Fatema Ahmmed
Fisheries and Marine Resources Technology, School of life science, Khulna University, Bangladesh
Mirja Kaizer Ahmmed
Feculty of Fisheries, Chittagong Veterinary and Animal Sciences University, Bangladesh
Md. Saifuddin Shah
Fisheries and Marine Resources Technology, School of life science, Khulna University, Bangladesh
Ghausiatur Reza Banu
Fisheries and Marine Resources Technology, School of life science, Khulna University, Bangladesh
Lactobacillus, Antagonistic effect, Aquaculture
Batiaghata upazilla, Khulna, Bangladesh
Crop-Soil-Water Management
The present study was conducted on three ghers of Batiaghata upazilla, Khulna. The ghers were randomly selected for collecting shrimp samples. Nine (9) specimens of shrimps, Paeneus monodon (8.10cm- 12.4cm in length and 13.12g-22.6g in weight) were collected from three ghers of the study area. The target organs (gill and intestine) of the samples were separated aseptically and weighed in electric balance. Organs (gills weight: 0.22g-0.34g and weights of intestinal tracts: 0.13g-0.25g.) were taken into eppendorf tubes with peptone water for isolating Lactobacillus spp. and alkaline saline peptone water was used for isolating Vibrio spp. Then they were homogenized using tissue homogenizer. Then homogenized solutions were centrifuged at 3000 rpm for 3 minutes. After centrifugation the supernatant liquid portion was collected with a micropipette and taken into eppendorf tube and preserved in deep freeze. Lactobacillus spp. was isolated from gills and intestinal tracts of the collected shrimp samples. The stock solution was diluted (tenfold dilution) with peptone water. 0.1 ml suitable dilution of each stock solution was inoculated in MRS Lactobacillus agar media and incubated at 370C temperature for 24-48 hours. After incubation at 370C temperature for 24–48 h, cream-colored colonies with yellow halos were collected and preserved for further experiment. Vibrio spp. was isolated from the gills and intestinal tracts of the collected shrimp samples from study area. ISO method was followed for isolating Vibrio spp. 0.1ml stock solution of each gill and intestinal tract was taken and mixed with 0.9ml alkaline saline peptone water (ASPW) in sterilized test tubes. Then the mixture was incubated at 37 °C for 6 ±1 hr. After that, whole culture of each test tube obtained in first selective enrichment was taken and transferred into other test tubes each containing 10 ml ASPW. Then the solution was incubated at 41.5 °C for 18 ± 1 hr. This was the second selective enrichment. Then serial dilution (tenfold) was done and 0.1ml suitable dilution of each culture was inoculated in thiosulfate citrate bile and sucrose (TCBS) agar plates. Then the inoculated TCBS agar plates were incubated at 37 °C. After 24 h ± 3 h of incubation, the plates were examined for the presence of typical colonies of presumptive Vibrio spp. (ISO/TS 21872-1, 2007). The identification of the Vibrio harveyi colonies was done by performing various biochemical tests viz, motility test, methyl red test, VP test, arginine dihydrolase, indole ring test, Salt tolerance test (0%, 1%, 3%, 5%, 7%, and 10%) and growth at ( 4°C, 28°C, 37°C, 55°C). The isolates were identified at the species level with the use of biochemical key. Gram stain was done to identify the gram positive and gram negative bacteria (Cowan and Steel’s, 1993). The Indole test was performed in a 48h culture in peptone water adding about 1ml ether; shake; run 0.5 ml Ehrlich’s reagent down the side of the tube. A red color in the solvent indicates positive reaction. The VP test was performed after completion of the methyl red test adding 0.6ml 5% α-naphthol solution and 0.2 ml 40% KOH aqueous solution; shake well, slope the tube, and examine after 15 min & 1 h. A positive reaction is indicated by a strong red color. The MR test inoculated the isolated bacteria with buffered glucose broth & incubated at 37°C for 48h. After incubation adding a few drops of methyl red solution to the culture, read immediately. A red color represents a positive test. The motility test stabbed inoculates tubes of motility medium to a depth of about 5 mm. Incubate at or below the optimum growth temperature. Motile organisms migrate throughout the medium, which becomes turbid; growth of non-motile organisms is confined to the stab inoculum. This test was done on nutrient agar media supplemented with varying amounts of NaCl (0%, 1%, 3%, 6%, 8%, and 10%). This was performed to study the salt tolerance range of the isolated species and the optimum concentration, once determined was supplemented in various media required to test their biochemical properties. The isolated Vibrio spp. was kept in incubator after inoculation at subsequent interval at temperature 40C, 280C, 370C, 550C and checked for their survivability and colony formation obtained at 280C and 370C. After identification of Vibrio harveyi by biochemical test one colony of V. harveyi was taken into eppendorf tube by isolating loop into 0.9ml peptone water. Prepared stock solution of V. harveyi (ISO/TS 21872-1, 2007). The stock solution was diluted (tenfold dilution) with peptone water (James and Hirsch, 1960) and prepared test solution of V. harveyi. Then 0.5 ml isolated probiotic solution was separately mixed with 0.5ml of test solution (V. harveyi). 0.1ml suitable dilution of the mixer solution was inoculated in TCBS agar media after at subsequent intervals of 4 hour up to 12hour. This procedure was done for 2 times. Test solution of V. harveyi ; without probiotic was also inoculated in TCBS agar media at 0 hour, 4th hour, 8th hour and 12th hour subsequently. All the inoculated TCBS agar plates were incubated at 370 C for 24± 3 hours. Standard plate count was done after incubation. Collected data were stored, explored and analyzed using Microsoft Excel (Microsoft office, 2007) and Statistical Package for the Social Sciences (SPSS version 16.0; SPSS, Inc., Chicago, IL). Independent sample t-test was applied to address the differences between the treatment and control at 1% significance level using SPSS (version 16.0).
Res. Agric. Livest. Fish. Vol. 5, No. 1, April 2018 : 127-135.
Journal