This document provides an overview of Mycobacterium tuberculosis laboratory diagnosis. It discusses the general characteristics of tubercle bacilli and various diagnostic tests including microscopy, culture, and newer molecular techniques. Microscopy remains the most rapid and cost-effective method for tuberculosis diagnosis but has limitations in sensitivity. Proper sputum sample collection and quality are important for maximizing diagnostic yields from microscopy and culture.

1. MYCOBACTERIUM : DIAGNOSIS
AND LABORATORY TEST
9/4/2013
1
Dr. Azura Hussin
Pathology Department
HRPZ II

2. Contents
9/4/2013
2
 Introduction
 General characteristics of tubercle bacilli
 Lab diagnosis of tuberculosis
 - current technologies
 - newer technologies
 - problems in lab diagnosis
 - quality control
 Summary

3. Introduction
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3
 Tuberculosis appears to be a disease as old
as human history
 Tuberculosis is a social disease with
medical implications and it is a major public
health problem
 It remains the leading cause of death by
infectious disease.
 Tubercle bacillus discovered - more than a
hundred years ago
 First discovered in 1882 by Robert Koch -
"Koch's bacillus

4. Introduction
12/09/14
4
 According to the WHO, there are approximately
20 million active cases in the world today, and
they infect 50-100 million people largely children
annually.
 The mortality is approximately 3 million annually
(at least 80% from developing countries)
 Malaysia, the number of cases detected per year
has not declined substantially either.
 Since 1989, 11,500 to 12,000 cases per year
were detected
 In 1995, 11,778 cases detected of which 6,500
cases are sputum positive and therefore are
infectious.

5. 9/4/2013
5
 Why so high?
 HIV/AIDS
 Emergence of drug resistance TB
 Migration
 ? Early diagnosis of tuberculosis
 initiating optimal treatment - enable a cure
 curb the transmission of infection and
disease to others in the community.

6. 9/4/2013
6

7. 9/4/2013
7

8. DIAGNOSIS
 Clinical :
- Symptoms (prolonged cough, fever, night sweats, LOA / W) and
- Signs (can be subtle as in minimal cases or obvious such as
consolidation, fibrosis or stony dullness due to pleural effusion)
 Radiological and/ or
 Bacteriological evidence
 The tuberculin or Mantoux test
12/09/14
8

9. 9/4/2013
9
 TB is diagnosed by finding My c o ba c te rium
tube rc ulo s is bacteria in a clinical specimen
taken from the patient.
 Other investigations may strongly suggest
tuberculosis as the diagnosis, they cannot
confirm it.
 A definitive diagnosis of tuberculosis can only be
made by culturing My c o ba c te rium tube rc ulo s is
organisms from a specimen taken from the
patient (most often sputum, but may also include
pus, CSF, biopsied tissue, etc.).
 A diagnosis made other than by culture may only
be classified as "probable" or "presumed".

10. Transmission
10 9/4/2013

11. Lineage of the agents of Mycobacterium
Kingdom Bacteria
Phylum Actinobacteria
Class Actinobacteria
Subclass Actinobacteridae
Order Actinomycetales
Suborder Corynebacterineae
Family Mycobacteriaceae
Genus Mycobacterium
unique genus
Species M. tuberculosis
M. bovis
M. africanum
M. microti
"M. canettii”
M. caprae
M. pinnipedii
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12. Intro duc tio n - Members of Genus My c o ba c te rium
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13. Mycobacterium
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13
 Mycobacterium Tuberculosis Complex
(MTC)
 species are closely related genetically
 differ in host, geographic range, certain
phenotypes and pathogenicity.
 Among MTC – Mycobacterium Tuberculosis
(MTB) is the most significant pathogen for
human.

