The document outlines the procedures and requirements for performing light emitting diode fluorescence microscopy (LED-FM) for tuberculosis diagnosis using auramine staining. It includes details on preparing necessary solutions, performing the staining technique, and examining stained smears, emphasizing the advantages of LED-FM over traditional methods. Additionally, it describes the properties and preparation of reagents, as well as the steps involved in the staining process.

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
1. Place slides smear upwards on a staining rack
at least 1 cm apart over a sink.
2. Flood slides with filtered 0.1% auramine,
leave for 20 minutes.
3. Gently wash slides with clean water. Tilt each
slide to drain off excess water.
4. 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.