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AGRICULTURAL BACTERIOLOGY

AGRICULTURAL
BACTERIOLOGY

THEORETICAL AND PRACTICAL

BY

JOHN PERCIVAL, M.A., F.L.S.

PROFESSOR OF AGRICULTURAL BOTANY, UNIVERSITY COLLEGE, READING

SECOND EDITION

LONDON

DUCKWORTH & CO.

3 HENRIETTA STREET, COVENT GARDEN, W.C.
1920

1

First Edition, 1910
Second Edition, 1920

All Rights Reserved

Printed in Great Britain
hv Turnbull <5r» Spears, EdinburgJt

PREFACE

The science of bacteriology, which has been so much
the domain of the student of disease, is now being
applied to the elucidation and solution of many of the
problems which confront the farmer, gardener, and dairy-
man in their daily life.

The extensive development of the dairying industry
has been greatly assisted by the bacteriologist, and very
material advances may be expected in the near future in
the application of the science to the study of the soil
and the phenomena connected with the economical and
efficient use of fertilisers for the nutrition of farm and
garden crops.

For some time a knowledge of bacteriology has been
demanded of candidates for examinations in agriculture,
dairying, and horticulture, but experience has shown that
few of them have had any practical acquaintance with
the subject, mainly, we believe, from lack of suitable
opportunities for proper training and absence of a text-
book dealing with the subject in a practical way.

It is to supply the needs of such students that the
present text-book has been written. It is hoped also
that it will be useful and interesting to agriculturists and
horticulturists generally who are anxious to obtain an
insight into the causes and methods of control of many

I'U

VI PREFACE

of the natural processes with which they come into
contact daily.

The work embodies the results of much experience
in the scientific training of students, and deals with most
of the points which the author considers of fundamental
importance in the study of bacteriology, so far as the
requirements of the farmer and gardener are concerned.

I shall be grateful for any suggestions for improving
the work.

My thanks are due to Miss Grace Heather Mason
for assistance in carrying out the work of the practical
exercises and also for help with the index and proofs.

JOHN PERCIVAL

May 1910.

CONTENTS

I. Introduction ...... i

Relationship of bacteria to other organisms in the Vegetable
Kingdom, 3 ; Methods of staining, 6.

II. Bacteria: their forms and reproduction . ii

The forms of bacteria, 11; Size, 13; Movement, 15; Reproduc-
tion (i) Vegetative, 18 ; (ii) by spores, 20 ; Classification of
bacteria, 23.

III. General conditions which influence the

growth of bacteria .... 29

The action of light, 29 ; Heat, 30 ; Water, 31 ; Oxygen, 31 ;
Food, 32 ; Sterilization, 35 ; Pasteurization, 38 ; Disinfectants,
antiseptics, and preservatives, 39.

IV. The methods of isolation and pure culture

OF BACTERIA ..... 43

Method of isolation, 44 ; Pure cultures, 51 ; Media, 54 ; Identi-
fication, classification, and registration of bacteria, 59 ; Cultiva-
tion of anaerobic bacteria, 64.

V. Fermentations and the action of enzymes . 67

Carbohydrates, 72 ; Fats and Oils, 78 ; Lipase, 78.

VI. Fermentations and the action of enzymes

{continued) . . . . ,81

Proteins, 81 ; Colour tests for proteins, 85 ; Salting out of pro-
teins, 86 ; Hydrolysis of proteins, 89 ; Proteolytic enzymes, 90 ;
Proteolytic enzymes of bacteria, 96.

vii

Vlll CONTENTS

CHAPTER PAGE

VI I. Fermentations and the action of enzymes

{continued) . . . . . joo

Putrefaction and decay, loo ; Putrefactive organisms, 103 ;
Oxidizing enzymes or oxidases, 114.

VIII. Bacteriology of the soil . . . .117

Numbers of bacteria in the soil, it8 : Kinds of bacteria in tiie
soil, 122 ; Action of antiseptics and heat upon the soil, 130.

134

IX. Nitrification . . . " .

Nitrification, 134.

X. Denitrification . . . . .152

Denitrification and reduction of nitrates and nitrites, 152.

XI. The fixation or assimilation of nitrogen . 169

The fixation or assimilation of the free nitrogen of the air, i68 ;
Clostridium pastorianum, 171 ; Species of Azotobacter, 175.

XII. The fixation or assimilation of nitrogen

{continued) . . . . .187

The fixation of nitrogen by bacteria in symbiosis with legu-
minous plants, 186.

XIII. Bacteriological examination of soils. . 206

Comparison of the fermentative action of soils, 206,

XIV. Farmyard manure . . . . .211

Nature and composition of farmyard manure, 211 ; Bacteria in
farmyard manure, 215; (i) The ammoniacal fermentation of urine,
217; (ii) Putrefaction in the manure heap, 225 ; (iii) Nitrification
and Denitrification, 226 ; (iv) The fermentation of cellulose, 226.

XV. Milk, its origin and composition . . 232

Secretion of milk, 232 ; Composition of milk, 234 ; Colostrum,
241 ; The enzymes of milk, 242 ; Action of heat on milk, 245.

CONTENTS IX

CHAPTER PAGB.

XV^I. Bacteria in mit.k, and their sources . . 24&

Number and source of origin of bacteria in miik, 248 ; Influence
of temperature and lime upon the numbers of bacteria in milk, 260 ;
Methods of reducing the numbers of bacteria in milk, 262.

XVII. Fermentations in milk . . . . 266

Lactic fermentation, 266 : Slimy or ropy fermentations in milk,
278; Development of colour in milk, 281; Bitter milk, 283;
Butyric acid fermentations, 285.

XVII I. Filtration ; Cooling; Pasteurization and

sterilization of milk ; Preservatives . 292^

Filtration, 292 ; The effect of low and high temperatures, 293 ;
Preservatives, 297.

XIX. The examination of milk : Milk standards . 300-

Dirt in milk, 300, Blood, leucocytes, and pus -cells, 302 ; Bac-
teria, number and kind, 304 ; Milk standards, 309.

XX. Sour milk beverages and foods . . . 315

Kefir, 314 ; Koumiss, 315 ; Mazun, 316 ; Joghurt and Lacto-
bacilline, 317.

XXI. Milk and disease ..... 32a

Tuberculosis, 321 ; Method of checking or stamping out bovine
tuberculosis, 326 ; Foot-and-mouth disease, 327 ; Typhoid fever,
327 ; Epidemic summer diarrhoea, 329 ; Diphtheria and septic sore
throats, 331 ; Scarlet fever, 332.

XXII. Cream and cream ripening

Cream, 333 ; Natural starters, 339 ; Pure culture starters, 340.

XXIII. Butter

Bacteria in butter, 346 ; Flavour and aroma of butter, 348 ; Taints
and defects of butter, 349 ; Salt in butter, 355.

333

344-

X CONTENTS

CHAPTER PAGE

XXIV. Cheese . . . . . .357

Rennet, 357; Cheese, composition and ripening, 359; Some
cheese defects, 374.

XXV. Moulds and yeasts . . . . .378

Moulds, 378 ; Yeasts, 390.

TABLES 397

INDEX . . . . . .401

AGRICULTURAL BACTERIOLOGY

CHAPTER I.

INTRODUCTION.

I. Take some hay and chop it into short pieces about

an inch long. Then place it in a glass beaker or cup,

pour cold water over it, and

leave in a warm room for a

day or two. The cold water

dissolves various substances

from the hay and becomes

brownish in colour, rather like

weak tea. If a drop of the

solution is taken from the

cup or beaker and examined

with a microscope, many

living things will be found Ficx.-Drop of hay infusion, ^z Protozoa;

swimming about in it. Fig. I ^ bacteria (x 200).

gives the appearance of what is generally seen in such
a drop examined under a moderate magnification (an
ordinary microscope with a f objective). The most
obvious living beings are small oval transparent organisms
which glide in all directions across the field of view
(Fig. I, a). Sometimes there are few of them, but in the
majority of trials with a hay infusion they will be met

2 INTRODUCTION

with in considerable numbers, and frequently several
kinds are visible. These all belong to the Animal King-
dom, and are included in the Protozoa, a division which
embraces the simplest forms of animal life.

Small pieces of leaves may also be observed in most
instances, as well as other bits of dead material. In
addition to these there will always be visible hundreds
of exceedingly small rod-shaped bodies distributed about
in the water. They are so minute, however, that unless
the attention is specially directed to them they may
easily be overlooked. By careful observation they are
seen to be of variable lengths and capable of independent
movement, travelling end on backwards and forwards
through the liquid.

Fig. 2 is a more highly magnified view of a portion of
a drop of a hay infusion, an J -inch objective being used
in this case. The large oval body, ^, is one of the
Protozoa referred to above ; ^ is a piece of mould fungus,
and some of its spores or reproductive bodies are seen at
c. They are non-motile. The living, rod-like organisms
are now easily seen, and their movements more readily
followed. Some of the longer ones are depicted in the
figure at d^ and others of a different form and size are
shown at e.

2. These minute living things are examples of
organisms known as bacteria. They are distributed all
over the world in incalculable numbers — in the air, in
water, in the soil, and wherever dust can be carried.
Their importance can scarcely be over-estimated, as they
are responsible for some of the most striking phenomena
in nature. They carry on useful work in preparing the
soil for the growth of crops, and render manures which
are applied to the land efficient for the nutrition of

INTRODUCTION 3

plants. They act as natural scavengers, getting rid of
all kinds of dead organic matter through the process of
fermentation and decay which they initiate and maintain.
Several industries, such as the manufacture of butter and
cheese, could not flourish without their aid. On the
whole their action is beneficial. Certain kinds are

Fig. 2. — Drop of hay infusion, a Protozoon ; h hypha of a fungus ;
c spores of fungus ; d and e bacteria. (700).

however, the cause of some of the most fatal diseases
affecting man and the lower animals.

3. Relationship of these Organisms to others in the
Vegetable Kingdom. — As bacteria are generally spoken
of as minute unicellular plants, it is of interest to indicate
the position in which they are usually placed among the
divisions of the Vegetable Kingdom,

4 INTRODUCTION

Living things are generally classified into plants and
animals, and there is little difficulty in placing the higher
forms, such as cows, birds, thistles, or cabbages, into one
or other of these two groups. Flowering plants are
characterized by the production of special structures
which botanists call seeds ; these contain rudimentary
plants. Flowerless plants, on the other hand, never pro-
duce seeds, but grow from unicellular structures termed
spores. Of flowerless plants three divisions are generally
recognized, viz.: (i) Ferns and their allies; (2) Mosses and
allies ; and (3) Thallophytes, which differ from the others
in possessing no definite root, stems, or leaves. The
Thallophytes are subdivided into (i) Algce and (2)
Fungi. The Algce, to which belong sea- weeds and their
relatives in fresh water, possess the green colouring
matter, chlorophyll, by the aid of which they can utilize
the carbon dioxide of the air for the production of carbon
compounds of their bodies when they are exposed to
daylight. Fungi, on the other hand, contain no chloro-
phyll, and, with the exception of a small group of
bacteria, they are unable to make use of carbon dioxide
for the formation of their carbon compounds. They
require to be fed with organic food, such as starch, sugar,
fats, proteins, and other similar substances directly derived
from other living or dead plants and animals.

The true fungi or Euniycetes, of which some common
examples are described In Chapter XXV., generally consist
of branching, thread-like cells, and are reproduced by
spores. Bacteria are usually associated with this division of
the Vegetable Kingdom, chiefly because of their want of
chlorophyll and general mode of nutrition, but in other
respects they exhibit little relationship with the true fungi,
and their real position in a natural scheme of classification,

INTRODUCTION

based upon what may be termed blood relationship or
descent, is uncertain. However, for practical purposes this
is of little moment, and they may be left as a division of
the Fungi, as indicated in the following scheme : —

I. Vege t-
able King-
dom.

A. Flowering
Plants or
Phanero-
gams.

B. Flowerless
Plants or
Crypto-
gams.

1. Pterido-jF^f"f'Ho''^e:
Dhvtes i tails, and
" ^ ■ ( Lycopods.

2. B r y o-/ Mosses and
phytes. \ Liverworts. ^]

3. T h allo-jAlgse.

. phytes. \ Fungi.

. (True
fungi or
E u m y-
cetes.)
. (Bacteria
or Schizo-
mycetes. )

2. Animal
K i n g-
dom.

Ex. 1. — To clean Slides and Cover-Slips. — Before any satis-
factory microscopic work can be done, it is necessary to clean
thoroughly all glass slides and cover-slips or cover-glasses.

(i) Place them in a hot solution of caustic soda (caustic soda
about 10 grams, water 100 c.c.) for an hour.

(2) Take them out and wash under water-tap : wipe and dry
them.

(3) Place them, one by one, in a beaker containing strong
nitric acid, and leave them for one or two hours.

(4) Take them out, wash under tap, wipe and dry with a
clean handkerchief.

(5) Store them in a beaker or specimen jar containing
absolute alcohol until wanted. When required for use, take out
and wipe clean.

Ex. 2. — Heat a platinum wire loop (Fig. 3) in a Bunsen flame ;
dip it just below the surface of an infusion of hay which has been
prepared in the manner described above and left in a warm room

6 INTRODUCTION

or incubator for four days. Transfer the drop on the wire loop
to a clean slide : put on cover-glass and examine with low power,
and then with ^-inch objective. Make drawings of bacteria seen.
Before and after using a platinum loop or needle always heat
it to redness in Bunsen flame : when not in use keep it on a
stand (Fig. 3) ; do not place it on the laboratory table or bench.
Ex. 3. — Make infusions of the leaves of cabbage, cereals,
clover, and other plants. Pour off the liquid, after it has
been on the leaves for three or four hours, into a clean beaker.
Leave in a warm room for four days, and examine drops as in
preceding exercise. Make drawings of bacteria seen.

Ex. 4. — Take some peas and soak them in water all night.
Then boil them for five minutes, and leave them to stand in the

boiled water for a day
or two in a warm room.
Take a drop of the
liquid and examine for

Fig. 3. — Copper stand for platinum needles baCtCria.

and loops. _ _ „ . . . -

Ex. 5.— Staining of
Bacteria. — Since bacteria are transparent and not readily seen,
they are frequently killed and stained, when their form is
rendered more easily visible.

(i) Prepare a saturated solution of gentian violet in alcohol,
using

Gentian violet 4 gr.

Absolute alcohol ..... 50 c.c.
Shake well, and allow it to stand twenty-four hours ; then filter
twice into a stoppered bottle ; label it, and keep for future use.

(2) Add three drops of the above alcoholic solution to a
watch-glass containing distilled water.

(3) Take single drops of the infusions from Exs. 3 and 4 and
spread them with the platinum loop asthinyf/wi- over the surface
of clean cover-glasses. Cover with a beaker to keep off dust,
and allow the films to dry. The moisture evaporates and leaves
the bacteria behind on the glasses.

INTRODUCTION 7

(4) Hold the cover-glass with Cornet forceps (Fig. 4) and
fix the bacteria by passing the glass, bacteria side up^ three times
through Bunsen flame. In doing this the hand should describe
a circle about one foot in diameter, at the rate of once round
every second, the path of the glass passing through the flame.

(5) Place the cover-glass, bacteria side down, on the surface
of the watery solution in the watch-glass (2), and allow it to
remain there for three to five minutes, during which time the
bacteria will become stained. Wash it in water and dry between
filter paper.

(6) Clean the side of the cover-glass on which there are no
bacteria, and place it on a drop of water on a clean slide.
Examine and make drawings of the stained bacteria.

Fig. 4. — Cornet spring forceps.

(7) If not suflficiently stained, float off the cover-glass with
water and stain again ; re-examine it after washing with water,
and, if satisfactory, dry it again between filter paper and leave
it in a warm place until all traces of moisture are gone ; then
mount it permanently in a drop of xylol balsam (Canada balsam
dissolved in xylol).

Ex. 6. — Prepare a solution of methylene blue in alcohol in
the same manner as the gentian violet solution in Ex. 5,
using : —

Methylene blue 4 gr.

Absolute alcohol ..... 50 c.c.
Label it and store in a stoppered bottle. Try its staining power
upon cover-slip preparations obtained as in Ex. 5 (3).

8 INTRODUCTION

Ex. 7. — Prepare the following solution : —

Loffler's Methylene Blue.

(i) Make up a i per cent, solution of caustic potash.

(2) Take i c.c. of it and add it to 100 c.c. of distilled water.

(3) Mix weak solution (2) with 30 c.c. of a saturated alcoholic
solution of methylene blue (Ex. 6). Shake, filter into a
stoppered bottle, and label it.

This is an excellent stain, not very deep, but it often brings
out delicate features of bacteria; it loses some of its staining
power on keeping. Try it on cover-glass preparations as
described in Ex. 5, allowing it to act for 3-10 minutes.

Ex. 8. — Prepare the following solution : —

Ziehl-Neelsen's Carbol-fuchsm.

Fuchsin . . . . . , . i gr.

Absolute alcohol 10 c.c.

Carbolic acid (crystals) . . . . 5 gr.

Distilled water . . . . . .100 c.c.

Dissolve the fuchsin in the alcohol in a mortar or porcelain
dish, breaking the fuchsin if necessary with a pestle or a glass
rod. Then dissolve the carbolic acid in the water and add half
of this solution to the dye in the mortar or dish ; stir well, and
pour into a stoppered bottle. Wash out the mortar with the
rest of the carbolic-acid solution, adding it to the mixture in the
bottle. Shake well, and after twenty-four hours filter it into an
amber-coloured stoppered bottle or ordinary glass bottle covered
with brown paper to exclude the light.

This gives an intense red stain. It is best to use a freshly
prepared weak solution made by adding i c.c. of the above
concentrated solution to 10 c.c. of distilled water; filter. Try
it on cover-glass films as described in Ex. 5, allowing it to act
for 1-3 minutes.

Ex. 9. — Gram's Method of Staining. — Prepare the following
solutions : —

INTRODUCTION 9

{a) Saturated alcoholic solution of gentian violet, 30 c.c.

Aniline water, 100 c.c.
As this solution does not keep well, only a small amount
should be made up.

The aniline water is obtained by thoroughly shaking up 4 c.c.
of aniline with 100 c.c. of water, and then filtering through moist
filter paper until the liquid is clear and free from any oily drops ;
any aniline which remains in the bottom of the flask or tube
after shaking should not be poured on the filter.

(d) Iodine i gr.

Potassium iodide .... 2 gr.

Water . ... . . 300 c.c.

Dissolve the potassium iodide in about 10 c.c. of the water
and add the iodine : when the latter is dissolved add the rest of
the water.

In using the above solutions for staining purposes, the follow-
ing procedure should be adopted : —

(i) Float the cover-glass, ^/m downwards^ on aniline-gentian-
violet solution in a watch-glass, and allow the stain to act for
five minutes.

Take up with forceps and drain off the superfluous stain by
dipping the edge of the cover-glass on blotting-paper.

(2) Then, immerse the cover-glass for 1-2 minutes in the
iodine solution in a watch-glass, film side up. The purple
colour of the gentian violet should change to a yellowish brown
tint.

(3) Now, remove the cover-glass, taking care to note on
which side the bacteria are ', drain off the superfluous liquid and
dip the cover-glass into a vessel containing absolute alcohol for
10 seconds, and into a second vessel of absolute alcohol for
10-15 seconds ; when no more of the gentian violet stain is seen
to wash off, take it out and wash it in water, and examine either
in water or after drying in Canada balsam. Too long immersion
in alcohol must be avoided.

10 INTRODUCTION

Bacteria may be divided into two groups, viz.: —
i. Tliose which stain by Gram's method (Gram positive, 4 )
ii. Those which do not retain the gentian-
violet stain when washed a short
time with alcohol . . (Gram negative, - )

In using the test upon a doubtful organism of coccus form, it
is sometimes useful to mix the latter with a bacillus^ such as
Bs. subtilis, known to stain by Gram's method ; or, if a bacillus
is to be tested, mix it with a Gram-staining coccus, such as
Sir. lacticus.

The following organisms are Gram positive : —
Most species of Micrococcus.

„ „ Sarcina.

Bs. subtilis.
Bs. megatherium.
Bs. mesentericus.
Bs. mycoides.
B. {Proteus) mirabilis,
B. {Proteus) Zenkeri.
Bad. acidi lactici.
Str. lacticus.
Most yeasts and fungi.
The following are Gram negative : —

Most species of Spirillum,
Bad. coli.

Bad. ladis aerogenes.
Bad. fluorescens.
Bad. prodigiosum.
Bad. pyocyaneum.
Try Gram's stain upon a series of cover-glass films of
organisms obtained from the hay, cabbage, and pea extracts
of Exs. 3 and 4.

CHAPTER II.

BACTERIA : THEIR FORMS AND REPRODUCTION.

I. The Forms of Bacteria. — The body of a bacterium
consists of a single cell, into the minute structure of

Fig. 5. — Forms of bacteria, i, Coccus ; 2, bacillus ; 3, vibrio ; 4, spirillum.

which it is not necessary to enter here. It is sufficient
to note that there is a central body of protoplasm which
stains readily with aniline dyes of various kinds, and
a skin or capsule, usually so thin as to be practically
invisible except by the aid of the highest powers of the
microscope. The capsule, or cell-wall, is not composed
of cellulose as in the higher plants, but of a material
apparently allied to the protein substances. The whole
body or cell is met with in many forms, as indicated in
Fig. 5. Some kinds of bacteria are spherical, each
individual being then termed a coccus. When much
longer than broad, the bacterium is known as a bacillus ;
a comma-shaped form is spoken of sometimes as a

12 BACTERIA : FORMS AND REPRODUCTION

vibrio ; a rigid spiral form in shape like a corkscrew is
a spirillum.

The terms bacterium and its plural bacteria are fre-
quently used in a general sense when speaking of one or
more or any of these minute organisms without reference
to their form. The word bacterium is, however, used
also in a limited sense to denote a
particular non-motile rod-like cell which
is shorter than a bacillus. Under certain
circumstances bacterial cells may become
distorted and irregular in shape. Such
Fig. 6.— Involution are known as involution-forms (Fig. 6),

forms of a bacterium , , . , , i • /-i i .^

and are met with chiefly where the
medium in which they are living is becoming exhausted
of its nutritive substances, or where there is an accumu-
lation of compounds which are detrimental to the
vitality of the organisms : they are diseased, degenerate
forms.

The capsule or skin surrounding the central body
of a bacterium as a rule is very thin,
and as it does not readily absorb dyes
it is rarely visible. In some species,
however, the capsule is of a mucil-
aginous or slimy nature, and swells up
with water to a thickness of several
times that of the rest of the cell (Fig. 7). pio. 7.-^!^ showing
It may then be faintly stained in several weii-deveioped capsules.
ways, and can be seen as a pale cell-wall surrounding
the darker central body of the bacterium. Very
frequently the swollen envelopes of large numbers of
bacteria adhere to each other, and form a gelatinous
mass or zoogloea colony of variable size, which is
often met with as a slimy skin-like " scum " on

SIZE 13

stagnant water, or in solutions containing decayed
organic matter.

2. Size. — Bacteria are among the smallest of all
living things. Their dimensions are usually stated in
thousandths of a millimetre. One thousandth of a
millimetre (.001 mm.), known as a micron, is denoted
by the sign i /x, and is equal to about ytJito of an inch
(.00004 inch). Cocci range from less than .5 /x up
to 2 /A in diameter ; bacteria and bacilli from .5 /x
or less in breadth to 10 /a in length; the majority,
however, are much shorter than the latter figure. On
account of their minute size they are, when dry, readily
blown about by the slightest breeze, and find their way
into any crevices wherever dust can penetrate. They
are carried in streams of water, and are to be found
in and about all foods, milk and other liquids, in
houses and stables and in the soil, on the skin and
in the stomach and intestines of all animals. They are,
however, never met with in the blood or tissues of
healthy animals or plants, and are rare in water which
has come from deep wells or springs, or has been filtered
through great depths of soil and rocks.

Ex. 10. — To Measure the Size of a Bacterium or other
Organism.

{a) A simple method is by means of a stage micrometer and
camera lucida. .

The stage micrometer consists of a scale of tenths and
hundredths of a millimetre accurately ruled in fine lines on a
glass plate.

Place it on the stage of the microscope, and make drawings
of its scale on a sheet of paper with the camera lucida, using in
turn the | in., Jin., \ in., and ^ in. objectives, with a No. 3, 4,
or other eyepiece.

14 bacteria: forms and reproduction

Take away the stage micrometer, and in its place put the
slide of the bacterium to be measured ; make a drawing of the
organism with the camera lucida. Now measure the dimen-
sions of the drawing with the scale prepared as above from
the micrometer scale, taking care that the eyepiece and objective
used in making the paper scale and the drawing of the bacterium
to be measured are the same.

(i) Measure any bacteria which you have mounted and

stained,
(ii) Mount a little yeast in water and measure the size of

the cells,
(iii) Measure the spores of any mould which you can

obtain.

{b) A bacterium or other organism may be measured directly
by means of an eyepiece scale and a stage micrometer. The
eyepiece scale consists of a disk of glass, which fits into the
interior of the eyepiece ; upon it a fine scale of equi-distant
lines is ruled.

Place the micrometer, ruled in yV^hs and y^oths of a milli-
metre, on the stage of the microscope, and examine it with
the eyepiece having the scale 'in it.

Bring the divisions of the latter into line with those of the
stage micrometer, and find the value of one division of the eye-
piece scale in yVhs or xJyths of a millimetre.

Use the various objectives successively, and record the results
for each.

Place the bacterium or other object to be measured on the
microscope stage and determine how many divisions of the
eyepiece scale it covers : it is now easy to calculate its real
dimensions, as the value of each division of the eyepiece scale
is known.

(i) Measure the objects mentioned above in (i), (ii), (iii)
of section {a) in this way, and compare the results
obtained by the previous method.

MOVEMENT

15

3, Movement. — In the first chapter it was noticed
that some bacteria are endowed with the power of
spontaneous movement ; they can rove from one part of
a drop of water to another as readily as fish in a pond.
This is brought about by the lashing action of one or more
exceedingly delicate hair-like cilia or flagella which are
attached to the outer coat or capsule of the bacterium.
Some organisms pos-
sess only one flagellum
at one Gnd{monotrichic),
others two or more in
a tuft at one or both
ends {lophotrichic), while
many bacilli have many
flagella distributed all
over the cell - wall,
and are spoken of as
peritrichic (Fig. 8).
Numbers of species of
bacteria are, however, O
devoid of motile cilia,
and are incapable of
spontaneous movement
in liquids. Very
frequently non - motile
forms exhibit a peculiar oscillatory movement when
put in water or other liquids. This motion back-
wards and forwards over a very short distance or
round and round in a circular or oval path is a purely
physical phenomenon known as Brownian movement,
and is exhibited by any kind of exceedingly small
particles, either living or dead, when they are suspended
in water.

Fic,. 8.— Arrangement of flagella on bacteria.
I, Monotrichic ; 2, lophotrichic ; 3, peritrichic.

i6 bacteria: forms and reproduction

Ex. 11. — staining of Flagella. — The flagella or motile organs
of bacteria are of extraordinary fineness and delicacy, and
appear to dissolve or become thrown off and disorganized
when the water in which the bacteria are living dries up.

Under certain conditions they may be rendered visible by
staining, but the operation is one of much difficulty and
uncertainty. The chief elements of success are extreme clean-
liness of the cover-glasses on which the organisms are placed,
and a culture of the bacteria in the right state of development,
the best results being obtained from growths eight to sixteen
hours old upon an agar medium.

Various staining methods have been devised, those of Loeffler
and Van Ermengem being as useful as any.

Loeffler's Method.

(i) Take an absolutely clean slide, cleaned as in Ex. i ;
wipe it with a clean handkerchief, and handle it with the
fingers as little as possible. Place upon it three small drops
of water.

(2) With a fine platinum loop remove a small portion of a
six to eighteen hours old agar culture of Bs. subtilis, Bad.
{Proteus) vulgare, or other motile species, and place it in the
first drop of water : this will become slightly turbid. Now
pass the platinum loop through the flame, and, when cool
transfer a part of the first drop to the second on the slide ; in
the same way add a small part of the second to the third drop.
By diluting in this manner the bacteria become separate from
each other.

(3) Take one or two absolutely clean cover-glasses. Now dip
the end of a platinum needle, the top of which has been bent at
right angles, into the third drop, and with one stroke gently rub
the water which adheres to it on to the surface of the cover-
glass ; repeat the operation on another part of the cover-glass.
Dry the cover-glass in an incubator at 37° C. for three or four
minutes. The dilutions, spreading of the drops and smears, and

MOVEMENT 17

the drying of the latter should be carried out as rapidly as
possible.

(4) Place the cover-glass in Cornet forceps, and cover the
surface of the film with a few drops of the following mordant,
which must be filtered : —

A 20 per cent, solution of tannin . . . 10 cc.

Saturated solution of ferrous sulphate . . 6 ,,

Saturated alcoholic solution of fuchsin . . i „
Warm the cover-glass, and allow the mordant to act for four
to six minutes; then wash with water from the tap, and before
the glass dries cover the surface with the following stain : —

Fuchsin saturated alcoholic solution . . 6 cc.

Aniline water (see Ex. 9) . . . . 20 „
Warm, and allow the stain to act for three minutes.

(5) Now wash the cover-glass with water; dry it in an
incubator at 37° C. ; mount it in a drop of xylol, and examine
with a Y2 i"- objective.

Van Ermengem's Method.

(i) Prepare the cover-glass films as in (i), (2), and (3)
above.

(2) Place the cover-glass films in the following solution, which
must be three or four days old : —

Osmic acid 2 per cent, solution . . . 10 cc.
20 per cent, tannin solution . . . ^ 20 „
Glacial acetic acid ..... i drop

Allow the solution to act for one hour at room temperature, or

five minutes at 50° to 60° C.

(3) W^ash first in distilled water, then in absolute alcohol, and
finally in distilled water again.

(4) Dip the cover-glass for thirty seconds in a .5 per cent,
solution of silver nitrate ; move it about gently in the liquid.

(5) Drain off the superfluous liquid, and then dip the cover-
glass for thirty seconds in the following solution : —

B

1 8 BACTERIA I FORMS AND REPRODUCTION

Gallic acid 5 gr.

Tannin 3 „

Fused acetate of soda 10 „

Distilled water 350 cc.

(6) Drain and dip it again in the silver nitrate solution (4),
moving it about until the film becomes blackish.

(7) Now wash it thoroughly in distilled water; dry it; when
quite dry mount it in xylol balsam, and examine with a high
power.

2, O CD 00

CO 00 qo5)

Fig. 9. — Vegetative reproduction of bacteria.

1. Bacillus with successive divisions.

2. Coccus giving rise to chain 3 {Streptococcus^, pairs 4 {Diplococci),

irregular group 5 {Staphylococci).

6. Coccus dividing in two directions {Micrococci).

7. Coccus dividing in three directions {Sarcina:).

4. Reproduction, (i) Vegetative Reproduction. —

When bacterial cells reach their adult normal form if the
conditions of temperature and nutrition are satisfactory,
a partition or septum is developed across the cell which
divides it into halves. The two halves, or daughter-cells,
then grow to an adult size and undergo similar division
(Fig. 9). The process is repeated, and vast numbers

REPRODUCTION 19

may arise in this way. It is this peculiarity which
has led to the name Schizomycetes, which means
splitting or fission fungi, being applied to this group of
organisms.

The dividing partition in the case of elongated forms
of bacteria is placed across the shortest diameter. Very
frequently several generations produced by such division
remain attached to each other instead of separating into
individual, isolated bacteria. Many round or coccus forms
divide regularly in one direction and give rise to longer
or shorter chains included under
the term streptococci. Cocci occur-
ring in pairs are termed diplococci.
Sometimes the divisions are irregular
although in one plane, and groups
are produced which are often spoken
of as staphylococci. In some cocci
division of the cell into four
daughter cells occurs by two par-

..,. . • 1 . 1 , 1 Fig. 10. — Chains of bacilli.

titions at right angles to each

other ; such are termed pediococci. In other kinds
separation into eight cells takes place by division in
three directions of space, ancj as in this class the cells
of successive generations usually remain attached to each
other, cubical packets or bundles of cocci are produced ;
they are classed under the name sarcincB (Fig. 9). As
previously stated, bacteria and bacilli which are longer
than broad in the majority of cases separate by division
plates which are placed across the cell transversely. If
the cells produced remain attached, long thread-like
filaments are formed (Fig. 10). Longitudinal division
also occurs among bacteria, but only very exceptionally.
The rate at which vegetative reproduction takes place

20 BACTERIA : FORMS AND REPRODUCTION

is dependent upon temperature, air supply, nutrition,
and other circumstances, but under favourable conditions
division may occur once in every hour or less, so that
from one individual many millions of descendants can
be produced in a day when placed in milk or other
suitable nutritive liquids.

(ii) Reproduction by Spores. — Under certain con-
ditions, and especially those which lead to unfavourable
or unhealthy growth, such as unsuitable temperature,
dryness, excess or deficiency of air supply, exhaustion
of nutrient materials and accumulation of waste pro-
ducts, the vegetative division of bacteria is retarded and
stopped. The protoplasm within the cell contracts and
forms an oval body known as a spore or endospore (Fig.
1 1 ), which, on account of its high refraction, appears as a
bright glistening spot surrounded by an apparently empty
shell of the mother-cell. The latter sooner or later
swells up and dissolves, leaving the free spore behind.
The protoplasm of the spore is denser than that of the
original mother-cell, and the coat of the spore is a some-
what thick membrane which is not readily permeable to
dyes or other solutions. The number of spores which
originate in each bacteriym is generally one, though
in one or two kinds two or more spores are seen in
each cell. The spore occupies various positions in the
cell arising in some species at one end, while at others
it is formed near the middle of the bacterium. In the
genus, Clostridium, the mother-cell assumes a peculiar
spindle-shaped form, swollen in the middle and narrow
at the ends when spore formation takes place : it may also
be shaped like a drumstick as in the bacilli which cause
tetanus (Fig. 1 1). In some species of bacteria endospores
are not found, but their vegetative cells cease to divide, be-

REPRODUCTION 21

come surrounded by a thick wall and assume the structure
and character of spores ; these are designated artliro-
spores. The spores of bacteria are capable of resisting ad-
verse conditions which would instantly destroy the vitality
of the organisms in an active vegetative state. Some of
them can withstand boiling in water for an hour, and
are not damaged by chemical solutions which would kill
off all growing bacterial cells. They are also able to
retain their power of germination for many years in a

Fig. II. — I. Spore formation in a bacillus.

2. Chain of bacilli with spore in each cell ; isolated spores below.
3 and 4. — Forms oi Clostyidiufn cells with spores.
5. Drumstick-shaped cells with spores.

dry state. When placed under favourable conditions of
temperature, and provided with a suitable moist nutrient
material and a proper air supply, the spores germinate.
They absorb water, enlarge and become less refractive.
In the anthrax bacillus the whole spore becomes gradually
transformed into a new elongated bacterium or bacillus,
the thin spore membrane is absorbed in the surrounding
liquid, and the protoplasm gradually assumes the form
of an actively-growing vegetative rod. In Bs. mega-
therimn^ after the spore has absorbed a certain amount
of water its cell wall opens at one end and the proto-
plasmic contents escape and grow into a typical rod-

22 BACTERIA : FORMS AND REPRODUCTION

shaped bacillus. This is spoken of as /^/<^r germination.
In Bs. mycoides the protoplasm of the spore grows out
through an opening which arises in the side of the spore
{equatorial germination), the time taken for complete
germination being in many cases about three or four
hours at a temperature of 30" C, (Fig. 12).

Fig. 12. — I. Equatorial germination of spore.
2. Polar germination of spore.

Ex. 12. — Staining of Spores. — Prepare cover-glass films of
"scum" from an old hay decoction (par. i, Chap. I.) and from
other old cultures likely to contain bacteria with spores. Fix
by passing through flame three times. Wash for two minutes in
chloroform to get rid of any drops of fat and other materials.
Wash with water and dry with filter paper. Pass through Bunsen
flame again.

Place now in a 5 per cent, solution of chromic acid for twenty
to thirty seconds (in some cases ten seconds is enough, in others
better results are obtained if left for two to three minutes or
longer ; further trials must be made if twenty to thirty seconds
give poor results). Wash in water. Dry and fix again.

Place now in carbol-fuchsin (Ex. 8) and heat for three minutes.
Wash with water and dip into 5 per cent, sulphuric acid for five
seconds to get rid of the fuchsin from all parts of the bacteria
except the spores. Wash again in water and then stain for a
minute with Loeffler's methylene blue (Ex. 7). Each spore
will be stained red and the rest of the bacillus blue.

Ex. 13. — Apply films to cover-glasses, leave to dry in air with-
out heat. When dry, place in hot J per cent, hydrochloric acid
for three to four minutes. Wash with water, dry and fix in
flame. Then stain with carbol-fuchsin ; wash in acid and counter
stain with methylene blue as in above Exercise.

CLASSIFICATION OF BACTERIA 23

Ex. 14. — Observe the germination of spores on an agar
"hanging block."

Take 10 c.c. of melted agar medium and pour it into a sterile
Petri dish about 4 inches in diameter. After cooling cut out of
it some square pieces of the solidified agar about 8 to 10 mm.
across with a sterile knife. Lift up a piece or block and place
it on a sterile glass slide ; cover it with a bell jar to keep off dust
until wanted.

Take a small amount of a culture containing spores from agar
or other medium and mix it with i c.c. of sterile broth in a sterile
test tube ; heat the mixture for ten minutes in a water bath at
75° C. to kill all growing organisms ; the spores remain uninjured.
Spread a small loopful on the surface of the agar block and
cover with a thin sterile cover-slip. Now run a needle under
the lower surface of the agar and lift the cover-slip with the
block attached to it; place it over a culture cell (made by
cementing a rubber, metal, or cardboard ring to an ordinary
slide with Canada balsam or other cement) so that the agar
block hangs down into the cell.

Before putting on the cover-slip a small amount of vaseline
may be smeared round the edge of the cell to prevent evapora-
tion of the agar medium.

Place the whole now in a large Petri dish in an incubator
at 25° to 30*. Make observations of the germination of the
spores and subsequent growth of the bacteria through the cover-
slip at intervals of three to six hours.

Ex. 15. — Try and observe the germination of the spores of
bacteria introduced into sterile broth as above, a drop of the
mixture being arranged as a hanging drop as figured and
described in Ex. 35.

5. Classification of Bacteria. — The bacteria form a class
of the sub-kingdom Schizophyta or Fission-plants, thus: —
Sub- Kingdom. Schizophyta.
Class I. SchizophycecB or Blue-green Algae.
Class II. Schizomycetes or Bacteria.

24 BACTERIA: FORMS AND REPRODUCTION

The former class usually contains chlorophyll or an
allied colouring material, and its representatives are very
commonly distributed in water or on moist soil, wood,
rock, and stones, where they form patches, usually of a
green or bluish-green colour. Schizomycetes or bacteria
resemble the Schizophyceae in the simplicity of their
organization, but contain no chlorophyll, except possibly
in a few instances. Many species, however, have the
power of forming purple, pink, yellow, and other colour-
ing material, and are not uncommon on decaying meat,
bread, and other organic substances, where they form
red, purple, or yellow spots.

The grouping of bacteria into families, genera, and
species is a matter of great difficulty. Various methods
have been proposed, based on the forms of the cells, the
presence and absence of spores, the possession or want
of flagella, and the power of producing disease and set-
ting up chemical changes. Unfortunately, a coccus may
lengthen into a bacillus form, or a bacillus may develop
in the form of a coccus under certain altered conditions
of growth and nutrition, so that the shape of the cell
cannot be used as a perfect basis of classification ; nor
are the presence or absence of cilia and the possession of
spores very satisfactory for the purpose, since the same
organism may be unable to form spores or develop cilia
at one time, and yet may do so when other external con-
ditions prevail. Many of the chief generic names of
bacteria are based on the form which the organisms most
commonly assume. We thus have the genera, Micrococcus,
Streptococcus, Sarcina, Bacterium, Bacillus, and Spirillum^
characterized by the cell-forms described on pages 1 1,
1 2, and 1 9. There is a tendency among some authorities
to restrict the generic term Bacillus to rod-like bacteria

CLASSIFICATION OF BACTERIA 2$

which possess flagella distributed all over the cell-wall
and are motile, the genus Bacteiduni consisting of those
rod-like forms which are devoid of motile organs. The
presence or absence of cilia is found, however, to be a
variable character, even in the same species, an organism
at one time appearing to be without them, while, when
grown on another medium and at a different temperature,
it may become motile and possess cilia. The scheme,
if rigidly carried out, would necessitate much alteration
of existing names, and would be likely to lead to con-
fusion. It is perhaps best at present to include the
non-sporing, rod-shaped organisms in the genus Bac-
terimn, those with genuine endospores being placed in
the genus Bacillus. The genus Pseudomonas includes rod-
like forms with flagella attached only to the ends or
poles of the cell. Planococcus embraces motile micro-
cocci ; Planosarcina^ motile sarcinae.

The Naming of Species. — Botanists use two Latin
words when speaking of a plant, the first of which
denotes the genus and the second the species to which it
belongs : thus the botanical name of red clover is
Trifolium pratense ; of white clover, Trifolium repens,
both species being different, yet belonging to the same
genus. The same obtains when speaking of bacteria :
thus we have Bacillus subtilis for the hay bacillus.
Bacillus anthracis for the organism causing anthrax.
Unfortunately, this scheme of nomenclature has not been
strictly adhered to by many irresponsible workers at
bacteriology, but it is hoped that some day the chaos
existing in the naming of these organisms will disappear
and order take its place. Hundreds of species have
been described, and named by various writers in different
parts of the world, but much comparative work must be

26 bacteria: forms and reproduction

done before it is known what are the differences and
relationship of these so-called species and how many are
really distinct. There is little doubt that many of them
are the same, and a reduction of names must take place
in the future. Classification and registration, accord-
ing to some scheme such as that suggested on p. 60,
would facilitate rapid and easy reference and comparison
of all bacteria, and students of bacteriological science
should adopt a plan of this kind in describing new
forms.

6. In addition to the simple types of bacteria already
described there is a large family of organisms resembling
bacteria in the shape of their individual cells and mode
of reproduction, but the cells are enclosed in long,
thread-like, tubular sheaths which may be simple or
appear branched. Some of these, such as Cladothrix
dichotoma (p. 128) and species of Crenothrix^ are not
uncommon in impure water and in soil.

7. The following scheme of classification adopted by
Migula, although imperfect in some respects, is as satis-
factory as any : —

Order I. EUBACTERIA. — Cells colourless, or occa-
sionally containing chlorophyll.

Family I. COCCACE^. — Cells globular when free.
Division occurs in one, two, or three planes with
the formation of chains, plates, or cubical packets.
Spores rarely formed.

Genus i. Streptococcus. — Cell-division in one plane,
the cells usually remaining attached in
the form of a string of pearls. (Fig. 9.)
, 2. Micrococcus. — Cell-division in two planes.

CLASSIFICATION OF BACTERIA 27

Genus 3. Sarcina. — Cell-division in three planes ;
cubical packets produced. (Fig. 9.)
„ 4. Planococcus. — Motile micrococci.
„ 5. Planosarcina. — Motile sarcinae.

Family II. Bacteriace^e. — Cells more or less
elongated, cylindrical, and straight, without an
enclosing sheath. Division at right angles to
long axis of cell.
Genus i. Bacterium. — Longer or shorter rods, non-
motile. Without endospores.
„ 2. Bacillus. — Longer or shorter rods, motile.
Flagella distributed all over the cell :
forms endospores. (Fig. 8.)
„ 3. Pseudonionas. — Longer or shorter rods,
motile. Flagella polar, i.e. arranged
at ends of the cell. (Fig. 8.)

Family III. SPIRILLACE^. — Cells elongated, curved,
or spiral, without enclosing sheath. Usually
motile.
Genus i. Spirosoma. — Cells rigid, not motile.

„ 2. Microspira. — Cells rigid, motile usually

with one polar flagellum.
„ 3. Spirillum. — Cells rigid, screw form (Fig.
5), motile with bunch of polar flagella.
(Fig. 8.)

Family IV. Chlamydobacteriace^:. — Cells rod-
shaped, in chains, surrounded by a common
gelatinous sheath. (Fig. 28.)

Several ill-defined genera distinguished, e.g. Creno-
thrix, Sphcerotilus {Cladothrix), and others.

Order II. Thiobacteria. — In this group the cells

28 BACTERIA I FORMS AND REPRODUCTION

are colourless, or rose, red, or violet — never green.
The cells contain granules of sulphur.

Family I. BEGGIATOACEi€i. — Colourless filamentous
forms.

Genus i. Thiothrix.
„ 2. Beggiatoa,

Family II. Rhodobacteriacecs. — Cells spherical, rod-
shaped, or spiral, containing rose, red, or violet
pigment.

CHAPTER III.

GENERAL CONDITIONS WHICH INFLUENCE
THE GROWTH OF BACTERIA.

The growth and multiplication of bacteria are checked
or increased by various external conditions, the chief
of which are the presence of light, heat, and oxygen,
and an adequate supply of water and food-materials ;
certain chemical substances mentioned later also have a
marked influence on the vital activity of these organisms.
I . The Action of Light. — Although a small group of
bacteria appear to need exposure to light in order to
carry on their physiological functions, the majority of
them only thrive in very feeble light, or in darkness, and
their growth and development are retarded by exposure
to diffuse daylight. The action of direct sunlight, even
for a short time, results in the weakening of their vital
powers, and if continued destroys them altogether. The
blue, violet, and ultra-violet rays of light are most
destructive to bacteria. The deleterious effect of light
appears to be, partially dependent upon the presence of
atmospheric oxygen, since under anaerobic conditions
many bacteria resist sunlight for some time. In certain
cases the formation of hydrogen peroxide takes place in
the medium in which organisms are growing, and in
such instances the death of the bacteria may be attributed
to the oxidizing action of this substance.

30 GROWTH OF BACTERIA

2. Heat. — Like other living organisms, bacteria can
only grow and carry on their functions between certain
temperatures. The minimum or lowest temperature is
that below which growth and multiplication ceases, the
maximum or highest being the point on the thermometric
scale above which growth is checked. Between these
extremes is the optimum or best temperature, at which
growth and division is most active; it is usually .some-
what nearer the maximum than the minimum point.
The minimum for many common species of bacteria lies
between i to io° C, the optimum 25 to 35° C, and the
maximum between 42 to 50° C. Certain kinds fre-
quently met with in soil, well water, and in the .sea,
tolerate low temperatures and grow freely at a few degrees
above freezing-point. On the other hand, the species
Bacillus tkennophilus, sometimes present in sewage and in
the intestinal tract of animals, can develop and multiply
at 70° C, a temperature sufficient to kill all animal cells.
Many kinds which grow well at 60° C. are also met with
in garden soil, manure heaps, and milk, and on the
surface of potatoes, straw, hay, and various cereal grains.
Although the vital activities of bacteria are reduced or
checked altogether by cold, these organisms are not
killed even when subjected to temperatures much below
the freezing-point of water. Certain forms have, indeed,
been immersed in liquid air for some time without show-
ing any loss of vitality. Temperatures of 5 or i o degrees
above the optimum, if continued for a time, act dele-
teriously on bacteria in a vegetative state of growth ;
their physiological powers are diminished, and at a few
degrees above their maximum death takes place in
actively growing cells of most species when they are kept
moist. The spores of bacteria, however, resist high

OXYGEN 31

temperatures for a considerable time without losing
their germinating power, those of the hay bacillus {Bs.
subtilis) being uninjured by boiling in water for half an
hour or longer. The spores of certain forms isolated
from soil have been found to be capable of development
after exposure for an hour to a dry heat of 130° C, but
a few minutes at 150° C. is sufficient to destroy all kinds
of spores.

3. Water. — Water is essential for the manifestation
and continuance of the phenomena of life in all forms of
living beings ; without it active life is suspended, and
prolonged dryness results in death. The power of resist-
ing drought varies with the kind of organism and with
its stage of development ; many kinds of bacteria in a
vegetative state become weakened or lose their vitality
altogether when dried for a {^v^ days, while their spores
will often withstand drying for many months, or in some
cases even for several years.

4. Oxygen. — Bacteria differ very much from each
other in regard to the need of free oxygen for their
growth and maintenance of their vital functions. Some
of them are dependent upon a supply of free oxygen in
the medium in which they are grown, and are spoken of
as aerobic species ; examples of this class are Bs. subtilis
and Bad. fluorescens. Forming another group are those
species which thrive only when free oxygen is excluded,
their vital powers being suspended or destroyed by
exposure to air for a few hours ; these are termed
anaeivbic bacteria, good examples of which are the
organisms responsible for butyric acid fermentations.
Between these obligate aerobes, which soon die unless
well supplied with air, and the obligate anaerobes, which
are checked by the presence of less than .5 per cent, of

32 GROWTH OF BACTERIA

the oxygen, there are large numbers of species which are
indifferent to the admission or exclusion of air, thriving
more or less satisfactorily under both conditions. Those
kinds, such as certain forms of lactic acid bacteria, and
many of the organisms causing putrefaction, which are
usually aerobic, but which can still grow and multiply
when the supply of atmospheric oxygen is diminished,
are described as facultative anaeivbic bacteria ; they can
live as anaerobes when necessity arises. The species
which generally live as anaerobes, but are yet capable of
existing under conditions which admit of the access of
fresh air, are \.QY\n&d facultative aerobic bacteria.

5. Food. — Food is essential to the life of all forms
of living things. Out of it they are able to construct
the material used in building up their bodies and in
repairing the wear and tear which goes on within them.
Moreover, the energy needed to maintain their vital
activity is derived chiefly from chemical changes induced
in the food which they consume. The substances
utilized as food by bacteria are always taken from
watery solutions, the dissolved materials diffusing into
the interior of the organism through the surrounding
cell-walls, although there are no visible openings in' the
latter. Such passage or diffusion of dissolved sub-
stances through membranes which have no apparent
openings in them is termed osmosis. While a few species
of bacteria are known which are able to develop when
supplied with very simple chemical compounds, such
as carbon dioxide, nitrogen, water, and inorganic salts,
the majority of them are unable to grow and multiply
unless they are provided with more complex organic
compounds, such as sugar, proteins, and similar sub-
stances. In respect of the source from which the

FOOD 33

organic compounds are derived, two classes of bacteria
may be recognized, viz., saprophytes, which, obtain them
from the dead and decaying bodies of plants and
animals, or the lifeless organic matter in soil, milk, water,
and other common materials ; the parasites , which feed
upon materials present in the tissues of living organisms,
their so-called hosts, and produce abnormal physiological
disturbances or diseases in the latter. Bacteria which
produce disease are spoken of as pathogenic organisms ;
examples are the bacilli which cause tuberculosis, anthrax,
diphtheria, and enteric fever in man and the lower
animals.

The elements composing the food of bacteria are
practically the same as those utilised by plants, viz.,
carbon, hydrogen, oxygen, sulphur, phosphorus, and
certain metallic elements, the chief of which are sodium,
calcium, magnesium, and iron. The first five enter into
the composition of the proteins, which form much of
the body substance of bacteria. The metallic elements
appear to be necessary for the growth and physiological
functions of these organisms, but how they are utilized
is not clearly known. It is said that pure cultures of
certain species of bacteria have been grown without
calcium, sodium, or potassium, but the experiments on
which these statements are made are not altogether
conclusive ; exceedingly minute traces are difficult to
eliminate from culture media, and these small amounts
are often quite sufficient for the needs of bacteria.

The food elements are obtained from many sources.
Some of them, e.g. oxygen and nitrogen, may be absorbed
in a free state ; usually, however, the latter can only
be used when presented in a combined form. Potassium,
calcium, and magnesium are taken from sulphates,
C

34 GROWTH OF BACTERIA

phosphates, nitrates, and other inorganic salts of these
metals. The sulphates and phosphates provide sulphur
and phosphorus respectively, and both elements are also
derived in part from proteins and other organic compounds.

Carbon can be obtained from a number of substances.
The nitrifying organisms are able to procure what they
require from the carbon dioxide of the atmosphere, and
it is probable that certain sulphur bacteria use this gas
as a source of carbon also. Many organic carbon com-
pounds, such as lactic, acetic, tartaric, citric, and other
acids or their salts, as well as sugars, alcohols, and their
allies, provide carbon ; additional sources are proteoses,
peptones, and other proteins.

The element nitrogen is obtained by Clostridium
pasteurianum and species of Azotobacter from the free
nitrogen of the air, and the nodule organisms associated
with the roots of leguminous plants can also make use
of free uncombined nitrogen. Bs. subtilis, Bad. fluorescens,
and many other kinds can obtain it from nitrates,
especially if the carbon which they need is supplied in
the form of sugar. Some species use ammonium com-
pounds, while the majority procure their necessary
nitrogen from more complex nitrogenous organic com-
pounds such as the amino-acids and proteins.

For the artificial nutrition of bacteria various liquid
media are employed, such as milk, whey, beef-broth,
blood-serum, and decoctions prepared from various
animal and vegetable substances. Solid jelly-like media
are also extensively used ; these consist of the liquids
just mentioned, or solutions of peptone, sugar, and other
nutrient materials, to which is added a certain amount
of gelatine or agar-agar. The preparation and use of
culture media are described in Chapter IV,

STERILIZATION 35

6. Sterilization. — Many of the most serious diseases
of man and domestic animals are caused and spread by
bacteria ; these minute organisms are also responsible
for the souring of milk and the putrefaction of meat,
eggs, and other food materials. It is therefore im-
portant to study the means by which their activities can
be checked or their vitality destroyed altogether.

Milk, beer, flesh, and all substances from which all
living germs have been removed or destroyed, are said
to be sterile^ and will keep indefinitely if, after steriliza-
tion, further access of bacteria is prevented.

Various methods of sterilization are in daily use, the
chief of which are based upon the destructive action of
heat, or upon the poisonous nature of certain chemical
compounds.

By maintaining a low temperature, food-stuffs may be
kept for an indefinite time without change. In a frozen
state meat, milk, and fruit of all kinds are sent long
distances without damage, and it is well known that
fruit, jam, milk, butter, bread, and meat keep best in
cool, dry situations. In such cases it must be borne in
mind that the low temperature merely checks the
development of the bacteria present, and does not
destroy them ; a sterile condition cannot be produced
in this way, and when the temperature is raised the
organisms present begin their growth and work of
destruction with unabated vigour.

The keeping qualities of foods of all kinds are greatly
increased by roasting, baking, boiling, and steaming, for
most bacteria are killed by these processes.

The most certain and complete sterility is obtained by
the application of heat. A temperature of 150^ C, main-
tained for an hour, is sufficient to destroy all forms of

36

GROWTH OF BACTERIA

hours.

bacteria, their spores included. Even the boiling of
liquids, such as water, milk, and broth at iOO° C, kills
all living things in them if continued long enough, but
certain spores resist this temperature for several
Usually it is only needful to heat most liquids
to 1 00° C. for fifteen or twenty
minutes to destroy all bacteria
in active growth, and a great
many of their spores also.

In the case of many liquids
it is not possible to heat them
in an open vessel above 1 00° C,
for when this temperature is
reached steam forms, and
evaporation occurs without a
further rise in temperature.

If, however, steam is pre-
vented from escaping, the liquid
may be heated above 100° C.
L^ An apparatus for effecting this
is the autoclave (Fig. 13). It
consists of a strong gun-metal
or brass boiler fitted with a lid
which can be screwed down.
In the latter a safety-valve is
provided, as well as a stopcock,
by means of which the steam can be allowed to escape
when necessary. Attached to the apparatus is a gauge
to indicate the pressure of the enclosed steam.

In using the autoclave for the sterilisation of water, milk,
or other media used in the cultivation of bacteria, the
following instructions may be adopted : Introduce a
small quantity of water into the autoclave, and after the

Fig. 13.— Autoclave.

STERILIZATION

37

vessels containing the media to be sterilized have been
placed within it, screw down the lid. The burners for
heating the apparatus should now be lighted. Leave
the stopcock open until steam begins to escape freely,
then close it, and allow the pressure to rise until the
gauge indicates i 5 lb., or a temperature of 1 1 5° to i 2 5°C.
is reached. Lower the gas-
burners, and allow the tempera-
ture to remain at the figure just
mentioned for half an hour ;
then turn off the gas, and cool
the autoclave to below 100° C.
before opening the stopcock.

Where the use of the auto-
clave is prohibited on account of
expense or for other reasons,
liquids may be rendered sterile
by the process of intermittent
sterilization at a temperature of
100° C. or less. This is effected
by heating to 1 00° C. for twenty
minutes on three or four succes-
sive days. The first heating
kills all bacteria which are in f^g. i4.-steain sterilizer,
an active growing state, only spores present retaining
their vitality. The latter are supposed to germinate
in the cool intervals, the organisms arising from them being
destroyed by the subsequent heating which they receive ;
possibly the alternate heating and cooling on successive
days may destroy ungerminated spores.

For intermittent sterilization of media ^ steam sterilizer is
used (Fig. 1 4). It consists of an iron or copper cylindrical
vessel, covered on the outside with felt, asbestos or other

38

GROWTH OF BACTERIA

non-conducting material. On the inside, some little way
above the bottom, is a perforated partition upon which
the glass flasks, tubes, and other vessels containing the
media to be sterilized can rest.

When used water is poured into the sterilizer, and the
latter then placed over a Bunsen burner; steam soon
forms, and fills' the space surrounding the tubes of cul-
ture media.

An inexpensive sub-
stitute for this appar-
atus is an ordinary
large saucepan fitted
with a potato steamer.
For the sterilization
of glass tubes, flasks,
and other laboratory
apparatus, which are
uninjured by high tem-
peratures, a hot-air
sterilizer (Fig. 15) is
generally used.

This is a cubical

Fig. i5.-Hot-air sterilizer. , shcet-iron boX, with a

door in front, the whole having double walls, with an air
space of about an inch between them.

A temperature of 150'' C. or higher is easily main-
tained in the interior by means of a Bunsen burner
placed beneath it ; all tubes, flasks, Petri dishes, and
similar apparatus should be subjected to this or a higher
temperature for not less than half an hour before being
used for bacteriological work.

7. Pasteurization, or heating for about twenty minutes
at 60° to 85° C, followed by rapid cooling to about 10°

DISINFECTANTS 39

C, is a process frequently adopted for the preservation
of foods for a limited time. It is largely employed in
the dairy industry, and is effective in destroying the
sporeless forms of bacteria without materially altering
the flavour or composition of the liquids. For its
application to milk and cream, see pp. 295-6.

8. Disinfectants, Antiseptics, and Preservatives. —
The vitality and work of bacteria can be checked or
destroyed by various chemical substances. Surgical
instruments and premises infected with disease germs
are generally sterilized by the application of these
compounds. Chemical substances which completely
annihilate bacteria are termed disinfectants^ while
those which merely retard the growth of these
organisms are known as antiseptics. No hard line of
separation, however, can be drawn between these two
classes, for the effect of any chemical compound depends
upon the strength of its solution and the time that it is
allowed to act ; weak solutions of disinfectants may have
an antiseptic action only. Moreover, what would be
sufficient to destroy or impede the growth of one species
of bacterium will not be equally effective upon another
kind.

The following are among the most widely used dis-
infecting and antiseptic agents : —

Mercuric chloride (HgClg), in a . i to . 2 per cent, solution,
is one of the most certain of disinfectants, but its
excessively poisonous quality precludes its general use
except in the laboratory under proper, control. It is
extensively used in surgical practice.

Sulphur dioxide gas (SO2), or solutions of it in
water (sulphurous acid), are effective destroyers of
bacteria and fungi. The vapour of burning sulphur,

4o GROWTH OF BACTERIA

which consists chiefly of sulphur-dioxide gas, has been
employed for a long time for the disinfection of
rooms in which fever patients have been nursed. The
sulphites of calcium and sodium are also valuable
disinfectants, and much used in breweries for the
cleansing of foul casks and other utensils.

The so-called ** chloride of lime " is frequently employed
as a disinfecting agent, its effectiveness in this respect
depending upon its power of giving off free chlorine
gas, which latter very rapidly destroys living bacteria
and their spores, especially when moisture is present.

A lo per cent, solution of carbolic acid or phenol
(CgHgOH) has disinfecting properties, but weaker
solutions do not effectively destroy the spores of bacteria.

Formaldehyde (CHgO) during the last few years has
been utilized for the destruction of micro-organisms. It
is very active, solutions containing .i to .2 per cent, of the
compound being sufficient to kill the spores of most
bacteria in less than an hour.

In weak solutions all the above-mentioned sub-
stances are good antiseptic agents. To this list of
chemicals may be added thymol^ toluene, chloroform,
salicylic acid, boric acid, borax, lime-wash, and alcohol,
all of which may be employed for retarding putrefaction
and other changes induced by bacteria.

One-half to one per cent, of toluene (CgHgCHg) is
frequently utilized to check the development of bacteria
in various solutions whose enzyme action it is desirable
to study. Chloroform (CHCI3), thymol, and salicylic
acid (CgH^OH.COgH) may be used for a similar
purpose.

Freshly -burnt quicklime (CaO), when slaked and made
into a " whitewash," is a cheap and valuable disinfectant

DISINFECTANTS 41

for application to cattle trucks, the walls and stalls
of cowsheds, stables, dairy premises, and other farm
buildings, which are liable to become infected with
germs of disease, or with bacteria which pollute milk
and do damage to dairy products generally.

Soda or sodium carbonate (Na2C03) is also useful
for the cleansing of domestic utensils of all kinds, its
alkaline character having a similar action upon micro-
organisms to that of lime.

For the preservation of foods of all kinds from decay
and putrefactive changes, the use of poisonous substances,
such as mercuric chloride, carbolic acid, and formaldehyde
is, of course, prohibited. Much, however, can be done
by drying, smoking, heating, and the addition to the
food of harmless materials, such as sugar, salt, and
saltpetre.

Fruits of many kinds, which often contain 80 to 90
per cent, of water, may be preserved by drying them,
the low water-content of the dried product being in-
sufficient to allow of the free growth of bacteria, especially
when sugar is present, the latter acting in some degree
as an antiseptic agent.

In the case of jam, the addition of sugar is combined
with boiling of the material, and a similar practice is
adopted in the preparation of condensed milk, 10 or 12
per cent of sugar being added to the milk, the water-
content of which has been reduced by evaporation to 2 5
per cent, or less.

In the " canning " or " tinning " industry, which deals
with vast quantities of fish, vegetables, and other perish-
able foods, reliance is placed upon the destruction of
bacteria contained in them by means of heat. The
sterilization of the tins and their contents was formerly

42 GROWTH OF BACTERIA

carried out in open tanks at a temperature of ioo° C,
the small opening in the centre of the lid being closed
with molten solder after the heating had been sufficiently
prolonged to destroy all living germs and their spores.

In order to ensure more certain sterility and reduce
the time necessary for the process, the tins are now
generally heated in large closed autoclaves under pres-
sure to 1 1 5 ° C. or more.

Common salt (NaCl) is one of the most useful sub-
stances for the preservation of food. The addition of a
small amount assists the keeping quality of butter and
cheese ; larger quantities are employed in the " curing "
of bacon, and a strong brine is capable of preventing the
bacterial decomposition of beef, pork, fish, and other
kinds of meat.

Boric acid (H3BO3) and boj'ax (Na2B407 + loHgO)
have also an inhibiting effect on the development of
bacteria, and have been extensively employed for the
temporary preservation of butter, cream, and other dairy
products. It is, however, doubtful if these compounds
are altogether harmless ; if used in sufficient amount to
check bacteria, they are liable to produce derangements
of the digestive system when the " preserved " food is
consumed by human beings.

CHAPTER IV.

THE METHODS OF ISOLATION AND PURE
CULTURE OF BACTERIA.

Method of Isolation. — In nature various species of
bacteria are met with mixed together, much as we find
different kinds of plants distributed in a natural pasture
or wood. But just as it is possible to separate from a
meadow any single flowering plant and grow it so as to get
a large collection of plants all of the same species, so it
is possible to isolate and cultivate a single species of
bacterium. A collection of bacteria consisting of indi-
viduals all alike, i.e, belonging to the same species, and
free from admixture with any other kinds is termed a
pure culture. It is only by the study of bacteria in pure
cultures that their form, mode of growth, physiological
activity, and other features can be determined.

The general principles underlying the separation of

bacteria and their pure culture may now be described,

but in order to obtain a thorough acquaintance with the

work the student must carefully work through the

^practical exercises appended.

The bacteria to be isolated will perhaps most
frequently be distributed in solutions of some sort such
as milk or water : where they are present in a solid
material, say cheese, butter, or soil, a small portion of
the material must be broken up and shaken with pure
sterilized water.

43

44 PURE CULTURE. OF BACTERIA

In a cubic centimetre or even a drop of the liquid
there will usually be hundreds or thousands of bacteria,
numbers far too large to be effectually dealt with. A
small portion of the milk or other liquid is, therefore,
transferred to a large bulk of water, which must, of
course, be first rendered free from living bacteria by boiling
for some time, and the whole shaken to distribute the
organisms evenly. (If the amount taken is a cubic centi-
metre containing, say, lOOO bacteria, and this is placed
in 99 cubic centimetres of water, the diluted mixture

will now contain on an average
ten of these organisms in every
cubic centimetre, a number
more easily dealt with.) A cubic
centimetre or smaller quantity
. T, . ,. . is now taken from such a mix-

1? iG. i6. — Petri dish.

ture and introduced into a test-
tube containing 8 to lo cubic centimetres of sterilized
liquid 7tutrient medium or solution of substances upon
which bacteria can feed and multiply, to which has been
added a small percentage of gelatine, agar-agar, or other
material possessing the property of solidifying to a jelly
at ordinary temperatures. The nutrient gelatine medium
is kept sufficiently warm to render it liquid, and the
whole is carefully shaken to separate and distribute the
bacteria which have been added to it. The contents of the
test-tube are then poured into a glass Petri dish (Fig. 1 6) or
on to the surface of a glass plate : the medium is allowed
to cool and solidify, protected from dust. In the thin
solid layer of nutrient jelly thus obtained the bacteria
are imprisoned, and although they can grow and flourish
they cannot move far or become mixed with each other
as would have been the case if the nutrient medium had

METHOD OF ISOLATION 45

remained permanently liquid. The dish or plate is
placed in an incubator or in a warm room, and after
a few hours each individual organism grows and
multiplies in the nutrient gelatine, its descendants form-
ing a small aggregation of similar bacteria termed a
colony (Fig. 1 7). The colonies produced by the various
kinds of bacteria vary considerably in form and colour

• ♦#

Fig. 17. — Common forms of bacterial colonies, i, Round; 2, lobed ;
3, amoeboid; 4, mycelioid.

and in other ways. The differences must be carefully
studied, as many species are characterized by the kind of
colony which they produce on the various media described
subsequently.

Ex. 16. — In the practical isolation of bacteria the first step
is to clean and sterilize the test-tubes, beakers, and flasks to be
used.^

(i) Test-Tubes. — New test-tubes (6 in. by fin.) should be
cleaned by rubbing the inside with a cotton-wool plug which has
been tied with strong thread to the end of a glass rod and
dipped in strong nitric acid. After cleaning with acid wash
thoroughly with water and allow them to drain until nearly dry,
then wash out with strong or absolute alcohol, and when dry
put into the mouth of each a cotton-wool plug ; place a number

^ Slides, test-tubes, beakers, stains, and chemicals, as well as incubators
and other bacteriological apparatus needed for the practical exercises, can be
obtained from Messrs Baird & Tatlock, Cross Street, Hatton Garden,
London, E.G.

46 PURE CULTURE OF BACTERIA

of the dried plugged tubes in a wire cage and heat in the hot-air
sterilizer to 150° C. for one hour (Fig. 15).

(2) Test-tubes which have been used and contain nutrient
gelatine or other media must be dipped in a saucepan of hot
water to liquefy the gelatine. Pour out the contents and then
place the tube in a saucepan containing a weak solution of
caustic potash, tilting them so that they may be completely
filled with the solution without imprisoning air bubbles. Boil
for an hour. Take out the tubes and wash them in water several
times, clean with nitric acid and alcohol and sterilize as explained
above, plugging with cotton-wool as with unused new tubes.

(3) Flasks, beakers, Petri dishes, pipettes, and other glass
apparatus should be cleaned and sterilized in the hot-air oven in
similar manner. All smaller apparatus and pairs of Petri dishes
should be wrapped separately in paper before sterilization.
They should be stored in a cupboard or drawer away from dust.

Ex. 17. — The next step is the manufacture of a sterilized
nutrient medium with addition of gelatine or agar-agar. This
requires great care if reliable and consistent results are to be
obtained. Bacteria are very sensitive to acids and alkalis ; unless
the media are suitable in this respect the organisms will not
grow. It is found that most bacteria grow satisfactorily when
the medium contains i per cent, of free normal acid, as ex-
plained below, though some commonly met with in the soil
require a neutral medium or one which is slightly alkaline. The
process of the manufacture of the nutrient gelatine may be con-
veniently divided into four stages.

1. Prepare the nutrient medium (bouillon or broth).

2. Add gelatine.

3. Neutralize carefully and completely, using a solution of
phenolphthalein in alcohol as indicator.

4. Add sufficient aqid to bring the whole to the proper state
of acidity.

Preparation of Standard Nutrient Gelatine.
I . The broth or nutrient medium.

METHOD OF ISOLATION 47

The broth is made thus : — Weigh out 5 grams of Lemcds
extract of meat and 10 grams of fresh Witte's peptone. Dissolve
these ingredients in a large flask containing 1000 c.c. of distilled
water at ordinary room temperature.

2. Addition of Gelatine.

Add to the flask of broth just prepared loo grams of best
sheet gelatine, and allow it to soak for half an hour. Place the
flask in hot water in a saucepan and heat slowly until the gelatine
and other materials are dissolved, shaking the flask from time to
time to mix the contents.

3. Neutralization process*

For neutralization the following solutions are required : —
(i) Normal hydrochloric acid.

(2) i-2oth normal acid.

(3) Normal caustic soda.

(4) I -20th normal caustic soda.

(5) 2 gram of phenolphthalein in 100 c.c. of 50 per cent.

alcohol.
Take from the flask 5 c.c. of the nutrient gelatine as prepared
above (i and 2), add it to 45 c.c. of distilled water in a porcelain
dish. Boil three minutes to expel any COg and add to it i c.c.
of the phenolphthalein solution. Run into the hot solution from
a burette just sufficient of the i-2oth normal caustic soda solution
to produce a clear bright pink colour, noting how many cubic
centimetres of the soda solution are needed. Repeat again
and find the average number of cubic centimetres of soda
needed to neutralize 5 c.c. of the nutrient medium. Then
calculate how many cubic centimetres will be needed to
neutralize the whole of the medium left in the flask. Divide
by twenty, this will give the number of cubic centimetres of the
normal caustic soda solution which will be needed to neutralize
the whole of the medium. Add this amount of normal soda
to the medium in the flask and test if it is just neutral, as it
ought to be, by taking out a few cubic centimetres and
adding to it a drop or two of the phenolphthalein. If it is

48

PURE CULTURE OF BACTERIA

still acid repeat the above process until the medium is just
neutralized.

4. Standard acidity.

After neutralization add ic c.c. of normal hydrochloric acid
to every 1000 c.c. of medium, i.o per cent, free normal acid is

now present, which leaves the
medium just sufficiently acid for
the growth of most bacteria.

When working on bacteria com-
mon in soil add only 5 c.c. of
normal hydrochloric acid instead
of 10, as many of these refuse
to grow in the more acid
medium.

It is usual to indicate these
standards of acidity by placing
the + sign before the number of
cubic centimetres of normal acid
used, e.g. + 10 and + 5 in the
cases mentioned. Where the
medium is neutral o may be used,
and in cases of alkaline media
the - sign is employed before
the number of cubic centimetres
of normal caustic soda or sodium
carbonate added to the medium
after neutralization.

5. To clarify. ♦

Cool the medium to below 40° C- and add slowly the white
of an egg well beaten up with a little water ; heat in the steam
sterilizer or a water bath, and then filter the hot solution through
filter paper moistened with boiling water into a sterilized flask :
use a hot-water funnel (Fig. 18) when filtering the medium.

6. Sterilization.

Take twenty or thirty clean sterilized test-tubes and pour into

Fig. 18.

Hot-water funnel for filtration
of gelatine and agar media.

METHOD OF ISOLATION 49

each about 8 or 10 c.c. of the medium, taking great care that none
of it touches the sides of the tube near the mouth. Plug each
tube with cotton wool and heat them all in the steam sterilizer
on three successive days for twenty minutes at a time, taking
care to have the water boihng before the tubes are placed in
the steriUzer. (The medium will keep a long time if stored away
from dust, especially if the plugged mouths of the tubes are
covered with indiarubber caps.)

Ex. 18. — Pour 250 to 300 c.c. distilled water into a flask,
lightly plug the opening with cotton wool, and boil for twenty
minutes on three successive days.

When the water is cool put into it i c.c. of the liquid contain-
ing the bacteria which are to be isolated. Take J c.c. or less
of the dilute mixture with a steriHzed pipette and transfer it to a
nutrient gelatine tube, the contents of which have been liquefied
by placing the end of the tube in warm water ; mix thoroughly,
not by shaking but by turning the tube and tilting it so as to
avoid bubbles of air in the medium, and then pour it into a
sterilized Petri dish. Cool rapidly by placing the Petri dish on
a glass plate or on ice for a minute or two ; then leave it in
a warm room and examine every day for colonies of bacteria.
They will appear as spots of varied colour and form on the
surface or within the substance of the gelatine medium.

Ex. 19. — Instead of making a dilute solution, as explained
above, of the liquid in which the bacteria to be isolated are
found, the following plan may adopted. Melt three or four
tubes of nutrient gelatine in warm water at 30° to 35° C. ;
label them i, 2, 3, and 4 respectively. Take up a drop of
the liquid containing the bacteria with a sterile platinum loop
and transfer it to the tube labelled i. Mix carefully and
transfer a drop of the mixture to tube 2 ; from tube 2 transfer
a drop to tube 3, and from 3 to 4 ; mix carefully each time
and sterilize the platinum loop each time before use by heating in
the flame. Then pour the contents of each tube into four sterilized
Petri dishes and leave them in a warm room to incubate.

50 PURE CULTURE OF BACTERIA

It will be found that many commonly distributed
bacteria have the power of liquefying nutrient gelatine
when they begin to grow." When this occurs the
colonies soon mix and isolation becomes impossible.
Another disadvantage connected with a solid gelatine
medium is that, as ordinarily prepared, it melts at 22° to
24° C, and, therefore, cannot be used for growth of
organisms at higher temperatures. To avoid these
troubles a nutrient medium is used containing agar-
agar in place of gelatine.

Agar-agar, or agar as it is generally called, is not
a protein like gelatine but a carbohydrate substance,
which is derived from certain species of Japanese and
eastern sea-weeds. It is not liquefied by bacteria, and
has the advantage of remaining solid at temperatures up
to nearly 80° C.

The substance is sold usually in bars, sheets, or
powder, the latter being the most generally used. A
small amount (1.5 per cent.) is sufficient when dissolved
in water to form a solid moist " jelly " suitable for the
growth of many micro-organisms.

Agar is very difficult to dissolve, requiring a tempera-
ture of near the boiling point of water to melt it, but
when dissolved its watery solution has the remarkable
property of remaining liquid and not gelatinizing until
the temperature has fallen far below the melting point,
namely, to near 39° C.

