consists of a cylindrical copper case, 16 cm. high and 9 cm. in
diameter, provided with a "pull-off" lid, containing a milk dipper, also
made of copper; and inside this, again, a wide-mouthed, stoppered glass
bottle of about 250 c.c. capacity (about 14 cm. high by 7 cm. diameter),
having a tablet for notes, sand-blasted on the side. The copper cylinder
and its contents, secured from shaking by packing with cotton-wool, are
sterilised in the hot-air oven (Fig. 26).
When collecting a sample,
1. Remove the cap from the cylinder.
2. Draw out the cotton-wool.
3. Lift out the bottle and dipper together.
4. Receive the milk in the sterile dipper, and pour it directly into the
sterile bottle.
5. Enter the particulars necessary for the identification of the
specimen, on the tablet, with a lead pencil, or pen and ink.
6. Pack the apparatus in the ice-box for transmission to the laboratory
in precisely the same manner as an ordinary water sample.
"Whole" milk may with advantage be collected in the sterile bottle
directly since the mouth is sufficiently wide for the milker to direct
the stream of milk into it.
~Condensed milk~ must be diluted with sterile distilled water in
accordance with the directions printed upon the label, then treated as
ordinary milk.
_Apparatus Required_:
Case of sterile capsules (25 c.c. capacity).
Case of sterile graduated pipettes, 10 c.c.
(in tenths of a cubic centimetre).
Case of sterile graduated pipettes, 1 c.c.
(in tenths of a cubic centimetre).
Flask containing 250 c.c. sterile bouillon.
Tall cylinder containing 2 per cent. lysol solution.
Plate-levelling stand.
Case of sterile plates.
Tubes nutrient gelatine or gelatine agar.
Tubes of wort gelatine.
Tubes of nutrient agar.
Water-bath regulated at 42 deg. C.
Bunsen burner.
Grease pencil.
METHOD.--
1. Arrange four sterile capsules in a row; number them I, II, III, and
IV.
2. Fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon
into each of the three remaining capsules.
3. Remove 1 c.c. milk from one of the bottles by means of a sterile
pipette and add it to the bouillon in capsule I; mix thoroughly by
repeatedly filling and emptying the pipette.
4. Remove 0.1 c.c. of the milky bouillon from capsule I, add it to the
contents of capsule II, and mix as before.
5. In like manner add 0.1 c.c. of the contents of capsule II to capsule
III; and then 0.1 c.c. of the contents of capsule III to capsule IV.
Then 1 c.c. of dilution I contains 0.1 c.c. milk sample.
1 c.c. of dilution II contains 0.001 c.c. milk sample.
1 c.c. of dilution III contains 0.00001 c.c. milk sample.
1 c.c. of dilution IV contains 0.0000001 c.c. milk sample.
6. Melt the gelatine and the agar tubes in boiling water; then transfer
to the water-bath and cool them down to 42 deg. C.
7. Number the gelatine tubes consecutively 1 to 12.
8. Inoculate the tubes with varying quantities of the material as
follows:
To tube No. 1 add 1.0 c.c. of the milk sample.
2 add 0.1 c.c. of the milk sample.
{ 3 add 1.0 c.c. from capsule I.
{ 4 add 0.1 c.c. from capsule I.
{ 5 add 1.0 c.c. from capsule II.
{ 6 add 0.1 c.c. from capsule II.
{ 7 add 0.5 c.c. from capsule III.
{ 8 add 0.3 c.c. from capsule III.
{ 9 add 0.2 c.c. from capsule III.
{ 10 add 0.5 c.c. from capsule IV.
{ 11 add 0.3 c.c. from capsule IV.
{ 12 add 0.2 c.c. from capsule IV.
9. Pour plates from the gelatine tubes; label, and incubate at 20 deg. C.
10. Liquefy five wort gelatine tubes and to them add 1.0 c.c. of the
milk sample and a similar quantity of the diluted milk from capsules I,
II, and III and IV respectively.
