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Presented to the
LIBRARY of the
UNIVERSITY OF TORONTO
by
PROFESSOR P. BYER

(M°AINSHACO)

LIMITESO

THE EXAMINATION OF MILK

FOR

PUBLIC HEALTH PURPOSES

JOSEPH RACE, F.I.C.

City Bacteriologist and Food Hxaminer, Ottawa; Chairman of Committee on
Standard Methods of Analysis, Canadian Public Health Association,
Member of Committee on Municipal Food Administration,
American Public Health Association

FIRST HDITION

NEW YORK
JOHN WILEY & SONS, Inc.

Lonpon: CHAPMAN & HALL, Limitep
1918

Copyright, 1917
BY
JOSEPH RACE

“LIBRARy
JAN 22 1997

) Levan 0610S

’

PREFACE

Tuts volume is primarily intended as a practical handbook
for those engaged in the chemical and bacteriological exami-

nation of milk for public health purposes, but it is also hoped

that it will be of material assistance to students and others who
have previously assimilated the fundamentals of bacteriologi-

. eal technique.

The control of milk supplies was formerly confined to a
chemical examination for adulteration, but since the beginning
of the 20th century the bacteriological examination has been
regarded as a ‘“‘ sine qua non,” and in America the present
tendency is to have both examinations made under the super-
vision of the Public Health Authorities. For this reason no
apology is necessary for the inclusion of chemical methods and
the data which will enable the examiner to interpret the results
obtained.

In the bacteriological section an attempt has been made to
include all methods that have been proved to be reliable and in
some instances the details of the standard methods of the
American Public Health Association have been given; in other
cases the report as published by the A.P.H.A. should be con-
sulted. |

The tables of bacteriological results have been added in
the hope that they will lead to the standardisation of records.
At present the results reported by many laboratories are not
comparable because of the form in which they are issued.

JosEPH RACE.
Orrawa, ONT.,
December, 1917.

CONTENTS

CHAPTER
Di GINO OF BAPE io is oc acd a ss uew aioe ss
Fat. Lactose. Proteids. Salts. Gases. Enzymes. Immune

bodies. Physical constants.

II. Norman CoMposiTION OF MILK.........5....0cccceececces
Average composition. Influence of brood, food, season,
milking interval, and stage of lactation on milk constituents.
Colostrum. Abnormal milk. Influence of disease. Milk
adulteration. Milk standards.

BEE, CoemICAL ERAMIN ATION (0) 2). i yk eee eee ee at
Fat. Total Solids. Ash. Specific Gravity. Solids Not-
fat. Lactose. Proteids. Acidity. Aldehyde value. Min-
eral constituents. Refraction of serum. Preservatives.
Coloring matter. Milk products. Cream. Enzymes.

er eeaT A FR OMIT aap ee ae was ada eye 5 Mente d
Intra-mammary milk. Efforts to obtain sterile milk. Fore
milk and strippings. Influence of washing, brushing, dust,
food, vessels, coolers, and storage conditions. Germicidal
action. Development of various organisms in milk.

V. Tae Enumeration or Bacteria IN MILK..................
Reasons for determination of total count. Relation of count
to toxicity. Plating methods. Gelatine. Agar. Compari-
son of media. Acidity. Accuracy of counts. American
standard method. Direct methods of Slack, Stewart, and
Breed. Indirect methods. Methylene blue test. Acidity.

Mra URCIOMEDOT AR: CORAMIOMIM, 55.0555 hos aba d cielacle vip ounce vce'e
B. coli. Occurrence of B. coliin milk. Estimation of B. coli.
Enrichment methods. Plate methods. Classification of
type. B.enteritidis sporogenes. Streptococci.

aes PETS AIMGA IIB niles Cie a he oie cele ede cobb be
Streptococci. Septicsore throat. B.diphtheriz. Diphther-
oid bacilli. B. typhosus. Gaertner group. Morgan’s Ba-
cillus No. I. B. tuberculosis. Pseudo tuberculosis.

Vv

66

93

113

135

150

vi CONTENTS

CHAPTER PAGE
VIII. Cutzs, Dint and. DBBRis) s 5.60.6 ick said oe eae kee eis 171
Cells. Epithelial cells. Blood cells. Estimation of cells.
Centrifugal methods. Direct methods. Significance.
Standards. Dirt and Debris. Nature of. Sedimentation
and centrifugal methods. Filtration methods. Significance
of dirt.

X: Misch. canmoue coc oi ike iebieva ea eatin wins as 5 ER 185
Pasteurised and Heated Milk. Effect of heat on cream line,
peroxidases, reductase, albumin, and rennin coagulation.

B. abortus. Acid producing organisms. Aciduric bacilli.
Fermentation test. Collection of samples. Recording

results.
fe a1 1 © OR SURE IRIN oe Ee, A 4 NYT ORR yn Mad I kf a aR SEU AM 207
Composition of special media. Useful tables.
Nase Dome 6. a i ee ees ais ba Ea yepo 217

EXAMINATION OF MILK
FOR PUBLIC HEALTH PURPOSES

CHAPTER I
CONSTITUENTS OF MILK

MILK is the opaque white fluid which is secreted by the
mammary glands. It consists essentially of an emulsion of
fat and a colloidal solution of caseinogen in water containing
lactose and traces of mineral matter. |

“Milk fat, with which is associated small quantities of
cholesterol, lecithin, and a trace of colouring matter, consists
of a mixture of triglycerides of various fatty acids. These
acids are mixtures of the straight chain series CnH2on41.COOH
and CaHon-1COOH, the less saturated acids being, accord-
ing to the best information, entirely absent. The relative
proportions of the various acids are by no means constant, being
dependent upon various factors such as foodstuffs, seasonal
variations, breed of cattle, and climatic conditions.

The fat is present in milk as enormous numbers of
small globules. and it is the reflection of light from these par-
ticles and those of caseinogen that produces the character-
istic white opaque appearance of milk. Although it was for-
merly held that the fat globules were surrounded by albuminous
membranes which preserved the form, it is now generally
accepted that this is due to surface tension and that the size
of the globules can be altered by physical methods.

The size of the fat globules in milk varies from 0.8u to 20u
with an average of about 2.74 and the number of globules
from 19X10* to 60X10® per cubic centimeter. Although no

2 CONSTITUENTS OF MILK

definite relation has been established between the breed of
cattle and the size and number of globules there are a number
of results which indicate that during interrupted milking the
size of the globules increases with the fat content and also that
as the lactation period proceeds the globules decrease 1 in size
and increase in number (see p. 43).

The origin and method of formation of milk fat have not
been discovered although many hypotheses have been proposed.
The normal process seems to be the formation of milk fat,
directly or indirectly, from nutritive fat, but when this source
is eliminated the formation of milk fat proceeds, though dimin-
ished in activity, by drawing upon the body fat. Even when
the body fat is exhausted, milk fat can be formed: this is attrib-
uted to proteids acting as the source of fat.

The various analytical and physical constants of milk fat
are:

Specific gravity oe APE ey 0.9094—0.9140
Refractive index, 35°C......... 1.4550-1.4586
Melting-point.................. 28° C.-36°-C.
Solidifying point............... 21° C.-27° C.
Reichert-Wollny value.......... 25-27

Iodine absorption.............. 31-35

The calorific value of butter fat, according to Stohmann,
is 9.231 calories per gram and according to Atwater, from 9.320
to 9.362 calories. A value of 9.3 is usually employed in cal-
culating the calorific value of milk fat. The molecular weight
of fat, as calculated from the amount of alkali required for
saponification and assuming that all the acids present are mono-
basic, is from 720-740, whilst the direct determination by the
eryoscopic method points to values from 696-716. The
presence of dibasic acids would harmonise these two sets of
results, but such acids have not been isolated from butter fat.

Lactose. Although there is some evidence of the presence
of traces of a monosaccharide in milk, the carbohydrate secreted

LACTOSE | 8

under normal conditions ee ee Lactose is a
disaccharide of the empirical formula Ci2H2201; and is found
in the milk of most mammals. Lactose is secreted in the gland
and is found only in the milk, though, if suckling is interrupted,
it may appear in the urine, from which is it eliminated on re-
moval of the lactating gland: if the gland is removed before
the lactation period commences it may not appear at all. The
fact that the blood in the mammary vein before parturition and
during lactation contains less dextrose than the blood of the
jugular vein (Kaufman and Lagne) suggests either dextrose, or
the constituents from which dextrose is formed, as the source
of lactose.

Two forms of laetose exist, and are known as the alpha and
beta varieties. When lactose is obtained by pause, ster

from water, the alpha modification, which crystallises in the
rhombic form, is formed: this modification exhibits the phe-
nomeno irotation, 1e., shows a decreasing specific
rotation with lapse of time after solution in water. For a
short period of time, the length of which depends upon the
temperature, the solution of alpha lactose shows a specific
rotation of fa]Jp>=+84.0, but this gradually diminishes until a
value of +52.5 is reached, this being the specific rotation of the
stable variety of lactose containing one molecule of water.
The corresponding value of the anhydrous lactose is +55.3.
Anhydrous lactose, obtained by heating the hydrated carbo-
hydrate to 130° C., does not produce multi-rotation in aqueous
solutions. The beta modification, produced by rapid evapora-
tion of aqueous solutions of lactose in metal vessels, has a
specific rotation [a]p+32.7 and shows the same birotation

initial rotation
” final rotation
This shows that the reaction is mono-molecular in character.

The density of the alpha variety is 1.545 ee and that of a
solution containing 10 grams per 100 c.cms., 1.0391 ee The
specific rotation is [a]p=52.5 at 20° C. and is lowered 0.075 for

each degree rise in temperature. The refractive index pp?°° of

ratio, Le as the alpha modification, viz., 1.6.

4 CONSTITUENTS OF MILK

a solution containing 10 grams per 100 c.cms. is 1.3461 and of a
5 per cent solution 1.3395.

Lactose is_not fermented by ordinary yeast (Saccharo-
mycetes cerevicie) and is not affected by € ordinary enzymes.
The enzyme lactase, which is capable of hydrolysing lactose
into dextrose and galactose, is found as an endo enzyme in
Torula kefyr and T. tyrcola and also as an exo enzyme in
Kefyr grains.

C12H22011 + H20 = CeHi206+CeHi20c.

Lactose Dextrose Galactose

Lactase is also widely distributed in the animal, kingdom,
being present in the mucous membrane of the stomachs of
infants and also in the expressed juices of muscle, liver, lungs,
and pancreas.

The action of acids generally is similar to that of lactase,
though the mineral acids are much more effective than those
of the organic series. Dextrose and galactose, according to
Fischer, have the following constitutional formule:

COH COH
n—O_OH nO_OH
HO—C—H HOO
n_b_on Ho—6_H
H-O_On n_O_OH
éH.0H non
Dextrose Galactose

These formule show both sugars to be isomeric aldoses of
the monose type. Their specific rotatory powers [a]p are

Dextrose. Galactose.
aitlineiins tote sia ..5-0 5 alan oka a ae 52.7 80.3
Alpba mogitication .o5:) 5 6s Ge a 105. 120.

Broan Taw no. We eae 2. 1.5

LACTOSE 5

The most important products derived from lactose, in connec-
tion with the bacteriological examination of milk, are the lactic _
acids. Lactic acid (CaHsOs) exists as four different isomers,
three having the constitutional formula CH3-CH(OH)-COOH
or alpha hydroxy propionic acid, and one CH2(OH) -CH2-COOH
hydracrylic acid or beta hydroxy propionic acid. As the latter
is not produced during the bacterial decomposition of lactose
no further description of this acid is necessary in this work.
Alpha hydroxy propionic acid, or lactic acid as it is usually
known as, contains an asymmetric carbon atom

H

:
CH3—C—COOH

|
OH.

and exists, therefore, in three different forms, viz., dextro,
levo, and racemic or inactive lactic acids. The dextro and
lzvo rotatory acids are both produced by micro-organisms, but
unless pure cultures are employed the majority of the acid
produced is of the racemic (d+) variety.

The density of lactic acid is 1.2485 ss and the refractive
index yg20° 1.4469. On evaporation of aqueous solutions of
lactic acid dehydrolactic acid CeH1005 is produced, and, ulti-
mately, at higher temperatures, lactide CeHgO4 is formed. The
boiling point of lactic acid is 83° C. at 1 mm. pressure and 119°
C. at 12 mm. pressure. Lactic acid, though insoluble in petro-
leum ether, is soluble in, and miscible with alcohol and ether
in all proportions.

Lactic acid forms well-defined salts with various metals
and these may be used for the separation of the acid. The
calcium salt which crystallises with 5 molecules of water is
soluble to the extent of 9.5 per cent in cold water: zinc lactate
(ZnCe6Hi904-3H20) is less soluble, 1.3 per cent in cold water
and 13 per cent in hot, and forms well-defined monoclinic
prisms.

6 CONSTITUENTS OF MILK

Proteids. The proteids of milk are:

Per Cent.

Caseinogen, 0°65 sod atens approximately 2.0-3.0

Lactalbumin.:: . 2 5.0 ,es5% approximately 0.3-0.8
Lactoglobulin............ a trace
‘Mucoid proteid.......... a trace

Caseinogen* is a distinctly acid phospho proteid which does
not contain purine or pyrimidine derivatives. Lactalbumin, as
its name implies, is one of the albumins and, therefore, soluble
in water and coagulated by heat. Lactoglobulin is insoluble
in water. but soluble in salt solutions.

According to Richmond the proteids of milk are characterised
by the following reactions: Caseinogen is precipitated by adding
sodium chloride, magnesium sulphate, or ammonium sulphate

to saturation: globulin is soluble in a saturated solution of ©

sodium chloride but is precipitated by magnesium and ammo-
nium sulphates: albumin is soluble in saturated solutions of
sodium chloride and magnesium sulphate but is precipitated
_by ammonium sulphate. Albumin, however, may be precip-
itated by magnesium sulphate in slightly acid solutions but is
redissolved on neutralisation of the solution. These reactions
are relative rather than specific and cannot be relied upon for
quantitative separation of the various proteids: they may,
however, be used for preparing the pure proteids by redissolving
and reprecipitating the various fractions. Other methods may
also be used for the separation of the proteids. For example,
the caseinogen may be removed by the action of chymase, the
lab ferment of rennet, or by filtration through coarse porcelain:
filtration through fine porcelain or boiling with a small quan-
tity of acid followed by filtration will remove all the proteids.
- Lactalbumin is slowly coagulated by heating at 70° C., but

very little is precipitated when the acidity is normal. Casein- _
* Caseinogen is used in these pages to designate the mother substance —

and paracasein the rennet transformation product: this nomenclature,
though not strictly logical, eliminates the ambiguity that arises from the
difference in the prevailing English and American phraseology.

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.

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airy” Sia

4a FRR! Gartte

CASEINOGEN 7

ogen and albumin may also be precipitated by the addition of a
_ solution of calcium chloride if the milk is previously heated to
35° to 45° C. All three proteids are soluble in alkalies and

insoluble in alcohol and ether: their copper, mercury, and other
salts of the heavy metals are insoluble, and all the lacto proteids
are completely precipitated by tannin and phosphotungstic acids.

Caseinogen, when pure, is a white, amorphous, odourless,
and alae i tatarterctehictt ie pratically coluhic in water:
The specific gravity is 1.257. Owing to the stability of the
additive compound which calcium caseinogenate forms with
calcium phosphate, in which form it is present in milk, the
preparation of pure caseinogen is a matter of considerable dif-
ficulty, and it is probable that at least a portion of the differ-
ences in composition found by various observers is due to this
factor. Repeated precipitation and solution remove the
greater part of the calcium but the last traces are extremely
difficult to eliminate (Van Slyke and Bosworth!). Caseinogen
is easily precipitated by the addition of a few drops of glacial
acetic acid to milk diluted with an equal volume of water, and
the precipitate may be redissolved by the addition of caustic
alkalies, alkaline earths, ammonia, carbonates, bicarbonates, or
phosphates, even in minute quantities. Schryver? has shown
that if the caseinogen produced by precipitation with acetic

- acid is allowed to remain in contact with the excess of acid

-

‘

(1 in 1000) at room temperature, or is heated with water to
37° C., a product is formed the solubility of which in lime water
is only about one-third that of natural caseinogen. This has
been designated as “metacaseinogen,” the solution of which in
half saturated lime water is opalescent but not opaque. Meta-
caseinogen can be reconverted into caseinogen by solution in
sodium hydrate and precipitation with acetic acid providing
that the contact with the acid is not unduly prolonged. Meta-
caseinogen is identical in composition with caseinogen: the
following are some of the more authentic analyses of caseinogen.

Most of the analyses given were obtained from material
prepared by Hammerstein’s method, i.e., by repeated precip-

8 CONSTITUENTS OF MILK

TABLE I

Q
es
oO
Z
wa
ae)

Hammerstein (1883-1885)........... 52. 96)7 .05/22 .73)15.65/0.76 0.85
Chittenden and Painter (1887)....... 53 .30]7 .07)22.03/15.91/0.82 |0.87
Lehmann and Hempel (1894)........ 54.00/7.04]..... 15.60|0.77 |0.85
Ellenberger (1902).................. 53 .07|7 .13/21.74/15.64/0.76 |0.80
Lacqueur and Sackur (1903).........}..... ret Siege 15.45|0.76 |0.77
Bary (TOE) ois is cdi cc's cco Cape eae as 52.82/7 .09/22 .92/15.64/0.72 10.81
RADE (LOO). iis soon k's Ck eee wa 52 .69/6.81/23.14/15.65/0.83 10.88
Van Slyke and Bosworth (1913) mean.|53.17|7.09/22.48/15.67/0.77 |0.82
Geéake (1913) 355.5 SUG 53. 20/7 .09)/22 .34)15.63/1.015/0.73

itation with acid and solution in alkali, and it is possible that
during this process a portion of the sulphur was removed as
sulphides as the sulphur portion of the molecule is slightly un-
stable. Lehmann’s material was obtained by filtration through
porous plates and probably contained a portion of the lime salts
which constitute part of the caseinogen complex in milk. From
the percentage composition, Richmond has calculated the em-
pirical formula for caseinogen to be Ci62He5sN4iSPOse2, and in
support of this he quotes experiments * in which he found that

N ; i :
100 potassium and sodium carbonate solutions, when treated

with an excess of caseinogen, dissolved 1.83 and 1.86 parts per
100 c.cms., respectively. The above formula, according to Rich-
mond, would give 1.84 parts per 100 c.cms. The author in
some unpublished experiments, determined the solubility of
caseinogen in 100 KOH and obtained a value of 1.83 grams per
100 c.cms. at room temperature (67° F.): other temperatures,
however, gave different values, so that these results cannot be
regarded as having any bearing on the constitution or weight
of the molecule. Various compounds of caseinogen with bases
have been reported. Soldner* separated compounds of casein-
ogen and lime containing 1.11 and 1.67 per cent of Ca., re-

CASEINOGEN 9

spectively. Lehmann’s material as separated by filtration
contained 1.02 to 1.25 per cent of Ca. Van Slyke and Bos-
worth! report four compounds with lime, containing 0.22, 0.44,
1.07, and 1.78 per cent of Ca. They also prepared compounds
| with ammonia, sodium, and potassium, containing 0.20 per
cent NH:, 0.26 per cent Na, and 0.44 per cent K. h

The acidity of caseinogen has been determined by many
observers with fairly good agreement. The more important
_ results are:

N 1 Casei
1 c.c. Preise equals sain OE a

equals
3 N
_ Lacqueur and Sackur...... 0.1138 gr. caseinogen | 8.81 c.c. joNa0H
_ Mathaiopoulos............ 0.11315 8.84
Na aciele «. si0: oS s:0'n 0 « - 0.1124 8.90
Van Slyke and Bosworth...| 0.1111 9.00

From the analysis of the lime salts, Van Slyke and Bosworth
regard caseinogen as an octobasic acid and classify these salts as
follows: :

Grams PER 100
GRAMS REACTION TO

CASEINOGEN. Valencies

Name of Compound. Satisfied.
: Phenol
Ca CaO Litmus. Phthalein |

0.22 | 0.31 |Monocalcium caseinogenate | ......]|...... 1

0.44 | 0.62 |Di calcium caseinogenate | ......|...... 2

1.07 | 1.50 |Neutral calcium caseinogenate| Neutral| Acid 5

1.78 | 2.50 |Basic calcium caseinogenate | ...... Neutral 8

From a consideration of the dissociation values of caseino-
genates in dilute solutions, Lacqueur and Sackur® regarded
caseinogen as either a penta or hexabasic acid but a later inves-
tigation of the physical properties by Robertson® shows that it is

10 CONSTITUENTS OF MILK

octobasic. This would give a molecular weight of approxi-
mately 8900.
Caseinogen, when dissolved in dilute alkali, has a pronounced.
levo rotatory action on polarized light, but the specific rota-
tion is not constant, varying from —94.8 to — 111.8, according ;
to the concentration and nature of the alkali used as the solvent
(Long). The soluble salts of caseinogen may be divided into
two classes (1) salts of the alkaline earths, and (2) salts of the
alkalies. According to Osborne7 these are distinguished by
the inability of the former to pass through the film of the
Martin gelatin filter and by the formation of opalescent solu-~
tions. The solutions of the second class filter through gelatin
membranes and are translucent. Both classes of salts are
neutral to phenolphthalein when the valency of caseinogen is
entirely satisfied, but when litmus is used as the indicator no
definite change is indicated and the point of neutralisation
varies with the concentration of the solution (Schryver).
Salts of copper, mercury, and lead, precipitate caseinogen
from neutral solutions, and mercury salts are also effective in the
presence of acid: the precipitates so obtained are not constant
in composition but vary with the conditions obtaining. The
insolubility of the compounds with the heavy metals is utilised
in milk analysis in the preparation of protein free milk serum _
for use in the polarimeter and refractometer. Caseinogen also —
exhibits basic properties and combines with acids with the |
formation of clear solutions. Long* found that 1 gram of

caseinogen combined with about 7 c.cms. of a acid in the form

of sulphuric, hydrochloric, hydrobromic, hydriodic, and acetic
acids to form soluble salt like compounds. Some observers —
have stated that precipitated caseinogen also combined with
acids but L. L. Van Slyke and D. D. Van Slyke® have shown
that the observed loss of acid on precipitation was due to sur-
face adsorption and depended upon the nature and concen-
tration of the acid, the temperature, the duration of contact,
and the degree of agitation.

CASEINOGEN 11

When caseinogen is acted upon by formaldehyde, the amino
groups condense with the H-CHO to form methylene deriva-
tives. The resultant compounds are not digested by trypsin
but can be decomposed by steam and the formaldehyde quan-

| titatively recovered in the distillate. On the formation of
| methylene derivatives, the alkalinity due to amino groups dis-

appears, and the caseinogen salt, which before condensation ©

| reacted neutral to phenolphthalein, becomes acid and can be
_ quantitatively titrated with alkalies. This reaction is the

basis of the aldehyde value (vide p. 75).
Caseinogen, on hydrolysis by pepsin, trypsin, or dilute
acids, undergoes proteoclastic digestion with the formation of

_ easeinogen proteoses or caseoses, as they have been called, which

are soluble in water. ‘These caseoses have been subdivided
into proto and deutero caseoses by their solubility in ammo-
nium sulphate solutions of certain concentration.

The ultimate products of the hydrolysis of caseinogen have
been extensively investigated and the results of various ob-
servers, obtained with caseinogen from various sources, are
given in Table II.

Caseinogen exists in milk as a salt combined with phos-
phate of calcium, and although the composition of this com-
plex has been investigated by many chemists during the last
sixty years, it is impossible even yet to state that it is defi-
nitely established. Richmond, from an analysis of the mate-
rial separated by filtration through a porous cell, assumes that
caseinogen exists in milk as a double calcium sodium caseino-
genate combined with half a molecule of tricalcic phosphate.
Cie2H255N418PO52-Ca-Na3z(CagP20g). The quantity of acid
required for the displacement of the sodium atom in this formula
by hydrogen, would be 8.3 c.cms. of normal acid per litre of
milk, and Richmond found that on adding 8.6 c.cms. N. hydro-
chlorie acid or sulplturic acid, the caseinogen was precipitated
on boiling, and that the acidity of the serum was equal to that
of the milk after boiling. L. L. Van Slyke and Bosworth
have pointed out that deductions based on the acidity of milk

12 CONSTITUENTS OF MILK

TABLE II
CASEINOGEN HYDROLYSIS PRODUCTS
Cow’s MILK. ‘Goat’s Miix. | Human MIxx.
r er,
Products of Hydrolysis. if PEI HOM re ag 9 .
iedaadaa ase Schittenhelm.) | Schittenhelm.)

pCR? koe 5 Cca's ok 0 0
Aa SO. ork cee 0.90 1.50
Wee ie ss3 ies 1.00
Leaeme.'.'). 2 as 10.50 7.40
Phenylalanine......... 3.20 2.75
Fyrosine s+ vgs nav ee 4.50 4.95 4.71
BOT. soo ks so ehh Ook 0.45
COMO oi o's ed ek es 0.06
Prodi, ..o Va ee 6.70 4.60
Oxyprolitie «.s4.436 Cui 1.50
Aspartic acid......... 1.20 1.20
Glutamic acid......... 11.00 12.00
Tryptophane.......... 1.50
ATGADIDO os die cents es 4.84
pee ee las el 5.80
Hiptidine 3.205 eee8t 2.59
Diaminotrioxy-

dodecanic acid...... 0.75
Aminovaleric acid... .. 7.20
ATMO: 4 o4 F< ees 1.60

and milk serum, as determined in the usual way by direct titra-
tion with alkali, may be entirely fallacious because of the errors
introduced by titrating phosphoric acid in the presence of lime
salts. Cameron and Hurst!!! have shown that the following
reactions may occur. |

(1) CaHPO, +2H20 =Ca(OH)2+H3POu.
(2) 2CaHPO,+Ca(OH)2=Ca3P20s +2H20. |

These result in the presence of free phosphoric acid in place
of neutral dicalcium phosphate and the acidity is, therefore,

CASEINOGEN | 13

apparently higher. When milk is filtered through porcelain,
the acidity of the serum is usually approximately half that of
the original milk on direct titration with alkali, but Van Slyke
and Bosworth have shown that, if before determining the acidity,
the lime salts are previously removed by precipitation with
neutral potassium oxalate, the acidity of the serum is equal to
that of the milk: in other words, the caseinogen calcium phos-

‘phate complex in milk is not acid to phenolphthalein but neu-
_ tral. Van Slyke and Bosworth !? filtered milk through porcelain
but instead of analysing the precipitate, compared the serum and

the original milk. This eliminates errors caused by the absorp-
tion of soluble salts if the first filtrates of serum are rejected.
Their results show that caseinogen exists in milk as neutral
calcium caseinogenate (caseinogen, Ca4) and neutral dicalcium
phosphate. These are not in chemical combination as they
could be almost completely separated by mechanical methods.

The reaction of caseinogen with rennin, a lab ferment, is
of considerable importance because of the information it yields

_ regarding the constitution of caseinogen, and also on account

of the presence of this ferment in the mucous lining of calves’
stomachs and the similarity of its action to that of the gastric

_ juices of the human stomach. Although this reaction has been

the subject of probably more investigations than any other sub-
ject in biological chemistry the modus operandi and the nature
of the reaction products are still comparatively obscure.

It has long been known that fresh milk coagulates in the
stomachs of the higher animals. An aqueous extract of the
inner lining of the stomach of the calf causes curdling and clots
milk producing a semi-solid mass. These facts have been

utilised since an early date in the manufacture of cheese.

The earlier views concerning the nature of this change need
not be considered in. detail as they have since been proved to
be entirely erroneous. The one most commonly accepted

_ regarded the action as one of decomposition of the milk sugar

into acids, which directly or indirectly produced the phe-
nomenon observed. The first important advance was made

14 CONSTITUENTS OF MILK

when Heintz ‘* found that the muscosa extract of stomachs
possessed the property of clotting milk of an alkaline reaction.
Hammerstein,!* and Schmidt,'> first showed that the coagulation
of milk by rennin was due to a soluble ferment which was named
“labferment.”’ or ‘‘ chymosin.””’ Hammerstein thoroughly in-
vestigated the nature of the reaction and his conclusions met
with fairly general acceptance until a few years ago. He
showed that caseinogen was not in true solution in milk but in a
state of colloidal suspension, and that the presence of a certain
quantity of calcium phosphate was necessary for the reaction
to occur: also that during the reaction the caseinogen was so
altered that it was unable to remain in colloidal suspension
and was precipitated in the presence of calcium phosphate as
paracasein calcium phosphate. He further found that the
caseinogen was split into at least two other proteids, casein (der
Kase) better described as paracasein, and whey proteid (Mol-
keneiweiss). These were distinguished by the insolubility in
water of the calcium salts of the former compared with the
smaller molecule of the latter and the solubility of its calcium
salts. The composition of these proteins according | to Koster
is shown in Table ITI.

TABLE III |
Paracasein. Whey Proteid.
CPUC bi sso ser epee EUR Mle CRM aa 52.88 59.33
Fi yarowen i sail. 0s feo eae ee 7.00 7.00
NRDOMERL Ss. 6.2655 5463 2 ee eee 15.84 13.25
Phosphorous (Richmond)............ 0.99

From these figures Richmond has calculated the approximate
formule for these substances to be.

Paracanein : 232052 oe ees C140 H222N36PO44.
- Whey protetd. ic. c¢s sea oe Ceo2H37N5Q0j0.

Hammerstein concluded that the conversion of caseinogen into

CASEINOGEN 15

paracasein was independent of the calcium salts present and
this has been confirmed by later observers. Some chemists
(Loevenhart?® and Briot!”), have claimed that an essential part
of the rennin reaction is a modification of the mineral con-
stituents, but Harden and Macallum!® have recently shown that
if caseinogen solutions are treated with sufficient rennin
(1 : 1000) no addition of calcium salts is required: Schryver ?
found that clot formation could be obtained in the entire absence
of calciumions. Duclaux!® was the first to find that no proteo-
clastic cleavage is produced by the action of rennin and this has
been confirmed by Van Slyke and Bosworth,?° Geake,?! and
Harden and Macallum.!® Loevenhart !® suggested that caseino-
gen and paracasein were chemically identical and that the differ-
ences in behaviour were due to changes in molecular association
or aggregation. This view is supported by Van Slyke and Hart?
and Van Slyke and Bosworth (vide supra) who suggested that
calcium caseinogenate is split by the action of rennin into two

molecules of calcium paracaseinate which is identical in per-_
centage composition with the’ original substance. Liwschiz?3
attempted to differentiate caseinogen and paracasein by biolog-
ical methods. Three methods were tried, precipitation, com-
plement binding, and anaphylaxis, and of these only comple-
ment binding gave positive results under certain conditions.
The other two methods entirely failed to distinguish between
the two substances. Schryver? has suggested that all the
substances necessary for clot formation pre-exist in milk and —
that aggregation is prevented by the absorption of simpler
molecules from the system. He formed the conception that a
ferment, for which the colloidal substances could act as a sub-
strate, could clear the surface of such substances of adsorbed
bodies and thus allow aggregation (clot) formation to take
place. He found that milk serum, Witte’s peptone, or glycine,
inhibited clot formation by rennin, and also that apparently
typical milk clots could be formed by the addition of calcium
chloride to calcium caseinogenate solutions and warming.
These differ from rennin clots, however, in their ability to pro-

16 CONSTITUENTS OF MILK

duce clottable solutions on dispersion by acidification after
solution in alkali. Clots produced by the action of rennin
cannot be redispersed, a fact that suggests some alteration in
structure. Schryver found that calcium caseinogenate solu-
tions on warming, and sodium caseinogenate solutions after
treatment with carbon dioxide in the cold, would produce clots
with rennin and suggested that these observations point to the
formation of caseinogen by the action of heat in the former, and
carbon dioxide in the latter, and that clot formation is produced
by the action of rennin on the free caseinogen or metacasein-
ogen (see p. 7).

Some observers have stated that a change in reaction occurs
during the action of rennin but Hewarden *4 found that hydrogen
ions were not necessary for the coagulation of milk or solutions
of caseinogen containing calcium. The author has found that
the curd produced from milk by rennin usually has an acidity
equivalent to 8.3 to 8.8 c.cms. of normal acid per litre of milk,
an amount which is identical with the acidity of the caseinogen
in the solution from which it is produced.

Caseinogen is also clotted by the action of trypsin and other
enzymes, but in the case of trypsin there is definite evidence of
proteoclastic cleavage with the formation of soluble com-
pounds Containing nitrogen and phosphorous.

Heating milk to 70° C. and upwards, retards the velocity
of the rennin reaction by partial destruction of the enzyme and
precipitation of the calcium salts:—refrigeration-also prevents
the formation of the characteristic curd but this property is
regained on heating to 37° C. (Morgenrath).

The optimum reaction temperature for rennin is about 40° C.
and at temperatures exceeding this it is gradually weakened and
finally destroyed: the destruction by heat follows the law of a
monomolecular reaction. The velocity of the rennin reaction
follows the usual laws until 40° C. is reached when the observed
values become smaller than the calculated values owing to
partial weakening of the enzyme by heat. Some of the results
obtained by Field on this subject are given in Table IV.

LACTALBUMIN ' 17

TABLE IV
T K K
10,000
Temperature. Time in Seconds. Observed p Calculated.
25 54 185 185
30 32 312 327
35 17 588 574
40 10.2 980 980
44 9 1111 1491
50 14.7 680 2742

The time required for the coagulation of milk by rennin,
other conditions being equal, is inversely proportional to the
concentration of the enzyme. Acids and salts of the alkaline
earths accelerate the reaction, while alkalies, albumoses, and
large amounts of neutral salts, retard it: the fat content also
influences the velocity of the reaction. The reaction can be
inhibited by the addition of normal horse serum and a similar
effect is produced by the anti-rennin prepared by. Morgenrath *°
by repeated injection of rennin into the blood stream of rabbits.
As the inhibitory action of horse serum can be prevented by
neutralisation with acid (Raudnitz and Jakoby) it seems prob-
able that both horse serum and anti-serum act by fixation of
the calcium ions.

Lactalbumin. This constituent of milk has, according to
Sebelien, the following composition:

Carbon. |Hydrogen.| Nitrogen. | Sulphur. Oxygen.

Lactalbumin........}| 52.19 | 7.18 15.77 1.73 23.13

These results show that the essential difference in compo-
sition between the albumin of milk and the phospho proteid
(caseinogen) lies in the absence of phosphorus in the former and
its markedly higher content of sulphur.

Lactalbumin follows the general reactions of other albumins
in being soluble in neutral saturated solutions of magnesium

18 CONSTITUENTS OF MILK

sulphate, but is precipitated by the addition of small quantities
of acetic acid. It is stated that lactalbumin may be obtained
in a crystalline form by diluting the saturated magnesium sul-
phate solution with an equal volume of water and setting aside
after the addition of acetic acid until permanently turbid.

Lactalbumin is also precipitated by sodium and ammonium
sulphates when added to saturation. Tannin, phosphotungstic
acid and other general reagents also precipitate lactalbumin:
the salts of the heavy metals are insoluble in water. Lactal-
bumin is insoluble in alcohol and this reagent may be employed
for the precipitation of lactalbumin from aqueous solutions:
the precipitate so obtained is easily soluble in water.

Lactalbumin is a white powder possessing neither taste nor
odour. It coagulates at 70° C. but the precipitation is never
complete. The specific rotatory power, according to Béchamp,
is[a]p = —67.5, but Sebelein obtained values varying from
—36.4to —38.0. Lindet 7° obtained a value of only —30.0, so
that apparently the preparations of both Béchamp and Sebelein
were mixtures of lactalbumin with some other substance, prob-
ably caseinogen [a]»=—119, having a much higher rotatory
power.

Lactoglobulin. Comparatively little is known regarding
the globulin constituent of milk. It is precipitated by neutral
sulphates such as magnesium sulphate but is quite soluble in
sodium chloride solutions even after acidification. It is not
clotted by rennin but coagulates under the action of heat: alone
at a temperature of 72° C. (Hewlett).

Probably not more than 0.1 per cent of lictoglobull is
present in normal milk although considerably more may be
found in colostrum.

Mucoid Proteid. This substance, according to Storch,
contains 14.76 per cent of nitrogen and 2.2 per cent of sulphur.
It is a greyish white powder which is slightly soluble in dilute
sodium and potassium hydrates though insoluble in ammonium
hydrate, acetic, and hydrochloric acids. Mucoid proteid gives
the usual proteid reactions with Millon’s reagent (red), and

SALTS 19

iodine (brown), and the xantho proteic reaction. On hydrolysis
with hydrochloric acid it yields a quantity of a substance capa-
ble of reducing Fehling’s copper solution.

This proteid is probably identical with the 6 casein of
Strewe who separated it from a casein (caseinogen) by dissolving
out the latter with ammonium hydrate.

Salts. In addition to the various acids and bases which
form part of the caseinogen complex, the serum of milk contains:
various salts_in solution. The average percentage of ash in
SE ae shout O75 per cent but fluctuates considerably. The ©
average composition of the ash of milk, as obtained by igni-
tion is given in Table V.

TABLE V
COMPOSITION OF ASH OF MILK (Ricumonp)

Per Cent.

IMT rg ook RO dhs GWU yc Lieto daha leis os 20.27
i oe SA She WS eh eet ro as yah ahee 2.80
AS a ES Say ne ee egress: AS, otal SEIN AF 28.71
ME oe iets ay, Bas Pm lek Ng oe ie ane? 6.67
NE 05 k.as Gul eee Siew ak aes oh ook 29.33
SER SRR aR gery ae get 0 eine NE Ue ENE ee 14.00
IU i lone hy. Si ure: Aug uke CL 0.97
TION tio d oo £55. soa Gey OMEN OR eH ors Trace
SE EER A PO PEED 1G a) Od 0.40
103.15

MEM. Fain a pies sib!o ped Ab AY need oles as fi 3.15
100.00

Distribution of the phosphoric acid.

Grams per 100 c.cms.

P.O; as caseinogen combined with NaCa....... 0.0605
P.O; as Ca,P2O0, coi Bite adi oo eared a tol @ 310 tar aia aA ite! Siwel'e 0.0625
P.O; as R,HPO, VAT IE WS re aC NM Ree 0.0770
y O98 Bi 15 8) ah I Sa SEE CRIES a AR Sea 0.0200

20 CONSTITUENTS OF MILK

The following results of Van Slyke and Bosworth show the
composition of milk serum as separated by filtration through
porcelain candles.

TaBLE VI
COMPOSITION OF MILK AND MILK SERUM
Ae $e Percen of
eee iw: (ae Gontaiedioe
in Serum.
ee sh 5 2 ao. ae ee ee 5.75 5.75 100.0
Cascinogen .... ists. cca eee 3.07 0.00 Nil
AIDGHNIN . 06 ta5 Ve erie dee 0.506 0.188 37.1
Nitrogen in other compounds... ... 0.049 -| 0.049 100.0
Chitke G6... i455 eA sli aha 0.237 0.237 100.0
Phosphorus (organic and inorganic)| 0.125 0.067 53.6 *
Phosphorus (organic)............ 0.087 0.056 64.4
Calera o.4 46s vs sau ceca yes 0.144 0.048 33.3
PEneORN. ss oe te 6 ok a eS 0.013 0.007 53.8
POLINA 4 5.5 esas se reek SEER 0.120 0.124 100.0
Sonam) 3A ee ees bees 0.055 0.057 100.0
Chlorine, «93:i iw Rea yas Sess 0.076 0.081 100.0
Adie. 4 oe haaeete peek bets 0.725 0.400 55.2

* Not obtained on same sample.

Van Slyke and Bosworth suggest that the various combina-
tions of acids and bases in milk are:

Proteins combined with calcium
Di-calcium phosphate (CaHPO,)

Calcium chloride

Mono-magnesium phosphate (MgH4P20s)
Sodium citrate (NasCeHsO7)... 0. eee ec ce een
Potassium citrate (K3Ce6H;07)
Di-potassium phosphate (K2HPQ,)

©¢ 6€ 646 8 2: 2 84.9 Be 8 BE 2 9 OC. Tae

40. 8. 6:9 le

Other constituents which have been found in minute traces
_are fluorine, iodine, silica, acetates, and thiocyanates.

Lecithin.
tities

CasHo009NP also exists in milk in minute quan-

ENZYMES 21

Gases. There is no definite evidence of the existence of
gases in milk as drawn from the udder, but, during this process,
it absorbs the normal constituents of the air. Two analyses of
milk gases by Winter Blyth are given in Table VII.

TABLE VII
COMPOSITION OF GASES IN MILK

Milk after Standing

Fresh Milk. ives Mean:

Cubic centimeters per 1000 c.cms. of milk

Carbon dioxide............. 0.06 60.47
PRINS i as. ok os sis > 19.13 9.30
ara a 77 .60 30.21

Blyth found that, on standing, the oxygen usually dis-
appeared in about twenty-four hours and that the carbon dioxide
content increased until it finally reached over 95 per cent of the
total gases, the residue being nitrogen.

Enzymes. It has been indubitably proved that fresh milk
contains a number of the substances known as enzymes, bodies
which are remarkable on account of certain properties which
they possess. Small quantities appear to be capable of pro-
ducing radical chemical changes without themselves under-
going alterations, although their activity is diminished by the
transformation products.

Enzymes are specific in character, i.e., only certain specific
enzymes are capable of acting upon certain compounds, and
this property has led to the adoption of a nomenclature which
classifies the enzyme in accordance with the nature of the com-
pound acted upon or the nature of the action produced. For
example, the enzyme acting upon amylose is known as amylase,
whilst lactase, glucase, and protease, act upon lactose, glucose,
and protein, respectively: oxidases and reductases oxidise
and reduce, and catalase acts as a catalytic agent.

Enzymes are thermolabile, have optimum temperatures of

22 CONSTITUENTS OF MILK |

reaction, and are injuriously influenced by toxins and various
salts. As they have never been isolated in a pure condition,
comparatively little is known as. to their composition and it is
by their properties rather than differences in apeheiire a that
enzymes are recognised.

Amongst the various enzymes that have been discovered
in milk are amylase, galactase, lipase, lactokinase, peroxidase,
reductase, and Catalase.

Amylase. Béchamp 2’, in 1883, prepared an amylase from
human milk that converted soluble starch into sugar as readily
as amylases from other sources. The presence of amylase in
cows’ milk has been denied by Moro, der Velde, Landtsheer,
and Kastle and affirmed by Zaitschick, Koning, Seligman,
Jensen, and others. The author has invariably found amylase
to be present, although only in minute quantities.

Galactase. This protease was first found in milk by Bab-
cock and Russell in 1897 78. They found that fresh centrifuge
slime showed proteolytic properties even when all bacterial
activity was checked by the presence of antiseptics. Wender 2°
has shown that the galactase prepared from centrifugal slimes
is not a pure enzyme but a mixture of galactase with peroxidases
and catalase. The presence of catalase in milk has, however,
been confirmed by von Freudenreich, Jensen, Spolverini, and
others. The action of galactase on proteids is very similar
to that of trypsin, proteoses and peptones being the inter-
mediate, and amino acids the final products.

Lactokinase, a kinase similar to enterokinase, and a fibrin
ferment have also been found in minute quantities.

Lipase, the enzyme capable of hydrolysing glycerides of
fatty acids such as monobutyrin, was found in milk by Marfan
and Gillet 9°. Cows’ milk was found to have a lipolytic activity
of 6-8 on Hanriot’s scale as against 20-30 for human milk.

Salolase. That human and asses’ milk have the property
of hydrolysing phenyl salicylate (salol) was observed by Nobé-
court and Merklen.*! The existence of this ferment in milk
was disputed by Désmouliérs and also by Mule and Willem,

CATALASE. 23

who found that the hydrolysis was really a saponification
effected by the presence of alkali and that only alkaline milks
showed the presence of salolase. Rullman, in 1910, proved
that milk obtained with aseptic precautions did not give the
salol splitting reaction. It has been suggested that salolase is
of bacterial origin, although this view is unsupported by experi-
mental data.

Peroxidases. Although Rullman has found traces of sub-
stances in milk capable of effecting oxidation by utilisation of
atmospheric oxygen (true oxidases), the peroxidases are much
more important. These ferments decompose hydrogen perox-
ide in accordance with the equation H202=H20+0. The
presence of nascent oxygen is ascertained by the addition of
some substance which undergoes a colour change on oxidation
(a chromogen). Benzidine, guiacol, ortol, amidol, p. pheny-
lenediamine, and phenolphthalin have been employed for this
purpose. Kastle and Porch *? showed that the power of milk
to induce the oxidation of phenolphthalin and other leuco
bases by hydrogen peroxide is greatly intensified by the addi-
tion of certain substances of the phenol type.

Catalase. Catalase (Loew) or superoxidase (Raudnitz)
like peroxidase has the property of decomposing hydrogen
peroxide, but, instead of atomic oxygen being produced and
absorbed by some compound present, molecular oxygen is
formed and may be collected in the gaseous form.

2H202 =2H20+ Os.

Some authors have included catalase with the reductases in
accordance with the view that the oxygen liberated is utilised in
an oxidation process and that the reaction is essentially one of
the reduction of hydrogen peroxide to water. There is, how-
ever, as little basis for the inclusion of catalase with the reduc-
tases as with the peroxidases, for, although its action is inter-
mediate between the two, it is entirely independent of them
and well-defined in character.

24 CONSTITUENTS OF MILK

Reductases. The ferments which cause the abstraction of
oxygen from compounds without the production of gaseous
oxygen, have been termed reductases. The essential differ-
ence between this reaction and that produced by catalases is
in the utilisation or transference of the oxygen removed.

Two types of reductase have been recognized and are dif-
ferentiated by their action on methylene blue. One type,
which appears to be of cellular origin and is present in fresh
milk, rapidly decolourises methylene blue solutions in the
presence of a trace of formaldehyde, whilst the other is capable
of effecting the reduction in the absence of formaldehyde and
is of bacterial origin.

BIOLOGICAL

Immune Bodies. Although the examination of milk for the
presence of immune bodies is but infrequently required in con-
nection with public health work, a general consideration of
these bodies and their significance is of interest. Before con-
sidering these in detail it will be advisable to review briefly the
theory of immunity.

After an attack of disease-producing organisms, animals
usually possess, for a varying length of time, an immunity
against a further attack, and this immunity is ascribed to the
presence of substances known as immune bodies. The re-
searches of Ehrlich and others have established that these
immune bodies, or anti-bodies as they are generally described,
are produced by external agencies. In addition to living and
dead bacteria, other substances such as animal and vegetable
proteins, animal cells, and toxins, may act as antigens. Ehr-
lich’s theory of immunity hypothecates the existence, in the
molecules constituting both the antigen and body cell, of
binding groups or haptophorie receptors which fit “as a key
fits the lock” and which anchor the antigen to the body cell.
In the case of toxins, other receptors are also assumed to be
present, viz., toxophores, which are responsible for the toxic
effects produced after the antigen has been anchored to the cell.

IMMUNE BODIES 25

The cell molecules may be destroyed as the result of this com-
bination or it may be stimulated by defensive action to the
production of receptors; continued excitation results in the
production of more receptors than are necessary for the func-
tions of the cell and it is assumed that these receptors are set
free in the fluids surrounding the cells, and that they possess
a greater affinity for the antigen than the same receptors of the

cell molecule. These free receptors constitute the antibodies.

Three varieties of antibodies are known.

(1) Uniceptors, such as antitoxins, which are regarded
as comparatively simple and which combine
directly with the antigen.

(2) Uniceptors, which have an enzyme-producing group
in addition to the haptophoric ‘iu as (agglu-
tinins, precipitins).

(2) Amboceptors, which require the presence of a third
substance before combination with the antigen can
be effected; this third substance is known as com-
plement.

Antigens, and uniceptors produced by them, are specific
in their action, and this applies equally to the amboceptor-
complement-antigen system of the third order of receptors.
For instance, tetanus antitoxin acts on tetanus toxins and on
no others, and typhoid serum agglutinates only B. typhosus.
This statement, however, is not absolutely true, as antigens
produced by allied groups of organisms possess receptors which
are common to all, but as the specificity becomes more definite
with increased dilution of the antibody, the affinity between
the specific receptors must be considered to preponderate.
The amboceptors of thé third order of antibodies also show
relative rather than absolute specificity.

The antibodies generally are distinguishable from comple-
ments by their resistance to heat. The uniceptors and ambo-
ceptors are thermostabile, i.e., are not destroyed by heating to

i CONSTITUENTS OF MILK

6° C. for thirty minutes, whereas complement is destroyed by
this treatment; complement is, therefore, thermolabile.

Antibodies, like enzymes, are of unknown chemical constitu-
tion and are usually designated by. the nature of the action pro-
duced; thus, antitoxins neutralise toxins, cytolysins dissolve
animal cells, hemolysins dissolve erythrocytes, bacteriolysins
dissolve bacteria, agglutinins agglutinate cells and bacteria, and
precipitins produce precipitates from solutions.

Immunity, by which is understood the existence of a cer-
tain resistance toward deleterious influences, may be either
acquired or natural. The apparent immunity of individuals,
races, and species to various diseases under normal conditions
is known as natural immunity, and very little is known of the
etiological factors involved. Acquired immunity may be acci-
dental, as in the case of the immunity acquired by an attack of a
disease, or artificially acquired by the introduction into the ©
system of either antigens or antibodies. When antibodies are
employed, the immunity is but of short duration compared
with the several years of immunity obtained by the use of anti-
gens. The former process is known as passive immunity and
the latter as active immunity.

