Section IA standard bacteriological test for molluscan shellfish should fulfil certain requirements stated: briefly, the test should show the degree of pollution, be accurate and rapid, and self-sufficient, not requiring subsequent confirmation. Review of the subject leads to recommendations, first, to overcome the technical difficulties of preparing samples for testing; secondly, on the nature of the test. In preparation: external shell sterilization can usually be omitted, shell water should be discarded and replaced by sterile water to make a total volume three times that of the body tissues. Pooling of individual shellfish into one sample is acceptable in routine examinations. In the test: a solid medium is preferable to a liquid medium, giving more accurate results, and review of existing tests leads to the conclusion that the use of roll tubes of MacConkey agar incubated at 44° should meet the requirements of a standard test.Section IIA modification was found necessary in the MacConkey agar: a mixture of 2 % gelatin and 5 % agar is used instead of the normal 2 % agar. Mechanical rolling devices for tubes are described and figured.Among other critical experiments, 1000 roll-tube colonies grown at 44° from shellfish included 969 with + + − − ‘IMViC’ reactions and 979 acid and gas producers at 44°. The coefficient of variation among replicate tests of samples of shellfish and water in roll tubes was not seriously greater than that for colony counts in Petri dishes at 37° with ordinary MacConkey agar.Colonies in roll tubes incubated at 44° can be counted as conveniently and accurately as those on Petri dishes, and, in general it is concluded that the new method is more satisfactory for estimation of faecalcolithan other methods at present in use.Section IIIDirections are given for the preparation of shellfish and inoculation into roll cultures, both for individual and for pooled examination, and the method of determining results is described.The interpretation of results is discussed, and it is suggested that shellfish which in four out of five samples from the same source are free from faecalcoliin 1 ml. quantities of body tissue should be regarded as satisfactory for food. The presence of more than two or three faecalcoliper ml. of body tissue in any one sample calls for appropriate action according to the number present.

1. The authors concluded in a previous paper that the most reliable single test for the detection ofBact. coliwas incubation in MacConkey's broth at 44° C, such a test forBact. colialone being necessary in shellfish because of occasional multiplication of other coliforms in purified mussels. The present investigation is an attempt to verify this finding on a broader basis.2. A description is given of the isolation of 1600 cultures of coliform organisms from polluted mussels, sewage, and faeces (human, cow, sheep and sea birds). Their classification according to ‘IMViC’ reactions and the production of gas in MacConkey's broth at temperatures of 37, 41, 44, 45 and 46° C. is given.3. Of the + + − − type, 1036 out of 1065 cultures (97·3%) were found to produce gas in MacConkey's broth at 44° C. A balance is noted between the + + − − type which does not produce gas at 44° C. (irregular I) and the two irregular types which do so (irregular II and irregular VI). Such a balance was reported by Bardsley (1938).4. From the cultures examined it is confirmed that incubation at 44° C. is the most suitable temperature for use with MacConkey's broth for permitting the maximum number ofBact. colito produce gas while inhibiting the maximum number of other coliforms.

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1. The use ofEsch. colialone as an index of faecal pollution for shellfish, and the correlation between the 44° C. MacConkey test and citrate tests are discussed.2. The mercury-toluene thermo-regulator used in these experiments, which gives a maximum variation of ± 0.1° C., is discussed briefly and illustrated.3. Experiments are described in which 522 colonies from polluted shellfish were isolated, inoculated into MacConkey's broth and incubated at temperatures of 37° C. and at successive 1° intervals from 41 to 46° C., in accurately controlled water-baths. An almost perfect negative correlation was found to exist between 44° C. incubation and the citrate test.4. It appeared that temperatures above 44° C. are detrimental to the growth ofEsch. coli.5. Certain cultures of citrate-negative lactose fermenters at 37° C., which were inhibited at 44° C., were found on further investigation to be mostly intermediate types.