Nt1310 Unit 3 Lab Report

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3.1.5.3. Coverslip culture technique
Coverslip culture technique was used for reviewing the morphological characteristics of the marine actinomycetes isolates (Pridham et al., 1958). Sterile glass cover slips were inserted at an angle of 450 into solidified SCA medium in petri plate. A loopful of inoculum of each isolate was streaked laterally to the line where the coverslip come across the agar and then the plates were inoculated at room temperature (28±200C) for 7 days. After the development of the mycelia on the coverslip, it was detached and placed on a glass slide for complete observation of structure and arrangement of the spore chain on aerial mycelium, spore morphology, spore bearing hyphae, using a phase-contrast microscope and photographed.
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Gram staining
A thin smear of selected marine actinomycetes were air-dried and heat fixed to a clean glass slide. The slide was then flooded with crystal violet for about a min and rinsed with tap water. Gram’s iodine solution was added and kept for a min. The slide was rinsed with tap water. The smear was decolorized with gram’s decolorizer and immediately washed with tap water, blot dried and identified under a light microscope.
3.1.5.5. Acid fast staining
The actinomycetes colony was studied by acid fast staining procedure. To a clean glass slide, a thin smear of the isolate was made on and heat fixed and then added with carbol fuchsin. It was heated gently till the steam raised and the process was repeated for three to four times in the course of 5 min, decolorized with acid alcohol for 15-20 seconds, then the slide was rinsed with water and the smear was counter stained with methylene blue for 30 seconds. Lastly, the smear was washed with running water and air dried.

3.2. BIOCHEMICAL CHARACTERIZATION (Shirling and Gottlieb,