2. Add a loopful of sterile vaseline to a tube of nutrient bouillon.

Prepare a subcultivation of the B. pestis in this and incubate at 37° C.

Note the formation of stalactites depending from the fat globules.

3. Make hanging-drop preparations from the bouillon and agar cultures, after forty-eight hours' incubation. Examine microscopically and compare.

4. Prepare cover-slip film preparations of each from all the media after twenty-four, forty-eight hours' and three days' incubation.

Stain carbolic methylene-blue, carbolic thionine blue, Gram's method, and compare.

5. Prepare smear cultivations on the surface of very dry agar and incubate at 37° C. for forty-eight hours. Make cover-slip preparations from the growth and stain with carbolic thionine blue.

Note the involution forms of B. pestis.

6. Examine the agar streak cultivations, after seven days' incubation, for spores.

7. Sterilise some salt, in a glass capsule, in the hotair oven.

Add about 0.5 gramme to a tube of nutrient bouillon and incubate at 37° C. for forty-eight hours, to determine its sterility.

8. Inoculate this bouillon with the B. pestis and incubate at 37° C. for twenty-four hours.

Examine the resulting growth microscopically.
Note the plasmolysed bacilli.

9. Stain the sections of bubo (containing B. pestis) with (a) Löffler's methylene-blue and (b) carbolic thionine blue.

10. Make a complete post-mortem examination of the cadaver of the rat infected intraperitoneally with the B. pestis.

Compare the naked-eye appearances of the cultures from day to day.

2. Prepare cover-slip film preparations of each from all the media after twenty-four hours' and three days' incubation.

Stain carbolic methylene-blue, carbolic fuchsin, Gram's method. Examine microscopically and compare.

3. Double stain sections of human liver (affected with actinomycosis) with picrocarmine and the GramWeigert method.

4. Stain the section of cow's tongue (infected with actinomyces) by Gram's method and counterstain with dilute aqueous solution of fuchsin.

5. Examine the pus from a breaking-down nodule (from cow's tongue) with a hand lens, pick out some of the minute yellowish granules with a sterile platinum loop, and transfer to a sterile capsule.

6. Crush the selected granules with a sterile glass. rod. Make cover-slip preparations and stain by Gram's method.

7. Inoculate four tubes of glycerine agar in series with some of the material.

Observe the resulting growth macroscopically and microscopically.

Note that growth only occurs in serum and blood media.

2. Prepare subcultivations from each:

Serum bouillon,

and

and incubate at 20° C.

Blood agar,

Observe from day to day for four days.
Note that no growth takes place.

3. Transfer the subcultivations (section 2) to the incubator at 37° C.

Note that no growth takes place the bacilli are dead.

4. Prepare cover-slip films of each bacillus from all the media upon which growth takes place, after sixteen, twenty-four, and forty-eight hours' incubation. Stain carbolic methylene-blue, very dilute aqueous solution of fuchsin, Gram's method. Examine micro

scopically and compare.

5. Prepare cover-slip preparations of the yellowish, more solid masses of the specimen of sputum from a case of influenza.

Stain in dilute fuchsin solution for twenty minutes. Examine microscopically.

6. Wash some of these selected portions of sputum in several changes of sterile distilled water.

7. Inoculate four blood agar tubes in series from the washed sputum, and incubate at 37° C.

Observe the resulting growth macroscopically and microscopically.

8. Prepare cover-slip films of pus from a case of acute muco-purulent conjunctivitis.

Stain carbolic methylene-blue, dilute fuchsin solution. 9. Inoculate four blood agar tubes in series from the pus from cases of acute conjunctivitis and incubate at 37° C. Observe the resulting growth macroscopically and microscopically.

Observe the naked-eye appearances of the cultures from day to day.

2. Make hanging-drop preparations from the bouillon and agar cultivations.

3. Prepare cover-slip films from the agar and bloodserum cultivations, after eighteen hours' and three days' incubation.

Stain carbolic methylene-blue, Neisser's method, and by Gram's method. Examine microscopically and compare.

Also compare with corresponding preparations of the B. diphtheria.

4. Stain sections of the testicle of the guinea-pig (containing B. mallei), by overstaining with Nicollé's gentian violet (vide page 83), and subsequently decolourising with 1 per cent. acetic acid.

5. Stain the sections of horse's lung (containing glanders bacilli) with Löffler's methylene-blue.

6. Make a complete post-mortem examination of the guinea-pig killed by infecting subcutaneously with the B. mallei.

Observe the naked-eye appearances of the cultures from day to day.

Note the extremely scanty growth on gelatine and agar at 20° C.

2. Make hanging-drop preparations from the bouillon and agar cultivations.

3. Prepare cover-slip films from the growth on each of the media and stain carbolic methylene-blue, dilute solution of fuchsin, by Gram's method; examine microscopically and compare.

Note the bacillary forms on agar.

4. Examine the agar cultivations, after seven days' incubation, for spores. Result negative.

5. Test a forty-eight-hour-old bouillon culture, filtered through a sterile filter paper, against the serum of a patient suffering from Malta fever.