11. Pour plates from the wort gelatine; label, and incubate at 20 deg. C.
12. Inoculate the liquefied agar tubes as follows:
To tube No. 1 add 0.1 c.c. of the milk sample.
2 add 0.1 c.c. from capsule I.
3 add 0.1 c.c. from capsule II.
4 add 0.1 c.c. from capsule III.
5 add 1.0 c.c. from capsule IV. }
6 add 0.1 c.c. from capsule IV. }
13. Pour plates from the agar tubes; label, and incubate at 37 deg. C.
14. After twenty-four hours' incubation "inspect," and after forty-eight
hours' incubation, "count" the agar plates and estimate the number of
"organisms growing at 37 deg. C." present per cubic centimetre of the
sample of milk.
15. After three, four, or five days' incubation, "count" the gelatine
plates and estimate therefrom the number of "organisms growing at 20 deg.
C." present per cubic centimetre of the sample of milk.
16. After a similar interval "count" the wort gelatine plates and
estimate the number of moulds and yeasts present per cubic centimetre of
the sample of milk.
NOTE.--Many observers prefer to employ gelatine agar (see
page 193) for the quantitative examination. In this case
gelatine-agar plates should be poured from tubes containing
the quantities of material indicated in step 8, incubated at
28 deg. C. to 30 deg. C. and after five days the "total number
of organisms developing at 28 deg. C." recorded.
~Qualitative.~--The qualitative bacteriological examination of milk is
chiefly directed to the detection of the presence of one or more of the
following pathogenic bacteria and when present to the estimation of
their numerical frequency.
Members of the Coli-typhoid group.
Vibrio cholerae.
Streptococcus pyogenes longus.
Micrococcus melitensis.
Staphylococcus pyogenes aureus.
Bacillus enteritidis sporogenes.
Bacillus diphtheriae.
Bacillus tuberculosis.
Some of these occur as accidental contaminations, either from the water
supply to the cow farm, or from the farm employees, whilst others are
derived directly from the cow.
In milk, as in water examinations, two methods are available, viz.:
Enrichment and Concentration--the former is used for the demonstration
of bacteria of intestinal origin, the latter for the isolation of the
micro-organisms of diphtheria and tubercle. The first essential in the
latter process is the concentration of the bacterial contents of a large
volume of the sample into a small compass; but in the case of milk,
thorough centrifugalisation is substituted for filtration.
_Apparatus Required_:
A large centrifugal machine. This machine, to be of real
service in the bacteriological examination of milk, must
conform to the following requirements:
1. The centrifugal machine must be of such size, and should
carry tubes or bottles of such capacity, as to enable from
200 to 500 c.c. of milk to be manipulated at one time.
2. The rate of centrifugalisation should be from 2500 to
3000 revolutions per minute.
3. The portion of the machine destined to carry the tubes
should be a metal disc, of sufficient weight to ensure good
"flank" movement, continuing over a considerable period of
time. In other words, the machine should run down very
gradually and slowly after the motive power is removed, thus
obviating any disturbance of the relative positions of
particulate matter in the solution that is being
centrifugalised.
4. The machine should preferably be driven by electricity,
or by power, but in the case of hand-driven machines--
(a) The gearing should be so arranged that the requisite
speed is obtained by not more than forty or fifty
revolutions of the crank handle per minute, so that it may
be maintained for periods of twenty or thirty minutes
without undue exertion.
(b) The handle employed should be provided with a special
fastening (e. g., a clutch similar to that employed for
the free wheel of a bicycle), or should be readily
detachable so that, on ceasing to turn, the handle should
not, by its weight and air resistance, act as a brake and
stop the machine too suddenly.
One of the few satisfactory machines of this class is shown
in figure 212.
[Illustration: FIG. 212.--Electrically driven centrifugal machine, with
flexible (broken) spindle encircled by the field magnets of the motor.]
Sterile centrifugal tubes, of some 60-70 c.c. capacity,
tapering to a point at the closed end, plugged with
cotton-wool.
Small centrifugal machine to run two tubes of 10 c.c.
capacity at 2500 to 3000 revolutions per minute preferably
driven by electricity, of the type figured on page 327 (Fig.
