II _Experimental._

_Tissue._--

1. Feed rats and mice on portions of the sample and observe the result.

2. If any of the animals die, make complete post-mortem examinations and endeavour to isolate the pathogenic organisms.

_Extract._--

1. Introduce various quant.i.ties of the bouillon extract into the stomachs of several rats, mice and guinea-pigs repeatedly over a period of two or three days by the intragastric method of inoculation (see page 367) and observe the result. Guinea-pigs and mice are very susceptible to infection by B. botulinus by this method; rabbits less so.

2. Inoculate rats, mice, and guinea-pigs subcutaneously into deep pockets, and intraperitoneally with various quant.i.ties of the bouillon extract, and observe the result.

3. Filter some of the extract through a Chamberland candle and incubate the filtrate to determine the presence of soluble toxins.

4. If any of the animals succ.u.mb to either of these methods of inoculation, make careful post-mortem examinations and endeavour to isolate the pathogenic organisms.

THE EXAMINATION OF OYSTERS AND OTHER Sh.e.l.lFISH.

On opening the sh.e.l.l of an oyster a certain amount of fluid termed "liquor" is found to be present. This varies in amount from a drop to many cubic centimetres (0.1 c.c. to 10 c.c.)--in the latter case the bulk of the fluid is probably the last quantum of water ingested by the bivalve before closing its sh.e.l.l. In order to obtain a working average of the bacteriological flora of a sample, ten oysters should be taken and the body, gastric juice and liquor should be thoroughly mixed before examination. The examination, as in dealing with other food stuffs, is directed to the search for members of the Coli-typhoid group, sewage streptococci and perhaps also B. enteritidis sporogenes.

_Apparatus Required_:

Two hard nail brushes.

Liquid soap.

Sterile water in aspirator jar with delivery nozzle controlled by a spring clip.

Sterile oyster knives.

Sterile gla.s.s dish, with cover, sufficiently large to accommodate ten oysters.

Sterile forceps.

Sterile scissors.

Sterile towels or large gauze pads.

Sterile graduated cylinders 1000 c.c. capacity, with either the lid or the bottom of a sterile Petri dish inverted over the open mouth as a cover.

Gla.s.s rods.

Corrosive sublimate solution, 1 per mille.

Bile salt broth tubes.

Litmus milk tubes.

Surface plates of nutrose agar.

Case of sterile pipettes, 1 c.c. (in tenths of a c.c.)

Case of sterile pipettes, 10 c.c. (in tenths of a c.c.)

Case of sterile gla.s.s capsules.

Erlenmeyer flasks, 250 c.c. capacity.

Double strength bile salt broth.

METHOD.--

1. Thoroughly clean the outside of the oyster sh.e.l.ls by scrubbing each in turn with liquid soap and nail brush under a tap of running water.

Then, holding an oyster sh.e.l.l in a pair of sterile forceps wash every part of the outside of the sh.e.l.l with a stream of sterile water running from an aspirator jar; deposit the oyster inside the sterile gla.s.s dish.

Repeat the process with each of the remaining oysters.

2. Before proceeding further, cleanse the hands thoroughly with clean nail brush, soap and water, then plunge them in lysol 2 per cent.

solution, and finally in sterile water.

3. Spread a sterile towel on the bench.

4. Remove one of the oysters from the sterile gla.s.s dish and place it, resting on its convex sh.e.l.l, on the towel. Turn a corner of the sterile towel over the upper flat sh.e.l.l to give a firmer grip to the left hand, which holds the sh.e.l.l in position.

5. With the sterile oyster knife (in the right hand) open the sh.e.l.l and separate the body of the oyster from the inner surface of the upper flat sh.e.l.l. Bend back and separate the flat sh.e.l.l, leaving the body of the oyster in and attached to the concave sh.e.l.l. Avoid spilling any of the liquor.

(Some dexterity in opening oysters should be acquired before undertaking these experiments).

6. Cut up the body of the oyster with sterile scissors into small pieces and allow the liquor freed from the body during the process to mix with the liquor previously in the sh.e.l.l.

7. Transfer the comminuted oyster and the liquor to the cylinder.

8. Treat each of the remaining oysters in similar fashion.

9. Mix the contents of the cylinder thoroughly by stirring with a sterile gla.s.s rod. The total volume will amount to about 100 c.c.

10. Use 0.1 c.c. of the mixed liquor to inseminate each of a series of three nutrose surface plates.

11. Inoculate 0.1 c.c. of the mixed liquor into each of three tubes of litmus milk.

12. Add sterile distilled water to the contents of the cylinder up to 1000 c.c. and stir thoroughly with a sterile gla.s.s rod and allow to settle. The bacterial content of each oyster may be regarded, for all practical purposes, as comprised in 100 c.c. of fluid.

13. Arrange four gla.s.s capsules in a row and number I, II, III, IV.

Pipette 9 c.c. sterile distilled water into each.

14. To capsule No. I add 1 c.c. of the diluted liquor, etc. from the cylinder, and mix thoroughly. To capsule II add 1 c.c. of dilution in capsule I and mix thoroughly. Carry over 1 c.c. of fluid from capsule II to capsule III, afterwards adding 1 c.c. of fluid from capsule III to capsule IV.