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Se 
Senet 


PRED 


New York 
State College of Agriculture 


At QGornell University 
Sthaca, N.Y. 


Library 


tant 
i 7 


06 
RIAG 
Ar 


THE EXAMINATION OF MILK 


FOR 


PUBLIC HEALTH PURPOSES 


JOSEPH RACH, F.I.C. 


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


FIRST EDITION 


NEW YORK 
JOHN WILEY & SONS, Ine. 


Lonpon: CHAPMAN & HALL, LiwitTep 
1918 


@ 
EY Gos 
fess 


Cop. | 


Copyright, 1917 
BY 
‘JOSEPH RACE 


ae 187 


PRESS OF 
BRAUNWORTH & CO, 
QOOK MANUFACTURERS 
BROOKLYN. N. Y. 


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- 
cal 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. 
iti 


Cornell University 


Library 


The original of this book is in 
the Cornell University Library. 


There are no known copyright restrictions in 
the United States on the use of the text. 


http://www.archive.org/details/cu31924002934267 


CONTENTS 


CHAPTER PAGE 
I. ConSTITUBNTS OF MILK........... 0. ccc cece ete eens 1 
Fat. Lactose. Proteids. Salts. Gases. Enzymes. Immune 
bodies. Physical constants. 


II. Normat ComposiTIon OF MILK.............00 cee eee eens 34 
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. 


TIT. Caemican EXAMINATION. 0.0.0.0... c cece eee eee ee nee 66 
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. 


IV,. Bacterra, in) Mites e i secen cee eens Pes vee ia ae eeeeee oe 93 
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. Tas Enumeration or Bacterta IN MILK.................. 113 
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. 


VI. EXcreMENTAL ORGANISMS........0.00. 0.00 c cece eee eee eees 135 
B. coli. Occurrence of B. coliin milk. Estimation of B. coli. 
Enrichment methods. Plate methods. Classification of 
type. B. enteritidis sporogenes. Streptococci. 

VII. ParHoGEntc ORGANISMS... . 0.2.00. 0.0 c ccc e eee e eee 150 
Streptococci. Septicsore throat. B.diphtheriz. Diphther- 
oid bacilli. B. typhosus. Gaertner group. Morgan’s Ba- 
cillus No. I. B. tuberculosis. Pseudo tuberculosis. 

Vv 


vi CONTENTS 


CHAPTER PAGE 
VITI. Cetus, Dirt anD DEBRIS........-00 00sec cece ee een eee eeas 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. 


TX.. MISCELLANEOUS). .oc5 occce ee ens e cane se rne eae ia a owas 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. 
PAPE ND 156 sgt 2.2 aad hu iser soy cubgscigah oe Sieg enaive ds Aieibaleeebnd Soeteandnde a nen HRS a Res 207 
Composition of special media. Useful tables. 
Name INDEX............... sss Scab cas teed i Ploaee tld czas 217 


EXAMINATION OF MILK 
FOR PUBLIC HEALTH PURPOSES 


CHAPTER I 
CONSTITUENTS OF MILK 


Mix 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 CaHon+1COOH 
and CaHen—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 very 
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 19108 to 60108 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 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 ao ee ee 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.............. 31-385 


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 
cryoscopic 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 3 


under normal conditions is lactose or milk sugar. Lactose is a 
disaccharide of the empirical formula Ci2H22011 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 lactose exist and are known as the alpha and 
beta varieties. When lactose is obtained by crystallisation 
from water, the alpha modification, which crystallises in the 
rhombic form, is formed: this modification exhibits the phe- 
nomenon of multirotation, Le., 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 [a]p=+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 [e]p+32.7 and shows the same birotation 

initial rotation 

18 final rotation 

This shows that the reaction is tend=nelerulae in character. 
1 


The density of the alpha variety is 1.545 722 and that of a 


solution. containing 10 grams per 100 c.cms., 1.0391 Pe. 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 up2° of 


ratio, 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 cerevicize) and is not affected by the 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. 

Ci2H22011-+H20 = CeH1206+CeHi206. 


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 
non HOH 
no-}_H HO_6_# 
HOH HO_b—H 
H_¢_on H_¢_on 
buon buon 
Dextrose Galactose 


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


Dextrose. Galactose. 
Equilibrium form....................... 52.7 80.3 . 
Alpha modification..................... 105. 120. 


Birotation ratio..................0000.. 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 (C3H6O3) 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 


i 
a 
OH 


and exists, therefore, in three different forms, viz., dextro, 
levo, and racemic or inactive lactic acids. The dextro and 
levo 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 By and the refractive 
index yg20° 1.4469. On evaporation of aqueous solutions of 
lactic acid dehydrolactic acid CeHi00s is produced, and, ulti- 
mately, at higher temperatures, lactide CsHsO4 isformed. 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 
(ZnCegHi004-3H20) 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.......  .---- approximately 2.0-3.0 
Lactalbumin......  ..... 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. lLactoglobulin 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. 


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 tasteless substance which is practically insoluble 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 [ 
Cc |H| 0 N i) P 
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 10.80 
Lacqueur and Sackur (1903)....... sdlewiees orshagel ecemiets 15.45]0.76 |0.77 
Burow (1905). 2.5 vezdesauidacs ders de 5282/7 .09]22 .92/15.64/0.72 |0.81 
Tangl (1908)..................0005. 52.69/6.81/23.14/15.65/0.83 |0.88 
Van Slyke and Bosworth (1913) mean.|53.17/7.09|22.48]15.67|0.77 |0.82 
Geake: (1918) «a... cecscee sea ecsvere; acs neces 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 Cig2HessN4i8POse, and in 
support of this he quotes experiments * in which he found that 


N : : ; 
700 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 700 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 NHaz, 0.26 per cent Na, and 0.44 per cent K. 

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


N. , 1 gram Casei 
lec. jo Na0H equals sue EEE Se 


equals 
4 N 
Lacqueur and Sackur...... 0.1138 gr. caseinogen | 8.81 e.c. i0N aOH 
Mathaiopoulos............ 0.11315 8.84 
ene secret ened 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 
GRAMB 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 Osborne? 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 
m 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 - 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, tr¥psin, or dilute 
acids, undergoes proteoclastic digestion with the formation of 
caseinogen 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 IT. 

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. 
Cy62Hes5sNa1SPOs2-Ca-Na3(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- 
chloric acid or sulphuric 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 1° 
have pointed out that deductions based on the acidity of milk 


12 CONSTITUENTS OF MILK 
Tase II 
CASEINOGEN HYDROLYSIS PRODUCTS 
Cow's MILs. Goat’s Mix. | Human Mix. 
Abderhalden, Fischer, 
Products of Hydrolysis. ‘ Osborne and Greed, ao daa ae al 
psec page hres Schittenhelm.) | Schittenhelm.) 
Glycine.............. 0 0 
Alanine.............. 0.90 1.50 
Valine............... 1.00 
Leucine.............. 10.50 7.40 
Phenylalanine......... 3.20 2.75 
Tyrosine............. 4.50 4.95 4.71 
Serine@y e543 hse Sais ee 0.45 
Cystine.............. 0.06 
Proline............... 6.70 4.60 
Oxyproline........... 1.50 
Aspartic acid......... 1.20 1.20 
Glutamic acid......... 11.00 12.00 
Tryptophane.......... 1.50 
Arganine............. 4.84 
Lysine............... 5.80 
Histidine............. 2.59 
Diaminotrioxy- 
dodecanic acid...... 0.75 
Aminovaleric acid..... 7.20 
Ammonia............. 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) CaHPOs +2H20 =Ca(OH)2+H3PO,. 
(2) 2CaHPOs+Ca(OH)2=Ca3P20g +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, Caz) 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 }3 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 
“Jabferment ” 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 III. 


Tasie ITI 
Paracasein. Whey Proteid. 
Carbone: ciai2s cetescaveed ned bein vad 52.88 59.33 
Hy dropenisic 2 iciaciad sine sea sean 7.00 : 7.00 
Nitrogens sscc33 gaa ari ebhinea aed aes 15.84 13.25 
Phosphorous (Richmond)............ 0.99 , 


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


Paracasein.................. Cy40H222N36PO44. 
Whey proteid................ Ceo2H37Ns5Oi10. 


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!8 have recently shown that 
if caseinogen solutions are treated with sufficient rennin 
(1: 1000) no addition of calcium salts is required: Schryver 2 
found that clot formation could be obtained in the entire absence 
of calcium ions. 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,2° Geake;?! and 
Harden and Macallum.!8 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?% 
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 


Tasie IV 
T K K 
Temperature. Time in Seconds. Observed oe 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 25 
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, 
isla]p = — 67.5, but Sebelein obtained values varying from 
—36.4to —38.0. Lindet 26 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 lactoglobulin 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 8 casein of 
Strewe who separated it from @ 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 cf milk contains 
various salts in solution. The average percentage of ash in 
milk is about 0.75 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. 

TAMG 8 ot hk a ddes BE AUS nd RN Ne AAO, ted rho aes 20.27 
Maenesiaiy sc asccceca tonic ean an aes Soe ee tes 2.80 
PO GASH G83 2 a. che recs eee oaks vind Mianlonn ated a audi eames audi 28.71 
DOGS Sa eee en ee ae a eS ee ae 6.67 
Phosphoric acid. .............-2002 cee ee eee eee 29.33 
Chlorine........ Riv Baa eb aare stele ole, Goh ad conn ue sche 14.00 
Carbon dioxide... 2.0.0... 0.0. c cece eee ee eens 0.97 
Sulphuricsacidss ctcvtes sera hess meeaieeee ees Trace 
Ferric oxide...............- Fi hogy oedonitnc ey apes tives mates 0.40 
108.15 

Less:oxygen' = Cl w.0¢ occ st ees pa hee cate a nee 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;P208 satis pctlbe, avgartelieeta vw aleatlta Arey tev enee seein 0.0625 
P.O; as R;HPO, Bisgk OER iain eri Ra SE tae AE a ES Be Ce TAD a Se 0.0770 
P.O; as RH2POQ,......- SY eath teense neces seas ae 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. 


TaBLeE VI 
COMPOSITION OF MILK AND MILK SERUM 
nese . Percentage of 
eg pet Milk Consatiteati 
in Serum. 

