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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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