Mycobacterium under Fluorescence Microscope: Introduction, Working Mechanism and Reporting System
Mycobacterium under Fluorescence microscope
Mycobacterium under Fluorescence microscope pictures of auramine-phenol stained slide under fluorescence microscopy having following features-
Bright yellow rods, variable in length, often curved or also fragmented, glowing against a background.
The working mechanism of Fluorescence stain
The fluorochrome dye, auramine-phenol, forms a complex with mycolic acids found in the acid-fast cell wall of test organisms which resist decolorization by acid-alcohol. Potassium permanganate ( counterstain), renders tissue and its debris non-fluorescent, therefore reducing the possibility of artifacts. The cells visualized under ultraviolet light appear bright yellow against a dark background ( but the above image missing may be due to improper use of counterstain). Mycobacterium tuberculosis pictures of auramine-phenol stained slide under fluorescence microscope having following features-
Bright yellow rods, variable in length, often curved or also fragmented, glowing against a background.
Reporting sputum smear
According to IUATLD (International Union against Tuberculosis and Lung Diseases) and WHO guidelines-
Report using 40 x objective and 10 x eyepiece
Scanty AFB 1-19 AFB in 40 fields
+ 20-199 AFB in 40 fields
++ 5-50 AFB per filed (examine 20 fields)
+++ >50 AFB per field ( examine 8 fields)
To say No AFB seen: Examine 40 fields that should be free from AFB
Report
According to IUATLD (International Union against Tuberculosis and Lung Diseases) and WHO guidelines-
AFB seen (++)
Advantage
Fluorochrome staining (Fluorescence microscopy) is highly sensitive for Mycobacterium tuberculosis than Ziehl-Neelsen staining because of observing at 40X objective whereas in Ziehl-Neelsen at 100X objective i.e. oil immersion field.
Further Readings
- Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
- Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
- Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
- Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
- Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
- Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
- Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
- Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
- District Laboratory Practice in Tropical Countries – Part-2- Monica Cheesebrough- 2nd Edn Update