Z-N staining method (AFB stain) -- part-1

Sample: sputum
Procedure:
1. Take small amount of the purulent part of the sputum sample using a wire loop
2. Make oval shape smear on a grease free slide . During the smear preparation the slide should be held near the flame.
3. The slide is heat fixed and leave for drying .
4. Smear covered with Cabol fuchsin and heated till fume appears in stain. Leave for 5 minutes.
5. Wash  the stain in running tape water and covered the smear with 20% sulphuric acid for 1-2 minutes.
6. Wash the slide and put methylene blue on the smear. Leave for 2 minutes.
7. Wash the slide in tape water and allowed to air dried.
        Examine the slide under oil immersion.

Sputum AFB smear (100×)

Appearance of AFB slide under the oil immersion:-
AFB:-Red , curved or straight continuous bead like rod
cells:-green or blue
background:-Green or Blue
Reporting of the sputum smear:-
0 AFB/300 field--------------No AFB seen
1-2  AFB/300 field-----------doubtful;repeat sample
1-9 AFB/100 field------------1+
1-9 AFB/10  field-------------2+
1-9 AFB/ field  ---------------3+
>9 AFB/ field  ----------------4+