Kinyoun Stain Method – Principle, Procedure and Results

Kinyoun stain method involves staining a prepared smear with Kinyoun carbol fuchsin at room temperature without heating the staining solution. This method is also known as the Kinyoun cold method or cold method of acid-fast staining.

In this method, a smear is first prepared on a glass slide and fixed. The smear is then stained with carbol fuchsin and decolorized using acid-alcohol. A counterstain is added to the smear, and the stained slide is examined under a microscope.

The term cold refers to the absence of heating during primary staining. However, the smear may still be heat-fixed during preparation, depending upon the laboratory protocol. In the conventional Ziehl-Neelsen method, carbol fuchsin is heated during staining.

What is the Kinyoun Stain Method?

The Kinyoun stain method is a differential acid-fast staining technique in which carbol fuchsin is used without heating the stain during its application. It is also known as the Kinyoun cold method or cold acid-fast staining method.

The Kinyoun method is used to demonstrate the presence of acid-fast microorganisms, particularly bacteria belonging to the genus Mycobacterium. The conventional method is mainly used for strongly acid-fast organisms. Modified Kinyoun staining is used for organisms that are weakly acid-fast, such as Nocardia species.

Kinyoun staining is one of the methods of acid-fast staining. Other methods include Ziehl-Neelsen staining.

Purpose of Kinyoun Stain Method

The following are the purposes of Kinyoun staining:

1. It is used to detect the presence of acid-fast microorganisms in prepared samples.
2. It helps to differentiate acid-fast cells from non-acid-fast cells based on their response to acid-alcohol decolorization.
3. It is used for microscopic examination of samples suspected to contain acid-fast microorganisms.
4. The method demonstrates the acid-fast property of microorganisms. However, a positive stain does not identify the exact species or confirm tuberculosis.

Principle of Kinyoun Staining

The principle of Kinyoun staining is based on the acid-fast property of bacteria having a cell envelope rich in mycolic acids. These mycolic acids form a waxy layer which makes it difficult for ordinary stains to enter the cells. Once stained with carbol fuchsin, the acid-fast cells retain the dye even after treatment with acid-alcohol.

In the Kinyoun cold method, a higher concentration of basic fuchsin and phenol is used in the primary staining solution. Phenol acts as a mordant and helps carbol fuchsin penetrate the waxy cell envelope. The higher concentration of the staining solution allows the dye to enter the cells without heating.

The retention of carbol fuchsin is due to the resistance of the mycolic-acid-rich cell envelope to decolorization by acid-alcohol. Non-acid-fast cells do not have this property. They lose the primary stain during decolorization and take up the counterstain.

Reagents and Materials Required for Kinyoun Staining

The following are the reagents and materials required for Kinyoun staining. The reagent composition given below is for the conventional Kinyoun method.

Kinyoun Carbol Fuchsin

Kinyoun carbol fuchsin is the primary staining solution. It consists of the following components:

1. Basic Fuchsin– 4 g of basic fuchsin is used as the primary dye. It stains the acid-fast cells red.
2. Phenol– 8 g of phenol crystals are required. Phenol acts as a mordant and helps the dye enter the waxy cell envelope.
3. Ethanol (95%)– 20 mL of ethanol is used to dissolve basic fuchsin.
4. Distilled Water– Phenol is dissolved in 100 mL of distilled water. The two solutions are then mixed to prepare Kinyoun carbol fuchsin.

Acid-Alcohol Decolorizer

The following reagents are used to prepare the acid-alcohol decolorizer:

1. Hydrochloric Acid– 3 mL of concentrated hydrochloric acid is required.
2. Ethanol (95%)– 97 mL of ethanol is mixed with hydrochloric acid. The acid-alcohol solution is used to remove the primary stain from non-acid-fast cells.
3. Modified Decolorizer– In the modified Kinyoun method, a weaker decolorizer such as 1% sulphuric acid may be used for weakly acid-fast organisms.

Counterstain

The following counterstains are used in Kinyoun staining:

1. Methylene Blue– 0.3 g of methylene blue chloride is dissolved in 100 mL of distilled water to prepare a 0.3% solution. It stains the decolorized cells and background blue.
2. Brilliant Green or Malachite Green– These are used as alternative counterstains in some Kinyoun staining protocols. They stain the non-acid-fast cells and background green.

