Current Laboratory Practice Series

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Current Laboratory Practice Series
Use of Fluorochrome Staining for Detecting
Acid-fast Mycobacteria

• Laboratory Practice Training Branch
• Division of Laboratory Systems
• Public Health Practice Program Office

U.S. DEPARTMENT OF HEALTH AND HUMAN
SERVICES Public Health Service
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Program Content

• Part I
• Fluorochrome Staining Procedure
• Part II
• Examining and Reporting
• Fluorochrome-stained Smears

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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

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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

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Overview of Acid-fast Microscopy

• Preparing and Fixing Smears
• Staining Smears
• Examining Smears
• Recording and Reporting Results

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The Testing Sample

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

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Primary Stains

• Auramine O
• Auramine O-Rhodamine-B
• Counter Stains
• Potassium Permanganate
• Acridine Orange

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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

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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

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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
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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
gt90/F
gt36/F
number of acid-fast bacilli observed per
microscopic field
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Achieving reliable results depends on -

• Obtaining quality specimens
• Preventing contamination of testing samples
• Following established procedures
  recommendations and
• Ensuring accurate record keeping

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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
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Current Laboratory Practice Series
Use of Fluorochrome Staining for Detecting
Acid-fast Mycobacteria

• Part II
• Examining and Reporting
• Fluorochrome-stained Smears

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• Primary Stains
• Auramine O
• (A-F organisms appear yellow-green)
• Auramine O-Rhodamine B
• (A-F organisms appear yellow-orange)
• Counter Stains
• Potassium Permanganate
• (Background appears dark)
• Acridine Orange
• (Background appears yellow-orange)

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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