Acid Fast Stain – Principle, Reagents, Procedure and Results

Acid-fast stain is included in microbiology laboratory practicals involving the staining and microscopic examination of bacterial smears. These practicals are usually carried out using bacterial cultures or prepared slides.

Acid-fast bacilli (AFB) smear microscopy is also used in clinical laboratories during investigations for suspected tuberculosis (TB). Sputum specimens are commonly examined using acid-fast staining as part of the laboratory investigation.

What Is an Acid-Fast Stain?

Acid-fast staining is a differential staining technique used in microbiology to distinguish the acid-fast bacteria from the non-acid-fast bacteria. The bacteria which retain the primary stain after decolorization with acid or acid-alcohol are called acid-fast bacteria. The bacteria which do not retain the stain are the non-acid-fast bacteria.

This ability to retain the primary stain is known as acid-fastness. It is due to the lipid-rich cell wall of the mycobacteria, which contains mycolic acids.

The term acid-fast refers to the resistance of the bacteria to decolorization by acid, and not to the use of an acidic dye. The primary stain used in this technique is a basic dye. The rod-shaped acid-fast bacteria are known as acid-fast bacilli (AFB).

Ziehl–Neelsen is one widely used method of acid-fast staining. Other methods of acid-fast staining are also based on the same property of these bacteria.

Objectives of Acid-Fast Staining

The objectives of acid-fast staining are as follows:

• To differentiate acid-fast bacteria from the non-acid-fast bacteria.
• To detect the presence of acid-fast bacilli in smears.
• To detect Mycobacterium tuberculosis and Mycobacterium leprae in clinical samples.
• To help in the diagnosis of tuberculosis and leprosy.

Principle of Acid-Fast Staining

Acid-fast staining is based on the mycolic acid-rich cell envelope of the mycobacteria. The cell envelope of these bacteria is waxy and lipid-rich. This waxy layer prevents the ordinary stains from entering the cell.

The primary stain used in acid-fast staining is carbol fuchsin, which contains phenol. The phenol helps the dye to enter the waxy cell envelope of the bacteria. In the Ziehl–Neelsen method, the smear is heated along with the stain. The heat makes the waxy layer porous, so that the stain can enter the cell.

The carbol fuchsin is not removed from the cell wall of the acid-fast bacilli even after decolorization with acid or acid-alcohol. The non-acid-fast bacteria do not contain this waxy layer in their cell wall. The primary stain is removed from these cells during decolorization. These cells then take up the counterstain.

In the Kinyoun method, heating is not required. The carbol fuchsin used in this method contains a higher concentration of phenol, which helps the dye to enter the cell wall without heating.

Types of Acid-Fast Staining

Acid fast staining can be done in a number of ways depending on the primary stain, the requirement for heating and the decolouriser employed. The two conventional carbol fuchsin methods are Ziehl-Neelsen and Kinyoun. Modified acid fast staining and fluorochrome staining are also used in the examination of acid fast organisms.

1. Ziehl–Neelsen Method (Hot Method)

Ziehl–Neelsen staining is also known as the ‘hot’ method of acid-fast staining. Carbol fuchsin is applied to the smear with a little heating. The heat is used to assist the dye in penetrating the waxy cell wall of the mycobacteria.

Acid or acid-alcohol is used for decolourisation, followed by counterstaining with methylene blue. The acid-fast bacilli will appear red against a blue background under the bright field microscope.

The method is named after Ziehl and Neelsen who worked with earlier staining methods in the 1880s.

2. Kinyoun Method (Cold Method)

The Kinyoun method is also referred to as cold acid-fast staining. It’s a variation of the Ziehl-Neelsen stain in that it uses carbol fuchsin, but the carbol fuchsin used in the Kinyoun method contains a higher proportion of basic fuchsin and phenol.

Heating is not necessary with this method. The higher concentration of the staining components facilitates entry of the dye into the bacterial cell wall without the need for heat.

The decolourisation and counterstaining is analogous to the conventional Ziehl–Neelsen method. The acid fast bacilli appear as red rods on a blue background under bright field.

3. Modified Acid-Fast Staining

Modified acid-fast staining is used for the detection of organisms which are only weakly or partially acid-fast. This involves modification of the decolourisation conditions used in the standard acid-fast stain.

A weaker decolourising solution (e.g. 1% H2SO4) is used in the modified Kinyoun method for Nocardia. They may appear as red branching filaments. The acid fastness of Nocardia is variable.

Modified acid-fast staining is also employed for the detection of intestinal coccidian oocysts (Cryptosporidium, Cyclospora and Cystoisospora). Modified Kinyoun and modified Ziehl–Neelsen staining are the methods used.

The concentration of the decolourising solution and the need for heating varies between these modified methods. The oocysts can appear pink to red under bright field. Cyclospora oocysts will stain variably.

