2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (_vide_ page 257), and expose to a temperature of 80 C. for twenty minutes.

3. Inoculate a _young_ white rat subcutaneously (on the inner aspect of one of the hind legs) with 1 c.c. of the emulsion. Observe during life, and, if the animal succ.u.mbs, make a complete post-mortem examination.

4. Melt three tubes of nutrient agar in boiling water and cool to 42 C.

5. Number the tubes 1, 2, and 3. To No. 1 add 0.2 c.c., to No. 2 add 0.3 c.c., and to No. 3 add 0.5 c.c. of the suspension, and pour plates therefrom.

6. Incubate at 37 C. for twenty-four or forty-eight hours.

7. Pick off any colonies resembling those of anthrax and subcultivate on all the ordinary laboratory media.

8. Inoculate another young white rat as in 3, using two loopfuls of the agar subcultivation emulsified with 1 c.c. sterile bouillon. Observe during life, and if the animal succ.u.mbs, make a complete post-mortem examination.

~B. Tetani.~--

1. Proceed as detailed above in steps 1 and 2 for the isolation of the B. anthracis.

2. Add 1 c.c. of the suspension to each of three tubes of glucose formate broth, and incubate anaerobically in Buchner's tubes at 37 C.

3. From such of the tubes as show visible growth (with or without the production of gas) after twenty-four hours' incubation inoculate guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1 c.c. of the bouillon cultivation as a dose. Observe carefully during life, and, if death occurs, make a complete post-mortem examination.

4. From the same tubes pour agar plates and incubate anaerobically in Bulloch's apparatus, at 37 C.

5. Subcultivate suspicious colonies on the various media, incubate anaerobically, making control cultivations on glucose formate agar, stab and streak, to incubate aerobically and carry out further inoculation experiments with the resulting growths.

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 a.n.a.lysis should include the examination (both qualitative and quant.i.tative) 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 quant.i.ty 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 quant.i.ties 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.

[Ill.u.s.tration: FIG. 211.--Milk-collecting bottle and dipper in case.]

Quant.i.tative.--

_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 gla.s.s 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.