A Wet mount technique for staining bacterial flagella is simple and is useful to identify the number and arrangement of flagella.

1. FLAGELLA STAINING
SUBMITTED BY:
M.Monisha,
I M.Sc.Applied Microbiology,
Sacred Heart College(Autonomous),
Tirupattur.
SUBMITTED TO:
Dr.P.Saranraj, M.Sc.,M.Phil.,Ph.D.,NET,
Head of the Department Microbiology,
Sacred Heart College (Autonomous),
Tirupattur.

2. FLAGELLA STAINING
AIM:
To visualize the presence and arrangement of flagella for the presumptive identification of motile
bacterial species by Wet mount method (Ryu method).
PRINCIPLE:
• A wet mount technique for staining bacterial flagella is simple and it is useful when the number and
arrangement of flagella are critical in identifying species of motile bacteria. The wet mount method is also
called the “Ryu method” because it uses Ryu flagella stain to examine the arrangement and number of
flagella.
MATERIALS REQUIRED
24 hours culture of Bacteria
Bunsen burner

3.  Microscopic slide
 Cover slip
 Ryu stain
 Microscope
PREPARATION OF RYU STAIN: It Involves The preparation of two solutions (Solution I-And II).
SOLUTION-I Includes the following components in a defined amount:
• 5% Aqueous solution of phenol: 10 ml
• Tannic Acid: 2 g
• An aqueous solution of aluminum potassium sulphate-12 hydrate: 10 ml.
SOLUTION-II Contains a saturated ethanoic solution of crystal violet, in which 12g of crystal violet is mixed
in 100 ml of 95% ethanol.
• The final stain is prepared by mixing solution-I and II in a ratio of 10:1. then, separate the coarse precipitated
particles from the stain via filtration using filter papers, and finally store the reagent at room temperature.

4. PROCEDURE OF FLAGELLA STAINING:
1. Grow the organisms to be stained at room temperature on blood agar for 16 to 24 hours.
2. Add a small drop of water to a microscope slide.
3. Dip a sterile inoculating loop into sterile water.
4. Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the
droplet of water).
5. Touch the loopful of motile cells to the drop of water on the slide.
6. Cover the faintly turbid drop of water on the slide with a cover slip. a proper wet mount has barely
enough liquid to fill the space under a cover slip. Small air spaces around the edge are preferable.
7. Examine the slide immediately under 40x for motile cells.

5. 8. If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes.
9. Apply 2 drops of Ryu flagella stain gently on the edge of the cover slip. the stain will flow by
capillary action and mix with the cell suspension.
10. After 5 to 10 minutes at room temperature, examine the cells for flagella.
11. Cells with flagella may be observed at 100x.
12. Finally, note down the results by examining the presence, number and arrangement of the flagella.
RESULT:
Observe the slide and note the following:
• Presence or absence of flagella
• Number of flagella per cell
• Location of flagella per cell

6. Types of flagella
a)Monotrichous
Example: Vibrio cholerae
b)Lophotrichous
Example: Spirillum
c)Peritrichous
Example: Salmonella typhi
d)Amphitrichous
Example: Alkaligens faecalis

7. REFERENCE
• FLAGELLA - HTTPS://MICROBIOLOGYINFO.COM/FLAGELLA-INTRODUCTION-TYPES-
EXAMPLES-PARTS-FUNCTIONS-AND-FLAGELLA-STAINING-PRINCIPAL-PROCEDURE-
AND-INTERPRETATION/

8. THANK YOU