Phenolic Auramine is used as primary staining solution in fluorescent staining. It is a staining technique mostly used because of it high sensitivity to detect mycobacterium tuberculosis in sputum,csf and other specimen as compared to ziehl neelsen.
It is a fluorochrome technique which enables a smear to be examine at lower magnification thereby shortening the time for microscopic examination
In cases where acid fast bacilli are few,they are more easily detected in a fluorochrome stained smear using a fluorescence microscope
Principle of Auramine Phenol technique
This differential staining technique is used for the identification of tubercle bacillus, other Mycobacteria, and Nocardia, which depends on the chemical composition of the bacterial cell wall.
- Auramine binds to the mycolic acid found in mycobacterial cell wall.The smear is illuminated with blue violet or ultraviolet light and auramine fluorescence indicating the presence of Acid fast bacilli
- Acid alcohol or phenol removes the dyes from the background of the smeared slide
- The weak solution of potassium permanganate is the counterstain which darken the background.
- Blue violet or Ultraviolet light enables auramine dye in the Acid fast bacilli cell wall to fluoresce bright yellow indicating the presence of Acid fast bacilli against a dark background
Composition of Auramine Phenol stain
|Auramine O||0.300 gm|
|Distilled water||100.000 ml|
Appearance of Auramine Phenol stain
It is a Yellow coloured solution.
Materials required for Auramine Phenol stain
- Auramine Phenol stain
- 1% acid alcohol
- 1g/l Potassium permanganate
- Suspected sample
- Clean slide
- Bunsen burner
Procedure for Acridine Orange stain
- Start by thoroughly flaming one side of a clean glass slide to be used for the smear and allow the slide to cool before smearing
- Now prepare a smear from the suspected specimen on the flamed side of the slide and allow to air dry.
- Heat-fix the smear by passing the slide through a flame
- Flood the slide with phenol auramine stain
- Heat to steaming for 10-15 minutes with a low flame and avoid boiling the stain or permit drying of the smear.
- Rinse the slide with distilled water and drain off excess liquid.
- Now flood the slide with alcohol decolorizer for 2-3 min. Slide will still appear pink.
- Rinse the slide thoroughly with distilled water then drain off excess.
- Cover the slide with potassium permanganate counterstain for 3-4 min and do not allow slide to dry.
- Rinse thoroughly with distilled water,wipe the back of the slide clean and allow the slide to air dry
- To prevent fading of the fluorescence,protect the stained smear from sunlight and bright light
- Examine the slide with 24 hours by fluorescence microscopy using the 10X to focus the smear the systematically switch to 40X objective to examine the smear for Acid fast bacilli.
Observation and interpretation
Microscopic Examination of Auramine phenol Fluorescent staining is observed under fluorescence microscope. Slides are screened under high power (400X) and verified under oil immersion lens.
|Acid Fast Bacilli||Appears bright yellow rod with variable length which may often be curved or fragmented glowing against a dark background|
How to report AFB in Auramine stain
|Scanty AFB||1-10 AFB in 40 fields|
|+||20-199 AFB in 40 fields|
|++||5-50 AFB per field (Examin 20 fields)|
|+++||＞ 50 AFB per field (Examin 8 fields)|
In case where no AFB are seen,examine 40 fields and report the smear as “No AFB Seen”