Reliable Detection of Viable Salmonella Enterica from Inoculated Chicken Samples by Fluorescent in Situ Hybridization (FISH) with Vivification Step

Abstract

Inspection of food for the presence of Salmonella is a major concern in food industry. Many rapid, reliable and sensitive novel methods have been developed to detect Salmonella from food samples. However a method to detect live bacterial cells separately from dead cells yet to be developed. Although fluorescent in situ hybridization (FISH) has been identified as promising method in this regards, the viable count could be overestimated by FISH technique due to dead cells with some amount of non degraded rRNA. Some times it could be underestimated because of inactive but viable cells which not produce detectable amount of rRNA. The possibility of using antibiotic treatment step in FISH technique to overcome this problem was studied. Chicken samples inoculated by differently prepared mixtures of live and heat killed Salmonella enterica cultures were used to isolate bacterial cells with and without antibiotic treatment (Nalidixic acid 10μg/ml and Ciprofloxacin 1μg/ ml for 2h at 370 C). FISH was performed with Salmonella specific 23S rRNA probe Sal3, 5’-AATCACTTCACCTACGTG-3’ labeled with fluorescein isothiocyanate (FITC) at 5’ end. Cells observed with high intensity under fluorescent microscope were identified as live cells. Results of statistical analysis for antibiotic treated and untreated samples indicated that the introduction of antibiotic treatment step in FISH technique permitted a successful application to over come the problem associated with viable Salmonella enterica detection and quantification.