Flagella Staining - Principle, Procedure and Types | Leifson Method

By Momina Shehzadi | BS Zoology Graduate, Pakistan

In this article, we will learn Flagella Staining - Principle, Procedure, Leifson Method and Types - in simple words with diagram. This topic is very important for BS Zoology and Microbiology students.

INTRODUCTION

Flagella staining is a special staining technique used to visualize bacterial flagella. Flagella are long, thin, hair like appendages that help bacteria in motility.

Flagella are very thin, only 20 nanometer in diameter, so they cannot be seen with light microscope by simple staining or Gram staining. Electron microscope can see flagella but electron microscope is expensive and not available in routine laboratory.

Therefore special flagella staining methods are used which increase thickness of flagella by depositing stain around flagella, making them thick enough to be visible under light microscope.

Flagella staining is important to identify motile bacteria and to differentiate bacteria on basis of flagellar arrangement which is important in classification.

WHAT IS FLAGELLA

Flagella are organelles of motility in bacteria. They are made of protein flagellin.

Structure of flagellum has three parts:

1. Filament: Long outer part made of flagellin protein, responsible for motility

2. Hook: Curved portion connecting filament to basal body

3. Basal body: Embedded in cell wall and membrane, acts as motor with rings, rotates flagellum using proton motive force

Size: Flagella are much longer than bacterial cell, about 5 to 10 micrometer long but very thin 20 nm diameter.

Arrangement of flagella:

a) Monotrichous: Single flagellum at one pole. Example Vibrio cholerae

b) Amphitrichous: Single flagellum at both poles. Example Alcaligenes faecalis

c) Lophotrichous: Tuft or cluster of flagella at one pole. Example Pseudomonas aeruginosa

d) Peritrichous: Flagella all around cell body. Example Escherichia coli, Salmonella Typhi, Proteus mirabilis

e) Atrichous: No flagella, non motile. Example Staphylococcus, Corynebacterium

Function: Motility, chemotaxis movement towards nutrients, role in virulence because motile bacteria can reach target tissue quickly, formation of biofilm.

PRINCIPLE OF FLAGELLA STAINING

Principle is based on increasing diameter of flagella by deposition of mordant and stain around flagella.

Flagella are too thin to be seen under light microscope. In flagella staining, a mordant like tannic acid is used which coats flagella and makes them thicker. Mordant precipitates around flagella and increases its diameter.

Then basic stain like basic fuchsin or crystal violet stains this thickened structure, making flagella visible as thick, wavy lines under microscope.

The staining is very delicate because flagella are easily detached from cell by shaking or heat. So special precautions are required: use young cultures, handle gently, do not heat fix strongly.

REQUIREMENTS FOR FLAGELLA STAINING - LEIFSON METHOD

Leifson method is most commonly used method for flagella staining.

Requirements:

Leifson stain which contains:

Solution A: Sodium chloride 1.5g, Distilled water 100ml

Solution B: Tannic acid 3g, Distilled water 100ml

Solution C: Basic fuchsin 1.2g, Ethanol 100ml - 95 percent

Mix equal volumes of Solution A and B, then add Solution C. This combined stain contains mordant tannic acid and sodium chloride plus stain basic fuchsin.

Other requirements: Young bacterial culture 16 to 18 hours old, clean grease free slides, microscope

Other Methods:

Gray method: Uses tannic acid, potassium alum and crystal violet

Bailey method: Uses crystal violet and ammonium oxalate

PROCEDURE OF LEIFSON FLAGELLA STAINING

This is very delicate procedure and needs careful handling.

Step 1: Preparation of Bacterial Suspension

Take very young culture 16 to 18 hours old grown on blood agar or nutrient agar. Young cultures have long, well developed flagella. Do not use old cultures because flagella may break.

Make very dilute suspension of bacteria in distilled water or saline. Do not centrifuge or vortex vigorously because flagella may detach.

Step 2: Preparation of Smear

Place a small drop of bacterial suspension on clean, grease free slide. The slide must be very clean, washed with soap, alcohol and dried. Grease free slide helps flagella to spread.

Allow drop to spread and air dry. Do not heat fix strongly. Only gentle warming or air drying. Strong heat will cause flagella to detach and coil.

Step 3: Staining

Flood smear with Leifson stain for 10 to 15 minutes. The stain contains mordant which deposits around flagella and makes them thicker. Keep slide undisturbed.

Step 4: Washing

Wash gently with water. Do not wash harshly. Air dry.

Step 5: Observation

Observe under oil immersion 100X lens. Focus carefully. First locate bacterial cells, then look for thin wavy lines extending from cells which are flagella.

