This document discusses sputum smear microscopy for the diagnosis of pulmonary tuberculosis. Sputum smear microscopy is the most confirmatory test but requires ensuring the sputum is from the lungs. It can miss 25% of positive cases with a single smear. When performed correctly it is simple, inexpensive, and provides timely results. Sputum smear microscopy is used for early diagnosis, confirming the acid-fast nature of the organism, monitoring treatment effectiveness, and determining if other tests are needed. The document outlines procedures for collecting and examining sputum samples via Ziehl-Neelsen staining under a microscope.

2.  Demonstration of AFB by sputum microscopy is the
most confirmatory test for pulmonary
tuberculosis, but one has to be sure that sputum has
come from lungs and it is not just saliva.
 Single direct smear
miss about 25% of sputum
positive cases
 Simple, inexpensive, appropriate technology which is
relatively easy to perform and to read, yields timely
results with very high sensitivity of detection of
tubercle bacilli.

3.  Early diagnosis of mycobacterial infections.
 Confirm acid fast nature of organism.
 Monitor effectiveness of treatment.
 May be determinant of performing other tests such
as culture or susceptibility testing.

4.  Severe cough for 2 or more weeks (often involving
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blood loss)
Weight loss
Night sweats
Weakness or fatigue(tiredness)
Chills
Fever
Pain in the area infected.

5.  Under NTP conditions, the IUATLD recommends
collecting 2 sputum samples “on the SPOT-early
MORNING” preferably within 2 days, from each
person presenting at health centre, with respiratory
symptoms of more than 3 weeks duration.
 Volume of sputum: 3ml-6ml(minimum acceptable
amount:2ml)
 SPOT specimen
when TB suspects present at
the health centre.
 MORNING specimen
sputum produced within
1 or 2 hours after rising.

6.  If sputum are collected within the same 24 hour
period, a minimum of 8 hours between specimen is
required.
 Sputum smear positive tuberculosis
a person
presenting with respiratory symptoms with at least
one positive sputum smear microscopy examination.
 Detects about 80% of TB suspects ultimately positive
on sputum smear examination with the 1st
specimen, and additional 15% with the 2nd
specimen .

7.  5-10 ml
 Thick and mucous, but may be fluid with pieces of
purulent material.
 Colour varies from opaque white to green, but
reddish to brown when blood is present.

8.  Primary stain binds cell wall mycolic acid.
 Intense decolorization does not release the primary
stain from the cell wall.
 Colour of AFB based on primary stain.
 Counter stain provides contrasting background.

9.  Transmitted light: Carbol fuchsin staining.[contrast
red colour AFB on blue background]
 Fluorescence : Auramine staining[contrast
light/dark]

10. PROCEDURE
 Make the smear on a clean glass slide, air dry and keep it
fixed.
 Add conc. Carbol fuchsin, kept for 5-7 minutes, with
intermittent heatings (till fumes arise), wash with water.
 Add 20% H2SO4, kept for 5 minutes, wash with water.
 Add methylene blue, kept for 3 minutes, wash with water.
 Air dry and observe under oil immersion objective of
microscope.

11. ZN STAINING

12. ZN STAINING

13. AURAMINE
STAINING

14.  A well stained ZN smear shows strong red AFB
against a weak blue background.
 Examine 100 fields with the bright field
microscope, 1000X magnification.
 Declare the smear negative if no AFB are found.
 For high positives examination of 20-30 fields may
suffice.

15. No.
Examination(No. of
bacilli)
No. of fields
Grading
1
0
300
-ve
2
2-3
300
Doubtful, repeat
smear.
3
1-9
100
1+
4
1-9
10
2+
5
1-9
1
3+
6
>10
1
4+

16.  Acid fast particles and other micro-
organisms(waxes, precipitate of
stain, nocardia, environmental mycobacteria, spores
of bacillus subtilis, fibres and pollens, food
particles, yeast and artifacts like scratch on slide)

17.  Inadequate sputum collection, failure to select
suitable sputum particles for making smear, poor
smear preparation and inadequate examination of
smears.

18.  Specific and confirmatory test for pulmonary
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tuberculosis.
It is the test for infectiousness of the patient.
Sputum examination is important to monitor
progress during chemotherapy.
It helps to detect drug failure and development of
resistance in patients in anti tubercular
treatment(fall and rise phenomenon)
It is most cost effective investigation.

19.  It is less sensitive investigation compared to
radiology. Since number of bacilli required for
demonstration of AFB in sputum is quite
enormous(10,000 bacilli/ml3), the cases excreting
less number of bacilli may not be diagnosed by
microscopy alone. Similarly cases of pleural effusion
and hilar lymphadenitis may also be missed.
 Does not differentiate between living and dead
bacilli.
 Typing of bacilli is not possible.
 False positive result can mislead in diagnosis.

20.  Sensitivity can be increased by concentration
method, processing sample with NALC-NAOH
method require expertise, but can detect 15-20%
more cases.