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Full text of "Standard methods for the bacterial examination of milk and the bacterial examination of air"

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LIBRARY 

NEW YORK STATE VETERINARY COLLEGE 

[THACA, N. Y. 




Standard Methods for the Bacterial 
Examination of Milk 

Standard Methods for the Bacterial 
Examination of Air 








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/cu31924003598780 



Standard Methods for the 
Bacterial Examination of Milk 



Standard Methods for the 
Bacterial Examination of Air 



By Committees of the 
Laboratory Section 
American Public 
Health Association 



Cornell University Library 
SF 253.A51S 



Standard methods for the bacterial exami 



3 1924 003 598 780 



Standard Methods 



for the 



Bacterial Examination of Milk 



and the 



Bacterial Examination of A 



lr 



By Committees of the Laboratory Section 
American Public Healtb Association 



Reprinted from the American Journal of Public Hygiene, 
Vol. VI, No. 3, August. 1910. 



s r-* 






REPORT OF THE COMMITTEE ON STANDARD METHODS 
OF BACTERIAL MILK ANALYSIS. 



The Committee on Bacterial Milk Analysis respectfully 
submits the following report for the consideration of the Labo- 
ratory Section of the American Public Health Association. 

I. 
Historical. 

At the meeting of the Laboratory Section of the American 
Public Health Association in Boston in 190O, at the suggestion 
of Prof. S. C. Prescott, of the Massachusetts Institute of Tech- 
nology, a Committee was appointed to study the various methods 
used for the bacteriological examination of milk and to recom- 
mend a uniform procedure. This committee consisted of Prof. 
S. C. Prescott, Chairman; Dr. W. H. Park, Dr. F H. Slack, Prof. 
H. L. Russell, Prof. C. E. Marshall, Prof. H. C. Harrison and 
Dr. E. C. Levy. 

A circular letter asking for information as to existing methods 
and technique used in bacteriological milk examinations was 
sent to leading bacteriologists of the United States and Canada. 
Many of these did not make such examinations, and the replies 
of others who did, showed wide variations of procedure on most 
important points, such as plating, composition of media, incuba- 
tion temperature, length of incubation, etc. 

At the Mexico City meeting in 1906, Prof. H. L. Russell, of 
the University of Wisconsin, was appointed Chairman of the 
Committee, but in June, 1907, he asked to be relieved of the 
chairmanship, and by the vote of the Council of the Laboratory 
Section, Dr. F H. Slack, of the Boston Board of Health Labora- 
tory, was appointed Chairman. 

At the Atlantic City meeting in 1907 the Committee presented 
a preliminary statement 1 , going into the subject matter in 
considerable detail. Dr. E. C. Levy withdrew as a member 
of the Committee and Dr. B. H. Stone was appointed in his 
stead, otherwise the Committee was continued unchanged. 



4 STANDARD METHODS 

At the Manitoba meeting in 1908 a report of progress was 
submitted 2 covering some points on which no recommendations 
were made in the preliminary statement; the Committee was 
continued without change. The two preliminary reports of the 
Committee have been favorably received and the technique 
recommended has been generally adopted. This final report is 
practically but a restatement of methods hitherto tentatively 
recommended. 

Acknowledgments are due to, and the Committee wishes to 
express its appreciation of the hearty co-operation and aid given 
by, the following persons. 

B. L. Arms, Boston, Mass. A. P. Norris, Cambridge, Mass. 

D. H. Bergey, Philadelphia, Pa. C. E. North, New York, N. Y. 
S. S. Buckley, College Park, Md. Z. Northrup, E. Lansing, Mich. 
W. M. Campbell, Boston, Mass. M. E. Pennington, Phila., Pa. 
H. W. Conn, Middletown, Conn. S. C. Prescott, Boston, Mass. 

F. R. Eilinger, Rochester, N. Y. B. R. Rickards, Columbus, Ohio. 

B. Farrand, E. Lansing, Mich. L. A. Rogers, Washington, D. C. 
L. W. Fetzer, College Park, Md. M. J. Rosenau, Washington, D. C. 
R. G. Freeman, New York, N. Y. W. G. Savage, Colchester, Eng. 
H. A. Harding, Geneva, N. Y. W. O. Scott, Providence, R. I. 

E. G. Hastings, Madison, Wis. T. Smith, Boston, Mass. 

P. G. Heinemann, Chicago, 111. L. P. Sprague, Burlington, Vt. 

H. W. Hill, Minneapolis, Minn. W. A. Stocking, Ithaca, N. Y. 

C. Hoffman, Madison, Wis. W. R. Stokes, Baltimore, Md. 

D. D. Jackson, Brooklyn, N. Y. L. VanDerLeck, McDonald Coll., 
H. Moak, Brooklyn, N. Y. P. Q. 

A. R. Ward, Berkeley, Cal. F.F.Wesbrook, Minneapolis, Minn. 

C. E. A. Winslow, Boston, Mass. H. L. Wilcox, New York, N. Y. 

Signed, F. H. Slack, Chairman. 

W. H. Park, C. E. Marshall, 

B. H. Stone, F. C. Harrison, 

H. L. Russell. 

II. 

Collection of Samples. Technique and Apparatus. 

Quantities of Milk Required for Analysis. The mini- 
mum quantity of milk necessary for making the ordinary bac- 
teriological examination is ten cubic centimeters. When mak- 
ing examinations for certified milk, if possible a pint or quart 
bottle should be taken and brought to the laboratory unopened. 



BACTERIAL EXAMINATION OF MILK O 

Collecting Apparatus. In collecting samples for bac- 
teriological examination it is essential that the sample be taken 
and kept in such a manner as to prevent either any addition of 
bacteria from without or multiplication of the bacteria originally 
present. 3 Bottles, tubes, pipettes, etc. used in the collection 
of samples, besides being washed, shall be sterilized with dry 
heat for an hour at about 150° C, or to the charring point of 
cotton. 

In the selection of "certified milk" samples it is recommended 
wherever possible that an unopened bottle be taken, placed in 
a suitably iced case and brought at once to the laboratory. 

Samples of "market milk" may be collected in the same 
manner as water samples, in sterile, wide-mouthed, glass- 
stoppered four ounce bottles; the case in which they are carried 
being well iced. The principal difficulty encountered in this 
method is in transferring the sample from the original container 
to the bottle, and the various string and wire devices by means 
of which the bottle is immersed in the original container are 
objectionable both on account of the labor of preparing such an 
outfit and also on account of the coating of milk left on the 
outside of the bottle when the sample has been taken. 

An apparatus designed for the use of test tubes as con- 
tainers is recommended as superior to one designed for bottles. 4 

Such a case for carrying the samples may be made of copper 
with double walls, interlaid with half inch felting, outside 
measure 19 x 9 x 5 inches, inside IS x S x 4 inches, divided into 
three compartments, the central one 6 x S x 4 inches for the 
samples, the others each 5x8x4 inches for ice. When iced 
and closed a constant temperature of 34° to 36° F. is main- 
tained; salt should not be used with the ice or the samples will 
be frozen. A layer of absorbent cotton will protect the ice 
from the air when the box is opened for a few minutes. Bits of 
this cotton are also useful when taking temperatures for quickly 
wiping the adhering milk from the thermometer. 

The samples are carried in cotton-stoppered test tubes 
6 x % inches, the compartment holding eight racks of four tubes 
each. Holes in the bottom of the partitions allow the water to 
circulate freely about the lower ends of the tubes. 



STANDARD METHODS 

The test tube racks are made of copper tubing weighted with 
a strip of lead and padded with rubber. When collecting or 
plating but one rack or a single tube should be removed at 
a time and the box closed so the other samples will not be 
exposed to the outside temperature. 

Sterile, straight sided, glass pipettes 18 x 3-8 inches with 
blunt pipette openings 1-8 inch in diameter at the lower ends are 
used in transferring the samples from the bottles, cans or coolers 
to the test tubes; these are carried under the sample case in a 
detachable copper case 19 x 4 x 3J^ inches, adapted for ster- 
ilizing and divided into two compartments, the upper one for 
clean sterile pipettes, the lower for pipettes after use, a sterile 
pipette being used for each sample. 

The use of the test tube for the collection of milk samples is 
recommended instead of bottles for the following reasons, 
dependent in most cases on the long, slender shape of the tube. 

1. Economy of floor area in the collecting case. 

2. The facility for maintaining low temperature by the cir- 
culation of the ice water about the lower ends of the tubes, thus 
giving uniformity in the treatment of the specimens. 

3. The case with which all the usual washing, sterilizing and 
general handling of test tubes can be done, since the test tube is 
a regular piece of apparatus involving no departure from the 
routine in all the ordinary manipulations. 

With samples kept properly iced in this particular form of 
case there is practically no change in the bacterial content even 
for 24 hours, the counts varying hardly more than might be 
expected in duplicate plates. It is recommended, however, that 
examination of the samples be proceeded with as quickly as 
possible after the collections are made. 

Identification of Samples. When bottles are used identi- 
fication numbers should be etched on both bottle and stopper. 
Test tubes should be labelled or etched and numbered. 

A complete record of the samples taken, giving date, time, 
place, name of party from whom sample is taken, name of col- 
lector, temperature of milk, character of original container, 
(tank, can, bottle), etc., should be written opposite duplicate 
numbers in a blank book or pocket card catalog, or this informa- 



BACTERIAL EXAMINATION OF MILK / 

tion may be written on small tags and tied or wired to the cor- 
responding test tube or bottle. 

Temperature. The temperature should be taken imme- 
diately after taking the sample for analysis, while the milk is 
still thoroughly mixed. 

If it is desired to take the temperature of "certified milk" this 
should be done when the sample is taken but from another bottle. 

A floating thermometer graduated to the Fahrenheit scale is 
most convenient and the temperature should be expressed to the 
nearest degree. It is necessary to standardize the thermometer 
for at least ten degrees on each side of the legal temperature 
limit. A quickly registering thermometer should be left for at 
least one minute in the milk and read as soon as removed. 

Representative Samples. — Care should be taken to secure a 
sample which is truly representative of the milk to be examined. 

One of several methods for mixing the milk may be used, com- 
parison having shown the results to be practically the same. 3 

1. Pouring the milk into a sterile receptacle and back. 

2. Shaking the milk thoroughly with receptacle turned up- 
side down (this may be done where the can or bottle is tightly 
stoppered or capped and is not so full as to prevent thorough 
agitation.) 

3. In open tanks in stores it is allowable to stir thoroughly 
with the long handled dipper generally found in use. 

4. Where the test tube collecting case is used thoroughly 
reliable results are secured by first shaking the can or bottle and 
then stirring with the large pipette before taking the sample, 
care being taken to close the upper end of the pipette with the 
finger so that no milk enters until after mixing, or the pipette 
may be emptied after stirring before the sample is taken. 

5. For certified milk samples it is recommended that on 
arrival at the laboratory the bottle be opened with aseptic pre- 
cautions and the milk thoroughly mixed by pouring back and 
forth between the original bottle and a sterile bottle. Another 
method is to mix as thoroughly as possible by agitation for two 
minutes in the original container before opening same. 

The interval between collection and analysis. — Generally 
speaking the shorter the time between collection and examina- 
tion of milk samples the more accurate will be the results. For 



8 STANDARD METHODS 

routine work the attempt should be made to plate within four 
hours of the time of collection. 

Too much stress cannot be laid on keeping the samples prop- 
erly iced during this interval. They should be kept below 40° F., 
but care should be taken that they are not frozen. 