On account of the sensitive nature of many kinds of
bacteria in regard to heat it is important to remember
that melted agar media should not be inoculated with
bacteria until the temperature has fallen to between
40' to 41° C. On the other hand, the temperature
should not be allowed to fall much lower than this, or

PURE CULTURES 5 1

solidification may set in and prevent the even distribu-
tion of the bacteria in the medium. These precautions
should be borne in mind when agar media are used,
especially where they are employed for quantitative
determination of the number of organisms in solutions.

Preparation of Nutrient Agar Medium.

Ex. 20. — Make a nutrient broth, using —

(i) Lemco's extract of meat, 5 grams.
Witte's peptone, 10 grams.
Water, 500 c.c.

(2) Take 15 grams of agar-agar and scak it for twelve hours
in 500 c.c. of water. Mix (i) and (2) and boil or heat in the
steam sterilizer twenty to thirty minutes until the agar and other
materials are dissolved. Neutralize and bring the hot solution
to proper acidity, as in Ex. 17 (3 and 4). Cool to 4o°-5o° C, and
clarify with white of an egg, as in Ex. 17 (5). Then heat in
steam sterilizer for i| hours, and filter when hot in a steamer or
through Chardin filter paper in a hot water funnel, first wetting
the filter paper with boiling water.. If not clear and free from
opaque spots when cool, reheat again, clarify, and pass through
a Chardin filter paper. Run 8 to 10 c.c. into test-tubes, and
sterilize as in Ex. 1 7 (6). Agar media filter very slowly. Where
an autoclave is available, place the neutralized solution, with the
white of an egg added, in the apparatus, and heat for half an
hour at two atmospheres pressure or for 45 minutes at 120° C.
Turn off the flame, and when cooled to below 100° C. open the
autoclave, and the agar medium will be found to filter clear
through a Chardin paper in fifteen to twenty minutes.

Ex. 21. — Repeat Ex. 18 and Ex. 19, using the above agar
medium instead of nutrient gelatine.

2. Pure Cultures. — After the bacteria have been
isolated, and the separate colonies allowed to grow on
the nutrient gelatine or agar medium, minute portions of
each distinctive colony are removed with a fine sterilized

52

PURE CULTURE OF BACTERIA

platinum needle and transferred to various liquid media;
e.g. to broth or milk, in which an abundant pure growth
of the particular organism can be obtained. The form
and other features of the bacterium can then be studied,
as well as its effect on the medium.

In addition to their development in liquids, it is usual
to make an examination of the growth of bacteria on the

Fig. iq. — Diagram of common forms of stab-cultures.

I, Filiform; 2, beaded ; 3, rhizoid or arborescent ; 4, beaded slab-culture

of anaerobic organism.

surface or deep within the substance of the solid gelatine
or agar media. For the latter purpose tubes of solid
media are inoculated by means of a platinum needle
dipped in a pure culture in broth, or in a colony isolated
on gelatine or agar, the needle being pushed deeply into
the substance of the solid medium and then withdrawn.
The colonies of the bacteria develop along the track of
the needle, forming a stab-culture (Fig. 19). The needle
may, on the other hand, be drawn lightly along the sur-
face of solid media in straight lines, and a streak-culture
obtained. The medium for the latter purpose may be

PURE CULTURES 53

melted in its tube and poured out into a sterile Petri
dish, where it solidifies into a broad level surface.

Gelatine or agar slants may be used for streak cultures
instead. These are obtained by melting the medium in
its tube, then placing the tube in a sloping direction, and
allowing it to remain in this position until the medium
cools and solidifies again. In this way a sloped surface
is obtained in the tube, on which streak-cultures can be
made (Fig. 20).

Although the characters of bacteria as exhibited by

Fig. 20. — Mode of preparing gelatine or agar "slants."

their colonies on solid media are not absolutely constant,
it is useful to note points of difference in this respect
between the various species.

Colonies are variously coloured, some being white and
opaque, others white and translucent, while many kinds
are distinctively coloured red, yellow, green, black, and
other tints.

The shape of the single colonies may be circular or
irregular in outline, with entire or variously indented
edges (Fig. i 7), and the surface may be flat and smooth,
or raised into prominences of various form. All these
characters must be carefully observed. In the case of
stab-cultures the extent and form of the growth along

54

PURE CULTURE OF BACTERIA

and away from the track of the needle must be noted
(Fig. 19), as well as the presence or absence of lique-
faction of the solid medium (Fig. 21),

Ex. 22. — Make stab-cultures in (i) gelatine and (2) agar tubes
of the bacteria which you have isolated.

Incubate and make drawings to illustrate the development of
the colonies both on the surface and in the substance of the

Fig. 21. — Diagram of types of liquefaction following stabs in gelatine media.

media. If liquefaction occurs, note the extent to which it pro-
gresses, and the form of space occupied by the liquefied medium.

Ex. 23. — Prepare some gelatine or agar " slants," and make
streak-cultures on these. Make drawings of the form of the
colonies along the streak, and note any peculiarities of colour,
dryness, form of growth, and any other points.

Ex. 24. — Make streak-cultures on solid media in Petri dishes.

3. Media. — There is no universal medium in or upon
which all kinds of bacteria will grow equally well. Some
of the organisms thrive best in beef broth, others in milk
or in solutions containing sugar, their requirements in
respect of nutrition being very varied. It is, therefore,

MEDIA 55

necessary to prepare a number of kinds of media before
any useful study can be made of the various species of
bacteria which are commonly met with.

The way in which the colonies develop in or upon the
different media and the form which they assume, as well
as the chemical action which the organisms have upon
the media, are matters of the greatest importance in
assisting us to distinguish one species from another. It
is essential that scrupulous care be taken in the prepara-
tion of the various kinds of media, and for each kind
some definite method should always be adopted and
adhered to, since slight modifications in their acidity and
composition or in their treatment in the sterilization
process often extensively influence the resulting growths
of bacteria upon them.

The same species of organism may exhibit many
points of difference when grown upon two media, which
are very nearly but not quite alike in acidity and com-
position. The multiplicity of so-called different species
of bacteria, and the great difficulty there is in comparing
the work and conclusions of those who have carried on
investigations in the subject, is due in a measure to the
want of uniformity in the media which have been used.

The following are among the most useful media
needed by the student. Those required for the study of
special organisms present in soil, manure, or dairy pro-
ducts will be mentioned later. In the examination of
new or unrecognized species its growth upon a variety of
media must be studied and recorded.

In the following exercises instructions are given for
the preparation of the kinds of media most frequently
needed for the growth and examination of common
bacteria.

$6 PURE CULTURE OF BACTERIA

A stock of each should be prepared for future use.

Ex. 25.— I. Nutrient Beef Broth (Bouillon). — Take i lb. of
lean beef free from fat, chop it up very fine, with a knife or
mincing machine.

Put it in a porcelain dish or glass beaker and pour on it
I litre of water. Leave it to soak all night in a cool place,
covered from dust. Strain the extract through muslin into a
saucepan and boil it for one hour. Filter through filter paper
into a large flask and make up the volume to i litre again. Add
to it-
Sodium chloride, 5 grams.
Witte's peptone, 10 grams.
Heat the whole in a steam sterilizer or water-bath for an hour,
shaking occasionally until all is dissolved.

Neutralize as in Ex. 17. Heat in steam for quarter of an hour.
Neutralize again if necessary and acidify to + 10 as in Ex. 17.
Filter into sterile flasks or test-tubes. Sterilize in flasks or in
tubes for three successive days for twenty minutes each time.

2. The following varieties of beef broth are occasionally
needed :—

Nutrient Beef Broth with Sugars. — Add to the above before
final filtration —

(i) Milk sugar or lactose, 20 grams, or
(ii) Grape sugar or glucose, 20 grams.
Ex. 26. — Lemco's Extract Broth (Bouillon). — Dissolve in
1 litre of water at room temperature —

Lemco's extract of meat, 5 grams.
Witte's peptone, 10 grams.

Neutralize as in Ex. 17. Boil for fifteen minutes, make up to
I litre, neutralize again if needed, and acidify to + 10 as in
Ex. 17. Boi], filter into sterile flasks or tubes, and then sterilize
on three successive days for twenty minutes each time.

Ex. 27. — Nutrient Gelatine. — Prepare as in Ex. 17, using —
(a) Nutrient beef, from Ex. 25, for nutrient medium, or
(d) Lemco's extract, as given in Ex. 17 and Ex. 26.

MEDIA 57

Ex. 28. — The following modifications are frequently needed
for special purposes : —

Nutrient Sugar Gelatines. — Add to the ordinary gelatine
medium of previous exercise before final filtration —
(a) Lactose sugar or lactose, 20 grams, or
(d) Glucose sugar or glucose, 20 grams.

Ex. 29. — Litmus-Grlucose Gelatine. — Before sterilization add
to a neutral gelatine medium 2 per cent, of glucose and enough
Kubel-Tiemann's neutral litmus to colour it a purple tint.

Colonies producing acids in this medium become surrounded
by a pink halo.

Ex. 30, — I. Nutrient Agar. — For its preparation see Ex. 20.

2. Milk-sugar or Lactose Agar. — Add to the ingredients
mentioned in list (i) Ex. 20 in the preparation of nutrient agar —

Milk-sugar or lactose, 20 grams.

3. Grape-sugar or Glucose Agar.^ — To nutrient agar add —

Glucose, 20 grams.

4. Glucose-chalk Agar. — To a neutral agar add enough
sterilized precipitated chalk to render the medium white and
opaque. When grown on plates of this medium, colonies which
produce acids dissolve the chalk and make the medium clear.

5. Litmus-glucose Agar. — Before sterilization add to a neutral-
ized agar enough Kubel-Tiemann's litmus to give the medium
a purple colour.

Ex. 31. — Milk. — Take some perfectly fresh milk and pass it
through a separator.

If the milk is acid, make it just neutral to litmus by adding
normal caustic soda solution or sodium carbonate solution.

Place the separated milk in sterilized tubes, and sterilize
in steam on three successive days for twenty minutes each time.
In order to be quite certain that the milk is free from spores
of bacteria, the tubes should be placed in an incubator for
twenty-four hours before being inoculated with the bacteria to
be studied : if organisms are present curdling or other changes
in the milk will usually be noticed.

58 PURE CULTURE OF BACTERIA

If separated milk cannot be obtained, take ordinary fresh
whole milk; leave it to stand in a beaker in a cool place
all night, and in the morning pipette or siphon off the " skim-
milk " from below the cream layer.

Ex. 32. — Litmus Milk. — Before tubing the milk, add
enough Kubel-Tiemann's neutral litmus to tinge it a purple
colour ; then sterilize as advised in previous exercise.

When inoculated with an acid-forming organism, the purple
colour becomes changed to pink.

Ex. 33 — Whey as Medium. — Take a litre of separated or
skim-milk in a dish or bowl, and pour into it lo c.c. of distilled
water, to which 8 to lo drops of commercial rennet 'extract have
been added. Stir briskly for a few seconds, so as to mix the whole
and allow the milk to coagulate. When the curd is formed, cut
it with a knife and leave it for an hour so that the whey may
separate. Then filter the whey through muslin. Add the white
of an egg whipped up with loo c.c. of water. Heat in steam
sterilizer for an hour. Filter into test tubes and sterilize in the
usual way.

Ex. 34. — Potato. — Procure some long sound tubers. Wash
them in water, and scrub off all soil with a nail brush. Cut off
the two ends, and with a cork-borer cut cylinders from the
potatoes. Divide the cylinders diagonally with a sterile knife,
and wash the pieces twelve hours in running water, or wash
them in several changes of water until no milkiness is observed.

Place each of the washed halves, thick end downwards, in
sterilized Buchner or potato tubes (Fig. 25), the bulbs of which
are half-filled with distilled water. The tubes can be tilted so that
the water touches the bases of the slices and keeps them moist
when bacteria are being cultivated upon them. If no special
tubes are at hand, place a piece of wet cotton wool at the bottom
of ordinary wide test-tubes before putting in the potato shoes.

Sterilize on three successive days in steam for thirty minutes
each time. It is very necessary to observe great cleanliness in
preparing the potato slices, as the soil upon the tubers frequently

REGISTRATION OF BACTERIA 59

contains spores of bacteria which are highly resistant to heat
and not readily killed by steaming.

4. Identification, Classification, and Registration
of Bacteria. — In determining the specific character of
a bacterium, its morphological and physiological features
must be thoroughly examined, and in describing a new
organism these features must be exhaustively studied
and the results accurately recorded in order to facilitate
future comparison and reference. No description should
be considered complete which does not contain full
information respecting the following points : —

(a) The form, size, and motility of the organism.

(d) The presence or absence of spore formation ; the
shape, size, and position of the spores in the parent cells.

(c) Staining quality by Gram's and other methods.

(d) The form and colour of the surface and deep
colonies in gelatine and agar media. Is the gdatine
liquefied ?

{e) The aerobic or anaerobic character and the growth
and behaviour at low and high temperatures (20'' and
2,7° C. respectively).

(/) Growth on potato.

(g) Action upon milk : coagulation and acid formation.

(k) Its action in broths containing various sugars ;
the power of forming acids and gas in these media. Its
relative growth in broth with +10, neutral and - 10
reactions.

(i) The power of reducing nitrates with the produc-
tion of gas.

The practical testing of the various characters of the
organisms is dealt with in subsequent exercises.

A simple and convenient method of classifying and
registering the character of bacteria which is especially

6o PURE CULTURE OF BACTERIA

useful in the case of nearly allied forms is that adopted
by the Society of American Bacteriologists. It is an
adaptation of the Dewey system of classifying books — a
record of the characteristic features of the various organ-
isms being kept in accordance with the numerical system
given below.

The registration of organisms in this manner would
greatly facilitate the comparison of most forms met with
in the course of research with those already known, and
it is hoped that some plan of this kind will be adopted
by all workers.

Numerical System of recording the chief Characters
of a Bacterium.

Group number.

lOO.

Endospores produced.

200.

„ not produced.

lO.

Aerobic.

20.

Facultative anaerobic.

30-

Strictly anaerobic.

I.

Gelatine liquefied.

2.

„ not liquefied.

.1

Acid and gas from glucose.

.2

„ and no gas from „

.3

No acid from glucose.

.4

No growth with „

.01

Acid and gas from lactose.

.02

„ and no gas from „

•03

No acid from lactose.

.04

No growth with „

.001

Acid and gas from saccharose.

.002

„ and no gas from „

.003

No acid from „

.004

No growth with „

CHARACTERS OF A BACTERIUM

6i

Group number.

.0001 Nitrates reduced with evolution of gas.

.0002 „ not reduced.

.0003 „ reduced without gas production.

.0000 1 Fluorescent.

.00002 Violet chromogen or colouring matter.

.00003 l^lue

.00004 Green „

.00005 Yellow „

.00006 Orange „

.00007 Red „

.00008 Brown „

.00009 Pink „

.00000 Non-chromogenic.

The genus is indicated by a symbol or contracted
term preceding the number, e.g.

Bacterium colt, Esch., is recorded thus : — Bad. 222.1 1 1 10
Streptococcus lacticus, Kr., „ „ Str. 222.22220

Other features not included in the group numbers
may be dealt with in tabular form as shown below, the
presence or absence of a feature being indicated by a
plus ( + ) or minus ( - ) sign. For example, the
characters of

c

'6
t
0

U

X,

1

0

+
+

Gelatine
plate.

Milk.

+

.1
0

1

3
0

+
+

1

"6
1

'x-
02

<
+
+

a

<

1

+
+

•0

•a

OA

s

>

Str. lacticus, Kr., are: — Str. 222.22220
Bact. acidi lactici, Hiippe, Bact. 222,11000

+

:

62 PURE CULTURE OF BACTERIA

The growth upon agar and potato or in broth and
other media can also be given in a small space by
judicious selection of terms for such a table.

Ex. 35. — Determine if the organisms forming the colonies on
any of the agar or gelatine plates which you have prepared are
motile or non-motile.

To do this, take a hollow-ground " hanging drop " slide and
sterilize it by passing it four or five times through the Bunsen
flame. Now smear a thin ring of vaseline round the edge of the
hollow in the slide.

Then take a cover-glass and, after passing it through the flame
two or three times, transfer a drop of sterile broth to the centre

with a platinum loop,
taking care that the drop

Fig. 22.— Section of hanging-drop slide. doCS UOt floW tO the

margin of the glass. Now introduce a small portion of the
culture to be tested into the drop with a platinum needle. Lift
the cover-glass with forceps and turn it over very rapidly so that
the drop is hanging on the under surface ; then lower it on to
the ring of vaseline. The drop should now be hanging in the
enclosed space without touching the slide, as in Fig. 22. As
communication between the hollow space and the outside air is
prevented by the vaseline, the drop does not readily dry up.

Keep the whole at room temperature or 22° C, and examine
with the microscope every half hour for two or three hours.
If the organisms are motile their movement will be readily
perceived, especially if a small diaphragm opening is used.

Care should be taken to avoid bringing the objective down on
the cover-glass : if difficulty arises in focussing, make a small
dot on the surface with ink and focus this first.

Ex. 36. — Test some of the organisms which you have
isolated for gas production —

{a) With Durham's Tubes.

First add about 10 c.c. of nutrient beef broth containing
glucose (see 2, Ex. 25) to several Durham's fermentation tubes

CHARACTERS OF A BACTERIUM

63

(Fig. 23); put in the small tubes open end downwards; plug

with cotton wool and sterilize for half an hour
on three successive days : the small inner tube
in each becomes completely filled with the
broth.

Now inoculate the medium with small portions
of the different colonies to be tested and incubate
at 37** C. If fermentation occurs the small
tube becomes partially filled with gas.

{b) With Saccharimeter Tubes.

The closed end of the tube should be filled
completely and half the bulb also with glucose
beef broth (Fig. 24, a). After sterilization,
inoculate and incubate at 37° C.

If gas is produced some of it will collect
near the closed end of the tube (Fig. 24, b).

It is useful to have the limb of some of the
tubes graduated. Note the amount collected

,--<^i2\^

^

r^

after
three
then
or 2

two or

days;

add I

c.c. of

strong caustic fermentation tubT/

potash solution.

Some of the gas is ab-
sorbed, the diminution re-
presenting the amount of
carbon dioxide present ; what
is left is in most cases
hydrogen, so that the ratio
of CO2 to H can be calcu-
lated.

If the medium is cloudy
only in the bulb, the organism is aerobic ; where the upper
end of the tube is cloudy and the medium below remains

Fig. 24. — Saccharimeter tubes.
a Full; b after inoculation and production of gas

64 PURE CULTURE OF BACTERIA

clear, the organism is anaerobic ; facultative anaerobes usually
render the whole contents of the tube turbid.

Inoculate a specimen of both the above tubes of media with
Bad. colt and note the results.

Ex. 37. — Melt a few tubes of nutrient beef broth gelatine con
taining glucose (see Ex. 25), and inoculate them with various
organisms to be tested for gas production.

Allow them to cool and solidify, then incubate at 20° C. j if
gas is produced it collects in bubbles throughout the medium.

Try a tube with Bact. coH. and note the results.

Cultivation of Anaerobic Bacteria. — In the cultiva-
tion of anaerobes it is necessary to devise some means
for excluding the oxygen from, or for removing it from,
the space surrounding the medium.

The air may be expelled from the medium by boiling ;
it may be extracted with the air-pump, or it may be
displaced by hydrogen, nitrogen, or some other gas.

The simplest arrangements for the growth of anaerobes
are the following : —

(i) Cultivation in a deep stab in agar or gelatine.
(2) Cultivation in a Buchner's tube.

Ex. 38. — (i) Anaerobic Cultivation in a Deep Stab. — Take a
tube which is more than half full of agar or gelatine medium
and place it in a beaker of warm water; heat to boiling for five
minutes so as to expel the dissolved air from the medium.

Cool the tube in water, and when the agar or gelatine is
solidified, make a deep stab to near the bottom of the tube with
a long platinum needle, upon which are the organisms to be
grown ; rotate the needle and then withdraw it. In the recently
melted medium the track of the needle closes up.

Now gently melt the upper layer of the medium by rotating it
rapidly near a Bunsen flame ; this more effectually closes the
track,

CULTIVATION OF ANAEROBIC BACTERIA 65

Another plan is to pour into the tube two or three centi-
metres of melted agar at a temperature not higher than 40° C.

After inoculation plug the tube and cover the opening with a
tight-fitting rubber cap washed in a dilute solution
of mercuric chloride. ———^^

Or the tube may be placed in a Buchner tube, f_ /

described below, without the rubber cap.

Under these conditions most anaerobic
organisms readily form colonies in the lower part
of the medium.

(2) Growth of Anaerobes in a Bucliner Tube.
— This is the most generally adopted method.

The Buchner tube consists of a strong wide-
bored test-tube, near the base of which is a
constriction (Fig. 25).

Make a streak of the organism to be grown
on an agar or gelatine slant or potato-slice.

Then take i to 2 grams of pyrogallic acid and
dissolve it in 2 to 3 c.c. of water; to this add 10
to 15 c.c. of a 20 per cent, solution of caustic
potash.

Pour the mixture into the Buchner tube and
also place the test-tube containing the bacteria
inside it as soon as possible.

Plug the opening of the Buchner tube im-
mediately with a close-fitting rubber stopper,
slightly greased with vaseline : to make the
joint gas tight it is advisable to cover it with

, , -_ Fig. 25. — Buchner

melted pararnn wax. tube, arranged

The mixture of pyrogallic acid and caustic o7anaerobkor°
potash absorbs the oxygen in the tube, leaving s^n'sms.
only the nitrogen of the air with a small trace of carbon monoxide
which is produced from the mixture. In this atmosphere most
anaerobic organisms grow satisfactorily.

(3) In Yeast Flask in Hydrogen or other Inert Gas. —

^ PURE CULTURE OP BACTERIA

Anaerobic organisms when grown in broth, milk and other
liquid media, very frequently give rise to the evolution of much
gas, which, in a completely sealed vessel, is likely to lead to
dangerous explosions.

On this account it is best to arrange for their culture in liquid
media in yeast flasks, arranged as indicated in Ex. 140, where
a simple mercury valve allows of the escape of gas without
permitting the entrance of air.

CHAPTER V.

FERMENTATIONS AND THE ACTION OF
ENZYMES.

I. When barley grains are supplied with an adequate
amount of water and kept warm they begin to germinate.
The embryo plant within develops and its root and
stem break their way out of the grain. At the same
time the starch of the floury part or endosperm is
changed into sugar, the sweet taste of which can be
recognized in the sprouted grain. The change of the
starch into sugar is brought about by the action of a
peculiar nitrogenous compound secreted by the cells of
the embryo plant. This substance, known as diastase
is an example of an extensive class of bodies termed
ferments or enzymes^ which are produced in various organs
of living plants and animals. It is found in all germinat-
ing seeds, tubers, and other parts of plants which contain
starch, and serves the purpose of changing this solid
reserve-product into soluble sugar and a gum-like sub-
stance, dextrin, which are able to diffuse through the
body of the pla:nt to wherever nutrition and growth are
going on.

2. A great many kinds of enzymes are known : some
of them have a specific action on certain kinds of sugars,
others are concerned with chemical changes in proteins,
urea, cellulose, and many other organic compounds.

While some of them, such as pepsin and trypsin men-

67

68 THE ACTION OF ENZYMES

tioned later, can act upon several different kinds of
compounds of the same class, many examples are
known of enzymes which are very specially concerned
with the decomposition of one definite kind of chemical
substance only and are inactive upon any others even of
very similar or allied composition. They are generally
soluble in water, and although their solutions may be
obtained in a concentrated form by extracting the
tissues of plants and animals containing them, they
have not yet been isolated in a pure state. One of
their most striking properties is the power which very
small quantities have of decomposing or changing large
amounts of the particular chemical compounds upon
which they work, and that without suffering much
diminution or loss during the process. Enzymes are
active only within a limited range of temperature.
Those obtained from the tissues of warm-blooded animals
carry on their work best at about 37° C, while those of
vegetable origin are generally most effective about 2 5 ° C.
At low temperatures their action is checked but not per-
manently destroyed ; they are, however, destroyed when
their solutions are heated to 70° or 80° C. for a short
time. In a dry state they resist a temperature of
120° C. or more without damage. The majority act
only in neutral or faintly acid solutions, but a few are
effective in an alkaline medium.

As already indicated enzymes are very sensitive com-
pounds and much influenced by external conditions.
They are readily destroyed by high temperatures and
strong acids or alkalis, but neutral salts are less injurious
and often precipitate the enzymes along with proteins
and other substances with which they are associated in
living tissues of plants and animals.

THE ACTION OF ENZYMES 69

Sunlight only affects them to a slight extent. Many
poisons, such as chloroform, phenol, thymol, salicylic
acid, sodium fluoride, and alcohol, which check the vital
activity of organisms or destroy them altogether, have
comparatively little effect on enzymes.

By adding one or other of these substances in small
amounts to liquid cultures of bacteria it is possible to
destroy or check the latter without materially affecting
any enzymes they may have formed and set free in the
medium.

3. When fruit juice or a solution containing sugar is
exposed to the air in summer it very frequently loses its
sweet taste and small bubbles of gas arise within it.
The juice becomes frothy, and a chemical examination
shows that the sugar is decomposed with the formation
of alcohol and carbon dioxide gas, the latter escaping in
small bubbles and producing an effect resembling the
boiling of water. The peculiar chemical change is found
to be due to the presence in the liquid of vast numbers
of living yeast-cells, which have grown and multiplied
from a few which have fallen in from the air or which
were adhering to the fruit when the juice was first pre-
pared. The iGxra fermentation {ixora ferv ere, to boil) has
been applied for a long time to decompositions like this
in which the frothing of the liquid is a characteristic
phenomenon ; it is, however, now extended to include
chemical changes brought about by enzymes such as the
diastase, even where no gas is produced, so that its
original meaning has disappeared.

The living yeast plant is sometimes spoken of as an
organized ferment in contradiction to the non-living
unorganized ferments or enzymes, of which diastase pre-
viously mentioned is an example. All early attempts to

70 THE ACTION OF ENZYMES

extract an enzyme or lifeless compound from yeast
capable of decomposing sugar into alcohol and carbon-
dioxide failed, and the alcoholic fermentation was
assumed to be the result of the activity of living yeast
cells only. About 1896, however, Buchner ground up
yeast with very fine sandy material in order to rupture
the cells, and then subjected the dough-like mass to
heavy pressure. By this means he extracted a juice
which, in the complete absence of living cells, decom-
posed small amounts of sugar into alcohol and carbon-
dioxide : the juice was found to contain an enzyme
which he named zymase. This result brings the
alcoholic fermentation by yeast into line with the
changes produced by unorganized ferments or enzymes.
More recently, lifeless products have also been obtained
from lactic bacteria which are able to produce lactic acid
from sugars, and, similarly, enzymes have been obtained
from the acetic bacteria which are able to oxidize alcohol
to acetic acid just as the living organisms do, except
to a smaller extent. The view, therefore, is gradually
gaining ground that fermentations of all kinds, whether
associated with living cells or not, are due to structureless
non-living enzymes, and that it is perhaps better to speak
of intra-cellular or endo-enzymes and extra-cellular ferments
or ecto-enzymes, the former term being applied to enzymes
which ordinarily carry on their work within the cells in
which they are produced, while the latter is reserved for
those which are excreted and are able to act independ-
ently of the cell. It must, however, be noted that there
are still many fermentations which do not take place
except in the presence of living organisms ; with many
of these we shall be specially concerned later.

4. It will be useful here to notice briefly some of the

THE ACTION OF ENZYMES 71

most important types of fermentation and the different
varieties of enzyme action ; those of agricultural interest
will be dealt with in greater detail in subsequent chapters.
Enzymes usually change or decompose the substances
upon which they act, forming from them bodies of simpler
constitution. The chemical reactions in the majority of
cases are examples of hydrolysis^ a form of decomposition
or dissociation which involves the fixation of the elements
of water. One molecule of a substance is split into two
with the addition of water (HgO) : for example, under
the influence of the enzyme invertase, which is secreted
by yeast, cane sugar is split into two simpler kinds of
sugar, according to the following equation : —

C12H22O11 + H2O = C6H12O6 + C6Hi206
cane sugar water glucose fructose

A small number of ferments are known which affect the
oxidation of compounds ; examples of this type are the
vinegar plant and Bacterium aceti, which convert alcohol
into acetic acid. Some few ferments function as reducing
agents. The fibrin ferment of blood and rennin in the
stomach of animals bring about the coagulation or
" clotting " of proteins in blood and milk respectively :
these are termed ''clotting'' enzymes.

In addition to those which effect simple hydrolysis,
oxidation or reduction, a large number of ferments are
known which induce chemical reactions of a much more
complicated character. Many obscure processes, such as
those of putrefaction and the ripening of cheese, are due
to the activity of ferments, but the various stages of the
transformations which occur are imperfectly understood.

In naming the enzymes it is usual to add the suffix -ase
to the root of the name of the substance upon which the

7^ THE ACTION OF ENZYMES

ferments were first observed to act ; for example, Duclaux
named one of the enzymes which attacks the casein of
cheese, casease ; that affecting maltose sugar is termed
maltase, the ferment which changes amylum or starch is
an amylase, the urea ferment urease, and so on. Similarly
the proteid-destroying class are described as proteases.
This nomenclature, however, is not strictly adhered to,
since it is customary to retain the names trypsin and
pepsin for two common proteases, and the terms emulsin,
ptyalin are still in ordinary use.

From many points of view it is convenient to classify
the enzymes according to the nature of the substance
which they are able to ferment. The most important
organic compounds which undergo fermentation are the
carbohydrates, proteins and fats. These form the greater
part of the body substance of all animals and plants, and
enter into the composition of food stuffs used by many
kinds of living organisms. It will be useful to refer
briefly here to the general character of the three classes,
and give a short account of the more commonly occurring
representatives in each.

I. Carbohydrates. — To this class belong cellulose,
starch, various kinds of sugar, and many other widely
distributed natural products. They all contain carbon,
hydrogen and oxygen, the two latter elements being
present in the same proportion as they exist in water :
hence the name hydrate of carbon or carbohydrate.
They may be divided into three groups : —

(i) The polysaccharoses, amyloses or starch group.

(ii) The disaccharoses, sometimes known as the sac-
charose or cane-sugar group.

(iii) The monosaccharoses, termed glucoses or the
grape-sugar group.

POLYSACCHAROSES 73

( I ) Poly saccharoses, (a) Starch. — The chief Poly sac-
charose is starch, a carbohydrate havings the empirical
formula, CgHjoOs, but really possessing a much
higher molecular weight. It is a reserve food very
commonly distributed in the cells of tubers, roots, seeds,
and other parts of plants, and occurs in the form of solid
grains built up of a layer of material arranged round a
more or less central nucleus or hilum. The substance of
the grain consists of starch or amylose, a compound
which is insoluble in cold water but swells up and
partially dissolves in hot water, forming " starch paste.''
A solution of iodine in potassium iodide turns it a
characteristic deep blue colour. When boiled for some
time with dilute acids it is finally converted into grape-
sugar, intermediate products, such as maltose and gummy
substances being first produced.

The Hydrolysis of Starch by Ferments. — When thin
starch-paste (prepared by boiling about a gram of starch
in 50 c.c. of water) is mixed with an extract of malt and
kept warm, the liquid gradually loses its opalescent
appearance and finally becomes clear. If small portions
of the mixture are removed and tested at short intervals,
with a solution of iodine and potassium iodide, the first
portion containing unaltered starch is coloured blue, the
second purple, later a reddish brown colour is observed,
and finally the solution when tested remains colourless.
The changes going on in the starch-paste are due to the
action of the enzyme diastase (amylase) contained in the
malt extract. At a temperature of 50° to 60° the starch
is hydrolysed into maltose and dextrin, a gum-like
substance.

SQHioOs + H2O = Q^H^.On + CeHioO,
starch maltose dextrin

74 THE ACTION OF ENZYMES

About 8 I per cent, of maltose and 1 9 per cent, of dex-
trin are found when the reaction is at an end. Under
certain conditions diastase will convert the dextrin into
maltose, so that the complete and final hydrolysis of
starch by this enzyme results in the formation of maltose
only. In all cases, however, a number of intermediate
dextrins are first produced, and it is to the presence of
these that the colour changes, previously mentioned, are
due, the purple colour being given by starch and a cer-
tain amount of one or more kinds of dextrin, the reddish
brown tint by the latter in the absence of starch, the
final products, namely, maltose and dextrin, being
uncoloured by iodine.

Many pathogenic bacteria are able to produce amylo-
lytic or starch-splitting ferments ; Bs. siihtills, Bs. mega-
therium, Clostridium butyricum, and other butyric bacteria,
as well as species of Proteus and bacteria isolated from
cereal grains and soil, have been found to yield enzymes
of this class also.

Ex. 39. — Cut thin sections from a piece of potato tuber and
from a wheat grain : examine with a low power and make draw-
ings of the starch-grains within the cells observed.

Ex. 40. — Make a strong solution of potassium iodide in water
and add to it a few crystals of iodine. Allow the mixture to
stand for twelve hours, and shake occasionally in order to
facilitate the solution of the iodine. When the latter is all
dissolved, add more water until the whole is the colour of dark
sherry.

When examining the starch-grains place a drop of this solution
near the edge of the cover-slip so that it may run under the
latter and come in contact with the starch-grains. Note the
change in colour of the starch-grains.

Ex. 41. — Make an extract of malt diastase as follows : Shake

CELLULOSE 75

up five grains of ground malt with 50 c.c. of cold water, and
after allowing it to stand for four hours, filter so as to get a clear
solution.

Next grind some starch with water in a mortar and pour a little
of the mixture into a 200 c.c. flask of boiling water. When cool
pour about 20 c.c. of this thin starch paste into three test-tubes :
show the presence of starch by adding a few drops of the solu-
tion of iodine mentioned in Ex. 40 to one tube, and to the other
two tubes add 3 or 4 c.c. of the diastase extract, and warm
them to 60° C. Test for the presence of starch in one of these
two tubes by taking out at intervals of five minutes a few drops
with a pipette and adding them to weak solutions of iodine kept
in a series of test-tubes.

After a time the starch is changed into sugar and dextrin :
When this has happened show the presence of the sugar by
means of Fehling's solution (Ex. 44).

See if Fehling's solution is acted upon by the thin starch
paste when no diastase is added.

(J?) Cellulose. — Somewhat allied to starch is cellulose
and its derivatives, which form the solid cell walls of
plant tissues : they are produced by the activity of the
protoplasm or living material within the cells. What
may be regarded as typical cellulose may be obtained
from cotton wool, paper, and flax fibre. It has an
empirical formula near CgHioOg, is insoluble in water,
dilute acids or alkalis, but dissolves in ammoniacal cupric
oxide and other solvents. Cellulose usually stains blue
when treated with strong sulphuric acid and iodine.
Several complicated compound celluloses, such as the
ligno-celluloses of timber and the adipo-celluloses of
cork, are known, but these cannot be mentioned further
here.

Hydrolysis and Fermentation of Cellulose. — The cellu-
lose walls of the endosperm of many seeds are hydrolysed

yG THE ACTION OF ENZYMES

apparently with the formation of sugars by an enzyme
named cytase, and a cellulose - dissolving enzyme is
possessed by many fungi, both parasitic and sapro-
phytic, which enables them to penetrate the epidermis,
wood and other tissues of plants.

In a later chapter (Chap. XIV.) is discussed the fermen-
tative decomposition of the cellulose of leaves, stems, straw,
paper, and similar materials by means of certain species
of bacteria present in manure heaps, the mud of swamps,
and the intestinal tract of certain herbivorous animals.

(ii) Disaccharoses. — The disaccharoses have the
formula C12H22O11. Three of the best known repre-
sentatives are cane-sugar, milk-sugar, and malt-sugar.

id) Cane-sugar or Saccharose is a very sweet crystalline
carbohydrate found in abundance in the cell sap of the
sugar cane, beetroot, and other plants.

When boiled with dilute acids it is transformed into
grape-sugar and fruit-sugar, the mixture of these two
being sometimes spoken of as invert-sugar.

An inverting enzyme, invertase, which effects a similar
chemical change in cane-sugar, is widely distributed in
nature : it is met with in yeast, and is possessed by Bs.
megatherium, Bs. vulgatus, and other bacteria.

Cane-sugar does not reduce Fehling's solution, and
cannot be fermented directly by yeast.

Ex. 42. — Dissolve some ordinary sugar in water and

(i) Test some of the solution for a " reducing " sugar as in

Ex. 44.

(ii) Take 10 c.c. of the solution and add to it three or four

drops of strong hydrochloric acid : boil for twenty minutes, and

after neutralizing the acid with a solution of sodium carbonate,

boil and test again with Fehling's solution.

MONOSACCHAROSES yj

{b) Milk Sugar or Lactose, C12H22O11 + HgO, is
found only in the animal kingdom, being an important
constituent of the milk of various animals. It occurs in
whey obtained in cheesemaking, and is a crystalline com-
pound with only a faint sweet taste. Milk-sugar is not
so soluble in water as cane-sugar : it reduces Fehling's
solution.

When treated with hot dilute acid it suffers hydrolysis,
and splits up into glucose and galactose thus : —

C12H22O11 + H2O = CgH^gOe + QHioOq

glucose galactose

The enzyme capable of effecting this change is termed
lactase. It is apparently possessed by many kinds of
bacteria.

In the lactic fermentation it is probable that the first
action of the bacteria is a hydrolytic process, the two sugars
produced being subsequently converted into lactic acid :
the chemical changes in lactic fermentation, and the
bacteria to which they are due, are discussed at greater
length in Chap. XVII.

Ex. 43. — Dissolve some milk-sugar in water and
(i) Test its reducing power with Fehling's solution (Ex. 44).
(ii) Taste some of the sugar.

{c) Malt- Sugar or Maltose is a compound with the
same empirical formula, C12H22O11, as the two preceding
sugars, and is produced by the action of diastase and
other enzymes upon starch. Hot dilute sulphuric acid
changes it into grape-sugar. Yeast is able to ferment it,
with the production of alcohol and carbon dioxide.

(iii) Monosaccharoses. — The most commonly occurring
monosaccharoses are glucose, or grape-sugar (formerly

78 THE ACTION OF ENZYMES

termed dextrose), and fructose^ or fruit-sugar (sometimes
called laevulose). Both have the empirical formula,
CeHjgOe. A mixture of these two sugars are formed
when the cane-sugar is " inverted " by boiling with acids,
or by the action of certain enzymes.

(a) Glucose or Grape-sugar is found in company with
an equal amount of fructose in most ripe fruits, and can
be produced by the hydrolysis of starch, cellulose, and
other polysaccharoses, the change being effected by
boiling with dilute sulphuric acid. It is not very sweet.

{b) Fructose or Fruit-sugar is met with in fruits along
with grape-sugar, as stated above, and in " invert-sugar."

{c) Galactose is another monosaccharose of the same
empirical composition as the two preceding compounds,
and is prepared by boiling milk-sugar or certain gums
with dilute sulphuric acid.

These sugars reduce Fehling's solution, and are directly
fermentable by yeast and certain species of bacteria.

Ex. 44. — Dissolve 35 grams of copper sulphate in 500 c.c. of
water, label this solution A: then dissolve 160 grams of caustic
potash and 173 grams of sodium potassium tartrate in 500 c.c.
of water and label the solution B. By mixing equal quantities
of A and B, Fehling's solution is produced. (The solution A
and B should be kept separate and only mixed when needed, as
the mixture does not keep long.)

Squeeze a few drops of grape juice into a test-tube containing
10 c.c. of the Fehling's solution : heat over a Bunsen flame and
note the reddish precipitate of cuprous oxide (CugO).

Test the juice of ripe plums and other fruits in^the same way.

2. Fats and Oils : Lipase. — Fats and oils contain
the same elements as carbohydrates, namely, carbon,
hydrogen, and oxygen, but the oxygen is in smaller pro-
portion to the hydrogen than in the latter compound.

FATS AND OILS 79

They are abundant in many animal and vegetable
tissues, generally in the form of round liquid globules.
They are present in stems and leaves, in flax seeds and
the seeds of rape, cotton, and other plants, where they
are reserves of food for use in the early nutrition of the
seedling. Milk contains a considerable amount of fat ; it
is also found stored up in the animal body, and is re-
absorbed into the blood when occasion requires, its
oxidation assisting in the maintenance of the body
temperature.

Fats and oils, as usually found in nature, are not
single pure substances, but mixtures of several chemical
compounds termed glycerides, which are formed by the
union of glycerin with a fatty acid ; e.g.-^ —

C3H,(OH)3 + 3C,H,CO,H = C,H,(C3H,CO,)3 + 3H,0

glycerin butyric acid butyrin

(a fatty acid) (a fat)

These compounds are chemically analogous to salts, and
are spoken of as ethereal salts of glycerin, the glycerin in
them playing the part of a base.

When treated with caustic soda or potash, the alkalis
turn out or set free the glycerin, and unite with the fatty
acid to form soaps^ which are potassium or sodium salts
of the fatty acids ; e.g. —

C3H,(C,,H3iCO,)3 + 3KOH = C3H,(OH)3 + 3Q,H3,CO,K

Palmitin caustic glycerin potassium palmitate

(a fat) potash (a potash soap)

Such chemical action is termed saponification.

In many seeds and other parts of plants, as well as in
the secretions of the pancreas in animals, an enzyme
named lipase occurs, which has the power of decom-
posing fats ; the change is a hydrolytic process, the fat3

8o THE ACTION OF ENZYMES

being split up into glycerin and the corresponding free
fatty acid, thus : —

C,H,iC,,K,,CO,% + 3H,0 = C3H,(OH)3 + (C^H^^CCH),

Stearin water glycerin stearic acid

(a fat) (a fatty acid)

In many bacteria and fungi a lipase is present.

CHAPTER VI.

FERMENTATIONS AND THE ACTION OP
ENZYMES — ( Continued),

I. Proteins. — Proteins are among the most important
compounds present in the tissues of animals and plants.
The majority are exceedingly complex in chemical consti-
tution, and although our knowledge of them has been
greatly advanced during the last few years by study of
their decomposition products, much remains to be done
before the exact nature of these substances is understood.
Protoplasm, the peculiar material in the cells of living
organisms, with which the phenomena of life are
mysteriously associated, consists of an unstable mixture
of proteins. They are also found in solutions, or in a
colloidal state in blood, lymph, milk, and similar liquids
obtained from organic beings. Some proteins are met
with in a dense semi-crystalline form in the storage
tissues of seeds and tubers, and others have been
obtained in a true crystalline state from blood, egg-
albumen, and certain seeds ; most of them, however, are
slimy, non-crystalline substances, like the white of an
egg) and contain nitrogen, and sulphur in addition to the
elements carbon, hydrogen and oxygen ; certain proteins,
such as the caseinogen of milk, contain phosphorus also.
The amount of carbon present is usually between
5 2 to 5 5 per cent. ; nitrogen, 1 6 to 1 8 per cent. ; hydrogen,
6.5 to 7.5 per cent. ; sulphur, from 2.5 to 5.3 per cent.

F 81

82 THE ACTION OF ENZYMES

Many proteins are precipitated as a coagulum or
insoluble clot when their solutions are boiled.

They are also peculiar in behaving both as acids or
bases. Some of them, such as casein, have the acid
character most strongly marked, and form neutral salts
with alkalis, while in others the basic properties are most
obvious. In aL, however, both properties are present in
greater or less degree.

It is necessary to note here that much confusion exists
in regard to the nomenclature used for the proteins and
their derivatives. The term proteid is very often used in
this country as equivalent to protein, but in Germany it
has a more restricted meaning, being applied to a special
group of proteins mentioned below ; if used at all it
should be applied ic the latter sense, and brought into
line with continental asage. Proteins are not infrequently
spoken of as albumens by some writers, and in France
they are described as " substances albuminoides." The
older school of chemists have kept the word albuminoid
as a term for proteins in general, speaking of albuminoids
of milk, feeding cake, etc., while physiologists have
habitually used it for a small class of proteins, of which
gelatine of bone and keratin of horn are representatives.

2. The classification and nomenclature of the proteins
is still in a transition state, and will need revision from
time to time to bring it into harmony with the advances
made in the knowledge of the chemical and physical
constitution of these substances. The following classes
and names are those at present recommended by a
committee of the Chemical Society of London : —

(i) Protamines. — These are strongly basic bodies,
obtained chiefly from the spermatozoa of fishes, and are
among the simplest proteins. They are not coagulated

THE ACTION OF ENZYMES 83

by heat, and are unaltered by pepsin, although they
break down under the influence of trypsin. Protamines
contain neither phosphorus nor sulphur, but have a high
nitrogen content (20 to 25 per cent.)

(2) Histories are also basic substances, not coagulated
by heat, very soluble in acids, but precipitated by ammonia.
They are mostly obtained from spermatozoa of fish, the
thymus glands of some animals, and from red blood-cells
of birds.

(3) Albumins.

(4) Globulins. — These two groups include the com-
monest and most representative proteins met with in
animal and vegetable tissues. Both are coagulated by
heat, and contain sulphur but little phosphorus. Albu-
mins present in egg-white, blood serum and milk are
soluble in water, and can be salted out from their solu-
tions by saturating the latter with ammonium sulphate.
Globulins are more numerous than the albumins, and
are present in eggs, blood, milk, most animal and vege-
table tissues, and in many seeds. They are insoluble in
distilled water and weak acids, and are readily precipi-
tated from solutions by half saturation with ammonia
sulphate ; magnesium sulphate, which does not throw
down albumins, precipitates globulins. They are feebly
acid substances, and dissolve easily in alkaline solutions.

(5) Sclero-proteins. — This ill-defined class includes
gelatine, the most abundant insoluble protein in bone and
cartilage and keratin, the chief constituent of wool, hair
and hoofs of animals. To these substances the term
" albuminoid " is sometimes restricted by physiologists.

(6) Phospho-proteins. — The chief representatives of this
class are caseinogen of milk, and vitellin, a constituent of
the yolk of eggs. They contain from \X.o \\ per cent, of

84 THE ACTION OF ENZYMES

phosphorus, and are acid substances, capable of forming
definite salts with alkalis. The pure phospho-proteins
are insoluble in water, and are thrown down when acids
are added to solutions of their salts.

(7) Conjugated proteins. — For these bodies German
writers adopt the generic term " proteid." They are
compounds which contain a protein united with a radical
or molecular group, termed a '•' prosthetic group." The
latter is either (i) a nucleic acid, (2) a carbohydrate, or
(3) a coloured product. As the peculiar characters of
the conjugated proteins are chiefly dependent upon the
kind of prosthetic group which they contain, they may
be conveniently divided into three sub-classes, viz. : {a)
Nucleoproteins, {b) Glucoproteins, and {c) Chromoproteins.
On hydrolysis by acids or enzymes a conjugated protein
splits into its prosthetic group and a protamine, histone,
or other protein.

(8) Derivatives of proteins. — When proteins are acted
upon by weak acids or enzymes they undergo hydro-
lysis, some of the products of decomposition being sub-
stances which give the reaction of proteins, but are
simpler in construction than the original body from
which they have been obtained. The chief of these
derived proteins are : —

{a) M eta-proteins, the so-called acid-albumins and alkali-
albuminates, formed by the gentle action of dilute acids
and alkalis respectively on proteins, belong to this class.

{h) Proteoses. — A group which embraces albumoses,
globuloses, gelatoses, etc. These substances are simpler
transformation products of proteins than the meta-pro-
teins. They can be salted out of acid solutions by
ammonium or zinc sulphate, but are not coagulated by heat.

{c) Peptones. — Further hydrolysis of the proteoses

COLOUR TESTS FOR PROTEINS 85

produce simpler bodies. These are the peptones, very
soluble substances, which cannot be salted out of their
solutions, but are precipitated by phosphotungstic, phos-
phomolybdic, or tannic acids.

{d) Polypeptides are a group of products of protein
cleavage beyond the peptone stage. The majority are
synthetical substances, which cannot here be dealt with.

3. Colour Tests for Proteins. — (i) The biuret reaction.
— A solution of a protein, when made strongly alka-
line with caustic potash, gives a red, pink, or violet
colour when a few drops of copper sulphate is added.
With many albumins the colour is blue or violet, with
proteoses and peptones red or pink.

(2) Milton's reagent (a solution of mercurous nitrate
in nitric acid) gives a pink or dark-red colour when
boiled with a protein which contains the oxyphenyl or
tyrosine group. The latter is absent from gelatine and
certain proteoses and peptones.

(3) The Xantho-pr oleic reaction. — Strong nitric acid,
added to a solution of protein and heated, gives a yellow
solution or a yellow precipitate. If ammonia or caustic
soda is added afterwards, a deep orange or reddish brown
colour is produced.

(4) Proteins containing sulphur, when boiled with a
solution of caustic soda containing lead acetate, gives a
black or brownish precipitate, or the solution becomes
dark-coloured owing to the formation of lead sulphide.

(5) Reaction of Molisch. — When a few drops of an
alcoholic solution of a-napthol is added to the solution
of a gluco-protein, and strong sulphuric acid added to
the mixture, a violet colour is produced. The sulphuric
acid splits off a carbohydrate from the gluco-protein,
and converts it into furfurol. The latter then gives a

86 THE ACTION OF EN2YMES

violet colour with a-naphthol. With thymol the colour
is carmine-red.

(6) Reaction of Hopkins and Cole, — Prepare a solution
of glyoxylic acid by adding sodium amalgam to a strong
solution of oxalic acid ; filter after the gas bubbles
disappear. To a solution of a protein add a little of
the above ; shake, and then add strong sulphuric acid.
A bluish violet colour is produced, which is due to the
presence of tryptophane, one of the dissociation products
of most proteins, except gelatine and the protamines.
Glacial acetic acid which has been exposed to sunlight
for some time often contains a small amount of glyoxylic
acid ; when such acid is added to a solution of a protein,
and strong sulphuric acid then poured in, a violet colour
is seen at the junction of the two liquids.

4. " Salting out " of Proteins. — Most proteins are
colloidal bodies, and can be precipitated from their solu-
tions by adding solutions of various inorganic crystalline
salts. Some of them are easily salted out in this way,
while others are thrown out of solution with difficulty.
By using salts of different nature in varying strengths
of solution, mixtures of the proteins may be more or
less completely separated into their constituents. The
method of salting out has been of great service in
obtaining pure unaltered proteins for chemical and
physiological study. The substances most frequently
used for salting out proteins are : —

Sodium chloride Potassium acetate

„ sulphate
„ acetate Ammonium sulphate

„ nitrate
Magnesium sulphate

Zinc sulphate

SALTING OUT OF PROTEINS 87

They are used in variable degrees of concentration,
the strength of solution necessary for the precipitation
of a particular protein being constant for that body.
The most efficient of the substances named are ammo-
nium and zinc sulphate, either of which, in saturated
solutions, will salt out all proteins and their derivatives,
except the peptones.

Certain acids (so-called alkaloidal reagents) precipitate
many proteins when used in excess ; those most com-
monly employed are : —

Phosphotungstic acid
Phosphomolybdic „
Tannic „

They are extensively used for the precipitation of
proteoses and peptones, tlie latter of which cannot be
salted out from their solutions.

Ex. 45. — Break an egg across the edge of a cup ; open the crack,
and allow the white to escape into a beaker, keeping back the
yolk. Transfer the white to a flask ; mix it with about ten
times its volume of water, and shake vigorously so as to dis-
solve as much as possible of the proteins. Use small quantities
of the solution of egg-white obtained to illustrate the colour
tests for proteins.

{a) Biuret test. — To i c.c. of the egg-white solution add 5 c.c.
of strong caustic potash solution and one or two drops of a
I per cent, solution of copper sulphate ; heat slightly, a violet
colour is seen.

{b) Millofis test. — Weigh out 10 grams of strong nitric acid
(sp. g. 1.4), add to it 10 grams of mercury ; heat, and allow the
metal to dissolve ; then dilute the liquid with twice its volume of
distilled water ; mix,, and after allowing it to stand for a time,

88 THE ACTION OF ENZYMES

pour off the clear liquid and label it Miilon's reagent. Add a
drop or two of fresh white of egg to 5 c.c of Millon's reagent,
and boil ; a brick red precipitate is obtained.

(c) Xantho-proteic reaction. — Add i c.c. of the egg-white
solution to 5 c.c. of concentrated nitric acid, and boil ; a yellow
precipitate is obtained. Pour off the excess of nitric acid and
add ammonia, the precipitate is coloured an orange or brown
tint.

{d) Hopkins and Cole's reaction. — Add about | a c.c. of the
egg-white solution to 3 to 5 c.c. of glacial acetic acid ; pour in care-
fully some strong sulphuric acid ; if no deep purple colour is
obtained use a dilute solution of glyoxylic acid instead of the
acetic acid.

Ex. 46. — Pour some undiluted white of egg into a test-tube,
and immerse the tube in a beaker of boiling water for a minute
or two ; notice the coagulation.

Try the same experiment with white of egg diluted and shaken
with twenty times its volume of tap water. Is the protein
coagulated ?

Ex. 47. — Break up a piece of ripe Stilton or Camembert
cheese in a mortar with a little water.

Wash out the whole into a beaker with distilled water and
filter.

Try the colour tests for proteins in this extract by methods
described in Ex. 45.

In what respects do the colour reactions differ from those
given with egg-white?

Ex. 48. — Dissolve a gram or two of gelatine in 50 c.c. of
boihng water ; cool and try the protein colour tests with small
portions of this solution.

Compare results with those obtained with egg-white and
cheese extract.

Ex. 49. — Try the colour tests with a solution of Witte's
peptone and compare results with those obtained with other
substances used in previous examples.

HYDROLYSIS OF PROTEINS 89

Ex. 50. — Shake up 10 grams of flour in a flask with 50 c.c
of water and allow it to stand for six hours ; filter.

Heat some of the filtrate and note the coagulation of a
vegetable albumin. In other portions of the watery extract
try the colour tests for proteins.

5. Hydrolysis of Proteins. — When proteins are
boiled with acids or alkalis, fused with caustic potash or
subjected to the action of superheated steam, they are de-
composed into simpler substances which do not give the
biuret reaction. By the hydrolysing action of boiling
hydrochloric or sulphuric acids the protein molecule is
split up into a great variety of degradation products, the
majority of which are amino-acids. Of these some
twenty or more different kinds have been isolated, the
chief of which, mentioned below, are found among the
dissociated products of practically all except the simplest
proteins.

(i) The monamino acids most frequently obtained are :
alanine, phenylalanine, leucine, glutamic, and aspartic
acids. They are all crystalline bodies with a sweetish
taste.

(2) Certain diamino acids are constantly found,
namely : arginine, lysine, and histidine. These are
strongly basic substances.

(3) Tyrosine, a hydroxy amino acid, is present in
nearly all proteins except gelatine and some protamines,
and

(4) Tryptophane also. The two last are easily
detected in a free or combined state by their colour
reactions (see pp. 85-86).

(5) In most cases the sulphur in the molecule is split
off as cystine or cysteine.

90 THE ACTION OF ENZYMES

(6) Ammonia (or its compounds) are also generally
met with in the mixture of dissociation products.
Possibly in some instances it is a secondary product
derived from the decomposition of some of the amino-
acids.

The relative amounts of all these substances vary
considerably. For example, over 22 per cent, of leucin
is obtained from the hydrolysis of egg - white, from
casein 10 per cent, while gelatine yields only about
2 per cent, of this substance. The amounts of glutamic
acid from casein is about 10 per cent, from gelatine
14 per cent., from serum globulin about 2 per cent

6. Proteases or Proteolytic Enzymes. — Soon after
food is taken into the stomach of an animal a colourless,
acid liquid, the gastric juice, is poured out from the gastric
glands. This juice contains among other substances two
enzymes, namely, pepsin and rennin^ and a small amount
(.2 to .5 per cent.) of hydrochloric acid. The complex,
insoluble proteins present in the food are very soon acted
upon by the pepsin, with the result that they are broken
down, step by step, into a series of soluble proteins of
simpler chemical constitution which can readily diffuse
through animal membranes and be transformed into
materials subsequently used in building up the body and
repairing its waste. Such chemical decomposition is
spoken of as proteolysis : the various stages of the process
are indicated in a general way below.

PROTEOLYTIC ENZYMES

91

With Pepsin in acid solutions.
Protein.

Meta-protein.

Acid-albumin.

Primary proteoses.

Prot-albumose. Hetero-albumose.

Secondary proteoses. Deutero-albumose. Deutero-albumose.

Peptones. Peptones.

The primary proteoses derived from the acid albumin
can be salted out by saturating the solution with sodium
chloride or half saturation with ammonium sulphate.
Of these two types exist, namely, proto- and hetero-
compounds ; the former are very soluble in pure salt-free
water, while the latter are only very slightly soluble in
water and begin to coagulate and separate from the proto-
albumoses when the mixture is dialysed to get rid of the
salt. The hetero-albumoses are precipitated from salt
solutions by dilution with water.

The secondary proteoses, representing products of
further hydrolysis, may be precipitated by complete
saturation of their solutions with ammonium sulphate.

From the proteoses peptones are ultimately formed.
These are bodies of simpler constitution which cannot be

92 THE ACTION OF ENZYMES

salted out of their solutions, but are precipitated by
phosphotungstic acid and other alkaloidal reagents,
and give a pure red biuret reaction. They are acid
subtances, very soluble in water, and contain no sulphur.
Only very small amounts of ammonia and amino-acids,
if any, are produced even by prolonged peptic digestion
of proteins, but in many instances dissociation products
intermediate between peptones and amino-acids are
formed in considerable amounts : these are probably
peptides, bodies which on hydrolysis with acids give
leucine and other amino-acids.

Ex. 51. — Take three large test-tubes, label them A, B, and
C respectively.
Half fill—

A with a dilute solution of hydrochloric acid (i c.c. of

strong acid in 150 of water).
B with dilute solution of hydrochloric acid, to which a

few drops of pepsin solution has been added.
C with dilute solution of pepsin only.
Into each tube add a small piece of fibrin which has been
washed and allowed to soak for half an hour in distilled water.

Place all three tubes in a water bath kept at 40° C. for twenty
to thirty minutes. Examine them at intervals and note that in
A and C, in which the ferment and acid act singly upon the
fibrin, the latter is undissolved : in B, where the ferment and
acid act together, the fibrin is digested and dissolved.
With the solution in B try the following experiments : —

{a) To a small portion of the liquid add a few drops of
htmus and then neutralize with very dilute caustic
soda : acid-albumin is precipitated.
{b) To another small quantity of the liquid add a drop of
strong nitric acid : a white precipitate of 2i proteose or
albumose appears, which dissolves with yellow colora-
tion on heating but reappears when the liquid is
cooled.

PROTEOLYTIC ENZYMES 93

(c) To a third portion of the liquid add a drop of a i per
cent, solution of copper sulphate and a few drops of
strong caustic soda solution : a pink colour shows
the presence oi peptone (the biuret reaction).

Ex. 52. — Repeat the above Ex., using pieces of the coagu-
lated protein of a hard-boiled egg instead of fibrin.

Ex. 53. — To 10 c.c. of milk in a test-tube add a few drops of
pepsin solution and a few drops of dilute hydrochloric acid
(see Ex. 51). Keep the whole for two hours in a water bath
at 40° C. Note that the milk is at first curdled ; after a time
the curd is gradually digested and dissolved, the liquid becoming
yellowish and bitter.

Filter some of the liquid and test it for peptones (the biuret
test).

The enzyme pepsin, which can be easily extracted from
gastric juice, acts only in an acid solution.

Proteins which escape or are not changed by it, as
well as those of simple constitution produced by its
action, pass along the intestinal tract and become sub-
jected to the action of the pancreatic juice poured out by
the pancreas into the duodenum. This secretion con-
tains several different enzymes, one of them being trypsin,
a proteolytic ferment, which breaks down proteins best
in a faintly alkaline solution. It not only forms soluble
proteoses and peptones from proteins but carries on the
digestion or splitting of all these compounds further, the
peptones being decomposed into several kinds of amino-
acids (the chief of which are glycocoll, alanine, phenyla-
lanine, leucine, aspartic, and glutamic acids and tyrosine),
a characteristic substance named tryptophane and small
quantities of ammonia : none of the latter products are
of protein nature, and consequently do not give the
biuret reaction.

94

THE ACTION OF ENZYMES

It is found that trypsin cannot dissociate the whole
of the protein into the amino-acids and other simple
substances just mentioned ; a certain amount of a
resistant residue remains after the enzyme has acted
for many months.

The action of trypsin is more rapid and complete
than pepsin ; few or no primary proteoses are obtained,
secondary compounds being apparently produced directly.

The action may be represented thus : —

Trypsin {in alkaline solution).
Protein.

alkali-albumin.

Secondary proteoses deutero-albumose.

/

peptone
(antipeptone).

peptone.

{a)

amino-acids

(leucine, tyrosine,

aspartic, and
glutamic acids).

il,) (c) ^

ammonia. hexone bases
(lysine, arginine,
histidine).

PROTEOLYTIC ENZYMES 95

The amount and kind of each of the decomposition
products produced by these proteolytic enzymes are
dependent upon the nature of the protein attacked and
on the temperature, acidity, or alkalinity, and other factors
of the reaction.

From the peptic digestion of casein a proteose (caseose)
is obtained which contains all the phosphorus of the
casein, later peptones arise, as well as paranuclein and
paranucleic acids.

With trypsin it is broken down more rapidly into
caseose, caseone (an antipeptone residue), tyrosine, and
some other simple products.

Gelatine is converted into primary and secondary
gelatoses, gelatine peptones, and, according to Nencki,
a certain amount of leucine, glycocoll, and ammonia.

Ex. 54. — Take three large test-tubes and label them A, B, C
respectively.

Half-fill each with a i per cent, solution of sodium carbonate
and add five or six drops of Benger's Liquor pancreaticus, which
cojitains trypsin.

Boil A, and then cool it.

Add a drop or two of hydrochloric acid to B, so as to make
it acid instead of alkaline.

Leave C alone.

Then add to each a piece of fibrin which has been washed and
soaked for half an hour in i per cent, sodium carbonate solution.

Place the tubes in a water bath at 40° C. for half an hour.

Examine them at intervals and note that in A no change is
observable, the ferment having been destroyed by boiling.

In B digestion does not occur, the solution being acid.

In C the fibrin is eaten away and dissolved without swelling
up, as in pepsin digestion.

(a) Filter the liquid in C and test a small portion for alkali-

96 THE ACTION OF ENZYMES

albumin by neutralizing it with a few drops of very dilute hydro-
chloric acid ; a precipitate indicates alkali-albumin.

ip) Filter off the precipitate and test the filtrate for peptone by
the biuret reaction.

{c) Carefully neutralize the solutions in A and B, then filter
and examine the filtrates for peptones.

Ex. 55. — Pour 2 c.c. of milk into a test-tube and dilute with
about 10 c.c. of water ; then add two or three drops of Benger's
Liquor pancreaticus. Immerse the tube in a water bath at
40° C. for half an hour.

Note that the milk is first curdled ; the curd is soon digested
and dissolved more rapidly and completely than when pepsin is
used (compare Ex. 53).

Test the yellowish liquid for peptones by the biuret test.

7. Proteases or Proteolytic Enzymes of Bacteria. —

In 1887 Bitter found that the cholera bacillus vv^hen grown
in a liquid nutrient medium produced and set free in the
liquid an enzyme which was capable of digesting fibrin
and gelatine. Since that date a large number of kinds
have been found to secrete or form proteases which break
down gelatine and other proteins into simpler com-
pounds. In the ordinary cultivation of bacteria the
presence of these proteolytic enzymes is indicated by
the liquefaction of the solid gelatine media upon which
the organisms are grown. The gelatine is decomposed
into peptone, amino-acids, and other simpler substances
which dissolve in the water present in the jelly. Where
extracellular proteases are produced, that is, where they
are manufactured by the living cells and set free into
the surrounding medium, liquefaction proceeds at a rapid
rate. Common " liquefiers " are, Bs. subtilis, Bad. pro-
digiosum, Bact. vulgare, Bs. vulgatus, and Bad. fluorescens.
A number of species are known which only liquefy

PROTEOLYTIC ENZYMES OF BACTERIA 97

gelatine in cultures which have been growing for a
considerable time. These form intracellular enzymes
which diffuse very slowly, if at all, from normal living
cells, but escape readily from unhealthy involution forms
or cells which are dead.

The enzymes manufactured by bacteria chiefly
resemble trypsin. Like the latter, their action is gen-
erally most energetic in neutral or alkaline media, and
the majority of them break down proteins into peptones,
amino - acids, ammonia, and other basic substances.
They all peptonise and liquefy gelatine, some of them
even when the medium is faintly acid. Emmerling and
Reiser investigated the action of Bact. fluorescens lique-
faciens on gelatine, and found a peptone present in the
" liquefied " medium along with methylamine, trimethyl-
amine, choline, and betaine ; ammonia was also set free.
Streptococcus longus cultivated under anaerobic con-
ditions in a medium containing fibrin produced tyrosine,
leucine, trymethylamine, methylamine, ammonia, and
some pyridine bases, as well as acetic, propionic, butyric,
and succinic acids.

The production of enzymes by bacteria and the
amount obtained depend upon the kind of organism,
the composition of the nutrient medium, temperature,
and other factors. A few species, such as Bs. subtilis,
Bact. prodigiosum, and Bact. pyocyaneum^ can form them
in nutrient solutions devoid of organic nitrogenous com-
pounds, but the majority only give rise to enzymes
when grown in meat juice, broth, milk, and other media
containing proteins. Most carbohydrates hinder the
formation of enzymes more or less completely without
checking the growth and development of the bacteria.
Some of the sugars, such as glucose, may inhibit their
G

98 THE ACTION OF ENZYMES

production altogether ; on the other hand, lactose has
little effect. A small amount of quinine can prevent
enzyme formation by Bad. pyocyaneum without inter-
fering much with the growth of the organism.

Although the formation of enzymes may be said to
be characteristic of certain species, many bacteria lose
this power when cultivated for a long time under some-
what abnormal conditions.

To determine the presence of a enzymes in a culture
of any organism grown in broth, milk, or other liquid
medium, it is necessary first to remove the living organ-
isms or check their development, after which the action
of the solution may be tested upon gelatine, fibrin, or
other protein.

By filtration through a Chamberland porcelain filter
the bacteria can be removed without destroying the
enzymes which they may have secreted ; this method
is very generally adopted in the study of bacterial
enzymes.

Another plan is to destroy the vitality of the organ
isms by heat ; but as most enzymes are decomposed or
their powers greatly diminished at 65 to 70° C. — a tem-
perature which is insufficient to kill many forms of
bacteria — the method is unsatisfactory or only of limited
application.

A simpler device, and one which is of general use, is
to treat the culture with an antiseptic substance of
sufficient strength to inhibit the growth of bacteria
without affecting the enzyme present. The compounds
best serving this purpose are toluene, phenol, thymol,
and salicylic acid ; chloroform is also useful, but is
volatile at somewhat low temperatures.

Px. 56. — Inoculate two or three tubes of nutrient broth with

PROTEOLYTIC ENZYMES OF BACTERIA 99

Bs. subtilis^ Bad. prodigtosum or Bad. liquefadens and in-
oculate for three days at 30° C.

To the cultures add about .25 c.c. of toluene and a very
small amount of barium sulphate powder so as to render the
liquid turbid : shake thoroughly.

Pour some of this culture into a tube containing ordinary
nutrient gelatine which has been made slightly alkaline with
sodium carbonate.

Allow the whole to stand and when the insoluble barium
sulphate has fallen on the surface of the solid gelatine mark the
point on the outside of the tube with a piece of gummed paper.

Keep the tube at 20° C.

If the solution contains a proteolytic enzyme, the solid
gelatine will be slowly dissolved and the thin layer of barium
sulphate will descend in the tube.

Try the experiment with Bad. colt and Str. ladicus and note
the results.

Ex. 57. — Cultivate one of the bacteria mentioned in the
previous Ex. in nutrient broth.

Add .25 c.c, of toluene, shake thoroughly and then suspend
in the solution a small piece of Griibler's " Karmin fibrin."

If the culture contains a enzymes the fibrin dissolves and the
carmine with which it is stained diffuses and colours the solution-
Try the experiment with Bad. coli^ Bad. acidi ladid, and
Str. ladicus, and note the results.

Ex. 58. — Melt a tube of nutrient agar in a beaker of boiling
water. Pour into it some sterilized skim milk which has been
heated to about 50° to 60° C. : the proportion of milk to agar
medium should be about one of milk to three of agar. Colo
to about 40° C, inoculate with Bs. subtllis, Bad. vulgare, or
other organism which possesses proteolytic powers. Pour into
a small Petri dish and inoculate at 30° C.

The casein of the milk is peptonised when the colonies
develop, the medium at those points becoming clear.

Try the experiment with Bad. colt and Str. ladicus.

CHAPTER VII.

FERMENTATIONS AND THE ACTION OF

EJSZYTl/LES— (Continued).

I. Putrefaction and Decay. — After the death of
animals or plants the materials of which their bodies
are composed soon begin to undergo decomposition
unless special precautions are taken to prevent it by
cooking or the application of antiseptics.

A host of bacteria of many different kinds set up
fermentations in the proteins, carbohydrates, fats, and
other organic compounds present, and step by step all
these substances are broken down into bodies of simpler
constitution, some of which have a disgusting odour.
Changes go on in all the different classes of compounds,
and the whole process is of a very complicated character ;
it is found, however, that the foul odour is particularly
associated with peculiar decompositions taking place in
the nitrogenous compounds. To the fermentations of
proteins, which are accompanied by the evolution of
evil-smelling products, the term putrefaction is applied.
Changes of this kind not only occur in the organic
compounds forming the dead bodies of plants and
animals, but also in various products, such as cooked
flesh, tinned foods, cheese, milk, and in dung and other
materials containing proteins. Putrefaction is most
evident when access of air is partially or entirely pre-
vented, i.e. when the bacteria causing it are living under

PUTREFACTION AND DECAY 101

more or less anaerobic conditions ; where an abundant
supply of air is maintained, as in porous soil or loosely-
compacted compost and manure heaps, the substances
possessing an objectionable odour are either not pro-
duced at all, or are rapidly oxidized into odourless
compounds, in which case the organic material is said
to decay. The end-products of the processes of putre-
faction and decay are the same, the proteins being
finally broken down into simple gaseous bodies such
as methane, carbon dioxide, hydrogen, ammonia, and
sulphuretted hydrogen, which diffuse into the atmosphere
or are washed into the soil by rain.

A great amount of investigation has been carried on
to determine the character of the changes involved in
the putrefaction process not only in nitrogenous materials
exposed to the air, but in food constituents passing
through the alimentary canal of animals. Experiments
have been made with pure cultures of bacteria upon
single proteins, and the general nature of putrefactive
degradation is fairly clear. In the first stages the pro-
cess is similar to that which takes place in proteins
under the influence of trypsin or boiling acids, albumoses,
peptones, and amino-acids being formed. The splitting,
however, proceeds in such a manner that only small
amounts of the two former kinds of compounds are met
with at any stage of the decomposition. The enzyme
trypsin is unable to bring about further change in the
amino-acids which it produces, but the putrefactive
bacteria can utilize these in their nutritive processes, and
carry the degradation much further. From some of the
amino-acids ammonia is eliminated, and the correspond-
ing acids without nitrogen are obtained, in the same
manner as if they were decomposed by alkalis or

102 THE ACTION OF ENZYMES

oxidizing agents. Probably in this way are produced
the formic, acetic, propionic, butyric, valerianic, caproic,
and other similar acids so frequently found in a free
state or as ammonium salts in putrefying substances.
Cinnamic and oxalic acids are sometimes present.

Two aromatic bodies, p-cresol and phenol, are often
obtained also, and are doubtless derived from tyrosine.

The most characteristic substances produced in the
putrefaction of proteins by bacteria are indole and skatole,
two compounds to which the unpleasant odour of faeces
is due. These are derived in some instances directly
from the proteins, but they are also produced from
tryptophane. The amount obtained is very variable,
and is dependent upon the species of bacterium causing
the decomposition, the nature of the medium in which
it is grown and certain factors which are not understood.
Some bacteria, such as the cholera organism and Bact.
vulgare, manufacture considerable amounts of indole when
grown in peptone solutions, while others give rise to little
or none. The presence of sugar generally interferes with
its formation. Bact. colt is able to form indole in broth
or peptone solutions so long as the liquid is neutral, but
none is obtained when the medium is acid, or when
much sugar is present. In view of the fact that one
species may produce it, and another nearly allied form
be devoid of that power, the presence or absence of indole
in a culture medium may be used as a valuable diagnostic
character in the identification of bacteria. Methods of
testing for it are given in Ex. 63.

From among the products of putrefaction a number
of weak bases have been isolated, some of which are
crystalline compounds, resembling the vegetable alkaloids :
they are known as ptomaines. Those of common occur-

PUTREFACTIVE ORGANISMS lO^

rence are various methylamines, ethylamines, certain
diamines, such as cadaverine and putrescine, and choline
and betaine. Most of them are harmless compounds or
only poisonous when taken in large amounts. Many,
however, are known which are exceedingly dangerous
even in minute doses, and the consumption of putrid
or decomposing foods containing them not infrequently
leads to fatal results. Muscarine, which has been
obtained from putrefying fish, neurine from decomposing
flesh, and tyrotoxicon from rotten cheese, are examples
of this poisonous class. Similar bodies have been
isolated from decaying shell-fish, tinned sardines, and
other foods which have undergone putrefactive changes.

It may be noted here that certain pathogenic bacteria,
such as the bacilli of tetanus and diphtheria, when
grown in culture solutions, are able to produce or secrete
much more deadly substances than the ptomaines just
mentioned. These are termed toxins^ but little is known
about their chemical nature : some of them appear to
be proteoses, while others resemble enzymes.

Sulphuretted hydrogen and methylmercaptan are
generally present in putrefying substances, and are
derived from organic sulphur compounds such as cysteine,
although in some instances bacteria are able to reduce
sulphates with the formation of hydrogen sulphide.
What becomes of the phosphorus of proteins is not
clear. It is said to be given off from decaying fish and
other organic materials as phosphoretted hydrogen, but
there is some doubt about this statement.

2. Putrefactive Organisms. — Although experiments
with pure cultures have shown that a great many species
of bacteria are able to bring about putrefactive decom-
position in materials containing protein, many of them

104 THE ACTION OF ENZYMES

are rare and not likely to occur in putrefying substances
under ordinary conditions. A small number, however,
are very widely distributed in nature, and are almost
invariably concerned in the destruction of animal and
vegetable organic matter. The chief of these are
described below.

I. Bacterium vulgare, L. and N. {Proteus vulgaris, Hauser). —
An examination of a piece of putrid meat which has been freely
exposed to the air reveals the presence of immense numbers of
minute bacteria which were formerly spoken of as Bacterium termo.
Investigation by Hauser and others showed that these organisms
were remarkable in being able to assume very many different
growth-forms, occurring sometimes as short rods almost like
cocci, and under other conditions as long straight rods or
spiral threads. On account of its variability the genus was
given the name Proteus^ and three more or less distinct forms were
described under the names Proteus vulgaris^ P. mirabilis, and
P. Zenkeri. More recently these have been grouped together as
varieties of the species Bacterium vulgare, L. and N. {Bacillus
vulgaris, Mig.). They are very common in putrid flesh, and of
frequent occurrence in water contaminated with decomposing
animal matter. They are also common inhabitants in the
intestines of man and other animals.

