AFB Stain

Sample for AFB stain

1. This can be done on any sputum sample, fluids, tissue, and urine.
2. For the sputum, advise consecutive three samples to rule out Tuberculosis.
   1. Early morning sputum is preferred, and the minimum volume accepted is 2mL.
3. Bronchial washing may be used, and the minimum volume needed is 2 mL.
4. Pleural fluid may be used.

Purpose of the test (Indications)

1. This is a special stain for the diagnosis of Mycobacterium tuberculosis in various specimens like sputum, caseous material, and tissue.
2. The AFB smear stain was used to monitor the treatment of Tuberculosis.
3. The AFB stain on sputum is indicated in a patient with:
   1. A cough.
   2. Night sweating.
   3. Anorexia.
   4. Fever.
   5. Hemoptysis.
   6. Unexplained weight loss.
   7. Fatigue and weakness.
   8. Chest pain.
4. Advise AFB stain in high-risk patients like immunocompromised patients, alcoholics, or have recent exposure to TB patients.

Precautions for AFB stain

1. Brush the teeth and remove the denture if it is there.
2. Rinse the mouth and take a deep cough sample.

Epidemiology of Mycobacterium Tuberculosis

1. Mycobacterium Tuberculosis is a very common disease throughout the world.
2. Roughly 1.86 billion people are infected with TB.

Spread of pulmonary tuberculosis

Mycobacterium Tuberculosis Signs and Symptoms:

Mycobacterium tuberculosis signs and symptoms

The principle of AFB stain

1. Acid-fastness is related to the composition of the bacterial outer capsule.
   1. The outer capsule (cell wall) is lipid-laden.
   2. These lipids consist of:
      1. Mycolic acid is a large fatty acid.
      2. Mycosides. Mycolic acid is bound to carbohydrates and forms glycolipid.
      3. Cord factor. It is formed by two mycolic acids and attached to the disaccharides molecule. It is only found in virulent strains.
      4. Sulfatides. It is a mycosides attached to sulfates and disaccharides.
      5. Wax-D. It is a complex mycoside, and its function is as an adjuvant. It increases the antibody formation to the antigen.
2. Once this capsule takes up the dye fuchsin, it is not decolorized by acid alcohol that is acid-fastness.

AFB stain principle

Reagents of AFB (Ziehl-Neelsen) method:

1. Carbol fuchsin
   1. Basic fuchsin = 10 grams.
   2. Ethanol or methanol = 100 mL.
   3. Phenol = 5 grams.
   4. Distilled water = 1000 mL.
   5. Method to prepare: Mix the ethanol with basic fuchsin. Try to mix until it is completely dissolved.
   6. Dissolve phenol carefully into the water.
   7. Mix no: 5 into no: 7. add the rest of the water and mix it well.
   8. Can store at room temperature for an indefinite time.
2. Acid alcohol 3% V/V.
   1. Absolute Ethanol or methanol 680 mL.
   2. Hydrochloric acid concentrated = 30 mL.
   3. Distilled water = 290 mL.
   4. Method to prepare: Mix alcohol with water.
   5. Now add HCL slowly to the above solution.
3. Malachite green 5 g/L (0.5% W/V).
   1. Malachite green = 5 grams.
   2. Distilled water = one liter.
   3. Mix the malachite green in the water till it is completely dissolved.
   4. This solution has been stable for several months.

AFB stain  (Ziehl-Neelsen acid-fast) staining procedure:

1. The tissues/sputum are smeared on the slides.
   1. Always take deep cough sputum and avoid saliva.
2. Then smear from the sputum is fixed by heat or alcohol.
   1. Heat fixes cells on a glass slide.
3. Flood the slide with carbol fuchsin stain.
   1. Heat the slide gently (around 60 °C) until it steams (5 min).
   2. You can see green scum.
   3. Avoid overheating.
   4. Pour off the carbol fuchsin.
4. Wash slides thoroughly with water.
5. Decolorize with acid-alcohol (5 min).
   1. Wash slides thoroughly with water.
6. Flood slide with methylene blue (or malachite green 1to 2 minutes) counterstain for 1 min.
   1. Wash with water.
   2. Blot excess water and dry in hand over bunsen flame.

AFB stain procedure

Result of AFB stain

The positive result is:

1. The acid-fast bacilli are red color rods, and the background is blue.
2. When these rods are seen, they indicate active tuberculosis.
3. The culture may confirm a positive AFB stain.

Positive sputum for Mycobacteria bacilli

The negative AFB stain is:

A negative result means that:

1. There is no infection.
2. The number of bacteria was not sufficient to be seen under the microscope.
3. Symptoms may be due to some other cause.

Reporting of the AFB stain.