Moni Krishno Mohanta
Department of Zoology, Genetics and Molecular Biology Laboratory, University of Rajshahi, Rajshahi-6205, Bangladesh.
Preonty Mallick
Department of Zoology, Genetics and Molecular Biology Laboratory, University of Rajshahi, Rajshahi-6205, Bangladesh.
Md. Fazlul Haque
Department of Zoology, Genetics and Molecular Biology Laboratory, University of Rajshahi, Rajshahi-6205, Bangladesh.
Md. Ariful Hasan
Department of Zoology, Genetics and Molecular Biology Laboratory, University of Rajshahi, Rajshahi-6205, Bangladesh.
Ananda Kumar Saha
Department of Zoology, Genetics and Molecular Biology Laboratory, University of Rajshahi, Rajshahi-6205, Bangladesh.
Antibiotics; Enterococcus; Macrobrachium rosenbergii; Micrococcus; Probiotic bacteria; Vibrio, Prawn
Avaynagar Thana under Jessore District, Bangladesh.
Animal Health and Management
In this study, the healthy and moribund prawn samples were collected from Avaynagar Thana under Jessore District, Bangladesh and recycled as sources of inocula for the isolation of probiotics as well as pathogenic bacteria. The collection of prawn samples, isolation of bacteria, and subsequent experiments were done from March to November 2019. The healthy and moribund giant freshwater prawn, Macrobrachium rosenbergii (40-50 g) were collected from Avaynagar Thana under Jessore District, Bangladesh. During transport to the laboratory, the samples were kept on ice and taken into sterile bags. They were then used as a source of inocula for the isolation of probiotic and pathogenic bacteria. Under sterile conditions, healthy prawn was dissected, the whole intestine was carefully removed and suspended separately in1ml in physiological saline. Then suspended intestine was homogenized with Glass Pestle Tissue Grinders (PYREX™) by 50 strokes for each sample in a sterile condition. One loopful of each suspension was separately inoculated into the nutrient broth which was incubated for 24h at 37°C with shaking at 120rpm (revolution per minute) using a shaker. Control flasks deprived of inoculums and also ready for incubation at 37°C with a shaker. The cultures that were found turbid after a period of 0 up to 2 days were used as inocula in further experiments. For isolation of pathogenic bacteria, the affected prawn was dissected firstly. Secondly, muscle and antenna were collected for pathogenic bacteria isolation. Each sample was diluted from 10-1 to 10-7 and 0.1 ml of diluted sample was taken from 10-5, 10-6, 10-7 and spread on nutrient agar plate separately. One plate preserves as the control without spreading the sample. At 37°C for 24 to 48 hours the plates were incubated. One loopful of primary enrichment culture was streaked on nutrient agar plates (Hi-media) and incubated for 24h at 37°C temperatures. The single colonies were found to grow on the medium. Four isolates were found from primary screening and further grown on a nutrient agar plate. The single colony was selected and again streaking was done on nutrient agar plates for subculture under germ-free environments. Pure cultures of probiotic and pathogenic isolates which were obtained by a repeated subculture of the single colony were grown on slants by stab and streak method for storage purposes and subsequently for identification and biochemical characterization. For the identification of the bacteria, observations of morphological characters with a binocular light microscope (Labomed, USA), growth characteristics, antibiotic sensitivity test, and biochemical tests viz. Triple Sugar Iron (TSI) test, Citrate utilization test, Sulfur Indole Motility Media (SIM) test, MacConkey agar test, Hydrogen Sulfide (H2S) Production Test, Potassium hydroxide (KOH) test, Catalase test, Oxidase test and Carbohydrate utilization tests were performed. The microorganisms were identified using Bergey’s Manual of Systematic Bacteriology [17]. For the determination of optimum pH on bacterial growth, the culture medium was adjusted to pH 5.0, 6.0, 7.0 and 8.0 by adding concentrated HCl or NaOH. Incubation temperature varied from 25°C to 42°C. The bacterial cell density of liquid cultures was determined at different time intervals by measuring optical density at 660nm with a photoelectric colorimeter (AE-11M, Erma Inc., Tokyo). An antibiotic sensitivity test of the isolates was performed as described by Saha and their colleagues [19]. Briefly, 1 ml of a fresh broth culture of the isolate was spread uniformly on a nutrient agar plate with a sterile glass spreader. The plate was air-dried for few minutes and then antibiotic discs were placed on inoculated nutrient agar plates which were incubated at 37°C for 24 hours. After incubation, clear zones indicated inhibition of growth of the isolate. Three strains of probiotic bacteria (RCB, WCB, YCB) and pathogenic bacteria (PB1, PB2, PB3) were cultured on nutrient agar and incubated at 37ºC for 24hours. Inhibitory activity tests were done on the nutrient agar plates by the cross-streak method. Pathogenic bacteria viz. PB1, PB2, PB3 were streaked in a line separately, and then probiotic bacterial strains viz. RCB, WCB, YCB were streaked separately in a perpendicular line. Every species pair was incubated at 37ºC for 48 hours and was cross-streaked in triplicate. After 48 hours of incubation, the inhibitory activity was observed through the linear zone of the plate and measured zone with the helping of mm scale.
Asian Journal of Fisheries and Aquatic Research- 6(3): 30-40, 2020
Journal