.

7. Kephir. — Kephir is an effervescent alcoholic sour drink
made from the milk of the cow, sheep or goat. This is also a
Bulgarian preparation. The kephir granules or seeds are simply
more or less dry residues of a previous fermentation and may be
obtained in the market. These granules are composed of the
organisms which give rise to the fermentation products, principally
Dispora {Bacillus) caucasica and several species of streptococci.
These several organisms are supposed to form a mutualistic as-

FERMENTED MILKS

227

sociation and cause alcoholic and lactic acid fermentation in the
milk.

8. Koumiss. — This drink is similar to kephir, made from
mare's milk, by the inhabitants of southern Russia and of Siberia.
The active organisms in the ferment are a yeast, a lactic acid
bacillus and a second species of bacterium which is characteristic
of the koumiss and which appears to be active only in association
with the other organisms, thus also indicating a mutualistic
association. The fermented milk contains lactic acid and
alcohol.

9. Soja Sauce. — This Chinese sauce or relish is made from
the fermented soja bean {Glycine hispidus). The beans are boiled

-t'VC

«=^

&:

N

:fV^,...

/.\ a ''jck.* ; \-_\)

•\

% \/

Fig. 81. — Bacteria of slimy wine. A, B, C, pure cultures of various forms; D,
mucilaginous sheath of slime bacteria. {After Kayser and Manceau.)

and mixed with parched flour and then exposed to the ferment
Aspergillus oryzce. Salt and water are added and the mixture
is allowed to ferment slowly, sometimes for years. The final
product assumes a rich brown color and a characteristic aroma.
It is then put in bags and almost a clear juice is expressed which
is then further clarified and pasteurized. In the second or long
process of fermentation several organisms are active along with
the Aspergillus, as Saccharomyces soja. Bacillus soja and Sarcina
hamayuchia.

10. Mazun. — This, like the kephir and koumiss, is a fermented
milk, usually of the cow and of the goat, which is much used in
16

228

BACTERIOLOGICAL METHODS

Armenia. The active organisms in the ferment are a bacillus
which appears to be identical with Bacillus subtilis and also several
different kinds of lactic acid bacteria.

ii. Leban. — This sour aromatic drink is very closely similar to
mazun and is made from boiled buffalo's, cow's and goat's milk.
It is of Egyptian origin. It is said to contain less alcohol than does
kephir. Leban fermentation is due to a streptobacillus which
coagulates milk and forms lactic acid. A diplococcus is also
present which ferments glucose, saccharose and maltose. A strep-

Fig. 82. — Sarcina ventriculi. — (McFarland, after Migula.)

tococcus hydrolyzes lactose and another organism is capable of
fermenting glucose and maltose but not lactose.

12. Ginger Beer. — This is a fermented sugar solution to which
ginger has been added. The essential fermenting organisms are
a saccharomyces (S. pyriformis) and Bacillus vermiforme. My co-
derma aceti is also present. The two essential organisms are
evidently in close mutualistic relationship. The drink produced
is acid and effervescing. The so-called ginger beer plant is simply
a mass or matrix of the active organisms and is used for the purpose

GINGER BEER AND BEBEES

229

of starting the fermentation. The ferment is evidently closely
related to the following.

13. Bebee Wine. — Bebees or California Bees, also known as
Japanese Beer Seeds, is a ferment composed largely of dried
yeast cells which when added to solutions of sugar or molasses
causes a quick alcoholic fermentation, resulting in a pleasant alco-
holic drink (bebee wine). The bebees or bebee granules resemble
dried peas somewhat, though they may be quite variable in
size. Some 15 years ago this ferment was quite common in the
United States, having been reported from California, Minnesota,

Fig. 83. — Vinegar organisms. A, Bacterium (Mycoderma) acetl; B, Bacterium
pasteurianus; C, Bacterium kiitzingianum; D,B. pasteurianum, showing the mucilagi-
nous sheath. This mucilaginous material causes the cells to stick together in large
masses, forming the so-called "mother of vinegar." — (Marshall.)

Kentucky and other states. It is evidently of Japanese origin.
Kebler and Lloyd made brief reports on this ferment several years
ago, and it is reported that the ferment has disappeared from the
American market.

Additional products of fermentation are vinegar, sauerkraut.
pickled cucumbers, apple cider, yeast cakes, sour dough and a
host of other substances used as food or employed in the prepara-
tion of foods. These may occasionally come to the notice of the
food bacteriologist. Vinegars, yeast cakes, sauerkraut and pickles,
in particular, may be attacked by objectionable organisms.

230 BACTERIOLOGICAL METHODS

Diseases may enter the pickling vats and ruin the entire contents
within a short time. "Hard cider" is apple wine or cider in
which the alcohol has been largely changed into acetic acid by the
Mycoderma aceti, forming cider vinegar. Cider vinegar in turn
may be invaded by bacteria which decompose the acetic acid
(Bacillus xylenum).

22. Standardization of Disinfectants

The success in modern surgery, preventive medicine and
sanitation is based upon the use of disinfectants. This state-
ment indicates the importance of disinfectants as articles of com-
merce, suggests the necessity of adequate supervision of the manu-
facture and commercial handling of these substances and points
out the necessity of guarding against adulteration and misrep-
resentation. A vast array of so-called antiseptics have been
placed on the market, the manufacturer claiming therefor
properties which they do not possess. These fraudulent and
exaggerated claims have impelled investigations of the marketed
disinfectants with a view to determining their true merit.

Methods for standardizing disinfectants on the basis of
their power to kill or destroy bacteria have been proposed by
various investigators, some of which have proven quite satis-
factory. The Rideal- Walker method and the Lancet method of
England and the Anderson-McClintic method of the U. S. Public
Health Service appear to find most favor, the latter method
being a modification of the two former. In the Rideal-Walker
and Anderson-McClintic methods the test organism used is the
typhoid bacillus, exposing definite quantities of pure cultures of
this organism to varying quantities of the disinfectant to be
tested in order to ascertain the killing strength as compared
with the standard which is pure phenol. The method is rather
complicated and demands great care and precision in technique
in order that the results may be reliable and uniform in the dif-

STANDARDIZATION OF DISINFECTANTS 23 1

ferent laboratories. A simplified method will no doubt be sub-
stituted for the Anderson-McClintic method. A method has
been proposed based on the percentage of bacteria killed within a
unit of time by a unit quantity of the disinfectant when added
to a unit quantity of a typhoid bacillus culture known to contain
a definite number of organisms. The Ohno-Hamilton method
is simpler than the Anderson-McClintic method and is included
for purposes of comparison.

An efficient disinfectant for general purposes should comply
with certain requirements which may be stated as follows:

1. Should be highly potent as destroyers of bacteria.

2. Should be readily soluble in water and should readily permeate or penetrate
solutions of organic substances.

3. Should be comparatively nontoxic to man, when applied externally or when
taken internally.

4. Should have a minimum albumen coagulating power, and conversely should
be capable of penetrating organic substances readily.

5. Should be comparatively cheap and should be readily usable by those of aver-
age ability and intelligence.

The ideal disinfectant, that is, one which is highly potent,
readily soluble in all organic solutions and capable of penetrating
such substances readily, and at the same time nontoxic and
cheap, does not exist. There is no disinfectant which is highly
efficient as a destroyer of bacteria and at the same time non-
toxic, notwithstanding all claims to the contrary by manu-
facturers. It is true, however, that disinfectants vary greatly
regarding the essentials above stated. Our present means for
testing the efficiency of disinfectants may be summarized as
follows:

1. John F. Anderson and Thomas B. McClintic1 of the United
States Public Health Service have worked out a method for de-
termining the comparative germ-destroying power of disinfectants,

1 John F. Anderson and Thomas B. McClintic. A Method for the Bacteriolog-
ical Standardization of Disinfectants. The Journal of Infectious Diseases, Vol.
VIII, No. i, Jan. 3, 191 1.

232 BACTERIOLOGICAL METHODS

which method is generally designated as the " Hygienic Laboratory
Method for Determining the Phenol Coefficient of Disinfectants."

2. Worth Hale1 of the United States Public Health Service
has worked out a method for determining the comparative toxicity
of coal tar disinfectants.

3 . A method for determining the albumen coagulating power
of disinfectants has been worked out in the bacteriological labora-
tories of the California College of Pharmacy.2

The rate and amount of solubility of disinfectants in water
is generally known or can be ascertained very readily. The
solubility of disinfectants in organic substances (as sputa, ex-
creta, pathological secretions, sewage, etc.), is of the greatest
importance and is conversely indicated by the albumen coagu-
lating power. Certain disinfectants not only do not coagulate
albuminous substances but have the power of penetrating such
substances. As is generally known most disinfecting solutions do
not penetrate or permeate coagulated albumen. Certain tests
which have been made at the California College of Pharmacy
would indicate that some of the coal tar disinfectants, such as
lysol, cresols with alkali, and others do actually penetrate coagu-
lated albumen, though very slowly. A better knowledge of col-
loids and of colloidal solutions would throw much light on the
behavior of disinfectants when added to organic substances and
would no doubt greatly modify the practical use of disinfectants.
It is generally known that the solubility or penetrability of
disinfecting solutions in the presence of organic substances is
increased by the addition of certain substances, thus very ma-
terially increasing the efficiency of such disinfectants.

It is most desirable to adopt a practical method for rating
disinfectants according to their efficiency value. In other words,

1 Worth Hale. A Method for Determining the Toxicity of Coal Tar Disinfec-
tants. Bull. No. 88, United States Public Health Service, April, 1913.

2,-Albert Schneider. An Albumen Coagulation Coefficient for Disinfectants.
The Pacific Pharmacist, Vol. V, No. n, March, 191 2.

STANDARDIZATION OF DISINFECTANTS 233

it is desirable to know what are the most efficient and cheapest
disinfectants for general use. It is suggested that such a method
of rating be in terms of comparison with phenol.

U. S. Public Health Service Phenol Coefficient

The following is a detailed description of the method for
determining the comparative (phenol) germ (bacteria) destroy-
ing power of disinfectants as given by John F. Anderson and
Thomas B. McClintic of the Hygienic Laboratory. In order
that the results by different workers may be uniform the details
must be followed out exactly.

Media. — Standard extract broth is used, both for the culture
to be tested and for the subcultures made after exposure to the
disinfectant. The broth is made from Liebig's extract of beef
and is in exact accordance with the standard methods adopted
by the American Public Health Association for water analysis.
Ten cc. of the broth are put into each test-tube. This amount
of broth has been found sufficient to avoid any antiseptic action
of the disinfectant carried over. It is important that the reaction
of the media is just +1.5.

Organism. — For the test organism, a 24 hr. broth culture in
extract broth of the B. typhosus is used. Before beginning a
test the culture should be carried over every 24 hr. on at least
3 successive days. For carrying over the culture one loop-
ful of a 4 mm. platinum loop is used.

Before being added to the disinfectant the culture is well
shaken, filtered through sterile filter paper, and placed in the
water bath in order that it may reach a temperature of 200 C.
before being added to the disinfectant.

Temperature. — A standard temperature of 200 C. has been
adopted for all experiments. This temperature is obtained by
the use of a specially devised water bath. The culture and
dilutions of the disinfectant are brought to this temperature be-
fore beginning the test.

234 BACTERIOLOGICAL METHODS

Proportion of Culture to Disinfectant. — One-tenth cc. of the
culture is used, added to 5 cc. of the disinfectant dilution. The
amount of culture is measured with a pipette graduated in tenths
of a cubic centimeter.

Inoculation Loops. — For making the transfer of the culture
after exposure to the disinfectant a platinum loop 4 mm. in diam-
eter of 23 U. S. standard gauge wire is used. We have found it
of advantage to have at least four, and preferably six, loops. In
order to save time in flaming the following method was devised:

A block about 3 in. wide, 10 in. high, and 1 2 in. long, containing
four or six grooves, spaced 2 in. apart, is used. Into each of the
grooves the platinum loop is laid so that the ends of the loops
extend about 5 in. beyond the side of the block. The first step
in the operation is to sterilize each loop by flaming with a fantail
Bunsen burner before beginning the experiment.

When ready to begin the operation the loop farthest from the
operator is taken in the right hand and the inoculation made.
It is then replaced in the groove with the right hand and the
Bunsen burner (fan tail) placed under it with the left hand. The
next loop is then used, replaced in its groove, and the Bunsen
burner placed under it with the left hand, the first loop having
been heated to redness while the second loop was in use. This
procedure is then continued until all the inoculations have been
made. The time required in making the inoculations and in
replacing the loop is short, it being found that 15 sec. is ample.

Incubations. — The subcultures are incubated 45 hr. at 370
C, and the results then read off and tabulated.

Dilution. — Capacity pipettes for the original dilutions are
invariably used. For the phenol controls a standard dilution
of pure phenol (Merck) is made and standardized by the U. S.
P. Method (Koppeschaar) to contain exactly 5 per cent, of pure
phenol by weight. From this stock solution the higher dilutions
are made fresh each day for that day's test.