162).
Sterile centrifugal tubes of 10 c.c. capacity with the
distal extremity contracted to a narrow tube and graduated
in hundredths of a cubic centimetre (Fig. 213).
Sterilised cork borer.
Case of sterile pipettes, 10 c.c. (in tenths of a cubic
centimetre).
Case of sterile pipettes, 1 c.c. (in tenths of a cubic
centimetre).
Sterile teat pipettes.
Flask of sterile normal saline solution.
METHOD.--
1. Fill 50 c.c. of the milk sample into each of four tubes, and replace
the cotton-wool plugs by solid rubber stoppers (sterilised by boiling),
and fit the tubes in the centrifugal machine.
NOTE.--One or two cubic centimetres of paraffinum liquidum
introduced into the buckets of the centrifuge before the
glass tubes are inserted will obviate any risk of breakage
to the latter.
[Illustration: FIG. 213.--Milk sedimenting tubes.]
[Illustration: FIG. 214.--Milk in centrifuge tube.]
2. Centrifugalise the milk sample for thirty minutes at a speed of 2500
revolutions per minute.
3. Remove the motive power and allow the machine to slow down gradually.
4. Remove the tubes of milk from the centrifuge. Each tube will now show
(Fig. 214):
(a) A superficial layer of cream (varying in thickness with different
samples) condensed into a semi-solid mass, which can be shown to
contain some organisms and a few leucocytes.
(b) A central layer of separated milk, thin, watery, and opalescent, and
containing extremely few bacteria.
(c) A sediment or deposit consisting of the great majority of the
contained bacteria and leucocytes, together with adventitious matter,
such as dirt, hair, epithelial cells, faecal debris, etc.
5. Withdraw the rubber stopper and remove a central plug of cream from
each tube by means of a sterile cork borer; place these masses of cream
in two sterile capsules. Label C^{1} and C^{2}.
6. Remove all but the last one or two c.c. of separated milk from each
tube, by means of sterile pipettes.
7. Mix the deposits thoroughly with the residual milk, pipette the
mixture from each pair of tubes into one sterile 10 c.c. tube
(graduated) by means of sterile teat pipettes, then fill to the 10 c.c.
mark with sterile normal saline solution and mix together. Label D^{1}
and D^{2}.
8. Place the two tubes of mixed deposit in the centrifuge, adjust by the
addition or subtraction of saline solution so that they counterpoise
exactly, and centrifugalise for ten minutes.
NOTE.--Each tube now contains the deposit from 100 c.c. of
the milk sample and the amount can be read off in hundredths
of a centimetre. The multiplication of this figure by 100
will give the amount of "Apparent Filth," in "parts per
million"--the usual method of recording this quality of
milk.
9. Pipette off all the supernatant fluid and invert the tube to drain on
to a pad of sterilised cotton-wool, contained in a beaker. (This wool is
subsequently cremated.)
10. Examine both cream (C^{1}) and deposit (D^{1}) microscopically--
(a) In hanging-drop preparations.
(b) In film preparations stained carbolic methylene-blue, by Gram's
method, by Neisser's method, and by Ziehl-Neelsen's method.
Note the presence or absence of altered and unaltered vegetable fibres;
pus cells, blood discs; cocci in groups or chains, diphtheroid bacilli,
Gram negative bacilli or cocci, spores and acid fast bacteria.
11. Adapt the final stages of the investigation to the special
requirements of each individual sample, thus:
~1. Members of the Coli-typhoid Group.~--
1. Emulsify the deposit from the second centrifugal tube (D^{2}) with 10
c.c. sterile bouillon and inoculate three tubes of bile salt broth as
follows:
To Tube No. 1 add 2.5 c.c. milk deposit emulsion
(=25 c.c. original milk.)
To Tube No. 2 add 1.0 c.c. milk deposit emulsion
(=10 c.c. original milk.)
To Tube No. 3 add 0.5 c.c. milk deposit emulsion
(= 5 c.c. original milk.)
