Current Laboratory Practice Series

Laboratory Practice Training Branch Division of Laboratory Systems

Public Health Practice Program Office

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICESPublic Health Service

Use of Fluorochrome Staining for Detecting Acid-fast Mycobacteria

Program Content

Part IFluorochrome Staining Procedure

Part IIExamining and Reporting

Fluorochrome-stained Smears

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

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

• Preparing and Fixing Smears

• Staining Smears

• Examining Smears

• Recording and Reporting Results

Overview of Acid-fast Microscopy

The Testing Sample

• All types of specimens can be evaluated– sputum, tissue, body fluids, etc.

• Viable or killed organisms

• Concentrated specimens are best

Auramine OAuramine O-Rhodamine-B

Counter StainsPotassium Permanganate

Acridine Orange

Primary Stains

Steps in the Staining Procedure

1. Add fluorochrome stain2. Rinse with water3. Decolorize with acid-alcohol4. Rinse with water5. Add counter stain6. Rinse with water7. Air-dry

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

Magnification b Number of Fields

200x250x400x450x

a The minimum number of fields to examine before reporting a smear as negative for acid-fast organisms.

bThis final magnification represents the objective lens magnification multiplied by the eyepiece magnification

30305570

Number of Fields to Examine at Selected Magnifications a

Examining and Reporting Acid-fast Smears

Number of AFB Observed

Report 200x,250x 400x,450x

No AFB seen 0 0Doubtful: repeat 1-2/30F* 1-2/70F

1+ 1-9/10F 2-18/50F2+ 1-9/F 4-36/10F3+ 10-90/F 4-36/F4+ >90/F >36/F

* number of acid-fast bacilli observed per microscopic field

Achieving reliable results depends on -

• Obtaining quality specimens• Preventing contamination of testing

samples • Following established procedures &

recommendations and• Ensuring accurate record keeping

CreditsThis 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

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

Billie Ruth Bird, B.A., and Bereneice M. Madison, Ph.D.

Technical Reviewers

Part II: Examining and Reporting

Fluorochrome-stained Smears

Current Laboratory Practice Series

Use of Fluorochrome Staining for Detecting Acid-fast Mycobacteria

Primary Stains

Auramine O (A-F organisms appear yellow-green)

Auramine O-Rhodamine B (A-F organisms appear yellow-orange)

Counter StainsPotassium Permanganate

(Background appears dark)Acridine Orange

(Background appears yellow-orange)

Credits-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

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

Billie Ruth Bird, B.A., and Bereneice M. Madison, Ph.D.

Technical Reviewers