Flagella Staining – Principle, Reagents, Procedure and Results

In flagella staining, a suitable bacterial sample is prepared on a slide and stained with the required reagents. The stained preparation is then examined under a microscope. Bacterial flagella are delicate and may get detached from the bacterial cells during rough handling. The bacterial culture should be suitable and transferred carefully to the slide.

Ryu stain and Leifson stain are two methods used for flagella staining. The Ryu method can be carried out using a wet mount. It uses crystal violet along with mordant components. In the Leifson method, a pararosaniline-based dye is used to stain an air-dried smear.

The preparation of the sample and the staining steps depend upon the method used. A wet mount and an air-dried smear cannot be prepared by the same procedure.

What is Flagella Staining?

Flagella staining is a special microbiological staining technique used to make the thin bacterial flagella visible under a microscope. The flagella are thread-like structures present on the surface of some bacterial cells. This technique helps to observe the presence, number and arrangement of flagella on bacterial cells.

Flagella staining is also known as flagella stain or flagellar stain. Ryu stain and Leifson stain are two methods used for staining bacterial flagella. These are specific staining methods and not other names for flagella staining.

Flagella staining is used to examine bacterial flagella, but it does not directly show the movement of bacteria. Bacterial motility is examined separately by observing living bacterial cells.

Purpose of Flagella Staining

The main purposes of flagella staining are as follows:

• It is used to detect the presence of flagella on bacterial cells.
• It helps to determine the number of flagella present on a bacterial cell.
• The arrangement and position of bacterial flagella can be examined under a microscope.
• It is used to study the morphological characteristics of bacteria based on their flagella.
• The number and arrangement of flagella help in the identification and differentiation of bacteria. These characteristics alone cannot confirm the identity of a bacterial species. Other characteristics and tests are also required.

Principle of Flagella Staining

The principle of flagella staining is based on increasing the thickness of bacterial flagella by depositing staining material around them. Bacterial flagella are very thin, about 10–30 nm in diameter. They cannot be resolved as individual filaments under a conventional bright-field microscope because their diameter is below the resolving power of the microscope.

In flagella staining, mordants such as tannic acid help in the deposition of staining material around the flagella. The deposited stain forms a coating around the flagella and increases their apparent thickness. The dye also provides contrast against the background. The stained flagella then become visible under the light microscope.

The precipitation of staining material depends upon the reagents used in the staining method. Different methods may involve different staining and precipitation reactions.

Materials and Reagents Required for Flagella Staining

The following materials and reagents are required for flagella staining. The reagents are different for Ryu and Leifson staining methods.

Materials Required

• Glass Slides and Coverslips – Clean glass slides are used for preparing bacterial samples. Coverslips are required for the wet-mount method.
• Compound Microscope – A microscope with suitable objectives is required to examine the stained flagella. Immersion oil is used with the oil-immersion objective.
• Bacterial Culture – A suitable young bacterial culture grown in broth or on solid medium.
• Inoculating Loop or Needle – These are used to transfer bacterial samples from the culture to the slide.
• Distilled Water – It is used for preparing bacterial suspensions and staining solutions.
• Pipettes and Test Tubes – These are used for preparing and transferring bacterial samples and reagents.
• Staining Rack and Wash Bottle – The staining rack is used to hold the slides during staining. The wash bottle is used for washing the slides.
• Other Materials – Bibulous paper, glass droppers, wax pencil and slide-cleaning materials. A centrifuge may also be required depending upon the method used.
• Protective Equipment – Laboratory coat, gloves and safety goggles are required for handling the staining reagents. Chemicals should be handled with suitable ventilation and disposed of according to laboratory safety guidelines.

Reagents for Ryu Flagella Staining

The following reagents are used in the modified Ryu formulation described in Collins and Lyne’s Microbiological Methods.