When antibodies are present in the blood, certain quantities
are excreted by the milk glands and may be found in the milk.
Ehrlich has demonstrated that offspring may, through suckling,
obtain a passive immunity from either an actively or passively
immunised mother. The antibody content of milk is usually
very much weaker than that of the blood from which it is
derived. Uniceptors of the second order are also transferable
to the milk and may be less than, equal to, or even greater, than
the amounts found in the blood. The evidence regarding the
transfer of the third order of antibodies is somewhat conflicting.
Amboceptors and complement derived from the blood may
appear in the milk, but this is unusual and various experi-
menters have stated that complement is not present in normal
ripe milk except in minute traces. In colostrum and milk
derived from udders affected with mastitis, however, both

OPSONINS 27

amboceptor and complement may be present. The applica-
tion of the complement fixation test for the detection of colos-
trum is only of scientific interest and mastitis can be much
more readily detected by an examination of the sediment of the
milk.

Opsonins, bodies which prepare bacterins for phagocytosis,
the process by which a cell (phagocyte) absorbs bacterins and
other particulate matter, have also been demonstrated in milk.

It is possible that anaphylactins, which induce the phenome-
non known as anaphylaxis or hypersensitiveness, may occur in
milk as it has been shown by Otto that the progeny of hyper-
sensitised guinea pigs were anaphylactic to homologous antigens.
The transmission, however, may have been either intrauterine
or through the milk. Mention might also be made of the bene-
ficial effect upon children suckling from mothers being treated
with ‘ 606,” although whether the results are due to the pass-
age of antibodies or arsenic is still in dispute. Considering the
indubitable proof of the passage of various classes of. anti-
bodies from the blood stream to milk, it is reasonable to assume
that aggressins, bodies which inhibit the protective power of the
cells, and toxins are also transferable. This hypothesis has
been experimentally established, but, like the antitoxins, the
amounts found in the milk are considerably smaller than in the
blood. If it is assumed that the gastro-intestinal tract of infants
is penetrated by proteids, the question of the transference of
toxins assumes practical importance. Even in individuals
showing severe symptoms, by far the greater part of the antigen
is anchored to the cell leaving but little in the free or labile
condition in the system, and, as only a fraction of this is trans-
ferred to the milk, the total amount assimilated by the off-
spring is probably negligible; & posteriori observations confirm
this deduction.

Since milk contains various proteid substances, it is capable
of acting as antigen and on injection produces a number of
antibodies. The lactoserum obtained by the use of cows’
milk contains precipitins, amboceptors, and hzemolysins, which

28 CONSTITUENTS OF MILK

are specific in their reactions and may be used as qualitative
tests for milk. Bauer succeeded in detecting as small a quan-
tity as 1 c.cm. of cows’ milk per litre of human milk by the
complement fixation method. The various proteids of milk,
caseinogen and albumin, etc., also produce specific antibodies
which may be recognised by the precipitin method. The
specificity of lactoserum, like those of sera in general, is relative
rather than absolute, the lactosera of closely related animals
being differentiated by the intensity of the reactions. The
phenomenon of anaphylaxis may also be induced by the injec-
tion of milk. Arthus and Besredka state that boiled milk, as
well as the raw product, is capable of producing the requisite
conditions, though Miessner found that a larger number of
injections were necessary before sensitisation was satisfac-
torily established. Caseinogen and albumin also produce
specific anaphylactins which may be used as a basis for differ-
ential tests.

Physical. The characteristic appearance of milk is pro-
duced by the colloidal suspension of caseinogen complex and
the emulsion of fat globules. When milk is allowed to remain
quiescent, the_fat globules, being of beech
surface and form a rage of cream w inetly yellowish
in tint, the residuat ‘milk being bluish white in colour. The
opacity is diminished by the addition of alkali, which dissolves
the caseinogen, and is increased by any process that reduces the
size of the fat globules. Heat alone, at different temperatures,
is capable of reducing the diameter of the fat globules, but it
may be more effectively accomplished by forcing milk heated
to 60° C. through very small orifices under high pressure.

The specific gravity of milk bears a definite relation to the
total solids it contains (see p. 70), being decreased by the fat
content and increased by the solids other than fat. The specific
gravity or density varies considerably with variations in season,
period of lactation, breed, and character and quantity of food,.
but 1026.4 to 1037.0 (water2-6 oe “"=1000) may be regarded as
the extreme limits. When milk, freshly drawn from the udder,

PHYSICAL 29

is allowed to stand for one hour to eliminate air bubbles, it
will be found to have a density somewhat lower than that
taken subsequently (Recknagel’s phenomenon). This pecu-
liarity has been investigated by several observers. Vieth con-
firmed Recknagel’s results and found the average rise to be
+1.3° (water=1000). H. Droop Richmond *? reports that in
70 per cent of his experiments the rise varied from 0.3° to 1.5°,
averaging 0.6°, and that in 30 per cent of the observations no
rise ‘in density was indicated; also that the rise was more
rapid at low temperatures than at high temperatures. H. D.
Richmond, from consideration of experiments made in con-
junction with 8. O. Richmond on the effect of heat upon the
density and specific heat of milk, regards the phenomenon as
largely due to the increase in density of the fat on solidification.
Changes in the milk sugar, cessation of expansion of the case-
inogen, absorption of gases, and enzyme action have also been
suggested as causes of this phenomenon but cannot be regarded

TaB.LeE VIII
EFFECT OF TEMPERATURE ON VOLUME

See Pakeoabeds, |: Volume. Dion rlaaide: ha monine
31 1.00000 60 1.00229
35 1.00016 65 1.00298
40 1.00041 70 1.00372
45 1.00074 75 1.00451
50 1.00114 80 1.00549
55 1.00164

as satisfactory. Various data confirming Richmond’s hypoth-
esis were obtained by Toyonaga, and Fleishmann and Weig-
ner.*4 The latter observers found that the change in density
was proportional to the amount of butter fat present. Micro-
- scopical examinations also showed that the solidified globules
were of greater density than the liquid globules at the same
temperatures.

30 CONSTITUENTS OF MILK

Although milk contains considerable quantities of water
(85-90 per cent), the maximum density is found at a tempera-
ture near to the freezing point and not at 4° C. as in the case of
water. The changes in the volume of milk due to temperature

alterations are somewhat variable, being dependent upon the
composition; the preceding table, due to Richmond, shows the
expansion in glass of milk containing 3.8 per cent of fat and
having a density of 1032.0.

The viscosity of milk, according to Taylor, is not prdpor-
tional to the percentage of total solids, but is a function of the
fat and the solids-not-fat content. He found that the relation
is expressed by the formula:

(viscosity —fat percentage X 0.0665)
0.177 ;

and that the viscosity temperature coefficient was

percentage solids-not-fat =

m=""+-0.00723t—0.0001560.

Taylor’s determinations of the viscosity of milk raised from 20°
to 60° C. and subsequently cooled, support the hypothesis of
Richmond regarding the explanation of Recknagel’s phenome-
non. Weigner*® found that homogenisation of milk slightly
increased the viscosity. Two samples having viscosities of
1.941 and 1.862, as determined with an Oswald viscosimeter,
were increased by homogenisation to 1.967 and 1.889, respec-
tively. Weigner thought that this was caused by increased
adsorption, especially of caseinogen.

The freezing point of milk is slightly lower than that of water,
being usually —0.54 to —0.57° C. and is especially influenced
by the mineral content other than that associated with the
caseinogen. As the salts are not subject to wide variation in
the milk of healthy cattle, the freezing point is usually fairly
constant. This forms the basis of the cryoscopic methods for
the detection of milk adulteration. Aitkens*’ shows that a
consideration of the osmotic pressure of the blood of animals
and that of the milk secreted points to the conclusion that the

(
|
f
-
:
|
:
:
;

, Tree

PHYSICAL ol

freezing point of milk will never fall below that of blood. He
found the freezing point of the blood of the cow to be —0.62° C.
and that of cows’ milk 0.55° C.-0.06° C.

In contrast with the relative constancy of the depression of
freezing point of cows’ milk, the specific conductivity shows
greater variations, although milk produced under normal con-
ditions does not show very marked differences.

The following results are given by various observers:

TaBLe [X
CONDUCTIVITY OF MILK
LL SGGE aaa aie cag eaeY nae K at 25° C. =0.00430-0 .00560
a 0 SEIS aS SR arate oan et arta a 0.00487-0 .00551
RINE cd. pov au a es iaiaie Si we Saele ee 0.00485
RRS ker vr eS aD 0.00494-0 .00517
Jackson and Rothera (1914)............. 0 .00493-0 .00641
Jackson and Rothera Herd milk (1914)... =0.00549-0 . 00587

Jackson and Rothera 3° point out that, owing to the osmotic
pressure of milk being controlled by that of the blood, the sub-
stances chiefly responsible for this manifestation, viz., the
milk sugar and soluble salts, cannot vary independently, but
must be inter-related. If the lactose is high the salts must be
low, and conversely, if the lactose is low the salts must be high
or the osmotic pressure would be lower than normal. Jackson
and Rothera found experimentally that the electrical conduc-
tivity of milk, which is mainly due to the soluble salts, is in-
versely proportional to lactose content. This inverse propor-
tionality was especially observable in milk produced under
pathogenic conditions, as shown by the following example:

Depres-

Conduc- | Lactose, sion of Sol. Ash, |Insol. Ash,

Quarter. tivity, K | Per Cent. | Freezing- | Per Cent. | Per Cent.
point. A

Left anterior (abnormal).| 0.0114 1.50 0.580 0.615 | 0.440
Right anterior (normal) .| 0.00569| 5.40 0.575 0.285 0.625

32 CONSTITUENTS OF MILK

As the proteins of milk obstruct the carriage of electricity
by the moving ions, the conductivity of whey or of serum is
greater than that of the milk from which it is prepared. Each
1 per cent of protein reduces the conductivity by 2.75 per cent
(Rothera and Jackson). The surface tension of milk is lower
than that of water, 0.053. as against 0.075 and the specific: heat
of milk containing 3.17 per cent of fat is, according to Fleish-
mann, 0.9457.

The refractive index of milk cannot be determined on account
of its opacity, but that of the serum, after removal: of the case-
inogen and fat, has been determined on a large number of sam-
ples by various observers and is now regarded as a valuable
aid in the detection of adulteration by the addition of water.

This method is of special value on account of the removal
of the constituents of milk that are most variable in amount,
viz., fat and caseinogen, leaving a serum containing the lac-
tose, mineral matter, and albumin which are generally the least
variable. Various methods, which vary somewhat in the
completeness of precipitation of caseinogen attained, have been
employed, 394° and normal values established for each. The
refractive index of fresh milk serum, prepared by filtration
through porous plates, varies from (u»20°C.) 1.34200 to
1.34275. The specific gravity of milk serum is equally as valua-
ble as the refractive index (see p. 79) but on account of the
longer time required for its determination it is not generally
used as a routine method. The ash of the serum also affords
valuable information for the detection of added water. (Lyth-
goe,*° and Burr and Berberich.*").

BIBLIOGRAPHY

. Van Slyke and Bosworth. Bull. 26, N. Y. Expt. Sta. Geneva, 1912.
Schryver. Proc. Roy. Soc., B. 86, 460-481.

. Richmond. Dairy Chemistry. London, 1914, p. 30.

Soldner. Landw. Versuch. Stat. 1888, 35, 351.

. Lacquer and Sackur. Beitr. Chem. Phys. u. Path. 1902, 3, 193.
Robertson. Jour. Phys. Chem. 1911, 15, 179.

. Osborne. Zeit. Physiol. Chem. 1901, 33, 240.

NOP ON

FSSSASRESSRSS SASRESL

BIBLIOGRAPHY 33

. Long. Jour. Amer. Chem. Soc. 1907, 29, 1334.
. L. L. Van Slyke and D. D. Van Slyke. Jour. Amer. Chem. Soc., 1907,

38, 383.

. Van Slyke and Bosworth. Bull. 37, N. Y. Expt. Sta. Geneva, 1914.
. Cameron and Hurst. Jour. Amer. Chem. Soc. 1904, 26, 905.

. Van Slyke and Bosworth. J. Bio. Chem. 1915, 20, 135.

. Heintz. Jour. f. Prakt, Chem. n. F. 6, 33.

. Hammerstein. Maly’sJahresb. 1872, 1118, cbid. 1874, 135; ibid. 1877,

158.

. Schmidt. Beitrage zur Kenntniss der Milch. Dorpat, 1871.

. Loevenhart. Zeit. f. Physiol. Chem. 1904, 41, 177.

. Briot. Etudes sur la pressure et l’antipressure. Thése de Paris, 1900.
. Harden and Macallum. Biochem. Jour. 1914, 8, 90.

Duclaux. Traité de Microbiologie. Paris, 1899, II, 291.

. Van Slyke and Bosworth. Jour. Biol. Chem. 1913, 14, 203.
21.

Geake. Biochem. Jour. 1914, 8, 30.

Van Slyke and Hart. J. Amer. Chem. Soc. 1905, 33, 461.

Liwschiz. Diss. Miinchen. 1913. Z. Kinderheilk, Ref. 8, 345.

Hewarden. Zeit. f. Physiol, Chem. 1907, 52, 184.

Morgenrath. Centr. f. Bakt. Abt. I, 26, 271.

Lindet. Bull. Soc. Chim. 13, 929.

Béchamp. Compt. Rendus. 96, 1508.

Babcock and Russell. Centr. f. Bakt. u. Par., Abt. II, 1900, 6, 17-22.
and 79-88.

Wender. Oesterr. Chem. Zeit. 6, 13.

Marfan and Gillet. Monatschr. f. Kinderheilk. 1902, I, 57.

Nobécourt and Merklen. Compt. Rend. Soc. Biol. 1901, 53, 148.

Kastle and Porch. Jour. Bio. Chem. 1908, 4, 301.

Richmond. Dairy Chemistry. London, 1914, p. 76.

Fleishmann and Weigner. Jour. Landw. 61, 283.

Taylor. J. Proc. Roy. Soc. N.S. W. 47, II, 174.

Weigner. Kolloid. Z. 1914, 15, 105.

Aitkens. Chem. News. 1908, 97, 241.

Jackson and Rothera. Biochem. Jour. 1914, 8, 1.

Arb. Gesundheits. 40, Heft. 3.

. Lythgoe. J. Ind. and Eng. Chem. 6, 904.
. Burr and Berberich. Chem. Zeit., 32, 617.

CHAPTER II
THE NORMAL COMPOSITION OF MILK

_ ‘THE average composition of cows’ milk as compared with
the milk of various other mammals is shown in Table No. X.
(Bunge ').

TABLE x °
COMPOSITION. OF MAMMALS’ MILK

Fat Caseinogen.| Albumin. | Lactose. Ash
Human (1)...:.... BoE Wt Gx toe. atees sues 5.9 0.2
Human (2) 23.5.5... 3.8 1:3 0.5 6.0 0.2
Human (3)........ Dias A Ve Poe Oho aes 6.5 0.3
DORs oct Petree 12.5 5.2 1.9 3.5 1,3
OMB a tisies ckcvenen 3.3 3.1 6.4 4.9 0.6
Rabies soos cat 10 Bac aeeee 2.0 2.6
Guinea pig........ CB .B ol sive deckie me Rear 1.3 0.6
Bam ate C.0 od ee aa as 3.8 1.1
Elephant.......... 79 60° 4s a ARS eke 8.8 0.7
BOUNDS § 538 Sci ditwdts 1:2 0.8 5.7 0.4
MBN SU hoi ua tated are 1.6 0.7 1.6 6.0 0.5
CMO elaz's's oy mle ttniace > fy mn ye | 0.9 4.9 0.7
RUNG Cate oye cane 4.8 3.2 1.1 4.5 0.8
oR Uy hag YP 6.9 5.0 1.6 4.5 0.9
Reindeer.......... 17:1 —~8.4 2.0 2.8 1.5
CAIN io uss eles re MD LER ea ype 5.6 1.8
RAR es FoR 3.2 3.0 0.9 5.6 0.8
Porpoise..:......... 54.8 7.6 0.5

Apart from the very varying amounts of fat the similarity
in the composition of the milk of these various mammals is very
remarkable.

34

AVERAGE COMPOSITION : 35

Various observers have recorded the results of thousands
_. of analyses of cows’ milk and some of the most authentic are
given in Table XI. .

) TaBLE XI

COMPOSITION OF COWS’ MILK

Average of Water. | Fat. ee carte opal Ash.
nogen.| min. | tose.

* 280,000 analyses, Aylesbury Dairy

_ Co., London, Richmond......... 87.35 |3.74| 3.0 | 0.4 | 4.7010.75

_ 5552analysesin U.S.A. Van Slyke} 87.10 |3.90] 2.5 | 0.7 | 5.10/0.70
Cheese factory milk. New York

| ‘State. MaytoNov. VanSlyke.| 87.40 |3.75) 2.45) 0.7 | 5.00/0.70

_ 800 analyses by Koenig........... 87.27 |3.64| 3.02] 0.53] 4.88/0.71

The essential difference between the European and Amer-
_ ican results lies in the ratio of lactose to proteids and the rela-
tive amounts of caseinogen and albumin that make up the total
| proteids. Numerous analyses by the author of Canadian milk
show that the average ratio of lactose to proteid in that country
is distinctly higher than those recorded by Richmond and
- Koenig. The figures of Lythgoe? for milk in Massachusetts,
_ confirm this view. At least a portion of the differences between

the relative amounts of caseinogen and albumin in the analyses

TaBLE XII
MAXIMUM VARIATIONS IN COMPOSITION
Fat. Solids Not-fat.
Per cent. Per cent.
SIE a3, 6 «0166.0 o-0\¢' ¢ 4 6.'# bo 14.67 13.76
SS Field dos ck esas seicaae. 1.04. 4.90

_ recorded in the above table is probably due to errors in the
- yarious methods used for the determination of these constit-

36

THE NORMAL COMPOSITION OF MILK

962°0.\-°"* °° €6Z'0 | 998°0 |062°0 |IT8°O |§¢2Z°0 |FO8'O | 98L°0 |'°°°* °° * ‘erp sed suey
: ‘Unies yur anos jo ysy
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Cis. ORE +. O28 2S OL. Se ee o's ee eee ee ‘uinses 1addog
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LIMITS AND VARIATIONS 37

uents. Later American analyses have shown that the normal
albumin content of 0.7 per cent, as recorded by Van Slyke,
is too high and that 0.5 per cent is much nearer the correct
value. |

Limits and Variations. The variation in the composition
of milk obtained from herds is not usually very great, but that
of individual cows may vary between very wide limits. The
following figures show the maximum and minimum that have
been recorded, the former by Cook and Hills of milk from a
Jersey cow just before going dry, and the latter by Richmond.

The fat content of milk is very variable and depends upon a
number of factors, the chief of which are breed, food, season,
interval between milkings, and stage of lactation.

The breed of the cow has a very important bearing upon the
quality of the milk produced, some (Jersey and Guernsey) giv-

ing milk containing 60 per cent more fat than others (Holstein).

Results of analyses of milk from various breeds are recorded
in Tables XIII, XIV, and XV.

TABLE XIV
FAT AND SOLIDS NOT-FAT IN MILK FROM VARIOUS BREEDS
(VIETH)
Tora. Souips. Fart. Souips Not-FratT.
Breed.
Aver- | Maxi-| Mini- | Aver- |Maxi- | Mini- | Aver- | Maxi-| Mini-
age. |mum.|mum.} age. |mum.{mum.| age. | mum. | mum.
Dairy shorthorn. .|12.90|18.70|10.2 | 4.03/10.2 | 1.3 | 8.87|10.6 | 7.6
Pedigree ‘‘ ..|12.86)16.8 |10.5 | 4.03] 7.5 | 1.9 | 8.83] 9.8 | 7.6
ae 14.89/19.9 {11.0 | 5.66) 9.8 | 2.0 | 9.23/10.4 | 8.1
IGS oral S e's 13.70|18.6 {10.6 | 4.72)10.5 | 1.8 | 8.98/10.6 | 4.9
Red Polled......|13.22)16.2 | 9.7 | 4.34) 6.6 | 2.5 | 8.88]10.2 | 7.1
ie 14.18|17.4 |11.5 | 4.87] 7.6 | 2.9 | 9.31]10.3 | 8.4
Montgomery.... ./12.61|16.1 |10.2 | 3.59) 6.5 | 1.4 | 9.02|10.0 | 7.9
BG och shea ee 14.15)17.6 {11.9 | 4.91] 8.3 | 3.0 | 9.24] 9.6 | 8.9

38 THE NORMAL COMPOSITION OF MILK

The figures in Table XV are compiled from results published
by the various American Experimental Agricultural Stations.

TABLE XV
Ratio.
Breed. Fote Fat. Lactose. | Proteid.
Solids. Lactose | Proteid
Proteid Fat

JOTSCY.. 52.55. 14.70 5.14 5.04 3.80 |! 1.32 0.74
Guernsey. 14.49 4.98 4.98 3.84 1.30 0.77
Ayrshire...... 12.72 3.85 5.02 3.34 1.50 0.87
Holstein...... 12.00 3.45). 4.65 3.15 1.47 0.91
Shorthorn 12.57 3.63 4.89 3.32 1.47 0.91
Red Pots. Roba Gee 4.03

The influence of breed upon the chemical characteristics of
the fat was investigated by Eckles and Shaw ? and their results
are summarised in Table XVI.

TaBLE XVI
EFFECT OF BREED ON CHARACTERISTICS OF FAT. (Ecxkizs
AND SHAW)
Relative Size 5 Reichert- Melting-
Breed. of Fat Rec ay ante Meissl point,
Globules. i r Value. Centigrade.
Jersey......... 328 30.5 228.9 26.7 32.9
Ayrshire....... 150 31.6 228 .2 25.9 33 5
Holstein....... 142 34.2 229.1 25.5 32.9
Shorthorn. .... 282 34.4 227.6 26.3 33.2

It is evident from the results recorded that the breed of cow
has a marked effect upon the composition of the milk obtained
and that certain constituents are more affected than others.
The fat is the most variable constituent, though the total

ee ae eae

LIMITS AND VARIATIONS 39

i amount of fat yielded by the various breeds is far less so and is
due to the quantity of milk being usually inversely propor-
tional to the fat percentage in the milk. The proportion, how-
ever, is not a direct one and it has been proved on many occa-
sions that the breeds giving the low fat percentages yield the
largest total weight of fat. For this reason the Dutch, Frisian,
and Holstein breeds are very popular for dairy purposes.
Concerning the effect of food upon the composition of milk,
numerous investigations have been made but the results ob-
tained are apparently somewhat contradictory. This is prob-
ably partially due to the conditions under which the experi-
ments were conducted being not strictly comparable. Earlier
observers failed to appreciate the fact that a certain weight of
‘ fat, proteid, and carbohydrates is necessary for providing body
_heat and for the repair of waste tissue in the cow, and that this
_ amount is proportional, though not directly so, to the weight of
the animal. If the food ration is only slightly in excess of this
' quantity, the effect of stimulants, such as oil cake, would be to
immediately increase both the percentage and total quantity
of butter fat secreted; on the other hand, if the ration is suf-
ficient for the body maintenance and milk secretion, additional
food would probably not increase either the percentage or the
quantity of butter fat, and it is conceivable that they may even
be somewhat reduced by this over-feeding process.
__ Of the more reliable investigations, those of Morgen, Beger,
Fingerling, Doll, Hancke, Sieglin, and Zielstorff* might be
“mentioned. They found that food free from fat sufficed for the
‘maintenance of animals in a healthy condition and increased
the live weight of the animal, but was totally unsuitable for
milk production. The addition of food fat in quantities
equivalent to 0.5 to 1.0 gram per kilo of the animal weight
favoured the production of milk fat. Later, the first three
_ observers, in a series of experiments extending over six years,
_ obtained results which showed that of all foods, fat alone exerts
a specific action on the production of milk fat and that, within
certain limits, fat is the most suitable food for butter fat pro-

tmp Pe rea

Se ie

ee, ae are

AO THE NORMAL COMPOSITION OF MILK

duction. Malméjac ® reports the following comparative figures
obtained in Algeria from cattle feeding on poor and rich
forage. |

Poor Dry Grass. Rich Forage.
Total aoliday \\ Fe 00.5 sas Sens oe 11.62-14.25 13.76-14.90
Babes d 5's ibe ke 4 Poe aes eee ee Bea 3.33-— 3.50 4.05-— 4.90
LAGHOED 5 as: eds Boda kok eos bbe es 4.53-— 5.64 4.47-— 5.55
PTCA. 6 <i i ols daeiew ea eae eae 3.13-— 4.46 3.33- 4.54
BR ooo oC Pea eR ALON Ce 0.60-— 0.90 0.82-— 0.93

Brewery and distillery waste grains in the wet condition
have often been fed to cows on account of the low price of this
material, but this procedure ultimately proves to be false
economy, as both the relative and absolute amount of milk fat
produced is reduced. During the last decade there has been ~
a decided tendency towards scientific feeding of dairy animals
with a well balanced ration which is just sufficient for the main-
tenance of body weight and also for the production of a definite
quantity of milk containing a specified amount of butter fat.
In this ration, digestibility, palatability, and proportion of
roughage to concentrates, are considered and calculated. An
example of this rational feeding is seen in the herds of the
Minnesota Experimental Station, as compared with the other
herds of the state. The common cows, i. e., cows with no dairy
heredity, of the Experimental Station yielded 5000 lbs. of milk
equal to 222 lbs. of butter per head as against 4000 lbs. of milk
equal to 175 lbs. of butter per head for the whole State of
Minnesota.

Stable or byre conditions, fatigue, and temperature, also
have slight effects upon the fat content of the milk produced.

The seasonal variation in the amount of butter fat in milk,
according to Droop Richmond’s figures, is well marked and
always occurs; he finds that the fat content usually decreases
during the spring and summer months, reaching a minimum
about midsummer, and then gradually rises to a maximum

LIMITS AND VARIATIONS © 41

during the winter. The fat content of the milk of Massa-
-chusetts and Ontario shows several modes during the year,
although the average values for the summer months are less -
_ than those for the winter months. Diagram No. I shows the
_ monthly variations in England for 1897-1913, as compiled
by Richmond, in Massachusetts as reported by Lythgoe of
State Board of Health, and in Ontario as calculated from the
_ Ottawa analyses of the author. Lythgoe has suggested that

DracraM No. I

EFFECT OF SEASON ON FAT CONTENT

2 BN
Fa P 6 N A ge i
fp Ate Pact oe oor me
4.0 ~ YW a / 3
ey Oo wes
- 3.9 oF x JS . iy f i VA Rea
P ff PRAY
poe
fu
3.7
By /
2 in a a a
ee cautious vatiearue
Canada— - —— -——

3.5
Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dee.

he irregularities in the curve for the Massachusetts supply, as
compared with Richmond’s results, are due to the larger number
of samples examined by the latter. Lythgoe’s results, however,
are calculated from approximately 13,000 samples examined
during three years, and the similarity of the curve to that

_ gests that the number of modes in the curves is not fortuitous,
_ but is due to seasonal variations together with variations caused
by changes of food peculiar to local climatic conditions.

Richmond also found that there were slight daily variations

42 THE NORMAL COMPOSITION OF MILK

in the quality of milk, the fat content of Monday’s milk being
usually slightly lower than that of the other days, but this is
. apparently due to the usual intervals between milkings being
slightly disturbed during the week-end.

The intervals elapsing between milkings have been shown by
various observers to have an influence on the percentage of —
fat, though relatively little on the absolute amount. Fleisch- ~
mann © found morning milk slightly richer than evening milk ~
and decided that the fat content varied with the intervals
between milking. Richmond? as the result of oyer 100,000

analyses made during sixteen years, gives the figures for the _

fat content of morning and evening milk as 3.56 and 3.93 per |
cent, respectively; the intervals being 10.8 and 13.2 hours. | |
His results also show that the difference is more marked during
the summer months. Eckles and Shaw § found that with equal
intervals between milkings, the morning milk was slightly higher
in fat content than the evening milk. The Reichert-Meissl
and Koettstoffer numbers of the butter fat were usually lower,
and the iodine number usually higher in the evening milk, while
no appreciable constant variation could be detected in the
physical characteristics. With animals milked more than
twice daily, the variations in the fat content of the milk were ~
larger and the highest value was usually found in the milk ~
drawn near the middle of the day. The explanation of this is ~
probably connected with the interval between feeding and 7
milking. |

The influence of the stage of lactation upon the fat content
of milk has been the subject of much experimental work, and
although some of the data is slightly contradictory, it has been ~
generally established that the percentage of fat usually de- ©
creases during the first three months of lactation, then remains
fairly constant for four to five months, and, finally, rises rapidly ~
to a maximum. This process is well illustrated by the results ©
of Eckles and Shaw ® which are given in Table XVII.

The chemical and physical characteristics of the butter fat
obtained in these experiments are recorded in Table XVIII.

LIMITS AND VARIATIONS
Taste XVII

43

[ AVERAGE PERCENTAGE OF FAT BY FOUR-WEEK PERIODS

PHYSICAL CONSTANTS OF FAT

AVERAGE DETERMINATIONS BY FouR-wEFx PERIODS

Jeisey. Shorthorn. Ayrshire. Holstein.
First period............. 5.20 4.08 3.94 3.14
MS ek 4.91 3.88 3.68 — 2.87
AU RS eran 5.02 3.71 3.60 2.78
ENS eae 4.79 - 3.54 3.59 3.11
a 4.88 3.56 3.70 3.11
I er 4.98 3.58 3.52 2.98
Mpeventh ............. 4.93. 3.69 3.63 3.08
aS eas ee 4.83 3.73 3.74 3.09
SS eee 4.84 4.19 3.71 3.05
eS oe doa. vs as 4.88 4.19 4.05 3.31
a ae 5.23 4.11 4.92 3.39
Ea Se. yi. NC,” aS OR eae 3.96 3.70
MME...) 5 0.--..-- Seca SANS IROL alee 4.18 4.48
Fourteenth ............. 2 7 AUR (SSUES DaG ERI Ee nn 3.68
TaBLE XVIII

i RELATION OF LACTATION PERIOD TO CHEMICAL AND

: Relative Melting . s Reichert- | Saponifica-
Period. gs a Size of cry Seca Meissl tied ,
Globules. | Centigrade. : Number. Number.
1 4.00 357 31.7 33.3 29.1 223.7
2 3.85 307 32.9 31.6 27.5 230.4
3 3.79 249 32.8 32.2 27.1 231.0
4 3.77 256 33.1 30.8 26.4 229 .6
5 3.82 200 33.3 31.4 26.6 229.2
6 3.79 204 33.2 31.7 26.4 228.9
a 2 3.83 201 33.3 32.9 25.5 225.7
8 3.85 192 33.4 33.3 22.2 226.7
. 9 3.97 180 33.5 34.6 24.2 225.6
10 4.11 152 33.9 35.4 22.5 223 .4
11 4.22 162 34.7 35.5 22.2 223.8
12 4.54 166 33.8 35.2 20.3 220.6
13 4.66 110 36.5 39.2 17.2 216.6

44 THE NORMAL COMPOSITION OF MILK

It was found that the size of the globules at the commence-
ment of lactation was about twice the average size for the whole
period; the size sharply diminished during the first six weeks —
and then, after remaining fairly constant for some months,
rapidly declined. The iodine value varied directly with the
fat content, and the saponification value, after a preliminary
rise, declined slowly, but gradually, with the constantly decreas- _
ing proportion of volatile fatty acids. The melting point ~
remained comparatively steady until the last periods when a
perceptible rise occurred; the refractive index showed no appre- _
-ciable variations. ; j

Non-fatty Solids. The non-fatty solids of milk are subject —
to variations from causes similar to those which determine ~
the variation in the fat content. The influence of breed is |
shown in Tables XIII, XIV, and XV, and that of season in |
Table XIX (Richmond "°). . tf

|
|

TABLE XIX
INFLUENCE OF SEASON ON SOLIDS-NOT-FAT
Month. Fat, [eee Tp ceues 4 Wate: 1 Aan
not-fat.

JADUAPY, . . kemcnteuylee 3.80 | 8.95 | 4.62 | 3.57 | 0.76 —
February.............- 3.70 | 8.97 | 4.70 | 3.62 | 0.75
DAATORGS 6 ou eee cates 3.62 9.91 4.72 3.45 0.74
Ap ae oe co 3.62 8.83 4.66 3.42 0.75
Mavis ot Sean 3.47 | 8.85 | 4.64 | 3.47 | 0.74
RS ee a ee 3.44 8.82 4.68 4.42 -| 0.74
Faby cy vem 3.59 | 8.67 | 4.69 | 3.23 | 0.75
BURR Ca eine ee 3.72 8.55 4.59 3.25 0.71
September............. 3.88 8.63 4.63 3.25 0.74
Cee oe 2 baa ei ies 3.91 8.76 4.63 3.38 0.75
Novembe?.) 3.) ocua 34) 3.94 8.81 4.63 3.42 0.76
December... vaweees’ 3.80 8.81 4.56 3.50 0.75

These results show that the solids-not-fat decline sympa-
thetically with the fat during the spring and summer months, ~

NON-FATTY SOLIDS 45

and increase during the autumn and winter seasons. The sep-
aration of the constituents forming the non-fatty portion of
solids makes it apparent that the decrease in the summer
months is due chiefly to the smaller proteid content, the lac-
_ tose and ash remaining fairly constant. The author’s results
for Ottawa milks also show a tendency towards a decline in the
non-fatty solids during the summer months, though the varia-
tions are more irregular than in the series of Richmond given
above. In these results the proteid was also the greatest variant
~ and usually accompanied the variations in the fat content.
) Richmond ™ found no difference between the non-fatty
solids of evening and morning milk on calculating the average
results for a number of years. Eckles and Shaw 8 also found
_ no appreciable difference in the total amount of non-fatty solids
_ in morning and evening milk, but their results show that this
is due to an increase of proteid in the morning milk with an
equivalent reduction in the lactose content.

The effect of the stage of lactation upon the solids-not-fat
i has also been reported upon by Eckles and Shaw.? The lac-
| tose remained comparatively constant (vide Table XIX) during
_ the greater portion of the period with a slight decline during
i the last two to three months. The ash was constant and the
| proteid decreased and increased sympathetically with the fat
a though not in direct proportion to it.
, This sympathetic relation between the amount of fat and
_proteid in milk has led to the introduction of several formule
) for the calculation of the proteid content from that of the fat.
) Timpe suggested the formula P=2+0.35F in which P and F
Tepresent the percentages of proteid and fat, and gave many
analyses in support of it, but Richmond has pointed out that
i when the series is extended, the agreement practically disap-
| pears. Van Slyke’s formula’? P=0.4(F—3.0)+2.8, is to be
| ferred to that of Timpe but cannot be considered as entirely
tisfactory. These formulz are calculated from the averages
of many analyses and represent the average relation between
| fat and proteid in normal milk. Whilst this is of considerable

46 THE NORMAL COMPOSITION OF MILK

TABLE XX

INFLUENCE OF STAGE OF LACTATION ON COMPOSITION —
BY FOUR-WEEK PERIODS

Period No. Fat Lactose Proteid. Total Solids.
1 4.00 4.87 2.68 12.74
2 3.85 4.84 2.36 12.26
3 3.79 4.94 2.49 12.29
4 3.77 — 4.82 2.49 12.24
5 3.82 4.80 2.62 12.35
6 3.79 4.75 2.68 12.50
7 3.83 4.88 2.68 12.61
8 3.85 4.83 2.74 12.70
9 3.97 4.62 2.87 12.78

10 4.11 4.55 3.06 13.16
1l 4.22 4.74 3.19 13.46
12 4.54 4.91 3.38 14.04
13 4.66 4.70 3.64 14.23
14 5.08 5.01 3.70 15.29

scientific interest, it is of comparatively little value to the milk —
examiner who is required to give an expression of opinion upon | ;
analytical results with reference to sophistication. Such sam-
ples may be derived from many sources and their composition __
influenced by many factors concerning which he has little or
no information; the examiner is, therefore, more vitally inter-
ested in the natural variations from the average than in the
average itself. 7
Reference has previously been made to various factors which _
produce variation in the composition of milk but it is advisable
to discuss in more detail their effect upon the relative propor-
tions of the various constituents. The effect of breed upon the
proteid lactose
fat an proteid
in the total solids is shown in tables XXI, XXII, and XXIII.
Although some of these results are somewhat discordan
the general tendency is usually in the same direction. Whe

ratios together with the percentage of fat

NON-FATTY SOLIDS 47

TABLE XXI
PROTEID
——_—_——_— T
FAT RATIO
Freed: Van Bivke. | Lythgee. pir sien Phil oe
Holstein. Frisian. ...... 0.87 0.86 0.95 0.93
ED CRO ee eee, 0.83
CRETE aa 0.82 0.75 0.88 0.93
American Holderness..... 0.83
: ear ead Fi, SABA Baie ND fe 0.96 0.89
ge oe a es 0.80
“2 RAP raed JASE ga lags 0.66 0.71 Bt As ¢ 0.78
aud odo al’ 0.64 0.61 0.74 0.83
TABLE XXII
LACTOSE
re TIO
PROTEID a
Eckles and New Jersey
Breed. Lythgoe. Shaw. Expt. Stat.
ED 1.60 1.54 1.43
NEN a ag aa 1.67
I en a 1.63 1.51 1.39
SNS a GOR Ee RS, SE AAR 1.39 1.47
2 eee 1.30 Pate eae pre 22
MSGI ecluu% a oho © 1.43 12) 1:22

the figures are considered in relation to the fat content yielded
_ by each breed, it will be found that, with an increasing per-
| proteid lactose
entage of fat, the fat 82 soit
hat the percentage of fat in the total solids increases.
Seasonal variations are also shown by the various ratios as
1 be seen from Table XXIV, the figures in which are cal-
sulated from those in Table XIX.

ratios decrease and

48 THE NORMAL COMPOSITION OF MILK

| Taste XXIII
PERCENTAGE OF FAT IN TOTAL SOLIDS
ARE Eckles and Ameri
/ Breed. - Vieth. Lythgoe. pods bach: Bek
fos), SP RRREEE OP IW 38.0 38.3 39.2 34.9
CHeRHECY . i 5s fons eee ike peo eo el Uubakee 34.9
WU RABE: 6 6 s.0 So! ae 34.7
DOE v5.5 dS ee 34.4
HOTU, 5 5. jole ois: 5g 2 amas ae 34.5
Dairy shorthorn......... 31.3
Pedigree shorthorn...... - $81.4
Partie 6, oe ata UPR ry Reale As 29.4 29.3 |
Riad. POUMAK 6). so eca'v slates 32.8
Ayres oo ds:3 veo op he aie 31.8 29.6 28.7
Duteh belt. oe Se ee ane 30.9
Montgomery.........-.- 28.5
Foleteins 6.25 i a a een 29.2 27.2 28.1
TaBLeE XXIV
SEASONAL VARIATIONS IN PROPORTIONS OF CONSTITUENTS
Proteid L . |
Month. “Sa Erowid: | in Total olds

JANUBTYE | coils scones 0.94 1.29 29.8
oS 5, Ae Gea io PRE 0.95 1.33 29.2
PARI Shs oie ike ainda 0.95 1.37 28.8
BD SS SEG ew a aS eT 0.95 1.33 29.1
DROW sl) pale esinie means 1.00 1.31 28.4
fi Re ERG Ouse et of e 0.99 1.34 28.1
PU se al cecaitluri ah tia ee 0.90 1.42 29.3
Pe Sa aa Gace p etircHhly 0.87 1.41 30.3
Sentemiber i. oi/ck isis tale te 0.84 1.42 31.0
Qetoer ool Se iek ein dited 0.86 1.37 30.9
Noveinber coer ide lee 0.87 1.35 30.9
Deoetiet oie Se eee 0.92 1.30 30.2

NON-FATTY SOLIDS 49

: The influence of the stage of lactation upon the various ratios
_ is shown in Table XXYV, which is based on the results recorded
in Table XVII.

TABLE XXV

INFLUENCE OF STAGE OF LACTATION ON PROPORTIONS OF
| CONSTITUENTS

By Four-week Periods

qi Proteid Lactose Percentage of Fat
pees NO. ~ Fat Proteid in Total Solids,

1 0.67 1.47 31.4

2 0.61 1.58 31.4

3 0.66 1.62 —6 30.8

4 0.66 1.55 30.8

5 0.69 1.50 30.9

6 0.71 1.46 30.3

7 0.70 1.50 30.4

8 0.71 1.46 30.3

9 0.72 1.32 31.1
10 0.74 1.22 31.2
11 0.76 1.23 31.4
12 0.74 1.22 32.3
13 0.78 1.13 32.8
14 0.73 1.23 - 33.2

The above results show that the general tendency during
the period of lactation is for the proteid to increase with the
fat, though at a slightly higher rate. This increased ee
ratio with increase of fat percentage, however, is not capable
_ of general application as the results show that the reverse is
the case when the increase in fat is due to the breed of the cow.
_ The lactose content being comparatively constant, its ratio to
_ that of the proteid is reduced with increase of percentage of
fat owing to the increased proteid content. The percentage
of fat in the total solids increases with the fat as the extra incre-

50 THE NORMAL COMPOSITION OF MILK

ment of proteid is more than counterbalanced by the constancy
in the lactose and mineral matter.

The percentage of ash in milk is comparatively constant but
small variations are observable and depend upon variations in
the proteid content, as a portion of the ash is combined with the
caseinogen to form the caseinogen complex. Richmond has ~
deduced the formula A =0.36+0.11P, in which A and P rep- —
resent the percentages of ash and proteid, for the calculation of
the ash content. |

It is upon the above basic relations between the amounts of
the various constituents in milk that the formule of Van Slyke,
previously referred to, and that of Olsen '%, P=T. s.-S
are based. Lythgoe has suggested that lactose may be cal-
culated from the following formule.

, ee P
L=T. S.- | F+0.7+ (7. s.-Fa)|

from Olsen’s formula and
L=T. S.—[F+0.7+ {0.4(F—3)} +2.8],

from Van Slyke’s formula. The ash in these formule is
assumed to be 0.70 per cent, but it would be preferable to sub-
stitute Richmond’s formula of A =0.36+0.11P for the assumed ~
value.

All the foregoing refers only to whole milk, that is, the mixed
milk obtained by continuous milking until the udders are dry.
The variations due to partial milking are very striking and ©

TABLE XXVI
(BoussINGAULT)
_ Portion. 1 g 3 4 5 6
Total solids.......... 10.47 | 10.75 | 10.85 | 11.23 | 11.63 | 12.67
tg SEARS Grate Ee pone NT 1.70 | 1.76| 2.10] 2:54] 3.14} 4.68
Solids-not-fat.........| 8.77] 8.99 | 8.75 | 8.69 | 8.45] 8.59

NON-FATTY SOLIDS" 51

may be much greater than those caused by the various factors
previously discussed. Analyses showing the composition of
milk obtained at various stages are given in Tables XXVI,
XXVII and XXVIII. |

TaBLE XXVII
AYRSHIRES (AutHor)
Fore Milk. Middle Milk. — Strippings.
Re es ee Sa 1.40 5.90 9.80
Bie teras Pee rere 4.95 4.94 4.87
Ee ec cb ak oo 3.17 2.98 2.78
Sig Sas ba we kes 54 0.80 0.74 0.71
y ctose
Ratios Proteid‘**°°"" 1.57 1.66 | 1.75
Proteid
Far 2.26 O81 0.28

TaBLE XXVIII

AVERAGE OF JERSEYS, SHORTHORNS, AND HOLSTEINS
(EcKLES AND SHAW)

Total Relative Size
Fat. Lactose. | Proteid. Ash. Solids of Fat
y Globules.
Fore milk...| 1.87 5.30 3.58 0.75 10.47 139
Strippings..| 6.28 | 5.33 | 3.38 | 0.70 | 14.86 215

The physical and chemical characteristics of the butter fat
as determined by Eckles and Shaw were as follows:

" TaBLe XXIX
irae Iodine Saponification Melting Yellow
la aetil Number, Number. point. Colour.

Fore milk... 27.2 34.1 230.1 33.9 39
Strippings. . 26.3 33.8 228.3 33.9 39

52 THE NORMAL COMPOSITION OF MILK

All these results show that the chief variation in the com-
position of milk at various stages of milking is due to fat, and
that the relative proportions of the plasma constituents remain
comparatively constant. The proteid is the most variable
component of the plasma and this fact is reflected in the in-

ee lactose
i 6 proteid

ratio is entirely different to those previously stated and

ratio and the decreasing ash percentage. The

proteid
fat

shows the entire lack of organic relation between these two
constituents. This points to the variation in the fat content
being due to mechanical causes and not to changes in meta-
bolism. This is also the view of Kirchner,'* who considered
that the fat globules are mechanically retained in the fine
ducts of the udder and escape in the strippings. Eckles and
Shaw point out, in support of this, that the larger the pro-
duction of milk the greater the increase in fat as the milking
proceeds; which is explained by the hypothesis that, in the
heavier milking cows, the udder is more congested and the
- openings of the ducts reduced by compression. The relative
size of the fat globules at various stages of milking also
supports this view.

Colostrum. The name “ onleataeinn ”” is applied to the udder
secretion before, and immediately after, parturition. A yellow
viscous secretion, not unlike that produced by pathological

conditions, is often formed, but this is replaced several days |

before parturition by the colostrum proper. Colostrum is a
yellowish, sometimes reddish (due to the presence of blood),
slimy liquid with an acid reaction and which shows a tendency
to separate. Compared with ripe milk the quantity of proteids
in colostrum is very high and is due more to increases in albumin
nuclein, and globulin than to an excess of caseinogen. This
points to glandular inflammation as a result of physiological
irritation. Cholesterol, lecethin, creatinine, tyrosine, and urea
are also present. Dextrose is present in addition to lactose,
which is slightly diminished in quantity, and the ash is higher

i

wees FO Om, oe ee

or.

a

COLOSTRUM

53

than in normal milk. The microscopical appearance of colos-
trum is characterised by the presence of glandular epithelium

TABLE XXX
COMPOSITION OF COLOSTRUM (Soruurst)

cert Hees Aa Late 1 Bager. ae ions Gisbelin, | Albuinia,
1 22.87 | 1.03 | 2.30 | 2.74 | 12.23 | 4.86 §.32 1.45
2 16.23 | 0.87 | 2.49 | 2.85 6.97 | 3.35 |. 2.04 1.01
3 15.16 | 0.86 | 3.41 | 3.37 5.82 | 3.09 1.45 0.75
4 15.19 | 0.82 | 4.74 | 3.62 4.69 | 2.70 0.66 0.78

5 15.74 | 0.82 | 5.10 | 3.63 4.01 | 2.61 0.55 0.52

6 15.75 | 0.82 | 4.55 | 3.86 4.04 | 2.56 0.48 0.49
7 15.72 | 0.80 | 5.49 | 3.92 a.46 12:2) 0.31 0.62
8 15.62 | 0.80 | 5.47 | 4.57 SO. oO. | Bole 0.27 0.61

9 15.47 | 0.82 | 5.62 | 4.22 | 3.35 | 2.15 0.25 0.59
1l 15.97 | 0.84 | 5.04 | 3.82 3.52 |: 2.52 0.22 0.59
14 16.55 | 0.84 | 5.15 | 5.00 3.21 | 2.20 0.20 0.56
16 16.28 | 0.83 | 4.90 | 5.01 3.32 | 2.34 0.19 0.55
17 16.06 | 0.81 | 4.79 | 4.87 3.24) 2.25 0.19 0.56

TABLE XXXI
COMPOSITION OF COLOSTRUM (Enatin@)
ary nian After After After After
_ A 10 Hours. | 24 Hours. | 48 Hours. | 72 Hours.
Calving.

Specific gravity...... 1.068 1.046 1.043 1.042 0.035
Total solids.......... 26.83 2): 26 19.37 14.19 13.36
Caseinogen.......... 2.65 4,28 4.50 3.25 3.33
Albumin and globulin.| 16.56 9.32 6.25 2.31 1.03
Rs a Ra) yaa 3.53 4.66 4.75 4.21 4.80
BN So a ss, See Fate 3.00 1.42 2.85 3.46 4.10
OS ey aaa aes 1.18 1.55 1.02 0.96 0.82

in the form of foam cells and signet ring-shaped cells with so-
called moons and caps, and in albuminophores. Numerous

54 THE NORMAL COMPOSITION OF MILK

leucocytes are present and also, during the first few days, large
numbers of erythrocytes. |

The composition of colostrum is shown in Tables XXX and
SXXI. |

According to Jensen the amylase and catalase content is
increased during the colostral period but reductase is absent.

ABNORMAL AND ADULTERATED MILK

Influence of Disease. Although the chemical examination
of milk produced under pathological conditions is of but little
practical importance owing to the infrequency with which such
conditions exist and the improbability of this milk being sold
. unmixed with normal milk, it is, nevertheless, of interest to
consider the general changes that occur. Acute diseases asso-
ciated with great pain and fever are usually characterised by a
rapid diminution in the quantity of milk secreted. In general
and specific infections the fat may be either increased or de-
creased with similar fluctuations in the ash and lactose contents.
According to Schnorf, most of the internal infections, even when
the udder is not involved, produce a diminution in the lactose
and proteid content as a result of increased metabolism. Cata-
lase, especially in peritonitis and tuberculosis, may be consid-
erably increased and changes in taste and coagulability may
result from general infections.

Although it is weli known that the composition of milk
changes with alterations in the function and condition of the
secreting organs, comparatively little is known regarding the
influence of diseases of the udder upon the various constituents
of the milk. Many analyses have been made and various ob-
servers have obtained what are apparently discordant results,
but this may be attributed to factors such as intensity and
duration of the disease being different.