For the dilutions of the disinfectant a 5 per cent, solution is

STANDARDIZATION OF DISINFECTANTS 235

made by adding 5 cc. of the disinfectant to 95 cc. of sterile
distilled water. A standardized 5 cc. capacity pipette is used
for this and after filling the pipette all excess of the disinfectant
on the outside of the pipette is wiped off with sterile gauze.
The contents of the pipette are then delivered into a cylinder
containing 95 cc. of sterile distilled water and the pipette washed
out as clean as possible by aspiration and blowing out the con-
tents of the pipette into the cylinder. The contents of the cylinder
are then thoroughly shaken and the dilutions up to 1 : 500 made
from it, using delivery pipettes for measuring. For those dis-
infectants which do not readily form a 5 per cent, solution we
make a 1 per cent, stock solution and from this make the dilutions
greater than 1 : 100 in accordance with the second table of dilu-
tions. If greater dilutions than 1 : 500 are to be made, a 1 per cent,
solution is made from the 5 per cent, solution, and the higher dilu-
tions made from this.

We had adopted the following scale for making dilutions:
For dilutions up to 1 : 70, increase or decrease by a difference
of 5 (i.e., 5 parts of water).

From 1:70 to 1:160 by a difference of 10
From 1 : 160 to 1 : 200 by a difference of 20
From 1:200 to 1:400 by a difference of 25
From 1 : 400 to 1 : 900 by a difference of 50
From 1:900 to 1: 1800 by a difference of 100
From 1: 1800 to 1:3200 by a difference of 200

and so on if higher dilutions are necessary.

It is important that the cylinders used for making the dilutions
be correctly graduated, as we have found disregard of this factor
an important source of error. It is preferable to use standardized
cylinders and pipettes, and we recommend that they be used
whenever possible. They of course should be perfectly clean.
For making the dilutions in accordance with the above scheme we
have found the following table of much service:

236

BACTERIOLOGICAL METHODS

Table I. Stock 5 Per Cent. Solution, (for Dilutions)

(5 cc. disinfectant + 95 cc. distilled water)

Solution A

cc. of A

cc. Dist. Water |

cc. of A

:c. Dist.

Water

cc. of A

1

:c. Dist. Water

1: 20

20 + O

or

IO +

0

1

or

4 +

O

1:25

20 + 5

or

10 +

2-5

or

4 +

I

1:30

20 + IO

or

10 +

5

or

4 +

2

i:35

20 + 15

or

10 +

7-5

or

4 +

3

1:40

20 + 20

or

10 +

10

or

4 +

4

1:45

20 + 25

or

10 +

12.5

or

4 +

5

1:50

20 + 30

or

10 +

15

or

4 +

6

i:55

20 + 35

or

10 +

17-5

or

4 +

7

1:60

20 + 40

or

10 +

20

or

4 +

8

1:65

20 + 45

or

10 +

22.5

or

4 +

9

1: 70

20 + 50

or

10 +

25

or

4 +

10

1: 70

20 + 50

or

10 +

25

or

4 +

10

1:80

20 + 60

or

10 +

30

or

4 +

12

1:90

20 + 70

or

10 +

35

or

4 +

14

1: 100

20 + 80

or

IO +

40

or

4 +

16

1: no

20 + 90

or

10 +

45

or

4 +

18

1:120

20 + IOO

or

10 +

50

or

4 +

20

1:130

20 + no

or

10 +

55

or

4 +

22

1 : 140

20 -f- 120

or

10 +

60

or

4 +

24

1:150

20 + 130

or

10 +

65

or

4 +

26

1 : 160

20 + 140

or

10 +

70

or

4 +

28

1: 160

20 + 140

or

» +

70

or

4 +

28

1:180

20 + 160

or

10 +

80

or

4 +

32

1: 200

20 + 180

or

IO +

90

or

4 +

36

1: 200

20 + 180

or

4 +

36

or

2 +

18

1:225

20 + 205

or

4 +

41

or

2 +

20.5

1:250

20 + 230

or

4 +

46

or

2 +

23

1:275

20 + 255

or

4 +

5i

or

.2 +

25-5

1:300

20 + 280

or

4 +

56

or

2 +

28

1:325

20 + 305

or

4 +

61

or

2 +

30.5

1:350

20 + 330

or

4 +

66

or

2 +

33

i:375

20 + 355

or

4 +

7i

or

2 +

35-5

1:400

20 + 380

or

4 +

76

or

2 +

38

1:450

20 + 430

or

4 +

86

or

2 +

43

1:500

20 + 480

or

4 +

96

or

2 +

48

STANDARDIZATION OF DISINFECTANTS

237

Table II. Stock i Per Cent. Solution (for Dilutions)

(1 cc. disinfectant; 99 cc. distilled water)

Solution A

cc. of A

1

cc. Dist. Water

cc. of A

cc. Dist.

Water

cc. of A

cc. Dist. Water

1 : 100 =

IOO + O

or

IO+ 1

0

1: no =

IOO + IO

or

IO +

1

1: 120 =

IOO + 20

or

IO +

2

1:130 =

IOO + 30

or

IO +

3

1 : 140 =

100 + 40

or

IO +

4

1:150 =

100 + 50

or

IO +

5

1: 160 =

100 + 60

or

IO +

6

1: 160 =

IOO + 60

or

IO +

6

1:180 =

IOO + 80

or

IO +

8

1 : 200 =

IOO + IOO

or

10 +

10

1 : 200 =

IOO + IOO

or

IO +

10

or

4 +

4

1:225 =

100 + 125

or

IO +

12.5

or

4 +

5

1:250 =

100 + 150

or

IO +

15

or

4 +

6

1:275 =

100 + 175

or

IO +

17-5

or

4 +

7

1:300 =

IOO + 200

or

IO +

20

or

4 +

8

1:325 =

100 + 225

or

IO +

22.5

or

4 +

9

1:350 =

100 + 250

or

IO +

25

or

4 +

10

i:375 =

100 + 275

or

IO +

27-5

or

4 +

n

1 : 400 =

100 + 300

or

IO +

30

or

4 +

12

1:400 =

10 + 30

or

4 +

12

or

2 +

6

1:450 =

10 + 35

or

4 +

14

or

2 +

7

1:500 =

10 + 40

or

4 +

16

or

2 +

8

1:550 =

10 + 45

or

4 +

18

or

2 +

9

1 : 600 =

10 + 50

or

4 +

20

or

2 +

10

1:650 =

10 + 55

or

4 +

22

or

2 +

n

1 : 700 =

10 + 60

or

4 +

24

or

2 +

12

1:750 =

10 + 65

or

4 +

26

or

2 +

13

1:800 =

10 + 70

or

4 +

28

or

2 +

14

1:850 -

10 + 75

or

4 +

30

or

2 +

rs

1:900 =

10 + 80

or

4 +

32

or

2 +

16

1:900 =

5+40

or

4 +

32

or

2 +

16

1 : 1000 =

5+45

or

4 +

36

or

2 +

iS

1 : 1 100 =

5+50

or

4 +

40

or

2 +

20

238

BACTERIOLOGICAL METHODS

Table II. Stock i Per Cent. Solution (for Dilutions)

(i cc. disinfectant; 99 cc. distilled water)

Solution A

cc. of A

cc. Dist. Water

cc. of A

cc. Dist

Water

cc. of A

cc. Dist. Water

i: i 200 =

5 + 55 or

4 +

44

or

2 +

22

1:1300 =

5 + 60 or

4 +

48

or

2 +

24

1 : 1400 =

5 + 65 or

4 +

52

or

2 +

26

1:1500 =

5 + 70 or

4 +

56

or

2 +

28

1: 1600 =

5 + 75 or

4 +

60

or

2 +

30

1 : 1 700 =

5 + 80 or

4 +

64

or

2 +

32

1: 1800 =

5 + 85 or

4 +

68

or

2 +

34

1: 1800 =

5 + 85 or

4 +

68

or

2 +

34

1:2000 =

5 + 95 or

4 +

76

or

2 +

38

1: 2200 =

5 + 105 or

4 +

84

or

2 +

42

1: 2400 =

5 + 115 or

4 +

92

or

2 +

46

1 : 2600 =

5 + 125 or

4 +

100

or

2 +

50

1 : 2800 =

5 + 135 or

4 +

108

or

2 +

54

1 : 3000 =

5 + 145 or

4 +

116

or

2 +

58

1:3200 =

5 + 155 or

4 +

124

or

2 +

62

Seeding Tubes. — The seeding tubes are glass test-tubes 1 in.
in diam. and about 3 in. long, with round bottoms. In order to

^fKCZ^

"'"•"Wtfi'TIf

Fig. 84. — Block for holding the subculture tubes. — (Anderson & McClintic, Hygiene
Laboratory Bulletin No. 82, U. S. Public Health Service )

measure the disinfectant into them they are placed in a suitable
wooden stand to receive them. We found it convenient to use a
wooden block containing six rows of fifteen holes each for the
disinfectant to be tested and a separate stand for the phenol con-
trols. The tubes are placed in the stand and each marked with
the strength of dilution it is to contain. The rows of tubes run-

STANDARDIZATION OF DISINFECTANTS

239

ning crosswise represent the same strength dilution, while the rows
running lengthwise represent the different strengths to be used in
the experiment.

Starting with the lowest
dilution (i.e., the strongest),
the cylinder is shaken, then 5
cc. are measured into the
tubes of the row to receive
that strength, using a 5 cc.
delivery pipette. In order to
economize glassware, the
same pipette is used for
measuring out the next dilu-
tion, first blowing out as
much of the remaining liquid
as possible, then drawing a
pipette full of the next dilu-
tion to be used and discard-
ing that, then filling the
pipette a second time, which
is then emptied into the seed-
ing tube.

The measuring out being
completed, the tubes are
placed in the water bath and
allowed to stand a few minutes
in order that the disinfectant
solution may reach the stand-
ard temperature. We have
not found it necessary to use
cotton plugs in the seeding
tubes. They are sterilized in paper-lined wired baskets, with the
closed end of the tubes up.

Subculture Tube Racks. — Wooden racks, with five rows of

L H WUDER

Fig. 85. — Water bath showing position
of holes for the thermometer and theseeding
tubes. — (Anderson &■ McClintic, Hygiene

Laboratory Bulletin No 82, U. S. Public
Health Strvice.)

240

BACTERIOLOGICAL METHODS

fourteen holes each, are used for holding the subculture tubes,
and as plants are made from each mixture of culture and dis-
infectant every 2^2 rain, up to 15 min., six tubes are required for
each dilution. Thus in each rack we have ten rows of six tubes
each with two empty cross rows of holes left, which are utilized
by placing over in the next row each tube as it is planted. This
makes it easy to keep run of the tubes that are planted. It is

fa'/////)j///v////////f})//'/ //{///////////////////////A

Fig. 86. — Cross section of water bath showing seeding tubes in position. —
{Anderson & McClintic, Hygiene Laboratory Bulletin No. 82, U. S. Public Health
Service.)

well also always to plant from the seeding tube in a certain hole
in the water bath into a certain row of tubes in the rack. This,
after a little practice, will help to avoid errors in planting.

Method of Conducting the Test.— If there are in one experi-
ment more than ten dilutions of the disinfectant, including the
phenol controls, the stronger solutions of the disinfectant and

STANDARDIZATION OF DISINFECTANTS 241

phenol are tested first, as it will not be necessary to plant them
after 7^ min. The weaker solutions are then immediately done
and are planted every 2% min. for 15 min.

For keeping the time a stop watch can be used, but an ordinary
watch will serve the same purpose by simply starting on the 2 J^ or
5 min. periods.

When everything is in readiness the culture is added to the
disinfectant solutions with a sterile pipette in tenths of a cubic
centimeter .

To add the culture, the seeding tube containing the disinfectant
is removed from the water bath with the left hand and slanted at
an angle of about 450, and with the right hand the end of the
pipette containing the culture is introduced and lightly touched
against the side of the tube where the liquid has run away on
account of slanting. At the proper time the culture is allowed to
run into the disinfectant solution, the pipette removed, the tube
straightened up, gently shaken three times, and replaced in the
water bath. The other tubes are done the same way in succession,
and it will be found that 15 sec. is ample time for each tube. By
adding the culture to the disinfectant with a pipette touched
against the side of the seeding tube, accurate measurements can
be made and each tube receive exactly the same amount of
"seeding," which is not the case when the culture is added by the
"drop."

If ten tubes are to be inoculated, only a few seconds will remain
after inoculating the last tube before a plant from the first tube
will have to be made.

The mixing tubes are not removed or disturbed in making the
planting except to insert the loop or spoon into them, touch the
bottom, withdraw, and then make the plant in broth. Every
effort is made to insert and withdraw the loops and spoons in a
uniform manner. The loops and spoons are bent to an angle of
about 450 where they are joined on to the shank, and therefore are
always filled with the mixture when withdrawn from the seeding

242

BACTERIOLOGICAL METHODS

tubes. After making the plants, the loops or spoons are flamed as
already described.