UBER. pcisaxect nai eto onianahow-tuaeteneenes 5.75 5.75 100.0 
CaseiO BED eines capone vadivicnassa icine 3.07 0.00 Nil 
AlbUM IMs ciscanan opus seo mcte eres 0.506 0.188 37.1 
Nitrogen in other compounds...... 0.049 0.049 100.0 
Cltticacides oe eerie ee ee 0.237 0.237 100.0 
Phosphorus (organic and inorganic)}| 0.125 0.067 53.6 * 
Phosphorus (organic)............ 0.087 0.056 64.4 
CBI GIIIAS cscs cecsusicenacantierdadsenedette anna 0.144 0.048 33.3 
Magnesium..................... 0.013 0.007 53.8 
PPOUASSIUI coin soscenncvive: anes avd emetic 0.120 0.124 100.0 
SOdUM yas casera tween mews 0.055 0.057 100.0 
Chlorine isis nejnsea’seomauie’s ayadinre ee me 0.076 0.081 100.0 
AS iisntus cs heyie ride epartee sees rae 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...................... 3.20 

Di-calcium phosphate (CaHPO,)................ .... 0.175 
Calcium chloride........0..0.0.0.. 000 ccc cece cece eens 0.119 
Mono-magnesium phosphate (MgH4P20s)............. 0.103 
Sodium citrate (NagsCeHsO7).. 2.200000 0 0. cece eee eee 0.222 
Potassium citrate (KsCeHsO7)... 2.0.20... 0. eee eee 0.052 
Di-potassium phosphate (KeHPOQ,)................0.. 0.230 


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


Lecithin. CssHg909NP also exists in milk in minute quan- 
tities. 


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. 


TaBLeE VII 
COMPOSITION OF GASES IN MILK 


Milk after Standing 


Fresh Milk. Two Hours. 


Cubic centimeters per 1000 c.cms. of milk 


Carbon dioxide............. 0.06 60.47 
OXY GON cs og seep ec kee aia 19.13 9.30 
Nitrogen...) ses cs ee gene: 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 composition 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 189775. They found that fresh centrifuge 
slime showed proteolytic properties even when all bacterial 
activity was checked by the presence of antiseptics. Wender 29 
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+0O. 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+02. 


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 haptophoric 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 receptor (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 arnboceptors of the 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 


26 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; 4 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 hemolysins, 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 smaller density, rise to the 
surface and form a layer of cream which is distinctly yellowish 
in tint, the residual 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 (water ue €- =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 S. 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 


TaBLe VIII 
EFFECT OF TEMPERATURE ON VOLUME 


; i Temperature i 

ee eee Volume: ene Gaienrigiy Volume. 
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.24 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 depefident 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 propor- 
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 X0.0665) 
percentage solids-not-fat = 0.177 . 


and that the viscosity temperature coefficient was 


="7+-0.00723t—0.000156#. 


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 


PHYSICAL 31 


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 
Koeppe (1898)................502 chert K at 25° C.=0.00430-0.00560 
Lehnert: (1897) 9.00% ssciatacsahaahosd oe es aces at 0.00487-0.00551 
Schnorf (1905)..............0...0000000- 0.00485 
Benaghi (1910)...............-...0005- 0.004940 .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, 
tivity, K | Per Cent.| Freezing- | Per Cent. } Per Cent, 
point. A 


Quarter. 


o 


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 (up20°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,4° 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. 


NOoar OD 


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, 38. 

. Hammerstein. Maly’s Jahresb. 1872, 1118, ibid. 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. 
.'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, 138. 

. 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 


Tum average composition of cows’ milk as compared with 
the milk of various other mammals is shown in Table No. X. 
(Bunge 1). 


TABLE X 
COMPOSITION OF MAMMALS’ MILK 

Fat. Caseinogen.| Albumin. | Lactose. Ash. 
Human (1)........ SUT | wesw eee! obits 5.9 0.2 
Human (2)........ 3.8 1.2 0.5 6.0 0.2 
Human (3)........ 3.3 Siemicaleniineac. | i Orb 0.3 
DORs: a2 ohae ner oes 12.5 5.2 1.9 3.5 1.3 
Caibiicord caikeon. econ 3.3 3.1 6.4 4.9 0.6 
Rabbit... ......... O85: ||) 2o-s40c58 |Psmcearse 2.0 2.6 
Guinea pig........ AEB” SP cotcemani Il weeaeees 1.3 0.6 
SowWese:svaleees nee GOL aestaratean Ih Dahawa trees 3.8 1.1 
Elephant.......... TOG. | esses || a ekernss 8.8 0.7 
OY S6 «0. 2 cites es aes 1.2 1.2 0.8 5.7 0.4 
AGB! isiccldue alsetthcewr aoe 1.6 0.7 1.6 6.0 0.5 
COW ices Saiearsien 3.7 3.0. 0.9 4.9 0.7% 
Gostnscsccacans 4.8 3.2 1.1 4.5 0.8 
Sheep....:........ 6.9 5.0 1.6 4.5 0.9 
Reindeer.......... 17.1 8.4 2.0 2.8 1.5 
Camel............ ek” |Illl » sovees Gee || eee 5.6 1.8 
Llama............ 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.{C28er Bibus|. ae. Ash. 
nogen.| min. | tose. 


280,000 analyses, Aylesbury Dairy 


Co., London, Richmond......... 87.35 |3.74| 3.0 | 0.4 | 4.70/0.75 
5552 analyses in 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 8.64) 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. 
Maximum..........-....+--+5+ 14.67 13.76 
Minimum. ¢ 2. sis:4c eecare nucle 1.04 4.90 


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


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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) 
Torta Souips. Fat. Soxips Not-rat. 
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 
JeTSCY i. nce viwes 14.89]19.9 |11.0 | 5.66] 9.8 | 2.0 | 9.23/10.4 | 8.1 
IKRORryi cs sleeecns 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 
Sussex.......... 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 
Welsh........... 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. rot al Fat. Lactose. | Proteid. 
Solids. Lactose | Proteid 
Proteid’ Fat ° 
Jersey........ 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 Poll.....) ....... 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. (Eckies 
AND SHAW) 
Relative Size ¢ 2 Reichert- Melting- 
Breed. | of Fat | Jodine | Saponificay | Meisal point, 
Globules. 7 : Value. Centigrade. 
Jersey......... 828 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 


LIMITS AND VARIATIONS 39 


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 proces, 

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- 


40 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 solids»... :25ce03-33s02e0%4 Sheen 11.62-14.25 13.76-14.90 
Be ee sic neiene cence sg 3.33- 3.50 4.05- 4.90 
TERCTOBE 55 2 diel 7,208 aut ad. nee antndaaeuatueed ane 4.538- 5.64 4.47-— 5.55 
Protei@. scsi dimwleseeuwadeeed ces 3.13- 4.46 8.33- 4.54 
Ash....... Fede idce aiaad Bana eben Bes 0.60- 0.90 0.82- 0.93 


a 


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 Al 


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 


Diacram No. I 


EFFECT OF SEASON ON FAT CONTENT 


41 fo _fasgo On = 
"| \ 2 Pat 
x Sot a 
4.0 Ua N “™ coe kanedl ~O, 
o’ 7 Pa oa ty SO 
2 
Baa eS IN. S| ff VA a) 
g a a / — af 
5 3.8} M 
13} 
& 
fu 
3.7 
va V 
3.6 NA j 
N\ Va England 
husetts---—-— 
. No lees 


“Jan. Feb, Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. 


the 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 
plotted from the author’s analyses of over 9000 samples sug- 
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 over 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 18.2 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 
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 9 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 43 


TaBLe XVII 
AVERAGE PERCENTAGE OF FAT BY FOUR-WEEK PERIODS 


Jeisey. Shorthorn. Ayrshire. Holstein. 
First period............. 5.20 4.08 3.94 3.14 
Second. ....... se 4.91 3.88 3.68 2.87 
Whird,| —iai/ox see's Shes 5.02 3.71 3.60 2.78 
Fourth = ............. 4.79 3.54 3.59 3.11 
Fifth dbo elec aes 4.88 3.56 3.70 3.11 
“Bixth  adiaeocueenncs 4.98 3.58 3.52 2.98 
Seventh ............. 4.93 3.69 3.63 3.03 
Highth =... 4.83 3.73 3.74 3.09 
Ninth  segeidia snd che 4.84 4.19 3.71 3.05 
Tenth —-wassiieadivetencn ens 4.88 4.19 4.05 3.31 
Eleventh ............. 5.23 4.11 4.92 3.39 
Twelfth  ............. 5.68- | ex doe sens 3.96 3.70 
Thirteenth ............. ORAS leat vies 4.18 4.48 
Fourteenth ............. GLA ee cscveseat lr eeekeca ae 3.68 


Taste XVIII 


RELATION OF LACTATION PERIOD TO CHEMICAL AND 
PHYSICAL CONSTANTS OF FAT 


AVERAGE DETERMINATIONS BY FouR-wEerx PERIODS 


: Percentage Relative Melting Iodine Reichert- Saponifica- 
Period. Size of point, Meissl tion 
ot hat: Globules. | Centigrade. Number. Number. Number. 
1 4.009 357 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 
7 3.83 201 33.3 82.9 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 85.4 22.5 223.4 
ll 4,22 162 34.7 35.5 22.2 223.8 
12 4.54 166 - 33.8 35.2 20.3 220.6 
13 4.665 110 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. 

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 1°). 


TaBLE XIX 

INFLUENCE OF SEASON ON SOLIDS-NOT-FAT 

Month. Fat. ea a Lactose. | Proteid. Ash. 
January............-.. 3.80 8.95 4.62 3.57 0.76 
February.............. 3.70 8.97 4.70 3.52 0.75 
March..............--. 3.62 9.91 4.72 3.45 0.74 
Aipriliscva aeons wea toacoayaie ds 3.62 8.83 4.66 3.42 0.75 
Mia Vesna gunt\sdecwtatete 3.47 8.85 4.64 3.47 0.74 
JUNC): os eee ress 3.44 8.82 4.68 4.42 0.74 
JUVicdnc angst eeasy 3.59 8.67 4.69 3.23 0.75 
AUIBUSt 235. < cgaas dice ekecas es 3.72 8.55 4.59 3.25 0.71 
September............. 3.88 8.63 4.63 3.25 0.74 
October............... 3.91 8.76 4.63 3.38 0.75 
November............. 3.94 8.81 4.63 3.42 0.76 
December............. 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 ® 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 
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 
the last two to three months. The ash was constant and the 
proteid decreased and increased sympathetically with the fat 
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 
represent the percentages of proteid and fat, and gave many 
analyses in support of it, but Richmond has pointed out that 
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 
preferred to that of Timpe but cannot be considered as entirely 
satisfactory. These formule 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 
11 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. 

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 proteid 
in the total solids is shown in tables XXI, XXII, and XXIII. 

Although some of these results are somewhat discordant, 

the general tendency is usually in the same direction. When 


ratios together with the percentage of fat 


NON-FATTY SOLIDS 47 


TaBLe XXI 
PROTEID 
———— RATIO 
PAT 
Breed. Van Slyke. Lythgoe. re pied 
Holstein. Frisian. ...... 0.87 0.86 0.95 0.93 
Dutch belt. ............) 0.00002. 0.83 
Ayrshire................ 0.82 0.75 0.88 0.93 
American Holderness... . . 0.83 
Shorthorn .............. O80 |) esacecse 0.96 0.89 
Devoniescscueosiaaeses 0.80 
Guernsey............... 0.66 O71). | seatssans 0.78 
DOTSCY ss sis) cance da diet dw 0.64 0.61 0.74 0.83 
TaBLeE XXII 

LACTOSE 

—— RATIO 

PROTEID # 

Eckles and New Jersey 
pred Lythgoe. Shaw. Expt. Stat. 

