Acid Fast Staining

Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease. The disease affects 1.8 billion people/year which is equal to one-third of the entire world population.

In the United States TB is on the decline. In 2007 a total of 13,293 cases were reported. The TB rate declined to 4.4 cases per 100,000 population, the lowest recorded rate since national reporting began in 1953. Despite this overall improvement, progress toward TB elimination has slowed in recent years; the average annual percentage decline in the TB rate slowed from 7.3% per year during 1993–2000 to 3.8% during 2000–2007. Also, since 1993 there has been a gradual decline in the number of TB patients with coinfection with HIV, and the number of cases of multiple drug-resistant TB has gradually dropped.

On the other hand, the proportion of TB cases contributed by foreign-born persons has increased each year since 1993. In 2007 the TB rate in foreign-born persons in the United States was 9.7 times higher than in U.S.-born persons. In many states, especially in the West, the upper Midwest, and the Northeast, most new cases of TB now occur in individuals who are foreign born.

Mycobacterium tuberculosis

Mycobacterium tuberculosis is the etiologic agent of tuberculosis in humans. Humans are the only reservoir for the bacterium. Mycobacterium bovis is the etiologic agent of TB in cows and rarely in humans. Both cows and humans can serve as reservoirs. Humans can also be infected by the consumption of unpasteurized milk. This route of transmission can lead to the development of extrapulmonary TB, exemplified in history by bone infections that led to hunched backs. Other human pathogens belonging to the Mycobacterium genus include Mycobacterium avium which causes a TB-like disease especially prevalent in AIDS patients, and Mycobacterium leprae, the causative agent of leprosy. General Characteristics

Mycobacterium tuberculosis is a fairly large nonmotile rod-shaped bacterium distantly related to the Actinomycetes. Many non pathogenic mycobacteria are components of the normal flora of humans, found most often in dry and oily locales. The rods are 2-4 micrometers in length and 0.2-0.5 um in width. Mycobacterium tuberculosis is an obligate aerobe. For this reason, in the classic case of tuberculosis, MTB complexes are always found in the well-aerated upper lobes of the lungs. The bacterium is a facultative intracellular parasite, usually of macrophages, and has a slow generation time, 15-20 hours, a physiological characteristic that may contribute to its virulence. Two media are used to grow MTB Middlebrook’s medium which is an agar based medium and Lowenstein-Jensen medium which is an egg based medium. MTB colonies are small and buff colored when grown on either medium. Both types of media contain inhibitors to keep contaminants from out-growing MT. It takes 4-6 weeks to get visual colonies on either type of media.

Colonies of Mycobacterium tuberculosis on Lowenstein-Jensen medium. CDC. Chains of cells in smears made from in vitro-grown colonies often form distinctive serpentine cords. This observation was first made by Robert Koch who associated cord factor with virulent strains of the bacterium. MTB is not classified as either Gram-positive or Gram-negative because it does not have the chemical characteristics of either, although the bacteria do contain peptidoglycan (murein) in their cell wall. If a Gram stain is performed on MTB, it stains very weakly Gram-positive or not at all (cells referred to as “ghosts”). Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria due to their impermeability by certain dyes and stains.

Despite this, once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds. One acid-fast staining method for Mycobacterium tuberculosis is the Ziehl-Neelsen stain. When this method is used, the MTB. smear is fixed, stained with carbol-fuchsin (a pink dye), and decolorized with acid-alcohol. The smear is counterstained with methylene-blue or certain other dyes. Acid-fast bacilli appear pink in a contrasting background. In order to detect Mycobacterium tuberculosis in a sputum sample, an excess of 10,000 organisms per ml of sputum are needed to visualize the bacilli with a 100X microscope objective (1000X mag). One acid-fast bacillus/slide is regarded as “suspicious” of an MTB infection.

Mycobacterium tuberculosis. Acid-fast stain. CDC.

Cell Wall Structure
The cell wall structure of Mycobacterium tuberculosis deserves special attention because it is unique among prokaryotes, and it is a major determinant of virulence for the bacterium. The cell wall complex contains peptidoglycan, but otherwise it is composed of complex lipids. Over 60% of the mycobacterial cell wall is lipid. The lipid fraction of MTB’s cell wall consists of three major components, mycolic acids, cord factor, and wax-D. Mycolic acids are unique alpha-branched lipids found in cell walls of Mycobacterium and Corynebacterium. They make up 50% of the dry weight of the mycobacterial cell envelope. Mycolic acids are strong hydrophobic molecules that form a lipid shell around the organism and affect permeability properties at the cell surface.

