, Diagnosis an-&& of Shrimp Diseases - Central Institute of ...

Alkaligens: etc. are resistant. The test is done by using 0/129 impregnated discs e~nployingconventional disc diff~ision method of Bauer et a1 (1966).Prepnrtrtion of 01'129 discs: Prepare 7500 and 500 pgIm1 (7.5 rng and 0.5 mg respectively) insrerile glass double distilled water. Spot 20 p1 of these stock solutions onto sterile antibioticdiscs to obtain discs containing 150 or 10 pg 01129. Dry the discs in a desiccator underaseptic conditions at room temperature. Store at 4OC till use.Procedure: Test sensitivity of the bacterial isolates as the protocol given for antibioticsensitivity testing.Interpretation: Zone of inhibition of growth around the 01129 impregnated discs indicatessusceptibility of bacterial isolate to 01129.Antibiotic / Drug sensitivity testingThe antibiotic I drug sensitivity testing method employed is Kirby-Bauer's discdiffusion technique. Co~nmercially available antibiotic discs are used for this purpose. Theculture medium used for antibiotic sensitivity testing is Mueller-Hinton agar supplementedwith 1 % sodium chloride.Preparation of the Irloculum:Inoculate pure culture of bacteria into 5 ml Zobell's marine broth tubes with the helpof sterile inoculation loop. Incubate for 2 to 8 h at 30°C till moderate growth is obtained.Note: Obtain turbidity of broth culture (by diluting the culture using sterile sea water or sterilephosphate buffered saline, pH 7.4) equivalent to 0.5 ml of 1.175% BaC12.2 Hz0 solutionadded to 99.5 ml of 0.36 N sulphuric acid.Inoculation:9Dip a sterile swab into the inoculum and squeeze off the excess fluid by pressing theswab against the inside wall of the tube. Streak the entire agar plate thoroughly on the surface.Application of antibiotic discs:Apply discs onto the plates aseptically using sterile forceps. Press the discs firmly onthe agar to enable smooth diffusion of antibiotic. Place the antibiotic discs at least 20 mmapart. Incubate the plates at 30°C.