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 quant.i.ty 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 ident.i.ty 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 succ.u.mb, make complete post-mortem examination and endeavour to isolate the pathogenic organisms from the local lesion. Confirm their ident.i.ty 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 quant.i.ty 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.

[Ill.u.s.tration: 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 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 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 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 C. for fifteen minutes--that is, until at least some of the ice cream has become liquid.

~Qualitative and Quant.i.tative Examination.~--Treat the fluid ice cream as milk and conduct the examination in precisely the same manner as described for milk (_vide_ page 443).