2.1 Sample collection Three types of commercially valuable dried fish were selected. Dried fish LOITTA (Harpodon nehereus), CHINGRI (Penaeus monodon) and CHURI (Trichiurus haumela) were collected in sterile plastic bags separately and the bags were teighed after collection to prevent extraneous contamination. Then the collected samples were carried out to the laboratory and preserved at 4°C.
2.2 Black cumin seeds collection About 500 g black cumin seeds (Nygella sativa) were bought from the spice market and sorted for separation of dirt and unwanted materials. The seeds were washed thoroughly with clean water and air dried at room temperature. Then the seeds were brought to the laboratory and preserved at room temperature in sterile plastic bag.
2.3 Preparation of extracts from black cumin seed (N. sativa) The black cumin seed was heated in oven at 50o C for 15 minutes and grinded by using mixer grinder (Capacitor start motor, WUHU motor factory, China). Approximately 300 gm of powdered cumin seed was taken into two sterile conical flasks containing 150 gm each. Then 350 ml of 100% ethanol and acetone were added into flasks separately at a ratio of 1:3. The mixtures were kept at room temperature for 72 hours. The mixtures were stirred every 24 hour using sterile glass rod. Then the mixtures were filtered through Whatman® No.1 filter paper. Filtration procedures were done further twice for complete extraction of the bioactive compounds. The filtrates were then collected in separate beaker and concentrated by evaporating the solvents. Approximately after 96 hours stock solution of the extract was ready to experiment. The liquid extracts were kept in a refrigerator (-20oC) for further study. The process was followed by Yasni., et al, 2009 with slight modification.
2.4 Preparation of extract at different concentrations 10%, 15% and 25% concentration of extract in both solvent (ethanol and acetone) was made separately by adding 100µl, 150µl and 250µl both extracts into 900µl, 850µl and 750µl of their relevant solvents (acetone and ethanol) respectively. After adding to the solvent, mixing was done in a unidirectional manner by a vortex mixer. A serial two-fold dilution of ethanol and acetone extracts ware done to get concentrations of 50%, 25%, 12.5%, 6.25% and 3.12% for MIC (minimum inhibitory concentration).
2.5 Isolation and Identification of Vibrio cholerae To isolate and enumerate specific pathogen of V. cholerae dried fishes were cut into small pieces with a sterile scissor and weighted in electric balance (HR-200). About 2 grams of samples were collected from each dried fish. The sample was then minced and grinded properly with alkaline peptone water using mortar and pestle. The mixture was taken into eppendorf tubes with alkaline peptone water used for isolating V. Cholerae. Two successive selective enrichments were done using alkaline saline peptone water (ASPW) for 6hr at 37°C followed by 18hr at 41°C. The plates were examined for the presence of typical colonies of presumptive Vibrio sp. The enumeration was done in Thiosulfate Citrate Bile and Sucrose sugar (TCBS) agar medium after incubation of 24-48hr at 37°C.). The standard plate was selected and counted the colonies. To identify V. cholera various biochemical tests viz gram staining, motility, triple Sugar Iron (TSI) agar test, indole test, methyl red (MR) test, oxidase test. salt tolerance test with varying amounts of NaCl (0%, 1%, 3%, 6% and 8% were conducted. All cultures of bacteria subsequently grown from stored stocks were streaked to get single colony prior to use. The bacteria were cultured on TCBS plates, nutrient agar plate and incubated at 37°C.
2.6 Experimental Design for In-vitro Challenge Test of black cumin against Vibrio choleae. At first the bacteria enrichment stock solution of each sample was done. Then 0.15 ml of 50%, 40%, 35%, 30%, 25% and 15% extracts (acetone and ethanol) of black cumin was separately mixed with 0.85 ml of test solution. 0.1ml suitable dilution of the mixture was inoculated in petri dishes that containing TCBS Cholera agar media after a subsequent interval of 2 hour up to 6 hour. This procedure was done 2 times. Test solution of sample without extract also inoculated TCBS cholera agar media at 0 hour, 2 h, 4 h and 6 h subsequently. All the inoculated TCBS agar plates were incubated at 37° C for 24 ± 3 hours. Standard plates count was done after incubation and compare the plate with and without extract.
2.7.2 Process B: Determining the Zone of Inhibition of black cumin extracts against V. cholerae Antibacterial activity of the black cumin extract was evaluated using disc diffusion method with slight modification. The discs 4mm of Whatman® paper were prepared for negative control. Standard antibiotic discs were used as a positive control to compare the antibacterial activity of black cumin. TCBS Cholera agar media was prepared and raised temperature up to 100°C in hot plate. Then the media transferred into the petri dishes and kept for cooling. 0.1ml of bacterial stock solutions were placed on separate petri dishes and spread throughout the plate by spread plate technique. The antibiotic and the discs that’s were loaded with 20μl of different concentrations (50%, 40%, 30%, 20%) of both ethanolic and acetonic extracts separately were placed on the bacterial solution inoculated plate with help of sterile forceps carefully with adequate spacing between each other. The plates were kept at room temperature for 30min, which helps to diffuse the extract on the medium. Later the plates were then incubated at 37°C for 24hrs in an incubator to determine the antibacterial activity of ethanolic and acetonic extracts of black cumin. Karamycin was used for Positive control and sterile Whatman® paper was used for negative control. After incubation, zone of inhibition in diameter was measured by a slide caliper (Tricle Brand) and recorded.