The following are the chief characters of typical Bad.
vulgare : —

The organisms are usually slender, very active motile rods,
from I to 4 /it long and .4 to .7 /a thick. As stated above they may
occur as rods 40 /a long or as long spiral threads. They
generally stain by Gram's method. Spores are not formed.

Bact. vulgare grows best under aerobic conditions, though
it is able to thrive as an anaerobe. From proteins it pro-
duces indole and sulphuretted hydrogen, and its cultures in
gelatine or bouillon have a foul odour.

Gelatine. — The colonies are at first thin, grey, and semi-

PUTREFACTIVE ORGANISMS 105

transparent ; later the gelatine is rapidly liquefied, the colonies
producing shallow cup-like depressions in twenty-four hours or
less at 22° C. The stab is thread-like at first; afterwards a
cylinder of turbid liquefied medium is seen, with a cup-shaped
depression at the surface.

Agar. — The colonies on agar are thin, bluish-grey, and semi-
transparent ; at first round, but irregular later and extending over
the whole surface.

Potato. — Little growth, yellowish-white.

Bouillon. — Turbid throughout, with an abundant sediment in
twenty-four hours.

Sugars. — Ferments grape and cane sugars with the production
of CO2 and H2 (i : 2); no gas from lactose.

Milk. — Casein is coagulated in two or three days, and after-
wards dissolved, with the production of a slightly acid yellow
liquid.

Proteus mirabilis, Hauser, is a form of Bad. vulgare which
readily produces pear-shaped or rounded involution forms 4 to 7 /x,
in diameter. The colonies on gelatine and agar are similar to
those of the typical species, but the former medium is more
slowly liquefied.

Proteus Zenkeri, Hauser, is also a variety of Bad. vulgare ; it
does not liquefy gelatine and grows best at 30° C. The surface
colonies on gelatine spread out in star-like forms, and the stab
shows characteristic thread-like growths all round the track of the
needle.

2. Very closely resembling Proteus Zenkeri is Bacterium
Zopfii, Kurth, originally isolated from the dung of fowls, and
since found commonly in putrid substances and impure water.
It is motile and varies much in form, very short rods and long
threads being obtained. Under certain unfavourable conditions
the threads break up into -round coccus-like cells, which develop
again into rods when placed in suitable nutrient media. The
colonies do not liquefy gelatine but grow outwards in delicate
irregular branches, the whole looking like the mycelium of a

io6 THE ACTION OF ENZYMES

mould. The gelatine stab resembles a miniature inverted spruce
fir tree. Bouillon is rendered slightly turbid and a small amount
of indole is produced. Milk is not coagulated, and on potato
the growth is usually very slight.

3. Bacterium coli, Escherich. — Another organism which is
usually present in decaying flesh and other organic substances is
Bad. coli commune, or Bad. co/t (Esch.) (Fig. 26). . It is a con-
stant inhabitant in the intestines of all mammals, and has
been isolated from the digestive tract of birds and fishes. The
excreta of animals contain it in enormous numbers, and it may
be considered as one of the most widely distributed of all
bacteria. It is not infrequently met
with in milk and other dairy products
which have been contaminated with the
dung of cattle, and from manured soils
and those containing decaying organic
debris it finds its way into well and river
water. A very large number of allied
varieties and forms are known which
differ so very slightly from each other
Fig. ■zd.— Bacterium coli, Esch: that their description and classification
*°°° ' is a matter of great difficulty.

Typical Bad. coli is a short, stumpy bacterium with rounded
ends, motile when young, and does not form spores nor stain by
Gram's method. It is generally single or united in pairs, each
cell being about 1.3 to 3/0, long, and .4 to .5 /a broad It grows
best as an aerobe at 37° C, but will thrive under anaerobic
conditions, especially when cultivated in media containing sugar.
Gelatine. — The surface colonies on gelatine are moist, trans-
lucent, whitish, flat and thin, with an irregular wavy outline, the
deep ones being round and yellowish-brown : there is no
liquefaction of the medium. In gelatine stab it forms a thick
white opaque growth along the track of the needle.

Agar. — On agar the colonies are smooth, shining, greyish-
white, elevated and not so irregular in outline as on gelatine.

PUTREFACTIVE ORGANISMS 107

Potato. — A yellowish-brown growth with puckered elevated
outline.

Bouillon. — The whole liquid is soon turbid and a dense
sediment is deposited. In peptone water indole is produced and
much sulphuretted hydrogen gas.

Milk is rendered acid at 37° C. and coagulated in less than
seven days, the curd containing more or less gas.

Sugar. — Grape sugar and lactose are fermented with the pro-
duction of acids and much gas, about one-third of which is carbon
dioxide, the rest hydrogen and nitrogen. It ferments dulcite also,
but most forms do not attack saccharose. A variety which
ferments and produces gas from the latter sugar is known as
Bad. coli communior.

4. Associated with Bad. coli in the alimentary canal of many
animals is Bacterium lactis aerogenes or Bacterium aerogenes,
Escherich, which was first isolated from the small intestine and
faeces of infants fed on cows' milk. It is found throughout the
intestinal tract and in the stomach, while Bad. coli is chiefly
confined to the colon and rectum.

Typical Bad. aerogenes is a short, thick bacterium very
similar in form and dimensions to Bad. coli^ usually i to 2 /a
long and .5 /* broad, though much longer cells are not
uncommon : it is Gram negative.

Like Bad. coli it does not form spores, and is a non-
liquefier ; it is, however, never motile at any stage of its
development.

Gelatine. — On gelatine the surface colonies are rounder in
outline, opaque, porcelain - white and convex. The deep
colonies are greyish-brown and granular.

Agar. — Thick white shining colonies.

Potato. — Whitish or yellowish brown growth, with elevated
warty edges and cheesy odour.

Bouillon. — Turbid, with much sediment, and usually a pellicle
or scum on the surface. It forms indole.

Milk. — Milk soon becomes strongly acid, and is coagulated,

lo8 THE ACTION OF ENZYMES

the curd having small gas bubbles in it. The organisms when
grown in this medium, generally develop capsules.

Sugars. — Glucose, lactose, and saccharose are all fermented
with the production of acid and much gas, but it does not
ferment dulcite. When Bacf. lactis aerogenes is grown for three
days in 2 per cent, glucose bouillon in a fermentation tube, and
a strong solution of caustic potash then added, a pink or reddish
fluorescent colorisation is developed after twenty-four hours :
this is known as the Voges-Proskauer reaction. (Ex, 65.)

5. Another "coliform" organism frequently met with is
Bact. cloacae, Jordan. It is one of the common bacteria in
sewage, has been isolated from the intestinal canal, and some-
times occurs in impure water and dirty milk. This bacterium
is motile, and in general morphological characters closely
resembles B. colt. The surface colonies in gelatine are thin,
whitish, and translucent, with darker centre and wavy outline ;
the deeper ones round and yellowish. Gelatine is slowly lique-
fied in four to thirty days. Bouillon is rendered turbid in twenty-
four hours, generally with slight scum formation. Milk is
acidified and coagulated in ten to eighteen days. It ferments glu-
cose and saccharose rapidly with acid and gas production, and
lactose, also, when the cultivation is prolonged. Dulcite is not
fermented by it, and it does not produce indole in peptone solu-
tions; like B. aerogenes, however, it gives the Voges-Proskauer
reaction.

Other organisms closely allied to B. colt are Bact. Griinthal,
Bact. neapolitanus, Bact. oxytocus perniciosus, and Bact. coscoroba.

For the biological characters of Bact. coli and " coliform "
glucose-fermenting organisms, see table on p. 306.

6. Bacterium fluorescens liquefaciens, Fliigge, is one of the
commonest species of bacterium in water, soil, and sewage ; it
also occurs in milk and other food materials sometimes, and
assists in the putrefaction of organic substances. It is a motile
organism from 1.4 to 5 a long, .4 to .5 ,a broad, with a bundle of
three to six polar flagella, on account of which it is occasionally

PUTREFACTIVE ORGANISMS 109

included in the genus Fseudomonas. B.fiuorescens is markedly
aerobic, stains very slightly or not at all by Gram's method, and
is somewhat difficult to stain with any dye except hot carbol-
fiichsin. It grows rapidly at 20° C., and does not form spores.

Gelatine. — On gelatine the surface colonies are round at first,
later somewhat irregular in outline, and producing rapid lique-
faction ; the deep colonies are pale yellow or greenish yellow.
The liquefied material exhibits a characteristic yellowish or
bluish-green fluorescence ; in it the bacterial colonies are found
as flocculi or crumbly masses, which settle to the bottom of the
liquid. The gelatine stab liquefies in the form of a cylinder,
with funnel-shaped depression at the surface.

Agar. — Colonies moist, rounded, yellowish or greenish, with
fluorescent border.

Potato. — Growth moist, slightly raised, yellowish at first, later
pale sepia brown.

Bouillon. — Becomes very turbid, with a pellicle on the surface,
and shows a striking yellowish green fluorescence ; usually only
traces of indole are found.

Milk is gradually changed to a yellowish-green alkaline liquid,
generally without previous coagulation ; any curd which is pre-
cipitated soon dissolves.

Sugar. — Glucose bouillon is rendered acid without develop-
ment of gas.

A number of allied forms are known, which are possibly
varieties of one species. The " green " and " blue " pus organ-
ism, Bact. pyocyaneum, Fliigge, a pathogenic species or form
commonly associated with pus of wounds which heal unsatisfac-
torily, is very nearly related, or merely a modification of Bact.
fluorescens liquefaciens. The two organisms are morphologically
alike, and in many physiological characters very closely resemble
each other also.

Another organism, Bact. fluorescens non-liquefaciens, Eisenberg
{Bact. immobile^ Kruse), resembles the type, but liquefies gelatine
very slightly. It is nearly related, if not identical, with Bact,

no THE ACTION OF ENZYMES

Jluorescens piitidus, Fliigge, frequently isolated from putrefying
substances, to which it gives a greenish tint and an odour of
trimethylamine. The latter is a motile organism, 1.6 to 5 [l long,
.4 to. 6 //. broad, with one or two flagella only. The surface
colonies on gelatine are transparent of coli form, but yellowish-
green in colour, and do not liquefy the medium ; in other
respects the organism agrees with B act. Jluorescens liquefaciens.

7. A common inhabitant of putrefying material is Bs.
prodigiosus, a description of which is given on page 282. It
sometimes develops bright red blood-like spots on bread, potato,
and other starchy foods.

8. In addition to the organisms previously mentioned certain
species of cocci, namely : (i) Micrococcus pyogenes (L. and N.),
var. aureus {Staphylococcus pyogenes aureus^ Rosenbach) ; (2)
Streptococcus pyogenes, Rosenbach ; (3) Micrococcus flavns,
Fliigge, are very frequently present in the early stages of the
putrefaction of organic substances. All are widely-distributed
organisms. The two former are of special importance to the
pathologist, since they are responsible for several inflammatory
and suppurative diseases in man.

(i) M. pyogenes is a coccus from .9 to i /x in diameter, and
occurs in irregular clumps or in pairs. It is non-motile, and
stains by Gram's method. Growth goes on well at tempera-
tures between 27 to 37° C., especially as an aerobe, although it
will thrive under anaerobic conditions. The colonies in
gelatine are small, whitish or pale yellow, and liquefy the
medium. On agar and potato they are whitish at first, but
ultimately become a bright orange yellow colour. Milk is
coagulated and made acid in a week or less. Bouillon soon
becomes cloudy, but finally clears, and deposits a yellow sedi-
ment.

Several varieties are met with, some of them differing in
the production of lemon-coloured (var. citreus) or white colonies
(var. albus).

(2) Streptococcus pyogenes occurs in the form of long chains,

PUTREFACTIVE ORGANISMS III

each coccus being about i /x in diameter, and readily stains by
Gram's method. It is a facultative anaerobe, and grows very
slowly at 20° C., the optimum temperature being 37° C. The
colonies on gelatine and agar are always very small, round, and
white or yellowish white, almost flat, and do not liquefy the
medium. On potato there is little or no growth. Milk is
generally coagulated in twenty-four hours or less, and glucose
bouillon becomes acid without separation of gas. A great
many allied forms or species are known, differing chiefly in
length of " chains " and in pathological virulence.

(3) Micrococcus flavus is a coccus .5 to i /a in diameter,
occurring usually in pairs, or three or four together. It is an
aerobic species, which grows well at 20° C. The gelatine colonies
are small, round, yellowish-white ; they liquefy and sink into the
medium. The liquefied gelatine stab is funnel-shaped, with
yellowish sediment. On potato its growth is distinct, thin, and
yellowish or greenish yellow. Milk is only partially coagulated,
and rendered acid in two or three weeks. This species is said
to be identical with or closely allied to Sarcina hitea^ Fliigge,
but typical sarcina " packets " are not formed.

9. The spore-producing species, Bs. subtilis (page 124) and
Bs. vulgatus (page 126), are not uncommon bacteria in decaying
organic matter.

10. The organisms already described are those chiefly found
in the early stages of the putrefactive process, when there is
abundant access of air. The production, however, of some of
the most characteristic foul-smelling substances in decomposing
materials is dependent upon anaerobic conditions, and bacteria,
which carry on their work best in the absence of oxygen, are
always • present in putrefying substances. In fact, the proteins
of organic matter appear to be more rapidly and more
effectually broken down by anaerobic species than by the
aerobes.

The chief of these anaerobes is Bacillus putrificus, Bienstock,
which is found in dung and sewage, and is very common in

112 THE ACTION OF ENZYMES

the soil. It is a long, motile, rod-shaped organism, 5 or 6 /a
long and .8 /x broad. Very resistant spores are formed at 35° C.
which arise at one end of the bacillus, the whole becoming
the shape of a drum-stick. Bs. putrificus produces a tryptic
enzyme, which splits up proteins with the formation of amines,
a solution of gelatine being changed into a foul-smelling liquid,
with the evolution of much gas, when access of air is pre-
vented.

Ex. 59. — Show the production of ammonia from proteins.
Prepare the following solution :—

Di-potassium phosphate . . 5 gr.
Magnesium sulphate . . 2.5 gr.
Sodium chloride . . . 2.5 gr.
Water ..... i litre.

Take three 200 c.c. flasks and pour into each 100 c.c. of the
solution.

Add to one . i per cent, of gelatine,
„ another . i per cent of fibrin,
„ a third . i per cent, of peptone,
and sterilize.
Inoculate each with Bs, mycoides and incubate at 30° C. for
ten to fifteen days.

{a) Test for ammonia with Nessler's solution.
{b) Heat with magnesia, and test the gas given off with
litmus paper.
Control tubes uninoculated should be tested also.

Ex. 60. — Add to a litre of the inorganic solution in previ-
ous exercise —

Glucose . . . . . 2 gr.
Sodium nitrate . . . 2 gr.

Sterilize: inoculate 100 c.c. of it with Bs. mycoides. Incubate
at 30° C, and every second day : —

(i) Test for nitrites (Ex. 74).

PUTREFACTIVE ORGANISMS 113

(2) Test for ammonia with Nessler's solution.

(3) Find out when the nitrite disappears.

Ex. 61. — To a very dilute solution of egg albumin add a
dilute solution of ferrous sulphate to prevent coagulation;
sterilize at 100° C. Inoculate with pure cultures of —
(a) B. mycoides^
{b) B. fluorescens liquefaciens^
(c) B. (Proteus) vulgare}
and incubate at 30° C. for ten to fifteen days.

(i) Test for ammonia with Nessler's solution.
(2) Heat with magnesia and test the gases evolved with
litmus paper. Compare with uninoculated sterilized
control tubes.

Ex. 62. — Chop in pieces a small piece of beef and place it in
a beaker of water.

Leave in a warm place for twenty-four to forty-eight hours until
putrid.

Examine a drop of the putrefying liquid for Proteus bacteria.
Note their lively motion.

Stain some with gentian-violet. Inoculate gelatine and agar
plates with a drop of the putrid liquid diluted with sterile water
and observe the form of the colonies obtained.

Ex. 63. — The Indole Test. — Prepare a 2 per cent, solution of
peptone; add to it \ per cent, of common salt and make it
slightly alkaline with a drop or two of sodium carbonate solution.

Place about 10 c.c. in a number of tubes and sterilize.

Inoculate tubes with —

1. Bad. vulgare,

2. Bact. coli,

3. Bact. lactis aerogenes,

4. Bact. fluorescens^

and incubate for six to seven days.

^ Pure cultures of these and other organisms can be obtained from the
Author.

114 THE ACTION OF ENZYMES

Test for indole in each —

{a) With sodium nitrite. — Make a small stock 5 per cent,
solution of sodium nitrite. Take i c.c. of this and dilute it to
100 c.c. Then add i or 2 c.c. of the dilute .05 per cent, solution
to the tube to be tested, and pour a few drops oi pure concentrated
sulphuric or hydrochloric acid down the side of the tube.

The acid liberates nitrous acid, which reacts with any indole
present, producing a pink or purple colour. If pink colour is
absent or not distinct, place the tube for thirty to forty minutes
in an incubator at 37° C.

Control tests should be made in uninoculated tubes to show
that the reaction is due to the growth of the bacteria.

{b) With paramidobenzaldehyde.

i. Marshall's test.

Dissolve 6 gr. of para-dimethylamidobenzaldehyde in 100 c.c.
of water and add 1 2 c.c. of concentrated sulphuric acid.

This reagent gives a rose or cherry-red colour when added to cul-
tures containing indole, and is more reliable than the former test.

ii. Ehrlich's test.

Solution A. Dissolve i gr. of para-dimethylamidobenzaldehyde
in 100 c.c. of 96 per cent, alcohol, and add 20 c.c. of concentrated
hydrochloric acid.

Solution B. Saturated solution of potassium persulphate in
water. To 10 c.c. of the culture to be tested add 5 c.c. of each
of the solutions A and B ; shake and allow to stand for five
minutes : a red colour indicates the presence of indole.

Ex. 64. — Grow the organisms mentioned in the previous
exercise in slightly alkaline sugar-free bouillon.

Incubate for eight to ten days. Then test for indole, as above.

Ex. 65. — The Voges-Proskauer Reaction. — Prepare a 2 per cent,
glucose bouillon : place it in fermentation tubes and sterilize.

Inoculate tubes with —

1. Bact. colt,

2. Bact. lactis aerogenes,

3. Bact. cloacce,

and incubate at 22° C. for three days.

OXIDIZING ENZYMES OR OXIDASES 115

Then add 2 or 3 c.c. of strong caustic potash solution.

The development of a pink colour on standing exposed to the
air for a time indicates the Voges-Proskauer reaction. Which of
the above give it ?

3. Oxidizing Enzymes or Oxidases. — When certain
fungi are cut open and the cut surfaces exposed to the
air the colourless juice or cell-sap of the plant becomes
blue, red, brown or otherwise colourized. A brown
colour also develops on the cut parts or in the expressed
juices of apples, pears, and many other fruits and vege-
table tissues. These colour changes are due to the
oxidation of certain compounds through the agency of
enzymes, which have been termed oxidases. They are very
widely distributed in the cells of plants and in milk and
other animal secretions : they are also produced by many
kinds of bacteria as well as by yeasts and other fungi.

Like other enzymes, they act in a specific and restricted
manner upon certain definite substances only. All of them
behave as carriers of oxygen, yielding the latter element to
the substances which are oxidized, and reoxidizing them-
selves from the air or from other easily reduced compounds.
In many cases the " browning " of plant tissues has been
shown to be due to reddish or brown compounds, resulting
from the oxidation of tannin or tyrosine by oxidases.

Some of them, which may be spoken of as direct
oxidases^ can utilize the free oxygen of the air ; others
are only able to effect oxidation in the presence of a
peroxide, such as peroxide of hydrogen, which readily
yields active nascent oxygen ; such are X.^xvci^^ peroxidases.

The presence of oxidases is readily tested by means of
an alcoholic solution of gum guaiacum, which when
oxidized gives a rich blue colour.

The addition of a direct oxidase results in a blue

Ii6 THE ACTION OF ENZYMES

colour immediately, but the colour does not develop until
hydrogen peroxide is added if a peroxidase only is present.

Ex. 66. — Make a tincture of gum guaiacum by adding a
few lumps of this substance to a test-tube or flask containing
alcohol; boil for some time, and to the yellow solution add
water cautiously until near the point at which a permanently
milky emulsion is formed. If too much water is accidentally
added, and the solution remains milky, it may be cleared by the
addition of a few drops of alcohol.

(a) Cut thin slices of apple, potato, and mangel, and place
them in small portions of the guaiacum solution.
Note the production of a blue colour.
(If) Try the experiment with slices which have been boiled for

five minutes.
(c) Add the juice from raw beef to some of the solution, and
note if a blue colour is produced; if not, try the
addition of a few drops of hydrogen peroxide.

An enzyme termed catalase is very widely distributed
in the protoplasm of all living animals and plants, and
is also found in many bacteria. It has the power of
liberating oxygen from hydrogen peroxide, but the
oxygen set free does not give a blue colour with tincture
of guaiacum.

Ex. 67.

(a) Pour some hydrogen peroxide solution over the surface

of an agar plate culture of several different kinds of

bacteria, and note the evolution of bubbles of oxygen.

Where are the bubbles most abundant ?
(^) Try the same experiment, using the tincture of guaiacum

and hydrogen peroxide mixture of Ex. 66. Is any

blue colour produced?

CHAPTER VIII.

BACTERIOLOGY OF THE SOIL.

I. The bacteriological examination of the soil is one of
the most recent branches of scientific study. A good
beginning has been made, but a very extensive unex-
plored field yet lies before the soil bacteriologist.

The work carried on by certain groups of organisms
has been examined in considerable detail, and the
processes of putrefaction and decay of organic substances
so far as relates to the nitrogen in them are fairly
understood. Much is known also of the nature and
causes of nitrification, denitrification, and the fixation or
assimilation of the free atmospheric nitrogen by soil
organisms ; but many of the problems connected with
the formation and decomposition of humus, the chemical
changes induced by bacteria and fungi in the mineral
constituents of the soil, and the influence of biological
processes upon the physical character of the latter await
solution.

Not only is further research needed respecting indi-
vidual processes going on within the soil, but systematic
study of the various kinds of organisms present in it —
moulds, algae, and protozoa, as well as bacteria — is
greatly wanted. The relationship of the work done by
each group must be exhaustively investigated, and the
relative importance of the various biological processes

in the production and maintenance of the fertility of

X17

Ii8 BACTERIOLOGY OF THE SOIL

cultivated land are problems which have yet to be dealt
with.

2. Numbers of Bacteria in the Soil. — It is a difficult
matter to determine the number of bacteria in a sample
of soil, and no really reliable method exists at present
for the accurate numerical estimation of all the various
kinds which may be present in it.

The counting of the colonies on agar or gelatine plates,
inoculated either with a definite amount of soil or with a
measured volume of sterilized water mixed with the soil
to be examined, is generally adopted. However, many
kinds pf soil organisms, and especially those concerned
with the fixation of nitrogen and the process of nitrifica-
tion, do not grow at all on these media, and unless
special precautions are taken to exclude air no colonies
of the anaerobic forms appear upon the plates.

The method adopted to free the bacteria from the soil
particles, the nature and chemical reaction of the media
employed, and many other factors influence the result.

The figures given below are comparative only, and
have mostly been determined by plate cultures on neutral
or slightly alkaline gelatine or agar media.

Very little is known in regard to the relationship
between the number of organisms in a soil as determined
by the plate methods in ordinary agar and gelatine
media and its fertility. That an increase or decrease in
the number of bacteria present has an important bearing
on the material available for the nutrition of crops is
certain, but much research must be prosecuted before the
connection is clearly understood.

The soil, containing as it does considerable amounts
of water and stable organic matter, is an excellent
medium for the development and multiplication of many

NUMBERS OF BACTERIA IN THE SOIL 119

kinds of bacteria. The number present in it varies very
much, being dependent on a great many factors, the
chief of which are the kind of soil, the depth at which
the sample is taken, the amount of moisture in it, its
physical condition, the time of year at which the
examination is made, and the kind of crop grown upon
the land and the manuring it has received.

Remy found in one gram of arable soil in fair con-
dition from 6 to 10 millions of bacteria and half a million
moulds, while in a poor sandy soil there were only 200
to 450 thousand bacteria and from 45 to 168 thousand
moulds.

Other investigations have shown that from i to 5 or
6 millions may be expected in most ordinary soils.

The bacteria are chiefly met with near the surface of
the ground, i.e. within the first 9 to 12 inches, where
good aeration and a suitable degree of moisture and
temperature prevail. Below this the conditions favour-
able for bacterial life are absent, and the numbers
diminish rapidly with increasing depth until a point is
reached where the soil is sterile.

Houston found about 1,688,000 in a gram of surface
soil, at a depth of i foot 1,100,000, at 3 feet deep
174,000, and at 6 feet below the surface only 410.
Other examinations have shown that in some soils few or
no living micro-organisms were met with below about 3
or 4 feet. Where the soil is of open texture and contains
large amounts of organic compounds the number in-
creases, and more bacteria are able to penetrate and live
in the deeper layers than where the soil consists of poor
compact clay.

The number of bacteria appears to go up and down
with variations in temperature at different seasons of the

120 BACTERIOLOGY OF THE SOIL

•
year, but careful observations have shown that this is
more due to alterations in the degree of moisture of the
soil than to temperature changes. The experiments of
Engberding and others indicate that the optimum
development of the majority of organisms occurs when
the soils contain 70 to 80 per cent, of their maximum
water-holding capacity.

Dry soils contain {gw bacteria, and dryness has been
shown by Lohnis to have an injurious effect on nitrifica-
tion and nitrogen assimilation.

Acid peaty soils and marsh land are poor in bacteria.
After drainage the number increases only slightly, but by
cultivation and the application of lime, sand, clay, and
dung the bacterial content soon approximates to that of
ordinary cultivated soils.

. The following figures of the change in the number of
bacteria after draining and cultivation of " ling, cotton
grass, and sphagnum moor " in Sweden are given by
Fabricius and von Feilitzen : —

Virgin moor.

Drained.

With addition of
sand and lime.

In May, .

56,000

204,000

4,797,000

July, .

225,000

384,000

8,129,000

Sept, .

144,000

64,000

7,771,000

Farmyard dung and other organic manures contain
enormous numbers of bacteria and large amounts of
compounds upon which bacteria thrive. After these
are applied to the soil the numbers, especially of putre-
factive and denitrifying organisms, rapidly rises for a
time. Heavily manured soils, or those to which sewage
has access, may contain from 50 to 120 millions per
gram. Later, however, the number falls to the original

NUMBERS OF BACTERIA IN THE SOIL 121

figure, the normal soil bacteria finally getting the upper
hand in the struggle which ensues.

No doubt the food material suited to B. coli and
other allied putrefactive forms soon diminishes, and
there is probably also an accumulation of organic acids
and other compounds inimical to the growth of this class
of bacteria.

The ploughing in of weeds, stubble, and " green
manures," which contain organic nitrogenous and carbon
compounds, provides pabulum for soil organisms and
assists their development.

Certain forms of Streptothrix and many moulds are
increased by the addition of straw, and probably the
organisms which " fix " free nitrogen are increased also
by straw residue.

Ammonium sulphate and nitrate of soda can be
utilized by many soil bacteria for nutritive purposes,
and potash phosphates and lime have been found to
assist in the development of the nitrogen assimilating
group of bacteria.

The growth of certain crops favour the development
of bacteria in the soil more than others. Stohlau and
Ernest in one case found where clover had been grown
seven or eight millions present in a gram of the soil,
but only one to two millions per gram of soil from an
adjoining plot after barley.

Ex. 68. — Prepare the following '* Heyden-Agar " medium : —
Agar, . .12.5 gr.
Albumose, . 7-5 gr.

(Heyden)
Distilled water, . i litre.
Dig up a block of soil with a spade ; break the block, and from
the centre take out a small sample and place it in a sterilized bottle.

122 BACTERIOLOGY OF THE SOIL

From this weigh out 15 or 20 gr. and add it to 300 or 400 c.c.
of sterilized tap water in a J-Htre flask. Shake well, and if
necessary break up the soil with a sterilized glass rod.

Take up with a wide bored sterilized pipette 25 c.c. of the
turbid water, and add it to another flask containing 300 or 400
c.c. of sterilized tap water.

From this second flask take 25 c.c. and transfer to a third, and
from the third to a fourth flask.

A. Inoculate .a Petri dish of the Heyden agar medium with
I c.c. of the water from the fourth flask and incubate at 20° C.
Count the colonies which appear in ten days, and calculate the
number of bacteria present in i gr. of the original soil.

B. Inoculate a Petri dish of —

(i) Nutrient gelatine.

(ii) Nutrient agar neutralized or rendered very slightly
alkaline with a drop or two of scKiium carbonate
solution, with i c.c. of the " soil-dilute," as in A.
Incubate at 20° for ten days.
Examine every day and count the colonies. Use a Pake's
disk (Fig. 45) beneath plate to facilitate counting.

The colonies of liquefiers on the gelatine medium may be
killed by touching them with a " caustic pencil " or stick of silver
nitrate.

Compare the results obtained with the three media.

Ex. 69. — Repeat the above with —

(i) Soil from a well- manured garden,
(ii) „ an arable field,
(iii) „ an old pasture.

3. Kinds of Bacteria in the Soil. — A large number of
kinds of bacteria have been isolated from the soil. Some
of them are added to it in the manures which are applied
to the land, others find their way into it in drainage water,
sewage, and rain. Many of the species are casual introduc-

KINDS OF BACTERIA IN THE SOIL 123

tions, whose presence is dependent more or less upon
accident. Others there are which must be considered as
normal inhabitants of the soil. To the latter class belong
many of the putrefying and denitrifying organisms, as
well as the nitrifying and nitrogen-fixing bacteria which
are described in the succeeding pages. The number of
these found by Lohnis in samples of soil taken from a
field in January and July respectively were : —

Jan.

July.

Putrefactive bacteria, .

. 3,750,000

5,000,000

Urea-splitting „

50,000

50,000

Nitrifying „

7,500

2,500

Denitrifying „

50,000

50,000

Nitrogen-fixing „

1,125

750

Certain anaerobic spore-bearing forms are frequently
met with also. Among the bacteria of especially common
occurrence in most field and garden soils may be men-
tioned : — Bs. mycoideSj Bs. subtilis, Bs. vulgatus^ Bs.
megatherium^ Bad. fluorescens liquefaciens^ Bad. fl, 7zon-
liquefacienSy Bs. putrificus, Micrococcus candicans^ various
forms of Proteus, and certain species of Cladothrix.

Bact. coli is commonly present in soils which are polluted
with sewage, or to which dung and similar organic excreta
have been recently added, but is not usually found, or
only in relatively small numbers, in virgin soils.

In addition to these, certain pathogenic species, such
as Streptococcus pyogenes, Bs. tetani, and Bs. cedematis
maligni, are not uncommonly met with in the soil.

Gottheil made an extensive study of the spore-bearing
bacteria attached to the roots of carrot, beet, radish,
turnip, parsley, cabbage, and other plants, and found

124 BACTERIOLOGY OF THE SOIL

Bacillus asterosporus^ Bs. ellenbachensis, Bs. graveolens,
Bs. pumiluSy Bs. ruminatus, Bs. tumescens, and a form of
Bs. subtilis were very frequently associated with roots of
the plants mentioned.

Whether a form of symbiosis exists between the higher
plants and these organisms is not at present clear.

Below are descriptions of the chief common organisms
present in almost all soils.

Bacillus subtilis, Cohn (Fig. 27), commonly known as the
*'hay bacillus," is found in hay and in almost all soils. It
is a stout rod-shaped organism, from 3
to 9 /-t long, .8 to I iJj broad, rounded
at the ends, motile, and often united
into long chains. Bs. subtilis is strongly
aerobic, and produces oval spores 1.7
to 2 /A long and .8 broad, which
germinate from the side (equatorial
germination).

The organism stains by Gram's
method.

Fig. 27. — Bacillus subtilis, Cohn /^ i ,' 't-.i r i

( X 850). Gelatine. — 1 he surface colonies soon

liquefy, producing a cup-shaped de-
pression, filled with greyish liquid. Examined with a low power
( X 60), very young colonies are yellowish and round, the edges
usually fringed with hair-like extensions.

In gelatine stab the liquefied portion is cylindrical, greyish-
white, with a firm white skin on the surface.

Agar. — The surface colonies are small, greyish-white, and,
with a low power ( x 60), seen to have very ragged irregular edges,
these parts resembling tangled threads of cotton. The deep
colonies generally have a more tangled appearance than those
on the surface.

The stab is thread-like, with one elevated round, moist growth
at the surface of the medium, greyish in colour.

KINDS OF BACTERIA IN THE SOIL 125

Po/afo.— Thick, white or yellowish growth, dull, becoming
mealy in older cultures.

Bouillon. — The liquid soon becomes turbid, and a skin forms
on the surface or becomes attached to the sides of the tube.

Milk. — Milk is rendered strongly alkaline, and is coagulated,
the curd eventually dissolving : a pellicle forms on the surface.

Sugar. — Solutions of dextrose and saccharose are made
acid.

Nitrate bouillon. — The nitrates are rapidly reduced to nitrites
and a pellicle forms.

Bs. mycoides, Fliigge, sometimes spoken of as the root
bacillus, is a very common inhabitant of most soils.

It is a stout rod-shaped bacillus, 6 to 12 /a long and about
1.2 /A broad, with almost square ends, only slightly motile as a
rule. It forms chains, is aerobic, and stains by Gram's method.

The spores are oval, 1.5 to 2 /x- long, .8 to .9 broad.

Gelatine. — The colonies appear to be formed of fine extra-
ordinarily tangled threads, sometimes in the form of a circular
patch, but soon ramifying outwards in all directions, and very
much like a growth of mould. The medium is, however, soon
Hquefied.

The stab is whitish and characteristic, the growth extending
from the liquefied track in the form of delicate hairs arranged
parallel to each other. On the surface a felted membrane is
formed.

Agar. — The surface colonies in agar are at first round and
greyish, tangled at the edges. The stab has hair-like extensions,
like those in gelatine.

Potato. — The growth on potato is abundant and very closely
resembles that of Bs. subtilis.

Bouillon soon becomes turbid, with a thick pellicle on the
surface ; later a sediment is formed and the liquid clears.

Milk is made alkaline and the curd peptonized.

Bs. megatherium, De Bary. — This organism was originally
isolated from infusions of cabbage leaves and is commonly

126 BACTERIOLOGY OF THE SOIL

present in soil. It is a large bacillus, 5 to 20 /^ long, 1.5 to 2 /a
broad, sometimes slightly bent and united in chains ; the ends
are not rounded.

It moves with a somewhat sluggish motion, is aerobic, and
produces large spores, 2 to 2.7 a^- long and i At, broad, which
exhibit polar and equatorial germination.

Stained by Gram's method.

Gelatine. — The colonies are round at first, later kidney-
shaped, generally without the fringed edge so frequently seen in
Bs. subtilis. The medium is liquefied.

The stab shows tube-like liquefaction, the liquid being turbid.

Agar. — Greyish elevated colonies, somewhat granular, with
short ramifications round the edges, capable of being drawn into
threads, and more slimy than those of Bs. subtilis.

Potato. — Like Bs. subtilis^ but usually yellower and more
mealy.

Bouillon is made turbid.

Milk. — Curd precipitated with very slight acid reaction at
first ; later slightly alkaline with little peptonization of the casein ;
no pellicle is formed.

Nitrate bouillon. — The nitrates are not reduced, and no pellicle
is formed.

Bs. ellenbachensis, «, Caron. — The " Alinit-bacillus " (see
p. 183). The bacillus closely resembles B. megatherium^ and
according to some authorities is a form or variety of this organism.
It is rod-shaped, 3 to 6 /x long, 1.25 to 1.5 ju, broad, often single
or united in pairs. Few or no long chains are formed. It is
motile, produces spores which germinate at the poles, and is
stained by Gram's method. Its behaviour on the various media
is very similar to that of Bs. subtilis.

Bacillus vulgatus, or Bs. mesentericus vulgatus, Fliigge. —
This organism is ordinarily termed the "potato bacillus," growths
of it being frequently found on imperfectly sterilized potato
slices. It is a common soil bacteruim, usually shorter and more
slender than B. subtilis^ being 2 to 7 /a long and .8 /x broad,

KINDS OF BACTERIA IN THE SOIL 127

frequently united into short threads. It is aerobic, motile, stains
by Gram's method, and forms small spores i to 1.2 /x long and
.6 broad.

Gelatine. — Surface colonies round, with greyish-white crumpled
skin, which soon sinks in the liquefied medium. The edges of
the colonies are not finely radiated as in Bs. subtilis^ but are
seen to be irregularly lobed when magnified ( x 50).

The stab liquefies in the form of a narrow tube, the surface
being covered with a greyish membrane.

Agar. — Surface colonies greyish, round, moist, and slighdy
elevated, becoming drier and wrinkled ; deep colonies, round or
spindle-shaped.

Potato. — The growth is abundant, usually greyish or yellowish,
glistening, and very much crumpled and twisted. Some varieties
produce a pink, brown or blackish colour.

Bouillon. — Slightly turbid, with a strong whitish film on the
surface.

Milk is generally coagulated slowly, and rendered alkaline and
slimy ; the curd is peptonized after a time.

Micrococcus candicans, Fliigge, is a coccus very widely dis-
tributed in the air, water, and soil. The cocci are i to 1.2 /x in
diameter, usually single or collected in small irregular clumps.
It is an aerobic organism, grows well at room temperature, and
is stained by Gram's method.

Gelatine. — The surface colonies are non-liquefying, round, in
about a week 2 to 3 mm. in diameter, raised, moist, and milk-white.

The deep colonies are small, .3 to .5 mm. in diameter, round
or lens-shaped, and opaque.

The stab is thread-like and beaded.

Agar. — The colonies are similar to those in gelatine, but whiter
and more raised.

Potato. — A dense white, oily growth with wavy margin, extending
little.

Bouillon. — Generally very turbid at first, ultimately becoming
clear with sediment and a surface pellicle.

128

BACTERIOLOGY OF THE SOIL

Milk. — Becomes slightly acid after two or three weeks without
coagulation.

Cladothrix dichotoma, Cohn. — This organism belongs to
Chlamydobacteriacese, a family of organisms included among the
bacteria, but showing much affinity with the blue-green Algae. It
is met with in the form of long cylindrical threads from i to

3 //- in diameter and i to 2 mm.
long, attached at one end to
bits of sand, earth, or vegetable
debris, in water and damp soil.

Each thread consists of a
gelatinous sheath, which en-
closes rod-shaped cells, 3 to 8 /a
long and i to 2.5 /a broad,
arranged end to end throughout
its whole length (Fig. 28),

The free ends of the thread
appear to be branched dicho-
tomously; this arises not through
a true division of the growing
point, but is brought about by
some of the cells in the sheath
dividing and growing out later-
ally into new filaments.

Some of the cells leave the
sheath, becoming flagellated or-
ganisms, which swim about for a time ; later these settle down
and begin to divide and form new threads.

C. dichotoma is not infrequent in water pipes and drains,
and one or more closely related kinds are sometimes found in
the soil: the "Bismarck brown" Cladothrix of Houston, is
probably identical with this species.

Gelatine. — The colonies are white, small, from . 5 to 2 mm. in
diameter, slow in growth, usually with a brown centre, and stain-

FlG. 28. — Cladothrix diclwtoma, Cohn.

1. As seen under low magnification

( X 100).

2. Highly magnified ( X 750), show-

ing sheath enclosing cells of
organism.
a Cells free from sheath.

KINDS OF BACTERIA IN THE SOIL 129

ing the surrounding medium a brown colour. The borders have
a fringe of filaments round them.

Slight liquefaction of the medium occurs in a week or ten days.

It grows well on gelatine medium (4 to 4.5 per cent, gelatine),
to which a very small amount of Lemco has been added ; also
in dilute bouillon.

Agar. — Whitish colonies, with wrinkled skin-like surfaces ; the
agar stained brown.

Potato. — Thick grey growth, with brown staining of the potato.

Bouillon. — White flaky colonies, slowly produced in it; the
medium is coloured a rich deep brown tint.

Milk. — Slowly made acid ; probably peptonized also.

Other species isolated from soil are Cladothrix rufula, Wright,
which colours media pinkish ; CI. profundus, Rav., and CI.
intestinalis, Rav., which stain the media brownish ; C. fungi-
formis, Rav., which does not stain the medium.

Ex. 70. — Dig up a spadeful of soil from—

1. A sandy field.

2. A field of medium loam.

3. A field of stiff clay.

4. A well manured garden.

5. A peaty heath.

Transfer untouched portions of the soil dug up to sterilized
wide-mouthed bottles.

From each of these samples take about 10 grams, and drop
them into separate flasks containing 250 c.c. of sterile tap water;
shake well.

Then remove io c.c. of the turbid water with a sterilized
pipette, and add it to another flask containing 250 c.c. of sterile
tap water : shake well.

Repeat the dilution once again, i.e. lo c.c. of the latter mixture
into 250 c.c. of water in a third flask.

From this third dilution take i c.c. and inoculate an agar plate
with it.
I

130 BACTERIOLOGY OF THE SOIL

Incubate at 22° C, and when the different colonies are well
developed examine the organisms of each. Make sketches and
describe the colonies.

Try and determine the species by cultivation on gelatine, agar,
and potato, and in Bouillon, milk, and other liquid media.
(Compare, if necessary, with type cultures, obtainable from the
Author.)

4. Action of Antiseptics and Heat upon the Soil. — ^Soil
to which carbon disulphide has first been added and then
allowed to evaporate is found to be capable of growing
a larger crop than a similar untreated soil. The same
increased fertility is observed when soil is treated with
chloroform, ether, toluene, xylol, and other poisonous
volatile compounds. Nitrification and the organisms
which produce it are destroyed, but partial sterilization
by means of antiseptic agents give rise after a short time
to a large increase of ammonium compounds, to which
latter the greater fertility of the soil is chiefly due. The
ammonium salts may be directly absorbed and assimi-
lated by plants, but partially sterilized soils rapidly
become reinfected with living nitrifying bacteria through
access of dust, unless special precautions are taken to
prevent this from happening, and the crop then receives
its nitrogen in the usual manner as nitrate.

Many investigations have been made to determine the
nature of the beneficial effect produced by the action of
antiseptics upon soil.

It is probable that part of the improvement observed
is caused by direct chemical action of those substances
upon the insoluble soil constituents, some of the latter
becoming available for plant food in the process.

It has been suggested also that these poisonous com-
pounds stimulate absorption by the roots and other physio-

ACTION OF ANTISEPTICS

131

logical functions of the crop, and thus lead to an increased
yield, but the fact that the good effect lasts for some time
after the compounds have evaporated, militates against
the acceptance of this view.

The investigations of Hiltner and Stormer, Pagnoul,
Hentze, Russell and Hutchinson show that the beneficial
action of antiseptics is connected with alterations in the
nature and number of the living organisms of the soil.
Hiltner and Stormer showed that the bacterial content
was reduced at first by the application of carbon disul-
phide, but later the numbers increased enormously.
Untreated soil, containing 9 J millions of bacteria per
gram, contained comparatively few immediately after
treatment, but after the lapse of a month or less, the
number rose to about 50 millions per gram. They
found that the nitrifying and denitrifying organisms
were destroyed almost completely, and did not again
develop to any extent unless fresh inoculation occurred.
Species of Streptotkrzx, which composed about 20 per
cent, of the total bacterial flora, were reduced, but the
non-liquefying organisms increased very rapidly.

Similar marked increase in numbers of bacteria in
partially sterilized soil was observed by Russell and
Hutchinson, and found to be correlated with proportional
increase in the production of ammonium compounds.

Number of Organisms per Gram of Soil.

Untreated Soil .
Toluened Soil .
Heated to q8° C.

At Beginning.

6,693,000

2,608,000

393

After 9 Days.

9,814,000

40,620,000

6,294,100

132

BACTERIOLOGY OF THE SOIL

Nitrogen present as Ammonia. Parts per
Million of Dry Soil.

Untreated Soil . .
Toluened Soil . .
Heated to 98° C. .

At
Beginning

After
7 Days.

After
15 Days.

2.2
4.2
8.6

1.9
27.9
27.0

2.7

34.3
32.6

i

The total nitrogen-content is not altered, the ammonia
being derived from humus, and the bodies of algae,
protozoa, mites, worms, and other living things which
have been killed by the antiseptic substance used.

They conclude that in ordinary soils amoebae and
other protozoa devour and keep down the numbers of
bacteria ; after treatment with toluene or other anti-
septics these larger organisms are destroyed, the more
resistant bacteria and their spores being then free to
develop without competition.

Soil which is heated to 100° C. or over becomes
sterile, and when the process is carried out thoroughly,
there is, of course, no subsequent development of
bacteria unless re-infection occurs. Moreover, heating
results in the formation of toxic substances which
prevent the germination of seeds and the growth of
plants for some time. After the disappearance of the
toxic compounds the soil is more fertile than unheated
soil of the same kind, the heating process having set free
some of its plant food.

Soils which have been partially sterilized by heating
to 90° or 95° C. for two or three hours resemble those

ACTION OF ANTISEPTICS 133

which have been treated with antiseptic agents. There
is at first a great decrease of bacteria, but this is soon
followed by a rapid development of organisms and the
production of considerable amounts of ammonium com-
pounds which increase the immediate fertility of the
soil. Nitrifying bacteria added to soils which have been
heated are checked for a time by the toxic substances
produced, whereas they develop freely and at once when
added to a soil which has been partially sterilized by
treatment with toluene.

CHAPTER IX.

NITRIFICATION.

Nitrification. — When substances such as dung, urine,
blood, flesh, or other debris of plants, containing organic
nitrogen compounds, are incorporated with the soil putre-
faction and decay (p. lOO) break them down little by
little, the nitrogen sooner or later becoming oxidized and
a nitrate is formed. It was by taking advantage of this
oxidizing process that nitre or saltpetre (potassium
nitrate) was formerly obtained in considerable quantities
from so-called " nitre-beds," which were carefully con-
structed heaps of animal and vegetable refuse, kept moist
with urine and other liquids ; the nitrogen was oxidized
and the potassium nitrate formed was subsequently
dissolved out and purified.

This natural production of nitrate, which occurs in
varying degrees in most soils, is termed nitrification. It
was formerly considered a purely chemical process, but
in 1877 Schloesing and Miintz, while investigating the
chemical changes in sewage, discovered that the oxida-
tion of ammonium compounds into nitrates was the
result of the vital activity of living organisms. The
process was readily checked by applications of chloroform
vapour to the soil, and by heating the latter to a
temperature of 100° C. They were not able to isolate
the special nitric " ferments," but they determined the
134

NITRIFICATION I35

limits of temperature between which the organisms
carried on their work, and also the conditions of aeration
and other factors which influenced the resulting nitrifica-
tion. At 50° C. the process is scarcely recognisable,
and at 55° C. it is destroyed : the optimum temperature
lies near 37° C. Warington, at a later date, showed that
nitrification usually takes place in solutions of ammonium
salts, with the formation of varying amounts of nitrites
and nitrates, and found that in some instances the oxida-
tion of ammonium compounds may proceed as far as the
formation of nitrites and there stop, no nitrates being
produced. He, moreover, proved that in the nitrification
of organic materials, such as milk, urine, and asparagin,
the formation of ammonium compounds first takes place.
Although it was found an easy matter to set up
nitrification in ammoniacal solutions by the addition of a
small amount of garden soil, all the early attempts to
isolate and prepare pure cultures of organisms capable
of oxidizing ammonium compounds led to unsatisfactory
results until 1890, when Frankland and Winogradsky
succeeded in the task. They found that the real agents
do not grow in bouillon and similar media containing
soluble organic substances, and stubbornly refuse to
develop on the ordinary gelatine and agar plates.
Winogradsky cultivated the organisms in a solution
containing ammonium sulphate, potassium phosphate,
and a small amount of basic magnesium carbonate.
From a medium of this kind devoid of all organic com-
pounds, and undergoing active nitrification, he inoculated
plates of gelatinous silica, impregnated with the inorganic
salts mentioned, and succeeded in isolating the two
kinds of bacteria now known to be the active agents of
the nitrification process.

ts^ NITRIFICATION

The transformation of complex organic nitrogenous
material which occurs in the soils takes place in three
stages, namely : —

1. The production of ammonium compounds from

the proteins, amino-acids, and other organic
bodies present.

2. The oxidation of the ammonium salts thus formed

into nitrites.

3. A further oxidation of nitrites into nitrates.
The term nitrification is usually restricted to the two

latter stages, that is, to the oxidation of ammonium
compounds into nitrates. No organisms are known
which will directly nitrify proteins, amino-acids, urea, or
similar nitrogenous substances ; these must be decom-
posed first and ammonium compounds prepared from
them, the latter being subsequently acted on by the
specific nitrifying bacteria.

The preliminary breaking down of the complex
organic matter with the formation of ammonium salts is
effected by a large number of different kinds of bacteria.
Most of the putrefactive group are concerned in the
work ; bacteria commonly present in soil which are
specially active in this direction are Bs. mycoides,
Bs. putiduSj Bs. mesentericus vulgatus, Proteus vulgaris,
Bact. fluorescens liquefaciens, and Micrococcus candicans.
Marchal found that Bs. mycoides liberated 46 per cent,
of the nitrogen of egg-albumen as ammonia, Proteus
vulgaris 36 per cent., and Bs. vulgatus 30 per cent, in
twenty days. In addition to bacteria, which are most
efficient in neutral or alkaline soils containing little
organic matter, certain iungi, such. d,s Penicillium glaucum,
Mucor Mucedo, M. racemosus, and species of Botrytis
and Torula, are also responsible for the breaking down

NITRIFICATION I37

of organic nitrogen compounds, particularly in the acid
soils of woods and meadows rich in humus and vegetable
debris.

The first step in the oxidation of ammonium salts is
brought about by the agency of bacteria, included by
Winogradsky in the genus Nitrosomonas. They produce
nitrites, and -pure cultures of them are imable to carry the
oxidation further. After being introduced into a nutrient
solution of ammonium sulphate (p. 144), an incubation
period lasting five or six days occurs, during which little
chemical change is effected ; after-
wards the formation of nitrites goes
on rapidly, as much as 90 mg. or
more per litre of solution being
produced in a day. In the early
stages of nitrification of such a
solution the nitrite organisms are
usually found in a zoogloea state,
attached to the particles of maff- Fig. ^^.—mtrosomonas curopcea,

y : y ' ^Z Win. (X 600).

nesmm carbonate lymg on the

bottom of the flask. The individual cells are difficult
to observe at first, but after eight or ten days they
frequently separate and become motile for a time before
forming a zoogloea again. The organisms isolated from
western European soils, and known as Nitrosomonas
europcea^ Win., are oval, from 1.2 to 1.8 /a long and
.9 to I /o. thick (Fig. 29), and in a motile state have a
single long polar flagellum. The colonies on silica jelly
are round, very minute, and at first colourless, highly
refractive with a brown edge ; lat^r they become dark
brown. Several apparently permanent modifications or
varieties have been described by Winogradsky ; some of
them from eastern Europe, Brazil, aud Australia being

138

NITRIFICATION

coccus forms i to 2 /^ in diameter. A very minute
variety, ,5 /x- in diameter, from Java possesses an
extremely long flagellum.

Associated with the nitrite organisms in the soil are the
nitrate bacteria, which oxidize the nitrites into nitrates
and are unable to attack ammonium compounds. They
are non-motile rod-shaped bacteria, about i //- long, .3
to .4 /x broad, and are included in the genus Nitrobacter
(Fig. 30) ; probably several forms exist, but owing to
the difficulty of isolating and cultivating them little is
known of their morphology. Nitro-
bacter Q-axi be grown on nitrite agar,
but the colonies are minute and
develop very slowly. In suitable
media, containing a small amount
of sodium nitrite, the formation
of nitrates by Nitrobacter begins
in forty-eight hours or less, but
goes on slowly for five or six days,
after which oxidation proceeds
more rapidly until all the nitrite is completely changed
in twelve or fourteen days. In nature both groups of
organisms are associated and live together. They are
abundant in well and river water, and are distributed
through the upper layers of the soil, being most abundant
from 4 to 9 inches below the surface, and not usually
found at a greater depth than 18 or 20 inches. When
both kinds of bacteria are allowed to act upon solutions
of ammonium salts the latter are completely changed
into nitrites before the formation of nitrates commences,
and nitrites can be readily recognized in the solution
by the usual tests. Under the conditions which prevail
in the soil, however, only nitrates — the end products of

Fig. 2P.— Nitrobacter, sp.
( X 600).

NITRIFICATION 139

their combined action — can be detected, the nitrites
which are formed being immediately oxidized as fast as
they are produced ; the two organisms appear to act
symbiotically and simultaneously.

Certain conditions are essential for the nitrification
process besides the presence of ammonium salts and the
specific nitrifying bacteria. The chief of these are an
adequate temperature and suitable degree of moisture,
abundant free oxygen and carbon dioxide, a basic
substance, such as calcium or magnesium carbonate,
darkness, and the absence of soluble organic matter and
free ammonia. At freezing point and during winter,
when the temperature is below 5"^ or 6° C, nitrification
is checked, but it begins in early spring and continues
more and more actively during the warm summer
months. At 45° C. the nitrite organisms are destroyed
in five minutes, but the nitrate bacteria are not killed
below about 55° C>: the optimum temperature for both
is near 37° C. ^Excessive dryness retards nitrification;
the best results, according to Coleman's experiments, are
obtained in the case of loamy soils, when the latter
contain about 1 6 per cent, of water ; when i o per cent,
is present the process is very slow, with 26 per cent, it
is stopped or greatly checked. The nitrifying organisms
are strongly aerobic, and a good supply of oxygen is
essential for the maintenance of their vital functions.
On this account the nitrification is most energetic in
well-drained, well-aerated porous soils ; in clays and
water-logged areas the organisms sometimes die out
altogether.

Experiments with pure cultures have shown that
a basic substance, such as carbonate of sodium,
magnesium or calcium, is necessary for the oxidation

140 NITRIFICATION

of ammonium compounds ; in the soil it is usually
supplied by calcium carbonate. Where lime is deficient
a dressing of chalk or quicklime must be added before
the nitrogen present can appear as a nitrate and be
made available for the nutrition of crops. Ammonium
salts of organic acids undergo as rapid nitrification as
ammonium sulphate or chloride when a basic carbonate
is available. The necessity for a basic carbonate of the
type mentioned, which reacts with ammonium salts to
form ammonium carbonate, suggests that the latter is
the only compound which can be directly nitrified.

Godlewsky showed that the nitrite bacteria obtain the
carbon which which they need for the synthesis of those
carbon compounds used in their growth from free carbon
dioxide gas ; possibly the same holds true for the nitrate
forms also. When grown in the presence of insoluble
carbonates the organisms do not develop if the atmo-
spheric air supplied to them is first passed through a
solution of caustic potash, which absorbs carbon dioxide.
It is probable that the carbon dioxide of the air is
necessary at first, but later the CO2 of the
carbonate present may be sufficient. They carry on
their work best in the dark, bright light being injurious
to them. As previously mentioned, soluble organic
substances act deleteriously upon both species of nitri-
fying organisms, the nitrite bacteria being more sensitive
in this respect than the nitrate - formers, especially
towards nitrogenous substances, such as asparagin or
peptone ; it is not possible to cultivate either of them
in bouillon or solutions of peptone, sugar, and other com-
pounds, which serve well for the growth of most of the
ordinary kinds of bacteria. These compounds act upon
the nitrifying organisms as phenol, salicylic acid, and other

NITRIFICATION

141

antiseptics do upon most bacteria, inhibiting or destroy-
ing their growth altogether when present in very small
quantities. One part in 4000 of glucose or peptone will
retard nitrification.

The amounts of various organic substances which were
found by Winogradsky and Omeliansky to retard or
completely check the two kinds of nitrifying bacteria are
given below. —

Nitrosomonas.

Nitrobacter.

Percentage amount which

Percentage

amount which

Retards.

Stops.

Retards.

Stops.

Glucose

.

.025 to '05

.2

•05

.2 to .3

Peptone ,

.

.025

.2

.8

1-25

Asparagin .

.

•05

•3

.05

.5 to I

Urea

.2

?

•05

I

Sodium butyrate

•5

1.5

•5

I

Beef broth

.

10

20 to 40

10

60

Ammonia .

.

—

—

•0005

.015

According to Boullanger and Massol the production
of nitrites is checked when the amount of ammonium
sulphate in the nutrient medium exceeds 4 or 5 per
cent., or when .5 per cent, or less of potassium or
sodium nitrite is present; more than 1.5 per cent, of
calcium or magnesium nitrite retards it. Almost all
nitrites are oxidized to nitrates so long as the
amount present does not exceed .05 to .1 per cent.,
the base having little influence on the process. The
development and physiological activity of the nitrate
bacteria are diminished in the presence of more than 2
per cent, of nitrite, and also when sodium or potassium
nitrate exceeds 2 or 2.5 and calcium nitrate more than
about 1.5 per cent. In manure heaps little nitrification
occurs owing to the inhibiting action of the excess of
organic material present and the want of good aeration.

142 NITRIFICATION

A very small quantity of free ammonia dr of ammonium
carbonate, which dissociates easily, also stops the growth
and multiplication of the nitrate organisms, but does not
much influence the oxidizing power of those already in
existence. Any nitrite formed in the soil may be
oxidized immediately to nitrate, even in the presence of
a certain amount of free ammonia, where species of
Nitrobacter are abundant. Generally, however, free
ammonia is absorbed at once and rendered inert by the
constituents of the soil.

The nitrifying organisms are remarkable in possessing
the power of manufacturing the organic compounds needed
for growth and multiplication of their bodies from simple
inorganic salts and carbon dioxide. In this respect they
resemble green plants, but they carry on their work
without the assistance of chlorophyll or sunlight. The
energy necessary for the reduction of carbon dioxide is
derived from the oxidation of ammonium compounds ;
from 33 to 'X^y grams of nitrogen are oxidized for each
gram of carbon assimilated, a fact which to some extent
explains the extremely slow development of these
organisms.

Every endeavour should be made by agriculturists to
assist the nitrifying organisms in their work of preparing
available nitrates for the needs of crops. The applica-
tion of lime, the removal of superfluous water from the
soil, and the provision of good aeration by thorough
cultivation, especially during summer, are among the
most important practices which can be adopted by
farmers for the stimulation of these useful soil organisms.

Sulphate of ammonia and manures, which must be
nitrified before the nitrogen is available to plants, are
best applied to soils of light porous character in which

NITRIFICATION 143

nitrification is especially energetic, while for soils of
damper close texture, in which the activity of the
nitrifying organisms are retarded or checked, nitrate of
soda or calcium nitrate are more suitable nitrogenous
fertilizers.

Ex. 71. — Show the production of ammonia from proteins.
Prepare the following solution : —

Di-potassium phosphate . . . 5 gr.

Magnesium sulphate . . . . 2.5 gr. '

Sodium chloride 2.5 gr.

Water i litre.

Take three 200 c.c. flasks and pour into each 100 c.c. of the
solution.

Add to one . i per cent, of gelatine.

„ another . i per cent, of fibrin.
„ a third . i per cent, of peptone,
and sterilize.

Inoculate each with Bs. mycoides and incubate at 30° C. for
ten to fifteen days.

{a) Test for ammonia with Nessler's solution.
{b) Heat with calcined magnesia and test the gas given off
with litmus paper.
Control tubes uninoculated should be tested also.

Ex. 72. — Add to a litre of the inorganic solution in previous
experiment.

Glucose . . . ■ . . . 2 gr.

Sodium nitrate 2 gr.

Sterilize; inoculate 100 c.c. of it with Bs. mycoides. Incubate
at 30° C, and every second day —

(i) Test for nitrites (Ex. 74).

(2) „ ammonia with Nessler's solution.

(3) Find out when the nitrate disappears.

Ex. 73, — To a dilute solution of egg albumin add a dilute

144

NITRIFICATION

• •

2gr.

I gr.

2gr.

•5gr-

•4 gr.

. I litre.

solution of ferrous sulphate to prevent coagulation : sterilize at
1 00° C. Inoculate with pure cultures of
(a) Bs. mycoides ;
{b) Bad. fluorescens liquefaciens ;
(c) Bad. (^Proteus) vulgare ;
and incubate at 30° C. for ten to fifteen days.

(i) Test for ammonia with Nessler's solution.
(2) Heat with calcined magnesia and test the gases evolved
with litmus paper.
Compare with uninoculated sterilized control tubes.

Ex. 74. — Prepare the following solution : —

Ammonium sulphate
Di-potassium phosphate
Sodium chloride .
Magnesium sulphate
Ferrous sulphate .
Distilled water

Pour 50 c.c. into three or four conical flasks and to each add
.5 gr. of basic magnesium carbonate. Then add to each about
I gram of garden soil taken 4 or 5 inches below the surface of
the ground, and keep at a temperature of 30° C. Withdraw
5 c.c. from each flask every three or four days and test one-third
of each 5 c.c. :■ —

(i) For ammonia with Nessler's solution.

(2) For nitrites by means of zinc iodide or cadmium iodide

starch paste and sulphuric acid.
Dissolve a small quantity of starch in water by heating ; cool
and add a crystal of Cdl and heat till dissolved, then add
1 to 2 c.c. of dilute H2SO4 (yq- per cent.) : a blue colour indicates
the presence of a nitrite.

(3) For nitrates after the nitrites have disappeared with

diphenylamine and sulphuric acid.
Dissolve a small crystal of diphenylamine in i or 2 c.c. of
strong HgSO^, then add a few drops of the nitrifying solution ;

NITRIFICATION 145

the development of a violet blue colour shows the presence of a
nitrate. Since the same colour is given with a nitrite, this test,
if intended for detecting nitrates, can only be applied when a
previous test for a nitrite has failed.
Note the time —

(i) Taken for the complete disappearance of ammonium
reaction.

(2) When nitrite first appears.

(3) When nitrate first appears.

(4) When the nitrite disappears.

Repeat the above experiment, using soil from —

(i) An ordinary arable field. (3) A peat bog.

(2) An old pasture. (4) A wood or forest.

Also with soil from —

(i) The surface of the ground. (3) 1 2 inches below ground.

(2) 6 inches below ground. (4) 3 feet „ „

Ex. 75. — ^To test the influence of temperature on nitrification,
repeat Ex. 74, keeping some of the flasks —
(i) In a cool place.

(2) At a temperature of 20° C.

(3) At a temperature of 30° to 35° C.

(4) Add to one of the flasks soil which has been heated to

a temperature of 1 00° C. for half an hour.

Ex. 76. — To test the inhibiting effect of soluble organic com-
pounds on nitrification, repeat Ex. 74, adding to a series of
flasks .01, .03, .3, I, and 3 per cent, of —

(i) Dextrose,

{2) Peptone,

(3) Urea
respectively.

Ex. 77. — Take six or eight conical flasks with bases about
4 or 5 inches in diameter : pour into each 50 c.c. of the
solution given in Ex. 74, and add .5 gr. of basic magnesium
carbonate. Inoculate one of the flasks with about i gram of

K

146 • NITRIFICATION

garden soil, and keep at a temperature of 30° C. Test a small
amount of the solution every fourth day with Nessler's solu-
tion until no reaction occurs. Then add i c.c. of a 10 per
cent, solution of ammonium sulphate and allow this to nitrify,
testing every three or four days until the ammonium salt is
completely changed. Then transfer some of this nitrifying
solution by means of a platinum loop to a second flask. When
the solution in the latter gives no reaction with Nessler transfer
some of it to a third flask, and similarly some of the third to
the fourth, and so on. By cultivating several generations in a
medium devoid of organic matter, in this way it is possible to
eliminate most of the non-nitrifying bacteria present in the soil
and obtain fairly pure cultures of the nitrifying organisms.

Try and obtain a pure culture which will produce nitrites or
nitrates when introduced into fresh ammoniacal solution, and
yet give no growth or turbidity when a drop is transferred to
bouillon tubes and incubated at 30° C.

Ex. 78. — To obtain pure cultures of the nitrifying organisms
their isolation must be carried out on "silica jelly" plates.

(a) For the preparation of " silica jelly " Winogradsky and
Omeliansky recommend the addition of a solution of pure
" water glass " (sp. gr. 1.05 to 1.06) to an equal quantity of hydro-
chloric acid (sp. gr. i.i).

Pour the solution of sodium silicate slowly into the dilute
hydrochloric acid (not the acid into the silicate solution), shaking
the mixture gently. The following reaction takes place, and a
solution of silicic acid is obtained, mixed with sodium chloride ; —

NaaSiOg + 2HC1 = H2Si03 + NaCl
Silicic Sodium
acid chloride

To get rid of the sodium chloride pour the mixture into a
parchment dialyser and float or suspend the latter in distilled
water ; the sodium chloride diffuses through the parchment into
the water, which should he changed twice a day : the silicic acid
is a colloidal gelatinous body and does not pass through, but

NITRIFICATION 147

remains in the dialyser. (Great care must be taken to procure
a sound piece of parchment or dialyzing membrane, or it will
not be possible to obtain a solution of silicic acid of the right
strength to form a firm solid jelly.) Test small portions of the
solution taken from the dialyzer with silver nitrate from time to
time, and when no milkiness is given with this reagent the solution
may be poured into a stoppered flask and sterilized : it does not
keep well and is best used in a day or two, since it is liable to
lose its clearness and become unfit for use.

{b) When required for the preparation of " silica jelly " plates,
for the cultivation of the nitrite organism, the silicic acid ob-
tained by the above method is mixed with the following solution,
which must be sterilized : —

Ammonium sulphate . . . . -3 gr.

Di-potassium phosphate . , . -i gr.

Sodium chloride 2 gr.

Magnesium sulphate 05 gr.

Ferrous sulphate 04 gr-

Water (twice distilled) . , . .100 c.c.
To 50 c.c. of the silicic acid solution add 2.5 c.c. of the above
and enough of a sterilized mixture of fine magnesium carbonate
and water to make the whole milky.

{c) (i) Inoculate as rapidly as possible with a loopful of the
purest culture obtained by the successive cultivation of the
nitrifying organism, as in Ex. 77, and after shaking gently pour
at once into a sterilized Petri dish and allow the liquid to
solidify ; incubate at 30° G., or

(ii) Pour the mixed silicic acid and ammoniacal solution into
a Petri dish, and after the "jelly" has "set" place a drop of
inoculating medium on the surface and very gently spread it in
streaks with a sterilized bent glass rod, taking care not to tear
the surface of the jelly.

As the silicic acid solution gelatinizes in a short time when
mixed with the ammonium sulphate solution, the mixing and
inoculation must be carried out rapidly. Incubate at 30° G.,

148 NITRIFICATION

placing the dish cover downwards so that the exuded water will
not remain on the surface of jelly. The deep colonies of the
nitrate organisms are very small and appear as refractive points
with dark rims, and as a rule gradually become surrounded by a
small clear space, from which the magnesium carbonate is dis-
solved as growth and nitrification proceeds : examined with a
low-power microscope they appear granular. Remove colonies
or small portions with a fine capillary tube, obtained by melting
a piece of ordinary thin glass tube and drawing it out rapidly
when soft ; transfer to flasks containing the ammonium sulphate
solution (Ex. 74). The tip of the tube conveying the nitrite
organism can be easily broken off and left in the flask by pushing
against the bottom of the latter. Incubate the inoculated solution
at 30° C. and

(i) test for nitrate at intervals;

(2) examine it with the microscope and note the form, size, and

motility of the organisms present ; stain some ;

(3) test for the presence of foreign, non-nitrifying species by

introducing some of the solution into bouillon : note if
the latter remains clear and without signs of growth for
twelve to fourteen days at least.
The nitrite organisms stain with carbol-fuchsin and are
roundish or oval, 1.25 to 1.8 /^ long and .9 to i {l thick, some-
what irregular in size.

Ex. 79. — Stevens-Temple Silica Jelly. — The following method
of preparing a " silica jelly " is due to Stevens and Temple, and
has the advantage of avoiding the process of dialysis, which in
the hands of beginners is troublesome and uncertain in its
results. The method leaves a certain amount of sodium chloride
in the "jelly," but this has not been found to interfere with the
cultivation of nitrifying organisms.

{a) Take 3 to 4 c.c. of a solution of sodium silicate (water
glass). Place in a porcelain dish and add 5 to 10 c.c. of con-
centrated hydrochloric acid to precipitate the silicic acid.
Evaporate to dryness : moisten again with hydrochloric acid

NITRIFICATION 149

and evaporate to dryness a second time. Then wash and
transfer to a Swedish filter paper, the weight of whose ash is
known. Wash the precipitate until the water gives no cloudi-
ness with silver nitrate solution. Burn the filter with the pre-
cipitate upon it in a weighed platinum or porcelain crucible and
heat to redness. Allow to cool, and weigh the silicic anhydride
(SiOg). Calculate the amount of silicic anhydride in i c.c. of
the original solution.

{b) Dilute the amount of water glass which you wish to use for
preparation of the jelly until the solution contains 4 or 5 per
cent, of silicic anhydride. Prepare hydrochloric acid of such
strength that i c.c. just neutralizes i c.c. of the "water glass"
solution, using methyl orange as an indicator. Take 104 c.c. of
the hydrochloric acid and add to it 100 c.c. of the "water glass "
solution: the excess of acid prevents coagulation during the
subsequent sterilization. The solution should be poured into
tubes or flasks and sterilized at 120° C. for fifteen minutes, or at
100° C. for twenty minutes, on three successive days : 10 to 20
c.c. are convenient amounts to place in tubes.

Since the medium cannot be liquefied after once it has " set,"
tubes for " slants " must be put in position as soon as possible
after mixing.

{c) Inoculate the tubes from the flasks containing nitrifying
organisms (Ex. 77) and add to each lo c.c. of sterilized solu-
tion, .5 c.c. of the ammonium sulphate solution (in Ex. 74) and
a drop or two of sodium carbonate solution, enough to make the
whole alkaline. Pour into Petri dish at once and allow to
solidify.

{d) Inoculation may be made on the surface of the solidified
medium as in Ex. 78 {c. ii).

Ex. 80. — Isolate and grow the nitrate organism. Prepare
the following solution : —

Sodium nitrite (puriss) . . . . i gr.
„ carbonate (calcined) . . • i gr.
„ chloride 5 gr.

ISO NITRIFICATION

Di-potassium phosphate (K2HPO4). . -5 gr.
Ferrous sulphate . . . . . .4 gr.

Magnesium sulphate 3 gr.

Distilled water i litre.

Take six or eight conical flasks with wide bases, 4 to 5 inches
in diameter : pour into each 50 c.c. of the solution, and add
.5 gram of basic magnesium carbonate. Inoculate one of the
flasks with about i gram of garden soil and keep at a tempera-
ture of 30° C. Test the solution for nitrites every two or three
days with zinc or cadmium iodide starch paste, and when this
reaction ceases test for nitrates with diphenylamine and sulphuric
acid (Ex. 74). Then add i c.c. of a 10 per cent, solution of
sodium nitrite and test again similarly until the added nitrite dis-
appears. Transfer a loopful from this flask into a second flask
of the solution, and when nitrate is formed in the second transfer
again to a third, and from the third to a fourth.

The nitrate organisms may be grown on silica jelly impregnated
with the above nitrite solution and magnesium carbonate, in the
same manner as that described for the nitrite bacteria, Ex. 78.

They grow, however, on the following agar medium, which is
more easily prepared : —

Sodium nitrite (puriss) .

. 2 gr.

„ carbonate (calcined) .

. I gr.

Di-potassium phosphate .

. .2 gr.

Agar

• 15 gr.

Tap water . .

. I litre

From the last of several successive generations of cultivations
in the nitrite solution, already given above, inoculate —

(i) The melted agar medium in the usual manner with a
loopful of the solution: pour into a Petri dish, turn it upside
down in order to allow exuded water to run ofl" the surface of the
medium.

(ii) Make a streak culture on solidified nitrite agar in a Petri dish.
Incubate for three or four weeks at 30° C. The surface colonies

NITRIFICATION 151

look like very small watery drops with granular contents : the
deep ones are exceedingly small, brown, kidney or spindle
shaped. The larger colonies observed are generally impurities.
Transfer some of the smallest colonies by means of capillary
tube (Ex. 78 ^. ii) to flasks of the nitrite solution, using low-
power microscope for selection of the colonies, and

(i) test for disappearance of nitrite and appearance of nitrate ;

(2) examine a drop of the solution and note the form and size

of organisms observed ;

(3) -inoculate ordinary slightly alkaline bouillon and note if

turbidity and growth occurs; carry on the incubation at

30° C. for two weeks at least.
From cultures which are bouillon sterile the organisms may
be obtained and stained with carbol fuchsin : they are usually
rod-shaped, about i /x long and .3 to .4 /x thick, or in
some instances somewhat oval and i to 1.5 /x long and up to
.8 'M thick.

CHAPTER X.

DENITRIFIOATION.

Denitrification and Reduction of Nitrates and
Nitrites. — In the process of nitrification ammonia or its
compounds are first oxidized to a nitrite, and finally to a
nitrate by certain specific organisms, as already described
in the previous chapter. Many bacteria are known
which carry on processes of reduction the reverse of
this, oxygen being removed from nitrates to a greater
or lesser extent. The reduction may be of several
kinds, namely : —

1. The nitrates may be reduced to nitrites and no

further.

2. The oxygen may be taken from nitrates and

nitrites, with the ultimate formation of
ammonia.

3. Nitrates and nitrites are sometimes reduced with

the evolution of nitric (NO) and nitrous
oxides (N2O).

4. The nitrates and nitrites may be reduced with

the final production of free nitrogen.

Although the term denitrification has been sometimes

loosely employed to denote all these forms of reduction,

it is now more strictly applied to the decompositions of

nitrates and nitrites, which result in the evolution of free

NITRATES AND NITRITES 153

nitrogen. The ability to reduce nitrates to nitrites is
possessed by a large number of different kinds of
bacteria: out of 32 forms isolated by Frankland from
air and water more than half of them had this
property, and of 109 examined by Maassen about 90
showed the same power. Many of these, and others
equally commonly distributed, are apparently able to
carry the reduction further and give rise to ammonia ;
the latter, however, may be derived from proteins and
other nitrogenous organic compounds often present where
bacterial action is going on. It is in cases where the
nitrates are decomposed with the formation of the
gaseous oxides or free nitrogen that the process becomes
of special interest to the agriculturist, since these changes
involve a loss of valuable fertilizing material from the
soil or from manure heaps. The conditions essential for
denitrification are ; —

a. The presence of certain species of bacteria.

b. A supply of nitrates.

c. A considerable amount of easily assimilated organic
substances.

d. Total absence or a very limited access of free
oxygen.

