The document provides instructions for sputum smear microscopy for acid-fast bacilli (AFB) detection. It describes sample collection and processing including centrifugation. Methods for smear preparation, staining using carbol fuschin and decolorization are outlined. The importance of laboratory safety, quality assurance, and the role of microscopy in diagnosis are briefly discussed.

7. SPUTUM CONCENTRATION NEBULISED SPUTUM BRONCHIAL WASH CENTRIFUGE BRONCHOALVEOLAR LAVAGE 3000 RPM × 15MINS GASTRIC L AVAGE NEUTRALISATION

8. less laborious less robust higher concentration fuchsin longer staining time errors !! NOT RECOMMENDED for low-income countries

10. 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

11. 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

12. Note: Color of AFB may vary with filter system on microscope

14. Counter Stain Background

20. 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.

21. 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

22. 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

23. 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

24. 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

26. 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

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

28. 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?

29. 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

30. 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

38. Thank you for your kind attention