Mycobacteriaceae – Part IVMicroscopic Examination

Created by: Michele L. Jurgensmeier MT(ASCP)Edited by: Kathy Talmon MT(ASCP)SMVoice over lecturer: Carol Larson MSEd, MT(ASCP)Clinical Laboratory Science Program

Mycobacteriaceae Microscopic Examination

• Direct specimen detection important due to slow growth rate of organisms

• Purposes– Determine acid fast characteristic– Detect new cases– Monitor progress of disease / treatment– Criteria for patient release from hospital

Mycobacteriaceae Microscopic Examination

• Fluorochrome stains (Auramine O)– Primary stain:

fluorescent dyes– Counter stain:

potassium permanganate

– AFB fluoresce yellow/gold against a black background

Enlarged view

Mycobacteriaceae Microscopic Examination

• Fluorochrome stains (Auramine O)– Screening procedure– Advantages

• More sensitive than carbolfuchsin stains• Examine on lower magnification

– View more fields in shorter time

– Disadvantages• Rapidly growing mycobacteria do not stain • Less specific

Mycobacteriaceae Microscopic Examination

• Carbolfuschin stains (Ziehl-Neelsen / Kinyoun) – Primary stain:

carbolfuschin– Counter stain:

methylene blue– AFB appear red

and background material blue Photo courtesy of CDC public image library

Content provider Dr. George P. Kubica

Mycobacteriaceae Microscopic Examination

• Carbolfuschin stains (Ziehl-Neelsen / Kinyoun)– Advantages

• More specific than fluorescent stains• Stains all mycobacteria

– Disadvantages• Longer examination time • Less sensitive

Mycobacteriaceae Microscopic Examination

• Interpretation of smears– Examine 300 oil immersion fields– Observe slides for morphology consistent

with mycobacteria– Other organisms may stain partially acid fast– Quantify organisms

• Follow disease state

Mycobacteriaceae Microscopic Examination –

Correlation With Culture Results• Extensive disease

– Good correlation• Minimal or less-advanced disease

– Less correlation• Antibiotic treatment• Unsatisfactory laboratory procedures

Mycobacteriaceae References:

1. Mahon, C.R. & Manuselis, G., Textbook of Diagnostic Microbiology, 2nd Ed., W.B. Saunders, 2000

2. Forbes, B.A., Sahm, D. F., & Weissfeld, A.S., Bailey and Scott’s Diagnostic Microbiology, 11th Ed., Mosby, 2002.

3. Koneman, Color Atlas and Textbook of Diagnostic Microbiology, 5th Ed., J.B. Lippincott Co., 1997

4. Murray PK, et al., Manual of Clinical Microbiology, 7th ed., ASM Publishing, Washington DC, 1999.