In acute forms of mastitis, caused by organisms of the colon
group, or streptococci, or in mixed infections, the milk may
have a bloody discolouration, liter becoming more like colos-

—

2. Ee he, me Sep OE eI

INFLUENCE OF DISEASE 55

trum in appearance and finally changing to a thick yellowish
secretion containing many dark flakes in a clear serum.

In chronic infections the changes are gradual and the ap-
pearance and composition of the milk may be almost normal
for a time; sooner or later, however, the cell content is increased
with a consequent increase in the albumin, and erythrocytes
cause a discolouration of the sediment on standing.

In udder infections the fat usually decreases but may fluc-
tuate rapidly within rather wide limits; the lactose and casein-
ogen usually decrease slightly, but the decrease in the latter
constituent is more than counterbalanced by a marked increase
in the albumin, resulting in an abnormally high proteid content.
proteid lactose
——— and ———

fat proteid
ratios as is shown in Table XXXII. On account of the bac-
terial origin of these infections, the enzymes in the milk are very
much increased.

These changes result in very abnormal

TABLE XXXII
EFFECT OF DISEASE ON COMPOSITION OF MILK

(SCHAFFER AND BENDZYNSKI)

‘ Total Proteid |Lactose
Character of Disease Solids. Fat. |Lactose.|Proteid.| Ash Saag pare

Non infectious garget...| 7.17 |0.82} 0.53 | 4.01 |0.79| 4.89 | 0.13
Yellow garget.......... 10.66 |1.99) 1.84 | 6.00 0.83] 3.01 | 0.31
Parenchymatous mastitis! 9.74 |2.16| 1.01 | 4.21 |0.99| 1.99 | 0.24

Another cause of abnormal composition of milk is the cessa-
tion of the lactation period. This has already been discussed
on page 49 where it was shown that during the last stages of
lactation, the Jactose

proteid
increased proteid percentage.

Milk Adulteration. Artificial abnormalities in the com-
position of milk produced by the addition of extraneous sub-

ratio decreased considerably owing to the

56 THE NORMAL COMPOSITION OF MILK

stances or by the abstraction of the natural constituents, gen-
erally by human agency, is usually conveyed by the term milk
adulteration, and this, strictly speaking, has no reference to
any standard that may be adopted.

For the detection of adulteration, a complete determination
of the various constituents of the sample should be made and the
amounts of fat, lactose, proteid; and ash so found compared
with the percentages as calculated from the formule of Van

Slyke, Olsen, and Richmond. The proteid lactose

and ————

fat proteid
should also be calculated. The addition of water does not
give proteid values which are materially different from those
calculated by the Olsen formula but are invariably less than
those calculated by the Van Slyke formula, the difference being
proportional to the amount of water added. The PVS (proteid
calculated by the Van Slyke method) in this case, is greater
than the P. O. (proteid calculated by the Olsen method). The
proteid lactose
ag ae proteid

ratios

ratios un-

addition of water leaves the

changed. _ !
The amount of proteid found by direct estimation in the
case of abstraction of fat would be greater than either of the
calculated values, and in this case P.O. would be greater than
PVS. This is due to the Van Slyke formula being based on the

constituent which has been abstracted. The sacnone ratio
| proteid
would be normal and the sce ratio abnormally high. In

lactose
proteid
is valuable in distinguishing between naturally abnormal milks
and those rendered abnormal by external agencies. High
lactose
proteid
duced by the various causes previously mentioned.

The refractive power of the serum should also be considered

both of these instances the ratio is unaltered and this —

ratios are extremely rare but low ones may be pro-

MILK ADULTERATION 57

in connection with samples suspected of being adulterated.
The index of refraction is reduced by the addition of water but
is unaltered by fat abstraction. The following are the mini-
mum figures for genuine milks when prepared by the usual
methods.

TaBLE XXXIII
REFRACTOMETER VALUES FOR MILK SERUM

Reading on Zeiss Immersion

Method of Preparation of Serum. Refractometer.

Copper sulphate..............0...0000- nS ANY 36
MINE TOT he il tha ea: k 520 cata vis! x as aie.'s'e 40
I TT ie oi ge BN a'eh eibia whens ie 38

Although the above methods are capable of detecting the
abstraction of small quantities of fat, their possibilities regarding
the indication of added water are more limited, and it is doubtful
if they could be relied upon to detect additions smaller than
would be necessary to reduce the total solids or solids-not-fat
below the requirements of any reasonably high standard.
Even though these methods are reliable for the detection of the
abstraction of small amounts of fat, the advisability of using
them as a basis for the certification of adulteration, when the
fat exceeds the standard, is extremely doubtful owing to the
difficulty of securing a conviction. ‘Those whose duties embrace
the analysis of public milk supplies meet many of these examples
and have, unfortunately, no option but to report them as gen-
uine, although they are undoubtedly sophisticated. This. is
one of the inherent disadvantages of minimum standards.

The addition of cane sugar or dextrin to watered milk for
the purpose of increasing the non-fatty solids is indicated by a

y é lactose
low proteid value, an abnormally high aotand
deficiency of ash. Methods for the detection and estimation
of cane sugar are given on page 88. Glycerine and starch

ratio, and a

58 THE NORMAL COMPOSITION OF MILK

have also been employed as counterfeits for non-fatty solids
reduced by the addition of water.

CALCULATION OF ADULTERATION

Added Water. The probable amount of water added to
milk may be calculated from the formula

Added water 100505 100 in which SNF represents

snf

the amount of solids-not-fat found, and snf the average amount
of solids-not-fat found in genuine milks during the same season.
If such records are not available a value of 8.8 may be assumed.
Where minimum standards are in force the value in the standard
is substituted in the above formula, whether it be for solids-
not-fat or total solids. Thus

Added water = 100 -—— he Hl x 100,
minimum svf allowed
i 100— T. S. found < 100.

minimum 7’. S, allowed

The added water calculated by this latter method is usually
stated in the certificate of analysis as “ at least . . . per cent.”’
Another formula for calculating the added water is
ss G+F
_ Added water = 100— 345
or lactometer reading, and F = the percentage of fat. The prob-
able amount added may be obtained by substituting 36.0 for
34.5.
Fat Abstraction. The removal of cream is indicated by
an abnormally low fat content and the minimum amount of
fat abstraction may be calculated from the formula.

Fat abstracted = 100—4X 100 where f, and F, are the

37 ~ X 100 where G = degrees of gravity

amounts of fat found in the sample and the minimum required
by the standard, respectively. The probable amount removed
may be obtained by substituting the average value for the
month in which the sample is taken.

MILK STANDARDS 59

MiLK STANDARDS

For the regulation of the sale of milk, various standards
have been established which the mixed milk of a herd of cows
might reasonably comply with, and it is, at least, this mini-
mum quality that a purchaser expects to be supplied with. In
England no specific standard has been adopted by statute but a
standard of 3.0 per cent of fat and 8.5 per cent of. solids-not-fat
was adopted many years ago by the Society of Public Analysts
as a guidance for analysts for milk that is of the nature, sub-
- stance, and quality that might reasonably be demanded by the
purchaser. The onus of proof regarding this contention, how-
ever, was upon the analyst, and it was not until 1901 that this
was transferred to the vendor by an order of the Board of Agri-
culture which stated that milk containing less than 3.0 per cent
of fat or 8.5 per cent of solids-not-fat shall be presumed to be
not genuine until the contrary is proved. This has led to the
“ appeal to the cow ” or the “ stall” or “‘ byre ” test in which
the cows are completely milked in the presence of a witness
or witnesses and the milk afterwards analysed for comparison
with the previous sample. If the results agree, the sample is
to be regarded as genuine and to comply with the provisions of
the Food and Drugs Act. It is obvious that great care should
be taken in obtaining the test sample by insisting upon all the
cows being thoroughly stripped of milk and, if possible, making
the test on the same day of theweek and at the same milking from
' which the first sample was obtained. Such a procedure evidently
regards milk as the secretion of healthy cows without having
regard to the breed, nature and quantity of food supply, and
treatment of the cow, and this is apparently also the view of the
Scottish High Court of Judiciary as expressed during the appeal
of Scott v. Jack. Lord Johnston expressed the opinion that
“ milk in the sense of the statute is milk drawn from the cow,
not milk in the process of formation in the chyle, in the blood,
in the glands of the cow. . . .” This decision that milk is to be
regarded as the secretion of healthy cattle leaves much to be

60 THE NORMAL COMPOSITION OF MILK

desired, as any breed may be used and the ration adjusted to
secure quantity rather than quality and so lead to a diminution
of both the average and minimum composition of the normal
secretion. |

The breeding of dairy cattle on scientific principles has led
to the introduction of strains which secrete large quantities of
milk of comparatively poor quality; the total weight of butter
fat produced is at a maximum and when such milk is to be used
for butter making this method of breeding must be regarded as
legitimate and commended as a step forward in intensive
breeding. When such produce is intended for sale as milk
a very different view must be taken of such methods for, as
regards the ultimate effect, there is no difference between
this process and the deliberate addition of water to milk of
superior quality. If milk is to be regarded as the secretion of
cows, without additions or abstraction, it is evident that a
premium is placed upon quantity regardless of quality, with the
consequence that the water content of milk will become in-
creasingly greater. It might be argued that such a course of
reasoning is merely hypothetical inasmuch as the average
composition of milk shows no definite tendency to deteriorate
from decade to decade. Unfortunately there are compara-
tively few reliable records of data covering considerable periods.
The records of the Aylesbury Dairy Co., London, as published
by Droop Richmond, show that the milk supplied in 1912 was
but very little different in composition to that supplied in 1900.
The intervening period is marked by a rise in quality in 1902 and
1903 after which there is a steady decline. The results are set
' out in Diagram II.

The conditions in New York City present an entirely differ,
ent aspect of this question. Prior to 1910 the standard de-
manded at least 12 per cent of total solids, but in that year the
interests representing the Holstein breeders were strong enough
to effect a reduction of the standard to 11.5 per cent. When
this new standard became operative, no “ quid pro quo” in
the shape of a reduction in price was received by the consumer,

MILK STANDARDS 61

although the report of the Health Department states that
“the reduction is a stimulus to adulteration and that the
records of the department show that certain dealers, who,
under the old-law, were just within the standard of 12 per cent,
are now selling milk, which repeated analyses have shown to be
just within the lowered standard of 11.5 per cent of total solids.”’
In this case it is evident that the quality of the milk supplied,
-by at least a portion of the producers, followed the standard,

DracraM II
YEARLY VARIATION IN COMPOSITION OF MILK (DROOP RICHMOND)

3.8 \ 12.8
&
of Is
‘a? \ 33
/ ¥ ‘ + es OH “ Paice 3
é

> i ty ; n
Zs} sg Sn erg
r) ? ‘“ “ i 5
§ f Se ye re a sue A h =
g : , Cian Se ‘. o
8 g ‘ tp
2 #4 bo) §
3.6 7 s 12.6 a
> / \ iS)
/ cy 5
g ‘ a

Fat \ f ae

Total Solids.....-.-- \e7
3.5 12.5

1900 1901 1902 1903 1904 1905 1906 1967 1908 1909 1910 1911 .1912. .

and it seems inevitable that the other producers will be driven
to the adoption of similar measures by stress of competition.
In both the United States and Canada, milk standards are
of an entirely different legal nature to those obtaining in Great
Britain; the minimum limits of composition are clearly defined
by ordinance or statute and admit of no appeal to the cow.
These standards are to be regarded as specifications of what is
required to be sold as milk and not the minimum quality that
might reasonably be expected by the purchaser. This is
equitable, as the purchaser, for a given price, should receive

62 THE NORMAL COMPOSITION OF MILK

an article of definite quality and not something that may be the
minimum quality produced by natural variations. To achieve
this, the dairyman must so grade his herd that the mixed milk
will at all times comply with the standard. It may be argued
that a rigid interpretation of a standard may inflict unnecessary
hardship on producers by reducing what is usually but a com-
paratively small margin of profit, but it is surely preferable
that the economic balance between producer and consumer
should be adjusted by an increased price rather than by a deter-
ioration in quality. The adjustment by price is understood by
everyone whereas the maintenance of the balance by a reduction
in quality is an invidious one only capable of being correctly
appreciated by experts.

Rigid enforcement of standards is also necessary in the
interests of dairymen in order to prevent unfair competition,
as it is obviously unfair to allow some to breed for quantity
and supply a quality which is, perhaps, only occasionally just
below the standard, whilst others are supplying milk which is
invariably above the standard. One typical example of this
unfair competition which the author experienced was the case
of producer X, who kept pure-bred Holsteins, which produced
milk of the required standard, 12 per cent of total solids and
3.0 per cent fat, during the greater part of the year, but just
failed to meet it during the season when the cows “ freshened.”
An examination of the herd during this period showed that nine
cows, out of the 22 head comprising the herd, secreted a low
quality of milk and were giving an abnormally large quantity,
one cow producing as much as 73 gallons per day. This pro-
ducer had an obvious advantage over others whose herds were
' graded with Ayrshires and other breeds giving a higher quality
but a smaller quantity.

The standards prescribed in various countries show but
small differences; those prevailing in States, provinces and
cities, which have power to make local regulations unfor-
tunately show iarger variations and these often conflict with
those of contiguous authorities. Table XXXIV gives a fairly

MILK STANDARDS 63

complete list of the standards for milk and cream obtaining in
English-speaking countries.

TABLE XXXIV
MILK AND CREAM STANDARDS
SKIM
MILK. eres CREAM,
Country, State or .
Province.
Total Solids- Solids-
Salida. Fat. Se Nuts: Fat.
Great Britain......|...... 3.00 | 8.50
Australia. “Full” “ Half”
New South Wales.}...... 3.20 | 8.50 8.80 35.0 25.0
“Double” “*Single”’
South Australia. .| 12.00) 3.25 | 8.50 8.80 35.0 25.0
“Cream”: « geauced
WAGOTIA......--- 12.00) 3.50 | 8.50 8.80 35.0 25.0
Cream.
Queensland...... 12.00} 3.30 | 8.50 8.80 35.0
“Double” ‘‘Single”’
Western Australia| 11.70) 3.20 | 8.50 8.80 35.0 25.0
Cream.
Tasmania....... '12.00| 3.30 | 8.50 8.80 35.0
Canada. Dominion: |...... 3.25 | 8.50 8.50 18.0
ae 12.00) 3.00 | 9.00 8.50 ,
British Columbia.| 11.75) 3.25 | 8.50
‘Manitoba.......|. Oe iets 3.25 | 8.50 | 8.50 18.0
New Brunswick..| No (|Stand |ards
Nova Scotia..... No |Stand {ards
(SS 12.00} 3.00 18.0
Quebec.......... 12:00. ' 3:00:17: Oi) orcs; 16.0
Saskatchewan... .| 12.00) 3.50 | ......]....... 20.0
"ad Ae 5:00) O20 eee 8 25.0
ie Veillend......1....-. 3.25 | 8.50 | 8.80 { i
India.
OS a ae 11.50| 3.00 | 8.50
Bombay......... 12.00) 3.50 | 8.50

64 THE NORMAL COMPOSITION OF MILK

TABLE XXXIV—(Continued)

MILK AND CREAM STANDARDS

MILK. hay! CREAM.
Country, State or
Province.
Total. Solids- Solids-
Solids. Fat. Not-fat. Not-fat Fat
United States. Federal.| ....... LS ame 8.50 9.25 18.0
MORNE. oui rae B we deen 3.00 8.50 8.80 18.0
COlOVSO scion oa Fe ie edie MOE Mt ies oi Pa a es 16.0
Connecticut.......... 11.75 3.25 Site dk h ee 16.0
District of Columbia...| 12.50 3.50 9.00 9.30 20.0
Pores ase SP be re tee 3.25 8.50 9.25 18.0
ey) APTA EE EY Ley eles 3.25 8.50 9.25 18.0
LERRA = widts Bea bitsiens 11.20 3.00 8.00 9.30 18.0
REO Sail a ae ite ae 3.00 8.50 9.25 18.0
TRGIORE'). seek ual. tet aoa 3.25 8.50 9.25 18.0
tf eRe ae pes Tae 12.00 oe iais SiS w PA ced be bes 16.0
SM ORs Cvieh wk e er eee eee. lta es LN. ote 18.0
RORMIOEY 0620 ee ee a eee 3.25 8.50 9.25 18.0
Louisiana. . oo. 6s hea .75 3.50 8.50 8.00
DAA oe eras Cade > eee 3.25 DO Le eee. 18.0
Darya 3 is ve hee 12.50 Ee Pe 9.25 18.0
Massachusetts........ 12.15 BGO Verh a 9.30 15.0
MICA: 35 ian tos 12.50 BO Tee ES Bore 18.0
Minnesota............ 13.00 3.25 With. do sou 20.0
nt 5 INO ae ae tetas Ga 12.00 3.25 8.75 9.25 18.0
Montane? 3.03 8d so ecek 11.75 3.25 Sa Sees 20.0
Nebraska. ki Se a BOD MAS ee ce Pics 18.0
New Hampshire....... TS. heed ee Fe RE Rene See 18.0
New Jersey........... 11.50 Fl! ie Sees sar 9.25 16.0
MGVSGR io bios bs elke 11.75 3.25 8.50 9.25 18.0
ew Y otie2 5 3 HU 11.50 B28 Lie ee ee 18.0
North Carolina........ 11.75 3.25 8.50 9.25 18.0
North Dakota........ 12.00 B.G6 bccn. Se aa ess 15.0
5 |S BREE Ni ail, aces Slee 12.00 3.00
APRON Eh Sica epry agn y Dye 5 3.20 Scie cates sa 18.0
Pennsylvania......... 12.00 3.25 SOOO Wingtea:s 18.0
Rhode Island......... 12.00 3.50
South Dakota. 245.00. [oe eae 3.25 8.50 9.25 18.0
DONDOREBR. i... 3 Nias 2:2 12.00 3.50 8.50 9.00 20.0
EMM oooh are a thane 12.00 3.25 8.50 9.25 18.0
Viikki hee Fa 12.00 3.20 ee BH tera e hese 18.0
Vermonecscs sicko. 2e 0s 11.75 3.25 8.50 9.25 18.0
VinWinitsiaste as ea ae 3.25 8.50 9.25 18.0
Washington .......... 12.00 3.25 8.75 9.30 18.0
Wisconsin..:.........- Sten 3.00 8.50 9.00 18.0

BIBLIOGRAPHY 65

BIBLIOGRAPHY

. Bunge. Path. and Phys. Chemistry. 2d oacl Edition trans. by

Starling. 1902, 104-105.

. Lythgoe. Ind. and Eng. Chem. 1914, 6, 901.
. Eckles and Shaw. Bull. 156 U.S. A. Dept. of Agr.
. Morgen et al. Landw. Versuch. Stat. 1904, 61, 1-284, ibid., 1906,

64, 93-242.

. Malméjac. J. Pharm. 1901, vi, 14, 70-74.
. Fleichmann. Untersuchung der Milch von sechszehn Kiihen. Landw-

schaftliche Jahrbiicher. Vol. 20, sup. 2, Berlin, 1891.

. Richmond. Dairy Chemistry. London, 1914.

. Eckles and Shaw. Bull. 157 U.S. A. Dept. of Agr.

. Eckles and Shaw. Bull 155 U.S. A. Dept. of Agr.

. Richmond. Analyst. 37, 300.

. Richmond. Dairy Chemistry. London, 1914, p. 160.
. Van Slyke. Jour. Amer. Chem. Soc., 30, 1166.

. Olsen. Ind. and Eng. Chem., I, 256.

. Kirchner. Handbuch. der Milchwirtschaft. 1898, 58.

CHAPTER III
CHEMICAL EXAMINATION

ALTHOUGH the extent of the chemical examination of milk
required in public health work is usually confined to the deter-
mination of the fat and total solids and the detection of pre-
servatives, a brief description of reliable methods for the esti-
mation of other constituents will also be given in this chapter
as they are invaluable for the correct diagnosis of sophistication.

As the great majority of ordinances and statutes regulating
the sale of milk contain no reference to constituents other than
fat and total solids, these will be considered first.

Estimation of Fat. The various methods introduced for
the determination of fat in milk may be divided into three
groups.

(1) Volumetric estimation of the fat brought to the surface
by centrifugal force after liberation by the addition of chemicals.

(2) Ethereal extraction of the fat liberated by the addition
of chemicals.

(3) Ethereal extraction of the dried milk.

The methods which comprise the second group, though
invaluable for dealing with milk products, are not in general
use for the examination of fresh milk and will not be given in
detail.

The mechanical methods of group one are now in almost
universal use and are capable, in practised hands, of yielding
accurate results. The three chief mechanical methods are the
Leffmann-Beam, Babcock, and Gerber. In England, the Leff-
man-Beam and the Gerber are almost exclusively used whilst
in America, although both the Babcock and Gerber processes
are Official, the former is more generally employed.

66

GERBER METHOD 67

Leffmann-Beam Process. 15 c.cms. of the sample are
transferred by means of a pipette into a flat-bottomed bottle
provided with a narrow neck graduated into 80 divisions, 10 of
which correspond to 1 per cent of fat by weight. 9 c.cms. of
concentrated commercial sulphuric acid are then added in three
portions with thorough admixture after each, and finally,
3 c.cms. of a mixture of equal volumes of concentrated hydro-
chloric acid and amyl alcohol. After shaking, the bottle is
filled to the zero mark with hot dilute sulphuric acid (1 in 2)
and whirled in the centrifuge for 3 to 4 minutes. The fat rises
to the top of the liquid as a yellowish coloured layer and the
percentage is read off by deducting the reading at the junction
of the fat and acid from the reading at the extreme top of the
fat, not the bottom of the meniscus.

Babcock Method. This method differs from the Leffmann-
Beam process in but a few details. The bottle neck is divided
into 50 divisions each representing 0.2 per cent of fat by weight
of the 17.6 c.cms. employed. The procedure is as follows:
the milk having been placed in the bottle 17.5 c.cms. of com-
mercial sulphuric acid are gradually added with constant agi-
tation until the caseinogen is dissolved. The bottle is then
placed in a centrifuge and whirled for four minutes at 690 to
1200 revolutions per minute, according to the diameter of the
machine; hot water is added until the bottle is filled to the
lower end of the neck, whirled for one minute, then filled to the
zero mark with hot water and whirled for one further minute
to bring the fat layer into the graduated neck. The per-
centage of fat is then read off as in the Leffman-Beam method,
care being taken that all readings are made between 130° and
150° F. when the fat is quite liquid. The author has found that
the indistinct line of demarkation between the fat and the acid
occasionally found with this process can be obviated by the
addition of 1 c.cm. of amy] alcohol after the addition of the acid.

Gerber Method. This differs from the modified Babcock
described only in the size and type of bottle, and quantities of
acid and milk employed. 11 c.cms. of milk, 1 ¢.cm. of amyl

68 CHEMICAL EXAMINATION

alcohol, and 10 c.cms. of sulphuric acid are mixed in the usual
way, rotated for three minutes, then immersed in a water bath
at 140° F. for a minute and the percentage of fat read off on the
graduated neck.

Skim milk is treated exactly as ordinary milk except in the
Gerber process in which two to three “minutes shaking are
required previous to whirling and a longer period is given in the
water bath to bring the temperature to 140° F.

For cream, special bottles are provided in the Babcock
method, but the ordinary ones may be used, as in the Leffmann-
Beam method, with a reduced quantity of sample. An appro-
priate weight of the sample is washed into the bottle with suf-
ficient water to bring the total volume to the normal volume of
the bottle, and the determination carried out as in the case of
milk. The result is multiplied by the ratio of the normal.
weight of the method (Leffmann-Beam 15.5 grms., Babcock
18.0 grms.) to the weight of the sample taken. In the Gerber
process (normal weight 11.35 grms) 0.5 gram of cream, 6 c.cms.
of hot water, 1 c.cm. of amyl alcohol, and 6.5 c.cms. of acid
are used with a further addition of 6 c.cms. of hot water pre-
vious to rotation.

GRAVIMETRIC METHODS

Gottlieb’s Method. In this method, which is probably
the best known one of group two, the caseinogen is dissolved in
ammonia and the liquid then extracted.with ether and petro-
leum ether. The solution of fat is evaporated and the residue
weighed. For further details of this process Richmond’s
Dairy Chemistry (Chas. Griffin & Co., London, 1914) should
be consulted.

Adam’s Method. 5 grams of milk are weighed out in a
porcelain or glass dish and absorbed on a coil of fat free paper
(special strips of fat-free paper are manufactured for this pur-
pose by various firms). The dish and coil are placed in the
water oven until thoroughly dry when the coil is placed in a
Sohxlet extraction cone and the residue in the dish extracted

——
a pr one DS

a ie

+ FO GENET. ME TNT sO Sik See I Ig ge

eo emer”

SPECIFIC GRAVITY 69

several times with absolute ether. ‘The ether so used is poured
over the coil and cone, previously placed in the extraction
apparatus, and, after the volume of solvent has been increased,
the apparatus is connected with a condenser and heated in a
water bath at about 45° C. After four or five hours extraction
the ether is distilled off and the fat dried to constant weight.
The removal of the ether is facilitated by drawing a current of
air through the flask by means of a vacuum pump. It is nec-
essary that the ether used in this process should be perfectly
dry, as otherwise small quantities of milk sugar and salts are
extracted with the fat.

This is the official method of the Society of Public Analysts
of Great Britain and one of the official methods of the Amer-
ican Official Association of Agricultural Chemists.

Total Solids. These may be determined either directly

_by drying to constant weight or indirectly by calculation from

the fat content and the specific gravity.

Direct Method. Five grams of milk are weighed into a
shallow platinum or quartz dish and after all visible liquid has
been driven off on the water bath, the dish and contents are
dried to constant weight in a steam oven. Ignited sand or
asbestos may be used to facilitate the drying process.

Ash. The residue from the determination of the total
solids may be ignited at a low temperature until white and the
residue weighed, or a fresh portion of 20 c.cms. evaporated
with the addition of 6 c.cms. of nitric acid, and ignited until
free from carbon at a temperature just below redness. The
former method is the more convenient and the latter the more
accurate one. :

Specific Gravity. This is determined either by a lac-
tometer, a Westphal balance, or the ordinary specific gravity
bottle. The lactometer method is the simplest and quickest,
but, owing to the comparatively short space occupied by each
graduation (usually 1°) and the opalescence of the liquid the
degree of accuracy obtained is low.

The gravity is usually expressed as the excess weight of

70 CHEMICAL EXAMINATION

1000 c.cms. of milk at 60° F. over an equal volume of water at
the same temperature. Thus, a Specific Gravity of 1032.2
(water = 1000) is usually expressed as 32.2 or, 32.2° lactometer
scale.

Lactometers indicate the specific gravity at a temperature
of 60° F. and it is, therefore, necessary to either bring the sample
to this temperature or to correct the reading. It is much more
convenient to ascertain the temperature of the sample imme-
diately before taking the specific gravity and to correct this
result to 60° F. by means of Table LX VIII, which will be
found in the appendix.

It is important that the specific gravity of milk should not be
determined within a short period of milking as, during the first
four hours, there is a decided increase often amounting to 1 to
1.5° (Recknagel’s phenomenon). The gravity should also
never be taken immediately after violent agitation of the sample
as the air entrapped by the fat globules during such a process
may lead to serious errors. If violent agitation is necessary for
any purpose, it is advisable to allow the sample to remain quies-
cent for two hours before proceeding with the specific gravity
determination. No attempt should be made to take the spe-
cific gravity of a sample that has commenced to curdle.

Total Solids, by Calculation. As the fatty and non-fatty
portions of milk are comparatively constant in composition,
it is evident that the specific gravity of milk can be calculated
from the percentages of these constituents. Fat tends to reduce
the gravity, and non-fatty solids to increase it. Hehner and
Richmond found that the following formula expressed with a
fair degree of accuracy the quantitative relation between these
constituents:

F=0.859 T. S.—0.2186G.

Where F=percentage of fat, 7. S. the percentage of total
solids and G the specific gravity expressed as mentioned above.
From this formula 7’. S.=1.164F+0.2546G.

A simplified form of this formula that has come into general

MILK SUGAR 71

use is 7. 8.=1.2F+0.25G. This is, with very slight modifi-
cations, the basis of Babcock’s tables which are official in Amer-
ica. Richmond now prefers the formula 7. S.=1.2F+0.25G
+0.14 and this was used in the preparation of the slide rule
which so greatly facilitates the calculation of the total solids
from the fat and specific gravity determinations. It is ad-
visable to remember that the differences between the results
obtained by use of the various formule are within the limits of
experimental error and that a direct determination should be
made when great accuracy is required.

Richmond’s and Babcock’s tables are given.in the appendix
on pages 210-213.

Solids Not-fat. These are estimated by deducting the per-
centage of fat from that of the total solids or they may be cal-
culated directly from the gravity and the percentage of fat.

Milk Sugar. Milk Sugar, or Lactose, may be estimated by
either the polarimetric, volumetric, or gravimetric methods.
When a polarimeter is available, this method is almost invari-
ably employed as but little time is required for the examination
of several samples. In the absence of this instrument, and
when only occasional determinations are required, the gravi-
metric method should be used.

Polarimetric Methods. These are based upon the exam-
ination of the milk serum in a polariscope after the separation
of the fat and proteids. A solution of mercuric nitrate, pre-
pared by dissolving mercury in twice its weight of nitric acid
(1.42) and diluting with an equal volume of water, is the most
suitable reagent for this purpose. As the removal of proteids
and fat reduce the volume of the lactose containing solution, it is
necessary to correct the readings for the percentages of these
constituents, but Richmond and Boseley (Dairy Chemistry)
point out that these calculations can be simplified by the use
of the following method. |

To 100 c.cms. of milk add

(a) A quantity of water in c.cms. equal to 75 the lactometer
reading or excess gravity over 1000.

72 CHEMICAL EXAMINATION

(b) A quantity of water in c.cms. equal to the fat1.11.

(c) A quantity of water in c.cms. to reduce the scale readings
to percentages of milk sugar.

(d) 3 c.cms. of acid mercuric nitrate.

After thorough agitation, filter through dry papers and
polarise the filtrate. The percentage of milk sugar can be read
off directly in scale readings.

The values of (c) are:

(a) For polariscopes reading angular degrees.

With 198.4 mm. tube 10.0 c.cms.

With 200 mm. tube 10.85 c.cms.

With 500 mm. tube 10.85 c.cms. (divide readings by 2.5).

(b) For the Laurent sugar scale (100° =21.67 angular degs. )

With 200 mm. tubes 2.33 c.cms. (divide readings by 5)
With 400 mm. tubes 2.33 c.cms. (divide readings by 10).
With 500 mm. tubes 2.33 c.cms. (divide readings by 12.5)

(c) For the Ventzke scale (100° =34.64 angular degrees).

With 200 mm. tube 6.65 c.cms. (divide readings by 3).
With 400 mm. tube 6.65 c.cms. (divide readings by 6).
With 500 mm. tube 6.65 c.cms. (divide readings by 7.5).

Gravimetric Method. Dilute 25 c.cms. of milk with 400
c.cms. of water in a 500 c.cm. flask, add 10 c.cms. of No. 1,
Fehling solution and 4.4 c.cms. of N-NaOH solution; make
up to 500 c.cms., shake, and filter through a dry paper. The
filtrate should be acid and contain copper in solution. Place
25 c.cms. each of Fehling’s solutions Nos. 1 and 2 in a beaker
and heat to the boiling point. When boiling briskly add 100
c.cms. of the milk serum and boil for six minutes. Filter imme-
diately through asbestos, supported by a platinum cone in a
hard glass filtering tube, with the aid of a suction pump, wash
thoroughly with boiling water and finally with alcohol followed
by ether. After drying, connect the tube with an apparatus
for supplying a continuous current of hydrogen and gently °
heat until the cuprous oxide is completely reduced to the

TOTAL PROTEIDS Ray 73

metallic state. Cool in an atmosphere of hydrogen and weigh.
The weight of copper is calculated to lactose from Table
LXXI in the appendix.

The weight of lactoseX20 gives the percentage per 100
c.cms. of sample. As an alternative method of weighing the
reduced oxide, a Gooch crucible may be used in which a layer
of asbestos about one-quarter of an inch in thickness has been
placed. Wash the asbestos thoroughly with hot water and
then with 10 c.cms. of alcohol followed by 10 c.cms. of ether.
Dry for thirty minutes in the steam oven and weigh. The pre-
cipitate of cuprous oxide is collected as above, washed with
water, treated with 10 c.cms. of alcohol and ether, successively,
and ‘dried for thirty minutes at 100° C. The weight of CuzO
multiplied by 0.8883 gives the weight of metallic copper.

PROTEIDS

Total Proteids. 5 gms. of milk are placed in a Kjeldahl
flask of about 150 c.cms. capacity and 20 c.cms. of pure conc.
sulphuric acid added. The mixture is heated over a small flame
until excessive frothing has ceased, and after cooling, 8-10 grms.
of acid potassium sulphate and a drop of mercury are added.
After placing a sealed funnel containing water in the mouth
of the flask to prevent excessive evaporation, the contents of
the flask are gradually heated and the flame slightly increased
- as frothing ceases. When the liquid becomes colourless the
flask is allowed to cool and the contents washed with the aid of
distilled water into a flask. This flask should be provided
with a stopper having two holes, one containing a trapped bulb
tube connected with a water condenser, and the other a tapped
funnel reaching almost to the bottom of the flask. After the
contents of the Kjeldahl flask have been transferred, a few pieces
of pumice, unglazed porcelain, or granulated zinc, are added
to prevent bumping and the distillation apparatus connected
up with the outlet of the condenser dipping into a beaker con-

taining 50 c.cms. of = acid. Through the funnel add 100 c.cms.

74 CHEMICAL EXAMINATION

of 30 per cent caustic soda, followed by 10 c.cms. of a 10 per
cent solution of potassium sulphide. The flame is placed under
the flask, and the distillation continued until about 200 c.cms.
have passed over. Before taking away the flame, the tap of
the funnel should be opened to prevent creating a partial vac-
uum and so drawing back the distillate into the flask. The
end of the condenser is washed with water, and the washings
mixed with the distillate which is finally titrated with = caustic
alkali using sensitive methyl orange or, preferably, methyl red

as the indicator. Each c.cm. of = acid neutralised =0.0014

grm. nitrogen or 0.028 per cent of nitrogen when 5 grms. of milk
are used. The percentage of nitrogen multiplied by 6.38 gives
the percentage of total proteids.

In all determinations of nitrogen by the above method, it is
essential that a blank determination should be made on all the
reagents and this amount deducted from all subsequent results.

Caseinogen. Dilute 10 gms. of the sample with about 90
c.cms. of water at 40° to 42° C. and add at once 1.5 c.cm. of a
10 per cent acetic acid solution. Stir with a glass rod and allow
to stand for about five minutes. Decant on to a wet filter,
wash several times with cold water by decantation and then
transfer the precipitate completely to the filter. Wash once
or twice with cold water. If the filtrate is not bright it should
be refiltered until that condition is attained. The nitrogen in
the precipitate is then estimated as above by the Kjeldahl
method. The percentage of nitrogen multiplied by 6.38 gives
the percentage of caseinogen. This method is only applicable
to fresh milk. |

Albumin. The filtrate from the precipitation of caseinogen
is first exactly neutralised with caustic alkali and then acidified
by the addition of 0.3 c.cm. of a 10 per cent solution of acetic
acid. After heating to boiling over a flame, the precipitate is
digested on the water bath for fifteen minutes. The liquid is
filtered through paper, the precipitate washed and finally used

. ay me ATS ee

ia

ALDEHYDE VALUE 75

for a nitrogen determination by the Kjeldahl method. Nitrogen
6.38 = Albumin.

Total Acidity. Lactic Acid. 10 c.cms. of milk are placed
in a white porcelain basin, a few drops of phenolphthalein

iticn added ‘and titrated with = at) Wau a Tue pink

colour is obtained. As the acidity of fresh milk is chiefly due
to phosphates, the expression of the acidity in terms of lactic
acid is somewhat misleading, although this is often done, 1 c.cm.

of ba alkali being equivalent to 0.009 grm. lactic acid. It is

I ccshie to express the acidity i in degrees, i.e., the number of
cubie centimeters of normal alkali required tor the neutralisa-

tion of 1 litre of milk. The number of cubic centimeters of = |

alkali required for the neutralisation of 10 c.ems. of milk, mul-
tiplied by 10 gives the required result in degrees. It is unfor-
tunate that in Germany the same term is used for a unit having
a very different value. The Sohxlet-Henkel degree usually
used throughout Germany is exactly 2.5 times greater than the
degree used in England and America.

Aldehyde Value. Richmond and Miuiller’s modification
(Richmond’s Dairy Chemistry) of Steinegger’s method is as
follows: 10 c.cms. of milk are made neutral to phenolphthalein

with = strontia, 2 c.cms. of 40 per cent formaldehyde added,

and again titrated to the same degree of neutrality. The
amount of the second addition of alkali less the amount re-
quired for the neutralisation of the formaldehyde added (pre-
viously determined), multiplied by 10 gives the aldehyde value.

This method is dependent upon the fact that the proteid
radicle is quantitatively converted to an acid by the aldehyde.
Richmond states that the strontia aldehyde figure is 1.1 times

greater than that given with = soda and that the former value
multiplied by 0.170 will give a close approximation to the total

76 CHEMICAL EXAMINATION

proteids. It is also pointed out that as caseinogen and albumin
do not give the same aldehyde value, the factor is only applica-
ble when the ratio of caseinogen to albumin is normal.

Mineral Constituents. The estimation of the mineral
constituents in milk is but infrequently required in connection
with public health work but on these occasions, the following
method, due to Droop Richmond, will be found advantageous
as it secures fairly accurate results with a minimum expenditure
of time and labour.

Fifty grams of milk are evaporated and charred to a black
ash: the mass is extracted with hot water and filtered, the insol-
uble portion, together with the paper (after washing) being
ignited until white; this gives the insoluble ash. Evaporation
of the filtrate and cautious heating gives the weight of the sol-
uble ash.

The soluble ash, after solution in water, is made up to a
known volume and aliquot portions used for the determination

of the alkalinity by titration with = acid with methyl orange

as indicator, and chlorine by titration with = silver nitrate,

using potassium chromate as indicator. 1 c.cm. of = acid

=(.0031 grm. NagO and 1 c.cm. = AgNOs =0.00355 grm. Cl.

The insoluble ash is dissolved in a slight excess of dilute
hydrochloric acid, and the solution (rearly neutralised if nec-
essary) heated to boiling; a cold saturated solution of ammo-
nium oxalate is dropped in slowly until further addition pro-
duces no further precipitate. After standing at least two hours,
the precipitate is filtered off, washed, and ignited at a low tem-
perature to vonvert the oxalate into carbonate; it is advisable
to moisten the ignited precipitate with ammonium carbonate
solution and reignite at a very low temperature. The precipi-
tate, after weighing, is dissolved in dilute hydrochloric acid,
keeping the bulk small, ammonia is added to alkaline reaction,

MINERAL CONSTITUENTS 77

and the small precipitate of calcium phosphate collected, ignited,
and weighed. Its weight is subtracted from the previous
weight, and the difference gives the weight of calcium carbonate,
which, multiplied by 0.4, gives the calcium, or by 0.56, the lime
(CaO) contained in it; the weight of calcium phosphate mul-
tiplied by 0.3871 gives the calcium (Ca), or by 0.5419, the lime
(CaO) contained in it. The total calcium or lime is the sum of
the two.

The filtrate is made strongly ammoniacal by the addition of
strong ammonia (0.880) and allowed to stand twenty-four hours.
The precipitated magnesium ammonium phosphate is filtered
off, washed with dilute ammonia, ignited, and the magnesium
pyrophosphate (Mge2P207) weighed. Its weight multiplied
by 0.2162 will give the magnesium (Mg), or by 0.3604, the
magnesia (MgO) contained in it.

To the filtrate from this, magnesia mixture is added, and
the precipitate, after standing twenty-four hours, is treated
as above. From the total weight of the two quantities of mag-_
nesium pyrophosphate, the phosphoric anhydride is calculated
by multiplying by 0.6396; to this is added the phosphoric anhy-
dride in the calcium phosphate, calculated by multiplying
the weight by 0.4581. This method takes no account of the
traces of iron present, which are precipitated with the calcium
phosphate and the magnesium-ammonium phosphate. If
desired, this may be estimated by dissolving the precipitate of
calcium phosphate and the first magnesium-ammonium phos-
phate precipitate in dilute hydrochloric acid, and determining
the iron colorimetrically as thiocyanate.

To estimate alkalies, another portion of milk is ignited as
before, and the total ash dissolved in dilute hydrochloric acid
and boiled; a few drops of barium chloride solution, containing
not more than 0.1 grm. of barium to 100 grms. of milk are
added, and the boiling continued for some minutes. After some
hours, the precipitate of barium sulphate is filtered off, washed,
ignited, and weighed; its weight multiplied by 0.3433, will
give the sulphuric anhydride (SOz) in the milk. If an excess

78 CHEMICAL EXAMINATION

of barium chloride has been added, a little phosphoric acid, or
ammonium phosphate, may now be added to the filtrate,
although it is not necessary if the quantity of barium chloride
given above has been employed. A quantity of ferric chloride
solution, sufficient to colour the solution brown, is added and
the filtrate made alkaline with ammonia. After boiling, the
precipitate is filtered off and well washed: the filtrate is evap-
orated and cautiously ignited: this weight represents the alka-
line chlorides. When the residue is dissolved in hot water, the
solution should be perfectly clear; if this be not so, a little
ammonium carbonate solution is added, the liquid evaporated
to dryness and the residue cautiously ignited; the residue is
again taken up with water, the solution filtered and evaporated,
and the residue cautiously ignited and weighed. This puri-
fication of the mixed alkaline chlorides is often found necessary
and it is essential, in order that accurate results may be obtained,
that the process should be carried out with great care, always
bearing in mind that alkaline chlorides are volatilised at com-
paratively low temperatures.

The chlorine in the mixed chlorides may be estimated by

titration with = silver nitrate, using potassium chromate as

indicator. Each cubic centimeter of x AgNOsz is equivalent

to 0.00355 grm. chlorine. The potassium and sodium are cal-
culated from the formule.

The weight of sodium =2.997C—1.4254W,

The weight of potassium = 2.4254W —3.987C.
in which W =the weight of the mixed alkaline chlorides,
and C=the weight of chlorine therein.

Examination of Milk Serum. As the fat and proteids are
the most variable constituents of milk, an examination of the
milk serum often affords valuable assistance in determining

EXAMINATION OF MILK SERUM 79

whether a sample is adulterated by the addition of water, or is
merely abnormal in composition. The principal constituents
of the serum are milk sugar and mineral matter, and a deter-
mination of these on the milk direct affords the same evidence as
an indirect examination of the serum, but as the latter can be

TABLE XXXV

RELATION OF REFRACTIVE INDEX TO SPECIFIC GRAVITY
(LYTHGOE)
1i falas a Specific Gravity.
rae snoxansll 20° C. ap AC: ee

28.0 1.33820 1.0149
29.0 1.33861 1.0160
30.0 1.33896 1.0170
31.0 1.33934 1.0180 ©
32.0 1.33972 1.0190
33.0 1.34010 1.0200
34.0 1.34048 1.0211
35.0 1.34086 1.0221
36.0 1.34124 1.0231
37.0 1.34162 1.0242
38.0 1.34199 1.0252
39.0. 1.34237 1.0262
40.0 1.34275 1.0273
41.0 1.34313 1.0283
42.0 1.34350 1.0293
43.0 1.34388 1.0303
44.0 1.34426 1.0313
45.0 1.34463 1.0323

performed more expeditiously, it is often included in the rou-
tine examination of milk. The serum is prepared by adding
2 c.cms. of 25 per cent acetic acid (Sp. Gr. 1.035) to 100 c.cms.
of sample at a temperature of 20° C., covering with a watch-
glass and heating to 70° C. for twenty minutes. After cooling
in ice water for ten minutes, the curd is separated by filtration

80 CHEMICAL EXAMINATION

through paper and 35 c.cms. of the filtrate, which should be
bright, are transferred to one of the beakers which accompany
the Zeiss immersion refractometer. The refraction is then
determined at exactly 20.0° C. A reading between 39.0 and
40.0 is suspicious whilst one less than 39.0 indicates the addition
of water.

Lythgoe! after determining the value of K in the Lorenz and

Lorentz formula

n—1 ad K

nW4t21 9’
which expresses the relation between the refractive index (n)
and the specific gravity (d), has calculated the values of d for
the various scale readings of the immersion refractometer, and
in the absence of this instrument, the specific gravity deter-
mination will achieve the same object after reference to Lyth-
goe’s table. (Table XXXYV, p. 79.) ,

DETECTION AND ESTIMATION OF PRESERVATIVES

The addition of preservatives to milk is usually absolutely
prohibited because it has been found perfectly feasible to market
this product in a sound condition without their use. No legit-
imate excuse, therefore, for the addition of any substance which
retards or inhibits bacterial development. Although the exig-
encies of certain branches of trade in milk products have, in
some cases, led to the adoption of regulations which permit
the addition of certain specified preservatives in quantities
not exceeding a specified limit, this practice should not be
encouraged, for, until it can be proved beyond reasonable
doubt that such preservatives are non-toxic, the public should
be safeguarded against these substances: public health should
be paramount to commercial interests and not sacrificed to
them. Unfortunately many statutes regarding the sophis-
tication of foodstuffs are even yet so framed as to place the onus
of proof as to damage to health upon the prosecutor and so give
the defendant the benefit of all doubts that may exist, but it is
pleasing to note that these are decreasing and that the present

FORMALDEHYDE 81

tendency is to prohibit the entire use of particular preservatives
and to restrict them generally.

The preservatives in most general use are boric acid, borax,
or mixtures of these two, and formaldehyde. For milk the last-
mentioned is the favourite owing to its potency and general con-
venience. The presence of boric acid or borax is allowed in
cream in England when declared on the label attached to the
container and in quantities not exceeding 0.25 per cent when
calculated as boric acid. Harden has shown that the addition
of an alkali (7 grms. of Na2O per 100 grms. of boric acid) in-
creases the efficiency of boric acid as a preservative, and it is
now customary to employ such a mixture for the preservation of
cream. Such mixtures also contain cane sugar or traces of
saccharin, the object of which is to mask incipient sourness.

Formaldehyde. Formaldehyde may be detected by any
_ of the following tests, but on account of its reliability and del-

- ieacy, the author recommends the Shrewsbury and Knapp
process.

Hehner Method. About 10 c.cms. of sample are placed
in a test tube and concentrated commercial sulphuric poured
carefully down the side so as to form a layer beneath the milk.
In the presence of formaldehyde, a violet ring is formed at the
junction of the two liquids. Richmond and Boseley modified
the test by adding an equal volume of water to the milk and
using acid of 90 to 94 per cent strength. One part in 200,000
produces a violet colouration which is permanent for several
days. In the absence of formaldehyde, a greenish ring is pro-

duced and a brick-red colouration in the acid layer.

_ Leonard? points out that the presence of a mild oxidising
agent is essential for the success of this test and that such an
agent, preferably a trace of ferric chloride, must be added if
pure acid is used. Droop Richmond? points out that the test
is dependent upon the reaction of formaldehyde with the
tryptophane of the caseinogen and that other aldehydes, e.g.,
vanillin, give similar reactions.

Hydrochloric Acid Test. 10 c.cms. of commercial hydro-

82 CHEMICAL EXAMINATION

chloric acid, containing 0.2 grm. of ferric chloride per litre,
are added to 5 c.cms. of milk in a porcelain basin and the mix-
ture heated to boiling with constant stirring. The presence of
formaldehyde is indicated by a violet colouration.

Shrewsbury and Knapp Test.4 The reagent for. this
test consists of a freshly prepared mixture of pure concentrated
hydrochloric acid with 0.1 per cent of pure nitric acid. 5 c.cms.
of the sample are placed in a test tube and vigourously shaken
with 10 c.cms. of the reagent, the mixture is heated in a water
bath to 50° C. for ten minutes and finally rapidly cooled to
about 15° C. A violet colouration denotes the presence of
formaldehyde, and a rose pink colouration, its absence. The
depth of the colouration, between 0.2 and 6 parts per million,
is approximately proportional to the amount of formaldehyde
present, so that this method may also be used for the estimation
of the preservative. When the amount exceeds six parts per
million, the milk should be suitably diluted.

Estimation of Formaldehyde. In addition to the method
previously mentioned, various others have been devised for the
estimation of formaldehyde, but not one as yet can be relied
upon to give accurate results. Most of these are based upon the
volatilisation of the aldehyde by distillation of an acid soluticn,
and subsequent volumetric estimation. Probably the most
useful is the following. To 100 c.cms. of sample contained in a
500 c.cm. Kjeldahl flask add 1 ¢.cm. of 1 : 3 sulphuric acid and
distil over 20 c.cms. (care is necessary if frothing is to be
avoided). The formaldehyde in the distillate, amounting to
approximately one-third of the total, is estimated iodometrically. —

25 c.cms. of Es iodine solution are added to the distillate and

normal caustic soda is added, drop by drop, until the liquid
becomes a clear yellow. After standing for fifteen minutes,
dilute sulphuric acid is added in excess to liberate the uncom-
bined iodine. The solution is then titrated with = sodium

thiosulphate, using a starch solution as the indicator in the end

BORIC ACID AND BORATES 83

reaction. Each cubic centimetre of . iodine solution absorbed

equals 0.0015 grm. of formaldehyde.

Monier-Williams, in a report to the Local Government
Board, states that a preservative is on the market which con-
tains a nitrite in addition to formaldehyde: the nitrite masks
the usual reactions but its effect may be destroyed by the
addition of a little urea.