4. Fluorochrome Acid-Fast Staining

Fluorochrome acid-fast staining is a fluorescent staining technique for the detection of acid fast bacilli. It uses auramine O or auramine-rhodamine as the primary stain rather than carbol fuchsin.

Heating is not required during staining and acid-alcohol is used for decolourisation. The acid-fast bacilli are bright yellow to yellow green in fluorescence against a dark background.

The stained smears are examined under a fluorescence microscope (or an LED fluorescence microscope). They can be looked at at a lower magnification than the conventional carbol-fuchsin stain under bright field microscopy, enabling a much larger area to be examined in a shorter time.

Reagents and Materials Required for Acid-Fast Staining

Reagents Used in Acid-Fast Staining

• Carbol fuchsin solution (primary stain)
• Acid-alcohol or sulfuric acid solution (decolorizer)
• Methylene blue solution (counterstain)
• Clean water or distilled water

Materials and Equipment

• Clean glass slides
• Bacterial sample or prepared smear
• Inoculating loop or sterile applicator sticks
• Slide marking pencil
• Staining rack
• Slide holder or forceps
• Bunsen burner, spirit lamp or suitable slide heater
• Wash bottle
• Staining tray
• Filter paper and funnel
• Microscope with 100× oil immersion objective
• Immersion oil
• Timer
• Laboratory coat, gloves and safety goggles
• Laboratory disinfectant
• Biohazard waste container
• Biological safety cabinet for infectious specimen preparation

Procedure of Acid-Fast Staining (Ziehl–Neelsen Method)

The procedure of Ziehl–Neelsen acid-fast staining using 0.3% carbol fuchsin, 3% acid-alcohol and 0.3% methylene blue is given below.

For laboratory practicals, a noninfectious instructional specimen should be used. Clinical sputum and other potentially infectious specimens require approved laboratory biosafety procedures during smear preparation and fixation. Heat fixation does not reliably inactivate Mycobacterium tuberculosis.

1. Take a clean, grease-free glass slide and label one end.
2. Prepare a thin smear on the slide using the instructional specimen.
3. Allow the smear to air-dry completely. Do not dry the smear over a flame.
4. The dried smear is heat-fixed by passing the underside of the slide through a flame for 2–3 seconds, 3–4 times. Allow the slide to cool.
5. Place the slide on a staining rack with the smear facing upwards and cover the smear with filtered 0.3% carbol fuchsin.
6. Heat the underside of the slide gently until steam appears. Keep the stain steaming for 5 minutes. Do not allow the stain to boil or dry.
7. Rinse the slide gently with clean water and drain off the excess water.
8. The smear is decolorized with 3% acid-alcohol for 2–3 minutes. Repeat the decolorization if the smear is not completely decolorized.
9. Rinse the slide with clean water and drain.
10. Cover the smear with 0.3% methylene blue for 1 minute.
11. Wash off the counterstain gently with clean water and allow the slide to air-dry. Do not wipe or blot the smear.
12. Place a drop of immersion oil on the dried smear and examine the smear under the microscope using the 100× oil-immersion objective.

Results and Interpretation of Acid-Fast Staining

In Ziehl–Neelsen staining the acid fast bacilli are red/pink against a blue background and the stained smear is examined under the bright field microscope using the 100x oil immersion objective.

Positive Acid-Fast Staining Result

Acid-fast organisms take up the carbol fuchsin stain and appear red or pink. On a conventional mycobacterial smear the bacilli are typically slender, straight or slightly curved rods. Some of the bacilli will be beaded. They can be seen singly or in small groups.

An AFB positive result is reported if acid fast bacilli are present on a clinical smear. It’s important to note that an AFB stain does not allow you to identify Mycobacterium tuberculosis specifically as other mycobacterial species will also be acid fast. Culture or molecular testing is required for species identification.

Negative Acid-Fast Staining Result

Non-acid fast bacterial cells will lose the carbol fuchsin stain during the decolourising stage and will be blue after counterstaining with methylene blue.

A known non-acid fast organism will give a negative acid fast stain. In a clinical AFB smear, a negative result means that no acid fast bacilli were seen in the requisite number of fields.

A negative AFB smear does not rule out TB as the number of bacilli in the specimen may be below the level detectable by microscopy.

The differences between positive and negative acid-fast staining results are given below:

Grading of AFB Smear Results

AFB smear grading is based on the number of acid-fast bacilli observed under the microscope. The bacilli are counted in the oil-immersion fields and the result is reported according to the grading system used by the laboratory.