After an experiment is finished the date and any necessary
details can be marked on one of the broth tubes and the rack
placed in the incubator at 370 C. for 48 hr. At the end of this
time the results are recorded on a chart especially devised for the
purpose. (See Table III.)

Determining the Coefficient. — After a large number of experi-
ments, we have concluded that the method employed by theLancet

Fig.

87. — Device for holding and flaming inoculating loops. — (Anderson &° McClintic,
Hygiene Laboratory Bulletin No. 82, U. S. Public Health Service.)

Commission, with certain modifications, is the best one for de-
termining the coefficient, i.e., the mean between the strength and
time coefficients.

In performing the test, plants are made every 2% min. up to
and including 15 min. To determine the coefficient, the figure
representing the degree of dilution of the weakest strength of the
disinfectant that kills within 2}^ min. is divided by the figure

STANDARDIZATION OF DISINFECTANTS

243

representing the degree of dilution of the weakest strength of the
phenol control that kills within the same time. The same is done
for the weakest strength that kills in 15 min. The mean of the two
is the coefficient. The method of determining the coefficient will
be seen in Table III.

Date: May 18, 1913.

TABLE III
Name, "A."
Temperature of medication, 200 C.
Culture used, B. typhosus; 24 hr.; extract broth filtered.
Proportion of culture and disinfectant, 0.1 cc. + 5 cc.
Organic matter, none; kind, none; amount, none.

Subculture media, standard extract broth. Reaction, + 1.5; quantity in each tube,
10 cc.

Time Culture Exposed to Action of Disinfectant

for Minutes

Sample

Dilution

Phenol Coefficient

2V2

5

7W

10

12^

15

Phenol

1:80

—

_

_

1:90

+

-

-

-

1: 100

+

+

+

-

-

-

1 : no

+

+

+

+

X

-

Disinfec-

1:350

—

—

—

tant "A"

i:375

-

-

1 : 400

+

So)375(4-69

1:425

+

+

-

-

-

110)650(5.91

1:450

+

+

-

-

-

2)10.60

1:500

+

+

—

—

-

5-30

,

1:550

+

+ +

-

—

—

1:600

+

+

+

+

-

-

1:650

+

+

+

+

+

-

1 : 700

+

+ +

+

+ '

+

5.30 = phenol

1:750

+

+ + +

+

+

coefficient

To Determine the Comparative Cost per Unit of Efficiency.—

When bids are solicited for supplying disinfectants they should
be required to be made so as to show the comparative cost per
100 units of efficiency of the disinfectant as compared with 100
units of pure phenol. It is manifestly cheaper to purchase a
17

244

BACTERIOLOGICAL METHODS

disinfectant that sells for 60 cents a gallon than one that sells for
30 cents a gallon, if the former has four times the efficiency of the
latter.

The true cost of a disinfectant can be determined only by
taking into consideration the phenol coefficient and the cost per
gallon of the disinfectant.

The following table (IV) is a good illustration of the value
of a determination of the comparative cost per 100 units of
disinfectant in terms of 100 units of pure phenol:

Table IV

It will be seen that the substance Chi has a higher coefficient
than any of the others in th eatable, butfits^high cost per gallon
results in its being placed second in cost per 100 units.

The cost per 100 units of efficiency as compared with pure
phenol is obtained by first dividing the cost per gallon of the
disinfectant by the cost per gallon of pure phenol; this gives
the price ratio between the disinfectant and pure phenol; the
cost ratio is then divided by the phenol coefficient, which gives us
the cost per unit of efficiency as compared with pure phenol = 1 .
The cost per unit is then multiplied by 100 to give the cost per
100 units.

The Ohno-Hamilton Phenol Coefficient

Tatsuzo Ohno and H. C. Hamilton of the Parke, Davis Re-
search Laboratory have proposed a method for the bacteriological

STANDARDIZATION OF DISINFECTANTS 245

standardization of disinfectants, which is a decided simplifica-
tion of the Anderson-McClintic (U. S. Public Health Service)
method, and it is hereby given in somewhat abbreviated form
(American Journal of Public Health, May, 191 2).

I. The organism used is a vigorous culture of B. typhosus
grown for 24 hr. in standard bouillon culture medium at 380 C.
It is taken from the incubator at least Y^ hr. before using, to
allow gradual adjustment to changed conditions of temperature
before exposure to the germicide. The culture and germicidal
agent should always be at the same temperature before interac-
tion takes place.

The 24 hr. bouillon culture is removed from the incubator
and kept at room temperature without agitation for about half
an hour. Then, without shaking the culture, as is usually done,
it is decanted into a specially constructed cotton filter, thus leav-
ing scum and large clumps on the filter and filtering the individual
bacteria in a practically isolated state. It is then filtered into
a sterile test-tube, which is subsequently shaken in order to ob-
tain a homogeneous filtrate and make it ready for use.

The cotton filter for the filtration of bacterial cells is an or-
dinary test-tube drawn out at one end like a centrifugal tube, the
small end cut open and the edges smoothed with a flame. Into
the large end a small pledget of good quality ordinary cotton is
introduced as far as the constricted portion of the tube, pushed
gently with the forceps, taking care not to form any fissure in
the cotton or to leave any spaces between the cotton and tube.
The open end of the tube is then plugged with cotton and the
whole wrapped in cotton and parchment paper and sterilized by
dry heat as usual. Before using the cotton filter after steriliza-
tion, the cotton in the tube should be gently pushed back to the
proper place by means of a sterile pipette, so that it is in exactly
the same position as before sterilization. During sterilization the
cotton is pushed up by the tension caused by the heat and its own

246 BACTERIOLOGICAL METHODS

elasticity, producing an undesirable space between the cotton and
the constricted part of the tube.

II. Culture Medium.

500 grams chopped beef.
20 grams peptone (Witte).
5 grams sodium chloride.
1000 cc. water.

The beef is digested at 500 C. for J^ hr., then boiled, strained,
the other ingredients added, then boiled again, filtered and ad-
justed to + 1 reaction.

III. The dilutions of sample and standard are made either by
weight or volume, depending on the character of the disinfectant
to be tested. In the case of liquids such as the coal-tar disin-
fectants, both sample and standard should be diluted by volume.

The Sample. — Dilutions of an emulsive coal-tar product
should be made by adding water gradually to the measured
quantity of disinfectant. The reason for this is that in some
cases the character of the emulsion is greatly altered by the
method of making the dilution.

An emulsion is less likely to break if it is made as follows:
To make a 1 per cent, solution, moisten the measuring flask or
cylinder with about 2 cc. water. With a capacity 1 cc. pipette
measure the disinfectant and mix it with the water, using this
mixture for a partial cleaning of the pipette. Then add more
water, stirring just enough to mix but not to make the mixture
foam. Wash out the pipette by drawing up and expelling the
dilution, then make up to the mark.

This method requires more care in measuring the final dilu-
tion if the meniscus is obscured by the emulsion. If the last
addition of water is made by carefully running it down the side
of the container, the surface liquid will not greatly obscure the
reading.

This 1 per cent, solution is further diluted to the desired ex-

STANDARDIZATION OF DISINFECTANTS 247

tent by mixing with distilled water in proper proportion, in each
case adding the measured disinfectant to the measured quantity
of water to make the desired dilution.

The Standard. — Merck's pure phenol is diluted by volume
by weighing out any desired quantity, dividing by the specific
gravity, 1.08, and dissolving in distilled water, diluting to twenty
times the volume of the carbolic acid used, to make a 5 per cent,
solution by volume in volume. Further dilutions can be made
from this as desired, since the solution is practically permanent.

The dilutions of carbolic acid ordinarily used with the results
to be expected are as follows:

Minutes
Dilutions 12345

1-110 _(_____

1-120 + + + — —

1-130 + + + +

The sample is usually diluted in such a way that not more
than a half unit in the coefficient lies between two dilutions.
For example

1-480 1-540 1-600 1-660 1-720

the intention being to compare with the carbolic acid dilution
1-120. A disinfectant value based on growth at 1 min. only
would be far from exact. Much more accurate results can be
obtained where the dilutions compared are those which kill the
organism in from 3 to 5 min.

IV. The proper mixture of culture and disinfectant is carried
out as follows: five drops of bacterial filtrate are introduced into
5 cc. of germicidal solution, contained in test-tubes 5^ in. long
by % in. in diam. This is added drop by drop in rapid suc-
cession, by means of a sterile pipette, 8H in- l°ng and 0.219 in.
in diam., the narrow end measuring about 0.075 in. in diam.,
held vertically at the mouth of the test-tube containing the
germicidal solution, so that the pipette with the germs will not

248 BACTERIOLOGICAL METHODS

come into direct contact with any part of the test-tube con-
taining the germicidal solution. If any of the organisms adhere
to the sides or to threads of cotton in the mouth of the tube they
might be inoculated into the bouillon without being exposed to
the germicide. As soon as the last one of the five drops is added
to the germicidal solution, they are mixed thoroughly for 15 sec.
or longer by holding the test-tube in the left hand and shaking it
with the fingers of the right hand; a formation of air bubbles
shaped like a long funnel extending from the bottom of the tube
toward the surface of the liquid shows that the mixing is effi-
ciently done, thus bringing every bacterial cell into direct contact
with the germicidal solution.

V. The subcultures are taken each minute for 5 min. by means
of a 23 platinum wire loop of 4 mm. inside diam.

Tests of two dilutions can be carried on at the same time by
one person, as a half-minute is more than is ordinarily necessary
for taking out a subculture and planting into the tube of culture
medium. Tests of five dilutions of a sample and three of the
standard can therefore be made in about 25 min.

To obtain a loopful of the mixture, the test-tube should be
tilted so as to prevent getting the foam formed on the surface of
the mixture during shaking instead of getting a liquid portion of
the mixture. The wire loop should be plunged almost to the
bottom of the tube before withdrawing.

These loopfuls are inoculated into test-tubes containing about
6 cc. of the standard culture medium above described. They
are placed in the incubator at 380 C. for 48 hr. or for such a
time as it is found that no further development of bacteria takes
place.

VI. Conclusions as to the value of a disinfectant from a test
conducted as described are drawn by comparing the dilutions of
the sample and the standard, which are equally efficient. While
in general one dilution of each is used for comparison, it is often
necessary to take the mean of a number, since it is not uncommon

STANDARDIZATION OF DISINFECTANTS 249

for two dilutions to give practically identical results. Note, for
example, such results as these:

Carbolic acid 1-120 + + — — —

1-130 + + + - -

Sample 1-720 + + — — —

1-780 + +

1-840 + + + +

In this case if no other dilutions of the sample are tested the
value is determined by comparing both 720 and 780 with carbolic
acid dilute 120 and the result is 6.25, the average of the two
values.

It will be noted that in the foregoing description no mention
is made of the temperature at which the test is made. While
temperature affects very vitally the process of disinfection, the
changes in temperature of an ordinary working room rarely
exceed io° C, while the average change would not exceed 50 C,
the year round; and since the standard is affected to practically
the same degree by these changes it seems an unnecessary com-
plication to carry out the test at a rigidly defined temperature.

It will also be noted that the time of contact between organism
and disinfectant is only one-third as long as is recommended in
most tests. While it may form in some cases a better picture of
the value of a disinfectant to find its efficiencies at two periods such
as 2}^ niin. and 15 min., practically no material change in its
value results from such a course. The taking of a subculture
each minute rather than at 2^ min. intervals makes for greater
accuracy, but this does not materially affect the results.

The Worth Hale Toxicity Coefficient

Worth Hale of the U. S. Public Health Service Hygienic
Laboratory has worked out a method for determining the com-
parative toxicity of disinfectants of which the following is a
briefly summarized outline.

250 BACTERIOLOGICAL METHODS

Test Animals. — The animals upon which the substance in
question is to be tested shall be white mice of not less than 15
nor more than 30 grams weight.

Dilutions and Dosage. — The dose is to be calculated per gram
of body weight and should when diluted equal between 0.03 and
0.04 cc. per gram weight; that is, 0.6 to 0.8 cc. for a 20 gram mouse.
The diluent is to be distilled water and primary dilutions are to
be made of such strength that the dose is easily measured with
a 1 cc. pipette graduated into hundredths. This is most easily
accomplished by the use of the substance in greater concentra-
tion than is required to kill in the above volume doses.

Administration of the Test Solutions. — After the required
dose of the diluted disinfectant has been estimated it is meas-
ured into a suitable dish and is then diluted further to the re-
quired volume by adding sterile distilled water in sufficient quan-
tity. A series of mice are then injected subcutaneously with
varying amounts of the substance until the least fatal dose (L.
F. D.) is determined, the mice being kept under observation.

Time Limit of the Observation.— After the animals have been
inoculated they are kept under observation for a period of 24 hr.
unless death results in a shorter period of time.