III. 

Media. Method of making. 5 

Agar. The standard medium for determining the number 
of bacteria in milk shall be agar 1%, reaction +1.5, Fuller's 
scale, made as follows: 

1. Boil 10 grams of thread agar in 500 cc. of water for half 
an hour and make up weight to 500 g. or digest for 10 minutes 
in the autoclave at 110° C. Let this cool to about 60° C. 

2. Infuse 500 g. finely chopped lean beef for twenty-four 
hours with its own weight of distilled water in the refrigerator. 

3. Make up any loss by evaporation. 

4. Strain infusion through cotton flannel, using pressure. 

5. Weigh filtered infusion. 

6. Add Witte's peptone 2%. 

7. Warm on water bath, stirring until peptone is dissolved 
and not allowing the temperature to rise above 60° C. 

8. To the 500 grams of meat infusion (with peptone) add 500 
grams of the 2 per cent, agar keeping the temperature below 
60° C. 

9. Heat over boiling water (or steam) bath thirty minutes. 

10. Restore weight lost by evaporation. 

11. Titrate after boiling one minute to expel carbonic acid. 

12. Adjust reaction to final point desired +1.5 by adding 
normal sodium hydrate. 

13. Boil two minutes over free flame constantly stirring. 

14. Restore weight lost by evaporation. 

15. Filter through absorbent cotton or coarse filter paper, 
passing the filtrate through the filter repeatedly until clear. 

16. Titrate and record the final reaction. 

17. Tube (10 c. c. to a tube) and sterilize in autoclave 1 hour 
at 15 lbs. pressure or in the streaming steam for twenty minutes 
on three successive days. 



BACTERIAL EXAMINATION OF MILK 9 

All variations from agar media made as described shall be 
considered as special media. The above medium is recom- 
mended as giving the highest and most uniform counts so far as 
our comparative work has extended. Methods by which the 
other media mentioned in this report were made are as follows: 

Bouillon. Infuse 500 g. finely chopped lean meat 24 hours 
with 1,000 c. c. distilled water in refrigerator. Restore loss by 
evaporation. Strain infusion through cotton flannel. 

Add 1% peptone. Warm on water bath, stirring until 
peptone is dissolved. 

Heat over boiling water, or steam bath thirty minutes. 
Restore loss by evaporation. 

Titrate, adjust reaction to +1% by adding normal sodium 
hydrate. 

Boil two minutes over a free flame, constantly stirring. 
Restore loss by evaporation. 

Filter through absorbent cotton, passing the liquid through 
until clear. Titrate and record final reaction. Tube, using 10 
c. c. to each tube. Sterilize. 

Aesculin Bile Salt Medium. (Liquid). 6 

Weigh out. 

1 or 2% of Witte's peptone. 
.5% Sodium taurochlorate (commercial). 
.1% aesculin. 
.05% Ferric citrate. 
100 c. c. tap water. 

After steaming 15 to 30 minutes the medium is filtered and 
filled into test tubes and sterilized (fractional sterilization). 

Preparation of Aesculin Bile Salt Agar. 8 

The directions for making a liter of aesculin bile salt agar 
are as follows: Boil until dissolved 15 grams of agar, 2.5 grams 
commercial bile salt, and 10 grams peptone (Witte) in 1,000 c. c. 
of distilled water. Neutralize with a normal solution of sodium 
hydrate. Cool below 60° C, add the whites of two eggs or a 
sufficient quantity of a solution of albumen, bring to the boil 
and filter as soon as the albumen has coagulated properly. Try 
the acidity and neutralize if necessary, and then add to the clear 



10 STANDARD METHODS 

hot filtrate — 1 gram aesculin (Merck) and 1 gram iron citrate 
scales (Merck). After these substances are dissolved test the 
acidity with decinormal soda solution. It will be found to be 
about +0.6, as a solution of 1 gram iron citrate scales in 1,000 
c. c. water gives an acidity of +0.56. In case the acidity is too 
high add alkali until the reaction is +0.6, and if the acidity is 
too low add more iron citrate until the reaction is +0.6. By 
following these directions exactly satisfactory and even results 
will be obtained. We have to emphasize here the different 
manner of neutralization from that recommended for ordinary 
media by the Committee on Standard Methods of the American 
Public Health Association, as the procedure outlined above is 
absolutely necessary. The main point of our reaction is the 
forming of the black colored salt in sufficient quantity to form 
as easily visible field. 

Lactose (or Dextrose) Litmus Agar, made as ordinary agar 
with the addition of 1% lactose (or dextrose) to the medium 
just before sterilization. Reaction shall be made neutral to 
phenolphthalein. If the medium is to be used in tubes the 
sterilized azolitmin solution shall not be added until just before 
the final sterilization. If the medium is to be used in Petri 
dishes the sterilized azolitmin shall not be added to the medi- 
um until it is ready to be poured into the dishes. 

Whey Agar. A liter of fresh skimmed milk at 41° C. is 
loppered by adding sufficient rennet (about 1 c. c. of liquid 
rennet in 20 c. c. of distilled water). After the curd is firm it is 
cut in fine pieces and placed in steam for forty minutes. It is 
then strained through muslin to remove the curd. The reaction 
of the whey is adjusted to +1.5 acid with the standardized 
NAOH and 1% of dry peptone and 1.5% of finely shredded agar 
is added. It is then placed in the steam for 1 hour. The acidity 
is readjusted to +1.5%. It is then cooled to 60° C. and clarified 
with egg. Counterpoised and boiled over a free flame for 5 
minutes. Filtered through cotton or a hot, washed plaited 
filter paper, tubed, sterilized 15 minutes for 3 successive days 
in steam. 



Commercial bile-salt may be obtained from Baird & Tatlock, Cross Street, Hatton 
Garden, London, England, costing about S2.50 per lb. 



BACTERIAL EXAMINATION OF MILK 11 

IV. 

Plating. Apparatus. Technique. 

Plating apparatus — For plating it is best to have a water 
bath in which to melt the media and a water jacketed water bath 
for keeping it at the required temperature; a wire rack which 
should fit both the water baths for holding the media tubes; a 
thermometer for recording the temperature of the water in the 
water jacketed bath, sterile one c. c. pipettes, sterile petri 
dishes, and sterile dilution water in measured quantities. 

Dilutions — Ordinary potable water, sterilized, may be used 
for dilutions. Occasionally spore forms are found in such water 
which resist ordinary autoclave sterilization; in such cases dis- 
tilled water may be used or the autoclave pressure increased. 
With dilution water in eight-ounce bottles calibrated for ninety- 
nine cubic centimeters and in test tubes calibrated for nine cubic 
centimeters, all the necessary dilutions may be made. 

Short, wide-mouthed "Blakes" or wide mouthed French 
square bottles are more easily handled and more economical of 
space than other forms of bottles or flasks. 

Eight ounce bottles are the best, as the required amount of 
dilution water only about half fills them, leaving room for 
shaking. Long-fibre, non-absorbent cotton should be used for 
plugs. It is well to use care in selecting cotton for this purpose 
to avoid short fibre or "dusty" cotton, which gives a cloud of 
lint-like particles on shaking. Bottles and tubes should be 
filled a little over the 99 c. c. and 9 c. c. marks to allow for loss 
during sterilization. 

The dilutions recommended are 1-10, 1-100, 1-1,000, 1-10,000, 
1-100,000 and 1-1,000,000. 

For certified milk the 1-100 dilution should be used, while 
1-100 and 1-10,000 will usually be found best for market milk. 

The 1-10 dilution is prepared by shaking the milk sample 
twenty-five times and then transferring 1 c. c. of the milk to a 
test tube containing 9 c. c. of sterile water. 

The 1-100 dilution is prepared in the same way, except that 
a bottle with 99 c. c. of sterile water is substituted for the test 
tube. 



12 STANDARD METHODS 

The 1-1,000 dilution is prepared by first making the 1-100 
dilution, shaking twenty-five times and transferring 1 c. c. of 
the dilution to a test tube containing 9 c. c. of sterile water. 

The 1-10,000, 1-100,000 and 1-1,000,000 dilutions are made 
in the same manner by dilutions of 1-100, 1-1,000 and 1-10,000 
dilutions, 1 c. c. to 99 c. c. of sterile water. 

It is recommended that that dilution be used which will pro- 
duce about 200 7 colonies to a plate, ranging from 40 to 200; 
where a 1-10 dilution exceeds this number the 1-100 dilution is 
more accurate, etc. The number of bacteria present, may, if 
desired, be approximately estimated before dilutions are made 
by direct microscopic examination of a properly prepared sedi- 
ment. Otherwise, it is necessary to make a range of dilutions, 
thereafter selecting for record the count obtained on that plate 
which yields between 40 and 200 colonies. 

Plating whole milk is unreliable, whatever quantities be used, 
since the bacteria are not so well separated as in the dilutions, 
and often, owing to the crowded conditions, only a portion of the 
bacteria present will develop into visible colonies. Moreover if 
a cubic centimeter of the milk is used, the turbidity of the jelly 
due to the presence of the milk hides the colonies present from 
the eye. 

Porous earthenware Petri dish covers are recommended as 
superior to glass since they absorb the excess moisture." They 
also have the advantage of being cheaper and more durable than 
glass; they are easily marked with ordinary lead pencil. With 
long incubation a tendency of plates with these covers to dry out 
has been observed by some workers ; for ordinary routine work 
however they are perfectly satisfactory using 10 c. c. of media 
to the plate and incubating in a saturated atmosphere. These 
covers should never be washed but always thoroughly dry 
sterilized before use. 

Another method of preventing spreaders is by inverting the 
dishes and placing in the glass cover of each a strip of sterile 
filterpaper moistened with one large drop of glycerine. Plates 
so treated do not dry out as quickly as with the porous tops and 
the glass ware does not become scratched. 



BACTERIAL EXAMINATION OF MILK 13 

Pipettes. Straight sides 1 c. c. pipettes are more easily 
handled than those with bulbs ; they may be made from ordinary 
3-1 'J inch glass tubing and should be about 10 inches in length. 

Plating Technique. 9 The agar after melting should be 
kept in the water jacketed water bath between 40° C. and 45° C. 
for at least fifteen minutes before using to make sure that the 
agar itself has reached the temperature of the surrounding water. 
If used too warm the heat may destroy some of the bacteria or 
retard their growth. 

For routine work in cities in order to bring down the actual 
number of colonies in a plate to about the standard of two hun- 
dred, it is well to use a dilution of 1-10,000. To make this dilu- 
tion use two bottles of sterile water each containing 99 c. c. 

Shake the milk sample twenty-five times, then with a sterile 
pipette transfer 1 c. c. to the first dilution water and rinse the 
pipette by drawing dilution water to the mark and expelling; 
this gives a dilution 1 to. 100. 

Shake the first dilution twenty-five times, then with a fresh 
sterile pipette transfer 1 c. c. to the second dilution water, rinsing 
the pipette to the mark as before; this gives a dilution 1-10,000. 
Shake the second dilution twenty-five times, then with a sterile 
pipette transfer 1 c. c. to the Petri dish, using care to raise the 
cover only as far as necessary to insert the end of the pipette. 

Take a tube of agar from the water bath, wipe the water from 
outside the tube with a piece of cloth, remove the plug, pass the 
mouth of the tube through a flame, and pour the agar into the 
plate, using the same care as before to avoid exposure of the 
plate contents to the air. 