The WHO/IUATLD grading system for conventional Ziehl–Neelsen staining is given below:

The following terms are used for reporting AFB smear results:

1. AFB Negative– No acid-fast bacilli are observed in 100 oil-immersion fields.
2. Scanty Positive– The smear contains 1–9 acid-fast bacilli in 100 fields. The exact number is reported. For example, scanty 3 means that three AFB were observed in 100 fields.
3. AFB 1+– The number of acid-fast bacilli ranges from 10 to 99 in 100 oil-immersion fields.
4. AFB 2+– An average of 1–10 acid-fast bacilli is observed per oil-immersion field.
5. AFB 3+– More than 10 acid-fast bacilli are observed per oil-immersion field.

Some laboratories use a different grading system with categories from 1+ to 4+. The number of bacilli required for each grade depends on the reporting standard.

Fluorescent AFB microscopy also uses different field counts and grading thresholds because the smears are examined at a lower magnification.

Examples of Acid-Fast and Non-Acid-Fast Bacteria

Acid-fast bacteria include Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium smegmatis. Their cell envelopes contain a large amount of mycolic acids and other lipids. M. tuberculosis and M. smegmatis retain carbol fuchsin during conventional Ziehl–Neelsen staining and appear red or pink.

M. leprae is also an acid-fast bacterium, but it may not be detected by routine Ziehl–Neelsen staining. Modified Ziehl–Neelsen or Fite–Faraco staining is used for its examination, particularly in tissue sections.

Nocardia species, such as Nocardia brasiliensis and Nocardia farcinica, are partially acid-fast bacteria. Their cell walls contain shorter-chain mycolic acids. These bacteria may appear as red, branching filaments when a modified acid-fast stain with a weaker decolorizing solution is used. The staining reaction is variable.

Escherichia coli and Staphylococcus aureus are examples of non-acid-fast bacteria. E. coli has a thin peptidoglycan layer and an outer membrane containing lipopolysaccharide. S. aureus contains a thick peptidoglycan cell wall with teichoic acids. Both lack the mycolic acid-rich envelope found in mycobacteria. They lose the carbol fuchsin stain during decolorization and appear blue when methylene blue is used as the counterstain.

The examples of acid-fast and non-acid-fast bacteria are given below:

Differences Between Acid-Fast Staining and Gram Staining

Acid-fast staining and Gram staining are two differential staining techniques used in microbiology. Acid-fast staining is used to distinguish acid-fast bacteria from non-acid-fast bacteria, whereas Gram staining is used to differentiate Gram-positive and Gram-negative bacteria.

The differences between acid-fast staining and Gram staining are given below:

Mycobacteria contain mycolic acids and other lipids in their cell envelope, which prevent the proper penetration of dyes used in routine Gram staining. These bacteria may appear weakly or irregularly Gram-positive, and some cells remain poorly stained.

Acid-fast staining is used for the examination of mycobacteria because they retain carbol fuchsin even after treatment with acid-alcohol. Gram-positive bacteria such as Staphylococcus aureus do not possess the same mycolic acid-rich envelope and are generally non-acid-fast.

Precautions of Acid-Fast Staining

The following are the precautions of acid-fast staining:

• Prepare a thin and evenly spread smear on a clean glass slide. A thick smear may prevent proper decolorization and make the acid-fast bacilli difficult to observe.
• Allow the smear to air dry completely before fixation. Do not heat the wet smear. Excessive heating during fixation may distort the bacterial cells and damage the smear.
• Cover the entire smear with carbol fuchsin for the required staining time without allowing the stain to dry on the slide.
• Heat the carbol fuchsin gently until steam appears during the Ziehl–Neelsen method. Avoid boiling or overheating the stain, as it may damage the smear and produce staining deposits. Insufficient heating may also give poor staining.
• Decolorize the smear for the specified time. Over-decolorization may remove carbol fuchsin from acid-fast bacilli and give a false-negative result. Under-decolorization may cause non-acid-fast cells to remain red.
• Use staining reagents of the correct concentration. Old or contaminated reagents may affect the staining results. Filter the carbol fuchsin if precipitates are present.
• Wash the slide gently with clean, filtered or distilled water. A strong stream of water may remove the smear from the slide.
• Keep the slides separate during staining and use clean staining racks to prevent cross-contamination.
• Stain precipitates, dust and other particles may resemble acid-fast bacilli under the microscope. Use clean slides and properly filtered staining solutions to reduce these artifacts.
• Allow the stained smear to dry completely. Do not wipe or rub the smear with tissue paper.
• Examine the slide systematically under the 100× oil-immersion objective. The microscope lenses should be clean, and the immersion oil must be removed from the objective after examining an AFB-positive slide.
• Use positive and negative control slides with each staining batch. The positive control is used to check the staining reaction, while the negative control helps to detect improper decolorization and contamination.
• Carbol fuchsin contains phenol, which is toxic and can cause severe skin and eye burns. Wear protective gloves, a laboratory coat and suitable eye protection. Avoid contact with the skin and inhalation of vapours.
• Acid-alcohol contains corrosive acid and flammable alcohol. Handle it carefully and keep it away from flames and other ignition sources.
• Clinical specimens suspected of containing Mycobacterium tuberculosis must be handled by trained personnel under approved tuberculosis laboratory biosafety procedures. Use appropriate containment, personal protective equipment and aerosol-control measures. Heat fixation does not reliably kill all tubercle bacilli.
• Do not use an open flame inside a biological safety cabinet, as it may disturb the airflow. Contaminated slides, staining materials and chemical wastes must be discarded according to the laboratory’s biosafety and chemical waste disposal procedures.