Boric Acid and Borates. These may be detected by adding
a few cubic centimetres of normal alkali to not less than 10
cubic centimetres of milk and evaporating to dryness over a
small flame. The flame is increased until a black ash results:
this is acidified with a few drops of hydrochloric acid. After
lixiviation with a few cubic centimetres of hot water, the ash is
removed by filtration through paper. A turmeric paper is
placed in the filtrate in such a manner that only a portion of it
can be wetted, and the liquid evaporated to dryness. <A red-
dish-brown colouration of the wetted portion, due to the for-
mation of rosocyanin, indicates the presence of boron com-
pounds. A drop of caustic soda changes the colouration to
various shades of green and purple which can be restored to
the original colour by the addition of hydrochloric acid.

A useful routine method for the detection of boron com-
pounds consists in heating about 10 c.cms. of milk in a porce-
lain dish with a few cubic centimetres of methyl alcohol and a
few drops of tincture of turmeric. The heating is conveni-
ently carried out in a water bath and the presence of boron
compounds is indicated by the formation of a reddish ring
round the basin.

The estimation of boron compounds is most conveniently
carried out by Thomson’s method.® One or two cubic centi-
metres of N- NaOH are added to 100 c.cms. of milk and the
whole evaporated to dryness in a platinum dish. The residue
is ignited to a black ash, heated with 20 c.cms. of water and
concentrated hydrochloric acid added, drop by drop, until
frothing ceases. The solution containing the carbonaceous

84 CHEMICAL EXAMINATION

matter is washed with a few cubic centimetres of water into a
100 c.cm. flask and 0.5 grm. dry calcium chloride added. After
the addition of a few drops of phenolphthalein, a 10 per cent
- golution of caustic soda is added until a faint pink colour per-
sists and finally 25 c.cms. of lime water. The object of this is
to precipitate the phosphates as calcium phosphate. Make up
the volume to 100 c.cms., mix thoroughly and filter through a
dry paper. To 50 c.cms. of the filtrate add N. sulphuric acid
until just colourless, then add a few drops methyl orange and
continue the titration until the yellow colour changes to pink.

= soda is now added until the reaction is just alkaline and the

liquid boiled to expel the carbonic acid liberated. The solu-
tion is cooled, a few drops of phenolphthalein solution and
sufficient neutral glycerine to amount to 40 per cent of the total
volume is added. The solution is finally titrated with a soda
until a permanent pink colouration is produced. Each cubic
= soda equals 0.0062 grm. of boric acid.

Benzoic Acid. The proteids are precipitated by the addi-
tion of 5 c.cms. of dilute hydrochloric acid and shaking: then
shake with several portions of ether, taking care to avoid the
formation of an emulsion. If this should occur, resort must be
made to a centrifuge in order to separate it. The ethereal
extract containing the benzoic acid and fat is shaken with water,
rendered alkaline by the addition of ammonia, and the aqueous
extract evaporated nearly to dryness. After all the ammonia
has disappeared, a few drops of ferric chloride are added and
the presence of benzoic acid is indicated by the formation of a
flesh-coloured precipitate. If any ammonia is left in the solu-
tion, a reddish-brown precipitate of ferric hydrate is obtained,
so that it is essential that all traces of this disturbing sub-
stance are removed before applying the final test.

Salicylic Acid. This is detected in exactly the same manner

as is described above for the detection of benzoic acid. On addi

centimetre of

wey see

ee ee ee en et ee es

- ut

DETECTION OF ADDED COLORING MATTER 85

tion of ferric chloride, a solution of salicylic acid produces a
characteristic violet colour, the intensity of which is somewhat
proportional to the amount of salicylic acid present.

Hydrogen Peroxide. As hydrogen peroxide decomposes
into free oxygen and water soon after its addition to milk, it is
impossible to detect this substance by means of the usual
reagents. The oxygen liberated, however, considerably mod-
ifies the enzymes present, and it is upon this fact that several
inferential tests for detecting hydrogen peroxide are based.
The immediate reductase reaction (see p. 89) is destroyed by
hydrogen peroxide, and the catalase (see p. 91) destroyed in
proportion to the amount added.

Before the hydrogen peroxide has decomposed it may be
detected by the peroxidase reaction (see p. 91).

Hypochlorites. Although hypochlorites have been sug-
gested as milk preservatives they have not been extensively
used as the amount required to produce any appreciable effect
also adversely affects the taste and odour. Milk containing
hypochlorites does not give the usual starch-iodide reaction
even with as large a quantity as 50 parts of available chlorine
per 100,000.

Detection of Added Colouring Matter. The following are
the provisionally official methods of the American Association
of Official Agricultural Chemists.

Warm about 150 c.cms. of milk in a basin over a flame and
add about 5 c.cms. of acetic acid, after which slowly continue
the heating almost to the boiling point whilst stirring. Gather
the curd, when possible, into one mass by means of the stirring
rod, and pour off the whey. If the curd breaks up into small
flecks, separate from the whey by straining through a sieve or
muslin. Press the curd free from adhering liquid, transfer to a
small flask, and macerate for several hours (preferably over-
night) in about 50 c.cms. of ether, the flask being tightly corked
and shaken at intervals. The ether is finally decanted from the
curd and is examined for annatto, the curd being reserved for
the detection of aniline orange and caramel.

86 CHEMICAL EXAMINATION

Annatto. After evaporation of the ether, the fatty residue
is made alkaline with caustic soda and, whilst still warm,
poured upon a very small wet filter paper. After the solution
has passed through, wash the fat from the paper with a stream
of water and dry the paper. If, after drying, the paper is
coloured orange, the presence of annatto is indicated. This
may be confirmed by adding a drop of stannous chloride solu-
tion, which, in the presence of annatto, produces a character-
istic pink on the orange-coloured paper.

Aniline Orange. ‘The curd of an uncoloured milk is per-
fectly white after complete extraction with ether, as is also
that of a milk coloured with annatto. If the extracted curd is
distinctly dyed an orange or yellowish colour, the presence of
aniline orange is indicated. To confirm this, treat a lump of
the fat-free curd with a little strong hydrochloric acid. If the
curd turns pink, the presence of aniline orange is assured.

Aniline orange may also be detected by Lythgoe’s method
which consists of the addition of 10 c.cms. of concentrated
hydrochloric acid to an equal volume of milk in a. porcelain
dish and imparting a rotary motion to the contents. If any
appreciable amount of aniline orange is present, a pink colour
is at once imparted to the curd particles as they separate.

Caramel. If the fat-free curd is coloured a dull brown,
caramel is suspected. Shake a lump of the curd with concen-
trated hydrochloric acid in a test tube and heat gently. In
the presence of caramel the acid solution will gradually turn a
deep blue, as will also the white fat-free curd of an uncoloured
_ milk, while the curd itself does not change colour. It is only
when this blue colouration of the acid occurs in conjunction with
a brown-coloured curd, which itself does not change colour,
that caramel can be suspected, as distinguished from the pink
colouration produced by aniline orange under similar circum-
stances.

a

CREAM 87

ANALYsIS OF MiLK Propucts

Cream. The normal constituents can be determined by
employing the usual methods of milk analysis after suitable
detection with water (vide p. 66). The amount of cream
used for dilution, however, should be weighed and not measured
volumetrically. The total solids should be determined by
evaporation, and Richmond recommends the addition of an
equal volume of alcohol to accelerate drying. Richmond also
finds that the total solids and fat bear the relation expressed by
the formula:

Fat =1.102 Total Solids— 10.2

Thickening agents are sometimes added to cream for the
purpose of increasing the viscosity and thus produce the appear-
ance of a cream of high fat content. The usual agents employed
are gelatine, starch, and saccharate of lime (viscogen). _

Small quantities of gelatine may be detected by Stokes’
method. Mercury is dissolved in twice its weight of con-
centrated nitric acid (1.42) and the solution diluted with
twenty-five times its volume of water. To 10 c.cms. of cream
add an equal bulk of mercuric nitrate solution and about 20
c.cms. of cold water. Shake vigourously and filter after stand-
ing for a few minutes. Inability to obtain a clear filtrate indi-
cates the presence of gelatine and this may be confirmed by
adding an equal volume of a saturated solution of picric acid.
A yellow precipitate is produced by gelatine in a cold solution.

Starch is detected by the formation of a blue colouration on
addition of a solution of iodine in potassium iodide.

Saccharate of lime may be detected by the estimation of
either the lime in the ash or by the lactose determination. The
lime in normal samples averages about 22.4 per cent of the ash
and any perceptible increase over this amount is suspicious.
Similarly an abnormally high polarimeter reading, equivalent,
when calculated as lactose, to more than 52.5 per cent of the
solids not fat, should also be regarded with suspicion.

88 CHEMICAL EXAMINATION

Skim Milk. The usual methods of milk analysis may be
applied.

Condensed Milk. About 30 grms. of milk are weighed out
and, after boiling with 50 c.cms. of water, the solution is cooled
and made up to 100 c.cms. The methods of analysis described
above under milk may then be applied, but longer extraction
should be given if the Adams process is used for the estimation
of the fat.

In sweetened samples the cane sugar is determined by sub-
tracting the sum of the fat, lactose, proteids, and ash, from the
total solids.

ENZYMES

Although the presence of enzymes in milk has been an
established fact for many years, it is only comparatively recently
that the origin of these ferments has been seriously considered:
The nature and characteristics of these bodies suggests that
they are derived from the blood and the results of various
experimenters show that they are largely associated with the
cells invariably found in milk samples. Whilst the greater
portion of the enzyme activity of milk is anchored to the cells
and may, consequently, be removed by filtration, there is also
present a smaller quantity of extra cellular activity. This is
not surprising when the rapid metabolic changes taking place
during the secretion of milk are considered. Certain enzymes,
such as Schardinger’s reductase, occur in amounts which vary
directly with the fat content and, unless, this enzyme is almost
entirely extra cellular, the cells should also vary somewhat with
the fat content. Although various hypotheses have been ad-
vanced as to the effect of enzymes in milk, the author believes
that too much importance has been attached to the qualitative
and too little to the quantitative tests for these substances.
The amylase content of normal milk is equivalent to about 0.4
grm. of starch per 100 c.cms. of milk per hour. The catalase
in 100 c.cms. liberates from hydrogen peroxide 10 ¢.cms. or
0.014 grm. of oxygen in two hours, whilst Babcock and Russell’s

ENZYMES 89

figures show the galactase activity to be capable of digesting
approximately 1 per cent of proteids in milk in twenty-four
hours. Compared with the activity of the normal secretions
of the alimentary tract, these quantities are so small as to pos-
sess but little, if any, physiological significance. Pathological
conditions such as mastitis, which involve inflammatory pro-
cesses of the udder, increase the cell content and, consequently,
also the enzyme activity of milk, whilst heating of the milk to
temperatures of 75° C. and over, weaken and finally destroy
the enzymes. As an aid to the diagnosis of such conditions and
for the control of pasteurisation, the determination of the fer-
ment activity may be found desirable and for this purpose the
following methods have been proved to be satisfactory. The
determinations that can be most conveniently carried out in
routine work and which do not require special apparatus are
the reductase and peroxide tests: the catalase and amylase
follow next in order of facility whilst the others are of more
scientific interest than practical utility.

Reductase. To 10 c.cms. of milk, add 1 ¢.cm. of Schar-
dinger’s reagent (190 parts water and 5 parts each of formalin
and a saturated alcoholic solution of methylene blue) and heat
to 43°-45° C.: the time required for decolourisation is noted.
The reoxidation of the surface layers by the air may be entirely
prevented by adding a small quantity of paraffin, but the
cream layer usually gives the. necessary protection.

Any desired temperature, not exceeding 60° C., may be
used for carrying out this test, but whatever temperature is
chosen must be adhered to in order that the results may be
strictly comparative. In most laboratories, a temperature of
43°45 C. will be found convenient as the water bath employed |
for liquid agar media is usually maintained at this temperature.

This ferment is not present in every sample of milk from
individual cows, being frequently absent from animals whose
offspring are still suckling and in animals whose lactation period
is just commencing (Schern) but the author has invariably
found it to be present in mixed market samples. Romer and

90 CHEMICAL EXAMINATION

Sames have found that it does not decolourise, or only com-
pletely so, in the fore milk and that the time required for
decolourisation becomes less as the milking proceeds. This
corresponds to the relative frequency of the fat content and
on this connection the following figures calculated from some of
the author’s results are of interest:

TABLE XXXVI

RELATION OF BUTTER FAT TO REDUCTASE CONTENT
Butter Fat Content. Average Time for Reduction.
Minutes

Less than 3.4 15

3.4 to 3.6 17

3.7 to 3.9 16

4.0to 4.2 14

4.3to 4.5 13

4.6 to 4.8 10

More than 4.9 7

The following results of the author show that there is no
relation, between the bacterial content of milk and the reductase
test or hastened reductase test as it is sometimes known as
(ef. p. 24):

TABLE XXXVII
RELATION OF BACTERIAL COUNT TO REDUCTASE CONTENT
Bacterial Count per C.cm. Average Terms of Reduction:
Agar 48 Hrs. at 37° C. Minutes.

Less than 10,000 13

10,001 to 50,000 16

50,001 to 100,000 14

100,001 to 200,000 17

200,001 to 300,000 17

300,001 to 400,000 19

——

me 2

= -apemeepenmnane RR SEE ee mane ah er a t.
.

ee

AMYLASE | 91

Peroxidases. The detection of this ferment may be carried
out by any of the following methods, all of which are reliable.

Rothenfusser’s Method. Two solutions are required: (1)
a 6 per cent solution of pure para phenylenediamine hydro-
chloride, and (2) a 1.8 per cent solution of crystallised guiacol
in 96 per cent alcohol. 15 c.cms. of No. 1 are added to 135
c.cms. of No. 2 and the mixture preserved in an amber-coloured
bottle. To 10 c.cms. of milk add 0.5 c.cm. of the reagent and
3 drops of hydrogen peroxide (8 per cent). A blue violet colour-
ation indicates a positive peroxidase reaction.

Wilkinson and Peter’s Method. To 10 c.cms. of milk add
1 c.cm. of a 10 per cent solution of benzidine in 96 per cent alco-
hol, 3 drops of 30 per cent acetic acid and finally 2 c.cms. of
3 per cent hydrogen peroxide. Peroxidases produce a blue
colouration which is usually localised in the precipitated casein-
ogen. |

Bellei’s Method. ‘To 10 c.cms. of milk, add three drops of a
1.5 per cent aqueous solution of ortol and two drops of a 3
per cent hydrogen peroxide solution. A red colouration indi-
cates the presence of peroxidases.

Peroxidases, like reductase, are more concentrated in the
cream layer of milk though it is impossible to establish any
definite parallelism between the butter fat content and the
deusity of the peroxidase reaction.

Catalase. The activity of this ferment is estimated by
mixing 15 c.cms. of milk and 5 c.cms. of 2 per cent hydrogen
peroxide in a special tube devised for this purpose by Lobeck.
In this apparatus the oxygen liberated is collected and measured
in a graduated tube previously filled with water. The libera-
tion of the oxygen is accelerated by incubation at blood heat
for two hours. Fresh milk usually evolves one to three cubic
centimetres of oxygen and results materially higher than these
are usually indicative either of excessive bacterial contamina-
tion or of excessive amounts of cellular elements produced by
physiological or pathological irritations of the udder.

Amylase. Into each of 10 test tubes, 10 c.cms. of milk are

92 CHEMICAL EXAMINATION

placed and to these are added 0.1, 0.2, 0.3 up to 1 c.cm. of a 1

per cent solution of soluble starch prepared by boiling with dis-
tilled water and cooling. After shaking, the tubes are placed
in a bath at 43°-45° C. for one hour and then rapidly cooled.
To each is added 1 c.cm. of a solution of iodine in potassium
iodide (1 grm. iodine, and 2 grms. potassium iodide in 300 c.cms.
of water), and the colour noted immediately after shaking.
The recording of the tints admits of no delay, as the colours
rapidly fade and all the tubes may regain their original shades.
A yellow tint indicates total conversion of the starch to sugar,
and a blue one unchanged starch: the correct reading is where
the yellow just commences to take on a greyish tint. With
normal fresh milk this will usually be found between the third

and fifth tubes. The indications of this test are similar to —

those of the catalase test, both being based on the quantity of
cellular elements.

Galactase. The Babcock and Russell method is probably

the most reliable for the estimation of this ferment, but the time
required for its execution is so long that it is never carried out
in routine examinations. The milk is incubated at blood heat
for 53 days with the addition of sufficient thymol to prevent
bacterial development and an estimation of the soluble nitro-
gen then made. The difference between this result and that
originally present indicates the amount produced by the enzyme
activity. This is usually less than 1 per cent per day.

BIBLIOGRAPHY

. Lythgoe. Jour. Ind. and Eng. Chem., 1914, 6, 906.
. Leonard. Analyst. 1896, 21, 157.

. Richmond. Dairy Chemistry. London, 1914, 186.

. Shrewsbury and Knapp. Analyst. 1909, 34, 12.

. Thomson. Analyst. 1903, 28, 184. .

Stoke. Analyst. 1897, 22, 320.

Orr wnd-e

CHAPTER IV

BACTERIA IN MILE

Mitk, like other secretions, is sterile at the moment of
secretion but it is usually impossible to obtain it from the udders
of cows in this condition even though every precaution be
taken and all operations are conducted under strictly aseptic
conditions. Many have held that bacteria may be trans-
ferred to milk’ directly from the blood stream of healthy cows,
but this view is now generally regarded as erroneous.

Amongst the earliest investigators to doubt the sterility of
the udder were Balley and Hall! who concluded from their
experiments that the milk cistern might be the seat of bac- -
terial development and one source of bacterial contamination
of milk. - Ward? carefully examined the udders of 19 milch
cows from 5 dairies and found that although the animals were
tubercular, the udders were normal. He found that all the
lactiferous ducts of the cows were contaminated throughout
with bacteria of which the majority were cocci. From his
studies on the anatomy of the udder Ward concluded that
with the possible exception of the sphincter muscle, at the lower
end of the teat, no obstruction capable of excluding bacteria
from the milk cistern exists. This would indicate‘ that the
source of contamination of milk even in the udder is external
and that the portal of entry is the teat.

Henderson * examined a number of cultures from seven
normal udders and obtained growth in 76 per cent, but two
cases of unexpanded udders from heifers gave sterile cultures
from the milk cistern, ducts, and parenchyma.

The intra-mammary contamination of milk in healthy udders
is usually small, and, although in some exceptional cases counts

93

94 BACTERIA IN MILK

as high as 15,000 per c.cm. have been obtained, it is probable
that at least a portion of this number was due to external con-
tamination caused by faulty aseptic conditions of milk with-
drawal.

Sedgwick and Batchelder * found that with moderate pre-
cautions on the part of the milker, the organisms in fresh milk
may not exceed 500 to 1000 per c.cm., but if ordinary flaring
pails were used with more or less disturbance of the bedding
and shaking of the udder, the count may be 30,000 or even more.

Park® found the average count from six separate cows,
five hours after collection, to be 4000 per c.cm. (minimum 400
per c.cm.) and the average of 25 cows as 4550.

McConkey ® observed that, with ordinary care and cleanli-
ness, it was possible to obtain milk containing less than 1500
bacteria per c.cm. and that such milk should not contain gas
formers in less than 50 c.cms.

Von Freudenreich’ thought it would be easy to obtain
sterile milk by using strict asepsis but soon found otherwise.
Such milk invariably contained 250-300 bacteria per c.cm.
though the hands of the milkers and the teats of the cows were
washed with soft soap and sterile water, then with servatol soap
and sterile water, and, finally with sterile water and then dried
on a sterile towel. The milkers’ hands were smeared with lano-
line and the fore milk rejected. The bacterial content of the
mixed milk of 28 cows so milked varied from 65-680 per c.cm.
Von Freudenreich and Thoni ® from a further series of experi-
ments concluded that freshly drawn milk, even when every
precaution is taken against contamination, always contains
bacteria; they found that these were mostly cocci and were
derived from the udder. A summary of the more important
attempts to obtain sterile milk is as follows:

Von Freudenreich, 200-300 per c.cm.

Szasz, 2 samples sterile. Average of 11=2700 per c.cm.
Hesse, 1600 per c:cm.
Marshall, 295 per c.cm.

Lux, 0 to 6800 per c.cm.

ee a

——,s
——o

—

BACTERIAL FLORA OF INTRA-MAMMARY MILK = 95

Kolle, 80 to 15,000 per c.cm.
33 per cent less than 300.
50 per cent less than 500.
4.7 per cent 700-800
Willem and Minne, 1 to 5 per c.cm.
Willem and Miele, 0 to 37 and 4 to 218 per c.cm.
Siebald, (1) Without protective measures. Under 10 to
- several thousands.
(2) After soaping the udder. 0 to 85 per c.cm.
(3) After soaping the udder and disinfecting with
alcohol and milking through sterile tubes,
0 to 12 per c.cm.

All these numerous experiments prove conclusively that some
intra-mammary contamination of milk exists and it will be
advisable next to consider the nature of this.

Like Ward 2 Freudenreich ® found that udder contamination
in healthy cows was mostly caused by cocci, but Str. lacticus
(Heinemann) was only found in three cases out of a total of
fifteen. B. coli was never found. The organisms found by
Henderson ? were streptococci, staphylococci and pseudo diph-
therie and similar results were obtained by Bergey.!°

From these and other results it would appear that cocci,
some of a proteolytic nature, form the prevailing type found in
udders and that the lactic acid producing bacteria, both coli-
form and Str. lacticus, are usually absent. Some of the strep-
tococci and staphylococci found in milk produced under strictly
aseptic conditions are biochemically similar to those usually
associated with inflammatory processes but are commonly of
much lower virulence.

Experiments on the viability of various organisms in the
environment of milk ducts has shown that they rapidly die,
many bacteria disappearing within a few days. Savage !!
inoculated the teats of goats with streptococci of both bovine
and human origin and found that the infecting organism
usually died in a few weeks, although in one case the strep-
tococci persisted for over seven months. The streptococci
from human sources were usually less viable. __

96 BACTERIA IN MILK

Although the majority of the evidence available favours the
hypothesis that the source of intramammary contamination
is external it is difficult to establish this entirely on account of
the impossibility of putting the ducts and cisterns in a sterile
condition. Once infection of the udder has occurred, the
organism, finding the mammary secretion an excellent pabulum

for development, persists and the small quantity of milk re-

maining from one milking contaminates the next, the process
being repeated until the cow becomes dry. That the amount
of milk allowed to remain in the udder has a very material
influence upon the bacterial count of the milk obtained at the
next milking is shown by the experiments of Stocking,!? who
found as the average of ten experiments 6542 bacteria per c.cm.
in milk obtained after thoroughly stripping the udder as against
11,324 per c.cm. when this was neglected. The importance of
this factor is now well recognised in large dairies using milking
machines, for it is invariably the custom to take out the last
strippings by hand, owing to the impossibility of obtaining
this milk by means of the machine. This hand-milked secretion
often contains more bacteria than the portion immediately
preceding it, due, Stocking suggests, to more vigorous manip-
ulation of the udder dislodging bacteria from the ducts and
which remained there during the earlier part of the milking.
The contaminated milk left in the ducts is, of course, mostly
discharged in the fore milk and a decreasing count is obtained
as milking proceeds. Stocking!” reports the following results
in this connection as the averages of four experiments:

Bacteria per ¢.cm.

Stroame YT and Bo sic gash osicds ade heen 10,143
Streame 5 frid @io5 fs bis Ske Vis Beane 2,347
Streame 9 and 10.056 vive cebiowend oe aks 272
Streame 13 and 16.35.0565 Cae vive ok oe 382
Stripping. 200 sce kees keene Oren 204

The influence of the rejection of the contaminated fore
milk was shown by the following figures:

———————— CO

SOURCES OF BACTERIA IN MILK 97

BACTERIA PER C.CM.

Total. Acid. Liquefying.
Fore milk rejected............. 499 99 33
Fore milk retained............. 522 . 189 9

Backhaus }° reports 10,400 bacteria per c.cm. in fore milk
as against practically sterile strippings whilst the author in one
instance obtained 50,000 per c.cm. in the fore milk, 4000 in |
the middle milk and 500 in the strippings. The advantage
obtained by the rejection of the fore milk is usually much
greater than is indicated by Stocking’s results reported above,
but this factor is largely determined by the precautions observed
in other directions and may be but a minor one if the udders
are thoroughly stripped and kept clean between and during
milking operations. This so-called intramammary contam-
ination, which is really external contamination, though con-
veyed to the milk whilst in the udder, is, however, only a frac-
tion of the external contamination that reaches the milk directly;
this is especially true of ordinary market milk. The external
contamination increases at every stage between milking and
delivery to the consumer and is very diverse in character.
The chief sources of contamination are:

(1) During milking. Bacteria from dirty udders, flanks, and hands of
milkers: also aerial contamination with dust
of food or litter.

(2) During handling. Dirty containers, strainers and cooling apparatus.

The influence of bodily cleanliness of the cow on the
bacterial count of the milk obtained has been investigated
on several occasions. Backhaus !* found 20,600 bacteria per
c.cm. in the milk of brushed cows as against 170,000 per
c.cm. from unbrushed cows. Stocking !* reports the following
results: 3

98 BACTERIA IN MILK

BACTERIA PER C.CM.

Total. Acid. Liquefying.
rose oo sss cate 2268 381 117
Untrudined ..i.ot te oaks 1207 213 59

Wiping the udders with a damp cloth previous to milking
reduced the bacterial count from 7,058 to 716 per c.cm. Sim-
ilar results are also reported by Harrison. Orr exposed
plates of nutrient medium for two minutes during milking and
afterwards incubated them for four days at 20°C. The results
are given in Table XX XVIII.

TaBLE XXXVIII
No. of Average
Housing of the Cows. Conditions of the Cows. Experi- | Count per
ments. Plate.
Summer, all cows out. .| Untouched 7 440
Summer, all cows out. .| Udders and flanks washed
and brushed 3 170
Winter, cows indoors.. .| Untouched 3 4752
Winter, cows indoors. ..| Udders and flanks brushed
but not washed 3 1752
Winter, cows indoors...| Udders and flanks brushed
and washed and left
| moist 6 230
Winter, cows indoors...| Udders and flanks brushed,
washed and dried 3 444

The practice of moistening the hands of the milkers by the
first milk streams was shown by Backhaus to increase the bac-
terial count from 5600 to 9000 per c.cm. The effect of the
character of the litter and the food employed is very marked
as is also that of the influence of time of feeding. The ten-

dency of the litter to dust formation is a factor in this direction.

EFFECT OF LITTER AND FEED 99

TABLE -XX XIX
BACTERIA IN LITTER (Bacxuavs)

Litter. Organisms, Per Gram.
Ee Ue Vee Uilo ky bee edie ain tis ¢ 2,000,000
aS SE ESSN PAD ERG aod Mae eae 7,500,000
E> MRSA A SISOS rR 10,000,000

The milk obtained contained

Bacteria per C.cm.,

PRL MR MOC ss PG wi ies Wears) oe wes 3500
7s RE IS ea a 7330

Backhaus also found that oil cake averaged 450,000 bacteria
per gram and bran 1,362,000 per gram, and there is no doubt
that other dry foods also contain similar large numbers of
organisms. Moist foods such as ensilage would have no effect
if entirely consumed but would be equally objectionable as
other foods if allowed to dry.

Stocking !? reports the following results in connection with
experiments on the influence of feeding before and after milking.

Hay AND CoRN

Total. Acid. Liquefying.
Given after milking........... 2096 790 108
Given before milking.......... 3506 1320 196
Dry Corn
Total. Acid. Liquefying.
Given after milking........... 1233 297 118
Given before milking ....... re 3656 692 123

100 BACTERIA IN MILK

The results of Harrison‘’* are equally interesting. The
organisms falling on an area equal to a circle having a diameter
of 12 inches were found to vary from 12,210 to 42,750 during
bedding, feeding and cleaning up, whilst one hour later similar
tests gave only 483 to 2370 organisms.

Orr © by exposing plates of nutrient medium for five min-
utes and afterwards incubating for four days at 20° C. obtained
from 1260 to 4500 organisms per 113 square inches (area of
circle 12 inches in diameter). The author has found that in
clean, well-ventilated cow byres as low a germ content as 200

per 113 square inches could be attained when tested with plates —

of nutrient agar for five minutes and incubated at 37° C. for
forty-eight hours. Coliform bacilli, as shown by neutral red
lactose agar plates, were usually absent.

The influence of milk containers is also well marked. Back-
haus found that fresh milk which originally contained only 6600
bacteria per c.cm. was increased in germ content to 97,000 per
c.cm. by passage through six containers. Wooden pails were
the most objectionable in this respect as they averaged 280,000
germs as against 1690 for galvanized iron and 1105 for enam-
elled ware. Pails after rinsing contained 28,600 organisms and
sterilized pails only 1300. Harrison also investigated the
cleansing of cans; by rinsing the vessels with 100 c.cms. of
sterile water he obtained the following results:

BACTERIA PER C.CM,

Improperly cleaned cans...............e.ceecess 215,000-806,320 |
Washed with tepid water and scalding.......... 13,080-— 93,400
Washed with tepid water and steaming 5 mins... 355- 1,792

Cloth and absorbent cotton strainers may also be a source
of bacterial contamination unless proper precautions are taken.
Milk coolers of the open type may introduce contamination
from both the cooler itself and from the air. This is well exem-
plified by the results both of Orr ° and the author. (Table XL.)
Two other sources of milk contamination are water and cow
feeces. It is obvious that all the water used for cleansing and

——— ee ee a ee ee |

COOLERS AND PAILS 101

TABLE XL
EFFECT OF MILK COOLERS

AVERAGE oF Four ExprermMENnts (ORR)

BACTERIA PER C.cCM. IN MILK.
Agar 48 Hrs. Gelatine 96 Hrs.
at 37° C. at 20° C.
ENE MREEIIES S20, oi) C5 Fou w ahome’ #\ 0,515 26,000 39,000
Se oh ee ee 48,000 104,000
Author’s results: Coliform,
Se eer ene 25,000 4
EE INNO 55g esis c wissen oe x ods 400,000 3,500
After thorough cleansing of coolers:
REISS Re Ee 28,000 ne
ECE Oe ee eee ee 30,000 8

rinsing the various utensils that come in contact with the milk at
various stages cannot all be sterilised, so that milk will contain
a number of the bacteria usually found in water supplies.

Cow feces may also be conveyed to milk by falling into
milking pails after becoming dried upon the udders and flanks
of the cows. This danger may be eliminated as has previously
been pointed out by washing these portions of the beasts.
Savage 1® gives several analyses of fresh cow excreta. (Table
XLI.)

From this general consideration of the various sources of
milk contamination it is obvious that milk even whilst fresh
may contain large numbers of an almost infinite variety of
organisms. Before taking up the methods of examination for
these organisms it will be advisable to consider the effect of
storage, for milk samples are rarely taken of the product in a
fresh condition. This point is also important in considering
the conditions requisite for preventing bacterial multiplication

102 BACTERIA IN MILK
TABLE XLI
BACTERIA IN COW FAECES (Savace)
ORGANISMS PER GRAM.
Source. B. enteritiditis
B. coli. Streptococci. sporogenes
Spores.

Cow No. 1 100,000— 1,000,000 | 10,000— 100,000 100—1000
2 1,000- 10,000 |100,000-1,000,000 10- 100
3 || 1,000,000-10,000,000 | More than 10,000,000 10— 100
4 || 1,000,000—10,000,000 | 100,000—1,000,000 100-1000

in the interval that elapses between sampling and the labora-
tory examination.

Park !’ took two samples of milk, one containing 3000 organ-
isms per c.cm. (agar forty-eight hours at 37° C.) and the other
30,000 per c.cm. and stored portions at various temperatures.
After various intervals of time the bacterial counts were again
taken with the results shown in Table XLII.

The author has made similar tests but, in addition to the
total bacterial count, an estimation was made of the B. coli
group by plating on rebipelagar (neutral red bile salt agar) and
incubating at 37° C. for twenty-four hours. The total bacteria
were counted on +1.0 per cent nutrient agar after forty-eight
hours incubation at 37° C.

It will be noticed in both these series of experiments, and
especially in Park’s, that at the lower temperature there is at
first an apparent diminution in the total bacterial count and
that this phenomenon is more definite and more prolonged at
the lowest temperature used. These observations have been
confirmed by many experimenters and led to the hypothesis
that milk possessed a weak, though definite bactericidal action:
this is usually referred to as the germicidal action of milk.
M. J. Rosenau !® thoroughly investigated this phenomenon and
concluded that no true germicidal action took place, but that

cee, -

—

ee ee a

EFFECT OF TEMPERATURE 103

TaBLE XLII
Upper figures represent sample No. 1. Original count 3,000.
Lower “ o si No. 2. a “30,000.
TIME WHICH ELAPSED BEFORE MAKING TEST.
Temperatures,
°F.
24 Hours. 48 Hours. 96 Hours. 168 Hours.
32 2,400 2,100 1,850 1,400
30,000 27,000 24,000 19,900
39 2,500 3,600 218,000 4,200,000
38,000 56,000 | 4,300,000 | 38,000,000
42 2,600 3,500 | 500,000
43,000 210,000 | 5,760,000
46 3,100 12,000
42,000 360,000
50 11,600 540,000
89,000 1,940,000
55 18,800 3,400,000
187,000 38,000,000
om -: 180,000 28,000,000
900,000 | 168,000,000
68 450,000 | 25,000,000,000
4,000,000 | 25,000,000,000
86 1,400,000,000
14,000,000,000
94 25,000,000,000
25,000,000,000

fresh milk appeared to act as a weak antiseptic. Vigorous
shaking of the samples demonstrated that the reduction in
count was more apparent than real and suggested that the

BACTERIA IN MILK

104

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GERMICIDAL ACTION 105

organisms had aggregated into clusters under the influence of
agglutinins. The so-called germicidal action was also found to
be specific but the specificity of different samples was variable.
Further proof of the fact that this phenomenon must be attrib-
uted to agglutinins rather than to bacteriolysins was found in
the behaviour of heated milk. Heating to 56° C. for thirty
minutes, a condition which destroys bacteriolysins, weakens
but does not entirely inhibit the action; it is entirely destroyed
at 75° C.

St. John and Pennington !° found that milk, after heating
to 79° C. for twenty minutes, not only failed to show an ap-
parent diminution in the number of organisms but also showed
a much greater rate of bacterial development throughout the
period of observation. They point out that this is a serious
objection to pasteurisation as a reinfected heated product
exerts no restraining effect upon the invading organisms and
may, therefore, be more infective than raw milk receiving the
same original contamination.

Stocking,?° who investigated this question, concluded that
the apparent diminution of organisms capable of development
on solid media was really due to bacteria finding the milk a
pabulum to which they are unaccustomed and consequently
died at a faster rate than they could multiply; he found that
this resting stage was scarcely observable with common lactic
acid organisms which appeared to develop more or less rapidly
and continuously from the moment of their introduction into
the milk. The absence of a “ germicidal effect ”’ with common
lactic acid organisms was confirmed by Rosenau and others and
supports rather than impairs the validity of the agglutination
hypothesis by accentuating its specificity. The resting stage
pointed out by Stocking must also be a factor, but cannot
wholly account for it as it fails to explain the comparative
absence of the phenomenon in heated milk unless it is assumed
that heating has resulted in chemical changes that have pro-
duced a more favourable environment for bacterial develop-
ment. Once this resting period or germicidal phase has passed,

106 BACTERIA IN MILK

bacterial development sets in, the rapidity of which depends
upon the temperature at which the sample is stored. The
organisms that have gained admittance to the milk do not all
find that substance a suitable medium for reproduction, but
certain classes develop rapidly and ultimately one or more of
these classes predominates. The bacteria that reproduce most
rapidly may be roughly divided into three groups according to
their biochemical characteristics, viz., acid producers, pro-
teolytic, and inert organisms. Ayers and Johnson?! made a
fourth general division by separating the alkali producers, but
this group is usually included in the inert group. The classifi-
cation was based upon the behaviour of the organisms on litmus
lactose gelatine, the acid producers being those capable of
producing red colonies, the proteolytic being liquefiers, and the
balance, having no well-defined characteristics on this medium,
the inert group. The acid producers may be subdivided into
two further groups according to their ability to ferment lactose
with the production of gas. This separates the coliform organ-
isms, which produce hydrogen and carbon dioxide from lac-

tose in addition to lactic acid, and the ordinary lactic acid -

organisms which do not give any gaseous products.

Although different samples of milk will all show varying
rates of development of the various groups, a general dis-
cussion of this point will, perhaps, be facilitated by consider-
ation of a concrete example. Table XLIV shows the results
of a daily examination of a sample of milk kept comparatively
cool.

All three groups, in this example, developed rapidly, the
greatest relative increase being shown by the coliform organ-
isms, until a maximum was reached at the end of five days. At
this stage the acidity was 44° and this amount was evidently
sufficient either alone or in conjunction with the other products
of metabolism, to restrain the rate of production. The coli-
form organisms were the first to be affected, although the other
acid producers and to an even smaller degree, the liquefiers,
were restrained. On the tenth day the liquefiers commenced

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108 BACTERIA IN MILK

to gradually decrease and a few days later it was impossible
to make an accurate estimation of their number owing to the
overgrowth of acid producers. The inert group developed
well during the first period and, after a reduction at the tenth
day period, persisted to the end of the experiment. The sample
ultimately developed a prolific growth of torule.

In considering the relative development of various groups
in milk, due regard must always be given to the two important
factors, viz., temperature and initial content, that determine
the results.

The effect of temperature was carefully investigated by
Conn and Esten,”” who plated out practically fresh milk usually
containing 20,000 bacteria per c.cm. on litmus lactose agar and
found that they were able to distinguish no less than 15 different
groups merely by their macroscopic appearance. They made
two series of experiments, the first at 37° C., 20° C., and 10° C.
and the second at 20° C., 10° C., and 1°C. The plating inter-
vals were:

37°-C. at 2 hour intervals
20° C. at 6 hour intervals

10° C. at 12 hour intervals
1° C. at 1 day intervals.

The main conclusions, as summarised by Conn and Esten,
were:

(1) The effect of variations of temperature upon the devel-
opment of different species of bacteria in milk is not always the
same under apparently identical conditions. In spite of such
variations, there seems to be clearly discernible a normal
development of bacteria associated with different tempera-
tures.

(2) There is, in all cases, a certain period at the beginning
when there is no increase in the total number of bacteria.
During this period some species are multiplying whilst others
are apparently dying. The length of this period depends upon
the temperature. At 37° C. it is very short, while at 10° C.
it may last from six to eight days, since, at this temperature,

TEMPERATURE AND BACTERIAL FLORA 109

milk may, in six days, actually contain fewer bacteria than when

fresh.

(3) After this preliminary period, there always follows a
multiplication of bacteria; but the types that develop differ
so markedly, that samples of the same milk kept at different
temperatures are, at later periods, very different in their bac-
terial content, even though they contain the same number of
bacteria.

(4) The development of the ordinary lactic species Bact.
lactis acidi (Str. lacticus), in practically all cases checks the
growth of other species of bacteria, and, finally, kills them,
since the bacteria regularly decrease in actual numbers after
the lactic bacteria have become very abundant.

(5) In practically all samples of milk kept at 20° C., the
multiplication of the Str. lacticus * begins quickly and pro-
gresses with great rapidity. They grow so rapidly that they
produce acid enough to curdle the milk in about forty hours,
the growth of other species being held in check. Milk when
curdled at this temperature shows a smooth acid curd, with no
gas bubbles. |

(6) A totally different result appears in milk kept at 37° C.
The results are somewhat more variable than at 20°C. Occa-
sionally the Str. lacticus grows vigorously at this temperature,
but the common result is a development of the B. lactis zerogenes
type. It forms a curd full of gas bubbles. If B. coli communis
is in the milk, this also grows luxuriantly at 37° C.

(7) In milk kept at 10° C., neither of the types of bacteria
seems to be favoured. The delay in growth lasts two to three
days, after which all types of bacteria appear to develop some-
what uniformly. Sometimes the lactic bacteria develop
abundantly, sometimes only slightly. The neutral bacteria
always grow rapidly, and. the liquefiers in many cases become
abundant. In time, the milk is apt to curdle, commonly with

* Str. lacticus has been substituted for B. lactis acidi (Hueppe) in
order to avoid confusion with B. acidi lactici (Escherich).

110 BACTERIA IN MILK

an acid reaction, but it never shows the predominance of Str.
lacticus found at 20° C.

(8) From our experiments there seems to be no difference
between the effect of 10° and 1° upon the bacteria, except upon
the rapidity of growth. 1° C. very markedly checks the growth
of bacteria; but, later they grow in large numbers. As at
10° C., the lactic bacteria fail to outgrow the other species, so
that all types develop abundantly. A few species appear to be
particularly well adapted to this low temperature and are espe-
cially abundant at the end of the experiment.

(9) The curdling point appears to be quite independent of
the number of bacteria present. In one sample at 37° C., the
milk curdled with only 8,000,000 organisms per c.cm. while
in others there have been found 4,000,000,000 per c.cm. without
any curdling. These differences are apparently due to the
development of enzymes, and partly to the products of some
species neutralising the action of others. The amount of acid
present at the time of ordinary acid curdling does not widely
vary.

(10) Milk is not necessarily wholesome because it is sweet,
especially if it has been kept at low temperatures. At the
temperature of an ice box milk may remain sweet for a long
time and yet contain enormous numbers of bacteria, among
which are species more likely to be unwholesome than those
that develop at 20° C.

Although these results show that temperature exerts a
selective action on the bacterial flora it must not be forgotten
that this may be wholly or partially negatived by a predominance
of any particular species in the original milk. For example,
milk produced under good conditions and containing less than
10,000 bacteria per c.cm. will very rarely show a predominance
of coliform organisms even when incubated at 37° C. The
curd produced by this class of milk is almost invariably of the
smooth acid type produced by Str. lacticus and seldom gives
the gas-blown curd typical of the B. coli group. An examina-
tion of the type of curd produced on incubation at 37° C. has

EFFECT OF LOW TEMPERATURES 111

been suggested as a simple method of determining the pre-
vailing type of organisms and will be considered in detail on
p. 197.

The development of bacteria in milk at low temperatures
was especially studied by Revenal, Hastings and Hammer.”*
Two samples of milk differing widely in bacterial content were
stored at 0° C. and the count made at intervals on lactose
agar by incubating at 37° C.

TaBLE XLV
Age of Milk, Days. Dairy Milk. Barn Milk.

0 130,000 3,500

6 72,500 4,050

15 633,500 52,900

20 3,230,000 1,240,000

36 34,950,000 4,800,000

74 91,500,000 36,500,000

106 39,750,000 192,500,000

160 32,650,000 361,000,000

That profound modifications had occurred was shown by
the fact that at the end of the experiment over 70 per cent of
the caseinogen was digested. The total nitrogen decreased, due
to liberation of nitrogen in the free state. Pennington also
found a digestion of caseinogen when milk was stored at low
temperatures, over 50 per cent being digested in five to six
weeks at 29°-32° F.

The above results show the importance of storing milk at
as low a temperature as is practicable; although 50° F. may be
regarded as the critical point for bacterial development, efforts
should be made to lower the temperature of milk samples as
far as possible if more than a few hours (3-4) elapse between
collection and examination. If the samples are immediately
surrounded with ice they may be kept for twenty-four hours
without altering the significance of the results although the

112 BACTERIA IN MILK

bacterial count may vary slightly; the direction of this varia-
tion will depend upon the condition of the milk when sampled,
low counts tending to decrease and high counts to become still
higher, thus leaving the general significance unaltered.

ON AMR WN eS

BIBLIOGRAPHY

. Balley and Hall. Centralbl. f. Bakt. 1895, Abt. 2, 793.

Ward. Bull. 178, Cornell Expt. Sta. 1898.

Henderson. J. Roy. San. Inst. 1904, 25, 563.

Sedgwick and Batchelder. Boston Med. Jour. 1892, 126, 25.
Park. Jour. of Hyg. 1901, 1, 391.

. McConkey. Jour. of Hyg. 1906, 6, 385.
. Von Freudenreich. Centralbl. f. Bakt. 1901, Abt. 2, 8, 674.
. Von Freudenreich and Thoni. Centralbl. f. Bakt. 1903, Abt. 2,

10, 305.

. Von Freudenreich. Centralbl. f. Bakt. 1903, 401.

. Bergy. Bull. 125, Penn. Dept. of Agr. 1904.

. Savage. Milk and Public Health. London, 1915, p. 19.

. Stocking. . Rpt. Storr’s Expt. Agr. Sta. 1906, Bull. 42.

. Backhaus. Molkerei Zeit., 1898, No. 4.

. Harrison. Rpt. Ontario Agr. Dept. 1896, 109-113.

. Orr. Rpt. on Milk Contamination, 1908.

. Savage. Bact. Examination of Water Supplies. London, 1906, p. 35
. Park. Jour. of Hyg. 1901, 1, 398.

. Rosenau. U. 8S. A. Pub. Health and Marine Hosp. Service, Hyg.

Lab. Bull. 56.

. St. John and Pennington. Jour. Inf. Dis., 1907, 4, 647.

. Stocking. Storr’s Expt. Agr. Sta. Bull. 28, 1904.

. Ayres and Johnson. U.S. A. Dept. of Agr., Bull. 126. 1910.

. Conn and Esten. Rpt. Storrs Expt. Agr. Sta. 1904, 27.

. Ravenal, Hastings, and Hammer. Jour. Inf. Dis., 1910, 7, 38.
. Pennington. J. of Bio. Chem. 1908, 4, 353.

li

~~ —

|

CHAPTER V
THE ENUMERATION OF BACTERIA IN MILK

AN approximate determination of the total bacteria in
milk by plating on solid media has, for many years, been one
usually made in connection with the examination of milk, and,
although later work has shown that the number so obtained
is usually but a small fraction of the total number present,
these methods have been generally retained on account of their
convenience, and the results are usually described as the total
bacterial counts. ‘There has been considerable difference of
opinion amongst sanitarians regarding the value of this test,
for, whilst some regard the total number of minor importance,
others believe that much valuable information can be obtained
by this determination alone. The fact that the great majority
of regulations for the sale of milk, where regulations have been
enacted, contain no other clause with reference to bacteria
than a maximum number clause, is sufficient to show the trend
of opinion on this subject. Those who deprecate the value of
the total bacteria enumeration take the stand that the large
majority of the bacteria usually found in milk are harmless
saprophytes, and that their determination is more or less a
waste of time and labour. Whilst the former statement is
undoubtedly true, the latter must be emphatically denied.
Until bacteriological technique becomes so developed that
routine methods can be applied for the detection of pathogenic
organisms, those employed in milk examination must be con-
tent with the inferential tests olbtained by determination of the
saprophytes. As has been shown in the preceding chapter,
milk drawn with reasonable aseptic precautions from the
udders of cows contains but few bacteria, and, if properly

113

114 THE ENUMERATION OF BACTERIA IN MILK

treated, can be delivered in that condition to the consumer.
Laxity on the part of the producer or dairyman by the use of
dirty containers or lack of cooling facilities, produces conditions
favourable to the development of bacteria for which milk forms
an excellent nidus. Once the milk has become contaminated,
the organisms multiply very rapidly under favourable con-
ditions, and, by the time the milk reaches the consumer, have
become excessive in number. A low bacterial count is an “a
posteriori” argument that proper and reasonable care has
been exercised in the production of the sample examined, and it

TABLE XLVI
TOXICITY OF MILK (Detfprine)

Mrxep Mitx Comina More tTHan 40 Mines AND GENERALLY Kept

24-60 Hours
Mean Temp. in Shade, Manchester, during time specimens Percentage of Good
were kept, Degrees Fahrenheit. Specimens.

30-35 58

35-40 38.5
40-45 40

45-50 20

50-55 AL ip
55-60 Nil

MILK FROM SHORT DISTANCES (LESS THAN 20 MiLEs) usuALLY KeptT
Less THAN 10 Hours

Mean Temp. in Shade, Manchester, during time specimens Percentage of Food

were kept, Degrees Fahrenheit. Specimens.
50-55 100
55-60 88.8
60-65 73.2
65-70

70-75 50.0

_ ee, hcl ee

if \
i
:
.
~- '

might fairly be inferred that such milk is less likely to contain
pathogenic organisms then one produced by men of careless
and slovenly habits. Farmers who take a pride in their produce
are more naturally liable to prevent infection of the milk by
supervision of their employees, but even if this be not true, it
must be admitted that the conditions which tend to keep in
check the saprophytes also tend to minimise the relative
infectiveness, so that to this extent at least, must credit be
given to careful producers and dairymen. Other conditions
being equal, the total bacterial count is a measure of relative
infectiveness. This statement is supported by the work of
Delépine ! on the toxicity of the Manchester milk supply. He
found that ‘‘ mixed milk . . . showed an increase of virulence
on inoculation into guinea pigs in proportion to the mean
temperature in the shade in Manchester during the time the
specimen was kept.”’ The results are given in Table XLVI,
all tuberculous specimens being excluded.

Increased temperature and keeping period result in an
increased count so that the above statement can be reduced to
one stating that the virulence to guinea pigs was proportional
to the bacterial count. Further figures reported by Delépine
regarding the relative toxicity of cooled and uncooled milk
___ confirm this.

REASONS FOR DETERMINATION OF TOTAL COUNT 115

TaBLeE XLVII
TOXICITY OF MILK (De fprne)

Percentage of Toxic

No. of Samples. Gariples:

1896-1897. Unrefrigerated milk... 141 10.7
1898-1901. Refrigerated milk..... 1782 2.1

Delépine states that “ the difference would probably have
| been greater if the milk had been cooled immediately after
milking.”

