A two-step enrichment protocol for isolation of low count putative lactic acid bacteria from freshwater fish intestines

The present study reports a double enrichment cum selective isolation based protocol for successful isolation of lactic acid bacteria (LAB) from freshwater fish intestine where it exists in substantially lower population counts. The procedure involved enrichment of homogenized intestinal samples of freshwater fish in de Man, Rogosa and Sharpe (MRS) broth medium followed by further enrichment cum selection in Lactobacillus Anaerobic MRS with Vancomycin and Bromocresol green (LAMVAB) broth and final isolation in LAMVAB agar. Traditional isolation method using only MRS medium was also performed with the same samples. Individual colony morphology was recorded and biochemical identification was done. Comparison of the two isolation procedures showed that, total number of LAB along with total number of different individual species of LAB such as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus brevis, Pediococcus acidilactici, Weissella paramesenteroides and Enterococcus faecium was more in the double enrichment cum selective isolation procedure using LAMVAB medium than that recovered from isolation procedure involving only MRS medium. Besides LAB, undesired spore forming rods and yeasts were found in the procedure involving only MRS medium thus indicating its low selectivity. Hence, the double enrichment cum selective isolation based protocol using LAMVAB medium was found to be reliable for successful recovery and isolation of LAB from intestines of fish.