Phenol Comparative Test. — Mice of the same lot are similarly
injected with pure phenol properly diluted to make the meas-
urements of the dose easy and then further diluted in a small
dish to equal a volume dose of 0.03 to 0.04 cc. per gram of body
weight and the fatal dose determined as above. This least fatal
dose (L. F. D.) of phenol is unity and the least fatal dose of
the substance in question is estimated in per cent, of this.

Determining the Comparative Toxicity. — The phenol toxicity
of the disinfectant tested is to the toxicity of phenol as x is to 100.
The example given below would be represented in the following
proportions: 4.5 :i8::x:ioo = 25 per cent., that is disinfectant
"A" is one-fourth as toxic as is pure phenol.

STANDARDIZATION OF DISINFECTANTS

251

Table V

Name of Disinfectant

Mouse,
Weight

Dose per Gm.,
Body Weight

Result

Time, Hr.
Min.

Disinfectant "A

Pure Phenol.

21.13
20.64
1S.32
19-05

18.46
20. 10
19.23
18.90

0.0012
0.0016
0.0018
0.0020

0.0035
o . 0040
0.0045
0.0050

Survived

Survived

Died

Died

Survived

Survived

Died

Died

10:30
2:15

0:25

Valuable information regarding the comparative toxicity of
many substances used as disinfectants may be obtained from
a study of the comparative medicinal doses. For example, the
medicinal doses of phenol, betol, resorcinol and corrosive sub-
limate are 1 grain, 3 grains, 4 grains and }io grain respectively.
These doses are practically in proportion to the toxicity of the
substances named and stating the dosage in the terms of the
phenol toxicity coefficient as proposed by Hale, we would get the
following results:

Phenol 100 . 00

Betol 33.30

Resorcinol 25 00

Corrosive sub 3000 . 00

As a rule, however, the exact composition of many of the
proprietary disinfectants is either not made known to the users
or is not disclosed by the manufacturers and in such cases the
only thing to be done in. order to ascertain whether or not the
claims of the manufacturers are correct, is to make tests as above
outlined. However, in cases where the composition of the dis-
infectant is definitely known, whether a simple or compound
substance, its comparative toxicity can be determined by as-
certaining the toxicity of the several ingredients and rating in
comparison with the standard, namely, pure phenol.

252 bacteriological methods

The Albumen Coagulating Coefficient of
Disinfectants

As is well known to surgeons and pathologists, the action of
disinfectants and their value in tissue disinfection and in the
disinfection of organic matter such as sputum, excreta, etc.,
varies according to their albumen coagulating power. Some
disinfectants, as alcohol, mercuric chloride, silver nitrate, copper
sulphate and others, coagulate albumen very actively and this
property checks or prevents further penetration and action.
Furthermore, inert combinations between the coagulating dis-
infectants (metallic ions) and the albuminous substances are
formed which render a portion of the disinfectant unavoidable for
further action. This behavior explains why some disinfectants are
more active when diluted, as for example alcohol and carbolic acid.
Even high dilutions of copper sulphate (i : 50,000 to 1 : 4,000,000)
will gradually kill bacteria in water or in other nonorganic liquids,
due to a coagulation of the bacterial plasm, whereas solutions of
5 per cent, to 10 per cent, of the same substance are considered
rather unsatisfactory disinfectants. The stronger solutions
coagulate the albuminous matter in which the bacteria may be
imbedded, no doubt quickly killing the organisms in the exposed
outer layers of the albuminous particles or masses while the
layer of coagulum encloses many of the bacteria effectually pro-
tecting them against further action of the disinfectant. These
enclosed bacteria may become liberated after a time due to a
breaking up of the coagulated covering or coating and, if patho-
genic, may cause a single infection or an epidemic.

The following are some of the disinfecting agents which pre-
cipitate or coagulate albumen actively:

Alcohol. Chloral hydrate.

Ether. Phenol.

Salts of heavy metals. Picric acid.

Camphor. Mineral acids.

Volatile oils. Some organic acids.
Tannic acid.

STANDARDIZATION OF DISINFECTANTS 253

The following are disinfecting agents which do not precipitate
or coagulate albumen:

Acetic acid. Salts of light metals.

Phosphoric acid. Lysol.

Alkalies and soaps. Cresols.

While the albumen coagulating power of the different disin-
fectants varies greatly, it does not follow that a disinfectant which
coagulates albumen actively in strong solution will do so when in
weaker solution. For example, pure carbolic acid is a strong
coagulant but in solutions of 5 per cent, and less it is indeed a very
weak coagulant. It is therefore not exactly in accord with fact to
designate carbolic acid as a disinfectant having a high coagulating
power and hence comparatively unsuitable as a tissue (abscesses,
infected wounds, etc.), and organic matter (ejecta, excreta, etc.),
disinfectant, because in strengths of 2.5 per cent, and 5 per cent,
it has only a slight coagulating power, but is still very active as a
germ (nonsporebearing) destroyer.

It is not intended to imply that a disinfectant becomes useless
as soon as it begins to coagulate albumen actively, but the indica-
tions are that the noncoagulating disinfectants are more satis-
factory than those which are active coagulants. The coagulating
coefficients give that solution strength of the disinfectants tested,
which indicates or marks a retardation in disinfecting efficiency
due to the coagulation of albuminous matter. This albumen
coagulating coefficient is wholly independent of the germ destroy-
ing coefficient as well as that of the toxicity coefficient.

The following is an outline of the proposed method for de-
termining the comparative albumen coagulating power of disin-
fectants, at the same time also indicating the solution percentage
limit of optimum efficiency and usefulness as disinfectants.

Albumen Test Solution

The standard test solution shall be a 1 per cent, aqueous
(distilled water) solution of pure dried egg albumen.

254 BACTERIOLOGICAL METHODS

The following methods for making the albumen solution are
submitted.

i. Gravimetric Method A. — Place 2 grams of pure powdered
egg albumen in 100 cc. of boiled distilled water, shake and set
aside for 6 to 12 hr., shaking frequently. Filter through a
tared filter paper which has been dried (at ioo° C.) to constant
weight. Filtering is slow, requiring perhaps 1 hr. time.
When the last drop has filtered through, dry the filter paper,
with the unfiltered albumen residue upon it, to constant weight
and weigh. Deduct from this weight the weight of the dried filter
paper to obtain the weight of the albumen residue. From 1
gram of egg albumen dried to constant weight determine the
percentage of moisture. From the data thus obtained it is easy
to determine the amount of boiled distilled water which must be
added to the filtrate (100 cc.) to make 1 per cent, dried albumen
solution.

We will suppose that the dried filter paper to be used in filtering
the albumen solution weighs 1.570 grams and this same paper with
the undissolved albumen residue (also dried at ioo° C. to constant
weight) weighs 1.965 grams, then the weight of the undissolved
dried albumen residue equals 0.395 gram. We will suppose that
1 gram of albumen loses 0.126 gram on drying, or 12.6 per cent,
moisture. 0.395 gram raised to its normal air moisture (0.395
gram + 12.6 per cent of 0.395 gram = 0.444 gram) and subtracted
from 2.00 grams leaves 1.556 grams, the amount of albumen that
passed through the filter paper. 12.6 per cent, of 1.556 grams =
0.196 gram, and 1.556 grams less 0.196 gram = 1.360 grams which
represents the amount of albumen, dried to constant weight, that
passed into solution. Therefore to make a 1 per cent, solution it
is necessary to add enough boiled distilled water to the filtrate to
make 1 : 100. In this case add water up to the 136.00 cc. mark.
We now have a 1 per cent, solution sufficiently accurate for all prac-
tical purposes.

This albumen test solution is now ready for use but it must be

STANDARDIZATION OF DISINFECTANTS 255

kept in mind that it is readily attacked by microbes. However,
if carefully prepared with pure albumen, boiled distilled water, in
sterile vessels, and put on ice or in a cool place, it will keep for
perhaps 4 days.

Any quantity of albumen solution may be made, it merely
being advised not to prepare more than may be required for the
tests contemplated.

2. Gravimetric Method B. — In a dried and tared platinum dish
place 5 cc. of the albumen filtrate (2 grams in 100 cc. of boiled
distilled water), evaporate over water bath and dry to constant
weight, and from this determine the percentage of albumen in the
solution and the amount of water that must be added to the
albumen filtrate to make 1 per cent.

The Phenol Standards

The standard of comparison is the opacity produced in 5
cc. of the 1 per cent, egg albumen solution when 5 cc. of 5 per
cent, phenol solution are added (in a standard test-tube of about 15
cc. capacity). This phenol tube is placed against a black back-
ground. In making a test, varying dilutions of the disinfectant
are added to the egg albumen solutions in a series of test-tubes
until the opacity produced is the same as that in the phenol tube.
In each test 5 cc. of the dilution are added to 5 cc. of egg albumen
in a standard test-tube and the two tubes compared, placed against
a black background.

Dilutions of Disinfectants to be Tested

The phenol control solution (5 per cent.) is made as for the
Anderson-McClintic method of standardizing disinfectants,
using only pure phenol crystals.

Of the disinfectants to be tested, 10 per cent, and 1 per cent,
primary stock solutions are made; 10 per cent, solutions of liquid

256 BACTERIOLOGICAL METHODS

disinfectants as alcohol, formalin and acids, and 1 per cent,
solutions of the salts of heavy metals and of soluble substances
generally. From these primary stock solutions the following
secondary dilutions or 'substock solutions are made, always in
those amounts which will serve the purpose, that is, in amounts
for perhaps ten subdilutions for each and every disinfectant to
be tested.

1 : 10 (of liquids only.)

1 :ioo

1 : 1000

1 : 10,000

1 : 100,000

Method of Testing

1. Phenol Standard. — Pour 5 cc. of the egg albumen solution
in a standard test-tube, using a standard 5 cc. pipette having a
free outflow. Add to this 5 cc. of the phenol stock solution (5
per cent.). Set tube in the standard test rack (with black back-
ground made of cardboard covered with black tissue' paper).
The degree of opacity developed is to serve as the standard of
comparison.

2. Preliminary Testing. — The albumen coagulating power of
the disinfectant being unknown, much time and labor can be
saved by testing with the four or rive substock solutions, adding 5 cc.
to 5 cc. of the egg albumen test solution, in order to find that dilu-
tion of the disinfectant which fails to show any opacity. We will
suppose that the 1 :iooo substock solution shows very marked
opacity or precipitation, then the 1 : 10,000 solution might be tried,
which may also show quite marked opacity, then the 1 : 100,000
may be tried. If this gives negative results then we know that
the phenol standard lies between 1 : 10,000 and 1 : 100,000 with the
probabilities that it is nearer^ 1 : 10,000.

3. Concluding Testing. — Going back to the 1 : 10,000 dilution,
make ten subdilutions, increasing the dilutions by a difference of

STANDARDIZATION OF DISINFECTANTS 257

1000 by simply adding the required parts of distilled water,
using small quantities, thus:

10 parts of 1 : 10,000 + 1 part water = 1 : 11,000
10 parts of 1 : 10,000 + 2 parts water = 1 : 12,000
10 parts of 1 : 10,000 + 3 parts water = 1 : 13,000
Etc.

Any other quantity proportions may be used, however, as
10, 15, 20, etc., parts of the substock solution with the required
parts of distilled water. If the i : iooo substock solution is to
be used then the dilutions should be increased by 100, as follows:

10 + 1 = 1 : 1 100
10 + 2 = 1 : 1200
10 + 3 = 1 :i3oo
10 + 4 = 1: 1400

or any other equal proportion of stock solution and distilled water
may be used, as

5 + 0.5, 100 + 10, or 1000 + 100, etc.

If the highest stock dilution (i : 100,000) is to be used, then the
increase should be 10,000, thus:

10 + 1 = 1 : 110,000
10 + 2 = 1 : 120,000
10 + 3 = 1: 130,000

Determining the Phenol Coefficient

Having determined that dilution which gives the same coagula-
tion opacity as the 5 per cent, carbolic acid, it is a very simple
matter to determine the phenol albumen coagulating coefficient
by simply dividing the strength of the dilution of the disinfectant
tested by the phenol dilution (1:20).

The following are the coagulating coefficients of a few dis-
infectants:

258

BACTERIOLOGICAL METHODS

Name of Disinfectant

Reaction Limit

Phenol Coefficient

Phenol

Copper sulphate

Mercuric chloride. . . .

Silver nitrate f. . .

Alcohol (95 per cent.)

1 : 20
1 : 15,000
1 : 10,000
1 : 9,500
1 : 3

1 .00

750.00

500 . 00

47500

0.15

The following table gives the efficiency value of some dis-
infectants. It will be seen that this value is of necessity variable,
depending upon the variation in the market price of the disinfec-
tants. It will also be seen that in the proposed rating the re-
markably high coagulation coefficient of some of the more im-
portant chemical disinfectants lowers the efficiency value
greatly.