116 THE ENUMERATION OF BACTERIA IN MILK

Results reported by the Chicago Department of Health 2
on the relative toxicity of raw and pasteurised milk also confirm
this hypothesis. |

After this consideration of the ‘raison d’étre ” of the bac-
terial enumeration, the methods by which this is accomplished
will now be treated in detail. These may be divided into two
groups: (a) plating methods and (6) direct microscopical
methods. The former are based upon the ability of the indi-
vidual organisms to reproduce at such a rate upon the medium
employed as to produce a visible colony within the period of
incubation, and the latter upon suitable preparation for direct
enumeration under high magnification.

Until within the last few years the former method was the one
usually employed, and as it is still in universal use, it will be
convenient to discuss it first.

Plain nutrient gelatine prepared with fresh beef infusion
was first used with the plate method for the enumeration of
bacteria in milk and still enjoys considerable repute with many
workers for this purpose, the colonies being usually counted
after four to five days incubation at 20° to 22° C. In late
years, however, and especially in America, this method has
largely been supplanted by the substitution of agar for gelatine
and the incubation period reduced to forty-eight hours at blood
heat. Although the agar medium does not produce as many
visible colonies within the incubation period as the gelatine one,
it possesses certain advantages which more than offset this
drawback. In routine work it is very desirable that results
should be obtained in the shortest possible time, and in this
respect the agar medium is decidedly preferable as it reduces
the time required by 60 per cent. If necessary the colonies

may be counted after twenty-four hours incubation, but the’

results so obtained do not exhibit the sharp contrasts given by
the longer period. Some of the author’s results are given in
Table XLVIII2

The average of the ratio of the forty-eight hour count to the
twenty-four hour count is 3.4, but if the abnormal value of

INCUBATION PERIOD

Taste XLVIII

EFFECT OF INCUBATION PERIOD ON MILK COUNTS ON
STANDARD AGAR

117

INCUBATION PERIOD aT 37° C.

Sample No Ratio pe natn
24 Hours
24 Hours. 48 Hours.
684 64,000 140,000 2:2
685 1,500 21,000 14.0
686 55,000 94,000 1.7
687 11,600 16,000 1.4
688 8,500 18,000 2.1
689 44,000 105,000 2.4
690 500 1,600 3.2
691 20,000 63,000 3.1
692 2,300 4,800 2.1
693 2,500 7,000 2.8
695 11,000 21,000 1.9.

sample 685 is omitted, it becomes 2.1 with a variation of from
1.4 to 3.2. Conn‘? reports “ that in the averages in 28 series of
samples submitted to four laboratories, the forty-eight hour
count was the larger in 25 cases, smaller in one case, and the

TaBLE XLIX
BACTERIA PER C.CM. ON
Sample No.
Standard Agar 48 Hours Standard Gelatine 5 Days

at 37° C. at 20° C.
1 123,000 224,000
2 8,000 8,600
4 10,300 8,800
5 1,300,000 1,500,000
7 85,000 113,000
8 155,000 240,000
9 12,700 8,600

118 THE ENUMERATION OF BACTERIA IN MILK

same in two cases.”” The averages of the whole series (omitting
the samples counting in millions) were 299,000 for the twenty-
four hour count and 147,000 for the twenty-four hour count.
This gives a ratio of 2.03: 1. It is obvious that no constant
factor can be employed for the ratio of the twenty-four hour
count to the forty-eight hour count as this will vary with the
bacterial flora. For the same reason the results obtained with

the use of different media are not comparable although they —

usually vary in the same direction. This is well illustrated by
the results given in Table XLIX which shows a comparison
between standard agar and gelatine.

It will be seen that when the bacterial count is low, the dif-
ference between the gelatine and agar count is but small,
and, although the gelatine medium usually gives the higher
result, this is not an invariable rule; the agar occasionally gives
a higher count, but this, in the author’s experience, only occurs
in a small minority of cases and as the bacterial count increases,
the ratio of the gelatine count to the agar count usually becomes
greater.

That the addition of 1 per cent of lactose to both nutrient
gelatine and agar, favours more rapid nea.) is shown in
Table L.

Picks L
Lac
; Standard Agar] ‘Per Gent | Gelatine | Gelatng,
ample No. 48 Hours 48 Hours. 5 Days 5 Days
at 37° C.

at 37° C. at 20° C. at 20° C.

1 123,000 180,000 224,000 240,000

2 8,000 8,400 8,600 8,300

3 12,000 11,000 6,500 12,300

4 10,300 11,900 8,800 13,500

5 1,300,000 1,350,000 1,500,000 1,850,000

6 “600,000 60,000 65,000 84,000

7 85,000 140,000 113,000 156,000

8 155,000 230,000 240,000 500,000

9 12,700 12,800 8,600 14,000

5 WARY mg ep Veen

EFFECT OF SUGAR IN MEDIA 119

Heinemann and Glenn ® investigated the action of dextrose
and lactose-litmus agar at 20° C. and 37° C. and concluded
that incubation at 20° C. for three days was the most preferable
technique as this temperature is less selective in its action than
higher ones and so yields more information as to the original
flora. After twenty-four hours incubation they found the 37°
count to be the higher, but this was reversed after a further
twenty-four hours incubation and the difference was still more
marked after seventy-two hours. Dextrose and lactose litmus
agar gave but insignificant differences in the total count but
the former showed a decidedly higher percentage of acid col-
onies, due, it is suggested, to colonies of the B. aerogenes type
becoming red only temporarily and finally assuming a blue
colour. For this reason Heinemann and Glenn prefer dex-
trose to lactose. The high counts obtained by these observers
seem to indicate that the samples had been kept for some time
and that considerable reproduction had taken place. This
possibly had an effect on the results obtained. For example:
Str. lacticus, which is usually abundant in stale milk, grows well
at 20°, but at 37° produces colonies in forty-eight hours that
are barely visible even with the aid of a low-power magnifying
glass and are usually overlooked when the medium is tinted with
litmus.

The Committee on Methods of Milk Analysis appointed by
the American Public Health Association to investigate the
various details of the plate method using an agar medium
reported as follows (Am. J. of Pub. Hyg., 18, 431).

Acidity (to phenolphthalein at boiling point). Of the
acidities+0.5, +1.0, +1.5 and 2.0, an acidity of +1.5 per cent
gave the best results.

Lactose. 0, 1, 2, 3, and 4 per cent of lactose was tried
at incubation temperatures of 20° C. and 37°C. At 37°C.,
they found that the medium free from lactose was preferable,
but at 20° C. the one containing 1 per cent of sugar was the
best.

Whey, Plain, and 4 Per Cent Lactose Agar media were com-

120 THE ENUMERATION OF BACTERIA IN MILK

pared in 74 tests. In 28 tests ordinary agar gave the best re-
_ sults, whey agar in 24 tests, and lactose agar in 22 tests. They
found that whey agar favoured the growth of lactic acid organ-
isms and ordinary agar of organisms other than lactic acid
producers.

Agar and Gelatine. Litmus lactose agar at 37° C. was com-
pared with litmus lactose gelatine at 20° C. in 25 tests: of these
gelatine gave higher results in 18 tests and agar in 7. Where
gelatine showed the higher count the percentage difference was
much greater than where agar showed the higher numbers. It
was also found that the differentiation of species was much
better on gelatine but that there was a considerable loss of
plates with this medium.

Both media were used at 20° C. in 24 tests and in this series
gelatine was the better in 14 and agar in 10 samples. When
beef peptone gelatine at 20° C. with seventy-two hours incu-
bation was tried against beef peptone agar at 37° C. with
twenty-four hours incubation, gelatine gave the higher count
in 18 tests, agar in 4 tests, and in one test they gave identical
results. The total gelatine count, however, was more than
_ double that on the agar plates. The standard method for the
examination of milk as adopted by the American Public Health
Association in 1912 was the plate method with a plain agar
medium of +1.5 per cent acidity, made with beef infusion
and 1 per cent each of peptone and dried agar. The 1916
‘report recommended certain alterations; concentrated beef
extract, 3 gms. per litre, was substituted for beef infusion and
the acidity was reduced to +1.0 per cent: the quantity of
peptone was reduced to 5 gms. per litre and the agar in-
creased to 1.2 per cent of the dried material. Although the
author has not compared fresh beef infusion media with similar
media prepared with Lemco for the enumeration of bacteria in
milk, his experience with water was that the Lemco media in-
variably gave higher and more consistent results. The reason
for variable results with beef infusions or decoctions lies in the
difficulty in obtaining solutions of even approximately con-

ee Ee

ACIDITY OF MEDIUM 121

stant composition and in the variable quantity of alkali.
required for the adjustment of the acidity. .

Clark * has pointed out that the method of adjusting the
acidity of media, as recommended in the standard methods of
analysis, is not scientific in principle and that it does not ensure
a constant hydrogen ion concentration. Various batches of
media prepared by different workers and adjusted to an acidity

of +1 per cent by the standard method (titration of the boiling

medium with alkali using phenolphthalein) were found to have
very different H ion potentials when tested by the electrical
method. No results are given by Clark as to the effect of this
variation on the bacterial reproduction in these media but the
comparative experiments of a group of New York bacteriolo-
gists indicate that any variation due to this cause is insignificant
and can safely be ignored. In these experiments media were

_ prepared by four laboratories and supplied to Dr. Conn, of

Middletown, Conn., who plated out two samples of milk on
each medium in triplicate. The results were as follows:

Medium. Borden. North. Board of Health. Lederle.
Sample 1... 12,000 15,000 14,000 13,000
Sample 2... 305,000 290,000 280,000 279,000

Three of the above media gave an acidity of +1.0 per cent,
as determined by Conn, and the fourth +0.9 per cent. These
results show that media prepared in various laboratories accord-
ing to standard methods give results as close as can be expected
from a consideration of the technique.

The technique of bacterial enumeration in milk was care-

fully investigated by the New York group of bacteriologists
above referred to and the results summarised by Conn.
Samples of various grades of milk and cream were prepared by
Conn and duplicate samples forwarded to the various laborator-
ies partaking in the work. As the samples invariably included
duplicate samples under different numbers, each sample was

122 THE ENUMERATION OF BACTERIA IN MILK

not only examined in four laboratories but each laboratory
was unknowingly checking the accuracy of its own work.
The various points investigated were as follows:

1. Method of Inoculation. Three methods were employed:
(a) Measurement of the sample into plates and pouring the
agar from flasks, (b) measurement into plates but pouring the
agar from tubes, and (c) inoculation of the tubes and pouring
into plates after rolling. The results obtained show the slight
superiority of the tube inoculation method but the advantage
is so slight as to be of no real importance. In the few cases
where methods (a) and (b) were compared, (a) gave higher
results though there is no manifest reason why this should
occur. In laboratories where large numbers of samples are
examined the slight superiority of the tube inoculation method
is more than offset by the economy in material and labour
effected by the use of the flask method. The author’s experi-
ence has been that, although the time required for plating sam-
ples was not very much reduced, the preparation of media was
greatly facilitated and the cost reduced.

Composition of Media. In one series three different media
were used (a) standard agar (beef bouillon with the addition of
1 per cent agar and peptone and adjusted to +1.5 per cent
acidity), (b) standard agar with the substitution of Liebig’s
extract for beef infusion, and (c) agar prepared with beef
extract but containing only one-twelfth the quantity in (6) and
having an acidity of +0.3 per cent.

The results showed that

In 30 samples (a) medium gave the highest count.
In 27 samples (c) medium gave the highest count.
In 20 samples (b) medium gave the highest count.

So far as the actual numbers were concerned the differences
were of no real significance so that, in this respect, the media
were of equal value. The size of the colonies on (c) medium
was generally small and rendered accurate counting more dif-
ficult. Against this disadvantage must be placed the decreased

ACCURACY OF COUNTS 123

trouble experienced with spreaders. Observations for spreaders
indicated that 128 were found with (a) medium, 21 with (6)
medium and 23 with (c) medium. On the whole, it would
appear that the (6) medium was the most satisfactory.

Uniformity of Technique. The several series of compara-
tive examinations produced some interesting data on the influ-
ence of technique. In the first series when each laboratory
used the technique as previously developed in that laboratory,
the results on duplicate samples showed a variation factor of
from 1.3 to 43.2 with an average of 6.2. The variation factor -
was obtained by dividing the highest result by the lowest.
Duplicate analyses in each laboratory also showed variations,
the average factors varying from 2.1 to 4.8 with a general
average of 3.7.

In a second series of tests the various laboratories all em-
ployed identical technique as to shaking of sample, diluting,
pipetting, inoculating, and counting of plates. As it was found
in the first series that one laboratory employed a magnifying
lens for counting plates and another the naked eye, it was
decided to use a standard lens in all laboratories and to deter-
mine the personal error in counting by an exchange of incubated
plates. The results showed that the personal error may be a
serious one, for, although the variation in duplicate counts of
identical plates was usually small, the extreme variation was
nearly 100 per cent. In this series the average variation in
each laboratory was from 1.6 to 2.2 with a general average
of 1.8. .

A five-day count was also compared with the two-day count
and, although the results were usually higher they were not
uniformly so. There seems to be no apparent advantage attain-
able by prolonging the incubation period beyond the usual
forty-eight hour period.

In the third series the effect of agitation, amongst other
points, was determined, and although the results are not con-
clusive they indicate the importance of standardising this por-
tion of the technique. In the third and fourth series the plate

124 THE ENUMERATION OF BACTERIA IN MILK

method of enumeration was also compared with the direct
microscopical method of Breed but this will be dealt with later.

From a consideration of this work Conn pointed out that
variations in technique are much more important than the com-
position of the medium, and that variations in results may
reasonably be expected, even under the best conditions due
(1) to clumping of the bacteria, and (2) to the bacteria being
in non-uniform suspension and not in solution. These two
factors render it improbable that two small samples will contain
equal numbers of organisms, and the lower the total number of
bacteria the greater will this divergence become. Conn ex-
pressed the opinion that ‘ individual counts under the best
conditions are subject to considerable variation and that no
single individual count can be relied upon.” ... “It is not
possible to rely upon a greater accuracy than 100 per cent even
when the average of more than one sample is obtained, although
most of the results fall considerably below this limit.”

During 1915 the author made a series of duplicate examina-
tions of milk by plating one of the routine samples in duplicate
daily; in this series plates containing 73> ¢.cm. and yep ¢.cm.
were inoculated and counted with a low-power glass after forty-
eight hours incubation at 37° C. Porous covers were used to
prevent loss of plates by spreaders. In 142 samples the differ-
ence between duplicate determinations varied from zero to
464 per cent with an average variation of 24.7 per cent. Ex-
pressed as a variation factor the average was 1.25 (1.247) with a
maximum of 4.64. The bacterial count varied from 1600 per
c.cm. to 1,200,000 per c.cm. and it was with the best grade milks,
i.e., those containing less than 10,000 per c.cm., that the vari-
ations were the largest. This was anticipated from a consider-
ation of the frequency distribution in the largest amount of
sample plated and could have been reduced by inoculating
larger quantities. This was not done because the labour in-
volved in so treating all samples, when but very few were of
this grade, was not justified by the increased precision so ob-
tainable, for whether a sample contains 1600 or 5000 organisms

ae

AMERICAN STANDARD METHODS 125

per c.cm. has no real bearing on its hygienic quality. This
series of comparative results is not so important as that reported
by Conn because of the psychological factor; both the person
plating out the samples (A. J. S.) and the one counting the
plates (J. R.) were aware that these determinations were being
made, and although every endeavour was made to honestly
record the actual conditions found, it is recognised that the
results are subject to these limitations.

The detailed technique for the plate method as adopted by
the American Public Health Association in 1916 is as follows:

Dilutions. For samples of unknown character dilutions
of 1 to 100, 1 to 1000, 1 to 10,000 shall be made, using sterile
water and pipettes after the ordinary method. In case the
character of the milk is known, less than three dilutions may be
made; but in no case shall less than two plates for each sample
be made. Grade A,* or its equivalent, should be plated in

duplicate, and a dilution lower than 1 to 100 may be used.

Shaking. Samples must be shaken twenty-five times.
Shaking is defined as meaning a rapid up and down motion
with an excursion of not less than 1 foot. |

Pipettes. Pipettes must be made to deliver between grad-

’ uation marks, not simply to deliver.

Pouring Plates. The melted agar must be poured promptly
after measuring out the proper quantities of milk. Not more
than twelve plates must be allowed to accumulate after the
distribution of the milk into the plates before pouring the
agar.

Incubation and Counting. One standard temperature only
is recognised—forty-eight hour incubation at 37° C.

If possible count those plates containing between 30 and |
200 colonies. If there are none such, count those plates con-_
taining nearest to 200 colonies. The whole number of colonies
on the plate shall be counted where the plates contain less than
200 colonies. |

* Milk usually containing less than 10,000 bacteria per c.em.

126 THE ENUMERATION OF BACTERIA IN MILK

Counting Lens. The lens recommended by the Committee
in 1914 is more fully defined. It is known as Engraver’s lens
No. 146, Bausch & Lomb catalogue. It is designated as 3}X,
its magnification being 2} diameters. Persons who are near-
sighted should wear their ordinary glasses while using this lens.
Farsighted persons should use the lens without their glasses.

Direct Methods. The direct methods of enumeration of
bacteria in milk are of comparatively recent development; in
these the milk or centrifugalised sediment is smeared over a
slide, and, after suitable staining, examined under a high-
power objective and the bacteria counted. The direct method
as modified by Slack ® is as follows. Two c.cms. of the sample,
after thorough shaking, are inserted into special tubes with
rubber stoppers at each end, and centrifugalised for ten minutes
at 2500 revolutions per minute in a special apparatus. This
apparatus is a modification of the one used by Stewart of Phil-
adelphia for leucocyte estimation, and consists of an aluminium
disc and cover 10 inches in diameter and 3 inch in depth, fitted
to hold twenty tubes arranged radially. This apparatus is
manufactured by the International Instrument Co., of Cam-
bridge, Mass., and can be used with the usual electrical cen-
trifuge. After centrifugalising, the tubes are carefully removed,
and, to obtain the sediment with the least disturbance, the tube
is held with the cream end downwards, whilst the cream layer
is removed by means of a platinum loop. The milk is then
carefully poured out without permitting air bubbles to ascend
the tube, and finally, with the tube in the same position, the
other stopper is removed and the sediment is smeared on a glass
slide with the aid of a drop of sterile water. An area of 459
cms. is a convenient one and squares of this size may be marked
off on a strip of glass with a blue grease pencil. The smear is
dried, fixed by heat, and stained with methylene blue. The
specimen is then examined under a 7 inch oil immersion lens
and the organisms counted. Each coccus, bacillus, diploco¢cus,
or chain represents a colony.on the 1—10,000 plate of the same
sample when grown on agar for twenty-four hours at 37° C.

DIRECT METHODS 127

This factor of 10,000 was modified later to 20,000 in order to
correspond to the forty-eight hour incubation period. Whilst
it was not claimed that the whole of the bacteria are contained
in the sediment, it was asserted that in 99 per cent of the sam-
ples a representative number is so precipitated, and that this
number bears a fairly constant relation to the bacterial count as
determined by plating on agar.®

Slack, in a series of over 2200 samples, compared the results
obtained by the centrifuge and plate methods (twenty-four
hours at 37° C.) and an error of less than 1 per cent was made in
passing as below 500,000 bacteria to the cubic centimetre,
milks which the plates showed to be above this limit.

This method has also been examined by Gooderich !°
who reports very favourably upon it and remarks that very little
improvement can be made upon the factor 210* (20,000) for
converting the microscopical results to the forty-eight hour
count on agar. He reports the limits for the factor as being
from 0.66X10* to 6.010%. With a standard. of 50,000 bac-
teria per c.cm. he found that the direct method wrongly passed
8.6 per cent, and wrongly condemned 8.9 per cent, but that
when the standard was raised to 100,000 these figures were
reduced to 1.4 and 4.3 per cent, respectively. In considering
these results it is important to note that all the determinations
were made on samples secured from the University Stock Farm.
The variations in bacterial content of such samples would not
be nearly so great as is met with in routine work on various
market milks of unknown origin, with the consequence that the
errors would be minimised. The small variation in the counts
is clearly indicated by the fact of the mention of only a 1—1000
dilution being used for plating. Such a procedure is impossible
in routine work on market samples in which the count may vary
from a few hundreds to 5,000,000 or even more. In view of
the excellent results obtained by Gooderich, the writer experi-
mented with this method, although a consideration of the fun-
damental principles did not lead to an anticipation of a high
degree of accuracy *. If the results were to correspond with the

128 THE ENUMERATION OF BACTERIA IN MILK

usual plate count it was essential that a constant proportion of

the bacteria capable of development on agar in forty-eight hours *

at 37° C. must be precipitated during the process of centri-
fugalisation. A portion of the bacterial flora of milk, however,.
does not produce visible colonies on agar under the usual condi-
tions, so that either these organisms must remain in suspension
or the error due to them be counterbalanced by some other
factor.

No difficulty was found with the technique until the micro-
scopical examination was made. The representative field in
which the organisms were to be counted was difficult to find
owing to the widely differing content of various fields. In
order to minimise this source of error ten fields were taken at
random and the average calculated.

In a series of market samples, for which the standard was
500,000 bacteria per c.cm. not a single sample was condemned
which passed the plate method; on the other hand, 17 rer cent
were passed which were condemned by the plate method.
. According to these results the direct method outlined above
would not be oppressive on the milk producer, and its adoption
would be tantamount to lowering the standard. In this series
the factor (c) for the conversion of microscopic counts to plate
counts varied within very wide limits, viz., from 0.4104 to
33.0104, and the author is convinced that this is largely due to
the difficulty found in obtaining an even distribution of organ-
isms on the slide. Two observers obtained widely varying
results from the same slide; a condition fatal to accuracy.
Breed," in 1911, improved this method by making a direct
smear of the milk and thus eliminating the centrifuge with its
many unknown factors. Breed’s method consists essentially
in spreading a small volume of milk over a marked area and
examining under a high-power objective after washing out the
fat followed by suitable staining. Skar,!* in 1912, independ-
ently developed a similar method which differs only in the
manner of staining and in allowing the fat to remain in the
smears. Rosam’s method!* differs essentially from Skar’s

ee ee?

gue

2 PET 2 SpE ee

Ferre:

DIRECT METHODS 129

method only in the method of smear examination: these are
made on a cover glass and examined whilst wet.

In some of the comparative experimental work reported by
Conn and discussed on page 123, a series of bacterial counts
was made by Breed and this was supplemented in a further
series by the inclusion of Brew, a co-worker with Breed. These
experimenters made microscopical counts on the samples plated.
by other observers, and Conn? considered that when the
groups of organisms only were counted, the count agreed some-
what closely with the plate count. When raw market milk
was examined, the variations found were generally not greater
than the differences between the plate counts in various labora-
tories, but for high-grade raw milk and pasteurised products it
is comparatively useless. The details of Breed’s process are
as follows: 0.01 c.cm. of milk, from a well-shaken sample, is
measured out by means of an accurately calibrated special
pipette and deposited on a glass slide on which an area of 1
square centimetre has been previously marked out. The drop is
evenly smeared over this area with a stiff needle and gently
dried at about 50° C. The slide is then placed in a Coplin
staining jar containing xylol or gasoline to remove the fat, and,
after drying, fixed in alcohol (70 to 95 per cent). Immediately
afterwards the smear is stained with 1 per cent aqueous methy-
lene blue and, finally decolourised to a light blue in 95 per cent
alcohol. The microscopical examination is made with a 75
inch oil immersion objective. In order to find the factor for
converting the number of organisms per field into organisms
per cubic centimetre the diameter of the field is determined with
a stage micrometer. The factor is then calculated from the
formula:

A
ape 100=y,

where y is the factor sought, z, the area of the smear in square
millimetres, and FR the radius of the field.
In practice it is convenient to pull out the draw tube until

130 THE ENUMERATION OF BACTERIA IN MILK

the area of the field is of such a value as will give a value to
y having as many ciphers as possible. The following are the
most satisfactory.

When R=0.080 m.m., y = 500,000
When &=0.089 m.m., y =400,000
When R=0.101 m.m., y = 300,000

When the desired result is obtained the position of the draw
tube is noted and always set at this point in future examinations.
In order to get results comparable with the plate method, only
the groups or clumps, together with isolated bacilli are counted;
individual cocci, diplococcus or streptococcus chains, and rod
forms where the plane of division shows clearly, are counted as
individuals. The number of fields to be examined must be
determined by the frequency of the organisms. It is obvious
that with a factor of 300,000 to 500,000, this method is of the
greatest advantage when the count averages one clump or more
per field; with high-grade milks under 10,000 bacteria per
c.cm. the number of fields to be examined would be so large,
if reasonable precision is to be obtained, as to consume as much
time as the plate method. Dead bacteria are counted with the
living, so that this process is not applicable to pasteurised
products; it would, however, be of advantage in determining
the quality before pasteurisation. A collateral advantage of
this method is that in addition to the quantitative estimation of
the bacteria, a cell count can be made at the same time and
information obtained regarding the bacterial flora.

As an indirect method for estimating the number of bacteria,
Barthol,! in 1908, suggested the employment of methylene
blue. It was found by Barthol and confirmed later by Jensen
and Muller, that the time required to decolourise methylene
blue bears a relationship to the number of bacteria present.
Fred !6 showed that 21 of 23 species of milk bacteria were capa-
ble of reducing methylene blue and that each species has a

Ce ee

INDIRECT METHODS 131

different coefficient of velocity; the velocity of reduction was a
linear function of the temperature (up to 37° C.) and, finally,
ceased with exhaustion of the medium. It was formerly sug-
gested that the reduction of methylene blue in this “slow
reductase test’ as it is usually termed, was due to enzymes
present in the intramammary milk, but it is now generally held
that such milk does not contain reducing substances and that
the reduction is due to intra and extra cellular products of bac-
terial origin.

Fred !? in an examination of 200 samples of milk by this
method (adding 1 c.cm. of a 0.05 per cent solution of pure
methylene blue in 0.4 per cent saline to 10 c.cms. of milk and
holding at 40° C.) found that the time required for reduction
‘was proportional to the bacterial count. His figures are given
in Table LI, each group representing the average of 20 samples.

TABLE LI
Average Number of Bacteria Average Time of Reduction
Group Number. :
per c.cm. in Hours.
1 29,647 11.9
2 73,587 9.7
3 160,150 9.5
4 283,250 8.0
5 548,300 7.8
6 1,016,600 4.7
7 1,469,650 3.1
8 2,505,000 2.7
9 4,690,000 1.5
10 8,624,800 1.0

Barthol !* found that samples containing more than 10,000,-
000 bacteria per c.cm. and 50 per cent of those containing
4-10 millions per c.cm. reduced within one hour. He concluded
that 10 millions per c.cm. was the lowest limit that could be
estimated by this method and that below this limit there is no

Pp ttl
i~3
fi;
ee -
7 |
‘
i

132 THE ENUMERATION OF BACTERIA IN MILK

relationship between the number of bacteria and the time
required for decolourisation. |

The author examined a number of milks by this test in 1914
but was unable to find any merit in it. Almost all the samples
failed to decolourise in the six hours that were available ‘or
observation under ordinary laboratory conditions, and they had
generally showed reduction by the following morning (twenty-
one hours). As over 90 per cent of these samples contained
less than one million bacteria per cubic centimetre these results
are not inconsistent with Fred’s (vide swpra), but as the time
of reduction could only be determined within wide limits no
real information could be deduced as to the bacterial condi-
tion of the sample, except that it did not contain very excessive
numbers. Samples that were allowed to stand and develop
large numbers of organisms showed small reduction periods and
it would seem that it is in the detection of such milk that the |
chief value of the test lies.

A further rapid indirect method that has been suggested
for the approximate determination of the bacterial content of
milk is the estimation of the acidity. Milk almost invariably
contains acid-producing organisms, and as these find milk an
excellent medium for development it would seem to be logical
to assume that the determination of the products of bacterial
metabolism would bear some relation to the number of organisms
present. Fred (vide supra) determined the acidity of 200
samples of milk and arranged the results into groups of 20
according to the bacterial count. His results are given in
Table LILI.

Fred is of the opinion that the acidity determination ‘serves
a useful purpose in indicating to some extent the proper dilu-
tions to be used for the bacterial counts, and adds that “‘ the
relationship to the number of bacteria is only approximate.”
Russell and Hastings have also suggested using this test as a
guide to the dilutions to be made in the plate method and advise
10, 100, and 1,000 dilutions for acidities under 0.2 per cent and
1,000, 10,000 and 100,000 for acidities over 0.2 per cent.

RELATION OF ACIDITY TO BACTERIAL COUNT 133

TaB_eE LIT

RELATION OF ACIDITY TO BACTERIAL COUNT (Frep)
Group Number. Average srs as Lactic Number 4 Bec per

1 0.189 - 29,647

2 0.188 73,587

3 0.183 160,150

4 0.201 283,250

5 0.192 548,300

6 0.205 1,016,600

7 0.206 1,469,650

8 0.212 2,505,000

9 0.231 4,690,000

10 0.250 8,624,000

The author, during 1914 and 1915, determined the acidity
and bacterial count of a number of the samples received for
routine examination with the following results:

TasBie LIII

RELATION OF ACIDITY TO BACTERIAL COUNT (Auruor)
- ACIDITY.
Number of Bacterial Count
Samples. Devens. Tastle. hed 48 Hours at 37° C.
Per Cent.

34 14 0.126 203,000
67 15 0.135 332,000
102 16 0.144 282,000
144 17 0.153 289,000
186 18 0.162 232,000
185 19 A I 212,000
120 20 0.180 175,000
32 21 0.189 408,000
28 22 0.198 397,000
9 23 0.207 541,000

134 THE ENUMERATION OF BACTERIA IN MILK

These results show no definite relationship between the
acidity and the bacterial count until the acidity approaches
0.20 per cent (22°), and in this respect, are confirmatory of
Fred’s results. Only 9 samples out of a total of 917 exceeded
22° acidity and it became obvious that the acidity determina-
tion even as a guide to the best dilutions to employ in plate
work did not give information commensurate with the labour
involved. For pasteurised and heated milk the acidity estima-
tion is of even less value than for ordinary raw milk owing to
the change in acidity acused by the heating processes,

BIBLIOGRAPHY

. Delépine. Jour. of Hyg. 1903, 3, 68.

. Laboratory Rpt. of Chicago Dept. of Health. 1907-1910.

Race. Can. Jour. of Pub. Health. 1915, 6, 13.

Conn. Pub. Health Rpt. U.S.A.P.H.S., 1915, 30, 2390.

Heinemann and Glenn. Jour. Inf. Dis. 1908, 5, 412.

American Jour. of Pub. Hyg. 18, 481.

. Clark. Jour. Inf. Dis. 1915, 17, 109-136.

. Slack. Tech. Quart. 1906, 19, No. 1.

. Standard Methods for Bact. Exam. of Milk, Amer. Pub. Health.
Assoc., 1912, p. 25.

10. Goodrich. Jour. Inf. Dis. 1914, 14, 512.

11. Breed. Centrabl. f. Bakt., Abt. 2, 30, 337-340.

12. Skar. Milchw. Zentbl. 41, 454461, ibid., 705-712.

13. Rosam. Milchw. Centbl. 1913, 42, 333.

14. Conn. Pub. Health Rpt. U.S.A.P.H.S. 1915, 30, 2394.

15. Barthol. Zeit. Untersuch. Nahr. Genussm. 1908, 15, 385-405.

16. Fred. Zeit. f. Bakt. u. Parasitenk. 1912, 35, Abt. 2, 391.

17. Fred. Rpt. Virginia Agar. Expt. Sta. 1911-12, 206-240.

18. Barthol. Zeit. Untersuch. Nahr. u.Genussm. 1911, 21, 513-534.

CHNAMR WN

CHAPTER VI
EXCREMENTAL ORGANISMS

THE estimation of typical excremental organisms in milk is of
considerable value because of the general absence of these
bacteria in intra-mammary milk; they indicate, therefore, the
amount of care exercised in the production and handling of
the milk in a rather better manner than the determination of
the total number of organisms, but as milk drawn under the best
conditions is never absolutely free from excremental organisms,
this advantage is merely relative.

The estimation of the bacteria usually regarded as indica-
tive of manurial pollution has not in the past been developed
to full advantage because of the somewhat elaborate technique
involved, and also because some sanitarians have regarded the
excremental bacterial content as being more determined by
duration and conditions of storage than by the original pollu-
tion. It would, undoubtedly, be of great advantage if some
method could be found of determining the manurial pollution
of a sample at the time of milking, not only because it would
yield precise information as to the condition requiring correc- —
tion, but also on account of the possible association of tubercle
bacilli with the fecal bacteria. Tubercle bacilli grow so slowly
in milk in comparison with the typical excremental organisms
that any inferential value associated with the determination of
the latter is rapidly nullified by the conditions usually obtain-
ing in the marketing of milk.

The organisms commonly used as indicators of manurial
pollution are B. coli, B. enteritidis sporogenes, and Streptococci,
and of these B. coli is probably the most important and the most
easily estimated. English bacteriologists have, on the whole,

135

136 EXCREMENTAL ORGANISMS

devoted more attention to these estimations than their Ameri-
can confréres, but neither have studied them as fully as they
deserve and it is to be hoped that this condition will soon be
rectified.

These organisms will now be treated in detail.

1. B. Coli. The term B. coli in these pages is used to
signify the general group of aerobic, non-sporulating organisms
that ferment lactose with the production of acid and gas, and
not one particular member of the group, such as B. coli com-
munis, having certain specific characteristics in addition to the
generic ones just described. Many attempts have been made
to regard certain members of this group as being more sig-
nificant than others-but this has been a comparative failure
when viewed by the light of later experience.

MacConkey ! reported upon the biochemical characters of a
number of members of the B. coli group, isolated from milk and
from the feces of cows, and classified them into four groups
according to their action on saccharose and dulcite. The
results are given in Table LIV.

TasBLe LIV
Milk. Cow’s Feces.
Per Cent. Per Cent.
Saccharose +dulcite+.............2000005. 32.7 47.9
Saccharose—dulcite+.............-00005- 39.2 25.0
Saccharose +dulcite—..............-.0005. 19.6 12.5
Saccharose —dulcite—.............22000 eee 8.4 16.6

MacConkey suggested that these groups should be further
subdivided according to the ability to ferment adonite and
inulin, the Voges and Proskauer reaction, and the motility.
In 1909 he reported the characteristics of colon organisms
isolated from animal and human feces and arranged the group-
ing in accordance with the subdivision? As this further
division has not been generally adopted, the results have been

B. COLI | 137

rearranged into the four general groups in Table LV and Orr’s
results * added for comparison.

TABLE LV
MacConkeEy. ORR.
Milk Milk Milk
Human |} Animal from from
from Manure.
Feces. | Feces. Cow- : Con-
Retailer.
shed. sumer.

Per Cent.|/Per Cent.|Per Cent.|Per Cent.|Per Cent.|Per Cent.

Saccharose+dulcite+.| 32.2 | 48.1 28.5 26.5 26.1 18.7
Saccharose —dulcite+.| 27.0 34.3 13.8 10.4 12.8 35.4
Saccharose+dulcite—.| 4.5 | ..... 43.9 39.1 41.1 33.4
Saccharose —dulcite—.|} 28.0 8.4 12°56 31. 204 Ete 47 8.4
Other strains.........| 8.3 9.2 142 3.6 3.3 4.1

The results of Rogers et al.,f who investigated 107 colon
organisms obtained from milk products, and some unpublished
ones of the author on the biochemical characters of coliform
organisms obtained from 226 samples of milk, are given in
Table LVI. ~

Taste LVI
Rogers et al. Author.
Per Cent. Per Cent.
Saccharose+dulcite+........ oes (nat 43 24.3. 46.5
Saccharose —dulcite+................. 14.9 8.4
Saccharose+dulcite—................. 37.4 36.3
Saccharose —dulcite—................. 23.4 8.8

The author’s results, obtained with samples of the Ottawa
milk supply, are somewhat in accordance with Orr’s results as
regards the predominance of saccharose fermenters, but show a
larger proportion of dulcite fermenters. This predominance of
saccharose fermenters accords with the results recorded for

138 EXCREMENTAL ORGANISMS

animal feces and would seem to differentiate between animal
and human pollution, but as the difference is one of degree only
and is not specific, no definite significance can be attached to it.
Although a large amount of work has been done on the separa-
tion of the colon group of organisms, no test or combination of
tests has been evolved that would indicate that any one sub-
group is more typical than another, and it must, therefore, be
borne in mind that to designate any organism as being typical B.
coli because it possesses certain biochemical and morphological
characteristics is a purely arbitrary and empirical procedure.
Moreover, these organisms are not to be regarded as having
immutable properties like chemical compounds, but to form
involution and mutation varieties according to the environment.

Milk, even when produced under the best. conditions, is
never quite free from B. coli, but if reasonable precautions are
taken, this group should not be present in 25 c.cm. quantities
of byre milk. Even after bottling and delivery to the pur-
chaser milk can be produced that will average less than two
B. coli per cubic centimetre, even during the summer months.

This is exemplified in Table LVII.
TaBLeE LVII
BACTERIA AND B. COLI IN CERTIFIED MILK (Avrnor)
Abomah: Mean Bacterial Mean B. Coli
Count per c.cm. per c.cm.

OM Sr ESy cs ties alge ae aie 5,700 1
PUM Dot ee aay eae ee gas 10,900 2
PUY 5 Gr ol als pele De oe ae 5,000 0.1
AllgtaiS Si. 802059 Foch eee 4,500 0.8
SOpmee en ) sue a aate's Pages 5,500 1.4

When milk is kept at a temperature not exceeding 45° F.
the B. coli do not increase (vide p. 104) and this temperature
may, therefore, be regarded as the critical anabolic tempera-
ture. Above this point they multiply rapidly and in summer

TEMPERATURE AND BB. COLI 139

the B. coli content of milk must be regarded as due more to
reproduction than to original contamination. Diagram No.
III, which shows the B. coli content of the Ottawa raw milk
supply compared with the mean atmospheric temperature,
demonstrates very clearly the effect of temperature. In the
autumn months the curves do not correspond because the mode
of the B. coli curve is lowered during the hot summer months

DracraM No. III
EFFECT OF ATMOSPHERIC TEMPERATURE ON B. COLI CONTENT

OTTAWA
18,000 100
16,000 90
14,000 R 80 +
7)
a a a
12,000 Vi 70 2
LQ. \ FE
° ley “As, ss
; MHiude a
310,000 7. aa 60 g
= Ke ‘
° ? M A
i=) =e) ¥
ages a V4 50
eid E
L rs
6,000 / i 40 2
‘ |
Pa
4 + - ;
000 es f 4 B. coli -
‘ \ i os Temperature __
,000 . Po Va 20
on ae
— — 10
Nov. Dec.jJan. Feb. Mar. Apr. May June July Aug. Sept. Oct.
H—— 1914 1915

by artificial cooling of the milk and the temperature of the milk
is, consequently, not proportional to the atmospheric, but it is
evident that artificial cooling is abandoned before the natural
agencies become entirely operative. It is also interesting to
note that after the very cold winter weather the B. coli content
does not increase until the mean atmospheric temperature
exceeds the critical temperature.

140 EXCREMENTAL ORGANISMS

Estimation of B. Coli. The methods in vogue for the esti-
mation of B. coli fall into two groups, (1) enrichment methods
and (2) plate methods.

Enrichment Methods. In the enrichment methods, varying
quantities of the sample are inoculated into liquid media and
incubated, thé media being subsequently examined as to the
presence or absence of B. coli. In this test a carbohydrate is
usually employed that is fermented by B. coli with the pro-
duction of gas and special tubes are used in which this gas is
trapped and retained as visible evidence of fermentation. On
account of the economy of space a small inverted tube con-
tained in a larger ordinary culture tube (Durham’s tube) is
now in almost universal use in the fermentation process. As
in water examination, there are a number of points in connec-
tion with this test that require consideration. ‘The first is the
composition of the medium to be employed. If the results are
to be based on the presence or absence of gas in the tubes, it
is evident that lactose and not dextrose must be the carbo-
hydrate employed as there are other groups than B. coli that
ferment the latter sugar. The nitrogen requisite for bacterial
reproduction is usually supplied by the addition of peptone,
although this may be partially displaced by sugar-free beef
infusion or extract. Potassium chloride is also a desirable con- —
stituent (Chamot and Sherwood). Such a medium will give
gas formation even with attenuated B. coli,and, if only vigorous
forms are desired to be estimated the medium can be prepared
with a base of fresh ox bile instead of water. There is con-
siderable evidence, however, that the lactose ox-bile medium
inhibits the growth of a number of vigorous forms of B. coli in
addition to the attenuated ones and for this reason the fresh bile
medium is often regarded with disfavour. MacConkey’s me-
dium, containing 0.5 per cent of bile salt, may also be used and
in this case the results will usually be intermediate between
those obtained with lactose broth and lactose bile. “The main
objection to lactose broth is the excessive number of anomalies
caused by the overgrowth of other organisms. Aciduric bacilli

ESTIMATION OF B. COLI | 141

occasionally reproduce so rapidly in the lower dilutions as to
prevent the growth of the coliform bacteria and so give a
negative gas test when a much higher dilution of the same
sample shows copious gas formation.

The usual amounts of lactose and peptone employed in the
fermentation test are 1 per cent of each, but Chamot and Sher-
wood ® have shown that a lactose content of 0.6 per cent pro-
duces equally satisfactory results as 1.0 per cent. Under 0.6

~ per cent the results were irregular and the total volume of gas

small, whilst quantities much exceeding 1.0 per cent retarded
the rate of gas formation. With normal acidities they found
that the total gas volume was proportional to the concentration
of the nitrogen whether present as peptone, beef extract or
infusion. With increasing amounts of peptone the increase
in gas volume was rapid until 4.0 per cent was reached and when
both final volume and rate of production were considered,
it was found that a concentration of 3.0 to 4.0 per cent was the
optimum. Potassium chloride (0.6 per cent) hastened gas
formation and was found superior to phosphates and other
salts. The concentrations finally recommended were lactose
0.8 per cent, peptone 3 to 4 per cent, KCl 0.6 per cent, and the
reaction +1.0 per cent. With lactose bile the nitrogen content
should be sufficient with the addition of only 1.0 per cent of
peptone, but in other media the higher amount should be em-
ployed. For the concentration method the author uses ordinary
lactose broth or lactose bile salt broth in preference to lactose
bile on account of the irregularities often found with lactose
bile and due to the variations in composition.

The number of tubes to be employed in order to obtain
reasonably precise results is the second point for consideration.
It has been usual to use such dilutions of milk that the quan-
tities represent decimal fractions of 1 ¢c.cm. and to endeavour
to obtain at least one positive and one negative result. Al-
though, in many instances, no attempt has been made to con-
vert such positive and negative findings into mathematical
expressions, others have attempted to do so by taking the

142 EXCREMENTAL ORGANISMS

reciprocal of the lowest quantity showing a positive result as
representing the number of B. coli per cubic centimetre. Thus,
0.1 c.em.+, 0.01 c.em+, 0.001 c.cm.—, was expressed as 100 B,
coli per cubic centimetre. When the average of a number of
samples from one source is calculated by this method (Phelps ®)
an accurate result is obtained providing the series is fairly
large (about 25), but McCrady 7 has shown that for individual
samples such assumptions are far from accurate. McCrady
calculates from the theory of probabilities that the most prob-
able number of B. coli present per cubic centimetre, if the above
result were obtained, would be 230 and not 100 as assumed. It
is possible that any number of B. coli per cubic centimetre would
produce this result and, in order to reduce the range of possibili-
ties and sharpen the probability curve, it becomes necessary to
employ more than one tube of each dilution. The greater the
number of tubes used the greater is the precision obtained. With
a milk of unknown origin that may contain up to 100,000 B. coli
per cubic centimetre it is obvious that even if only three tubes of
each dilution are used the total number of tubes for each sample
becomes so great as to be cumbersome. For this reason the tube
method of estimating B. coli in milk cannot be recommended.

The third point for consideration is the method of recording
the results. If desired, all tubes showing gas may be plated out
on rebipelagar or litmus lactose agar and the red colonies so
obtained put through confirmatory tests, but as such a pro-
cedure requires much time and labour it will be found more
convenient and fairly accurate to record all tubes as positive
that show more than 5 per cent of gas. Anomalies at the
higher end of the series should be ignored as they are probably
the result of overgrowths, but those at the lower end should
be corrected by moving the lower positive results to the next
higher dilution; thus, 1.0 c.cm.—, 0.1 c.cm.+, 0.01 c.cm.+,
0.001 c.em.+, should be recorded as 1.0 ¢.cm.+, 0.1 ¢.cm.+,
0.01 c.cm.+, 0.001 c.cm.4+, but 1.0 ¢.cm.+, 0.1 c¢.cm.+,
0.01 c.em.—, 0.001 ¢.em.+, should be recorded as 1.0 c.cm.+,
0.1 c.cm.-+, 0.01 c.cm.+, 0.001 c.cm.—.

se eee ee ee

ESTIMATION OF B. COLI 143

Plate Methods. Quite a number of solid media have been
suggested for the isolation and enumeration of B. coli and allied
organisms and of these the most useful are Endo’s medium
(fuchsin sulphite agar), Drigalski and Conradi’s medium (nut-
rose agar), zsculin bile salt agar, and rebipelagar (neutral
red bile salt agar). On account of the difficulties connected
with the preparation and use of the first two media the
author prefers the latter two. These are easy to prepare (see
appendix p. 207) and may be used in exactly the same manner
as ordinary nutrient agar or gelatine. The Committee on
Standard Methods of Milk Analysis of the American Public
Health Association investigated the latter two media and
reported in favour of the esculin medium. They found more
bacteria of the B. coli group on rebipelagar in nearly every
instance but this was due to the difficulty in deciding which
were the coliform colonies on the esculin medium. Of more
than fifty colonies subcultured from the neutral red medium
only 67 per cent were found to be B. coli or B. erogenes (B.
lactis erogenes) whereas all the dark colonies from the ezsculin
medium were of the B. coli family. Savage %, from his expe-
rience with esculin agar and rebipelagar, as compared with
lactose bile salt broth, has expressed the opinion that both
media are equally useful but inferior to L. B. B. tubes on
account of the difficulty in arriving at accurate estimations
of the numbers by direct plating. The author has had very
little experience with esculin agar, but the extended observa-
tions that he has made with rebipelagar do not entirely agree
with the above results. A-series of comparative experiments
on 100 samples with rebipelagar and lactose bile salt broth
gave the following results, gas formation being regarded as
evidence of the presence of B. coli in the tube series without
confirmation.

Medium. B. coli per C.cm.
pe SE el aa a a 15,326
BU TMA, ie eb ac nee Wl oak eld 00's 10,182

144 EXCREMENTAL ORGANISMS

In 72 samples the two methods agreed, that is the plate
count was in approximate agreement with the reciprocal of the
smallest quantity of the sample showing gas formation. In 25
samples the results differed by one dilution (the dilutions being
decimal fractions of a cubic centimetre), in two samples by two
dilutions, and in one sample by three dilutions. The agree-
ment in the averages is very reasonable when the chance errors
of distribution inherent to the tube method are considered, and
the differences between individual samples can be shown to be
well within the limits calculated by the theory of probabilities.

The errors connected with rebipelagar are caused (1) by the
destruction of the characteristic colour of the B. coli colonies by
the diffusion of amines or other alkaline substances through the
medium and (2) by the development of red colonies by organ-
isms not of the B. coli group. When a dilution of the sample
is employed that prevents overcrowding of the colonies, the
first error is usually avoided unless there is a large excess of
alkali forming organisms present; this condition can be easily
recognised because either a yellow area is produced concen-
trically from a colony, or, as is usually the case, the whole of
the medium is yellow. The error due to organisms other than
coliform bacteria is small and can be largely eliminated by
experience. The characteristic forms produced by coliform
organisms on the surface of the plate may either be a colony
of deep red colour producing a haze in the surrounding medium,
or one with a red centre surrounded by a yellowish or pinkish
aureole of slimy consistency. The subsurface colonies are of
the former variety but may not invariably produce the haze
which is due to the diffusion of acid into the surrounding
medium. The author, during the examination of several
hundreds of coliform colonies from milk plated on rebipelagar,
has only met with two organisms, one a coccus and the other a
bacillus, that produced colonies resembling those typical of
B. coli, but many organisms that ferment lactose with the pro-
duction of acid may, especially after prolonged incubation,
produce colonies that bear a superficial resemblance to those

ee a a

ee a ee a SE

ee ee =.

CLASSIFICATION OF B. COLI TYPE 145

described above. There is also a danger of mistaking pin point
red colonies produced by acid-forming streptococci for those
produced by attenuated B. coli and it will be found advisable
to ignore all such colonies when examining the plates. By
this procedure, only organisms in a fairly vigorous state are
counted, and, although it is somewhat empirical in character,
it produces results that are of greater sanitary significance.
Of 271 red colonies fished from rebipelagar, the author found
that 236 (87 per cent) were of the B. coli group so that even if
all the red colonies are counted no serious errors will be intro-
duced.

One difficulty in connection with the use of rebipelagar is the
quality of the bile salt. Many brands of this salt are pur-
chasable but very few are satisfactory. Sodium taurocholate,
sodium glycocholate, and many brands of the commercial bile
salt are too restrictive in their action on B. coli and if the
amount is reduced to avoid this, the selective action is de-
stroyed. With bile salt of satisfactory quality, vigorous B.
coli will produce colonies 3 to 5 mm. in diameter in twenty-
four hours at 37° C. and all brands that fail to do this should
be rejected.

