lab2-细菌的芽孢染色.pptVIP

实验二　细菌的芽孢染色;Due to the resistant nature of the endospore coats, endospores are difficult to stain. Strong dyes and vigorous staining conditions such as heat are needed. Once stained, however, endospores are equally hard to decolorize. Since few bacterial genera produce endospores, the endospore stain is a good diagnostic test for species of Bacillus and Clostridium. ;三 材料与器材;1. Heat-fix a smear of Bacillus megaterium as follows: a. Using the dropper bottle of distilled water found in your staining rack, place a small drop of water on a clean slide by touching the dropper to the slide. b. Aseptically remove a small amount of the culture from the edge of the growth on the agar surface and generously mix it with the drop of water until the water turns cloudy. c. Burn the remaining bacteria off of the loop. d. Using the loop, spread the suspension over the entire slide to form a thin film. e. Allow this thin suspension to completely air dry. f. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix. ;2. Place a piece of blotting paper over the smear and saturate with malachite green. 3. Let the malachite green sit on the slide for one minute and proceed to the next step. 4. Holding the slide with forceps, carefully heat the slide in the flame of a bunsen burner until the stain just begins to steam. Remove the slide from the heat until steaming stops; then gently reheat. Continue steaming the smear in this manner for five minutes. As the malachite green evaporates, continually add more. Do not let the paper dry out. ;5. After five minutes of steaming, wash the excess stain and blotting paper off the slide with water. Dont forget to wash of any dye that got onto the bottom of the slide. 6. Blot the slide dry. 7. Now flood the smear with safranin and stain for one minute. 8. Wash off the excess safranin with water, blot dry, and observe using oil immersion microscopy. With this endospore staining procedure, endospores wi