Holstein. Frisian........... 1.60 1.54 1.43 
Dutch belt... .............. 1.67 
Ayrshitesc< ccc sniasaw anes 1.63 1.51 1.39 
Shorthornid sisiaenscceeecke| akheass eee 1.39 1.47 
Guernsey ss sci ceeds eeeues 1330} oe wks oes 1.22 
POLICY ea sides tesa sit co sees rst ruth 1.43 1.21 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- 
oteid lactose 
fat proteid 
that the percentage of fat in the total solids increases. 

Seasonal variations are also shown by the various ratios as 
will be seen from Table XXIV, the figures in which are cal- 
culated from those in Table XTX. 


centage of fat, the pr ratios decrease and 


48 THE NORMAL COMPOSITION OF MILK 


TaBLe XXIII 
PERCENTAGE OF FAT IN TOTAL SOLIDS 
Breed. Vieth. Lythgoe. ys ein co ace 
JOTSCY ices dees sme 38.0 38.3 39.2 34.9 
Gueriseyissacccaee cee] ae Rash oe B09!) || heres city 34.9 
Welsh...............05- 34.7 
SUSSEX! o.5 co cacawnene a se 34.4 
KOT se oe sens toner ees 84.5 
Dairy shorthorn......... 31.3 
Pedigree shorthorn...... 31.4 
Shorthort...c2c:e0bseee| Sooo een | teasaeses 29.4 29.3 
Red polled.............. 32.8 
AGTSHINEs» «wheres Aveda) os Bae PAS 31.8 29.6 28.7 
Dutch belt... ......005.] 00. ee eee 30.9 
Montgomery............ 28.5 
Holsteins sceg cs te aeesl| Aaee cae 29.2 27.2 28.1 
TaBLE XXIV 
SEASONAL VARIATIONS IN PROPORTIONS OF CONSTITUENTS 
Proteid L 
Month, Fat Prod : pee 
JANUAaryy id caidas e932 0.94 1.29 29.8 
February... ........5-0-005- 0.95 1.33 29.2 
Marchis ac cciveseu teenie 0.95 1.37 28.8 
Aoprilocs yi Shtes ages Peat 0.95 1.33 29.1 
Mays caccgeeek ete eneecee 1.00 1.31 28.4 
JUNG sierccigsugie eee eee tess 0.99 1.34 28.1 
DUTY: Secenstcvalnh stages Rance ade 0.90 1.42 29.3 
Aupust.. candies hades 0.87 1.41 30.3 
September..............-. 0.84 1.42 31.0 
October. ci. cose es ees we 0.86 1.37 30.9 
November.............5-5 0.87 1.35 30.9 
December..............-. 0.92 1.30 30.2 


ad 


NON-FATTY SOLIDS 49 


The influence of the stage of lactation upon the various ratios 
is shown in Table XXV, 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 
peiod Na | Put ee | ee a 
1 0.67 1.47 31.4 
2 0.61 1.58 31.4 
3 0.66 1.62 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 
proteid 

fat 
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- 


fat, though at a slightly higher rate. This increased 


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 =0.386+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.-tS 
are based. Lythgoe has suggested that lactose may be cal- 
culated from the following formule. 


7 Es 
L=T. 8.— [F+0.7+ (7. 8. Tag) | 


from Olsen’s formula and 
L=T. S.—[F+0.7+ {0.4(F—8)} +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 2 3 4 5 6 
Total solids.......... 10.47 | 10.75 | 10.85 | 11.23 | 11.63 | 12.67 
Pati gasscneamosvanes 1.70 1.76 2.10 2.54 3.14 4.08 
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. 


Taste XXVII 
AYRSHIRES (AvurtHoR) 
Fore Milk. Middle Milk. Strippings. 
Mati cddovncraiaee iiss 1.40 5.90 9.80 
Lactose: i22s exeiesa ts 4.95 4.94 4.87 
Proteid............... 3.17 2.98 2.78 
Ashi tcclleciteate canine 0.80 0.74 0.71 
. Lactose 
Ratios Proteid °°" 1.57 1.66 1.75 
Proteid 
Fat 2.26 0.51 0.28 
TasLeE XXVIII 


AVERAGE OF JERSEYS, SHORTHORNS, AND HOLSTEINS 
(EcKLEs anD SHaw) 


Relative Size 
Fat. Lactose. | Proteid. Ash. Ee ie of Fat 
: 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: 


TasLleE XXIX 
peu Iodine Saponification Melting Yellow 
Number. 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- 


eee, lactose 
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 “ colostrum ” 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 


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) 
Milking | Total Total | Casein- 2 F 
Number.| Solids Ash. Fat. | Sugar. Proteid.| ogen. Globulin. | Albumin. 
1 22.87 | 1.03 | 2.30 | 2.74 | 12.23 | 4.86 5.32 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 | 3.638 | 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 | 3.46 | 2.21 0.31 0.62 
8 15.62 | 0.80 | 5.47 | 4.57 | 3.36 | 2.17 0.27 0.61 
9 15.47 | 0.82 | 5.62 | 4.22 | 3.35 | 2.15 0.25 0.59 
11 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 (Enetine) 
Immediately) aster | After | After | After 
oe 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 21.23 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 
HAG seonnce adi enieoa acacnene 3.53 4.66 4.75 4.21 4.80 
Lactose............. 3.00 1.42 2.85 3.46 4.10 
Ashicc outs se wadousaele ss 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 
XXXI. 

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. 

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


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. 
oteid aan lactose 
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 pr 


TABLE XXXII 
EFFECT OF DISEASE ON COMPOSITION OF MILK 


(SCHAFFER AND BENDZYNSEI) 


Total 
Solids. 


Proteid |Lactose 


Fat. | Lact y s ‘ 
a Nea Fat '|Proteid 


Character of Disease. Proteid.| Ash. 


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


oo° 
RE & 


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 
lactose 
proteid 
increased proteid percentage. 

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


lactation, the 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 proterd and feces ratios 

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 d lactose 

fat proteid 


addition of water leaves the ratios un- 


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 
PYS. This is due to the Van Slyke formula being based on the 
lactose __.. 
proteid TanO 


ratio abnormally high. In 


constituent which has been abstracted. The 


proteid 
fat 
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 


would be normal and the 


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 
Method of Preparation of Serum. Gece ras 
Copper sulphate........... 0.0.0 cece ee ee eee 36 
Acetic aol densa 16 ston Gees ose ean aah os ones Bh cde XI 40 
Natural souring... 000. ice s.80.0. see. ebe sees 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 
lactose 
proteid 
deficiency of ash. Methods for the detection and estimation 
of cane sugar are given on page 88. Glycerine and starch 


low proteid value, an abnormally high 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 =100—SN? 100 in which SNF represents 


of 


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 


died water=ing = gy, 
minimum snf allowed 
or 100 Hes foun 200. 


~ minimum T. 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 

Added water = 100 StF x 100 where G = degrees of gravity 
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 = 100-4100 where f, and F, are the 


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 


Miik 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, 


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


x 12.8 
3.8 A 
on 
ae \ 


/ ki SS] in 


Percentage of Total Solids 


‘ep 
63.7 7 Ni 4 Po 419,7 
ay e Oe .. #13, 
° ’ De f .. 
& # ‘ a A oh, 
£ y F w . % 
3 / ‘ 
£36 7 x 12.6 
oe / \ 
’ s 
6 
Pati ST Al 
Total Solids-.___---- \7 
3.5 12.5 


1900 1901 1902 1903 1904 1905 1906 1987 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 7% 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 larger 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 
Sxim 
Mi1z. Mire. Cream. 
Country, State or 
Province. 
Total Solids- Solids- 
solids. | F#* | Not-fat. | Not-fat. 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” seduces 
Victoria......... 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} 3.80 | 8.50 | 8.80 35.0 
Canada. Dominion: ....| 8.25 | 8.50 | 8.50 18.0 
Alberta......... 12.00} 3.00 | 9.00 | 8.50 
British Columbia .| 11.75] 3.25 | 8.50 
Manitoba.......]...... 8.25 | 8.50 | 8.50 18.0 
New Brunswick..| No {Stand |ards 
Nova Scotia..... No [Stand |ards 
Ontario......... 12.00} 3.00 18.0 
Quebec.......... 12.00) 3.00 | 9.00 ;....... 16.0 
Saskatchewan....| 12.00) 3.50] ......]....... 20.0 
South Africa.......[...... 3.00 | 8.50 |....... 25.0 
25.0 
UG ie ey 205 | eine a 32 i : 
New Zeala 3.25 | 8.50 8.80 { 40.0 
India. 
Caleutta........ 11.50) 3.00 | 8.50 
Bombay......... 12.00) 3.50 | 8.50 


64 THE NORMAL COMPOSITION OF MILK 
TaBLE XX XIV—(Continued) 


MILK anp Cream STANDARDS 


Skim 


Minx. Cream. 
Country, State or isan 
Province. 