• Crystal Violet – It is used to stain the bacterial flagella. The staining solution contains 12 g of crystal violet in 100 mL of ethanol.
• Tannic Acid – It acts as a mordant and helps in the deposition of staining material around the flagella. The mordant solution contains 10 g of tannic acid.
• Aluminium Potassium Sulfate – It is used as a mordant. The formulation contains 50 mL of saturated aluminium potassium sulfate dodecahydrate solution.
• Phenol – It is used as a preservative. The mordant contains 50 mL of 5% aqueous phenol. Phenol is toxic and corrosive and should be handled carefully.
• Staining Solution – The mordant solution contains tannic acid, aluminium potassium sulfate and phenol. Ten parts of this solution are mixed with one part of the crystal violet solution. The mixture is kept for 3 days before use. Other Ryu formulations may require different preparations.

Reagents for Leifson Flagella Staining

The following reagents are used in the Leifson formulation described by the American Society for Microbiology.

• Pararosaniline Dye – It is used to stain the bacterial flagella. The dye solution contains 0.9 g of pararosaniline acetate and 0.3 g of pararosaniline hydrochloride in 100 mL of 95% ethanol. These dyes should be handled carefully.
• Tannic Acid – It is used as a mordant. The solution contains 3 g of tannic acid dissolved in 100 mL of distilled water.
• Sodium Chloride – It helps to maintain the salt concentration required for staining. The solution contains 1.5 g of sodium chloride in 100 mL of distilled water.
• Ethanol – It is used as a solvent for the dye components. The evaporation of ethanol helps in the precipitation of staining material. It is flammable and should be kept away from open flames.
• Distilled Water – It is used to dissolve tannic acid and sodium chloride.
• Leifson Staining Solution – Equal volumes of tannic acid and sodium chloride solutions are mixed. Two volumes of this mixture are then added to one volume of the dye solution.

Procedure of Flagella Staining

Flagella staining can be performed by Ryu and Leifson methods. The preparation of the bacterial sample and the staining procedure are different for these two methods.

Preparation of Bacterial Culture and Slides

1. Select a young bacterial culture grown on a suitable medium. The growth temperature and culture age depend upon the bacterial species and the staining method.
2. Clean the glass slides properly to remove dust, grease and other materials. Clean coverslips are also required for the Ryu wet-mount method.
3. Transfer a small amount of bacterial culture using a sterile inoculating loop. The bacterial cells should be handled carefully because rough handling may detach the flagella.
4. The bacterial suspension should be slightly cloudy. A thick suspension may interfere with the observation of flagella.
5. In the Ryu method, the bacterial suspension is examined as a wet mount. The Leifson method requires an air-dried smear without heat fixation.

Ryu Flagella Staining – Wet Mount Method

The following are the steps involved in the Ryu wet-mount method described by Heimbrook and coworkers:

1. Take a clean glass slide and place a small drop of sterile distilled water on it.
2. Dip a sterile inoculating loop into distilled water and touch the edge of a colony from a suitable young culture. Allow the bacteria to enter the water.
3. Transfer the bacteria to the water drop on the slide without vigorous mixing.
4. Place a clean coverslip over the bacterial suspension. The water should be just enough to fill the space under the coverslip.
5. Examine the wet mount under the 40× objective to check for bacterial motility.
6. If motile cells are present, leave the slide at room temperature for 5–10 minutes. During this time, some bacterial cells attach to the slide or coverslip.
7. Add two drops of Ryu flagella stain carefully at the edge of the coverslip. The stain enters under the coverslip by capillary action and mixes with the bacterial suspension.
8. Allow the stain to act for 5–10 minutes at room temperature.
9. Examine the stained preparation under the 100× oil-immersion objective of a bright-field microscope. The flagella can be observed in the region where the staining concentration is suitable, usually between the edge and centre of the coverslip. The cells attached to the coverslip may give a clearer view of the flagella.

The coverslip is not removed and the preparation is not washed during this wet-mount staining procedure.