Carefully and thoroughly mix the agar and diluted milk in the 
Petri dish by a rotary motion, avoiding the formation of air bub- 
bles or slopping the agar, and after allowing the agar to harden 
for at least fifteen minutes at room temperature place the dish 
bottom down in the incubator. 

Controls. Plating should always be checked by controls. 
A blank plate should be made with each series of milk plates for 
control on the agar, water, air, Petri dishes, pipettes, etc. 

For control of the technique of plating, it is recommended that 
for work on "market milk" duplicate plates be made each day 
on several samples. 



14 STANDARD METHODS 

"Certified milk" should always be plated in. duplicate and 
where it is possible it is well to have one man's work occasionally 
checked by another. 

Unless duplicate plates show as a rule approximately the same 
count the worker should see if there is error in his technique. 

Plating should always be done in a place free from dust or 
currents of air. 

In order that colonies may have sufficient food for proper 
development 10 c. c. of agar shall be used for each plate. In 
plating a large number of samples at one time, the dilution and 
transfer of diluted milk to the plates may be done for four or 
eight samples, then the agar poured, one tube to each plate, then 
another eight samples diluted, etc. 

V. 
Incubation and Counting. 

Two standard temperatures are recognized. 

1. 48 hour incubation at 37° C. 

2. Five day incubation at 21° C. 

Regulations governing the number of bacteria allowable in 
milk should direct the method to be used in examination and in all 
reports, papers, etc., on the bacterial count of milk this factor 
should be explicitly stated. 

Incubators should be carefully regulated. Whatever tem- 
perature of incubation may be used it is important that the incu- 
bator air should be saturated with moisture ; this may be accom- 
plished by either having a depression in the floor of the incu- 
bator filled with water or by setting a pan of water on one of the 
shelves. 

Counting. Expression of results. Since minor differences 
in milk counts are within the working error of the methods and 
are of no significance in practice, the following scale has been 
adopted for recording results of market milk examination. 

Counts below 50,000 are distinguished by five thousands. 

Counts between 50,000 and 100,000 are distinguished by ten 
thousands. 

Counts between 100,000 and 500,000 are distinguished by 
fifty thousands. 



BACTERIAL EXAMINATION OF MILK 15 

Counts between 500,000 and' 5,000,000 are distinguished by 
hundred thousands. 

Counts above 5,000,000 are distinguished by millions. 
Therefore only the following figures are used in reporting. 



5,000 




400,000 


10,000 




450,000 


15,000, etc. 


, to 50,000 


500,000 


60,000 




600,000 


70,000 




700,000 


80,000 




800,000 


90,000 




900,000 


100,000 




1,000,000 


150,000 




1,100,000, etc., to 5,000,000 


200,000 




6,000,000 


250,000 




7,000,000 


300,000 




8,000,000, etc., by millions 


350,000 







Counts on "certified" or "inspected" milk shall be expressed 
as closely as the dilution factor will allow. 

The whole number of colonies on the plate shall be counted, 
the practice of counting a fractional part being resorted to only 
in case of necessity, such as partial spreading. 

Various counting devices have been recommended by differ- 
ent workers. The more simple ones, where the whole plate can 
be seen at once, are more desirable on account of there being less 
likelihood of recounting colonies. Colonies too small to be seen 
with the naked eye or with slight magnification shall not be 
considered in the count. 

VI. 

Milk Sediments. 

It is probable that within the past five years more research 
work has been done in relation to the various sediment or centri- 
fuge tests for milk than any other method of examination. 

These tests were originally started with the idea of detecting 
mammitis by noting the increase in the polynuclear cells and 
have become amplified to such an extent that excepting for cer- 
tified milk a fairly satisfactory bacterial analysis of milk may 
be made by these methods alone. Roughly these tests now 
include (a) Estimation of leucocytes, (b) Estimation of number 
of bacteria together with morphology of same, (c) Estimation of 



16 STANDARD METHODS 

foreign matter, dirt, feces, etc. It is however impossible to 
group them separately under these headings since by some of the 
methods all of these determinations are made. For the most 
accurate leucocyte counting the Doane-Buckley test as modified 
by Russell and Hoffman is recommended, while the Stewart 
method as modified by Hill and Slack is recommended for routine 
inspection work. 

Leucocytes are present in all normal milks and their number 
occasionally fluctuates greatly without apparent cause. Milk 
from animals suffering from udder inflammations almost con- 
stantly shows a high leucocytic content and without question is 
unfit for human consumption. 10 

While there is no point in the milk from a single animal where 
we can say it passes from normal to abnormal in this respect, 
enough research has been made to prove that the mixed milk 
from several normal animals very seldom exceeds 500,000 
leucocytes to the cubic centimeter. While healthy cows with 
no distinguishable lesions may occasionally for short periods pass 
this limit, such variations are very transient in character and if 
the mixed milk from several cows shows such high content of 
leucocytes it raises a suspicion of some abnormal condition. 11 

While a leucocytic count of 500,000 or more to the cubic 
centimeter in the case of a single animal may be transient and 
negligible, when found in mixed milk it is sufficient evidence to 
warrant the exclusion of such milk from the market, until satis- 
factory veterinary inspection of the herd is made. 

Stokes 12 devised a microscopic examination of milk for pus 
cells and streptococci as a means of detecting the presence of 
mammitis among cows supplying the milk. Centrifugal sedi- 
ment from ten cubic centimeters of milk was stained and exam- 
ined with one-twelfth oil immersion lens. He regarded the pres- 
ence in the milk of an individual cow of five cells per field of the 
oil immersion lens as justification for excluding the animal from 
the herd. 

Bergey 13 modified Stoke's method and made extensive exam- 
inations of the milk of individual cows. Parallel bacteriological 
examinations of the milk for both species and numbers supple- 
mented his examinations for cells. 



BACTERIAL EXAMINATION OF MILK 17 

Tromsdorff 14 devised a method which consists in centrifu- 
galizing 5 c. c. of milk in a special centrifuge tube with a lower 
constricted portion so graduated as to permit of reading off 
directly the amount of sediment. The mixed milk of cows with 
sound udders, as a rule, shows sediment varying from traces to 
.5 c. c. per liter with 1 c. c. per liter as the maximum. Tromms- 
dorff recommends the test as an aid in the detection of chronic 
mammitis. 

Stewart 15 of the Philadelphia Bureau of Health further modi- 
fied Stoke's method so that it was practicable to use it for the 
examination of large numbers of samples of mixed herd milk. 
Stewart describes the apparatus and method as follows: 

"This apparatus consists of a circular pan about 12 inches in 
diameter and % inches deep, containing twenty small glass tubes. 
The tubes contain 1 c. c. of milk and are filled by means of a 
small bulb similar to that ordinarily used on medicine droppers. 
The end of the tube is closed by a small rubber stopper, and the 
tubes are held in the pan by clamps. This pan is fitted upon 
the ordinary Beckel water centrifuge and covered with a lid 
which is held down by a thumb screw. The pan covered in this 
way furnished a surface of very slight resistance to the atmos- 
phere during its revolution, somewhat on the principle of a child's 
top." 

By the old method the arms of centrifuge containing the milk 
encountered so much resistance in their revolution that the speed 
with 15 lbs. water pressure was not more than 1,200 revolutions 
per minute, while the speed obtained with the new apparatus is 
from 2,500 to 3,000 revolutions per minute with 15 lbs. pressure. 
This rapid speed causes sedimentation to occur in less than five 
minutes. When this is completed the centrifuge pan can be 
lifted from the motor and the per cent, of cream measured by a 
graduated scale marked upon the tube. The heavier matter, as 
the insoluble dirt, pus cells and bacteria, is thrown to the peri- 
pheral end of the tub where it adheres to the rubber cork in the 
lumen of the tube. To examine this sediment the cork is care- 
fully removed and a spread made by rubbing the cork containing 
the sediment over an area of a square centimeter on a 3-inch by 
(i-inch glass slide. The proper area of the smear is obtained by 
placing underneath the slide a scale of circles having an area of 



18 STANDARD METHODS 

a square centimeter. After the smears are dried in the air 
without fixation of heat, the preparation is stained by the Jenner 
blood stain for two minutes, keeping the stain in constant motion. 
The excess of stain is washed off in water and the preparation is 
dried in the air. By this blood staining method the pus and 
blood cells are stained perfectly and the ordinary micro-organisms 
take the blue stain well. 

The stained specimens are examined with a one-twelfth oil 
immersion objective and a No. 3 eye piece. The character of the 
bacteria is noted and the average number of pus cells per field is 
counted. This average number is multiplied by 4,400 since there 
are about 4,400 fields to a square centimeter as estimated by the 
stage micrometer. This result is approximately the number 
of pus cells per cubic centimeter. 

Hill & Slack 16 modified the Stewart method by using tubes 
of a larger bore containing two cubic centimeters, stoppered at 
each end, centrifugalizing at a speed of 2500 revolutions a minute 
for 10 minutes and smearing the sediment evenly over 4 square 
centimeters with a drop of sterile water. The advantages claimed 
over the Stewart method are: 

The tubes are so large that there is practically no capillary 
action. The time of centrifugalizing is increased. The use of 
water with the smear allows of a thin even smear, the small 
amount of material of the same dilution left on the stopper being 
negligible for all practical purposes. 

The number of leucocytes per 1-12 oil immersion field multiplied 
by 20,000 gives the approximate number per cubic centimeter. 

So little of the sediment is seen at once with the high power 
that it is well to confirm the diagnosis of pus by making a 
thorough examination of the whole surface with a low power 
lens, to determine how uniform a smear has been made. By 
using an eye-piece micrometer ruled in squares, the relation of 
one square to that of 1-12 immersion lens being previously 
calculated, a count may be made with the low power lens. 

The Doane-Buckley 17 quantitative method of estimating 
leucocytes is described by them as follows: 

"With this method ten cubic centimeters of milk are centri- 
fuged for four minutes in graduated sedimentation tubes, at 
an approximate speed of 2,000 revolutions per minute. The 



BACTERIAL EXAMINATION OF MILK 19 

cream is lifted out with a cotton swab, care being taken to get as 
much as possible of the fat. It is then centrifuged one minute 
more and the cream again removed with a cotton swab. Any 
fat remaining in the milk interferes seriously with the counting, 
as, if there are more than a few globules they form a layer on 
the top of the liquid in the counting chamber, and as the leuco- 
cytes settle to the bottom of the chamber, it is difficult to see 
through the fat. It is only with cows giving milk difficult of 
separation where this trouble is experienced, and with such 
animals considerable care is necessary in removing all the cream 
gathered at the top of the sedimentation tube. The method of 
removing the fat with cotton is the best one that has occurred 
to us, and it is the only part of the process that does not operate 
with entire satisfaction in every instance. 

Following the removal of the cream, after the second centri- 
fuging the bottom of the tube will contain a portion of the 
sediment which is easily seen. This sediment may, in extreme 
cases of cows suffering from garget amount to as much as one 
cubic centimeter. Ordinarily it will be considerably less than 
one-half cubic centimeter. The amount varies considerably 
with the number of leucocytes, but not absolutely. The milk 
above this sediment is removed with a small siphon, which can 
be easily arranged with bent glass tubes drawn to a fine point 
and supplied with a small rubber end pinch cock. In using the 
siphon it is better to keep the point near the surface of the 
milk in the tube in order not to agitate the precipitated leuco- 
cytes and draw a number of them off with the milk. The milk 
in the tube may be siphoned within an eighth of an inch of the 
sediment in the tube. This will usually be below the J^ c. c. 
mark. Two drops of saturated alcoholic solution of methylene 
blue are then added, thoroughly mixed with the sediment by 
shaking, and then set in boiling water for two or three minutes 
to assist the leucocytes in taking the color. The contents of the 
tube can be boiled by holding it directly in the flame, but it has 
no advantage over the use of the water bath, and it is very 
likely to break the glass. After heating, some water is added to 
the tube to render the color less dense. Ordinarily filling the 
tube to the 1 c. c. mark will be sufficient, and this quantity gives 
an easy factor for calculating the final results. 