Total Solids- Solids- 
Solids. Fat. | Not-fat. | Not-fat. | Tat 
United States. Federal.|........ 3.25 8.50 9.25 18.0 
Calitorn ities did cose ace) iene 3.00 8.50 8.80 18.0 
Colorado ic evo taees | wae sad BOO | | eres ie Sex oun r 16.0 
Connecticut.......... 11.75 3.25 8.50 | ...... 16.0 
District of Columbia...} 12.50 3.50 9.00 9.30 20.0 
Florida...............] .-..40. 3.25 8.50 9.25 18.0 
GeOFP1A.. . cineuty oRulsas all Said 8.25 8.50 9.25 18.0 
dao: : 2.4 cede) decane 11.20 3.00 8.00 9.30 18.0 
WNING1S is sss aciree Vaan | Gow e-ada 3.00 8.50 9.25 18.0 
Mn dianais cic eis voceehskealy eee bebe 3.25 8.50 9.25 18.0 
POWade eo cues Bla 12.00 OO. ok ka eta il ata cae 16.0 
AC ANS ES vo ec.s tes ak Be PS 20!) | Bee we kas-clll an seeaia 18.0 
Kentucky............] ....... 3.25 8.50 9.25 18.0 

Louisiana............. 75 3.50 8.50 8.00 
AINE! 55. os, seats eadaticton 11.75 3.25 BisDO™ fi ecars darn 18.0 
Maryland............ 12.50 33:50! jl) sernes4 9.25 18.0 
Massachusetts......../ 12.15 SOD il gents 9.30 15.0 
Michigan............. 12.50 SOO. Nf sexes nll seees 18.0 
Minnesota............ 13.00 3.25 OS |) Scacceascs 20.0 
Missouri. eves cocaine 12.00 3.25 8.75 9.25 18.0 
Montana............. 11.75 3.25 B.50) | ceeded 20.0 
Nebraska.............[ 2.2.00. BOO?) sacs ducks | ccgeeen s 18.0 
New Hampshire.......| 12.00 | ...... 8250) | gees 18.0 
New Jersey........... 11.50 S.00" |) gia vad 9.25 16.0 
INGWad a fice de tees eas 11.75 3.25 8.50 9.25 18.0 
New York............ 11.50 BOO | Geawaw | Sa dein 18.0 
North Carolina........ 11.75 3.25 8.50 9.25 18.0 
North Dakota........ 12.00 B008 4 eclees Il swe ou 15.0 

OWIG oka seeder 12.00 3.00 
Oregon... .........4., 11.70 3.20 8300) |. a zutss 18.0 
Pennsylvania......... 12.00 3.25 82500 | eeesde 18.0 
Rhode Island......... 12,00 3.50 

South Dakota.........] .....-. 8.25 8.50 9.25 18.0 
Tennessee..........-. 12.00 3.50 8.50 9.00 20.0 
TEXAS a orsisc re! sna ndhecnig lace 12.00 3.25 8.50 9.25 18.0 
Uitaasces cee scvranteinontalate 12.00 3.20 B00! | eeeiems 18.0 
Vermont............5 11.75 3.25 8.50 9.25 18.0 
Vitginias ¢ecncaei ed: peace ds 3.25 8.50 9.25 18.0 
Washington .......... 12.00 3.25 8.75 9.30 18.0 
Wisconsin. .........26{ ce eee ee 3.00 8.50 9.00 18.0 


BIBLIOGRAPHY 65 


BIBLIOGRAPHY 


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


Starling. 1902, 104-105. 


. Lythgoe. Ind. and Eng. Chem. 1914, 6, 901. 
. Eckles and Shaw. Bull. 156 U. 8. A. Dept. of Agr. 
. Morgen et al. Landw. Versuch. Stat. 1904, 61, 1-284, zbid., 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 


AutuoueH 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 600 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 c.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 e.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 


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.cems. 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 


Py 


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 LXVIII, 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, T.S. the percentage of total 
solids and G the specific gravity expressed as mentioned above. 
From this formula T. S.=1.164F'+0.2546G. 

A simplified form of this formula that has come into general 


MILK SUGAR 71 


use is T. S.=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 JT. 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 fat X1.11. 

(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 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 Cuz0 
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 a 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 x caustic 
alkali using sensitive methyl orange or, preferably, methyl red 
as the indicator. Each c.cm. of x 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 


ALDEHYDE VALUE 75 


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

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


solution added and titrated with a alkali until a faint 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 x alkali being equivalent to 0.009 grm. lactic acid. It is 


preferable to express the acidity in degrees, i.e., the number of 
cubie centimeters of normal alkali required for the neutralisa- 
tion of 1 litre of milk. The number of cubic centimeters of x 
alkali required for the neutralisation of 10 c.cms. 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 Miller’s modification 
(Richmond’s Dairy Chemistry) of Steinegger’s method is as 
follows: 10 e.cms. of milk are made neutral to phenolphthalein 


with x 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 x 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 x acid with methyl orange 
as indicator, and chlorine by titration with a silver nitrate, 


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


=0.0031 grm. NazO and 1 ¢c.cm. x AgNO =0.00355 grm. Cl. 


The insoluble ash is dissolved in a slight excess of dilute 
hydrochloric acid, and the solution (nearly 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 (MgeP207) 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 (SO) 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 a silver nitrate, using potassium chromate as 


indicator. Each cubic centimeter of = 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) 
eading Immersion Specific Gravity. 
serene 20° C. tip 20" & iz 
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 


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 XXXV, 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 NagO 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- 
icacy, 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. 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 c.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 x 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 x sodium 
thiosulphate, using a starch solution as the indicator in the end 


BORIC ACID AND BORATES 83 


reaction. Each cubic centimetre of x 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 methy! 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 
solution 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.ems., 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. 
a 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 
centimetre of x 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 


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. 


CREAM 87 


ANALYsIS OF M1LK PropuctTs 


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 
e.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.ems. 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 c.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 c.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 48°-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 
offsprin ing 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: 


TaBLeE 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 

4.0t04.2 14 

4.3to 4.5 13 

4.6to 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 
(cf. 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 a 


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 
..8 drops of hydrogen peroxide (3 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. 

Bellet’s Method. To 10c.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 
density 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. 


OapRwnwe 


CHAPTER IV 


BACTERIA IN MILK 


Mix, 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. 


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- 
therize 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 1! 
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 c.cm. 


Streams 1 and 2............... cece eee 10,143 
Streams 5 and 6............. 222.00 c cues 2,347 
Streams 9 and 10....................00. 272 
Streams 13 and 14....................0. 382 
Strippinigss: ees owee rs theeses het aeons 204 


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


SOURCES OF BACTERIA IN MILK 97 


BactTERIA PER C.CM. 


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


Backhaus 8 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: 


98 BACTERIA IN MILK 


BaAcTERIA PER C,CM. 


Total. Acid. Liquefying. 
Brushed................ 2268 381 117 
Unbrushed............. 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 XXXIX 
BACTERIA IN LITTER (Bacxuavs) 


Litter. Organisms, Per Gram. 
MPOAG iansc ie sehat nas Quaolap de hta cued idedm as ienausyene Gusausiangee 2,000,000 
Good! StPaw..csosa nas es anea onward sateen 7,500,000 
‘Bad Stra Weces sexes wetie seine wale Gee's aa seam 10,000,000 


The milk obtained contained 


Bacteria per C.cm. 


With peat litters cca. ceecnscawvesishes ceres 3500 
With straw litter...............--.---005. 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 1? reports the following results in connection with 
experiments on the influence of feeding before and after milking. 


Hay anp 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 ......... 3656 692 123 


100 BACTERIA IN MILK 


The results of Harrison1* 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 5 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...................0005 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 
feces. It is obvious that all the water used for cleansing and 


COOLERS AND PAILS 101 


TaBLE XL 
EFFECT OF MILK COOLERS 


AVERAGE OF Four EXPERIMENTS (ORR) 


BacTERIA PER C.cCM. IN MILK. 
Agar 48 Hrs. Gelatine 96 Hrs. 
at 37°C. at 20° C. 
Before cooling ................-.-- 26,000 39,000 
After cooling................2.008. 48,000 104,000 
Author’s results: Coliform. 
Before cooling................... 25,000 4 
After cooling.................... 400,000 3,500 
After thorough cleansing of coolers: 
Before cooling................... 28,000 2 
After cooling.................... 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 '© gives several analyses of fresh cow excreta. (Table 
XLL) 

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 


TasLe XLI 
BACTERIA IN COW FECES (Savace) 
OnrGANISM8 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 1” 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 1* thoroughly investigated this phenomenon and 
concluded that no true germicidal action took place, but that 


EFFECT OF TEMPERATURE 103 


TaBLte XLII 
Upper figures represent sample No. 1. Original count 3,000. 
Lower “ - “No. 2. i “30,000. 
TIME wich EvapseD BEFORE Maxine Test. 
Temperatures, 
ce os 
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 
60 180,000 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 


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“DO oL8 98 “SIY HZ IUsepediqoy pus ‘D .2E 8 “SIY QP IvSY 


SLIOSAY s,AOHLAY 


TIVIX Fav 


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 !9 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 cemmon 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 


107 


BACTERIAL DEVELOPMENT IN MILK 


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(“OHLOY) 


AITX Fav 


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 11°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. 

(8) 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 erogenes 
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 (8-4) 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. 


ONRaARONE 


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.S. 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. 


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 obtained 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 (Detfépine) 


Mixep Miitx Comina More tHan 40 Mites anp GreneraLtty 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 ee 
55-60 Nil 


Mitk From Suort Distances (LESS THAN 20 MiLEs) usuaLLy Kepr 
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 sda 
70-75 50.0 


REASONS FOR DETERMINATION OF TOTAL COUNT 115 


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, 


TasLe XLVII 
TOXICITY OF MILK (DeE.f&prne) 


Percentage of Toxic 


No. of Samples. Samples. 


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 ? 
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 


TaBLe XLVIII 


EFFECT OF INCUBATION PERIOD ON MILK COUNTS ON 
STANDARD AGAR 


117 


IncuBaTion Periop at 37° C. 


Sample No. Ratio - i. 
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 reproduction is shown in 
Table L. 


TaBLe L 
Standard Agar| Maree Ar | Suandad | Leste 
Sample No. fe ours 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 


EFFECT OF SUGAR IN MEDIA 119 


Heinemann and Glenn 5 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, 8, 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- 


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 Conn4 
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 (6) 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 exchagge 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 74> c.cm. and zo ¢.cm. 
were inoculatediand 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 


‘ 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. 8.) 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 ¢.cm. 


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 33X, 
its magnification being 24 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 +5 inch oil immersion lens 
and the organisms counted. ach coccus, bacillus, diplococcus, 
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 0.66 X10* to 6.0104. 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 3. 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 0.4X10* to 
33.0 X104, 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 


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: 
x 


7k? 


x100=y, 


where y is the factor sought, x, the area of the smear in square 
millimetres, and & 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=0.080m.m., y=500,000 
When R=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 


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 
Group Number. Average Number of Bacteria Average Time of Reduction 
per c.cm. in Hours. 
1 29,647 11.9 
2 73,587 97 
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 18 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 


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 supra), 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 LII. 

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 


TasiLe LIT 

RELATION OF ACIDITY TO BACTERIAL COUNT (Frep) 
Group Number. Average ee as Lactic | Number e oe 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 

x. Se 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: 


Taste LIII 
RELATION OF ACIDITY TO BACTERIAL COUNT (Auvrsor) 
AcIDITY. 
Number of ‘ - Bacterial Count 
Samples. Degrees. Lactic Acid, ao Honrs etry? 
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 0.171 212,000 
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, 4381. 

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, 454-461, 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. 


POONA AR wr 


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. 


Tasle LIV 
Milk. Cow’s Faces. 
Per Cent. Per Cent. 
Saecherode +dulcite+.... 0... eee ee 32.7 47.9 
Saccharose—dulcite+................005. 39.2 25.0 
Saccharose +dulcite—..............-00000. 19.6 12.5 
Saccharose —dulcite—...........-..0-0000. 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.2 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. 


Taste LV 
MacConkey. 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 12.6 | 20.4 16.7 8.4 
Other strains.........| 8.3 9.2 1.2 3.6 3.3 | 4.1 


The results of Rogers et al.,4 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+................. 24.3 46.5 
Saccharose —dulcite+................. 14.9 8.4 
Saccharose+dulcite—................. 37.4 36.3 
Saccharose —dulcite—...... seaeepngens 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. 


