Isolation of Rhizobium From a Nodule

Date 21/4/23 No. 4 Expt. Page No. 8 ISOLATION OF RHIZOBIUM FROM A NODULE AIM To iddate rhigobiim from the root nodules of the leguminous plant PRINCIPLE Rhigobium is a symbiotic N2 fixing organism which is found in the red nodules of legume plants The againsm is a gran negative aerobic Ef becomes pleomorphic under adverse conditions the root nodules were leaned 4 reface steeliged using 0.1% Mgll2 EP 70% ethand solution before the rhigobium all isdates 4 grown on YEMA a specific media for growth of Rhigobium. MATERIALS REQUIRED Test-tube conical flask + sterilized pipette , spreader, petei- plates, glats rod , yeast extract mannitd agar root nodules noriliged distilled water 70% 4 ethanol sodium hypochlari to mercusic chloride. PROCEDURE 1 wash ed thoroughly to remove soil. 2. collect about 10 nodules from each plant 3. while moving the nodule . use toe ceps on the root appenda- yes to reduce the risk of damaging the nodule. 4. Immerse intact, undamaged nodules for 500 10 " conds in 95% ethand or isopropanol 4 then transfer the nodules to a 2.5 - 3% solution of sodium hypochlaile 4 soak Teacher's Signature

Date 9 Expt. No. Page No. to 2- 4 min 2 Kinse in Morill water 41 then desiccate the nodules 4 place the inner substance of the nodules in a stellined distilled water 6 Nix it well with the help of glass rod. 7. Pipette 100ml of sample in the solidified agas plade al then with the help of L shaped glass end person the spread plate method After plating place the plates in the incubator at 37% td 24 hes. OBSERVATION After 72 hours of incubation isdated small medium El large sized orange daved olonies were observed on the plates Upon been staining of all isdated organisms pink colored and shaped bacteria was observed under the oil immersion misoscope RESULT The isdated organisms from the nodules all gean negative rods. (bacillus) Teacher's Signature