Examination of Milk

"One-cow" or "whole" milk, if taken from the apparently healthy animal (that is, an animal without any obvious lesion of the udder or teats) with ordinary precautions as to cleanliness, avoidance of dust, etc., contains but few organisms. In dealing with one-cow milk, from a suspected, or an obviously diseased animal, a complete analysis should include the examination (both qualitative and quantitative) of samples of (a) fore-milk, (b) mid-milk, (c) strippings, and, if possible, from each quarter of the udder. "Mixed" milk, on the other hand, by the time it leaves the retailer's hands, usually contains as many micro-organisms as an equal volume of sewage and indeed during the examination it is treated as such.

It is possible however to collect and store mixed milk in so cleanly a manner that its germ content does not exceed 5000 micro-organisms per cubic centimetre. Such comparative freedom from extraneous bacteria is usually secured by the purveyor only when he resorts to the process of pasteurisation (heating the milk to 65° C. for twenty minutes or to 77° C. for one minute) or the simpler plan of adding preservatives to the milk. Information regarding the employment of these methods for the destruction of bacteria should always be sought in the case of mixed milk samples, and in this connection the following tests will be found useful:

1. Raw Milk (Saul).

To 10 c.c. milk in a test tube, add 1 c.c. of a 1 per cent. aqueous solution of ortol (ortho-methyl-amino-phenol sulphate), recently prepared and mix. Next add 0.2 c.c. of a 3 per cent. peroxide of hydrogen solution. The appearance of a brick red color within 30 seconds indicates raw milk. Milk heated to 74° C. for thirty minutes undergoes no alteration in color; if heated to 75° C. for ten minutes only, the brick red color appears after standing for about two minutes.

2. Boric Acid.

Evaporate to dryness, 50 c.c. of the milk which has been rendered slightly alkaline to litmus, then incinerate.

Dissolve in distilled water, add slight excess of dilute hydrochloric acid and again evaporate to dryness.

Dissolve the residue in a small quantity of hot water and moisten a piece of turmeric paper with the solution. Dry the turmeric paper. Rose or cherry-red color = borax or boric acid.

3. Formaldehyde (Hehner).

To 10 c.c. milk in a test tube add 5 c.c. concentrated commercial sulphuric acid slowly, so that the two fluids do not mix. Hold the tube vertically and agitate very gently. Violet zone at the junction of the two liquids = formaldehyde.

4. Hydrogen Peroxide.

To 10 c.c. milk (diluted with equal quantities of water) in a test tube add 0.4 c.c. of a 4 per cent. alcoholic solution of benzidine and 0.2 c.c. acetic acid. Blue coloration of the mixture = hydrogen peroxide.

5. Salicylic Acid.

Precipitate the caseinogen by the addition of acetic acid and filter. To the filtrate add a few drops of 1 per cent. aqueous solution of ferric chloride. Purple coloration = salicylic acid.

6. Sodium Carbonate or Bicarbonate.

To 10 c.c. of the milk in a test tube add 10 c.c. of alcohol and 0.3 c.c. of a 1 per cent. alcoholic solution of rosolic acid. Brownish color = pure milk; rose color = preserved milk.

Quantitative.—

Collection of Sample.—

The apparatus used for the collection of a retail mixed milk sample 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° 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° 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° 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° 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° 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° 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° 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° C. to 30° C. and after five days the "total number of organisms developing at 28° 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 choleræ.
Streptococcus pyogenes longus.
Micrococcus melitensis.
Staphylococcus pyogenes aureus.
Bacillus enteritidis sporogenes.
Bacillus diphtheriæ.
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.

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.

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, fæcal débris, 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 and C.

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 and D.

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) and deposit (D) 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) 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° 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 Choleræ.—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° 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° C. for ten minutes and then incubate anaerobically at 37° 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. Diphtheriæ.—

(A) 1. Plant three sets of serial cultivations, twelve tubes in each set, from (a) cream C, (b) deposit D upon oblique inspissated blood-serum, and incubate at 37° 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 toxæmia, 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-Lœffler 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 or D).

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.

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 into each of three tubes of glucose formate bouillon, and incubate anaerobically (in Buchner's tubes) for twenty-four hours at 37° 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. diphtheriæ 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° 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 or D.

2. Spread several sets of surface plates upon nutrose agar, each from one drop of the original milk sample.

3. Incubate aerobically at 37° 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.