These, it will be noticed, are practically opposite con-
ditions to those required for nitrification. The exclusion
of any one of them checks denitrification. A large
number of kinds of bacteria have been isolated which are
capable of decomposing nitrates with the evolution of
free nitrogen. Many of them are found in water and in
the soil. They are also especially abundant upon straw
and in the dung of the horse, cow, and other herbivorous
animals ; in faeces of man and carnivorous animals they
are rarely present. All these organisms are aerobic

1 54 DENITRIFICATION

species and grow well enough in bouillon when supplied
with free oxygen, but do not reduce nitrates to more
than a slight extent under such circumstances. When
cultivated anaerobically or with a diminished supply of
air they take oxygen from nitrates or nitrites, using it
for the respiratory combustion of comparatively large
amounts of organic carbon compounds. That the want
of oxygen is the chief cause of denitrification may be
deduced from the cultivation of Bad. pyocyaneum^ or a
similar organism with and without nitrite in the presence
and absence of air respectively. Growth and multiplica-
tion take place in alkaline nitrite bouillon under aerobic
conditions without decomposition of the nitrite, but when
cultivated as an anaerobe the nitrite disappears rapidly
with the evolution of free nitrogen. Without the nitrite
under anaerobic conditions little or no growth occurs,
and respiration is stopped even when suitable oxidizable
material is available. The power of utilizing organic
compounds apparently as the source of energy for the
reduction of nitrates which takes place in the denitrifica-
tion process, varies considerably with different species
of bacteria. Some of these can oxidize certain carbo-
hydrates and acids which are not attacked by other
species. For instance, Salzmann found that Bad.
Hartlebii made use of xylan, glucose, lactose, and
maltose, but none of these were of service for the growth
oi^Bact. Stutzeri.

The amount of nitrate decomposed is proportiorial
to the amount of easily assimilated carbon compounds
available. Those substances which serve best for
the denitrifying species of bacteria are neutral salts
of organic acids, especially butyric, lactic, and citric
acids ; acetic, propionic, valerianic, and other acids are

NITRATES AND NITRITES 155

useful also. Mixtures of these with glucose, starch, and
other carbohydrates are utilized also, but not usually the
latter alone. Many of the alcohols, such as ethyl, propyl,
and isobutyl alcohols and glycerine are oxidized by them,
and hippuric and uric acids are utilized also by some
species. Many organic compounds, which are not found
to be of service to pure cultures of denitrifying bacteria,
are indirectly utilized when the latter are associated with
putrefactive organisms. A number of the compounds
so readily utilized by denitrifiers are produced by the
action of putrefactive organisms upon the complex in-
soluble compounds in animal and vegetable tissues,
humus and the undigested materials in faeces. Some
organisms, such'ss'Bac^. denitrificans, L. and N., are known
which can set free nitrogen from nitrites ; they are
unable to attack nitrates directly, but when grown sym-
biotically or along with B, coli and other species which
are able to carry out for them the preliminary reduction
of the nitrate to nitrite, free nitrogen is evolved. In
manure and compost heaps there is little or no denitri-
fication with its consequent loss of nitrogen in the
gaseous form, since the necessary nitrates are generally
missing, the excess of soluble organic matter and absence
of aeration preventing their formation except in the upper
layers of the heaps. In well-aerated composts, however,
and in farmyard manure loosely put together nitrification
may occur and be followed by denitrification when the
heaps become saturated with water through exposure to
heavy rains. In ordinary arable soils loss of nitrogen
by denitrification is uncommon, for where nitrates are
abundant the easily assimilated carbon compounds upon
which denitrifiers depend are wanting. -That it may
occur in poorly aerated, water-logged soils under anaerobic

156 y DENITRIFICATION

conditions, was shown in an experiment carried out by
Warington. A column of soil was saturated so as to
drive out the air from the small spaces within it, the
water being allowed to percolate through so as to extract
all the nitrate present. After no reaction for nitrate was
obtained, a certain amount of nitrate of soda was then
placed on the surface of the soil and watered into it
daily. It was found that only 2 1 per cent, of the
amount applied to the soil could be recovered, 79 per
cent, being lost by denitrification : transverse fissures
developed across the soil at some depth below the
surface, these being due to the evolution of nitrogen and
carbon dioxide gas.

In 1897 Wagner and Maercker drew attention to
some remarkable result? obtained from the growth of
oats, mustard, and other plants in pots, to the soil of
which fresh undecomposed farmyard manure had been
applied alone or along with nitrate of soda. They found
that where fresh horse or cow dung were added in large
amount the crop yield was less than where no manure
at all was used, and also discovered that under certain
conditions the application of dung greatly depressed the
produce from soil which had received nitrate of soda as
well. The following are typical results : —

Oats {Maercker).

Yield of Grain and Straw.

Soil

without manure .

,

44.82

grams.

J)

with nitrate of soda (i

•5

grams N.)

128.37

)>

(1867.

^ increase).

»

with horse dung (2.

25/

35-91

(i) ,-

(^0%

decrease),

grams N.)

I

23-33

(2) „

(487,

decrease).

NITRATES AND NITRITES 1 57

Mustard {Wagner).

Yield of Grain and Straw.
Soil without manure . . 1.6 grams.

„ with nitrate of soda (2

grams N.) . . 36.6 „

„ with horse dung (2 grams N.) .5 „
„ with cow dung ( „ ) .4 »

The amount of nitrogen recovered in the crop when
nitrate of soda was used alone in one case was '/y per
cent., whereas only 52 per cent, was recovered when the
nitrate was applied in conjunction with horse dung.
That the difference was due to the destructive action of
the horse dung on the nitrate was concluded from a
determination of the nitrates extracted with water from
soil treated with nitrate and nitrate plus dung in cases
where no crop was grown. A pot of soil to which
. 5 gram of nitrate of soda was added was compared with
one to which dung and the same amount of nitrate were
applied and left for an interval of six weeks. While
93 per cent, of the nitrate in the former appeared in the
extract, the pot receiving the dung in addition yielded
only 42 per cent. ; moreover, the filtrate from soil to
which no addition of nitrogenous manure had been made
was richer in nitrate -than that to which horse dung had
been applied, the latter having not only destroyed the
added nitrate, but also that naturally present in the soil
as well.

The addition of chopped straw in large quantities
to soil containing nitrate has a similar effect to that
produced by fresh dung.

The correct interpretation of the results of Wagner's
and Maercker's work is of importance since casual con-
sideration might lead to the conclusion that fresh horse

158 DENITRIFICATION

and cow manure are practically valueless for the nutrition
of plants, and that their addition to the soil is dangerous
on account of the consequent destruction of nitrates
which follows such practice. It must be noted that in
all the experiments very large amounts of fresh dung
were used, far in excess of what would be used in farm
practice. Wagner attributed the bad effect of dung and
straw chiefly to the large numbers of denitrifying bacteria
which they contain. No doubt these materials add
denitrifying bacteria to the soil, but the loss of nitrogen
observed is due more particularly to the large quantity
of easily assimilated organic matter which the dung
contains, for without this material denitrification cannot
proceed. Well rotted dung or farmyard manure which
has been ploughed into the land some time is found to
have little or no destructive effect upon nitrates added
subsequently, and does not reduce the yield of crops in
the same way as fresh dung does : the numbers of
denitrifying organisms are not reduced by rotting of the
manure, but the easily oxidized organic matter which
they need for their special denitrifying effect is greatly
diminished by the putrefactive changes and decay which
takes place in an aerated heap or in the soil.

The reduction of crop under the conditions of these
pot experiments, where large amounts of fresh dung or
straw are used, is due in part to the destruction of
nitrates and loss of gaseous nitrogen ; but some of it is
due to another cause, namely, the assimilation of nitrates
by certain species of bacteria added by the dung and
straw, or stimulated by these materials into active
growth, and there occurs a locking up of nitrogen in
an organic form in the bodies of these organisms.
Berthelot and others have shown that the organic

NITRATES AND NITRITES 159

nitrogen compounds of the soil may be increased at the
expense of the nitrate in it. Such syntheses of insoluble
nitrogenous material temporarily reduce the amount of
nitrogen available for the growth of ordinary field crops :
after death of the bacteria, however, nitrification sets
in, and the nitrogen becomes again of service for the
nutrition of green plants. Experience of farmers and
experiments at Rothamsted and elsewhere have shown
that under ordinary conditions of farming a good return
is generally obtained from the nitrogen of nitrate of
soda, even when applied with dung, so long as the latter
is used in moderate amounts. It is only in gardens
where 50 to 100 tons of fresh dung per acre are
occasionally employed that denitrifying effects such as
those observed in Wagner and Maercker's pot experi-
ments are likely to be encountered.

A large number of species of bacteria are known to
possess the power of reducing nitrates to nitrites ; a
much smaller number, however, are found capable of
destroying nitrates with the evolution of free nitrogen.

From the recent investigations of Beijerinck and
Minkman, it would appear that the denitrification of
a nitrate in the soil usually results in the formation of
considerable amounts of carbon dioxide and nitrous
oxide gases, from the latter of which free nitrogen is
ultimately evolved. The decompositions occurring in
the process may be expressed thus : —

2KNO3 + C... = 2KNO2 + CO2
2KNO2 + C... = 2K2CO3 + N2O
2N2O + C... -C02 + 2N2

where C... represents an organic carbon compound.

They found that in some instances the mixed gases
given off from bouillon with 5 to 1 2 per cent, of nitrate

l6o DENITRIFICATION

in it contained as much as 80 per cent, of nitrous
oxide.

Many denitrifying bacteria, such as the forms examined
by Gayon and Dupetit, Warington, Giltay, and Aberson,
are insufficiently known to warrant inclusion here ; the
chief of the forms capable of setting free nitrogen
which have been isolated and accurately described are
mentioned below : —

Bact. Stutzeri, L. and N. {Bad. denitrificans II., Burri and
Stutzer). — A feebly motile organism isolated from straw, soil,
and the air ; grows best with small supply of oxygen. Generally
rods with rounded ends and non-motile, grown on agar, 1.5 to
2.5 fjj long, 0.5 to I fj^ thick. Sometimes spindle shaped.
In nitrate bouillon the rods are thinner (.25 to .3 /x in
thickness). Stains more at poles than in middle, except in
very young cultures.

Gelatine. — Not liquefied. Surface colonies dull white, i to
2 mm. diameter, with characteristic radial rib-like markings
and raised rims : deep colonies are small, white, round or
spindle form with ragged edge. Stab beaded, white, slimy after
a time.

Agar. — Surface colonies somewhat similar to those of gelatine
at first ; later they become flatter and less distinct in character.

Potato. — Growth ribbed ; pale flesh colour on potato feebly
alkalinized with .05 per cent, sodium carbonate.

Nitrate bouillon (.3 per cent.). — Denitrification in one to two
days or less, with abundant frothing and sediment.

Bact. denitrificans, L. and N. {Bac. denitrificans /., Burri and
Stutzer). — An aerobic species obtained from horse dung, straw,
and soil. On agar 1.5 to 2.5 /x long, .75 ft thick; in bouillon
2 to 3 /A long. Rods mostly non- motile, stains uniformly with
methyl-violet, but more at poles with carbol-fuchsin.

Gelatine. — Surface colonies are not usually found to develop in

NITRATES AND NITRITES i6i

spite of the aerobic character of the organism. When present
are white, somewhat transparent and sUmy later, with ragged
edge. Deep colonies small, rounded, and yellowish white.
Stab threadlike, very delicate growth all over surface.

Agar. — Surface colonies are produced on agar; roundish
edge ; they are very thin often, with a whitish centre, and from
.5 to 3 cm. across.

Potato. — On alkalinized potato (.05 per cent. NagCOg) little
growth at room temperature : at 30° C. in two days, dirty brownish
red : with shining surface.

Nitrate Bouillon. — Denitrifi cation of nitrate only in presence
of B. coli or other reducing organism. Denitrifies a nitrite
bouillon alone, with frothing.

Bact. agile, Amp. and Gar. — A facultative anaerobic organism
isolated from cow dung; very short, motile rods, i to 1.5 /a long,
.1 to .3 ju, thick.

Gelatine. — Not liquefied. Surface colonies, round, white,
scarcely visible (150 to 170 ft diameter), and grow slowly. In
nitrate gelatine small gas bubbles surround them. Stab thin and
slightly beaded, with small growth at surface, i mm. diameter,
greyish.

Agar. — White, slimy colonies, which remain small.

Potato. — On alkalinized (.05 per cent. NagCOg) potato,
growth is very slow, thin, yellow or dirty in brown colour.

Nitrate Bouillon. — Grows well in this medium, denitrifying
and producing froth with fine gas bubbles and turbidity.

Bact. Hartlebii, Jensen. — A motile organism isolated from
soil. Rods, 2 to 4 fji long, .75 /* thick; stains easily and
generally uniformly.

Gelatine. — Not liquefied. Surface colonies very small, i to 3
mm. in diameter, slimy, semi-transparent and white, usually with
entire border. Stab beaded, with a white slimy growth at the
surface, 3 to 6 mm. diameter.

Agar. — Surface colonies slimy, translucent; later more watery ;
deep colonies small white and entire.
L

l62 DENITRIFICATION

Nitrate Bouillon. — Turbid in twenty-four hours ; denitrification
and frothing in two to three days.

Bact. centropunctatum, Jensen. — Isolated from cow dung and
faeces of guinea-pig. On agar under aerobic conditions is coccus-
like, .3 to .5 ju, in diameter, with a large slimy capsule 3 /x dia-
meter. In nitrate bouillon is rod-shaped, motile, and like Bact.
Stutzeri. Under anaerobic cultivation are large, oval organisms
5 ft long, I /x thick, which stain chiefly at the ends. On
account of thick capsule is difficult to stain unless washed in
xylol and alcohol first on cover sHp.

Gelatine. — Not liquefied. Surface colonies thin, whitish trans-
lucent, ragged borders ; deep colonies, small white entire border.
Stab beaded, with white spreading growth on surface.

Agar. — Surface colonies thick, moist, not slimy, dirty grey in
colour.

Nitrate Bouillon. — Turbid in one to two days, with frothing
and denitrification.

Bact. Schirokikhi, Jensen. — A feebly, motile, aerobic organism,
isolated from horse dung. Rods with rounded ends, usually three
or four together in a chain when grown in bouillon ; in pairs on
solid media. Spores are abundant, and produced in two days.

Gelatine. — Liquefied to a pale bluish liquid in which are yellow
grains. . Surface colonies are slow in developing ; on the third
day, at 20° C, are about i to 2 mm. in diameter, in five days or less
colonies run together. Stab ; rapid, funnel-shaped liquefaction,
liquid pale grey at first, yellowish afterwards, with sediment.

Agar. — Surface colonies develop sooner than on gelatine, and
are star-shaped in twenty-four hours at 37° C, with a raised centre
surrounded by pale blue rays. In three or four days the colonies
become rounded, 2 to 4 cm. in diameter, with a yellowish brown
zone round each.

Potato. — On alkalinized potato (.05 NagCOg) growth is light
brown.

Nitrate Bouillon. — (25 per cent. KNO3). Denitrification com-
plete in five to eight days, at 30° to 35° C.

NITRATES AND NITRITES 163

Bact. filefaciens, Jensen. — A non-motile organism isolated
from an old impure culture of Bad. Stutzeri. Rods usually
.5 to 1.5 \i long and .5 to .75 thick; older cells stain irregularly.

Gelatine. — Not liquefied. Surface colonies thin, dull and some-
what slimy; deep colonies very small, white, round. Stab beaded,
with well developed white surface growth.

Agar. — Surface colonies dirty white, slimy, can be drawn out
into long strings.

Nitrate Bouillon. — Denitrification in one to two days at 30° C. ;
liquid pretty clear with a stringy sediment.

Bact. nitrovomm, Jensen. — A non-motile organism isolated
from horse dung. Rods very small, .5 to i /a long, .5 /x thick.

Gelatine. — Not liquefied. Surface colonies very small, .2 to
5 mm. in diameter, white, slimy, and round; deep colonies scarcely
visible. Stab slender and beaded, its surface growth 6 to 10 mm.
diameter, with concentric rings sometimes.

Agar. — Surface colonies, dirty, white, slimy, 2 to 4 mm. in
diameter.

Nitrate Bouillon. — Slightly turbid in twenty-four hours ; deni-
trification in two or three days, with little froth ; sand-like
sediment.

Vibrio denitrificans. Sew. — A peculiar variable bacterium
isolated by Sewerin from horse dung. In bouillon comma and
spirillum forms occur as well as rods i to 10 ft long; branched
and triangular involution forms are also found after four or five
days. On solid media usually rods 2 to 4 /x long, .5 /a thick.

Gelatine. — Not liquefied. Small, round, white surface colonies,
slow in development. Stab beaded.

Agar. — In two days at 30° C. small (i to 2 mm.), bluish- white,
slimy colonies.

Nitrate Bouillon. — Denitrification occurs in two or three days
with turbidity and only a slight frothing.

Vibrio denitrificans, Hoflich. — A motile vibrio isolated from
horse and cow dung, straw and soil. Both rods and vibrio forms

l64 DENITRIFICATION

occur, the latter 1.4 to 2.3 fi long, .4 to 5 /a thick, often many
together in zoogloea.

Gelatine. — Not liquefied. Surface colonies slightly raised,
whitish, smooth at first, later puckered, and 2 to 3 mm. in diameter;
deep colonies, small (r to 2 mm.), bluish- white, blackberry form,
with opaque yellowish brown centre.

Agar. — Surface colonies, rounded in forty-eight hours at 35° C,
I to 2 mm. diameter, centre milk-white, with whitish cleaner space
round.

Potato. — At 35° C. in two to three days, a yellowish brown,
slightly raised, growth not spreading much.

Nitrate Bouillon (3 per cent.). — In twelve hours turbid; in
twenty-four to thirty-six hours much froth, with complete deni-
trification and little sediment; remains turbid after frothing
ceases. Differs from the preceding in form of deep colony,
more uniform staining, want of sliminess, and abundant frothing
in nitrate bouillon.

Bacillus (Proteus) denitrificans,Hoflich. — An aerobic "proteus-
like" bacterium, isolated from horse and cow dung, straw and
soil. In bouillon the organisms are motile, 5 to 10 ft long and
.3 to .4 ja thick ; they grow into long threads (100 /a long or
more), which often break up in three or four days into rods,
vibrio, and even coccus forms, all without spores.

Gelatine. — Not liquefied; the surface colonies are round or
irregular at first, and gradually extend over 2 to 3 sq. cm. of
surface as a filmy growth, bluish-white and semi-transparent.
Deep colonies are whitish, and remain small. Stab a delicate
whitish line of very small colonies.

Agar. — In twenty hours, at 35° C, the surface colonies
are bluish-white, transparent, very thin, i cm. in diameter.
They spread very rapidly, and cover the plate in about two
days.

Potato. — No visible growth.

Nitrate Bouillon (3 per cent.). — In twenty hours, at 35° C,
diffuse turbidity with denitrification and formation of froth and

1 litre.

logr.

5gr.

3gr.

5gr.

lo gr.

3gr-

I litre.

NITRATES AND NITRITES 165

fine bubbles. Later liquid clears and froth disappears, leaving
faint iridescent scum and whitish light sediment.

Ex, 81. — Prepare the following media : —

1. Nitrate Bouillon.

Extract of lean beef (see p. 56)

Peptone

Sodium chloride ....
„ nitrate ....

(^)

Lemco's Extract ....

Peptone .....

Sodium nitrate ....

Water ......

No. I usually gives best results. Make both just neutral with
sodium carbonate and sterilize as usual.

2. Nitrate Gelatine.

Nitrate bouillon as above . . . i litre.

Gelatine too gr.

Neutralize with sodium carbonate; place 10 to 15 c.c. in tubes
and sterilize.

3. Nirate Agar.

Nitrate bouillon as above . . . i litre.

Agar-agar 15 gr.

Neutralize as above, tube and sterilize.

Ex. 82. — (i) Add 5 grams of fresh horse dung to —

Water . . . . . . 100 gr.

Sodium nitrate 3 gr.

Incubate about 30° C. : notice frothing, and test for nitrate
with diphenylamine and sulphuric acid, as in Ex. 74, every two
days for a fortnight. Nitrate usually disappears in six or eight
days.

(2) Repeat above, using 5 grams of chopped straw instead of
dung.

i66 DENITRIFICATION

(3) Boil the solution after adding the dung ; incubate and test
for nitrate as before. Does boiling check denitrification ?

Ex. 83. — Isolate denitrifying organisms from dung, soil,
ditch water, and straw.

(a) Inoculate tubes containing 10 to 15 c.c. of neutral nitrate
bouillon (Ex. 81) with small pieces of fresh dung, soil, or
other material to be examined, or with watery extracts of them.
Incubate at 30° to 35° C. and when frothing is well developed
inoculate ordinary agar and gelatine plates from the fermenting
medium. Transfer small portions of the colonies which appear
on the plates into tubes of nitrate bouillon ; observe frothing and
test for disappearance of nitrate with diphenylamine (Ex. 74)
during a fortnight, or longer if necessary.

It is generally better to incubate the medium inoculated
directly with the dung or its extract anaerobically in a yeast
flask at 35° C. (Ex. 140), as this checks the development of
aerobic, liquefying, and non-denitrifying species and makes the
subsequent isolation of denitrifying species less laborious.

(d) Similar results are obtained thus : Shake up small pieces
of dung, soil, etc., in sterilized water and inoculate agar and
gelatine plates with a drop of this extract in the usual way.
Test the denitrifying power of the several colonies obtained by
transference to nitrate bouillon as before.

Ex. 84. — Take a glass cylinder 2 feet long, 2 or 3 inches
in diameter, and close one end with a cork through which a
small piece of glass tube is passed. Place in an upright position
and half fill the cylinder with garden soil closely packed, first
covering the cork on the inside with coarse calico so that the
soil does not pass through the small glass tube : close the lower
end of the latter with a cork. Now pour water into the wide
open end of the cylinder and wait until the soil is saturated;
then add enough to fill the empty space above the surface of the
soil. Remove the cork from the small glass tube and allow the
water to percolate through : test for nitrates with diphenylamine
(Ex. 74) every second day. The water which drains through

NITRATES AND NITRITES

167

should be poured back again on the soil so as to keep the latter
saturated the whole time. The nitrates are ultimately destroyed
by denitrification, when the water coming through shows no
reaction with diphenylamine.

Ex. 85. — Inoculate tubes of nitrate bouillon (Ex. 81) with
a gram of soil or fresh horse or cow dung, and
(i) Incubate at room temperature as an aerobe.

(2) „ 35° C.

(3) „ 35° C. „ anaerobe in yeast flask.

Note (a) time taken to develop froth ; {b) pre-
sence or absence of turbidity and sediment ; {c)
disappearance of nitrate (diphenylamine test).

Ex. 86. — Melt some tubes of nitrate gelatine
(Ex. 81) in warm water, and introduce into each
a loopful of nitrate bouillon containing any deni-
trifying species isolated in Ex. ^:^. Gently shake
the tubes to distribute the bacteria in the medium :
allow the latter to solidify and incubate at 18°
to 20° C. Note the development of gas bubbles
in the gelatine (Fig. 31).

Ex. 87. — Prepare the following solution : —
Calcium tartrate
Potassium nitrate
Di-potassium phosphate
Tap water
Sterilize.

Fill several 50 c.c. sterilized flasks to within an
inch of the top with the solution, and introduce fig. 31.— gL bub-
into each i to 2 grams of soil, dung, or other ^Irr'afe'^^^^Pradi:
material. Then completely fill to the brim and ^^ denitrifying

^ ■' _ organism.

close with a cork, through which is passed a small
piece of glass tube ; some of the solution will run out of the tube,
but the flask will be full : inoculate at 28° C. Denitrification soon
begins, and the gases (CO2 and N) evolved drive out some of

10 gr.
10 gr.

•25 gr-
500 c.c.

i68

DENITRIFICATION

the solution, but the space in the flask is filled with inert gases
and the culture grows under practically anaerobic conditions.
From this flask introduce some of the fermenting liquid into
another filled completely in the same manner with the culture
solution. When frothing has gone on some time in the second
flask inoculate a third from it. In this way a culture can be
obtained containing denitrifying species almost entirely, the
aerobic non-denitrifying species present in the soil or dung being
suppressed. Inoculate agar and gelatine plates from the last
flask, and isolate and examine the organisms which develop.
Ex. 88. — A good medium in which to grow denitrifying species

Giltay's solution.

Sodium nitrate

2 gr.

Citric acid ....

5gr.

Magnesium sulphate .

2 gr.

Di-potassium phosphate

2 gr.

Calcium chloride

.2gr.

Water

I litre

Neutralize with a solution of sodium carbonate, and add a
drop of ferric chloride.

Cultivate denitrifiers in flasks or tubes of this solution, and
note increasing alkalinity as growth proceeds for three or four
weeks. Oxygen removed from the sodium nitrate gives rise to
caustic soda (NaOH), some of which unites with the COg pro-
duced from the oxidation of the citric acid, and forms sodium
carbonate (NagCOg). Both the NaOH and Na2C03 increase
the alkalinity of the solution, which may be quantitively estimated
with decinormal acid.

CHAPTER XI.

THE FIXATION OR ASSIMILATION OF
NITROGEN.

The " Fixation " or Assimilation of the Free Nitrogen
of the Air. — In the processes of decay, putrefaction, and
denitrification some of the world's combined nitrogen is
destroyed, the nitrogen being set free in the gaseous
elementary form. Without the existence of an opposite
synthetic process, by means of which the free nitrogen of
the atmosphere is brought into combination again, it is
obvious that the stock of nitrogen compounds would
decrease. To some extent such synthesis or chemical
union of nitrogen and oxygen takes place in the air
through the agency of lightning and other electric dis-
charges, the nitrous and nitric acids so formed being
eventually brought down in the rain. The amount,
however, of the combined nitrogen added to the soil
annually in this country by showers and dew is very
small, being only 4 or 5 lbs. per acre, and a large pro-
portion of it is not derived from free nitrogen, but
consists of compounds of ammonia, which have escaped
or evaporated into the air from the soil and decaying
organic matter.

Berthelot, in some experiments carried out about 1885,
showed that bare uncropped soil exposed to the air in-
creased very considerably in combined nitrogen content,

even when careful notice was taken of the addition of

169

I/O FIXATION OR ASSIMILATION OF NITROGEN

nitrogen compounds from the atmosphere. After seven
months 50 kilograms of soil showed an increase of 12.38
grams of nitrogen, the amount due to the ammonia and
nitric acid in the rain during this time being less than
half a gram. Indirect evidence of similar and larger
increments has been obtained also from analysis of the
soil and of the produce from it during several years,
when the crops were grown without nitrogenous manures.

In the first 6 inches of soil of an arable field from
20 to 30 lbs. or more of combined nitogen per acre is
added annually, the only source of which is the free
nitrogen of the air. From the fact that no gain of nitrogen
was found in soils which were first heated to 1 20' C. in
a current of superheated steam, Berthelot concluded that
the addition was in some way connected with the exist-
ence of minute living organisms in the unheated soil,
although he was not able to isolate them satisfactorily.
However, through the pioneer work of Hellriegel, Wil-
farth, Winogradsky, and Beijerinck, several organisms
capable of " fixing " or assimilating the free nitrogen
are now well known : the chief of them are species of
Clostridium^ Azotobacter^ and Pseudomonas. There seems
little doubt that there are other forms of bacteria besides
these which are able to build up nitrogen compounds from
the free nitrogen of the air, and evidence exists which
appears to indicate that some of the lower fungi are
similarly endowed.

The power to utilize gaseous nitrogen is not possessed
equally by all the organisms named. Pseudomonas rddi-
cicola in association with leguminous plants is able to
add indirectly large amounts of complex nitrogenous
compounds to the land, and these, as well as those
manufactured by the other bacteria mentioned, ulti-

CLOSTRIDIUM PASTORIANUM 17 1

mately become of service in the nutrition of ordinary-
green plants through the processes of decay and nitri-
faction. The species of Azotobacter add considerable
quantities of combined nitrogen also, and a small increase
is due to organisms belonging to the genus Clostridium,

It is not too much to say that the practical application
of the forces possessed by these and similar organisms is
a matter of the utmost importance for the production of
crops of all kinds, and a complete determination of the
conditions under which they carry on their functions to
the greatest advantage, is certain to lead to far-reaching
effects upon the world's agriculture.

I. Clostridium pastorianum, Win. — In 1895 Wino-
gradsky isolated from the soil of St Petersburg an
organism which was capable of assimilating free nitrogen
when grown under anaerobic conditions in a medium con-
taining dextrose and certain inorganic compounds. A
solution containing 40 grams of dextrose was inoculated
with a pure culture of the bacillus, and a stream of pure
nitrogen was kept passing through it. After twenty
days the sugar had disappeared, and analysis showed an
increase of 53.6 milligrams of nitrogen in a combined
form in the solution. The dextrose was decomposed,
42 to 45 per cent, of it appearing as butyric and acetic
acids, the rest being split up with the formation of carbon
dioxide and hydrogen. Usually small traces of isobutyl
or propyl alcohol and lactic acid are found also.

Winogradsky named the organism Clostridium pas-
torianum. It is a strict anaerobic bacillus, and can
assimilate nitrogen in media free from nitrogenous com-
pounds when oxygen is eliminated. It can also fix free
nitrogen in solutions even when air is admitted if aerobic
species are present to remove the free oxygen and create

i;2 FIXATION OR ASSIMILATION OF NITROGEN

suitable anaerobic conditions for its development and
growth. A mixture of the Clostridium and some aerobic
bacteria from the soil when grown in a medium contain-
ing 20 grams of dextrose and .01 gram of ammonium
sulphate, gave an increase of 24.4 mg. of combined
nitrogen, after the dextrose had all disappeared ; during
the experiment there was free access of air, from which
carbon dioxide and nitrogenous compounds had been
removed by passing through caustic soda and sulphuric
acid.

Clostridium pastorianum belongs to the group of
bacteria which produce butyric acid, and resembles its
associates in morphology and in many of its physio-
logical powers. It can utilize as a source of energy for
its nitrogen-fixing process not only dextrose but levulose,
cane-sugar, galactose, and some other sugars. How it
obtains the necessary carbohydrate in the soil has not
been determined. Two sources appear to be possible,
namely, the residues from decaying plant tissues, or the
carbohydrate manufactured by minute green algae so
frequently met with in soil. The latter hypothesis is
the more likely, and is supported by evidence mentioned
later. According to Winogradsky, it does not form
butyl alcohol nor ferment mannite, starch, lactose, or
calcium lactate, in these respects differing from the
common representatives of the " butyric " group. In an
active state of growth the organism is a short cylindrical
bacillus 1.5 to 2 fj. long and 1.2 to 1.3 /^ broad, with
little or no motile power, and stains yellow with iodine
solution. After further development it becomes an oval
or spindle-shaped organism of the usual " Clostridium "
form, with granular contents ; in this stage it contains
granulose, a carbohydrate which closely resembles starch,

CLOSTRIDIUM PASTORIANUM 173

and like the latter substance stains a violet colour with
a solution of iodine (Ex. 40).

Spores arise at the end of the mother-cells, but when
fully formed they occupy the middle of the swollen cell,
which becomes transformed into a kind of spore capsule.
The ripe spores are about 1.6 ^ long and 1.3 /// broad,
and readily germinate when placed in a sugar medium
under anaerobic conditions. Grown on potato or carrot
in a vacuum or in nitrogen, the bacillus produces raised
greyish-yellow colonies about i mm. across, and gives
rise to a cheesy odour. The individual bacilli are
longer and thicker than they are when cultivated in
liquid media, and sometimes develop vibrio or coccus-like
involution-forms incapable of spore-formation or nitrogen
fixation : long sporeless threads are formed in solutions
containing peptone, asparagin, or other nitrogenous com-
pounds, but these die out rapidly. It does not thrive well
on sugar agar medium, and refuses to grow on gelatine.

C. pastorianum is widely distributed, but is not
present in all soils ; it is generally met with in associa-
tion with aerobic species.

Winogradsky found in soils of South Russia and Paris
other forms of species of Clostridium with nitrogen-fixing
powers. One of these was thicker than C. pastorianum
(1.6 to 1.8 thick), with spores 1.9 long and 1.5 thick:
they all proved difficult to isolate, and were not further
studied.

Freudenreich also obtained from Swiss soils an
organism which closely resembled Winogradsky's Clos-
tridium in all its characters, and had the power of
fermenting ,mannite, which the latter is said not to
possess.

Another nitrogen-fixing species is Clostridium ameri-

174 FIXATION OR ASSIMILATION OF NITROGEN

canum isolated by H. Pringsheim. It is a motile
organism of the " butyric " group which can carry on its
work under less stringent anaerobic conditions than
those necessary for C. pastorianum. In pure cultures in
open flasks it sets up carbohydrate fermentation with
nitrogen fixation, and in an atmosphere of hydrogen it is
able to ferment starch, mannite, and lactose.

Benecke and Keutner described a large Clostridium
{C. giganteum) from the soil, but its nitrogen fixing power
was not tested. The cells are pointed at the ends, and
some of them contain two spores. The spores of this
species are very large, being 2.5 im long and 1.5 [m
broad.

The power of nitrogen fixation appears to be a
common function of many organisms belonging to the
class which cause butyric-acid fermentations, but the
amount of combined nitrogen added to the soil by the
activity of these bacteria is probably small.

For every gram of carbohydrate fermented from 1.2
to 3.7 milligrams of free nitrogen are assimilated.

Ex. 89. — Prepare the following solution : —

Di-potassium phosphate . . , . i gr.

Magnesium sulphate 5 gr.

Sodium chloride \

Ferrous sulphate \ A very small crystal of each.

Manganese sulphate J

Dextrose . . . . . . • 30 gr.

Precipitated chalk 30 gr.

Water . i litre.

Place 250 c.c. in four separate conical flasks, and inoculate
each with . 5 gr. of soil from four different localities. Close the
flasks with cotton wool plugs, and place them in the incubator

SPECIES OF AZOTOBACTER 175

at 22° C. or 30° C. In a few days notice the active production
of gas bubbles.

Suck up some of the chalk from the bottom of the flasks with
a pipette, spread out on a slide and examine with the microscope :

(i) Are Clostridium forms present?

(2) Run under cover slip some iodine solution. Do the
bacteria stain with a blue colour ?

(3) If spores are present stain as in Ex. 12.

(4) Try and obtain growths of any of the organisms present
on potato under anaerobic conditions. (Buchner's tube
method.)

2. Species of Azotobacter. — In 1901 Beijerinck
isolated from soil and canal water two species of
organisms which are able to assimilate the free nitrogen
of the air and build up proteins and other nitrogen
compounds needed for the growth and development of
their bodies. These he included in a genus which he
named Azotobacter. By their multiplication they are
able to add considerable amounts of combined nitrogen
to the soil, which ultimately becomes available to farm
and garden cro^^^-fehfough the processes of decay and
nitrification, bin ce Beijerinck's discovery in 1901 much
attention has been devoted to the determination of the
distribution, growth, and conditions essential for nitrogen
fixation by these organisms. The different species are
readily isolated from almost every kind of soil except
those which are acid or of dry, sandy character. They
are also commonly present in well and river water and in
sea water, and are found on the surface slime of species
of Fucus, Laminaria, and other algse. Freudenreich and
Thiele found them in the soil down to a depth of 50 or
60 cm. ; at 100 to 190 cm. deep Clostridium pas-
torianum was met with, but no species of Azotobacter.

1/6 FIXATION OR ASSIMILATION OF NITROGEN

As in the case of Clostridium, these organisms obtain
the energy they need to bring the inert free nitrogen
into chemical combination from the combustion of carbon
compounds in the soil. The presence of phosphates and
lime is necessary for their growth, and possibly potash
and other inorganic salts are useful also ; according to
Fischer's investigations they die out or become inert in
soils containing less than .i per cent, of lime. All the
species of Azotobacter are strongly aerobic and need an
abundant supply of air. An adequate amount of
moisture is also essential for their welfare, the best results
being obtained when the soil contains between 5 to i 5
per cent. ; more than this leads to imperfect aeration.
They only thrive satisfactorily when nitrogen compounds'*
are present in small amount or absent altogether. • ij^^y
carry out their work most effectively at a tem^rature
between 25° C. and 30° C, but growth is active at 20° C, /
and A. Koch states that he found^ nitrogen fixation going
on in the soil during winter. At temperatures above
30° or 35^ C. they produce involution forms and become
weakened in vitality and nitrogen-fixing power. To
avoid complications with Clostridium pastorianum present
in the soil upon which he experimented, Beijerinck
used mannite and calcium, sodium or potassium salts of
propionic acid as the necessary carbon food for these
organisms, since these substances do not readily undergo
the " butyric " fermentation. He found Azotobacter also
capable of utilizing glucose, levulose, galactose, cane
sugar, and salts of several organic acids as sources of
carbon.

The nutrient medium in which the cultures are made
is placed in small amounts in wide-bottomed flasks, so as
to allow a free access of air to a large surface of the

SPECIES OF AZOTOBACTER

177

solution. With 20 grams of glucose or mannite in a
litre of nutrient solution Freudenreich found that Azoto-
bacter chroococcum assimilated or " fixed " from 1 2 to
24 mg. of nitrogen in two or three weeks. When grown
on gypsum plates with less liquid present and abundant
air supply 160 mg. of nitrogen were assimiliated in the
same time. By cultivating it upon agar mannite-
phosphate plates A. Koch obtained an increase of
180 mg. of combined nitrogen per litre of medium.

Gerlach and Vogel found that the amount of nitrogen
assimilated is influenced largely by the amount of glucose
present in the nutrient solution. From i to 12 grams
of sugar per litre proved beneficial, and the amount of
nitrogen assimilated increased proportionally ; i 5 grams
checked the action of the organisms to some extent.
The following are some of their results after five weeks'
growth : —

Milligrams of
Nitrogen fixed.

gr. of grape sugar present . . 7.4

17.3
39.4
91.4
. 127.9
62. 9

They also showed that the power of nitrogen fixation
decreases with the age of the culture, as indicated below,
and is reduced by cultivating the organisms for several
generations in nutrient media : —

Milligrams of N.
in I litre.

A culture 18 days old " fixed " . , 127.9 i" 5 weeks*

no „ „ . . 54-9

» 328 „ „ . . 23.4 „

M

With I gr

))

3 »

»>

5 „

j>

10 „

)»

12 „

»

15 «

1/8 FIXATION OR ASSIMILATION OF NITROGEN

All the organisms belonging to the genus Azotobacter
are extremely variable in size and shape. The typical
form is a short oval cell from 4 to 6 /x long and 3 to 4 a&
broad (Fig. 32). Under certain conditions these cells
become thicker and rounded into a coccus, two of which
are usually found united — a diplococcus. They appear
also as streptococci, and can divide
in two or three directions so as
to form sarcina - like " packets " ;
the latter are especially abundant
in old cultures. Spores are not
known. The cell walls are often
thick and slimy and stain well with
methylene blue or gentian violet.
Fig. -ii.-AZtabacter chroo- The protoplasm within contains
....«;;., Beijk.(x 500). ^^^jj refractive granules and usu-
ally a vacuole. Iodine stains the organisms a yellowish
tint. Four or five species or varieties have been de-
scribed. Although they differ in nitrogen fixing power,
they are probably modifications of one organism. The
following are the best known : —

A. cliroococcum, Beijk. — This, the first species isolated, is very
widely distributed in field and garden soils. It produces after a
time a brown or blackish coloration on solid substrata and on
the surface of liquid media. In young cultures the cells are
motile, short, plump rods, 4.5 to 7 /a long, and 3 to 4 //< broad,
generally in pairs. On agar-mannite plates (Ex. 91) at 30° C.
the colonies are pasty at first, round, white, usually with dark
concentric rings in the middle : later they become dark brown,
and may grow to be 5 or 6 mm. in diameter The cells in three
or four days are short rods with rounded ends, often in pairs,
4 to 5 //- in diameter; others are longer oval rods, 3 to 6 ^a
long and 2 to 3 ^ thick. In a week or ten days they may

SPECIES OF AZOTOBACTER 179

become smaller and arranged in short chains, three to eight or
more together ; the " sarcina" form appears in abundance when
the colonies become brown. In liquid media the pigmentation
appears usually in four or five weeks from the time of inoculation
where the medium is kept at 20° to 25° C.

A. agilis, Beijk., originally isolated from canal water, is a large
actively motile organism with a bundle of polar cilia. The
cells are oval, transparent, with distinct cell-walls, granular
protoplasm, and vacuoles. It grows well on an agar medium
containing glucose in place of mannite (Ex. 91). On such
medium with J per cent, of calcium propionate in place of
the sugar, the colonies of A. agilis becomes surrounded by a
greenish yellow fluorescent zone which is characteristic of the
species.

A. vinelandii, Lipman, a form isolated from some North
American soils, produces a thick white membrane on mannite
solutions, which becomes yellowish. Ultimately the liquid
becomes yellowish-red in colour. On mannite agar the colonies
are semi-transparent at first, rounded, and 2 to 4 mm. in
diameter : they produce a yellowish pigment, which diffuses
through the medium.

A. Beijerinckii is another species or form isolated by Lipman
from American soils. The organisms are spherical, non-motile,
and much larger than the preceding kinds ; in mannite solutions
they occur singly or in short chains.

When grown in mannite solutions a white deposit is produced,
and small white colonies appear on the surface of the liquid and
on the walls of the flask.

The colonies oh mannite agar are well developed in four or five
days, somewhat rounded, white, and moist, with netted surfaces.

Ex. 90. — Grow and isolate species of Azotobacter from soil.
Prepare the following solution : —

Di-potassium phosphate . . . . .2 gr.
Mannite . . ' . . . . . 20 gr. ,
Tap water i litre

i8o FIXATION OR ASSIMILATION OF NITROGEN

Add loo c.c. of the solution to a conical wide-bottomed flask,
and inoculate with .i to .2 gr. of garden soil. Incubate at
28° to 30° C. in the dark. In two or three days a scum or
pellicle forms which consists of a mixture or zoogloea of small
bacteria and Azotobacter with amoebae and infusoria. Examine
a small portion of the scum for stumpy or oval rods of
Azotobacter. Transfer a drop or a small portion of the scum
from this flask to another flask of the sterilized nutrient solution
and incubate : later transfer a drop from the second to the third
flask. In this way a tolerably pure culture of Azotobacter may
be obtained in three or four generations. From it quite pure
cultures may b.e prepared by isolation on mannite agar as
indicated in the next Exercise.

Ex. 91. — Prepare the following mannite agar medium : —

Di-potassium phosphate 02 gr.

Mannite ....... 2 gr.

Agar . . 2 gr.

Distilled water ...... 100 c.c.

Sterilize. Add 10 to 20 c.c. to a Petri dish and slowly cool,
turning the dish upside down, so as to allow the water of
condensation to collect on the lid and not on the surface of the
medium. Add a small portion of the membrane or a drop of
the solution from the flasks in the previous Exercise to a small
quantity of sterile distilled water, and pour some of it over the
surface of the plate. Incubate at 28° C, and note the colonies
which grow : examine the size and shape of the organisms from
each kind of colony.

Test the action of the organisms from the different colonies in
" bouillon " at 20° C This medium remains sterile or clear
when inoculated with pure Azotobacter^ but generally becomes
turbid when other organisms are added.

Ex. 92. — Azotobacter colonies grow well on gypsum plates
which are permeated by a mannite phosphate solution.

Take some plaster of Paris (gypsum) and mix it with water to

SPECIES OF AZOTOBACTER l8l

the consistency of thick cream (about eight volumes of gypsum
powder to three of water are needed) ; pour it on a plate of
glass so as to be about a quarter of an inch thick, and
before it has set cut it into narrow strips 3 inches long by
J in. to f in. broad, or into circular pieces which will fit easily
inside an ordinary Petri dish.

Prepare the following solution : —

Di-potassium phosphate 05 gr.

Sodium chloride 05 gr-

Mannite . . . ' . . . . 2 gr.

Water ........ 100 cc.

Add about 5 cc. to some test-tubes, and place in each of
these a strip of gypsum, so that the lower end dips into the
solution : or pour a few cc. into a Petri dish, and put in a
circular plate of the plaster : do not wet the upper surface of the
latter. Then place a drop or two of a culture of Azotobacter
on surface of the gypsum plate, and incubate at about 28° to
30° C. Observe the greyish-white growth, which becomes dark
brown generally in three or four days.

Ex. 93. — Isolate Azotobacter from soil by the following
method (Gerlach and Vogel) : —

Take 20 grams of fresh garden soil and place in a large
Petri dish : pour on it 100 cc. of the following solution: —

Grape sugar. .....

Di-potassium phosphate . i

Sodium chloride

Calcium carbonate ....

Ferrous sulphate

Water

Keep it in the dark at 28° C. for two or three days. Examine
the scum which arises for cells of Azotobacter : these are usually
abundant and crowded into a slimy zoogloea.

(i) With a platinum needle transfer a small portion of the
scum to a drop of water on a glass slide, cover with a slip, and
press the latter so as to break up the zoogloea.

I gr.
•25 gr-
•25 gr-
•25 gr.
a trace
500 cc.

1 82 FIXATION OR ASSIMILATION OF NITROGEN

(ii) Run under the cover slip a drop of iodine solution, or
mount a small piece of the scum in the latter. Observe the
yellow colour of the Azotobacter cells, and look 'out for violet-
stained Clostridium cells, which are generally present,
(iii) Inoculate a plate of the following agar medium : —

Agar 2 gr.

Grape sugar 2 gr.

Di-potassium phosphate . . . . -2 gr.

Water 100 c.c.

The colonies of Azotobacter are at first colourless and shining ;
later they become elevated and yellow, and finally, in five or six
days, turn a deep brown colour.

If no Azotobacter organisms are seen on first examination of
the scum as above, add i c.c. of the liquid from the mixed soil
and culture solution to 50 c.c. of the nutrient solution, and
incubate for two or three days at 28° C. Another grape-sugar
plate can then be inoculated with a drop of this culture, and the
Azotobacter colonies afterwards obtained.

3. It is hardly likely that the only organisms capable of
assimilating the free nitrogen of the air are those already
mentioned belonging to the genera Clostridium and Azoto-
bacter, although Winogradsky and others obtained negative
results with many bacteria which they specially investi-
gated from this point of view. There is indeed some
evidence that a number of species of soil organisms are
able to effect the synthesis of nitrogen compounds from
the free nitrogen of the air in slight degree, but the diffi-
culties connected with the investigation of the problem
are great. In most cases the increase in nitrogen con-
tent due to nitrogen fixation is necessarily very small,
and frequently lies within the limits of experimental
error, so that much caution must be exercised in accept-
ing or rejecting results.

ALINIT 183

Alinit. — About 1895 Caron isolated from the soil a
bacterium, Bacillus ellenbachensis a, and which in pure
cultures was offered for sale under the name "Alinit." It
was claimed that this organism when applied to poor soils
was capable of " fixing " the nitrogen of the air, which
ultimately became of service for the nutrition of cereal
crops. Experiments to test the beneficial influence of
"Alinit" have led to conflicting results, Stoklasa, Grandeau,
Malpeaux, and others maintaining that infected plots
frequently produced much larger crops than plots which
were untreated, while many experiments on barley and
other crops carried out in different parts of Europe
showed no increase in yield after the application of
" Alinit " cultures. Bs. ellenbachensis belongs to a
common group of soil bacteria characterized by their
large size, aerobic nature, and power of spore production ;
the group includes the hay bacillus {Bs. subtilis), Bs.
mycoides, Bs. megatherium^ and some others closely
resembling these. It is a putrefactive species, which
decomposes proteins with the formation of peptones,
amino-acids, and other compounds, and under certain
conditions brings about the reduction of nitrates to
nitrites. According to Breal and Bokorny, leucine,
tyrosine, and other similar nitrogenous substances pro-
duced in putrefactive processes can be utilized for nutri-
tive purposes by green plants of various kinds, so that
the observed beneficial effects of the " alinit " bacterium
may be due in some cases to its power of breaking down
the insoluble nitrogen compounds and rendering them
available to crops. In addition to the power just
described, Stoklasa maintains that the " alinit " bacillus
(which he considers is identical with Bs. tnegatherium,^
De Bary) is able to assimilate free nitrogen when living

1 84 FIXATION OR ASSIMILATION OF NITROGEN

in media or soil containing little or no combined nitrogen,
provided that xylose, galactose, or other carbon compounds
are supplied.

The " Alinit " Bacillus (Bs. ellenbachensis^ a^ Caron) is a large
rod-shaped organism, which does not form very long threads as a
rule. Each cell has rounded ends, and is from 3 to 6 ja long
and 1.25 to 1.5 \i thick; it is stained by Gram's method. The
organism is motile, and forms central oval spores, which exhibit
distinct polar germination, the young rod escaping from the end
of the spore. The optimum temperature for growth is about
28° to 30° C.j and it is aerobic.

Gelatine. — The colonies are at first roundish, somewhat dark
in the centre, with radial filamentous outgrowths ; later the gela-
tine is liquefied. The " stab " soon shows funnel-shaped lique-
faction, with a pellicle on the surface and a flocculent deposit.

Agar. — Surface colonies yellowish-white, roundish or slightly
granular and puckered, surrounded by hair-like extensions ; deep
colonies with irregular thread-like projections, which resemble
the hyphae of fungi.

Potato. — Greyish-white growth, soft and flat.

Milk. — Milk is coagulated, and becomes alkaline, with a
surface scum ; the precipitated casein is peptonized and slowly
dissolved.

3. An aerobic spore-forming bacterium capable of
assimilating free nitrogen was isolated from the soil around
the nodules of Vicia villosa and other leguminous plants
by Lohnis and Westerman ; they named it Bad. danicus.
It is very variable in form and size, appearing in mannite
solutions as a large coccus or as a rod 3 to 5 /a long and
I ih thick. On mannite-agar it produces spores some-
times in " clostridial " cells, the colonies on this medium
being round, shining greyish-white 2 to 4 mm. in diameter
in five days at 20° C.

ALINIT 185

The growth on potato very closely resembles that
produced by Bs. vulgatus.

The organism stains irregularly with fuchsin.

4. Frank, Hellriegel, and others observed that unsteril-
ized soils which become covered with minute green algae
frequently increase very considerably in nitrogen content,
but when protected from the light, or when the surface
is covered with sand or other material to prevent the
growth of these small plants, such soils do not become
richer in combined nitrogen. Schloesing and Laurent
found that free nitrogen of the air was absorbed and fixed
when the latter was enclosed in a vessel containing soil
covered with a coat of green algae and exposed to light.
The accumulation of nitrogen compounds produced under
these conditions may amount to .8 gr. for i kg. of soil,
and occurs chiefly in the thin layer near the surface.
Such observations led to the belief that minute green
algae belonging to Cyanophyceae and Chlorophyceae are
able to utilize free atmospheric nitrogen, but Kruger and
Schneidewind and Kossowitch,by careful experiments with
pure cultures of Cystococcus, Stichococcus and Chlorella in
media from which combined nitrogen is eliminated have
proved that this view is incorrect. No growth occurs
when free nitrogen is supplied to such cultures, but there
is luxuriant development when nitrates are provided.
Bouilhac obtained similar results with cultures of Nostoc
punctiforme. It has been found, however, that when soil
bacteria are added to pure cultures of these algae nitrogen
fixation goes on extensively. Between the algae and the
bacteria there is no doubt a kind of symbiosis ; the latter
organisms fix the nitrogen, deriving the organic com-
pounds essential for the process from products of carbon
assimilation carried on in the light by the green algae,

1 86 FIXATION OR ASSIMILATION OF NITROGEN

the algae in turn obtaining some or all the nitrogen they
need for nutrition from their associates,

Stoklasa states that the " Alinit " bacillus fixes atmo-
spheric nitrogen in considerably larger amounts in the
presence of species of Stichococcus and Nostoc than when
these algae are absent. He also notes that soils contain-
ing an abundance of algae are richer in bacteria than
those which are poor in algae, and that Pleurococcus and
Nostoc contain 6 to 7 per cent, of pentose, 70 per cent, of
which is soluble in water, and easily available for the
nutritive requirements of bacteria.

CHAPTER XII.

THE FIXATION OR ASSIMILATION OP
NITROGEN — ( Continued),

I. The Fixation of Nitrogen by Bacteria in Symbiosis
with Leguminous Plants. — It has been known among
agriculturists from the earliest times that the growth of
a leguminous crop, such as beans or clover, leaves the
land in a high condition for the growth of a subsequent
crop of wheat or other cereal. It was recognized that
in some way or another a leguminous crop differed very
much from any other class in its effects upon the land,
and its cultivation was to a large extent found to be
equivalent to the application of a considerable amount
of dung or other manure. Experiments carried out in the
open field at Rothamsted have shown that the growth
of a leguminous plant leaves the ground richer in nitrogen
than it was before, and this in spite of the fact that the
crop itself is highly nitrogenous in composition. Below
are given the average annual amounts of nitrogen per acre
obtained in the several crops mentioned, grown on the
land every season during a period of about twenty-four
years : the soil was manured with phosphate, potash, and
other mineral manures, but no nitrogen was applied : —

Average amount of Nitrogen
per Acre per Annum.

Wheat . . . .22.1 lbs.

Barley . . . . 22.4 lbs.

Beans ... * 45.5 lbs.

Clover (22 years only) . 39.8 lbs.

187

1 88 laXATION OR ASSIMILATION OF NITROGEN

From this table it is seen that the annual amount of
nitrogen in a crop of beans or clover is more than double
that in a cereal crop grown under similar conditions side
by side on the same field. In Little Hoos Field, Rotham-
sted, barley was grown in 1873 adjoining a plot of
clover : both received the same treatment, and the soil
was uniform in character. The amounts of nitrogen
obtained in the two crops were : —

Lbs. of Nitrogen
per Acre.

In the barley. . . . 37.3

In the clover . . . . 15 1-3

In the following season barley was sown on both plots,
the nitrogen obtained in the crop being : —

Lbs. of Nitrogen
per Acre.

In the barley after barley . 39.1

In the barley after clover . 69.4

Difference . 30.3

From the figures given above it is seen that the clover
contained more than four times as much nitrogen as the
barley growing side by side with it in the same field :
moreover, the soil of the plot from which the clover was
cut was left so much richer in nitrogen than that on
which barley had been grown that another barley crop
in the following year was able to obtain over 30 lbs.
more of nitrogen from it than from the latter plot.
Chemical analysis also showed an increased nitrogen-
content in the soil which had grown a clover crop over
that from which barley had been harvested.

'Herr Schultz obtained results of a similar nature on a

FIXATION OF NITROGEN BY BACTERIA 189

large scale on his estate at Lupitz in Germany. The
land there is of a light sandy character, and about half a
century ago was too poor to grow a profitable crop of
oats or rye without an expensive application of nitro-
genous and other fertilizers. However, after the growth
of lupins or serradella, manured with superphosphates
and kainit, the barren soil has improved so much that
large yields of cereals as well as leguminous crops
have been obtained annually from it for over fifty years
in succession, and the land has become three times as
rich in nitrogen as it was at first, although no nitro-
genous manure has been applied during this long period.

Results such as these created a special interest in the
investigation of the sources from which leguminous plants
obtain their stock of nitrogen, and many distinguished
workers applied themselves to the subject during the
last^falf of the nineteenth century.

2. When wheat, mustard, or other non-leguminous
plants are grown in plots containing field or garden
soil of known chemical composition, the total amount of
nitrogen in the fully developed plants and the soil at
the end of the experiment is equal to that contained
originally in the soil and the seeds which were sown. In
the case of beans, however, and vetches or other legu-
minous plants grown in the same soil under similar
conditions, it is found that the nitrogen present in the
soil and the plants when fully grown is very much
greater than that which was supplied at the beginning
of the experiment in the soil and seed. They are able
to accumulate large quantities of nitrogen in a manner'
not possessed by cereals or other crops, and from a
source which is evidently not available to the latter.
Acquaintance with facts of this kind suggested the

190 FIXATION OR ASSIMILATION OF NITROGEN

atmosphere as the source from which leguminous plants
draw their store of nitrogen. Since the amount present
in the form of ammonia and oxides of nitrogen was
found to be very small and quite insufficient to account
for the quantity collected by leguminous crops, chemists
and physiologists were driven to consider the free
nitrogen of the air as a possible nutrient substance,
although on a priori grounds it appeared unlikely that
plants could assimilate and bring into chemical com-
bination an element of such inert and indifferent nature.

The careful investigations of Leibig and Boussingault
in the first half of last century appeared to support
the view that plants could not utilize free nitrogen ;
but between 1850 and 1857 Georges Ville in France
carried out some experiments with leguminous plants
grown in natural soil in closed glass cases, to which only
air which had been carefully deprived of any combined
nitrogen had access, and found that they grew luxuriantly
under these conditions and contained more nitrogen
than was originally present in the soil and seed used.
He concluded that the plants were able to assimilate
free nitrogen, and although it Was suggested that small
amounts of ammonia had been inadvertently supplied to
the plants in the distilled water used for watering them,
Ville convinced himself by repeated experiments that
his conclusion was correct.

Later, Lawes and Gilbert in England carried out
experiments upon peas, vetches, and lupins, growing
them in carefully sterilized sand, to which were added
phosphates, potash, and other necessary mineral food-
constituents, but no combined nitrogen : air, however,
with its free nitrogen was admitted to the cultures. In
all cases their results were negative ; the plants under

FIXATION OF NITROGEN BY BACTERIA 191

these conditions soon suffered from nitrogen hunger and
remained small and stunted in growth. It was not until
about 1886 that the question of the assimilation of free
nitrogen by leguminous plants was solved by the investi-
gations of Hellriegel and Wilfarth in Germany. Any
doubt about the problem was removed, and a satisfactory
explanation was furnished of the diametrically opposite
results obtained by Ville and Lawes and Gilbert. It
was discovered that peas grown in sterilized soil are
dependent on combined nitrogen and die of nitrogen
hunger when the amount supplied is small, just as cereals
do ; neither peas nor wheat are able to make use of
the free nitrogen of the air under these conditions.
They learnt, however, that after an addition to the
sterilized soil of a few cubic centimetres of a watery
extract of soil from a field which had grown peas, the
failing wheat plants were in no way benefited, but the
drooping pea plants recovered rapidly and increased
greatly in nitrogen-content, the free nitrogen of the
atmosphere supplying all their needs in respect of this
element. Upon the roots of the peas to which the
extract of the soil had been applied were seen a number
of warty excrescences termed nodules (Fig. 33), but on
those grown entirely in sterilized soil none were found.
A direct relationship was found to exist between the
possession of root nodules and the assimilation of free
nitrogen by leguminous plants. With them the plants
thrive luxuriantly, and nitrogenous compounds accumu-
late in all their tissues, even when no other source is
available for nitrogen than that present in a free state in
the atmosphere : without them growth is limited unless
combined nitrogen is supplied. The fact that steriliza-
tion of the soil or heating the watery soil extract to

192 FIXATION OR ASSIMILATION OF NITROGEN

70° C. or over entirely checked the formation of nodules
suggested that living organisms were in some way-
responsible for the appearance of these peculiar struc-
tures, and since the researches of Hellriegel and Wilfarth

the soil bacteria which stimu-
late the production of root
nodules have been isolated and
extensively studied. |

The evidence obtained by
Hellriegel and Wilfarth of the
assimilation of free nitrogen
was indirect : they showed a
large gain of nitrogen in plants .
grown under conditions in
which no other source than
that of the free nitrogen of
the air was available. In
1890, however, direct proof of
the removal of free nitrogen
from the air and its fixation
in the plant was obtained by
Schloesing and Laurent. They
grew leguminous plants in
closed vessels and made an-
alyses of the air at the be-
ginning and end of the

Fig. 33 -Root of runner bean (^Phaseo- experiment I the amOUUt of

/«.) showing nodules. nitrogen taken from the air

was found to correspond very closely with the increase
of nitrogen in the crop.

3. The form, number and arrangement of the root-
nodules upon the different species of leguminous plants
varies considerably. Those of lupins are large, com-

FIXATION OF NITROGEN BY BACTERIA 193

paratively few in number and attached by wide bases
to the tap root and stronger lateral roots. In the case
of many species the nodules are found in large numbers
on the smaller roots to which they are attached by
narrow necks. Some of them, such as those upon
Robinia, Trifolium^ Onobrychzs, and Medtcago, are elon-
gated and hot infrequently branched : while those of

a b

Fig. 34.— « Root hair of pea showing infection-thread.

b Infection-thread passing through cell of young nodule.

Anthyllis, Seii^adella, and Phaseolus are usually small
round structures.

The nodules are caused by the soil organism Pseudo-
monas radicicola (Beijk.), Moore {Bacillus radicicola^
Beijk.), which penetrates into the young roots chiefly
through the root hairs : in lupins and some other species
infection apparently takes place through the older parts
of the roots as well. After entry the organism multiplies
and forms a filamentous zoogloea, — the infection-
thread — which resembles a fungus hypha so closely that
it was at first confused with the latter. The infection-
thread consists of small rod-shaped bacteria imbedded in
a gelatinous substance from which they are difficult to
differentiate. It can be traced along the interior of the
root-hairs, and through the cells of the cortex. Where
N

194 FIXATION OR ASSIMILATION OF NITROGEN

it passes through the cell-walls it generally exhibits
peculiar trumpet-shaped expansions (Fig. 34). Here and
there upon it are minute swellings from which bacteria
appear to escape into the cell-cavity which they subse-
quently fill by repeated division and growth.

The nodules arise endogenously from division of the
infected cortical cells near the endodermis of the rootlets.

i-^^%

ij

Fig. 35. — a Transverse section of nodule from red clover root showing
dark bacteroidal tissue in centre, with cortical tissue and
vascular bundles round it ( X 20).
b Single cell of bacteroidal tissue ( X 250).

Occupying the central part of a fully developed specimen
is a mass of parenchyma filled with bacteria or bacteroids
(Fig. 35). Surrounding the bacteroidal tissue, which is
often pinkish or greenish in colour, are small vascular
bundles connected with those of the root, upon which
the nodule is growing : outside is the cortex and
epidermis. The bacteroidal tissue is a meristem, which by
rapid division in all directions alike results in the pro-
duction of a more or less spherical nodule : frequently,
however, division goes on chiefly at the end of the tissue
farthest away from the root, in which case the nodule

FIXATION OF NITROGEN BY BACTERIA 195

becomes elongated. The infection-threads soon extend
into the new tissue, and although they do not always
come into direct contact with the nuclei of the cells
through which they pass, the latter appear to be in-
fluenced by the presence of the bacteria in the thread,
since they increase in size and develop an abnormal
number of nucleoli, after which they finally degenerate.
In clover and some other plants the infection-threads
remain visible for some time
before becoming obliterated.

The organisms present in the
infection-threads and youngest
parts of the nodules are short
straight rods. In the older parts
they become distributed through
out the interior of the cells,
grow considerably in size and

thickness, and undergo change FiG. 36.— Bacterolds from red clover
. ^ , I , , rj, nodule ( X looo).

mto branched, curved or i

and Y shaped forms, commonly termed bacteroids, in
which state they stain irregularly, and appear to consist
of vacuolated protoplasm (Fig, 36). The bacteroids
soon lose their power of division, and after a time are
dissolved by a proteolytic enzyme secreted from the
surrounding protoplasm, the compounds produced being
transferred to the flowers and ripening seeds of the
leguminous plant. The nodules eventually shrivel and
decay, but some of the small rods remain unchanged
and find their way into the soil ready to infect a
subsequent crop.

The relationship between the bacteria and the legumi-
nous plant is one of symbiosis or mutual assistance.
At first the bacteria live as parasites, deriving all their

196 FIXATION OR ASSIMILATION OF NITROGEN

food from the leguminous plant as well as the carbo-
hydrates essential for the supply of energy needed in
the nitrogen fixing process. Starch is generally present in
the tissues of the nodules, especially in autumn and during
the resting period of the plant. It is possible that the
organisms in some cases live altogether as parasites with-
out offering anything in return for the nutrient substances
which they receive from their host, under which circum-
stances the plant may exhibit little or no increase in
nitrogen content. Usually, however, the nodules grow
in size, vast numbers of bacteroids are produced, and
large amounts of nitrogen are obtained by them from
the air, and used for the formation of nitrogenous com-
pounds, of which their bodies are mainly composed.
After a time the bacteroids become disorganized and
dissolved, the compounds in solution being absorbed
and utilized by the leguminous plant for its own
nutrition, and especially for the formation of its
seeds, the plant thus becoming parasitic upon the
bacteria.

The facts of the assimilation of the free nitrogen of
the air are clear, but the manner in which the process is
carried out by the combination of plant and bacteria is
still obscure. There is apparently no increase of nitrogen
in the soil itself on which leguminous plants are growing,
the excess obtained on analysis of the soil after the growth
of a crop of beans or clover being due to the root residues
left in the ground after the plants are cut and harvested.
Nobbe and Hiltner and others have shown that the
gaseous nitrogen is absorbed by the nodules themselves,
and not by the other parts of the plant : plants grown in
water culture with the root nodules immersed gain no
nitrogen, and not until the latter are brought above the

FIXATION OF NITROGEN BY BACTERIA 197

surface of the liquid into the air are they of service to
their symbiotic partner.

The greatest amount of nitrogen assimilation takes
place at the time when the formation of bacteroids has
reached a maximum, at which stage the plants are just
beginning to flower ; during the ripening of the seed the
organic nitrogenous compounds are removed from the
nodules, and their nitrogen content becomes reduced to
near that of the rest of the plant. Stoklasa found that
lupin nodules in which the bacteroidal tissue was fully
developed contained 5.2 per cent, of nitrogen, most of
which was present in the form of proteins : the plants
were then in flower. Later, when fruit and seeds
were ripe, the nodules contained only 1.7 per cent., an
amount about equal to that normally present in the
unaltered parts of the roots. Analyses of the nodules of
peas and dwarf beans by Frank showed a nitrogen content
of about 7 to 7 J per cent.

Maze and Golding have shown that the nodule
bacteria are able to assimilate the free nitrogen of the
air to a limited extent when grown in pure cultures apart
from the leguminous plant, and it is possible that under
certain favourable conditions, where a suitable supply of
carbohydrates or other organic carbon compounds is
available, they may be able to do the same during their
existence as separate isolated agents in the soil. But
very little is known of their life and nutrition in the soil.
Nodule-forming bacteria are ubiquitous, and it is ex-
ceptional to find soil in which nodules are not formed on
one or another species of leguminous plant, even in
cases where the land has carried no crop of this class for
forty or fifty years. Certain poor soils, however, are met
with in which the specialized organisms adapted to lupins,

198 FIXATION OR ASSIMILATION OF NITROGEN

sefradella, and some other species of leguminous plants
are missing, and until they are supplied either in soil
from a field which has grown a good crop of these
plants or in pure cultures, such plants grow very
unsatisfactorily.

4. As already indicated, there is considerable variety
in the morphology of the nodule organisms, but all the
forms are doubtless modifications of a single species
produced by variation in environment. Many of the
forms exhibit striking differences also in regard to their
power of producing nodules. The most luxuriant growth
of these root excrescences upon any particular plant is
brought about in the speediest and most effective manner
by inoculation with cultures derived from nodules of the
same species of plant. Thus upon the roots of peas they
are most easily produced by organisms derived from pea
nodules, although their formation can be stimulated by
those obtained from beans and vetches. Those taken
from the roots of Robinia are active upon beans and
certain clovers. Several varieties, such as those obtained
from lupins, clovers, and serradella, are somewhat ex-
clusive in their behaviour, and do not affect plants
belonging to other genera. The bacteria from peas
produce nodules upon beans, but not upon lupins, nor
upon plants belonging to the genera Trifolmm, Medicago,
Anthyllis^ or Ornithopus. Although what may be
termed the virulence of the organism, or its prompt
and unfailing power of nodule production, is largely
dependent upon the origin of the bacterium, it is also
greatly influenced by nutrition ; pure cultures which have
been grown for some time upon gelatine or media
containing considerable amounts of combined nitrogen
lose much of their power in this respect. The com-

FIXATION OF NITROGEN BY BACTERIA 199

position of the soil and its chemical reaction influence
nodule formation, and affect the vitality and virulence of
the nodule bacteria. The latter are rarely abundant
in acid soils, and for the healthy exercise of their
physiological powers they need an adequate supply of
mineral substances, especially compounds containing
lime, phosphates, and potash ; without these their
virulence is reduced and their ability to assist the
leguminous crop is diminished. These facts are of
great importance in the preparation and use of pure
cultures for agricultural purposes.

5. Soil inoculation. — From what has been said pre-
viously it is obvious that the growth of a good legu-
minous crop is of the greatest importance to the
farmer. In itself it provides valuable food for farm
stock, and at the same time leaves the land well
supplied with nitrogen, which vastly assists the growth
of cereals and other crops succeeding it. The certain
production of a good leguminous crop is therefore
very desirable. In most cases the sowing of the
proper seed is enough to ensure success, the nodule
bacteria being very abundant in the majority of soils.
In some soils, however, certain kinds of leguminous
plants have been found to grow very poorly : no nodules
have appeared upon their roots, and the land has been
no more benefited than it would have been by the growth
of a cereal crop. In such instances the right nodule-
organism is missing from the land, and its introduction
is necessary before satisfactory results can be expected.
Experiment has shown that this can be achieved by
taking soil from a field which has grown a good crop of
the particular plant in question, and spreading it over the
land on which the crop has failed. Such a process of

500 FlXAtiON OR ASSIMILATION OF NITROGEN

soil inoculation very frequently has given good results,
but it is a laborious and expensive process. To avoid
these disadvantages, Nobbe, in Germany, conceived the
idea of inoculating the soil or the seed before sowing
with pure cultures of organisms from the several legu-
minous crops usually cultivated on the farm. The pure
cultures were grown upon agar and gelatine media
impregnated with decoctions of the leaves of the various
legumes, and sold under the name of Nitragin. The
usual method of using the latter was to transfer the
culture to water or milk, and wet the seeds with the
liquid. When dry the seeds were sown, the right
organisms being necessarily supplied to the soil at the
same time. Hundreds of trials were made with
" Nitragin " in most European countries, and although
in a small percentage of cases the inoculated seed pro-
duced larger yields of crop than untreated samples, the
majority of the trials proved futile : the soil contained
plenty of the nodule organisms for infection of the young
leguminous seedlings, and to add more was superfluous.

In 1904, Dr G. T. Moore, in America, came to
the conclusion that the failures of Nobbe's " Nitragin "
were largely due to the loss of vitality or virulence
of the nodule organisms contained in it, a result
brought about by cultivating them upon media rich in
nitrogen. There is little doubt that the nitrogen-fixing
power and power of infecting the roots of legumes is
greatly reduced by the presence in the soil of nitrates
and other nitrogenous compounds, and it is likely that
the virulence of the organisms is also checked by cultiva-
tion on artificial media containing considerable amounts
of combined nitrogen. Moore grew the organisms in
liquid media containing very small amounts of nitro-

FIXATION OF NITROGEN BY BACTERIA 201

genous compounds. Pieces of cotton wool were soaked
in the culture and then dried ; in a dry state attached to
cotton wool the organisms retain their vitality for a year
or two. Along with the infected cotton wool two
packets of nutrient salts were supplied, one containing
sugar, magnesium sulphate, and potassium phosphate,
the other ammonium phosphate. The first packet was
dissolved in water, and the cotton wool then added. In
this solution the common bacteria of the air and water do
not grow freely, but the nodule organisms readily multiply
in it. After twenty-four hours the ammonium phosphate
is added, to encourage further development of the nodule
bacteria. If growth is satisfactory the liquid becomes
opalescent or milky, after which it is used to sprinkle
over the seeds ; these are then allowed to dry, and are
sown afterwards in the ordinary way.

More recently cultures similar to those of Moore have
been issued under the name Nitro-bacterine.

The practical utilization of pure cultures of the
" nodule-forming " organisms is still in the experimental
stage, in spite of the large amount of attention which
has been given to the problem. It is quite certain
that in certain cases seed inoculation in the manner
described above has been very beneficial and remunera-
tive, but exact knowledge of the conditions which ensure
an increased yield after such inoculation is still wanting.
The evidence seems to point to the conclusion that on
much of the cultivated land in Europe the use of
" nitragin " or " nitro-bacterine " is quite unnecessary for
the growth of good crops of the ordinary leguminous
plants, the soil being already well supplied with
organisms of the right kind for the adequate infection
of the roots of the crop. Failure to obtain increased

202 FIXATION OR ASSIMILATION OF NITROGEN

returns after inoculation seems to occur where the land
is supplied with an excess of easily assimilated nitro-
genous compounds which check the growth of the nodule
organisms, and also on acid soils and those deficient in
phosphates and potash.

Inoculation is likely to be effective upon soils poor in
nitrogen, especially where leguminous crops have not
been grown previously, and also on land which has
given meagre crops of this class, with roots devoid of
nodules.

Much careful and continued experiment is needed to
determine the best kinds of leguminous crop to grow,
how to grow them, and how to use them, so as to bring
to the farm the greatest amount of nitrogen at the least
expense. These are problems which should be con-
sidered along with the cultivation and application of
pure cultures of the various nodule organisms if the
work is to be of economic interest.

Pseudomonas radicicola (Beijk.), Moore. — Ps, radicicola is an
aerobic bacterium without spores, and grows best at about
15° C, being easily killed at a temperature of 60° to 70° C. As
previously indicated, the size and form of the organisms present
in the nodules of the Leguminosae vary with the condition of
development of the nodule, and the kind of plant from which
they are taken. In the infection threads and young parts of the
nodule, the bacteria are always short non-motile rods about
I to 2 A^ long and .5 to .6 ^ broad. The bacteroids differ con-
siderably in different plants. Those of Fhaseolus, Hedysaruni,
and Gefiista are thickish rods, straight or curved, but rarely
branched : in Vicia^ Pisum and Trifolmm, when fully developed,
they become T and Y shaped, 2 to 4 fjt> long, .5 to .8 //. broad,
while those of Lupinus are often extensively and irregularly
branched.

FIXATION OF NITROGEN BY BACTERIA 203

When grown on artificial media the nodule organisms are at
first minute rods, single or in pairs, actively motile, each posses-
sing a single polar flagellum, hence their inclusion in the genus
Pseudomonas. Later they lose their motile power and change
into bacteroids, becoming irregular in form, curved, and some-
times more or less branched. They often excrete a mucilaginous
substance ; the protoplasm is of unequal density and does not
stain uniformly.

Wood-Ash-Maltose solution. — In this solution the organisms
flourish, the medium becoming turbid and ropy with a whitish
growth in it which accumulates on the bottom of the flask.

Agar. — On Wood-ash-maltose agar (Ex. 99) plates the surface
colonies are round, raised, with a translucent appearance very
like drops of melted paraffin wax. They may grow to a diameter
of 2 or 3 mm. in five to ten days, and at first are watery, but
after a time develop a slimy substance which can be drawn out
into long threads. The deep colonies are smaller, round, oval
or triangular, granular and whitish.

Ex. 94. — Dig up well-grown specimens of beans, red clover,
alsike, kidney - vetch, birds' -foot trefoil, sanfoin, and other
leguminous plants just before they flower : wash the roots care-
fully, and make observations upon the number, position, size,
form and colour of the nodules present.

Ex. 95. — Examine the nodules on the roots of beans and
peas (i) when the plants are a month old; (2) when they are
just coming into flower ; and (3) when the seeds are almost ripe.
Cut sections of the nodules, and note the difference in the ex-
ternal and internal colour and texture of each at the different
stages of growth of the plants.

Ex. 96. — Cut transverse and longitudinal sections of a bean
nodule. Examine with a low power, and make drawings indi-
cating the position and form of the various tissues. Then
examine with a high power, and make detailed drawings of the
several parts.

204 FIXATION OR ASSIMILATION OF NITROGEN

Ex. 97. — Cut transversely across a nodule of a bean root : dig
out a small portion from the central part exposed, and crush it
in a few drops of distilled water. Then take a small drop of the
latter, and spread it over the surface of a clean cover-slip.

When dry float it, bacteria side downwards, on a very weak
solution of gentian violet, and leave to stain all night.

Excellent stained specimens of the nodule organisms are
obtained in this way.