Classification of B. Coli Type. It has been indicated earlier
in this chapter (page 136) that an attempt to regard one par-
ticular type of B. coli as having more sanitary significance
than others has been a comparative failure. The present
problem is not the definition of the properties of a distinct
biotype such as B. coli communis or B. acidi lactici but the
correlation of properties with the immediate previous environ-
ment. The fecal types of B. coli can apparently be distin-
guished from those occurring on grain by the hydrogen ion
concentration produced in dextrose broth containing 0.5 per
cent of dextrose, 1.0 per cent of peptone, and 0.2 per cent of acid
potassium phosphate. This can best be determined by the
methyl red reaction of Clark and Lubs !* which Levine !* has
shown to be correlated with the Voges and Proskauer reaction.
The precise sanitary significance of these so-called grain types

146 EXCREMENTAL ORGANISMS

has yet to be determined but the present trend of opinion is
towards the view that the methyl red negative, Voges and
Proskauer positive types (grain types) are harmless sapro-
phytes. The members of the B. coli group derived from human
and bovine hosts can be partially distinguished by the usual
reactions in sugar broths, the proteoclastic cleavage of gelatine,
and the production of indol from peptone, but these reactions
are not sufficiently specific for routine work although they have
a limited application for research purposes.

2. B. Enteritidis Sporogenes. As the spores of B. enteri-
tidis sporogenes are present in considerable quantities in
manure and do not multiply in milk, the estimation of these
would constitute an admirable test for original pollution if all
otherf sources of these spores could be eliminated. The spores,
however, may be derived from dirty vessels and in practice it
is found that milk cans form a most fruitful source of these

organisms. Milk cans, unless thoroughly sterilised with live _

steam, are very liable to contain large numbers of spores of
various organisms as the treatment given, though usually
sufficiently severe to kill the non-sporulating organisms, is not
drastic enough to kill the spores. The usual temperature at
which milk is pasteurised (143°-145° F.) is also not sufficiently
high to kill the spores, so that the spore test is of considerable
value in arriving at an opinion as to the bacteriological condi-
tion of pasteurised milk previous to pasteurisation. This test
is, however, of much smaller value than the direct microscopical
test previously described.

For the estimation of B. enteritidis sporogenes spores,
various quantities of the milk are measured out into sterile
test tubes, heated in a water bath at 80° C. for fifteen minutes,
cooled, and incubated anerobically at 37° C. To obtain
anerobic conditions the tubes may be placed in an air-tight jar
containing alkaline pyrogallic, but satisfactory results may
be obtained by covering the surface of the sample in each tube
with paraffine; it is rather doubtful whether even this precau-

tion is necessary, as the butter fat which rapidly rises and seals

—— ~ “s

a ae ee

ee ee ee ee

ee

STREPTOCOCCI 147

the surface usually produces the necessary conditions. The
method of Savage !° is the most suitable with regard to the
quantities of the sample to be tested. He suggests using ten
tubes and placing 2 c.cms. in each tube, but this quantity may
of course be varied in accordance with the nature of the sample.
It is decidedly preferable to use a number of tubes containing
small amounts of milk than only a few tubes containing larger
amounts (vide supra). After two days incubation the tubes are
examined for the ‘ enteritidis change ”’ which is indicated by a
complete separation of the curd and the production of acid;
the latter being easily detected by litmus solution. As other
organisms, such as B. butyricus, give this reaction, it is not to be
entirely relied upon, but these organisms are mainly non-
pathogenic and may be differentiated by injecting 1 ¢c.cm. of
the whey subcutaneously into a guinea pig.

Using ten tubes containing 2 c.cms. each, the most probable
number of spores present in 100 c.cms. of sample for each pos-
sible result is given in the Table LVIII, which is adapted from
McCrady’s results.’

A

TasiE LVIII

Result. Positive Tubes. Most Probable Number of
Spores per 100 c.cms.
to 0
to 5
to 11
to 17
to 25 .
to 34
to 45
1 60
to 80
to 114
to Over 114

3. Streptococci. Cow manure contains 100,000 to 10,000-,
000,000 streptococci per gram, and the estimation of these

148 EXCREMENTAL ORGANISMS

organisms in milk was long ago suggested as a means of deter-
mining manurial pollution, but, after considerable work had
been done on the nature and significance of the streptococci
usually found in milk this test fell into general desuetude. It
was found that milk drawn under the best aseptic conditions
contained streptococci which found milk an excellent nidus for
reproduction and that it was practically impossible by simple
tests to distinguish these organisms from those derived from
manure. The examination of milk for Str. lacticus and Str.
pyogenes will be discussed later, but it may be stated here that
the identification of these organisms is far from being reliable
and that their significance is still an open question.

For the estimation of streptococci, varying dilutions, as in
the enrichment method for B. coli, are inoculated into neutral
red dextrose broth tubes and incubated at 37° C. for two days.
The sediment is then examined microscopically for long chains
by means of a hanging drop preparation and all doubtful cases
confirmed by stained’smears. If desired, the streptococci may
be isolated in pure culture, and the morphological and bio-
chemical characteristics determined by spreading the diluted
sediment over ordinary nutrient agar or whey agar and fishing
off the isolated colonies after incubation. The properties of
Str. bovis, Str. equinus and Str. feecalis are given in Table LIX
on page 155. The criticism made above with regard to the
tube method for expressing a numeral value for B. coli applies
equally to this method for estimating streptococci. As prob-
ably only excessive numbers of fecal streptococci have any
sanitary significance, the examination of a direct smear as in
the Breed method for estimating the total number of bacteria
or of a smear from a centrifugalised deposit, will give equally
good results with less expenditure of time and labour.

ae

On =

alll cell eel oma
WN SOON DO

BIBLIOGRAPHY 149

BIBLIOGRAPHY

. McConkey. Jour. of Hyg. 1906, 6, 385.
. McConkey. Jour. of Hyg. 1909, 9, 86.
. Orr. Rpt. on an investigation as to the contamination of milk.

London, 1908.
Rogers etal. J. Inf. Dis. 1914, 14, 411-475.
Chamot and Sherwood. J. Amer. Chem. Soc. 1915, 37, 1949-59.

. Phelps. Amer. Pub. Health Assoc. Rpt. 33, 9.

McCrady. J. Inf. Dis. 1915, 17, 183-212.

. Rpt. of Amer. Pub. Health Assoc., Amer. J. of Pub. Health. 18, 481.
. Savage. Milk and the Public Health. London, 1914, 10, 163.

. Savage. Ibid., p. 189.

. Rogers et al. Jour. Inf. Dis. 1915, 17, 137.

. Clark and Lubs. Jour. Inf. Dis. 1915, 17, 160.

. Levine. Jour. Inf. Dis. 1916, 18, 358.

CHAPTER VII
PATHOGENIC ORGANISMS

Streptococci. Although the etiological relation of septic
sore throat to infected milk has been noted on many occasions
in Great Britain during the past thirty years, it is only during
the past decade that any systematic investigations have been
carried out and the bacteriology of this pathological condition
developed. Probably the first bacteriological examination of
any note was made in connection with the Angelsey outbreak
of 18971 when it was reported that Staphylococcus pyogenes
and Streptococcus pyogenes were found in the milk but no B.
diphtheriz. Examination of the patients’ throats gave similar
results. Some of the most important contributions to the
bacteriology of septic sore throat are those of Savage.? Of the
36 cases of mastitis investigated, 21, or 68 per cent were due to
streptococci, 5, or 16 per cent to staphylococci, and the re-
mainder to B. coli, B. tuberculosis and unclassified causes.
On cultivation of the streptococci in the usual Gordon test
media, it was found that a large percentage was of one type,
called by Savage, Streptococcus mastiditis. This type tended
to long chain formation and grew luxuriantly in broth forming a
flocculent deposit above which the supernatant liquid remained
clear. Lactose, dextrose, and saccharose were invariably fer-
mented with the production of acid, and occasionally salacin,
raffinose, and inulin. Mannite was never fermented. In milk
acid was produced and a clot formed within three days; gelatin
was not liquefied and no neutral red reaction was produced.
It was non-pathogenic to mice. In 16 cases of sore throat
Savage found the two chief varieties of streptococci to corre-
spond to Andrewes and Holder’s Str. anginosus and Str. pyo-

150

a

ee ee eee ee

SE Eg ITT TE RAR SS Al ihe ENERO SE GPF ANG make

he

STREPTOCOCCI 151

genes types with the former predominating (vide p. 155).
The bovine type Str. mastiditis, and the human type Str.
anginosus he was unable to distinguish either morphologically
or biochemically, but a marked difference in virulence was
found on animal injection. By auto inoculation on the tonsils
Savage was unable to produce either local or general symptoms
with Str. mastiditis even when massive doses were employed,
and, in general, the organisms could only be recovered with
difficulty even after such a short period as two to three days.
The author has been unable to find any record of any tests being
made by Savage as to the hemolytic properties of the organisms
isolated by him; this is of considerable importance, as hemolysis
is now generally regarded as characteristic of the pathogenic
types Str. pyogenes and Str. anginosus.

Until 1911 septic sore throat seems to have been passed
unrecognised in America, but the Boston epidemic in that year,
with over 2000 cases, gave an impetus to the study of this disease,
and since then it has proved to be one of the most fertile fields
for research work. In the Boston epidemic, as in the later ones
at Chicago, Baltimore, Concord (N. H.) and other places, the
origin was traced to the milk supply and it was circumstantially
established that the specific cause was a hemolytic strepto-
coccus of the pyogenes variety.

Krumwiede and Valentine* investigated an outbreak of
septic sore throat on Long Island in 1914 and reported that it
was caused by the transfer of pathogenic streptococci from a
case of sore throat on a farm to one of the cows in the herd. An
examination of the herd showed that five cows were giving milk
containing a moderate number of streptococci from one or more
quarters and that one of these gave physical evidence of mas-
titis. All these streptococci, however, were non-hemolytic,
but one other cow was found in which were moderate numbers
of hemolytic streptococci in two quarters and enormous num-
bers in a third quarter. The milk from this quarter was floc-
culent. These streptococci were morphologically and_bio-
chemically identical with those isolated from the throats of the

152 PATHOGENIC ORGANISMS

sufferers in the epidemic and from the probable original case.
These organisms were of the Str. pyogenes type and fermented
salicin but not raffinose or mannite.

Another link in the chain of evidence in favour of the
streptococcal origin of these outbreaks, was founded by Jack-
son,* who showed that experimental arthritis could be pro-
duced in rabbits by the intravenous injection of hemolytic
streptococci. This is important on account of the frequency of
joint infection as a sequel to septic sore throat as noted by
many observers in the various epidemics.

Davis and Capps ° endeavoured to produce an experimental
infection of milk by smearing the uninjured teats of a cow with
typical hemolytic streptococci recently isolated from a case of
streptococcal tonsilitis; this was unsuccessful, but on repeating
the experiment after previously abrading the end of the teat
near the meatus, an infection occurred and streptococci and
leucocytes were found in abundance in the milk of the infected
quarter. Similar results were produced by injecting the cul-
ture into the udder.

In view of the strong evidence that milk-borne streptococci
were causative agents of septic sore throat it became imperative
that a study should be made of the streptococci which are
invariably found in milk, even though produced under the best
conditions, in order to ascertain if there were any relation be-
tween these facts. Heinemann ® has shown that Str. lacticus
occurs constantly in milk and that the morphological and bio-
chemical characteristics of this organism on ordinary media
are identical with those of Str. pyogenes. Later’ he found
that by repeated passage through rabbits, he was able to exalt
the virulence of Str. lacticus to such an extent that compara-
tively small doses were fatal. The lesions produced were very
similar to those produced in human beings by Str. pyogenes.
Miiller ® found that milk streptococci and pathogenic strep-
tococci showed no material difference in their agglutination and
hemolytic properties but differed widely in the rapidity with
which they coagulated milk. Heinemann in 1915 ° reported

~s Pe ee tee eh r

EXAMINATION FOR STREPTOCOCCI 153

the results of further experiments on the pathogenicity of Str.
lacticus and these in general confirm his earlier work. Two
strains, one only of which was hemolytic, but both capable of
fermenting a variety of the usual test substances, were exalted
in virulence by animal passage, and it is important to note that
the fermentative capacity gradually decreased until finally one
strain fermented only dextrose, and the other dextrose and

_ saccharose. The non-hemolytic strain became hemolytic and

both showed an increased tendency to chain formation. From
these results Heinemann suggests that the determination of the
fermentative ability of the streptococci might be of value in
determining the previous envircnment of the organisms. If
in contact with an animal lesion a low fermentative capacity
would result whilst a high capacity would indicate a medium
rich in carbohydrates.

Although the questions of the variability of streptococci
in mastitis and the relation of mastitis to septic sore throat,
are still far from being satisfactorily solved, it has been fairly
definitely established that the great majority of the strep-
tococci ordinarily found in milk are non-pathogenic and do
not indicate a pathological condition of the udder. Str. lac-
ticus, which may be found in almost every sample of milk, is
used industrially in cheese manufacture and is also employed
as a therapeutic agent. This streptococcus is typical of the
group characterised by high fermentative capacity and low
pathogenicity. The pathogenic streptococci, on the other
hand, ferment but few of the Gordon test substances and pro-
duce low acidities in the media that are fermented; the mor-
phological appearance is characterised by the picket fence
(stalkett) formation but the chain may be either short or long;
hemolysis is marked.

Examination for Streptococci. Probably the most satis-
factory method of examination for excessive numbers of strep-
tococci resulting from mastitis, is the direct miscroscopical
method of a smear prepared either by the Stewart-Sloan method
described on page 126 or the Breed method described on page

154 PATHOGENIC ORGANISMS

129. In the microscopical examination, the streptococci having
the typical form of Str. lacticus (elongated cocci, usually in
pairs) should be ignored and a search made for the picket fence
variety only. These, on staining with methylene blue, usually
appear in chains with solidly stained portions at right angles
to the longitudinal axis; capsules are usual but are not invari-
ably found. Some observers attach more significance to the
long-chain types, but in view of the numerous cases in which the
short-chain types have been associated with pathological con-
ditions, it would appear to be good policy to attach equal
significance to both varieties. The property of chain forma-
tion is undoubtedly a variable one and is profoundly modified
by the composition of the medium and general environment.

In the indirect method, the sample is diluted as in the exam-
ination for fecal streptococci and the various dilutions seeded
into dextrose broth. After incubation for forty-eight hours
at 37° C., the cultures are examined for chain formation by
making a smear or a hanging drop preparation; from the
smallest quantity containing typical chains the approximate
number of streptococci can be calculated. If desired, the broth
cultures can be plated out on nutrient agar or gelatine, and the
organisms isolated in pure culture. The quickest and most
satisfactory method of examination for pathogenic streptococci
is by plating on blood agar. MRuediger*! as early as 1912
suggested the differentiation of Str. pyogenes from Str. lacticus
by the hemolytic properties of the former and since that date
several workers have demonstrated that hemolysis is a usual
property of the pathogenic streptococci. All hemolytic strains,
however, are not pathogenic.

The best technique is to add various dilutions of the sample
to 10 c.cms. of meat infusion agar containing 1 c.cm. of horse
blood and then pour into Petri plates. These are incubated
at 37° C. and examined after twenty-four and forty-eight hours
for hemolysis. Those colonies showing a clear, transparent,
colourless zone are transferred to broth and finally inoculated in
the usual Gordon test media, viz., dextrose, saccharose, raf-

NE aS

EXAMINATION FOR STREPTOCOCCI 155

finose, mannite, lactose, and salicin broths for determination of
acidity, in milk for coagulation, and to blood agar plates for
hemolysis. A virulence test is also desirable, but in considering
the results obtained due regard must be given to the dosage and
method of inoculation. A quantity of broth that is sufficient to
kill the test animal in three days when injected intravenously
might not produce more than local symptoms when given sub-
cutaneously, and similar conditions apply to the dosage. For

- guinea pigs 1 c.cm. of a forty-eight hour broth culture and for

mice 0.5 c.cm. of a twenty-four hour culture have been found
to give satisfactory results when injected into the peritoneal
cavity. ‘i

The biochemical characteristics should be determined
quantitatively by Winslow’ s method ®° if the best results are to
be secured.

Taste LIX

BIOCHEMICAL CHARACTERS OF PRINCIPAL TYPES OF
STREPTOCOCCI. (Broapuurst)

d
. elou a | 3
. ‘ o oO n es
Name of Variety. 3 3 5 g x g 3 E 2 Type sorta by
a ie ie A ek ee
= ar ee ee a fe == Fd
ees 1 1 0 | & 101 0 1 ® || + | Andrews and
Str. mitis...... xX|X/]xX/O;}O0}]xX|{-|]- Horder
Str. pyogenes..| X | X | X|O}O|;X|]+]- ce
Str. salicarius..| X | X | X | ®| Oj] O ae ‘6
Str. anginosus..| X | X | X|@®@/{/O|Oj]+]- cs
Str. gracilis....| X | X|O]}]Oj]X]xX]—jJ|+ 66
? Bie POO hey we ~ 7
Str. fecalis....| X | X | X|O;, X |X] —|] —- <6
Str. versatilis..| X | X | X | X | X | X | — |] — | Broadhurst
Str. bovinus...| X | X |X} X|O |; xX] -— J] — | Winslow

X indicates that test substance is fermented sump production of acid and without
gas formation.
@ indicates that test substance is occasionally fermented.

156 PATHOGENIC ORGANISMS

The fermentation and hemolytic reactions of the best-
known types of streptococci, excepting Str. lacticus, are shown
in Table LIX.

B. DipHTHERLE

Milk has, on several occasions, been proved to be a vehicle
for B. diphtheriz and responsible for epidemics of diphtheria,
and it is consequently sometimes necessary for the bacteriolo-
gist to examine milk for this organism.

There is no satisfactory evidence that diphtheria organisms
may invade the udder and so cause infection of the milk, but
it is more than probable that milk has become accidentally in-
fected from human sources and that the organisms have rapidly
increased in number. Milk is not an ideal medium for the
development of B. diphtheriew but fairly rapid multiplication
does occur until checked by the metabolic products of the acid
producers.

The number of authentic cases in which B. diptheriz has
been isolated from milk are comparatively few. Bowhill,!°
in 1899, isolated diphtheria organisms from milk and prepared
broth cultures that were fatal to guinea pigs in forty-eight hours.
The same year Eyre !! isolated a virulent diphtheritic bacillus
from milk and, later, cases were reported by Klein,!* Dean and
Todd 1° and Marshall.'*

For the isolation of the organisms, Bowhill directly inocu-
lated Loeffler’s blood serum with the sample. Eyre, and Dean
and Todd concentrated the organisms by centrifugalising and
afterwards streaked the sediment over a number of tubes of
blood serum. The cream layer was treated in a similar man-
ner. Characteristic colonies were fished and those mor-
phologically resembling B. diphtherie isolated as pure cultures
and tested for pathogenicity. Klein and Marshall used the
animal inoculation method. The former inoculated two guinea
pigs with one sample, one subcutaneously in the groin, and the
other intraperitoneally. The latter pig remained well, but the
former, on the fifth day, showed swollen inguinal glands sur-

B. DIPHTHERLE: 157

rounded by soft cedematous tissue. On autopsy the sub-
cutaneous tissue in the region of the seat of inoculation was
cedematous and streaked with blood. The inguinal glands
were enlarged, firm, and deeply congested. Film preparations
from the juice of the incised gland showed numerous diphtheritic
organisms. A pure culture was obtained which was proved to
be B. diphtheriz by the virulence test and also by the antitoxin
_ test.
For the examination of milk for B. diphtheriz, the serum
method undoubtedly offers the best chance of obtaining a posi-
tive result. 50 c.cms. of sample are centrifugalised at 2000
revolutions per minute for twenty minutes and the cream layer
removed to a sterile dish. The milk layer is withdrawn by
means of a suction pump connected to a fine bore glass tube
until only 1-2 c.cms. remain. The sediment, and cream
layer, are used for inoculating either blood serum plates or
tubes. If tubes are used, one loopful is employed for smearing
the surface of a number of tubes in succession so that at least
one tube will be obtained in which the colonies are well isolated.
In this manner a total of from 40 to 50 tubes is used for one
sample and examined after sixteen or eighteen hours incubation
at 37° C. From the tubes containing well-isolated colonies,
subcultures are made of all colonies in any way resembling B.
diphtheriz and examined as to their morphological character-
istics and biochemical properties. B.. diphtherie is usually
found in fresh serum preparations as a slender rod about 3 in
length and exhibiting well-defined polar granules when stained
with Loeffler’s methylene blue or Ponder’s stain (see appendix).
The club-shaped bacillus is sometimes found, and also beaded
and barred varieties but the bipolar type (type c, Westbrook
classification) is the most typical. B. diphtheris does not
liquefy gelatine, is Gram positive, and ferments dextrose,
levulose, galactose, arabinose, and maltose without formation
of gas but not saccharose and mannite. Older cultures some-
times produce acid in lactose and glycerine. The bacillus is
non-motile and does not form spores. |

158 PATHOGENIC ORGANISMS

The organisms that pass the morphological and biochemical
tests must be tested for virulence to guinea pigs. Two pigs
are used, one for a subcutaneous or intra-peritonial injection of
the twenty-four hour broth culture alone (1 ¢.cm.) and the other
for a mixture of the culture with 1 c.cm..of a diphtheritic anti-
toxin of high titre. The unprotected pig usually dies within
thirty-six hours, and almost invariably within forty-eight hours,
if the culture is one of typical B. diphtherie. The protected
animal should show no definite symptoms and remain alive.

Diphtheroid Bacilli. On many occasions bacilli have been
found in milk having the characteristic granular staining prop-
erties of some forms of B. diphtherie but sharply differentiated
from this organism by the absence of virulence. Bergey !°
investigated a number of these organisms which were apparently
identical with B. diphtherie, and divided them into three
groups according to their biochemical properties. Two groups
showed fermentative activity markedly different to the diph-
theritic group and that of the third was identical but non-
pathogenic. Savage !® investigated a number of the diph-
theroid organisms found in milk sediments. These were

apparently identical and closely resembled B. diphtheriz in
staining properties and microscopical appearance except for an
absence of blue granules in preparations stained with Neisser’s
stain. The bacilli were Gram positive, non-motile, and devel-
oped on nutrient agar as small, discrete, translucent colonies.
On serum they were slightly coloured and such organisms did
not give the typical microscopical appearance found with the
growths on agar. Litmus milk was unaffected and, except for
a trace of acid in lactose, neither gas nor acid was produced
in the usual test media. They were non-pathogenic to mice.
Klein !7 found a bacillus in milk which he called B. diphther-
oides. This organism differed morphologically from B. diph-
theriz, Hoffmann’s bacillus, and the xerosis group. No
' growth was observed on gelatine at 21° C. or on agar at temper-
atures less than 25° C. On agar at 37° C. the growth was slow
and no colonies appeared until the third day when they devel-

B. TYPHOSUS 159

oped as small grey dots. Milk was coagulated at 37° C. with
_acid formation and a separation of the milk constituents into
a cream layer at the top, curd at the bottom, and whey in
between. On blood serum the colonies appeared on the third
day as depressions due to liquefaction of the medium. On
injection into guinea pigs, well-developed local abscesses ap-
peared in one to two weeks. Intra-peritoneal injection pro-
duced abscesses on the omentum and on the pancreas or around
the kidney. The author has, on several occasions, isolated
bacilli from milk that resembled B. diphtheriz, but the majority
of these could be distinguished from the typical pathogenic
variety by the size. The most usual type was about 5y in
length and slightly pointed at both ends; they retained the
stain when treated by Gram’s method and gave a typical
barred appearance with Loeffler’s methylene blue and Ponder’s
stain. On agar, and on blood serum, the organisms developed
as small white opaque colonies. Gelatine was not liquefied.
Dextrose, lactose, saccharose, mannite, and dulcite were not
fermented and no visible change was produced in litmus milk.
They were non-motile and did not form spores; broth cultures
were non-pathogenic to guinea pigs when treated by the intra-
peritoneal method.

No etiological connection has been discovered between
these diphtheroid bacilli and any pathological condition and
they must, therefore, be regarded as harmless saphrophytes
that are of no importance or significance in public health work.

B. Typuosus

There are on record several hundreds of epidemics of
typhoid fever that are definitely attributed to milk as the
immediate source of infection, but there is, so far as the author
can ascertain, not a single authentic case recorded in which B.
typhosus has been isolated from milk suspected of causing an
epidemic. Typhoid infection of milk is of external origin and
whether it is due to a carrier, or to a person having the dis-

160 PATHOGENIC ORGANISMS

ease, or water, it is almost invariably intermittent or transitory
with the consequence that by the time an outbreak has oc-
curred and can be traced to the milk supply it is almost hopeless
to expect to isolate the infecting organism. This, however,
should not deter those responsible for the investigation of such
cases from attempting the isolation of B. typhosus.

Isolation of B. Typhosus. Jackson and Melia!® recommend
inoculating the sample into lactose bile and incubating at 37° C.
The cultures are to be transplanted in varying dilutions into
Hesse agar and examined after twenty-four hours at 37° C.
On this medium B. coli forms small succinct colonies; B.
typhosus is most characteristic on plates containing but few
colonies; colonies of a large size are then formed, often several
centimetres in diameter, and consisting of a broad translucent
or scarcely turbid zone between a white opaque centre or nucleus
and the perfectly circular narrow white edge. Tonney et al.!9
found that lactose bile is inhibitory to B. typhosus as well as to
the colon group of organisms and this is confirmed by the au-
thor’s experience.

The following method, which is an adaptation of Browning
and Thornton’s method *° for the .isolation of typhoid bacilli
from feces, can be recommended for the isolation of B. typhosus
from milk. Centrifugalise 50 c.cms. of the sample for twenty
minutes at 2000 to 2500 revolutions per minute. Remove the
cream layer to a sterile tube and place it in a water bath at
37° to 40° C. Draw off the skim milk by means of a fine glass
tube attached to a suction pump until about 3 c.cms. remain.
After thoroughly distributing the sediment throughout the
liquid it is inoculated into three brilliant green peptone’, tubes,
one cubic centimetre being placed in each tube. The molten
cream layer should be similarly treated as a proportion of the
organisms may be trapped by the rising fat globules during the
centrifugalising process. The brilliant green medium is pre-
pared by steaming a 2 per cent peptone solution, containing
0.5 per cent of sodium chloride, for forty-five minutes and
filtering after making the reaction slightly alkaline to litmus.

GAERTNER GROUP 161

The medium is sterilised under pressure either in bulk or in
10 c.cm. quantities in tubes. The brilliant green (Hochst) is
kept as a stock 1 per cent solution which is made into a 1 in
10,000 solution just before use by diluting 0.1 c.em. to 10 ¢.cms.
Before inoculating the 10 c.cms. of peptone saline medium with
the suspected material, 0.5 c.cm. of the 1 in 10,000 brilliant green
solution isadded. The tubes are incubated at 37° C. for twenty
_to twenty-four hours and then plated out on neutral red bile salt
agar or Endo’s medium, preferably the former. The colourless
characteristic colonies are fished and put through the usual
agglutination and biochemical tests. Using this method, the
author has been able to isolate B. typhosus from the sediment of
milk to which had been added 28 typhoid bacilli per 100 ¢.cms.

Paratyphoid-enteritidis or Gaertner Group. The organisms
of this group may be isolated by the same method as is given
above for B. typhosus or, if no examination is required for B.
typhosus, the sediment and cream may be inoculated into meat
peptone dextrose broth (neutral to phenolphthalein) containing
0.15 c.cm. of a 1 per cent solution of brilliant green per 10 c.cms.
of broth. (Tonney.*°) This strength of brilliant green (1 in
6600) inhibits the growth of the Escherich and Eberth groups,
and enables the Gaertner group to predominate the broth cul-
tures. The broth cultures are subsequently plated out on
neutral red lactose bile salt agar and the non-lactose fermenters
worked out in the usual way.

Morgan’s Bacillus No.1. During the last few years the
attention of sanitarians has been directed to the etiological
relationship between milk supplies and epidemic summer
diarrhcea. It has been evident for many years that artificial
feeding of infants was a contributing factor but no definite
cause was assigned for this phenomenon. Defective feeding has,
no doubt, contributed to the excessive infantile mortality that
occurs each summer, but there is a rapidly accumulating mass
of evidence that the epidemic variety of summer diarrhea is
primarily or secondarily dependent upon the activity of micro-
organisms. The substitution of a clean milk supply or the

162 PATHOGENIC ORGANISMS

pasteurisation of the old supply has, in many cases, led to an
abatement of infantile diseases and this would indicate that an
excessive number of bacteria of all kinds and not any particular
group is responsible for the effects observed. (Park and Holt.2)

Scholberg and Wallis ?* suggest that the prejudicial effect
is due to physical and chemical changes produced by bacterial
contamination. ‘They found that the products of proteoclastic
digestion appear in milk as the atmospheric temperature in-
creased and that the albumoses and peptones so produced may
be toxic to infants.

Morgan and Ledingham,?? in 1909, iisib an investigation
of the bacteriology of summer diarrhea and concluded that a
non-lactose fermenting, non-liquefying organism which they
isolated and which is now usually known as Morgan’s Number 1
Bacillus, bore a close relationship to the disease.

_Lewis,24 Ross,?° O’Brien 76 and Orr,?’ made numerous exami-
nations of the feces of infants and, although they found that
the non-gelatine liquefying, non-lactose fermenters were ab-
normally prevalent in the cases of diarrhoea, they could not
establish any definite causal relationship. In 1911, Lewis 28
and Alexander? made further observations on this group and
showed that Morgan’s No. 1 Bacillus was conspicuously fre-
quent in the feces of infants having epidemic diarrhoea. In
the same year Graham Smith *° found that the non-gelatine
liquefying non-lactose fermenters were especially prevalent
in flies during the seasonal prevalence of diarrhcea and that
Morgan’s No. 1 Bacillus, whilst rarely present in flies from
houses not containing diarrhceal cases, was frequently found in
houses associated with this disease.

Lewis *! pointed out the importance of applying the agglu-
tination test to the various organisms which gave the usual
fermentation reactions for Morgan’s No. 1 Bacillus.

The etiological relationship of Morgan’s No. 1 Bacillus to
epidemic summer diarrhcea is not yet fully established, but the
evidence in favour of this hypothesis is undoubtedly strong and
points to the infection of the milk supply in the home by flies.

‘osuvyo OU =— ‘SBS ynoyIIM uorjonpoid ploev=X ‘uolonpoid se¥=+ :puoseT
‘BIPeul Y}OIG puB oyeBipAYoqiey

163

FA et a ee ee ef a aa me ns SoudSTVYTe sIpRoBy “g
IV | PPV | Fp | Xx ee pe Pe mK Ka KY Xf | = | * couxey gq) asseyucedp -g
ee gk BS Soe Coe Cet ee Soa re ee Spe Ses eae Geel ed PS EE eos ae ee (B3IYQ) wliojueshp “g
poy poy = ae eG eS Ie =. pers oe eee 4. pes x 4 x x ES + Si wVie oR ee eT snsoydAy J
:dnoi6 y430Qq 9Y,L
“MIV 2-000 ot oh at SY ee Se i! Enea Se eget + + =f es fs SS A ae ee I ‘ON S$ UBBIOJY
Bs “HIV poy Etcaes: Picts + + + ETS ear: AEBS See Leccges te + a af + SE + Teen hee ie ee Jayysedins ‘g
5 "IV poy - + eer AS AT ae <a = —|- 5 Mie + + + + ae + Oe 2) uinns eiapoyo as
ma MV | PPV | — Ft it] tym ley xXx yy ym lt iti t+i]4t]—-t4ttoc q ‘snsoydAyered -g
O PPV |-— FT IT Iti O KI - k IH lH lH litte iti titties V ‘snsoydAyered *g
a IV se ee ae cL fe + pat’: aes. pasa so ee — = + + + + Se + alec bn es eee aha é-e Le ek SIPIQlI0}Ue g
= [ANOLD 19URLIVH) AY,
jen + + 4+ a + me we + a oe + oh ot 4 4 -+ - ee, A a a ee a BIIvoyo ‘d
: poy | pry cS a ie Racecar Cees oes eres ie [ake faa Po bbe | Seasons ghia TON] Iplwe “gq
ee wd 2 ad Gel ig we ee ee a cee ele PRaeei ecas a Bac ie ig at ee gs OS fs Eee a ee soudsOIav SILT “GT
4 poy |peyit+/+i+/+)+{—-|-]+/+]4+]+/+]4]+l4+l-J4hooce JOTUNUITIOD T[Oo “g
PPV PPV TPE Ee TF pm meee pm lee eit le edt pee STUNUIULOD T[O9 “gf
s ANOig) YORtIYIST OY],
‘oyeyT | Aue |) St] P| 2] Oo] Oo] 2] SS S) Ae ee ee 2 el =, Q 2
SI EIEI SIS FIEI ALB Bi/S/ EIFS alele
“TBIEISTELP IP ele els) ai slel 3] elé
‘MII SAWLI'T ° a 218 es ee Ss ee

SWSINVOUO AO dNOUD CIOHdAL-ITOO AO SHUALOVUVHO TVOINATHOOIA
XT Fav,

164 PATHOGENIC ORGANISMS

Examination. Morgan’s No. 1 Bacillus is very suscep-
tible to the action of brilliant green and will not appear on the
rebipelagar plates in the enrichment method for isolating the
organisms of the Gaertner group. The best procedure is to
inoculate the centrifugalised deposit from about 40-50 c.cms.
of milk into a number of tubes of neutral red lactose bile salt
agar and incubate for twenty-four to forty-eight hours after
mixing and pouring into petri plates. All colourless colonies
are fished into dextrose broth and those organisms producing
acid and gas in this medium are afterwards tested in the usual
media for biochemical reactions and also with a specific serum
in low dilution. Morgan’s No. 1 Bacillus invariably ferments
dextrose and lzvulose, and usually also arabinose and galactose,
with the production of acid and gas. Mannite is also usually
fermented but not saccharose, dulcite, maltose, dextrin, or
salicin. Indol is produced in peptone water and milk becomes
alkaline in about ten days. Gelatine is not liquefied.

B. TuBERCULOSIS

For the detection of B. tuberculosis in milk two processes
have been employed: (a) the microscopical and (6) the inocula-
tion method.

Microscopical. In very rare cases the presence of B. tuber-
culosis in milk may be demonstrated by the examination of
stained films of the milk without previous concentration, but
the percentage of positive results so obtained is so small as to
render the process valueless for public health work. When
the organisms are comparatively numerous they may be found
in the deposit obtained by centrifugalising 50 to 100 c.cms. of
milk at 2000 to 3000 revolutions per minute for thirty minutes.
For the preparation of cover-slip films Delépine *? recommends
spreading small portions of the sediment over cover slips which,
when dry, are placed in a covered capsule containing equal
parts of absolute alcohol and ether for two hours. At the
expiration of this period the capsule is placed in a dish con-

INNOCULATION METHOD 165

taining water at 80° to 90° C. The mixture of alcohol and
ether boils at once and after ten to fifteen minutes the cover
slips are removed and washed with absolute alcohol. The
films are then stained with carbol-fuchsine and counterstained
with methyline blue according to the Ziehl-Neelson method
which is as follows:

(1) Stain in hot carbol-fuchsine for five to ten eae being
- careful to avoid over-heating.

(2) Decolourise by dipping in 25 per cent sulphuric acid.

(3) Wash in water.

(4) Wash in alcohol until no more stain is removed.

(5) Wash in water.

(6) Counterstain for one minute with methylene blue.

(7) Wash in water, dry, and mount.

Delépine found that when this method of preparation was
carefully followed, very clear films were obtained and no dif-
ficulty was caused by other. acid fast bacilli when sufficient
attention was paid to the morphological characteristics of the
organisms.

Inoculation Method. The inoculation method is the only
one that can be relied upon for the detection of very small
numbers of B. tuberculosis in milk, but the time required to
obtain reliable results is not less than three weeks as com-
pared with the few hours required for the completion of the
microscopical method. It is good routine practice to make
microscopical preparations of all sediments obtained by cen-
trifugalisation and to inoculate those yielding negative or doubt-
ful results.

To prepare the sediment, 100 c.cms. of milk are centrifu-
galised at 2000 to 3000 revolutions per minute for at least
thirty minutes, and, after removing the cream layer with a
sterile spatula or spoon, the separated milk is drawn off through
a small bore glass tube attached to a suction pump until about
4 c.cms. of milk remain. This milk is thoroughly mixed with
the deposit and subsequently used for the inoculation of two
animals. If the milk is known to be “ clean” the milk may

166 PATHOGENIC ORGANISMS

be reduced to 2 c.cms. and only one animal used for the deposit,
the other being reserved for a portion of the cream layer.

On account of its sensitiveness to tuberculosis, the guinea
pig is the most suitable animal for inoculation and the best
results are obtained with animals weighing from 200 to 300
grams.

Two methods of inoculation are in general use: (a) subcuta-
neous injection at the inner side of the left hind leg and (6)
intraperitoneal injection through the belly wall. Delépine
prefers to inoculate at the inner aspect of the left leg at the level
of the femoro-tibial articulation on account of the comparative
results obtained by the uni-lateral development of the lesions.
This, he found, was especially noticeable in the early stages
with small amounts of infectious material and by noting the
extent of the lesion development in two pigs killed after twenty-
one and thirty-five days, a rough estimation of the degree of
infectivity was procured. In the very early stages the
lesions were limited to the subcutaneous tissue and the
four groups of lymphatic glands (the popliteal, superficial
inguinal, deep inguinal, and the sacro-lumbar) on the same
side of the body as the seat of inoculation. Later the retro-
hepatic gland and spleen were involved followed by the liver,
lungs, bronchial suprascapular, and cervical glands on both
sides of the body. Finally there was a more complete invasion
of the lymphatic glands in front of the diaphragm on both sides
of the body and an involvement of the superficial and deep
inguinal and other glands behind the diaphragm on the right
side of the body.

With the intra-peritoneal inoculation the lymphatic glands
of the peritoneum and mysentery are first involved, followed
by the liver and spleen. The cervical, bronchial, inguinal, and
popliteal follow, but the lesion development is bilateral through-
out.

In order to accelerate the development of the disease when
the subcutaneous method is used, Block 3* suggested that the
inguinal glands on the inoculation side should be slightly dam-

a ae

INNOCULATION METHOD 167

aged by squeezing them. This procedure reduces the resistance
of the glands and enables an earlier diagnosis to be made.
Dodd,** and Joannovico and Kapsammer *° carefully studied
this technique and found it entirely successful. They found
that even doubtful cases could be diagnosed within fourteen
days.

The microscopic appearance of the lesions is usually suf-
ficient to enable a trained observer to make an accurate diag-
nosis, but in all doubtful cases cover slips preparations should be
made and supplemented if necessary by histological sections.
For cultures, nodules are squeezed between two sterile slides
and the contents smeared over glycerine agar slopes. The
cultures are incubated at 37° C.

For the differentiation of tubercular from other infections,
Anderson *© suggested the subcutaneous injection of 2 ¢.cms.
of tuberculin. In a healthy animal a slight febrile reaction
occurs and passes off in a few hours, but this quantity of tuber-
culin is sufficient to cause death in less than twenty-four hours in
a guinea pig showing well developed tuberculosis. When the
lesions are slight the animal will become sick but may not die.
This method may be used as an addition to the usual autopsy
but should not be substituted for it. :

Even when the best technique is used, it is often found that
the experimental animals may die from acute infections within
a few days of inoculation. This is due to “ dirty ” milk and
can be partially eliminated by the treatment of the sediment
with 5 per cent antiformin for thirty minutes and finally
washing with physiological saline. Eastwood and Griffith 3”
found that 10 per cent antiformin slightly weakened the tubercle
bacilli and that a 20 per cent solution almost destroyed them.

Death of the inoculated animals after ten days, from infec-
tions other than generalised tuberculosis, is largely due to
improper attention to the housing conditions of the guinea
pigs. These must be kept isolated in clean cages with not more
than two animals to a cage and housed in well-ventilated
rooms.

168°. PATHOGENIC ORGANISMS

Pseudo-tuberculosis. Milk occasionally contains organisms
capable of producing chronic lesions which partially simulate
those of B. tuberculosis and to which the designation of pseudo-
tuberculosis has been given. Delépine found that amongst
these infections was one resembling chronic pyemia, but in his
opinion the resemblance is superficial and no experienced pathol-
ogist could mistake such lesions in the guinea pig for true
tuberculous lesions; also that an experimenter with scanty
pathological experience could not make a mistake if the or-
ganisms in the lesions are microscopically examined. The
finding of the giant cells, characteristic of true tuberculosis, in
histological sections would also clear up doubtful microscopic
diagnoses.

In pigs that have been kept for five to six weeks the chronic
lesions due to B. abortus may be found, but as this organism is
not acid fast there is no difficulty in eliminating this possible
source of error.

Bovine and Human Types of B. Tuberculosis. The differ-
ence in the cultural and other characteristics of these types is
essentially relative rather than absolute and this fact must
always be kept in mind when attempting to classify cultures of
B. tuberculosis.

Eastwood and Griffith ** classified cultures as dysgonic or
eugonic according to the luxuriance of the growth on glycer-
inised agar and they found that the dysgonic type was usually
of high virulence for rabbits and corresponded to the bovine
type. The human type grew well on glycerine-agar but pos-
sessed much lower virulence for rabbits. The chief differences
in the two types may be summarised as follows:

Bovine. HuMAN.

Morphology. Only slight differences can be found, the bovine organisms
being usually shorter, straighter, and thicker.

Cultural characteristics. ;

Glycerine-agar. Grows feebly and Grows. luxuriantly and _ usually
with development of discrete col- without difficulty. Growth
onies. often wrinkled.

BIBLIOGRAPHY 169

Bovine serum. Grows slowly and Grows fairly rapidly.
appears as a fine, filmy, non-pig-
mented growth after two to three
weeks. ;
Glycerine broth 2 per cent acid.
Acid reaction diminishes and may Remains permanently acid.

finally become alkaline.
Pathogenicity.
Calves. Highly pathogenic. Non-pathogenic.
Rabbits. Highly pathogenic. — Slightly pathogenic. The lesions

Subcutaneous inoculation with 10 are often localised in the lungs
m.gr. causes an acute generalised and kidneys or scattered.
fatal tuberculosis.

In the preparation of cultures from lesions for differentiation
of type the primary ones should be made on Dorset’s egg medium
(see Appendix) and subcultivated to blood serum or glycerine-

agar.
BIBLIOGRAPHY |

1. Coleman. Rpt. of M. O. for Angelsey. 1897.
2. Savage. Rpt. of M.O.toL.G.B. 1906-07, 228-252, ibid., 1907-08,
359-424, ibid., 1908-09, 294-315. |
3. Krumwiede and Valentine. Rpt. 36 New York City Health Dept.
4. Jackson. Jour. Inf. Dis. 1913, 12, 364-385.
5. Davis and Capps. Jour. Inf. Dis. 1914, 15, 135-140.
6. Heinemann. Jour. Inf. Dis. 1906, 3, 175.
7. Heinemann. Jour. Inf. Dis. 1907, 4, 87-92.
8. Muller. Arch. f. Hyg. 1906, 56, 90.
9. Heinemann. Jour. Inf. Dis. 1915, 16, 221-240.
10. Bowhill. Jour. State Med. 1899, 705-710.
11. Eyre. Brit. Med. Jour. 1899, 2, 586.
12. Klein. Jour. of Hyg. 1901, 1, 85.
13. Dean and Todd. Jour. of Hyg. 1902, 2, 194-205.
14. Marshall. Jour. of Hyg. 1907, 7, 32.
15. Bergey. Jour. Med. Research. 1904, 11, 445.
16. Savage. Rpt. of M.O.to L.G. B. 1906-07, 224-225.
17. Klein. Jour. of Hyg. 1901, 1, 78.
18. Jackson and Melia. Jour. Inf. Dis. 1909, 6, 194.
19. Tonney et al. Jour. Inf. Dis. 1916, 18, 243.
20. Tonney. Jour. Inf. Dis. 1913, 13, 263-272.
21. Park and Holt. Arch. of Ped. 1913, 20, 881.

170

22.
23.
24.
. Ross. Rpt. of M. O. to L. G. B. 1910-11, 366.

. O’Brien. Rpt. of M. O. to L. G. B. 1910-11, 373.

. Orr. Rpt. of M.O. to L. G. B. 1910-11, 386.

. Lewis. Rpt. of M.O.to L. G. B. 1911-12, 286.

. Alexander. Rpt. of L.G. B. 1911-12, 303.

. Graham Smith. Rpt. of M. O. to L.G.B. 1911-12, 319.

. Lewis. Rpt. of M.O. to L. G. B. 1912-13, 375.

. Delépine. Rpt. of M. O. to L. G. B. 1908-09, 370.

. Bloch. Berlin. klin. Wochenschrift. 1907, 40, 511.

. Dodd. Jour. Roy. Inst. Pub. Health. 1909, 17, 360.

. Joannovico and’ Kapsammer. Berlin. klin. Wochenschrift. 1907,

PATHOGENIC ORGANISMS
Scholberg and Wallis. Rpt. of M. O. to L. G. B. 1909-10, 504.

Morgan and Ledingham. Proc. Roy. Soc. Med. 2, 1909, 133.
Lewis. Rpt. of M. O.to L.G. B. 1910-11, 346.

44, 1439.

. Anderson. U.S. A., P. H. and M. H.8., Hyg. Lab. Bull. 46, 183.

. Eastwood and Griffiths. Rpt. of M.O. to L.G. B. 1912, 303.

. Eastwood and Griffiths. Rpt. to L. G. B., Pub. Health Series, No. 88.
. Winslow. Jour. Inf. Dis., 1912, 10, 285.

. Browning and Thornton. Brit. Med. Jour. 1915 (Aug. 14), 248-250.
. Ruediger. Science. 1912, 35, 223.

CHAPTER XIII
CELLS, DIRT AND DEBRIS

- Cells. For nearly a century it was recognised that cells
or cell fragments were present in the secretion as formed in
the alveoli, but it is only comparatively recently that any
efforts were made to ascertain if any cells were present in the
discharged milk. In 1897 Stokes and Wegefarth! directed
attention to the presence of leucocytes in milk and, since then,
considerable study has been given to this subject. These
observers differentiated the leucocytes from the epithelial cells
by the form of the nuclei but, unfortunately, designated the
former as pus cells, a nomenclature that was perpetuated by
many later writers. This designation is no longer accepted
and the cells are regarded as constituents of normal milk. There
is still some diversity of opinion regarding the nature of these
cells, some experimenters, including Winkler, Hewlett, Villar,
and Revis, holding that they are predominantly of epithelial
origin, whilst others, amongst whom are Bergey, Doane, Miller,
Breed, Ernst, and Savage, regard them mixtures of blood cells ©
and epithelial cells.

Hewlett, Villar, and Revis? support the contention of Wink-
ler and Michaelis that the celis in normal milk are chiefly young
epithelial cells which have become detached. In a later paper
they find that in the milk of healthy cows in full milk and
which do not give a high cell count, the majority of the cells
appear to be “large uninuclears ” with a small admixture of
other cells. At the beginning and end of lactation and when
the cell count was high from other causes, whether physiological
or pathological, the ‘‘ multinuclears”’ predominated. Scan-
nel * pointed out that epithelial cells are mononuclear and that,

171

172 CELLS, DIRT AND DEBRIS

although on dividing, they may appear as polymorphonuclears
' it is inconceivable that they should divide at such a rate as to
produce 500,000 per c.cm. There are also certain histological
characteristics that differentiate nucleated epithelial cells and
mononuclear leucocytes.

The views of those who regard the cells found in milk as
mixtures of blood and epithelial cells, which is the more gen-
erally accepted explanation, are well set forth in a recent book
by Ernst * in which the histological characters of the cells are
treated ‘‘ in extenso.” 3

According to Ernst the cells are of dual origin. (a) Epi-
thelial cells derived from the tissue lining the ducts and from the
secretory glands and,

(b) Leucocytes which have passed through the walls of the
capillaries and lymphatics and finally obtained access to the
gland secretion. This would appear to be normal process in
all secretory glands. Under special stimulation, either from
mechanical or pathological causes, the number and nature of
the cells may undergo radical changes depending upon the
nature and extent of the stimulation. This affords a rational
explanation of the diversified cells found in milk and alterations
in their relative proportions under varying conditions. A
general description of the cells usually found in milk follows.

Epithelial Cells. (a) From compound epithelium: these
are found as small platelets often folded in so many various ways
that the original shape of the cell is entirely obscured. They
are most numerous during the early period of the lactation and
- are due to the mechanical stimulation of the teats by milking.

(6) From the milk cistern; usually oval or rectangular in
shape, frequently elongated to a point along the longitudinal
axis and having an oval nucleus. In normal milk they are
usually found singly but increased desquamation produced
by stimulation may cause masses of cells to appear arranged like
the petals of a flower round a common centre.

(c) From secretory ducts and alveoli: these vary in size accord-
ing to the number of fat globules they contain (5 to 45 yw) and

BLOOD CELLS 173

when very distended they are known as ‘foam cells.” The
nucleus is usually well marked when unmixed with fat and only
surrounded with a narrow margin of protoplasm; the presence
of fat produces the characteristic honeycombed appearance of
the colostral bodies and such cells are only found in patho-
logical conditions and at the beginning and end of the lacta-
tion period. Some observers report that these large cells may
‘contain several nuclei, but Ernst never found more than one
and suggested that the apparent multiplication of nuclei was
due to mononuclear cells becoming superimposed.

Blood Cells. (a) Red blood cells or erythrocytes appear as
biconcave discs or as thorn-apple shaped cells containing meta-
chromatic granules.

