This document provides guidance on examining stained sputum smears under a microscope to detect acid-fast bacilli (AFB) and report tuberculosis (TB) infection status. It describes the proper technique for examining smears at 100x magnification by observing at least 300 fields. Smears should be graded based on the number of AFB observed per field as no AFB, scanty (1-9 AFB/100 fields), 1+ (10-99 AFB/100 fields), 2+ (1-10 AFB/50 fields), or 3+ (more than 10 AFB/20 fields). Inaccurate grading can lead to false negatives or false positives, impacting patient treatment and epidemiological analysis

1. MICROSCOPY SMEAR
EXAMINATION
RECORDING & REPORTING

2. CCoonntteenntt OOvveerrvviieeww
•Required materials
•Reading the smear
•Appearance of AFB
•Malaysia grading scale
•WHO/IUATLD grading scale
•Cleaning of objectives
•Storage of smears

4. SSyysstteemmaattiicc EExxaammiinnaattiioonn
ooff SSmmeeaarrss
2 cm
3 cm

5. OOvveerrvviieeww ooff MMiiccrroossccooppyy
• Place smear facing upwards onto stage
• Focus using 10x objective
• Apply immersion oil and focus 100x
objective
• Examine at least 300 fields
• Quantitate AFB
• Record and report results
• Store all smears

6. SSttaaiinneedd SSmmeeaarr -- 11

7. AAFFBB iinn SSiinnggllee AArrrraannggeemmeenntt

8. AAFFBB -- iinn VVaarriioouuss AArrrraannggeemmeennttss

9. AAFFBB -- iinn VVaarriioouuss AArrrraannggeemmeennttss

10. EExxcceessssiivvee HHeeaatt FFiixxiinngg ooff SSmmeeaarr

11. GGllaassss SSlliiddee SSccrraattcchh aanndd AAFFBB

12. WWhhaatt iiss tthhee iimmppoorrttaannccee ooff SSmmeeaarr
ggrraaddiinngg ??
-To know the disease status
-Quality Control

13. AApppplliiccaattiioonn ooff OOiill

14. OBSERVATION OOFF SSTTAAIINNEEDD SSMMEEAARR
(oolldd MMaallaayyssiiaann ssttaannddaarrdd))
3 cm = 100 visual field
•Examine the smear
under x100 objective
with the 10 eye piece
lens
•Read at least 300 visual
fields to give a report a
negative

15. RReeccoorrddiinngg aanndd rreeppoorrttiinngg
-- mmaaxxiimmuumm 5500 bbaacciillllii iiss ccoouunntteedd
Old Malaysian Standard
If 50 or more bacilli found in the first line > 50 /L
11 to 49 bacilli seen at the end of first line
 Write the actual no. of AFB seen
35/L, or
11/L
If 50 bacilli seen in any part of the slides 50/ >L
Number of bacilli seen after 3 lines
examined
 Write the actual no. of AFB seen
3/3L or
49 /3L
No AFB seen 0/3L

16. OOBBSSEERRVVAATTIIOONN OOFF SSTTAAIINNEEDD SSMMEEAARR
--IInntteerrnnaattiioonnaall SSttaannddaarrdd--
3 cm = 150 visual field
•Examine the smear
under x100 objective
with the 10 eye piece
lens
•Read at least 300 visual
fields to give a report a
negative

17. Number of AFB Number of fields*
examined
What to report
No AFB in 300
fields 300 fields No Acid Fast Bacilli seen
1–9 AFB in 100
fields 100 fields
Record exact figure
(1 to 9 AFB per 100 fields)
10– 99 AFB in
100 fields 100 fields
1 +
1– 10 AFB in
each field 50 fields
2 +
More than 10
AFB in each
field
20 fields
3 +
* Oil immersion fields

18. RReeccoorrddiinngg aanndd
RReeppoorrttiinngg
Interpretation of Smears

19. SSmmeeaarr CCoonnttaaiinnss NNoo
AAFFBB iinn 330000 FFiieellddss wwiillll
bbee ggrraaddeedd aass
No AFB Seen

20. 00 -- NNoo AAFFBB sseeeenn iinn 330000 vviissuuaall
ffiieellddss
1VF 2VF 3VF 4VF................................ 148VF 149VF 150VF