Leifson Flagella Staining – Smear Method

The following are the steps involved in the Leifson smear method described in the American Society for Microbiology protocol:

1. Take a suitable young bacterial culture. The described protocol uses cultures grown for about 16–20 hours, although the culture conditions may differ for other bacteria.
2. Prepare a slightly cloudy bacterial suspension in distilled water. If a broth culture is used, the cells are washed by two rounds of centrifugation and resuspended in distilled water to remove the culture medium.
3. Take a clean glass slide and place a small drop of the bacterial suspension near one end.
4. Spread the suspension carefully to prepare a thin smear on the slide.
5. Allow the smear to dry completely at room temperature. Do not heat-fix the smear because heat may damage the flagella.
6. Draw a rectangular boundary around the dried smear using a wax pencil and place the slide on a staining rack.
7. Cover the smear with Leifson staining solution.
8. Allow the stain to remain on the slide for 7–15 minutes at room temperature. A golden film and fine precipitate appear during staining. The required staining time may differ depending upon the staining solution.
9. Remove the stain carefully using a gentle flow of water. The film should be washed off without disturbing the bacterial smear.
10. Allow the slide to air dry.
11. Examine the dried slide under the 100× oil-immersion objective of a bright-field microscope, at approximately 1000× total magnification.

Results and Interpretation of Flagella Staining

The stained bacterial cells and their flagella are examined under a microscope. In the crystal violet Ryu method, the bacterial cells and flagella appear violet or purple. In the Leifson method, they appear red. The flagella are seen as thin filaments extending from the bacterial cells.

Positive and Negative Staining Results

1. Positive Result – The stained flagella are visible around the bacterial cells. The number and position of the flagella can be determined when they are clearly visible. The presence of flagella does not directly show active bacterial movement.
2. Negative Result – No flagella are visible around the bacterial cells. This should be recorded as “flagella not visualized”. A single negative field does not confirm the absence of flagella because the flagella may be detached or the staining may not be sufficient. Other microscopic fields should also be examined.

Interpretation of Flagellar Arrangement

The arrangement of bacterial flagella can be determined by observing their number and position on the stained bacterial cells. The following arrangements may be observed:

• In monotrichous arrangement, a single flagellum is present at one pole of the bacterial cell.
• In lophotrichous arrangement, a tuft of flagella is present at one pole of the bacterial cell.
• In amphitrichous arrangement, flagella are present at both poles of the bacterial cell.
• In peritrichous arrangement, flagella are distributed around the bacterial cell.

The arrangement may be difficult to determine when the bacterial cells overlap or stain precipitates are present. Only clearly visible flagella attached to the bacterial cells should be used to determine the arrangement.

Precautions and Quality Control of Flagella Staining

The following precautions should be taken during flagella staining:

• The glass slides should be clean and free from dust and grease. These materials may also take up the stain.
• A young bacterial culture is used for staining. Old cultures may have fewer flagella or may lose their flagella.
• The bacterial cells are transferred carefully to the slide. Flagella are delicate and may get detached from the cells during vigorous shaking or mixing.
• Thick bacterial smears are not suitable for the clear observation of flagella.
• In Leifson staining, the bacterial smear is allowed to dry at room temperature. Heat fixation is not done as it may damage the flagella.
• The staining time depends upon the method used. Excessive staining may produce dye precipitates on the slide. Old or improperly stored staining solutions may also give poor results.
• The Leifson-stained slide is washed gently to remove the excess stain. Strong washing may remove the smear or damage the flagella. No washing is done in the Ryu wet-mount method.
• Dye precipitates may appear as thin filaments. These may be confused with bacterial flagella. The flagella should be clearly attached to the bacterial cells before determining their arrangement.
• A known flagellated bacterial strain is used as a positive control. The flagella should be visible in this preparation. If the positive control does not show flagella, the staining should be repeated.
• A known non-flagellated bacterial strain is used as a negative control. No flagella should be present in this preparation. The presence of stained filaments may indicate contamination or stain precipitates.
• Several microscopic fields should be examined before recording the result. The absence of visible flagella in one field does not mean that the bacterial cells have no flagella.
• Proper aseptic techniques should be followed during the handling of bacterial cultures. The biosafety precautions depend upon the organism used. Contaminated materials should be disinfected or disposed of properly.
• Laboratory coats, gloves and safety goggles should be used during staining. Phenol is toxic and corrosive. Phenol-containing staining solutions should be handled in a chemical fume hood with suitable protective gloves.
• Ethanol is flammable and should be kept away from open flames. The safety data sheets should be followed for handling, storage and disposal of the staining chemicals.