Ex. 98. — Prepare the following solution : —

Mono-potassium phosphate (KHgPOJ i.o gr.
Magnesium phosphate ... .05 gr.
Distilled water . . . . . 500 c.c.

To one 100 c.c. add .5 gr. sodium succinate.
„ another 100 c.c. „ i gr. glycerine.
„ „ 100 c.c. „ I gr. peptone.
„ ,, 100 c.c. ,, 2 gr. mannite.
Heat each to boiling, and filter: then pour 10 to 15 c.c.
of each into sterile tubes, and Sterilize on three successive days.

Inoculate a tube of each with organisms taken from the interior
of nodules of pea, runner bean, red clover, and broad bean, with
a sterilized knife and needle.

Incubate for fourteen to twenty-one days, and then examine a
drop of the culture for " bacteroids " in a fresh state and stain
with dilute gentian violet as in the previous experiment.

In which medium do the organisms grow least satisfactorily ?
Ex. 99. — Grow the nodule organisms on Harrison and Barlow's
wood-ashes-maltose agar.

Add 8 gr. of well-burnt wood ashes to 500 c.c. of distilled
water, and boil for one minute : filter through two sheets of
paper.

To 400 c.c. of the filtrate add : —

4 gr. of agar,
and 4 gr. of maltose.
Heat until dissolved: then filter, tube, and sterilize in the
usual way, leaving some to solidify for agar slants.

FIXATION OF NITROGEN BY BACTERIA 205

Now isolate and prepare pure culture of the nodule organisms
from beans, peas, red clover, and other leguminous plants.

Dig up the plant and wash the roots. Remove a clean nodule
with forceps, taking care not to squeeze it too much, and im-
merse it for one minute in mercuric chloride solution : —
Mercuric chloride . . . . -25 gr.
Hydrochloric acid, sp. g. 1^2 . . .5 c.c.

Water ...... 250 c.c*

Take it out with sterile forceps, and place it on blotting-paper
to absorb the solution. Cut it open with a sterile knife : dig
out with the flamed point a small portion of the bacteroidal tissue
from the centre of the nodule, and transfer it to a heated watch-
glass containing a drop or two of sterile water.

Inoculate a tube of melted agar with a loopful of the liquid on
the watch-glass, pour into a Petri dish, and incubate at 20° C.

In two or three days note the white parafifin-like shining
colonies ; examine and stain organisms from them.

Make streaks on agar slants.

Ex. 100. — Prepare wood-ash-maltose solution (Harrison and
"Sarlow).

Take 8 grams of good, well-burnt wood ashes, and put them in
a flask with 500 c.c. of distilled water : boil for a minute, and
then filter.

To 400 c.c. of the filtrate, add 4 grams of maltose, and boil.

Then pour about 10 c.c. into a number of sterile tubes, and
sterilize in the usual way.

In these tubes grow the nodule organisms obtained from
nodules in the manner described in the previous experiment.

CHAPTER XIII.
BACTERIOLOGICAL EXAMINATION OF SOILS.

Comparison of the Fermentative Action of Soils. —

The determination of the numbers of bacteria in soils of
different fields has hitherto not given very reliable indi-
cations of the relative fertility of the latter. Somewhat
better results have been obtained, however, by a com-
parison of the fermentative activity of the different soils
when introduced in similar amounts into various nutritive
solutions, and no doubt it is in the improvement of this
kind of method of soil examination that our knowledge
will be increased of the relationship between the fertility
of the soil and the biological phenomena which occur
within it.

What is required are reliable methods of determining
the kinds and relative proportions of bacteria in the soil,
and the work which each kind is capable of performing,
rather than means of determining their total number.

Remy, Lohnis, Buhlert, Fickendey, and others have
attempted to compare the bacteriological character of
soils in respect of their power of decomposing proteins
by introducing similar amounts of the different soils into
peptone solutions, and estimating the amount of ammonia
produced in a given time in each case. In a similar way
the relative power of nitrification, denitrification, and
nitrogen fixation possessed by various soils have been
compared.

FERMENTATIVE ACTION OF SOILS 207

The work is at present of a tentative character : there
is great difficulty of obtaining small samples of soil
which are strictly comparable and representative of the
whole field or garden, and further research is necessary
to ascertain the most suitable solutions to be used in the
work.

It is hoped, however, that investigations along these
lines may ultimately lead to the discovery of a rapid
means of comparing the fertility of soils.

Ex. 101. — Dig up a block of soil with a spade : break it, and
from the untouched portion remove about a lb. with a knife or
trowel sterilized in the field with a spirit flame. Store the sample
in a wide-mouthed sterilized bottle.

Take 300 gr. of the soil, and add it to a flask containing
300 C.C. of sterile tap water : shake the contents for five minutes.

(i) Test the putrefactive power or peptone-destroying power
of the soil.

Prepare a i|^ per cent, solution of peptone in tap water, and
sterilize it. Place 10 c.c. of this solution in a test-tube, and add
to it by means of a wide-bored pipette 5 c.c. of the above turbid
soil and water mixture.

Incubate at 25° C. for four days. Then pour the contents of
the tube into a flask to which is added i or 2 grams of calcined
magnesia, and a drop or two of paraffin, to prevent subsequent
frothing of the liquid.

Distil off the ammonia from the compound produced by the
action of the soil bacteria on the peptone into —^ normal
sulphuric acid.

(ii) Test the urea-splitting power of the soil.

Take to c.c. of Beijerinck's urea solution (p. 225). Add to
it 10 CO. of the above soil and water mixture, and incubate at
22° C. or three days.

Estimate the amount of ammonium carbonate produced by
titration with normal hydrochloric acid.

208 BACTERIOLOGICAL EXAMINATION OF SOILS

(iii) Find out if the soil possesses denitrifying power.
Prepare the following solution : —

Grape sugar

1-5 gr.

Sodium nitrate .

•3 gr-

Di-potassium phosphate

.igr.

Tap water ....

lOO c.c.

Add lo c.c. to test-tube, and sterilize. Then add 5 c.c. of the
soil and water mixture, and incubate at 25° C.

Test for nitrate every day with diphenylamine and sulphuric
acid as in Ex. 74 until the reaction disappears.

(iv) Test the nitrifying power of the soil.

Prepare the following solution : —

Ammonium sulphate . . . .4 gr.
Di-potassium phosphate (KgHPO^) .2 gr.
Tap water 100 c.c.

Take 25 c.c. and add i gr. basic magnesium carbonate.

To this add 20 c.c. of the soil and water mixture, and incubate
at 20° C.

After six weeks estimate the nitrate present
(a) by Schloesing's method, or
(if) by the colorimetric method of Grandval and Lajoux.

The latter depends on the principle that picric acid is formed
when nitrates are treated with an excess of phenol-sulphuric acid,
and this compound gives a strong yellow-coloured solution of
ammonium picrate when dissolved in weak ammonia.

An empirical colorimetric scale is first prepared as follows : —

Dissolve 7.22 gr. of potassium nitrate in i litre of distilled
water. Take 50 c.c. of this, and add it to i litre of water; from
the latter measure out 100 c.c, and add it to a third litre of
distilled water, i c.c. of the last dilution contains .005 gr. of
nitrogen.

FERMENTATIVE ACTION OF SOILS 209

Add to watch-glasses or small beaker the following amounts of
the third solution : —

I c.c. (which corresponds to .005 gr. of Nitrogen).

2 C.C.

5J >

, .01

4 c.c.

J) 5

, .02

6c.c.

>> 5

.03

8 c.c.

J) J

.04

10 c.c.

>) J

•05

15 c.c.

)J >

.075

20 C.C.

J> >

5 .10

and evaporate all to dryness over a water bath.

Then pour over the dry matter on each glass i c.c. of a
solution of

4 gr. phenol in

36 gr. sulphuric acid.

Dilute with 5 c.c. of water, and wash off each into separate
narrow colorimetric cylinders with 15 c.c. of a 5 per cent,
solution of ammonia, and fill up to the 50 c.c. mark.

Add to the series a cylinder of distilled water.

After preparation of the standard colour scale take the nitrified
solution to be tested, and filter it. Wash the sediment in the
filter with distilled water, and make up the volume of the filtrate
to 100 or 150 c.c. Of this solution take 50 c.c, evaporate to
dryness, and treat as indicated in the preparation of the scale.

Make up the solution of ammonium picrate obtained to 50 cc,
and place it in a cylinder alongside those of the scale for com-
parison of tint.

From the result. calculate the amount of nitrate present in the
nitrified solution.

(v) Test the nitrogen-fixing power of the soil.

Place the following solution in a large Erlenmeyer flask : —

Dextrose 4 gr.

Di-potassium phosphate . . .2 gr.

Sodium chloride . , , . .2 gr.
0

210 BACTERIOLOGICAL EXAMINATION OF SOILS

Calcium carbonate . . . i gr.

Tap water ..... 200 c.c.

Add 20 c.c. of the soil and water mixture and incubate at 20° C.

After four or five weeks estimate the total amount of nitrogen
in the flask by Kjeldahl's method, and deduct from it the average
nitrogen content of 20 c.c. of the soil mixture which should be
ascertained at the beginning of the experiment.

Ex. 102. — Repeat the above experiments with : —

1. a light sandy soil,

2. a peaty soil,

3. an ordinary arable soil,

4. a richly manured garden soil,

and compare the soils in respect of their powers of nitrification,
denitrification, putrefaction, urea-decomposition and nitrogen-
assimilation.

CHAPTER XIV.
FARMYARD MANURE.

I. Nature and Composition of Farmyard Manure. —

A large amount of food consumed by a horse, cow, or
other animal undergoes digestion in the stomach and
intestines, being changed in the process into various
chemical substances, which are absorbed into the blood
stream, and carried to different parts of the body where
growth is going on, or where the repair of tissues is
needed. As a result of the vital activity of the organism,
its substance is continually being broken down into waste
products which must be removed or excreted from the
body if healthy life is to be preserved. The fats and
carbohydrates taken in are oxidised to water and
carbon dioxide gas, the latter escaping into the air from
the lungs in the act of breathing. The nitrogen of the
proteins in the food and wearing tissues ultimately
appears as urea and allied soluble nitrogenous substances
which are excreted in the urine. The sulphur and phos-
phorus in nutrient materials are oxidized, and leave the
body as alkaline sulphates and phosphates partly in the
solid faeces and partly in the urine along with other
dissolved salts. The undigested material passes along
the intestinal canal, and is evacuated as faeces or dung.

Farmyard manure is composed mainly of three con-
stituents, namely : —

(i) The faeces or dung, and,

(2) The urine,

212 FARMYARD MANURE

of horses, cattle and other farm animals along with a
certain amount of,

(3) litter used in bedding for the animals.
The dung in a fresh state contains from 70 to 80 per
cent, of water, and is composed of the insoluble and
undigested residue of the food with certain materials
derived from the digestive juices of the intestinal canal.
It is an exceedingly complex mixture, the chief substances
present in it being woody fibre, undigested cellulose, fat and
starch, mucus and bile pigments, together with certain de-
composition products such as indole and skatole produced
by putrefactive bacteria as well as fatty acids, alkaline
soaps, and small amounts of magnesium and calcium
phosphates. In the putrefactive and other fermentative
changes occurring in the intestines considerable quantities
of carbon dioxide, methane and hydrogen gases are
produced, and these are found in freshly voided dung
with free nitrogen derived from air which has been
swallowed with the food.

The urine is a watery solution of chemical compounds
derived from the digested food and from waste products
arising in muscular and other tissues. It contains about
96 per cent, of water and 4 per cent, of dissolved
materials. The most important substance present in it
is urea^ a nitrogenous crystalline compound originating
from proteins absorbed by the blood, and in part ap-
parently from leucine and tyrosine produced by tryptic
digestion in the intestines. Urea is also a product of
the disintegration or wear and tear going on in various
organs. It amounts to about one half of the total solids
of the urine. Other nitrogenous waste materials are uric
and hippuric acids ; these are met with chiefly as sodium
salts, sodium and potassium hippurate being especially

COMPOSITION OF FARMYARD MANURE 213

characteristic of the urine of herbivorous animals. About
I per cent, of sodium chloride is found in urine also, and
small amounts of acid sodium phosphate, phosphates of
calcium and magnesium, and the sulphates of sodium and
potassium.

The litter which is used to absorb the liquid excretions
and provide a dry bed for horses, cattle and other animals
kept under cover is usually the straw of some of the
cereals ; but peat, bracken fern or dried leaves are used
sometimes in place of straw. All these contain large
amounts of cellulose with small quantities of other carbo-
hydrates and nitrogenous bodies, as well as the ash of the
vegetable tissues concerned.

Under ordinary conditions 'on a farm the faeces, urine
and litter become more or less intimately mixed together
to form farmyard manure. This mixture is very compli-
cated in character and very varied in composition. The
amount and nature of the food, and the kind and age of
the animal consuming it, as well as the work done by the
animal, influence the composition of the manure. The
richer the food in proteins the richer will be the excreta
in nitrogen : fattening animals extract and retain less of
the food constituents than young growing animals, and
so on. Careful experiments have shown that, as a rule,
about 45 per cent, of the organic matter, 85 to 95 per
cent, of the nitrogen, and 95 to 99 per cent, of the
mineral constituents of the food are recovered in the
faeces and dung where careful arrangements are made
for the collection of the latter. The following analyses
by Stoeckhardt indicate the chief differences in composi-
tion between the faeces and urine of the different farm
animals.

214

FARMYARD MANURE

F^CES.

',

Per cent

Water.

Nitrogen.

Phosphoric Acid. Alkalis.

Horse

. 7^

.5

.35 -3

Cow

. 84

•3

.25 .1

Pig

. 80

.6

.45 .5

Sheep

. 58

•75
Urine.

.6 .3

Water.

Nitrogen.

Phosphoric Acid. Alkalis.

Horse

. 89

1.2

trace. i . 5

Cow

• 92

.8

trace. 1.4

Pig

. 97

.3

.13 .2

Sheep

. 87

1.4

.05 2.0

The urine is rich in nitrogen and alkalis and poor
in phosphates. Over 98 per cent, of the potash and
soda of the food is found in the urine, while most of
the phosphorus, magnesium, and lime compounds are
obtained in the solid excreta.

The various substances in the liquid and solid excreta
soon undergo chemical changes. The urine begins to
ferment while it is in the stable, and further extensive
decomposition occurs throughout the whole mass of the
farmyard manure soon after it is placed on the heap, or
allowed to accumulate in the yards. These changes are
brought about by the agency of vast numbers of bacteria
and fungi. Heat is developed mainly as the result of
the oxidation of organic carbon compounds, and the
heap is seen to " steam " in autumn or winter when the
temperature is low enough to condense the water vapour
which is given off. Much of the organic substance dis-
appears as carbon dioxide and other gases, and the bulk
of the manure is soon visibly decreased and its weight

BACTERIA IN FARMYARD MANURE 215

reduced. Voelcker's analyses of a heap of farmyard
manure composed of a mixture of the excreta of horses,
cows, and pigs, with the usual straw litter, showed a loss
of 20 per cent, of its weight when the straw was half
rotted ; at a later stage, when the heap was " fatty " or
" cheesy " in texture, it was reduced 40 per cent., and
when the fermentations or decompositions were practically
complete the heap was found to have lost 50 to 60 per
cent, of its original weight.

2. Bacteria in Farmyard Manure. — Normal urine
when it is excreted is free from living organisms, but the
solid faeces of all kinds of animals contains vast numbers
of bacteria. Wiiterich and Freudenreich examined the
fresh dung of two cows fed on grass, and found that in
one case the number present in a gram of dung was
10 and 12 J millions respectively on two separate days,
in the other the numbers were 1.8 and 4 millions on the
same days. Later, after feeding the same animals with
hay the number of the bacteria in the dung rose in the
case of one cow to 187 millions, and in the other to
387 millions per gram. The numbers found by Savage
in the dung of three horses examined was from ij to
2 millions, in four cows from i to i o millions, and in
three pigs examined the solid excreta contained 10
to 100 millions per gram. The most characteristic
bacterium present in especially large numbers in the
intestines and in the fresh faeces of all kinds of animals
is Bad. coli^ of which there are several very slightly
modified forms differing in their power of fermenting
carbohydrates. Wiiterich and Freudenreich found Bad.
coll chiefly in the faeces of cows along with a smaller
number of Bs. subtilis and Bad. Schafferi. Savage found
many streptococci as well as Bad. coli in the faeces of

2i6 FARMYARD MANURE

farm animals, together with the spores of Bs. enteritidis
sporogenes, the latter being especially abundant in fresh
pig dung. Heinick showed that the excreta from pigs
often contains Bad. lactis aerogenes, as well as B. coli^
and large numbers of the former organism have been
found in the intestinal canal and faeces of other animals
also.

The bacteria mentioned may be considered natural
inhabitants of the digestive tract : others are introduced
in the food, and under certain circumstances may live
and increase in number within the animal, and be found
in the dung. A number of species also gain access to
the manure while it is in the stable and farmyard, and
multiply rapidly in it, the mixture of liquid and solid
excreta proving an excellent nutrient medium for them.
Some of the kinds are introduced in the litter and in the
fodder ; others are conveyed by the dust in the air and
the water used about the premises.

Sewerin isolated from horse dung thirty-two species
or varieties, the majority of which in manure one month
old were rod-shaped organisms : cocci were compara-
tively uncommon until the manure had been stored three
months. In an old sample of stable manure Lohnis and
Kunze found forms of Proteus and the uro-bacteria most
abundant with large numbers of Bad. fluorescens (liquefy-
ing and non-liquefying forms) as well as Bad. putzdum,
Bs. mesentericus^ Bs. subtilis, and M. candicans : many
denitrifiers were isolated also.

3. While many of the chemical changes which take
place in the dung heap are imperfectly understood, and
will require much research before they are completely
elucidated, some of the fermentations which occur
have been extensively investigated and their nature

AMMONIACAL FERMENTATION OF URINE 217

explained. Of these the following are the most
important : —

(i) The ammoniacal fermentations of urea and
allied nitrogenous compounds present in the
liquid excreta.

(ii) The breaking down of the proteins in the
putrefactive process (see pp. 100-103).

(iii) Nitrification and denitrification (see pp. 134-160).

(iv) The fermentation of cellulose.

(i) The Ammoniacal Fermentation of Urine. —

Freshly excreted urine of man is slightly acid due to
the presence in it of acid sodium phosphate, that of
most herbivorous animals being neutral or slightly
alkaline. However, on being exposed to the air for a
few hours the urine of all animals becomes very strongly
alkaline and develops an odour of ammonia, which is
often prevalent in stables. The urea, uric and hippuric
acids, also undergo fermentation with the production of
ammonium carbonate, from which gaseous ammonia is
set free. In the case of urea the change is one of
hydrolysis, thus : —

CO(NH2)2 + 2H2O = (NH4)2C03

Urea Ammonium Carbonate

A number of different species usually spoken of as " uro-
bacteria " are capable of effecting this change. They
are very widely distributed in the air, in dirty water, in
dust and soil, and in manure heaps. Some of them are
coccus forms while others are bacilli : the latter appear
to prevail in soil. All are aerobic, and grow best at a
temperature of 30° to 32° C. in 2 to 5 per cent, solutions
of urea made slightly alkaline by adding .2 per cent, of

2i8 FARMYARD MANURE

ammonium carbonate. A number of species form spores
which resist the temperature of a 95° to 100° C. for an
hour or more ; others do not produce spores, and are
easily destroyed by heating to 70° C. for a short time.
Some of them grow an ordinary beef gelatine media,
while others only develop on gelatine or agar media
containing urea. They differ very considerably in their
power of hydrolysing urea ; Urobacillus Pasteurii under
favourable conditions at 30° C. is able to decompose
3 gr. per litre per hour, while many of the Urococci are
only able to ferment this amount in twenty-four hours.
When grown on solid media containing urea a character-
istic whitish ring or halo appears, consisting of minute
powdery crystals of calcium carbonate mixed possibly
with calcium phosphate.

The ammoniacal fermentation of urea by the uro-
bacteria is due to the action of an enzyme, urease,
which, according to Miquel is excreted into the sur-
rounding liquid nutrient medium in which the organisms
are cultivated. From such medium Miquel stated that
an active solution of urease can be obtained by filtra-
tion through a porcelain filter, but Beijerinck, Moll,
Leube, and others have not been able to confirm this.
Beijerinck has shown that in the passage of a liquid
culture of the uro-bacteria through filter paper many of
the organisms are kept back and the filtrate has little
fermentative action on urea, which would not be the case
if the urease was in solution. A precipitate can be
obtained by the addition of absolute alcohol to a ten to
fourteen days old culture, which when dried at 30° to
35° C. and added to a solution of urea sets up an ener-
getic ammoniacal fermentation at 48"" to 50° C, specially
in the presence of small amounts of glycerine and

AMMONIACAL FERMENTATION OF URINE 219

saccharose. Possibly the urease may diffuse from dead
cells, but it is most active as an intracellular enzyme of
the living bacteria.

Comparatively little is known of the bacterial decom-
position of uric and hippuric acids. The nitrogen in
both appears to become changed into ammonium com-
pounds, but the stages of the process and the organisms
connected in it are very imperfectly known. In some
experiments carried out by F. and L. Sestini, a solution
of uric acid infected with decomposing urine and exposed
to the air was changed into ammonium bicarbonate and
carbon dioxide, and Gerard showed later that some
organisms hydrolyse the compound into urea and tar-
tronic acid, the former being acted upon later by the
uro-bacteria and changed into ammonium carbonate.

Hippuric acid can be converted by acids and alkalis into
its constituents benzoic acid and glycocoll (amino-acetic
acid), and Van Tieghem states that the same change can
be induced by Urococcus urecB. Burri considers that
calcium hippurate is fermented by certain bacteria with
the production of ammonium carbonate, and Schellmann
isolated a number of organisms from sewage and soil
which broke down hippuric acid and glycocoll to com-
pounds of ammonia, but not much is known of their
morphology or relationship.

Micrococcus ureae. — Cohn {M. Van Tieghemi^ Miq.) is one of
the most prevalent of the urea bacteria in the air, water, soil, and
decomposing urine. It is a small coccus, .8 to 1.5 mm. in dia-
meter, met with usually singly or in pairs and tetrads, never in
chains. It does not form spores. Unlike most of the allied
uro-bacteria, it grows on ordinary nutrient media containing no
urea.

Gelatine. — The surface colonies on gelatine are non-liquefying,

220 FARMYARD MANURE

white, round, semi-transparent, and somewhat slimy; some of
them grow and reach a diameter of about lo mm. in a week or
ten days at 22° C. The "stab" is threadlike, white, and raised
at the surface.

Urea gelatine (p. 224).^On this medium the colonies are
surrounded by a ring of small crystals.

Agar. — Surface colonies are white with a somewhat pearly
iridescence.

Bouillon. — This medium is rendered turbid in a day or two
and a precipitate is produced.

Urea bouillon. — It grows rapidly in 2 per cent, urea bouillon,
and the urea soon disappears.

M. uresB liquefaciens, Fliigge, is a uro-coccus, 1.25 to 2 il in
diameter, single, or three to ten cells in a chain. On gelatine its
colonies are round, dark grey at first, yellowish-brown later, with
a dark centre and wavy border. The " stab " liquefies in
funnel-shape, the liquefied part becoming turbid, and deposits a
yellowish-white precipitate.

Urococcus Dowdeswelli, Miquel, is an aerobic organism
common in air, water, and the soil, oval, 2 to 3 /a long and i /a
broad, usually met with singly or in pairs, but may grow into
short chains. It does not form spores.

Gelatine. — The surface colonies are non-liquefying at first,
later yellow; the "stab" is nail-like with a hemispherical
head.

Agar. — On agar its colonies are yellow and soon extend to
considerable size.

Potato. — Yellow growth.

Bouillon^ becomes turbid with yellow deposit.

Urosarcina Hansenii, Miquel, is a coccus occurring in tetrads,
or irregular heaps.

Gelatine. — The surface colonies are non-liquefying, small and
yellow.

Bouillon^ is rendered only slightly or not at all turbid, but a
yellow sandy precipitate appears in a few days.

AMMONIACAL FERMENTATION OF URINE 221

Urobacillus Miquelii, Beijk. — A motile bacillus 3 to 4 yw, long,
I ^ broad, sometimes in pairs ; no spores are produced. It was
isolated from soil and grows in ordinary media. It is frequent
in soil and has little urea fermenting power.

Gelatine. — The colonies are yellowish-white or sometimes pale
rose, of irregular branched outline, closely resembling those of
B. Zopfii. After growing for a time on gelatine it loses its
power of hydrolysing urea, but becomes more energetic in
liquefying gelatine.

Urobacillus Leubei, Beijk. — A motile red-shaped organism
isolated from soil. It is 3 to 5 /w, long, 1.5 (l thick, and forms
oval spores, .8 to i ^a long : some of the spore-bearing cells are
occasionally Closfridiu7n-\\ke. It grows on ordinary gelatine
media.

Gelatine. — The spore-bearing surface colonies are 2 to 3 /a in
diameter, dull, yellow and thin. The colonies of organisms
which have not produced spores are brighter and more trans-
parent. The gelatine is not liquefied even in old cultures. On
ammonium carbonate gelatine the colonies are much larger and
closely resemble those of U. Pasteurii; the latter, however, does
not grow on ordinary beef gelatine, so that doubtful colonies
may be distinguished by transference to this medium.

Planosarcina ureas, Beijk.— A motile sarcina found in garden
soil. It occurs in the form of packets of 4 to 8 or more cells, each
cell about .7 to 1.2 /a in diameter. It produces round spores
.6 /A in diameter. The power of fermenting urea is not so
marked as that of the other coccus forms. It grows on ordinary
beef gelatine.

Gelatine. — The colonies are flat, non-liquefying, pasty and
yellowish.

Urobacillus Pasteurii, Miq., is an aerobic rod - shaped
organism, 2 to 5 /^ long and i to i J /Ct broad, sometimes in pairs
or longer chains. It produces spores usually near the end of
the cells and not infrequently appears as a Clostridium form.
It is motile and is Gram positive. The optimum temperature

222 FARMYARD MANURE

for growth is about 32° C. It will not grow on ordinary media,
but thrives when provided with urea and ammonium carbonate.
It is commonly present in manured soils, and is one of the most
energetic of all uro-bacteria, being able to ferment large amounts
of urea in a short time.

Urea Gelatine (p. 224). — The surface colonies are round,
moist, greyish-white, slow in growth, often not more than 3 mm.
in diameter after a week. The centre after a time becomes
somewhat concave and the medium partially liquefied. Deep
colonies are at first small, yellowish or brownish in colour :
they may come to the surface and grow out into a thin trans-
parent film \ ih Qx more across.

Urea bouillon {y^. 223). — The liquid " becomes slightly turbid
and develops an odour of ammonia. No growth occurs on
ordinary gelatine, potato or agar media, or in bouillon or milk.

Urobacillus Schutzenbergii, I., Miquel. — A motile, short,
rod-shaped organism, without spores, i /-o long, .3 to .5 /-t thick,
generally occurring in pairs.

Gelatine. — The surface colonies are round, semi-transparent,
milky, and cup-shaped. The gelatine is liquefied.

Urea-gelatine, (p. 224). — On 2 per cent, urea-gelatine the
colonies are i to 2 mm. in diameter, and surrounded by a halo
of crystals ; no liquefaction occurs.

Agar. — On agar the colonies are greenish white.

Urobacillus Schutzenbergii, II., Cambier, is a motile aerobic
organism, 3 to 5 /-o long, .6 il thick, without spores.

Gelatine. — The colonies are white, and produce rapid liquefac-
tion of the medium.

Agar. — The surface colonies are white, small, with a slightly
pearly lustre.

Bouillon is rendered turbid, and remains so for some time,
with little precipitate.

Urobacillus Duclauxii, Miquel. — A common uro-bacillus in
river and canal water, and a facultative anaerobe. The cells are
2 to 10 /A long, .6 to .8 ^ thick, and motile in alkaline media,

AMMONIACAL FERMENTATION OF URINE 223

It forms oval spores, which resist a temperature of 95° for two
hours. It only grows satisfactorily in media containing urea or
ammonium carbonate, and at a somewhat high temperature,
namely, about 40° C.

Urea-gelatine (p. 224). — On this medium the colonies are very
small ; no liquefaction occurs.

Bouillon containing ammonium carbonate becomes turbid and
ropy, with an unpleasant odour.

Urobacillus Maddoxii, Miquel, is a motile bacillus, 3 to 6 , a
long, I /ic thick, isolated from river water and sewage. Oval
spores are found. It grows very poorly in bouillon or on gelatine
media ; even where the latter contains urea the development is
small, but crystal formation is recognizable. On ammoniacal
agar plates it grows better ; the colonies are thick and white.

Urobacillus Freudenreichii, Miquel. — A motile aerobic
bacillus, 5 to 6 /A long and i /^ thick : it forms spores, and was
isolated by Miquel from soil, cow manure, and river water.

Urea gelatine (p. 224). — The colonies are round, white, non-
liquefying, and surrounded by a ring of crystals.

Bouillon. — In two to three days the medium becomes turbid,
later the cloudiness disappears, and a white precipitate is thrown
down.

Ex. 103. — Prepare the following urea-bouillon : —
Water . . . . . ico c.c.
Lemco's beef extract . . , 5 gr.

Witte's peptone . . . i-o gr.
Urea ..... lo.o gr.

Boil ; cool. The medium should be slightly alkaline ; if not,
add a drop or two of ammonium carbonate solution. Place
in a 250 c.c. flask and infect with a gram of garden soil, and
incubate at 20° to 30° C. for ten days.

{a) Examine a drop of the fermenting medium from day to
day, and determine if cocci or bacilli are present. Stain with
gentian violet. At first Urococci or Planosarcina may be present.

224 FARMYARD MANURE

but later an almost pure culture of Urobacillus Pasteurii is
obtained, the large amount of urea (lo per cent.) inhibiting
the growth of most of the other organisms met with in the
soil.

ip) Transfer a drop to a fresh flask of the medium, incubate
for ten days at 30° C, and examine as before.

{c) Repeat the above, using garden soil which has been heated
to 90° C. for one hour instead of a fresh sample.

Ex. 104. — Prepare 250 c.c. urea-bouillon as above ; infect with
garden soil and incubate at 22° C.

Remove 20 c.c. of the solution with a pipette, and titrate with
normal hydrochloric acid. Repeat the titration of 20 c.c. drawn
from the flask every three days, and note the increasing amount
of acid needed for neutralization. The increased alkalinity is
due to the formation of ammonium carbonate. 22 c.c. of
normal HCl neutralizes about i gram of ammonium carbonate.

Ex. 105. — Expose some urine in a cool, shady place to the
open air in a shallow vessel. Examine, after two or three days,
for bacteria present ; stain with gentian violet.

Ex. 106. — Prepare (i) 2 per cent, urea-gelatine; (ii) 2 per
cent, urea-agar.

(i) Water ..... 1000 c.c.
Lemco's extract (or beef broth) 5 gr.

Witte's peptone . . . 10 gr-

Gelatine 100 gr.

Urea, 20 gr.

(ii) Same as above, using 1 5 gr. agar in place of the gelatine.
If acid neutralize with ammonium carbonate ;
sterilize and tube as usual.
{a) Inoculate a tube of each with a drop of the liquid from
Exs. 103 and 105. Pour into Petri dish and incubate —
(i) The gelatine medium at 22° C.
(ii) The agar medium at 30° C.
{V) Examine the organisms from the several kinds of colonies
on the plates.

AMMONIACAL FERMENTATION OF URINE 225

(c) Observe the urea fermenting colonies which are surrounded
with small white crystals of calcium carbonate.

(d) Transfer portions of the different colonies into flasks of
the following nutrient medium suggested by Beijerinck : —

Water ..... 1000 c.c.

Sodium acetate . . . . 10 gr.
Mono-potassium phosphate

(KH2PO,) . . . . 25gr.

Urea So gr-

Incubate each at 22° or 30° C.
Note and compare the production of ammonium carbonate by
titrating 10 c.c. of the solution from the different flasks at
intervals of one to three days.

Ex. 107. — Add about 2 grams of garden soil or decomposing
urine to the following solution : —

Di-potassium phosphate . . -25 gr.

Ammonium sulphate . . . i-o gr.

Urea 5.0 gr.

Tap water . . . . .100 c.c.
Incubate at 3° C, ; after forty-eight hours the solution contains
urea-splitting organisms chiefly.

With a drop of this culture inoculate tubes of the following
protein-free agar medium suggested by Sohngen.

Calcium malate . . , . 5-o gr-
Di-potassium phosphate , . -5 gr.

Urea 10 gr.

Agar 15 gr.

Tap water ..... 1000 c.c.
Repeat the observations (d) to (d) of previous experiment
on the colonies which appear on this medium.

(ii) Putrefaction in the Manure Heap. — Although a

large amount of the protein substances present in the food of

an animal is digested and absorbed from the intestinal

tract, some remains and passes out in the faeces, either

?

226 FARMYARD MANURE

little changed or broken down to a considerable extent
into simple compounds.

The putrefactive decomposition which begins in the
intestines of the animal is continued in the manure heap :
its nature and the organisms which carry out the work
have been discussed in a previous chapter (pp. loo-i 14),
which should be referred to again in this connection.

Ex. 108. — Take 5 or 10 grams of fresh manure of the horse,
cow, and pig. Shake each portion in a flask containing 200 c.c. of
sterile water: transfer 10 c.c. of the turbid liquid to another
similar flask containing 200 c.c. of sterile water. From this
second flask inoculate a tube of gelatine or agar medium and
transfer to Petri dish. Incubate at 20° to 25° C. : note the kind
of colonies obtained and examine the organisms producing
these. Prepare pure cultures of those which appear to be dis-
tinct. Cultivate in various media and endeavour to determine
their species, comparing with authentic pure cultures of B. colt,
B. aerogenes, Proteus sp., B. fluorescens^ etc.

Ex. 109. — Repeat the above experiment, using old decayed
manure.

(iii) Nitrification and Denitrification. — The nature of
both these processes and the bacteria to whose activity
they are due have already been treated in detail
previously (see pp. 135-168). Under ordinary conditions
little nitrification occurs in the manure heap except per-
haps in the outer layers, and it is not until the manure has
become incorporated with the soil that its nitrogenous
compounds become rapidly changed into nitrates.

(iv) The Fermentation of Cellulose. — The solid fabric
of the body of a plant consists chiefly of cell-walls, which
in the main are composed of a carbohydrate termed
cellulose, having the empirical formula C^HjoOg. It is

THE FERMENTATION OF CELLULOSE 227

a compound insoluble in water or weak acids and alkalis,
and under certain conditions can be hydrolysed into a
hexose sugar. Through mixture or combination with
other substances the cellulose of young cells becomes
greatly modified in its chemical and physical properties,
and a series of " compound celluloses " are formed. Thus
we have the pectocelluloses of cotton and flax fibres, and
the parenchymatous tissues in the flesh of turnips and
mangels which contain cellulose mixed or combined with
pectose : the adipocelluloses forming the cell-walls of cork
containing a waxy or fatty material suberin, and the
lignocelluloses of timber and lignified vessels and fibres in
the vascular tissues of all the higher plants. Paper is
composed of one or other of these celluloses obtained
from wood, straw or cotton and linen fibre.

After the death of the plants, many of the celluloses
in them resist decomposition for a time, but sooner or
later they are hydrolysed to soluble carbohydrates, which
under aerobic conditions are finally oxidized to carbon
dioxide and water, the latter ultimately finding their way
back into the air and soil from which they were origi-
nally derived. This breaking down of cellulose is carried
out by certain bacteria and fungi, the end products of the
process being carbon dioxide and water where free and
abundant access of air is permitted. In situations where
the air-supply is restricted, as in the mud of marshes and
stagnant ponds, the cellulose is fermented by a number of
anaerobic bacteria and methane, or marsh gas (CH4),
hydrogen and carbon dioxide are produced together with
small amounts of butyric and other fatty acids. The
gases are seen to rise in bubbles when the mud and
rotting vegetation at the bottom of a pond is stirred
with .a pole or stick, and vessels completely filled with

228

FARMYARD MANURE

nutrient solutions containing straw, paper or dead leaves
in " suspension " soon begin to evolve methane after
inoculation with a small amount of such mud.

Henneberg and Stohmann found that from 50 to 70 per
cent, of the cellulose present in the hay, straw, and other
food consurAed by horses and cattle disappears in its
passage through the alimentary canal of these animals
and is not recovered in the faeces. The extent of the
loss depends on the kind of cellulose, but even the
resistent forms met with in paper and wood shavings are
partially decomposed when taken into the bodies of farm
animals. It was at first assumed that these compounds
were digested by the enzymes of the stomach and
intestines, but the experiments of Tappeiner and others
have shown that the greater portion of the decomposition
is due to the action of cellulose- fermenting bacteria
existing in the first stomach of cattle and in the colon
and other parts of the large intestines of herbivorous
animals generally. The organisms break down the
cellulose into methane, hydrogen, and carbon dioxide, and
small amounts of other substances, which are used as
food or excreted in the urine and faeces. Tappeiner
showed that paper, straw, and other vegetable debris can
be fermented by material taken from the stomach and
intestines of cows and horses, the products obtained
being the gases just mentioned, and small quantities of
acetic and isobutyric acids.

In manure heaps fermentation of the cellulose of the
litter and undigested residue of the food goes on and
the bulk of the organic material rapidly diminishes. In
the upper well-aerated parts of the heap complete oxida-
tion occurs, and carbon dioxide and water are the end-
products of the change ; but in the interior where

THE FERMENTATION OF CELLULOSE 229

anaerobic conditions prevail, methane, hydrogen, and
carbon dioxide and other substances are formed.
Deherain and Dupont made analyses of the gases within
a large heap of mixed farmyard manure. Below are
some of the results : —

August 24, 1899 Temp.

COo

CH4

H

N

0

Top of the heap 7 1 °

50

17-4

3-1

29.5

0

Middle 6f

6S

23.9

7-4

•7

0

Bottom 63°

49

40.8

3.9

6.0

0

About a month later (Sept.

20 1899)

Top of the heap 66°

42.7

52.4

0

9.8

I.I

Middle 65°

49-5

48.3

0

2.2

0

Bottom 52°

47.8

51.2

0

I.O

0

Van Tieghem, Hoppe-Seyler, and the earlier workers
at cellulose fermentation, concluded that the chief
organisms concerned in the process were Bacillus amylo-
bacter and allied species of butyric bacteria. According
to Van Senus, Bs, amylobacter works symbiotically with
an aerobic species in the decomposition of cellulose.
Certain it is that many of the butyric forms which stain
a blue colour with iodine are present when the cellulose
tissues of plants are undergoing fermentation and dis-
organization under anaerobic conditions, but further
experiments are needed to determine the particular part
which they play.

The isolation of two forms of bacteria capable of
breaking down cellulose with the formation of methane
and hydrogen was accomplished by Omeliansky in 1895.
Pieces of filter paper and cotton wool were placed in
flasks of nutrient solutions containing potassium phos-
phate, magnesium sulphate, ammonium phosphate and

230 FARMYARD MANURE

chalk, and a small quantity of fresh horse dung or river
mud was added. The cultures were kept at 35'' C. for
some months, and the gases evolved were collected at
short intervals over mercury and analyzed. When
fermentation was complete about 2 grams of filter paper
were found to have yielded 552 c.c. of mixed gases, 1 9 1
c.c. being methane, and 361 c.c. carbon dioxide. At
the same time, acetic and butyric acids were produced in
the proportion of 9 molecules of the former to i of the
latter. The bacteria giving rise to
this type of fermentation were
found to be anaerobic, rod-shaped
bacilli, slightly bent and about 5 /x
long and .4 />& thick. They form
spores I /A in diameter at the ends
of the cells, the latter then re-
sembling miniature drumsticks
(Fio;. S7).

Fig. 37. — Organism which gives ^ & ^/ /
rise to methane fermentation of Omelianskv dlSCOVCrcd alsO

cellulose (X 750). ^ ^ ■^

that in river mud and fresh
horse dung another very closely allied organism was
present, which in pure cultures ferments cellulose with
the production of carbon dioxide and hydrogen, no
'methane being formed. Under the action of this
bacterium about 3.5 grams of cellulose gave 810 c.c. of
gas, 154.3 ^'^' of which were hydrogen and the rest
carbon dioxide. Acetic and butyric acids were produced
as in the methane fermentation but in a different propor-
tion, there being 1.7 molecules of the former to i of
the latter. The organism responsible for the hydrogen
fermentation of cellulose is similar in form, but some-
what larger than that which gives rise to methane, being
4 to 8 //. (or sometimes 10 to 15) long, and .5 /a thick;

THE FERMENTATION OF CELLULOSE 23!

its spores are round and 1.5 [i in diameter, produced
at the end of the cells as in the allied species. Neither
kind stain blue with iodine and are therefore different in
this respect from Bs. amylobacter. They have not yet
been cultivated on solid media and to separate them
is a tedious operation.

Ex. 110. — Prepare the following solution: —

Water ..... 1000 c.c.
Mono-potassium phosphate (KH2PO4) i gr.
Ammonium phosphate . . . t gr.
Magnesium sulphate . . . -5 gr-
Sodium chloride, , . a small crystal.

Pour into a 250 c.c. flask, and add 5 grams of calcium car-
bonate. Introduce about 3 grams of filter paper or blotting
paper cut into small pieces. Then add a small piece of fresh
horse dung or some mud from the bottom of an old pond. Fill
the flask completely with the solution, and insert a cork through
which passes a bent glass tube, arranged so as to collect any
gas given off. Keep in a warm room. Observe the evolution
of bubbles of gas which begins in three or four weeks in an
inverted test-tube over mercury.

(i) When the test-tube is nearly full force into it with a syringe
or pipette, some concentrated caustic potash solution. COg is
absorbed by the latter. Notice the diminution in the volume of
the gas.

(2) Place the thumb over end of the tube, turn the tube
mouth upwards and apply a light ; CH4 burns.

(3) Remove a bit of the filter paper after two or three months,
and tear off small pieces with needles; mount on a glass slide or
cover slip, and examine for bacteria. Stain with carbol fuchsin
and methylene blue.

CHAPTER XV.

MILK: ITS ORIGIN AND COMPOSITION.

I. Secretion of Milk. — The milk of animals is a
peculiar secretion manufactured in special glands known
as mammae. The udder of the cow con-
sists of such glands, with which are
associated four teats. Each teat has a
small single orifice through which the
milk escapes when pressure is put upon
it as in the act of suckling or milking.
Just inside the orifice of the teat and for
some distance inwards is an open cavity,
the lactiferous sinus or " milk-cistern," in
which .the secretion is stored when the
animal is " in milk." From the reservoir
there branch off into the substance of the
udder a series of tubular channels or
lactiferous ducts, irregular in diameter and
distribution, but becoming smaller and
smaller as they are traced backwards.
The finest branches, of which there

Fig. 38.— Diagram il-
lustrating origin of
milk in udder of cow. i 1 • i •

a Alveoli in which are thousands, ultimately end m exceed-

secretion takes place; . , 1 i-i 1

b lactiferous duct; mgly Small pouch-like Structures termed

c orifice of teat.

the actual
alveolus is
consists of

alveoli (Fig. 38). It is
manufacture of milk takes
about 2ffo of ^i^ iJ^ch in
a hollow cavity lined with

m

these that

place. Each

diameter and

a single layer

SECRETION OF MILK

^33

of secreting cells. When the gland is inactive and not
secreting milk these cells are cubical or somewhat
flattened, and contain granular protoplasm with a
single oval nucleus (Fig. 39). When milk is being
produced they under-
go peculiar changes.
They increase in
length very con-
siderably and extend
into the cavity of the
alveoli, thus reduc-
ing its empty space.
Each cell may now
possess two or more
nuclei, the basal one
normal in form and
size, the other near
the free end of the
cell being more or
less degenerate and fig. sg-Transverse
irregular in outline
and structure. With-
in the cells are pro-
duced numbers of globules of oil or fat, as well as
other materials, and from them water, salts of various
kinds, sugar, and probably some of the oil, is dis-
charged into the cavity of the alveolus and tend
to fill it. Sooner or later the elevated portions of
the secreting cells, with their degenerate nuclei, pro-
toplasmic contents and oil, are cast off into the
cavity of the alveoli and float in the liquid already
secreted into it, the whole mixture forming what is
known as milk. The detached solid parts of the cells

39. — iransverse sections of alveoli ot milk glands
of cow.

1. Inactive alveolus.

2. Alveoli during milk secretion ; a secreting epi-

thelium ; b fat globule in cavity of alveolus.
( X 300).

234 MILK : ITS ORIGIN AND COMPOSITION

undergo disintegration and partial solution in the more
liquid portion of the secretion. From the above it will
be gathered that milk is not merely a secretion like
saliva or gastric juice exuded from the secreting cells,
but consists of a certain amount of a liquid secretion,
together with solid portions of the secreting cells them-
selves, which have undergone transformation and a kind
of fatty degeneration and then been cast off into the
previously accumulated liquid in the alveoli. The
materials from which the secreting cells manufacture
milk are derived through the lymph from the blood
stream which circulates in an extensive network of
capillary blood vessels surrounding the alveoli. Few
if any materials are passed on into the milk from the
blood stream direct without transformation, but white
blood corpuscles or leucocytes often make their way
between the secreting cells from the blood into the milk,
in which they may be easily recognized.

2. Composition of Milk. — From the foregoing account
of the formation of milk it may be inferred that its com-
position must necessarily be complex. It contains a
large number of organic compounds both solid and in
solution. When freshly drawn it possesses a so-called
amphoteric reaction^ i.e. it is acid to blue litmus and
alkaline to turmeric paper, a peculiarity associated with
the presence in it of acid phosphates, citrates and certain
proteins. The chief constituents present in cows' milk
are water, proteins, fats, sugar and mineral materials,
with a very minute trace of colouring substance, the
following representing the general average composition
according to Richmond : —

Water .... 87.10

Proteins .... 3.40

PROTEINS 235

Fat ..... 3.90

Milk sugar .... 4.75

Mineral matter . . . .75

Freshly drawn milk also contains a small quantity of
carbon dioxide gas and takes up oxygen and nitrogen of
the air during the process of milking. The fat and some
of proteins are suspended in the water, the rest being in
solution. The term milk-sermn is applied to the
liquid portion of the milk in which the fat and solid
materials float. It will be useful to deal with each of
these constituents separately.

(i) Proteins. — These are highly complex organic
substances, containing carbon, hydrogen, oxygen, and
nitrogen with a small amount of phosphorus and
: sulphur. There are several kinds present in milk, the
following being the chief: — Caseinogen, lact-albumin^
with traces of lacto-globulin^ and certain enzymes, the
nature of which is imperfectly known. More than 80
per cent, of the proteins in milk is in the xorm of a complex
phosphoprotein, which we shall speak of as caseinogen.
It is not present in the blood of the cow, but is a
product manufactured in and by the secreting cells of
the mammary glands : it is in combination with calcium
as explained below.

Unfortunately the term casein has been and is still
used for this compound as well as for the two curds
thrown down when acids or rennet are added respectively
to milk, although these curdy substances are quite
different from each other and from the original protein
in milk.

While it is not possible in the present state of know-
ledge to define exactly the relationship of these " caseins "
to each other, to prevent needless confusion it is

236 MILK : ITS ORIGIN AND COMPOSITION

essential that three separate terms with limited conno-
tation should be used for them.

The protein caseinogen is an acid body, capable of
uniting with soda, potash, and lime to form salts, those of
the two alkalis being soluble in water. The condition
in which it exists in milk is a matter of dispute, but the
most reliable evidence seems to support the view that it
is combined with calcium as calcium caseinogenate along
with a certain amount of calcium phosphate.

From the fact that caseinogen or its compounds do
not pass through a porcelain filter, and can be collected
as a slime by centrifuging milk, it would appear that these
bodies are present in the form of minute particles sus-
pended in the milk serum.

Calcium caseinogenate can be thrown down as a
precipitate by saturating milk with sodium chloride,
magnesium sulphate, or other agents used for "salting
out " proteins (p. Z(>).

When lactic, acetic, or other acids are added to milk,
a reaction takes place, which may be represented
thus : —

^^ '-'^-^ . rif^^v . . ^-'-^

Calcium caseinogenate + lactic acid = calcium lactate -I- caseinogen

the free caseinogen being a substance insoluble in'
water, appears as a precipitate which we may call
acid-curd. Such a curd, of course, is simpler than the
caseinogenate from which it was derived ; in a pure
state it contains no calcium, although as usually obtained
it has entangled within it some of the milk-serum and
its dissolved calcium phosphate, as well as the fat-
globules of the milk.

Rennet contains a coagulating enzyme, rennin^ which,
when added to warm unboiled milk, precipitates a

PROTEINS 237

coagulum or curd, named by some authorities casein, but
often spoken of as paracasein : it may also be termed
rennet-curd. The exact nature of the action is not
thoroughly understood, but the coagulation apparently
takes place in three distinct stages. The first action of
the rennet is to change the caseinogen into a soluble
body, which we may name " soluble casein " ; almost coin-
1 cident with this, is the production of soluble calcium
! compounds ; the latter then throw down the casein as
a dense coagulum which differs from the curd produced
by acids in containing practically all the calcium
phosphate of the milk. In it is entangled much of the
fat originally diffused through the milk. In the absence
of soluble calcium compounds, the " soluble casein "
remains in solution or suspension, giving no visible
evidence of its existence.

Soluble calcium salts do not precipitate a curd from

i fresh unrenneted milk, nor will rennet coagulate milk,

from which the calcium salts have been removed by the

action of ammonium oxalate until a soluble calcium salt

is added.

As already indicated, caseinogen behaves towards the

alkalis soda, and potash as an acid body, uniting with

; them to form salts which dissolve readily in water ; these

salts, although they are decomposed by acids with the

precipitates of caseinogen, are not coagulated by rennet.

The following summary of the names in use for the
I phosphoprotein of milk and its derivatives may be
I useful : —

(i) In milk calcium caseinogenale (often loosely termed
casein) is present.

(2) Acids precipitate free caseinogen (sometimes called
casein).

238 MILK : ITS ORIGIN AND COMPOSITION

(3) Rennet changes caseinogen into a soluble body,
" soluble casein " (sometimes termed paracasein), which
remains in solution if no soluble calcium salts are
present.

Soluble calcium salts precipitate it as casein (or
paracasein).

Lact-albumin is another protein of milk, but, unlike
caseinogen, it coagulates on heating and can be obtained
as a slimy substance by boiling the residual " whey "
after the curd thrown down on the addition of an acid
has been removed by filtration. The skim formed on
the surface of fresh milk when it is boiled consists of
coagulated lact-albumin with a certain amount of dried
caseinogen.

(^ii) Fats. — These consist of mixtures of the common
solid animal fats, palmitin and stearin (compounds of
glycerine with palmitic and stearic acids), with variable
proportions of olein and other liquid fats, compounds of
glycerine with oleic, butyric, caproic and other acids
(see p. jZ). The fats are present in the form of small
spherical globules of variable sizes, the diameter of some
being as high as .01 mm. while in others it is as low as
.0016 mm. Like the caseinogen they do not appear
to be merely collected from the blood-stream as such
and transferred to the milk, but are formed by the
chemical activity of the secreting cells from proteins and
possibly from fats supplied to the gland.

(iii) Milk Sugar. — Milk sugar or lactose is present to
the amount of 4 to 5 per cent, in solution in the milk and
can be obtained by evaporating whey. It is a crystalline
material with a moderately sweet taste, and is easily
changed into lactic acid by many species of micro-
organisms. Like the other constituents of m.ilk it is

GASES OF MILK 239

formed in and by the secreting cells of the mammary
gland and does not exist in the blood of the animal.

(iv) The inorganic salts or mineral constituents of
milk are many and variable, the chief being calcium
phosphate. Without the presence of soluble calcium
compounds, rennet cannot precipitate a " curd." Phos-
phates of potassium and magnesium are also present, as
well as chlorides of sodium and potassium, iron, silica,
and traces of other materials. In fresh milk there are
considerable proportions of compounds of citric and other
organic acids.

(v) Gases of Milk — Milk drawn fresh from the cow
contains up to 5 or 6 per cent, of its volume of gases,
the chief of which are carbon dioxide, oxygen, and
nitrogen, the former being in largest amount.

Ex. 111. — (a) Place a drop of milk on a slide and cover with
a thin slip.

Examine with a f in., J in., and vi in. objective. Note the form
and size of the fat globules. Measure (Ex. 10) and make drawings
of them.

If possible examine and compare the fat globules of Jersey,
Shorthorn and Ayrshire or Dutch cows.

(d) Add a drop of osmic acid solution to a drop of the milk
and examine again as above : note the black colour of the fat
globules.

Ex. 112. — Dip a piece of blue or neutral litmus paper into
fresh milk and notice reddening or acid reaction. Dip a piece
of turmeric paper into it and note.

Take 50 c.c. of fresh milk and add to it 2 c,c. of a 2 per cent,
solution of phenol-phthalein in alcohol. Run in slowly from a
burette a decinormal solution of caustic soda ; stir and note how
many c.c. are needed before a permanent pink colour is produced.

Ex. 113. — (a) Add a few drops of dilute hydrochloric acid to
some fresh milk.

240 MILK : ITS ORIGIN AND COMPOSITION

{b) Add I c.c. of commercial "rennet" to lo of water: then
pour I CO. of the mixture into lo of milk : casein is precipitated.

Note the difference in the texture of the curds obtained in
the two cases.

Ex. 114. — Heat 20 c,c. of fresh milk in a wide test-tube to 35
or 40° C. and add to it 5 c.c. of a weak solution of commercial
rennet (i in 10 of water).

Note the formation of curd or precipitation of casein in a minute
or less.

Ex. 115. — Try the previous experiment, using boiled rennet
extract.

Ex. 116. — Take 50 cc of fresh milk in a flask warmed to 40° C
and add a few drops of acetic acid.

Filter off the curd and dissolve : —

{a) One part of it in a weak solution of caustic soda ;

(b) another part in lime water.

Add a dilute solution of rennet to both and keep them at 40° C. :
in which of them is a curd reproduced ?

Ex. 117. — To some fresh milk in a test-tube add one or two
drops of a saturated solution of ammonium oxalate : then add a
few drops of a solution of rennet : keep at 40° C, for an hour and
note absence of coagulation. Now add a few drops of a 2 per
cent, solution of calcium chloride : coagulation occurs rapidly.

Ex. 118. — {a) To 20 c.c. of milk add 10 c.c. of water : heat to
60 to 70° C. and add a few drops of 20 per cent, acetic acid.

Caseinogen is precipitated.

Filter twice.

(b) Test a portion of the filtrate for lact-albumin by Millon's
reagent (Ex. 45).

{c) Boil the rest and note the coagulation of the lact-albumin.

{d) Filter and test the filtrate for sugar. Add a few drops of
copper sulphate solution and strong caustic potash until a blue
clear solution is obtained and then boil : a yellow precipitate
indicates the presence of sugar,

COLOSTRUM 241

3. Colostrum. — This is the term applied to the secre-
tion which is produced in the alveoli of the udder just
before and for a few days after parturition of the animal.
It is a yellow opaque liquid, alkaline in reaction, and
sometimes contains traces of blood. On heating it
becomes coagulated. A microscopic examination reveals
the presence in it of a number of colostrum corpuscles
from .005 to .025 mm. in di-
ameter, some of which closely
resemble leucocytes or white
blood corpuscles with irregular
nuclei, at the same time ex-
hibiting living amoeboid move-
ment. Many of them contain
fat globules while others appear
to be more or less disintegrated

cells, free from fat and nuclear pro. 4o.-c;;^^corpuscie
bodies (Fig. 40). The aver- (X500.)

age composition is near the following —

Water . . . . 71.7 per cent.

Fat . . . . 3.4 per cent.

Caseinogen and other proteins 20.7 per cent.

Sugar . . . . 2.5 per cent.

Mineral matter . . 1.8 per cent.

On comparison with the average composition given on
pp. 234-5 it is seen that colostrum differs very considerably
from milk, in containing six or seven times as much
proteins, less water, less sugar, and more mineral matter.
The fat in it is of a more solid character than that of
ordinary milk and the proteins consist of a little
caseinogen with a larger proportion — 12 to 14 per cent. —
of albumin and globulin which are usually present in

242 MILK : ITS ORIGIN AND COMPOSITION

very small amount only in normal milk. Dextrose as
well as milk sugar is present. It curdles very slowly
with acids and rennet : the change from the manufacture
of colostrum to the formation of ordinary milk by the
udder takes place gradually, but colostrum corpuscles do
not disappear entirely for two or three weeks. The milk,
although not unwholesome, is not generally used for dairy
purposes until the lapse of three to five days after
parturition, since cream containing it does not make good
butter or cheese, and the milk cannot be so readily
** separated." Before lactation the alveoli are found to
consist of solid masses of cells, between which there are
no alveolar cavities. Later the central cells of the
alveoli with their nuclei and other contents are discharged,
and according to some authorities become " colostrum
corpuscles " alveolar cavities at the same time being
established ; others consider that the " colostrum
corpuscles" are simply leucocytes which have absorbed
fat as an amoeba absorbs its food.

Ex. 119, — Obtain some fresh colostrum : place a drop of it
on a slide and cover with a clean slip.

Examine with a f in., J in., and ^ in. objective. Note the
form and make drawings of the fat globules and colostrum
corpuscles.

Spread a drop of colostrum on a cover-slip and allow it to dry :
pass through flame and dip it in ether for three minutes and
then in absolute alcohol for one minute, by means of which
treatment the fat is removed from the film : dry and stain
with Lofiler's methylene blue, after which examine with J and
^ in. objective.

Make drawings of bacteria and other objects which have become
stained a blue colour.

4. The Enzymes of Milk. — In 1897 Babcock and

THE ENZYMES OF MILK 243

Russell discovered that the proteins of milk underwent
proteolytic disintegration even in the presence of ether,
chloroform and other compounds which inhabit the growth
of bacteria. Milk which contained on an average 20
per cent, of its proteins in a soluble form after being
kept for three or four weeks in vessels to which antiseptics
had been added to prevent bacterial action, was found
to contain ;^S per cent, of its proteins as albumoses and
peptones. Milk which has been heated to a temperature
sufficient to destroy enzymes does not undergo such
changes. Subsequent investigation showed that the
proteolytic decomposition of milk obtained and kept
under aseptic conditions was due to the presence in it
of a trypsin-like enzyme, to which the name oi galactase
has been given.

The amount in milk is very small, and although at
first it was thought that it played a major part in the
ripening processes of cheese, its influence in this direction
is now known to be comparatively slight.

Traces of lipase, diastase, and other enzymes are said
to occur in milk, but the evidence for the statement is
not very convincing.

Positive results are readily obtained with tests for
peroxidases (p. 115), but whether they are caused by
galactase only or by other types of enzymes is not
known with certainty.

Since the enzymes in milk are destroyed when the
latter is heated for a short time to 80° C. the presence
or absence of reaction with tincture of guaiacum or
paraphenylendiamine and hydrogen peroxide affords a
ready means of distinguishing boiled from fresh milk.

In some countries where regulations are enforced with
a view of checking tuberculosis among farm stock, the

244 MILK : ITS ORIGIN AND COMPOSITION

separated milk from public creameries must be pasteurized
at a temperature not less than 80° C. before being returned
to the farms for the feeding of calves and pigs ; these
tests assist in determining if the regulation has been
carried out.

Ex. 120. — Put some lumps of gum guaiacum in a test-tube
containing alcohol and boil for a short time. Filter the yellow
solution, and gently add water until near the point at which a
permanent milky emulsion is obtained.

(a) Add a small portion to some fresh milk, and then pour in
a few drops of hydrogen peroxide solution. Slightly warm the
milk : the production of a blue colour shows the presence of an
oxidizing enzyme — peroxidase.

(d) Try the experiment with boiled milk.

Ex. 121. — Make a 2 per cent, solution of paraphenylendia-
mine " hydrochlorate."

(a) Take 10 c.c. of fresh unboiled milk, warm slightly, and add
to it 10 or 12 drops of a 3 per cent, solution of hydrogen peroxide.

From a pipette slowly run into it down the side of the tube
I c.c. of the paraphenylendiamine solution. Avoid shaking or
mixing the contents, and note within five minutes the production
of a blue colour showing the presence of peroxidase in the milk.

(d) Try the experiment with milk which has been heated for
five minutes to 60°, 70°, 80°, 90°, and 100° C, and then cooled
to about 50° or 55° C.

Determine the temperature at which the oxidizing enzymes
are destroyed.

Milk contains a reducing enzyme, i.e. an enzyme which
possesses the power of " reducing " or taking away
oxygen from a compound in which oxygen is only in
loose combination. Some reductases are direct reducers ;
others, however, such as that found in milk, can only
effect reduction when a reducing agent, such as formal-

ACTION OF HEAT ON MILK 245

dehyde, is present also. Methylene blue and indigo-
carmine, which, lose their colour when reduced, are
frequently used in testing solutions for these enzymes.
The reductase of milk is called aldehyde catalase. It
is probably associated with the fat globules of the milk,
reduction taking place more rapidly with strippings than
with the poorer foremilk.

Ex. 122. — Prepare the following solution : —

Schardinger's reagent.

Saturated alcoholic methylene blue solution . 1 c.c.

Formalin f c.c.

Water 38 c.c.

{a) Add three drops to 10 c.c. of fresh milk, and pour in a
small amount of melted or liquid paraffin to exclude oxygen.
Keep the whole at 45° to 50° C, and note the disappearance of
the blue colour in four hours or less.

{b) Try the experiment with (i) foremilk, (ii) midmilk, and
(iii) strippings. Note the time taken to destroy the colour in
each case.

5. Action of Heat on Milk. — When milk is boiled
even for a few minutes it acquires a characteristic taste
which is disliked, especially by children accustomed to
the fresh liquid.

Its properties are considerably changed owing to
alterations in the physical and chemical nature of several
of its constituents.

The cooked flavour is acquired if a temperature of
160'' F. is maintained for more than about ten minutes,
and in much shorter time as the boiling point is
approached. The objectionable taste, however, is
removed to a large extent both from the cream and the

246 MILK : ITS ORIGIN AND COMPOSITION

separated milk if the heated milk is passed through the
separator.

Little change, if any, occurs in the chemical constitu-
tion of the fats, and cream can be heated to a higher
temperature than milk before the cooked flavour is pro-
duced. The small groups or clusters of fat globules
which are found in fresh milk are largely disintegrated
into separate globules by heating, and the cream, which
rises slowly, is thin.

The milk sugar is slightly oxidized, and acids are
split off from it, the alkaline phosphates of the serum
assisting the change ; the milk becomes distinctly acid
if it is boiled for three or four hours, and yellowish or
brownish in colour, the altered tint, however, being more
likely due to alteration in the proteins than in the
sugar.

Very striking changes are made in the proteins of
the milk by heating. The soluble albumin becomes
coagulated and probably decomposed to some extent
with the evolution of a small quantity of sulphuretted
hydrogen at temperatures from 70° C. to 100° C. The
pellicle or skin which forms when milk i? heated in an
open vessel consists of coagulated albumin and dried
caseinogen, with fat and other constituents also.

The power of curdling by rennet and acids is very
much reduced, a point of great importance to the cheese-
maker. For the coagulation of the caseinogen more
rennet is required and the curd is too crumbly and soft
for the manufacture of hard pressed cheeses. The altered
character of the caseinogen appears to be chiefly con-
nected with the precipitation of insoluble calcium phos-
phate, and although some improvement in the curdling
property can be made by the addition of dissolving agents

ACTION OF HEAT ON MILK 247

such as carbon dioxide, hydrochloric, and other acids, or
by the addition of soluble calcium salts, no really char-
acteristic Cheddar, Cheshire, or other hard cheese has yet
been made from strongly heated milk.

As stated previously heating destroys the normal
enzymes of milk. There is little doubt that the coagula-
tion of the albumen, the changes in the solubility of the
casein and calcium salts, and the destruction of the
enzymes render the milk less digestible and reduces its
nutritive qualities.

Ex. 123. — Mount a drop of fresh milk and one of the same
sample after being boiled. Examine with a f in. and ^ in. objec-
tive and compare the arrangement of the fat globules in each.

Ex. 124. — Take 200 c.c. of fresh milk and divide it into two
parts : boil one of these and then cool it. Taste both.

Add 10 c.c. of a weak solution of commercial rennet (5 of
rennet in 50 of water) to the boiled and the unboiled samples in
small beakers.

Note the time taken to curdle each : the exact point can be
determined by scattering some pieces of charcoal scraped from
the end of a burnt match on the surface of the milk and stirring
with a glass rod : the floating particles stop at once when
coagulation occurs.

Examine the curd produced : which is the firmest in texture ?

CHAPTER XVI.

BACTERIA IN MILK AND THEIR SOURCES.

I. Number and Source of Origin of Bacteria in
Milk. — In the last chapter the method of secretion of
milk was discussed. In the present one it will be useful
to deal with the contamination of milk by bacteria and
the methods by which these organisms obtain access to
it. As secreted by the alveoli of the healthy udder milk
is quite free from bacteria, and it is possible to draw off
from the interior of the udder a sterile sample by means
of special apparatus. Moreover, it is very doubtful if
bacteria, present in the blood or lymph, or causing local
disease in the animal not involving the udder, ever gain
access to the milk. However, where tubercular disease
of the udder exists it is possible for the secretion to
become infected with Bacillus tuberculosis. In various
inflammatory processes in the substance of the udder and
its milk ducts and ulceration in the teats, the milk may
become contaminated with certain forms of streptococci,
which are connected with the disease. Leucocytes, which
are practically the same structures as pus-cells and from
which the latter are derived, are probably met with in all
normal healthy milk in small numbers as mentioned in
the previous chapter ; but when pus-cells are abundant,
and especially if they are accompanied by many strepto-
cocci, the milk should be treated as diseased, and should

not be used in the dairy or for domestic purposes. By some

24B

NUMBER AND SOURCE OF BACTERIA IN MILK 249

authorities it is suspected that a relationship exists between
certain types of streptococci of milk and scarlet fever, as
well as sore throats and intestinal troubles, in persons
consuming milk containing these forms of bacteria.

In addition to these pathogenic bacteria, which are only
met with where the udder or parts of it is affected with
some form of disease, milk contains certain kinds of non-
pathogenic bacteria which have obtained an entrance to
the lactiferous sinus or milk cistern and some of the ducts
by way of the orifice in the teats. Generally speaking,
they are most abundant in the milk accumulated in the
lowest parts of the udder, and therefore obtained in
quantity in the first milk drawn from it in the milking
process. . The number of species present in the milk in
the udder are very few, the commonest being forms of
streptococci, which are apparently innocuous and present
in many cows which are quite healthy. They may be
looked upon as more or less normal inhabitants of the
milk ducts. Probably others obtain an entrance in the
same way, but find the conditions unfavourable for their
further growth and development, and consequently die out.

Many investigations have been made to determine
the bacterial content of the milk which is drawn from
healthy cows, the outsides of whose udders have been
carefully brushed and washed and the milker's hands
washed with antiseptic solutions to prevent the addition
of bacteria to the milk from these sources. The milk
first drawn is found invariably to contain considerable
numbers of bacteria ; Harrison obtained in one c.c. of
such "foremilk" from 18,000 to over 50,000, Schultz
as many as 97,000, while the investigations of Orr and
Moore showed an average content of 3000 to 8000
per c.c.

250 BACTERIA IN MILK AND THEIR SOURCES

As milking proceeds, the contaminated foremilk exist-
ing in the teat and milk cistern is soon removed, and
the milk obtained later has very few bacteria in it. The
" strippings," or last drawn milk, rarely contains more
than 400 to 500 per c.c.

According to Swithinbank and Newman it is possible
to draw quite sterile samples from the udders by means
of a " milking tube " inserted through the teat after the
" foremilk " has been milked out, if careful precautions
are taken to sterilize the apparatus used and wash the
udder and teats with an effective germicide before the
operation.

On the other hand, it may be noted that Schultz,
Freudenreich, Boekhout, De Vries, and others failed to
obtain sterile milk from the udder of healthy cows, even
where careful precautions were observed. There is little
doubt that at the point of secretion, that is, in the fine
alveoli of the mammary gland, milk is free from living
organisms ; the extent, however, of the invasion of the
milk ducts in the lower portion of the udder is not
clearly known. It probably depends to a large extent
on the physiological condition of the cow and the kind
of bacteria concerned ; some organisms may be able to
penetrate a considerable distance into the udder from
the orifice of the teat, while others may be unable to
exist or multiply at all under the conditions prevailing
in the milk cistern and ducts.

As soon as milk is drawn from the udder it becomes
liable to further pollution from various sources. Cows
which are not carefully groomed have on their flanks,
and on the udder to some extent, bits of dried dung,
mud, and other materials, with which they are sure to
come in contact when lying down in the cowshed,

NUMBER AND SOURCE OF BACTERIA IN MILK 25 1

yard or fields. During milking, portions of these are
shed or rubbed off, and fall into the milk with hairs of
the animal. An enormous number of organisms may be
introduced in this manner, since old dried dung fre-
quently contains more than 1,500,000,000 per gram,
and many hundreds of thousands have been found
attached to single hairs on the flanks of dirty cows. Orr
obtained an average of 4752 colonies on agar plates
exposed underneath the udder for two minutes during
milking in the cowshed in winter. Where the udders
and flanks of the cows had been brushed, 1752 colonies
per plate were obtained, and in the case of four cows
which were well-groomed and the udders washed and
left slightly moist, similar plates left for the same period
of time showed an average of only 230 colonies.

The milk from cows with brushed and washed udders
was found by Russell and Orr to contain 330 to 472 per
c.c, while the bacterial content of the milk of the mixed
ungroomed herd averaged from 1 1,000 to i 5,000 per c.c.

Many of the bacteria in milk are derived from the
milkers. On many farms the milkers have no special
garment or overall to slip on when milking is to be
done, but milk in everyday clothing, which is frequently
the reverse of clean. The filthy, disgusting habit of
dipping the hands, often unwashed, into the milk when
milking is going on is a very common practice which leads
to bacterial contamination. The introduction of germs
of disease brought from the homes of the milkers is
likely to occur, and there is little doubt that scarlet
fever, and possibly the bacteria of typhoid fever and
diphtheria, have been transmitted to milk by the milker
and other human beings concerned with the management
of the cows and the handling of milk on the farm.

252 BACTERIA IN MILK AND THEIR SOURCES

-Plate exposed one minute in cowshed before
milking commenced.

More attention should be given to the cleanhness of

the milker, and his
freedom' from ex-
posure to possible
disease which can
be transmitted
through milk, than
is generally the
case.

Another source
of contamination ot
milk is the floating
matter of the air in
the cowshed. This
consists of dry par-
ticles of dung, dust

from hay and other fodder, as well as many similar

materials which are

bearers of bacteria.

Harrison found that

the number of bac-
teria falling on a circu-
lar surface one foot in

diameter (about equal

to the opening of an

ordinary milk pail)

was from about 1 3,000

to 40,000 per minute

in a cowshed where

the cows had just been

bedded. About an

hour later much of the

dust had settled, and the number deposited on a similar

Fig. 42. — Plate exposed one minute while milking
was being done.

NUMBER AND SOURCE OF BACTERIA IN MILK 253

area was found to be from about 450 to 2400 per
minute. The use of
milk-pails with nar-
rower mouths than
those in common
use has been found
to reduce contami-
nation and improve
the keeping quality
of the milk (Figs.
41-44).

Flies also carry
many objectionable
bacteria from putre-
fying material upon Fig 43.— Plate exposed one minute in cowshed after
which they settle, giving the cows hay.

and are responsible for the introduction of these

into milk in which
they drown.

Milking cans,
pails, milk coolers,
and other vessels
on the farm, un-
less very carefully
cleaned, are also
fruitful sources of
contamination.'
The cansorchurns
in which the milk
is despatched by
rail are respon-
sible for a very
large share of the

Fig. 44. — Plate exposed one minute after cowshed was
brushed out.

254 BACTERIA IN MILK AND THEIR SOURCES

contamination which occurs at the farm. These are often
returned to the farmer without being cleaned properly,
often with small amounts of sour milk in them, the
latter sometimes containing as many as 300,000,000
per cubic centimetre. Even where steam had been used
to clean the cans before being returned washings with
100 c.c. of sterile water were proved to contain from
400 to 1 800 bacteria per c.c. Both Stocking and Orr
have shown also that the use of milking machines
increases the bacterial contamination very considerably :
this is due to the difficulty of thoroughly cleaning the
connecting tubes and also to the sucking in of dust laden
air when the cusps fall off the teats of the animals.

The water to which the cows have access, and which
is used for washing and rinsing the cans, churns, and
other vessels in the cowshed and dairy, often contain
bacteria, which find their way into the milk.

The cow, the milker, the air of the cowshed, flies,
water supply, and imperfectly cleaned farm dairy utensils,
are the main sources from which milk becomes con-
taminated with bacteria on the farm, and, without doubt,
it is here that the chief troublesome forms become
associated with it.

As already indicated, the number found in any par-
ticular sample will depend upon the amount of care taken
to remove the causes which lead to the introduction of
bacteria into it. Where dirty conditions prevail the
milk may contain vast numbers of organisms ; where
careful methods are adopted the bacterial content may
be greatly reduced. Freudenreich, Knopf, Orr, and others
found an average of 50,000 to 80,000 bacteria per c.c.
of milk collected in the cowshed soon after milking. In
some few instances the number was as low as 5000, and

NUMBER AND SOURCE OF BACTERIA IN MILK 255

in others as high as 1,048,000, the variation being due
to differences in the season at which the samples were
taken, the cleanliness of the cows, the method of milking,
and other factors.

With reasonable care it should be possible at most of
the farms in this country to produce milk containing not
more than about 10,000 to 20,000 bacteria per c.c. when
it leaves the farm premises ; samples taken at the farm
and found to contain more than 50,000 per c.c. should
be considered as unnecessarily dirty and indicating
negligence and disregard of precautions against con-
tamination.

In addition to the pollution of milk which takes place
at the farm, there is additional contamination during
transit to the consumer. Bacteria are added during the
railway journey, at the retailer's premises, on the milk-
man's rounds, and, finally, at the consumer's house.

Very many of the milk churns sent by rail are faulty
in design, the lids allowing of easy access of dust
and rain into the interior. The vans in which they
travel are often dirty, and no special care is taken to
unload or store the churns at properly cleaned depots,
away from dust, foul odours, and other damaging
influences.

When the churns are opened at the railway termini
and small amounts transferred by means of imperfectly
cleaned ladles or measures to the dairyman's cart, con-
tamination occurs ; the same happens when the milk
is poured over the rims of churns on which dust has
collected or which have been handled by workmen whose
personal cleanliness is not of the highest order.

Hewlett and Barton found from 20,000 to 8,390,000
bacteria per c.c. in milk samples taken at various railway

256 BACTERIA IN MILK AND THEIR SOURCES

stations in London : Orr's figures for similar samples
at several Yorkshire stations were 19,800 to 3,620,000,
with an average of 232,600 per c.c.

At present milk is sold in all sorts of shops, some
of them clean, but the majority unsatisfactory from the
point of view of a clean milk supply. The vessels in
which the milk is stored is, in many instances, open to
the visits of flies and to the access of dust blown in from
the street, the measures are not always clean, and, where
the milk is stored in the living room of a house, there is
considerable risk of infection with pathogenic organisms.

