Rapid surface colony counts determination with three new miniaturised techniques.

Abstract

Three different miniaturised methods for the rapid surface viable counting are described. The methods were tried in parallel to seven different existing methods (Table 1) for viable counts and were found to be easier, quicker and insome cases more accurate. The techniques require about 10% of the material and time needed for conventional spread-plates method and the results were in no way inferior to that (Table 1 and 2). Mini agar discs were cut aseptically with an especially designed stainless steel agar disc cutter (25 mm internal and 28 mm external diameter, Fig. 1b) or with a test tube of similar diameter. The area of the resulted mini-agar-disc of 25 mm diameter was kept such (about 1/10th of the normal plate) that the ratio of the colony-bearing area to the inoculm remained the same as on big plates in spread-plate-method (Table 2). In normal Petri dishes (about 90 mm diameter) up to seven mini agar discs were possible to cut. Each small agar disc was seperated from the other mini-disc by a distance of at least 6 mm (Fig. 1a). The empty place around the disc was still enlarged during over drying of the plates and during incubation. This created complete isolation from the neighbouring disc. For micro-determination of surface viable counts 10 micronl from each dilution was delivered on a well-dired mini-disc with a piston micropipette. The inoculm was immediately spread on the whole mini-disc with a specially designed flame sterilizable platinum-Mini-spreader (Fig. 2a). No spinning of the plate was needed. Alternatively the dropping pipette and spreader was replaced by a calibrated platinum wire Loop-spreader (Fig. 2b). A loop of 3 mm internal diameter made from a platinum-iridium wire of 0.75 mm thickness proved most useful and carried a drop of 10 micronl. Differences especially in surface tension of various diluting fluids did not influence to drop of this size and no recalibration was needed for water and nutrient broth. The loop was further shaped to Loop-spreader form. From each bacterial suspension 10 micronl were carried and spread on each mini-disc. The method is useful for pathogenic organisms as the loop can readily be flame sterilized. For routine purposes where only approximate numbers of bacteria need to be known a still rapid semiquantitative method was deviced making use of a calibrated stainless steel Stamping-disc (Fig. 2c). A disc of 25mm diameter and 1 mm thickness delivered approximateyl 10 microlitres of supensions and was found to be most useful to stamp seven dilutions on a single plate. In collections and bacteriology laboratories where by conventional methods large number of plates are to be plated and counted the presented techniques could prove most convenient, rapid and economical.