Rapid identification of bacterial agents in fecal samples of rodents by flow cytometry

Abstract

Experiments were performed to determine whether flow cytometer could be used for rapid identification of bacteria based on their light scatter properties. Fresh fecal samples were collected from mice and were cultured on nutrient agar. Prominent colonies from the samples were sub-cultured again on differential media (MacConkey’s and Eosin Methylene Blue) and further subjected to both biochemical and FACS (Fluorescent Activated Cell Sorter) analysis. In conventional method, samples were cultured and organisms were isolated using appropriate media and further identified by using specific biochemical methods. For flow cytometric analysis, 20,000 cells were acquired from each sample (n=5) and subjected them for analysis by using both forward scatter and side scatter properties. The dilution of microbial samples, cytometer used and the voltage of forward and side scatter on the cytometer were maintained constant throughout the study. These characters were observed together on two dimensional Dot plots, Density plots and Contour plots. Significant differences in individual characteristics were observed in forward and side scatter properties among E. coli, Salmonella typhimurium, Staphylococcus aureus and Pseudomonas aerogenosa. Further, bacterial populations were quantified and also mixed populations of microorganisms were prepared and they were further sorted as individual population using FACS Aria II sorter and subjected to usual microbial and biochemical analysis. In the present study, results obtained from conventional method and flow cytometric analysis were compared and found to be correlating each other very well. This rapid detection of microorganisms and their quantification by flow cytometry method in few hours would certainly benefit the animal facilities in adopting effective, preventive or curative measures in short span of time.