Current Laboratory Practice Series

1. Use of Fluorochrome Staining for Detecting Acid-fast Mycobacteria Current Laboratory Practice Series Laboratory Practice Training Branch Division of Laboratory Systems Public Health Practice Program Office U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service

2. Program Content Part I Fluorochrome Staining Procedure Part II Examining and Reporting Fluorochrome-stained Smears

4. Acid-fast microscopy is - • A rapid method to screen for the most infectious cases of presumed tuberculosis • Used to make early decisions regarding respiratory isolation of patients • Used to initiate treatment and monitor response to therapy • A determinant for performing other tests

5. Fuchsin-stained smears require - • Use of 1000x magnification • Use of oil immersion • Examination of 300 microscopic fields • About 15 minutes to examine one negative smear • Examination by an experienced microscopist

7. Overview of Acid-fast Microscopy • Preparing and Fixing Smears • Staining Smears • Examining Smears • Recording and Reporting Results

8. The Testing Sample • All types of specimens can be evaluated • sputum, tissue, body fluids, etc. • Viable or killed organisms • Concentrated specimens are best

16. Primary Stains Auramine O Auramine O-Rhodamine-B Counter Stains Potassium Permanganate Acridine Orange

19. Steps in the Staining Procedure 1. Add fluorochrome stain 2. Rinse with water 3. Decolorize with acid-alcohol 4. Rinse with water 5. Add counter stain 6. Rinse with water 7. Air-dry

22. Control Slides • Assess the quality of the reagents • Determine if the staining is performed properly • Determine if the microscope is working properly • Detect environmental contaminants • Help find the plane of focus

32. Number of Fields to Examine at Selected Magnifications a b Magnification Number of Fields 200x 250x 400x 450x 30 30 55 70 a The minimum number of fields to examine before reporting a smear as negative for acid-fast organisms. b This final magnification represents the objective lens magnification multiplied by the eyepiece magnification

33. Examining and Reporting Acid-fast Smears Number of AFB Observed Report 200x,250x 400x,450x No AFB seen 0 0 Doubtful: repeat 1-2/30F* 1-2/70F 1+ 1-9/10F 2-18/50F 2+ 1-9/F 4-36/10F 3+ 10-90/F 4-36/F 4+ >90/F >36/F * number of acid-fast bacilli observed per microscopic field

35. Achieving reliable results depends on - • Obtaining quality specimens • Preventing contamination of testing samples • Following established procedures & recommendations and • Ensuring accurate record keeping

36. Credits This Program was developed by the Division of Laboratory Systems, Public Health Practice Program Office, Centers for Disease Control and Prevention Billie Ruth Bird, B.A., and Bereneice M. Madison, Ph.D. Special Thanks to Georgia Public Health Laboratory, Georgia Department of Human Resources Technical Reviewers Yvonne Hale, M.S. Florida Department of Health Ron Smithwick, M.S. Beverly Metchock, Dr.P.H. Centers for Disease Control and Prevention Nancy G. Warren, Ph.D. Laboratory Corporation of America

37. Use of Fluorochrome Staining for Detecting Acid-fast Mycobacteria Current Laboratory Practice Series Part II: Examining and Reporting Fluorochrome-stained Smears

38. Primary Stains Auramine O (A-F organisms appear yellow-green) Auramine O-RhodamineB (A-F organisms appear yellow-orange) Counter Stains Potassium Permanganate (Background appears dark) Acridine Orange (Background appears yellow-orange)

49. Credits- Billie Ruth Bird, B.A., and Bereneice M. Madison, Ph.D. This Program was developed by the Division of Laboratory Systems, Public Health Practice Program Office, Centers for Disease Control and Prevention Special Thanks to Georgia Public Health Laboratory, Georgia Department of Human Resources Technical Reviewers Yvonne Hale, M.S. Florida Department of Health Ron Smithwick, M.S. Beverly Metchock, Dr.P.H. Centers for Disease Control and Prevention Nancy G. Warren, Ph.D. Laboratory Corporation of America