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Unformatted text preview: Experiment 3-7: Acid-Fast Staining: Ziehl-Neelsen Method(pgs.91-94)
Purpose: To learn the proper procedure for acid-fast staining.
The Acid-Fast stain is a differential staining technique that it used to detect the presence of mycolic acid. Mycobacterium are rod-shaped, gram-positive bacteria which like to grow at ~30oC. The species of this genus have varied rates of growth. Organisms of the genus Mycobacterium have a unique waxy lipid layer called mycolic acid which makes the colonies look waxy and crusty. Mycolic acid binds to the peptidoglycan of the cell wall (without mycolic acid, the cell stains Gram-positive) creating a waxy, hydrophobic layer. They are thus resistant to dehydration. The waxy layer poses problems for staining: 1. The bacteria require special staining methods - Gram stains or other stains do not penetrate the waxy layer except if it is removed; waxy layer repels aqueous stains - staining the bacteria requires a mix of phenol and carbolfuchsin (primary stain) - phenol helps the stain penetrate the waxy layer of the lipid - carbolfuchsin is lipid soluble so it can enter the waxy layer - steam and heat act as a mordant and are required to drive the stain into the cell - acid-alcohol is used as a decolorizer of non acid-fast cells. - Mycobacterium are considered to be acid-fast since it is difficult to decolorize them - Other non acid-fast bacteria will be decolorized and lose the red color of the stain - Methylene blue is used as a counterstain in order to stain the non acid-fast bacteria Acid-fast bacteria thus appear red; while non acid-fast bacteria appear blue. Examples of diseases caused by Acid-fast bacteria:
Mycobacterium tuberculosis - causes tuberculosis; an infection of the lungs where the patient is very sick or dies because of the resulting immune response. Left unchecked, an acute pulmonary infection occurs destroying lung tissue and spreading the disease to other parts of the body resulting in death. - grows slowly and takes 8 weeks to call a culture negative or positive - still a prevalent disease in the world Mycobacterium leprae - causes leprosy; an infection of the skin where lesions form on the extremities (face, hands, feet, etc) causing a patient to become very sick or die due to the resulting immune response - can't be grown on artificial media, only has been cultured on armadillo feet - still a prevalent disease in parts of the world 1 Materials and Methods:
TSA slant cultures of: Mycobacterium smegmatis TSB cultures of: Staphylococcus epidermidis Stains: Carbolfuchsin Acid Alcohol Methylene blue Procedure: 1. Prepare smears for each of the organisms individually and one mixed smear For M. smegmatis you don't need a lot of bacteria; don't dig into the agar slant; try to break up the clumps of bacteria 2. Setup a Bunsen burner on your ring stand with the ring clamp and wire gauze over it. 3. Fill the can (from your student drawer) with water from the tap until it is about half-full. Place the can on top of the wire gauze on the ring clamp. 4. Turn on the Bunsen burner to heat/boil the water. (Let steam start to appear before putting the slide on the can.) 5. Place the slide with smear(s) on it on top of the can 6. Place a piece of paper towel on top of the smear on the slide and saturate the paper with Carbolfuchsin. 7. Steam the smear over the boiling water for 5 minutes. NOTE: If the Carbolfuchsin evaporates off, then add more - Keep the paper wet. Use forceps to take the slide off the can when finished - it will be hot!! 8. Allow the slide to cool, remove the paper, and decolorize with acid alcohol for 15-20 seconds. 9. Rinse with water to stop the action of the decolorizer 10. Add Methylene blue to the slide for 1 minute and then rinse with water 11. Blot the slide between sheets of Bibulous paper. 12. Examine the slide under oil immersion. 2 ...
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This note was uploaded on 04/17/2008 for the course BIO 2200 taught by Professor Choong-minkang during the Fall '08 term at Wayne State University.
- Fall '08