microbiology practical

1. General Microbiology-605
Practical
Sheeba Batool

2. Flagella Stain
Objectives:
• Identify flagella on bacterial cells.
• Differentiate among different types of flagellation.

3. Principle of Flagella
Stain
The basic point about the
flagella stain is that the
combination of chemicals
produces a thickened coat
around the flagella, making
them more easily seen with a
light microscope. Flagella are
extremely thin and of small
diameter, so they are below the
resolution of the light
microscope if unstained.

4. • We will not be making our own flagella stains for a variety of
reasons:
• producing a good flagella is rather like making good art
• flagella are very delicate and tend to break off the cells easily
when manipulated
• environmental factors such as temperature, pH, age of culture
can affect the stability of the flagella (notice that lots of cells
have lost their flagella)

5. Categories of flagellation
•monotrichous = single flagellum
•peritrichous = flagella all around
•amphitrichous = flagella at both ends
•lophotrichous = tuft of many flagella at one end or both ends
•atrichous = without flagella, nonmotile

6. Motility
Motility can be identified in a couple of different ways:
• the hanging drop wet mount
• motility agar media (SIM and tetrazolium motility agars used later)

7. Apparatus Required
Glass Slides
Cover Slips
Microscope
Loop
burner

8. Chemicals
Flagella Stain(RYU flagella
stain (Remel, Lenexa,
Kansas)
(Crystal violet+ Alcohol)
Fresh Culture

9. Preparations:
1.These stains are
bought and ready
to use.
01
1.Although they
have cover slips,
you still use oil
when on 100X
magnification.
02
1.Be sure to
remove the oil with
the lens paper.
03

10. The procedure of Flagella Stain
(Wet Mount Technique)
• Grow the organism to be stained at room temperature on blood agar for 16 to 24
hours.
• Add a small drop of water to a microscope slide.
• Dip a sterile inoculating loop into sterile water.
• Touch the loopful of water to the colony margin briefly (this allows motile cells to
swim into the droplet of water).
• Touch the loopful of motile cells to the drop of water on the slide. Note: Agitating
the loop in the droplet of water on the slide causes the flagella to shear off the cell.
• Cover the faintly turbid drop of water on the slide with a coverslip. A proper wet
mount has barely enough liquid to fill the space under a coverslip. Small air spaces
around the edge are preferable.
• Examine the slide immediately under 40× to 50× for motile cells. If motile cells
are not seen, do not proceed with the stain.

11. Continued…
• If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes. This
allows the bacterial cells time to adhere either to the glass slide or to the coverslip.
• Gently apply 2 drops of RYU flagella stain (Remel, Lenexa, Kansas) to the edge of the
coverslip. The stain will flow by capillary action and mix with the cell suspension. Small
air pockets around the edge of the wet mount are useful in aiding the capillary action.
• After 5 to 10 minutes at room temperature, examine the cells for flagella.
• Cells with flagella may be observed at 100× (oil) in the zone of optimum stain
concentration, about halfway from the edge of the coverslip to the center of the mount.
• 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.

12. Results
• a

13. References
https://bio.libretexts.org/Courses/Portland_Community_College/Cascade_Micro
biology/02%3A_How_We_See_the_Invisible_World/2.4%3A_Staining_Microsco
pic_Specimens