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Acid fast staining SOP

The document outlines the acid-fast staining procedure used to detect Mycobacterium tuberculosis, detailing the purpose, method, performance characteristics, and types of samples required. It includes patient preparation guidelines, necessary equipment, quality control measures, and potential sources of variation. Results are reported as positive or negative for acid-fast bacilli, with specific instructions for interpretation and reporting.

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40 views

Acid fast staining SOP

The document outlines the acid-fast staining procedure used to detect Mycobacterium tuberculosis, detailing the purpose, method, performance characteristics, and types of samples required. It includes patient preparation guidelines, necessary equipment, quality control measures, and potential sources of variation. Results are reported as positive or negative for acid-fast bacilli, with specific instructions for interpretation and reporting.

Uploaded by

dmanneme
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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ACID FAST STAINING

1. PURPOSE OF EXAMINATION: The purpose of acid-fast staining is to detect and identify Mycobacterium
tuberculosis and other acid-fast bacilli in clinical specimens.

2. PRINCIPLE & METHOD:


 The principle of acid-fast staining is based on the ability of acid-fast bacilli to resist decolorization by
acid-alcohol.
 The method used is the Ziehl-Neelsen stain.

3. PERFORMANCE CHARACTERISTICS :
 Sensitivity: ≥ 50% for sputum smears and ≥ 80% for tissue sections.
 Specificity: ≥ 95%.
 Positive Predictive Value (PPV): dependent on prevalence of tuberculosis.
 Negative Predictive Value (NPV): ≥ 95%.

4. TYPE OF SAMPLE:
 Sputum.
 Tissue sections.
 Other clinical specimens (e.g. urine, stool).

5. PATIENT PREPARATION:
 Patients should be instructed to provide a sputum specimen early in the morning.
 Patients should be instructed to avoid eating or drinking before providing a specimen.

6. TYPE OF CONTAINER AND ADDITIVES:


 Sputum containers with a tight-fitting lid.
 No additives are required.

7. REQUIRED EQUIPMENT AND REAGENTS:


 Microscope.
 Ziehl-Neelsen stain.
 Acid-alcohol decolorizer.
 Methylene blue counterstain.

8. ENVIRONMENTAL AND SAFETY CONTROLS:


 The laboratory should be well-ventilated.
 Personnel should wear gloves and a lab coat.

9. CALIBRATION PROCEDURES:
 The microscope should be calibrated regularly.
 The Ziehl-Neelsen stain should be checked for quality and expiration date.

10. PROCEDURE STEPS:


 Prepare a smear on clean grease free slide.
 Allow it for air dry and fix it by giving a gentle heat.
 Flood the slide with Carbol Fuchsin stain until it get spread all over the slide.
 Heat the slide for 5 minutes on a very low flame (temperature may go up to 60°C.
 Then the slide is kept undisturbed for 5 minutes and allows it to cool.
 Rinse the slide with distilled water and wash the stain.
 Washing is followed by Acid Fast Decolourizer (3% v/v acid alcohol) treatment for about 2
minutes.
 Remove the Acid-fast Decolourizer with water.
 Flood the slide with Methylene Blue (counterstain) for about 2 min.
 Wash the counter stain, allow it dry in air and then observe it under oil immersion objective.

11. QUALITY CONTROL:


 Use of positive and negative controls.
 Regular maintenance of equipment.
 Training and competency assessment of personnel.

12. INTERFERENCE:
 Contamination.
 Staining artifacts.
 Poor smear quality.

13. CALCULATION OF RESULTS:


 NA
 Results are reported as "positive" or "negative" for acid-fast bacilli.

14. BIOLOGICAL REFERENCE INTERVALS:


 NA

15. REPORTABLE INTERVAL OF EXAMINATION RESULTS:


 Results are reported as soon as possible after completion of testing.

16. INSTRUCTIONS FOR DETERMINING QUANTITATIVE RESULTS WHEN A RESULT IS NOT


WITHIN THE MEASUREMENT INTERVAL:
 NA

17. ALERT/CRITICAL VALUES:


 Positive results for acid-fast bacilli.

18. LABORATORY CLINICAL INTERPRETATION:


 Non-Reactive Results: No acid-fast bacilli detected.
 Reactive Results: Acid-fast bacilli detected.
 Invalid Result: Result cannot be interpreted due to contamination or staining artifacts.

19. POTENTIAL SOURCES OF VARIATION:


 Poor smear quality.
 Contamination.
 Staining artifacts.

20. REFERENCES:
1. World Health Organization. (2018). Laboratory diagnosis of tuberculosis.
2. Centers for Disease Control and Prevention. (2020). Acid-Fast Staining.
3. Baron, E. J., L. R Peterson, and S. M. Finegold. 1994. Bailey and Scott’s diagnostic microbiology, 9th ed.
Mosby-Year Book, Inc., St. Louis, MO.
4. Beck, R. W. 2000. A chronology of microbiology in historical context. ASM Press, Washington, DC.
5. Benson , H. J. 1985. Experiment 12 - Smear Preparation, Microbiological Applications: A laboratory manual in
general microbiology, 4th ed. Wm. C. Brown Publishers, Dubuque, IA.
6. Bishop, P. J., and G. Neuman. 1970. The history of the ZiehlNeelsen stain. Tubercle 51:196.
7. Delisle, G., and L. Tomalty. 2002. Mycobacterium tuberculosis. MicrobeLibrary, American Society for
Microbiology, Washington, DC.
8. Gerhardt, P., R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips. 1981.
Manual of methods for general microbiology. ASM Press, Washington, DC.
9. Gerhardt, P., R. G. E. Murray, W. A. Wood, and N. R. Krieg. 1994. Methods for general and molecular
bacteriology. ASM Press, Washington, DC.
10. Kinyoun, J. J. 1915. A note on Uhlenhuth’s method for sputum examination for tubercle bacilli. Am. J. Public
Health 5:867.

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