1.) The safranin staining at the end.

You could look at the slide after the discoloration step (alcohol), and see if you had a clear bacteria or the pinkish (pinkish would be negative of course) and the clear would be the uncounterstained gram positive. 2.)The electron microscope uses a beam of electrons much like the light microscope uses light waves. Unlike light, electrons do not pass through air without heating it. This means that the tube, lenses and specimen that the electron beam passes through must be in a vacuum. Living things don't do so good in a vacuum. Also to see the structures well in a specimen it must be stained with a heavy metal or coated in a vacuum with carbon or metal. Acid Fast Stain The primary stain is: carbol fuschin The mordant is: heat (heating enhances penetration and retention of the dye) or detergent (cold method; turgitol) accompanying primary stain The mordant helps to force the stain go through wax coats on cells The decolorization is: acid alcohol 3% HCI in 95% ethanol Acid-fast bacteria will hold the primary stain because the carbol fuchsin is more soluble in the cell mycolic acid than in the acid-alcohol solution The non acid-fast bacteria will decolorize The counterstain is: methylene blue Red = acid-fast bacteria These organisms will appear red in color under the microscope through this staining technique Blue = nonacid-fast bacteria These organisms will appear blue in color under the microscope through this staining technique PROCEDURE (Ziehl-Neelsen Method) 1. Heat-fix a smear of a bacterium as follows: a. Using the dropper bottle of distilled water found in your staining rack, place 1/2 a drop of water on a clean slide by touching the dropper to the slide. b. Aseptically remove a small amount of the bacterium from the agar surface and mix it with the water. Flame the loop and let it cool. c. Using the loop, spread the mixture over the entire slide to form a thin film. d. Allow this thin suspension to completely air dry. e. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix. 2. Cover the smear with a piece of blotting paper and flood with carbol fuchsin. 3. Steam for 5 minutes by passing the slide through the flame of a gas burner. 4. Allow the slide to cool and wash with water. 5. Add the acid-alcohol decolorizing slowly dropwise until the dye no longer runs off from the smear. 6. Rinse with water. 7. Counterstain with methylene blue for 1 minute. 8. Wash with water, blot dry, and observe using oil immersion microscopy.

Acid-fast bacteria will appear red; non-acid-fast will appear blue. Gram Stain: Procedure Four reagents are used to perform a gram stain: crystal violet, Gram's Iodine, acetone - alcohol, and safranin.

Direct Smear Preparation If the specimen is received on a swab, gently role the swab on a clean glass slide to avoid rupturing host cells. Allow to air dry. Direct Smear Pap If the specimen is a fluid, place a drop of fluid on a clean glass slide and allow to air dry. *In both cases, the specimen is fixed to the glass slide by passing it a few times over a flame. Control Slide Fishers Band Gram Slide has control Gram positive cocci and Gram Negative rods. Staining Procedure Step 1. Flood the slide with crystal violet for 1 minute. Rinse with water. Step 2. Flood the slide with Gram's iodine for 1 minute. Rinse with water. Staining Procedure Step 3. Decolorize the slides by gently rinsing with an acetone - alcohol solution for 1 to 10 seconds dependent on content of acetone in solution. Rinse with water. step4. Flood the slide with saffranin, the counterstain, for 1 minute. Rinse with water and dry air. 1. Phenol is a component of the carbolfuchsin reagent for the Ziehl-Neelsen and Kinyoun methods for acid fast staining. Phenol is a dangerous chemical if not handled carefully. It is poisonous, corrosive and combustible. If you must handle the phenol crystals to prepare the carbofuchsin stain, you must be familiar with the safe handling requirements demanded by your institutions safety protocols. Use an MSDS (material safety data sheet) along with your departmental standard operating procedures to become familiar with the proper handling, disposal procedures, as well as the precautions to take to prevent hazards due to inhalation, skin or eye contact or ingestion of phenol.

At a minimum you should know that you should wear gloves when handling phenol and that heating the phenol for melting increases the risk of exposure through inhalation. Be sure that procedures are in place to prevent such exposure. Phenol must be disposed of as a hazardous waste. Even small amounts on paper towels or disposable containers must be confined and processed. Consult your departmental operating procedures for specific instructions. 2. In courses for non majors nonpathogenic mycobacteria are typically selected to illustrate the acidfast property attributed to a few groups of organisms. In advanced courses and in clinical laboratories however, cultures and specimens are likely to contain pathogens. Since the mycobacteria can be spread in the air, creating an inhalation exposure hazard, care should be taken to confine cultures and specimens to areas in the lab designed to minimize the exposure risk. You should receive specific instructions for handling such specimens to protect yourself and to avoid generating aerosols with the specimens that would increase the risk of dispersing the mycobacteria in the air.