CONTROL: Any tissue containing acid-fast organisms. Use Millipore filtered water in the waterbath and staining procedure.
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1 SURGICAL PATHOLOGY - HISTOLOGY Date: STAINING MANUAL - MICROORGANISMS Page: 1 of 3 ACID-FAST BACTERIA - ZIEHL-NEELSEN STAIN (AFB) PURPOSE: Used in the demonstration of acid-fast bacteria belonging to the genus 'mycobacterium', which include the causative agent for tuberculosis. PRINCIPLE: The lipoid capsule of the acid-fast organism takes up carbolfuchsin and resists decolorization with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such high molecular weight that it is waxy at room temperature and successful penetration by the aqueousbased staining solutions (such as Gram's) is prevented. CONTROL: Any tissue containing acid-fast organisms. Use Millipore filtered water in the waterbath and staining procedure. FIXATIVE: 10% formalin TECHNIQUE: Cut paraffin sections at 4-5 microns. EQUIPMENT: Rinse all glassware in DI water, coplin jars, microwave oven. REAGENTS: Ziehl-Neelsen Carbol-Fuchsin Solution: ml Mix well, filter into brown bottle. Label bottle with date and initials, solution is stable for 1 year. 1% Acid Alcohol: Hydrochloric acid 10.0 ml 70% Alcohol ml Mix well, label with date and initials, stable for 1 year. CAUTION: Corrosive. CAUTION: Carcinogen, toxin.
2 MICROOGANISMS ZIEHL-NEELSEN-AFB Page: 2 of 3 Stock 5.0 ml 50.0 ml 45.0 ml Mix well, filter into bottle. Label with date and initials, stable for 1 year. Pour into coplin jar, stable for 2 months. SAFETY/PPE: Wear nitrile gloves, goggles and lab coat. Keep hot, uncapped, solutions in the exhaust hood. Avoid contact and inhalation. : carcinogen. Phenol: toxic by ingestion, inhalation and skin absorption. Readily absorbed through skin, causing increased heart rate, convulsions and death. Will burn eyes and skin, analgesic action may cause loss of pain. Target organ effects in digestive, nervous and urinary systems. Hydrochloric acid: strong irritant to skin, eyes and respiratory system. Target organ effects via inhalation on skin, respiratory, reproductive and fetal systems. : produced deleterious effects on fertility in rats. PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. *Carbol-fuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. Filter solution once a week. 3. Wash in running tap water. 4. 1% Acid alcohol until light pink and color stops running. 5. Wash in running tap water for 5 minutes.. 6. Rinse in distilled water. 7. Working methylene blue for 30 seconds. 8. Rinse in water. 9. Dehydrate, clear, and coverslip. *Conventional Method: 60 C oven for 1 hour.
3 MICROOGANISMS ZIEHL-NEELSEN-AFB Page: 3 of 3 RESULTS: Acid-fast bacilli bright red Background blue REFERENCES: Sheehan D,Hrapchak B, Theory and practice of Histotechnology, 2nd Ed, 1980, pp ,battelle Press, Ohio Carson F, Histotechnology: A Self-Instructional Text, 1990, pp ,ascp,ill Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech Prepared: Approved: By: By: Downloaded from WebPath: Internet Pathology Laboratory
4 PROCEDURE CARD ACID-FAST BACTERIA - ZIEHL-NEELSEN STAIN (AFB) CONTROL: Any tissue containing acid-fast organisms. Use Millipore filtered water in the waterbath and staining procedure. PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. *Carbol-fuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. Filter solution once a week. 3. Wash in running tap water. 4. 1% Acid alcohol until light pink and color stops running. 5. Wash in running tap water for 5 minutes.. 6. Rinse in distilled water. 7. Working methylene blue for 30 seconds. 8. Rinse in water. 9. Dehydrate, clear, and coverslip. *Conventional Method: 60 C oven for 1 hour. RESULTS: Acid-fast bacilli Background bright red blue SAFETY/PPE: Open hot solution within the exhaust hood. Phenol will cause burns, wear nitrile gloves. : carcinogen. Ziehl-Neelsen Carbol-Fuchsin Solution: ml Mix well, filter into brown bottle. Label bottle with date and initials, solution is stable for 1 year. CAUTION: Carcinogen, toxin. 1% Acid Alcohol: Hydrochloric acid 10.0 ml 70% Alcohol ml Mix well, label with date and initials, stable for 1 year ml Mix well, filter into bottle. Label with date and initials, stable for 1 year. Stock 5.0 ml 45.0 ml Pour into coplin jar, stable for 2 months. CAUTION: Flammable, corrosive.
5 METHYLENE BLUE 50.0 ml Mix well, filter into bottle, add a crystal of thymol. Stable for 1 year. Stock 5.0 ml 45.0 ml Pour into coplin jar, stable for 2 months. EXPIRATION: METHYLENE BLUE ZIEHL-NEELSEN CARBOL FUCHSIN ml Mix well, filter into brown bottle. Stable for 6 months. CAUTION: Carcinogen, toxin, avoid contact and inhalation. EXPIRATION: CARBOL FUCHSIN 1% ACID ALCOHOL: FOR AFB 70% alcohol ml Hydrochloric acid 10.0 ml Stable for 1 year. CAUTION: Flammable, corrosive
CONTROL: An infected appendix, or any tissue containing both negative and positive gram rods.
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