PLATES IV AND VD URING an investigation of Cryptococcus neoformans isolated from clinical specimens, we observed that growth of the fungus was considerably reduced in the vicinity of colonies of Pseudomonas aeruginosa. This phenomenon was first noted by Fisher (1954), who found that the inhibitory effect was exhibited only by living cultures of certain strains of P. aeruginosa. His efforts to isolate the inhibitor were unsuccessful and no further report on this phenomenon has since appeared. We report here a study of factors affecting the production of inhibitor by P. aeruginosa and an initial attempt to isolate and characterise the substances involved. MATERIALS AND METHODSStrains tested. Of 44 strains of P. aeruginosa tested for their inhibitory activity on C. neoformans, 11 (nos. 8, 10, 17, 26, 39, 52, l0/55, 8/39, 283, 577 and 584) were obtained from Dr M. T. Parker, Colindale, England; 13 (Gillies and Govan's type strains 1, 2, 3, 4, 5, 6, 7, 8 and A, By C, D, E) were obtained from the Public Health Laboratory, Edmonton, Canada; and 20 were freshly isolated from clinical specimens of wound and burn infections in Hong Kong. Fourteen strains of C. neoformans were tested for their sensitivity to the inhibitory effect of P. aeruginosa. These included 13 strains isolated from the cerebrospinal fluid of cryptococcal meningitis cases in Hong Kong and one strain, no. 1499.22, obtained from Dr Margarita Silva, Columbia University, NY, USA.The media used for this study were: Sabouraud Dextrose Agar (Oxoid), Tryptone Soya Agar (TSA, Oxoid), Diagnostic Sensitivity Test Agar (DST, Oxoid), and King's media A (without CuC12) and B. The latter two media were prepared according to the published formulae (King, Ward and Raney, 1954). Glucose was added to TSA, DST and the two King's media to final concentrations of 0.2, 0.5, 1.0, 2-0 and 3.0% to study its effect on the production of inhibitors.Methods for testing the inhibitory action of P. aeruginosa on the growth of C. neoformans. In preliminary studies, two methods were used to demonstrate the inhibitory effect. (1) By means of the technique described by Fisher (1954)' plates of TSA medium were initially lawned with C. neoformans, and after drying were inoculated centrally with P. aeruginosa (lower row, fig. 1). Small zones of inhibition were obtained. (2) With a method based on that described by Gillies and Govan (1966), P. aeruginosa from a 24-h broth culture was inoculated centrally in a circle of 1-cm diameter on TSA plates and incubated for 2 days. Growth was then removed, the residual cells were killed with chloroform vapour, and the surface of the plate was lawned with C. neoforrnans (top row, fig. 1). Zones of inhibition were larger, and it was apparent that inhibition did not depend on the presence of living P. aeruginosa.Media.
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