Taste LVII 
BACTERIA AND B. COLI IN CERTIFIED MILK (Aurnor) 
Month. Mean Bacterial Mean B. Coli 
* Count per c.cm. per c.cm. 

Mae siticeow sould eters waeeedgy en ee en Aas 5,700 1 
JUNC sss cy x aria mae ote ieawars xe ee 10,900 2 
Jy oiot age ee ee 5,000 0.1 
AUGUSTA venta chien bce Sed ew PR AS 4,500 0.8 
September.............-....-- 005 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 B. 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 ray 80 
512,000 S| é} 70 


‘e) 
s 


-Temperature Degs. Fahrenheit 


B. Coli per c-cm. 


fey 
10,000 EVA oR 
_ heed ye) 


4,000 30 
~ ! fe Belt 
a ‘\ |-o Temperature __ __ 
000 - “zy 20 
Ol Sf 4 
PNOs teu 
Nov. Dec.;Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. a0 
+— 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, the 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 ¢.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 ¢.cem+, 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 ” 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.cm.+, should be recorded as 1.0 e.cm.+, 0.1 c.em.+, 
0.01 c.cm.+, 0.001 c.cm.+, but 1.0 c.cm.+, 0.1 c.em.+4, 
0.01 c.cm.—, 0.001 c.cm.+, should be recorded as 1.0 c.cm.+, 
0.1 c.em.+, 0.01 ¢.cm.+, 0.001 c.cm.—. 


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), xsculin 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 xsculin 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 eerogenes) whereas all the dark colonies from the zsculin 
medium were of the B. coli family. Savage 9, 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. coliin the tube series without 
confirmation. 


Medium. B. coli per C.cm. 
Rebipelagar. 2.0. escecea eet es ew aed 15,326 
Lactose broth. ..............0.20005. 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 


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 
other 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 
anzrobic 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 


STREPTOCOCCI 147 


the surface usually produces the necessary conditions. The 
method of Savage 1° 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.” 


TasLe LVIII 


Result. Positive Tubes. see elec etait of 
i 0 
vo 5 
a 11 
ts 17 
as 25 
or 34 
& 45 
0 60 
=e 80 
ae 114 
1 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. fecalis 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. 


_ 


BIBLIOGRAPHY 149 


BIBLIOGRAPHY 


. McConkey. Jour. of Hyg. 1906, 6, 385. 


2. McConkey. Jour. of Hyg. 1909, 9, 86. 


ow 


. Orr. Rpt. on an investigation as to the contamination of milk. 


London, 1908. 


. Rogerset al. 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, 431. 
. 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 


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 ease 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. 
Miller ® 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 


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. Ruediger*! 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- 


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. 

The biochemical characteristics should be determined 
quantitatively by Winslow’s method 29 if the best results are to 
be secured. 


Taste LIX 


BIOCHEMICAL CHARACTERS OF PRINCIPAL TYPES OF 
STREPTOCOCCI. (Broapuurst) 


E 
Name of Variety. E g E i : q 3 E 3 Type Named by 
alaial2ia|2lale- 

Str. equinus...| X | O} X | O/]O/] X|—J| — | Andrews and 
Str. mitis...... xX/X}]xX;]oO;};o;]xX{-4]—- Horder 
Str. pyogenes..| X | X | X]O]}]O|] xX] +] — es 
Str. salicarius..| X | X | X | ®/]O]O - te 
Str. anginosus..| X | X |X] @/O]O]+] —- os 
Str. gracilis....| X | X]O]O/}]X]X]—] 4+ ee 

? xX/|xXx;O;O|; xX |] xX{]—-]—- a 
Str. fecalis....)] KX | KX} KX] O, X| xX] -—]—- ue 
Str. versatilis..| X | X | X | KX | X |X] — 1] — | Broadhurst 
Str. bovinus...| X | X | X/]Xj]O!X | —]| — | Winslow 


X indicates that test substance is fermented with 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. DieHTHERIEZ 


Milk’ has, on several occasions, been proved to be a vehicle 
for B. diphtherize 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. diphtheriz but fairly rapid multiplication 
does occur until checked by the metabolic products of the acid 
producers. 

The number of authentic cases in which B. diptherie 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 #8 and Marshall.!* 

For the isolation of the organisms, Bowhill directly inocu- 
lated Loefiler’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. diphtheriz 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. diphtherix, 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 indubation 
at 37° C. From the tubes containing well-isolated colonies, 
subcultures are made of all colonies in any way resembling B. 
diphtherie 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 3y 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. diphtherize 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 c.cm.) and the other 
for a mixture of the culture with 1 ¢.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. diphtheriz but sharply differentiated 
from this organism by the absence of virulence. Bergey 1° 
investigated a number of these organisms which were apparently 
identical with B. diphtheriz, 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. TypHosus 


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 faeces, 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.cm. 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 is added. 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 23 typhoid bacilli per 100 c.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.2°) 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 
diarrhoea. 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 diarrhcea 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.?!) 

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, made 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,”* Ross,?° O’Brien 7° 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 diarrhoea and that 
Morgan’s No. 1 Bacillus, whilst rarely present in flies from 
houses not containing diarrhoeal 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 diarrhea 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. 


163 


COLI-TYPHOID GROUP 


‘aduvyo ou =— ‘sed ynoyyIM uoKNposd ploe=X ‘uoKnonpoid seJ=+ :pueseTy 
“eIpew YJOIq pus a}vIpsyoqrey 
mIV | IV] —~|}—}-—}-}-]-y-]-t-}]-J]-J]-]- ]-t-J-J4 loc: SouesT]VY[e speowy “gy 
SIV | PPV JFL] xX) —}- |} } ao] a ]r [rx px yx px) ] = [+ Geuxepg) ereyuesip -g 
IV | PPV | —}]—-—|—-}-J]-}]-J-]—-]-l-l]-l-IxKIxIx]-]-— Joc: (e3Iqg) wtuajuesdp "g 
poy | poy | — SSS pic: [ee ee ee es fi xIxIlx|x]— se RSE ote ek epee ES snsoyd (£4 “g 
idnos6 yj4aQy UL 
“HIV see + + eke = =, peren tee os ae sss ee! oa + 4 + _ + er) I ‘ON S,UBBIOT 
IIV poy rn) ee + + + os.|1)! awk paw! | Pperree | eae eed + +4 + +4 en + Se eee eee Jaytysedins ‘d 
MIV | poy | += gee lees [ee | eas) | | Ss -|-J-]- A abe feb Ps [ee Spi ta ee esas Ne uinns exepoya “g 
MIV | PPV |-— | + yt} t+]—f/—l-]xX}]-—}—-}—ttits4+]4+}—-f4 touch q@ ‘snsoydéyered ‘gq 
pPey }—}+}/+)}/+)/-]K}]-|x]—-}]-J-J]4+t]4t]4]4+]-J+ttoocc: Vv ‘snsoydAyered -g 
> Dc ln ae eV abe ce cali cal ee ced ld a SIPI}itoyae “q 
SdNOsg) saURLIDD) OY J, 
+ + + ised + Senne pce + + + 4+ + + + + + + Se ee ee ee #a0vopo ‘g 
prey | PPV Jt] t+ [t+] —-]—-—}-}-]-s]—)—{+it+it+]4+]4+)-]+ [pv os eed cee POR] Iplow “g 
poy | PPV ;Fi+)/+)-—]/-—}-—}-]+]}]Fl+ti+i4]4)4 ]4+]-|F poe saueso1ev Soe] “g 
poy | pev |+]/+i+)/+/+}/-|-]+]4+]4]4+]4+]4t]4tl4l-j4locce IOTUNTIALOD TTO9 “gq 
poy | pey|+/+)/+)/+/4+]}/—-/-|]4+]-J-J4+/]4]4]4]4]-J4 occ STUNUIUIOD 109 “gf 
dnoig yo.ayosy oy L 
i “eh m | mw m| ~a)/te|z|o)le Qa 
FIelal S/S) S/F) Ele) F)/e}s|slel es] ea) 
PP [deme | Aebe Y Mees ss 5] 8 a/12%)o}]/9o) 8) a Bl cer 
‘HTI SQWLIT : ® @ & tia Vien esa aca J 


SWSINVOYO FO dNOUD GIOHdALTITIOO JO SUALOVUVHO TVOINGHOOIA 


XT Wavy, 


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 levulose, 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 32 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 86° to $0° 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 minutes, 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 (0) 
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 *? suggested that the 
inguinal glands on the inoculation side should be slightly dam- 


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 c.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 
@ 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. Homan. 
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 


Bovine serum. Grows slowly and 
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 
finally become alkaline. 

Pathogenicity. 

Calves. Highly pathogenic. 

Rabbits. Highly pathogenic. 

Subcutaneous inoculation with 10 
m.gr. causes an acute generalised 
fatal tuberculosis. 


169 


Grows fairly rapidly. 


Remains permanently acid. 


Non-pathogenic. 

Slightly pathogenic. The lesions 
are often localised in the lungs 
and kidneys or scattered. 


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- 


1897. 


agar. 
BIBLIOGRAPHY 
1. Coleman. Rpt. of M. O. for Angelsey. 
2. Savage. 


. Krumwiede and Valentine. 
. Jackson. Jour. Inf. Dis. 

. Davis and Capps. 
Heinemann. Jour. Inf. Dis. 
. Heinemann. Jour. Inf. Dis. 
. Muller. Arch. f. Hyg. 
. Heinemann. Jour. Inf. Dis. 
. Bowhill. Jour. State Med. 
. Eyre. Brit. Med. Jour. 
. Klein. Jour. of Hyg. 

. Dean and Todd. Jour. of Hyg. 
. Marshall. Jour. of Hyg. 
. Bergey. Jour. Med. Research. 
. Savage. 
. Klein. Jour. of Hyg. 
. Jackson and Melia. 

. Tonney et al. Jour. Inf. Dis. 
20. Tonney. Jour. Inf. Dis. 
21. Park and Holt. Arch. of Ped. 


oN ell eel cll ool oe ee 
OO OND Oo PWN OO OND OP & 


Jour. Inf. Dis. 
1906, 3, 175. 
1907, 4, 87-92. 
1906, 56, 90. 
1915, 16, 221-240. 
1899, 705-710. 
1899, 2, 586. 
1901, 1, 85. 
1902, 2, 194-205. 
1907, 7, 32. 
1904, 11, 445. 
Rpt. of M. O. to L. G. B. 
1901, 1, 78. 
Jour. Inf. Dis. 
1916, 18, 243. 
1913, 13, 263-272. 
1913, 20, 881. 


Rpt. of M. O. to L. G. B. 1906-07, 228-252, ibid., 1907-08, 
359-424, ibid., 1908-09, 294-315. 

Rpt. 86 New York City Health Dept. 
1913, 12, 364-385. 


1914, 15, 135-140. 