2. Inoculate tube of bile salt broth No. 4 with 1 c.c. of the original
milk.
3. Inoculate further tubes of bile salt broth with previously prepared
dilutions (see page 445) as follows:
To tube No. 5 add 1.0 c.c. from capsule I.
To tube No. 6 add 0.1 c.c. from capsule I.
To tube No. 7 add 1.0 c.c. from capsule II.
To tube No. 8 add 0.1 c.c. from capsule II.
To tube No. 9 add 1.0 c.c. from capsule III.
To tube No. 10 add 0.1 c.c. from capsule III.
To tube No. 11 add 1.0 c.c. from capsule IV.
To tube No. 12 add 0.1 c.c. from capsule IV.
and incubate anaerobically (in Buchner's tubes) at 42 deg. C. for a
maximum period of forty-eight hours.
4. If growth occurs complete the investigation as detailed under the
corresponding section of water examination (see pages 428 to 431).
NOTE.--The B. coli communis, derived from the alvine
discharges of the cow, is almost universally present in
large or small numbers, in retail milk. Its detection,
therefore, unless in enormous numbers, (when it indicates
want of cleanliness), is of little value.
~2. Vibrio Cholerae.~--Inoculate tubes of peptone water by using the same
amounts as in the search for members of the Coli-typhoid groups (_vide
ante_ 1-3); incubate aerobically at 37 deg. C. and complete the
examination as detailed under the corresponding section of water
examination (see page 439).
~3. B. Enteritidis Sporogenes.~--Inoculate tubes of litmus milk with
similar amounts to those used in the previous searches, omitting tube
No. 1 (_vide ante_ 1-3) place in the differential steriliser at 80 deg.
C. for ten minutes and then incubate anaerobically at 37 deg. C. for a
maximum period of forty-eight hours. Complete the investigation as
detailed under the corresponding section of water examination (see
page 438).
~4. B. Diphtheriae.~--
(A) 1. Plant three sets of serial cultivations, twelve tubes in each
set, from (a) cream C^{2}, (b) deposit D^{1} upon oblique
inspissated blood-serum, and incubate at 37 deg. C.
2. Pick off any suspicious colonies which may have made their appearance
twelve hours after incubation, examine microscopically and subcultivate
upon blood-serum and place in the incubator; return the original tubes
to the incubator.
3. Repeat this after eighteen hours' incubation.
4. From the resulting growths make cover-slip preparations and stain
carbolic methylene-blue, Neisser's method, Gram's method. Subcultivate
such as appear to be composed of diphtheria bacilli in glucose peptone
solution. Note those in which acid production takes place.
5. Inoculate guinea-pigs subcutaneously with one or two cubic
centimetres forty-eight-hour-old glucose bouillon cultivation derived
from the first subcultivation of each glucose fermenter, and observe the
result.
6. If death, apparently from diphtheritic toxaemia, ensues, inoculate two
more guinea pigs with a similar quantity of the lethal culture. Reserve
one animal as a control and into the other inject 1000 units of
antidiphtheritic serum. If the control dies and the treated animal
survives, the proof of the identity of the organism isolated with the
Klebs-Loeffler bacillus becomes absolute.
7. Inoculate guinea-pigs subcutaneously with filtered glucose bouillon
cultivations (toxins?) and observe the result.
(B) 1. Emulsify the remainder of the deposit with 5 c.c. sterile
bouillon and inoculate two guinea-pigs, thus: guinea-pig a,
subcutaneously with 1 c.c. emulsion; guinea-pig b, subcutaneously with
2 c.c. emulsion; and observe the result.
2. If either or both of the inoculated animals succumb, make complete
post-mortem examination and endeavour to isolate the pathogenic
organisms from the local lesion. Confirm their identity as in A5 and 6
(_vide supra_).
~5. Bacillus Tuberculosis.~--
(A) 1. Inoculate each of three guinea-pigs (previously tested with
tuberculin, to prove their freedom from spontaneous tuberculosis)
subcutaneously at the inner aspect of the bend of the left knee, with 1
c.c. of the deposit emulsion remaining in one or other tube (D^{1} or
D^{2}).
