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Afb microscopy and quality assurance copy

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    Afb microscopy and quality assurance copy Afb microscopy and quality assurance copy Presentation Transcript

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      • SPUTUM CONCENTRATION
      • NEBULISED SPUTUM
      • BRONCHIAL WASH CENTRIFUGE
      • BRONCHOALVEOLAR LAVAGE 3000 RPM × 15MINS
      • GASTRIC L AVAGE NEUTRALISATION
          • less laborious
          • less robust
          • higher concentration fuchsin
          • longer staining time errors !!
          • NOT RECOMMENDED for low-income countries
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      • SMEAR PREPARATION
      • With applicator stick from the thick/yellowish part make 15 mm by 20 mm oval smear
      • FIXATION
      • Once dried in air pass the slides 2-3 times over flame or by sprit for 3 mins
      • STAINING
      • Cover the slide with Carbol fuschin 0.3% after filtering through Whatman filter paper no.1
      • Heat the slide until it steams and continue it for 5 minutes (don’t allow the stain to dry)
      • Wash it under tap water
      • Cover the slide with 3% acid alcohol (HCl and 95% alcohol or 20% Sulphuric acid (1 minute)
      • Counterstain with malachite green for 1 minute
      • Each lab staff must know that the reagents for staining are highly corrosive substances and must ensure personal protection
      • Efforts must me made to minimize and control lab operations that induce potentially infectious aerosols
      • Opening of container/preparing smears on slides/flaming
      • Use safety cabinet always!!
      • Sterilize the loop after smear
      • Note: Color of AFB may vary with filter system on microscope
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    • Counter Stain Background
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    • Accuracy : The closeness of measurements to the true value. Precision : The amount of variation in the measurements. Bias: The difference between the expectation of a test result and an accepted reference value.
    • Location Category False Negative False positive Pre-laboratory Administrative
      • Specimen quality
      • Specimen labeling
      • Patient identification
      • Transport conditions
      • Specimen
      • Labeling
      • Patient identification
      • Specimen container
      Laboratory Administrative
      • specimen handling
      • specimen registration
      • recording and/or reporting result
      • specimen registration
      • recording and/or reporting result
      Technical
      • smear preparation
      • stain formulations
      • staining technique
      • microscope performance
      • smear examination technique
      • smear preparation
      • stain formulations
      • staining technique
      • smear examination technique
      • What is a good sample?
        • What is saliva?
        • Good sample = yellow? mucous fluid?
        • Discharge from the bronchial tree
        • May contain solid or purulent substances
        • Minimal amounts of oral/ nasal material
        • May contain macrophages and other cells indicative of infectious disease
      • Leakage– ask for new sample
      • Mucopurulent or purulent required; NOT SALIVA
      • Purulent:thick sticky greenish
      • Mucopurulent: thick brown or green
      • Mucoid:viscous and clear
      • Mucosalivary: mucoid
      • Minimum 2 ml
      • Correct labeling
      • Smearing
        • delay: no problem (keep away from sun)
        • "good particle“ homogenization may be more reliable
        • standard size: for ease of quantification
        • thickness, evenness: find a balance
        • Sensitivity to light, counter-stain
      • Are they worth the effort?
        • NaOH digestion + centrifugation : CULTURE
        • Hypochlorite (NaOCl) digestion & concentration
          • homogenization, easy background
          • oxidation: staining easier
          • co-flocculation with proteins ??
          • concentration by sedimentation, centrifugation, filtration or flotation
        • Contradictory reports on efficiency
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      • Carbol fuchsin staining
        • Uses higher fuschsin concentration
        • Dissolve well !!!
          • IUATLD/WHO : 0.3%
        • Heat well-apply long enough
        • Cold staining!!
        • Batch staining- great potential for cross- contamination
      • Decolorization
        • must be complete
        • not possible to de-stain too much
        • repeat as needed
        • use strong acids
        • alcohol not absolutely needed
        • Provides good contrast for observation of AFB
        • background for focusing, not too strong
        • methylene blue 0.3% ?
          • diluted or < 1 min
          • use of malachite green?
        • Red slender rods on blue background
          • accept only typical shape, at least some
        • depends condition of microscope!
        • light! binocular, mechanical stage, good optics,100x oil immersion objective, 10x eyepieces
        • Requires: patience, sincerity
          • AFB microscopy is not difficult but tough
      • 1-9 AFB in 100 fields Exact number
      • 10-99 in 100 fields AFB seen +
      • 1-10 in 50 fields AFB seen ++
      • > 10 in 20 fields AFB seen +++
      • NO bacilli No AFB seen
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    • Thank you for your kind attention