Principle:
Most of the bacteria can be stained with aqueous solutions of para rosaniline dyes but certain organisms especially which belongs to family mycobacteriaceae resist entry of these weaker dyes and hence these weak solutions are unsuitable for staining them.The bacteria can also be stained with strong solutions which contain phenols which help in penetration of the in to the cell. Heating further enhances entry of the dye in to the cell. These organisms once stained resist decolorization with acids such as H2SO4, HCL, HNO3 etc. and even with alcohol. Those organisms which resist decolorize with acid retain primary stain and are called acid fast organisms and those which are decolorized by acid will takee up counter stain and are called non acid fast organisms. Reason for acid fastness is due to the presence of long chain fatty acid ,mycolic acid in the cell wall of these organisms.
2+: 1-10 AFB/OIF in at least 20 visual fields
3+: More than 10 AFB/OIF in at least 20 visual fields
Most of the bacteria can be stained with aqueous solutions of para rosaniline dyes but certain organisms especially which belongs to family mycobacteriaceae resist entry of these weaker dyes and hence these weak solutions are unsuitable for staining them.The bacteria can also be stained with strong solutions which contain phenols which help in penetration of the in to the cell. Heating further enhances entry of the dye in to the cell. These organisms once stained resist decolorization with acids such as H2SO4, HCL, HNO3 etc. and even with alcohol. Those organisms which resist decolorize with acid retain primary stain and are called acid fast organisms and those which are decolorized by acid will takee up counter stain and are called non acid fast organisms. Reason for acid fastness is due to the presence of long chain fatty acid ,mycolic acid in the cell wall of these organisms.
Procedure:
- Fix the smear by passing the slide over a flame for 3 times.
- Cover the smear with CARBOL-FUCHSIN stain for 5-10 minutes.
- Decolorize the smear with until the smear appears colorless and rinses with tap water.
- Cover the smear with METHYLENE BLUE stain for 5 minutes and rinses with tap water.
- Examine the slide under the oil immersion lens.
Interpretation:
- Positive AFB: Presence of pink-red rods
- Negative AFB: Absence of pink- red rods
- If the result is negative (-), report as NO AFB SEEN.
- If positive (+):
2+: 1-10 AFB/OIF in at least 20 visual fields
3+: More than 10 AFB/OIF in at least 20 visual fields
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