14.  Caused by a bacteria -
Mycobacterium tuberculosis
 Rod shaped – slender or
slightly curve
 Size : 2-4 um x 0.2-0.5 um
 Non-motile
 Non-spore forming
 Obligate aerobe - lung
 Intracellular parasite
(monocytes/macrophages)
 Weak Gram positive
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Grows slowly (15-20 hrs)
Optimum T : 37 C
Colonies take 4-6 weeks to become
visible on LJ media.
can withstand weak disinfectants and
can survive in a dry state for weeks

15. 9/4/2013
15
 Stains poorly by Gram
Stain-”Ghosts”
 Use heated Ziehl-Neelsen Carbol-fuchsin
 Resists decolorizing by Acid-Alcohol
→ Acid-Fast

16. Colony Morphology
 Growth on LJ medium,
 colonies are rough, flat
with a raised centre,
wrinkled
 buff coloured, younger
colonies are paler
 Organisms tends to
grow in parallel groups
producing the colonial
characteristics of
serpentine coding
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17. 17
# Colonies of Mycobacterium tuberculosis on Lowenstein
Jensen medium. Colonies are small and buff coloured.
# In vitro-grown colonies often form distinctive serpentine
cords. This observation was first made by Robert Koch who
associated cord factor with virulent strains of the bacterium.
# It takes 4-6 weeks to get visual colonies 9/4/2013

18. Gram-negative organisms Mycobacteria
MYCOLATE
arabinogalactan
acyl lipid + LPS
lipids lipid bilayer peptidoglycan
lipid bilayer
Mycobacteria produce a thick mycolate-rich outer covering which
functions as an exceptionally efficient barrier.
Cell wall
Unique cell wall
More than 60% lipid
Mycolic acids are hydrophobic
Cord Factor is toxic to mammalian cells
9/4/2013 inhibits migration of Polymorphic Neutrophils
18

19. Mycobacterium Cell Wall
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 High concentration of lipids cause:
Impermeability to stains/dyes
Resistance to many antibiotics
Resistance to kill by acidic and
alkaline environments, even
intracellularly
Resistance to osmotic lysis by
Complement Fixation
Resistance to lethal oxidants

20. MMaakkmmaall aaddaallaahh kkoommppoonneenn
uuttaammaa ddaallaamm --TTBB CCoonnttrrooll
TIADA MAKMAL
TIADA DIAGNOSIS
TIADA PENGUBATAN
TIADA DOTS
TIADA TB CONTROL20 12/09/14

21. PPeerraannaann MMaakkmmaall
• Mengesan penyakit berjangkit
• Memantau kesan perawatan
• Memastikan kesembuhan pesakit
21 12/09/14

22. Network Of TB Laboratory
(Organization of TB Laboratory Services)
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District Hospitals
General Hospitals (State)
Institute of Respiratory Medicine
(MKAK Sungai Buloh)
Specimen collection centers/
some are examination centers
Examination centers (D/S)
Examination centers (D/S)
AFB Culture centers
TB Reference Laboratory
D/S, C/S, ID, BCG Viability
Tests
Data compilation
Training / Set procedures
Health Center

23. Laboratory diagnosis
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23
 There are numbers of diagnostic tests :
 simple AFB microscopy to complex molecular
techniques;
 The diagnostic modalities desirable features:
 sensitivity,
 specificity,
 reproducibility,
 cost effectiveness,
 safety,
 simplicity and easy application for wider use
 Quantitative - so that the infectiveness of the
individual cases can be measured

24. Specimen Collection - TB
24
What is a good sample and how to obtain it?
 Best specimen comes
from the lung.
 Saliva or nasal
secretions are
unsatisfactory.
 Collect in open
space- not toilets /
dark unventilated
rooms
 Remove dentures and
rinse mouth with
water.
 Inhale deeply 2–3
times, breathe out
hard each time.
 Cough deeply from
the chest.
 Place the open
container close to
the mouth to collect
the specimen.
9/4/2013

25. Samples for TB smear and
culture  Types of samples
 Sputum*
 CSF
 Gastric washing*
 Lymph nodes
 Tissues
 Stool
 urine
* Induction sputum : smear
negative / unable to produced
sputum
* Gastric lavage/Bronchoscopy
: may considered in unsuitable
sputum induction
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Number of samples
At least 2 or 3 fresh
purulent samples from lower
respiratory tract collected as
spot – morning - spot Transportation
Ideally within one
working day

26.  Three samples
required (Different
day):
Clinic patient:
Spot– morning –
spot
Hospitalized
patient: 3 morning
specimens (better)
Yield increases
rapidly after three
specimens
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Examples of containers

27. Reduction of number of smears for the
diagnosis of pulmonary TB, 2007
WHO recommends the number of specimens to
be examined for screening of TB cases
Can be reduced from three to two,
 in places where a well-functioning external quality
assurance (EQA) system exists,
 where the workload is very high and
 human resources are limited.
12/09/14
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28. Tests offered - KKM
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 Smears microscopic – ZN / IF
 Culture, sensitivity and identification
 PCR
 Line Probe Assay

29. Quality sample = Quality smear
Quality smear = Quality lab result
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30. Specimen Quality
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Saliva/ Induced Sputum Blood Stained

31. Specimen Quality
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Purulent Mucoid

32. Contents of a Sputum Specimen
Sputum cup
Saliva (soluble in
saline)
Mucous (from
trachea &
bronchus)
Matters from
pulmonary lesion
(++++ bacilli)
Cheesy
Composition of a good -yellowish
Sputum Specimen
32 9/4/2013

33. Distribution of AFB in the
sputum specimen
Quality of Sputum % AFB
SALIVA 4.9
SALIVA + MUCUS 7.7
MUCOPURULENT 19.2
PURULENT 39.1
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34. 9/4/2013
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SMEAR EXAMINATION
Z-N Stain
Fluorescent Stain
Light Microscope
Specimen
Fluorescent Microscope

35. Systematic Examination of
Smears
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3 cm
2 cm

36. Uniform Smear Size
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37. Why We Do Sputum Smear
Examination?
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 Diagnosis
-sign and symptoms
- Monitoring
 Positive sputum –smear = Infectious case
 This in not a sensitive technique- but rapid and
specific

38. OBSERVATION OF STAINED SMEAR
-International Standard-
 Examine the smear
under x100 objective
with the 10 eye piece
lens
 Read at least 300 visual
fields to give a report a
negative
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38
1
2
3 cm = 150 visual field

39. Microscopy
39  the simplest and most rapid procedure
currently available to detect AFB in clinical
specimens by ZN staining method.
9/4/2013

40. Microscopy
9/4/2013
40 Albert e t a l., 2002.
 Sensitivity: 61.3-63.4%
 Highest – patients with cavitary disease
 Lowest – patients with weak cough / less
advanced disease
 Specificity: 97.3-97.4%
 AFB smear microscopy in symptomatic
patients has high specificity
(98%)
 Concentration of sputum sample by
cytocentrifugation has been found to enhance
the sensitivity to almost 100%.

41. Microscopy - limitation
9/4/2013
41
 impossible to distinguish different
mycobacterial species.
 Sputum : 5 000 – 10,000 tubercle bacilli/ml –
smear positive.
 Smear negative on expectorated sputum
→ Cannot rule-out TB (ie extra pulmonary TB)
 Smear negative/ unable to produce sputum:
 Sputum induction
 Fiberoptic bronchoscopy
 Gastric washing

42. Good Quality Staining of AFB
9/4/2013
42

43. AAFFBB iinn SSiinnggllee AArrrraannggeemmeenntt
43 9/4/2013

44. AFB - in Various Arrangements
9/4/2013
44

45. AAFFBB iinn CClluummppss
45 9/4/2013

46. AFB Stain-LED Fluorescence Microscope
46 12/09/14

47. LED Fluorescent microscope
 Advantages:
 Covers 15 times as many fields
compared to ZN
 Same area can be covered at
a shorter time
 Examination of slides for 1.41
min by FM equivalent to 2.48
min by ZN
 Heating not required
 More sensitive - low bacillary
content
 Increase sensitivity of
conventional light microscope
of about 10%
 Less expensive ( maintenance,
dark room)
 Disadvantages:
 Handling & maintenance of
optical equipment require
advanced skill
 Periodic replacement of
bulbs
 Continuous supply of power
 High cost of microscope and
maintenance
 Doubtful smears to be re
examined by ZN
12/09/14
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48. METHOD ADVANTAGES DISADVANTAGES
Techniques;
Ziehl-Neelsen
Kinyoun
Flourochrome
 Rapid
 Simple/Easy to read
 Specificity >98%.
 Minimal infrastructure
required to set up
 Indicator of infectious
case
 Treatment
monitoring
 Retrospective
checking – cultures
growing are AFB
 Tedious
 Less sensitive
(45-60%)
(>100,000 bacilli
/ml)
 Trained &
experience
microscopist
 Presumptive
diagnosis of
mycobacterial dz
 Can’t
differentiate
between species
 Can’t be use for
DST
Microscopy
48 9/4/2013