Limitations of Flagella Staining

The following are the limitations of flagella staining:

• The staining of bacterial flagella is difficult because they are very thin and delicate.
• Flagella may break or get detached from the bacterial cells during sample preparation and washing.
• A young bacterial culture is required. Old cultures may have fewer flagella or may lose them. The number of flagella also depends upon the growth conditions of the bacteria.
• Thick smears are not suitable for the clear observation of flagella.
• There is a chance of confusing dye precipitates with bacterial flagella.
• The results depend upon the staining time and the preparation of staining reagents.
• Overlapping bacterial cells make it difficult to determine the number and arrangement of flagella.
• Sometimes, the flagella are not visible even after staining. This does not mean that the flagella are absent.
• It cannot be used to determine the active movement of bacteria.
• The number and arrangement of flagella alone cannot identify a bacterial species.
• Some methods require more time and use hazardous chemicals such as phenol.

Uses of Flagella Staining

The following are the uses of flagella staining:

• It is used to detect the presence of flagella in bacterial cells.
• The number and arrangement of bacterial flagella can be determined by staining.
• It helps to differentiate between bacteria having polar and peritrichous flagella.
• The arrangement of flagella is used as an additional characteristic in the identification of certain bacterial species.
• It is used to study bacterial flagella under a light microscope.
• The stained preparation can be used to compare the flagellar arrangements of different bacterial cultures.
• It helps to examine the flagella of motile bacteria. However, the staining does not directly show the movement of bacteria.
• Used in microbiology practical classes to demonstrate different types of flagellar arrangements.

Advantages of Flagella Staining

The following are the advantages of flagella staining:

• It helps to make the thin bacterial flagella visible under a light microscope.
• The number and arrangement of bacterial flagella can be determined by staining.
• It is used to differentiate between bacteria having different flagellar arrangements.
• The technique does not require an electron microscope.
• The arrangement of flagella is an additional characteristic used in the identification of certain bacterial species.
• The Ryu wet-mount method is simple and can be performed using ordinary glass slides and coverslips.
• In the Ryu method, the bacterial cells are examined without preparing a heat-fixed smear.
• The Ryu staining solution remains stable at room temperature for a long period.
• It can be performed using equipment commonly available in microbiology laboratories.

References

1. Breakwell, D. P., Moyes, R. B., & Reynolds, J. (2009). Differential staining of bacteria: Flagella stain. Current Protocols in Microbiology, 15(1), A.3G.1–A.3G.5. https://doi.org/10.1002/9780471729259.mca03gs15
2. Clark, W. A. (1976). A simplified Leifson flagella stain. Journal of Clinical Microbiology, 3(6), 632–634. https://doi.org/10.1128/jcm.3.6.632-634.1976
3. Collins, C. H., Lyne, P. M., Grange, J. M., & Falkinham, J. O. (Eds.). (2004). Collins and Lyne’s microbiological methods (8th ed.). Arnold. https://search.worldcat.org/title/424037460
4. Heimbrook, M. E., Wang, W. L., & Campbell, G. (1989). Staining bacterial flagella easily. Journal of Clinical Microbiology, 27(11), 2612–2615. https://doi.org/10.1128/jcm.27.11.2612-2615.1989
5. Leifson, E. (1951). Staining, shape, and arrangement of bacterial flagella. Journal of Bacteriology, 62(4), 377–389. https://doi.org/10.1128/jb.62.4.377-389.1951
6. Leifson, E., & Hugh, R. (1953). Variation in shape and arrangement of bacterial flagella. Journal of Bacteriology, 65(3), 263–271. https://doi.org/10.1128/jb.65.3.263-271.1953