DEVELOPMENT OF A SPOT-TITER CULTURE ASSAY FOR QUANTIFYING BACTERIA AND VIRAL INDICATORS

ABSTRACT

The spread-plate and double agar layer (DAL) methods are common for the enumeration of bacteria and viral indicators (bacteriophages). However, they may become cumbersome in large matrix experiments or when the titer of the organism varies by several orders of magnitude. A bacterial spot-titer assay has been available for decades but has not been adapted to bacteriophages and has rarely been applied to the analysis of environmental samples. In this study, a spot-titer culture-based method was investigated for bacteria and bacteriophages. The method involves spot-plating replicate 10-µl volumes of several sample dilutions on a single plate, incubating, and counting colonies or plaques. Parallel assays of laboratory cultures and environmentally isolated organisms show that the spot-titer method is equally straightforward and statistically comparable to the spread-plate and DAL methods (R² = 0.989 for laboratory strains and R² = 0.972 for environmental samples), while more cost- and labor-efficient.

PRACTICAL APPLICATIONS

The spread-plate and double agar layer (DAL) methods currently used for enumeration of bacteria and viral indicators, may become labor- and resource-intensive (culture media, plates, technician time and incubator space) in large matrix experiments, which are often needed in the laboratory to evaluate environmental conditions. The spot-titer method has several advantages over the spread-plate and DAL methods: (1) it requires less time to dispense spots than to spread the microbe; (2) it uses fewer materials (15–20% of the laboratory supplies as the traditional methods); (3) it requires less effort; and (4) since the sample is distributed in distinct spots, colony/plaque counting is faster and less labor intensive. The spot-titer method was found to economize resources without sacrificing accuracy or precision, and is a practical method for routine use in large matrix experiments (e.g., survival or disinfection studies) and enumeration of high-titer environmental samples.