49. Culture
 Advantages
For susceptibility
testing
Able to identify the
species of
mycobacterium
9/4/2013
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Disadvantages
Slow grower : delay in
the lab diagnosis /
treatment
Laborious
Experience staff
Special equipment
 Expensive

50. 9/4/2013
50
Culture should be attempted
1. For Surveillance of drug resistance as an
integral part of evaluation of control program
2. Smear negative pulmonary & extra
pulmonary
3. Childhood tuberculosis
4. Follow up of Tuberculosis cases who fail to
respond to standard treatment
5. Investigations of high risk individuals who
are symptomatic
– HIV +ve cases
– History of exposure to MDR cases
– Laboratory workers

51. CULTURE
Mycobacterial culture can be
performed on:
 Conventional : egg
based solid medium
 Lowenstein-Jensen
medium
 Ogawa medium
 Automated : Liquid
based medium
 Kirschner™s or
Middlebrook 7H9 broth.
 Middle brook 7H12
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BACTEC MGIT 960 system
BacT/Alert 3D
VersaTREK

52. Automated Mycobacteria Culture System
9/4/2013
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1. Mycobacteri
a Growth
detection.
2. Antimicrobi
al
Susceptibilit
y Testing
(AST)

53. My c o ba c te rium tube rc ulo s is colonies on Middle Brook Agar.
9/4/2013
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54. 9/4/2013
54

55. BIOCHEMICAL IDENTIFICATION FOR
MYCOBACTERIUM TUBERCULO SIS
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Biochemical Test Result
1 Niacin Test Positive
2 Catalase 68º C/pH 6.8 Negative
3 Nitrate Reduction Test Positive

56. 9/4/2013
56
Nitrate Reduction Test
Catalase Test 68ºC/pH 7

57. New technologies - Molecular techniques
9/4/2013
57
 Conventional PCR
 Real time PCR – direct detection from
samples
# identification / sensitivity
# detecting MDR / mutants gene
associated with antibiotic resistant
 Detection and identification of
mycobacteria directly from clinical
samples - an alternative for smear
microscopy

58. 9/4/2013
58
 Line Probe Assay (LPA) - can be used to
screen smear-positive sputum specimens for
resistance to rifampicin and isoniazid in 1-2
days.
 has the potential to substantially reduce the
turnaround time of DST results.
 training, supervision and adherence to
stringent laboratory protocols to ensure high
quality results during routine implementation

59.  Tests take only hours to perform and may provide
a rapid diagnosis, within 1-2 days of receipt of a
sputum sample
 Cannot replace culture and drug sensitivity tests
 Too expensive to be use routinely
 sensitivity : < 60% - > 95%
 <60% in smear negative (but positive for TB
culture)
 > 95% samples acid fast smear positive sputum
samples,
12/09/14
59

60. 9/4/2013
60

61. 9/4/2013
61

62. Lane 1: DNA Ladder Marker
Lane 2: Sample 1
Lane 3: Sample 2
Lane 4: Sample 3
Lane 5: Sample 4
Lane 6 Sample 5
Lane 7: PCR Pos Con
Lane 8: PCR Neg Con
Lane 9: Pos Ext Con
Lane 10: Neg Ext Con
9/4/2013
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63. Serological diagnosis of
Tuberculosis
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63
 Should allow a rapid diagnosis, simple, cost
effective
 Most of the serological tests :
 low turn around time,
 high negative predictive value and are useful as
screening tests.
 Variable or low sensitivity :
o 76% in smear positive cases, 59% in smear
negative cases, 64% in LN TB and 46% in pleural
TB
 expensive, require trained personnel and often
have difficulty in distinguishing between
M. tube rc ulo s is a nd NTM.
WHO (2011) : Patient safety- commercial
serological assay should not be used to diagnose
pulmonary or extra pulmonary TB