20 STANDARD METHODS 

In putting this liquid containing the leucocytes into the 
"blood counter considerable care is necessary, owing to the 
tendency of the leucocytes to sink to the bottom. At this place 
a capillary tube is used, and the cover glass was held in one 
hand ready to cover the chamber as soon as the drop was trans- 
ferred to the counting counter. After placing the glass cover 
over the chamber, about a minute is allowed the leucocytes to 
settle to the bottom of the chamber. There are very few 
foreign bodies likely to be mistaken in counting for leucocytes. 
Ordinarily the polynuclear leucocytes predominate and the 
stained nuclei with the unstained surrounding cell show up very 
distinctly. A few small leucocytes with large nuclei may be 
found and these may be confounded with yeast cells until the 
worker becomes familiar with the distinction. 

As regards counting we have taken a standard with a cubic 
centimeter as a basis quantity of milk, though we are of course 
aware that the corpuscles in the blood are enumerated with a 
cubic millimeter basis. We adopted the centimeter largely for 
two reasons. In counting bacteria in the milk the cubic centi- 
meter is always the basis employed. Simply because the 
leucocytes were derived from the blood seemed to be no reason 
why the same basis for counting should be employed as was 
used with the blood, while to the ordinary bacteriological 
worker to whom this work will fall, if ever adopted to any 
extent, the cubic centimeter standard would be a little more 
easily comprehended because more frequently used. The blood 
counter holds one-tenth cubic millimeter and one-ten-thousandth 
cubic centimeter. If ten cubic centimeters of milk are used and 
the 1 cubic centimeter of fluid is in the tube after siphoning, and 
the coloring matter and the water used to dilute has been added, 
then the resulting number of leucocytes in the counting multi- 
plied by 1,000 will be the total number of leucocytes per cubic 
centimeter in the milk. If a total of 75 leucocytes was counted 
in the chamber there would be 75,000 leucocytes per cubic 
centimeter in the milk. 

In the actual counting under the microscope a square milli- 
meter of the counting chamber will be found to be ruled off into 
400 smaller equal squares. This facilitates an accurate and 
rapid count. Where the number of leucocytes is not great the 



BACTERIAL EXAMINATION OF MILK 21 

entire field can be counted in a short time. Where there is a 
great number of leucocytes a few squares or sets of squares in 
different parts of the ruled surface will give approximately the 
number. 

There are occasionally a few variations desirable from these 
rules, but it may be well to state that the details have been 
pretty carefully and thoroughly worked over and compared, 
and it is seldom that short cuts can be made if correct results are 
desired. The time and speed of centrifuging are placed as low 
as possible for accurate work. When there is one-half c. c. or 
more of sediment, it is necessary to use more of the methylene 
blue for staining, as there will be too great a number of leuco- 
cytes to make a satisfactory count in the counting chamber, it 
is better to add water until there are two cubic centimeters, or 
sometimes even more in the sedimentation tube. 

This method of counting, while long in explaining is in reality 
short and simple in application. Moreover, it is based on accurate 
measurements in every detail, and the results are correspondingly 
reliable." 

Savage 18 devised a method similar to that of Doane & Buckley 
which, like theirs, shows large numbers of leucocytes in the 
milk of normal cows. 

Russell & Hoffman 10 working farther with the Doane & 
Buckley method have made several modifications and recom- 
mend the following procedure: 

Collection of Samples. Samples for analysis should be 
taken from the entire milking of the animal, as the strippings con- 
tain a somewhat larger number of cells than other portions of the 
milk. For the purpose of examination take 200 c. c. in 
stoppered bottle. 

Time Interval between Collection and Analysis. To 
secure satisfactory results, milk must be examined in a sweet 
condition. 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 preserved 
for satisfactory microscopical examination by the addition of 
formalin at the time of collection — a proportion of 1 c. c. to 250 
c. c. of milk. Formalin has been found the best preservative 
to use although it causes contraction of the cells to some extent. 



22 standard methods 

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- 
genous 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 — Ten c. c. of milk Are 
placed in an ordinary sedimentation tube, and after heating as 
above directed and subsequently shaking, the milk is centrifu- 
galized twenty minutes at 1,200 revolutions per minute. A hand 
centrifuge may be employed for this purpose; where available a 
steam turbine Babcock milk tester may be found more prac- 
ticable. 

Note. — This speed maintained for the time mentioned, is sufficient 
to sediment practically all the cell elements suspended in the milk. In 
our experience we have found the number of cells in supernatant milk to 
average only 3J^%. 

3. Preparing the sample for examination — After centrifu- 
galizing, the cream and the supernatant milk are removed, with 
the exception of the last Yi c. c, 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 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 differ- 
entiation of the cells from the small fat globules, in the liquid rise 
so that a distinct microscopic observation can be made. 

Examination of Material. The preparation is examined 
in an unstained condition. 



BACTERIAL EXAMINATION OF MILK 23 

Note. — Most observers have usually stained the sediment prior to 
examination, but we have found with the above treatment that the cells 
may be enumerated quite as well in an unstained condition as a stained 
condition. 

The count is made with a one-inch eye piece and 1-6 objective. 
Where the number of cell elements exceed 12 or 15 per micro- 
scopic field, above referred to, one-fourth of the entire ruled 
area of the counter, equivalent to 100 of the smallest squares of 
the cell, are counted. Where the cell elements are less abundant, 
one-half of the entire area (two to four hundred squares) are 
enumerated. 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; mul- 
tiplied by four million we have the number of cells per cubic cen- 
itmeter in the sediment examined. As the sediment represents 
the concentration of cells into one-twentieth of the original 
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 centi- 
meter in the original milk. 

Note. — The above factor of 4,000,000 is obtained as follows: The 
cubic content of the blood counter represents one-tenth of a cubic centi- 
meter. This volume is divided by means of the ruled scale into 400 
small cubes each equal to one four-thousandth of a cubic millimeter, or 
one four-millionth of a cubic centimeter. 

Expression of Results. All results should be expressed in 
number of cells per cubic centimeter of the original milk, and in 
order to avoid fictitious accuracy, should be given in accordance 
with the method adopted by the Committee on Standard Methods 
of Water Analysis, as reported in the report in the Laboratory 
Section of the American Public Health Association for 1905, 
page 94. 

Stone & Sprague have devised a centrifuge tube for quantita- 
tive and qualitative analysis of milk sediment which is practi- 
cally a combination of the Stewart and Trommsdorff tubes, being 
a 15 c. c. tube, the lower portion of which is drawn into a finely 
graduated tube about 1-16 inch in diameter and closed with a 
rubber stopper. 

For this method of examination they claim the following 
advantages : 



24 STANDARD METHODS 

"First, it measures more accurately the sediment. Second, 
smears of the sediment can be made in the same manner as with 
the Slack tube. Third, the column of sediment tends to become 
stratified so that the different elements can be easily made out. 
Fourth, the tubes are much more easily cleaned. With this tube 
we have been able to detect without microscopical examination, 
the presence of cases of acute mastitis when the milk was diluted 
thirty times with normal milk. For ordinary routine work we 
think that centrifugalization in these tubes, using one-tenth 
cubic centimeter of a leucocyte sediment to fifteen cubic centi- 
meters of milk, in a filled tube, as a maximin limit, will be of as 
much value as a count. The character as well as the quantity 
of the sediment should of course be taken into consideration. A 
sediment from cases of even slight mastitis practically always 
having a yellowish or pinkish tinge of pus, mixed with a smaller 
or larger amount of blood. Microscopical examination usually 
shows red cells as well as pus cells. This test should of course be 
supplemented by an actual examination of the herds in every 
case." 

The Microscopic Estimate of Bacteria. (Slack). 20 "The 
apparatus and the method for making the microscopic estimate 
are as follows: The special apparatus for centrifugalizing the 
milk, modified from one used for leucocyte estimation by Stewart 
of Philadelphia consists of an aluminum disk and cover, 10 inches 
in diameter and 5-8 inch in depth, fitted to hold twenty small 
glass tubes arranged radially. These tubes hold about 2 c. c. 
each and are closed at both ends with rubber stoppers. 

The milk samples are thoroughly shaken, the tubes filled, stop- 
pered, inserted into their proper numbered receptacles in the 
disk, and centrifulgalized for ten minutes at a speed of from two 
to three thousand revolutions per minute. Thus in each tube the 
whole sediment from a known quantity of milk is obtained, and 
may be spread over a given area. A space about 4 sq. cm. is 
most convenient, being the right size to allow thorough emulsion 
of the sediment with a drop or two of sterile water, and to permit 
drying into' a thin even smear. It is convenient to smear a num- 
ber of samples consecutively on a long glass slide which has 
previously been correctly spaced with a blue pencil. 



BACTERIAL EXAMINATION OF MILK 25 

To obtain the sediment with the least disturbance, the 
stopper is first removed from the inner, or cream end, then the 
tube is held with the cream end downwards, the cream removed 
with a platinum loop and the milk poured out; lastly, still hold- 
ing the cream end down, the other stopper is carefully removed 
with the adhering sediment and the sediment smeared evenly 
with a drop of sterile water over the space on the glass slide, the 
stopper being rubbed directly on the glass until the sediment 
has been transferred. When this is properly done the amount 
of diluted sediment remaining on the stopper is practically negli- 
gible. The smear is then dried with gentle heat and stained 
with methylene blue. 

The microscopic examination of a milk sediment thus easily 
prepared reveals more than any other single test. It shows the 
character of the milk, the approximate number and morphology 
of the bacteria, and the presence of pus or streptococci. 

It is not claimed that all the bacteria in the milk subjected to 
centrifugalization are precipitated into the sediment; but it is 
claimed that in 99 per cent, of the samples a representative 
number, is so precipitated, and that this number bears a fairly 
constant relation to the 1-10,000 dilution plate culture when 
grown in a saturated atmosphere at 37° C* for twenty-four 
hours, 1 per cent, agar being used with a reaction of +1.5. 

We may say as a rough estimate, that each coccus, bacillus, 
diplococcus, or chain in the 1-12 oil immersion field represents 
one colony in the 1-10,000 plate from the same sample. In most 
cases the count of a representative field multiplied by 10,000 gives 
approximately the number of bacteria per cubic centimeter. By 
the use of this method a good idea of the condition of a single 
sample of milk can be obtained in less than twenty minutes. 
Thirty samples can be examined in an hour. At the contrac- 
tor's receiving station one can easily examine 100 to 200 samples 
daily, thus keeping close watch over the dairies. 

In ordinary routine city inspection only those samples need 
be plated which are doubtful or above the limit established. In 
this work the plate would corroborate the microscopic findings 



* The method « as originally worked out in relation to the 24 hour count at 37° C. The 
exact factor would have to be figured in accordance with the area of the 1-12 immersion 
field of the individual microscope. The value of the test lies, however, not in accurate 
counting so much as in the decisive picture obtained of the character uf the milk. 



26 STANDARD METHODS 

and strengthen the evidence of the court cases. Where plates 
are to be made the microscopic estimate gives an indication of 
the proper dilution to use. 

