Module

AFB MICROSCOPY

Content Overview

Collection of sputum specimens

Quality of sputum specimens
Processing of sputum specimens
Registration of sputum specimens
Sputum smearing and staining
Smear examination
Recording and reporting
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Collection of Sputum Specimens

Collect 1-3 sputum specimens for diagnostic

purposes according to NTP policy

Follow NTP policy for declaring or excluding a

smear-positive case

Follow NTP policy for follow-up smears

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Instructions to Patients

Give clear instructions to patients on how to

collect the sputum:

Importance of sputum examination for TB

diagnosis
Need for real sputum, not saliva
How to produce good sputum
How to open and close the container
How to avoid contamination of outside of the
container
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Characteristics
of Good Sputum Specimen

3-5 ml
Usually thick and mucous, but may be fluid with
pieces of purulent material
Color varies from opaque white to green, reddish to
brown when blood is present
Clear saliva is not suitable; but examine saliva if a
better specimen can not be produced, especially for
follow-up examinations
A specimen mainly containing blood should not be
examined; patient should see a doctor for immediate
management
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Processing Sputum Specimens

Process specimens as soon as possible for

the benefit of the patients

However, for microscopy time between
collection and staining matters little

a refrigerator is not needed to keep samples

After preparation of a fixed smear, further

processing can wait

Collection and Transportation

of Sputum

Options if the health facility attended by the

patient does not perform smear microscopy

Refer patients to nearest microscopy facility
Have the specimens taken by assigned person or
sent by public transport to microscopy facility
Have the specimens collected, smears prepared
and fixed and taken to microscopy facility by
assigned person

Registration of Sputum Specimens

Register all specimens for AFB microscopy in the TB

Laboratory Register
One line for the diagnostic smears of each patient
One line for each follow-up
One or more results may be registrered on one line

Label the sputum containers on the side, not on the

lid, with laboratory serial number and the column
number or code for diagnostic smears
Label the slides with the laboratory serial number
and the column code
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Sputum Smearing (1)

Select new, clean, grease-free, unscratched

slides, free of fingerprints

Use a pencil in case of a frosted slide and a
diamond pencil in case of unfrosted slides to
write the serial number
Ensure that the number on the slide is the
same as on the sputum container

Sputum Smearing (2)

Select and pick up yellowish (or whitish)

particles with an applicator stick, e.g. bamboo,

or a wire loop
Prepare the slide in oval shape in the center
of the slide, 2 (length) by 1 (width) cm or 3 by
2 cm
Crush thick particles with the stick/wire loop,
spread for a sufficiently long time to obtain
even smears
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Sputum Smearing (3)

Place the used stick in a discard container;

use a new stick for each specimen

Dip the wire loop in a sand-alcohol bottle;
move the loop up and down to remove excess
sputum; heat in flame until red-hot
Air-dry the smear at room temperature
If the smear is completely dry, pass the slide
2-3 times, 2-3 seconds over a flame, holding
the slide upwards
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Smear Staining
with ZIehl-Neelsen Stain (1)

A well stained ZN smear shows strong red AFB,

against a weak blue background
Background should not show much remaining red
and no red artifacts
Proposed technique is to use 1% carbol fuchsin
(strong red) and 0.1% methylene blue (weak blue) as
a counter stain
Destaining with 3% hydrochloric acid in alcohol or 2025% sulphuric acid in water
must be complete
can be repeated to ensure complete absence of red color
from the background
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ZN Stained Smear

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Smear Staining
with Ziehl-Neelsen Stain (2)
STEPS:
Arrange slides in a serial order on a staining bridge, smear
side up, avoid slides touching each other
Cover slides completely with carbol fuchsin solution (through
filter paper)
Gently heat to steam
Leave for 10-15 minutes
Rinse with water and drain
Cover with decolorizing solution, 3-5 min, rinse with water,
drain. Repeat if needed, rinse and drain
Apply methylene blue solution for 1, at most 2 minutes
Rinse with water and drain
Air dry on a slide rack