64. Some commercially available antibody tests for
diagnosis of pulmonary TB
9/4/2013
64
Name of the assays Antigen used
MycoDot (Dot-blot) Lipo arabino mannan
(LAM)
Detect-TB (ELISA) Recombinant protein Peptide
Pathozyme Myco
38 kDa (recombinant Ag)
(ELISA)
and LAM
Pathozyme TB(ELISA) 38 kDa (recombinant)
Antigen A60 (ELISA) Antigen -60
ICT diagnostics
38 kDa (recombinant)
(membrane based)

65. Serological test - FOR LTBI
Lancet 2006;367:1328-1334.
9/4/2013
65
 Blood tests : T-SPOT.TB , QuantiFERON-TB Gold
 393 consecutive with suspect TB had both tests –
blood and skin test
Positive result:
 T-SPOT.TB of 38% vs QuantiFERON-TB Gold 26%
(p < 0.0001).
 Both blood tests showed similar overall agreement
with the skin test
 Blood tests were better than the skin test in
distinguishing BCG-vaccinated individuals from
those with true positive results.
Indeterminate results
 11% with QuantiFERON-TB Gold
 3% with T-SPOT.TB (p < 0.0001).

66.  Detection of
activated T-cells –
producing gamma
interferon
 enable more people
to be diagnosed and
treated while their
infection is still
dormant / immune
deficient
 ? thus a powerful
new tool to help
health authorities
curb and control the
TB epidemic
 faster (results within
24 hours)
 detecting antibodies
induced by an
infection
 Painful, scarring
 False positive :
- exposure / contact
with TB case
- Post vaccination
 3- 7 days to read the
induration
9/4/2013
66
TB Spot-TB
Skin Test

67. High sensitivity (~90%) has been shown in culture confirmed TB patients
Specificity is also very high (>98%) even in BCG vaccinated individuals.
67 9/4/2013

68. Additional procedure /diagnostic
tests
 Extra pulmonary TB : histology or cytology
 TB lymphadenitis : FNA – cytology
 Pleural TB : thoracoscopy – histology/culture/
Adenosin Deaminase (ADA)
 TB meningitis : PCR / ADA
9/4/2013
68

69. Identification
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69
Only done in MKAK Sg. Buloh
Conventional
NAAT ( Nucleic acid amplification
test)
Hybridisation gene probe

70. QC
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70
 Internal QC –
 staining materials
 External QC
Slides rechecking
 Within same lab
 Within labs from same district / state
MKAK Sg. Buloh
RCPA Australia

71. Problems in Lab. Dx
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71
 The bacteria – slow growers
 Sample quality / criterion
Wrong technique – STM not available
 Poor staining material
 Staff competency & attitude
 Equipment problem – microscope

72.  Poor samples
 Improper
techniques
 Number of
organisms too
small
 Partially
suppressed by
antibiotics
 Mycobacterial
contamination of
water or
solutions
 Transfer from
+ve smears
during staining or
examination
 Staining artefacts
 Insufficient
destaining of non
AFB organisms
 Other Acid fast
orga9n/4/i2s01m3 s
72
False negative False positives

73. Impact of smear negative TB
Delays of up to 2-3 weeks in starting anti-
TB treatment is common
•Late intensive care admissions
•In-hospital mortality
•Nosocomial transmission
•Inappropriate treatment
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74. Tuberculosis Reference
Laboratory
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74
1. National Public Health Laboratory,
Ministry of Health Malaysia,
Sungai Buloh, Selangor.

