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SUMMARY: The familiar resazurin test used in milk testing has been adapted to the estimation of nisin, using a fast-growing strain of Streptococcus cremoris as test organism. The reproducibility and accuracy of this test compares favourably with the many other assay methods for nisin which use Strep. agalactiae as test organism. Results may be obtained in less than an hour as compared with 15-48 hr. in the other tests and are a measure of bactericidal power only. Aseptic technique during the actual assay procedure is unnecessary. Methods were devised to ensure a suitable standard inoculum, which is imperative for this test. As little as 0.5 unit nish/ml. may be estimated. Inhibition of reductase activity and acid production by bacteria in milk have been used for the assay of penicillin and streptomycin. Reid & Brewer (1946) used Bacillus subtilis as test organism and added methylene blue as indicator system at the beginning of incubation. Approximate readings were possible after 4 hr. and final readings were taken after 18 hr. Sanchez & Lamensans (1947) used Lactobacillus bulguricus as test organism. Their indicator system was a solution containing methyl red and methylene blue, drops of which were added every 30 min. to control tubes. The test could usually be completed within 24 hr. Hirsch (1950) quotes a similar test as a rough guide for nisin activity? again using methylene blue as indicator, the test organism being a fast-growing strain of Strep. cremoris (1P5). The use of such a very fast- growing and sensitive organism (about four times as sensitive as Strep. ugulactiue; Hirsch, 1950) makes it difficult to obtain a stable end-point; this is easily overshot if a reasonably accurate and therefore narrow dilution range is chosen. A graded response as obtainable with resazurin as indicator is therefore preferable to the all-or-nothing response of methylene blue. Moreover? methods must be devised to ensure a reasonably constant inoculum with regard to growth phase and number of bacteria. We have attempted to embody these requirements into a method which could be used on a routine scale with a minimum of labour, apparatus and experience. MATERIALS Cultures. Fast-growing strains of Strep. cremoris were used. The original N.I.R.D. strain ' 1 P5 ? became somewhat sluggish and was eventually replaced by 'T17', a cheese-starter organism recently isolated from a culture from a Cheshire cheese factory. Doubtless many other strains would prove suitable. Subculture (0.1 yo ? v/v) was carried out every 24 hr. in the sterile milk medium given below. Incubation was at 30'. Medium. Spray-dried skim milk (120 g.; Benger's Ltd., Coleraine) was reconstituted as a 12 yo (w/v) solution in tap water. Volumes of 800 ml. were

The Assay of the Antibiotic Nisin by Means of a Reductase (Resazurin) test