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Smear Staining with Auramine (1)

STEPS
Arrange slides in a serial order on a staining bridge, slide side
up, avoid slides touch each other
Apply filtered auramine solution, leave for at least 15, max. 30
minutes
Rinse with water and drain
Apply decolorizing solution, 1-3 minutes, rinse with water and
drain, repeat if needed, rinse with water and drain
Apply counter stain for max. 1 minute
Rinse with water and drain
Air dry on a slide rack
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Smear Staining with Auramine (2)

AFB show yellow or green fluorescence

against a more or less dark background,

depending on the lamp and filters used
Destaining solution must have alcohol; acid
usually 0.5% HCl
Usual counter stain is 0.5% potassium
permanganate
Keep stained slides in the dark, if examination
cannot be done immediately after staining
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Auramine Stained Smear (1)

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Auramine Stained Smear (2)

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Examination of ZN Smears

Examine 1 length of the smear (2 cm) or 100 fields with

the bright field microscope, 1000x magnification
Declare the smear as negative if no AFB are found
If <10 AFB are found, count the number of AFB
If >= 10 AFB are found, grade the smear 1+, 2+, or 3+
For high positives examination of 20-30 fields may
suffice

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Examination of
Auramine Stained Smears

Examine 1 length of the smear (2 cm) with FM

best use a 20x objective: 1 length is 30 fields with this
objective, but 3 times the area seen through a 100X
objective
if a 40x objective is used, one length counts 40-50 fields or 2
times the area seen through a 100X objective

If necessary, confirm AFB, typical curved rod shape,

turning to a higher power objective (40X if screening
with 20X)
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Grading of
Auramine Stained Smears

Examine one length if no or few AFB are seen

declare negative if none at all are seen
if less than 30 AFB are found using 20x objective, or less than
20 AFB using 40x objective, the result is scanty. Report the
exact number of AFB per length
if AFB per length count 30 to 299 using 20X objective, or 20 to
199 using 40X objective, grade the smear as 1+

Examine about 10 fields if many AFB are seen

if on average there are more than 100 AFB per field with 20X
objective, or more than 50 with 40X, grade the smear as 3+
if per field between 10 and 100 AFB are seen with 20X
objective, or between 5 and 50 with 40X objective, grade as 2+
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Recording and Reporting

Write the smear results in the TB Laboratory

Register

Write the smear results on the sputum

examination request forms

Forward the filled sputum request form to the

person requesting the sputum examinations

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Grading of ZN Smears
IUATLD/WHO scale

ZN 1000x,
1 length=100 HPF

Negative

0 AFB / 1 length

Scanty

1-9 AFB /
1 length

1+

10-99 AFB /
1 length

2+

1-10 AFB /
1 HPF on average

3+

>10 AFB /
1 HPF on average
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Grading of Smears in FM
Using 20X Objective
IUATLD/WHO scale

FM 20x,
1 length = 30 fields
1 length = 300 HPF

Negative

0 AFB / 1 length

Scanty

1-29 AFB /
1 length

1+

30-299 AFB /
1 length

2+

10-100 AFB /
1 field on average

3+

>100 AFB /
1 field on average

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Grading of Smears in FM
Using 40X Objective
IUATLD/WHO scale

FM 400x,
1 length = 40-50 fields
1 length = 200 HPF

Negative

0 AFB /1 length

Scanty

1-19 AFB /
1 length

1+

20-199 AFB /
1 length

2+

5-50 AFB /
1 field on average

3+

>50 AFB /
1 field on average

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Laboratory Register for Sputum Smear Microscopy

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Request and
Reporting
Form for
Sputum
Smear
Examination

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Key Messages

Good quality specimens, smearing, staining and reading

are all essential for correct diagnosis of tuberculosis
Ziehl-Neelsen and fluorochrome methods are both
suitable, but fluorescence is better in case of high
workload and/or few AFB
Grading scales for quantification of AFB in smears vary
with the magnification system and thus objective used
The specific request form and the laboratory register
must always be used to record and report smear
examination results
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