The estimation of the amount of contamination which
takes place after the milk has left the farm, and due to
the several causes enumerated, cannot be accurately
made on account of the increase brought about by the
growth and multiplication of organisms during transit to
the consumer, this disturbing influence being especially
active in summer, when the temperature is comparatively
high.

According to Swithinbank and Newman's investiga-
tions, the milk retailed in London usually contains from
340,000 to nearly 5 millions per c.c. ; in New York,
Park found from i to 5 millions per c.c.

In the private houses of the consumer additions are
made to the bacterial content, the amount varying with
the place of storage, the cleanliness of the receptacles
in which the milk is kept, and other factors.

Orr concluded from his investigations in Yorkshire
that the farmer was responsible for about 40 per cent,
of the organisms present in the consumer's milk, the rest,
or 60 per cent., being contributed in approximately equal
amounts by the dairyman, the consumer himself, and by
pollution during the railway journey.

NUMBER AND SOURCE OF BACTERIA IN MILK 257

Ex. 125.— Determine the number of bacteria in a sample of
milk. — Since the number of bacteria is usually very large in an
ordinary sample of milk, it is necessary to dilute the latter in
order to avoid too much crowding of the colonies on the plates
which are inoculated by it.

For the purpose of dilution sterile water must be used.

It is usual to dilute the milk 10, 100, 500, or 1000 times.

Proceed as follows : —

From a burette run into a series of test-tubes 9 c.c. of water,
and mark the level of the water in each tube with a file ; add a
few drops more, so that the water stands a little above the mark.
Plug with cotton-wool, and sterilize by heating on three suc-
cessive days for thirty minutes. (It will be found useful to
have a series of small conical flasks marked at the 49 c.c. level,
and a similar lot marked at the 99 c.c. level.)

Now take the sample of milk to be examined and shake it
vigorously to obtain uniform distribution of its contents.

Remove with a sterile i c.c. pipette i c.c. of milk, and add it
to the test-tube containing 9 c.c. of water. The milk is now
diluted 10 times (i in 10), and i c.c. of the mixture of milk and
water contains -j^^. of a c.c. of milk.

Gently shake the mixture for a short time, and then remove
with another sterile pipette i c.c. from this first dilution and
add it to another tube containing 9 c.c. of sterile water. The
second dilution now obtained is i in 100, and t c.c. of it
contains JJ^ c.c. of the original milk. Shake and transfer i c.c.
of this to 9 c.c. of sterile water, the dilution in the third tube is
I in 1000, I c.c. containing xoVo c-^- of the milk.

If a dilution of 500 times (i in 500) is required, add i c.c.
to 9 c.c. of water, and then i c.c. of this to a flask containing
49 C.C. of water.

Having prepared a suitable dilution, say i in 1000, add .5 c.c.
of it to a tube of melted lactose gelatine from a sterilized
graduated pipette, and, after gentle mixing, pour the inoculate^
medium into a Petri dish and allow it to solidify.

258 BACTERIA IN MILK AND THEIR SOURCES

Inoculate three more plates in a similar manner, and incubate
all at 1 8° to 20° ; count the colonies appearing in seventy-two
hours, and calculate the average number on each.

The counting can be facilitated by using a Pake's disc (Fig. 45)
placed beneath the plates.

From the average number of colonies on a plate, the dilution

or 6

Fig, 45. — Pake's counting disc.

of the milk, and the amount of the latter used for inoculation,
the number of the organisms in i c.c. of the original sample can
be calculated thus : —

No. on plate x dilution x —i—. — — ^ = no. of organisms

r amount used for inoculation c

in the original sample.

£.g. If 420 colonies are found on a plate inoculated with
.5 c.c. of the milk diluted 1000 times, the number present in
I c.c. of the original sample is 420 x 1000 x -J = 840,000.

Ex. 126. — Repeat the above but use a lactose agar medium
instead of gelatine, and incubate at 37° C. for forty-eight hours.

Ex. 127.— Milk 50 c.c. of "foremilk," "midmilk," and "strip-
pin^s " from the same quarter of the cow's udder into three sterile

NUMBER AND SOURCE OF BACTERIA IN MILK 259

flasks. Keep them cool, and after arrival at the laboratory
determine as soon as possible the number of organisms in i c.c.
of each by the method described in Ex. 125, using both
lactose gelatine and agar media.

Ex. 128. — Take four sterile Petri dishes and pour into each
the contents of a tube of lactose gelatine medium. Cover and
allow the medium to solidify.

Prepare a similar number of lactose agar plates.

Take them to the farm ; remove the covers and expose the
surface of one of the gelatine and one of the agar plates for one
minute : —

(i) In the open air.

(2) In the cowshed before the cattle are brought in for

milking.

(3) Under or near the uader of the cow during milking.

(4) In the cowshed after it has been swept out.

After the stipulated time of exposure of the plates put on the
covers, wrap them in paper and take them to the laboratory.
(a) Incubate the gelatine plates at 20° C.
(d) Incubate the agar plates at 35° C.

Count and compare the number of colonies appearing on the
two media in 12, 24, 48, and 72 hours, noting the number which
liquefy the gelatine.

Ex. 129. — Note the number of colonies on the plate which
has been exposed for thirty seconds under or near the udder
during milking, as in the preceding experiment. Calculate the
area of the exposed surface of the plate by multiplying the square
of half its diameter by 3.14. Find the area of the opening of the
milking pail in the same way, and from this and the number
of bacteria found to fall on the estimated surface of the plate
in one minute calculate the number which fall into the pail
during the whole time of milking.

• Ex. 130, — Prepare Petri dishes of lactose gelatine and agar
media.

26o BACTERIA IN MILK AND THEIR SOURCES

Catch a fly in the cowshed, place it under the cover of the
Petri dish and allow it to run over the surface of the gelatine
or agar medium for one minute : then let it escape.

Incubate the plate at 20° C. and 37° C. respectively.

Count the number and note the kind of colonies which grow.

Try the experiment with flies caught in other places.

Ex. 131. — Place a cow's hair on the surface of a sterile lactose
gelatine or agar plate.

Incubate for forty-eight hours, and note the growths which take
place where the hair touches the medium.

2. Influence of Temperature and Time upon the
numbers of Bacteria in Milk. — Although milk is an
excellent medium for the nutrition of bacteria, their
development in it is dependent upon suitable tempera-
tures. Park found that when kept at freezing point
(0° C.) for four to seven days the bacteria in it decreased
slightly in number, and even at temperatures up to
6° C. growth was checked and little or no increase
occurred. At higher temperatures, up to the optimum
of 25° C. {j'j'' F.), at which division goes on most
rapidly, bacteria increase at an enormous rate, and milk
in which only a small number are found initially may
contain many millions in a i^^N hours if kept in a warm
place. This becomes obvious when it is remembered
that a single bacterium which divides once every half hour
can produce in twelve hours 16,777,216 offspring, pro-
vided that the temperature is suitable and the amount
and nature of the medium will allow of such increase.

The following figures indicate the effect of time and
temperature on the bacterial content of milk ; the
samples taken at the farm contained 22,000 bacteria
per c.c.

NUMBER AND SOURCE OF BACTERIA IN MILK 261

Number of Bacteria after

Milk kept at

3 hrs.

6 hrs.

9 hrs.

24 hrs.

15° c.
25° C.
35° C.

27,000

48,000

751,000

66,000

476,000

2,000,000

I 10,000
2,530,000
8,360,000

3,850,000
643,000,000
112,500,000

Several observers have noted that in many cases the
number of bacteria in milk decreases for a time after it is
drawn from the cow. The length of time during which
the decrease continues depends upon the kinds of
organisms present and the temperature at which the
milk is kept. When the temperature is low — say, about
10° C. — the decrease may continue for thirty-six hours
or more ; at 30° C. it lasts only for two or three hours.
This peculiar fall in numbers and' cessation of bacterial
development, which occurs only during the first few
hours in samples of fresh milk, has been attributed by
some to the presence in the milk of a bactericidal sub-
stance which is soon used up or decomposed after it has
left the udder. It may, however, be due to the death of
many of the organisms which have come from the udder
of the animal or from the surrounding air, owing to the
altered conditions of existence to which they are exposed ;
those unaccustomed to live in milk die off or take time
to become adapted to it.

After the bacterial content has reached its lowest
point a steady rise sets in and proceeds more or less
rapidly, according to the temperature, until a maximum
is attained, about the time the milk becomes strongly
acid and curdled. From this point the numbers begin to
decrease again, and little by little the bacteria die off until

for 6 hours.

262 BACTERIA IN MILK AND THEIR SOURCES

the milk, if kept for several weeks, is left in an almost
sterile condition, or containing only moulds and similar
fungi capable of tolerating the acidity of the medium.

Ex. 132. — Take about 250 to 300 c.c. of milk at the cowshed,
and after thorough mixing pour 50 to 60 c.c. into four small
flasks ; label them Nos. i, 2, 3, and 4 respectively.
Keep No, i cool with ice.

„ 2 at a temperature of 10° to 15° C.
„ 3 „ „ 20° to 25° C.

>J 4 5J 5) 35 C' >

After this, determine the number of bacteria per c.c. in each
of the four samples by method described in Ex. 125.

Ex. 133. — Determine the numbers of bacteria in a sample of
milk taken at the farm into a sterile flask as soon as possible
after being drawn ; then, at the following intervals, using both
agar and gelatine media for the work : —
2, 4, 6, 12, 24 hours.
4, 8, 12, 24, 40, 80, 120 days.

The milk to be kept all the time in a tightly-corked flask at
ordinary room temperature.

Note the appearance of mould colonies on the plates inocu-
lated with the older milk.

3. Methods of reducing the Numbers of Bacteria
in Milk. — To obtain a supply of milk with the least
possible number of bacteria in it, necessitates constant
attention to cleanliness on the part of all concerned in
its production and distribution, and the maintenance of
low temperatures so that the organisms unavoidably
present in it cannot multiply to an objectionable
extent.

It is not within the region of practical dairy farming
to obtain a commercial supply of milk absolutely free

REDUCING BACTERIA IN MILK 2^3

from bacteria, but it need not entail much trouble nor
expense to provide wholesome milk which shall remain
fresh and sweet under proper conditions of storage for
twenty-four hours or so, within which time all ordinary
supplies for domestic use ought to be consumed.

Although considerable increase in the bacterial
content usually takes place after the milk has left the
farmer's premises, this is probably more owing to the
growth and multiplication of those introduced at the
farm than to further additions made while the milk is
on its way to the consumer. The investigations of
Stevens and others upon market milk have shown that
initial contamination has greater influence upon the
bacterical content of milk delivered to the consumer than
age and temperature. The primary infection at the
farm and dairy should be checked, therefore, as far as
possible, and this can only be accomplished by strict
attention to the following suggestions.

In the first place, it is absolutely essential that only
milk from healthy animals should be sent from the farm.
All cows suffering from disease should be isolated from
the milking herd, and no milk should be used for human
consumption 'from tuberculous animals or those in any
way affected with udder troubles.

A supply of good clean water is of great service on a
farm. Not only is the health of the animals improved
by it, but many of the worst milk troubles are avoided.
Where cows are watered at dirty ponds in which they
wade and stir up mud, various parts of their bodies
become splashed with liquid containing bacteria, many
of which sooner or later find their way into the milk and
cause persistent trouble. Taints and bad keeping
quality of the milk are sure to be encountered where

264 BACTERIA IN MILK AND THEIR SOURCES

these conditions prevail, especially if the water used for
washing the cans and other utensils is also impure.

The cows should be regularly groomed between the milk-
ing hours, all pieces of dung and loose hair being brushed
off their udders, flanks, and haunches. Just before milk-
ing, the udders should be washed with tepid boiled
water and wiped with a clean boiled cloth ; the use of
dirty cloths which have been allowed to lie about for
sometime must be prevented.

The milkers should put on clean overalls and be
compelled to wash their hands before milking. On
no account should the filthy habit of " wet milking " be
tolerated ; the dipping of the hands into the fresh milk
is a disgusting practice likely to lead to serious disease,
and cannot be too strongly condemned.

The rejection of the first few jets from the teats,
and the early removal of the milk from the cowshed to
a clean cool place free from dust and away from the
manure heap, greatly assist in keeping down the
bacterical content.

Since dust and dirt are the chief carriers of bacteria
every effort should be made to reduce these to a
minimum. From the pail the milk should be passed
through closely woven linen fabric or cotton wool
strainers to take out all the larger particles of dung, straw,
hair, and other materials which fall into it. The cow-
sheds should be white-washed at least twice a year, and
the floors must be kept clean. The dung should be
taken out as soon as possible and adequate means
provided for the draining away of urine.

The brushing out of the stalls and the feeding of the
cows with hay or dusty fodder should take place after
milking — not just before — so as to avoid filling the air

kfiDUCiNG BACTERIA IN MlLK 265

with particles of dust which soon fall into and con-
taminate the milk

Too much attention cannot be given to the cleaning
of the milk pails, churns, and other utensils, both by the
farmer and the dairyman. The latter not unfrequently
returns unwashed churns containing sour milk swarming
with bacteria, to effectually get rid of which involves
much trouble to the farmer.

All vessels should first be rinsed with cold water and
then treated with live steam or boiling water. Rusty
and badly indented cans, from the crevices of which it is
difficult to clean out all the milk, should be discarded.
Ample arrangements should be made for keeping clean
cans in a cool clean part of the farm buildings, preferably
in the dairy or cooling room.

The keeping qualities of the milk can be greatly
improved by cooling it to 10° C. (50° F.) directly after
it is drawn. If a cooler is used for this purpose it should
be kept in a clean, dust-free place, and after use must be
carefully cleaned: in any drops of milk left on it,
bacteria increase enormously and may be washed off into
the next milk which is passed over it.

Churns or cans in which milk is sent by rail should be
locked or sealed, and those patterns in which the lids allow
some of the contents to splash out and run back again
into the churn should be abandoned. The close-fitting,
overlapping form of lid effectively prevents this from
happening and at the same time does not allow rain to
enter.

The provision of specially constructed refrigerator vans
and cool, clean depots at the railway termini would do
much to reduce contamination.

CHAPTER XVII.

FERMENTATIONS IN MILK.

I. Lactic Fermentation. — When milk is first drawn
from the cow it possesses a sweetish taste, although when
tested by chemical means it is ' found to be slightly acid.
If it is allowed to stand in a vessel for a day or two
during warm summer weather, it becomes much more
sour or acid and at the same time curdles. The curdling
is due to the precipitation of the casein of the milk
by the action of an acid which chemical examination
has shown to be produced from the milk sugar.
The acid is known as lactic acid (or oxypropionic acid,
C2H4(OH)COOH), its production from the sugar being
generally indicated by the following equation : —

C12H22O11 + H2O = 4(C3H603)

milk sugar lactic acid

or lactose

It is probable that the milk sugar is first hydrolysed
by an enzyme, lactase, the hexose sugars produced being
subsequently acted on with the formation of lactic acid,
thus : —

Ci2H220n + HoO = CgHiaOg + C6Hi206=:4C3H603
lactose glucose galactose lactic acid

The change is brought about by the activity of

bacteria which gain access to the milk after it is drawn

from the udder of the animal. Theoretically the loss of
266

LACTIC FERMENTATION 267

sugar in the fermentation process should be replaced by
a definite amount of lactic acid, but the amount of acid
produced is alwiays less than would be expected from the
above equation. Some of the sugar is used by the
bacteria for nutrition and respiration with the formation
of several compounds, such as acetic, formic, and succinic
acids, and the liberation of the gases, carbon dioxide,
hydrogen, nitrogen, and occasionally methane. The
quantity of lactic acid used, and the amounts and nature
of the by-products obtained, is dependent upon the
species of organisms present in the milk and on the
temperature and access of air. Under the most favour-
able conditions the yield of lactic acid is not more than
about 89 per. cent, of what would be expected from the
sugar which is lost, and is usually much less than this.
At a temperature of 10° C.(50° F.) the lactic organisms
grow very slowly and the amount of acid produced by
them is very small. The best temperature for their
physiological activity lies between 20° C. (70"^ F.) and
32° C. (90° F.). They are very sensitive to the action of
free acids, .15 per cent, of hydrochloric or .03 per cent,
of sulphuric acids completely check their work. More-
over, the fermentation of the sugar in milk generally
ceases when the lactic acid present is over .8 per cent,
before all the sugar is used up. The process can be
made to advance further by neutralizing the acid with
calcium carbonate as fast as it is produced.

• Ex. 134. — Into one flask pour 500 c.c. of fresh milk and to
another similar flask add the same quantity of milk and boil it
for one minute : cool.

Leave both in a warm place and test the acidity of samples
taken from each flask every twelve or twenty-four hours until
the milk is curdled ; shake it well before each test.

268 FERMENTATIONS IN MILK

To do this, measure out with a pipette 25 or 50 c.c. into a
porcelain dish. Add to it five drops of the following solution of
phenolphthalein : —

Phenolphthalein 5 gr.

Alcohol, 90 per cent 150 c.c.

(This solution turns pink when an alkali is added to it and
remains colourless when acid : try it.)

Now run into the milk a decinormal solution of caustic soda
from a graduated burette, carefully noting the point at which the
caustic soda solution stands in the burette before beginning the
experiment.

At first when the caustic soda solution touches the milk a pink
coloration is produced, which disappears on stirring with a glass
rod.

Run in more, drop by drop, until the pink colour is per-
manent, stirring vigorously after each addition of the soda ;
when the pink colour remains for ten to twenty seconds after
stirring, all the acid has been neutralized.

Read off on the scale of the burette how many c.c. of the soda
solution has been used.

Calculate what percentage of acid was present in the milk,
assuming that each c.c. of the soda solution corresponds to .009 gr.
lactic acid.

Draw up a table showing the amount of acid in each sample
of milk from day to day.

Lactic acid exists in three forms, all having the
same chemical composition, yet differing from each
other in molecular constitution and physical properties.
A solution of one of these, known as dextro-lactic acid,
has the power of affecting a beam of polarized light when
passed through it, the plane of the light being rotated
towards the right. Another form rotates the light
equally to the left, when passed through solutions of the
same strength ; it is termed laevo-lactic acid. When

LACTIC FERMENTATION 269

mixed in equal proportions the resulting solution has no
action on polarized light, one neutralizing the effect of
the other. The acid in such a solution is spoken of as
inactive lactic acid. Many kinds of the lactic bacteria
produce the inactive acid in milk and solutions
containing sugar and other carbohydrates. " Formerly
this substance was spoken of as fermentative lactic acid.
Some organisms, however, are now known which produce
dextro-lactic acid, others the laevo-lactic variety. Perhaps
the majority produce the former, although the power
of a certain kind of bacterium of producing a definite
kind of lactic acid is dependent on the carbohydrate
fermented as well as on the character of the medium
and other factors. The acid produced under similar
conditions may be used as a diagnostic character to assist
in the discrimination of varieties or races of nearly allied
kinds.

Over a hundred so - called species or kinds of
bacteria have been described which have the power of
producing lactic acid from various sugars and other
carbohydrates. Some of them have not been found in
milk, nor are they ever likely to occur in it. A very
large number, however, have been obtained from milk
and dairy products ; and bacteriologists, who have been
concerned with their investigation, have often bestowed
on them different specific names in such a way as to
create the impression that they were distinct and unre-
lated. There is little doubt, however, that many of them
are closely related but slightly modified forms of the
same organism, and that the number of fundamentally
different species is comparatively few. Indeed, among
organisms whose form, physiological power, and other
diagnostic characters are so readily modified by their

2/0 FERMENTATIONS IN MILK

environment, the term species as used when deahng with
the higher plants and animals cannot be applied with
any degree of precision. Well-marked, permanent, or
constant differences which the term implies do not exist
among the lactic bacteria. In the present state of
knowledge it is, therefore, perhaps best to place those
which have been described in groups according to the
presence or absence of certain characters which are
subject to comparatively little variation, the name of
each group being that of one of the most distinct forms
represented in it.

General Characters of lactic bacteria — Most of
the lactic bateria met with in milk and its products are
cocci or short-rods, and are non-motile. They are with-
out the power of spore formation, and consequently
liquids containing only these forms are easily rendered
.sterile by heating to boiling point. Some forms of the
lactic bacteria grow well enough on gelatine, while others
exhibit feeble growth upon this or any other solid
medium. Their colonies are generally either white or
yellowish-white, and do not liquefy gelatine. They pro-
duce lactic acid in milk, especially at elevated tempera-
tures, and usually curdle it sooner or later. -Some grow
well in the presence of oxygen, others, perhaps the
majority of kinds, are facultative anaerobes.

The following groups suggested by Lohnis may be
recognized, viz.: — .

Group i. Streptococcus lacticus, Kruse.
„ ii. Bacterium acidi lactici, Hiippe.
„ iii. Bacterium casei, Freudenreich.
„ iv. Micrococcus lactici acidi, Marpmann.

The typical bacteria of groups i, and ii, are the most

CHARACTERS OF LACTIC BACTERIA 271

frequent, being found in almost • all samples of cows*
milk.

(a) Group i. — The typical bacterium of this group,
is that which is known as Str. lacticus, Kruse, and is
the commonest of all forms occurring in milk. To it
most of the souring in the dairy is due. It appears to
be the same organism as : —

Bactei'mm lactis, described by Lister.

„ Guntheri, L. & N.

„ lactis acidi, Leichmann.

„ lactis acidi y I and 2 of Conn.

Lactococcus lactis, Beijk.

The cells are usually slightly oval coc^i, .6 to i /x long,
and .5 ih broad, which before the process of division may
lengthen slightly, so that it is difficult to decide whether
they should be described as short bacteria or cocci.
They are mostly in pairs or short chains of four or five
together, hence the term streptococcus applied to them.
This species is somewhat anaerobic and forms very
minute colonies below the surface of gelatine plate
cultures. Under any circumstances the growth is very
slight on solid media, and the colonies are mostly round,
J to J mm. in diameter, about the size of a small pin's
head, but may be oval or irregular in outline sometimes.
The organism stains with Gram's stain, and does not
produce gas when inoculated into fermentation tubes
containing broth and sugar.

In milk it grows most rapidly at a temperature of
about 30° to 35° C, and the forms generally met with pro-
duce acid and curdling in six or eight hours, the curd being
dense and uniform, without gas bubbles and no separa-
tion of whey. Some varieties are slow in action at this

2/2 FERMENTATIONS IN MILK

temperature. Those isolated from fresh milk often give
rise to lactic acid, feebly at first, but their power in this
respect increases very considerably when they are culti-
vated in milk for some time. The soured milk has a
pure, clean taste, occasionally with a fine distinct aroma,
the acid being chiefly right handed lactic acid with
little or no acetic or volatile acid by-products. Sir.
iactzcus, or forms of it, are the most useful of all lactic
bacteria for dairy purposes, and the majority of the
" starters " utilized in manufacture
of butter and cheese are more or
less pure cultures of this type.

Streptococcus lacticus, Kruse (Fig.
46). — The following morphological and
biological features are characteristic of
the type : —

Size and form. — A streptococcus or
YiG. ^^.—streptococcus lacticus, short bacterium .5 /x to I /x in length.
Stains by Gram's method.
Gelatine. — The colonies are white and yellowish sometimes,
very minute, and grow only below the surface.

Stab growth is slight, usually beaded or granular ; no surface
growth.

Agar. — Very small, little or none on surface.
Potato. — Little or no growth.

Bouillon. — Very slight growth, with the faintest turbidity and
sediment.

Milk. — Acid without gas : curd dense, smooth, and firm, with-
out separation of whey. No peptonization of the casein.

Included in Group i., and very nearly allied to Strepto-
coccus lacticuSy are a number of other bacteria, differing
in some particular characters from the above description.
Streptococcals Kefir., Mig., from Kefir, and some others

CHARACTERS OF LACTIC BACTERIA 273

produce acid without coagulation of milk. Str, hollandicus,
Scholl, found in the Dutch " lange Wei " (see p. 279), and
Micrococcus mucilaginosus^ Mig., form slimy material.
There are others which set free slight amounts of gas
during fermentation, and are negative to Gram's stain.
To these may be added Str. mastitidis, Guill., a form
causing epidemic inflammation of the udder of cows, the
affected animals yielding small quantities of yellowish
milk : pure cultures coagulate milk and set free small
amounts of gas. Related to this group is the pathogenic,
Str, pyogenes, Rosenbach, which sets up several inflam-
matory diseases in animals.

{U) Group ii. — The typical organism of this group is
that described by Hiippe, under the name of Bacterium
acidi lactici. It is widely distributed in milk but rarely
in any abundance, its development being probably checked
by organisms of the Str. lacticus type. The chief char-
acter distinguishing it from the bacteria of group i. is its
power of fermenting milk sugar and other carbohydrates,
with the production of considerable amounts of carbon
dioxide gas, often mixed with a little hydrogen. The
organism is always rod-shaped, but varies much in
length. Perhaps the average dimensions are i to i -|/x long,
and I to i/x broad, the cells may, however, reach a length
of I o or 15 (L and i J or 2 /x broad, and in certain media
undergo peculiar changes in form. They are usually
united in pairs, but some forms may appear as chains of
stumpy cocci.

Bad. acidi lactici grows best aerobically and forms
rounded white, slimy colonies on the surface of gelatine
plates. They are often 5 or 10 mm. in diameter, much
larger and very different from the colonies of Str. lacticus.
The bacteria stain faintly with Gram's stain. When
s

274 FERMENTATIONS IN MILK

introduced into milk, souring takes place rather slowly. In
a day or two at a temperature of 30° to 40° C, coagulation
occurs, and the curd which is precipitated contains a few
small gas bubbles, the serum which separates being clear.
The taste and odour developed are generally disagreeable.
Lactic acid is formed and often with it occurs a fair
amount of acetic acid as well as traces of formic and
succinic acids and alcohol. Many troubles of the dairy-
man in butter-making and cheese-making can be traced
to organisms belonging to this group.

The following are the chief characters of typical Bact. acidi
accici, Hueppe.

Size and Form. — Rods i to i J ft long and | to i /x broad in pairs
and chains.

Gelatine. — Surface colonies round or lobed, white and moist
and slimy, very similar to those of B. coli. Deep colonies, small,
usually spindle-shaped.

Agar. — Irregular, greyish, shining ; deep colonies, small, round
or spindle-shaped.

Potato. — Abundant growth, greyish at first, then yellow or
brown.

Bouillon. — Turbid with abundant sediment.

Milk. — Acid produced and curd precipitated with a few small
bubbles in it.

Sugar Bouillon. — It ferments glucose and lactose with acid
and gas production, but not saccharose.

Indole is produced by it in peptone solution (Ex. 63), but it does
not give the Voges and Proskauer reaction (Ex. 65).

A form which appears to be almost as common as
the type already described and very closely allied to it,
is known as Bacterium lactis aerogenes, Esch. It was
first isolated from the milk-stools or faecal matter of

CHARACTERS OF LACTIC BACTERIA 275

infants, but is often met with in milk, although rarely in
any abundance. The colonies on gelatine plates are
round, shining, white like porcelain, with smooth, circular
margins, and more or less hemispherical, standing up
from the surface of the plate. The bacteria from typical
cultures do not stain by Gram's method (see p. 8). Some
forms in this group very closely resemble Bacterium colt
(p. 106), and are difficult to distinguish from the latter
with certainty. Eckles' Bacillus No. 8, which gives a
pleasant, ripe cheese aroma in milk, and is a useful
" starter " for the butter-maker, appears to be a form
near Bact. lactis aerogenes. The Bacilli Guillebeau,
a and b (Freudenreich), obtained from milk of cows
suffering from inflammation of udder, and giving rise
to " gassy " or " blown " cheeses, are closely related to
Bact. I. aerogenes. In the same group there are included
many other bacteria, isolated from milk by various
workers, which differ slightly from the typical form
described above. Bact. limbatum, Marpmann, coagulates
milk but produces no gas. Bs. lactis innocuus, Wilde,
and Bs. No. 41, Conn, give rise to a little acid only, but
do not coagulate milk or develop gas in it. The latter
variety gives rise to a pleasant aroma in unsterilized
milk. Several varieties which have been isolated from
cheese should be included here. Very closely resembling
Bact. acidi lactici, Hiappe, and therefore to be included
in the group is Bact. pneumonice of Friedlander, the cause
of pneumonia in man. It does not coagulate milk, but
forms gas. When taken from the body or from cultures
in milk the organisms exhibit well-developed gelatinous
capsules. The colonies on gelatine are round, moist,
and white, very similar to those of Bact. lactis aerogenes.
To the same group may be added Bact. lactis viscosum.

276 FERMENTATIONS IN MILK

Adametz, a variety which renders milk slimy and causes
trouble in dairies sometimes.

{c) Group iii. — In this group the typical form may
be taken as Bacterium casei, Freudenreich. The
organism is a thin, elongated rod, 2 to 3 /a long and
.5 to .7 /x broad. Most of the forms in this group grow
feebly and are usually more or less anaerobic. Their
colonies are small, about the size of a pin's head, white
or yellowish-white, and variable in. shape. The typical
Bact. casei produces inactive lactic acid in milk and
coagulates the latter in three^ days at a temperature of
30° C. with gas formation. Allied to it are Bact.
casei, Leichmann, and Bact. lactis acidi, Marpmann, which
do not produce gas in milk. Bs. caucasicus, Beijk.
(p. 315), one of the active fermentation agents in Kefir
grains, and^^. bulgaricus, Heupel.(p. 3 1 9), should probably
be included in this group. Other allied forms are known
which neither precipitate casein nor form gas.

{d) Group iv. — The type species or form of this
group is Micrococcus lactis acidi, Marpmann. It is a-
small coccus .6 to .8 /x in diameter, occurring singly or
in pairs and small heaps, but never in chains. In milk
it grows slowly, rendering it acid at 20" C, but not
coagulating it below 30° C. The colonies on gelatine
are small and round, yellowish-white in colour, growing
chiefly on the surface of the plate. No gas is formed.
Micrococcus /j/r?^^;^£7.9, Rosenbach, and its varieties, which
are associated with the formation of pus in various
inflammatory and suppurative diseases, belong to this
group. They coagulate milk and tend to produce
enzymes, which dissolve casein and liquefy gelatine. In
some varieties the colonies are white, in others different
shades of yellow or orange. The organisms producing

CHARACTERS OF LACTIC BACTERIA 277

bitterness in cheese, Micrococcus casei arnari, Freudrch.,
and Conn's Micrococcus of bitter milk, belong to this
group also.

Ex. 135. — Inoculate tubes of sterile milk with pure cultures
of:—

{a) Sir. lacticus, Kr.
{b) Bs. acidi lacHci, Hueppe.
(c) Bad. lactis aerogenes, Esch.
{d) Bad. coll, Esch.
{e) Bs. bulgaricus, Hpl.
Note the kind of curd thrown down by each, and the presence
or absence of gas bubbles in it.

Ex. 136. — Make cover-films from pure cultures of the organ-
isms mentioned in the previous experiment, and stain them
with : —

{a) weak gentian violet,
{b) Lceffler's methylene blue,
{c) carbol fuchsin,
{d) Gram's stain.
Examine with the highest power available and make drawings
of the organisms.

Ex. 137. — Mix a few drops of sour milk in a test-tube with
sterile water.

Inoculate tubes of melted litmus gelatine and litmus agar
with a loopful of the diluted milk, and pour into separate Petri
dishes.

{a) Incubate the gelatine plates at 20° C, the agar plates at
35° C, and note the number, form, and acidity of the colonies
obtained.

Do any of them liquefy the gelatine ?

{b) Inoculate tubes of litmus milk with small portions of the
different colonies observed.

Incubate at 20° C. and make notes on the following points
during seven to fourteen days : —

I . The presence or absence of curd.

2/8 FERMENTATIONS IN MILK

2. The texture of the curd thrown down.

3. The separation of whey.

4. The presence or absence of gas bubbles.

5. The odour of the fermented milk.

Do any of the organisms peptonize or soften and digest the
curd?

(^r) With portions of the different colonies isolated above —
Inoculate an agar slant.
„ a potato slice.
„ a gelatine stab.

„ a tube of glucose broth in a Durham's tube.
„ ,, lactose ,, ,5 „

„ „ saccharose „ „ „

Test their nitrate reducing power (Exs. 74 and 82).

„ indole production (Ex. 63).
Try Voges and Proskauer's reaction with each (Ex. 65).
Record the characters of each in accordance with the scheme
given on pp. 60-61. .

2. Slimy or Eopy Fermentations of Milk. — Many
liquids, such as grape juice, beer, cider, and various plant
infusions which contain sugar, sometimes become ropy
or slimy, so much so that they may be drawn into long
gelatinous strings. A similar production of a slimy
substance is not unfrequent in milk and cream, and is
brought about by the action of bacteria, most of which
appear to be introduced into the dairy in the water
supply or in dust from hay and other food materials.
The chemical nature of the ropy material has not been
exactly determined. It appears to arise, however,
either : —

(i) from the milk sugar which is changed into a gum-
like carbohydrate ;
(2) from the production of a slimy protein substance

SLIMY OR ROPY FERMENTATIONS OF MILK 279

from the casein, which substance may be set free
in the serum or watery part in the milk, or
(3) from the presence of swollen gelatinous capsules
round the organisms.

Possibly the slimy character of the medium is due
in certain cases to the enormous number of bacteria
developed in it.

Many species or varieties of bacteria are capable of
producing varying degrees of ropiness in milk and
cream. In some instances the milk or the milk serum
becomes ropy in a few hours, in which case the cream is
of normal consistency: in most samples, however, generally
the change occurs only after a considerable period, and
is therefore a commoner phenomenon in cream, and
especially when the latter is raised in pans. Ropiness
is less frequent in " separator " cream.

The slime-producing organisms which have been most
carefully investigated and described are referred to
below.

(a) Streptococcus hoUandicus, Scholl, known as the "Ropy
Whey" or "Lange Wei" organism, is a coccus which occurs in
pairs and longer chains. It is allied to the ordinary Str. lacticus,
and, like it, produces lactic acid, but has the additional
power of rendering milk stringy and capable of being drawn out
into long threads in from twelve to fifteen hours at a temperature
of 22° C. The slime appears to be a product of the albumin of
the milk. Ropy Whey is used as a " starter " in Holland in the
manufacture of Edam cheese. The colonies on milk-peptone
gelatine are very small : they do not liquefy the medium.

(b) Micrococcus Freudenreichii, Guillebeau, is a very large
aerobic coccus, often nearly 2 /z in diameter, which produces
stringiness in milk in a few hours at a temperature of 20° C. or
less. Its colonies on gelatine are very small, round, and white.

28o FERMENTATIONS IN MILK

the medium soon becoming liquefied : on potato they are pale
yellow.

(c) Bacterium lactis viscosum, Adametz, is a non-motile,
coccus-like bacterium, with a well-developed capsule, about i /a
in diameter, and producing sHmy whitish colonies on gelatine
and agar somewhat like those of Bac^. lactis aerogenes. It some-
times occurs in threads 2 to 5 ^a long. When introduced into
milk it renders it slightly alkaline and viscous in a day or two,
but the stringy condition does not develop until a much longer
time has elapsed. The milk sugar is not much altered, the slimy
substance being apparently produced at the expense of the casein
of the milk. Cream inoculated with it becomes slimy if kept some
time, and butter made from such cream is soft and does not keep
well. The bacterium is generally introduced to the dairy in the
water supply, and is one of the commonest causes of ropy milk.

Resembling this species is Bs. lactis pituitosi, Loffler, a thick
bacillus which breaks into coccus-like segments and renders milk
slightly acid.

{d) Bacterium Guillebeau, c, Freudenreich, is a type of
bacterium isolated from milk yielded by cows suffering from
inflammation of the udder. Milk is rendered slimy by it. It
produces colonies on gelatine like those of BacL lactis aerogenes.

(e) Bacterium Hessii, Guillebeau, a motile form 3 to 5 /a.
long, I to 2 /A thick, nearly related to the hay bacillus (Bs. sub-
tilis\ makes milk ropy, but the ropiness gradually disappears
when acidity develops.

(/) Bs. viscosus, I. van Laer, a bacillus 1.6 to 2.4 /x
long, .8 /x thick, isolated from beer, in which it produces
slimy material, can produce ropiness in milk. It forms small
white colonies on gelatine plates, which at first are round,
becoming slimy with irregular margins later.

{g) Streptococcus hornensis, Boek., a coccus isolated from
slimy milk in Holland, and found in separator slime, water, and
on flowers. It produces small white colonies on gelatine.
Introduced into milk containing cane sugar at 22° to 30° C.,

DEVELOPMENT OF COLOUR IN MILK 281

the liquid is rendered very ropy, the sHmy product being the
gummy carbohydrate dextran.

{h) An organism very closely resembling Sir. lacticus was found
by Burri to be the cause of slimy milk and whey in a Swiss dairy.
He traced it to the milk ducts in the udder of some of the
cows.

3. Development of Colour in Milk. — Many bacteria
possess the power of manufacturing colouring matter,
which may become distributed in the media in which
they are growing, colouring them red, pink, yellow, blue,
or some other tint. They may occur in milk, although
colour production through the agency of bacteria is
rarely met with in this liquid, and then only when it is
kept a considerable time. The following varieties of
coloured milk are best known : —

(i) Red Milk. — It is not very uncommon to meet
with milk having a reddish tinge or which deposits a
reddish sediment after standing for a time in a can or
other Vessel. The colour is due to the presence of
blood derived usually from the rupture of a small blood-
vessel in the udder of the animal. In such cases the red
blood corpuscles are readily detected by the microscope
(see p. 302). In some instances, however, the red colour
is not visible in the milk freshly drawn, but develops
slowly and increases slightly from day to day. Such
examples are. due to the growth of certain species ot
bacteria, of which the following have been isolated and
studied : —

{a) Bacterium erythrogenes, L. & N. {Bacillus lactis
erylhrogenes, Grotenfelt). The organism is non-motile and rod-
shaped, .8 to 2 [Jj long and .5 to .8 /a thick. When grown in
milk its coagulation occurs slowly, the serum separates from the

282 FERMENTATIONS IN MILK

casein and the cream becomes blood-red in colour and slightly
alkaline. The colonies liquefy gelatine and produce a red
coloration upon it, and upon agar when grown in the dark. In
the light the colonies are yellowish.

(b) Micrococcus roseus, L. & N., is a very common bacterium
found in the air. It grows readily in milk, forming a red sedi-
ment in the latter without otherwise effecting much change.
Nearly allied to this is Sarcina rosea, Schroter, which has been
found to give a red coloration to milk, with slow precipitation
and solution of casein.

[c) Bacillus prodigiosus, Fliigge (L. & N.) {Micrococcus pro-
dtgiosus, Cohn), is also a common air and water organism which
appears as a large coccus i /a in diameter on solid media and as
rods in liquid media. It is sometimes motile, and when grown
on potato and agar produces rose-red to reddish-purple colonies.
It liquefies gelatine and the colonies are often greyish-white
instead of reddish in colour. On the surface of milk or cream
it may produce its characteristic blood-red pigment, but in the
liquid it precipitates and dissolves the casein with the production
of a faint yellow colour only.

(ii) Yellow Milk. — A number of bacteria have been
found to give rise to a yellow or orange coloration in
milk, especially if the latter is kept for a long time. The
species, which has been most carefully investigated, is

Bacterium synxanthum, Ehr, a short, thin rod-like organism
obtained from boiled milk, in which it gives rise to a bright
yellow colour; the casein is dissolved, the solution becoming
alkaline. In acid milk the colour is not developed.

(iii) Blue Milk. — The development of a sky-blue colour
in milk has occurred from time to time in certain dairies,
especially on the Continent, and so far back as 1838
investigations were made to determine its cause. That
it was due to the work of micro-organisms was demon-

BITTER MILK 283

strated by Fuchs in 1841. The colour is due to the
activity of a bacterium named

Bacillus lactis cyanogenes, by Hiippe {Bad. syncyaneum^
L & N.), a motile aerobic rod-shaped form i to 3 /a long and .5 /a.
thick. After introduction into fresh milk it induces a blue
colour in from one to three days, or earlier if the temperature is
maintained at about 20° C. The colour appears first at the
surface of the milk in small spots or patches, and spreads slowly
into the deeper parts. The production of a rich deep blue tint
is dependent upon the simultaneous production of a certain
amount of acid by the lactic organisms, although the acid is
removed in some way almost as fast as it is formed, since the
coloured milk has an alkaline reaction. On keeping, the colour
changes to a dirty grey or brown. The colonies on gelatine are
usually yellowish- or greyish- white at first, but later become brownish
purple. It is easily checked by the presence of other bacteria,
and is destroyed at a temperature of 60° or 70° C. A greyish
tinge only is developed by the bliie milk bacillus in sterilized
milk or in fresh milk which has not become acid.

Ex. 138. — Prepare stained slides of the following organisms : —

Bad. ladis viscosus, Adamtz.
Str. hollandkus, Sch.
Micrococcus roseus, L. & N.
Bs. prodigiosus^ Fliigge.
Bs. lactis cyanogenes^ Fliigge.

a. Examine with high power, and make drawings of each.

b. Try their action upon (i) fresh milk, and (2) milk to which

sugar has been added.

c. Grow them upon gelatine and agar media, and note the

form and colour of the colonies.

4. Bitter Milk. — The bitter flavour of milk some-
times appears to be brought about through the con-
sumption by the cows of leaves of turnips and other
cruciferous plants, lupins, and vetches, as well as certain

284 FERMENTATIONS IN MILK

weeds, such as chicory, tansy, and wild chamomile. Cows
with udder disease also yield bitter milk occasionally.

In all these instances the bitterness is recognisable in
the milk as it is drawn from the animal, and it does not
increase with age of the milk.

There are, however, many cases recorded of serious
bitterness of milk which have originated through the
activity of bacteria.

In boiled milk the bitter flavour not infrequently
arises from the spore-bearing organisms, such as Bs.
subtilis and its allies, as well as some of the butyric acid
organisms : in unboiled samples these bacteria are
generally kept in check by the lactic acid organisms.

In fresh milk bitterness may arise ; the trouble in
many cases has been traced to the contamination with
various kinds of bacteria. Varieties of Bad. coli and
Bacf. lactis aerogenes have been found capable of inducing
the objectionable change in milk, the peculiar taste being
brought about in most cases by decomposition of the
protein of the milk. Freudenreich isolated from bitter
cheese a small coccus, which he named Micrococcus casei
amari. It liquefies gelatine, renders milk acid, and coagu-
lates the casein, producing at the same time a bitter taste.

The same writer described ^ motile bacillus obtained
from bitter cream which liquefies gelatine and gives rise
to an intensely bitter flavour in milk, and coagulates it
without rendering it acid.

Eckles has also described a small oval coccus capable
of giving rise to bitterness in milk, and rendering cheese
made from it very bitter (see p. 377).

Harrison traced the bitterness of milk in a cheese
factory to contamination with a species of Torula or
non-sporing yeast (see p. Z77\

BUTYRIC ACID FERMENTATIONS 285

5. Butyric Acid Fermentations. — When a mixture of
milk, old cheese, sugar, and chalk or calcium carbonate
is kept warm fermentation soon begins ; a great evolu-
tion of gas occurs, and butyric acid is formed, which,
when neutralized by the chalk, yields calcium butyrate.
In the process lactic acid or calcium lactate is first
produced, and this is fermented subsequently with the
formation of calcium butyrate.

The nature and cause of this and similar reactions
where carbohydrates are broken down with the pro-
duction of butyric acid were studied by Pasteur, Van
Tieghem, and others. They showed that the fermenta-
tions were brought about by the action of a group of
anaerobic bacteria which are widely distributed in water,
milk, cheese, soil, dung, and other materials.

A large number of butyric acid bacteria have been
described since Pasteur's time as if they were distinct,
but the recent investigations of Beijerinck, Schattenfroh,
and Grassberger have shown that most of those described
by earlier writers are varietal forms of a few distinct
species only.

In the fermentation of calcium lactate this substance
is changed into calcium butyrate, but at the same time
certain bye-products are formed in variable amounts ;
the gases carbon dioxide and hydrogen are given off,
and small quantities of ethyl and butyl alcohol are
frequently formed along with propionic and other acids.

The two stages in the change of milk-sugar into
butyric acid may be expressed thus : —

(I) C12H22O11 + H2O = 4C3He03

milk-sugar lactic acid

(2) 2C3H603 = C4H802+C02+H2

lactic acid butyric acid carbon hydro-
dioxide gen

286 FERMENTATIONS IN MILK

In addition to the indirect butyric acid fermentation
of milk sugar, direct fermentation of this and other sugars
without the preliminary change into lactic acid can be
carried out by several organisms ; other bacteria are
known which produce butyric acid from starch, citric,
malic, and other acids, and from glycerine.

It is usual to limit the term butyric acid fermentation
more particularly to the production of this acid from
carbohydrates and glycerine. Most of the organisms
possessing this power are anaerobic bacilli, which in
a vegetative state store up granulase, a kind of carbo-
hydrate giving a blue or violet colour with iodine, like
that given by starch. They form highly resistant
spores, which may be heated to the temperature of
boiling water without being destroyed. These often
appear in the middle of the bacterial cell, and when
fully developed bulge out the walls of the latter into the
form of a spindle ; a bacterial cell of this shape is usually
termed a Clostridium.

Besides the above typical butyric acid fermentations
in which a carbohydrate is broken down, with the forma-
tion of butyric acid as a chief product, many fermentations
are known in which butyric acid in larger or smaller
amounts arises in the decomposition of proteins, the
organisms concerned belonging mainly to the putrefactive
class, most of them being closely allied to Bs. vulgatus.

Butyric Acid Bacteria. — i. Great uncertainty exists
in regard to the relationship of the butyric acid organisms
described by many different writers ; Pasteur's vibrion
butyrzque, and Van Tieghem's Bacillus Ainylobacter^ were
considered by Prazmowski the same as his Clost7'idimu
butyricum. It is not feasible here to give an account of
the various forms which have been described. Most of

BUTYRIC ACID BACTERIA 287

them, although differing in name, are no doubt similar
organisms ; it will suffice to briefly describe the two
main species isolated and studied by Schattenfroh and
Grassberger, since these two species apparently include
most of the anaerobic forms which attack carbo-
hydrates.

a. Species L, described as "motile non-liquefying Granulo-
bacillus saccharobutyricus," is apparently the same organism as
B. saccharobutyricus of Von Klecki, Granulobacter saccharo-
butyricum of Beijerinck, B. amylozyma of Perdrix, and is
closely allied to the pathogenic organisms B. Chaiivoei, which
causes blackleg in cattle, and B. enteritidis sporogenes.

It is common in water, soil, meals of various cereals, and com-
l)aratively rare in milk.

The bacteria are rod-shaped, with peritrichous fiagella; they
grow best under strict anaerobic conditions at 35° to 37° C. Clos-
tridia are formed containing oval spores, 1.8 to 2.3 (Jj long and
1.3 to 1.7 [jj broad.

The organism converts milk-sugar almost completely into
butyric acid, carbon dioxide, and hydrogen, little or no lactic
acid being formed; from grape-sugar, however, it forms both
acids.

Proteins are not affected by it.

Gelatine is not liquefied. In grape-sugar gelatine media stab-
cultures may exhibit three types of growth, viz., {a) A beaded
track with the organisms in the Clostridium form ; {b) a root-like
track, with fine outgrowths ; ic) a turbid track, showing gas
bubbles, the organisms motile and without granulose.

Grape sugar agar. — In stab-culture much gas is produced,
and a strong smell of butyric acid.

Potato. — Extensive growth, white and frothy, with strong odour
of butyric acid. The bacteria contain much granulose.

Milk. — In milk butyric acid is produced, and casein, full of gas
bubbles, is precipitated, but not peptonized.

288 FERMENTATIONS IN MILK

Bouillon. — In ordinary peptone broth little development occurs,
but it grows rapidly in glucose peptone solution.

b. The second species, described as the "non-motile Granulo-
bacillus saccharobutyricus," is a common organism in milk,
dung of farm animals, and human faeces ; it is also abundant in
water, soil, and the meal of cereals.

Two varieties exist, one a long cylindrical bacillus with
rounded ends, often in chains three to six together ; the other
a smaller and shorter organism, rarely united in chains.

Both are strictly anaerobic.

In the fermentation of milk-sugar, and carbohydrates by these
organisms, lactic as well as butyric acid is formed, often in
larger quantity than the latter compound.

Granulose is present in the growing cells, but generally absent
from those in which spores are formed : this substance is most
abundantly produced when the organism is supplied with starch,
little or none being made when sugar is supplied.

The spores withstand boiling for an hour and a half.

Gelatine media are liquefied.

Agar. — Surface colonies on slightly alkaline agar, containing
I grain of starch or sugar per litre, are smooth and shining,
irregular in outline in the case of the long variety, rounder and
more watery in the short-celled form.

Milk is rendered acid : the casein is coagulated but not
peptonized; it becomes full of gas bubbles. ;

Bouillon containing 2 per cent, of starch or grape-sugar is a
good medium for the growth of this organism, but spores are not
produced in it.

Ex. 139. — To obtain Clostridium-forms of Granulobacillus
saccharobutyricus, the following receipt, suggested by Beijerinck,
gives successful results : —
Put in a flask : —

Cane-sugar . . . . . 10 gr.

Finely-ground fibrin . . . . 10 ,,

Precipitated calcium carbonate , , 6 „

BUTYRIC ACID BACTERIA 289

Sodium phosphate . . . . .1 gr.

Magnesium sulphate . . . . . i „

Sodium chloride . . . . . i „

Water ....... 200 c c.

Boil briskly for two or three minutes, and while boiling add
10 to 15 grams of fresh garden soil.

Plug loosely with cotton wool and transfer while hot to an
incubator kept at 35° C.

The heating destroys moulds and non-sporing bacteria and
drives out dissolved air : the spores of the butyric acid bacteria
resist boiling, and, as the liquid cools, germinate and grow, the
necessary anaerobic conditions being maintained to a certain
extent by resistant aerobic bacteria introduced in the soil.

Examine the sediment at the bottom of the flask daily for a
week as follows : —

(a) Tilt the flask gently and remove a small portion of the
slimy deposit with a platinum loop.

Add it to a drop of a dilute solution of iodine in potassium
iodide on a glass slide : put on a cover-sHp and examine with a
high power.

The butyric organisms containing granulose are stained a
violet or violet-blue colour.

Look out for clostridial forms with spores in them ; the latter
appear as unstained, bright, oval or round bodies in the clostridial-
cells.

ip) When spores are found mix a small amount of the slimy
deposit with two or three drops of water in a watch-glass : then
spread a drop or two of the mixture on two or three cover-slips
Allow to dry : then stain spores by method given in Ex. 12.

Ex. 140. — Half fill a small yeast flask with the solution men-
tioned in the previous experiment.

Close the neck with a rubber cork through which passes a
narrow bent glass tube arranged as in Fig. 47.

Now boil the solution for two to three minutes : and while
boiling add 10 to 15 grams of fresh garden soil,
T

290

FERMENTATIONS IN MILK

Connect with a short piece of rubber tube to a hydrogen
generator and pass the gas through the solution for a few
minutes : then clamp the rubber connecting tube and disconnect
the hydrogen apparatus.

Dip the open end of the side tube of the flask into a small

beaker or cylinder con-
.XH taining mercury and trans-
fer to the incubator at

37° C.

Since the oxygen has been
expelled the anaerobic organ-
isms can now grow in the
medium, and any gas which is
produced in the fermentation
process can escape by the mer-
cury valve without setting up
any great internal pressure.

Examine a drop of the cul-
ture after forty-eight hours and
note the character of the bac-
teria present.

Test for granulose with iodine
in potassium iodide.

If Clostridia with spores are
present, stain the latter by method given in Ex. 1 2.

2. Although it is advisable perhaps to limit the term
butyric acid fermentation to the production of this acid
from carbohydrates, lactic acid, or glycerine, it must be
noted that it is a frequent product in the decomposition
of proteins by bacteria.

In the former process butyric acid is one of
the chief substances obtained, while in the latter
the amount is usually small and of secondary import-
ance.

Fig.

-Yeast flask arranged for cultivation
of anaerobic organisms.

BUTYRIC ACID BACTERIA 291

Many of the organisms which give butyric acid when
breaking down proteins are aerobic.

(a) Belonging to this class is Bacillus butyricus, Hueppe, an
aerobic bacterium occasionally present in milk, and said to be
able to produce butyric acid from milk-sugar after this compound
has been hydrolysed by other organisms.

It is a rod-shaped bacillus 8 to 10 /^ long, 1 to 1.2 /x broad ;
stains by Gram's method and has large oval spores.

Gelatine. — The colonies are yellowish-white ; liquefying stab-
cultures soon show deep funnel-shaped liquefied portions.

Agar. — Yellowish-white slimy colonies, rounded and slightly
elevated.

Potato. — A slimy yellowish-brown growth.

Milk. — Coagulated and rendered alkaline, casein peptonized ;
neither gas nor indole are produced, but a small amount of
HoS is formed.

{[}) In this group should be included Bacillus vulgatus (p. 126)
and probably other putrefactive aerobic soil bacteria, though the
amount of butyric acid which they produce is small.

{c) Emmerling isolated an aerobic bacillus (Bacillus boocop
ricus) from cow dung which formed butyric acid from glycerine ;
gelatine was not liquefied by it.

CHAPTER XVIII.

FILTRATION ; COOLING ; PASTEURIZATION AND
STERILIZATION OF MILK; PRESERVATIVES.

The suggestions made in Chapter XVI., § 3, have
reference chiefly to the production of a clean milk —
that is, they aim at protecting it from becoming con-
taminated with bacteria. It is, however, important to
deal with another side of the question, namely, the
methods to be adopted in order to remove or destroy
those organisms which unavoidably gain an entrance
into it, or to check their development.

Three methods may be specially mentioned :
(i) The filtration of the milk ;
(ii) the application of low or high temperatures ,
(iii) the introduction of " preservatives " or soluble
chemical substances which inhibit the mul-
tiplication of bacteria.

(i) Filtration. — Strainers consisting of various com-
binations of cotton or linen fabrics placed over fine wire
gauze should always be used at the cowshed or dairy,
to remove the particles of dung, hair, hay, and other
refuse which fall into the milk. Milk may be filtered
through layers of gravel, sand, and cloth, which remove a
considerable proportion of the bacteria present, although
they do not take out all of them. The difficulties con-
nected with the cleaning and renewal of such filters, as
293

EFFECT OF LOW AND HIGH TEMPERATURES 293

well as the comparative slowness of the operation, have
prevented their general adoption. Germ -free milk for
laboratory purposes can be obtained by filtration through
porcelain filters, but much of the fat is removed in the
process as well as the bacteria.

(ii) The Effect of Low and High Temperatures, (a)
Cooling. — One of the most effective means of prevent-
ing milk from undergoing fermentative changes is to
cool it and keep it at a low temperature. The sooner
this can be done after the milk is drawn from the
cow the better. Most of the bacteria which damage
milk, from the consumer's point of view, grow very
slowly at 10° C. (50° F.), and where it can be cooled
immediately after milking, and maintained at this or a
lower temperature, it will keep without any recognizable
change for several days. Moreover, cooling does not
appreciably alter the flavour of the product.

It must be noted that the bacteria are not killed by
this treatment, nor can they be killed by the application
of the lowest temperatures attainable : they are merely
checked for the time, and will develop readily- enough
when the temperature is raised.

(d) Sterilization. — The sterilization of milk can be
secured by heating it under pressure to a temperature
of 1 10° C. or 1 1 5"" C. (230° to 240° F.) for half an hour.
Boiling it for twenty minutes destroys all the common
forms, but the spores of certain species which are con-
veyed into milk by particles of hay and soil resist the
treatment for some time, and may ultimately grow and
set up objectionable chemical changes in it when the
temperature is reduced. However, continuous boiling
for an hour or two will destroy the spores of all bacteria,
and render it sterile.

294 FILTRATION AND HEATING OF MILK

The sterilization of milk in this way has not become
a popular process, partly because of the trouble and
expense involved, but mainly on account of the altera-
tion in flavour which results. The taste of boiled milk
is very readily recognized, and is disliked by children as
well as by adults. Nevertheless, in spite of the dis-
advantages and objections to the use of boiled milk,
boiling for a short time is the simplest and safest means
of getting rid of pathogenic organisms, which, under
present conditions of production and distribution, too
frequently find their way into the milk supplied to the
householder.

Ex. 141. — Pour 250 c.c. of fresh evening's milk into a
sterile flask, and allow it to stand in a cool pantry until next
morning.

{a) Dilute a small amount of it to i in 1000 with sterile
water, and prepare two litmus agar plates from it,
using .5 c.c. for inoculation.
{d) Boil 100 c.c. in a flask for five minutes; cool, and
prepare two litmus lactose agar plates from it as
above.

(c) Plug the flask with cotton-wool and leave it in a room

at ordinary temperatures for twenty-four hours ;
then prepare two litmus agar plates from it as
before.

Incubate all the above at 35° C. for forty-eight
hours; note and compare the number of colonies,
their form and acidity, which appear on each plate.

Stain and examine specimens of the organisms
from the different colonies.

(d) Place the unboiled milk remaining in a 100 c.c. flask

by the side of the boiled sample.

Leave them until both are curdjed, noting which
curdles first, the nature of the curd, the separation

PASTEURIZATION 295

of whey, and any other features of difference between,
the two samples.

(c) Pasteurization. — The process of pasteurization
has been extensively applied to milk with a view of
increasing its keeping quality, without appreciably alter-
ing its natural flavour or nutritive property. It consists
in heating the milk to a temperature of 60° C. to 80° C.
( 1 40° to 1 8 5 ° F.) for twenty minutes or less, and then
cooling it rapidly to about lo"" C. (50° F.). There is no
definite standard of temperature for the process ; that
perhaps most generally adopted is 85° C. at which the
milk is kept for three to five minutes. At 70° C.
(158° F.), or lower, the heating should be continued for
twenty minutes. In all cases for efficient pasteurization
it is essential to keep the milk stirred or agitated so as
to secure uniform heating.

The heating destroys from 95 to 99 per cent, of
bacteria present in the vegetative state; and, if not too
prolonged, does not give the milk a cooked taste.
Cleaner milk is obtained, and at the low temperature to
which it is immediately cooled the few remaining or-
ganisms cannot grow ; the milk retains its freshness for
three or four days, provided that it is kept in a cool
place and free from dust and other contaminating sub-
stances. The lactic acid bacteria and many others are
destroyed at the temperatures indicated above, but the
spores and growing organisms of certain species remain
uninjured. According to Fliigge, the pathogenic or-
ganisms responsible for tuberculosis, diphtheria, and
typhoid fever are killed by heating the milk to 70° C.
(158° F.) for half an hour, although this treatment leaves
it with a slightly boiled taste.

296 FILTRATION AND HEATING OF MILK

The great reduction in numbers by pasteurization is
seen in the following table compiled from Professor
Stewart's experiments : —

Average No. of
bacteria per c.c. be-
fore pasteurization.

Time of
Heating.
Minutes.

Temp.

136,262
78,562
49,867
77,062

10
10
10
10

60° c.
65° c.
70° c.
75° C.

No. of Dacteria per

c.c, 24 hours after

pasteurization.

1722

.58
O
O

Flavour.

Unaltered
Little affected
Slightly boiled
Boiled.

It is important to bear in mihd that pasteurization is
a double process, namely, the application of a tempera-
ture sufficient to destroy many living bacteria, followed
by rapid cooling. To disregard the latter part of the
process is to render it useless ; for if this is neglected,
and the heated milk allowed to cool slowly, the remain-
ing uninjured organisms multiply rapidly when the
optimum temperature is reached, and the milk may
contain more bacteria after treatment than before.

Ex. 142. — Take 250 c.c. of fresh evening's milk and keep it
in the pantry until next morning.

Dilute a small amount of it to i in 1000 with sterile water,
and prepare two litmus lactose agar plates, using .5 c.c. for
inoculation.

Heat — (i) 50 c.c. of the milk to 50° C. for 10 minutes.

(2) „ „ „ 60° C. „ „

(3) »' » »' 70° C „ „

' Cool quickly.

Dilute a small amount to i in 100, and prepare two litmus
lactose agar plates from each sample, using .5 c.c. for
inoculation

PRESERVATIVES 297

Incubate all at 35° C. for 48 hours.

Count the colonies on the several plates, and note the effect
of the heating on the numbers of bacteria in the milk.
Observe which colonies are acid on each plate.

(iii) Preservatives. — The use of saltpetre, salt, and
other substances for the purpose of preserving all kinds
of food from putrefaction and other fermentative changes
is widespread. Many attempts have been made also to
check the souring of milk by the addition of certain
chemical compounds which in small doses are known to
inhibit bacterial growth. Such powerfully poisonous
bactericides as mercuric chloride or carbolic acid cannot
of course, be used for this purpose, but it is unfortunately
too often the case that antiseptics with deleterious
properties are added to milk by farmers and dairymen.
The substances most commonly employed are boric acid,
borax, salicylic acid, and formalin ; in most countries
their use is illegal, and infractions of the law should be
punished.

All materials which effectually check the development
of bacteria are certain to impair the digestive functions,
particularly of infants and young children, whose diet
consists largely of milk. There is little doubt that the
continued use of small amounts of these compounds
would result in damage to the health of adults also if
milk were extensively used as a food by such persons.

The addition of small quantities by the farmer has
been repeated by the dealer and dairyman, and in some
instances by the householder as well, so that before the
milk was consumed it contained pernicious doses of these
harmful substances.

Apart from the directly injurious effects of preserva-
tives upon the health of the community, their use

298 FILTRATION AND HEATING OF MILK

indirectly tends to foster negligence and uncleanliness
in the production and distribution of milk, and prevents
the spread of efforts to improve the conditions which
prevail at the farms and dairymen's premises.

Although boric acid is, perhaps, not so injurious in
small doses as formalin or salicylic acid, its addition to
milk should be prohibited, and the law strictly enforced.
These compounds do not preserve by eliminating or
destroying the bacteria, and they are only used to make
a dirty milk appear or behave as a clean sample.

For improving the keeping qualities of milk, it is best
to depend upon cleanliness of the farm and dairy, and
the maintenance of the product at a low temperature up
to the time that it is needed for consumption.

During the last few years many investigations have
been made upon the bactericidal power of hydrogen
peroxide, a compound with somewhat poisonous pro-
perties, but which under certain conditions is readily
decomposed into the two harmless substances, water and
oxygen, thus : —

H20.2 = H20 + 0.

The free oxygen is given off in an active " nascent "
state, in which condition it is able to destroy the vitality
of bacteria and their spores.

The addition of small quantities to milk has been
found to kill off the bacteria in it and render it sterile.

Its action is materially decreased by minute amounts
of acids, and at low temperatures the quantity it is
necessary to add to milk to destroy all bacteria present
in it, is too great to allow of the milk being used for
human consumption afterwards. However, at higher
temperatures, very small quantities suffice for sterilizing

PRESERVATIVES 299

purposes. In Budde's process of sterilization, the milk
is heated to 48° or 50" C, and to it is added .035 per
cent, (about i 2 c.c. of a 3 per cent, solution) of hydrogen
peroxide. After keeping the milk at this temperature
for half an hour, during which time it should be stirred
or gently agitated, the temperature is raised to 52' C,
and this degree of heat maintained for two or three
hours. The milk is subsequently cooled rapidly, and
sent out in sterile bottles.

The chief objections to the use of hydrogen peroxide
for this purpose are these : —

(a) A very small amount of the compound remains
undecomposed in the milk, and the effect of this upon the
health of small children is not known with certainty.

{b) Comparatively minute quantities give the milk a
taste which is disliked by some persons.

{c) Much of the hydrogen peroxide which is sold
contains traces of hydrochloric acid, 'arsenic, and barium
compounds, and the pure solution is too expensive at
present for general use.

CHAPTER XIX.

THE EXAMINATION OF MILE: MILK
STANDARDS.

I. Dirt in Milk. — A great many impurities are found
in milk, some of them, as already noticed, consisting of
inert matter not necessarily harmful ; others, however, are
of an objectionable or unclean character, and likely to
damage its nutritive value, or cause more or less serious
illness among those consuming it. Under present con-
ditions of production and distribution, it is not possible to
send to the consumer milk entirely free from pollution
of one kind or another.

Much of the milk before it leaves the farm or dairy is
passed through sieves or strainers of muslin, cotton-wool
and similar materials, but these only remove the larger
particles of foreign matter which fall int9 ^^ ^^ ^^^ ^^"^^
of milking or when it is in the cowshed.

The material collected by such strainers consists
chiefly of hair from the cow, pieces of dried dung, and
fragments of hay, straw, and other fodder. More rarely
are found parts of dead flies and other insects, cobwebs,
bits of feather and wood, woollen, linen, and cotton
threads and fibres from the garments of the milker,
skin from his hands, and hyphae of fungi.

Although the grosser material is removed, a variable
quantity of " dirt " passes through the ordinary strainers

used, and appears as a sediment covering the bottom of

300

DIRT IN MILK

301

the vessels in which the milk is allowed to stand for a
few hours. It may be more quickly collected, and its
amount approximately estimated by means of a centrifuge.

?

5oo(^3c c

y

I

\

/

Ex. 143. — Estimation of the sediment in milk.

Take a carefully mixed
sample of the milk to
be examined ; not less I

than a litre should be
used. Add 4 c.c. of
formalin to it to prevent
the growth of bacteria,
and pour the milk into
the apparatus (Fig. 48)
up to 500 c.c. or the
litre mark. Cover the
opening of the cylinder
with a glass plate, and
allow the whole to stand
for twelve hours. Push
the rubber plug at the
end of the brass rod
through the milk to the
bottom of the cylinder,
so as to block the outlet
at the bottom. Then
remove the measuring
tube and centrifuge at
two thousand revolu-
tions for three minutes. Remove the milk to just above the
graduated portion of the tube with a pipette, and fill up to the
10 c.c. mark with a .1 per cent, solution of sodium carbonate;
centrifuge again for two minutes.

Read off the volume of the deposit obtained, and calculate the
number of volumes of sediment in i million volumes of milk.

Fig. 48.— Apparatus for estimation of sediment
in milk.

302 THE EXAMINATION OF MILK

2. Blood ; Leucocytes ; " Pus-cells." — Red blood
corpuscles, and white corpuscles or leucocytes and " pus-
cells " are not uncommon in milk.

The presence of blood may arise through temporary
rupture of a small blood vessel in the udder, or may
come from more serious lesions connected with disease
of the gland. The red corpuscles in such cases may be
sufficient in number to colour the milk. When present
they may be easily recognized on microscopic examina-
tion of the sediment thrown down in the milk after it has
stood for a time. They are circular bi-concave disks,
without nuclei, from 6 to 8 /a in diameter, and of pale
yellowish tinge when viewed singly.

On account of their form, light is unequally transmitted
through them, the centre appearing brighter or darker
than the surrounding border, according as the focus of
the microscope is altered. When abundant they may be
seen piled closely together in " rouleaux " like coins.

The colourless corpuscles or leucocytes are larger
spherical protoplasmic structures, over lo /x in diameter
present in the blood and lymph of animals. They con-
sist of granular protoplasm and contain one or more
nuclei : in a living state they exhibit amoeboid move-
ment their outline constantly changing in form. Under
certain conditions many of the leucocytes pass through
the walls of the capillary blood vessels into the sur-
rounding tissues; this is especially the case at points
where there is inflammation. The pus or " matter "
which accumulates in wounded or inflamed areas con-
sists largely of dead, altered, or degenerate leucocytes, to
which the term " pus-cells " has been applied.

The exact significance of leucocytes in milk is a
matter upon- which there is much difference of opinion.

blood; leucocytes; ''pus-cells*' 303

" Pus-cells " cannot be distinguished at all times with
certainty from normal leucocytes unconnected with
inflammatory or diseased conditions. While it is certain
that in the case of mastitis and other diseases of the
udder, the number of leucocytes in the milk increases
very considerably, it is not possible to fix upon a
standard based upon number per cubic centimetre which
shall be indicative of pus, since in milk from normal
healthy animals the quantity is found to be subject to
great fluctuation.

Usually the presence of many leucocytes accompanied
by streptococci has been taken to denote suppuration
or unhealthy inflammatory state of the udder, and milk
in which both are found in abundance should be con-
demned as unfit for domestic use. The nuclei stain
easily with methylene blue and are readily observed in
stained cover-glass " films " prepared from sediment or
centrifuge deposit obtained from milk containing them.

Ex. 144. — Examine microscopically the deposit obtained in
the preceding Experiment.

First pipette off the sodium car-
bonate solution : then fill up with
distilled water and centrifuge.
Pipette off the water to near the
top of the deposit. Stir up the
latter with a platinum wire ; remove
a drop with a platinum loop and
spread it as evenly as possible over
a f inch square cover slip.

Allow it to dry in a place free ''™.„t7f''„;S^wTh^?'-•'^SJ.*'f•
from dust and then immerse the chains,

cover in ether for three minutes : transfer' to absolute alcohol for
a minute, and after drying stain for ten seconds with Loffler's
methylene blue.

304 THE EXAMINATION OF MILK

Wash, dry and mount in Canada Balsam.

{a) Examine with a \ in. objective and No. 4 eye piece and
count the number of leucocytes in ten separate fields (Fig. 49) ;
calculate the average.

(b) Note the number and forms of bacteria present. Are
chains of streptococci abundant ?

{c) Prepare another film and after treatment with ether and
alcohol, examine for presence of acid-fast organisms (see p. 325).
Stain for several minutes with hot carbol-fuchsin, wash and de-
colourize with 25 per cent, sulphuric acid and counterstain for
ten seconds with methylene blue.

Wash, dry, and mount in Canada Balsam, and examine with
t\ in. objective.

3. Bacteria : number and kind. — In addition to
blood, pus-cells and inert substances, the milk contains
various kinds of living bacteria, some of them derived
from the interior of the udder, others being introduced in
the particles of dung, fodder and other materials already-
enumerated.

The number of bacteria present at the time of delivery
to the consumer is partly controlled by the amount of
the initial contamination but is also dependent upon the
temperature at which the milk has been kept, and the
time which has elapsed since it was first drawn from
the cow.

The fixation of a standard based upon the number
of organisms is therefore difficult, and is of less moment
than the examination of milk in regard to the kinds
which it contains. Some of them render milk acid very
rapidly, or otherwise alter it so that it soon becomes
unfit for domestic use : others may set up decompositions
which result in the formation of poisonous compounds
or cause disease. An abundance of Streptococcus lacticus

BACTERIA : NUMBER AND KIND 305

would be less harmful than a similar number of ^. coli
or other organisms derived from dung, putrescent
substances or sewage-polluted water.

The bacteria of the greatest importance from a
hygienic point of view are the pathogenic species and
those which indicate contamination with dirty water,
sewage, faeces and similar objectionable materials. The
examination of milk for the germs of disease is beyond the
scope of this work. The exact and certain determination
of the pathogenic character of many of them involves
inoculation experiments upon animals, and no other
method will give really reliable evidence of the presence
or absence of B. tuberculosis.

(i) Acid-fast Bacteria. — It is however advisable to
test milk for acid-fast organisms in the manner indicated
in Ex. 150, and if found the further examination in
other ways may be undertaken by the proper authorities
if necessary.

(ii) B. enteritidis sporogenes. — As mentioned later
(p. 330), Klein and others believe that Bacillus enteri-
tidis sporogenes is responsible for epidemics of diarrhoea
among children, but the matter is still in dispute.

Apart from this aspect, tests for Bs. enteritidis are
useful, since it is a very common inhabitant of animal
faeces and its presence may be taken as an indication
of pollution for this source. According to Orr's investi-
gation, it would seem that the farmer and not the
dairyman is chiefly responsible for its occurrence in milk.
As it does not multiply in milk except under anaerobic
conditions its occurrence in i c.c. or less of any sample
may be taken to denote unclean conditions at the farm.

Ex. 145. — Examine samples of milk for the presence of Bacil-
lus enteritidis sporogenes by the method described in Ex. 151.

U

3o6

THE EXAMINATION OF MILK

(iii) Bacterium coli communis and allied organisms.

— The occurrence of Bact. coli in milk is one of the most
important indications of pollution by manure, sewage or
dirty water contaminated with faecal matter.

The characters of the bacterium are given on p. 1 06.

Besides true typical Bact. coli there are a number of
very closely allied organisms commonly met with in
faeces and water which is polluted by animal excreta.
They are spoken of as " coliform " bacteria, and are
regarded as more or less permanent modifications or
varieties of Bact. coli, differing only from the type in
certain biological characters such as the power of curdling
milk, fermentative action on various carbohydrates and
production of indole.

For all of these forms a search should be made in
milk : the following table, based on the admirable
researches of MacConkey is of great value in diagnosing
any organisms of this class which may be isolated.

CHARACTERS OF "COLIFORM" OROANISMS.

1
+

§

s

0
+

i

+

1 jj
1

"a
Q

«5

1

<

1

Bact. Griinthal

_

-

—

+

—

Bact. acidi lactici (Hiippe)

-

+

+

-

+

+

-

Bact. coli communis ....

+

+

+

-

+

-

+

~

Bact. neapolitanus ....

-

+

+

+

+

-

+

-

Bact. oxytocus perniciosus . .

-

+

+

+

+

+

+

+

Bact. lactis aerogenes . . .

-

+

+

+

-

+

-

+

Bact. cloaccc ......

+

+

+

+

-

-

-

+

Bact. coscoroba

-

+ 1

1

+

+

i

-

+

-

BACTERIA : NUMBER AND KIND 307

They all ferment glucose and lactose with the produc-
tion of acid and gas ; they acidify and throw down
casein in milk in three or four days ; none of them
liquefy gelatine except Bad. cloacce whjch induces lique-
faction only when the cultures are kept for some time
(four to thirty days).

Ex. 146. — To determine if acid and gas-producing organisms
are in a sample of milk.

Prepare the following medium : —

MacConkey's Bile-salt Peptone Water.
Sodium taurocholate (bile salt) . . 2.5 gr.
Glucose . . . . . . 2.5 gr.

Peptone . . . . . . lo.o gr.

Water 500 c.c.

Heat gently to assist solution of ingredients ; filter, and then
add sufficient amount of boiled Kubel-Tiemann's neutral litmus
solution to colour the medium.

Pour it into Durham's fermentation tubes, lo c.c. in each, and
steam for twenty minutes on three successive days.

Inoculate some of these tubes with i c.c. of the sample of
milk to be tested for acid and gas producing organisms ; also
inoculate with i c.c. of the milk diluted to yy, ~^, yoVo, ro Joo"*

Incubate at 37° C. for forty-eight hours.
Note in which dilutions acid and gas are produced.
Ex. 147. — Separation and identification of glucose- and
lactose-fermenting (coliform) organisms in milk.

Prepare the following medium : —

MacConkeys Bile-salt Lactose Agar.

Sodium taurocholate . . . . 2.5 gr.

Peptone . . . . . . 10 gr.

Agar 7.5 gr.

Water 500 c.c

3o8 THE EXAMINATION OF MILK

Heat in water bath for one hour, so as to dissolve the con-
stituents and when cooled to 50° C. add the white of an egg, and
heat again in water bath for ij hours; then filter. To the
filtrate add : —

per 100 c.c. of
the medium.

I gr, of lactose

I c.c. of a .5 per cent, solution of

neutral-red
I c.c. of a .1 per cent, solution of

" krystal-violet "

Pour the medium into sterile test-tubes, 10 c.c. in each, and
sterilize by heating twenty minutes on three successive days.

Melt some of the tubes in hot water, and pour the contents
into sterile Petri dishes.