Laboratory Materials

The following are the laboratory materials required for Kinyoun staining:

1. Glass Slides– Clean and grease-free glass slides are required for the preparation of smears.
2. Inoculating Loop or Pipette– A sterile inoculating loop or disposable pipette is used to transfer the sample onto the slide.
3. Smear Fixation Materials– A slide warmer or suitable chemical fixative may be required for smear fixation, depending upon the laboratory protocol.
4. Staining Rack– It is used to hold the slides during staining.
5. Wash Bottle– A wash bottle containing distilled water is required for washing the slides.
6. Staining Containers– Reagent bottles, droppers and suitable waste containers are required for handling the staining solutions.
7. Microscope– A compound light microscope with a 100× oil-immersion objective is used to examine the stained smear. Immersion oil is also required.
8. Protective Equipment– Laboratory coat, gloves and protective eyewear are required. Phenol is toxic, hydrochloric acid is corrosive and ethanol is flammable. Proper ventilation and laboratory containment are required while handling the chemicals and potentially infectious samples.

Procedure of Kinyoun Staining

The procedure of Kinyoun staining is given below:

1. Take a suitable specimen or laboratory-prepared sample for Kinyoun staining.
2. Suspected mycobacterial specimens are handled in a Class I exhaust protective cabinet in a Containment Level 3 laboratory under the specified protocol.
3. A thin and uniform smear of the specimen is prepared on a clean glass slide.
4. The smear is allowed to air dry completely and is then fixed with methanol. Methanol fixation alone should not be considered sufficient to inactivate mycobacteria unless the method has been validated.
5. Prepare a known acid-fast positive control and a proven negative control. These are stained along with the test smear.
6. The slide is placed on a staining rack and the smear is covered with Kinyoun’s carbol fuchsin.
7. The smear is stained for 5 minutes at room temperature. Heating is not required.
8. The excess stain is removed by washing gently with distilled or deionized water until the water runs clear.
9. Add acid-alcohol containing 3% hydrochloric acid in ethanol and allow it to act for 3 minutes. Wash the slide with water.
10. Repeat the decolorization for 1–2 minutes or until no more red colour comes off the smear.
11. The slide is washed again with distilled or deionized water and tilted to remove the excess water.
12. Cover the smear with methylene blue and allow it to stain for 3–4 minutes.
13. Wash the slide gently with distilled water and allow it to air dry.
14. The stained smear is examined first under the 40× objective and then under the 100× oil immersion objective.
15. The acid-fast organisms appear red, while the non-acid-fast organisms and background material appear blue.
16. Examine the positive and negative control slides. The positive control must show red acid-fast organisms, while the negative control should not contain any red acid-fast organisms. The test result is accepted only when both controls give the expected results.

Results and Interpretation of Kinyoun Staining

The results of Kinyoun staining are given below:

Acid-Fast Positive Result

• Acid-fast organisms retain the carbol fuchsin after treatment with acid-alcohol.
• They appear red, pink, or fuchsia under the microscope.
• Acid-fast mycobacteria generally appear as slender rod-shaped bacilli.
• The carbol fuchsin remains in the acid-fast cells even after decolorization.

Acid-Fast Negative Result

• Non-acid-fast organisms lose the carbol fuchsin during decolorization.
• When methylene blue is used as the counterstain, these cells appear blue.
• If a green counterstain such as malachite green is used, the non-acid-fast cells and background appear green.

Interpretation of Microscopic Findings

• The presence of red or pink bacilli after decolorization indicates an acid-fast-positive result.
• Acid-fast bacilli present in a clinical specimen are reported as AFB when required by the laboratory protocol.
• A positive acid-fast result does not identify the organism as Mycobacterium tuberculosis. Other mycobacteria can also give a positive result.
• Culture, molecular tests, and other identification methods are required for species identification.
• The number of acid-fast bacilli can be graded when grading is required by the selected clinical protocol.

Comparison of Acid-Fast Positive and Acid-Fast Negative Results

Modifications of Kinyoun Staining

The Kinyoun staining method is modified for certain organisms by changing the staining reagents or decolorizing solution. The following are the modifications of Kinyoun staining:

Modified Kinyoun Staining for Weakly Acid-Fast Bacteria

• In modified Kinyoun staining, 1% sulphuric acid is used instead of 3% hydrochloric acid for decolorization.
• The weaker acid removes less of the primary stain. It is used for staining weakly acid-fast bacteria such as Nocardia.
• Nocardia species are partially acid-fast and appear as red, branching filaments. Some parts of the filaments may remain unstained.
• This method is also used for certain species of Rhodococcus, Gordonia, Actinomadura and Tsukamurella. The degree of acid-fastness varies among these organisms.
• Malachite green or brilliant green may be used in place of methylene blue. The non-acid-fast cells then appear green instead of blue.
• In another modification, 20% sulphuric acid is used followed by 95% alcohol for decolorization. This is different from the weaker acid method used for partially acid-fast bacteria.

