Bacteria cells are collected and dispersed in a nontoxic solution such as phosphate buffered saline or physiologic water. The cell suspension is then diluted, normally in a series with a 10-fold dilution, in order to obtain plates with a countable number of bacteria. A certain amount from each dilution is plated, in replicates of 3 or 4, on petri dishes with nutrient agar intended for the bacteria in question. The petri dishes are incubated in suitable environments and for an appropriate time before colonies are counted. Each colony that can be counted is called a colony-forming unit (CFU), and the number of CFUs is related to the viable number of bacteria in the sample. The number of bacteria in the original sample is calculated mathematically, factoring in the amount plated and its dilution factor. The results are often presented as CFU/ml (colony-forming units per millilitre) for liquids, and CFU/g (colony-forming units per gram) for solids.
Spiral plating technique
The traditional method with diluting and plating is time-consuming, and therefore the number of variables in a test can be limited. NIOM’s microbiology laboratory is therefore using a spiral plating technique where an instrument, the WASP spiral plater (Figure 1), deposits a given volume of sample on to a rotating agar plate.
The volume of the sample deposited per unit area of plate decreases across the spiral resulting in a dilution effect, and allowing counting of isolated colonies (Figure 2) in respective sectors of known deposition volume.
For counting of bacteria, NIOM uses an automated colony counter, aCOLyte, and results are transferred directly to Excel for further analysis.
NIOM Newsletter May 2014