After the medium is set, place the dishes in the incubator
with the covers slightly open at- one side, so that the surface
may dry.

Then take a loopful of the cultures of the diluted milk of
Ex. 146, in which both gas and acid production are observed,
and mix it with 5 or 10 c.c. of plain sterile broth; from this
mixture take out a loopful of the liquid and deposit it on the
surface of the bile-salt agar in a Petri dish ; spread it over the
medium by means of a sterile glass rod, bent at right angles. *

Incubate at 37° C. for forty hours.

Make sub-cultures of the different colonies observed upon
ordinary agar slopes and incubate for twenty-four hours.

(i) Find if the organisms isolated are able to ferment
glucose, lactose, saccharose, dulcite, and adonite by adding a
loopful of the culture from the agar slope to Durham's fermenta-
tion tubes, containing the following broth : —

Lemco ...... 10 gr.

Peptone 10 gr.

Sodium bicarbonate .... i gr.

Water 1000 c.c.

to which has been added i per cent, of the several carbo-

MILK STANDARDS 309

hydrates, and a small amount of boiled Kubel-Tiemann's litmus
solution sufficient to colour the medium a violet tinge.

(ii) Apply the indole test to each (see Ex. 6;^).

(iii) Cultivate each in glucose bouillon and determine which
gives the Voges-Proskauer reaction (see Ex. 65).

(iv) Cultivate each kind of organism in plain broth at 37' C,
and, after six hours, examine a drop and determine which of
them are motile.

(v) To which species does each belong ; compare the results
with the table on p. 306.

Ex. 148. — Determine the relative percentage of liquefying
to non-liquefying kinds in several samples of ordinary market
milk.

Dilute the samples to i in 10,000 if twelve hours old; to i in
100,000 if twenty-four hours old.

Inoculate two or three litmus gelatine plates with .5 c.c. of
each, and incubate them at 22° C.

Observe the colonies from day to day ; count the " liquefiers "
and "non-liquefiers," and calculate the relative percentage
of each.

Milk containing from 30 to 50 per cent, of liquefying
organisms is suspicious and likely to have been contaminated
with filth or dirty water.

Ex. 149. — Test the acidity of market samples of milk by
the method described in Ex. 134.

How many fail to reach Newman's standard? (See p. 311.)

(100 c.c. of the milk should not require more than 22 c.c. of
decinormal caustic soda solution for neutralization.)

4. Milk Standards. — From time to time various pro-
posals have been made, chiefly at the instigation of the
medical profession, for the adoption of standards of
cleanliness, acidity, and bacteriological characters for
all milk supplied to the public. In some countries such
standards have been and are more or less rigidly enforced,

310 THE EXAMINATION OF MILK

along with regulations framed to secure the sale of milk
only from healthy cows, kept and milked under hygienic
conditions. In the British Isles at present the matter is
left largely to voluntary effort, and until the public can
be educated to demand a sounder, cleaner milk, there
will be no general improvement in the purity of this
important article of diet.

Many investigations have been made to determine the
amount of dirt, the acidity, and the number and kinds of
bacteria in milk as it is actually delivered by the farmers,
dairymen, and others who sell it ; these researches are
useful in giving a clue to the standard which might very
well be adopted without hardship to the farmer or retailer
of milk.

The imposition of certain regulations for the inspection
of cows and cowsheds, and the fixation of legal standards
of purity, all of which would be in the interests of public
health, would undoubtedly involve expense to the pro-
ducer and distributer, and this would have to be met by
the payment of a somewhat higher price for milk than at
present.

For farmers, dairy companies, and others who are
desirous of supplying their customers with clean milk,
the following standards should be kept in view : —

(i) Dirt. — As estimated by the method described in
Ex. 143, market samples of milk have been found to
contain from about 5 to over 120 volumes of dirt per
million of milk. Most of the impurity is introduced at
the farm, comparatively little being added during the
journey to the consumer. From the work of Houston
and Orr it would appear that the enactment of a regula-
tion that in any sample taken at the farm in this country
there should not be more than 30 or 40 volumes of

MILK STANDARDS 311

dirt per million of milk, would be a generous standard,
and one which would not entail unnecessary and burden-
some attention to cleanliness on the part of the farmer.

(ii) Number of bacteria and teicperature standard.
--Milk produced under clean conditions, if cooled to
10° C. or lower as soon as possible after it leaves the
cowshed, and maintained at that temperature, need not
contain more than 10,000 or 12,000 bacteria per c.c. in
summer, or half this number in winter ; some dairies are
able to supply milk of this quality to their customers all
the year round.

Park suggested that milk containing more than
100,000 per c.c. should not be sold for domestic use,
but this standard is easily exceeded in districts where
ice or other means of refrigeration are not available,
especially in the warmer seasons of the year.

In Boston the standard of 500,000 per c.c. is enforced
upon retailers, and milk for sale must be stored at tem-
peratures not higher than 10° C.

In this country the number of bacteria in samples
taken at the cowshed should not exceed 50,000 per c.c.
Milk taken at the railway termini of large towns does not
usually contain more than 500,000 per c.c, unless the
weather is very warm, and the railway journey has been
prolonged in exposed vans and waggons.

(iii) Acidity. — Milk which has not been kept cool,
or which has been stored in unclean vessels in dirty
shops, especially if obtained from a badly kept farm,
soon becomes acid and more or less toxic from the
action of " coliform " organisms in it.

Newman proposed that no milk should be sold 100
c.c. of which required for neutralization more than 22
c.c. of a deci-normal solution of caustic soda.

312 THE EXAMINATION OF MILK

(iv) B. enteritidis sporogenes. — Milk found to con-
tain this organism in i c.c. or less should be regarded
with suspicion or condemned, especially if it is likely to
be used for the feeding of infants and young children.

(v) Glucose-fermenting bacteria. — Where one-tenth
of a cubic centimetre or less of a sample of milk, taken
at the farm^ is found to contain organisms which fer-
ment glucose with the production of acid and gas, it
should not be sold or used for domestic purposes.

CHAPTER XX.
SOUR-MILK BEVERAGES AND FOODS.

I. In the manufacture of most alcoholic liquors,
malt sugar or cane sugar are fermented by yeasts,
alcohol, along with a certain amount of carbon dioxide
gas and other substances being produced.

Lactose, or milk sugar, although a disaccharide, and
similar in chemical composition to the sugars just men-
tioned, is not so readily fermented. A few yeasts are,
however, known which are able to form alcohol and
carbon dioxide from it, either directly, after hydrolysis,
by their own " inverting " enzymes, or indirectly, after
it has been decomposed into glucose and galactose by
bacteria. Similar alcoholic fermentation can be accom-
plished by certain bacteria.

Generally, it would appear that the development of
lactic acid organisms checks the fermenting activity of
yeasts ; but a few examples are known of the produc-
tion of alcohol in milk soon after, or simultaneously
with, the formation of lactic acid, the one process no
doubt modifying the other.

In various parts of the world alcoholic beverages or

foods containing lactic acid and alcohol are made from

the milk of domestic animals by ferments, the chief

of these being Kefir, Koumiss, and Mazun. Joghurt, a

sour-milk product, containing little or no alcohol, must

also be noticed here.

313

314 SOUR-MILK BEVERAGES AND FOODS

2. Kefir. — The name Kefir is given to an acid alco-
holic beverage prepared from the milk of cattle, sheep,
or goats by inhabitants of the Caucasus.

In the manufacture of it, milk is poured into skin
bags or " bottles," and fermented by means of Kefir-
grains. After two or three days, during which time it
is shaken occasionally, it is ready for consumption, and
forms a refreshing, foaming drink which, according to
some authorities, possesses considerable medicinal and
nutritive properties.

The chief chemical changes occurring in the milk are
concerned with the milk-sugar. This constituent yields
lactic acid, alcohol, and carbon dioxide. Casein is pre-
cipitated by the acid, and on shaking the liquid breaks
up into "fine flocculent particles which are readily
digested.

The Kefir-grains, or active fermenting agents, in a
dry state, are small yellowish tough bodies, about the
size of large millet seeds or small peas. After soaking
in milk or water, they swell up and grow into gelatinous,
elastic convoluted structures which may become as large
as a walnut.

They are found to consist of a zoogloea of yeast-cells
and several kinds of bacteria which apparently carry on
a symbiotic existence.

Many kinds of organisms are usually present, mostly
as unavoidable impurities ; according to Freudenreich,
only three or four are essential for the characteristic
fermentation of Kefir, namely, a non-sporing yeast or
Torula {Torula kefir) met with chiefly in the rind of the
grains. Bacillus caucasicus^ and two forms of Streptococcus.

The yeast has roundish-oval cells measuring 3 to
5 /x by 2 to 3 /A, and produces a dark rose-coloured

KOUMISS 315

growth on potato. It is incapable of inverting or fer-
menting milk-sugar by itself, but manufactures alcohol
from the products of the hydrolysis of this sugar, which
arise through the activity of a Streptococcus b. The latter
is a small oval coccus which gives rise to lactic acid
and gas in milk without precipitation of the casein.
Streptococcus- a. is a larger species, resembling Str.
I adieus in many of its properties, but produces gas
when grown in milk.

Bacillus caucasicus, Beijk. {Dispora caucasica, Kern).
—One of the constant components of the interior of
Kefir-grains is a non-motile organism 5 to 6 //- long,
.4 to .5 <^ broad, frequently curved or bent, and often
having a spore-like granule at each end. It coagulates
milk, forming much lactic acid but no gas.

Recently, Nicolaiewa states that she was able to
produce good Kefir by the combined action of pure
cultures of this bacillus and the Torula ; the others she
considers are impurities, as also Torula ellipsoidea^
Oospora lactis, and Sarcina lutea, which are usually
found in Kefir.

3. Koumiss is another alcoholic beverage prepared
from milk. Its manufacture has been carried on from
time immemorial by the nomadic tribes inhabiting the
Steppes of Russia. These people make it from the milk
of mares, asses, and camels.

To start the fermentation in fresh milk, a small
amount of old Koumiss, or some of the dried sediment
from an old sample is added ; in some districts " barm,"
or " liquid yeast " from a brewery, is used for the same
purpose.

The organisms at work in Tartarian Koumiss are not
well known.

3i6 SOUR-MILK BEVERAGES AND FOODS

Some of the milk-sugar is fermented by a yeast,
alcohol and much carbon dioxide gas being produced ;
at the same time lactic acid is formed, and the casein
precipitated in a finely divided state. A portion of the
casein is broken down into albumoses.

A good sample is a foaming, frothy beverage con-
taining about I per cent, of lactic acid and not more
than 2 per cent, of alcohol.

A form of Koumiss is manufactured in Europe from
cow's milk by adding to it cane-sugar and beer-yeast,
and allowing it to ferment for two or three days. This
and similarly prepared beverages have been much recom-
mended for the alleviation and cure of consumption,
diabetes, and anaemia.

4. Mazun. — Mazun is a kind of coagulated fermented
product made in Armenia and neighbouring countries
from the milk of the buffalo, sheep, and goat, and
occasionally from cow's milk. The milk to be fermented
is boiled and when cooled down to about blood-heat a
small amount of old Mazun is added ; and the whole
allowed to ferment for twelve to thirty-six hours.

The soured coagulum obtained is of variable consist-
ency being thickest when buffalo's milk is used and
thinnest when made from cow's milk.

It is eaten as a sort of junket, or can be mixed with
water to form a piquant aromatic drink containing lactic
acid and a small amount of alcohol.

The peculiar fermentation of Mazun, according to
Weigmann, Griiber, and Hess, is brought about by the
combined action of a yeast and two varieties of bacteria.

The yeast, a variety of Saccharomyces pastortanus, is
able to ferment milk-sugar, and at the same time gives
rise to a pleasant aroma.

JOGHURT AND LACTOBACILLINE 317

The two bacteria to whose action lactic acid is due
are, Bacterium mazun and Bacillus mazun^ the former a
non-motile organism producing much acid and a dense
curd, the latter a slightly motile spore-bearing bacillus,
which rapidly peptonizes casein when cultivated by itself
in sterilized milk.

Mazun can be used for souring cream for the manu-
facture of butter, and a kind of cheese is sometimes made
fronj the curd which it throws down in milk.

5. Joghurt and Lactobacilline. — Joghurt (pronounced
Yoh-oort) is a form of acid-coagulated milk used exten-
sively in Turkey, Bulgaria, and the Northern Balkans.

The milk of the buffalo, cow, sheep, or goat is thoroughly
boiled for a time, and after cooling to 45° C. is inoculated
with an old sample of Joghurt or some of the " ferment "
obtained from it, which is named " Maya." It is then
kept at a temperature of about 40° C. for nine to sixteen
hours when the fermentation is complete.

The thick coagulated milk which has an agreeably
acid taste and a pleasant aromatic odour, is then eaten
either alone or with bread, rice, or other foods.

Genuine Joghurt, obtained from districts where it is in
daily use, usually contains a considerable number of
different bacteria, yeasts, and moulds, many of them being
unavoidable impurities.

The characteristic organism xsBacillus ifulg-aricuSylieupel,
which according to Luerssen and Kiihn is frequently accom-
panied by two other bacteria briefly described below.

In the fermentation lactic acid is formed in abundance
with little or no alcohol, on which latter account this pro-
duct appears to be more beneficial than Kefir or Mazun.

During the last few years a great deal of attention
has been paid to this and similar acid-milk pro-

3i8 SOUR-MILK BEVERAGES AND FOODS

ducts on account of their supposed dietetic and
remedial value.

Several hardy, long-lived races in different parts of
Eastern Europe and Western Asia consume considerable
quantities of fermented milk, and it has been assumed
that their freedom from disease and longevity may be
directly connected with the use of this article of diet.
In the intestinal tract of man, especially in the colon,
putrefactive bacteria are abundant. Here they carry
on their work of decomposition and give rise to indole,
skatole and other foul-smelling, poisonous compounds.
According to Metschnikoff these are absorbed- into the
system and bring about premature old age.

With a view of checking or reducing such intestinal
auto-intoxication and changing the bacterial flora of the
colon, he has recommended the introduction of the lactic
acid organisms through the medium of sour milk.

As it is not safe to consume spontaneously-soured
samples on account of the possible contamination with
disease germs, pure cultures of
suitable lactic bacteria in sterilized
or pasteurized milk are used
instead.

The chief organism employed

for this purpose is Bacillus buU

garicus, Heupel, which has been

isolated from Joghurt or Maya by

Massol, Grigoroff, Luerssen, Kuhn,

and others (Fig. 50).

Pure cultures are on the market in the form of tabloids

and powders, and sold under various names, such as

" Lactobacilline," "Joghurt powder," and " Maya ferment."

Attempts have also been made to introduce them into

chocolate and other bon-bons.

Fig. 50.— The Joghurt bacillus
{^Bacillus bulgaricus, Heupel).

JOGHURT AND LACTOBACILLINE 319

Much care, however is needed in the preparation of
pure cultures on a commercial scale, and many of these
products, for which absurdly exaggerated claims have
been made, are found on examination to be impure, or to
contain few of the right organisms in a living condition.

The Joghurt bacillus {Bacillus bulgaricus, Heupel), is a long,
slender, rod-shaped bacillus, rounded at the end, occurring
singly or in chains and threads. Single cells are usually about
3 to 10 /A long, and i to 1.25 [m broad. It stains by Gram's
method, is aerobic or anaerobic, and difficult to cultivate. It
grows best at 45° to 50° C., little or no growth at 20° C., slow
at 25° C., fair at 30° C.

Whey gelatine. — Stab, filiform, beaded, not liquefied, and no
surface growth.

Whey agar. — Colonies roundish or irregular, greyish, .5 to
1.5 in diameter, with filamentous curly margins.

Potato. — No growth.

Milk is rendered acid and coagulated in eight to eighteen
hours at 44° C. There is no gas formation, and the casein is
not peptonized.

This organism is remarkable in being able to produce large
amounts of lactic acid in milk, some forms giving rise to over
3 per cent, in ten days when incubated at 35 to 36° C.

The Granular bacillus of Jog hurt is a longer organism, which
stains irregularly with Loffler's methylene blue. Its surface
colonies on sugar agar media are transparent and irregular ; deep
colonies, curly or radiate. It grows best at 37 to 45" C.

On potato no visible growth occurs.

Milk is coagulated and becomes acid very rapidly at 37 to
40° C. ; no gas formation.

The Streptococcus of Joghurt occurs usually in pairs, which stain
readily with Gram's gentian violet. It grows only on milk agar,
in small, yellowish-white, round colonies. Milk is made acid
and coagulated.

CHAPTER XXI.

MILK AND DISEASE.

I. A GREAT many of the epidemic diseases to which
mankind is subject have been spread by means of water
and milk. Fortunately, during the last century vast
improvements have taken place in the selection and
management of the public water supply : purer sources
have been tapped, and better regulations framed and
carried out in regard to the storage and distribution of
water for domestic purposes ; the disposal of sewage has
been supervised, and contamination of drinking water by
disease germs has been very greatly reduced.

A different picture is presented to view when the
question of the relationship of the milk-supply to disease
is examined,* and much remains to be done, both by the
farmer and the general public, before the standard of
purity and safety is as high for milk as for water.

Milk is one of the most important of all foods, especi-
ally for the young, and, unlike most of the materials
consumed, is very largely taken into the system in a raw,
uncooked, and unsterilized state.

Moreover, it is a liquid which is eminently adapted for
the nutrition of many kinds of bacteria, and disease pro-
ducing organisms which gain access to it, grow and
multiply in it at an enormous rate. Such infected
milk has not infrequently spread illness and death over

a wide area in a short time.

320

TUBERCULOSIS 321

Milk-borne diseases may be divided into two groups,
namely : —

( 1 ) Those which attack both the cow and man, such as
tuberculosis, foot and mouth disease, and possibly other
illnesses ; and

(2) Those which are rarely or never met with among
dairy stock, but whose germs find their way into the
milk after it is drawn : to this class belong typhoid fever,
diphtheria, scarlet fever, and certain obscure throat
troubles, diarrhoea, and intestinal ailments.

2. Tuberculosis. — One of the great scourges of the
human race is tuberculosis. The number of deaths per
annum in the British Islands due to various forms of the
disease is over 60,000, and probably more than ten times
this number suffer from it.

The disease is also found extensively distributed
among domestic animals, being specially prevalent in
cattle and pigs ; less of the trouble is met with in horses,
sheep, and goats. Cats and dogs are subject to it, and
a form of the disease is common among poultry.

There are from 2 J to 3 millions of cows and heifers
in Great Britain ; about 25 to 30 per cent. (600,000 to
800,000) are tuberculous ; according to M'Fadyean
about 2 per cent, of them, representing a total of over
50,000 cows, have the disease in the udder, and yield
milk containing the virulent germs of the disease.

In Islington (London) Dr Harris found, in 1899, over
14 per cent of the samples of milk examined contained
the tubercle bacillus ; from 28 to 38 per cent, of the
milk examined in Berlin in 1897 by two different
observers was found to be infected, and in Paris 33 per
cent, of the samples gave similar results. The un-
doubted presence of these bacilli in the milk supplied to

322 MILK AND DISEASE

the public makes the question of the transmissibihty of
the disease to mankind through the consumption of this
article of diet a matter of first-rate importance.

A great amount of research has been made into the
relationship between bovine and human tuberculosis, the
mode of infection of animals and man, and the morpho-
logical and physiological variations of the bacilli derived
from various sources ; many of the points raised in the
investigation of these problems are still unsettled.

Koch, the discoverer of the bacillus, put forward, in
1 90 1, the view that the organisms responsible for human
and bovine forms of the disease are distinct, at any rate
in infective power, and maintained that the bacilli of
tuberculosis in cattle are very rarely, if ever, responsible
for the disease in man.

Of course definite experimental proof by inoculation
is out of the question, but the Royal Commission ap-
pointed to investigate the problem, although its final
conclusions are not yet forthcoming, has obtained suffi-
cient reliable evidence to confute Koch's statement.

It has been found that the bacilli derived from bovine
tuberculosis infect calves, pigs, cats, dogs, the Rhesus
monkey, and the chimpanzee, when inoculated into the
system or supplied to the animals in food ; they are
therefore not specially adapted to attack cattle only.
The experiments have also shown that the bacilli derived
from human sources are able to produce the disease when
given in the food or introduced by inoculation into calves,
rabbits, monkeys, and apes.

The Commission has already reported that : " There
can be no doubt that in a certain number of cases the
tuberculosis occurring in the human subject, especially in
children, is the direct result of the introduction of the

TUBERCULOSIS 323

bacillus of bovine tuberculosis, and that in the majority
of these cases the disease is introduced through cows'
milk."

The dung and other excreta of tuberculous animals
frequently contain the bacilli of the disease, and in a dried
form is found floating in the air of cowsheds, barns,
and other farm buildings. Infection among cattle is
accomplished through the inhalation of dust of the infected
premises or of bacilli coughed into the air by diseased
animals, and also through the feeding of calves with milk
containing the organisms.

Jn Denmark and some other countries the separated
milk sent back from creameries to the farms for the
feeding of calves and pigs must first be pasteurized, so
as to destroy any virulent bacteria in it ; without this
precaution the milk from infected cattle on one farm
may be carried to another which is free from the disease.

Tuberculosis is spread among human beings chiefly,
perhaps, by organisms coughed into the air by con-
sumptives and the bacilli of dried sputum, which rise
with dust into the air and are breathed into the lungs.
Fliigge and other authorities consider that the disease is
conveyed to children mainly through the digestive tract
in milk and other food containing the bacteria, some
holding the view that tuberculosis of the lungs or
phthisis frequently arises from organisms entering the
system in this way.

The tubercle bacillus gains an entrance into milk
chiefly from cattle with tuberculous udders, but infected
milk may be given by cattle in which the disease cannot
be detected in the milk glands. It also enters in the
form of cowshed or stable dust, consisting of particles of
dung and other excreta from diseased animals. Possibly

324 MILK AND DISEASE

consumptive milkers may sometimes be responsible for
the infection of the milk.

It is only to be expected that butter, cheese, and
other dairy products prepared from unsterilized milk or
cream will not infrequently contain tubercle bacillus :
such has been found to be the case by many different
workers. Moreover, the organism may live in milk for
several months, and in cheese for a year or more.

To avoid danger from these sources it is advisable,
wherever possible, to manufacture dairy products only
from milk or cream which 'have been pasteurized.

Bacillus tuberculosis (R. Koch), is a very variable organism.
It is non-motile, usually rod-shaped, from 3 to 5 /x long, .5 //< broad,
sometimes curved, and in old cultures frequently appears in the
form of branching threads.

In stained specimens a number of unstained vacuole-like
portions are usually seen.

It stains easily with carbol-fuchsin and the stain is " acid-fast "
— i.e. it is not decolourized by 25 per cent, sulphuric or 30 per
cent, nitric acids : it also stains by Gram's method.

The bacillus is aerobic, and thrives best at 37° C. Its
thermal death-point is of great importance in regard to the destruc-
tion of living organisms in milk by the process of pasteurization.
Many experiments have shown that temperatures of 60° C.
maintained for twenty minutes, 65° C. for fifteen minutes, or
70° C. for ten minutes, are usually sufficient to destroy it, but
Bang considers that nothing less than 85° C, kept up for five
or ten minutes, suffices for absolutely reliable results.

Direct sunlight destroys the bacillus very soon, and diffuse
daylight acts in a similar way but more slowly.

A 5 per cent, solution of carbolic acid kills it in thirty to
sixty seconds, absolute alcohol in five minutes ^nd a i in
1000 solution of mercuric chloride in ten minutes.

TUBERCULOSIS 325

Growth in artificial media is always slow, and fortunately little
or no growth occurs in milk.

Glycerin Agar. — Bs. tuberculosis grows scantily or not at all
on ordinary agar or gelatine. On glycerine agar the growth in
ten to twelve days is small and crumbly ; later it extends, and
in six or eight weeks may form a wrinkled lichen-like patch,
whitish-yellow or brownish-yellow in colour.

Potato. — It grows in four or five weeks on potato placed in
a Roux's tube and kept moist with five per cent, glycerine ; the
growth is dull and yellowish.

Veal broth containing 5 or 6 per cent, of glycerine is a good
medium for it. Small cream-coloured flocculent masses are
produced in the liquid with surface film-formation occasionally.

£x. 150. — Examine a sample of milk for ''acid-fast" organisms.

{a) Half fill two centrifuge tubes with milk and rotate for five
minutes at 2000 revolutions per minute.

{b) Break up the cream and centrifuge again.

{c) Remove the cream and pipette oif the milk to just above
the deposit. Add water so as to half fill the tubes and rotate
for five minutes.

{d) Pipette off the water, and transfer a drop of the liquid and
the deposit with a platinum loop to a cover-slip. When air-dry
pass through flame.

{e) Determine if any " acid-fast " bacteria are present. To do
this take the cover-slip and place upon it 2 or 3 drops of weak
carbol-fuchsin (see Ex. 8). Gently heat to steaming - point
(60° C.) over flame for three minutes with aid of Cornet forceps,
adding fresh stain to supply the place of that which evaporates.
If preferred, the films may be heated while staining over an
asbestos board or copper plate, or floated in a watch-glass
containing the stain.

(/) Wash well in water and then immerse in a 25 per cent,
solution of sulphuric acid for twenty seconds : wash in water,
and if pink colour comes out repeat the treatment with acid and

326 MILK AND DISEASE

water alternately until no more colour washes out when placed
in the latter.

(g) After decolourization stain for one minute with Loffler's
methylene blue and wash well : dry and mount in Canada
balsam.

(/i) Examine with J and -^\ in. objective : the acid-fast organisms
and spores of certain species which may possibly be present
appear red, other bacteria, pus and mucus are blue.

Methods of checking or stamping out bovine tuber-
culosis.— It is outside our province to discuss the
question of the methods to be adopted to deal with
human tuberculosis ; the farmer, however, should en-
deavour to stamp out the disease from his herds and
make an effort to prevent tuberculous, milk from being
supplied to his patrons.

(i) All animals suffering from disease of tlie udder, and
" wasters " in an advanced state of disease, should be
destroyed immediately in order to prevent them from
infecting the healthy stock.

(ii) Much good can be done by isolation and sub-
sequent elimination of all animals which give evidence
of tuberculosis when tested with " tuberculin " : the test
should be applied at intervals for some considerable
period.

Two kinds of tuberculin are in use, namely : ( i ) the
old^ a filtered extract from a glycerine veal-broth culture
of Bs. tuberculosis ; (2) the new, a centrifuged extract of
a dried and thoroughly pulverized culture.

When injected in very small quantities into the blood
of an animal suffering from tuberculosis, even in a
clinically unrecognizable form, there is a very marked
rise of 1.5 to 2.5° C. in twelve to fifteen hours.

TYPHOID FEVER 327

Healthy animals when treated with tuberculin are not
affected by it.

(iii) Milk from suspected animals, or those reacting to
tuberculin, should be carefully pasteurized or sterilized
before being used for the feeding of calves or other farm
stock, for the tubercle bacillus is sometimes present in
milk from cows whose udders are apparently free from
the disease ; and where tuberculosis is present in the
system it may develop and involve the udder at a
rapid rate.

Rabinowitsch, Kempner, and Mohler showed that
from 20 to 50 per cent, of the cows which reacted to
tuberculin gave milk containing virulent bacilli capable
of setting up tuberculosis when inoculated into animals,
although the reacting cows showed no signs of udder
disease.

(iv) Separated milk from creameries and dairies
generally should be pasteurized before being used.

(v) Only animals which do not react with tuberculin
should be retained for breeding purposes.

(vi) Good ventilation and adequate lighting of the
buildings in which cattle are housed tend to reduce the
disease ; damp, dark, badly ventilated sheds reduce the
vitality of the animals and render them less resistant to
the attacks of the tubercle bacillus.

3. Foot and moutli disease of cattle may be con-
veyed, especially to infants and young children, by
infected milk, but occurrences of the disease among
human beings are comparatively rare.

4. Typhoid fever. — Many epidemics of this dangerous
illness have been traced to the consumption of milk
containing the typhoid bacillus {Bacillus typhosus). The
disease does not attack cattle, but the entry of the

328 MILK AND DISEASE

organisms responsible for it takes place through con-
taminated water used at the farm or dairy.

The bacilli, which very closely resemble Bad. colt,
are found in the blood and also in vast numbers in the
faeces and urine of typhoid patients. Even after
recovery in some cases virulent organisms continue to
be discharged in the excreta for months or years, and to
such apparently healthy typhoid carriers are attributed
the mysterious outbreaks and recurrences of the disease
which are recorded from time to time.

At cottages, farms, and dairies wherever the sanitary
arrangements are unsatisfactory, and the thorough disin-
fection of excreta from typhoid patients is not carried
out, there is great danger of the wells or other sources
of the water supply becoming contaminated with living
typhoid germs.

The use of such polluted water for the washing of churns,
milk-pails and other dairy utensils is the chief means
of introducing the bacilli into milk, and numbers of
outbreaks of the disease have been proved to originate
in this way.

There is evidence also to show that flies after settling
upon faeces containing typhoid organisms often carry
upon their head, feet, and wings the germs of the
disease, and transfer them to milk and food to which
they have access.

Dry infected excreta may blow about as dust and find
its way into milk.

B. typhosus thrives and multiplies in milk for many
days, but its development is considerably checked by the
lactic organisms ; Bassenge states that an acidity of .4
or .5 per cent, destroys the bacillus in twenty-four
hours.

EPIDEMIC SUMMER DIARRHCEA 329

Pasteurized and boiled milk constitute good media
for the growth of the bacillus, and when introduced into
these, the organism, having a field free from competition,
grows rapidly and remains virulent for a month or more.

It is, therefore, very necessary to use clean vessels for
storing pasteurized or boiled milk, and cover them so as
to prevent access of flies and dust, which might reinfect
the milk.

In cream the typhoid bacteria become more abundant
than in milk, especially if the acidity is abnormally low,
and butter prepared from it is infective.

The time during which they remain in a living or virulent
condition in butter appears to depend on the acidity of
the cream from which it is made ; in ordinary acid-cream
butter they probably die out in a week or ten days.

5. Epidemic Summer Diarrhoea is one of the most
fatal ailments to which infants are subject, the death-
rate among children up to five years of age from this
disease alone amounting generally to over i o per 1 000 ;
its elimination would very materially alter the vital
statistics of young children.

The fatality from this disease is highest in the
warmest part of the year, namely, in the months of July
and August, and is especially prevalent among children
of the working classes in urban areas.

Many investigations have shown that it is essentially
a trouble connected with the use of cows' milk, hand-
reared infants being most affected by it.

The disease is apparently due to poisonous compounds
in the milk, manufactured by swarms of bacteria introduced
from dirty surroundings, the production of the deleterious
substances taking place either before the milk is taken
or after it enters the stomach and intestinal tract.

330 MILK AND DISEASE

Hitherto no single specific organism has been found
to which the illness can be attributed with certainty ;
possibly the bad effects are brought about by the com-
bined action of two or more kinds.

There is little doubt, however, that the responsible
organisms are introduced in dust and dirt.

Booker came to the conclusion that the species most
actively concerned with the disease are Proteus vulgaiHs,
and a Streptococcus, probably 5. pyogenes, although Klein
attributes the complaint to Bacillus enteritidis sporogenes,
a widely-distributed anaerobic organism found in soil,
sewage-polluted water, and faecal matter, and frequently
present in milk.

Careful cooling and scrupulous cleanliness in the
production and distribution of milk in the summer, along
with care in the use of clean vessels for storage of milk
at home, would very greatly reduce infant mortality
from this cause.

Bacillus enteritidis sporogenes, Klein. — This is a common
anaerobic spore-producing organism isolated originally from
human excreta and milk, the consumption of which had led to
an epidemic of diarrhoea. It is frequently found in soil and in
animal faeces.

The bacilli are 2.5 to 3.5 /«< long, .8 to 1.25 /a broad, some-
times motile, and readily stain by Gram's method. The spores
are oval, usually placed in the middle of the bacterial cell. It
very closely resembles Bacillus Welchii and the non-motile
butyric acid bacillus (p. 288).

Gelatine. — Surface colonies, semi-transparent, no liquefaction.
In stab-cultures the colonies are small and spherical, and many
gas bubbles are produced.

Agar. — Surface colonies, round, grey, flat, with thin lobed or
indented margins.

DIPHTHERIA AND SEPTIC SORE THROATS 331

Milk. — The changes in recently boiled milk, inoculated with
this organism and cultivated anaerobically, are characteristic.
Much gas is formed ; the casein is coagulated and the surface is
covered with pinkish scum enclosing gas bubbles. The whey
below is clear and colourless, has a strongly acid reaction and
rancid smell, butyric acid being produced.

Ex. 151. — Test samples of milk for B. enterittdis sporogenes : —
(f) Add 10 c.c. of the milk to a sterile tube; plug and heat
at 80° C. for 15 minutes. Place the tube in a wide
Buchner tube (Fig. 25) containing pyrogallic acid and
caustic potash ; seal so as to secure anaerobic conditions
and incubate at 37° C. for two days. Pinkish frothy
scum appears if B. enterittdis sporogenes is present,
(ii) Try the same experiment, using 20 c.c. of milk,
(iii) Try the same experiment, using i c.c. of the milk, adding
it to a tube of sterile milk.

6. Diphtheria and Septic Sore Throats. — Several
outbreaks of true diphtheria have been traced to the
milk-supply, and in the majority of instances there has been
definite evidence to show that the bacillus of the disease
found its way into the milk from human sources and did
not come from the cow. At the farm or dairy supplying
the milk one or more persons were suffering from diph-
theria or had only recently recovered from it. In many
cases of mild throat epidemics similar evidence of the
human infection of the milk is generally forthcoming.

There are, however, many instances where epidemic
" septic " sore throats of a more or less diphtheritic
character have been set up by milk given by cows
suffering from various obscure forms of mastitis or
inflammatory diseases of the udder

332 MILK AND DISEASE

The exact nature of these illnesses, their cause, and
the relationship to the milk-supply are still obscure.

7. Scarlet Fever. — From its infectious character and
the peculiar course of its development it is inferred that
scarlet fever is due to a minute, but still undiscovered,
organism. 'The disease is disseminated very readily
from one person to another, and by means of linen, bed-
clothes, books, letters, and other objects which have been
in contact with patients suffering from it. Without
doubt milk can carry the contagion, and a great many
epidemics of the disease have been spread by infected
milk. In most of these cases it has been found that the
milk received its disease-producing power from human
sources, either the convalescent milkers or some one
employed about the farm or dairy suffering from the
ailment supplying the virus.

Some authorities, however, entertain the view that
cows may suffer from some form of the disease, and
convey the contagion to the milk.

CHAPTER XXII.

CREAM AND CREAM RIPENING.

Cream. — When milk is placed in a shallow vessel
and allowed to stand for a time, the globules of fat,
which have a smaller specific gravity than the serum in
which they are distributed, rise to the surface of the
liquid and form a layer of variable thickness. This
layer of fat globules, with a certain amount of milk
serum, is known as cream ; it can be poured off or
removed in other ways, leaving behind skim-milk, a
product comparatively poor in fat.

In addition to this method of obtaining cream, which may
take several hours to complete, cream may be taken from
fresh milk by the centrifugal separator in a few minutes.

The composition of cream is very variable : thick or
thin cream can be obtained at will by means of the
separator, and the amount of serum left in it even when
raised by gravitation processes can be regulated to some
extent by " raising " the cream in shallow or deep
vessels and varying the temperature at which the milk
is kept. The comparative composition of cream, " sepa-
rated/' and fresh milk is shown below : —

Per cent.

^

Water.

Fat.

Proteins.

Milk-Sugar.

Ash.

Thin cream

. 64

28

3-25

4.5

•65

Thick „

• 44

45

3-05

2.7

.60

Separated milk

. 90

.1

3-9

4.9

.76

Skim-milk(shallowpans) . . .

I-I-5

...

...

Fresh milk

• 87

3-9

3-4

4-75

•75

333

334 CREAM AND CREAM RIPENING

The bacterial content of fresh cream obtained by
means of the separator is usually low, especially if the
milk has been recently drawn from the cow under clean
conditions. It is generally lower than that of the milk
from which it is derived, since many of the bacteria in the
milk are driven to the sides of the separator bowl, and
are collected in the protein " slime " along with any
other solid particles which may happen to be present.

Cream of this kind is sweet, bacteria having had no
time to develop or bring about any change in it. In
fresh cream raised in deep or shallow pans in twelve to
eighteen hours by gravitation, bacteria are found in
considerable numbers. The organisms occurring in the
milk when it is " set " soon multiply, and by the time
the cream is a day old the numbers present vary from
about 250,000 to over 6,000,000 per cubic centimetre.

Although sweet, freshly-separated cream is sometimes
churned and made into butter, the greatest amount of
butter is made from cream which has been kept from
thirty-six to forty-eight hours and allowed to become
sour either spontaneously or after the addition of special
cultures of bacteria. This " souring " process is in reality
a complex kind of fermentation termed " ripening^

In the ripening of cream certain chemical and physical
changes occur, and there is a very extensive multiplica-
tion of bacteria.

Cream properly ripened and ready for churning pos-
sesses a clean acid taste due to the lactic acid fermen-
tation ; the amount of acid allowed to develop before
churning should not exceed .5 or .65 percent, calculated
as lactic acid.

In addition to the acid taste, ripening results in tne
production of a characteristic taste or flavour of butter

CREAM 335

and a pleasant odour or aroma ; to what these latter
characters are due is not known with certainty. Since
it is. found that lactic bacteria alone cannot properly
ripen cream, it is probable that aromatic compounds of
an agreeable flavour are produced by the action of
bacteria allied to the putrefactive organisms upon the
proteins of the serum, these being absorbed by the fat
and transferred ultimately to the butter.

Very little, if any, chemical changes occur in the fat,
but its physical characters are altered by ripening and
ripened cream churns more quickly than sweet cream ;
it also gives up more of its fat in the form of butter, and
less of the fat remains in the buttermilk than when sweet
cream is used.

The exact nature of the change in the fat which
enables the globules to coalesce readily when shaken in
the churning process is not understood.

Some authorities have put forward the view that the
globules are surrounded by a film of protein, which is
dissolved or decomposed during ripening, thus allowing
easy amalgamation of one globule with another. It is,
however, more likely that the phenomenon is connected
with alterations in the viscosity and surface tension of
the serum.

Ex. 152. — Mount a drop of cream on a slide, and examine
with a f and J in. objective.

Compare the number and appearance of the fat globules with
those of a similarly mounted drop of skim-milk.

As previously mentioned, unripened cream contains
from :| to 6 millions of organisms per c.c. ; in the
ripened product from thirty-six to forty-eight hours old, at
which period the bacteria reach their maximum develop-

336 CREAM AND CREAM RIPENING

ment, from 500,000,000 to 1,500,000,000 are present ;
few other natural liquid media contain so many.

After reaching this point the numbers decline with the
age of the cream ; in four or five days a few millions
only are found, and at the end of a week nearly all
living organisms have disappeared except the fungus
Oospora lactis.

There is thus a periodic rise and fall in the number of
bacteria in cream similar to that which has been observed
in milk when it is kept.

In freshly-raised cream there is a miscellaneous collec-
tion of different kinds of bacteria. Liquefying varieties
are common, as well as others which have little apparent
action on milk or gelatine media ; lactic acid forms are
comparatively rare.

During the first twenty-four hours after the milk is
drawn from the cow the growth of all the kinds goes on
simultaneously, and large numbers are carried by the
upward stream of fat globules into the cream layer
after the milk has been " set " for a time. About 2 to
20 per cent are liquefiers ; 10 to 75 per cent, neither
produce acid nor liquefy gelatine ; the rest are lactic
acid varieties.

As ripening proceeds the latter multiply enormously,
until when the cream is thirty-six to forty-eight hours old
they form 95 to 99 per cent, of the many millions present ;
all other varieties decrease to very small proportions, or
die out altogether.

Of the lactic acid bacteria found in ripened cream,
St. lacticus and a nearly allied form are far the most
abundant. Bad. lactis aerogenes is also invariably met
with at all stages of the process, but is never found in
large numbers when ripening runs its normal course.

CREAM 337

Later the lactic forms vanish also, and only fungi
remain.

Ex. 153. — Make a careful study of (i) the numbers and
(2) the kinds of bacteria in —
(a) Freshly separated cream.

(d) Well-ripened cream, thirty-six to forty-eight hours old.
(c) Cream left in a covered vessel away from dust for ten
days.
Dilute and plate out on litmus lactose gelatine and agar media
as described in Exs. 125, 126.

What percentage produces acid or liquefies gelatine ?
Examine and prepare permanent stained slides of the various
colonies found.

Grow pure cultures of the different kinds of organisms on agar,
slants, and

(i) Test the action of each upon : —
{a) Milk.

(d) Nitrate bouillon.

(c) Bouillon containing the various carbohydrates men-
tioned in Ex. 147 (i).
(ii) Test their power of producing indole (Ex. 6^).
(iii) See if any give the Voges and Proskauer reaction (Ex. 65).
Classify and, as far as possible, name the organisms found,
comparing them with authentic examples.

The ripening process is one of the greatest importance
from the dairyman's point of view : when it goes wrong
the butter frequently turns out to be objectionable in
flavour and aroma, and of poor keeping quality. En-
deavour should, therefore, be made to control it, and
although want of knowledge of many of its chemical and
bacteriological details makes exact direction impossible,
much can be done in a general way to guide it.

Temperatures between 18.5° and 21° C. (65° to 70° F.)

Y

338 CREAM AND CREAM RIPENING

have a salutory influence upon it. Below about 18.5° C.
(65° F.) the beneficial lactic acid bacteria are checked,
and the organisms likely to give rise to bitter flavours
and ofiensive odour are assisted : above 21° C. (70° F.)
gas-producing forms often flourish, and the cream and
butter become " oily."

Appearance and smell will sometimes guide an experi-
enced buttermaker in determining the ripeness of the
cream, but it is generally advisable to determine by
chemical means the degree of acidity acquired, for it is
found that when the cream contains .5 to .6 of acid
(calculated as lactic acid), the process has usually ad-
vanced as far as it is advisable to allow it to go. Care
should be taken to prevent the acid from becoming
greater than this, or precipitation of the casein in lumps
takes place, and these appear later as white specks in
the butter and seriously deteriorate its flavour and
keeping quality. Acidity in itself is not necessarily an
accurate gauge of correct ripening for acid products, and
the development of agreeable flavour and aroma are
different and distinct features of the process : never-
theless, under normal conditions experiment has shown
that the degree of acidity mentioned is generally
correlated with suitable ripeness for butter-making
purposes.

At the temperatures indicated above ripening should
be complete in one or two days in summer and three or
four days in winter.

Encouragement should be given to the aerobic bacteria
by frequent stirring of the cream so as to ensure an
adequate supply of air throughout the liquid ; bad
flavours are frequently associated with exuberant growth
of anaerobic species.

NATURAL STARTERS 339

The kinds of bacteria which develop in the ripening
cream are of primary importance in the manufacture of
good butter, since the preponderance of a single prejudicial
species in it will often ruin the flavour and cause financial
loss. As already pointed out, in normal ripening there
is a succession of waves of growth of different kinds of
bacteria, and hitherto no complete solution has been
obtained of the problem of how to secure the presence
of the right kind of organism at the right moment in
adequate numbers. No single species isolated from
cream has been found to be productive of completely
satisfactory results when introduced and grown in sterile
cream.

Tentative methods which go a long way towards
effective control of ripening are in use ; these consist in
the addition to the cream of what are known as " starters'^
which are more or less pure cultures of one or two varieties
of bacteria.

Two classes of starters may be recognized, namely : — -
(i) natural starters and (2) artificial or so-called ^^com-
mercial" starters.

Natural Starters consist of milk or cream which has
been allowed to turn sour and ripen spontaneously ;
buttermilk from a dairy which is producing butter of
fine flavour and aroma may be included in this class.

For the preparation of these home-made starters the
following procedure may be adopted. Select a healthy
cow in the herd, and after washing the udder and flanks
of the animal with soap and warm water, draw the milk,
rejecting the first few jets, into a clean pail which has
been sterilized by boiling water or steam. Cover up the
vessel to prevent the access of dust, and keep it in a
warm place, at a temperature of 65° to 70° F. in summer,

340 CREAM AND CREAM RIPENING

75° F. in winter, for twenty-four to forty-eight hours,
until it has become sour but not curdled.

The development of more than .6 to .7 per cent, of
acid should be avoided as likely to weaken the useful
lactic bacteria, and give an opportunity for the butyric
and putrefactive types to grow.

After removing the cream from the surface the skim-
milk may be used to inoculate the cream to be ripened.
If a larger quantity of starter is needed, that already
prepared in the above manner should be mixed with the
requisite amount of separated milk, which has been pas-
teurized at a temperature of 180° F. for half an hour,
and the whole then allowed to sour. Some prefer not
to use the spontaneously soured milk directly for in-
oculation, but the second or third subcultures of it in
pasteurized milk.

In any case starters of this kind are the result of
chance, and are not always satisfactory ; bad taints
difficult to get rid of may be propagated by them.
Trial alone will settle whether a sample prepared in this
way is good or bad.

Ex. 154. — Prepare a home-made starter by the method just
described.

Plate out and determine the kinds of organisms in it.
Try their action upon milk separately.

Pure Culture Starters. — With a view of reducing
failures to a minimum many attempts have been made
to prepare pure cultures of bacteria which, when intro-
duced in sufficient numbers into cream will lead to good
uniform ripening.

Very many kinds have been isolated from milk, cream,
and other dairy products and tested from this point of

PURE CULTURE STARTERS 341

view, and although no single species has been discovered
to possess all the qualities for the production of the
highest grade of butter, several varieties have been found
which are highly beneficial in the preparation of well
ripened cream. Single pure cultures of these, or mix-
tures of pure cultures of two or more kinds, have been
placed on the market, and large quantities of such com-
mercial starters are extensively used wherever dairying
is practised.

Jensen suggests that for reliable and satisfactory results
a culture should possess the following properties : —

a. It should render cream sour in a short time ;

b. The organisms in it should be able to grow at

a temperature of 60° to 65° F. ;

c. It should coagulate or "thicken" the cream uniformly,

giving it a clean, acid taste ; and

d. The butter prepared from the ripened cream should

possess an agreeable flavour and odour.

Pure culture starters are sent out either in a dry con-
dition mixed with some inert powder, or in a liquid
medium such as milk or bouillon.

They should be kept in a cool place and out of
contact with contaminating dust, and when once opened
the whole contents should be used immediately.

Since a tube does not contain enough bacteria to act
as a starter for any considerable volume of cream, it is
necessary for practical dairy purposes to build up a
large bulk of milk containing enormous numbers of the
organism. This is done by adding the pure culture to
a gallon or two of pasteurized separated milk, and keeping
it for about twenty-four hours at 65° to 75° F. until the
whole is sour.

If more is needed, or where a continuous supply is

342 CREAM AND CREAM RIPENING

wanted, some of this soured milk may be added daily to a
further amount of pasteurized milk. However pure they
are at first, cultures kept going from day to day sooner
or later become valueless ; the organisms degenerate or
slowly lose their vitality and ripening power, or the starter,
in spite of careful precautions, becomes contaminated with
objectionable organisms. This usually occurs in a week
or two, after which time it is necessary to begin again
with pure commercial or laboratory cultures.

Ex. 155. — Make a bacteriological analysis of any commercial
starter you can obtain. If a powder, dissolve or distribute in
sterile distilled water and plate out on litmus lactose gelatine
and agar. Isolate and make pure cultures of each kind found.
Examine and stain specimens and try the action of each on milk
and other media. How many are liquefiers ? Are any coliform
organisms present? (Ex.147). Are moulds abundant ?

The chief object of the addition of a starter to cream
is to immediately increase the number of organisms of a
desirable kind to such an extent that they may suppress
or overcome the activity of any bacteria of a deleterious,
nature, which may have found their way into the cream.
Usually from 8 to 10 volumes of starter are added to
every 100 volumes of cream to be ripened.
Starters may be added —

(i) to cream raised by gravitation, which is neces-
sarily several hours old and therefore contains
considerable numbers of bacteria.

(2) to freshly separated cream, in which also a

certain number of different kinds of bacteria
are present, and

(3) to cream which has been subjected to the

pasteurization process to get rid of all
organisms in a growing state.

PURE CULTURE STARTERS 343

In the first case it is possible that the bacteria already
there, may be of sufficiently vigorous stock to carry on
the fermentations independently of the added starter.
Such a state of affairs is likely to occur with compara-.
tively old cream, and it is obvious that the addition of a
starter in cases like this may have little or no appreci-
able effect : the operation is not worth the trouble it
involves. The butter obtained where this practice pre-
vails is not likely to be uniform at all seasons and may
sometimes be of very bad flavour.

In the second case there is perhaps less danger of
difficulty than where older cream is used, but irregular
ripening may arise from organisms which have come
from the farm and cowshed. The plan of adding the
starter to pasteurized cream, while involving more
expense, frees the starter from competition with adverse
organisms and allows those of the chosen, desired type a
free field for development. The keeping quality of the
butter is improved as most of the organisms capable of
giving rise to taints and bad flavours are destroyed by
pasteurization. A more uniform grade of butter is pro-
duced and can be maintained with comparative certainty
throughout the year. This method of using starters is
adopted in Denmark and other countries where there is
a progressive dairying industry, partly on account of the
economic value of a uniform product and partly because
the law requires the pasteurization of all milk which
passes through creameries, with a view of assisting in the
extermination of tuberculosis. The skim-milk and butter-
milk which may ultimately be returned to the farms and
consumed by susceptible young stock, is thus rendered
free from the bacilli of the disease.

CHAPTER XXIII.

BUTTER.

I. When cream is violently agitated in a churn or
other vessel, the fat globules in it coalesce into larger or
smaller grains or lumps which are removed from the
separated serum or buttermilk and kneaded subsequently
into an apparently compact mass of butter.

In this process it is as well to note that the fat
globules do not as a whole lose their separate identity,
but remain distinct and do not melt together as drops of
oil do when brought into contact with each other.

Ex. 156. — Mount small portions of cream taken from a churn
at various stages of the churning process up to the time when
the butter " comes."

Press down the cover-slip.

Examine with a | in. and \ in. objective and note the
coalescence of the fat globules into small clusters.

Ex. 157. — Squeeze a very small piece of butter between the
slide and cover-slip and examine with a f in. and \ in. objective.

Is the butter homogeneous ? ^

Make drawings of what you see.

It is practically impossible, by churning cream, to
obtain the milk fats, pure and free from serum, so that
butter always contains in addition to the fats a small
but appreciable amount of protein, milk-sugar, and mineral

substances as well as from 9 to 20 per cent, of water ;

344

BUTTER

345

butter for sale in this country must not contain more
than 1 6 per cent, of water.

The quantity of non-fatty material present varies con-
siderably with the kind of cream used, the method of
churning, and the amount of kneading or " working " to
which the butter has been subjected, with the object of
removing buttermilk. In most dairies a small quantity
of salt is also added.

The following may be taken as the average composi-
tion of fresh and salt butter respectively : —

/

Fresh Butter.

Salt Butter.

Water .

14

12.5

Fat

83.5

84.5

Protein .

.8

.5

Milk-sugar

1.5

.6

Ash .

.2

.1

Salt

1.8

Butter is composed of eight or more distinct kinds of
fats, which are of course present also in normal milk.
The chief of these are the fats palmitin, stearin^ and
olein, glycerides of palmitic, stearic, and oleic acids
respectively, and butyrin, caproin, caprylin, caprin,
and myristin, glycerides of butyric, caproic, caprylic,
capric, and myristic acids.

The last five acids are soluble in water, and are known
as volatile fatty acids, since they can be distilled over in
a current of steam without change : the first three are
insoluble and non-volatile.

Palmitin and stearin are solid white compounds, which
occur also in beef and mutton fats ; olein is a liquid fat
which only solidifies at or below about 40° F. ; it is found

346 BUTTER

in olive and cod liver oils. Butyrin, which is the
characteristic fat of butter, melts at about 77° F.

The proportion of each of these in butter is subject to
much variation, due to differences in the physiological
condition of the cows and the food supplied to them.
The amount of olein may rise to nearly half the total
weight of butter fat. It is greatest in the early period
of lactation, and may fall to about half the maximum at
a later date. With a decrease in the size of the fat
globules in the milk the olein decreases, and the butter
becomes harder on account of the higher proportion of
palmitin and stearin. Mangels, good grass, and green
fodder generally tend to produce butter rich in volatile
acids and of low melting point ; hay and cotton-cake
give harder butter, with higher melting-point.

Usually about 92 per cent, of the whole consists of
palmitin, stearin, and olein, the rest being made up of
the volatile fats, of which butyrin is the chief.

The approximate relative proportions are given in the
following table : —

Per Cent.

, Palmitin, stearin, and myristin . 49 to 52

Olein 37 „ 42

Butyrin . . . . • 6 „ 7.5

Caproin, caprylin, etc. . . . 2 „ 3

2. Bacteria in Butter. — The number and kinds of
bacteria found in butter depend upon the bacterial contents
of the cream from which it is made, the amount of added
salt or preservative, and the age of the samples. In
butter containing more than the usual amount of water,
milk-sugar, protein, and other constituents of the cream,
bacteria grow rapidly for a time, and i gram of a freshly-

BACTERIA IN BUTTER

347

made sample has been found to contain from 15 to 30
or 40 millions or more. The number, however, always
decreases quickly on account of the want of aeration in
the interior and the poor food-supply, for pure fats are
not readily attacked by bacteria.

Jensen gives the following table, which indicates the
decrease taking place in a marked sample of butter made
from ripened cream, and also the difference in number of
organisms on the surface and in the interior : —

In Surface Layer.

In the Interior.

Total.

Liquefying
Organisms.

Oospora
Lactis.

Total.

Liquefying
Organisms.

Oospora
Lactis.

Freshsample

1 week old

2 weeks old

4 M )>

I2,CXX),000

14,000,000

16,000,000

2,700,000

20,000

20,000

I

0

80,000
580,000
200,000
480,000

12,000,000

2,000,000

2,000,000

360,000

20,000
12,000

0

80,000
20,000

400

0

The numbers which have been found in butter are many
and of various species. Some of them may be considered
as accidental impurities introduced from the air or water-
supply of the farm and dairy ; others are more or less
characteristic of most samples of butter, and come into it
from the milk or cream out of which it is manufactured.
Among the organisms usually present the following may
be mentioned : —

(a) Lactic acid organisms, especially Sir. lacticus and

its allies.
{U) Bs. lactis aerogenes.

348 BUTTER

{c) Bs. fluorescens liquefaciens.
{d) Micrococcus acidi lactici (Kriiger).
{e) Oospora lactis.
(/) Lactose-fermenting yeasts.

(^g) Certain fungi, especially species of MiicoVy Penicil'
Hum, and Cladosporium.
Less frequent, but yet commonly found are : —
{a) Sarcina lutea,
{b) Bs. vulgatus.
{c) Species of Proteus,
id) True Bs. coli.
(e) Species of Streptothrix.

3. Flavour and Aroma of Butter. — In the manufacture
of butter great attention is paid to the development of
an agreeable flavour and pleasant aroma, since it is upon
these two qualities that the commercial value of the
product largely depends.

Flavour, which refers to taste, and aroma, which is
another term for smell or odour, being usually perceived
together when butter is eaten, are often confused.

They are, however, distinct qualities, and not neces-
sarily connected with each other, for butter may possess
an agreeable aroma with an objectionable flavour, and
vice versa ; nevertheless the same substance may influ-
ence both these qualities sometimes.

What the compounds are which give to butter its
characteristic odour and taste are not known. The
evidence hitherto obtained upon the matter points to the
conclusion that some of them are sweet-smelling esters
derived from the fermentation of milk-sugar, while others
are decomposition products of the casein of the butter.
Moreover, it is to the mixture of these that aroma and

TAINTS AND DEFECTS OF BUTTER 349

flavour must be attributed, and not to any one substance
only.

Much research has been carried on to determine the
kind of organisms which produce perfect flavour and
aroma. No single species of bacterium, however, has
been found to be able to give the desired result when
used as a pure culture and inoculated into sterilized,
pasteurized, or ordinary cream. Some of them isolated
by Conn, Storch, Weigmann, and others, have given a
good butter when used singly in this way, but not one
with all the good qualities of a first-class sample.

It is pretty well established that the organisms which
influence or induce flavour and aroma do not belong to
the class which are specially concerned with the lactic
acid fermentations, but rather to those which induce
alkaline and putrefactive changes.

On this assumption it is likely that they would ruin
the butter if allowed to get the upper hand or a free
field for development ; they are, however, ordinarily kept
in check by the lactic acid bacteria.

4. Taints and Defects of Butter. — Many of the faults
which reduce the flavour and market-value of butter are
due to want of skill in churning and general dairy
management, as well as to the nature of the food con-
sumed by the cows.

The fats of milk may be of low melting-point, and
the butter consequently soft and oily, owing to the use
of linseed-cake as food, or the butter may be too hard
from the consumption of too much cotton-cake by the
cows. Streakiness, or mottling, may be caused by
irregular salting or the presence of specks of casein
owing to over- ripening of the cream. Offensive flavours
may arise from giving the cows turnips or cabbages, or

350 BUTTER

from their eating garlic and other weeds of the meadows
and pastures.

Bad odours of the cowshed and dairy are also readily
absorbed by milk, cream, and butter.

With these faults we are not here specially concerned.
There are, however, many troubles of the buttermaker
due to the action of bacteria, and some of these require
notice ; those of comparatively common occurrence are
rancidity, tallowy flavour, turnip flavour, bitterness, fishy
taste and smell, and mustiness or mouldy odour.

(i) Rancid Butter. — There is no chemical change of
butter which has been so much investigated, or which
has given rise to so much controversy as that which
results in the condition usually spoken of as rancid. A
large number of factors are known to be concerned in
the production of rancid butter, but the exact influence
of each is still a matter of doubt.

It is difficult to define " rancidity," and many of the
conflicting results of investigation have been connected
with the want of exact knowledge of what is meant by
the term " rancid."

Butter, especially if not skilfully made, when kept for a
long time loses its pleasant aroma, and gradually takes on
a pungent or acrid smell and taste, which, though easily
recognized, is difficult to describe. Simultaneously with
the development of the rancid flavour the butter gener-
ally becomes more or less acid from the production of
free fatty acids within it. If left exposed to light, the
yellow colour disappears from the surface layers, which
become transparent at the same time ; little by little
these changes penetrate to the centre of the mass, the
progression reminding one of the ripening of a soft
cheese.

TAINTS AND DEFECTS OF BUTTER 351

In rancid butter the following substances are usually
found, few or none of which are present in fresh
butter : —

1 . Free fatty acids ;

2. Certain oxy-acids ;

3. Aldehydes and ketones ;

4. Esters or ethereal salts, especially of butyric and

volatile fatty acids.
The amount of each varies considerably with the age
of the sample and the extent of the action of the various
agents to which these products are due.

Duclaux and several of the earlier investigators attri-
buted the peculiar rancid taste and odour to free butyric
acid or some allied fatty acid, but that is not the case.

Very frequently it is found that high acid content
goes along with strong rancid conditions ; but stored
butter may become strongly acid and possess an objec-
tionable flavour sufficient to render it uneatable without
being rancid ; on the other hand, rancid butter may
contain little or no free acid.

The free fatty acids arise through the splitting of the
fats, glycerine being set free at the same time. Some
of the glycerine undergoes oxidation, and aldehydes and
ketones with rank taste and evil smell are produced.

Moreover, oxygen is absorbed by unsaturated com-
pounds, such as oleic acid.

None of these compounds, however, give the strictly
rancid flavour.

According to Amthor and Reinmann and others, the
characteristic rancid odour and flavour is that of the esters
of the volatile acids, and especially of ethyl-butyrate, the
ethyl ester of butyric acid.

Amthor believed that the ethyl group of the butyric

352 ^ BUTTER

ester was derived from milk-sugar by fermentation ; but
Jensen suggests that it more likely comes from an inter-
mediate product, monobutyrin, obtained in the decom-
position of tributyrin.

The conditions which govern and accelerate rancidity
are : access of air, light, warmth, and moisture, high
casein and milk-sugar content, and the presence of
various micro-organisms.

The splitting of the fats apparently arises in part
through the action of moisture, carbon dioxide, and
lactic and other acids produced by fermentation of the
milk-sugar, and also to a considerable extent through
the agency of micro-organisms, especially Bs. fluorescens
liquefaciens^ Oospora lactis^ as well as various yeasts and
" mould " fungi. Penicillium glaucum and certain species
of Aspergillus are able to hydrolyse fats by means of
lipase, an enzyme which they possess.

Free butyric acid arises through the decomposition of
butyrin in this way, and not as the result of butyric
fermentation : the latter is the work of anaerobic species
of bacteria (p. 285) which play very little or no part in
the development of rancidity in butter.

The oxidation of oleic acid and the production of
aldehydes and ketones is to a certain extent due to the
direct chemical action of the air under the influence of
light, and the butter may develop a tallow-like, but not
rancid, character and taste by this means alone. Butter
does not become rancid when oxygen is completely
excluded. However, sterilized butter does not develop
the rancid flavour and odour even when exposed to
light and air ; so that the action of air would appear to
be chiefly important because of its assisting the growth
of bacteria and fungi of the aerobic class.

TAINTS AND DEFECTS OF BUTTER 353

The recent researches of Reinmann and Jensen have
shown that rancidity is mainly the work of aerobic
bacteria and fungi, conditioned by the presence of air,
moisture and the other factors already mentioned. Its
full development is associated with a number of different
chemical reactions which occur among the decomposition
products of the fats, caseinogen, and milk-sugar ; the
changes are not quite so complex, perhaps, as those
which take place in a ripening cheese, but are in many
respects analogous to the latter.

There is much evidence to support the view that
micro-organisms are chiefly responsible for the initiation
and maintenance of the changes involved. A sample
of normal butter containing a considerable amount of
casein, sugar, and other materials upon which bacteria
thrive becomes rancid much more rapidly than a speci-
men containing a smaller quantity of these compounds.

Butter made from sterilized cream keeps fresh for a
long time, in spite of the fact that it contains an
abundant supply of food for bacteria ; however, when
inoculated with a small piece of rancid butter, the whole
becomes rancid very quickly.

Antiseptics, such as salicylic acid and formalin, check
it, and keeping the butter in a cool place tends to prevent
it. That the change is not entirely due to enzymes is
deduced from the observations that antiseptic agents
which allow these to act, but check the chemical activity
of living organisms, stop the development of rancidity.

No single species of bacterium or fungus which has
been isolated from rancid butter has been found capable
of inducing the rancid taste and flavour in a sterile
sample. The production of this objectionable taint is
the result of the interaction of several kinds of organisms
z

354 * BUTTER

of which the following appear to be the most important,
since they are practically always met with in abundance
in rancid butter : —

1. One or two forms of lactic acid bacteria ;

2. Oospora lactis\

3. Yeasts, some of which form rose or yellow colours

on gelatine and agar ;

4. Cladosporiuvt butyri, a species of hyphomycete

belonging to a widely distributed genus.

Less frequently certain " moulds " (species of Mucor
and Penicillium) are present, as well as Bs. lactis aerogenes
and Bs. fl. liquefaciens.

(ii) The Tallowy Flavour and appearance may be
brought about by the decomposition of the fats and the
production of free fatty acids. The cause of the change,
as already pointed out, is sometimes bacterial ; but in
other cases the tallowy taste and texture is the result of
purely chemical processes, namely oxidation and hydro-
lysis under the influence of light and moisture, especially
accompanied by somewhat high temperature. Storage
of the butter in a dark, cool, and comparatively dry
place will usually check this trouble.

(iii) Turnip Flavour in Butter. — When large quan-
tities of turnips are used for the feeding of cows the milk
becomes tainted, and the cream and butter obtained from
it possesses an objectionable flavour and odour of these
roots.

The exact cause of the turnipy flavour is not yet
understood perfectly. In some cases the milk as it is
drawn from the cow possesses the peculiar flavour or
may absorb it from the air of the cowshed where turnips
are stored, or used for food ; in most instances, however,
tb? trouble arises later ^nd is a bacteriological one,

TAINTS AND DEFECTS OF BUTTER 355

Weigmann and his pupils have shown that the dung
of cows fed with large amounts of turnips contain certain
varieties of Pseudomonas belonging to the c<?//-group of
bacteria which are able to give rise to the taste and
odour of turnips in varying degree when introduced into
milk, cream, and butter. Presumably these gain access
to the milk in particles of dung, for when the udders of
the cows feeding on turnips are brushed and washed and
the dung disinfected with lime the milk and the cream
and butter derived from it do not develop the turnipy taste.
These organisms produce the strongest development
of the objectionable taste when working in combination
with varieties of Streptothrix and certain fungi, especially
a form of Penicilliuin brevicaide.

Foul odours may arise in butter which is contaminated
with dirty water, dung, and other materials containing
Bad, mycoides^ Bact. vi^lgatiis, Bs. fluorescens liquefaciens^
and other organisms of the putrefactive group.

Bitterness may be due to food of the cow or the activity
of bacteria.

Fishy and cheesy odours and tastes have, in certain
instances, been traced to the work of moulds and species
of ToriUa (p. 394).

5. Salt in Butter. — The addition of salt has a very
beneficial influence upon the keeping quality of butter
and in small amounts assists in preserving it free from
taints.

The number of organisms present in salted butter
increases much more slowly^ and the ultimate decrease
which occurs begins at an earlier period than in unsalted
samples.

This is evident from the following table given by
Fettick ;—

356

BUriER

Number of Organisms in

I gram of same butter
I gram of with 3 per cent,
unsalted butter. of salt added.

Fresh .

29,303 25,408

lo days old .

72,782 23,539

20 „ „ .

. 400,178 242,588

70 „ „ .

. 559,771 327,938

98 „ „ .

. 643,040 302,430

Fettick also showed that the moulds of butter, of which
species of Penicillium, Oospora, and Mucor are most
prevalent, are very rapidly reduced in salted butter, but
continue to develop for a considerable time when the
material is left unsalted.

Number of Moulds in

I gram of same butter
I gram of with 3 per cent,

unsalted butter. of salt added.

784

750

241

O

This is an important result, since some of the objec-
tionable oily, rancid, fishy, and cheesy flavours of butter
have been traced to this class of organisms. More than
3 per cent, of salt would appear to be disadvantageous,
since larger amounts inhibit the lactic acid bacteria
without interfering much with the growth of Bad. coli,
Bad. I. aerogenes, Bs. subtilis, and Bad. putrificus. Highly
salted butter is liable to develop taints which do not
appear in samples containing less of this preservative, a
point which has to be borne in mind by the producer of
butter for the export trade.

Fresh

687

10 days old

9,375

20 „

. 99,125

70 „

. 263,429

CHAPTER XXIV.
CHCESE.

I. Rennet. — For the coagulation of the caseinogen of
milk in the manufacture of cheese the salted and dried
inner membrane of the so-called " fourth " or true digestive
stomach of very young calves is frequently employed
under the name of rennet.

Such material contains a coagulase or coagulating
enzyme, termed rennin or chymosm, and also a certain
amount of pepsin, the former precipitating paracasein
in milk, while the latter in acid solutions breaks down the
curd into simpler proteins.

Rennin is found in the stomachs of most young
mammals, and commercial extracts are prepared from
those of dogs and pigs, as well as calves.

A similar coagulase is present in certain seeds, in the

leaves and stems of Bedstraw {Galium verum, L.),and in

the flowers of the Artichoke {Cynara Scolynms, L.).

Fitz and Hueppe showed that it is possessed by Bacillus

butyricus ; Warrington found it in Bs. fluorescens lique-

■faciens, and Vignol proved its existence in Bs. vulgatus.

Certain varieties of Proteus^ Bs. prodigiostis, and some of

the lactic acid organisms have the power of coagulating

caseinogen and subsequently dissolving it by means of

a pepsin which they secrete, the former action being

most in evidence in cultures kept at 20 to 25° C, while

their peptonizing property is most active at 37° C.

357

358 CHEESK

Sometimes small pieces of the dried and salted
stomachs are added to milk in order to coagulate it but
most cheesemakers now use a brine extract of rennet
containing the active principle — rennin. Home-made
extracts are prepared by soaking the " veils " or stomachs
in .3 to .6 per cent, solution of salt for three or four
weeks, after which the liquid is filtered through charcoal.
The extract should be dark-coloured but clear, turbidity
indicating objectionable decomposition.

Commercial extracts in much stronger brine (16 per
cent, solutions) are available as well as rennet " powders "
and " tabloids " : these are more reliable, being more
regular in strength than home-prepared material, and
less likely to contain bacteria and other impurities which
give rise to tainted or faulty cheese.

Rennet is a very active substance, exceedingly small
amounts being able to coagulate large quantities of milk.
One part of ordinary extract, which is itself a very weak
solution of the active enzyme, will very frequently curdle
as much as 5000 to 6000 times its volume of milk ; it
has been calculated that one part of pure rennin will coagu-
late 30 million parts of milk.

The extracts are very sensitive to the action of light,
heat, and certain chemical agents. They should be kept
cool and in the dark, not in glass bottles, for the alkali
of the glass dissolves out and damages the enzyme.

Rennin is most active in neutral or in very dilute acid
or alkaline solutions ; strong alkalis or strong acids soon
destroy it.

Its coagulating power increases from about 10° C,
below which it ceases to act, up to the optimum, about
39 to 42° C. ; above this temperature it decreases until
at about 70° C. its strength rapidly diminishes even in

CHEESE 3^9

strong solutions which are much less sensitive than dilute
extracts.

Certain antiseptics, especially borax and formalin,
damage its curdling power, but chloroform, thymol (. i per
cent.), or carbolic acid (. 5 to i per cent.) have little effect
upon it, although they check the activity of bacteria.

In the stomachs of the young animals, from which
home-made rennet extracts are sometimes prepared,
several different species of bacteria are often found.
According to Thoni such bacterial flora consists of —

(i) A group of organisms, such as Bs, subtilis, Bs.
vulgatus^ species of Sarcina and Streptothrix^
which have little effect on the quality of the
extract and soon disappear ;
(ii) Some of the useful lactic acid organisms ;
(iii) Bs. coll and Bact lactis aerogenes, which may
multiply and ultimately contaminate the curd ;
this group is the most to be feared by the
cheesemaker.

Ex. 158. — Repeat Exs. 113, 114.

Ex. 159. — Make a bacteriological analysis of home-made and
commercial rennet extracts on the lines indicated in Exs. 125,
147, 155-

2. Cheese, (i) Composition and ripening. — When
rennet is added to milk which has been allowed to
become slightly sour, a solid curd or coagulum is
thrown down, and a greenish yellow liquid, the whey,
is separated. This curd is the raw material from which
various kinds of cheese are made. It consists chiefly of
a protein substance termed casein (sometimes paracasein,
see p. 237), along with a considerable proportion of the
fat, sugar, and mineral matter originally present in the

36o CHEESE

milk. About one-half of the solids in the milk is
recovered in the cheese. The fresh curd is manipulated
in a great variety of ways, and from it a correspondingly
large number of kinds of cheese are made, differing in
size, form, texture, and other physical characters. The
different kinds vary considerably in chemical composition.
The firm or hard cheeses obtained by coagulation at
temperatures between 30 to 35° C. contain from —

28 to 35 per cent, of water,

25 „ 35 » fat,

23 „ 35 „ proteins,

1 „ 3 „ sugar, lactic acid, and

2.7 „ 4 „ ash.

The soft cheeses prepared from curd precipitated at
temperatures below 30° C. contain from —

45 to 50 percent, of water,

20 „ 30 „ fat,

18 „ 20 „ proteins,

and small amounts of sugar, lactic acid, and mineral
matter.

Freshly made cheese is at first of a firm, elastic
texture, is slightly acid, practically insoluble in water,
indigestible, and possesses no very definite cheesy odour
or taste. Sooner or later, however, very extensive
physical and chemical changes occur within it, and the
cheese is said to ripen. Much of the insoluble material
becomes soluble and is then easily digested, at the same
time the different varieties of cheeses develop their own
peculiar characteristic aroma and flavour. The chemical
changes which take place during the ripening process
are very complex, and, in spite of much investigation,

CHEESE 361

are not very clearly understood. The milk-sugar which
can be detected in freshly made cheese generally dis-
appears in a few days, and a watery extract of a ripe
one contains none of it. The fat undergoes very little
alteration. Various acids which are not found in the
fresh cheese appear in the ripened product. Lactic acid
and its salts are met with, and small amounts of acetic,
formic, propionic, butyric, caproic, valeric, and other
acids are frequently present either in the free state or
united with ammonia or other bases. Ripe cheese
always contains a certain amount of free non-volatile
fatty acids, such as oleic, palmitic, and stearic acids.
The most important changes occurring during the ripen-
ing process are those which are concerned with the
cleavage or breaking down of the casein. This protein
undergoes gradual hydrolysis as ripening proceeds,
simpler compounds arising at each stage of the process.
At first the casein is changed into caseogluten, a sub-
stance closely resembling casein, but differing from it in
being soluble in a solution of salt. Later the more
soluble and non-crystalline proteoses (caseoses) are
formed ; from these less complex peptones (caseones)
are produced, and later the peptones give rise to the
simpler soluble crystalline amino-acids, the chief of
which are glycocoll, alanine, leucine, tyrosine, phenyl-
alanine, and tryptophane. From the amino-acids origin-
ate small amounts of various amines, such as putrescine
and cadaverine, to which may be attributed some of the
objectionable flavours and odours of old cheese. Eventu-
ally ammonia is formed, and in some instances may be
set free in very small amounts, as in the soft cheeses,
but it is more frequently present in combination with
various acid compounds.

362 CHEESE

Van Slyke and Hart found in Cheddar cheese
twenty-four hours old that only about 6 per cent, of
the total nitrogen in it was soluble in water, whereas
in a ripened one, nine months old, 53 per cent, was
soluble. The changes in the proteins during the
ripening of cheese resemble those observed in peptic
or tryptic digestion of proteins, and, as far as they go,
are similar to a process of putrefaction, proteoses, pep-
tones, and amino-acids being produced in both cases.
In ordinary putrefaction, however, certain compounds
are produced, especially oxyacids, phenol, indole, skatole,
and various gaseous substances which are never met
with in normally ripened cheese. Possibly the milk-
sugar, or the organic phosphorus compounds have an
inhibiting effect on the putrefactive bacteria, or the
organisms which utilize carbohydrates check or destroy
them.

The changes in the constituents of cheese, which
have just been described, take place slowly, and almost
uniformly, throughout the whole mass of hard pressed
varieties, such as Cheddar, Emmenthal, and Edam, the
time taken to obtain a thoroughly ripe condition being
several months. Most of the whey is removed from the
curd at the time of manufacture of this type of cheese.
On the other hand, in the soft cheeses a much greater
amount of vv'hey remains in the curd, and ripening takes
place at a rapid rate, the casein being broken down into
soluble compounds in a few days. Ripening begins on
the outside and proceeds inwards, a section of a partially
ripened specimen showing a distinct outer layer of soft,
ripened, creamy material, surrounding an interior of firm,
white, unaltered curd.

