Acidfast Smear Microscopy Details of Techniques

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Acid-fast Smear Microscopy Details of Techniques
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Acid-Fast Organisms

• Primary stain binds cell wall mycolic acids
• Intense decolorization does not release primary
  stain from the cell wall of AFB
• Color of AFB-based on primary stain
• Counterstain provides contrasting background

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Acid-Fast Stains

• Early diagnosis of mycobacterial infections
• Confirms acid-fast nature of organisms
• Monitor patients on antimycobacterial therapy
• May be a determinant for performing other tests
  such as culture or susceptibility testing

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AFB Microscopy Techniques

• Two basic techniques same basic principle
• Transmitted light Carbol fuchsin staining
• Contrast red color AFB/ on blue background
• Fluorescence auramine staining
• contrast light/dark

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Basic Fuchsin AFB Stains

• Zeihl Neelsens-hot stain
• Kinyouns-cold stain
• Modifications
• Most of this discussion will be spent on these
  techniques

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Fluorochrome AFB Microscopy

• Place of fluorescence in low-income ?
• more rapid and sensitive
• specificity same with sufficient experience
• equipment cost , bulbs, technical demands
• for busy labs (gt 25 slides daily)
• External quality assessment should be done if
  this method is performed
• Performing EQA PT and rechecking is a challenge

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Fluorescence and Bright-field Microscopy
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Fluorochrome AFB Microscopy

• Primary fluorochrome AFB fluoresces
• Auramine O Green
• Auramine O-Rhodamine B Yellow/orange
• Acridine Orange Yellow/orange
• Note Color of AFB may vary with filter system
  on microscope

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Auramine Stain
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Fluorochrome AFB Microscopy
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Auramine with Acridine Orange Counter stain
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Bright-field Techniques

• Hot Ziehl-Neelsen in practice most reliable
• more visible AFB
• stronger color
• Cold methods  Kinyoun, Tan Thiam Hok
• less laborious but also less robust
• higher concentration fuchsin, longer staining
  time
• errors !!
• not recommended for low-income countries

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Sputum Sample

• 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
• Follow-up examination samples?

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Preparation of AFB Smears

• 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
• light conditions, counter-stain

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Sputum Concentration Methods

• 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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Bleach Concentration Studies

• REFERENCE SM / CULT POS. SMEARS
• DIRECT BLEACH DIRECT BLEACH
• GEBRE 1995, centrif. 31 69
  125
• MIORNER 1996, sedim.
  25
• ALLWOOD 1997, centrif. 43 52
  10.5 14.8
• WILKINSON 1997, centrif. 43 44
  12.7 12.4
• ÄNGEBY 2000, centrif. 57 65
  9 12.9
• VAN DEUN 2000, sedim. 15.5
  16.6

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Danger of Cross Contamination

• If tubes re-used
• Contamination (water for dilution)
• Need a centrifuge
• Work specimen preparation or reading ?
• Useful with HIV ?

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Fixation of AFB Smears

• Fixation
• may kill some bacilli
• makes smear stick to slide
• by heat or alcohol
• do not overheat
• safe smear ?

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Primary Staining-ZN

• Carbol fuchsin staining
• Uses higher fuchsin concentration
• Dissolve well !!!
• IUATLD/WHO 0.3
• references??
• Heat well-apply long enough
• Cold staining!!
• Batch staining- great potential for cross-
  contamination

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Decolorization of Smears

• Decolorization
• must be complete
• not possible to de-stain too much
• repeat as needed
• use strong acids
• alcohol not absolutely needed

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Counter-staining

• Provides good contrast for observation of AFB
• background for focusing, not too strong
• methylene blue 0.3 ?
• diluted or lt 1 min
• use of malachite green?

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Microscopic Reading

• 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

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Recording and Interpretation

• follow NTP instructions no. of fields, sputa
• quantification2 cm 1 length 100 HPF
• IUATLD/WHO scale

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Zeihl Neelsen and Fluorochrome MicroscopyAFB
Quantification Scales