852 Concise Book of Medical Laboratory Technology: Methods and Interpretations

1. Place the specimen under test on a clean, scratchless

glass slide using a sterile plating loop.

2. Spread by tracing concentric circles well separated

over an area of 200 mm square (20 mm × 10 mm),

take care as to not to reach the edge of the slide.

Alternatively for dense mucoid specimen press the

specimen between two glass slides and pull apart

gently to form a thin film of mucous.

3. When the smear is completed, plunge the inoculating

loop into liquid disinfectant (2% Glutaraldehyde) and

shake to remove any sputum, then flame sterilize loop.

4. Air-dry the smear.

5. Fix the smear by passing the slide approximately three

times on the flame.

 (Note: While passing the smear slide on the flame see

that the side opposite the smear is facing the flame).

6. Mix well and add AFB stain (A) over the smear to cover

it completely (4–5 drops ≈ 0.2 mL, may be required).

7. Keep for 6 minutes and then rinse with plenty of

distilled water slowly to remove excess of AFB stain (A).

8. Tilt slide to drain, mix well and add AFB stain (B) over

the smear to cover it completely (4–5 drops ≈ 0.2 mL,

may be required).

9. Keep for 6 minutes and then rinse the smear once more

with distilled water to remove excess of AFB stain (B).

10. Air-dry and observe under oil immersion (magnification 100 X objective).

Interpretation of Results

1. Presence of pink to red colored slender Bacilli—Smear

Acid Fast Bacilli positive.

2. Absence of pink to red colored slender Bacilli—Smear

Acid Fast Bacilli negative.

3. Pus cells and other bacteria stain purple to blue color.

Grading of Results

After 5 minutes of examination covering about 100 fields.

Number of Acid Fast Report

Bacilli observed

No Acid Fast Bacilli Negative

1–10 Acid Fast Bacilli Actual Number

> 10 Acid Fast Bacilli +

Masses of Acid Fast

Bacilli in several fields } ++

Remarks