SPUTUM COLLECTION AND EXAMINATION
Three sputum samples are collected from all patients with symptoms of
tuberculosis. The three samples are collected over two days - spot on the
first day, and one early morning and one spot on the second day. This is
equally sensitive as the collection of early morning specimens on three days ;
which tests not only the patients sputum but also his patience !. Sputum
examination is of central importance in the RNTCP, and thus ensuring
proper collection ,transportation and examination of sputum is of critical value
in the success of the programme. Microscopy is more objective and reliable than
X-Ray. Inter-observer variation is much less with microscopy than with X-Ray.
AFB microscopy also provides information on infectiousness of the patient and
allows prioritization of cases. It is fair to say that sputum smear AFB
positivity provides "smoking gun" evidence of Tuberculosis while X-Ray
does not. A systematic evaluation by National TB Institute, Bangalore of well
functioning District TB centres found that nearly 70% of the cases diagnosed and
put on treatment on the basis of X-Ray, did not have tuberculosis at all.
Guidelines for collection of sputum
The person collecting the sputum demonstrates how to open and close the
container, takes the patient to an open space far away from other people
or a vacant room set out for this purpose , and demonstrates with actual actions
how to bring out sputum. The patient is instructed to inhale deeply 2-3 times
with his mouth open, cough out deeply from the chest, open the container and
spit the sputum into it [at least 2 ml is ideal]. This is the spot
specimen. The patient is then given a sputum container to collect an early
morning specimen. When he returns with the early morning specimen, a second
spot specimen is collected.
Ideally all three specimens should be collected within two days and sent
to the microscopy laboratory as soon as possible, and definitely within one week.
Specimens should not be discarded citing the reason that it is a few days old,
especially if it is a good quality specimen.
Key steps in the preparation and staining of smears
The Ziehl-Neelsen staining:
step1. Pick up large, yellow,purulent part of sputum and spread evenly
onto 2/3 of central
portion of the slide
step2. Air dry slide for 15 minutes
step3. Fix the dry slide by heating briefly 3-5 times for 3-4 seconds
each time.
step4. Stain the slide with 1% carbol fuchsin.
step5. Heat the slide from underneath till vapours arise.
step6. Let the slides stand for 5 minutes.
step7. Rinse the slides with tap water and drain off excess water.
step8. Decolourize with 25% sulphuric acid and let it stand for 2-4 minutes.
step9. Rinse away excess stain with tap water and drain off the water.
step10.Counterstain with 0.1% methylene blue and let stand for 30
minutes.Gently
rinse with tap water and allow the slide to dry.
Examine the slide
under the microscope.
Recording of results:
