Acid-Fast Stain Protocols - Ziehl-Neelsen, Kinyoun, Truant Methods

There are three common acid-fast staining methods, Ziehl-Neelsen (hot), Kinyoun (cold), and Auramine-Rhodamine Fluorochrome (Truant method). The Ziehl-Neelsen method has endured as a reliable and effective way to demonstrate the acid-fast bacteria.

In 1882 Robert Koch reported the discovery of the tubercle bacillus (4) and described the appearance of the bacilli resulting from a complex staining procedure. During the same time period several other researchers (Ehrlich, Ziehl, Rindfleisch, and Neelsen), intending to improve on Koch’s method, introduced modifications to the reagents and the procedure. Franz Ziehl was the first to use carbolic acid (phenol) as the mordant. Friedrich Neelsen kept Ziehl’s mordant, but changed the primary stain to the basic fuchsin (first used by Ehrlich in 1882). This method became known as the Ziehl-Neelsen method in the early to mid 1890s. In this method heat is used to help drive the primary stain into the waxy cell walls of these difficult-to-stain cells. The use of heat in this method has been the reason that this technique is called the “hot staining” method.

Ziehl-Neelsen method	Kinyoun method	Truant method
Carbolfuchsin stain: •Basic fuchsin, 0.3 g •Ethanol, 95% (vol/vol), 10 ml •Phenol, heat-melted crystals, 5 ml •Distilled water, 95 ml •Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water. •Mix and let stand for several days. Filter before use. Decolorizing solvent: •Ethanol, 95% (vol/vol), 97 ml •Hydrochloric acid (concentrated), 3 ml Counterstain: •Methylene blue chloride, 0.3 g •Distilled water, 100 ml	Kinyoun carbolfuchsin solution: •Solution A. Dissolve 4 g of basic fuchsin in 20 ml of ethyl alcohol. •Solution B. Dissolve 8 g of phenol (melted) in 100 ml of distilled water. •Mix solutions A and B together and allow to stand for a few days. Acid-alcohol decolorizing agent: •Ethanol, 95% (vol/vol), 97 ml •Hydrochloric acid (concentrated), 3 ml Methylene blue counterstain: •Methylene blue chloride, 0.3 g •Distilled water, 100 ml •Dissolve by shaking.	Fluorescent staining reagent: •Auramine O, CI 41000, 1.50 g •Rhodamine B, CI 749, 0.75 g •Glycerol, 75 ml •Phenol (heat melted crystals), 10 ml •Distilled water, 50 ml •Mix the two dyes well with 25 ml of the water and the phenol. Add the remaining water and glycerol and mix again. •Filter the resulting staining fluorescent reagent through glass wool and store at 4 oC or room temperature. Decolorizing solvent: •Ethanol, 70% (vol/vol), 99.5 ml •Hydrochloric acid (concentrated), 0.5 ml Counterstain: •Potassium permanganate, 0.5 g •Distilled water, 99.5 g

Basic Smear Preparation

1. Clean a glass slide (some labs will provide pre-cleaned slides; be sure to remove any dust or crushed glass debris) according to instructions provided by your instructor.
2. Prepare the sample according to instructions provided by your instructor. Make certain that an aerosol is not generated during this process.
3. Using a sterile pipet or microbiological loop, apply a small sample of the specimen to the slide by slowly spreading the liquid to make a thin film; If you are using solid matter from a colony, be sure to choose a very minute sample and spread it into a very thin film. Applying the cells before adding water (or other mixing fluid) will help the cells adhere to the slide. The size of the film should be about 1 cm in diameter. Avoid any actions that would splatter droplets of the sample in the surrounding area.
4. Allow the smear to dry completely.
5. Fix the smear at 80 oC for 15 minutes or for 2 hours on a hot plate set for 65 oC to 70 oC.
6. Proceed to the staining protocol of your choice.

Ziehl-Neelsen method for acid-fast staining

1. Heat fix an air dried smear at 80 oC for at least 15 minutes or for 2 hours on an electric hot plate at 65 oC – 70 oC
2. Place a slide with an air-dried and heat-fixed smear on suitable staining device. Cut a piece of absorbent paper to fit the slide and saturate the paper with the carbolfuchsin stain.
3. Carefully heat the underside of the slide by passing a flame under the rack or by placing the slide on a hot plate until steam rises (without boiling!). Keep the preparation moist with stain and steaming for 5 minutes, repeating the heating as needed.
4. (CAUTION: overheating causes spattering of the stain and may crack the slide).
5. Wash the film in a gentle and indirect stream of tap water* until no color appears in the effluent.
6. Holding the slide with forceps, wash the slide with the decolorizing solvent. Immediately wash with tap water*, as above. Repeat the decolorizing and the washing until the stained smear appears faintly pink and the fluid washing off the slide runs clear.
7. Flood the smear with the methylene blue counterstain for 20 to 30 seconds, and wash with tap water*, as above.
8. Gently blot, or air dry the smear.
9. Examine under oil immersion.** Acid-fast bacteria appear red, and non-acid-fast bacteria (and other organisms and cellular materials) appear blue.

* Most labs use deionized or distilled water for all lab procedures. See note in Tips and Comments section regarding the use of tap water.
** For clinical samples, examination of at least 300 fields is required before declaring a specimen negative for acid-fast bacteria.

Kinyoun method for acid-fast staining

1. Heat fix an air dried smear at 80 oC for at least 15 minutes or for 2 hours on an electric hot plate at 65 oC – 70 oC
2. Flood slides with Kinyoun’s carbolfuchsin reagent and allow to stain for 5 minutes at room temperature.
3. Rinse with deionized water and tilt slide to drain.
4. Decolorize with acid-alcohol for 3 minutes and rinse again with deionized water.*
5. Redecolorize with acid-alcohol for 1-2 minutes or until no more red color runs from the smear.*
6. Rinse with deionized water and drain standing water from the slide surface by tipping the slide.
7. Flood slide with methylene blue counterstain and allow to stain for 4 minutes.
8. Rinse with distilled water and allow to air dry.
9. Examine under high dry (400X) magnification, and confirm acid-fast structures under oil immersion (1000X).

*Most practitioners apply decolorizer until the fluid washing off the slide runs clear.

Truant method for acid-fast staining

1. Heat fix an air dried smear at 80 oC for at least 15 minutes or for 2 hours on an electric hot plate at 65 oC – 70 oC
2. Wash the slide with a gentle and indirect stream of distilled water until no color appears in the effluent.
3. Flood the smear with the decolorizing agent for 2 to 3 minutes, and then wash with distilled water as above.
4. Flood the smear with the permanganate counterstain for 2 to 4 minutes.
5. Wash the slide with distilled water as above, blot with absorbent paper, and dry.
6. Examine the slide with a fluorescence microscope equipped with a BG-12 exciter filter and an OG-1barrier filter. Acid-fast bacteria appear as brightly fluorescent, yellow-orange cells in a dark field; non-acid-fast cells appear dark.