History

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.

The Ziehl-Neelsen method has endured as a reliable and effective way to demonstrate the acid-fast bacteria. 

In 1915, Kinyoun published a method that has become known as the “cold staining” method because the heating step was removed in favor of using a higher concentration of the carbolfuchsin primary stain. 

Purpose 

The acid-fast stain is performed on samples to demonstrate the characteristic of  acid fastness in certain bacteria and the cysts of Cryptosporidium and Isospora.  Clinically, the most important application is to detect Mycobacterium tuberculosis in sputum samples to confirm or rule out a diagnosis of tuberculosis in patients.

Theory 

There are three common acid-fast staining methods, Ziehl-Neelsen (hot), Kinyoun (cold), and Auramine-Rhodamine Fluorochrome (Truant method).  The emphasis in this Atlas-Protocol project will be on the Ziehl-Neelsen and the Kinyoun methods because the slides produced by these methods can be visualized using a standard bright-field microscope.  The fluorochrome method is used by large laboratories that have a fluorescent (ultraviolet) microscope.  For comparison purposes, the recipe for the reagents and the protocol for all three methods are included below, but images for the fluorochrome method will not be a part of the Atlas at this time. 

Many bacterial cells are easily stained with simple stains or using the Gram stain.  A few types of bacteria, such as the mycobacteria and Nocardia species, do not stain using these techniques or, if stained, they produce a variable reaction because their walls are not permeable to the rosaniline dyes in common staining regimens (12).  The cell walls of the mycobacteria contain mycolic acids giving the cell walls a high lipid content.  This characteristic is thought to be the reason (5, 10) these bacteria are difficult to stain.  To view these cells in samples staining requires higher concentrations of the dye solution and/or a heating period (4). However, once a stain is introduced into the cell wall, removing it with a decolorizer is even more difficult. The expression “acid fast” is derived from the observation that even with the addition of hydrochloric acid to the alcohol decolorizer, some of the stained cells retain the primary stain (carbolfuchsin).  Cells that release the primary stain (carbolfuchsin) with decolorizing will be visible after the counterstaining step is complete.  Bacteria described as acid fast will appear red when examining specimens using bright-field microscopy.  Non-acid-fast cells and field debris will appear blue.   

Acid fastness is a characteristic that is shared by just a few organisms, so staining to determine if organisms possess this trait is useful in microbial identification schemes.   

RECIPES (6, 7, 15)

A.  Ziehl-Neelsen method for acid-fast staining (6, 7)

       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

B.  Kinyoun method for acid-fast staining (15)

           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.

C. Truant method for acid-fast staining (6, 7)

          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

PROTOCOLS (1, 6, 7) 

Smear Preparation 

Ordinarily, preparing a smear for staining involves applying a very small sample to the center of a carefully cleaned glass slide.  The microbial sample is usually taken from a broth culture or a suspension of microorganisms produced by mixing a tiny amount of solid matter from colonies with water.  The suspension can be made directly on the slide or it can be mixed in a tube and transferred to the slide. 

Since many bacteria cling to each other in culture (both broth and colonial form), vigorous manual or mechanical mixing may be required to produce a proper distribution of the organisms for microscopic evaluation.  Clumps of organisms make it difficult to observe the characteristics of individual cells. 

The quality of the staining for acid-fast bacteria will be affected by the quality of the smear. A good quality smear will have a thin film of the specimen or culture, allowing individual cells to respond to the staining protocol.  The waxy nature of the mycobacteria organisms causes them to repel water so fluids should be added to the slide after spreading the sample in a thin film over the slide.   

Organisms grown in media containing complex lipids will stain better, and usually, grow better. 

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. 

Note:  Reports of the survival of mycobacteria at these temperatures have been made.  Follow the proper handling precautions of your laboratory/institution.

A.  Ziehl-Neelsen method for acid-fast staining (1, 6, 7) 

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. 

         (CAUTION:  overheating causes spattering of the stain and may crack the slide).

4.      Wash the film in a gentle and indirect stream of tap water* until no color appears in the effluent.

5.      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.

6.      Flood the smear with the methylene blue counterstain for 20 to 30 seconds, and wash with tap water*, as above.

7.   Gently blot, or air dry the smear.

8.   Examine under oil immersion.**  Acid-fast bacteria appear red, and non-acid-fast bacteria (and other organisms and cellular materials) appear blue. 

        * NOTE:  Most labs use deionized or distilled water for all lab procedures.  See note in Tips and Comments section regarding the use of tap water.

        ** NOTE:  For clinical samples, examination of at least 300 fields is required before declaring a specimen negative for acid-fast bacteria.