Mycobacterium Tuberculosis:- Part 4 – AFB Stain (Acid Fast Bacilli Stain) Procedure and Interpretations
AFB Stain
Sample for AFB stain
- This can be done on any sputum sample, fluids, tissue, and urine.
- For the sputum, advise consecutive three samples to rule out Tuberculosis.
- Early morning sputum is preferred, and the minimum volume accepted is 2mL.
- Bronchial washing may be used, and the minimum volume needed is 2 mL.
- Pleural fluid may be used.
Purpose of the test (Indications) for AFB Stain
- This is a special stain for the diagnosis of Mycobacterium tuberculosis in various specimens like sputum, caseous material, and tissue.
- The AFB smear stain was used to monitor the treatment of Tuberculosis.
- The AFB stain on sputum is indicated in a patient with:
- A cough.
- Night sweating.
- Anorexia.
- Fever.
- Hemoptysis.
- Unexplained weight loss.
- Fatigue and weakness.
- Chest pain.
- Advise AFB stain in high-risk patients like immunocompromised patients, alcoholics, or have recent exposure to TB patients.
Precautions for AFB stain
- Brush the teeth and remove the denture if it is there.
- Rinse the mouth and take a deep cough sample.
Epidemiology of Mycobacterium Tuberculosis
- Mycobacterium Tuberculosis is a very common disease throughout the world.
- Roughly 1.86 billion people are infected with TB.
Mycobacterium Tuberculosis Signs and Symptoms:
The principle of AFB stain
- Acid-fastness is related to the composition of the bacterial outer capsule.
- The outer capsule (cell wall) is lipid-laden.
- These lipids consist of:
- Mycolic acid is a large fatty acid.
- Mycosides. Mycolic acid is bound to carbohydrates and forms glycolipids.
- Cord factor. It is formed by two mycolic acids and attached to the disaccharide molecule. It is only found in virulent strains.
- Sulfatides. It is a mycosides attached to sulfates and disaccharides.
- Wax-D. It is a complex mycoside, and its function is as an adjuvant. It increases the antibody formation to the antigen.
- Once this capsule takes up the dye fuchsin, it is not decolorized by acid alcohol, which is, acid-fastness.
Reagents of AFB (Ziehl-Neelsen) method:
- Carbol fuchsin
- Basic fuchsin = 10 grams.
- Ethanol or methanol = 100 mL.
- Phenol = 5 grams.
- Distilled water = 1000 mL.
- Method to prepare: Mix the ethanol with basic fuchsin. Try to mix until it is completely dissolved.
- Dissolve phenol carefully into the water.
- Mix no: 5 into no: 7. add the rest of the water and mix it well.
- It can store at room temperature for an indefinite time.
- Acid alcohol 3% V/V.
- Absolute Ethanol or methanol 680 mL.
- Hydrochloric acid concentrated = 30 mL.
- Distilled water = 290 mL.
- Method to prepare: Mix alcohol with water.
- Now add HCL slowly to the above solution.
- Malachite green 5 g/L (0.5% W/V).
- Malachite green = 5 grams.
- Distilled water = one liter.
- Mix the malachite green in the water till it is completely dissolved.
- This solution has been stable for several months.
AFB stain (Ziehl-Neelsen acid-fast) staining procedure:
- The tissues/sputum are smeared on the slides.
- Always take deep cough sputum and avoid saliva.
- Then smear from the sputum is fixed by heat or alcohol.
- Heat fixes cells on a glass slide.
- Flood the slide with a carbol fuchsin stain.
- Heat the slide gently (around 60 °C) until it steams (5 min).
- You can see green scum.
- Avoid overheating.
- Pour off the carbol fuchsin.
- Wash slides thoroughly with water.
- Decolorize with acid-alcohol (5 min).
- Wash slides thoroughly with water.
- Flood slide with methylene blue (or malachite green 1 to 2 minutes) counterstain for 1 min.
- Wash with water.
- Blot excess water and dry in hand over bunsen flame.
Result of the AFB stain
The positive result is:
- The acid-fast bacilli are red color rods, and the background is blue.
- When these rods are seen, they indicate active tuberculosis.
- The culture may confirm a positive AFB stain.
The negative AFB stain is:
A negative result means that:
- There is no infection.
- The number of bacteria was not sufficient to be seen under the microscope.
- Symptoms may be due to some other cause.
Reporting of the AFB stain.
Presence of AF bacilli | Positivity report | Another system |
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Questions and answers:
Question 1: What is the purpose of acid-fast stain?
Question 3: How you can report the AFB stain?
Nice notes
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