auramine_staining technique DavidManyiel.pptx

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1. LIGHT EMITTING DIODE FLUORESCENCE MICROSCOPY (LED-FM)

2. Objectives Students should be able to: Identify reagents for FM examination Prepare 0.1% auramine solution Perform auramine staining technique Examine auramine stained smears, grade and report results Trouble shoot auramine staining procedure

3. Background Sputum smear microscopy (SMS) is useful for 1. Diagnosing people with infectious TB 2. Monitoring progress of TB treatment 3. Confirming that cure has been achieved. • 5000 -10,000 AFB per ml of sputum are required to produce a positive

4. LED FM (LIGHT EMITTING DIODE FLOURESCENCE MICROSCOPE) Recommended by WHO in 2011 to replace light microscopy for detection of acid-fast bacilli in high TB burden countries. It detects more sputum smear positive TB cases than Ziehl-Nelseen method due to its higher sensitivity (+10%). Slides are examined at x200 or x400 magnification, thus same area that needs examination for 10 minutes with a light microscope is examined in 2 minutes.

5. Also no use of immersion oil as well as heat during staining.

6. Principle of FM The property of acid-fastness is based on the presence of mycolic acids in mycobacterial cell wall. The primary stain, auramine O, forms a complex with the cell wall mycolic acids. Intense decolourization with acid alcohol does not remove the primary stain from mycobacteria cell wall.

7. A counter stain, potassium permanganate, quenches (reduces) non-specific fluorescence in the background; however, it provides little contrast for focusing, therefore Methylene blue is often used to provide a contrasting background. AFB are stained bright yellow against a dark background, but with some filter system they appear green.

8. Smear Preparation Refer to ZN technique

9. Requirements for auramine staining 0.3% methylene blue Weigh 3g of methylene blue. Dissolve in 1000ml of D water Label with the initials, date of preparation and expiry. Methylene blue expires in 12 months

10. 0.5 Acid Alcohol( Decolourizer ) Measure 995 ml of alcohol Carefully add 5 ml of hydrochloric acid to the alcohol. Label the bottle as 0.5% acid alcohol, date and intial . Store in a dark cupboard in room temperature for 12 months

11. Preparation of 0.1% auramine Solution A (1 L of 1 % stock auramine in alcohol) Add 1000 mL of ethanol or methanol to a 1L glass flask Add 10.0 g of auramine powder, mix until dissolved completely Heating inactivates auramine , so DO NOT use heat.

12. Label this solution: 1.0% auramine , include preparation and expiry dates and initials Store in a dark bottle in a cupboard at room temperature (expiry 12 months) Solution B (1 L of 3% stock phenolic solution) Weigh and dissolve 30 g of phenol crystals in 1000 mL distilled water, mix Transfer to a storage container

13. Label the container 3% phenolic solution, include preparation and expiry dates and your initials Store in a cupboard at room temperature (expiry 12 months)

14. To prepare 500 mL of 0.1% auramine from solutions A and B Add 50 mL of solution A to a 500 mL dark glass bottle Add 450 mL of solution B and mix Label the bottle 0.1% auramine , include preparation and expiry dates and your initials • Store in a cupboard at room temperature (expiry 2 months) Filter auramine solution when applying to smears Perform quality control with + 1 (check for the number and color of AFB) and negative smears.

15. Auramine Staining procedure Place slides smear upwards on a staining rack at least 1 cm apart over a sink. Flood slides with filtered 0.1% auramine , leave for 20 minutes. Gently wash slides with clean water. Tilt each slide to drain off excess water. Decolorize with 0.5% acid-alcohol for 1-2 min.

16. 5. Wash as before with water. Tilt each slide to drain off excess water. 6. Counter stain with 0.3% methylene blue for 30-60 seconds. 7. Wash as before and tilt each slide to drain off excess water . 8 . Slope the slides on a drying rack to air dry away from direct sunlight.