SYNOPSIS. An agar overlay technique developed to investigate the effect of various substances on growth of a selected group of protozoa is described. Test systems employing Crithidia fasciculata (culex), Ochromonas malhamensis, Tetrahymena pyriformis W and Trichomonas foetus were devised to evaluate the effect of known compounds and to assay the activity of antibiotic fermentation beers. This disc‐plate method can be summarized as follows: preparation of a foundation layer; overlaying of a mixture of agar, nutrients, protective antibiotics, and inoculum; placing of paper discs with the test material on the overlay followed by incubation and subsequent observation of the plates for zones of inhibition of growth. The concentration of agar in the overlay sufficient to restrict movement of the protozoa without inhibiting their growth, nutrients required for adequate growth and continued viability of the inoculum, and the concentration of penicillin and streptomycin which will not inhibit the assay protozoa but beeffective against the microorganisms in the fermentation beers are discussed. This technique offers a method not only for primary screening for cytotoxic substances but also one which may be adaptable to other studies of protozoa.

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