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Annexure -2
Fluorescence staining procedure
Smear Preparation-
Mark a new, clean, grease free slide with laboratory number
Pick the purulent portion of the sputum using the crushed end of the broom stick
Prepare smear in an oval shape in the centre of the slide(3x2cm), for good
spreading of sputum firmly press the stick perpendicular to the slide and move in
small concentric circles
Thorough spreading of sputum is very important; it should be neither too thick nor
too thin. Prior to staining, hold the smear about 4-5 cm over a piece of printed
paper, if letters cannot be read, it is too thick.
Allow smear to air dry at room temperature
Heat fix by passing the slide over flame 2-3 times for about 2-3 seconds each
time. (Do not heat or keep the slide stationary over the flame or for too long or
else it will be scorched)
Staining
Arrange slides in serial order on staining bridge, with smear side up, at a distance of
at leastone cm between every slide
1. Flood the slide with filtered 0.1% Auramine solution
2. Do not heat
3. Keep the staining reagent for at least 20 min; make sure that the smear area
iscontinuously covered with Auramine by adding more if needed
4. Rinse with water and drain
5. Apply decolourising solution, 0.5% acid alcohol for 3 minutes
6. Gently rinse with water until the macroscopically visible stain has been washed
awayand drained
7. Flood smear with 0.5% potassium permanganate solution for 1 minute. Time
iscritical because counter staining for longer time may quench the acid fast
bacillifluorescence.
8. Gently rinse with water and drain
9. Air dry on a slide rack away from sunlight. If they are not read immediately
placethem in slide box.
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