1906-07, 224-225. 


1909, 6, 194. 


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. 

- 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. 1912-138, 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, 


44, 1439. 


. Anderson. U.S. A., P. H. and M. H.S., 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.” 

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, 

(6) 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 u) 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 uw 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 ») 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 u 
but may be 25 » 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 »), 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 19055 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 8 are as follows: 

Collection of Samples. Samples for analysis should be 


CONCENTRATION 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 ccm. 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 } 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 43-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.c. to one-half c.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 method 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.em. 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.em. 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 foliowing formula 
for calculating the number of cells per cubic m.m. 


cells per cubic m.m. of milk =", 


where y=the average number of leucocytes per field of vision, 
d=the number of squares which just spans the diameter. This 


approximation of se 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 contairled over 500,000 cells per c.cm. Stone and 
Sprague,!! using the Doane-Buckley method, examined two 
healthy cows during the whole milking period (1,167 samples) 
with the following results: 


SIGNIFICANCE 179 


Samples. Cell Count. 
12 per Cent yess os aes se vee os aes under 10,000 per c.cm. 
MQ ase Bree Se tate b eacs 10,000 to 20,000 
GEO! se gitlidegeae cons 20,000 to 100,000 
ZOO ane aansteececn teal 100,000 to 500,000 
DiS Sha avattedatan! tron edet over 500,000 


Breed and Stidger,!? using the direct method, found varia- 
tions ranging from 5000 to 20,000,000 cells per c.cem. 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 14 
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. As a 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 15 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, ete. 

(d) Vegetable and mineral matter derived either from food, dung or 
litter or from dirty utensils and wash water. 

(e) Alge, moulds, and bacteria from various sources. 


As the cells and bacteria are separately determined, the 


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 NazCOs). 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 disc of cotton wool followed by an inspection of 
the disc for visible dirt. 

Tonney 1® suggested the use of a small disc 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 19 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 normally and so affect what 
is commercially known as the “ cream line.” 

The effect of time and temperature, the two factors 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 


Taste LXI 
EFFECT OF HEAT ON ENZYMES IN MILK 


WEAKENED DESTROYED 
Enzyme. Authority. 
At Temp. in At Temp. In 
°C, Minutes. &C. Minutes. 
Galactase.....| Babcock and 
Russell 65-70 10 76-80 
Von Freudenreich | 65-70 30 75-80 
Hippius 65 30 
Amylase...... Koning i |......] ...... 68 30 
Hippius |......]...... 75-80 
Race 68 30 8&3 30 
Lipase....... GileG feeete Hl eee €5 
Lactokinase...| Hougardy | ......] ...... 75 30 
Oxidases..... Marian> 2 Ps saicanga PP adane 79 
Bippius ft tae Peace 76 
Peroxidases...] Wender | ...... | ...... 83 
Schardinger. | ......] ...... 80 
Ostertag = | ......] ...-ee 80 
Lythgoe 70 30 75 30 
Race 68 30 73 30 
Numerous others. Or) |) ged eces 79-80 
Catalase...... Van Italie | ...... |] ...... 63 30 
Wender —if sa... J we aeee 80 
Reductase....| Jensen (aessti‘(L«Cw«ws | we over 70 
Lythgoe 65 30 70 30 
Race 68 30 71 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 


180; sl 
Pen 
a Fat, Sugar, 
a Casein, Salts 
+ 160 
[Sy 
e 
Es Taste 
I 
B50 
Aa Albumin 
s Enzymes 
s 
3 
I 
co) 
7 
aq Cream Line 
4140 
Tuberculosis 
Typhoid 
130 Streptococci 
Diptheria 
120) 
10 20 30 40 50 


Time in Minutes 


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. 


TasBLeE LXII 
EFFECT OF HEAT ON PEROXIDASE TEST 


Buraden é BENZIDINE REACTION AFTER HEATING TO 
Heating in 
Miniites: 145° F. | 150° F. | 155° F. | 160° F. | 165°F. | 170° F. 
5 + + + ae F aE 
10 + t i + a + 
15 + + EZ = ai Faint 
20 + + + + Faint |Very faint 
25 abe + + Faint a ba 
30 + + + |Very faint} — = 


Taste LXIIT 
EFFECT OF HEAT ON REDUCTASE TEST 


Time (Minutes) Required for Discharge of Colour after Heating 
D ~ to (Sample less blank). 
y uration of. 
Heating in ms 
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 readily 
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. 
62:8° Cy (L446) ok wes cen ne ene Nil 
6516" Cs(150° Be) sg: sent cemne eee a 5.75 
68:3° C2 (155° BD) sce scavaie sexe eae 12.75 
VAS CLCIGOP EE) 52s cnicenteheuhe encase 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. 


TasLe 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. 


Mitx Heated ror Teirty MINUTES AT 


Bee Raw Milk. i 
55° C. 60° C. 65°C. 70° C. 75° C. 
131° F. 140° F. 149° F. 158° F. 167° F. 


Min. Sec.| Min. Sec.| Min. Sec.| Min. Sec.} Min. Sec.| Min. Sec. 


19 08;16 56)16 53/17 12 


2 19) 984. | ssscseet | eecteace # Gas dane 20 38] 36 30 
TQ QB: isso sesecees nigga ackee nee tgale canis 20 25 | 37 30 


190 PASTEURISED OR HEATED MILK 


Bacittus ABoRTUS 


Since 1897 when Bang and Stribald ? 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 0) 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 yougg 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 5 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 
_ 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- 


Nitrate, asparagin and 
urea are commonly 


pounds. decomposed. Gela- 
tine is not liquified. 
Action in litmus whole Rendered slightly alka- 
milk. line. 


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 
broth. 


in glycerine 


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 


(ArTER 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 


Similar to those from 


Similar to the cultures 


thin layer a few mm.| pathogenic sources. { from pathogenic 
beneath the surface.| Colonies sometimes | sources. 

scattered throughout 

the entire depth of 

agar. 

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 | The medium is usu- | of variety b. 
medium. Sediment| ally clouded, but 
is made up of little | sometimes the growth = 


granules. 


is precipitated, leav- 
ing a clear medium. 


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 x 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 


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.: 
Milk tubes............ 71.5 22.8 5.7 
L. L. G. plates......... 43.7 53.5 2.8 
After heating to 150° F.: 
Milk tubes............ 84.6 10.5 4.9 
L. L. G. plates........ 41.2 57.7 1.1 


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 anzr- 
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. 


Group. 
Action on 
A. B. Cc. 
Milken eer se eae es ae Clot Clot No clot 
Maltose.............. 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 Test In MitK 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.!® 

In 1914 the author compared the results 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 oF CuRD 
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. 


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 ezrogenes organisms. 


Type B ' 


Lactic acid in great numbers, few if any coli and erogenes 
organisms, some cocci and fluorescent bacteria. Gas formation 
indicates the presence of coli, erogenes, or butyric organisms. 


Type C 


Lactic, coli, and srogenes bacteria predominate with many 
cocci. 


Type D 


Lactic acid mixed with coli and zrogenes 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. 
Tn 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 is taken. 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 


066 | OO | P88 | FSI | Gel | Gul | O8F | €O2 | LOZ | LOS 6 er | 0009¢2 | esBIOAy 
zs6 | ovat | gos | sez | peo | tee | esctp | rer | aes | oevs z'6> | ooo'6se |**** "3290900 
2°86 | gst | 6:38 | 2°62 | aes | over | aise | 1's | Tor | oss'9 T'19 | O00‘0%S |‘ xequieydag 
o'26 | Fog | Tle | aise | pse | ecez | tor | eit | ae over | $°59 | ooo'sce |°°° °° 
eu6 | pic | gz | Fee | Fee | T¥e | sez | ocoe | Es oos'zt | 0°89 | ooo'zze |: 
v26 | g'st | gigs | est | 6:29 | s:zz | Ise | 9:2t | ¢:2z | ooo'or | 9:9 | ooo'sce |: 
£66 | 2:8 T'te | ger | 622 | ete | ges | ase | 62z | Fe0'9 91g | o00'6oz |° 
O-00r | 8's z16 | 9:6 91g | # st | 29 | Sze | zoe | eoe'z 06% | ooo'9Tz |: 

O°00L | IT 686 | 27S ze6 | GL zsg | es | prs | 202 %'9¢ | ooo'stt |: 

O°00L | #°T 9°36 | 0O°¢ 9°86 | #6 zs | F2e | s'19 | EHP 8°6t | o00'gor 

0'001 | 32 926 | SL zoe | eat | 622 | G08 | 92% | 61g est | ooo'get 

0'001} 9's ¥96 | eZ tes | Ter | o92 | ger | aze | seo't 891 | 000'PPT |*: *zequid00q 
P66 | OL 616 | sat | tes | wzt | 219 | ose | zo | ozs 208 | 000'8ZT |*** tequeaoN 
oogtoor | FRR" | goo'or | PO8PF | coor | “OBBE oot on OL |rworo sed} Ae, | -ano'o sod 

repuy i JepugQ reapuy) rapuy qepuy) qunoy _ yunog “qyuOyW 

100‘01 TOO! Ta I anes Souyy 
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18301, 
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et 1°86 ras 2°98 1:96 0 0F aa 8°22 1°¥Z ze get |“ -equraydeg 
€°9 2°86 TST 9°82 £799 £°£% Z'el T°OL 8°83 el 6ST 
8 9°16 % LL ZL 8°98 re 9°06 8°91 6°21 6'£ SST 
gh 2°26 6°ST 8°92 aaa eee | 3°81 Z'61 zST OF TST 
Ee €°96 1°8 Z'88 0.98 2°29 Z'91 0°98 £°28 Lg 981 
ce 3°96 St £788 €°0F O° P81 9°92 11 o"g aa 
9°0 +66 OF a 0°82 9 ¥ 61 0'8% 90°F re SLT 
TEN oor | 62 1°26 z'9% 6°IL 92 1°95 PE ara 6EI 
9'T 5°86 oT 8°96 6:98 6°6S £°1Z 9'8€ 1°98 ard ZeI 
2:0 £°66 6'¢ ¥°86 9°2% 8°02 9°83 Z°8P £°9F 6° - Ze1 [+ + -xequraveqy 
oe °96 6% 9°€6 6 FE 89 z°61 $'6E 8°28 LT LT |‘ -requiaaon 

