Kristina SimonovicSection O3 Feb 16Exercise ThreeTitle:Differential Staining Continued: Acid Fast & Endospore, Sterile/Aseptic Technique, Continue Purification of UnknownHypothesis:If the specimen is correctly stained, then it will appear clearly when looking at it through a microscope.Methods:For the acid-fast stain, prepare a heat-fixed dry mount of Mycobacterium smegmatis and include a non-acid-fast control on the slide. Cover the smear with filter paper that is the same size as the slide and saturate it with carbol fuchsin. Using a clothespin to hold the slide in a horizontal position, gently heat the slide from the bottom until steam rises from the slide for 5-7 minutes. Remove the paper and rinse the slide with water. Decolorize with acid alcohol for 5 seconds thenwas with water. Counterstain with methylene blue for 2 minutes. Wash with water and blot dry and then view the slide under the microscope. For the endospore stain, use Bacillus megaterium and follow the same procedures for the acid-fast stain expect instead of staining it with carbol fuchsin, stain this slide with malachite green. Remove the paper and rinse the slide with water for30 seconds. Counterstain with safranin stain for 2 minutes. Rinse with water and blot dry and then view the specimen under the microscope. Safety Concerns:1. Stains used on slides tend to stain anything they touch.2. Wear safety goggles when staining slides.3. Do not get oil on the 10 and 40 power lenses.4. Proper use of the microscope is necessary. Relevance:Learning how to correctly stain slides will make finding the specimen under the microscope much
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