VII. 

Special Bacteria and Tests for Same. 

Pathogenic Organisms. Typhoid and diphtheria bacilli 
are rarely sought for in milk because it is recognized that al- 
though these diseases are often conveyed in milk, the period of 
incubation is such that by the time the outbreak on a special 
route is noticed the contagion has usually disappeared from the 
the milk. 21 Typhoid may be isolated by the use of Lactose bile 
medium as recommended by Jackson and Melia. 22 

Tubercle Bacilli in Milk. 23 Collection of samples and 
technique. "Pint or quart samples of milk should be obtained, 
kept well iced, and delivered to the laboratory as soon as possible. 
The milk and cream should be well mixed by shaking vigorously. 
50 c. c. of the mixed milk are then transferred to a large centri- 
fuge flask and 100 c. c. of sterile water added. Centrifuge for 
one hour at 2,000 revolutions per minute. The milk was diluted 
with twice its volume of water with the idea that it would decrease 
the specific gravity of the milk and so permit of the easier sedi- 
mentation of the tubercle bacilli. Guinea pigs are then inocu- 
lated, subcutaneously in the belly wall, with 5 c. c. of the sedi- 
ment thus obtained. The guinea pigs not dying in at least two 
months are chloroformed, after being tested with tuberculin, 
and careful autopsies made. Smears, cultures and sections are 
made from the various organs of the animals that show any 
change from the normal. The smears are stained with carbol 
fuchsin and examined for acid fast bacilli. 

Cultures are made on glycerinized potato and glycerine agar 
to rule out Rabinovitch's quick growing acid fast butter bacillus. 

Sections are stained with carbol fuchsin for tubercle bacilli 
and also with haematin and eosin for histological appearances. 

Tuberculous guinea pigs may be differentiated from non- 
tuberculous by giving sufficient crude tuberculin (2 c. c.) sub- 
cutaneously to cause the death of the tuberculous animals in 
twenty-four hours. 



BACTERIAL EXAMINATION OF MILK 27 

Of about 250 guinea pigs tested in this way no animal that 
did not have tuberculosis died. Two or three that had slight 
lesions did not die but became sick. It was noted that all the 
animals died whose lesions had become caseated. 

The reaction seems of distinct service in eliminating infections 
with acid fast organisms and the suggestion is made that with 
some modification the procedure may have a distinct place as an 
aid in differentiating true tuberculosis from infections with other 
acid fast organisms which produce tubercular-like lesions." 
It is of course understood that the examination of milk for 
tubercle bacilli is by the very nature of the test limited. For 
the control of this disease in cattle we must rely upon the 
tuberculin test. 

Wisconsin Curd Test. 24 The Wisconsin curd test is con- 
ducted as follows: 

1. Sterilize milk containers so as to destroy all bacteria in 
vessels. This step is very important and can be done by heat- 
ing cans in boiling water or steam for not less than one-half hour. 

2. Place about one pint of milk in a covered jar and heat to 
about 92° F 

'.I. Add ten drops of commercial extract of rennet and mix 
thoroughly with the milk to quickly coagulate. 

4. After coagulation cut curd fine with case knife to facili- 
tate separation of whey ; leave curd in whey one-half hour to an 
hour, drain off whey at frequent intervals until curd is well 
matted. 

5. Incubate curd at 98° to 100° F. immersing jar in warm 
water. Keep jars covered to retain odors. 

6. After six to nine hours incubation open jars and observe 
odor, examine curds by cutting with sharp knife and observe. 

7. Very bad milk will betray the presence of gas-producing 
bacteria by the spongy texture of the curd and will have an off- 
flavor. 

s. If more than one sample is tested at the same time, dip 
knife and thermometer in hot water before each time used." 

Asa rule milks showing the presence of gas or bad odors in any 
considerable degree are milks that have been more or less pol- 
luted with extraneous organisms or carelessly handled, and as a 
consequence such milks show a curd filled with pin holes due to 



28 STANDARD METHODS 

gas. It is not intended that this test should be used for an abso- 
lute indication of the presence of gas-producing organisms, but 
rather it has been of service in the detection of the condition of 
market milk. It is possible that a milk containing but few bac- 
teria may give a very undesirable curd. In order to obtain a 
good curd we must either have a milk which contains almost no 
bacteria or one which contains large numbers of lactic acid organ- 
isms. While more valuable in testing milks for cheese making it 
is useful in the examination of market milk if used with judgment. 
Work done with this test for the detection of fecel matter shows 
that positive results can be obtained from other gas formers than 
B. coli. The advantage of the test is that it is simple to perform 
in the dairies and very quick in its results, determinations being 
made over night. The disadvantage is that while it is a valuable 
indicator it is by no means a sure test for fecal matter. 

Other methods of detecting gas-producing organisms in milk — 
Gas producing organisms may be tested for in milk, as in water, 
with glucose or lactose broth in fermentation tubes. Test sim- 
ilar to presumptive test for B. coli in water analysis may be 
made by inoculating into these broth fermentation tubes a c. c. 
each of the 1-100, 1-1,000 and 1-10,000 dilutions, or if B. coli 
organisms are to be numerically determined the milk may be 
plated in lactose litmus agar, red colonies counted and species 
tests worked out. Lactose-bile medium has also been used for 
the determination of B. coli in milk. 

The presence of these gas-producing organisms in abundance 
usually indicates dirty conditions of stables, cows or vessels. In 
small quantities they may be found in most milks. 

In Baltimore routine examinations are made for B. coli in 
milk in 1-1,000 c. c. of each sample. 

One c. c. of the 1-1,000 dilution is placed in ordinary bile con- 
taining 1% lactose in a fermentation tube and allowed to stand 
at 37° for 72 hours, at the end of which time if there is more than 
15% gas, plates are made, colonies isolated and run through spe- 
cies tests. Of many hundred examinations the colon bacillus is 
found in about 25% of the samples in winter and 75% in summer 
in 1-1,000 of a c. c. 



BACTERIAL EXAMINATION OF MILK 



29 



The following table prepared from the results of the routine 
examination of the Baltimore milk supplies for 1906 shows that 
the colon bacillus is more apt to be present in milk of high 
bacterial content. 



Number of Bacteria 


No. of 
Samples 


B. coli present 
1-1000 c. c. 


B. coli absent 
1-1000 c. c. 


10,000 and under 

500,000 and under 

500,000 to 1,000,000 

1,000,000 and over 


32 
92 

236 
64 

338 


15% 
20% 
30% 
66% 
72% 


85% 
80% 
70% 
34% 
28% 



Gas Production may be demonstrated by adding to a meas- 
ured quantity (1U c. c. ) of milk in a fermentation tube either 3 
c. c. of 5% solution of carbolic acid or 1 c. c. of a sterilized 2% 
solution of bile salt containing neutral red in sufficient quantity 
to give the milk a deep pink color. Incubate for 24 hours at 37° 
The chemicals evidently inhibit the growth of the lactic acid 
bacteria. Where neutral red is used if the gas producing bacteria 
are in large numbers the deep pink of the milk is changed to a 
canary yellow. 

Dextrose litmus agar and lactose litmus agar are of use in 
differentiating acid formers, the former giving better results. 

Whey agar favors the growth of lactic acid organisms, but is 
unfavorable for other types. 

In incubation at 21° C. the addition of 1% lactose to agar 
has given higher counts than agar without. 

Determination of Streptococci. 16 "Although by careful 
searching a few streptococci will be found in most sediments 
from pus milk they are seldom found to any great extent by 
direct microscopical examination. Occasionally a sample will 
be found crowded with long chains; more often, streptococci, if 
present, are in the form of diplococci or very short chains. 

Where streptococci, diplococci or cocci are found in the 
sediment and the plate from the same sample contains colonies 
resembling streptococci colonies, these colonies mav be grown 
in bouillon to see if chains will develop. 



30 STANDARD METHODS 

First make and record an estimate of the number of such 
colonies present, then transfer from 10 to 50 of them to bouillon 
and grow for 15-24 hours at 37° C. To examine the bouillon 
culture, spread a loopful on a glass slide, fix with heat, fix with 
alcohol while slide is still quite hot, stain with methylene blue, 
wash immediately, dry and examine. 

Streptococci in small numbers are present in most market 
milks as shown by Heinemann and many of the short chain 
varieties are undoubtedly at the time harmless, though by pass- 
ing through animals their pathogenicity may become marked. 25 

Long chain streptococci are more apt to indicate inflamma- 
tory reactions 28 and milk containing these in large numbers is 
certainly not a safe article of diet. 

A milk should not be condemned because a few chains are 
found together with large numbers of other microscopic organisms 
in a bouillon culture, but it is safer to exclude a milk from the 
market when these three tests agree. 

1. Microscopic examination of the sediment shows strep- 
tococci, diplococci or cocci. 

2. The plate from the same sample shows colonies resem- 
bling streptococci colonies exceeding a count of 100,000 to a 
cubic centimeter. 

3. The bouillon culture from these colonies shows long 
chain streptococci alone or in great excess compared with the 
other bacteria present." 

Milk showing in the stained sediment both abundance of long 
chain streptococci and pus should be condemned as unsafe. 

VIII. 

Laboratory Procedure on Routine Samples. 

The following procedure is recommended for routine work. 

1. Centrifugalize, make smeared sediment, stain and exam- 
ine microscopically for approximate number of leucocytes, 
approximate number of bacteria, types of bacteria, streptococci, 
etc. 

2. Plate at least those samples, as indicated by the micro- 
scopical examination which show bacterial content around or 
more than the number permitted by the regulation. 

3. Incubate 48 hours at 37° C. or 5 days at 21° C. 



BACTERIAL EXAMINATION OF MILK 31 

4. Count colonies. 

5. From plates showing numerous pin point colonies transfer 
ten or more to broth and grow 15-24 hours arid examine for 
streptococci. 

IX. 

Interpretation of Results. 27 

Bacterial Count. A high bacterial count in milk indicates 
lack of cleanliness in production, or lack of care after production. 
Age of the milk is also an important factor and in interpreting 
results the distance milk has to be brought, etc., should be taken 
into consideration. Thus a count of 100,000 bacteria to a cubic 
centimeter should be considered a serious contamination in milk 
which may be delivered to the consumer within a few hours of 
production, while a count of no higher than 100,000 in milk pro- 
duced at a distance and say 24 to 36 hours old is evidence of 
ordinarily, good care. To produce a milk averaging under 
10,000 bacteria to the cubic centimeter requires the utmost 
care and watchfulness of each detail. 

Leucocytes. — A leucocytic content of 500,000 or over to the 
cubic centimeter especially in testing mixed milk should be re- 
garded as suggestive of some inflammatory condition of the 
udder and the milk excluded until after satisfactory veterinary 
inspection. 

Indication of the presence of pus is more sure if the leucocytes 
are clumped. 

Streptococci. Long chained streptococci are sometimes 
found in the smeared sediment especially in pus milks, their 
presence in such smears or when found by the plate method in 
numbers of over 100,000 to the cubic centimeter should be con- 
sidered sufficient evidence for exclusion of the milk until after 
satisfactory veterinary examination of the cows. 

B. coli are present in most milks, their presence in large 
numbers in milk should be regarded as evidence of unsatisfactory 
conditions at the dairy. 



32 STANDARD METHODS 



Bibliography. 

