Flagella Stain- Principle, Procedure and Results Interpretation

 

Objective of Test

This test used to presumptive identification of motile bacterial species from non motile bacteria by presence and arrangement of flagella.

There are four types of flagellar bacteria based on presence and arrangement of flagella, such as Monotrichous, Amphitrichous, Lophotrichous and Peritrichous.

Principle

Most motile bacteria possess flagella, the shape, number, and position of which are important characteristics in the differentiation of genera and species identification, particularly when biochemical reactions are weak or equivocal. The Leifson staining technique (or modification) is most commonly used in clinical laboratories and is not difficult to perform, providing exact details that are followed in each step of the procedure.

Bacterial flagella can be stained by alcoholic solutions of rosaniline dyes that form a precipitate as the alcohol evaporates in the staining procedure. Basic fuchsin (pararosaniline acetate) serves as the primary stain with tannic acid added to the solution as a mordant. A counterstain, such as methylene blue, may be used to better visualize the bacteria in instances in which the primary stain is weak or does not react at all with the bacterial cell wall.

Media and Reagents

Leifson Method

1. Basic fuchsin, 1.2% in 95% alcohol

Commercial product must be certified for flagellar stain.

Pararosaniline acetate is preferred. If the dye solution is a mixture of pararosaniline acetate and pararosaniline hydrochloride, the hydrochloride compound must not compose more than two thirds of the solution.

The stain must have the odor of acetic acid to be satisfactory.

When new stain is prepared, at least 1 day must be allowed for the dye to enter completely into solution.

2. Tannic acid, 3% in water

The suspension must have a light yellow color to be satisfactory.

Add phenol in a 1:200 concentration to discourage emergence of molds during storage.

3. Sodium chloride, 1.5% in water

4. Final stain

Combine the three stock solutions in equal volumes. The stain is ready for use immediately after preparation and should be stored in a tightly stoppered bottle.

A precipitate will form during storage. This should not be disturbed; rather, remove the staining solution from the top with a pipette.

Shelf life of the staining solution is 1 week at room temperature, 1 month in the refrigerator, and indefinite if frozen.

Ryu Method

1. Solution I

5% phenol, 10.0 mL

Powdered tannic acid, 2.0 g

Saturated aluminum potassium sulfate 12-hydrate (crystals) (prepare 14g potassium alum in 100 ml distilled H2O), 10.0 ml.

2. Solution II

Saturated solution of crystal violet in alcohol (prepare 12 g in 100 mL of 95% ethanol)

3. Final stain

Ten parts of solution I (mordant) are mixed with one part of solution II and stored in a plastic bottle at room temperature.

Quality Control

The following bacterial species may serve as controls:

Positive control: Peritrichous, Escherichia coli; polar, monotrichous, Pseudomonas aeruginosa; multitrichous, Burkholderia cepacia

Negative control: Acinetobacter baumannii

Test Procedure

Leifson Method

Using a young culture on an appropriate agar medium, prepare a light suspension of the bacteria in water.

Place 2 drops of the bacterial suspension toward one end of an acid-cleaned microscope slide.

Allow to air-dry. With a wax pencil, draw a perpendicular line on the glass surface toward the opposite end from the dried suspension.

Place the slide on a tilted rack and overlay the dried bacterial suspension with a thin film of stain.

The wax pencil mark prevents the stain from running off the end of the slide.

Stain for 5–15 minutes, allowing a precipitate to form as the alcohol evaporates. The staining time is decreased if the stain is fresh, if the room temperature is high, if there are air currents in the laboratory, or if the layer of stain is thin. The opposite effects increase the staining time.

When a precipitate forms, rinse the slide gently with distilled water, drain off excess water, and allow to air-dry.

Ryu Method

Prepare the smear by picking up some young growth from carbohydrate-free culture medium on an inoculating needle and lightly touching the centre of each of 2 drops of distilled water on a new, pre-cleaned slide. Let the drops air-dry at room temperature.

Flood the air-dried smears with the staining solution for 1–5 minutes. Wash the staining solution off in tap water. (Avoids excessive debris in the background)

After the smears have dried, examine them under the oil-immersion objective of the microscope. Cell bodies and flagella stain violet.

Results Interpretation

Observe the stained slide under the oil-immersion (100×) objective of the microscope. Dark-staining red to blue-black flagella should be observed with number of flagella per cell and Location of flagella per cell.

Reference

Koneman’s Color Atlas and Text book of Diagnistic Microbiology.  Clark WA. A simplified Leifson flagella stain. J Clin Microbiol 1976;3:632–634.

Leifson E. Atlas of Bacterial Flagellation. New York, NY: Academic Press, 1960.

Ryu E. A simple method of staining bacterial flagella. Kitasato Arch Exp Med 1937;14:218–219.