(b) Leucocytes. These constitute a very considerable per-
centage of the total cells in normal physiological conditions
and may entirely predominate in pathological ones. All
varieties of leucocytes may be found but the usual frequency
of occurrence is in the following order: polymorphonuclears,
lymphocytes, large mononuclears, and transitionals.

The polymorphonuclear leucocytes, of which the majority
are neutrophylic in their staining properties, are usually 7.5 to
10 » in diameter and stain characteristically with methylene
blue as a deeply stained lobed or polymorphonucleus sur-
rounded by faintly coloured protoplasm. The lymphocytes are
usually considerably smaller (5.7 uw) than the ‘ polymorphs ”’
but vary very considerably in size. The nucleus is round and
occupies practically the whole of the cell. Mononuclear leu-
cocytes are much larger than the lymphocytes (usually 13-16 p
but may be 25 uw in diameter) and two to three times the size of
erythrocytes. The nucleus is large and oval and is eccentrically
situated in a relatively large amount of protoplasm. With
methylene blue the nucleus stains moderately well and the
cytoplasm contains fine amorphous particles which produce
the appearance of ground glass. With Leishmann’s stain the
nucleus is ruby coloured and the cytoplasm blue but containing
a few ruby granules. The transitional cells are about the size of

174 CELLS, DIRT AND DEBRIS

the large mononuclears. The nucleus shows varieties of transi-
tion between the indented mononuclear and the irregular poly-
morphonuclear cell. As a rule, it is indented, crescrentic in
shape, and not possessing the multiplication so characteristic
of the polymorphonuclear leucocytes.

Degenerated cells of various kinds may also be present in
milk. Cells may, under various influences, become partially
or wholly disintegrated and the contents dispersed in fragments.
The nucleus may split up and the chromatin spread through the
plasma as dust or flakes. These flakes are often designated as
“‘ Nissen’s Globules ” and present the appearance of a darkly
stained centre, with or without a lightly stained border. The
albuminophores of Bab and Shulz which they describe as lym-
phocytes (15 to 20 u), containing fat and one to four proteid
bodies, are regarded by Ernst as degenerated fat containing
cells which have been attacked by macrocytes and then further
degenerated until the nucleus is no longer visible.

Estimation of Cells. The first attempt to estimate the
number of cells in milk was that of Stokes and Wegefarth in
1897 ! and consisted in the examination under an oil immersion
lens of a stained film prepared from the sediment obtained by
centrifugal action. This method was adopted with but slight
modifications by Bergey, Stewart and Slack. ;

Doane and Buckley in 1905° devised what is known as
the ‘ volumetric method ” in which a counting cell, such as is
commonly used in the estimation of cells in blood, was used for
the enumeration of the cells in the centrifugalised deposit from
10 c.cms. of milk. Russell and Hoffmann® compared the
“smeared sediment ” and “ volumetric ” methods and found
an average variation of 112 per cent in the former as against
only 6 per cent in the latter. They found also’ that a pre-
liminary heating of the milk to 70° C. produced higher and more
consistent results. The details of this method, as adopted by
the Committee on Standard Methods of Bacterial Milk Analysis
of the American Public Health Association ® are as follows:

Collection of Samples. Samples for analysis should be

MQM a ve

—

FS Sr ig Eee oe = er rs rr

= aye xe

CE owen. st ane hcomeeg lama ta Reto

CONCEN TRATION OF CELLULAR ELEMENTS 175

taken from the entire milking of the animal, as the strippings
contain a somewhat larger number of cells than other portions
of the milk. For the purpose of examination take 200 c.cms.
in a stoppered bottle.

Time Interval between Collection and Analysis. To secure
satisfactory results, milk must be examined in a sweet condi-
tion. Development of acidity tends to precipitate casein

in the milk and thus obscure the examination of microscopic

preparations. Samples received from a distance can be pre-
served for satisfactory microscopical examination by the
addition of formalin at the time of collection—a proportion of
1 c.cm. to 250 c.cms. of milk. Formalin has been found the
best preservative to use although it causes contraction of the
cells to some extent.

PROCEDURE WITH REFERENCE TO PREPARATION OF SAMPLE

1. Heating Sample. To secure the complete sedimentation
of the cellular elements in the milk, it is necessary to heat the
same to a temperature which will break down the fat globule
clusters, or lessen the ordinary creaming properties of the milk.
Samples should be heated at 65° to 70° C. for not less than ten
minutes, or from 80° to 85° where very short periods of exposure
(one minute) are given. This treatment causes the more homo-
geneous distribution of the fat globules through the milk, and
when the sample is then subjected to centrifugal force, the
cell elements are not caught in the rising fat globules, but on
account of their higher specific gravity are concentrated in the
sediment by centrifugal force.

2. Concentration of Cellular Elements. After centrifugali-
sation the cream and the supernatant milk are removed, with
the exception of the last 3 c.cm., by aspirating with an exhaust
pump and wiping the walls of the tube with a cotton swab.
After thoroughly mixing the sediment with a glass rod, enough
of the emulsion is placed in an ordinary blood counter (Thoma-
Zeiss pattern) to fill exactly the cell. The preparation is then
allowed to stand for a minute or two to permit the cellular

176 CELLS, DIRT AND DEBRIS

elements to settle to the bottom of the cell while the few fat
globules in the liquid rise to the surface. This method permits
of the differentiation of the cells from the small fat globules in
the liquid so that a distinct microscopic observation can be
made.

Examination of Material. The preparation is examined
in an unstained condition. The count is made with a 1-inch
eyepiece and 4-inch objective. Where the number of cell ele-
ments exceed 12 or 15 per microscopic field, one-fourth of the
entire ruled area of the counter, equivalent to 100 of the smaller
squares of the cell, is counted. Where the cell elements are
less abundant, one-half of the entire area (two to four hundred
squares) isexamined. The average number of cells per smallest
square is then obtained, which when multiplied by 200,000 gives
the number of cells per cubic centimeter in the original milk:
multiplication by four million gives the number of cells per cubic
centimetre in the sediment examined. As the sediment repre- —
sents the concentration of cells into one-twentieth of the orig-
inal volume of milk taken (10 ¢.c. to one-half ¢.c.) this number
should be divided by twenty to give the number of cells per .
cubic centimetre in the original milk.

Expression of Results. All results should be expressed in
number of cells per cubic centimetre of the original milk, and,
in order to avoid fictitious accuracy and yet to express the
numerical results by a method consistent with the precision of
the work, the rules given below should be followed:

NUMBERS OF CELLS PER C.CM.

From 1,001 to 10,000 recorded to the nearest 100

10,001 ‘10,000 500
50,001 100,000 1,000
100,001 500,000 — 10,000
500,001. 1,000,000 50,000
1,000,001 10,000,000 100,000

Savage, in 1905, independently worked out a volumetric
method based upon the same principle as the Doane-Buckley

EXPRESSION OF RESULTS 177

method but differing radically in technique. This was pub-
lished in 1906.9 The méthod of Savage is the better one of
the volumetric methods, so full details will be given: 1 c.cm.
of milk is placed in a tube having a capacity of 15 c.cms. and
diluted with Toisson’s solution (see Appendix) until the tube
is almost filled. The tube used is of special shape having the
_ lower end about one-quarter the diameter of the general body
of the tube and accurately graduated at 1 c.cm. After well
mixing the fluids, the tube is centrifugalised at 1800 revolu-
tions per minute for ten minutes. After breaking up the cream
with a clean rod the tube is whirled for a further five minutes.
The supernatant liquid is removed through a fine tube by
means of a vacuum pump until just 1 c.cm. remains. After
distributing the cells as evenly as possible in the sediment, a
sufficient quantity is placed in the cell of a Thoma-Zeiss or
some other convenient form of hemocytometer and the cells
counted in a number of fields of vision. Savage recommends
drawing out the microscope tube until an exact number of
squares spans the field of vision and gives the following formula
for calculating the number of cells per cubic m.m.

cells per cubic m.m. of milk= eg

where y=the average number of leucocytes per field of vision,
d=the number of squares which just spans the diameter. This
approximation of oo is accurate to within 0.5 per cent.
The cells in the ruled squares can also be counted and the result
calculated as in ordinary blood work, but as these represent
but a small proportion of the total area of the cell, errors due
to unequal distribution of the cells would be proportionately
greater.

Hewlett, Villar and Revis add 6 drops of formalin to 60-70
c.cms. of milk in order to break down aggregations of cells and
to prevent the cells being entangled in the cream layer. The

178 CELLS, DIRT AND DEBRIS

heating of the diluted milk tubes to 70° in Savage’s method
before centrifugalising would possibly produce higher results.

In 1910 Prescott and Breed !° suggested the examination of
the milk directly by means of stained smears. They found the
results obtained by this method to be very much higher than
by the Doane-Buckley method and that they were also more
consistent. This was due to the varying number of cells
trapped by the rising fat globules. Breed afterwards devel-
oped the process given on p. 129 which is obviously as applicable —
to cell examination as to the enumeration of bacteria. As
previously mentioned, the accuracy of this method depends
upon the even distribution of the cells and, if this condition
does not obtain, a very large number of fields must be examined
in order to obtain a fair average. With a cell count over 500,000 —
per c.cm. the author has obtained good results with this method
but for smaller counts the method of Savage is to be preferred
on account of the factor for the conversion of the cells per field
to cells per unit volume being so much smaller.

Significance. Despite the numerous investigations that
have been made in Europe and America during the last seven-
teen years, the significance to be attached to presence of cells
in milk is still surrounded with difficulties. It has already
been pointed out that a large number of cells are to be expected
in the secretion of such an active organ as the udder even under
normal physiological conditions and that stimulus, whether
mechanical or pathological, results in an increase in numbers.
As might be anticipated under such conditions the difficulty
lies in establishing what might fairly be regarded as the normal
variation in the number of cells. Savage found variations
ranging from 50,000 to 1,000,000 cells per c.cm. Russell and
Hoffmann found counts as high as 1,800,000 in animals in which
there was no history of clinical disease while 33 per cent of the
samples contained over 500,000 cells per c.cm. Stone and
Sprague," using the Doane-Buckley method, examined two
healthy cows during the whole milking oe (1,167 samples)
with the following results:

SIGNIFICANCE 179

Samples. Cell Count.
pe Fe ae a aoa under 10,000 per c.cm.
7.0 LASS ate 10,000 to 20,000
Re eR See AE aie sbi Gia 20,000 to 100,000
NECA ok i a, DR Eee Zoey a ae 100,000 to 500,000
Bere rear yeh ee ate over 500,000

Breed and Stidger,!* using the direct method, found varia-
tions ranging from 5000 to 20,000,000 cells per c.cm. in milk
whcih they regarded as normal. Breed !? examined 122 cows
which averaged 868,000 cells per cubic centimetre; fifty-nine
gave counts under 500,000 per cubic centimetre, 36 between
500,000 and 1,000,000 per cubic centimetre, and 27 gave
counts over 1,000,000 per cubic centimetre.

Hewlett et al.2 found that a change of feed influenced
the cell count. As regards physiological influences, Savage #
found that the previous number of calves and the age of the
cow had apparently little or no effect; just after, calving the
leucocytes are increased, but after this condition has subsided
the period since parturition has no effect until secretion com-
mences to diminish. The cells at this period often show very
abnormal values though not invariably so (Breed). Regarding
the relative proportion of cells in the fore milk and middle milk
the evidence is inconclusive, but it is agreed that there is an
increase in the number discharged in the strippings. There
are marked daily variations in the number of cells discharged
and equally large ones in the product of the four quarters of one
cow, for which no adequate explanation has been offered.
Pathological conditions may increase the cell content very
materially. Savage '* obtained cell counts as high as 368,000,-
000 per cubic centimetre in cases of mastitis and in these con-
ditions he also found that the relative proportions of the cells
approximated to those found in pus. The increased count was
particularly due to polymorphonuclear leucocytes which rep-
resented 75 to 80 per cent of total number of cells. Even after
the clinical evidence of mastitis has disappeared the cell count
may continue to be excessive for a considerable period. Some

180 CELLS, DIRT AND DEBRIS

workers have endeavoured to find a relation between the cell
count and the number of streptococci and other bacteria but
with no marked success. Milk stasis has been shown by many ©
observers to have a profound effect on the cell count by mark-
edly increasing the number of leucocytes.

Whilst it is impossible to formulate any rigid standard for
‘individual cows the author believes that mixed milk con-
taining over 1,000,000 cells per cubic centimetre as determined
by the Savage or Breed methods should be regarded with sus-
picion and the supply at once investigated. An excessive cell
count is not sufficient, per se, to warrant condemnation of a
supply, but if other unsatisfactory conditions also exist, such as
large numbers of streptococci, the public should be protected
by the exclusion of the supply until the condition is abated.

The tentative working basis of 1,000,000 cells per cubic
centimetre is not so low as to prevent the possibility of passing
a sample of mixed milk from a herd containing one case of
garget but is sufficiently so to provide a reasonable safeguard
without being oppressive on the producer. Asa routine method
of milk examination, the cell count has little to commend it in
the case of herd milk, but in the examination of individual
cows it is often of great service.

Dirt and Debris. During the present century many
attempts have been made to ‘quantitatively determine the
amount of dirt and debris in milk. Several methods have been
used, but as there is no agreement as to what is to be regarded
as dirt these have given results which, although comparable
among themselves, bear no relation to each other.

The sediment from milk according to Delépine !° consists of

(a) Cells derived from the udders.

(b) Hairs and cells from the milker, or cows or other farm animals.

(c) Wool, cotton or other fibres from strainers, etc.

(d) Vegetable and mineral matter derived either from food, dung or
litter or from dirty utensils and wash water.

(e) Algze, moulds, and bacteria from various sources.

As the cells and bacteria are separately determined, the

ee

DELEPINE, BABCOCK, AND GERBER 181

estimation of the sediment somewhat overlaps in that direc-
tion and its amount, “ ceteris paribus,’ should bear some
relation to the number of cells and bacteria.

The methods that have been proposed for the estimation of
the sediment in milk may be divided into two main groups.

(1) Preparation of sediment by centrifugalisation.

(2) Preparation of sediment by filtration.

Group 1. One of the oldest methods of this type is that of
Houston © who added 1 c.c. of formalin to 1 litre milk and
allowed the mixture to stand in a long tube with a narrow lower
graduated extremity closed by a glass tap. A primary reading
was obtained after twenty-four hours by making a direct obser-
vation on the scale. The sediment was then flushed out into a
small graduated tube and the volume made up to 10 c.cms. with
slightly alkaline water (0.1 per cent NazCO3). After cen-
trifugalisation for two minutes, a further observation was
made. This was termed the ‘secondary reading.” On
account of the large volume of milk required, this method has
not been generally adopted.

Delépine, Babcock, and Gerber all adopted methods in
which the milk was centrifugalised for a specified time and the
volume of sediment read off directly on the graduated lower
extremity of the tube. Conn modified the usual centrifugal
method by washing the sediment with distilled water and,
finally, collecting it in tared filter papers which were after-
wards dried and weighed. To convert the dry weight to a
moist weight a factor was necessary and this was found to
average 7. This factor was somewhat variable and depended
upon the nature of the debris. Revis}’ uses a tube having a
capacity of approximately 70 c.cms.; to this is attached a small
glass cup by means of a ground-glass joint. Inside the con-
stricted lower portion of the larger tube a glass rod is ground in
to form a plunger valve.

In the determination, the lower glass cap is fitted and 50 .
c.cms. of milk placed in the tube which is then whirled for five
minutes at 2000 revolutions per minute. After inserting the

182 CELLS, DIRT AND DEBRIS

rod valve, the lower tube is detached, the contents rejected and,
after reconnecting with the lower tube, 50 c.cms. of distilled
water are added and the valve withdrawn. After stirring the
sediment thoroughly with a platinum needle, the tube and
contents are given a further five minutes in the centrifuge. The
supernatant liquid is removed as before but prior to the final
washing with distilled water, the sediment is treated with 1
c.cm. of Eau de Javelle (antiformin may be substituted) for
the purpose of dissolving the leucocytes and epithelial cells.
After the final washing, the valve is inserted, and the lower
cap removed and dried in the water oven with its contents.
From the weight so obtained the tare of the cap is deducted
and a correction made for a blank. determination on the mate-
rials used. The dirt may be used for a microscopical examina-
tion. According to Revis, the hypochlorite has no action on
dirt constituents, but in view of the well-known action of chlo-
rine on cellulose this statement must be accepted with reserve.

Group 2. The filtration methods included in this group
are practically all based on the filtration of a given volume of
milk through a dise of cotton wool followed by an inspection of
the disc for visible dirt.

Tonney !® suggested the use of a small dise of absorbent
cotton in a Gooch crucible and operated with reduced pressure —
obtained from a water pump. This is a fairly satisfactory pro-
cedure for laboratory examinations but is usually precluded by
an insufficiency of sample. This principle of filtration for the
purpose of demonstrating visible dirt has led to the manufacture
of many commercial types of apparatus which have been used in
dairies and creameries, and by milk inspectors, with more or less
success. The types now on the market are the Lorenz or Wis-
consin, Stewart, and Gerber, which use gravity filtration, and
the Lorenz improved and Wizard which employ pressure or
suction. A detailed account of these has been given by
Schroeder 1° of the Health Department of New York City,
but as these are of but very limited utility in laboratory work
they will not be discussed ‘‘ in extenso ” here.

BIBLIOGRAPHY | 183

Significance of Sediment. If no efforts were made by
producers and dairymen to remove sediment from milk, the
determination of the dirt and debris would be an invaluable
guide to the care exercised in the production and handling of
milk, but in view of the fact that strainers or slime separators
are in almost universal use, the amount of sediment may bear
no relation whatever to the general condition of the milk. It has
been shown by many sanitarians that the suspended debris
represents only a small proportion of the total dirt and if this
solid debris is removed by filtration or separation the general
physical appearance of the milk might be entirely fallacious.
The use of cotton disc filters by sanitary inspectors has accom-
plished much in the last few years by demonstrating to vendors
in an incontrovertible manner the dirtiness of their product, but
no real progress will be affected thereby if the farmer increases
the efficiency of his strainers instead of preventing the access of
dirt. There is a possibility that sanitarians may defeat their
own objects by the placing too much reliance on the disc test
and failing to correlate it with the bacterial count and other
tests. Such “ prima facie” evidence of cleanliness may be
nothing but a specious fallacy.

BIBLIOGRAPHY

1. Stokes and Wegefarth. Med. News. 1897,71,45-48. J. State Med.,
5, 439.

2. Hewlett, Villar, and Revis. J. of Hyg. 1909, 9, 271-278.

3. Scannel. Amer. Jour. Pub. Health. 1912, 2, 962.

4. Ernst. Milk Hygiene. Trans. by Mohler and Eighorn. Chicago,
1914.

5. Doane and Buckley. Md. Agr. Expt. Sta., Bull. 102, 205-223.

6. Russell and Hoffmann. J. Inf. Dis., Supple. 1907, 3, 63-75.

7. Russell and Hoffman. Amer. Jour. Pub. Hyg. 1908, 18, 285-291.

8. Amer. Jour. Pub. Hyg. 1910. 20, 315-345.

9. Savage. Jour. of Hyg. 1906, 6, 123-138.

10. Prescott and Breed. J. Inf. Dis. 1911, 7, 632-640.

11. Stone and Sprague. Jour. Med. Research. 20, 235.

12. Breed and Stiger. J. Inf. Dis. 1911, 8, 361-385.

13. Breed. New York Expt. Sta., Bull. No. 38. 1914.

184 CELLS, DIRT AND DEBRIS

. Savage. Rpt. of M.O. to L.G. B. 1906-07, 228-236.

. Delépine. Rpt. to Manchester Sanitary Committee. 1908.

. Houston. Rpt. to London County Council. No. 933. 1905.
. Revis. Jour. Roy. Inst. Pub. Health. 1908, 56, 734.

. Tonney. Amer. Jour. Pub. Health. 1912, 2, 280-281.

. Schroeder. Amer. Jour. Pub. Health. 1914, 4, 50-64.

CHAPTER IX
PASTEURISED OR HEATED MILK

In addition to the usual bacteriological tests it is occa-
sionally advisable to examine pasteurised milk with a view to
determining the nature of the heat treatment to which it has |
been subjected. Prolonged heating at temperatures exceeding
150° F. results in the destruction of the enzymes and the loss
of albumin and soluble phosphates; the fat globules may also
be so altered that they do not rise iahamack and so affect what
- is commercially known as the “ cream line.’’

The effect of time and temperature, the two iter con-
trolling the general effect, have been admirably expressed by _
Dr. North of New York, in a diagram which, with slight modi-
fications to bring it into harmony with the author’s results, is
reproduced on page 187..

For the detection of overheated milk, several methods are
_ available: (1) determination of the cream line, (2) enzyme
reactions, and (3) estimation of the albumin.

Cream Line. Place 100 c.cms. of the sample in a cream-
ometer or graduated cylinder and observe the percentage of
cream obtained after standing for six hours at 60° F. If less
than 2.5 per cent of cream rises for each 1 per cent of fat con-
tained in the original milk, the presence of heated milk must be
suspected. If less than 2.5 per cent of cream is found for each 1
per cent of fat, the sample may either be milk pasteurised at. a
temperature exceeding 150° F., or a mixture of sterilised and
fresh milk.

Enzymes. The effect of heat on milk enzymes has been
studied by many workers and the more important results are
given in Table LXI.

185

186 PASTEURISED OR HEATED MILK

TasBLE LXI
EFFECT OF HEAT ON ENZYMES IN MILK
WEAKENED DESTROYED
Enzyme. Authority. ;
At Temp. in At Temp. In
de Minutes. red, 0 Minutes.
Galactase.....| Babcock and
Russell 65-70 10 76-80
Von Freudenreich | 65-70 | 30 75-80
Hippius 65 30
Amylase...... Bowing oly. Fe Te cara ces 68 30
pee 0 eee sae Probe 75-80 |
Race 68 30 &3 30
LAAN 6S 6s 3). TRS oe eae Pe ah €5
Lactokinase...| Hougardy #§ | ...... | ...... 75 30
Oxidases..... Se SOE MUR SPRSALA ISL g Fie Foe 79
Ac its 1” CANNES, MEMES, GI (Y's cn 76
Peroxidases. ..| Wender 9 (8 "4a eh ae 83
Schardinger 3! S Gis. aaebiereeers 80
Onterter : 227 3)) Bie eae aie 80
Lythgoe 70 30. 75 30
Race 68 30 73 30
Numerous others. Yi: tats Mie paras: 79-80
Catalase...... Van Italie 3: hua Rake 63 30
Wenders soe ire ae eek eee 80
Reductase. ...| Jensen Wishcoele: Mideae a ie over 70
Lythgoe 65 30 70 30
Race 68 30 a i | 30

Although these results are slightly discordant, they all show
that thirty minutes treatment at temperatures less than 65° C.

ENZYMES 187

(149° F.) has no effect on the enzymes usually found in fresh
milk.

The tests most easily applied are the hastened reductase

DracraM No. IV

Time in Minutes

180 »
170 Meligturanren ee Fat, Sugar,
Casein, Salts

|

: 4160

.)

if 3

; é Taste
, =

if %
© abe Albumin
’ g Enzymes
i , 2
it a
| 5 uso Yu :, Nai Line

f |

i YW) ZA Taberculosis
| ' Typhoid
1 { 1304 Streptococci

§
‘ Diptheria

Ir
- 120

if 10 20 30 40 50

reaction by means of Schardinger’s reagent, and the peroxidase
reaction with benzidine (page 91). The intensity of the

188 _ PASTEURISED OR HEATED MILK

peroxidase reaction is inversely proportional to the intensity
of the heat treatment and a similar indication is given if more
than twenty to twenty-five minutes are required to discharge
the blue colour in the reductase test.

The results obtained by the author on the effect of heat on
the peroxidase and reductase tests are given in Tables LXII and
LXIII. |

TaBLe LXII
EFFECT OF HEAT ON PEROXIDASE TEST
E BENZIDINE REACTION AFTER HEATING TO
Duration of
Heating in
punate: 145° F. | 150° F. | 155° F. | 160°F. | 165°F. | 170°F,
5 + + + mo + +
10 + a 5 8 - T. +
15 + + + a + Faint
20 aa t “- “ Faint |Very faint
25 + + + Faint _ _
30 + + + |Very faint} — _
Taste LXIII
EFFECT OF HEAT ON REDUCTASE TEST
Time (Minutes) Required for Discharge of Colour after Heating
, to (Sample less blank).
Duration of
Heating in -
145° F. 150° F. 155° F. 160° F. 170° F.
5 0 1 1 3
10 1 2 2 9 Over 24 hr.
15 2 3 3.5 30
20 3 4 5 66 Over 24 hr.
25 3 4 6 204
30 4 6 7 Over 24 hr.

|
|
|
|
|

ESTIMATION OF ALBUMIN 189

If milk has been treated with an excess of hydrogen peroxide
or heated with a smaller quantity of this substance, the perox-
idases are destroyed and a negative reaction is obtained with
the usual reagents. Formaldehyde, in the quantities usually
employed for milk preservation, has no apparent effect on the
Schardinger test.

Estimation of Albumin. The estimation is most sain
performed in the manner described on page 74.

Rupp! obtained the following results with heated milk.

Milk Heated for Thirty Percentage of Albumin

Minutes at : Precipitated.
27 Soak Sat aE Sa nd) GRR a eae eae ea ate Nil
SIME Par) CRAM EER veo reve arsigwe vate 4 aes g 5.75
ATC I eh coda a ia avec as 12.75
(09 Gd OAW6 | | od BP PS as eR RA UE 30.87

The rennin coagulation may also be used for the detection
of sterilised milk or milk heated at temperatures exceeding
65° C. Rupp’s results (vide supra) in this connection are
given in Table LXIV.

TasBLE LXIV

Time required for rennin coagulation of raw and heated
milk. Milk 200 c.cms.: rennin solution (0.15; 100 c.cms. water)
5 c.cms.

MiLtkx Heatep ror Turrty MINUTES AT
Experi | paw Milk.
pest. 55° C 60° C, 65° C. 70° C. 75° C,
131° F 140° F, 149° F, 158° F. 167° ¥,
Min. Sec.| Min. Sec.| Min. Sec.| Min. Sec.| Min. Sec.| Min. Sec.
1 ie ee Fae: ae ae. A ae 22
19 08;16 56/)116 638117 12
2 RGR We err a 20 38) 36 30
Do) HEE Da eclled Sh Sol th MRED AER Nenu 20 25) 37 #30

190 PASTEURISED OR HEATED MILK

Bacittus ABORTUS

Since 1897 when Bang and Stribald 2 isolated B. abortus as
the causative agent of the infectious abortion in cattle, con-
siderable study has been given to this organism in various parts
of the world. McFadyean and Stockman? corroborated
Bang’s findings, but later work has resulted in the discovery
of several allied forms with the consequence that B. abortus is
now regarded as a species and not as a distinct biotype.

During the last decade several workers have found B. abortus
in milk by the inoculation method and in some instances as
many as 60 per cent of the samples gave positive results. The
lesions produced by these samples were not usually sufficient
to cause death.

Although the descriptions of B. abortus as given by various
workers showed considerable variations, it remained for Evans #
to classify the various forms and to indicate the relative fre-
quency of certain varieties in normal udders. By plating milk
on agar containing 10 per cent of bovine serum, Evans isolated
B. abortus from 45 (23.4 per cent) of the 192 samples exam-
ined. These samples were obtained from 5 dairies. Thirty-
three cultures exhibited a marked lipolytic action on milk fat
and were, consequently, designated as B. abortus variety
lipolyticus. Twelve cultures (variety 6) differed from the
pathogenic varieties in their ability to ferment the usual test
substances, and morphology. The reactions of the varieties
isolated by Evans are given in Table LXV, together with those
of the typical pathogenic varieties for comparison.

B. abortus in young cultures shows the typical slender rod
form but involution forms are often found in older ones and
foetal exudates often contain coccoid varieties. Ordinary
aniline dyes may be used for staining purposes, carbol fuchsin
followed by 1 per cent acetic acid, dilute carbol fuchsin, and
Loeffler’s methylene blue giving very satisfactory results.
The organism is decolourised during Gram’s method of staining.
For cultural preparations agar containing 10 per cent of serum

BACILLUS ABORTUS 191

may be used or an agar gelatine serum mixture (4 per cent
gelatine, 6 per cent agar and 1 per cent serum) the serum in
which is previously heated to 60° C. for one hour on 4 con-
secutive days to ensure sterility. This latter medium is very
satisfactory for shake cultures. In carbohydrate media slightly
variable results are recorded. Most workers report that neutral
carbohydrate broths remain neutral or are rendered slightly
alkaline, except for a few cultures which produce slight acidity
in dextrose. Good and Corbett ® report that B. abortus variety
equinus showed an average of 2 per cent of gas in lactose in 93
cultures and no gas in 23. In saccharose 58 gave a little less
than 2 per cent of gas and 28 were negative. Some cultures
also produced marked quantities of gas in xylose, dextrose,
arabinose, dulcite, sorbite, mannite, maltose, and raffinose. |
Duplicate and triplicate tests with lactose and saccharose gave
varying results but Good and Corbett are convinced that the
gas produced is the result of chemical action and not adven-
titious. From the results of the fermentation tests, these
workers place the equinus variety in the Gaertner group of
organisms. The great difference in fermentative ability be-
tween this variety and the other members of the B. abortus
group would appear to warrant a change in the nomenclature
of the equinus variety and its removal from the abortus group.

In guinea pigs, milk containing B. abortus often produces a
nodular condition of the spleen and liver, the macroscopical
appearance having a somewhat superficial resemblance to that
produced by B. tuberculosis. -In pregnant test animals, inoc-
ulation with cultures usually produces abortion in a few days
but in some cases the action is much delayed and in others the
gestation period may be quite normal.

ACID-PRODUCING ORGANISMS

Although the organisms found in milk capable of fermenting
lactose with the production of acid include such widely differ-
ing groups as diplococci, staphylococci, streptococci, and bacilli,

192 PASTEURISED OR HEATED MILK
TABLE
COMPARATIVE CHARACTERISTICS OF SEVERAL
Sh B. abortus from Original |B. abortus from Pathogenic
Descriptions. Sources.
Morphology. Small rods, the largest | Slender rods, 0.8 to

as long as the tuber-
cle bacilli. (Bang.)

1.5 microns in length.

Reaction in dextrose,
maltose, lactose, raf-
finose, mannite, and
glycerine broth.

Alkaline broth is given

an amphoteric or
slightly acid reac-
tion to Tournesol
paper. (Nowak.)

Neutral broth is ren-
dered slightly alka-
line, except that a few
cultures form a slight
acidity in dextrose.

Decomposition of ni-
trogenous com-
pounds.

Nitrate, asparagin and
urea are commonly
decomposed. Gela-
tine is not liquified.

Action in litmus whole
milk.

Rendered slightly alka-
e.

Growth in agar shake.

Growth in colonies is
confined to a zone of
from 10 to 15 mm.
This zone lies about
5 mm. under surface
of the agar. (Bang.)

Good growth on sur-
face. Sometimes a
growth throughout a
zone of several mm.
at the top. Rarely a
diaphragm growth.

Growth on plain in-
fusion agar slope.

Separate colonies re-
semble rose coloured
droplets reflecting a
greenish tinge.

Abundant compact
growth chamois and
cream buff in col-
our.

Growth in glycerine
broth. ;

A poor growth. A

fine sediment is
thrown down made
up of whitish grains.
(Bang.)

Good growth which
clouds the medium.

Effect of serum in the
agar.

Growth is greatly fav-
oured. (Bang.)

Abundant growth
without serum.

LXV

VARIETIES OF BACILLUS ABORTUS.

B. ABORTUS GROUP

193

(AFTER Evans)

B. abortus Lipolyticus.

B. abortus Variety b.

B. abortus Variety c.

Slender rods, 0.8 to 1.5
microns in length.

Slender rods, 0.8 to 1.5
microns in length.

Slender rods, 0.8 to
1.5 microns in length.

No change.

Dextrose and maltose
broths are rendered
acid. No change in
other broths.

Slightly alkaline.

Nitrate, and asparagin
not decomposed; urea
rarely. Gelatine not

Nitrate, asparagin, and
urea usually decom-
posed. Gelatine is

Nitrate, asparagin and
urea sometimes de-
composed. Gelatine

liquefied. not liquified. sometimes liquified.
Acid is developed in | Slightly alkaline in | No change.
the cream layer. most cases. No

change in others.

Colonies confined to a
thin layer a few mm.
beneath the surface.

Similar to those from
pathogenic sources.
Colonies sometimes
scattered throughout
the entire depth of
"agar.

Similar to the cultures
from pathogenic
sources.

A few cultures resem- | Similar to the cultures | Similar to cultures
ble those from patho- | from pathogenic | from pathogenic
genic sources. The|_ sources. sources.
growth scanty in
separate colonies.

Scanty growth which | Abundant growth. | Similar to the growth

does not cloud the
medium. Sediment
is made up of little
granules.

The medium is usu-
ally clouded, but
sometimes the growth
is precipitated, leav-
ing a clear medium.

of variety b.

Growth greatly fav-
oured.

Abundant growth

without serum.

Abundant growth
without serum.

194 PASTEURISED OR HEATED MILK

it is sometimes desirable, as in the study of the effect of heat or
chemical germicides upon the bacterial flora, to determine the
relative proportion of this group to the total bacteria, without
reference to the morphological characters of the individual
members.

This division of the flora into groups on an acid-producing
basis is necessarily an empirical one, but it is comparatively
simple and has proved useful on many occasions.

The development of this method and its application to the
examination of raw and pasteurised milk is largely due to
Ayers and Johnson of the United States Department of Agri-
culture. In their earlier work they grouped the flora into acid
forming, alkali forming, inert, and peptonising organisms ac-
cording to their action on litmus lactose gelatine. This was
effected by plating out the sample on this medium and counting
the various groups after incubation for five days at 18° C. By
this method it is often difficult to distinguish between the feeble
acid formers, the feeble alkali formers, and the inert group, but
fairly satisfactory results have been obtained with it in the
author’s laboratory © and it has the advantage of being much
quicker and simpler than the later developments. The first
modification made by Ayers’ was an effort to obtain a more
accurate count of the peptonising group by the elimination of
spoilt plates caused by the spread of the gelatine liquefiers. A
neutral lactose casein medium (see Appendix) was substituted
for litmus lactose gelatine and the peptonisers differentiated
by flooding the surface of the medium, after six days incuba-

tion at 30° C., with Bl lactic acid. The colonies of peptonis-

ing organisms became white owing to the precipitation of casein
by the acid. Ayers, in the same report (p. 227) also suggested
the division of the flora into five groups according to the action’
on litmus milk. The colonies developing on lactose casein agar
or infusion agar were fished into litmus milk tubes and incu-
bated for fourteen days at 30° C. According to the appear-
ance of the milk after this period the organisms were classified

al a

ee So

|

ACIDURIC BACILLI 195

as acid forming and coagulating, acid forming, inert, alkali
forming, and peptonising. A comparison of the milk tube
method and the litmus lactose gelatine plates was made by
Ayers and Johnson ® who obtained the following results as the
averages of four samples.

Acid. Alkali and Inert. Peptonising.
After heating to 140° F.:
Baile Gabes ee 71.5 22.8 5.7
BG: plates; 22. ss. 43.7 53.5 2.8
After heating to 150° F.:
PU COS o's kes ds Ss 84.6 10.5 4.9
AG SE Sar” eer aa 41.2 1.7 Rak

The milk tube method possesses the advantage of differen-
tiating those organisms having feeble fermentative ability and
also develops a larger proportion of peptonisers. The latter
result may be partially due to the nature of the nitrogenous
substance used for the test as it is exceedingly improbable
that proteolysis proceeds at the same rate with all test sub-
stances.

Aciduric Bacilli, Among the acid-producing organisms,
one sub-division, that of the aciduric or acidophylic bacteria,
is especially worthy of further mention because it contains the
commercially important B. bulgaricus. This organism has
achieved considerable repute during the last few years as a
therapeutic agent by reason of its influence on the flora of the
intestinal canal and it has, consequently, become necessary
to make bacteriological examinations of the tablets used for
this purpose. ‘

- Although the aciduric bacilli grow luxuriantly in dextrose
and lactose broth containing acetic or lactic acid they usually
grow very sparingly or not at all on the usual laboratory media.
They vary considerably in length (3 to 7 u) and occur singly or in
chains or threads. They develop under both erobic and aner-
obic conditions and, although typically Gram positive, old cul-

196 PASTEURISED OR HEATED MILK

tures may be Gram negative. Spore formation is never ob-
served and they ferment carbohydrates with the production of
acid but do not form gas. Milk coagulation is produced by some
members of the group and not by others.

For the isolation of this group there is no better method
than that used by Heyman in 1898, viz., the use of a meat pep-
tone broth containing 2 per cent dextrose and 0.3 per cent
acetic acid. After incubation at 37° C. for forty-eight hours,
a portion of the culture is seeded into another broth tube and
the process repeated until only aciduric bacilli remain. For
further isolation dextrose agar containing 1.5 per cent agar
and 2 per cent dextrose without any adjustment of the acidity
may be used. According to Rahe ® the addition of 0.2 per cent
of sodium oleate as recommended by Salge !° is productive of
good results. By this method Rahe (vide supra) investigated
a number of the aciduric bacteria, and divided them into three
groups according to their biochemical properties.

CGRovup.
Action on
A. B. C.
ENE sists ¥ 5s) Rn seh a Clot Clot No clot
DIOIGONS oot cic a bs cuseas Not fermented| Fermented Fermented

Group A, which is the B. bulgaricus group, is characterised
by a rapid clotting of milk and its usual inability to ferment
carbohydrates other than lactose and dextrose.

Group B also clots milk but ferments maltose, saccharose,
and levulose in addition to lactose and dextrose, and usually
also mannite and raffinose.

Group C does not clot milk and ferments maltose even
more vigorously than group B. Saccharose and levulose are
fermented and usually raffinose, but mannite is not acted upon.

FERMENTATION TEST 197

THE FERMENTATION TEstT IN MILK EXAMINATION

This test is performed by incubating the sample in sterile
vessels and observing the chemical and physical changes that
take place.

The earliest experimental work in this connection was prob-
ably that of Walter, cantonal chemist at Soleure. This ob-
server kept milk at 98° F., and stated that ‘‘ milk, if good, will
not curdle or undergo abnormal fermentation in ten to twelve
hours.” A special apparatus was devised for this purpose by
Schaffer," who recorded the amount of gas evolved in 100° F.
from a definite volume of milk. He found that good milk
formed no gas and remained fluid after twelve hours. This
test was chiefly used in connection with the suitability of milk
for cheese manufacture; milks that produced ‘“ heaving”
were detected by this test. :

The Wisconsin curd test !? was also evolved for cheese
manufacture and differs from the Swiss tests given above in the
use of rennet for the production of a definite curd which is
pressed and afterwards set aside for observation.

The Gerber fermentation test consists in incubating tubes of
milk at 104° to 106° F. for six hours and then observing the
odour, taste, and appearance for abnormal qualities. The
heating is then continued for a second six-hour period and any
abnormal coagulations, such as gas holes, are then noted.
Gerber stated that coagulation in less than twelve hours is
abnormal, and that milk that does not curdle in twenty-four
hours to forty-eight hours is open to suspicion regarding
preservatives. |

According to Jensen,!* the milk is heated to 30° to 35° C.
for eight to twelve hours and examined; replaced for a further
period and again examined. After the second period he found
that the clean samples are sour and curdled and form a homo-
geneous coagulum without much separation of curd and gas
formation. Frequently gas bubbles have split the coagulum
and considerable fluid has separated. This change, he states,

198 PASTEURISED OR HEATED MILK

does not necessarily signify that the milk was particularly rich
in bacteria of putrefaction. If curdling is accompanied by an
offensive odour or, if the coagulum is peptonised, the presence of
putrefactive bacteria is inferred. He continues, “ by boiling
milk a short time and then incubating, only spore formers
develop, and as these are not checked by the lactic bacteria,
they increase rapidly and cause the milk to curdle by the action
of ferments. Pasteurised milk does not sour, but no precipitate
conclusions should be drawn from the results of this test.”

Peter,!* Dugelli,© and Klein ' have used this test for milk
examination and find that it gives the prevailing types of micro-
organisms with a considerable degree of accuracy. A combina-
tion of the fermentation test with the methylene blue reduction
test has been recommended by Lohnis and Schroeter,!? and by
Fred and Chappelean.}8

In 1914 the author compared the sweatin obtained by this
test with the usual bacterial count on agar (forty-eight hours at
blood heat) and the B. coli count in rebipelagar. The samples
were transferred to sterile tubes plugged with absorbent cotton
and incubated at 37° C. (98.5° F.) for 20-24 hours. 787 sam-
ples of ordinary raw milk, 98 samples of pasteurised milk, and
69 samples of nursery milk were examined in this way and the
results recorded according to the classification of Dugelli
(vide supra). This classification, together with the bacterial
flora which Dugelli states is indicated by each type, is as follows:

Types or CurRD
Type A

Liquid. The sample does not show any marked change
except perhaps a slight deposit on the bottom of the tube.

1. Completely liquid, sweet or sour taste.

2. Somewhat coagulated at the bottom or on the walls.

3. A slight ring of curd under the cream, but otherwise
liquid and sour. |

ee ee

Pia a, Narn tas all iat

TYPES OF CURD 199

4, Completely liquid or with a slight separation of the solid
components of the curd. Taste strongly acid or bitter acid.

Type B

Gelatinous or Jelly-like. The sample is more or less
curdled and the casein is united into a gelatin-like mass without
any marked separation of the curd.

1. A beautiful, smooth gelatinous mass without curd sepa-
ration and a pure acid flavour.

2. Smooth but some gas bubbles and furrows.

3. Generally smooth, but with curd separation and marked
by gas bubbles and furrows.

4. Generally smooth, with curd separation, but with nu-
merous gas bubbles and furrows.

Type C

Granular. The milk curdles, but the curd, instead of being
smooth consists of many small grains. Between the more or
less fine curd grains, creamy cheese-like particles may be found.

1. Curd only partly granular and partly gelatin-like with
little cheese separation.

2. Curd of fine granular structure and uniformly divided so
that the curd looks white.

3. Curd shows a marked separation with mostly large grains.

4. Large granules and complete coagulation with a creamy
deposit.

Type D

Cheese Curd. The casein is flocculent or in clumps, and is
attached to the sides of the vessel. The curd is more or less
completely separated from the whey. :

1. Casein is a soft, united mass. The curd is greenish in
colour and slightly acid.

2. Casein is a firm mass, curd green, and slightly acid.

3. Casein pulled apart and divided, a greenish white,
strongly acid curd.

200 PASTEURISED OR HEATED MILK

_ 4, Casein entirely separated and attached to the sides of the
tube. A white curd, strongly acid.

Type E

Gaseous. The tube is well marked with gas bubbles.

1. Cream filled with bubbles.

2. Cream and curd filled with bubbles.

3. Bubbles so numerous that the curd floats on the whey and
forms a raised surface.

4. The gas development is so pronounced that the curd is
forced upwards in the tube, often forcing out the stopper.
Bacteria Flora, as indicated by Fermentation Test. (Dugelli.)

Type A

Bacteria present in very small numbers. Cocci predominate
with few lactic acid, coli and zrogenes organisms.

Type B

Lactic acid in great numbers, few if any coli and «#rogenes
- organisms, some cocci and fluorescent bacteria. Gas formation
indicates the presence of coli, erogenes, or butyric organisms.

Type C

Lactic, coli, and zrogenes bacteria predominate with many
cocci.

Type D

Lactic acid mixed with coli and erogenes organisms.

Type E

Coli and erogenes organisms abound if much gas is
formed; also lactic bacteria, cocci and B. vulgatus.

TYPES OF CURD 201

The author’s results showed that the type of fermentation
was determined by a combination of factors which varied in
different samples. The chief factors were the total and relative
numbers of the various groups of organisms which constituted
the bacterial flora.

When the total bacteria were very low the fermentation was
usually of the A type, i.e., very little visible alteration occurred
in the physical appearance of the sample, and a smooth acid
flavour was produced. The acid producers were so few in num-
bers as to be unable to produce, under the incubator conditions,
sufficient acid to coagulate the caseinogen. This is the dis-
tinguishing feature of type A. In types B, C, and D, there was
a distinct coagulation, but the character varied in each group
according to the organisms associated with the acid producers.
The acid producers in each case produced their effect, and if the
ratio of acid formers to gas formers were large, little or no evi-
dence of gas formation was observed. As this ratio decreased
furrows became evident and numerous gas bubbles were found
enclosed in the curd, whilst in extreme cases the gas formation
was so marked as to force the cream layer to the top of the tube.
As any gas formed previous to the production of a-firm curd
would be lost without leaving any evidence, it follows that any
gas observed must have been produced after coagulation and in
a medium of increased acidity. To effect this the proportion of
colon organisms must be considerable, as, otherwise, their
development would be retarded by the metabolic products of
the acid group. Very many samples, however, were observed
to produce gas bubbles in the fermentation test, and yet con-
tained originally less than one B. coli per cubic centimetre.
In these cases either the small numbers of the B. coli must have

_ increased very rapidly in proportion to the acid formers or be

of an acid resisting type. At ordinary temperatures (50° to
60° F.), the colon content usually continued to increase until
about 0.7 per cent of acidity, calculated as lactic acid, was
produced.

The results also showed that the same type of fermentation

202 PASTEURISED OR HEATED MILK

was produced by very widely differing B. coli contents, and it
was, therefore, impossible to form a definite opinion regarding
the B. coli content from the appearance of the fermentation |
test. The A type was almost invariably produced by milk ©
low in B. coli, whilst D5 pointed to excessive contamination
with this group, but with regard to the intermediate types,
which the majority of market milks produce, no definite con-
clusions could be deduced. The same remarks apply regarding
the relation of the total bacterial count to the type of fermenta-
tion, and, under these circumstances, it is difficult to attach
much value to this test. Some observers have a high opinion
of this test, because it is supposed to yield evidence as to bac-
terial flora and thus enable deductions to be made as to the
conditions under which the milk was produced and its subse-
quent treatment, but the author’s results do not substantiate
this claim.

The conditions of the test, viz., incubation, at blood heat,
are artificial, as milk is never, under ordinary circumstances,
kept at this temperature, and it is not logically sound to assume
that the biological and chemical changes are the same at dif-
ferent temperatures as a change of temperature always favours
the growth of one or more groups in preference to others.

COLLECTION OF SAMPLES

All milk sold in bulk must be thoroughly mixed before
samples are taken and every endeavour should be made to
obtain milk in the same manner in which the vendor supplies
the same to the consumer. The Committee of the American
Public Health Association, appointed for the standardisation
of bacteriological examination of milk, have recommended that
bacteriological samples should be obtained from bulk milk by
means of sterile pipettes, but this method samples milk which
is in the possession of the vendor and ignores possible contami-
nation in the vessel used for the transfer of such milk to the
consumer. The author has observed numerous instances in

COLLECTION OF SAMPLES 203

which this vessel has had very appreciable effects upon the bac-
terial count and the number of coliform bacteria. For the col-
lection of combined chemical and bacteriological samples the
author has used for several years rectangular, narrow-necked, —
six-ounce glass-stoppered bottles, 16 of which can be placed in a
tray, 10 by 63 inches. This tray is surrounded with ice and
water, and the whole contained in a water-tight galvanised-iron
box 143 by 103 by 7 inches. In cold climates the cooling
mixture can be dispensed with in winter and when there is
any possibility of the milk freezing, wide-mouthed bottles
should be used to prevent freezing of the sample and so blocking
the neck of the bottle during the transfer of the sample. All
milk retailed in bottle should be delivered to the laboratory
in the original container unopened as the only other method of
satisfactorily sampling such milk is to transfer the sample to a
sterile bottle and then back to the original container, this being
repeated several times. The sterile bottle necessary for the
success of this method cannot usually be obtained so that this
system should not be encouraged.

All samples should be labelled in such a way that there can
be no possibility of doubt as to the identity of each sample
and a complete record of the sampling data made immediately
after the sample istaken. This should include name of vendor,
date, time and place, temperature, character of container
and name of collector. The temperature of milk in bulk is
observed immediately after the sample has been taken whilst
that of bottled milk should be obtained from a second bottle.
A quickly reacting Fahrenheit thermometer is suitable for this
purpose.