75. Role of TB Reference
Laboratory
9/4/2013
75
 To perform DST and Identification tests
for AFB isolated from peripheral Laboratory
 Standardization of Lab Techniques-
 To conduct training to technical staff (MLT)
 Compilation and monitoring data

76. Why Drug Susceptibility Tests (DST) ?
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76
DST done for 3 main purpose;
I. As guidance in the choice of the first
course of chemotherapy.
II. To confirm emergence of drug resistance in a patient who
failed to show bacteriological response to treatment and
may guide the choice of further course.
III. May be employed to estimate prevalence of ;
- Primary Drug Resistance &
- Acquired Drug Resistance in the community

77. 77 9/4/2013

78. Reporting
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78
 TAT for slide smear shall be 24
hours from the time of specimen
receipt (to be reviewed later)
 TAT for identification : 3 days after
specimen receipt at MKAK Sg Buloh
 TAT for sensitivity :
 preliminary – 17 days
 final – 31 days

79. WHO Quantification scale Ziehl
Neelsen
Number of AFB Number of fields*
examined
What to report
fields 300 fields No Acid Fast Bacilli seen
No AFB in 300
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 +
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* Oil immersion fields

80. 80 9/4/2013

81. 81 9/4/2013

82. 82 9/4/2013

83. 83 9/4/2013

84. KELEMAHAN
PEWARNAAN
9/4/2013
84

85. Penyelengaraan Mikroskop yang baik
12/09/14
85

86. Slide artefact -- EExxcceessssiivvee HHeeaatt
FFiixxiinngg ooff SSmmeeaarr
86 9/4/2013

87. SSlliiddee aarrtteeffaacctt -- GGllaassss SSlliiddee
SSccrraattcchh aanndd AAFFBB
87 9/4/2013

88. SSoooott DDeeppoossiitt oonn UUnnddeerrssiiddee ooff
SSmmeeaarr
88 12/09/14

89. What Are the Causes of
Pale Staining AFB?
12/09/14
89

90. PPaallee SSttaaiinniinngg AAFFBB
90 12/09/14

91. Causes of Pale Staining AFB
 Concentration of CF <0.3%:
 Staining time less than 5 minutes
 Inadequate heating of carbol fuchsin on the
slide
 Excessive exposure time with an acid
alcohol decoloriser may remove the carbol
fuchsin from the AFB
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91

92. Time Effect of Carbol Fuchsin on Intensity of AFB
2 min
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92

93. 3 min
93 12/09/14

94. 5 min
94 12/09/14

95. Effect of NNoott HHeeaattiinngg tthhee CCaarrbbooll
FFuucchhssiinn ttoo SStteeaammiinngg
95 12/09/14

96. What Are the Causes of Excessive
Counterstaining ?
 Leaving counterstain on slide for longer
than one minute
 Methylene blue concentration > 0.3%
12/09/14
96

97. GGoooodd QQuuaalliittyy CCoouunntteerrssttaaiinn
97 12/09/14

98. EExxcceessssiivvee CCoouunntteerrssttaaiinn
98 12/09/14

99. Effect of Excessive CCoouunntteerrssttaaiinn--11
c
Nuclei too dark
Background too strong
99 12/09/14

100. Effect of Excessive CCoouunntteerrssttaaiinn--22
Background too strong
100 12/09/14

101. CCoommbbiinnaattiioonn ooff EExxcceessssiivvee
CCoouunntteerrssttaaiinn && PPoooorrllyy SSttaaiinneedd AAFFBB
101 12/09/14

102. What Are the Causes of
Insufficient Decolorisation?
12/09/14
102

103. AAsssseessssmmeenntt ooff SSmmeeaarr QQuuaalliittyy
Uneven smear preparation
and insufficient
decolorisation
103 12/09/14

104. IInnssuuffffiicciieenntt DDeeccoolloouurriissaattiioonn
104 12/09/14

105. IImmppoorrttaannccee ooff CCoorrrreecctt
DDeeccoolloouurriissaattiioonn
Insufficient Correct
105 12/09/14

106. Summary
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106
 Tuberculosis still stands as the most important
infectious disease in humans despite of the
advances in treatment.
 Early and accurate detection of active cases
remains an important objective for improved
implementation of chemotherapy and for reduction
in the spread of the disease.
 The identification of the mycobacteria will depends
on the quality of sample and the lab techniques
 Several limitations to the traditional techniques
used.
 New techniques / diagnostic tests : to improved
the diagnosis and shortened the testing time in the
laboratory.

107. 107 9/4/2013