AcidFast Bacillus AFB Gram stain Gram stain gives

Acid-Fast Bacillus AFB

Gram stain • Gram stain gives positive or negative reaction • It does not applied to all bacteria ex. Acid Fast Bacilli (AFB), which means acidresistance bacilli. • Not stained with Gram stain. • Samples: 3 successive early morning sputum samples.

Acid Fast Bacilli (AFB) • Why Gram stain is not used ? - AFB cell wall has ~ 60% lipid (waxy). So, it makes gram stains impermeable. • Ex. Mycobacteria spp. • Mycobacterium tuberculosis (MTB)

AFB stain • Two procedures : 1 - Light microscope - Ziehl-Neelsen and Kinyoun methods 2 - Fluorescent microscope - Fluorochrome method

1 - Ziehl-Neelsen stain The procedure utilizes heat and phenol (carbolic acid) to help the penetration of the dye, basic fuchsin, to the inside of mycobacterial cells, which are impermeable to basic rosaniline dyes in routin stains such as the Gram stain. • Procedure : 1 - Primary stain: carbol fuchsin , 5 minutes then wash 2 - decolorizer : acid alcohol , 1 -2 minutes then wash 3 - counterstain: methylene blue, 1 minutes then wash

• Under microscope : • red bacilli (AFB) • stained by ZN stain with blue background.

2 -Kinyoun Method (Cold ZN stain) • Does not require heating. • It is safer than ZN stain. • Reagents: 1 - Carbol fuchsin , 5 minutes, then wash 2 - Acid alcohol , 5 minutes, then wash 3 - Methylene blue, 5 minutes, then was and dry the slide.

3 - Auramine-Rhodamine Staining • Fluorescent staining (use UV light ) • More sensitive than ZN staining • Auramine-Rhodamine binds to cell wall ( mycolic acid) • Examination under fluorescent microscope: • Procedure : 1 - primary stain: Auramine-Rhodamine, 15 minutes 2 - Decolorizer : Acid Alcohol , 2 minutes 3 - Counterstain : Potassium Permanganate , 3 minutes

Yellow to orange bacilli glowing, stright or slightly curved, single or in group with black background.

• Medium • Lowenstein-Jensen Medium (LJ medium) • It is a selective medium for isolation of mycobacteria. It is egg-based medium (not agar- based medium) • Selective agent : malachite green which inhibits growth of gram positive, gram negative, and fungi. • Incubation: aerobically at 37 C for 6 -8 weeks because M. TB grow very slowly • Colonies: buff, rough and tough colonies.

• Medium • Colonies: buff, rough and tough colonies. • 50% of TB patients are negative with ZN stain. However, culturing sputum samples must be done.

Summary • Gram stain does not applied to all bacteria. • Acid Fast Bacilli (AFB) • Samples: 3 successive early morning sputum samples. • Two procedures : Light microscope (Ziehl-Neelsen and Kinyoun methods) Fluorescent microscope (Fluorochrome method) • 1 - Ziehl-Neelsen stain • 2 -Kinyoun Method (Cold ZN stain) • 3 - Auramine-Rhodamine Staining • Lowenstein-Jensen Medium (LJ medium) is a selective medium for isolation of mycobacteria.