Efficiency Values of a Few Disinfectants

Name of Dis-
infectant

Phenol
Coeff.

Tox.
Coeff.

Coag.
Coeff.

Comp.
Cost

Eff.
Value

Special
Properties

Phenol

Boracic acid

i .00

0.23

Chloro-naphtho- 6 . 06

leum
Copper sulphate . . 3 . 30

Lysol

Mercuric chloride.

Neko

Potassium per-
manganate
Silver nitrate

Trikresol

2

12

43

00

20

00

0

85

38

00

2

62

1 .00

0.05

o. 16
1 .00?

0.45
50

o. 20

0.50
3.00

0.90

750

650

475

0.15
1 .00
0.15
1 .00

0.15
1 .00

0. 20

1 . 20
0.65
4-33
1.27
8.46
0.50

3-33
0.25
1.66
5-64
37.60
0.40
2.66

5.22

Odor

Odorless

Odor

0.004 Odorless. Slight
color
Odorless

o.44
0.06
5.66
0.04

Odorless. Corrodes

metal
Odor

Odorless. Deodor-
ant. Stains
0.075 Odorless. Stains

o.73

Odor

STANDARDIZATION OF DISINFECTANTS

259

The efficiency value of any disinfectant is found by dividing
the phenol coefficient by the sum of the other coefficients, as
follows :

Phenol coefficient Efficiency

Tox. coefficient + coag. coefficient + comp. cost value

In the table the first figure in the comparative cost column is
the market price per pound of the disinfectant and the second figure
is the comparative cost (compared with phenol at 15 cents
per pound) .

The Toxicity and Germ Destroying Power of Some of
the More Important Disinfectants

The values given are obtained from various sources and in some
instances require further verification. The table will serve as a
guide to a valuation of the disinfectants for purposes of general
disinfection.

Germ Destroying Power and Toxicity of Disinfectants

Name

Germ Destroying Power
(Phenol as i)

Toxicity
(Phenol as ioo)

Alcohol

Alum

Ammonia

Ammonium chloride

Ammonium sulphate

Antozone

Arsenious acid

Arsenite of soda

Bacterol

Benetol

Bichloride of platinum. .

Boracic acid

Bromine

Cabot's sulpho-naphthol
Calcium chloride

18

5.00

10.00

15.00

10.50

5.00

5000

00

3000

00

45

00

33

00

5

00

11

00

3

50

260 BACTERIOLOGICAL METHODS

Germ Destroying Power and Toxicity of Disinfectants — Continued

Name

Camphor

Carbolene

Carbolic acid

Carbolozone

Car-sul

Caustic acid

Chinosol

Chloride of gold

Chlorine

Chloro-naphtholeum

Chromic acid

Copper sulphate

Corrosive sublimate

Cre-bol-you

Cremolene

Creo-carboline disinfectant

Creola

Creoleum (Dusenberry) . . .

Creolin (Pearson)

Creolol (Rudish's)

Creosol (saponified)

Creo-Sul

Creosoleum - . .

Cresylone

Crude carbolic acid

Cupric chloride

Cyllin

Dioxygen

Electrozone

Ether

Ferrous sulphate

Formacone liquid

Formaldehyde

Germ Destroying Power
(Phenol as i)

Toxicity
(Phenol as 100)

33-30

I.36

II .OO

I .OO

IOO.OO

I.48

6.40

2.00

16.OO

O. 17

I20.00

°-95

25.OO

12.50

12.50

6.06

16.OO

15.00

IOO.OO

3-3°

IOO.OO?

43.00

3000 . OO

9.00

1 . 26

4-03

30.00

0.52

12.80

1 .00

9.00

3-25

18.00

1.24

13.00

1.03

6.40

15.00

2 .90

11.00

56.00

2-75

90.00

4.20

80.00

11 .00

0.02

0.90

2.50

25 .00

0. 27

0.50

0.04

40.00

0.30

75.00

STANDARDIZATION OF DISINFECTANTS 261

Germ Destroying Power and Toxicity of Disinfectants — Continued

Name

Germ Destroying Power
(Phenol as 1)

Toxicity
(Phenol as 1 00)

Germol

Glycerin (sp. gr. 1.25) . . .

Hycol

Hydrate of chloral

Hydrocyanic acid

Hydrogen peroxide

Hygeno A

Iodine

Iron sulphate

Izal

Killitol

Kreosota

Kreotas

Kreso

Kresolig

Kretol

Lead chloride

Lead nitrate

Lincoln disinfectant

Liquid creoleum

Liq. cres. comp., U.S. P. .

Lisapol

Listerine

Lysol

Mercuric chloride

Mercuric iodide

Milkol

Mineral acids

Naphthalene

Naphthol phenoline

Neko

Nitrate of cobalt

Noncarbolic disinfectant.

2.12

16.00

0.015

0.50

12.30

32.00

0.32

10.00

7 -So

10,000 . 00

6.30

5.00

3-56

17.00

12.50

400 . 00

0. 27

40.00

8.00

0.02

1 .26

5.60

1 . 10

5.60

3-92

22.50

2.18

56.00

0.92

14 .00

1 -5o

200.00

0.83

300 . 00

1.48

17.00

9.00

3.00

56.00

0.01

50.00

0.20

2 . 12

45.00

43.OO

5000 . 00

I20.00

1000.00

II .00

I-I.SO

120.00

2.5O

7 50

6.4O

6.40

20.00

20.00

rS.°

7-5o

262 BACTERIOLOGICAL METHODS

Germ Destroying Power and Toxicity of Disinfectants — Continued

Name

Germ Destroying Power
(Phenol as 1)

Toxicity
(Phenol as 100)

IOOO

00

32

00

IOO

00

7

5o

80

00

4

50

19

00

9

00

10

00

Osmic acid

Phenaco

Phenol (pure)

Phenol disinfectant

Phenol, disinfecting and cleansing

Phenol liquid, U.S.P. (1890)

Phenol sodique

Phenosote

Phenotas disinfectant

Pi-ne-ex

Pino-lyptol

Piatt's chlorides

Potassium bichromate

Potassium cyanide

Potassium iodide

Potassium permanganate

Public health disinfectant

Pyxol

R. R. Roger's disinfectant

Salicylate of soda

Salicylic acid

Sanax

Sanitas

Saponified cresol

Silver nitrate

Sodium borate

Sodium chloride

Sulpho-naphthol

Tarola

Trikresol

20th Cent, disinfectant

Veriform germicide

Victor sanitary fluid

20.00

15.00

1 .00

0.61

1.77
0.01
3-43
i-37

o. 27
0.01

3.00
3.00

0.02

0.85
0.48

3-03
3.20

0.30

O. 22

O.3O

I.O3

38.OO

O.O4
0.02

3-87
3.12
2.62

0.13
o.43

3.20

500

00

1500

00

5

00

25

00

28

00

56

00

6

50

5

00

22

00

6

00

300

00

5

00

00

15

00

05

16.00
90.00

10.00

15.00
13.00

STANDARDIZATION OF DISINFECTANTS 263

Germ Destroying Power and Toxicity of Disinfectants. — Continued

Name

Wescol disinfectant. .
WorrePs disinfectant

Zenoleum

Zinc chloride

Zodane

Zodone (4).
Zonol

Germ Destroying Power .
(Phenol as 1)

Toxicity
(Phenol as 100)

22.00

O.OI

2.25

19.00

1-56

100.00

O.04

I.62

8.60

2-37

10.00

The Narcotic and Antiseptic Properties of the Essential

Oils

It is generally believed that the addition of spices to foods
serves to preserve them, that is, prevent decomposition changes.
In a general way this is in accord with facts. The antiseptic
properties of essential oils are quite marked and the antiseptic
properties of spices are largely due to the essential oils which they
contain. Important investigations in regard to the narcotic and
antiseptic properties of the more important essential oils have
been made by Martindale, Coupin and Geinitz. The following
is a summary of results by Geinitz as given in the Semi-annual
Report of Schimmel and Co., for Oct., 191 2.

The principal outcome of Geinitz' investigations is the establishment of the fact
that the narcotic and disinfecting properties of the essential oils do not correspond
with those of the active constituents of those oils; the sequence of the series differs
widely. For example, Russian anise oil and its active constituent, anethol, have
no antiseptic action whatsoever, but both have a pronounced narcotic action upon
cold-blooded animals. It would appear that the group which exerts an antiseptic
action and that which acts narcotically are not found in the same molecule of the
odoriferous bodies; nay, in many of these substances one of the groups is wanting
altogether. It is also necessary to abandon the theory that narcosis is determined
simply by the great solubility of lipoid in the cells of the nervous system, and that
the antiseptic action of essential oils depends upon solubility of the bacteria in the
lipoids. The explanation of the facts which have been observed is probably that
the organism of the bacteria with its peculiar metabolic process occupies in Nature a

264

BACTERIOLOGICAL METHODS

position wholly for itself. For the results of narcotic experiments which have been
obtained with essential oils in the case of coid-blooded animals and in that of the higher
plants arc altogether differenl from those which have been obtained with bacteria.

The results of narcotic experiments with fishes and tadpoles, of respiration
experiments with toads and of injection experiments with frogs are reproduced in
the form of tables arranged according to the degree of activity of the essential oils.

For the purpose of testing narcotic action on fishes, roaches (Lcuciscus rutilus) were
used. The limit of concentration taken was the dilution which produced perceptible
narcosis in the fish within a period of 24 hr., that is to say, a condition when the
animal, without displaying much spontaneous motion, floated in the water in an
atactic condition and altogether failed to respond to squeezing with hooked pincers.
Only those experiments were regarded as affording proof in which the fish recovered
when replaced in fresh water.

For the purpose of estimating the antiseptic action, Geinitz added in each case
to 10 cc. of fresh milk, placed in a test-tube of 16 to 18 cc. capacity, first as much
Sulphur depuratum as would lie on the point of a knife, and afterward the antiseptic.
After vigoro us shaking a piece of filtering paper soaked with solution of lead acetate
was hung up in the upper part of the test-tube in such a way as not to come in contact
with the milk, and the test-tube was closed with a wad of cotton- wool. The tubes
were then kept 24 hr. in a water plug bath at about 380 C. If, after that lapse of
time, the lead paper was found to be blackened, it was evident that the tube in
question did not contain a sufficient proportion of the antiseptic. As a series of test-
tubes was always being treated with an increasing quantity of antiseptic, it was easy
to determine exactly when the limit of concentration was reached at which the activ-
ity of the bacteria was impeded.1

Experiments in Narcosis, Made on Fishes

Substance

Dilution

Substance Dilution

Mustard oil (aMyliso-

sulphocyanate)

Cinnamon oil

1 : 1,320,000
180, occ
153,846
134,01c

133,333
125,918
116,327
111,836

104,587

Fennel oil

1 : 34;535
32,000

28,571

28,000
26,806
26,087
22,000
20,105

Terpineol (liquid)

Coumarin

Turpentine oil, fraction

containing /3-pinene

d-a-Pinene

Borneo camphor

Eucalyptol (Cineol)

/-a-Pinene

Citral

Carvacrol

Thyme oil

Carvone

Sandalwood oil

Eugenol

Anethol

1 Abstracted from the Sitzungsberichte und abhandlungen der naturforschenden
Gesellschaft zu Rostock, New Series, Vol. IV, 191 2. Rostock, 1913. The paper was
awarded a prize.

The evolution of sulphuretted hydrogen from milk diluted with sulphur is due to
bacterial action.

STANDARDIZATION OF DISINFECTANTS

265

Experiments in Narcosis, Made on Fishes — Continued

Substance Dilution Substance

Dilution

Benzaldehyde

18,096

Greek turpentine oil 86,405

French turpentine oil 79A93

Spanish turpentine oil 77>304

American turpentine oil 77>3°4

Calamus oil 71,862

German peppermint oil 70,000

Russian anise oil 67,000

Clove oil 67,000

Mitcham peppermint oil 66,666

Caraway oil 62,500

Safrol 60,560

Heliotropin 45,454

Heptylalcohol 45,000

Rose oil 41,7*5

Lavender oil 38,764

Mace oil 38,527

Octylalcohol 38,090

Geraniol 3 7,500

Oenanthol

Menthenone

Umbellulone

Eulimene (artificial

limonene)

Rosemary oil

Juniperberry oil

Cymene

Terpineol (cryst.)

Anisic aldehyde

Lemon oil

Chloroform

FenchyKso valerate. . .
Bornylwo valerate. . . .
Limonene

Chloral hydrate.

