Sputum is the liquid substance that is produced from the lower respiratory tract when one coughs. In addition to mucus, sputum contains many materials that are not visible to the naked eye. It often consists of bacteria, cellular fragments, blood, and pus.

1. PRESENTATION ON SPUTUM
TEST
By Gloria Asantewaa

2. CONTENT
• Definition of sputum
• What is sputum test?
• Acid-faststain sputum test
• Methodology and results
• Limitations and advantages of the test
• Precautionary measures
• Other tests
• Conclusion

3. WHAT IS SPUTUM?
• Sputum is the liquid substance that is
produced from the lower respiratory tract
when one coughs.
• In addition to mucus, sputum contains many
materials that are not visible to the naked eye.
It often consists of bacteria, cellular
fragments, blood, and pus.

4. SPUTUM TEST
• Sputum test is the general test ordered for by
a doctor for respiratory tract infections such as
pneumonia (caused by Streptococcus
pneumoniae) and tuberculosis (caused by
Mycobacterium tuberculosis)
• The types of sputum test differ by the types of
stain used and the bacteria being tested for.

5. SPUTUM TEST CONTD.
• The gram stain sputum test is used for
detecting gram positive and negative bacteria
• Whiles the acid-fast stain sputum test is used
to detect acid fast mycobacteria

6. ACID-FAST STAIN SPUTUM TEST
• Certain bacterial species like mycobacterium
tuberculosis have unusual lipids (mycolic acid)
in their cell walls. This substance renders the
cell wall very waxy and impenetrable by
aqueous stain solutions.
• They therefore have to be stained bythe acid
fast stain using carbol fuschin

7. ACID-FAST STAIN CONTD.
• There are three common acid-fast staining
methods, Ziehl-Neelsen (hot), Kinyoun (cold),
and Auramine-Rhodamine Fluorochrome
(Truant method).
• The slides produced by the Ziehl-Neelson and
Kinyoun methods can be visualized using a
standard bright-field microscope.
• The fluorochrome method is used by large
laboratories that use fluorescent microscopes

8. ACID-FAST STAIN CONTD.
• In the ‘hot’ Zn technique, the phenol-carbol
fuchsin stain is heated to enable the dye to
penetrate the waxy mycobacterial cell wall.
• In the ‘cold’ technique known as Kinyoun
Method, stain are not heated but the
penetration is achieved by increasing
concentration of basic fuchsin and phenol and
incorporating a ‘wetting agent’ chemical.

9. ACID-FAST STAIN CONTD.
• The stain binds to the mycolic acid in the
mycobacterial cell wall
• . After staining, an acid decolorizing solution is
applied. This removes the red dye from the
background cells, tissue fibres, and any
organisms in the smear except mycobacteria
which retain (hold fast to) the dye and are
therefore referred to as acid fast bacilli (AFB).

10. METHODOLOGY
• Collect the sputum sample.
• Spread the sputum evenly and thinly over the
central area of the slide with a sterile pippette
by a continuous rotational movement. The
recommended size of the smear is about 20
mm by 10 mm
• Allow the smear to dry completely.
• Fix the smear at 80°C for 15 minutes or for 2
hours on a hot plate set for 65°C to 70°C.

11. METHODOLOGY CONTD.
• The preferred method of staining(Ziehl
Neelsen) is then used.
• Cover the smear with carbolfuchsin stain.
• Heat the smear until vapour just begins to rise
(i.e. about 60°C).
• Allow the heated stain to remain on the slide
for 5 minutes.
• Wash the film in a gentle stream of clean
water until no color appears in the effluent.

12. METHODOLOGY CONTD.
• Holding the slide with forceps, wash the slide
with the decolorizing solvent(3% v/v acid
alcohol or 20% sulfuric acid) . Immediately wash
with clean water, as above.
• Repeat the decolorizing and the washing until
the stained smear appears faintly pink and the
fluid washing off the slide runs clear.
• Counterstain with malachite green stain for 1-
2 minutes.

13. METHODOLOGY CONTD.
• Wash well with clean water
• Wipe the back of the slide clean, and place it
in a draining rack for smear to air dry.
• Examine the smear microscopically, using the
100x oil immersion objective and scan the
smear systematically.

14. METHODOLOGY CONTD.
• Acid-fast bacteria appear red, and non-acid-
fast bacteria (and other organisms and cellular
materials) appear blue/green.

15. Mycobacterium tuberculosis
viewed under bright field
microscopeA. B.

16. RESULTS
• When no AFB are seen after examining 300
fields, it is reported as ‘No AFB seen’.(normal)
• When very few AFB are seen i.e. when only 1
or 2 AFB are seen after examining 100 fields, a
further specimen is requested to examine
(Those AFB might have came from tap water
(saprophytic mycobacteria), or it may be
scratch of glass slide or by the use of same
piece of blotting paper while drying.

17. RESULTS CONTD.
• When any red bacilli are seen, the smear is
reported as‘AFB positive’ (abnormal) and an
indication of the number of bacteria present is
given as follows:
• More than 10 AFB/field at least in 20 fields:
report as + + +
• 1-10 AFB/field at least in 50 fields: report as
++
• 10-99 AFB/ 100 fields: report as +
• 1-9 AFB/100 fields: report the exact number

18. LIMITATIONS OF SPUTUM TEST
• High bacterial load 5,000-10,000 AFB /mL is
required for detection
• Smear sensitivity is further reduced in those
with extra-pulmonary TB, those with HIV-co-
infection, and those with disease due to
nontuberculous mycobacteria (NTM).

19. LIMITATIONS CONTD.
• Limited specificity . All mycobacteria are acid
fast .Does not provide species
identification.Local prevalence of MTB and
NTM determine the predictive values of a
positive smear for MTBMicroscopy for acid-
fast bacilli (AFB) cannot distinguish
Mycobacterium tuberculosis from NTM,Viable
from non-viable organisms,Drug-susceptible
from drug-resistant strains.

20. ADVANTAGES OF THE SPUTUM
TEST
• Microscopy of sputum smears is simple and
inexpensive, quickly detecting infectious cases
of pulmonary TB; Sputum specimens from
patients with pulmonary TB – especially those
with cavitary disease – often contain
sufficiently large numbers of acid-fast bacilli to
be readily detected by microscopy.

21. PRECAUTIONARY MEASURES
• Sputum sample should be obtained in a well
ventilated area since TB is an airborne disease.
• Sputum sample should be coughed up from
deep inside the lungs
• Tap water should not be used in washing off
the stains since it may contain saprophytic
bacteria that may induce a false positive test
• Smear slide samples should not be blotted
dry.

22. PRECAUTIONARY MEASURES
CONTD.
• If the test is not carried out immediately after
sample collection, sample may be refrigerated
after collection for up to 24hours or kept at
room temperature for about 2 hours.
• Sample should be collected early inthe
morning after brushing no antiseptic should
be used

23. OTHER TESTS
• There are a number of ways to test for
tuberculosis apart from the sputum test.
Some are:
• Chest X-ray as a TB test
• Culture test
• Biopsy
• Skin test
• Interferon Gamma Release Assay (IGRA)

24. CONCLUSION
• Checking your sputum is one of the best ways
to find out if you have TB disease. If you are
already taking medicine for TB, checking your
sputum is the best way to tell if the medicine
is working.