APPENDIXES APPENDIX - I. Buffer, Reagent and Culture Media Preparation. 5 gm. 5 gm. 15 gm

Transcription

1 APPENDIXES

2 APPENDIXES APPENDIX - I Buffer, Reagent and Culture Media Preparation a. Tryptone soy broth Casein triptic digest NaCl Soy peptone 1 b. Tryptone soy agar Casein triptic digest NaCl Soy peptone Agar 1 1 c. Agarose media 1.8 gm of agar in 100 ml of distilled water e. Phosphate Buffer solution (PBS) (ph 7.4) 0.1M Na 2 HPO 4.12H 2 O 0.1M NaHPO 4.H 2 O gm gm f. Hank s Balanced Salt solution (ph ) HBSS 9.8 gm in 900 ml of distilledd water Sodium bicarbonate 0.3 Finally addd distilled water to the above mixture up to ml. Enhancement of Immune response in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) Pers. P mixed diet 162

3 g. Griess reagent (for 100 ml) 1 % Sulphanilamide 0.1% N-Naphthyl ethylene diamine 2.5 % Phosphoric acid 1 gm 0.1 gm 2. APPENDIX II a. Preparation of RaRBC A rabbit was bled from the ear vein and blood was collected into Alsever s solution in 1:1 ratio. The blood was centrifuged at 4000 g for 15 min. The supernatant was discarded and centrifuge with sterile PBS containing Ca ++ and Mg % (v/v) RaRBC suspension was prepared in same buffer. b. Preparation of SRBC Blood from sheep was collected and stored at 10 C in Alsever s solution (ph 6.1). Then, the cells were washed thrice in isotonic NaCl solution (0.15N) and resuspended to the required concentration. Enhancement of Immune response in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) Pers. mixed diet 163

4 APPENDIX - III Novel Technique que of Direct Heart Puncture for Collecting Blood lood from Fish 1. This new methodology ology is invented for our research work. 2. The soft tissue (between (betwe the gill region and the body cavity)) is seen se through opened operculum m for ddirect heart puncture. 3. Now, through thee soft tissue we can get to the heart by inserting ting a 24G needle towards downward rd lef left side to bulbous arteriosus (near the base to the body wall). 4. Care should be taken w while collecting the blood from heart without thout shaking and tearing the tissue 5. To expertise in this technique te and to gain confidence, a number er of practices in euthanized fish are re required. requ 6. This is a very safee and easy method to collect the blood from fish. Conventional method od Novel method Fig. App.1: Illustration ion sho shows the direct heart puncture method for the he collection col of blood from rom fish. fis Enhancement of Immune response nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.)) Pers. P mixed diet 164

5 APPENDIX IV Preparation of Micrococcus lysodeikticus suspension Micrococcus lysodeikticus inoculated in Luria bertani (LB) broth and incubate at 28 C. After overnight culture is centrifuge at 5000 rpm for 10 min. 20 mg of M. lysodeikticus mixed with 100 ml of 0.05 mm sodium phosphate buffer. Luria bertani (LB) broth Yeast extract NaCl Peptone 10 gm 10 gm Finally addd distilled water to the above mixture upto 100 ml. APPENDIX V a. Preparation of viable cell culture medium for leucocytes separation Blood Collecting medium RPMI-1640 supplemented with + 50 IU ml -1 Sodium heparin IU ml -1 penicillin and mg ml -1 streptomycin Leucocytes wash medium RPMI-1640 supplemented with + 10 IU ml -1 Sodium heparin, IU ml -1 penicillin and mg ml -1 streptomycin Leucocytes culture medium RPMI-1640 supplemented with 3% (v/v) of pooled tilapia serum IU ml -1 penicillin mg ml -1 streptomycin and + 4 mm L-glutamine Enhancement of Immune response in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) Pers. mixed diet 165

6 b. Trypan blue for vital staining (Dye exclusion method) Place a suitable volume of a cell suspension ( µl) in an appropriate tube add an equal volume of 0.4% Trypan blue and gently mix, let stand for 5 min at room temperature. Place 10 µl of stained cells in a haemocytometer and count the number of viable (unstained) and dead (stained) cells. Calculate the average number of unstained cells in each quadrant, and multiply by to find cells/ml. The percentage of viable cells is the number of viable cells divided by the number of dead and viable cells. APPENDIX VI Polyclonal Antibody Production Against A. Hydrophila in Rabbits Strain MTCC 646 was used to raise rabbit anticarp polyclonal antibody against A. hydrophila. The bacterium was grown in tryptone soy broth (TSB) overnight and harvested. The cells were adjusted to a concentration of ml -1 in PBS and were heat-killed by incubating in a water bath at 60 C for f 1 hr. The effectiveness of heat killing was determined by looking for growth on tryptone soy agar (TSA). After confirmation that there were no live bacteria in the suspension, it was emulsified with Hunter s Titermax adjuvant (Uptima, France) in equal e volume. About 1 ml of blood (pre-immune) was collected from the ear vein of a female New Zealand White rabbit, and then the rabbit was injected with 0.4 ml off the bacterial suspension subcutaneously into four sites using a 21G needle. Booster injections were given once in a week for a four week period. Test bleeds were collected 2 weeks after each injection and stored overnight at 4 C. The blood was centrifuged att 2000 g for 5 min and the serum collected and stored at -20 C for analysis. The antibody titre of the serum and the antigens recognised by the serum were analysed by ELISA respectively, for each serum sample. The final immunization was carried out with the same amount of the antigen diluted in PBS and administrated by an intravenous injection into the ear vein. The animal was bled after 10 days by cardiac puncture after anaesthesia. Serum was separated and stored at -20 C for further analysis. Enhancement of Immune response in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) Pers. mixed diet 166