Alcohol

Ether

16,788
16,000
15,000

13,000

13,333
11,320

10,965
10,554
10,164

9,157

8,070

6,345

6,300
1,040

286
190
166

23. Determining the Purity and Quality of Sera, Bacterins and

Related Products

Sooner or later the regulatory work under the pure drugs laws
of the land will cover the newer remedies which have come into
prominence within recent years, such as therapeutically active
sera, the so-called bacterial vaccines or bacterins, tuberculins,
smallpox vaccine, rabies vaccine, glandular extracts, etc. It
is, however, self-evident that such supervision on the part of the
bacteriologists in drugs laboratories will not be necessary as far
as the products manufactured under supervision of the U. S.
Public Health Service are concerned. State and city authorities
(inspectors) may perhaps find a supply of these products in the

266 BACTERIOLOGICAL METHODS

more remote drug stores which have exceeded the age limit or
which have become deteriorated in some manner, but even this
must be, in the very nature of things, rather a remote possibility.
It is therefore not likely that the drug bacteriologist will be
called upon to examine any of the standard products put up in
the Government inspected laboratories. There are, however,
numerous preparations placed on the market which are said to
have properties similar to the standard sera, etc., but which are
of a fraudulent character. It then becomes necessary to resort
to certain tests which will determine whether or not the article
under consideration possesses the properties claimed for it. Such
tests are both chemical and bacteriological. The chemical tests
are largely qualitative and include certain color reactions, pre-
cipitation reactions, etc. However, much remains yet to be done
in the way of devising methods which will prove practically useful,
Some of the very recent laboratory guides to the examination of
medicinal substances contain suggestions which will prove useful,
and these may be applied in special cases. For example, a number
of chemical tests have been suggested for determining the presence
of ductless gland products and of various animal secretions. The
absolute merit of these tests is seriously questioned by some authori-
ties; however, their confirmatory significance is generally admitted.

Biological products the activity of which depends upon the
presence of living germs are comparatively few and are not likely
to be brought to the attention of the drug bacteriologist. The bio-
logical products are intended for hypodermic, intravenous, intra-
muscular or some similar mode of use and must therefore conform
to certain specific requirements. They must be entirely free from
all undesirable foreign bacteria, dead or alive, and must not con-
tain undesirable foreign biological or toxicological products.

The complete examination of biological products comprises
standardization and certain so-called safety tests, and is carried
out in all of the laboratories operating under Government super-

BIOLOGICAL PRODUCTS 267

vision. These tests, as carried out in the laboratories of Parke,
Davis and Co., may be outlined as follows:

I. Standardization.

1. Potency. — Determining the number of units per cc.

2. Activity. — Ascertaining the power to produce the desired results. This
is simply a check on the potency test.

3. Serum Tests. — In some of the biological products certain tests are made
to determine the difference between anti-sera and the normal serum of the
same species.

II. Safety Tests.

1. Freedom from bacterial contamination in those products which are sup-
posedly free from living germs.

2. Determining the purity of the cultures in those products which are com-
posed of pure cultures of a given kind of germ.

3. In case of products which are supposed to contain dead bacteria only,
tests are made to determine the absence of all organisms capable of
multiplying.

In order to test biological products as to the absence of viable
or living organisms, about 2 cc. of the sample is cultured under
aerobic and anaerobic conditions. To determine the purity of a
product containing living bacteria, cultures are made in suitable
media and these are carefully studied as to specific cultural char-
acteristics and appearance under the compound microscope.

For the purpose of standardizing the products, a well-equipped
laboratory is necessary, including the necessary experimental
animals. The full routine followed out in the laboratory of the
factory need not be carried out in the regulatory drug laboratory.
In most cases the work will consist of making animal inoculation
tests to determine the potency of the marketed article in order to
ascertain whether or not it possesses the properties claimed for it.
In some instances it may be necessary to determine the presence
of toxic ingredients. Perhaps the most likely tests will be those
which come under the head of potency and safety tests. For the
present purpose the above outline will no doubt suffice. As to
what methods may become desirable and necessary, only time
and further experience will indicate.

268 BACTERIOLOGICAL METHODS

The bacterial contamination of smallpox vaccine has received
considerable attention on the part of American bacteriologists.
Such vaccines are rarely wholly free from extraneous bacteria, no
matter how carefully prepared. It is rather remarkable that the
method of manufacturing the vaccine is not modified in accordance
with modern progress in sanitation. Since the smallpox virus is
filterable it would seem possible to pass the dissolved material
through a porcelain or clay filter leaving behind the bacteria and
other undesirable foreign matter. The filtrate could be tested
for the possible presence of such bacteria as might have passed
through the filter and these destroyed by suitable agents (such as
will not interfere with the activity of the vaccine), and the
filtrate perhaps concentrated to the desired degree or perhaps used
in the liquid form. However, it is likely that the present method of
manufacture and use of the smallpox vaccine will continue for
some time. The marketed smallpox vaccine should contain but
few viable bacteria, not to exceed 200 per dry point or per gly-
cerinated tube. According to extensive tests made by Rosenau
in 1 902-1 903, dry points and glycerinated tubes contained as
high as 44,000 bacteria per point or tube, but tests made since
that time (Nelson and others) show much lower figures, ranging
from ten or fifteen to 300 bacteria per point or tube. Small-
pox virus should also be examined (occasionally at least) for the
presence of colon bacilli, streptococci, tubercle bacilli and the
tetanus bacillus.

24. Special Biological and Toxicological Tests

Arsenic in Foods and Medicines — Biological Test. — Arsenic
is widely distributed in nature and is extensively used in the arts
and industries. Medicinally it is a very popular tonic and is also
much used as an insecticide in the form of sprays and washes.
Animal hides are frequently preserved by arsenic which accounts
for the presence of this poison in gelatin made from such hides.

SPECIAL TOXICOLOGICAL TESTS 269

Fruits and vegetables which have been sprayed with arsenical
compounds for the purpose of destroying insect pests, may contain
enough of this substance to produce symptoms of poisoning.
Arsenic is occasionally added to alcoholic beverages to give them a
tonic effect. It has been demonstrated that very minute amounts
of arsenic are normally present in various organs of the human
body, as the thyroid gland, thymus gland and liver, although
some investigators question the correctness of this claim. However
these somewhat problematical traces of arsenic in organs of the
human body and also in the organs of other animals need not
concern the food and drug analyst as far as routine work is
concerned.

As a rule, the tests for arsenic outlined in the majority of text-
books are chemical and hence this work is usually relegated to the
chemical laboratory. Within recent years attempts have been
made to employ biological tests for determining the presence of
arsenic in food substances, based upon the discovery that certain
molds when growing in substances containing arsenic will give rise
to garlic-like odors.

Gosio demonstrated that certain molds which when grown in
and upon media containing very minute quantities of arsenic
gave rise to gaseous compounds characterized by a garlic-like
odor. Seven different kinds of molds have this power, more
especially Penicillium brevicaule, which Gosio isolated from air
and which he frequently found on decomposing paper. Crumbs
of bread (wheaten) form the culture medium for this mold and
the incubation is done at 280 to 320 C, a vigorous growth being
produced within 48 hr. In the presence of not more than
0.0000 1 gram of arsenic in such culture there will be noticeable
a distinct and very characteristic garlicky odor which may persist
for months, if the culture is not killed. These arsenic molds
do not produce garlic odors or gases with sulphur, phosphorus,
antimony, boron, and bismuth compounds but they do have the
power of converting selenium and tellurium compounds into

270 BACTERIOLOGICAL METHODS

volatile substances having the garlic-like odor. The following
procedure is recommended.

If the material to be examined is liquid, let the dry bread
crumbs (either white or graham) absorb it to saturation, and
then scatter a small quantity of fine crumbs over the surface. If
the material to be tested is solid, grind or cut it into small pieces
and mix with an equal amount of the bread crumbs and then
moisten with a little sterile distilled water. Place the prepared
material in sterile flasks of suitable size and plug with sterile
cotton. Sterilize the flask and contents by the usual fractional
method at ioo° C, or for 30 min. in the autoclave. Absolute
sterilization must be secured. There is no danger of volatilizing
the arsenic at these temperatures. As soon as flask and contents
are cold, inoculate with the mold, as follows. The mold cultures
may be grown on bread or on pieces of potato. Remove a small
quantity of the mold in the spore -forming stage and mix with
peptone salt solution or sterilized water. Add enough of this
mold suspension to just moisten the bread in the flask. Do not
add more of the spore-bearing material than the mass (bread and
arsenical substance) in the flask will absorb as too much moisture
will retard growth. Cover the inoculated flask with a rubber cap
and incubate at a temperature of 370 C, although the ordinary
room temperature will answer the purpose. As soon as the growth
is clearly visible to the naked eye, which may be in 24 hr., the char-
acteristic garlic odor will be noticed upon opening the flask. If
no odor is appreciable, again seal and incubate for another 24 hr.
period or even longer. In case the substances to be tested are
strongly acid, they may first be neutralized by means of calcium
carbonate. It must also be kept in mind that Penicillium brevi-
caule, as well as other molds, will convert tellurium and selenium
compounds into volatile substances having a garlic-like odor. The
arsenic and tellurium odors are very closely similar but that from
selenium is somewhat different in quality, more like that of mer-
captan. The test is extremely delicate, 0.00001 gram of arsenic

SPECIAL TOXICOLOGICAL TESTS 271

can be recognized with certainty. A solution of 0.00001 gram
of potassium tellurite in 10 cc. of mold infested gelatin medium
in a cotton plugged test-tube gave out a strong odor of garlic for
several weeks.

Biginelli ascertained that the gases formed by Penicillium
brevicaule in arsenical cultures were completely absorbed by solu-
tions of mercuric chloride with the formation of a double compound
of mercuric chloride and diethyl arsine which is quite easily decom-
posed accompanied by the reappearance of the garlic odor.

The test is unlimited in its application and will respond in the
presence of all manner of organic substances and bacterial contami-
nations. It is far more delicate than any of the chemical tests and
can be carried out in much shorter time.

Toxicity Tests with Defibrinated Blood. — The older physi-
ologists and toxicologists made the interesting observation that toxic
substances of various kinds produced certain changes in the blood.
Some poisons disintegrated the red corpuscles, some caused the
corpuscles to clump or to agglutinate and still others reduced or
even completely inhibited the coagulating power of the blood.
These phenomena have suggested the possibility of estimating or
measuring the toxicity of certain groups or classes of substances by
noting the effects which they produce when brought in contact
with red blood corpuscles. The more important groups of toxic
substances which give rise to marked reactions with red blood
corpuscles are the toxalbumins or toxins, the saponins and many of
the toxic chemical compounds. The following tests may prove of
value in the food and drugs laboratories.

Toxalbumins or Toxins. — Toxalbumins and toxins are poisonous
substances formed in plants and animals as the result of microbic
invasion and also as the result of metabolism in the plant or animal
itself. Of special interest are the vegetable toxalbumins which
possess the remarkable property of clumping, agglutinating and
finally precipitating red blood corpuscles and have therefore been
designated "vegetable agglutinins." A mere trace of these sub-

272 BACTERIOLOGICAL METHODS

stances, when added to defibrinated blood in a test-tube, causes
clumping into a mass resembling sealing wax. The most im-
portant vegetable agglutinins are abrin, ricin, robin and crotin.
Of these, ricin, abrin and crotin also cause the coagulation of milk.

To make the agglutination tests, defibrinated blood is used.
Whip the fresh blood (of ox, horse, guinea-pig or rabbit) by means
of twigs, bunch of thin wires, wire mesh egg beater, or run the
blood into Erlenmeyer flasks with iron filings and shake vigorously
for several minutes. The fibrin is deposited on the twigs, wires, or
on the iron filings, thus separating it from the corpuscles and the
serum. Removing the serum from the blood and displacing it by
physiological salt solution renders the reaction more pronounced,
thus pointing to the existence of antiagglutinins in the serum.
Ricin will agglutinate the blood of the guinea-pig in dilutions of
1 : 600,000. Abrin, crotin and robin react in a similar manner.

Saponins. — These substances are widely distributed in the
plant kingdom and have chemical properties linking them with the
glucosides. They have been designated nitrogen-free glucosides.
The dry powder causes violent sneezing when inhaled and the
aqueous solutions foam when shaken. Most of them are neutral
in reaction and are capable of holding many finely divided sub-
stances in suspension. They dialyze with difficulty and incom-
pletely. They dissolve in hot as well as in cold water but are in-
soluble in absolute alcohol and in ether.

Saponins have been found in many different species of plants.
The more important and better known are digitonin (in Digitalis
purpurea), saponin (Saponaria officinalis), githagin (Agrostemma
githago), senegin (Poly gala senega), saponin (Chlorogalum pomeri-
dianum), struthiin (Gypsophila struthium), sapotoxin (Quillaja
saponaria and Sapindus saponaria), and sarsaparilla-saponin
(Sarsaparilla species). Saponins are highly toxic when introduced
into the blood directly and some of them are well-known poisoning
agents. American Indians have long made use of the roots of
Chlorogalum for the purpose of stupefying fish. Most saponins

SPECIAL TOXICOLOGICAL TESTS 273

are however absorbed quite slowly which makes it possible for per-
sons in good health to take comparatively large quantities of weak
solutions without producing serious harm. They are protoplas-
mic poisons and it is due to this property that they hemolyze blood
causing it to become laky. It has been demonstrated experimen-
tally that saponins act more energetically upon blood corpuscles
separated from the serum because the serum contains cholesterin
which retards hemolysis. It is suggested that the t hemolytic
action of saponins is due to the removal of the lining membrane
of the corpuscles which consists of lecithin, forming lecithin-sa-
ponin. Saponins also combine with cholesterin (forming choles-
terin-saponin) and the affinities of any saponin being satisfied
by the cholesterin, it no longer acts upon the lecithin. This ex-
plains why cholesterin retards or checks the hemolytic action of the
saponins. The saponins also dissolve white blood corpuscles but
to a much weaker degree.