1. American Journal of Public Hygiene, Nov., 1907, page 331. 

2. American Journal of Public Hygiene, Nov., 1908, page 425. 

3. Report of Boston Board of Health, 1906, page 76. 

4. Winslow & Hill. The Production and Handling of Clean 

Milk, 1909, p. 252. 

5. A. P. H. A. Committee on Standard Methods of Water 

Analysis, pp. 104-109. 

6. Harrison & Van der Leek. Centralblatt fur Bacteriologie 

I. Abte. Originale 51, page 607. 

7. Hill. The Mathematics of the Bacterial Count. Am. Jour. 

of Public Hygiene, Aug., 1908, p. 300. 

8. Hill. Journ. Med. Research. Vol. XIII, No. I, Dec, 

1904, pp. 93-96. 

9. American Journal Public Hygiene, Nov., 1904, page 237. 

10. Jensens Milk Hygiene (Pearson), J. B. Lippincott & Co., 

1907, p. 93. 

11. Stone & Sprague. Journ. of Med. Research, Vol. XX, 

No. Ill, p. 235. 
Jordan. American Jour. Pub. Hygiene, Feb., 1909, p. 126. 

12. Stokes. Annual Report of Health Dept. of Baltimore, 

1897, p. 105. Journal of State Medicine, 1897, p. 439. 

13. Bergey. Bulletin No. 125, Dept. Agriculture, Comm. of 

Penn. 

14. Trommsdorff. R. Munch. Med. Woch., 1906, 53, p. 541. 

15. Stewart. American Medicine. Vol. IX, No. 12, p. 486. 

16. Slack. Journ. Inf. Dis. Sup. No. 2, Feb., 1906, p. 214.. 
Kendall. Studies from N. Y. Research Laboratory. 

Vol. Ill, 1907, p. 169. 

17. Doane & Buckley. Bulletin 102, Maryland Agricultural 

Experiment Sta. 
Ward, Henderson & Haring. 19th Biennial Report of the 

State Board of Health, California, 1904-06, p. 142. 
Russell & Hoffman, Journ. Inf. Diseases, Sup. No. 3, May, 

1907, p. 63. 

18. Savage. Journal of Hygiene (Cambridge, 1906) No. 2, 

p. 123. 
Abstract in Expt. Sta. Record, Vol. XVII, No. 10, p. 223. 



BACTERIAL EXAMINATION OF MILK 33 

19. Russell & Hoffman. Am. Journ. Public Hygiene, Aug., 

1908, page 285. 

20. Slack. Technology Quarterly. Vol. XIX. No. 1, Mar., '06. 
Wilcox. Studies from N. Y. Research Laboratory, Vol. Ill, 

1907, p. 169. 

21. Conn. Practical Dairy Bacteriology, p. 105, Orange, 

Judd & Co., N. Y., 1908. 

22. Jackson & Melia. Am. Jour. Pub. Hygiene, Feb., 1909, 

p. 83. 
Hill. Am. Jour. Pub. Hygiene, Feb., 1909, p. 135. 

23. Anderson. Bulletin No. 41. Hygienic Lab. P. H. & 

M. H. Service. 

24. Wiley. Foods and Their Adulterations. P. Blackiston 

& Son, 1907. 

25. Heinemann, Journ. Infect. Dis. Vol. IV No. 1, Jan., 1908, 

p. 67. 
Harris. Journ. Infect. Dis. Sup. No. 3, May, 1907, p. 50. 

26. Andrews & Horder. The Lancet. Sept. 15-22-29, 1906. 

27. Winslow & Hill. The Production and Handling of Clean 

Milk. 1909, p. 244. 
Ward. Pure Milk and the Public Health, Taylor & Car- 
penter, 1909, p. 132. 



REPORT OF THE COMMITTEE ON STANDARD METHODS 
FOR THE EXAMINATION OF AIR. 



I. Synopsis. 

The most important impurities of air, which it is possible 
to detect and measure in sanitary investigations, are physical, 
rather than chemical or bacteriological. The evil effects of heat 
and humidity upon the human organism are universally recog- 
nized. Dust particles injure the throat and lungs and play an 
important part in predisposing to tuberculosis. Bad lighting 
exerts an obviously harmful effect upon the eyes. Hence the Com- 
mittee believes that determinations of temperature, humidity, 
dust and intensity of light should be fundamental in all 
sanitary investigations. Standard procedures are recommended 
for all four of these tests. 

Chemical determinations of carbon dioxid in the air, while 
historically of supreme importance, are held by the Committee 
to furnish less direct evidence of unfavorable hygienic conditions 
than do the tests for temperature and humidity and dust, 
(Gilbert, 1909 ; Great Britain, 1909). In combination with these 
latter tests they may, however, be of value, and a standard pro- 
cedure is suggested. In certain special investigations the deter- 
mination of the number of bacteria present in the air may also 
be of interest and a standard procedure is recommended for this 
purpose. 

Other minor questions are discussed in the report, without the 
recommendation of standard procedures. 

II. Physical Determinations. 

The principal physical properties of air which it is desirable 
to take into consideration are temperature, humidity, pressure, 
dust, light and the velocity of air currents. It would be desir- 
able to include sound and odor, but at the present time it seems 
impracticable to bring these two important properties of air 
within the range of exact observation and record. 

For most practical purposes it is desirable that analyses of 
air should show average conditions, that is, conditions which 



BACTERIAL EXAMINATION OF AIR 35 

obtain over an appreciable period of time, as, for example, from 
thirty seconds to several minutes. In most cases the minute 
changes in the atmosphere which are constantly occurring are of 
small consequence to the analyst and can be neglected, except 
in unusually delicate researches, where special apparatus is 
required. Fortunately most types of physical apparatus 
intended for the analysis of air are adapted to register these 
average conditions, so that the records need no calculation to 
make them suitable for practical use. 

The reason why average conditions are recorded is that the 
instruments have a lag, which makes the reading occur some 
time after the occurrence of the conditions which produced 
it. For most purposes this lag or inertia is of little conse- 
quence, but in some instruments it is so great as to be seriously 
objectionable. For example, some types of thermometers take 
15 or 20 minutes to record the temperature when a decided 
change occurs. This lag may make the reading useless, where 
a thorough knowledge of the changes is important. 

1. Temperature. 

For most purposes the temperature of the air can most con- 
veniently be determined by means of mercurial thermometers. 
These are made in a great variety of forms depending upon the 
uses to which they are to be put. An accurate and convenient 
form of thermometer is a naked tube with an elongated bulb 
of mercury at one end and a ring at the other through which 
a cord can be tied. The scale in degrees and fractions thereof is 
etched upon the glass. Thermometers of this type may possess 
considerable accuracy. Generally they can be relied upon to 
about one-half to one-fifth of one degree. 

It is common to place rod thermometers upon a backing of 
metal, card or wood, the scale in this case not being etched upon 
the glass but painted upon the backing. It is perhaps unneces- 
sary to say that thermometers of this type are often more orna- 
mental than accurate. They usually possess a decided lag and 
are, for this reason, frequently unserviceable. When employed 
for careful air work thermometers should be suspended freely in 
he atmosphere or, at least, placed in a current of air sufficient 
to insure good ventilation about the mercury column. 



36 STANDARD METHODS 

Registering thermometers are of two principal types — those 
which record maximum and minimum temperatures and those 
which make a record of all the changes of temperature that 
occur. The latter instruments are provided with clock works 
which move sheets of paper under a pen by which the record 
is made. 

The maximum and minimum thermometer is constructed 
so as to have a small rod of metal free to move in the tube which 
holds the mercury. A rising or falling column of mercury pushes 
the metal rod before it, but leaves the rod upon receding again. 
When necessary the metal rod is brought to the point of contact 
with the mercury by means of a small horse-shoe magnet manipu- 
lated outside of the thermometer tube. Maximum and mini- 
mum thermometers of this type are almost invariably mounted 
upon a backing and consequently have a considerable lag. 
They are, nevertheless, serviceable where fluctuations in tem- 
perature are not rapid and can be recommended for determining 
the highest and lowest temperatures, under such circumstances. 

Standard Method for Temperature. For an intelligent under- 
standing of the sanitary condition of any room, car or other enclosed 
space neither single determinations nor maximum and minimum records 
are sufficient. Recording thermometers should be used, placed at various 
selected points and records should be obtained covering a period of several 
days. Such instruments are of several types. Instead of mercury the 
contracting and expanding medium is some rigid metal or combination of 
metals whose contraction and expansion causes a pen point to bear over a 
moving paper scale and so leave an ink trace. The clock work is gener- 
ally wound up for a week, for which period the paper scale is also adapted. 
Scales for recording thermometers are of two principal types — those 
which are printed upon circular discs of paper, the rising and falling 
temperatures being recorded by a line which moves at a greater or less 
distance from the centre, and those upon which the scale is approximately 
rectangular, with the rising and falling temperatures tracing a line which 
runs in the general direction of one edge of the paper. For most purposes 
the latter type of scale is preferable. Among the best of these instru- 
ments are those made by Jules Richard of Paris. Scales are printed in 
either centigrade or fahrenheit degrees. An instrument closely resem- 
bling that of Jules Richard is sold by Queen & Co. This type is sug- 
gested as a standard. 

2. Humidity. 

'"?*' Standard Method for Humidity. Although not always strictly 
accurate, especially at low temperatures, the most generally useful 
instrument for determining humidity is the psychrometer or wet and dry 
bulb thermometer. This instrument is made in several types, that 
employed by the United States Weather Bureau being simple, efficient 
and economical. The psychrometers employed by the Weather Bureau 
are of two principal kinds. In one case the two thermometers with their 



BACTERIAL EXAMINATION OF AIR 37 

wet and dry bulbs are whirled in a vertical plane by means of a small 
machine actuated by hand power. In the second, which is the most 
convenient for ordinary work, the thermometers are provided with a 
suitable handle by which the apparatus is whirled about by the hand of 
the investigator. The instrument suggested as standard is of the latter 
(1908) type modified slightly by Soper. It consists of two mercurial 
thermometers 24 centimeters long, graduated from —10 to 125 degrees 
Fahrenheit, fastened upon an aluminum back, 1.5 cm. apart center to 
center. The bulbs project beyond the aluminum back for 5 cm., one of 
the bulbs being' covered with cloth. The upper end of the aluminum 
back is connected by two loose wire links with a substantial handle by 
which it can be whirled. The whole is carried in a cylindrical aluminum 
case. This instrument may be obtained from Schneider Bros., 265 Green 
St., N. Y., or from Queen & Co. The manner of use is fully described m 
Bulletin N T o. 235 of the U. S. Weather Bureau, which contains the full 
tables necessary for calculating humidity from the wet and dry bulb 
readings, and is also described in Ward's Meteorology (Ward, 1899). 

Stationary wet and dry bulb thermometers mounted, as com- 
monly seen, with a heavy backing are not suitable for the deter- 
mination of relative humidity, owing to their lag and the like- 
lihood that the wet bulb will not be suitably moistened or ven- 
tilated. 

The hair hygrometer whose action depends upon the exten- 
sion and contraction of a suitably prepared hair under the influ- 
ence of moisture can be made an accurate instrument ; and some 
types are arranged for continuous record. Certain forms of the 
instrument are open to the same objection which has been 
raised against thermometers which have a backing ; there is diffi- 
culty in causing a sufficient current of air to come in contact 
with them. 

3. Dust. 

The simplest and one of the most useful methods of determin- 
ing the amount of dust and its composition is by means of suitable 
receptacles, such as Petri dishes, upon which the dust is allowed to 
settle for a sufficient period of time to enable a considerable 
quantity to accumulate. Particles are then examined under a 
microscope, or, if desired, they can be swept by means of a 
camel's hair brush upon a watch glass and weighed. 