(ii) Aroma and flavour. — During the ripening process

J

CHEESE 363

cheeses develop a characteristic aroma or odour, which
is well-marked and quite distinct from that of fresh
curd. The pleasant, sourish smell of a ripening cheese
is probably due, to some extent, to minute traces of
formic, acetic, and similar free volatile fatty acids present
in it. Propionic and butyric acids, which are generally
found in greater amount, have a penetrating and some-
what rancid smell. Isovaleric acid has a characteristic
old-cheesy odour, and the unpleasant smell of the rind
of certain overripened cheeses has been traced to the
presence of caproic and caprylic acids. The peculiar
odours of cheeses are not entirely due to free, fatty
acids ; nitrogenous compounds, such as amines and
ammonia, contribute their share. Putrescine and cad-
averine, both of which have a sharp, objectionable odour,
have also been isolated from cheese. The flavour or
taste of a good nutty cheese appears to be connected with
the amino-acids, most of which have a mild, sweetish
taste. According to Jensen, the delicate, nut-like flavour
of the best Emmenthal cheese increases with an increase
in the amount of those soluble nitrogenous compounds
which cannot be precipitated from their solutions by
phosphotungstic acid, a class which includes the amino-
acids. Many of the more complex protein substances
have each a characteristic taste and smell, but nothing
definite is known about their influence in the production
of good flavour and aroma in ripe cheese. In some
instances considerable amounts of bitter substances are
produced, but little is known of their nature or origin :
they only arise in cheeses undergoing putrefaction or
abnormal decomposition, or where little or no salt has
been used in the manufacture. Many of the peptones
have a bitter taste, but in normally ripened cheese they

364 CHEESE

are never at any time abundant, and their bitter flavour
appears to be removed by common salt in a manner not
yet understood.

(iii) Causes of the ripening of Cheese. — While the
general facts of the ripening process are fairly clear, the
causes which operate in the production of the chemical
and physical changes involved in the process are still
imperfectly understood, and much controversy has arisen
in regard to them. It is, however, agreed on all sides
that ripening is due chiefly, if not altogether to the
activity of bacteria and fungi. The process only
goes on when these are present, and cheese prepared
from sterilized milk, or kept under conditions which
destroy living organisms, do not ripen. Russell and
Babcock state that they found cheese would ripen when
immersed in chloroform, and suggested that the process
was due to the action of galactase, a trypic enzyme
normally present in milk and capable of hydrolysing
casein. It is very doubtful, however, if the action of
bacteria was precluded in their experiments, and careful
investigations by other workers do not support Russell
and Babcock's view. It is probable that galactase plays
some part in the breaking down of proteins in cheese,
but its influence is small. Another proteolytic enzyme,
namely pepsin, is introduced into cheese in the rennet
used for curdling the milk, and this no doubt also takes
a share in the ripening process. An increased addition
of rennet is followed by an increment in the amount
of those soluble nitrogenous compounds which are
characteristic of peptic digestion. Moreover, pure pepsin
added to cheese hastens the ripening process, more
particularly in Cheddar and similar varieties, where
considerable acidity is developed in the early stages of

CHEESE 365

manufacture. The ripening process appears to be a
peptic proteolysis at first ; later, when less acid or more
alkaline conditions prevail, it resembles tryptic digestion.
Although galactase and the pepsin of rennet are
probably concerned in some degree with the ripening
of cheese, it is to bacteria that the chief cause must
be attributed. Various kinds of organisms are present
in the milk as it comes from the teats of the cow, or
find their way into it from the air and utensils of the
stable and dairy. With the rennet large numbers are
added, since each c.c. of the extract ordinarily used contains
from 25 to 35 millions of bacteria.

Many elaborate investigations have been undertaken
to ascertain the number and kind of bacteria present in
cheeses at various stages in the ripening process. It
is usually found that in the fresh curd there are fewer
bacteria than in the slightly acid milk from which the
curd has been precipitated by rennet, large numbers being
of course removed in the whey. According to Russell
and Weinzirl, in a new Cheddar cheese there is a decrease
in the bacterial content during the first twenty-four to
forty-eight hours, due apparently to a reduced temperature
and to a further loss of whey in the press. From this
point a rapid rise in numbers takes place until a
maximum is reached, when a gram of cheese will contain
from 90 to 150 millions or more of bacteria. The time
taken for the development of the maximum number of
organisms depends upon the kind of cheese, the tempera-
ture at which it is kept, and other factors ; in Cheddar
it usually occurs between the tenth and twentieth day.
A steady decline in numbers now goes on for several
weeks or months until the cheese contains comparatively
few living organisms. By microscopic examination of

366 ' CHEESE

sections of cheese the bacteria in it can be seen in situ
and their distribution, development and form at various
stages in the ripening can be studied. Heaps or colonies
of larger or smaller extent are found distributed irregu-
larly throughout the mass, a fact which makes it difficult
to obtain concordant quantitative results regarding the
number of bacteria present in cheese. The colonies are
generally pure, but occasionally two or more kinds may
be intimately mixed. In the spaces between these
single organisms, small groups or isolated spores are
generally seen, but in some cheeses, especially those which
have undergone ripening for some time, the spaces are
free from living bacteria.

A very large number of species of bacteria have been
isolated from different varieties of cheese. Many of
them have been imperfectly described, and a list cannot
be given here. They may, however, be classified for
convenience into the following groups : —

(i) Organisms which are capable of fermenting
milk-sugar with the production of lactic
acid without gas formation, e.g. Streptococcus
lacticus, Kruse.
(ii) Those which give rise to lactic acid and con-
siderable amounts of free carbon dioxide and
hydrogen, e.g. Bs. acidi lactici^ Hiippe, and
Bact. lactis aerogenes, Esch.
(iii) Proteolytic bacteria, which digest or decom-
pose casein,
(iv) A group of miscellaneous kinds which appear
to be without any power of attacking the
carbohydrate or proteins in cheese, and which
die out or remain dormant in it.
Much of the controversy which has arisen in regard

CHEESE 367

to the ripening process has been concerned with a deter-
mination of the function or part played by these various
groups of bacteria, and how the several kinds affect the
different components of cheese. The chief views held by
the different workers at the problem are discussed below.
(a) Duclaux, who was the first to make a systematic
study of the ripening process, isolated from Cantal cheese
ten species of bacteria, the most important of which were
placed by him in the genus Tyrothrix. The latter were
all somewhat large bacilli, which grew readily in milk
and other liquid media into large threads sometimes
100 /a to 120 ^ long, and formed spores which were able
to resist a temperature of 100° C. for a short time.
They are now known to be very similar in many of
their characters to the hay bacillus {Bs. subtilis) and the
potato bacillus {Bs. vulgatus). When introduced into
milk Duclaux found they were able to decompose the
casein with the production of an albumose, amino-acids,
and other compounds met with in ripened cheese, and
concluded that they were the chief agents in the ripening
process. The power of these organisms to peptonize
casein Duclaux considered was due to the secretion by
them of a proteolytic enzyme, casease, which diffused
slowly throughout the substance of the cheese. Since
the peptonizing bacteria are checked by the presence of
much acid, they only begin their specific work after the
initial lactic fermentation is over, and the acid produced
has been destroyed or neutralized. This view has been
supported by Adametz, Von Klecki, Winkler and others,
and a pure culture of Bacillus nobilis^ an aerobic form of
a Tyrothrix obtained from Emmenthal cheese, has been
sold for cheesemaking under the name '^ tyrogenT Its
use, however, has not met with success. If the peptonizing

368 CHEESE

group of bacteria such as species of Tyroihrix and
allied spore-producing kinds were the chief cause of
ripening, it would be expected that they would be more
abundant in a ripened cheese than in one which has
undergone little change, but this is not the case. They
are never abundant in cheese at any time, and are
generally absent altogether in Cheddar and other hard
cheeses a few months old : even when added in large
numbers to the curd they fail to increase, and die out
usually before any sign of ripening can be detected.

{b) The view that the lactic bacteria are the chief
agents in the ripening of cheese was strongly supported
by Freudenreich, and many of his pupils still maintain
this opinion. The common lactic bacteria are by far the
most abundant organisms met with in cheese of all kinds,
and in the early stages of the ripening comprise more than
95 per cent, of the total bacteria present. Weinzirl
found that of the total bacteria present in Cheddar
cheese about 74 per cent, were lactic forms near Strepto-
coccus lacticus and 22 per cent, allied to Bs. acidi lactici
(Hiippe), leaving only 3 or 4 per cent, for all the other
species, and similar results have been obtained by
Percival and Mason in Stilton cheeses. The lactic
bacteria uphold their supremacy almost to the end of
the ripening period, and in practice it is found that
cheeses which contain large numbers of these organisms
are much superior in quality to those in which fewer are
found. Cheese made from milk from which they are
absent always ripens slowly and abnormally, whereas
when a culture or " starter " of lactic acid bacteria is
added such milk will produce a good cheese.

In hard cheeses, where the organisms other than
lactic bacteria are in the early stages in greater pro-

CHEESE 369

portion than 3 or 4 : 96 or 97, the ripening is likely
to be unsatisfactory and result in the production of a
cheese of poor flavour and aroma.

Freudenreich isolated from Emmenthal cheese several
cocci and rod-shaped bacteria which produced lactic acid
and curdled milk, and at the same time broke down the
casein into soluble proteins when the acid was neutral-
ized with chalk. Gorini states that some bacteria act
as lactic acid producers at higher temperatures, and
peptonize casein under the cooler conditions of the
cheese store-rooms.

{c) Weigmann and others consider that the ripening
of cheese is a much more complicated problem than that
which is suggested by Duclaux and Freudenreich. Many
workers hold that the specific lactic organisms do not
ripen cheese. It is impossible under ordinary conditions
of manufacture to avoid the access of these bacteria to
the milk and fresh curd, and they find the latter a suit-
able medium in which to grow and multiply extensively,
but their influence upon the ripening is indirect only. It
is suggested that they destroy or check some kind of
bacteria which are detrimental to the normal develop-
ment of taste and flavour, and produce an acid condition
which is essential to the growth of the peptonizing
group and the action of their enzymes. In some
instances sour whey or other acid starter is added during
the manufacture of the cheese in order to ensure that the
ripening shall proceed in a normal manner. Ripening
is assumed to be a kind of symbiotic fermentation in
which the lactic organisms use up the oxygen and pre-
pare the ground for the anaerobic species which are
supposed to be the bacteria chiefly responsible for the
degradation of the proteins in the cheese. Weigmann

2 A

370 CHEESE

obtained two species of anaerobic bacteria, Paraplectruin
foetiduni and Clostridium licheniforme, from cheese ; they
belong to a rod-shaped, spore-bearing class which produce
butyric acid, and are supposed to be concerned with the
ripening of the cheese and the development of aroma
and flavour in it.

{d) Boekhout and De Vries are inclined to believe that,
in the case of Edam cheese at any rate, the cause of
ripening is not to be found in the work of bacteria
during or after the lactic fermentation has taken place,
but is due to the enzymes produced by peptonizing
bacteria which get into the milk in the stable and in the
dairy. These organisms begin their work when the
cheese is made, although their activity is soon checked
by the development of the lactic bacteria. However,
the proteolytic enzymes already produced by them are
not destroyed, and these substances continue to break
down the paracasein and ripen the cheese.

{e) The author is of opinion that the lactic acid
organisms are chiefly responsible, in an indirect manner,
for the ripening of most cheeses, the proteolytic enzyme
upon which the process depends being apparently intra-
cellular, only coming into operation at or about the time
of death of the bacteria.

The organisms, of which many millions per gram can
be detected in unripe cheese, disappear as ripening
proceeds, their bodies being broken down under the
influence of their intracellular trypic enzyme ; after this
autolytic digestion the enzyme set free from the dead
bacteria continues its proteolytic action upon the sur-
rounding curd.

Ex. 160. — Take a small piece of cheese, smooth one side with

CHEESE ' 371

a razor, and press the smooth surface gently down on a clean
cover-slip, so as to leave an impression of the cheese upon it.

Pass the slip through the flame and dip it in ether for two
minutes and then in absolute alcohol for one minute to remove
the fat. Stain for ten minutes with Nicolle's carbol-thionin
solution (saturated solution of thionin in 90 per cent, alcohol,
5 c.c. ; I per cent, solution of carbolic acid, 50 c.c). Examine
with low and high powers.

Note the forms of the bacteria present. Are they cocci or
rods?

Are the organisms arranged in heaps or colonies, or distributed
singly ?

Ex. 161. — Try the above experiment with cheeses of different
ages. Make observations on the form and arrangement of the
bacteria in fresh, half-ripe, and fully ripe cheeses.

(iv) Ripening of Soft Cheeses. — The chief differences
betvi^een the hard and soft varieties of cheese have been
incidentally mentioned earlier in the chapter. To the
soft kinds no artificial pressure is applied, the whey in
the curd being merely allowed to drain away by gravita-
tion. Such cheeses, of which Camembert may be taken
as one of the best known examples, frequently contains
from 45 to 50 per cent, of water. On account of this
high moisture content, bacteria grow in them very
rapidly, and moulds soon cover their external surfaces.

The chemical changes go on quickly, progressing from
the outside inwards, a partially ripened specimen show-
ing a firm core of .acid curd, around which is a layer
of buttery, semi-liquid or digested casein of variable
thickness.

The casein is broken down into proteoses, peptones,
and amino-acids : the decompositions are similar in kind
to those found in the ripening of hard cheeses, but they

372 CHEESE

are carried further and at a more rapid rate, complete
ripening occupying not more than three or four weeks,
instead of as many months.

In normal examples tryptophane, indole, skatole, sul-
phuretted hydrogen, and other products of putrefactive
fermentations are absent.

Good Camembert cheese contains about .25 per cent,
of ammonia when ripe, mainly combined with acid
radicles ; only in over-ripe samples can free ammonia be
detected, its occurrence in considerable amounts indicating
putrefaction and decay.

The biological factors in the ripening of soft cheeses
are different from those of the hard varieties ; in the
latter, bacteria alone are mainly responsible for the
chemical changes, while the former are ripened chiefly
by means of fungi.

In both types, of course, the lactic acid organisms are
abundant, and assist in the preliminary changes occurring
in the cheeses ; but in the soft Camembert cheese Penicil-
Hum candzdum, Rogers (by some authors ndLxn^dPemcillium
Camembertii) is the active ripening agent, the mycelium
of this fungus secreting a proteolytic enzyme which
diffuses into and peptonizes the acid curd.

Always associated with it is Oospora lactis and various
yeasts, to which the pleasing aroma and flavour are in
part due, for cheeses ripened with pure cultures of P.
Camembertii do not develop the characteristic Camembert
taste.

For a successful result an abundant development of
Streptococcus lacticus and allied lactic organisms is
essential, otherwise foul-smelling decompositions are
certain to arise.

In three or four days the two fungi just mentioned

SOME CHEESE DEFECTS 373

appear as a white felt, covering the surface of the cheese.
The acidity decreases and an alkaline condition is estab-
lished in the rind and a thin layer immediately beneath
it, softening and digestion of the curd occurring at the
same time.

An excessive growth of Oospora lactis must be avoided,
as this leads to the production of an objectionable
flavour.

3. Intermediate between the hard and soft cheeses are
Stilton, Gorgonzola, and other varieties which are drier,
firmer, and much larger than the soft forms, but not so
dense as the Cheddar and hard types. Upon the rind of
these kinds moulds and yeasts grow abundantly, and
after the lactic acid organisms have reached their maxi-
mum development and begun to disappear, the whole
body of the loosely-packed curd becomes penetrated by
the mycelium of a Penicillium. In the fissures in the
interior of the cheese the mould develops its characteristic
blue-green conidiophores, a well-ripened Stilton showing,
when cut across, a series of branching blueish veins.

4. Some cheese defects, (i) Gassy or blown cheese. —
One of the most frequent and most troublesome difficulties
with which the cheesemaker has to contend is the pro-
duction of " gassy " or " blown " cheeses, which invariably
possess a disagreeable flavour. Throughout the sub-
stance of the cheeses large numbers of more or less
spherical cavities or " bubbles " are formed. In some
cases the holes are not larger than a pin's head, while in
others they may grow to a large size, and give rise to
cheeses which are cracked, swollen, and distorted in
shape.

The holes or bubble-like cavities are produced by
" gassy " fermentation of the milk-sugar, and develop

374 CHEESE

to the greatest and most serious extent in cheeses of the
Emmenthal and Gruyere types, which are manufactured
from somewhat sweetish curd.

Sometimes the holes appear in the early stages of
manufacture, the curd in such instances containing
enough gas to make it float in the whey. Frequently,
however, the trouble only shows itself when the cheese is
in the press or in the ripening room.

The organisms which are responsible for the majority
of *' gassy " cheeses are varieties of Bad. coli or Bad.
ladis aerogenes, especially those which give rise to much
gas when grown in milk-sugar media.

Bad. Schafferi and Bad. Guillebeau of Freudenreich
belong to this class, and other organisms closely allied
to Bad. ladis aerogenes have been isolated and shown to
be the cause of " blown " cheese and bubbly curd by
Peter, Schneebeli and others.

BoUey and Hall found a close relationship between
this trouble and contamination of the milk with cow-
dung, and this appears to be specially true when the
latter comes from animals suffering from digestive dis-
turbances, brought on by the consumption of wet grass,
clover, and other succulent green foods.

There is some evidence that some of the organisms
associated with mastitis or inflamed udder sometimes
have a causal connection with spongy curd. Bs. Guillebeau^
Freud., and Micrococcus Sornthalii, Adametz, are said to
possess this double character.

Certain varieties of the butyric acid organisms and
yeasts probably give rise to gassy cheeses also. Various
methods are adopted to get rid of gassy fermentation in
cheese.

Every effort should be made to reduce contamination

SOME CHEESE DEFECTS 375

with animal faeces ; cleanliness at the farm will go a
long way towards preventing it.

The lactic acid fermentation inhibits the organisms
which cause it, and the addition of sour milk " lange
wei," or a specially clean starter, is advisable where
sponginess of curd is expected.

Ripening at low temperatures is also useful. The
addition of . i per cent of potassium nitrate (saltpetre) to
the milk for cheesemaking is beneficial. Boekhout
and De Vries have shown that the gas-producing
bacteria break up the milk-sugar in the milk or cheese
with the evolution of carbon dioxide and hydrogen gases,
but when potassium nitrate is present they obtain the
oxygen which they need from this compound, leaving
the milk-sugar to be changed by the ordinary lactic
acid organisms.

Ex. 162. — Make a fermentation or Wisconsin curd-test with
clean milk, and one to which a small amount of cow-dung or
pure culture of Bad. lactis aerogenes has been added.

Take two glass jars or tin vessels each holding about a pint ;
they must have tin or glass covers or lids. Sterilize them in the
hot-air sterilizer ; when cool fill the vessels about two-thirds full
of the milks to be tested, and put on the lids.

Now place them in an incubator or water bath, kept at 38° C,
and when the milk has reached this temperature add to each vessel
ten drops of commercial rennet diluted with two or three cubic
centimetres of boiled water. Shake or rotate the vessels gently
so as to mix the rennet with the milk, and when curdling is
complete, allow them to stand twenty minutes. Cut the curd in
each into small pieces with a separate sterilized knife, or with
one which is sterilized each time before use by dipping in boiling
water, and cover up for half an hour. Then open the vessel and
smell ; note pleasant or disagreeable odour.

376 CHEESE

Pour off the whey, cover up, and leave for an hour ; then
note odour again, and pour off any whey still left.

Repeat observation every two hours.

After eight or twelve hours, take out the curd from each and
cut it in two with a sharp knife.

Note the presence or absence of oval or round bubbles, or
pin-holes, and the odour of the curd.

Large holes of irregular shape are due to want of close packing
of the curd after being cut.

Ex. 163. — Inoculate two tubes of whey-gelatine with Bad,
lactis aerogenes, or other organism isolated from spongy curd.

Incubate one of them at 22° C; keep the other in a cool
place at 15° C. or lower.

Note after twenty-four to twenty-eight hours the presence or
absence of gas bubbles in each.

Ex. 164. — Take two tubes of whey-gelatine ; to one of them
add . I per cent, of potassium nitrate.

Now inoculate both with BacL lactis aerogenes or similar gas-
producing organism.

Inoculate at 22° C.

Do colonies appear equally in both tubes ?
. In which are gas bubbles most abundant ?

Test both at intervals of six hours for nitrites by method
described in Ex. 74.

(ii) Bitter Cheese. — The production of bitterness in
cheese is usually due to the work of micro-organisms.

The putrefactive bacteria may cause this trouble to
arise, and some of the organisms noted as being respon-
sible for bitterness in milk (p. 283) have been found to
induce the objectionable flavour in cheese, although
bitter milk does not necessarily make bitter cheese.

Freudenreich's Micrococcus casei amari was found to
be one of the causes of the development of the objection-
able flavour in Emmenthal cheese.

SOME CHEESE DEFECTS 377

In 1 90 1 Harrison isolated a species of Torula
(p. 396) which he determined was the cause of serious
trouble in one of the leading cheese factories in Canada.
The organism, which he named Torula amara, produced
an unpleasant bitter flavour in milk and also in cheese
prepared from it.

It was traced to the leaves of certain species of maple
which grew near where the milk- cans were stored.
Grown in beer wort, the cells are oval, from 7.5 to 9 [j.
long, and, like most yeasts, occur singly or in short
chains and small clumps. Inoculated into sterile milk
the bitter taste develops in five or six hours at 37° C,
with the formation of considerable quantities of gas and
very slight acidity.

Cultures give the buiret-reaction of peptones.

Cheese made from milk inoculated with the Torula
were bitter.

Eckles in 1905 isolated a small bacterium which was
found to be the cause of bitterness in Cheddar cheese,
the objectionable taste often developing in a few days.

The organism is a short rod, .8 to i /x long and .6 (h
broad ; when grown in whey it assumes the form of an
oval coccus, .7 ih long, .6 ih broad.

It stains by Gram's method, and liquefies gelatine, the
colonies being small and white at first, afterwards
yellowish. Grown on agar at 37° C. for twenty-four
hours, the colonies are very small, resembling those of
Sir. lacticus ; deep colonies are yellow, those on the
surface almost colourless.

Milk is coagulated by it in twenty-four hours at 37° C,
the whey becoming straw-coloured and intensely bitter.

CHAPTER XXV.

MOULDS AND YEASTS.

Moulds, yeasts, and other minute fungi are almost as
widely distributed as bacteria, and the student of
bacteriology is constantly meeting with them both as
impurities in his cultures, and upon butter, cheese, and
other materials with the study of which he may be
largely concerned.

Some of these fungi are of great technical importance,
and there is no doubt that many of the yeasts and
moulds found in the soil, in manures, and in the interior
and on the outside of cheese and other dairy products
play considerable part in the characteristic chemical
changes which go on in these materials.

The complicated chemical fermentations occurring in
the putrefaction and decay of manure and organic matter
in the soil, as well as in the " ripening " of cheese and
other foods, are often the result of the symbiotic or com-
bined activity of moulds, yeasts, and bacteria working
simultaneously.

The present chapter deals with the chief morpho-
logical characters of the common kinds of moulds and
yeasts.

Moulds.

I. The genus Penicillium. — One of the most frequently

encountered small fungi, is common blue mould (P^;?/*:////?/;;/

glaucum, Brefeld). The body of the plant is composed of
378

THE GENUS PENICILLIUM 379

fine cottony filaments termed hyphco. Each hypha is a
transparent tube - like structure fi.lled with colourless
protoplasm, in which are vacuoles or spaces filled with
water containing various substances in solution. In the
younger parts of the hyphae the protoplasm, in which
small oil-globules are frequently abundant, fills the whole-
tube, but in the older portions it is found only as a thin
lining on the inside of the hyphal filament.

Growth goes on at the top of each hypha.

Placed across the hyphae of Penicillium at irregular
intervals are thin transverse partitions or septa, and these
are present in. all except the lowest orders of fungi.

In most of the common moulds the body of the plant
may be divided into two parts, narnely : —

(i) The vegetative portion, named the mycelium or
spawn, consisting of a tangled mass of hyphae, which is
generally embedded in the bread, cheese, or other
material from which the fungus obtains its food.

(2) A more or less specialized portion, which bears the
reproductive organs.

Reproduction among fungi is carried on by means of
single-celled structures termed spores.

In Blue Mould these are borne in chains on sterigmata,
which are arranged in a whorled pencil-like fashion at
the ends of erect fertile hyphae, as depicted in Fig. 5 i .
Spores produced at the end of hyphae and not enclosed
in spore cases are frequently spoken of as conidia.

Some, if not all, of the species of Penicillium, have
the power of forming fruiting bodies, inside which are
spores of another type (ascospores) : for further details
upon these structures the student is referred to special
text-books on fungi.

A number of apparently different species or varieties

38o

MOULDS AND YEASTS

of Penicillium are known, the following being, perhaps,
the most important : —

(i) P. glaucum, Brefeld. — This species is found on
bread, jam, fruit, and almost all kinds of decaying
organic material ; to its presence are due the character-
istic blue veins in a ripe Stilton or Dorset cheese.

Fig. 51. A common " mould," Penici/Hum £-laucum, Bref. A Hyphse form-
ing part of its mycelium ; a erect hypha bearing conidia c in chains, 6
detached conidia, at s germination of a conidium has taken place, /germ-
tube, the beginning of a new mycelium. (Enlarged 500 diameters.)

The mycelium and hyphae in a young state are white and
cotton-like ; later, when the conidia are formed, the
fungus becomes a greenish-blue colour.

The sterigmata are from 8 to 13 /a long and 3 to 4 /O-
broad. The conidia are round, smooth, and usually
from 3 to 4 /A in diameter, with a greenish tinge.

THE GENUS PENICILLIUM 381

The fungus grows best about 25 to 27° C.

(ii) P. camemberti, Thorn. — A form or species of
Penicillium, associated with the ripening of Camembert
cheese, appears to be closely related to P. glaucum, but
differs from it in some of its features. It is probably
the same as P. album of Epstein, and P, candidum
described by Rogers as occurring on Brie cheese.

At first its hyphae are very white ; in the early conidial
stages it has a greyish-green tinge, becoming greyish-
white later.

The sterigmata, 2 to 4 usually together, are 8 to 1 1 /a
long, 2 to 3 ,<A broad ; the conidia in a young state are
sometimes slightly elliptical, but ultimately round and
smooth, from 4.5 to 5.5 ^ in diameter and yellowish-
green when ripe.

The casein of milk is peptonized by it without
coagulation.

(iii) P. brevicaule, Sacc, differs somewhat from the
ordinary types of Penicillium in having its spores some-
times placed directly on the mycelial hyphae instead of
on special sterigmata. At first the fungus is white, but
later turns brownish-yellow or brown ; we have seen a
deep orange form on the " coats " of Stilton cheese.

The sterigmata are comparatively few, usually bearing
single terminal round conidia, which, when ripe, are
5 to 7 /x in diameter, with warted surfaces.

Weigmann and Wolff found a form of this fungus in
the dung of cows fed largely on turnips (see p. 355).

Ex. 165. — Place a piece of mouldy Stilton cheese, or mouldy
bread, on some damp blotting-paper in a deep Petri dish. Leave
for a day or two until active growth of the mould commences.

(a) Remove a portion of the blue mould with a pair of

382 , MOULDS AND YEASTS

forceps ; place it on a dry slide and examine it with a
low power of the microscope or a strong hand lens.
{b) Mount some in a drop of water, and after tearing the
hyphae apart with a couple of needles, examine it first
with a low power, noting the large number of
separate conidia and tangled hyphae.

Then examine it with a high power, and make
drawings of the conidia and the hyphae with their
contents and septa.
{c) Take a fresh piece of the mould and dip it in a watch-
glass containing absolute alcohol, to which a drop or
two of strong ammonia have been added.

Then transfer successively to a series of watch-
glasses containing 75 per cent., 50 per cent., 25 per
cent., and 10 per cent, alcohol respectively, leaving it
thirty seconds in each.

Mount it in a drop of water, and examine it for
conidiophores with unbroken brush-like sterigmata and
chains of conidia.

Make drawings illustrating the arrangement and
structure of the several parts observed.

Measure the conidia and sterigmata.

Ex. 166. — Place a piece of Camembert cheese under a bell-
jar, or in a Petri dish, on some damp blotting-paper. After a
few days, examine the moulds which grow on it, in the manner
described in the previous experiment.

Make drawings and measurements of the parts of the fungi
observed : compare with F. glaucum.

Ex. 167. — Examine the yellowish, orange, or brownish patches
on the outside of any Stilton cheese available for P. brevicaule.

2. The genus Aspergillus.

An almost equally ubiquitous group of fungi are
included in the genus Aspergillus. They are closely
related to the genus Penicilliuniy and, like the latter, the

THE GENUS ASPERGILLUS

383

hyphae and conidia of several species are grey or bluish-
green in colour.

The erect spore-bearing hyphae or conidiophores do
not branch, but end in swollen, rounded apices. The
sterigmata,* also, are usually simple and unbranched,
and bear chains of round or oval conidia (Fig. 52).
These fungi produce
small round yellow or
brownish perithecia or
fruits containing asco-
spores, which are more
frequently met with, per-
haps, than the corre-
sponding organs oiPeni-
cillium.

Common representa-
tives are : —

(i) A. glaucus, Link.,
is a very widely distri-
buted fungus on old -^ More highly magnifiedC X 300).

bread, jam, and all kinds of decaying organic sub-
stances. The creeping hyphae are cottony, branched,
indistinctly septate : the fertile hyphae are usually
without septa, inflated at the end to about 60 (l
in diameter, and covered with small sterigmata bearing
chains of round conidia about 8 to i o /a, in diameter.
The conidia are at first colourless, but later become
glaucous or greyish-green, and finally brownish in colour :
they frequently have very minute wart-like projections
on their outer surfaces.

The perithecia which are commonly found on many
organic substrata are round, bright-yellow bodies 100 to
200 /x in diameter, and containing asci with 5 to 8

Fig. 52. — Aspergillus glaucus^ Link.
Conidiophores under low magnification (X 60).

384 MOULDS AND YEASTS

colourless oval ascospores 7 to i o /* long and 5 to 8 ^a
broad : later they become brown.

The ascigerous or perithecial stage of the fungus is
known as Eurotium herbariorum, Link.

(ii) A. flavus^ Link., when fully developed, is a
yellowish or tawny-coloured fungus. It occurs on
bread, dried plants, and various organic substances, and
is found as a saprophyte in the human ear sometimes.

The apical inflated part of the conidiophore measures
30 to 40/0, across : the conidia are round, minutely
warted, yellowish, and 5 to 7 /x in diameter. It grows
best at about 37° C.

(iii) Aspergillus niger, Van Tieghem, is a common
mould with dark brown or black globose heads of
conidia. The hyphse are colourless, or practically so,
but the conidia are a smoky-brown tint, round, and 3 to
5 AC in diameter. The inflated end of the conidiophore
is about 80 (L across, 'and bears a large number of deli-
cate branched sterigmata, on which latter account the
fungus is more correctly included in the genus Sterig-
matocystis under the name 5. niger, Cramer.

(iv) A. Oryzae, Cohn, is utilized in Japan for the pre-
paration of Sak^, a kind of wine made from rice.

Ex. 168. — Procure a specimen of Aspergillus glaucus and
examine it both with low and high power according to the
method described in Ex. 165.

Note the shape and arrangement of the conidia : compare with
Penicillium glaucum.

Make drawing of the conidiophore, its inflated apex, and the
conidia, and measure the diameter of each.

3. Oladosporium herbarum, Link. — One of the dark-
coloured saprophytic moulds met with everywhere upon

OOSPORA LACTIS 385

decaying organic matter is Cladosporium herbarum. Link.,
a conidial stage of some ascomycetous fungus. It fre-
quently produces dark olive-brown patches upon damp
paper, wood, barley, and other grains, dead leaves of all
kinds, and vegetable tissues generally. A form of it,
Cladosporium butyri, occurs sometimes upon rancid
butter (p. 354) and other dairy products.

The hyphae, densely woven into a blackish-olive
stratum, often become divided into very short, almost
rectangular, lengths by transverse partitions, which pieces
may ultimately separate and germinate in the same
manner as a conidium or spore.

The true conidia arise singly or in very short chains
of two or three members at the tips of sparingly branched
hyphae : they are at first pale in colour but later become
a dark olive-brown colour. In size and form they are
extremely variable, some of them being oval and others
oblong or cylindrical, usually smooth, simple, or with
one or two septa : most commonly they are oval, from
12 to 20 yCA long and 5 to 8 //- broad.

Ex. 169. — Procure and examine a specimen of Cladosporium
herbarum.

Note the colour of the hyphae and their numerous septa.
Observe also the form and size of the conidia.

4. Oospora lactis, Sacc. — This organism, most com-
monly known by its earlier name Oidium lactis, Fres.,
forms a snow-white velvety covering upon cream and
butter and on the outsides of many of the softer kinds
of cheese. The sterile hyphae are closely interlaced with
each other and spread over considerable areas when the
conditions for nutrition are suitable. In liquid media
they frequently break up into short lengths very closely

2 B

386

MOULDS AND YEASTS

resembling the conidia of the fungus, except that the
ends preserve their rectangular outline for some time,

becoming rounded only when
they begin to germinate (Fig.

5)-

The fertile hyphae bear con-
idia, some of which are globose
or oval, others longer and more
or less cylindrical from 1 8 to
2 1 /A long and 5 to 8 ,<a broad.
Oospoi'a lactis grows well
upon acid media, especially
those containing lactic acid,
which it oxidizes readily.
Acid gelatine is liquefied and the casein of milk is pep-
tonized by it : the optimum temperature for its growth
is about 20° C.

Fig. ^z—Oospora lactis, Sacc.
(pidium lactis, Fres.).

Ex. 170. — Procure a piece of rancid butter, or keep a piece of
butter on damp blotting-paper in a Petri dish for three or four
weeks.

From the outside of either scrape off a small portion : work it
gently in a mortar with a little water and inoculate a tube of
melted lactose gelatine with a drop of the mixture. Pour into a
Petri dish and incubate at 20° C

Examine any white stellate colonies for Oospora lactis.

When found, make drawings of the conidia and hyphae.

Inoculate a tube of sterile milk with the fungus and incubate
at 20° for two days : examine a drop of the milk for the fungus
and note the rectangular outline of pieces of the hyphae.

Stain some with weak gentian violet.

The fungus may sometimes be obtained on cream when it is
left in a beaker undisturbed for a time.

Ex. 171. — Grow Oospora lactis in sterile litmus milk.

THE GENUS MUCOR

387

Does the latter become acid or alkaline ?
Is casein precipitated or dissolved ?
Note the odour of the milk.

5. The genus Mucor embraces another group of small
moulds, one of the commonest representatives being
Mucor MucedOy L.

(i) Mucor Mucedo, L., leads a saprophytic life, and
is very commonly found as a white fluffy mould upon
damp bread, jam, decaying fruit, and other organic
materials, especially those containing sugars and starch.

It is frequently seen on damp horse-manure, and
also occurs upon butter and cheese, in which it sets up
decomposition of the fats and proteins.

The mycelium differs from that of the previously
mentioned fungi in being unsep-
tate, or without divisions across
its hyphae. The fertile hyphae
are erect, unbranched, and bear
on their ends round structures,
100 to 200 /A in diameter, termed
sporangia^ inside which are pro-
duced a large number of smooth,
transparent, oval spores, usually
about 12 to 18 ^ long and 6
to 8 A^ broad (Fig. 54).

The apex of the sporangial
hyphae or sporangiophore extends
some way into the interior of
the sporangium as a cylindrical or ovoid bladder-like
projection termed the columella.

Most species of Mucor give rise to a sexually-produced
kind of spore — a thick-walled, black-warted zygospore —

.;

O

Fig. 54- — Sporangia of Mucor
Mucedo, L.
a Magnified 5 diameters.
b Magnified 400 ,,
c Spores ( X 500).

388

MOULDS AND YEASTS

which usually appears in the substratum on which the
fungus is growing. For further details of its origin
and structure the student must refer to text-books of
mycology.

(ii) Mucor pyriformis, Fischer, is another species
found on damp, bruised, and decaying apples and other
fruits, from the sugars of which it appears to be able to
produce small quantities of sweet-smelling esters and
citric acid.

It possesses large brownish sporangia 350 to 400 ^a
in diameter, in which are oval spores 6 to 9 /a long
and 4 to 5 /o, broad : the columella is pear-shaped.

(iii) Mucor racemosus, Fres., is a very widely dis-
tributed species upon fruit, bread, and vegetable tissues
of all kinds, and found also upon horse-dung, cheese,
and many dairy products.

Its sporangia are small, from 20 to 60 /a in diameter,
yellowish or brownish in tint, and borne
either upon simple unbranched hyphae
or on sporangiophores with short race-
mose branches (Fig. 55).

The spores are smooth and elliptical,
5 to 6 ^ long and about 4 i^ broad ; the
columella is egg-shaped.

rl I The fungus grows best at 20 to

I I 25° C, and is of especial interest on
^ " account of its power of fermenting
sugar solutions, with the production
of considerable amounts of alcohol.
This it largely accomplishes by means of sprouting
yeast-like cells which originate from submerged hyphae
or portions of the mycelium.

Ex. 172. — Place a piece of fresh horse-dung under a bell-jar

Fig. 5$.— Mucor
racemosus, Fres. (X5).

THE GENUS MUCOR 389

on wet blotting-paper ; leave until mouldy. Various species of
fungi make their appearance. Look especially for Mucor Mucedo
or M, racemosus, known by their round sporangia, which look
like small pin-heads on the ends of thin white stalks.

When obtained, take up with fine pointed forceps a very small
portion, and transfer to a drop of water on a slide. Cover with
cover-slip, and examine the hyphae bearing the sporangia ; note
the absence of septa in the hyphae.

The sporangia, with ripe spores in them, burst immediately
when placed in water.

Examine the oval spores with a high power.

To see the spores within the sporangia, take the hyphae bearing

Fig. 56. — Moist chamber for observing the germination and growth of spores in
hanging-drops. i Cardboard with circular hole punched in it, resting on ordinary
glass slide. B Section of A with cover-slip (c) and hanging-drop {r) in position.

them with forceps and mount in alcohol on a slide : then examine
quickly with a high power.

Ex. 173. — In order to observe the germination of these and
other spores, and watch their subsequent development for a time,
a moist chamber, prepared as follows, is necessary : —

Place fifteen or sixteen pieces of blotting-paper on one another
and punch out, or cut out, a round or square hole slightly less
than the size of a three-quarter inch cover-slip. Cut the blotting-
paper afterwards so as to fit on a slide, as in A, Fig. 56.

390 MOULDS AND YEASTS

A piece of stout cardboard, cut in a similar manner, may be
used instead of blotting-paper.

In the centre of a cover-slip place a small drop of water, or a
drop of a very dilute extract of French plums which has been
boiled. Shake or otherwise transfer the spore to be germinated
into the drop of water, and then place the cover-slip over the
hole in the cardboard with the drop hanging downwards, as in B^
Fig. 56.

Keep the whole on damp blotting-paper under a bell-jar.

The spores can be readily examined from day to day, even
with a high power, through the glass of the cover-slip without
moving or disturbing the latter.

Yeasts {^Saccharomyces and Toruld).

I . Saccharomyces. — The term yeast is applied to a large
series of simple unicellular fungi which have the power
of producing alcohol when grown in the juices of fruits,
malt-extract or " wort," and other solutions containing
sugars.

Those which are included in the genus Saccharomyces
reproduce themselves in two ways, viz : —

{a) By the process of sprouting, budding, or gemma-
tion.

{f}) By means of specialized spores (ascospores).

Certain unicellular fungi, closely resembling yeasts
in form and size, and in the sprouting process, but which
do not form ascospores, are classified as belonging to
the form -genus Torula.

In the vegetative state yeast plants consist of single
oval cells, which under certain conditions may become
considerably elongated so as to resemble hyphal fila-
ments, but no true mycelium is formed by these fungi.

The yeast-cell has an elastic cell-wall filled with
protoplasm in which are one or two large vacuoles.

SACCHAROMYCES 391

When grown in sugar-solutions multiplication takes place
by the process of budding, each cell sending out one or
more " sprouts " or protrusions which grow to the size
of the parent and then separate from the latter as new
individuals.

The true yeasts when grown upon slices of potato or
moist slabs of gypsum, with little nourishment, form
spores, the protoplasm of each cell dividing into two or

• ' }

Fig. 57. — Beer Yeast {Saccharomyces cerevisice, Meyer), showing
budding {a) and ascospore formation {b).

four portions which surround themselves with thin walls,
the whole yeast plant thus becoming transformed into a
spore-case termed an ascus^ the spores within being
spoken of as ascospores (Fig. 57).

Many species, races, and varieties of Saccharomyces
are recognized : they all break down sugars into alcohol
and carbon dioxide gas, forming at the same time small
quantities of glycerine, succinic acid and other organic
compounds. They hydrolyse or " invert " maltose and
other di-saccharose sugars by means of the enzyme
invertase which they secrete, but their power of producing
alcohol is due to an enzyme zymase which can only be
obtained from the cells by grinding them with sand or
other fine powder and then subjecting the dead cells to
high pressure.

392 MOULDS AND YEASTS

Most of the races of yeast used in the manufacture
of beer in this country carry on rapid fermentation at
temperatures between 1 2 to 24° C, and are lifted to the
surface of the Hquid as a frothy scum by the bubbles of
carbon dioxide set free : such are known as top-fermenta-
tion yeasts. Others, termed bottom-fermentation yeasts, and
utilized for the production of lager beer at 5 to 10° C.
produce alcohol more slowly, and accumulate as a muddy
layer on the bottom of the vessel in which the fermenta-
tion takes place.

A great number of species and varieties of yeasts
have been described : a few only need mention here.

(i) Saccharomyces cerevisiae, Meyer. Beer yeast. —
The typical form of this species, of which many varieties
exist, is roundish oval : usually i to 4 ascospores from
3 to 6 A*- in diameter, are produced in each cell the
optimum temperature for their formation when grown on
gypsum blocks is about 30° C. It ferments glucose,
saccharose and maltose, but not lactose.

(ii) S. pastorianus, Reess., possesses oval, round and

» long sausage - shaped cells

JL %^^^ (Fig. 58). The ascospores

X. m ^ are generally i to 4 in num-

am ml^ 9P m^ ^^^' ^^^ i'^om 2 to 4 ^a in

^% ^ ^ P ^ j^ diameter. They are formed

^ J ¥ ^ r § "^°st rapidly at 27 to 28° C.

^^F^ M W ^ m It is a wild yeast which gives

WW a bitter taste and unpleasant

¥10. ^z.-saccharo,^ces pastorianus, gmell to beer. It does not

ferment lactose,
(iii) S. ellipsoideus, Reess., is one of the species
or forms of yeast concerned in the manufacture of wines
from grape juice : it occurs on the exterior of grapes,

SACCHAROMYCES 393

plums, cherries and other fruits. The cells are usually-
elliptical in outline, but elongated forms are frequent.

The ascospores, 2 to 4 /x in diameter, are produced in
about twenty hours at 25° C.

Like the above species it does not ferment lactose.

(iv) S. flava lactis, Kruger. — This form produces
yellow colonies with rapid liquefaction on gelatine media :
it was originally isolated from a " cheesy " butter which
had a disagreeable odour of decomposing urine. Its
cells are ellipsoidal and about 4 ^a long. It ferments
lactose as well as maltose and cane-sugars.

(v) Saccharomyces fragilis, Jorgensen, obtained from
Kefir, ferments milk-sugar with production of a small
quantity of alcohol.

It is a small oval species, sometimes with elongated
cells : two to four oval ascospores are produced in about
twenty hours when the cells are grown on gypsum
blocks at 25° C.

Ex. 174. — Obtain a small piece of German yeast. Break it
in pieces and stir it up in water in a tea-cup. Allow the yeast
to settle and pour off the water. Mount in water a drop of the
creamy yeast left at the bottom of the cup, and examine with a
high power.

Draw a single cell and observe its cell-wall, protoplasm, and
vacuoles.

Ex. 175. — Half fill a small tea-cup with water and dissolve in it
a teaspoonful of sugar, put in a piece of German yeast about the
size of a broad-bean and stand the whole in a warm place.
When the yeast begins to accumulate on the surface of the water,
take a very minute portion and mount it in water. Examine
with a high power and sketch the budding or sprouting
cells.

Ex. 176. — Smear some of the creamy yeast from Ex. 174 on

394 MOULDS AND YEASTS

the cut surface of a slice of potato ; place the latter on damp
blotting-paper in a Petri dish and incubate at 20° C.

Scrape off daily a little of the yeast and mount in water.
Examine with an J objective for ascospores.

Ex. 177. — Take some plaster of Paris (or gypsum) and mix it
with water in the proportion of eight volumes of the powder to
three of water. Mould it rapidly before it sets into the form of
short cylinders or truncated cones about an inch in diameter
and j of an inch high, pressing the upper surfaces against a
sheet of glass to render them smooth. The material soon sets
into hard blocks which should be sterilized by heating when dry
in a hot-air sterilizer to 115° C. for one hour.

Smear some of the creamy yeast from Ex. 1 74 on the upper
surface of a block, and place the latter in a deep Petri dish.
Pour in boiled water until the block is half immersed : then
place the whole in an incubator at 20° C, and examine small
portions of the yeast in twenty-four to forty-eight hours for asco-
spores.

Ex. 178. — Repeat the above experiments, using ordinary
brewers' yeast instead of German yeast.

Ex. 179. — Inoculate gelatine plates with a drop of water in
which, yeast has been distributed : isolate the yeasts from any
accompanying bacteria and grow them upon gelatine, agar, and
potato, noting the form and character of the pure colonies.

Ex. 180. — Prepare cover-glass films of yeasts from cultures in
beer-wort or other liquid media, and stain with Loffler's methy-
lene blue or weak gentian violet ; wash, dry, and mount.

Ex. 181. — Mix some of the gypsum-block cultures showing
ascospores with a little water : spread a small drop on a cover-
glass, and after air-drying, fix and stain with carbol-fuchsin for
two minutes. Then wash in water and in alcohol to remove
colour from all parts of the cells except the spores : counterstain
with Loffler's methylene blue for three minutes.

2. Torula. — The genus Torula embraces single-celled

TORULA 395

budding or sprouting fungi, which form neither endospores
nor true hyphae or mycelia (Fig. 59). In morphology
and physiological function they are very closely allied to
the yeasts proper, differing
from the latter only in the
absence of ascospore forma-
tion. Some of them may ^t" ' *'^^
belong to the genus Saccha- ^^^ '"^^^^ ^^:x^Jy^
romyces ; others, which are
very imperfect, may possibly
be budding or sprouting ^'''- 59— t'^'-^^^ ceiis.
conidia of certain higher fungi.

The various forms of Torula have rounded, oval, or
somewhat elongated cells like the yeasts previously
described, and may occasionally be met with united into
chains of variable length. Many of them are colourless,
others are coloured a reddish-pink or rosy tint. Some
systematists classify the colourless and highly coloured
forms in the genus Oospora, reserving the generic name
Torula for those of brown, olive, and blackish hue.

These minute fungi are ubiquitous, being found in
water, in the dust of the air, in the upper layers of the
soil, and among decaying vegetable refuse. Some of
them are particularly abundant upon cheese, butter, and
other dairy products, and are able to grow and produce
acids in milk, with or without coagulation of the casein.
In Kefir and Mazun they are present, and there is some
evidence for the belief that they are of importance in the
development of the characteristic flavours of certain
kinds of cheeses and milk beverages.

Very few of them are known with a sufficient degree
of accuracy and completeness to warrant special descrip-
tion here.

396 MOULDS AND YEASTS

A form named by Harrison, Torula amara, was found
to be the cause of a bitter taste in milk and cheese, and
Rogers mentions a variety which gave an objectionable
flavour to butter which was packed in wooden casks.

Ex. 182. — Isolate and study the various kinds of Torula
occurring —

(yd) On the rind of Stilton or soft cheeses ;
{b) In soil ;

{c) In farmyard manure.
Grow them on gelatine and agar media, in plain and sugar-
bouillon, and in milk.

Note the form and colour of their colonies, their liquefying
action on gelatine, their power of coagulating or rendering milk
acid or alkaline, and their action on bouillon containing sugar.
Differentiate them from Saccharomyces.
Try growth on gypsum blocks.

Bacteriological apparatus of all kinds can be obtained from Messrs Baird
& Tatlock, Cross Street, Hatton Garden, London, E.G.

TABLES.

Measures of Length.

I inch
I foot
I yard
I chain
I mile

I millimetre =
I centimetre =
I decimetre =
I metre =

I decametre =
I hectometre =
I kilometre =

English.

12 in. =

3 ft.
2 2 yds. =

1760 yds. =

Metric System.

.001 metre =
.01 „ =

25.4 millimetres.
30.8 centimetres.
91.44

20.11 metres.
1.6 1 kilometres.

I

10

100
1000

.0393 in. or about
•393 » »
3-937 » »
39-370 „

f m.
4 in.
3ift.

metres.

. » = 1094 yds.

Measures of Area.
English.

.621 mile.

T square inch.

I „ foot = 144 sq. in.

I „ yard = 9 sq. ft. =

I acre =4840 sq. yds. =

Metric System.
I sq. centimetre = .155 sq. in.

= 1 19.6 sq. yds.

I are

I hectare

2.47

acres.

8 milliares.
40.46 ares.

= 3.9 perches.

397

398

TABLES

Measures of Capacity.

English.

I minim

I fluid drachm

I „ ounce

I pint

I quart

I gallon

60 minims
8 drachms

20 ounces
2 pints
4 quarts

.059 cubic centimetre.
3.55 „ centimetres
28.35 »
567-9 „ ..

1. 1 37 litres.

4-546 „

I fluid ounce weighs i ounce avoirdupois.

Metric System.

I cubic centimetre (i c.c.) = 16.89 minims.
I litre = 1000 c.c. = 1.76 pint = .2 2 gallon = 35.2 fluid ounces.
I gallon of water = 227.27 cubic inches and weighs 10 lbs. at
62° F.

Measures of Weight.
Avoirdupois.

I dram

=

1.77 grams.

I ounce =

16 drams

=

28.35 »

I pound =^

16 ounces

=

453-59 »

I hundredweight (cwt.) -

ri2 lbs.

=

50.8 kilograms.

I ton =--

20 cwts. = 22

40 lbs.

=

1016

I ounce =

437.5 grains.
Troy.

I grain

=

.0648 gram.

I pennyweight (dwt.) =

24 grains

=

1.55 grams.

I ounce =

20 dwts.

=

31-10

I pound =

12 oz.

=

373-2

I ounce =

480 grains

MEASURES OF CAPACITY AND WEIGHT 399

1 milligram
1 gram
I kilogram

Metric System.
.001 gram =

I
1000

grams

The circumference of a circle
The area of a circle
The surface of a sphere
The volume of a, sphere

.015 grain (Avoir).
15.43 grains „
2.2 pounds „

= 'Kd.

4'n'r^ or -rd^

3.1416.

diameter.

radius

400

TABLES

Comparison of the Centigrade and Fahrenheit
Thermometer Scales.

(i) To convert degrees Centigrade to Fahrenheit,
multiply by 9, divide by 5, and add 32.

(ii) To convert degrees Fahrenheit to Centigrade,
subtract 32 and multiply the remainder by 5 and divide

by 9.

•°c

°F.

°C.

°F.

0 =

= 32

26 =

= 78.8

I

33-8

27

80.6

2

35-6

28

82.4

3

37.4

29

84.2

4

39-2

30

86.

5

41.

31

87.8

6

42.8

32

89.6

7

44.6

33

91.4

8

46.4

34

92.2

9

48.2

35

95-

10

50.

36

96.8

1 1

51.8

37

98.6

12

53.6

38

100.4

13

55-4

39

102.2

14

57-2

40

104.

15

59-

41

105.8

16

60.8

42

107.6

17

62.6

43

109.4

18

64.4

44

1 1 1.2

19

66.2

45

113-

20

68.

46

114.8

21

69.8

47

II 6.6

22

71.6

48

1 1 8.4

23

73-4

49

120.2

24

75.2

50

122.

25

77'

°C.

y.

51 =

[23.8

52

[25.6

53

[27.4

54

[29.2

55

131-

56

132.8

57

1346

58

136.4

59

[38.2

60

140.

61

[41.8

62

f43-6

^l

t45.4

64

[47.2

65

[49.

66

[50.8

67 ]

[52.6

68 ]

[54.4

69 ]

[56.2

70 ]

[58.

71 ]

59.8

72 ]

61.6

•73 1

63-4

74 ]

[65.2

75 1

67.

°C.

°F.

76-

= 168.8

77

170.6

78

172.4

79

174.2

80

176.

81

177.8

82

179.6

83

181.4

84

183.2

85

185.

86

186.8

^7

188.6

88

190.4

89

192.2

90

194.

91

195.8

92

197.6

93

199.4

94

201.2

95

203.

96

204.8

97

206.6

98

208.4

99

210.2

100

212.

INDEX

gn

He

Acid albumins, 84, 91, 92.

curd, 236.

fast organisms, test for, 325.

Acidity of milk, 311.
Adipocelluloses, 227.
Aerobic, 31.
Agar-agar, 50.

albumose, Ex. 68.

glucose, 57.

jrape-sugar, 57.

Teyden, 121,

lactose, 57.

litmus-glucose, 57.

milk-sugar, 57.

nitrate, 165.

nutrient, 51.

Alanine, 89.
Albumens, 82.
Albuminoid, 82, 83.
Albumins, 83.
Albumose agar, Ex. 68.
Albumoses, 91, 92.
Aldehyde catalase, 245.
Algae, 4, 5, 185 ; blue-green, 23.
Alinit, 183 ; bacillus, 126, 186.
Alkaloidal reagents, 87.
Alveoli, 232 (Fig. 39).
Amino acids, 89, 93.
Amphoteric reaction, 234,
Amylase, 72, 73.
Amylolytic, 74.
Amy loses, 72.
Anaerobic, 31.

bacteria, cultivation of, 64.

Antiseptic, 39.
Antiseptics, 98.

action on soil, 130.

Arginine, 89.
Arthrospores, 21.
Artificial starters, 339.
Ascospores, 391,
Ascus, 391.
Aspartic acid, 89.
Aspergillus, 382.

Jlavus, 384.

glaucus, 383 (Fig. 52).

z c

Aspergillus niger, 384.

Oryz(B, 384.

Assimilation of nitrogen, 169.
Autoclave, 36.
Azotobacter, 175.

aoilisj 179.

Beijerinckii, 179.

chroococcuvi, 178 (Fig. 32).

vinelandii, 179.

Bacillus, ii, 24, 25, 27 (Fig. 5).

acidi lactici, 274.

amylobacter, 229, 286.

avtylozyma, 287.

asierosporus, 124.

boocopricus, 291.

bulgaricus, 276, 317, 318, 319.

butyricus, 291.

caucasicjis, 276, 315.

Chauvoei, 287.

colon, 106, 306.

cyanogenes, 283.

denitrificans , 164.

ellenbachensis, 124, 1*6, 183, 184.

enteritidis sporogenes, 216, 287

305. 330-

graveolens, 124.

Guillebeau, 275.

lactis eryihrogenes, 281.

mazun, 317.

megatherium, 125.

mesentericus vulgatus, 126.

mycoides, 125.

nobilis, 367.

cedematis maligni, 123.

pneumonicB, 27^.

prodigiosus, 282.

pumilus, 124.

putrificus. III.

rum-inatus, 124.

saccharobutyricus , 287.

subtilis, 124 (Fig. 27).

tetani, 123.

thermophilus, 30.

tuberculosis, 324.

tumescens, 124.

401

402

INDEX

Bacillus typhosus^ 327.

viscosus, 280.

vulgaris, 104,

vulgatus, 126.

Welchii, 330.

Bacteria, 2, 5, 12.

action of heat on, 30.

light on, 29.

oxygen on, 31.

water on, 31.

butyric acid, 286.

classification of, 23, 59.

food of, 32-34.

forms of, II.

identification of, 59.

in cheese, 366.

in farmyard manure, 215.

in milk, 248, 265.

in soil, 123, 136.

isolation of, 43.

lactic, 270.

movement of, 15.

naming of, 25.

• on roots, 123.

registration of, 59.

size of, 13.

staining of, 6,

Bacteriaceae, 27.
Bacterium, 25, 27.

acidi lactici, 273, 274, 306.

aerogenes, 107.

agile, 161.

casei, 2^6.

caucasicnm, 276, 315.

centropunctatum, 162.

cloaccB, 108, 306.

coliy 106 (Fig. 26).

commune, 106, 306.

commttnior, 107.

coscoraba, 108, 306.

danicus, 184.

denitri/icans,i.z^t^, 160.

erythrogenes, 281.

Jilefaciens, 163.

Jluorescens liquefaciens , 108.

non-liquefaciens, 109.

putidus, no.

Griinthal, 108, 306.

Guillebeau, 280, 374.

Giintheri, 271.

Hertlebii, i5i.

■ Hessii, 280.

immobile, 109.

lactis, 271.

— ^— acidi, 271,

Bacterium lactis aerogenes, 107, 216,
274, 306.

cyanogenes, 283.

erythrogenes, 281.

in7iocuus, 275.

pituitosi, 280.

viscosum, 275, 280.

limbatum, 275.

neapolita?ius, 108, 306.

nitrovornm, 163.

oxytocus pernicios2is , 108, 306.

prodigiosum, no, 282.

putidum, 216.

pyocyaneum, 109.

Schafferi, 215, 374.

Schirokikhi, 162.

Stutzeri, 160.

syncyaneum, 283.

synxanthum, 282.

termo, 104.

vulgare, 104.

Zopfii, 105.

Bacteroidal tissue, 194 (Fig. 35).
Bacteroids, 194, 195 (Figs. 35, 36).
Beef broth, nutrient, 56.
Beer yeast, 392.
Beggiatoa, 28.
Beggiatoaceas, 28.
Bismarck-brown Cladothrix, 128.
Bitter cheese, 376.

milk, 283.

Biuret reaction, 85, 87.
Blood corpuscles, 302.

in milk, 302.

Blown cheeses, 275, 373.
Blue milk, 282.
Borax, 42.
Boric acid, 42.
Bouillon, 56 ; nitrate, 165.
Brownian movement, 15.
Bryophytes, 5.
Buchner, 70.

tube, 65 (Fig. 25).

Butyric acid, 79.

bacteria, 286.

fermentations, 284.

Butyrin, 79, 346.
Butter, 344.

aroma, 348.

bacteria in, 346.

cheesy, 393.

composition, 345.

defects, 349.

fats, 345.

flavour, 348,

INDEX

403

Butter milk, 344.

rancid, 350.

salt in, 355.

taints, 349.

tallowy, 354.

turnip flavour, 354,

Calcium caseinogenate, 236, 237.
Cane sugar, 72, 76.
Capsules, 12 (Fig. 7).
Carbohydrates, 72.
Carbol fuchsin, 8.
Carbolic acid, 40.
Casease. 72, 367.
Casein, 235, 237,

soluble, 237, 238.

Caseinogen, 83, 235, 237.
Caseogluten, 361.
Caseones, 361.
Caseose, 95, 361.
Catalase, 116.

aldehyde, 245.

Cellulose, 75.

fermentation of, 75, 226.

hydrolysis of, 75,

Cheese, aroma, 362.

bacteria in, 366.

bitter, 376.

composition, 359.

defects, 373.

flavour, 363.

gassy, 275, 373.

ripening, 359, 364, 371.

Cheeses, blown, 275, 373.
Chlamydobacteriaceas, 27.
Chlorella, 185.
Chlorophyceas, 185.
Chromogen, 61.
Chymosin, 357.
Cilia, 15.
Cladosporium butyri, 354, 385.

herbarum, 384.

Cladothrix, 26, 27.

"Bismarck-brown," 128,

dichotoma, 128 (Fig. 28).

• fungifor7nis, "129.

intestinalis, 129.

profundus, 129.

rufula, 129.

Classification of bacteria , 23, 59.
Clostridium, 20 (Fig. ii).

americanum, 173.

butyricum, 286.

giganteum, 174.

lichenifonne, 370.

Clostridium pastorianum, 171.

Clotting enzymes, 71.

Coagulase, 357.

Coccacae, 26.

Coccus, II (Fig. 5).

Coliform organisms, 306.

Colony, 45 (Fig. 17).

Colostrum corpuscles, 241 (Fig. 40).

Colour in milk, 280.

Columella, 387.

Commercial starters, 339.

Conidiophore, 383.

Conjugated proteins, 84.

Conn's aroma bacillus, 275.

Cornet forceps, 7 (Fig. 4).

Corpuscles, colostrum, 241.

Cream, 332.

composition, 332.

ripening, 334.

Crenothrix, 26, 27.
Cryptogams, 5.
Culture, pure, 43, 51.
Curd, acid, 236.

rennet, 237.

Cyanophyceas, 185.
Cysteine, 89.
Cystine, 89.
CystocQCCus, 185.
Cytase, 76.

Decay, ioi

Denitrification, 152.
Dextrin, 73.
Dextro-lactic acid, 268.
Dextrose, 78.
Diarrhoea, summer, 329.
Diastase, 67.
Diphtheria, 331.
Dtplococcus, 19 (P'ig. 9).
Dirt in milk, 300, 310.
Di-saccharoses, 72, 76.
Disinfectants, 39.
Dispora caucasica, 315.
Dung, 211, 212, 214.
Durham's tubes, 62 (Fig. 23).

Eckle's bacillus, 275.
Ectoenzymes, 70.
Endoenzymes, 70.
Endospore, 20.
Enzymes, 67, 69.

clotting, 71.

proteolytic, 90.

of milk, 242.

Epidemic diarrhoea, 329.

404

INDEX

Equatorial germination, 22 (Fig. 12).

Eubacteria, 26.

Eumycetes, 4.

Eurotium herbariorum, 384.

Facultative, 32.
Faeces, 211, 213, 214.
Farmyard manure, 211.

bacteria in, 215.

Fats, 78.

of butter, 345.

of milk, 238.

Fehling's solution, 78.
Fermentation, lactic, 266.

of cellulose, 226.

of urine, 217.

67, 69.

butyric acid, 284.

tubes, 62, 63.

Fermentative lactic acid, 269.
Ferment, organized, 69.
Ferments, 67.

extracellular, 70.

intracellular, 70.

unorganized, 69.

Ferns, 4, 5.

Films, 6.

Filtration, 292.

Fixation of nitrogen, 169.

Flagella, 15 (Fig. 8).

■ staining of, 16.

Flowering plants, 4, 5.
Flowerless plants, 4, 5.
Food of bacteria, 32-34.
Foot and mouth disease, 327.
Foremilk, bacteria in, 249.
Formaldehyde, 40.
Fructose, 71, 78.
Fruit-sugar, 78.
Fungi, 4, 5.

in soil, 136.

Galactose, 78, 243.
Gases of milk, 239.
Gassy cheeses, 275, 373.
Gelatine, decomposition of, 95, 97.

glucose, 57.

lactose, 57.

litmus glucose, 57.

nitrate, 165.

nutrient, 46, 56.

Gelatines, nutrient sugar, 57.
Germination of spores, 21, 22, 23.
Giltay's solution, 168.
Globulins, 83.
Gluco-proteins, 84, 85.

Glucose, 72, "jj.

Glutamic acid, 89.

Glycerides, 79, 345

Glycerine, 79, 80.

GlycocoU, 219.

Gram's stain, 8, 10.

Granulobacter saccharobutyricus^ 287,

Granulose, 286.

Grape-sugar, 72, tj, 78.

Hanging block, Ex. 14.

drop, 62, 389 (Figs. 22, 56).

Hay bacillus, 124.

Hexone bases, 94.

Heyden agar, 121.

Hippuric acid, 212, 219.

Histidine, 89.

Histones, 83.

Hopkins and Cole, 86, 88.

Hosts, 33.

Hot-air sterilizer, 38.

Hot-water funnel, 48 (Fig. 18).

Hydrogen peroxide, 298.

Hydrolysis, 71.

Hyphas, 379.

Inactive lactic acid, 269.
Indole, 102.

test, 113.

Infection-thread, 193 (Fig. 34).
Inoculation, soil, 199-202.
Intermittent sterilization, 37, 48.
Invertase, 71, 76, 391.
Invert-sugar, 76, 78.
Involution forms, 12 (Fig. 6).
Isolation of bacteria, 43.

JOGHURT, 317.

bacillus, 318 (Fig. 50).

Koumiss, 315.

Lact-albumin, 235, 238.

Lactase, 266.

Lactic acid, 268, 269.

bacteria, 270.

fermentation, 266.

Lactiferous ducts, 232.

sinus, 232.

Lactobacilline, 317.
Lactococcus lactis, o.'jx.
Lacto-globulin , 235.
Lactose, jj.
Laevo-lactic acid, 268.
Laevulose, 78.

INDEX

405

Lange wei, 375.

Leguminous plants, 187-202.

Leucine, 89.

Leucocytes in milk, 248, 302 (Fig. 49).

Lignocelluloses, 227,

Lipase, 79, 80.

Liquefaction, 54, 96 (Fig. 21).

Liquefiers, 96.

Liquor pancreaticus, 95.

Litmus agar, 57; gelatine, 57; milk, 58.

Loffler's methylene blue, 8.

Lophotrichic, 15 (Fig. 8).

Lysine, 89.

MacConkey's bile-salt peptone water,

307-
Maltase, 72. ,
Maltose, 73, ^^.
Malt-sugar, 'jt-

Manure heap, putrefaction in, 225.
Marshall's Indole test, 114,
Marsh gas, 212, 227.
Maximum temperature, 30.
Maya ferment, 317, 318.
Mazun, 316.
Media, 54.
Meta-proteins, 84.
Methane, 212, 227.
Micrococcus, 26 (Fig. 9).

casei amari, 284, 376.

candicans, 127.

flavus, no, III.

Freudenreichii , 279.

lactis acidi, 276.

mucilaginosus , 273.

prodigiosus, 282.

pyogenes alb us, 1 10, 276.

aureus, no.

citreus, no.

roseus, 282.

Sornthalii, 374.

urecB, 219.

liquefaciens, 220.

Micron, 13.
Microspira, 27.
Milk, 233.

acidity of, 311.

action of heat on, 245.

and disease, 320.

bitter, 283.

blood in, 302.

blue, 282.

■ cistern, 232.

colour in, 280.

composition of, 234.

Milk, contamination of, 248-256.

cooling of, 293.

dirt in, 300.

enzymes, 242.

examination of, 300-308.

fats, 238.

gases of, 239.

inorganic salts of, 239.

leucocytes in, 302.

litmus, 57.

medium, 57.

minerals in, 239.

Pasteurization, 295.

preservatives, 297.

proteins of, 235.

pus cells in, 302,

red, 281.

reductase of, 244.

ropy fermentation, 278.

secretion of, 232.

serum, 235.

slimy fermentation, 278.

standard acidity, 311.

standards, 309.

sterilization, 293.

sugar, 77, 238.

to reduce bacteria in, 262.

yellow, 282.

Millon's reagent, 85.
— — test, 87.
Minerals of milk, 239.
Minimum temperature, 30.
Molisch's reaction, 85.
Mono-saccharoses, 72, 'jj.
Monotrichic, 15 (Fig. 8).
Mosses, 4, 5.
Moulds, 378.
Mucor Mucedo, 387,

pyriformis, 388.

racemosus, 388.

Mycelium, 379.

Naming of bacteria, 25.

Natural starters, 339.

Neutralization of medium, 47.

Newman's standard, 311.

Nitrate agar, bouillon, gelatine, 165.

Nitrogen, 200.

Nitrates, test for, 144, Ex. 74.

Nitrification, 134.

Nitrites, test for, 144, Ex. 74.

Nitrobacter, 138 (Fig. 30).

Nitro-bacterine, 201.

Nitrogen, assimilation of, 169-202.

fixation of, 169-202.

Nitrosomonas , 137 (Fig. 29).

4o6

INDEX

Nodules, 191 (Fig. 33).
Nostoc, 185, 186.
Nucleoproteins, 84.
Nutrient agar, 51.
Nutrient beef broth, 56.
Nutrient gelatine, 46, 56.

Obligate, 31.

Oidium lacHs, 385.

Oils, 78.

Oospora lactis, 315, 336, 347, 348, 354,

373' 385.
Optimum temperature, 30.
Organized ferment, 69.
Osmosis, 32.
Oxidases, 115.
Oxidizing enzymes, 114.

Pake's disk, 258 (Fig. 45).
Paraplectrum, 370.
Paracasein, 237, 238.
Parasites, 33.
Pasteurization, 38, 295.
Pathogenic, 33.
Pectocelluloses, 227.
Pediococci, 19.
Penicillium, 373.

brevicaule, 381.

• camembertii, 372, 381.

candidum, 372, 381.

glaucum, 380 (Fig. 51).

Pepsin, 90, 91, 93,
Peptides, 92.
Peptones, 84, 91, 93.
Peritrichic, 15 (Fig. 8).
Peroxidases, 115.
Petri dish, 44 (Fig. 16).
Phanerogams, 5.
Phenol, 40.
Phenylalanine, 89.
Phospho-proteins, 83.
Planococcus, 25, 27.
Planosarcina, 25, 27.
Planosarcina ui'ece, 221.
Pleurococcus, 186.
Polar germination, 2? (Fig. 12).
Polypeptides, 85.
Polysaccharoses, 72, 72.
Potato bacillus, 126

medium, 58,

Preservatives, 39, 297.
Prosthetic group, 84.
Protamines, 82.
Protease, 72, 96.
Proteid, 82, 84.

Proteins, 81.

colour tests for, 85.

conjugated, 84.

hydrolysis of, 89.

of milk, 235.

salting out of, 86.

Proteolytic enzymes, 90, 96.
Proteolysis, 90.
Proteoses, 84, 91, 92.
Proteus denitri/icans^ 164.

mirabilis, 104, 105.

vulgaris, 104, 330.

Z.enkeri, 104, 105.

Protozoa, 2.
Pseudomonas, 25, 27.

radicieola, 170, 193, 202.

Pteridophytes, 5.
Ptomaines, 102.
Pure culture, 43, 51.
Pus-cells, 302.
Putrefaction, 100.

in manure heap, 225.

Putrefactive bacteria, 136.
organisms, 103.

Rancid butter, 350.
Red milk, 281.
Reductase of milk, 244.
Reduction of nitrates, 152.

nitrites, 152.

Rennet, 236.

curd, 237.

Rennin, 90, 236, 357.
Reproduction by spores, 20.

vegetative, 18.

Rhodobacteriaceas, 28.
Ripening of cheese, 359, 364, 371.

cream, 334.

Roots, bacteria on, 123.
Ropy fermentation, 278.

Saccharimeter tubes, 63. "
Saccharomyces, 390.

cerevisicB, 392.

ellipsoideus, 392.

flava lactis, 393.

fragilis, 393.

pastorianus, 316, 392.

Saccharose, 72, 76.
Saponification, 79.
Saprophytes, 33.
Sarcina, 19, 27 (Fig. 9),
Sarcina lutea, iii, 315.
rosea, 282.

INDEX

407

Scarlet fever, 332.
Schardinger's reagent, 245.
Schizomycetes, 23.
Schizophyceoe, 23.
Schizophyta, 23.
Sclero-proteins, 83.
Septic sore throats, 331.
Silica jelly media, 146, 148.
Skatole, 102,
Slants, 53 {V\g. 20).
Slides, to clean, 5.
Slimy fermentation, 278.
Soaps, 79.

Soft cheese ripening, 371.
Soil, action of heat on, 130.

antiseptics on, 130.

bacteria, kinds of, 122.

numbers of, 118.

denitrifying power, 208.

fermentative action, 206.

fungi in soil, 136.

inoculation, 199-202.

nitrifying power, 208.

nitrogen-fixing power, 209,

putrefactive power, 207.

urea-splitting power, 207.

Soluble casein, 237, 238.

Sore throats, 331.

Sour-milk beverages, 313.

Spawn of fungus, 379.

Sphcerbtilus, 27.

Spirillaceas, 27.

Spirosoma, 27.

Spirillum, 12, 27 (Fig. 5).

Sporangia, 387.

Sporangiophore, 387.

Spore, 20 (Fig. 11).

Spores, germination of, 21, 22, 23.

staining of, 22.

Stab culture, 52 (Fig. 19).
Staining of spores, 22,
Standard acidity, 311.
Staphyloccoais, 19 (Fig. 9).
Staphylococcus pyogenes aureus, no.
Starch, 73.

hydrolysis of," 73.

Starters, 339.

natural, 339.

pure culture, 340.

Steam sterilizer, 37.
Stearic acid, 80.
Sjearin, 80.
Sterigmata, 379.
Sterigmatocystis niger, 384.
Sterile, 35.

Sterilization, 35, 48.

of milk, 293.

intermittent, 37, 48.

Stichococczis, 185, 186.
Streak culture, 52.
Streptococcus, 19, 26 (Fig. 9).

hollandicus, 273, 279.

hornensis, 280.

Kefir, 272.

lacticus, 271, 272 (Fig. 46).

longus, 97.

mastitidis, 273.

pyogenes, no, 273, 330.

StreptotliTix, 121.
Strippings, bacteria in, 250.
Summer diarrhoea, 329.
Symbiosis, 124, 185, 186, 195.

Tables, 397.
Temperature, maximum, 30,

minimum, 30.

optimum, 30.

Test, biuret, Ex. 45.

curd, Ex. 162.

Fehling's, Ex. 44.

for acid-fast organisms, Ex. 150.

acidity in milk, Ex. 134.

albumose, Ex. 51.

• Bs. enteritidis, Ex. 151.

— — dirt in milk, Ex. 143.

enzymes, bacterial, Exs.

56-58.

in milk, Exs. 120-122,

indole, Ex. 63.

nitrates, Ex. 74.

nitrites, Ex. 74.

oxidases, Exs. 66, 67.

peptone, Ex. 51.

proteins, Ex. 45.

proteose, Ex. 51.

reductase, Ex. 122.

of soil activity, Ex. loi.

Wisconsin curd, Ex. 162.

Thallophytes, 4, 5.
Thiobacteria, 27.
Thiothrix, 28. '
Torula, 390.

amara, 2,77^ 396.

ellipsoidea, 315.

Kefir, 314.

Toxins, 103.
Trypsin, 93, 94.
Tryptophane, 86, 89, 93,
Tuberculin, 326.
Tuberculosis, 321.

4o8

INDEX

Tuberculosis, bovine, 326,
Typhoid fever, 327.
Tyrogen, 367.
Tyrosine, 85, 89.
Tyrothrix, 367.

Unorganized ferments, 69.
Urea, 212, 217.
Urease, 72, 218.
Uric acid, 212, 219.
Urine, 212.

fermentation of, 217.

Urobacillus Duclauxii, i-z-z.

Freudenreichiiy 223.

Leubei, 221.

Maddoxii, 223.

Miguelii, 221.

Pasteurii, 221.

Schutzenbergii, 222.

Uro-bacteria, 217.
Urococcus Dowdeswellii, 220.
-^ — Hansenii, 220.

17.

Van Ermengem's method
Vibrio, 12 (Fig. 5).

denitrificans, 163.

Vibrion butyrique, 286.

Vitellin, 83.

Voges-Proskauer reaction, 108, 114.

Water-glass media, 146, 148.
Whey, as medium, 58.

Xantho-proteic reaction, 85, 88.

Yeasts, 590.

bottom fermentation, 392.

top fermentation, 392.

flask, 65, 290 (Fig. 47).

Yellow milk, 282.
Yoghurt (^Joghurt), 317.

ZOOGLOiA, 12.
Zygospore, 387.
Zymase, 70, 391.

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UNIVERSITY OF CAUFORNIA UBRARY