The Examination of Oysters and Other Shellfish

On opening the shell 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 shell. 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 glass 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.

Glass 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 glass capsules.

Erlenmeyer flasks, 250 c.c. capacity.

Double strength bile salt broth.

Method.—

1. Thoroughly clean the outside of the oyster shells by scrubbing each in turn with liquid soap and nail brush under a tap of running water. Then, holding an oyster shell in a pair of sterile forceps wash every part of the outside of the shell with a stream of sterile water running from an aspirator jar; deposit the oyster inside the sterile glass 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 glass dish and place it, resting on its convex shell, on the towel. Turn a corner of the sterile towel over the upper flat shell to give a firmer grip to the left hand, which holds the shell in position.

5. With the sterile oyster knife (in the right hand) open the shell and separate the body of the oyster from the inner surface of the upper flat shell. Bend back and separate the flat shell, leaving the body of the oyster in and attached to the concave shell. 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 shell.

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 glass 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 glass 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 glass 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.

15. Label tubes of bile salt broth and inoculate with the following amounts of diluted oysters:

No. 6 with 10 c.c. cylinder fluid = 0.1 oyster.
No. 5 with 1 c.c. cylinder fluid = 0.01 oyster.
No. 4 with 1 c.c. capsule I fluid = 0.001 oyster.
No. 3 with 1 c.c. capsule II fluid = 0.0001 oyster.
No. 2 with 1 c.c. capsule III fluid = 0.00001 oyster.
No. 1 with 1 c.c. capsule IV fluid = 0.000001 oyster.

16. Transfer 100 c.c. cylinder fluid (= 1 oyster) to an Erlenmeyer flask and add 50 c.c. double strength bile salt broth, and label 7.

17. Duplicate all the above indicated cultures.

18. Put up the tube cultures in Buchner's tubes and incubate anaerobically at 42° C.

If growth occurs in tube 1 the organism finally isolated, e. g., B. coli, must have been present to the extent of one million per oyster.

19. Complete the examination for members of the Coli-typhoid group and sewage streptococci, as directed under Water Examination, page 429 (steps 11-21).

20. Inoculate a series of 6 tubes of litmus milk with quantities of the material similar to those indicated in step 15; heat to 80° C. for ten minutes, and incubate under anaerobic conditions at 37° C. Examine for the presence of B. enteritidis sporogenes as directed under Water Examination, page 438 (steps 7-10).