21. 11--99 AAFFBB sseeeenn
iinn110000 VVFF wwiillll
bbee ggrraaddeedd aass
Scanty

22. Scanty - 1-9 AFB seen in 100 VF
(Write the actual number of AFB seen, e.g. 1/100VF,
3/100VF…9/100VF)

23. Scanty - 1-9 AFB seen in 100 VF
(Write the actual number of AFB seen, e.g. 1/100VF,
3/100VF…9/100VF)
7/100VF

24. AA ssmmeeaarr wwiitthh 1100
ttoo 9999 bbaacciillllii iinn
110000 ffiieellddss wwiillll bbee
ggrraaddeedd aass 1+

25. 11++ -- 1100--9999 AAFFBB sseeeenn iinn 110000 VVFF
1+

26. 11++ -- 1100--9999 AAFFBB sseeeenn iinn 110000 VVFF
1+

27. AA ssmmeeaarr wwiitthh 11
ttoo 99 bbaacciillllii iinn aatt
lleeaasstt 5500 ffiieellddss
wwiillll bbee ggrraaddeedd
aass 2+

28. 22++ -- 11--1100 AAFFBB//OOIIFF iinn aatt lleeaasstt
5500 VVFF
1VF=3 2VF=1 3VF=2................ 49VF=3 50VF=4
2+

29. AA ssmmeeaarr wwiitthh
mmoorree tthhaann 1100
AAFFBB//OOIIFF iinn aatt
lleeaasstt 2200 ffiieellddss
wwiillll bbee ggrraaddeedd aass
3+

30. 33++ -- MMoorree tthhaann 1100 AAFFBB//OOIIFF iinn aatt lleeaasstt 2200
VVFF
3+

31. RReeccoorrddiinngg RReessuullttss

32. CClleeaanniinngg tthhee OObbjjeeccttiivveess

33. HHooww ttoo ssttoorree tthhee sslliiddeess??

34. RReemmoovvee OOiill ffrroomm SSmmeeaarrss

35. SSttoorraaggee ooff SSmmeeaarrss

36. WWhhaatt wwiillll hhaappppeenn wwhheenn tthhee
ssmmeeaarr rreessuullttss aarree rreeppoorrtteedd
iinnccoorrrreeccttllyy ??

38. These variations can be minimized
by;

39. MMAAIINN CCAAUUSSEESS OOFF FFAALLSSEE RREEAADDIINNGGSS IINN SSMMEEAARR
PPRREEPPAARRAATTIIOONN
Check point Causes False Positive
F(+)
False Negative
F(-)
Size Too big
Too small
**
Evenness Uneven
Sloughed-off
**
Thickness Too thick
Too thin
**
Cleanliness Dirt
Artifact
**
*
Sputum quality Saliva *
Staining Overheating
Insufficient heating
Under decolourization
*
*
***

40. FFaallssee NNeeggaattiivveess aanndd
CCoonnsseeqquueenncceess

41. FFaallssee PPoossiittiivveess aanndd
CCoonnsseeqquueenncceess

42. EXERCISE
GGrraaddee tthhee FFoolllloowwiinngg
SSmmeeaarrss::

43. SMEAR 1

44. GGrraaddiinngg--11
66//110000VVFF

45. SSmmeeaarr--22

46. GGrraaddiinngg--22
11++

47. SSmmeeaarr-- 33

48. GGRRAADDIINNGG--33
2+

49. SSmmeeaarr--44

50. GGRRAADDIINNGG--44
88//110000VVFF

51. SSmmeeaarr--55

52. GGrraaddiinngg--55
33++

53. SSmmeeaarr--66

54. GGrraaddiinngg--66
22++

55. SSmmeeaarr--77

56. GGrraaddiinngg--77
11++

57. SSmmeeaarr--88

58. GGrraaddiinngg--88
11++