Ziehl-Neelsen stain | Acid fast stain | Principle | Steps | Interpretation

❖ The method of choice for sputum smear microscopy is Ziehl-Neelsen (Acid fast) stain because it is the only stain that consistently provides good results without requiring special equipment.

❖ Cold staining techniques such as the Kinyoun or Tan Thiam Hok methods are not recommended: it has been proven that they hardly allow the detection of acid-fast bacilli (AFB) in paucibacillary samples and that the coloring quickly fades.

A- Ziehl-Neelsen staining : preparation of stains

❶ Coloring agent solutions:

Ziehl's carbol fuchsin (solution A):

- 3% alcoholic fuchsin stock solution

         Basic fuchsin ........ 3 g
         Alcohol 95% ..... up to 100 ml 

- Place the required amount of fuchsin in a graduated flask (or cylinder) and add the amount of ethanol or methyl alcohol necessary to obtain a total volume of 100 ml; Shake well until completely dissolved.

- Small amounts of this solution must be filtered before coloring.

Aqueous solution of phenol (solution B):

        Phenol crystals ...................... 5 g
        Distilled water, if possible ..... up to 90 ml

- Before adding the water, liquefy the phenol crystals in a flask by heating them slightly.

Ziehl-Neelsen staining

To prepare Ziehl's carburized fuchsin working solution, mix 10 ml of solution A with 90 ml of solution B.

❷ Bleaching agent solutions:

Alcohol-acid solution:

        Alcohol 95% ............................ 970 ml
        Concentrated hydrochloric acid (35%) ..... 30 ml

- Or when alcohol is not available:

25% aqueous sulfuric acid solution:

        Water, distilled if possible ..... ........ 300 ml
        Concentrated sulfuric acid ............ 100 ml

- Place 300 ml of water in a 1 liter Erlenmeyer flask. Slowly add 100 ml of concentrated sulfuric acid, letting it run down the side of the flask. The mixture will heat up.

Never put water in concentrated sulfuric acid, this can cause explosive splashing.

❸ methylene blue counter-stain solution:

       Methylene blue chloride. . . . . . . 0.3 g
       Water, distilled if possible. .... up to 100 ml

C- Ziehl-Neelsen (Acid Fast) Staining procedure

- Spread the sputum evenly over the central area of the blade with a continuous rotating movement; we recommend a spread of about 20 mm by 10mm

- Place the slides on the dryer with the spreading surface facing up and allow to air dry for about 30 minutes

- Fix the dried slides by holding them with pliers and passing them over the flame 5 times for about 4 seconds, with the spreading side facing up. Do not heat-set wet boards and avoid excessive heating.

☲ Staining :

❶ Place the fixed slides on the staining rack according to their order number, spreading side up. The blades should be separated by an interval of 1 cm and never touch each other.

❷ Cover the slides one after the other with Ziehl's 0.3% carbolic fuchsin working solution, filtered

❸By placing a strip of absorbent paper such as filter paper or even newspaper, the staining solution will be retained and the deposit of fuchsin crystals on the smear will be avoided.

❹ Heat the slides from below using the flame of a Bunsen burner, alcohol lamp or cotton ball soaked in alcohol, until steam is emitted. The dye solution should never be boiled. Do not allow the dye to dry out

❺ Leave the slides covered with a hot and steaming solution of carbolic fuchsin for 5 minutes, ironing the flame if necessary

❻ Rinse the slides gently with water to remove excess carbol fuchsin

❼ Drain the excess rinsing water from the slides. Sputum smears are red in color.

☲ Discoloration :

❶ Cover the slides with 25% sulfuric acid or an alcohol acid solution and leave to act for 3 minutes, after that the red coloration should have almost disappeared. If necessary, repeat this sequence for two more minutes .

❷Gently wash off the sulfuric acid or alcohol-acid and excess food coloring with water. Drain excess rinse water from the slides.

☲ Counterstaining :

❶ Cover the slides one after the other with the counterstaining solution (0.3% methylene blue) and leave to act for 1 minute

❷ Rinse the slides with water individually

❸ Drain the water from the slides and let them air dry

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1) Cover the slides with carbolic fuchsin

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2) Heat the slides from below

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3) Rinse

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4) Drain off the excess rinsing water

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5) Cover the slides with sulfuric acid

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6) Rinse

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7) Drain off the excess rinsing water

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8) counterstain (0.3% methylene blue)

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9) Rinse

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Final result

"Ziehl-Neelsen" spreading and coloring quality::

☰ The appearance of a correctly stained smear is light blue under the effect of methylene blue. If the color is dark blue, the smear is too thick, under these conditions it is not possible to read a text through the slide