2. Introduce a small quantity of the cream into a subcutaneous pocket
prepared at the inner aspect of the bend of the right knee of each of
these three animals. Place a sealed dressing on the wound.
3. Observe carefully, and weigh accurately each day.
4. Kill one guinea-pig at the end of the second week and make a
complete post-mortem examination.
5. If the result of the examination is negative or inconclusive, kill a
second guinea-pig at the end of the third week and examine carefully.
[Illustration: FIG. 215.--Cadaver of guinea-pig experimentally infected
with B. tuberculosis.]
6. If still negative or inconclusive, kill the third guinea-pig at the
end of the _sixth_ week. Make a careful post-mortem examination.
Examine material from any caseous glands microscopically and inoculate
freely on to Dorset's egg medium.
NOTE.--Every post-mortem examination of animals infected
with tuberculous material should include the naked eye and
microscopical examination of the popliteal, superficial and
deep inguinal, iliac, lumbar and axillary glands on each
side of the body, also the retrohepatic, bronchial and
sternal glands, the spleen, liver and lungs (Fig. 215).
(B) 1. Intimately mix all the available cream and deposit from the milk
sample, and transfer to a sterile Erlenmeyer flask.
2. Treat the mixture by the antiformin method (_vide_ Appendix, page
502).
3. Inoculate each of two guinea-pigs, intraperitoneally, with half of
the emulsion thus obtained.
4. Kill one of the guinea-pigs at the end of the first week and examine
carefully.
5. Kill the second guinea-pig at the end of the second week and examine
carefully.
6. Utilise the remainder of the deposit for microscopical examination
and cultivations upon Dorset's egg medium.
NOTE.--No value whatever attaches to the result of a
microscopical examination for the presence of the B.
tuberculosis unless confirmed by the result of inoculation
experiments.
~6. Streptococcus Pyogenes Longus.~--
(A) 1. Spread serial surface plates upon nutrose agar. Also plant serial
cultivations upon sloped nutrient agar (six tubes in series).
2. If the resulting growth shows colonies which resemble those of the
streptococcus, make subcultivations upon agar and in bouillon, in the
first instance, and study carefully.
(B) 1. Plant a large loopful of the deposit D^{2} into each of three
tubes of glucose formate bouillon, and incubate anaerobically (in
Buchner's tubes) for twenty-four hours at 37 deg. C.
2. If the resulting growth resembles that of the streptococcus, make
subcultivations upon nutrient agar.
3. Prepare subcultivations of any suspicious colonies that appear, upon
all the ordinary media, and study carefully.
If the streptococcus is successfully isolated, inoculate serum bouillon
cultivations into the mouse, guinea-pig, and rabbit, to determine its
pathogenicity and virulence.
~7. Staphylococcus Pyogenes Aureus.~--
1. Examine carefully the growth upon the serial blood serum cultivations
prepared to isolate B. diphtheriae and the serial agar cultivations to
isolate streptococci after forty-eight hours' incubation.
2. Pick off any suspicious orange coloured colonies, plant on sloped
agar, and incubate at 20 deg. C. Observe pigment formation.
3. Prepare subcultivations from any suspicious growths upon all the
ordinary media, study carefully and investigate their pathogenicity.
~8. Micrococcus Melitensis.~--The milk from an animal infected with M.
melitensis usually contains the organisms in large numbers and but few
other bacteria.
1. Spread several sets of surface plates upon nutrose agar, each from
one loopful of the deposit in tube D^{1} or D^{2}.
2. Spread several sets of surface plates upon nutrose agar, each from
one drop of the original milk sample.
3. Incubate aerobically at 37 deg. C. and examine daily up to the end
of ten days.
4. Pick off suspicious colonies, examine them microscopically and
subcultivate upon nutrose agar in tubes; upon glucose agar and in litmus
milk.
5. Test the subsequent growth against the serum of an experimental
animal inoculated against M. melitensis to determine its
agglutinability.