It is a practicable and desirable procedure to filter air through 
cotton filters or filters of other material, the quantity of air being 
measured either by means of a gas meter or other device. What- 
ever the filtering medium the quantity of air should be large, in 
order that the quantity of dust may be appreciable in amount and 
fairly representative in quality. By weighing the filtering 



38 STANDARD METHODS 

material before and after passing the air through it the aggregate 
weight of dust in the quantity of air taken for examination can 
be determined. It is necessary, in most cases, to guard against 
increase in weight of the filtering material through the absorp- 
tion of water. This can be done by placing the filtering material 
in a desiccator before and after filtration and just before weighing 
in each case. 

Standard Method for Dust Determinations in Ordinary Air. 
For very careful work the number of dust particles in the atmosphere can 
be determined by an instrument invented by Professor John Aitken and 
called a dust counter. This instrument is expensive; and a somewhat 
smaller but more generally useful instrument, devised also by Aitken, and 
called the Koniscope is recommended for standard determinations. The 
dust counter and koniscope operate upon the principle that dust particles 
form nuclei upon which moisture condenses and precipitates from a sat- 
urated atmosphere. In the dust counter the droplets are counted, in the 
koniscope the opacity of cloud is estimated. There are not, apparently, 
many cases in which the dust counter can be turned to practical account 
in sanitary investigations. 

The Koniscope consists of two brass tubes connected at right angles 
and suitably fitted with stopcocks and a small air pump. By exhausting 
the air from one of the tubes, allowing the space to become saturated with 
water vapor by evaporation from wet blotting paper within, and then 
allowing this moisture to condense upon the dusty astmophere under 
examination, clouds of different degrees of density can be formed inside 
the tube. The density of the clouds can approximately be measured by 
looking through the tube from one end to the other, windows being pro- 
vided for this purpose. A table is supplied with the instrument to give 
the approximate number of dust particles corresponding to clouds of 
different degree of density. 

The koniscope can be obtained from Queen & Co. This instru- 
ment is easily handled and sufficiently delicate to merit wider use 
than has yet been made of it in sanitary investigations. It is capable of 
detecting with great delicacy different currents of air, where the only 
difference between them lies in the number of dust particles present. 

Standard Method for Dust Determinations in Air Heavily 
Laden With Dust Particles. One of the principal objections to filtra- 
tion methods in studying the dust in ordinary air lies in the fact that 
enormous volumes of air must be filtered in order to obtain appreciable 
results. In factories and other places where the dust is thick the follow- 
ing method is recommended. A measured volume of air is drawn through 
a filter of granulated sugar, and the sugar is dissolved and the dust 
suspended in a measured volume of distilled water. The volume taken 
must vary with the amount of dust present in the air. The sugar should 
be of the ordinary granulated type with grains between .25 and 1.00 mm. 
in diameter. The layer of sugar should be 1 cm. deep and may be held in 
place in a. glass tube of 1 or 2 cm. bore by a perforated stopper and square 
of bolting cloth or by a plug of cotton. The air sample should be col- 
lected rather rapidly ; for heavily laden air a suction cylinder of metal with 
a closely fitting piston may be used. Where larger volumes of air are to 
be examined a Roots blower, operating on the suction principle, can be 
used to advantage, the quantity of air being measured by a gas meter 
interposed between the blower and the filter. 



BACTERIAL EXAMINATION OF AIR 39 

The weight of dust present may be determined by filtering the water 
in which the dust has been suspended through a Gooch crucible. The 
number of dust particles may be found by the following method (Winslow, 
1908) : After thorough agitation, one c. c. of the suspension is placed in a 
Sedgwick-Rafter cell and the particles are counted under the microscope 
by the method used in the microscopical enumeration of micro-organisms 
in drinking water (Whipple, 1905). The cell is 50 mm. by 20 mm. in 
area and 1 mm. deep and the method employed consists essentially in 
counting the number of particles in representative mm. squares. Both 
the top and bottom of the cell must be examined to get dusts lighter and 
heavier than water. Glassware and sugar must be clean and control 
determinations should be made, to detect any chance pollution. 

4. Illumination. 
Two general methods are available for the practical deter- 
mination of the intensity of lighting. The first of these methods 
depends upon the distance at which print of a given size can be 
read by an investigator possessing average eyesight. A card of 
type of different sizes such as is commonly employed by oculists 
is taken to the point where the light is to be measured and some 
line of type is selected for the test. The distance at which this 
type must be held from the eyes in order to be legible is then 
measured and compared with the distance at which the same 
type can be seen in unobstructed daylight. The difference 
between the two distances is taken as a basis of difference in the 
strength of the illumination. 

Standard AIethod for Measuring Illumination. The second 
method, which is recommended as a standard procedure, depends on the 
use of photo-sensitive paper such as can be obtained from any dealer in 
photographic materials. By exposing the sensitized paper through a slot 
in a cardboard for :i sufficient period of time and noting the number of 
seconds or minutes consumed to match in depth a standard shade of 
color the intensity of light can be determined with much accuracy. If a 
fresh piece of paper is exposed to the direct rays of the sun for three 
seconds it will assume a shade which can be used as a standard for a given 
series of tests. The intensity of light at other points may be compared 
with this by noting the number of seconds required to color a fresh piece 
of paper from the same lot to the same shade. 

5. Velocity of Air Currents. 
The velocity of strong air currents is customarily measured 
by means of recording anemometers. There are so many of these 
instruments on the market and their use is so generally under- 
stood that it seems unnecessary to describe them. Anemometers 
require a considerable velocity of air and they should never 
be used without a carefully prepared table of corrections whereby 
their readings can be adjusted. 



40 STANDARD METHODS 

It often becomes desirable in sanitary investigations, parti- 
cularly in studies of ventilation, to determine the strength and 
direction of currents of air which are too delicate to be measured 
by means of anemometers. Lighted candles have sometimes 
been used to show the direction of such delicate air currents, the 
flame being deflected in the direction in which the current is mov- 
ing. More delicate than this is the method of noting the course 
taken by the smoke from a joss stick, cigarette or cigar. For a 
further discussion of the study of air currents reference may be 
made to Shaw (1907). 

6. Notes on Physical Determinations. 

Physical observations of the atmosphere to be of value must 
not only be made with accuracy and with instruments suitable 
to the particular tests made, but the observations should be 
sufficiently numerous to indicate representative or, at least, aver- 
age conditions at the place under inquiry. One determination 
of temperature or humidity, for example, is of little service unless 
it is known that the circumstances under which that determina- 
tion was made frequently occur. No instrument, of course, is 
mathematically exact. Each has its error and it is important 
to learn its error and allow for it whenever failure to do so would 
affect the value of the results desired. The difficulties of adjust- 
ment and uncertainty of results obtained with very delicate 
apparatus in the hands of unskilled workers make the recom- 
mendation of the most refined instruments seem unwise in this 
place, where practical rather than ultra-scientific methods are 
desirable. 

It is important in using any of the physical instruments 
referred to here that their accuracy be not over-rated. All 
instruments employed in sanitary investigations should be thor- 
oughly understood by the investigator using them, and where any 
considerable importance attaches to the results the instruments 
should be standardized. To standardize an instrument is to- 
compare it with some other instrument whose accuracy has been 
demonstrated and its error known. An extensive investigation 
should be carried on by the help of a special testing station, where 
all the instruments can be standardized and examined from time 
to time by a person especially assigned to this work. 



BACTERIAL EXAMINATION OF AIR 



41 



In the absence of a testing station or other convenient means 
of standardizing instruments, apparatus for the physical exami- 
nation of air can be sent to the Bureau of Standards, Washington, 
D. C. At that Bureau examinations can be made of thermome- 
ters and other instruments and the results reported upon at a 
nominal cost. Every laboratory and sanitary worker should 
have a few instruments which have been tested by this or some 
other laboratory and can be depended upon as accurate enough 
to be used for comparison. 

III. Chemical Determinations. 

1. Laboratory Methods for Determining Carbon Dioxide 
with a High Degree of Accuracy. 

Numerous efforts have been made to develop methods of 
analyzing air for carbon dioxide, applicable to the varying con- 
ditions under which the chemist, sanitary engineer or inspector 
must work. The chemist is called upon to make exceedingly 
accurate, careful analyses for scientific purposes, while the inspec- 
tor and engineer are called upon to make estimates and compari- 
sons. It is plain that no one system or method will satisfactorily 
meet the requirements of all these conditions and therefore in 
preparing a description of the most satisfactory processes for use 
as standard methods, the available methods have been classed 
either as accurate methods or as general tests. 

For accurate, scientific work, say, when accuracy to 1-10 of a 
part per ten thousand is required, the committee recommends as 
the standard the Patterson apparatus as modified by Sonden, 
one form of which has been used by Dr. F. G. Benedict of the 
Carnegie Nutrition Laboratory for over a year, with the great- 
est satisfaction* 

This apparatus measures a given volume of air, and absorbs 
the contained carbon dioxide in potassium hydroxide, afterward 
accurately measuring the remainder, thus giving the carbon 
dioxide present by volume. The air is measured in all cases at 
the same pressure and temperature and is measured accurately 
by means of the readings on a very finely graduated capillary. 
The principle is simple, but accurate operation requires con- 
siderable technique. 

This apparatus will shortly be described in print by Dr. Benedict. 



42 



STANDARD METHODS 



The apparatus may be had by applying to Sonden in Stock- 
holm at a cost of something less than one hundred dollars. 

For accurate inspection work, say, one-quarter of a part per 
ten thousand, the Eimer and Amend form of the Petterson 
Palmquist apparatus is recommended. This is very similar to 
the Sonden form but not as delicate. Its cost is about fifty-five 
•dollars. 



2. Practical Methods of Determining Carbon Dioxide 
for Sanitary Purposes. 

The time method of Cohen and Appleyard (1894), is recom- 
mended as combining practicability and reasonable accuracy 
in a degree suitable for practical sanitary work. 

Standard Method for Carbon Dioxide. If a dilute solution of lime 
water, slightly colored with phenolphthalein, is brought in contact with 
air containing more than enough C0 2 to combine with all the lime present, 
the solution will be gradually decolorized, the length of time required 
depending upon the amount of CO z present. The quantity of lime water 
and volume of air remaining the same, the rate of decolorization varies 
inversely with amount of carbon dioxide. The method is scientific in 
principle because it recognizes the fact that the absorption of C0 2 by Ca 
■ or Ba hydroxide solution is a time reaction. 

Collect samples of air in one-half liter glass-stoppered bottles by any 
of the methods of collection. Run in 10 cc. standard lime water, replace 
stopper, and note time. Shake bottle vigorously with both hands until 
color disappears. Note time required, and ascertain corresponding 
amount of C0 2 from table. 

TABLE. 



Time in Minutes 

to Decolorize 

Solution 


C0 2 per 
10,000 


Time in Minutes 

to Decolorize 

Solution 


C0 2 per 
10,000 


H 

1J 
1J 

2 
2* 

2| 
3i 


16.0 
13.8 
12.8 
12.0 
11.5 
8.6 
7.7 


3i 

4 

5 

Si 

61 

7i 


6.0 
5.3 
5.1 
4.6 
4.4 
4.2 
3.5 



BACTERIAL EXAMINATION OF AIR 



43 



3. Rough Methods of Determining Carbon Dioxide. 

For the sake of completeness a brief description of the shaker 
methods of determining carbon dioxide is here included, although 
their accuracy is not such as to warrant the committee in recom- 
mending their use. 