Result:

Bacterial cells appear dark red or purple.

Flagella appear as thin, wavy, hair like structures extending from cells, same color as cell but slightly lighter, much longer than cell.

In peritrichous bacteria like E. coli, many flagella all around cell visible.

In monotrichous bacteria like Vibrio, single flagellum at one end visible.

PRECAUTIONS FOR FLAGELLA STAINING

1. Use very young culture 16 to 18 hours old because flagella are well developed and not broken.

2. Slides must be absolutely grease free, cleaned with chromic acid or alcohol, otherwise flagella will not spread and stain will precipitate.

3. Do not heat fix strongly, only air dry or very gentle heat, because flagella are delicate and easily detached.

4. Handle suspension very gently, no vigorous shaking, no centrifugation.

5. Staining time should be 10 to 15 minutes, do not over stain.

6. Observe quickly because stain fades with time.

7. This staining is difficult and may need repeated attempts to get good result. So it requires practice.

ALTERNATIVE METHOD TO DEMONSTRATE MOTILITY

Because flagella staining is difficult, motility of bacteria can also be demonstrated by other simpler methods:

1. Hanging Drop Method: Live bacteria observed under microscope in hanging drop, motile bacteria show darting motility.

2. Motility Test Medium: Semi solid medium with 0.4 percent agar and TTC indicator. Motile bacteria spread away from stab line, non motile grow only along stab line.

3. Dark Field Microscopy: Motile bacteria observed as bright objects moving in dark background.

But these methods only show motility, they do not show arrangement of flagella. For arrangement, flagella staining is required.

IMPORTANCE OF FLAGELLA STAINING

1. Identification: Flagellar arrangement helps in identification and classification of bacteria. For example, Pseudomonas can be differentiated from E. coli on basis of flagella: Pseudomonas is lophotrichous, E. coli is peritrichous.

2. Differentiation of Motile and Non Motile Bacteria: Important to differentiate species like motile Proteus mirabilis which swarms on agar from non motile Klebsiella.

3. Pathogenicity: Motile bacteria are often more virulent because they can move through mucus and reach target sites. Example motility helps Vibrio cholerae to penetrate intestinal mucus.

4. Serology: Flagellar antigens are called H antigens and are important for serotyping. For example Salmonella Typhi has O antigen on cell wall and H antigen on flagella. Widal test detects antibodies against O and H antigens.

5. Taxonomy: Flagellar arrangement is used as taxonomic character in Bergey's Manual of Systematic Bacteriology.

COMPARISON OF FLAGELLA AND PILI

Flagella are long, thick, few in number, made of flagellin, responsible for motility, visible by flagella staining. Pili are short, thin, numerous, made of pilin, responsible for attachment and conjugation, cannot be seen by light microscope, seen by electron microscope.

CONCLUSION

Flagella staining is delicate special staining technique to visualize thin bacterial flagella by increasing their thickness with mordant. Leifson method is most common method using tannic acid and basic fuchsin. Although difficult to perform, it gives important information about presence and arrangement of flagella which helps in identification and classification of bacteria. For BS Zoology students, it is important to understand principle and types of flagellar arrangement.

FAQs for Students - Flagella Staining

Q1: Why flagella cannot be seen by simple staining?

Ans: Because flagella are very thin, only 20 nanometer diameter, which is below resolving power of light microscope for unstained structures. They need mordant to increase thickness to become visible.

Q2: What is Leifson method of flagella staining?

Ans: Leifson method is most common flagella staining method using mixture of sodium chloride, tannic acid as mordant and basic fuchsin as stain. Mordant deposits around flagella making them thicker and visible. Staining time is 10 to 15 minutes.

Q3: Why young cultures are used for flagella staining?

Ans: Young cultures 16 to 18 hours old are used because flagella are well developed, long and intact in young actively growing bacteria. In old cultures flagella may break and detach.

Q4: Why grease free slides are required?

Ans: Grease free slides are required because grease prevents spreading of flagella and causes precipitation of stain. Clean slides allow flagella to spread properly and give clear background.

Q5: What is difference between peritrichous and lophotrichous flagella?

Ans: Peritrichous means flagella present all around bacterial cell, example E. coli and Salmonella. Lophotrichous means tuft or cluster of flagella at one pole, example Pseudomonas aeruginosa.

Q6: What is H antigen?

Ans: H antigen is flagellar antigen made of flagellin protein. It is heat labile and used for serotyping of bacteria like Salmonella. Antibodies against H antigen are detected in Widal test for typhoid diagnosis.

Also Read: Endospore Staining - Principle, Procedure and Result

Written by Momina Shehzadi, BS Zoology Graduate from Pakistan.

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