000'000'T | o00‘000'r | °° SPOT! goo'oog | GP | ooo‘aat | PPT | ooo'oe | 08,08 goo‘or | -serdmes | 

eee Erosion: | PPO | spoon dP nao 'pe pad | too'ot | Gogemg | oe 


ONINIVLNOD SAIdNVG JO TOVINGOUGT “"WO'O UAd LNOAOD IVIUGLOVG NI NOLLVINVA 


VAMVLLO ‘SI6I ‘Ts YAAOLOO ONIGNA UVAA MIIN SUAWUVA NI IIOO ‘@ GNV VISHLOVE IVLOL 
TAXT Savy 


RECORDING RESULTS 


TaBLe LXVII 


PASTEURISED MILK YEAR ENDING OCTOBER 831, 1915. OTTAWA 
BacTERIOLOGICAL 


205 


VARIATION IN BACTERIAL Count. PERCENTAGE OF SAMPLES CONTAINING 


Month. Under 20,008 Under ma) 01 Under eee Under 
10,000 50,000 50,000 100,000 | 100,000 500,000 500,000 
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 ING | ecoeeaes Nil 
February....| 33.3 66.7 100.0 Sn ere Nil 
March...... 43.8 56.2 160.0 NUS Gh: csteuaetsd t8 Nil 
Aprile iicie cae 25.0 75.0 100.0 Nake 4h sepia Nil 
May. «| 81.8 18.2 100.0 Nil | #sssan-4 Nil 
JUNE oss cane 57.9 21.1 79.0 21:0). || siewsee Nil 
Pub yes cece eee 57.1 38.1 95.2 Nil 95.2 4.8 100.0 
August....../ 50.0 50.0 100.0 Ni |) re os Nil 
September...} 31.6 63.2 94.8 5.2 100.0 Nil 
October..... 33.3 60.1 93.4 6.6 100.0 Nil 
Average..... 45.0 50.4 95.4 3.7 99.1 0.9 100.0 
Mean Mean VARIATION IN B. COLI PER C.cM. PERCENTAGE 
ee Eacteriat B. coli or SAMPLES 
ont —— per Under | ll to | Under | 51 to | Under | Over 
Ber gers |, See 10 50 50 100 1 100 
November... 25,000 3 88.3 11.7 100.0 ING > | te eye ts Nil 
December. . . 19,000 15 56.1 31.2 87.3 12-7 100.0 Nil 
January.....| 16,200 9 76.9 15.4 92.3 7.7 | 100.0} Nil 
February....} 18,000 3 93.4 6.6 | 100.0 ING yh cece Nil 
March...... 14,400 35 75.0 6.2 81.2 Nil 81.2 | 18.0 
April........ 17,000 6 91.7 8.3 | 100.0 Nak | scsces Nil 
May........ 13,000 7 86.3 13.7 | 100.0 Nil i} setecese Nil 
June........ 22,600 55 42.1 42.1 84.2 15.8 100.0 Nil 
DULY? 35 ieescsees 28,000 141 40.0 20.0 60.0 | 10.0 90.0 | 10.0 
August...... 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 
Ostober..... 18,700 6 86.7 13.3 | 100.0 NAD ceca a Nil 
Average..... 19,600 102 69.5 18.8 88.3 5.7 94.0 6.0 
CHEMICAL 
SaMPLES BELOW STANDARD. Genuine MILgs. 
nen Percentage of a ; Average Composition. Number 
onth. . . elow 8.5 ro) 
Deficient | Deficient |'per Cent Total Solids |Samples. 
in Fat. Total Not-fat Fat Solids. | Not-fat 
Solids. Solids Solids. . 4 
November.. 4.03 13.02 8.99 17 
December 3.85 12.81 8.96 16 
January. 3.79 12.70 8.99 13 
February 3.73 12.63 8.90 15 
March 3.62 12.59 8.97 16 
April. 3.65 12.46 8.81 12 
May. 3.70 12.67 8.97 22 
June. 3.84 12.73 8.89 19 
July. 3.77 12.52 8.75 21 
August. 3.86 12.57 8.71 16 
ept..... 3.97 12.74 8.77 19 
October..... 3.93 12.97 9.04 15 
Total 
AVOTAZC cis5:5|) sasseniers, | se eis 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 bé 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 LX VI 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.S. 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. Ayers. 28th Rpt. Bureau Animal Ind., U.S. A. 228. 

8. 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 

12 

13 


i 


. Schaffer. Landw. Jahrbuch der Schweiz, 7, 72. 
. Wisconsin Expt. Stat. Annual Rpts. 1895 and 1898. 
. 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: 


GC. ea oO SC Oe 20 grams 
PEPtOne. aia nti COS atc uae Saee suede 20 grams 
Bile salt commercial............... een 2 5 grams 
Water ines cities Shwe weet cinerea nree 1¢C0O 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 c.ems. 
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: 


Methyl violet...............2....... 0.025 gram 
Sodium chloride..................... 1.0 gram 
Sodium sulphate..................... 8.0 grams 
Glycerine... 2.06... cece cece ee eee 30 ¢.cms 
Distilled water..................000, 160 c.cms 


The solution should be freshly filtered. 
207 


208 APPENDIX 


Ponder’s Stain. Kinyoun’s modification. 


Toluidine blue................20000- 0.1 gram 

Aare Dwovax et tiiaenaschin Os See hed a 0.01 gram 
Methylene blue..................... 0.01 gram 
Glacial acetic acid.:................. 1.0 c.cm. 

95 per cent alcohol................... 5.0 c.cms. 
Distilled water..............2.0--00. 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 threé 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 cent and, if the acidity is higher than this, a portion 
of the casein will be precipitated during sterilisation. 


209 


APPENDIX 


GL PLE | F'9E | SSE | CFE | SE | Toe | OTE | 66S | 68S | 8°24 | 8°9S | 24°S% | L°Fs | 2°es | 9°3s | 9'1s GL 
bL @ 46 | coe | Loe | I've | Oke | 6 le | 6°08 | 86a | 2°86 1 212 | 9°92 | O-es | O'Fe | Ose | Gag | o*1z 24 
€L O°LE | O'9E | BFE | 6 SE | Sse | Bi TE | 2°08 | L4°6% | 98S | GL | G'9S | G'St | F's | FES) F°2S | SIs eZ 
GL 8'9E | 8'SE | LE | Lee | 9°7E | 9'TE | GO | G°6S | F'8S | HLS’ | F'9S | SSS | EFS | Ses'| Ess | 3'°1z GL 
TZ 2°9§ | 9°GE | O'FE | OEE | GSE | STE | FOS | F'GS | ESS | 4s | G'9S | SSS | SHS | oss | Sse | Tis TL 
OL $'9€ | GSE | SPE | PEE | bse | ETE | EOE | Z'6S | J 8z | S'24e | 19s | 1'Ss | Tbs | Tes] Tae | Ole OL 
69 €'9€ | ESE | EVE | Eee | SSE | Sie | fos | 1'6s | T°8s | T'L4z | 0'9% | O'SS | OFZ | O'S | 0°22 | 6°02 69 
89 G'9E | SSE | SPE | See | TSE | Te | L'0e | O'6S | 0°82 | O'LZ.| 6'SS | 6'FS] | BES | 82S | BIZ | 2°02 89 
L9 T'9E | O'SE | O'FE | O'SE | O'SE | G'OE | 66S | 8°8S | B'Le | 89S | L4°SS | LFS | LES | 24°3S | L's | 9'0% 249 
99 6'SE | 6 FE | G'S | G'ce | STE | 8°08 | 86S | L4°8S | 2°22 | £°9S | 9'SS | 9'FS | 9'ES | 92s | 9'1Z | G'Oe 99 
g9 8'cs | BPs | See | L°se | LTE | LOE | 9'6S | 9'8S | 9'2% | 9°9% | F°Sa | G'FS | Ges | Sse | SIZ | FOZ go 
$9 Q'ce | O'FE | 9'e8 | Ose | STE | SOs | G'6z 1 G'8s | Gi24e | G'9S | FSS | PHS | FSS | FSS | PIs | S'0% $9 
29 GSE | GPE | Gee | Gos | FTE | FOS | F'6S | FSS | HLS | E'9S | ESS | SHS | E'S | E's | E's | 2°02 £9 
69 €°SE | € Fe | See | Ese | Sle | Oe | €°6S | €°8S | 4S | 9% | BSS | SPS | SES | T'3S | SIs | 2:0z (4)) 
19 T'Sé | T'#€ | Tes | L's | Tre | L'o0e | 1'6% | 1°8% | T2409 | 1°93 | T'S | T'hs | Tes | 173s | TIS | 170% 19 
09 O'SE | O'FE | OSE | O'SE | O'TE | O'OE | 0'6Z | O'8S | O'LZ | 0'9Z | O'SS | O'F!S | O'ES | 0'4S | O'TS | 0°02 09 
6¢ 8'PE | 6 ee | 6°SE | 6'1E | 6'OE | 6'6s | 68a | 62% | 69S | 6'SS | 6'FS | 6'ES | 6'SS | 61S | 6'0% | 6'6T 6g 
8¢ L'be | Lee | 2°se | 2°1e | 8'0E | 86s | 88S | 82S | 8°9S | 8°SS | 8'FS | 8'SS | 8'SsS | 61S | 6°02 | 6'6T 8g 
Lg | S'PE | 9°eE | 9'sE | OTE | 9°08 | L°6 | L°8S | L°2G | L'9S | LSS | LHS | L°SS | BSS | BIS | 80S | 8'6I Lg 
9¢ U'vE | GSE | GSE | G'Te | GOS | 96% | 98S | 9'Le | 9°93 | 9°SS | 9° FS | 9°ES | L°GS | LTS | 2°08 | 2°61 9¢ 
gg eve | b'SE | F'se | FTES | V'Ok | 6s | S'8s | GL] | S'9% | S'Ss | O'S | 9'ES | 9°% | 91s | 90% | 9'6T s¢ 
og GPE | ESE | ESE | ETE | SOE | S'6S | F'8% | F'LS | F'9S | FSS | G'FS | GES | G'es | GIs | $'0s | SET iat 
eg | O'Fe | Te | Tse | ZTE | s:08 | Z'6z | &'8a | SLs | '9% | SSS | HFS | SS | F'S% | FIs | FOS | FET es 
oo | 6'ee | OSE | O'SE | T'Te | TOS | 1'6S | F'8s | SLs | 9S | S'9S | S' FS | SSS | ESS | SIs | '0S | F'6T eg 
ig 8's | 6°3E | 6'Te | O'TS | O'OE | T°6s | T°8S | T'2e | 3°93 | BSS | B' Fs | SSS | ESS] | SIs | SOS | €'61 1g 
o¢ L°S€ | 8°ZE | STE | 60S | 6'°6Z | 0'6Z | 0'8Z | O'LS | T'9% | T°SZ] TFS | SES | BBS | STS | Z'0S | SET os 
6P 9'€€ | L°3E | LTE | LOS | 8°6S | 68S | 0'8S | O'2S | T°9S | T'SS | TFS | SES | BSS | SIS | Z'0S | ZG 60 
8P G'ee | 9°3E | 9 TE | 9°08 | 4°6S | 8°8S | 62S | 6'9S | 0'9 | O'S | O'FS | T'ES | 1'%s | TTS | 1°0% | T'6T 8F 
Lv bes | GSE | STE | GOS | 9°6Z | 2°83 | 8'LZ | 8'9Z | 6'SS | 6'FS | O'FS | O'SS | 0'ZS | O'TS | O'0% | T'6I Ld 
oF ESE | PSE | FE | GOS | 9'6S | 4°8% | 8'LZ | 8'9ST | 6'SS | 6'FS | 6'ES | 6°ZS | 0'ZS | OTS | 0'02 | O'6T 9F 
SP GE | €'SE | FTE | F'0E | G6 | 9'8% | LL] | 2°9% | BGS | 8 FS | B8'es | 63S | 6 TS | 6°03 | 6'6I | O'6T cP 
“qe ge FE ge se Té 0g 63 86 LE 9% go SG &@ (<4 1<4 0G “98 q 
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210 


APPENDIX 


TABLE 
FOR CALCULATION OF TOTAL SOLIDS 


ACCORDING TO BaBcock. 