The volume of air that must be brought into contact with a 
definite quantity of lime water, in order to neutralize all the lime, 
is taken as a measure of the C0 2 in the air. The quantity of lime 
water and the time of reaction remaining constant, the amount of 
C0 2 varies inversely as the volume of air. The apparatus con- 
sists of graduated shakers either Wolpert or Fitz, and a pipette 
for measuring 10 c. c. of lime water. 

Be sure the plunger of the shaker slides easily, then remove and 
run into the tube 10 c. c. of the lime water solution. Introduce the 
plunger, and press it to the top of the solution, then withdraw it to 
the higher graduation. Close the mouth of the small tube in the 
Fitz, or the stem of the plunger in the Wolpert with the finger and 
shake vigorously for 30 seconds. The volume of air brought in 
contact with the solution is 50 c. c. in the Fitz and 40 c. c. in the 
Wolpert. Remove finger closing small end, press inner tube or 
plunger again to top of solution in Wolpert or to T in Fitz, and 
draw it up as before, thus admitting 20 c. c. fresh air in the Fitz 
and 40 in the Wolpert. Shake for 30 seconds. Repeat until 
color is discharged. The first trial will probably give the approxi- 
mate result, and subsequent tests will aid in giving the correct one. 
From the volume of air used, the amount of CO^ can be deter- 
mined from the table. 

TABLE. 



Air in cc. 


C0 2 per 


Air in cc. 


CO, per 


Used 


10,000 


Used 


10,000 


30 


28 


91 


9 bad 


36 


•>•> 


103 


8 


46 


18 very bad 


117 


7 


58 


14 


13S 


6 


69 


12 


165 


5 good 


82 


10 


207 


4 



44 STANDARD METHODS 

Stoppers and vials should be washed and dried and kept sep- 
arate and parts of the shaker should be kept separate. In using 
the shaker see that the fingers are clean. Take care to avoid loss 
of liquid on addition of fresh air. 

4. Methods of Collection. 

In the case of the Cohen and Appleyard Method. 

Fully as important as the actual test is the method of 
collecting the sample. For this the committee recommends as 
standard for more accurate work, the method of collection by 
water siphon. 

Standard Method of Collection. The Water Siphon Method. 
Two bottles (diameter one-third the height), volume about one-half litre, 
of nearly equal capacity should be fitted with rubber stoppers carrying 
small glass tubing connected by several feet of rubber connector, with 
clamps. Fill one bottle completely with water, nearly free from carbon 
dioxide. 

The pair of bottles is taken to the place from which the air is to be 
collected. The inlet tube may be long to reach to near the ceiling, or 
short; if long, the first siphoning should be rejected, to secure filling the 
inlet tube with the air desired, the stoppers exchanged, and the sample 
taken. The air-filled bottle should be stoppered and taken to the lab- 
oratory; or the test solution at once added, and the bottle stoppered and 
shaken, noting minutes and seconds. One bottle of water with a small 
reserve will serve for a number of takings before absorbing a deleterious 
amount of C0 2 . 

The Steam Vacuum Method may be used as an alternative 
in less accurate work. The steam is supplied by a 500 c. c. flask 
serving as a boiler, with a bunsen burner to apply the heat. The 
flask should be fitted with a rubber stopper carrying a No. 6 glass 
tube so arranged that one end extends within 3^ inch of the bot- 
tom of the bottle when placed in position on the stand. The 
bottles should be of about 500 c. c. capacity, made for a ground- 
glass stopper but fitted with a rubber stopper. 

To prepare the jet, the water in the flask should boil for five 
minutes in order to expel completely the air in the water and the 
flask. The pressure should be sufficient to throw the vaporized 
steam at least 1 foot above the exposed end of the tube. 

Place the empty bottle on the stand in an inverted position 
and allow to remain for three minutes. In the meantime apply 
a thin coating of vaseline half way up the sides of the stopper. 
The vaseline acts as an unguent, reducing the coefficient of fric- 
tion to such an extent that the principal resistance is due to the 
reaction of the stopper against compression. This enables one 



BACTERIAL EXAMINATION OF AIR 45 

to force the stopper in far enough to bring the glass and rubber 
into intimate contact, which is essential. The vaseline also fills 
the interstices between the rubber and the glass, so as to make 
leakage impossible. 

Protecting the hand with a cloth, raise the bottle from the 
stand, and the instant it clears the end of the tube insert the stop- 
per while the bottle is still inverted. The stopper may be pushed 
in more securely by pushing it against the table with a few pounds 
pressure while the bottle is still in the inverted position. Keep 
the stopper in under this pressure for a few minutes until the 
vacuum begins to form, after which the atmospheric pressure will 
keep it in place. 

All the bottles required are treated in the same way. The 
rubber stopper should be at least one size larger than would 
ordinarily be used for the bottle, and should project three-eighths 
of an inch or more so as to be easily removed when the sample is 
to be taken. 

Sample bottles may be tested for completeness of vacuum 
by holding them in an inverted position under water at 70° F., 
and removing the stopper. After the water has replaced the 
vacuum, the stopper is inserted and the bottle removed. 

5. Solutions. 

Standard Lime Water for General Tests. To a litre of distilled 
water add 2.5 cc. of phenolphthalein (made by dissolving .7 grams of 
phenolphthalein in 50 cc. of alcohol and adding an equal volume of water) . 
Stand the bottle of water on a piece of white paper and add drop by drop 
saturated lime water till a faint color persists for a full minute. Xo'w add 
0.3 cc. of saturated lime water and quickly cork the bottle, or connect the 
pipette. 

IV. Bacteriological Determinations. 
The determination of the number of bacteria in air seems to 
the Committee to have less importance than was once believed. 
Disease spread through air is probably due most often to direct 
pollution with spray from the mouth ; and it does not seem possi- 
ble to measure such pollution in a quantitative way. The total 
number of saprophytic bacteria often corresponds with the 
amount of dust present. This is especially true when the dust 
is not of metallic or other industrial origin. In the examination 
of the air of barns, dairies, theatres, factories and streets bac- 
terial data may prove of value. 



46 STANDARD METHODS 

1. Quantitative Determinations: A large number of differ- 
ent pieces of apparatus have been devised which are, after all,, 
simply adaptations of three general methods, viz.: 

(a) Filtration of air; (b) Bubbling air through some liquid 
medium; (c) Precipitating the bacteria from a given volume of 
air. While each of these methods can be made to give fairly 
satisfactory quantitative data in the hands of competent workers, 
nevertheless the committee is of the opinion that the time has 
arrived when one of them should be adopted as a standard and 
the others preferably dropped. In adopting a method as stan- 
dard, the following principles should govern the selection: 

(a) Simplicity and inexpensiveness of apparatus. 

(b) Ease of operation. 

(c) Universal applicability. 

Basing judgment upon these considerations and upon num- 
erous comparative tests made for the purpose, (Weinzirl and 
Fos), the Committee is of the opinion that the filtration 
method comes nearer to the ideal than either of the other two, 
and, therefore, that it should be adopted as standard. The 
apparatus and procedure is described as follows: 

Standard Method for Enumerating Bacteria in Air. (Filtra- 
tion method of Petri). The filter tubes are glass tubes 1J cm. in diameter 
and 10 cm. long. In the end of each is placed a perforated cork stopper 
through which a glass tube 6 mm. in diameter is passed. The filter con- 
sists of a layer of sand which has been passed through a 100 mesh sieve, 
1 cm. deep supported by a layer of bolting cloth covering the cork. Two 
filter tubes are connected in tandem and a measured volume of air, 10 
litres or more, is drawn through at a constant rate by suction. The suc- 
tion is applied by means of an aspirator of known volume, preferably one 
of the double or continuous type. Either the Aspirator, Magnus (No. 
12,210, $7.50-19.00), or the Double Aspirator (No. 12,212, S20--J25), 
both made by Bausch and Lomb are suitable for this purpose. Before 
using a pair of filter tubes, a test for possible leakage is made by placing 
the thumb over the cotton stopper and applying the aspirator; if the 
suction is weak or absent, the corks must be tightened or the tubes dis- 
carded. All corks should be tightened and connections wired immedi- 
ately before using the filters. The collection of the sample should take 
from 1 to 2 minutes, per liter. 

After filtering a definite volume of air through the tubes, the sand is 
shaken out into 10 cc. of sterile water, thoroughly shaken and aliquot 
portions plated in ordinary nutrient agar, all plates being made in dupli- 
cate. The plates are incubated at room temperature for five days, when 
final counts are made. If petri dishes 9 cm. in diameter are employed, 
all plates showing a larger count than 200 colonies should be rejected to- 
eliminate inhibitive action. 



BACTERIAL EXAMINATION OF AIR 47 

A rough idea of the bacterial content of the air may be ob- 
tained by the method of exposing plates for definite periods of 
time and counting the colonies which develop from the germs 
falling upon them. This procedure is not, however, recommended 
by the Committee on account of the fact that results are notably 
affected by varying environmental conditions and are not related 
to any specific volume of air. 

2. Qualitative Determinations: In the study of the bac- 
teriology of sewer air the colon bacillus and the sewage strepto- 
coccus may conveniently be used as indices of contamination. 
Samples may be collected as for the quantitative determination 
and, after incubating the filtering sand in dextrose broth, must be 
kept for at least a week and examined daily after three days. 
Any streptococcus producing a faint growth on agar and coag- 
ulating milk may be considered as of human origin. 

Gordon (1904) has suggested the use of the similar strepto- 
cocci found in the mouth as indices of pollution by mouth spray. 
Your committee (Winslow and Robinson, 1909) has been una- 
ble to find such organisms in the air, even under extreme 
conditions, in sufficient numbers to warrant the recommendation 
of this test. 

C.-E. A. Winslow, Chairman. 

Ellen H. Richards, 

G. A. Soper, 

J. Bosley Thomas, 

John Weinzirl. 

References. 

Cohen and Appleyard. 1894. Popular Methods for Esti- 
mating Carbon Dioxide in Air. Chemical News, LXX, 111. 

Gilbert, R. M., 1909. An Improvement in Heating and 
Ventilating: the Use of Recording Thermometers and Hygrome- 
ters by the Ventilating Engineer. Industrial Engineering, I, 271 . 

Gordon, M. H., 1904. Report on a Bacterial Test for Esti- 
mating Pollution of Air, Supplement to the Thirty-Second Annual 
Report of the Local Government Board Containing the Report of 
the Medical Officer for 1902-03, 421. 



48 STANDARD METHODS 

Great Britain, 1909. Report of the Departmental Commit- 
tee on Humidity and Ventilation in Cotton Weaving Sheds. 
London. 

Shaw, W. N., 1907. Air Currents and the Laws of Ventila- 
tion. Cambridge. 

Soper, G. A., 1908. The Air and Ventilation of Subways. 
New York. 

Ward, R. de C, 1899. Practical Exercises in Elementary 
Meteorology. Boston. 

Whipple, G. C, 1905. Microscopy of Drinking Water. 
New York. 

Winslow, C.-E. A., 1908. A Method for Determining the 
Number of Dust Particles in Air. Engineering News, LX, 748. 

Winslow, C.-E. A., and Robinson, E. A., 1909. An Inves- 
tigation of the Extent of the Bacterial Pollution of the Atmos- 
phere by Mouth Spray, Journal of Infectious Diseases, VII, 17. 



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