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SPUTUM SMEAR MICROSCOPY DJD 2002Cebu
Sputum Smear Microscopy
in DOTS Programmes
David Dawson
WHO Collaborating Centre
The Prince Charles HospitalBrisbane Australia
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DOTS Strategy for TB Control
government commitment to ensuring sustained
comprehensive TB control activities
case detection by sputum-smear microscopyamong symptomatic patients
standardised short-course chemotherapy usingregimens of 6 to 8 months, for at least all
confirmed smear-positive cases. regular and uninterrupted supply of drugs
standardized recording and reporting systems
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The DOTS Partnership
N.T.P LABS
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TB suspects
MICROSCOPY
positive negative
NOT TB
TBcases
review
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Microscopy in DOTS
specimen collection and transport
laboratory facilities
smear preparation
Ziehl-Neelsen staining
microscopy
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Specimen
poor
poor
good
good
Lab Skill
poor
good
poor
good
Result
poor
poor
poor
good
Quality of Final Result
vs Specimen Quality vs Lab Skill
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Specimen Collection
(DOTS Programmes)
spot spot
Day 1 Day 2
early a.m.
(clinic) (clinic)(home)
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Specimen Collection and Transport
use appropriate container
ensure container is labelled, sealed
completed request form
2-5 ml sputum notsaliva
store in cool location
transport without delay
maintain specimen security
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Laboratory Facilities
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Laboratory Facilities
(for TB Microscopy)
defined, secure work area effective ventilation
electric microscope (x 1000) tidy uncluttered work bench
level staining sink microscopy bench
secure storage
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Biosafety in TB Microscopy infection risk in microscopy is LOW
smear preparation carries highest risk
effective ventilation removes aerosol
staff must be educated in biosafety
personal protective devices (?? value)
disinfectants vs MTB (phenolics)
staff health should be monitored
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TB Microscopy
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TB Microscopy
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Smear Preparation
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Lab Procedure for TB Microscopy
1. Check patient details
(specimen containervs
request form)2. Enter details in Laboratory Register.
3. Label specimen with lab ref number4. Check (and record) specimen quality.
5. Prepare smear.6. Stain, examine, report.
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QA Issues in Smear Preparation
1. Check, record, report specimen quality.
2. Keep lab register accurate, current.
3. Label slide before making smear.
4. Ensure smear prepared is representative
of the specimen.
5. Use gentle heat for fixing smear.(overheating destroys acid-fastness)
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Smear Preparation
(TB Microscopy)
adequate size 1-2 x 2-3 cm
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Smear Preparation for TB Microscopy
1. label slide 2. make smear
4. heat fix 3. air dry
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Ziehl-Neelsen Staining
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Ziehl-Neelsen Stain
General Format
1. Primary Stain (carbol fuchsin)
2. Decolouriser (acid +/- alcohol)
3. Counterstain (methylene blue)
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M tuberculosisM tuberculosis ProteusProteus spsp e.g.e.g.
carbol fuchsin
HEAT
Principle of ZN Stain - 1
complex waxy cell wall
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M tuberculosisM tuberculosis ProteusProteus spsp
carbol fuchsin
decolourise
Principle of ZN Stain - 2
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M tuberculosisM tuberculosis Proteus spProteus sp
carbol fuchsin
decolourise
Principle of ZN Stain - 3
counterstain
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M tuberculosisM tuberculosis Proteus spProteus sp
carbol fuchsin
decolourise
Principle of ZN Stain - 4
counterstain
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3% HCl in ethanolor ethanol + 5% H2SO4
or 5% H2SO4
or 25% H2SO4
ZN Decolourisation Conditions
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semi-uniform transparent film
2 cm by 1 cm
free of stain deposit, artefact
MTB stained deep red
counterstain gives contrast
Attributes of High Quality
Ziehl-Neelsen Preps
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Quality Assurance Issues
Labels on reagent bottles should show:
1. Name of reagent.
2. Preparation date.3. Where prepared and by whom.
4. Expiry date (6 months for ZN).5. Date/s of QC checks (for CF).
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Quality Assurance Issues
Labels on reagent bottles should show:
1. Name of reagent.
2. Preparation date.3. Where prepared and by whom.
4. Expiry date (6 months for ZN).5. Date/s of QC checks (for CF).
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QA Issues in ZN Staining
1. Use standard, documented method.
2. Use mycobacteria-free water for makingcarbol fuchsin.
3. Perform QC on all reagents before use.(Results of QC must be documented.)
4. Leave hot CF in contact 5-10 mins.
How often should control slides be run?
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Smear Examination
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Examination of ZN Smears
Under oil immersion, examine at
least 100, but up to 300,useful, representative fields
(minimum time 5 mins)
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Reporting ZN Results
No AFB No AFB found in 100 fields
xAFB 10 AFB per field
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Sources of Error in
Acid-fast Microscopy
False NEGATIVE poor specimen quality
faulty smear preparation
poor quality carbol fuchsin
carbol fuchsin exposure too short
technician inexperience
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False POSITIVE
AFB contamination container slide specimen carryover carbol fuchsin solution
artefact in specimen/smear
technician inexperience
Sources of Error in
Acid-fast Microscopy