In making the blood tests for the presence of saponins, isotonic
(to blood serum) or physiological salt solution (0.9 per cent.) is
added to the defibrinated blood, 100 parts to one of the deribrin-
ated blood. Dilute the suspected saponin bearing substance with
physiological salt solution and add it to the diluted blood suspen-
sion. If saponin is present the mixture at once becomes laky due
to hemolysis. Githagin will develop the hemolytic action in
dilutions of 1 : 50,000.

It must be borne in mind that a variety of substances will pro-
duce hemolysis, such as ether, chloroform, alkalies, gallic acid and
solanine. The lytic test above outlined may be employed as a
check or corroboration of the chemical and perhaps additional
biological tests.

Chemical Hemolysis. — Vandevelde has suggested a method for
determining the toxicity of chemical compounds by hemolysis.
Defibrinated ox blood is used in addition to the following solutions.
A solution of 0.9 per cent, of salt in 50 per cent, alcohol (by volume,
specific gravity of 0.9548 at 150 C); physiological salt solution

274

BACTERIOLOGICAL METHODS

(0.9 per cent.) and a suspension of 5 per cent, defibrinated ox
blood in 0.9 per cent, salt solution.

To make the experiments test-tubes are used and the compound
microscope is not required. In a series of standard test-tubes
place 2.5 cc. of the suspended blood (in the sodium chloride solu-
tion) and the same amount of the solution to be tested (in varying
amounts of physiological salt solutions, therefore different strength
solutions) in order to ascertain the exact point when hemolysis
takes place. A solution which does not produce hemolysis after
a definite period of time (3 hr.), but which does result in hemolysis
on the smallest further addition of the substance under examina-
tion is spoken of as a " critical solution." The time limit in these
tests is 3 hr. If after the expiration of this period of time, the trace
addition of the solution does not produce the hemolytic effect,
the test is negative and the next stronger or higher concentrate
must be tried. The term critical coefficient refers to the number
giving the concentration of the substance necessary to hemolyze or
kill the red corpuscles.

The following is the result obtained by Vandevelde regarding
the critical solution of ethyl alcohol.

Cc. of Sus-

Cc. of NaCl Sol.

Cc. NaCl

Alcoholic Sol. in

Reaction

pended Blood

in Alcohol

Solution

Volume Per Cent.

in 3 hr.

2-5

2 . 20

0.30

22.O

Hemolysis

2-5

2.15

0-35

21-5

Hemolysis

2-5

2 . IO

0.40

21 .0

Hemolysis

2.5

2.05

o.45

20.5

Hemolysis

2-5

2.00

0.50

20.0

Hemolysis

2-5

i-95

0.55

19-5

Negative

2.5

1 .90

0.60

19.0

Negative

From this table it will appear that the critical solution of ethyl
alcohol contains 19.5 cc. of absolute alcohol in 100 cc, or 15.489
grams of alcohol in 100 cc. According to Vandevelde, the addi-
tion of methyl alcohol diminished the toxicity of ethyl alcohol,
whereas the higher alcohols were found to be more toxic than the

SPECIAL TOXICOLOGICAL TESTS 275

latter. Giving the toxicity of ioo parts of ethyl alcohol as 100,
then 47 parts by weight of isopropylic alcohol, 29 parts of
isobutylic alcohol and 12.5 parts of amylic alcohol were found to
be isotoxic with that quantity of ethyl alcohol.

The term toxin, more accurately speaking, applies to poisonous
substances elaborated by bacteria and which require an incuba-
tion period before forming antibodies or antitoxins. The toxins
formed by the bacterial group appear to be intimately associated
with the life processes of the living cell, but their chemical com-
position remains thus far unknown. We know that they are very
readily destroyed by heating (6o° to 8o° C.) and that they are
chemically very unstable, and that they are among the most highly
poisonous agents known to science. They are far more toxic than
the potent vegetable alkaloids and animal toxalbumins, as is shown
in the following tabulation (Jordan):

Atropine, fatal dose to man 130 mg.

Strychnine, fatal dose to man 30-40 mg.

Cobra venom, fatal dose to man 4-375 nig.

Tetanus toxin, fatal dose to man o . 23 mg.

Various animals produce toxalbumins or toxins, as snakes
(crotalin, viperine), scorpions, tarantulas, the Gila monster and
other lizards. Rattle-snake venom evidently possesses a variety
of properties. It will agglutinate blood, neutralize the fibrinogen,
hemolyze red corpuscles, and is highly neurotoxic. Within recent
years antibodies have been produced against these several toxic
substances.

Muscarine, the toxic agent of Amanita muscaria (fly agaric), is
an alkaloid which acts very quickly, whereas the toxic agents of
Amanita phalloides and A. verna are toxin-like in that there is an
incubation period of from 10 to 14 hr. before the toxic symptoms
begin to manifest themselves. They are strongly hemolytic. It
is supposed that the pollen grains of certain flowers contain toxin-
like substances to which certain persons are peculiarly suscepti-
ble. All toxalbumins or toxins, whether derived from bacteria,
19

276 BACTERIOLOGICAL METHODS

fungi or higher plants, possess the very characteristic property of
forming antibodies or antitoxins. Thus there is antivenin used in
the treatment of snake-bite, also antibodies used in the treatment
of hay fever, etc., which products are more fully described in works
on medical bacteriology.

Frog Tests for the Presence of Alkaloids. — Lively small frogs
respond quite readily to the action of even high dilutions or very
minute quantities of vegetable alkaloids. It is suggested that
the food bacteriologists perform the following tests, which are
frequently desired as a check or corroboration of the findings of
the chemist and toxicologist.

The material used for the frog injections is the evaporated
ether extract dissolved in a small amount of sterilized distilled
water. The injections are made hypodermically in the lymph sac
on the back of the frog. It is advised that the tests be made in
duplicate and repeated as often as may be required to attain ab-
solutely conclusive results. Each test should be checked by in-
jecting approximately minimal fatal doses of the pure alkaloid it-
self, obtained from some reliable house. This will make it pos-
sible to note the toxic symptoms produced by the pure alkaloid, and
compare with the symptoms produced by the suspected alkaloid in
the substance under consideration. These check tests are gener-
ally omitted by the analyst who has had extensive laboratory ex-
perience and who is therefore in a position to recognize the nature
of the poison (alkaloid) from the symptoms manifested by the
inoculated frog. In many instances the frog alkaloidal tests may
serve as checks upon the blood tests already described.

In extracting the suspected substances it must be kept in mind
that alkaloids are very sparingly soluble in water, but when acidu-
lated (hydrochloric acid about 1 per cent.) water is used the acid
forms the salt (chloride) which is readily soluble in water. Alka-
loids are soluble in ether and in mixtures of ether and chloroform,
therefore these reagents should be used rather than the acidulated
water, especially since they are also antiseptic and the extractives

SPECIAL TOXICOLOGICAL TESTS 277

which they yield are freer from extraneous impurities, and also
because the residue which is injected into the frog (dissolved in the
water) represents the alkaloid and not its chloride. Should,
however, the evaporated acidulous extract be used for making the
frog test, then the check test should be made with the correspond-
ing pure alkaloidal salt.

If the inoculated frog shows no marked symptoms of poisoning
in the course of 3 or 4 hr., it is very likely that the residue under sus-
picion does not contain any very poisonous substance. The pri-
mary object of the tests is to ascertain whether or not dangerously
toxic substances (alkaloids) are present in foods, and not for the
purpose of determining the identity of the alkaloid nor to demon-
strate the presence of comparatively nontoxic alkaloids.

INDEX

Abrin, 272
Abscesses, 201
Acne, 201
Actinomyces, 184
Adjustment of media, 74
Agar, Hesse's, 135

litmus, 78

nutrient, 77

test, 14
Agglutination, 108
Agglutinins, vegetable, 271
Albumen coagulation, 252
Alcohol, 212
Aldehydes, 216
Algae, 14

in water, 114
Alkaloid tests, 276
Amanita muscaria, 275

phalloides, 275

verna, 275
Ampuls, 202
Analytical reports, 17
Anderson-McClintic, 231
Animal fat, 161
Animals, diseased, 26
Antiformin, 135

methods, 135
Antimony test, 16
Apparatus, bact., 83

counting, 41
Aquae, 198
Arachnodiscus, 14
Arrak, 225
Arsenic, 268

Arsenical test, 15, 268
Ash determination, 10
Aspergillus, 224, 227
Atropine, 198, 275
Autoclave, 73
Azolitmin, 74

B

Baby food, 5

Bacillus aerogenes, 132, 155

B. anthracis, 184

bifermentens, 153
botulinus, 158, 177, 183
bulgaricus, 139
californiensis, 206
eaucasica, 226
cholerae, 112

test for, 113
coli, 91, 94

in milk, 128
cyanogenes, 132
enteritidis, 101, 158
erythrogenes, 132
gelatinosum, 205
gummosus, 205
kutzingianum, 214
levaniformans, 203
liodermos, 203
mesentericus, 203
oxydans, 214
paratyphosus, 102
pasteurianum, 214
perfringens, 153
prodigiosus, 132
psittacosis, 102
soja, 227

279

280

INDEX

B. subtilis, 127, 153

suipestifer, 102

synxanthus, 132

tetani, 180, 183, 199

tuberculosis, 133, 136

typhi murium, 101

typhosus, 102
in water, 105

vermiforme, 228

viscosus, 219

vulgatus, 203

Welchii, 155

xylenum, 230
Bacon beetle, 140
Bacteria, 23

dead, 49, 50

examination of, 13, 35

in foods, 23, 58

in cream, 132

in ice creams, 138

in meats, 183, 153

in milk, 123

in water, 117

intestinal, 91

lactic acid, 63, 140

living, 49, 50

of cadaver, 184

of eggs, 195

of vinegar, 229

potato group, 203

sugar, 203
Bacterial dilutions, 86
Bacterins, 265
Bacteriological reports, 20

technique, 83
Bark tissues, PI. VI
Bartow, E., 25
Bast cells, PL II
Beaker sand test, 10
Bean tissues, PL V
Beebe wine, 229
Beef fat, 164
Beer, 218

diseases, 219

Beer, ginger, 228
Beetle, bacon, 140
Belladonna, 4
Benzoic acid test, 11
Berries, polluted, 26
Biginelli, 271
Bile, 93

lactose, 79
Binders, meat, 180
Biological products, 265
Bismuth test, 16
Bitter beer, 221

cheese, 141

milk, 132, 141
Bitting, 51
Black cheese, 141
Blanks, report, 18
Blood tests, 271
Blue cheese, 141

milk, 132
Body cell count, 125
Body cells, in milk, 124
Boehme, 100
Boiled milk, 131
Boils, 201
Boric acid test, 1 1
Botulism, 177
Bouquet, 217
Brandy, 216

rectified, 217
Brautegam, 167
Bread contamination, 25
Breed, 125
Broca, G., 49
Broth, dextrose, 77

liver, 79

nutrient, 76
Broths, sugar, 77
Buckwheat, PL III
Biirker ruling, 45
Butter, 167

fat, 121

renovated, 126
Buttermilk, 139

INDEX

281

Cadaver bacilli, 184
Candies, 209
Canned foods, 63

meats, 154
Carbuncles, 201
Carriers, typhoid, 27, 103
Cassia buds, PL IV
Castor oil, 160
Catsups, 54, 59
Centrifugal tube, 38
Cestoda, ova, 67
Cheese, 54

bitter, 141

black, 141

blue, 141

hopper, 140

mite, 140

Penicillium, 143

poisoning, 142

putrid, 141

ripening, 140

skipper, 140

spoiling of, 140
Chemical hemolysis, 273
Chemists, 1
Chinese eggs, 191

gardeners, 25
Chlorogalum, 272
Cholera, Asiatic, 112

germ test, 113
Cholesterol, 161
Cider, hard, 230
Cinchona test, 9
Citromyces glaber, 215

pfefferianus, 215
Cladosporum, 197
Clams, 151
Clove stems, PI. Ill
Coagulation coefficient, 252
Cobra venom, 275
Cocaine, 198
Coffee adulterants, PL IV