LacToMETER READING 


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APPENDIX 211 


LXIX 


FROM FAT AND LACTOMETER READING 
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212 APPENDIX 


TABLE 


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LacToMETER READING 


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APPENDIX 213 


LXxX 


FAT AND LACTOMETER READING 
Droop RicHMOND 


at 60° F. 


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214 APPENDIX 


Taste LXXI 


TABLE FOR CONVERSION OF CUPROUS OXIDE (Cu,0) AND 
COPPER TO LACTOSE 


MILLicRaMs 
Cu20 Cu Lactose Cu.0 Cu Lactose Cuz0 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 |} 208.8 | 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 
127.1 | 113 81.3 || 172.2 | 153 | 111.1 217.2 | 193 | 141.6 
128.2 | 114 82.0 || 173.3 | 154 | 111.9 || 218.3 | 194 | 142.3 
129.4 | 115 82.7 || 174.5 | 155 | 112.6 || 219.5 |] 195 | 143.1 
1380.5 | 116 83.5 || 175.6 | 156 | 113.4 || 220.6 | 196 | 143.9 
131.7 | 117 84.2 || 176.7 | 157 | 114.1 || 221.8 | 197 | 144.6 
1382.8 | 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 
185.1 | 120 86.4 || 180.1 | 160 | 116.4 |} 225.2 | 200 | 146.9 
186.2 | 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 
1388.4 | 123 88.7 || 183.4 | 163 | 118.6 || 228.5 |} 2038 | 149.2 
139.6 | 124 89.4 || 184.5 | 164 | 119.4 || 229.6 | 204 | 150.0 
140.7 | 125 90.1 185.7 | 165 | 120.2 || 280.7 | 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 | 123.2 |} 285.2 | 209 | 153.7 
146.4 | 130 93.8 || 191.4 | 170 | 128.9 || 236.4 | 210 | 154.5 
147.5 | 131 94.6 || 192.5 | 171 124.7 || 237.5 | 211 155.2 
148.6 | 132 95.3 || 198.6 | 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 || 243.1) 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 | 139 | 100.5 || 201.5 | 179 | 180.8 || 246.5 | 219 | 161.2 


APPENDIX 215 


Taste LXXI—Continued 


TABLE FOR CONVERSION OF CUPROUS OXIDE (Cu,0) AND 
COPPER TO LACTOSE 


MILLiGcRaMs 


Cuz0 Cu Lactose Cur0 Cu Lactose Cu0 Cu Lactose 


247.7 | 220 | 161.9 || 292.7 | 260 | 192.5 |) 337.8 | 300 | 224.4 
248.8 | 221 | 162.7 || 298. 261 | 193.3 || 338.9 | 301 | 225.2 
249.9 | 222 | 163.4 |) 204. 262 | 194.1 || 340.0 | 302 | 225.9 
251.0 | 223 | 164.2 || 296. 263 | 194.9 |) 341.1 | 303 | 226.7 
252.1 | 224 | 164.9 || 297. 264 | 195.7 || 342.2 | 304 | 227.5 
253.3 | 225 | 165.7 || 298. 265 | 196.4 || 343.4 | 305 | 228.3 
254.4 | 226 | 166.4 || 299. 
255.5 | 227 | 167.2 || 300. 
256.6 | 228 | 167.9 || 301. 
257.7 | 229 | 168.6 |} 302. 


8 
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258.9 | 230 | 169.4 Ae 270 | 200.3 || 349.0 | 310 | 232.2 
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3 
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7 
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260.0 | 231 | 170.1 || 305. 
261.1 | 232 | 170.9 || 306. 
262.2 | 233 | 171.6 || 307. 
263.3 | 234 | 172.4 || 308. 
264.5 | 235 | 173.1 |) 309. 
265.6 | 236 | 173.9 || 310. 
266.8 | 237 | 174.6 || 311. 
267.9 | 288 | 175.4 || 313. 
269.0 | 239 | 176.2 || 314. 


-270.2 | 240 | 176.9 || 315.3 | 280 | 208. 
271.3 | 241 | 177.7 |] 316.4 | 281 | 209. 
272.4 | 242 | 178.5 || 317.5 
273.5 | 243 | 179.3 || 318.6 
274.6 | 244 | 180.1 || 319.7 
275.8 | 245 | 180.8 |} 320.9 | 285 | 212. 
276.9 | 246 | 181.6 || 322.0 
278.1 | 247 | 182.4 |) 323.1 
279.2 | 248 | 183.2 || 324.2 
280.3 | 249 | 184.0 || 325.3 | 289 | 215. 


281.5 | 250 | 184.4 || 326.5 | 290 | 216. 
282.6 | 251 | 185.5 || 327.6 | 291 | 217. 
283.7 | 252 | 186.3 || 328.7 | 292 | 217. 
284.8 | 253 | 187.1 |) 329.8 | 293 | 218. 
286.0 | 254 | 187.9 || 330.9 | 294 | 219. 
287.1 | 255 | 188.7 || 332.1 | 295 | 220. 
288.2 | 256 | 189.4 || 333.2 | 296 | 221. 
289.3 | 257 | 190.2 || 334.4 | 297 | 221 
290.4 | 258 | 191.0 |] 335.5 | 298 | 222 
291.5 | 259 | 191.8 || 336.7 | 299 


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216 APPENDIX 


TaBLE LXXI—Continued 


TABLE FOR CONVERSION OF CUPROUS OXIDE (CuO) AND 
COPPER TO LACTOSE 


MILLIGRAMS 


Cu:0 Cu Lactose Cu0 Cu Lactose Cu20 Cu Lactose 


382.7 | 340 | 255.7 || 405.3 | 360 | 272.1 || 427.9 | 380 | 289.1 
383.8 | 341 | 256.5 || 406.4 | 361 | 272.9 || 429.0 | 381 | 289.9 
385.0 | 342 | 257.4 || 407.5 | 362 | 273.7 || 4380.1 | 382 | 290.8 
886.1} 343 | 258.2 || 408.6 | 363 | 274.5 |} 481.2 | 383 | 291.7 
387.2 | 344 | 259.0 || 409.7 | 364 | 275.3 || 482.3 | 384 | 292.5 
388.4 | 345 | 259.8 || 410.9 | 365 | 276.2 || 483.5 | 385 | 293.4 
889.5 | 346 | 260.6 |} 412.0 | 366 | 277.1 || 434.6 | 386 | 294.2 
390.6 | 347 | 261.4 || 413.1 | 367 | 277.9 || 435.8 | 387 | 295.1 
391.7 | 348 | 262.3 || 414.2 | 368 | 278.8 || 436.9 | 388 | 296.0 
392.8 | 349 | 263.1 || 415.3 | 369 | 279.6 || 438.0 | 389 | 296.8 


394.0 | 350 | 263.9 || 416.5 | 370 | 280.5 || 439.2 | 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 | 3859 | 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 

casein, 194, 208 
Ageressins, 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 
tate 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 


Cc 

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. diphtherie, 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 


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, ® 
table, 214 


219 


Lecithin, 20 
Leucocytes, 173 
Lipase, 22 

Litter, bacteria in, 99 


M 

Media, acidity of, 119 

esculin, 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 


oO 
Opsonins, 27 


P 

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 


8 
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 


T 
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 


Vv 
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 


Aitkens, 30 
Alexander, 162 
Anderson, 167 
Andrewes, 150 
Arthus, 28 
Ayers, 106, 194 


B 
Babcock, 22, 92, 181 
Backhaus, 97, 99, 100 
Balley, 93 
Bang, 190 
Barthol, 130 
Batchelder, 94 
Béchamp, 17, 22 
Beger, 39 
Bellei, 91 
Benzynski, 55 
Berberich, 32 
Besredka, 28 
Block, 166 
Blyth, 21, 
Borden, 121 
Boseley, 71, 81 
Bosworth, 7, 8, 11, 15, 20 
Boussingault, 50 
Bowhill, 156 
Breed, 128, 171, 178, 179 
Brew, 129 
Briot, 15 
Broadhurst, 155 
Browning, 160 
Buckley, 174 
Bunge, 34 


221 


Burow, 8 
Burr, 32 


Cc 
Cameron, 12 
Capps, 152 
Chamot, 140 
Chappelean, 198 
Chittenden, 8 
Clark, 121, 145 
Cook, 37 
Conn, 108, 117, 121, 124 
Corbett, 191 


D 

Davis, 152 
Dean, 156 
Delépine, 115, 164, 166, 168, 180, 

181 
Désmouliers, 22 
Doane, 171, 174 
Dodd, 167 
Doll, 39 
Duclaux, 15 
Dugelli, 198 


E 
Eastwood, 167, 168 
Eckles, 38, 42, 45, 50 
Ellenberger, 8 
Engling, 53 
Ernst, 171, 172 
Esten, 108 
Evans, 190 
Eyre, 157 


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 
McFadyean, 190 
Melia, 160 
Merklen, 22 
Michaelis, 171 
Miessner, 28 


Miller, 75, 180, 171 


NAME INDEX 223 
Monier-Williams, 83 Rueduger, 154 
Morgan, 161 Rullman, 23 
Morgen, 39 Rupp, 189 
Morgenrath, 17 Russell, 22, 92, 174, 178 
Moro, 22 
Mule, 22 8 
Muller, 152 Sackur, 8 
Salge, 196 
N Savage, 101, 143, 147, 150, 158, 171, 


Nobécourt, 22 
North, 121, 185 


O 
O’Brien, 162 
Olsen, 50 
Orr, 98, 100, 137, 162 
Otto, 27 

P 
Painter, 8 


Park, 94, 102, 162 
Pennington, 105, 111 
Peter, 91, 198 
Porch, 23 

Prescott, 178 


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 


176, 178, 179 
Schaffer, 55 
Schardinger, 89 
Schern, 89 
Schmidt, 14 
Schnorf, 54 
Scholberg, 162 
Schrewsbury, 82 
Schroeder, 182 
Schroeter, 198 
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 


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 


Ww 


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