1. Mycobacteriaceae – Part IV Microscopic Examination Created by: Michele L. Jurgensmeier MT(ASCP) Edited by: Kathy Talmon MT(ASCP)SM Voice over lecturer: Carol Larson MSEd, MT(ASCP) Clinical Laboratory Science Program

2. 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

3. 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

4. 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

5. 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

6. Mycobacteriaceae Microscopic Examination Carbolfuschin stains (Ziehl-Neelsen / Kinyoun) –Advantages More specific than fluorescent stains Stains all mycobacteria –Disadvantages Longer examination time Less sensitive

7. 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

8. Mycobacteriaceae Microscopic Examination – Correlation With Culture Results Extensive disease –Good correlation Minimal or less-advanced disease –Less correlation Antibiotic treatment Unsatisfactory laboratory procedures

9. 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, 5 th Ed., J.B. Lippincott Co., 1997 4.Murray PK, et al., Manual of Clinical Microbiology, 7th ed., ASM Publishing, Washington DC, 1999.