Cold storage eggs, 191
Colon bacillus in water, 117
test, 97

typhoid bact., 94
Color reactions, 10, 15, 160
Colors, of oils, 160
Concentrates, 35, 38
Condensed milk, 143
Condiments, 209
Congeners, 216
Conium test, 9
Conn's bacillus, 141
Contamination, 91

limits, 52

sewage, 97
Copper test, 16
Corn syrup, 208

tissues, PL V
Counting apparatus, 41
Counts, 44
Coupin, 263
Cream, bacteria in, 132

ripening, 54, 132
Crotalin, 275
Crotin, 272
Crystals, PL II

fat, 159,
Cultural methods, 68
Culture media, 69, 71

standard, 75
Cultures, mixing of, 85

plate, 85

tube, 87

types, 87, 89
Curcuma thread, 1 1

D

Davis, 50

Deposits, finger nail, 201
Dematium pullulans, 219
Desmids, 116
Dextrose broth, 77
Diatoms, 14

282

INDEX

Dilutions, 43

bacterial, 86
Dioxide test, 129
Diplococcus, of eggs, 191
Direct examination, 36
Dirt, 10
Diseases, 26
Disinfectants, 230, 259
Distillation, 217
Dried eggs, 190
Drigalski, no
Drinks, fermented, 210

medicated, 218
Drugs, classification, 2
Dusting powders, 201

E

Eber's test, 179

Edelmann, 167

Eels, vinegar, 55

Efficiency, of disinfectants, 258

Egg bacteria, 195

decomposition, 196

media, 195

membrane, 189

tests, 188
Eggs, 187

Chinese, 191

dried, 190

examination of, 192

frozen, 191

storage, 191
Ehrlich, 100
Ellis, 153
Emery, J. A., 164
Emich, F., 15
Endo medium, 80
Enzymes, 93
Equipment, 6
Ergot, 198
Essential oils, 263
Estimates, quantitative, 4
Evaporated eggs, 190

Evaporation, 40
Examination, direct, 35

of eggs, 193
Excrement, human, 25

Factory methods, 61
Fat crystals, 159

in milk, 121

tests, 127
Feces, human, 91
Fermentation, 212
Fermented drinks, 210

foods, 210
Ferments, acid forming, 214
Fertilizer, human, 25
Fillers, ice cream, 138

meat, 180
Filter, clay, 126
Filtering, 36
Filtration, fractional, 39
Finger-nail deposits, 201
Fish meats, 155

pickled, 158
Fitzgerald, 170
Flies, 61

Fluid extracts, 197
Food, contamination, 23

fermented, 210

laboratory, 32

poisons, 29
Foods, classification, 2, 3

investigation, 32

polluted, 25
Foot-and-mouth disease, 139
Formaldehyde test, 12
Fornet, 170
Frog tests, 276
Frozen eggs, 191

milk, 144
Fruit fresh, 52

juices, 203

rotten, 64

INDEX

283

Fruit, starch, 12

whole, 52
Fruits, acid, 62
Fusel oil, 216

Gaertner bacilli, 101, i$i
Gas formation, 98]
Gay, 170
Geinitz, 263
Gelatin, tests for, 72

moldy, 65

nutrient, 77
Gila monster, 275
Ginger beer, 228
Githagin, 273
Glassware, 70
Globules, fat, 121
Gluten tests, 13
Glycerin, 198
Glycine hispida, 227
Gmelin, 50
Gold test, 16
Goose fat, 160
Gosio, 50
Graham, 181
Grahe's test, 9
Grape sugar, 167
Greenlee, 190
Gum formers, 203

H

Hiss' medium, 79
Hog cholera, 184

bacteria, 101, 158
Hopper, of cheese, 140
Hops, 218
Horse meat, 167
Housewife, 52
Howard, B. J., 46, 48
Howell, Miss K., 25
Hydrogen dioxide test, 129
Hygienic lab., 32
Hypodermic syringes, 174

I

Ice-cream bacteria, 138

fillers, 138
Ice creams, 138
Incubation, 88
Indol test, 100
Infections, 24

skin, 201
Intestinal bacteria, 91
Iodine reaction, 12
Iron test, 16
Itch mite, 201

J

Jams, 58
Jordan, 275

K

Hamilton, H. C., 244
Hand gluten test, 13
Hanford epidemic, 27
Hard cider, 230
Hansen, 50, 213
Heat sterilization, 26
Hemacytometer, 36, 43
Hemolysis, chemical, 273
Herring, pickled, 57
Hesse's agar, 135

Kebler, L. F., 229
Kerr, R. H., 161
Kephir, 226
King, 50

Kitasato filter, 37
Knapp, 131
Koch, Robert, 113

cholera test, 113
Koenig, 17
Koumiss, 227

284

INDEX

Laboratory, equipment, 6

food, i

methods, 2, 30, 31
Lactose bile, 79

litmus agar, 78
Lafar, 204
Lancet method, 230
Lard, 164

oil, 160
Lead test, 16
Leaks, 62
Leban, 228
Leuconostoc, 205
Levan, 205
Lignin test, 9
Limits, of contain., 62

of organisms, 51
Linseed oil, 160
Liver broth, 79
Lloyd, J. U., 229
Loop, platinum, 85

tubes, no
Lumpy jaw, 184

M

Mace test, 9
Magpotine, 119
Mallow leaf, PI. V
Manufacturers, 52
Martindale, 263
Maya, 226
Meat bacteriology, 177

binders, 180

canned, 154

extracts, 69

fillers, 180

horse, 167

of fish, 155

organisms, 181

precipitins, 169

sausage, 177

Meat, spoiled, 154, 174

starch in, 181

toxins, 175
Meats, 152

dried, 157

smoked, 157

storage, 155

sugar in, 167
Media, reaction of, 74

standard, 75

tubing of, 84
Medicamenta, 198
Medicated drinks, 218
Medicinal syrups, 203
Medicines, 197
Mercury test, 16
Methods, bacteriological, 96

cultural, 68

laboratory, 2, 30, 32

tabulation, 31
MetschnikofT, 29
Meyer, Karl F., 169
Micro-analysts, 1, 3, 6
Micrococcus casei amari, 141
Micro-gluten test, 13
Microscope, 1, 6
Microscopical reports, 18
Miescher's bodies, 187
Milk, 26

artificial, 82

bacteria in, 124

bitter, 132, 141

blue, 132

boiled, 131

body cells, 124

condensed, 143

dried, 144

examination of, 120

fat count, 122
globules, 121

frozen, 144

medium, 81

powdered, 144

raw, 131

INDEX

285

Milk, red, 132
ropy, 132
sour, 139
standards, 123
tuberculous, 128
water in, 131
yellow, 132

Mineral waters, 118

Miquel, 117

Mite, of cheese, 140

Molasses, 203, 208

Mold counting, 47
in foods, 58
in gelatin, 65
in plums, 56
in tomato pulp, 51

Morphine, 198

Mucor, 204

mucedo, 211

M filler, 170

Mussels, 151

Mycoderma, 204, 214

N

Nelson, 268
Nematodes, 61, 66, 69
Neutral red test, 100
Nostoc, 114
Novy, 29
Nutrient agar, 77

broth, 76

gelatin, 77
Nutrose, 82

media, 135

Oberhefe, 213
Ohno, T., 244
Oidium lactis, 127, 147
Oils, essential, 263
Oleomargarine test, 126

Olive pits, PI. Ill
Organisms, identifL, 54

in food, 58

in meat, 182
Organoleptic tests, 58
Oscillaria, 114
Ova, 66
Oysters, 144

shucked, 151

Pancake flour, 5
Pancreatin, 93
Paramecia, 116
Parasites, 14

intestinal, 66
Pastes, tomato, 59
Pear pulp, PL II
Penicillium brevicaule, 269

glaucum, 140, 142
Peptone medium, 82
Percentage estimates, 4
Petri dishes, 83
Pfeiffer's phenomenon, 113
Pharmaceutical laboratory, 200

sanitation, 200
Pharmaceuticals, 197
Phenol coefficient, 233, 244
Phenolphthalein, 74
Phloroglucin, 9
Phytosterol, 161
Phytophthora, 197
Pickles, 54
Pilocarpine, 198
Pine tissues, PL III
Pink colonies, 99
Piophila casei, 140
Planting in media, 85
Plate cultures, 85
Platinum loop, 85
Podkoassa, 226
Poisons, of foods, 29

286

INDEX

Pollen grains, PI. I
Pollution, of foods, 26
Potassium hydroxide, 9
Powdered milk, 144
Powders, 201
Precipitin meat test, 168
Prescott, 105
Preserves, 58
Products, canned, 63
Proteus vulgaris, 153
Pseudo-trichinae, 187
Pulp, decomposed, 48

mold, 51

tomato, 46
Pus cells, in milk, 125

organisms, 198
Putrid cheese, 141

Qualitative determinations, 90
Quantitative methods, 68

Rating, of shellfish, 148
Rattle-snake, 275
Ravenel, 63
Raw milk test, 131
Reaction limits, 17

of media, 74
Red milk, 132
Report blanks, 18
Reports, analytical, 17
Rhamnus bark, PI. VI
Rhizopus, 197
Rice tissues, PI. IV

wine, 223
Ricin, 272
Rideal-Walker, 230
Ripening, of cream, 132
Robin, 272
Ropiness, of beer, 219

Ropy milk, 132
Rotten fruit, 64

Saccharomyces species, 213

anomalus, 215

ellipsoides, 216

hansenii, 215

mycoderma, 214

pasteurianus, 215

pastorianus, 221

ruber, 141

sake\ 225

soja, 227
Sak6, 223

Salicylic acid test, 1 1
Sand, 4

test, 10
Saponins, 272
Sarcina, 228

hamayuchia, 227
Sarcocystis, 187
Sauerkraut, 54
Sausages, 177
Savage, 158
Sawyer, W. A., 27
Scalp, 201

lotions, 201
Schneider, A., 232
Sclerenchyma cells, PI. II
Sea water, 95
Selenium, 269

test, 49
Sera, 265
Sewage, 91
Shellfish, 144
Siedentopf, 13
Silver test, 16
Skin infections, 201
Skipper, of cheese, 140
Slant cultures, 89
Smallpox vaccines, 268
Soda fountain, 202

INDEX

287

Soda bottling, 207
Sodium caseinate, 82
Soja bean, 227

sauce, 227
Soup stocks, 154
Sour milk, 139
Souring of beer, 220
Spaghetti, 27
Spiritus frumenti, 216
Spoiled meats, 174
Spore counter, 41
Spores, in catsups, 53

in pepper, 57
Sputum, 136
Stab cultures, 87
Standard media, 75
Standardization, of milk, 123
Starch, in fruits, 12

filler, 12

in meat, 168, 181

paper, 12

test, 12
Starches, PL I
Starkey, no
Sterilization, of foods, 26

of media, 73
Stiles, G. W., 33
Stitt, 137

Storage meats, 155
Streptococcus acidi lactici, 127

aureus, 130

hollandicus, 132

lacticus, 132

longus, 153

pyogenes, 130
Strychnine, 275
Sublimation tests, 1 1
Sugar, in meats, 167

broths, 77

grape, 167

test, in meat, 167
Sugars, invert, 203
Sulphurous acid test, 12
Syringes, hypodermic, 174
Syrup, corn, 208

Syrups, 198, 202

medicinal, 203
Swells, 62

Technique, bact., 83
Tellurium, 269

test, 49
Temperature differential, 99
Tests, blood, 271

quantitative, 36

tubes, 83
Tinctures, 197
Tomato pastes, 59

pulp, 46
Tonsillitis, 26
Torula, 204

amara, 132
Toxalbumins, 271
Toxicity coefficient, 249
Toxins, 271

in meat, 175
Treacle, 203
Trematodes, 66
Trichinae, 184
Trichomes, PI. V
Tube cultures, 87
Trioglyphis siro, 140
Tube, centrifugal, 37
Tuberculous milk, 128
Tubes, loop, no
Tubing media, 84
Turbid beer, 221
Turck ruling, 36, 44
Typhoid bacillus, 102

carrier, 27, 103

epidemics, 106

infection, 2$

medium, 79

methods, 105

U

Ultra -microscope, 13
Unsanitary methods, 61

288

INDEX

V

Vaccines, 265
Vacuum, partial, 40
Vandevelde, 274
Vaughan, 29, 138
Vegetable fat, 161
Vinegar bacteria, 229

eels, 55
Viperine, 275

W

Water, analysis of, 114

bacteria, 117

bottled, 118

mineral, 118

tests, 114
Watered milk, 131
Waters, polluted, 25
Weigmann's bacillus, 141
Wheat flour, 5

tissues, PI. IV

Whiskey, 216

rectified, 217

Widal test, 107

Wilson, 105

Wine, bebee, 229
slimy, 227

Wines, 222

diseased, 222

Yeast, upper, 213
Yeasts, 204

in foods, 58
Yellow milk, 132
Yoghurt, 226

Zappert ruling, 44
Zinc test, 16
Zsygmondy, 13
Zymases, 212

9

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