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blue, Gram's method, Neisser's method, Ziehl-Neelsen's method.

11. Adapt the final stages of the investigation to the special requirements of each individual sample, as follows:

Members of the Typhoid and Colon Groups.―
Bacillus Enteritidis of Gaertner.-

Bacillus Enteritidis Sporogenes.

Vibrio Choleræ.

When searching for any or either of these organisms, emulsify the deposit with 10 c.c. sterile bouillon and proceed with the examination as described under

water.

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.

B. Diphtheriæ.—

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

2. Pick off any suspicious colonies which may have made their appearance nine hours after incubation, and subcultivate upon blood-serum; 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.

5. Inoculate guinea-pigs subcutaneously with fortyeight-hour-old glucose bouillon cultivation derived from the first subcultivation, and observe the result.

6. Inoculate guinea-pigs subcutaneously with filtered

glucose bouillon cultivations (toxins ?) and observe the result.

(B) 1. Emulsify the remainder of the deposit with 3 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.

Bacillus Tuberculosis.-Add 5 c.c. sterile bouillon to the deposit in the tube and emulsify thoroughly.

(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 emulsion.

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. (Compare Fig. 158, page 307.)

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.

(B) 1. Place the tube containing the remainder of the emulsion in a water-bath at 56° C. for ten minutes. 2. Remove the tube from the water-bath and cool rapidly.

3. Inoculate each of two guinea-pigs, intraperitoneally, with 1 c.c. of the emulsion.

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.

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.

Streptococcus Pyogenes Longus.—

(A) 1. Plant serial cultivations from the deposit upon (a) oblique inspissated blood-serum (six tubes. in series) and (b) oblique 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 into each of three tubes of glucose formate bouillon, and incubate anaerobically (in Buchner's tubes) for twentyfour 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.

Staphylococcus Pyogenes Aureus.—

1. Prepare serial cultivations upon oblique nutrient agar (eight tubes in series).

2. Incubate at 37° C. for twenty-four hours.

3. Pick off any suspicious colonies, plant on oblique agar, and incubate at 20° C. Observe pigment formation.

4. Prepare subcultivations from any suspicious growths upon all the ordinary media and study carefully.

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.

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° 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 346).

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° C.

Case of sterile plates.

Case of sterile graduated pipettes, I c.c. (in hundredths). Tubes of gelatine-agar (+10 reaction).

Plate-levelling stand, with its water chamber filled with water at 42° 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° C. until the contents are liquid.

3. Liquefy eight tubes of gelatine-agar and place them in the water-bath at 42° 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., 0.1, 0.2, 0.3, 0.5, 0.01, 0.02, 0.03, and 0.05 c.c. 5. Pour a plate cultivation from each of the gelatineagar tubes and incubate at 28° 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. Scales and weights.

Sterilised spatula.

Water-bath regulated at 42° 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.

Centrifugal machine.

Sterile tubes (for the centrifuge) closed with solid rubber stoppers.

Case of sterile pipettes.

METHOD.

1. Weigh out 100 grammes of the sample in a sterile beaker.

2. Plug the mouth of the beaker with sterile cottonwool and immerse the beaker in a water-bath at 42° C. until the contents are completely liquefied.

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