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.

Effects of environmental storage conditions on survival of indicator organisms in a blend of surface water and dual disinfected reclaimed water

“…Concentrations of micro‐organisms were quantified over the 5‐day test period using the spot‐plate titre assay as described in Beck et al . () on the selective media described above. Briefly, 150 mm plates were prepared using the selective agars, and the samples were diluted using phosphate buffered saline (Higgins et al .…”

“…The following environmental conditions were used: samples storage at 4 and 20°C (average outdoor temperatures for NC), mixing on a Fisher Scientific Ocelot platform shaker (Hampton, NH) at speeds of 0, 60, and 120 rev min À1 , and holding time of 3 and 5 days. Concentrations of micro-organisms were quantified over the 5-day test period using the spot-plate titre assay as described in Beck et al (2009) on the selective media described above. Briefly, 150 mm plates were prepared using the selective agars, and the samples were diluted using phosphate buffered saline (Higgins et al 2003).…”

Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types

“…The filtrate titers were then determined for all 10 hosts. The phage titer was quantified by a spot titer culture assay (43). Accession number(s).…”

UV LED water disinfection: Validation and small system demonstration study

“…Before and after the year‐long demonstration study, the reactor was challenge tested with MS2 bacteriophage (ATCC 15597‐B1) in dechlorinated (granular activated carbon [GAC]‐treated) tap water across a range of flow rates and UV absorbances (UVA)/UVT that were modified using lignin sulfonate (LSA). UVT was calculated based on measured UVA (cm −1 ) (UV–Vis Cary 100, Agilent) using Equation : MS2 infectivity before and after treatment was enumerated using Escherichia coli F amp host (ATCC 700891) by the USEPA 1602 single‐layer agar method (USEPA, ), modified as a spot‐plating assay when viral titer was expected to be >10 2 plaque forming units (pfu)/mL (Beck et al, ). MS2 log 10 inactivation (log I ) was calculated as the log 10 ratio of pfu/mL before versus after UV irradiation.…”