Mycolic Acids are thought to be a significant determinant of virulence in MTB. Probably, they prevent attack of the mycobacteria by cationic proteins, lysozyme, and oxygen radicals in the phagocytic granule. They also protect extracellular mycobacteria from complement deposition in serum. Cord Factor is responsible for the serpentine cording mentioned above. Cord factor is toxic to mammalian cells and is also an inhibitor of PMN migration. Cord factor is most abundantly produced in virulent strains of MTB. Wax-D in the cell envelope is the major component of Freund’s complete adjuvant (CFA). The high concentration of lipids in the cell wall of Mycobacterium tuberculosis have been associated with these properties of the bacterium: Impermeability to stains and dyes

Resistance to many antibiotics
Resistance to killing by acidic and alkaline compounds
Resistance to osmotic lysis via complement deposition
Resistance to lethal oxidations and survival inside of macrophages The Ziehl–Neelsen stain, also known as the acid-fast stain, was first described by two German doctors: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898). It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group because it is responsible for tuberculosis (TB). Other important Mycobacterium species involved in human disease are Mycobacterium kansasii, Mycobacterium marinum, and members of the Mycobacterium avium complex. Acid fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids.

These acids resist staining by ordinary methods such as a Gram stain.[1] It can also be used to stain a few other bacteria, such as Nocardia. The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol, and methylene blue. Acid-fast bacilli will be bright red after staining. A variation on this staining method is used in mycology to differentially stain acid-fast incrustations in the cuticular hyphae of certain species of fungi in the genus Russula.[2][3] It is also useful in the identification of some protozoa, namely Cryptosporidium and Isospora. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because the eggs in an ovum and parasite sputum sample (OnP) can be dissolved by the stain, and is often used in this clinical setting because signs and symptoms of paragonimiasis closely resemble those of TB. Procedure…

1.Drop suspension onto slide
2.Air dry slide 10 minutes at 60 °C, heat-fix slide 10 minutes at 90 °C
3.Flood slide with Carbol Fuchsin
4.Hold a flame beneath the slide until steam appears but do not allow it to boil
5. Allow hot slide to sit for 3 to 5 minutes, rinse with tap water
6.Flood slide with 30% hydrochloric acid in isopropyl alcohol
7.Allow to sit 1 minute, rinse with tap water
8.Flood slide with Methylene Blue
9.Allow to sit 1 minute, rinse with tap water
10.Blot dry
11.View under oil immersion lens
Studies have shown that an AFB stain without a culture has a poor negative predictive value. An AFB Culture should be performed along with an AFB stain; this has a much higher negative predictive value. Modifications

•5% sulfuric acid is used for destaining Mycobacterium leprae instead of the 20% used for Mycobacterium tuberculosis. •Kinyoun modification (or cold Ziehl–Neelsen technique) is also available. •A protocol in which a detergent is substituted for the highly toxic phenol in the fuchsin staining solution.[4] •PROCEDURE (Ziehl-Neelsen Method)

•1. Heat-fix a smear of a bacterium as follows:
•a. Using the dropper bottle of distilled water found in your staining rack, place 1/2 a drop of water on a clean slide by touching the dropper to the slide. •b. Aseptically remove a small amount of the bacterium from the agar surface and mix it with the water. Flame the loop and let it cool. •c. Using the loop, spread the mixture over the entire slide to form a thin film. •d. Allow this thin suspension to completely air dry.

•e. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix. •2. Cover the smear with a piece of blotting paper and flood with carbol fuchsin. •3. Steam for 5 minutes by passing the slide through the flame of a gas burner. •4. Allow the slide to cool and wash with water.

•5. Add the acid-alcohol decolorizing slowly dropwise until the dye no longer runs off from the smear.
•6. Rinse with water.
•7. Counterstain with methylene blue for 1 minute.
•8. Wash with water, blot dry, and observe using oil immersion microscopy.

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 4oC 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 80oC for 15 minutes or for 2 hours on a hot plate set for 65oC to 70oC.

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 80oC for at least 15 minutes or for 2 hours on an electric hot plate at 65oC – 70oC

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. •
•
FIG. 1. Mycobacterium tuberculosis (5).

•B. Kinyoun method for acid-fast staining (1)

1. Heat fix an air dried smear at 80oC for at least 15 minutes or for 2
hours on an electric hot plate at 65oC – 70oC

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).

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

C. Truant method for acid-fast staining (1, 6, 7)

1. Heat fix an air dried smear at 80oC for at least 15 minutes or for 2 hours on an electric hot plate at 65oC – 70oC

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