Crystal Violet Staining Preparation of a smear and simple stain of the material between your teeth Take a sterile

terile toothpick, remove some solid material between your wisdom teeth and mix it into a drop of water so that you have suspension spread over the middle third of the microscope slide. NO COVER SLIP USED! Let the slide air-dry. Heat-fix the dry smear by running the slide quickly through the flame a few times. If your fingers get hot, you have heat-fixed TOO MUCH. Place the slide over the stain tray. Flood the smear with crystal violet: let sit for 1 minute. Wash the slide WELL with distilled water. Blot the smear slide with your bibulous paper pad. Focus on the sample using the 10X lens (BE SURE that you are on the bright field microscopy): you should see masses of purple material, most of it too small to see. USE COARSE ADJUSTMENT FIRST AND THEN FINE ADJUSTMENT KNOB to focus your view. DO NOT TOUCH THE COARSE ADJUSTMENT KNOB AGAIN, move to 40X and refine your view using the fine adjustment. Determine the shapes of the bacteria and take the picture using your camera under 40X. Ready to go to 100X now? DO NOT move the stage. Rotate the nosepiece so that an intermediate position between the 40X and 100X objectives is obtained. Place the cover slip on the specimen and add a drop of immersion oil on the center of the viewing area of the slide. Continue to rotate the nosepiece so that the 100X objective is rotated into the oil. DO NOT UNDER ANY CIRCUMSTANCES PLACE THE 40x OBJECTIVE IN THE OIL. Use only the fine adjustment to focus the specimen. Determine the shapes of the bacteria and take the picture using your camera under 100X Immediately after using the oil, remove any residual oil from the slide and from the front of the 100X objective by gently rubbing with lens paper dipped in alcohol. Once oil is placed on the slide, the 10X and 40X objectives will no longer be useful until the oil is removed. The oil will blur any image attempted with the lower magnifications. Use oil only when necessary and after completing all work at the lower magnifications. In order to return to work at the lower magnifications, the slide must be completely cleaned of any residual oil and dried. For this reason, oil immersion is employed only when necessary and only after thorough observations at the high dry magnification (40X). Clean the slides, microscopes and your lab station before wrapping up. Write the lab write up using the protocol given to you at the beginning of the year.

Discussion Questions: Why is it important to limit the quantity of cells used to prepare a smear? For preparation of a smear on a slide, what is the purpose of heat fixation? What problems can arise when the slide is heated in a flame? What causes a stain to adhere to bacterial cells? Why are all colored dyes not necessarily useful for simple staining?