THE SPREAD PLATE AS A METHOD FOR THE ENUMERATION OF MARINE BACTERIA1, 2

A comparison was made of agar plates spread with glass rods and poured agar plates for the enumeration of bacteria in the waters of Fisher’s Island Sound, salinity 30&. Spread plates were shown to he markedly superior. Highest counts were obtained by spreading, using rods treated with Desicotc (a silicone solution) and incubating plates at 25”C, rather than at 16”C, and in air rather than in air with CO2 content increased. INTBODUCI’ION For the enumeration of bacteria in marine waters, extensive use is made of the poured agar plate. The possibility that some organisms indigenous to waters at less than 20°C were killed by even short exposure to 45°C and might grow more promptly on the surface of an agar plate suggested a challenge of the pour plate enumeration against a spread plate technique. The variations in conditions commonly employed suggested also the comparison of the effect of incubation at different temperatures and in an atmosphere of increased COz. METHODS AND MATERIALS Water was obtained at various tidal conditions during the summer and fall of 1958, from one location at Latimer Reef, which is about 6 miles off the coast of Noank, Conneticut, and east of East Point, Fisher’s Island, New York. Top samples were collected in sterile 500 ml wide-mouth reagent bottles. Samples were iced until examination at the laboratory, within 2 hours. Preliminary studies with samples from Fisher’s Island Sound showed that counts could be obtained using decimal dilutions of 10-l to lo-“. Dilution blanks (9 and 99 ml of sea water) were sterilized in the autoclave. Pipettes were 1.1 ml (milk) pipettes, sterilized in the oven, and stored in cans. 1 Supported in part by Grand #E-706, National Institutes of Health. 2 Contribution #l from the Marinc Research Laboratory, University of Connecticut, Noank, Connecticut. The plating medium was that used at the Woods Hole Oceanographic Institute reported by ZoBell (1946) and had the following composition: sea water from the tap at the Noank Marine Research Laboratory ( salinity about 30X0 ) ; peptone ( Gelysate, Baltimore Biological Laboratory), 0.1%; glucose, 0.1%; K2HP0.*, 0.005%; Bacto-agar ( Digestive Ferments Company), 1.5%; final pH 7.6. It was necessary to filter through cheesecloth. All “platings” were made in duplicate. For the pour plates, the melted agar was tempered at 45”C, then mixed with the sample or dilution. For the spread technique, about 15 ml was poured into plates prior to use. Dilutions were made so that deposition of 0.1 ml on the surface resulted in the desired dilution. Using a separate rod for each, the inoculum was spread evenly over the entire surface of the agar by a rotary twirling motion of the plate under the rod. The rods were made of 5 mm Pyrex, formed into a spreading portion with handle as follows: a 90” bend was made near the center of a 25 cm length, about 2 cm from which another 90” bend was made, thus producing a crank with parallel but opposite legs; a third ‘upward” bend gave a spreading portion about 8.0 cm in length. The rods were oven-sterilized and stored in cans. Pour plates were incubated at 16”+-1°C; the spread plates at lS”*l”C and 25”*l”C. The lower temperature was chosen to approximate that of the waters sampled, while the 25°C is the upper limit suggested by ZoBell ( 1954). For incubation in an atmosphere of increased COZ, the plates were in78 ENUMERATION OF MARINE BACTERIA BY THE SPREAD PLATE TABLE 1. Comparison of colony counts (X 102) obtained by spread and pour plates at 16°C incubated in air and in air with CO2 79 Air nntc saE-plc Spread RiYs Air with CO2 Ratios of counts EllKl Pour Ratio . Tide (S) (1’) Spread Pour S/P S air/S CO2 P air/P CO2 1