Limitations of Acid-Fast Staining

The following are the limitations of acid-fast staining:

• Acid-fast smear microscopy has low sensitivity when the number of bacilli in a specimen is small. Conventional bright-field microscopy generally requires approximately 5,000–10,000 bacilli per mL of sputum for detection.
• A negative AFB smear does not exclude tuberculosis. The bacilli may not be detected in patients with early disease, childhood tuberculosis or extrapulmonary tuberculosis.
• Acid-fast staining cannot distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria. Additional tests are required to identify the species.
• The staining method cannot determine whether the observed bacilli are living or dead. Non-viable bacilli may also give a positive acid-fast staining result.
• Acid-fast staining cannot determine whether the bacteria are susceptible or resistant to anti-tuberculosis drugs.
• The accuracy of the staining result depends on the quality of the specimen and smear preparation. Very thick or thin smears may give false-negative results.
• Excessive decolorization may remove the primary stain from acid-fast bacilli, while incomplete decolorization may produce false-positive staining results.
• Contaminated slides, staining solutions and water may give false-positive results. Stain precipitates and other artifacts may also be mistaken for acid-fast bacilli.
• The results of microscopic examination depend on the experience of the laboratory personnel and the quality of the microscope. Small numbers of bacilli may be missed during examination.
• Some partially acid-fast organisms, such as Nocardia, may lose the primary stain when strong decolorizing solutions are used. Modified acid-fast staining methods are required for their detection.
• Conventional Ziehl–Neelsen staining requires examination under the oil-immersion objective and is generally less sensitive than fluorochrome acid-fast microscopy.

Applications of Acid-Fast Staining

The following are the applications of acid-fast staining:

• Acid-fast staining is used to detect acid-fast bacilli in sputum specimens collected from patients suspected of pulmonary tuberculosis.
• Respiratory specimens such as bronchoalveolar lavage samples can also be examined for the presence of acid-fast bacilli.
• It is used in the examination of lymph node aspirates, tissue sections and certain body fluid specimens in suspected extrapulmonary tuberculosis. The sensitivity is often low when the number of bacilli is small.
• During tuberculosis treatment, sputum smear examination is used to monitor changes in the number of acid-fast bacilli. The staining method cannot distinguish between living and dead bacilli.
• AFB smear grading helps in estimating the bacillary load and assessing the possible infectiousness of patients with pulmonary tuberculosis.
• Acid-fast staining is used for the detection of nontuberculous mycobacteria in clinical specimens. Further tests are required for species identification.
• Mycobacterial cultures can be stained to examine the acid-fast characteristics of the isolated bacteria.
• Modified acid-fast staining methods are used to detect Mycobacterium leprae in skin smears and tissue sections from patients with leprosy.
• Partially acid-fast bacteria such as Nocardia species can be detected using modified acid-fast staining with a weaker decolorizing solution.
• In microbiology laboratory practicals, acid-fast staining is used to differentiate acid-fast bacteria such as Mycobacterium smegmatis from non-acid-fast bacteria.

Advantages of Acid-Fast Staining

The following are the advantages of acid-fast staining:

• Acid-fast staining is a simple technique that can be performed using basic laboratory equipment and staining reagents.
• The method is inexpensive compared to culture and molecular diagnostic methods.
• It gives results within a short time. Acid-fast bacilli can be examined directly in a stained smear without waiting for bacterial growth in culture.
• The Ziehl–Neelsen method requires only a light microscope with an oil-immersion objective for examination of the stained smear.
• Acid-fast staining helps to differentiate acid-fast bacteria from non-acid-fast bacteria by their staining reaction.
• Acid-fast bacilli can be easily observed as red or pink rods against a blue background in properly stained smears.
• Sputum smear microscopy helps in the rapid detection of patients with a high number of acid-fast bacilli, including many infectious cases of pulmonary tuberculosis.
• The number of acid-fast bacilli can be reported by smear grading, which provides a semi-quantitative estimate of the bacillary load.
• It is used during tuberculosis treatment follow-up to observe changes in the number of acid-fast bacilli in sputum specimens.
• The technique can be carried out in peripheral laboratories where advanced diagnostic equipment is not available.

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