A PART from some phytopathogens, the fluorescent group of pseudomonads comprises Pseudomonas aeruginosa, P. fluorescens and P. putida. P. aeruginosa is the commonest and most clinically significant species. The other two species are occasionally associated with serious infections (Gilardi, 1972 and 1976;Martin, Maker and Washington, 1973) but, more significantly, they may be confused with P. aeruginosa in the laboratory (Falkiner, Keane and Taylor, Brown and Scott-Foster (1970) devised a simple milk medium to distinguish between P. aeruginosa and P. fluorescens. Colonies of P. aeruginosa were surrounded by a clear zone due to hydrolysis of the casein, and formed a green pigment that diffused into the medium. We report our experience of this medium for the differentiation of fluorescent pseudomonads.
1974).
MATERIALS AND METHODSBacterial strains. A study of pseudomonas infections in the seven hospitals served by this laboratory was started in 1971; 3800 isolates of P. aeruginosa were collected in 7 years. Eighteen strains of P. aeruginosa that did not produce pyocyanin on Diagnostic Sensitivity Test Agar (DST, Oxoid) and Cetrimide Agar (Pseudosel, BBL) were included in the present study because only these strains require further identification. Forty-six isolates of P. putida and 21 of P. fluorescens were collected from the same seven hospitals and one reference strain of P. fluorescens, strain NCTC10038, was included in this study.Culture media and methods. Antibiotic sensitivities were determined by a modified KirbyBauer method and Multodiscs (Oxoid) on DST agar. The ability of strains to grow at 42°C was determined by incubating a nutrient-broth culture, freshly-seeded from a colony on solid medium, at 42°C in a waterbath overnight and subculturing twice at 24-h intervals into nutrient broth held at 42°C.Lecithinase production was indicated by the development of a zone of turbidity around colonies on Nutrient Agar (Oxoid) containing 10% Egg Yolk Emulsion (Oxoid) within 5 days. Hydrolysis of gelatin was indicated by the release of charcoal from Charcoal-Gelatin Discs (Oxoid) in heavily-seeded Nutrient Broth No. 2 (Oxoid) after incubation at 37°C for 72 h. Ammonium Salt Sugars (Difco) containing ethanol, glucose and mannitol were used to confirm the identity of the fluorescent pseudomonads (King and Phillips, 1978).Fluorescein production was demonstrated by fluorescence on DST and Pseudosel Agar under an ultraviolet lamp and pyocyanin production was detected in King's "A" medium (Pseudomonas Agar P, Difco; King, Ward and Raney, 1954).Preparation and use of milk medium. The milk agar was prepared as described by Brown and Scott-Foster (1970) except that Dairy Bawn Defatted Milk Granules (Mitchelstown Creamery) were used instead of Marvel (Cadbury); the formulations of these products were identical (manufacturers' specifications). The plates were seeded in the normal manner and incubated at 37°C for 24 h and then at room temperature for another 48 h. The procedure was repeated with incubation at 37°C for 72 h, ...