6. If apparently M. melitensis, inoculate growth from a nutrose agar
culture after three days incubation intracranially into the guinea-pig.
ICE CREAM.
~Collection of the Sample.~--
1. Remove the sample from the drum in the ladle or spoon with which the
vendor retails the ice cream, and place it at once in a sterile copper
capsule, similar to that employed for earth samples (_vide_ page 471).
2. Pack for transmission in the ice-box.
3. On arrival at the laboratory place the copper capsules containing the
ice cream in the incubator at 20 deg. C. for fifteen minutes--that is,
until at least some of the ice cream has become liquid.
~Qualitative and Quantitative Examination.~--Treat the fluid ice cream as
milk and conduct the examination in precisely the same manner as
described for milk (_vide_ page 443).
EXAMINATION OF CREAM AND BUTTER.
~Collection of the Sample.~--Collect, store, and transmit samples to the
laboratory, precisely as is done in the case of ice cream.
~Quantitative.~--
_Apparatus Required_:
Sterile test-tube.
Sterilised spatula.
Water-bath regulated at 42 deg. C.
Case of sterile plates.
Case of sterile graduated pipettes, 1 c.c. (in hundredths).
Tubes of gelatine-agar (+10 reaction).
Plate-levelling stand, with its water chamber filled with water at
42 deg. C.
METHOD.--
1. Transfer a few grammes of the sample to a sterile test-tube by means
of the sterilised spatula.
2. Place the tube in the water-bath at 42 deg. C. until the contents are
liquid.
3. Liquefy eight tubes of gelatine-agar and place them in the water-bath
at 42 deg. C, and cool down to that temperature.
4. Inoculate the gelatine-agar tubes with the following quantities of
the sample by the help of a sterile pipette graduated to hundredths of a
cubic centimetre--viz.,
To tube No. 1 add 1 c.c. liquefied butter.
2 add 0.5 c.c. liquefied butter.
3 add 0.3 c.c. liquefied butter.
4 add 0.2 c.c. liquefied butter.
5 add 0.1 c.c. liquefied butter.
6 add 0.05 c.c. liquefied butter.
7 add 0.03 c.c. liquefied butter.
8 add 0.02 c.c. liquefied butter.
9 add 0.01 c.c. liquefied butter.
5. Pour a plate cultivation from each of the gelatine-agar tubes and
incubate at 28 deg. C.
6. "Count" the plates after three days' incubation, and from the figures
thus obtained estimate the number of organisms present per cubic
centimetre of the sample.
~Qualitative.~--
_Apparatus Required_:
Sterile beaker, its mouth plugged with sterile cotton-wool.
Counterpoise for beaker.
Scales and weights.
Sterilised spatula.
Water-bath regulated at 42 deg. C.
Separatory funnel, 250 c.c. capacity, its delivery tube
protected against contamination by passing it through a
cotton-wool plug into the interior of a small Erlenmeyer
flask which serves to support the funnel. This piece of
apparatus is sterilised _en masse_ in the hot-air oven.
Large centrifugal machine.
Sterile tubes (for the centrifuge) closed with solid rubber
stoppers.
Case of sterile pipettes, 10 c.c.
Case of sterile graduated pipettes, 1 c.c. (in tenths of a
cubic centimetre).
METHOD.--
1. Weigh out 100 grammes of the sample in a sterile beaker.
2. Plug the mouth of the beaker with sterile cotton-wool and immerse the
beaker in a water-bath at 42 deg. C. until the contents are completely
liquefied.
3. Fill the liquefied butter into the sterile separatory funnel.
4. Transfer the funnel to the incubator at 37 deg. C. and allow it to
remain there for four days.
At the end of this time the contents of the funnel will have separated
into two distinct strata.
(a) A superficial oily layer, practically free from bacteria.
(b) A deep watery layer, turbid and cloudy from the growth of bacteria.
5. Draw off the subnatant turbid layer into sterile centrifugal tubes,
previously warned to about 42 deg. C., and centrifugalise at once.