Modified Kinyoun Staining for Intestinal Coccidia

• The modified Kinyoun staining method is used to detect the oocysts of intestinal parasites such as Cryptosporidium, Cyclospora and Cystoisospora.
• A thin smear is prepared from fresh or formalin-preserved stool, usually after concentration. The smear is allowed to dry and fixed with methanol.
• In one modified method, the smear is stained with Kinyoun carbol fuchsin for 1 minute without heating.
• The smear is decolorized for 2 minutes using a solution containing 10 mL sulphuric acid and 90 mL absolute ethanol. It is then counterstained with 3% malachite green for 2 minutes.
• Some modified methods use 1–3% aqueous sulphuric acid without alcohol. The concentration and staining time depend upon the method used.
• The stained oocysts usually appear pink or red against a green background. When methylene blue is used, the background appears blue.
• Cryptosporidium oocysts are usually round and appear pink to red. Some oocysts may remain unstained.
• Cyclospora oocysts show variable staining. Some appear red or pink, while others remain pale or unstained.
• Cystoisospora oocysts may appear pink, with the internal structures staining more strongly. Their size and shape are also examined during identification.

Tergitol-Assisted Kinyoun Staining

• Tergitol No. 7 is used as a wetting agent in some modifications of Kinyoun staining.
• It reduces the surface tension and helps carbol fuchsin enter the waxy cell envelope without heating.
• In one formulation, 1 drop of Tergitol No. 7 is added to 30–40 mL of Kinyoun carbol fuchsin.
• This modification is used in certain staining protocols. Tergitol is not required in all modified Kinyoun staining methods.

Kinyoun Stain vs Ziehl-Neelsen Stain

Kinyoun staining and Ziehl-Neelsen staining are two methods used for staining acid-fast bacteria. Both methods use carbol fuchsin as the primary stain. The main difference between these methods is the use of heat during staining.

In Ziehl-Neelsen staining, heat is used to help carbol fuchsin enter the waxy cell wall. Kinyoun staining is carried out without heating. It uses a more concentrated carbol fuchsin solution to stain the acid-fast cells.

The differences between Kinyoun staining and Ziehl-Neelsen staining are given below:

The concentration of carbol fuchsin, phenol and other reagents may vary in different laboratory protocols. The staining time and decolorizing solutions also depend upon the method used.

Both methods demonstrate the acid-fast property of microorganisms. A positive staining result does not identify the exact species of the organism.

Applications of Kinyoun Staining

The following are the applications of Kinyoun staining:

• It is used to differentiate acid-fast bacteria from non-acid-fast bacteria.
• Kinyoun staining is used to detect acid-fast bacilli in sputum samples from patients suspected of pulmonary tuberculosis.
• It is used for the microscopic examination of mycobacteria present in culture smears.
• Modified Kinyoun staining is used to detect weakly acid-fast bacteria such as Nocardia and Rhodococcus species.
• It is used to detect the oocysts of Cryptosporidium, Cyclospora, and Cystoisospora in stool samples by the modified staining method.
• Kinyoun staining is also used in microbiology laboratories to demonstrate acid-fastness without heating the primary stain.

Limitations of Kinyoun Staining

The following are the limitations of Kinyoun staining:

• Kinyoun staining is less sensitive than fluorescent staining methods for detecting acid-fast bacilli.
• A small number of acid-fast bacilli in the sample may not be detected. A negative staining result does not exclude tuberculosis.
• It cannot differentiate Mycobacterium tuberculosis from other acid-fast mycobacteria.
• The staining method cannot distinguish between living and dead bacteria.
• Weakly acid-fast bacteria such as Nocardia may lose the primary stain during decolorization. A weaker decolorizing solution is required for their detection.
• Excessive decolorization may give false-negative results, while insufficient decolorization may give false-positive results.
• The staining results depend upon the preparation of the smear, staining reagents, and proper microscopic examination.
• Contamination of the staining reagents may give false-positive results.
• Kinyoun staining cannot be used to determine whether the bacteria are resistant to antibiotics.

Advantages of Kinyoun Staining

The following are the advantages of Kinyoun staining:

• Kinyoun staining does not require heating of the primary stain.
• It is a simple method and can be performed using basic laboratory equipment.
• The absence of heating reduces exposure to phenol vapours during staining.
• It is a rapid staining method and can be used for the detection of acid-fast bacilli in clinical samples.
• A large number of smears can be stained without heating the stain.
• The stained bacteria can be examined using an ordinary light microscope. No fluorescence microscope is required.
• Acid-fast bacteria appear red, while non-acid-fast bacteria take the colour of the counterstain.
• Modified Kinyoun staining can also be used to detect weakly acid-fast organisms such as Nocardia.

References

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