FLAGELLA STAINING

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 is useful when the number and arrangement of flagella are critical in identifying species of motile bacteria. Flagella are too thin to be visualized using a Bright field microscope with ordinary stains, such as the Gram stain, or a Simple stain. A Wet mount technique is used for staining bacterial Flagella, and it is simple and useful when the number and arrangement of Flagella are critical to the identification of species of motile bacteria. The Staining procedures require the use of a Mordant so that the stain adheres in layers to the flagella, allowing visualization.

MATERIALS REQUIRED

i.       24 hours culture of Bacteria

ii.     Bunsen burner

iii.   Microscopic slide

iv.   Cover slip

v.     Ryu stain

vi.   Microscope.

Ryu Flagella Stain

·       The Ryu stain has two components.

a)    Solution I, the Mordant, contains 10 ml of 5 % Aqueous solution of Phenol, 2 g of Tannic acid, and 10 ml of Saturated aqueous solution of Aluminum potassium sulfate-12 hydrate.

b)    Solution II, the stain, is a Saturated Ethanolic solution of Crystal violet (12 g in 100 ml of 95 % ethanol).

·       The final stain was prepared by mixing 1 part Solution II with 10 parts solution I and then filtering the mixture through filter paper to remove coarse precipitate

PROCEDURE

a)     Grow the bacteria to be stained at room temperature on Blood agar for 16 to 24 hours.

b)     Add a small drop of water to a Microscope slide.

c)     Dip a sterile Inoculating loop into sterile water.

d)     Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the droplet of water).

e)   Touch the loopful of Motile cells to the drop of water on the slide (Agitating the loop in the droplet of water on the slide causes the flagella to shear off the cell).

f)   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.

g)    Examine the slide immediately under 40 x for motile cells.

h)    If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes.

i)    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.

j)     After 5 to 10 minutes at room temperature, examine the cells for flagella.

k)     Cells with flagella may be observed at 100× (oil) in the zone of optimum stain concentration, about half way from the edge of the coverslip to the center of the mount.

l)    Focusing the microscope on the cells attached to the coverslip rather than on the cells attached to the slide facilitates visualization of the flagella. The precipitate from the stain is primarily on the slide rather than the coverslip.

OBSERVATION AND RESULTS

            Observe the slide and note the following: (i) Presence or absence of flagella, ii) Number of flagella per cell and iii) Location of flagella per cell.