If the object of the examination of samples is to obtain
figures representative of the total milk supply and from which
averages can be calculated which are strictly comparative from
month to month or from year to year, the collection of samples
must be carried out as scientifically as possible and not in the
usual haphazard fashion. The output of each vendor should
be estimated and the number of samples varied in proportion

PASTEURISED OR HEATED MILK

204

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86 | O'Zt | Z'98 | giz | #89 | Tee | ete | tet | zee | ers | ser | oo0'6sz |*****10qG0700
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“RECORDING RESULTS

TaBLeE LXVII

PASTEURISED MILK YEAR ENDING OCTOBER 31, 1915. OTTAWA
BACTERIOLOGICAL

205

VARIATION IN BACTERIAL Count. PERCENTAGE OF SAMPLES CONTAINING

Month. Under syd Under | ney I Under ey Under
November...| 41.2 47.0 88.2 5.9 94.1 5.9 100.00
December...| 31.2 62.5 93.7 6.3 100.0 Nil
January.....| 53.8 46.2 100.0 PG Bata e Nil
February....| 33.3 66.7 100.0 RS Se Ue Nil
March...... 43.8 56.2 160.0 Spel BEA ae Nil
eT ee ee 25.0 75.0 100.0 BONE GE Sate arenics-« Nil

oe ed SaaS 81.8 18.2 100.0 ERs os cre rotor: Nil
Jane: ese 57.9 3t 1 79.0 7 | ean Ae eae Nil
Sh eee | 57.1 38.1 95.2 Nil 95.2 4.8 100.0
August...... 50.0 50.0 100.0 Ly EAE HECTARE Runa Nil
September 31.6 63.2 94.8 §.2 100.0 Nil
October..... 33.3 60.1 93.4 6.6 100.0 Nil
Average..... 45.0 50.4 95.4 > 99.1 0.9 100.0
Mean Mean VARIATION IN B. COLI PER C.CM. PERCENTAGE
ONY Bacterial | B. coli OF SAMPLES
on ount pes Under | 11 to | Under | 51 to | Under | Over
Poe OCA) 0.6m 10 50 50 100 100 | 100
November 25,000 3 88.3 Ld. 7: 100.0 Ju} na a ae Nil
December 19,000 15 56.1 31.2 Seia Vy tece 100.0 Nil
January 16,200 9 76.9 15.4 92.3 (ues 100.0 Nil
February 18,000 3 93.4 6.6 100.0 183 CSR Rupee Nil
March...... 14,400 35 75.0 6.2 81.2 Nil 81.2 | 18.0
Ae. 35k 17,000 6 °91.7 8.3 100.0 fb Miah bar Saas Nil
See 13,000 Z 86.3 13.7 100.0 10) SI Dare al Nil
eis Ss iisss So 22,600 55 42.1 42.1 84.2 |] 15.8 | 100.0 Nil
Se Ae 28,000 141 40.0 20.0 60.0 | 10.0 90.0 | 10.0
7. 411 -| 11,000 46 50.0 25.0 75.0 | 12.5 87.5 | 12:5
September 33,000 896 47.3 31.6 78.9 | 10.4 89.3 | 10.7
October..... 18,700 6 86.7 13.3 | 100.0 a aN aie Nil
Average. 19,600 102 69.5 18.8 88.3 6.7 94.0 6.0
CHEMICAL
SAMPLES BELOW STANDARD. GENUINE MILKs.
Guat Percentage of scr ; Average Composition. Ac siwas
onth. . . elow 8.5 (o)
ay i Per Cent Fat. : Total Solids |Samples.
Solids z Solids Not-fat. if Solids. | Not-fat.
: Soli
POW CMRIOR G5 Bigot s'a bk: cadiets PP Gh) (eevee as tle 4.03 13.02 8.99 17
MEN GR 547d oe KAT ONT atalino in hd SOR eae 3.85 12.81 8.96 16
RIUM ION oc a cee A e'Siaisar S.A 6 Batarais ent 3.79 12.70 8.99 13
MN. BS) iodiew 6) Lew pe os OE eats Be 3.73 12.63 8.90 15
NE See PES So die hs A alee a OA” vical oats 3.62 12.59 8.97 16
Se oda SPD AT (OR Pes ee (ed Rea AP 3.65 12.46 8.81 12
YS Oe eee! Te Ee te eS yea) MES ae aoe ae 3.70 12.67 8.97 22
Aas ere OEN is wis seth peathitaes IN. as emis ss 3.84 12.73 8.89 19
EA Shah alate BE at cies AOL OP Eile Ts dials occas bs a iy 12.52 8.75 21
ENS TS OR Beate aA LIN 6.4 3.86 12.57 8.71 16
NGS Pak a eRe baad ase ee RDS adie o's bee 3.97 12.74 8.77 19
TE AEE, BOA Ee ea a pee 3.93 12.97 9.04 15
Total
Co ee! SEP Re Ay ree 0.5 3.81 12:72 8.91 201

206 PASTEURISED OR HEATED MILK

to the output. When various grades of milk are offered for
sale, the results should be separately recorded. The interval
between sampling and examination should be as short as pos-
sible although no appreciable alteration occurs even in twenty-
four hours if the samples are kept between 32° and 40° F.

Recording Results. The ordinary method of recording
results by expressing the average total bacterial count or the
average number of bacteria of some particular group of organ-
isms, may give a result which does not represent the quality of
the supply if the variations from the mean are large, or if the
number of variants is comparatively small. The median
would be more representative of the actual quality than the
mean but a better plan is to express variations in the counts in
the manner set forth in Tables LXVI and LXVII. The size of
the groups in the scheme is quite arbitrary, but where milk is
graded they should agree with the limits permitted in each par-
ticular grade.

BIBLIOGRAPHY

. Rupp. Bull. 166, U. 8. A. Dept. of Agr.

. Bang and Stribald. Zeit. f. Tiermidicin. 1897, 1, 241-278.

. McFadyean and Stockman. Rpt. of departmental committee to the
Board of Agr., Appendix to Part 1. London, 1909.

4. Evans. Jour. Inf. Dis. 1916, 18, 437-477.

5. Good and Corbett. Jour. Inf. Dis. 1916, 18, 586-596.

6. Race. Can. Jour. Pub. Health. 1915, 6, 490.

7

8

On =

. Ayers. 28th Rpt. Bureau Animal Ind., U.S. A. 228.
. Ayers and Johnson. Bull. 161, Bureau Animal Ind., U.S.A.
9. Rahe. Jour. Inf. Dis. 1914, 15, 148.
10. Salge. Jahrb. f. Kinderh. 1904, 59, 309.
11. Schaffer. Landw. Jahrbuch der Schweiz, 7, 72.
12. Wisconsin Expt. Stat. Annual Rpts. 1895 and 1898.
13. Milk Hygiene by Jensen. Trans. by Mohler and Eichhorn. Chicago,
1914.
14. Peter. Jahresb. d. Molkereischule Rutti. 1905-1906. 210.
15. Dugelli. Centralbl. Bakt., 11 Abt., Bd., 18, pp. 37, 224, 439.
16. Klein. Amer. Vet. Review. Oct., 1912, 25.
17. Lohnis and Schroeter, Centralbl. f. Bakt., II, Abt., Bd., 32. 1912, 181.
18. Fred and Chappelean. Virginia Agr. Expt. Stat., 1911-1912, 233.

APPENDIX

Rebipelagar or Neutral Red Bile Salt Agar:

MR sy 5 Ks 1 SR te Bele hadi, cogs Bid Oe or we w Whee 20 grams
NN BHI v9 es Silesia DOUG bib a Big 0s Wacela os 20 grams
Weine BOIL COMUMOCTCIAN, «o!. sos bs co ee oe cee ss 5 grams
WOME Ss ocaee b hte sic Sear dba cas cas 1000 c.cms.

Heat the ingredients in a double pan or autoclave until
completely dissolved; titrate with alkali and adjust the reac-
tion to +1.0 per cent to phenolphthalein. Cool to 45° C.,
coagulate with egg albumen (5 grams dissolved in water),
heat to boiling, adjust the weight and filter. Tube in con-
venient quantities, after adding 5 grams of lactose and 5 e.cms.
of a 1.0 per cent solution of neutral red. |

Aesculin Bile Salt. (Harrison and Vanderleck, Trans.
Roy. Soc. of Canada, 1909, Sec. IV, 147.)

Dissolve in water 1.0 per cent of Witte’s peptone, 0.25 per
cent of bile salt, and 1.5 to 2.0 per cent of agar. Neutralise
with alkali, coagulate with egg albumen and filter. Add 0.2
per cent of citrate of iron and 0.1 per cent of esculin. This
amount of citrate of iron should give a final acidity of +0.7 per
cent and produces a slight fluorescence in the medium. ‘

Toissons’s Solution:

BORNE VINE ee sa ae eh a rclelele 0.025 gram
ME MIINY CMOPUIR 36515565 Sine ae led Ssd be Gs 1.0 gram
SUURDCRND MEINE So gS cic. 3's, bd civic Cwae cs 8.0 grams
RINNE Sc Vulurint, Uolnule pa tide oc a 30 ¢.cms.
7” ROR aoe Sige Fa or AA 160 c.cms.

The solution should be freshly filtered.
207

208 APPENDIX

Tokuidine. bitie >: 6. s/s usce i eee 0.1 gram
Azure's. 3.5%: MERA gene eee DR 2 Pa 0.01 gram
Methylene Dhue viii 5 25690 a a eee 0.01 gram
Glacial acetic Aeid: 0.5 eo. a 1.0 c.cm
95 per cent alcohol.) ) 20000565 0004s. 5.0 c.cms.
Distalled Water: 6345 fie Bo iets 120 c.cms.

The films should be stained for two minutes or more,

Dorset’s Egg Medium. Take 12 fresh eggs, wash the shells
with water and then with undiluted formalin; allow to dry.
Break the eggs into a graduated cylinder and note the total
volume. Add one part of sterile saline solution (0.85 per cent
sodium chloride) to three parts of the mixed eggs. Pour into a
sterile beaker or basin and whip with an egg whisk; filter
through cheese cloth or muslin into a sterile flask and tube
10 c.cms. in the usual way. Inspissate at 75° C. for one hour in
a sloping position and then add 0.5 c.cm. of sterile glycerine
broth (physiological saline containing 6.0 per cent of glycerine)
to each tube to prevent drying. Incubate at 37° C. for forty-
eight hours and reject all contaminated tubes. Eyre recom-
mends adding sufficient alcoholic basic fuchsin to produce a
distinct colouration before the medium is tubed.

Casein agar. To 300 c.cms. of distilled water add 10 grams
of casein (C. P. Hammersten) and 7 c.cms. of N. NaOH. Heat
to boiling for several hours until thoroughly dissolved. Adjust
the weight and bring the reaction to 0.2 per cent acid. The
agar solution is prepared by dissolving 10 grams of agar in 500
c.cms. of water. Both solutions are filtered, mixed, tubed,
and sterilised under pressure. The final reaction should be
+0.1 per cenit and, if the acidity is higher than this, a portion
of the casein will be precipitated during sterilisation.

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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
ed 4
72
73
74
75

210

APPENDIX

TABLE

FOR CALCULATION OF TOTAL SOLIDS
AccorpING To Bascock.
LACTOMETER READING

SSB2SReursua| >

Fat. 26.0| 26.9 27.0 | 27.5 | 28.0 | 28.5 | 29.0 | 29 30.5 | 31
0.0 | 6 6.62} 6.75) 6.87) 7.00} 7.12) 7.25] 7 7.62) 7.
0.1 | 6.62) 6.74 .87] 6.99) 7.12) 7.24) 7.37] 7 7.74) 7.
0.2 | 6.74) 6.86) 6.99) 7.11] 7.24) 7.36] 7.49) 7 7.86) 7.
0.3 | 6.86) 6.98} 7.11] 7.23) 7.36) 7.48) 7.61) 7 7.98) 8.
0.4 | 6.98} 7.10] 7.23) 7.35) 7.48) 7.60) 7.73] 7 8.10} 8.
0.5 | 7.10) 7.22) 7.35) 7.47) 7.60) 7.72) 7.85) 7 2: 33 8.
.°0.6 | 7.22) 7.34) 7.47) 7.59) 7.72) 7.84) 7.97) 8 8.34) 8.
0.7 | 7.34) 7.46) 7.59) 7.71) 7.84) 7.96) 8.09) 8 8.46] 8.
0.8 | 7.46) 7.58) 7.71 7 Se 7.96} 8.08) 8.21] 8 8.58) 8.
0.9 | 7.58) 7.70) 7.83) 7.95] 8.08) 8.20) 8.33) 8 8.70) 8.
1.0 | 7.70) 7.82) 7.95) 8.07) 8.20) 8.32} 8.45) 8 8.82) 8.
1.1 | 7.82) 7.94) 8.07) 8.19} 8.32), 8.44) 8.57] 8 8.94) 9.
1.2 | 7.94) 8.06} 8.19) 8.31] 8.44) 8.56) 8.69] 8 9.06} 9.19
1.3 | 8.06) 8.18) 8.31} 8.43) 8.56) 8.68] 8.81] 8 9.18} 9.31
1.4 | 8.18} 8.30) 8.43] 8.55) 8.68] 8.80) 8.93) 9 9.30} 9.43
1.5 | 8.30) 8.42) 8.55) 8.67) 8.80] 8.92) 9.05) 9 9.42) 9.55
1.6 | 8.42) 8.54) 8.67) 8.79] 8.92) 9.04) 9.17] 9 9.54) 9.67
1.7 | 8.54) 8.66} 8.79} 8.91) 9.04) 9.16) 9.29) 9 9.66) 9.79
1.8 | 8.66) 8.78} 8.91) 9.03) 9.16] 9.28) 9.41] 9 9.78} 9.91
1.9 | 8.78) 8.90} 9.03} 9.15) 9.28] 9.40) 9.53) 9 9.90} 10.03
2.0 | 8.90) 9.02} 9.15) 9.27; 9.40] 9.52! 9.65] 9 10.02} 10.15
2.1 | 9.02) 9.14) 9.27] 9.39] 9.52) 9.64) 9.77) 9 10.14} 10.27
2.2 | 9.14 7. a 9.39} 9.51) 9.64) 9.76) 9.89) 10 10.26} 10.39
2.3 | 9.26) 9.38) 9.51) 9.63] 9.76) 9.88) 10.01} 10 10.38) 10.51
2.4 | 9.38} 9.50) 9.63) 9.75) 9.88) 10.00) 10.13} 10 10.50) 10.63
2.5 | 9.50) 9.62} 9.75) 9.87] 10.00) 10.12) 10.25) 10 10.62} 10.75
2.6 | 9.62) 9.74) 9.87) 9.99} 10.12) 10.24) 10.37] 10 10.74| 10.87
2.7 | 9.74) 9.86) 9.99) 10.11] 10.24) 10.36) 10.49] 10 10.86} 10.99
2.8 | 9.86] 9.98) 10.11) 10.23] 10.36} 10.48) 10.61] 10 10.98} 11.11
2.9 ) 9.98/10.10) 10.23) 10.35) 10.48} 10.60) 10.73} 10 11.10} 11.23
3.0 |10.10)10.22) 10.35) 10.47 10.60) 10.72} 10.85} 10 11.23] 11.36
3.1 |10.22)10.34) 10.47) 10.59} 10.72) 10.84) 10.97) 11 11.35} 11.48
3.2 |10.34/10.46}. 10.59] 10.71) 10 10.96} 11.09] 11 11.47| 11.60
3.3 |10.46/10.58) 10.71} 10.83} 10.96) 11.09] 11.21} 11 11.59} 11.72
3.4 |10.58/10.70) 10.83) 10.96) 11.09) 11.21) 11.34] 11 11.71) 11.
3.5*|10.70/10.82| 10.95) 11.09) 11.21) 11.33) 11.46] 11 11.83] 11.96
3.6 |10.82)10.95) 11.08) 11.20) 11.33) 11.45) 11.58] 11 11.95] 12.08
3.7 |10.94/11.07) 11.20) 11.32] 11.45) 11.57) 11.70} 11 12.07) 12
3.8 |11.06/11.19) 11.32) 11.44] 11.57) 11.69) 11.82] 11 12.19] 12.32
3.9 |11.18)11.31) 11.44) 11.56] 11.69) 11.81] 11.94] 12 12.31] 12.44
4.0 |11.30)11.43) 11.56) 11.68) 11.81] 11.93) 12 12 13.43} 12.56
4.1 |11.42}11.55) 11.68) 11.80) 11.93) 12.05) 12.18} 12 12.55) 12.68
4.2 |11.54/11.67) 11.80) 11.92) 12.05) 12.17} 12.30] 12 12.67} 12.80
4.3 |11.66)11.79) 11.92) 12.04) 12.17) 12.29) 12.42] 12 12.79] 12.92
4.4 |11.78)11.91] 12.04) 12.16} 12.29] 12.41) 12.54] 12 12.91) 13.
4.5 |11.90)12.03) 12.16) 12.28) 12.41] 12.53) 12.66} 12 13.03] 13.16
4.6 |12.03/12.15} 12.28) 12.40] 12.53] 12.65) 12.78] 12 13.15} 13.
4.7 |12.15}12.27) 12.40) 12.52) 12.65) 12.77) 12.90) 13 13.27) 13.40
4.8 |12.27|12.39| 12 52| 12.64) 12.77) 12.89) 13.02] 13 13.39] 13.52
4.9 |12.39)12.51) 12.64) 12.76) 12.89] 13.01} 13.14] 13 13.51} 13.
‘5.0 |12.51}12.63] 12.76] 12.88} 13.01] 13.13) 13.26) 13 13.63] 13.76
5.1 |12.63/12.75]} 12.88) 13.00] 13.13] 13.25) 13.38] 13 13.76| 13.89
5.2 |12.75)12.87} 13.00) 13.12] 13.25) 13.37) 13 13 13.88] 14.01
5.3 |12.87|12.99} 13.12] 13.24] 13.37] 13.49) 13.62] 13 14.00] 14.13
5.4 |12.99)13.11) 13.24) 13.36] 13.49) 13.61) 13.74] 13 14.12] 14.25
5.5 |13.11/13.23) 13.36} 13.48] 13.61] 13.73) 13.86] 13 14.24| 14.37
5.6 |13.23)13.35| 13.48) 13.60] 13.73) 13.86) 13.98] 14 14.36] 14.49
5.7 |13.35}13.47| 13.60} 13.72) 13.85) 13.98) 14.11] 14 14.48) 14.61
5.8 |13.47/13.59] 13.72) 13.84] 13.97] 14.10) 14.23] 14 14.61} 14.74
5.9 '13.59113.71' 13.841 13.97] 14.10! 14.22' 14.35! 14 14.73' 14.86

APPENDIX 211

FROM FAT AND LACTOMETER READING
AMERICAN STANDARD

at 60° F.

31.5 | 32.0 | 32.5 33.0 | 33.5 | 34.0 | 34.5 | 35.0 | 35.5 | 36.0] 36.5] Fat
7.87) 8.00} 8.12] 8.25] 8.37) 8.50] 8.62] 8.75) 8.87] 9.00] 9.12
7.99| 8.12) 8.24] 8.37) 8.49] 8.62] 8.74] 8.87] 8.99) 9.12] 9.24
8.11) 8.24) 9.36) 8.49| 8.61] 8.74] 8.86] 8.99] 9.11] 9.24) 9.36
8.23) 8.36) 8.48] 8.61] 8.73] 8.86] 8.98} 9.11] 9.23) 9.36] 9.48
8.35) 8.48) 8.60} 8.73] 8.85) 9.98] 9.10} 9.23) 9.35] 9.48] 9.60
8.47| 8.60) 8.72] 8.85| 8.97] 9.10) 9.22) 9.35] 9.47] 9.60] 9.72
8.59| 8.72) 8.84] 8.97) 9.09} 9.22} 9.34) 9.47} 9.59) 9.72] 9.84
eo 8.84] 8.96) 9.09) 9.21] 9.34} 9.46) 9.59] 9.71] 9.84) 9.96
8.83] 8.96) 9.08) 9.21) 9.33) 9.46] 9.58] 9.71] 9.83] 9.96]10.08
8.95| 9.08} 9.20) 9.33} 9.45) 9.58} 9.70] 9.83] 9.95/10.08|10.20
9.07} 9.20} 9.32} 9.45) 9.57) 9.70) 9.82] 9.95) 10.07|10.20]10.32
9.19} 9.32) 9.44) 9.57) 9.69) 9.82) 9.94] 10.07} 10.19]10.32/10.44
9.31| 9.44) 9.56) 9.69) 9.81) 9.94] 10.06] 10.19] 10.31]10.44/10.56
9.43) 9.56) 9.68) 9.81} 9.93) 10.06] 10.18] 10.31] 10.43/10.56/10.68
9.55| 9.68) 9.80) 9.93} 10.05] 10.18} 10.30] 10.43] 10.55)10.68)10.80
9.67) 9.80' 9.92} 10.05) 10.17} 10.30] 10.42] 10.55) 10.67/10.80/10.92
9.79| 9.92| 10.04} 10.17} 10.29) 10.42] 10.54] 10.67] 10.79]10.92\11.04
9.91) 10. 10.16} 10.29) 10.41] 10.54] 10.66] 10.79) 10.91/11.04]11.16
10.03} 10.16) 10.28] 10.41] 10.53] 10.66} 10.78] 10.91] 11.04]11.17]11.29
10.15} 10.28) 10.40] 10.53] 10.65] 10.78} 10.90} 11.03) 11.16]11.29]11.41
10.27} 10.40) 10.53] 10.66) 10.78] 10.91} 11.03] 11.16] 11.28]11.41]11.53
10.39]} 10.52) 10.65) 10.78} 10.90] 11.03] 11.15] 11.28) 11.40]11.53]11.65
10.51} 10.64] 10.77] 10.90} 11.02] 11.15) 11.27] 11.40] 11.52)11.65)11.77
10.63} 10.76) 10.89] 11.02) 11.14] 11.27) 11.39) 11.52) 11.64]11.77|11.89
10.75] 10.88) 11.01] 11.14] 11.26} 11.39] 11.51] 11.64] 11.76/11.89]12.01
10.87) 11.00} 11.13) 11.26) 11.38] 11.51] 11.63] 11.76} 11.88]12.01/12.13
10.99 11.12 11.25) 11.38) 11.50] 11.63) 11.75] 11.88] 12.00)12.13]12.25
OU. 001 10. 24) 1425371, 11. SO) 11. 62: EL. 26h 42, 87) 12: 12.12)12.25)12:37
11.23] 11.37) 11.49] 11.62| 11.74) 11.87] 11.99] 12.12] 12.24]12.37|12.49

12.47} 12.60] 12.72] 12.85) 12.97|13.10/13.22

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212 APPENDIX

TABLE

FOR sisi hesnasideht G TOTAL SOLIDS FROM |
ACCORDING TO

‘ LACTOMETER READING

wey

[>] ;

a 26.0 | 26.5 | 27.0 | 27.5 | 28.0 | 28.5 | 29.0 | 29.5 | 30.0 | 30.5 | 31.0
6.652) 6.776) 6.900) 7.025) 7.150) 7.274) 7.397) 7.522) 7.647| 7.771) 7.895
6.77 | 6.90 | 7.02 | 7.15 | 7.27 | 7.39 | 7.52 | 7.64 | 7.77 | 7.89 | 8.02
6.89 | 7.02 | 7.14 | 7.26 | 7.39 | 7.51 | 7.64 | 7.76 | 7.89 | 8.01 | 8.14
7.01 | 7.14 | 7.26 | 7.39 | 7.51 | 7.63 | 7.76 | 7.88 | 8.01 | 8.13 | 8.26
7.13 | 7.26 | 7.38 | 7.51 | 7.63 | 7.75 | 7.88 | 8.00 | 8.13 | 8.25 | 8.38
7.25 | 7.38 | 7.50 | 7.63 | 7.75 | 7.87 | 8.00 | 8.12 | 8.25 | 8.37 | 8.50
7.37 | 7.50 | 7.62 | 7.75 | 7.87 | 7.99 | 8.12 | 8.24 | 8.37 | 8.49 | 8.62
7.49 | 7.62 | 7.74 | 7.87 | 7.99 | 8.11 | 8.24 | 8.36 | 8.49 | 8.61 | 8.74
7.61 | 7.74 | 7.86 | 7.99 | 8.11 | 8.23 | 8.36 | 8.48 | 8.61 | 8.73 | 8.86
7.73 | 7.86 | 7.98 | 8.11 | 8.23 | 8.35 | 8.48 | 8.60 | 8.73 | 8.85 | 8.98
7.85 | 7.98 | 8.10 | 8.23 | 8.35 | 8.47 | 8.60 | 8.72 | 8.85 | 8.97 | 9.10
7.97 | 8.10 | 8.22 | 8.35 | 8.47 | 8.59 | 8.72 | 8.84 | 8.97 | 9.09 | 9.22

8.09 | 8.22 | 8.34 | 8.47 | 8.59 | 8.71 | 8.84 | 8.96 | 9.09 | 9.21 | 9.34
8.21 | 8.34 | 8.46 | 8.59 | 8.71 | 8.83 | 8.96 | 9.08 | 9.21 | 9.33 | 9.46
8.33 | 8.46 | 8.58 | 8.71 | 8.83 | 8.95 | 9.08 | 9.20 | 9.33 | 9.45 | 9.58
8.45 | 8.58 | 8.70 | 8.83 | 8.95 | 9.07 | 9.20 | 9.32 | 9.45 | 9.57 | 9.70
8.57 | 8.70 | 8.82 | 8.95 | 9.07 | 9.19 | 9.32 | 9.44 | 9.57 | 9.69 | 9.82
8.69 | 8.82 | 8.94 | 9.07 | 9.19 | 9.31 | 9.44 | 9.56 | 9.69 | 9.81 | 9.94
8.81 | 8.94 | 9.06 | 9.19 | 9.31 | 9.43 | 9.56 | 9.68 | 9.81 | 9.93 |10.06
8.93 | 9.06 | 9.18 | 9.31 | 9.43 | 9.55 | 9.68 | 9.80 | 9.93 /10.05 }10.18
9.05 | 9.18 | 9.30 | 9.43 | 9.55 | 9.67 | 9.80 | 9.92 |10.05 |10.17 [10.30
9.17 | 9.30 | 9.42 | 9.55 | 9.67 | 9.79 | 9.92 |10. 10.17 |10. 10.42
9.29 | 9.42 | 9.54 | 9.67 | 9.79 | 9.91 |10.04 |10.16 {10.29 |10.41 /|10
9.41 | 9.54 | 9.66 | 9.79 | 9.91 [10.03 |10.16 [10.28 |10.41 |10.53 |10.66
9.53 | 9.66 | 9.78 | 9.91 |10.03 |10.15 |10.28 |10.40 |10.53 [10.65 |10.78
9.65 | 9.78 | 9.90 {10.03 |10.15 [10.27 |10.40 |10.52 |10.65 |10.77 |10.90
9.77 | 9. 10.02 |10.15 |10.27 |10.39 |10.52 {10.64 |10.77 |10.89 |11.02
9.89 |10.02 |10.14 |10.27 |10.39 |10.51 |10.64 {10.76 |10.89 |11.01 |11.14

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APPENDIX } 213

LXX

FAT AND LACTOMETER READING
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214 APPENDIX

TaBLE LXXI
TABLE FOR CONVERSION OF CUPROUS OXIDE (Cu.0) AND
COPPER TO LACTOSE
MILLIGRAMS
Cuz0 Cu Lactose Cu20 Cu Lactose Cu20 Cu Lactose
112.6 | 100 71.6 || 157.6 | 140 | 101.3 || 202.7 | 180 | 131.6
113.7 |} 101 72.4 || 158.7 | 141 102.0 |} 203.8 | 181 132.4
114.8 | 102 73.1 || 159.8 | 142 | 102.8 || 204.9 | 182 | 183.1
115.9 | 103 73.8 || 160.9 | 143 | 103.5 |} 206.0 | 183 | 133.9
117.0 | 104 74.6 || 162.0 | 144 | 104.3 || 207.1 | 184 | 134.7
118.2 | 105 75.3 || 163.2 | 145 | 105.1 |} 208.3 | 185 | 135.4
119.3 | 106 76.1 || 164.3 | 146 | 105.8 || 209.4] 186 | 136.2
120.4 | 107 76.8 || 165.5 | 147 | 106.6 || 210.5 | 187 | 137.0
121.5 | 108 77.6 || 166.6 | 148 | 107.3 || 211.6 | 188 | 137.7
122.7 | 109 78.3 || 167.7 | 149 | 108.1 || 212.7 | 189 | 138.5
123.8 | 110 79.0 |} 168.9 | 150 | 108.8 || 213.9 | 190 | 139.3
124.9} 111 79.8 || 170.0 | 151 | 109.6 || 215.0 | 191 | 140.0
126.0 | 112 80.5 || 171.1 | 152 | 110.3 || 216.1 | 192 | 140.8
127,13.) 133 81.3 || 172.2 | 153 | 111.1 || 217.2 | 193 | 141.6
128.2 | 114 82.0 |} 1738.3 | 154 | 111.9 || 218.3 | 194 | 142.3
129.4} 115 82.7 || 174.5 | 155 | 112.6 || 219.5 | 195 | 148.1
130.5 | 116 83.5 || 175.6 | 156 | 1138.4 || 220.6 | 196 | 1438.9
1SE. 7.) -447 84.2 || 176.7 | 157 | 114.1 |} 221.8 | 197 | 144.6
132.8 | 118 85.0 || 177.8 | 158 | 114.9 || 222.9 | 198 | 145.4
133.9 | 119 85.7 || 178.9 | 159 | 115.6 || 224.0] 199 | 146.2
135.1 | 120 86.4 || 180.1 | 160 | 116.4 |} 225.2 | 200 | 146.9
136.2 | 121 87.2 || 181.2 | 161 | 117.1 || 226.3 | 201 | 147.7
137.3 | 122 87.9 || 182.3 | 162 | 117.9 || 227.4 | 202 | 148.5
138.4 | 123 88.7 || 183.4 | 163 | 118.6 || 228.5 | 203 | 149.2
139.5 | 124 89.4 || 184.5 | 164 | 119.4 || 229.6 | 204 | 150.0
140.7 | 125 90.1 || 185.7 | 165 | 120.2 || 230.7 | 205 | 150.7
141.8 | 126 90.9 || 186.8 | 166 | 120.9 || 231.9 | 206 | 151.5
143.0 | 127 91.6 |} 188.0 | 167 | 121.7 || 233.0 | 207 | 152.2
144.1 | 128 92.4 || 189.1 | 168 | 122.4 || 234.1 | 208 | 153.0
145.2 | 129 93.1 || 190.2 | 169 | 128.2 || 235.2 | 209 | 153.7
146.4 | 130 93.8 || 191.4 | 170 | 123.9 || 236.4 | 210 | 154.5
147.5 | 131 94.6 |] 192.5 | 171 | 124.7 || 287.5 | 211 | 155.2
148.6 | 132 95.3 || 193.6 | 172 | 125.5 || 238.6 | 212 | 156.0
149.7 | 133 96.1 || 194.7 | 173 | 126.2 || 239.7 | 213 | 156.7
150.8 | 134 96.9 |} 195.8 | 174 | 127.0 || 240.8 | 214 | 157.5
152.9 | 135 97.6 || 197.0 | 175 | 127.8 || 242.0] 215 | 158.2
153.1.} 136 98.3 || 198.1 | 176 | 128.5 || 2438.1 | 216 | 159.0
154.2 | 137 99.1 || 199.3 | 177 | 129.3 || 244.3 | 217 | 159.7
155.3 | 138 99.8 || 200.4 |} 178 | 180.1 || 245.4 | 218 | 160.4
156.4 | 1389 | 100.5 || 201.5 | 179 | 180.8 || 246.5 | 219 | 161.2

APPENDIX 215

TaBLE LX XI—Continued

TABLE FOR CONVERSION OF CUPROUS OXIDE (Cu,0) AND
COPPER TO LACTOSE >

MILLIGRAMS

Cu20 Cu Lactose Cu20 Cu Lactose Cu20 Cu Lactose

247.7 | 220 | 161.9 || 292.7 | 260 | 192.5 || 337.8 | 300 | 224.4
248.8 | 221 | 162.7 |} 293.8 | 261 | 193.3 || 338.9 | 301 | 225.2
249.9 | 222 | 163.4 || 294.9 | 262 | 194.1 || 340.0 | 302 | 225.9
251.0 | 223 | 164.2 || 296.0 | 263 | 194.9 || 341.1 | 303 | 226.7
252.1 | 224 | 164.9 || 297.1 | 264 | 195.7 |} 342.2 | 304 | 227.5
253.3 | 225 | 165.7 || 298.3 | 265 | 196.4 |} 343.4 | 305 | 228.3
254.4 | 226 | 166.4 || 299.4 | 266 | 197.2 || 344.5 | 306 | 229.1
255.5 | 227 | 167.2 || 300.5 | 267 | 198.0 |}. 345.6 | 307 | 229.8
228 | 167.9 || 301.6 | 268 | 198.8 || 346.7 | 308 | 230.6
229 | 168.6 || 302.7 | 269. | 199.5 || 347.8 | 309 | 231.4

230 | 169.4 |} 303.9 | 270 | 200.3 |} 349.0 | 310 | 232.2
231 | 170.1 |} 305.0 | 271 | 201.1 || 350.1 | 311 | 232.9
232 | 170.9 |} 306.2 | 272 | 201.9 |} 351.2 | 312 | 233.7
233 | 171.6 |} 307.3 | 273 | 202.7 || 352.3 | 313 | 234.5
234 | 172.4 || 308.4 | 274 | 203.5 || 353.4 | 314 | 235.3
235 | 173.1 |} 309.6 | 275 | 204.3 || 354.6 | 315 | 236.1
236 | 173.9 || 310.7 | 276 | 205.1 || 355.7 | 316 | 236.8
237 | 174.6 || 311.8 | 277 | 205.9 || 356.8 | 317 | 237.6
238 | 175.4 || 313.0 | 278 | 206.7 || 357.9 | 318 | 238.4
239 | 176.2 || 314.1 | 279 | 207.5 || 359.0 | 319 | 239.2

240 | 176.9 || 315.3 | 280 | 208.3 |} 360.2 | 320 | 240.0
241 | 177.7 || 316.4 | 281 | 209.1 |) 361.3 | 321 | 240.7
242 | 178.5 || 317.5 | 282 | 209.9 || 362.4 | 322 | 241.5
243 | 179.3 || 318.6 | 283 | 210.7 || 363.5 | 323 | 242.3
‘ : 211.5 || 364.6 | 324 | 243.1
245 | 180.8 || 320.9 | 285 | 212.3 || 365.8 | 325 | 243.9
246 | 181.6 || 322.0 | 286 | 213.1 || 366.9 | 326 | 244.6
247 | 182.4 || 323.1 | 287 | 213.9 || 368.0 | 327 | 245.4
248 | 183.2 || 324.2 | 288 | 214.7 || 369.1 | 328 | 246.2
249 | 184.0 || 325.3 | 289 | 215.5 || 370.2 | 329 | 247.0

250 | 184.4 || 326.5 | 290 | 216.3 || 371.4 | 330 | 247.7 .
251 | 185.5 || 327.6 | 291 | 217.1 || 372.5 | 331 | 248.5
252 | 186.3 || 328.7 | 292 | 217.9 || 373.6 | 332 | 249.2
253 | 187.1 || 329.8 | 293 | 218.7 || 374.7) 333 | 250.0
254 | 187.9 || 330.9 | 294 | 219.5 || 375.8 | 334 | 250.8
255 | 188.7 |} 332.1 | 295 | 220.3 || 377.0 | 335 | 251.6
256 | 189.4 || 333.2 | 296 | 221.1 || 378.1 | 336 | 252.5
257 | 190.2 || 334.4 | 297 | 221.9 || 379.3 | 337 | 253.3
258 | 191.0 || 335.5 | 298 | 222.7 || 380.4 | 338 | 254.1
259 | 191.8 || 336.7 | 299 | 223.5 || 381.5 | 339 | 254.9

SEUUNNNONY USSINNSSNY BUBBENNENY HE
TRWNEHOTDONAM WNHHOCHRAURWN COBRUWNHODSD US
s
2
2

216 APPENDIX

TABLE LX XI—Continued

TABLE FOR CONVERSION OF CUPROUS OXIDE (Cu,0) AND
COPPER TO LACTOSE

MILLIGRAMS

Cuz0 Cu Lactose Cu20 Cu Lactose

382.7 | 340 | 255.7 || 405.3 | 360 | 272.1
383.8 | 341 | 256.5 || 406.4 | 361 | 272.9
385.0 | 342 | 257.4 || 407.5 | 362 | 273.7
386.1 | 343 | 258.2 || 408.6 | 363 | 274.5
387.2 | 344 | 259.0 || 409.7 | 364 | 275.3
388.4 | 345 | 259.8 || 410.9 | 365 | 276.2
389.5 | 346 | 260.6 || 412.0 | 366 | 277.1
390.6 | 347 | 261.4 || 413.1 | 367 | 277.9
391.7 | 348 | 262.3 || 414.2 | 368 | 278.8

S BSESESESES| p

1

2

3

5

6

:
392.8 | 349 | 263.1 || 415.3 | 369 | 279.6 0} 389 | 296.8
394.0 | 350 | 263.9 || 416.5 | 370 | 280.5 2| 390 | 297.7
395.1 | 351 | 264.7 || 417.6 | 371 | 281.4 || 440.3 | 391 | 298.5
396.2 | 352 | 265.5 || 418.8 | 372 | 282.2 || 441.4 | 392 | 299.4
397.3 | 353 | 266.3 || 419.9 | 373 | 283.1 || 442.5 | 393 | 300.3
398.4 | 354 | 267.2 || 421.0 | 374 | 283.9 || 443.6 | 394 | 301.1
399.6 | 355 | 268.0 || 422.2 | 375 | 284.8 || 444.8 | 395 | 302.0
400.7 | 356 | 268.8 || 423.3 | 376 | 285.7 || 445.9 | 396 | 302.8
401.9 | 357 | 269.6 || 424.5 | 377 | 286.5 || 447.0 | 397 | 303.7
403.0 | 358 | 270.4 || 425.6 | 378 | 287.4 || 448.1 | 398 | 304.6
404.1 | 359 | 271.2 || 426.7 | 379 | 288.2 || 449.2} 399 | 305.4
450.4 | 400 | 306.3

SUBJECT INDEX

A

Abnormal milk, 54
Acidity, 75

and bacteria, 132

of media, 119, 121
Acid producing organisms, 191
Aciduric bacilli, 195
Adulteration of milk, 55

calculation of, 58
Agar media, 117, 120

whey, 119

lactose, 119

lactose bile salt, 143

eesculin, 143
- easein, 194, 208

Aggressins, 27
Air, bacteria in, 100
Albumin, 74

effect of heat on, 189
Aldehyde value, 75
Alkali-forming organisms, 194
Ambocepters, 26
Amylase, 22

detection and estimation, 91
Aniline orange, 86
Annatto, 86
Antibodies, 26
“Appeal to the cow” test, 59
Ash, 50, 76

estimation of, 69

B
B. abortus, 190
characteristics of, 192
B. bulgaricus, 196

B. butyricus, 147

B. coli, 136
appearance of colonies, 144
calculation of results, 142
effect of atmospheric tempera-

ture, 139

enrichment methods, 140
estimation of, 140
grain types, 145
liquid media for, 140
plate methods of estimating, 143
rate of development, 107
type, classification of, 145

B. diphtheria, 156
detection of, 157

B. enteritidis sporogenes, 146

B. lactis acidi, 109

B. lactis aerogenes, 109, 119

B. paratyphosus, 161

B. tuberculosis, 135
detection of, 164
inoculation method, 165
pseudo, 168
types, 169

B. typhosus, 159
isolation of, 160

Bacteria in milk, 93
acid-producing, 106
alkali-producing, 106
development of, 102
effect of brushing cows on, 98
effect of low temperatures on, 111
enumeration of, 113

Breed’s method, 129
by acidity, 132

217

218

Bacteria in milk, enumeration, of,
direct methods, 126
methylene blue test, 130
plate methods, 116
intra-mammary, 93
Bacterial counts, accuracy of, 117,
121
effect of sugars on, 118
Benzoic acid, 84
Borates, 83
Boric acid, 83
Breed of cattle, 37
effect on fat constants, 38
effect on milk composition, 47

C
~ Cane sugar, 88
Caramel, 86
Caseinogen, 7
composition of, 8
estimation of, 74
hydrolysis of, 11
meta, 7
para, 14
properties of, 10
reaction with rennin, 13, 16
Catalase, 23
estimation of, 91
Cells, 171
blood, 173
epithelial, 172
estimation of, 174
foam, 173
number in milk, 178
Certified milk, 138
Colonies, counting of, 125
Colostrum, 52
Colouring matter, 85
Complement, 26
Composition of milk, 34
limits of, 37
maximum variations, 35
variations, 37

SUBJECT INDEX

Condensed milk, 88
Conductivity, 31
Containers, Bacteria in milk, 100
Coolers, 100
Counting lens, 126
Cream, 87

line in pasteurised milk, 185
Curd test, 197

bacterial flora, 200

types, 198

D

Death points in milk:

B. diphtheria, 187

B. tuberculosis, 187

B. typhosus, 187
‘Debris, 161

estimation of, 180
Diphtheroid bacilli, 158
Dirt, 161

estimation of, 180

significance of, 183

testers, 182
Disease, effect on composition, 54

E

Enrichment methods for B. coli, 140
Enzymes, 21

effect of heat on, 186

estimation of, 88
Epithelial cells, 172
Erythrocytes, 173
Excremental organisms, 135

F
Fat, constants of, 2
estimation of, 66
globules, 1, 44, 52
nature of, 1
Fermentation test, 197
Food, effect on composition of
milk, 39
bacteria in, 99

ee ae a es

SUBJECT INDEX

Fore milk, 50

bacteria in, 96
Formaldehyde, 81
Freezing point of milk, 30

G

Galactase, 22

estimation of, 92
Gaertner group, 161
Gases in milk, 21
Gelatine, detection of, 87

media, 117, 120
Germicidal action, 102

H
Hemolysins, 27
Hemolytic streptococci, 151
Hoffman's bacillus, 158
Homogenised milk, 30
Hypochlorites, 85
Hydrogen ion concentration, 121
Hydrogen peroxide, 85

I
Immune bodies, 24
Incubation period, 117
Inert organisms, 194
Intra-mammary bacterial pollution,
93 ;

L
Lactalbumin, 17, 74
properties of, 18
Lactation stage, effect of, 45, 49
Lacto globulin, 18
Lactokinase, 22
Lactometer table, 209
Lactose, bile, 140
broth, 140
estimation of, 71
origin of, 3
properties, 5
specific rotation, 3
‘table, 214

219

Lecithin, 20
Leucocytes, 173

. Lipase, 22

Litter, bacteria in, 99

M
Media, acidity of, 119
eesculin, 143, 207
brilliant green, 160
casein, 208
. Drigalski and Conradi’s, 143
egg, 169, 208 |
Endo’s, 143
for B. coli, 141
rebipelagar, 143, 207
standard, 120, 122
Methyl red reaction, 145
Milk coolers, effect of, 100
Milking intervals, effect of, 42
Milk serum, 78 —

‘Mineral constituents, 76

Morgan’s bacillus No. 1. 161

O
Opsonins, 27

: Pp
Pails, bacteria in, 100
Paracasein, 14
Paratyphoid group, 161
Pasteurised milk, 105

cream line in, 185

enzymes in, 186

Ottawa results, 205
Peptonising organisms, 194
Peroxidases, 23

effect of heat, 188

estimation of, 91
Physical characteristics of milk, 28
Plating technique, 123, 125
Ponder’s stain, 208
Preservatives, 80
Precipitins, 27

220 SUBJECT INDEX

Proteids, 6
estimation of, 73
mucoid, 18
whey, 14

R
Recknagel phenomenon, 29
Reductases, 24
effect of heat on, 188
estimation of, 89
Refractive index, 32, 79
limits for, 57
Rennin, effect of heat on, 189
Results, calculation of, 142
recording, 206

S
Saccharate of lime, 87
Salicylic acid, 84
Salolase, 22
Salts, 19
Samples, collection of, 202
Schardinger’s reagent, 89
Seasonal variation in milk, 40
Septic sore throat, 150
Serum, 19, 57, 78
Skim milk, 88
Solids-not-fat, 44
Specific gravity, 28
determination of, 69
Specific heat, 32
Staphylococcus pyogenes, 150
Standards for milk, 59
tables, 63
Starch, detection of, 87

Streptococci:
biochemical characteristics, 158
fecal, 147
hemolytic, 151
pathogenic, 148, 153
Streptococcus lacticus, 109, 119, 152,
153
mastitidis, 150
pyogenes, 152
Strippings, 50
bacteria in, 96
Surface tension, 32

sy
Toisson’s solution, 207
Total solids, estimation of, 69
tables for calculating, 210-213
Toxicity of milk, 114
of pasteurised milk, 116

Toxins, 27
U
Udder, bacteria in, 95
influence of wiping, washing, etc.,
98
V
Viscogen, 87
Viscosity, 60
Voges and Proskauer reaction, 136,
145
Volume change with temperature,
29, 30
Z

Ziehl-Neelson method for tubercle
bacilli, 164

NAME INDEX

A Burow, 8
Aitkens, 30 Burr, 32
Alexander, 162
Anderson, 167 C
Andrewes, 150 Cameron, 12
Arthus, 28 Capps, 152
. Ayers, 106, 194 Chamot, 140

Chappelean, 198

B Chittenden, 8
Babcock, 22, 92, 181 Clark, 121, 145
Backhaus, 97, 99, 100 Cook, 37
Balley, 93 Conn, 108, 117, 121, 124
Bang, 190 Corbett, 191
Barthol, 130
Batchelder, 94 D
Béchamp, 17, 22 Davis, 152
Beger, 39 Dean, 156
Bellei, 91 Delépine, 115, 164, 166, 168, 180,
Benzynski, 55 181
Berberich, 32 Désmouliers, 22
Besredka, 28 Doane, 171, 174
Block, 166 Dodd, 167

- Blyth, 21 Doll, 39

Borden, 121 Duclaux, 15
Boseley, 71, 81 Dugelli, 198
Bosworth, 7, 8, 11, 15, 20
Boussingault, 50 E
Bowhill, 156 Eastwood, 167, 168
Breed, 128, 171, 178, 179 Kckles, 38, 42, 45, 50
Brew, 129 Ellenberger, 8
Briot, 15 Engling, 53
Broadhurst, 155 Ernst, 171, 172
Browning, 160 Ksten, 108
Buckley, 174 Evans, 190
Bunge, 34 Eyre, 157

221

222

F
Fingerling, 39
Fleishmann, 29, 32
Fred, 130, 198
Freudenreich von, 22, 94

G
Geake, 8, 15
Gerber, 181, 197
Gillet, 22
Glenn, 119
Good, 191
Gooderich, 127
Griffiths, 167, 168

H

Hall, 93
Hammer, 111
Hammerstein, 8, 14
Hancke, 39
Harden, 15
Harrison, 98, 100
Hastings, 111
Hehner, 81
Heidemann, 119, 152
Heintz, 12
Hempel, 8
Henderson, 93, 95
Hewarden, 16
Hewlett, 18, 171, 177, 179
Heyman, 196
Hills, 37
Hoffmann, 174, 178
Holder, 150
Holt, 162
Houston, 181
Hurst, 12

| J
Jackoby, 17
Jackson, 31, 152, 160
Jensen, 22, 54, 130, 197
Joannovico, 167
Johnson, 106, 194

NAME INDEX

K
Kapsammer, 167
Kastle, 22, 23

- Kaufman, 3
Klein, 156, 158, 198

Koning, 22, 35
Koster, 14
Krumwiede, 151

L
Lacqueur, 8
Lagne, 3
Landtsheer, 22
Lederle, 121
Ledingham, 161
Lehmann, 8
Leonard, 81
Levine, 145
Lewis, 162
Lindet, 18
Liwschiz, 15
Lobeck, 91
Loevenhart, 15
Loew, 23

Lohnis, 198

Long, 9, 10

Lubs, 145
Lythgoe, 32, 35, 86

; M
Macallum, 15
Malméjac, 40
Marfan, 22
Marshall, 156
Mathaiopoulos, 9
McConkey, 94, 136
McCrady, 142, 147
MeFadyean, 190
Melia, 160
Merklen, 22
Michaelis, 171
Miessner, 28
Miller, 75, 130, 171

fi
NAME INDEX

Park, 94, 102, 162
Pennington, 105, 111
Peter, 91, 198
Porch, 23

Prescott, 178

R

Race, 194
Rahe, 196
Raudnitz, 17, 23
Revenel, 111
Revis, 171, 177, 181
Richmond, H. D., 6, 8, 29, 30, 37,

44, 60, 71, 75, 81, 87
Richmond, 8. O., 29
Robertson, 9
Rogers, 137
Romer, 89
Rosam, 128
Rosenau, M. J., 102
Ross, 162
Rothera, 31
Rothenfusser, 91

220
Monier-Williams, 83 Rueduger, 154
Morgan, 161 Rullman, 23
Morgen, 39 Rupp, 189
-Morgenrath, 17 Russell, 22, 92, 174, 178
Moro, 22
Mule, 22 Ss
Muller, 152 Sackur, 8
Salge, 196
ON Savage, 101, 148, 147, 150, 158, 171,
Nobécourt, 22 176, 178, 179
North, 121, 185 Schaffer, 55
Schardinger, 89
O Schern, 89
O’Brien, 162 Schmidt, 14
Olsen, 50 Schnorf, 54
Orr, 98, 100, 137, 162 Scholberg, 162
Otto, 27 Schrewsbury, 82
Schroeder, 182
P Schroeter, 198
Painter, 8 Schryver, 7, 15

Sebelein, 18
Sedgwick, 94
Seligman, 22

- Shaw, 38, 42, 45, 50

Sherwood, 140

Sieglin, 39

Skar, 128

Slack, 126, 174

Slyke, L. L. Van, 7, 8,11, 15, 20
Slyke, D. D. Van, 10
Smith, Graham, 162
Soldner, 8 —

Sothurst, 53

Spolverini, 22

Sprague, 178

Stewart, 126, 174

St. John, 105

Stidger, 179

Stribald, 190

Stocking, 96, 97, 99, 105

Stockman, 190

Stohman, 2
Stokes, 87, 171

224

Stone, 178
Storch, 18
Strewe, 19

Tange, 8

Taylor, 30

Thoni, 94

Thomson, 83
Thornton, 160
Timpe, 45

Todd, 156

Tonney, 160, 161, 182

Vv
Valentine, 151
Velde der, 22

NAME INDEX

Vieth, 37
Villar, 171, 177

; W
Wallis, 162
Walter, 197
Ward, 93, 95
Wegefarth, 171
Weigner, 29, 30 —
Wender, 22
Wilkinson, 91
Willem, 22
Winkler, 171
Winslow, 155

Zaitschik, 22
Zielstorff, 39

rs