Flagella Dr Ashna Ajimsha PG resident Department of Microbiology SGMC and RF Venjaramoodu Kerala

1. Flagella
Dr.Ashna Ajimsha

2.  Long, filamentous, thread-like appendages, protruding from
the cell wall
 Composed of protein subunits called flagellin
 Organs of locomotion
 5-20 Micrometres long
 0.01-0.02 Micrometre thick

3. Arrangement of flagella
 Monotrichous (single polar flagellum)-e.g. Vibrio cholera
Pseudomonas
Campylobacter
 Lophotrichous (multiple polar flagella)- e.g. Spirillum.
 Peritrichous (flagella over entire cell surface)
e.g. SalmonellaTyphi
E.coli.
 Amphitrichous - e.g. Alcaligenes faecalis

5. Ultrastructure of flagella

7. Flagella composed of 3 parts
 Filament
 Hook
 Basal body
FILAMENT
o Longest portion of the flagellum
o Extends from the cell surface to the tip
o 13-17nm in Diameter
o Hollow, rigid cylinder
o Composed of parallel subfibrils of 30-40 kDa subunit of a single protein
called flagellin
o Ends with a capping protein
o Forms a left handed helix as it exits the cell

8. Flagellin
 Capacity to self assemble
 Monomers of flagellin are synthesized & passed through the
lumen of the cylinder
At the growing tip of the helix
Monomer undergoes conformational change
Added to the distal end of the flagellum

9. HOOK
o Short, curved, flexible segment
o Acts as a “sleeve” from which flagellar filament emerges
o Links filament to basal body
BASAL BODY
o Portion of flagellum embedded in the cell
o Most complex part of flagellum
o Made of 2-4 rings connected to a a central rod
o In GN bacteria,
-4 rings {L, P, S, M}
-10 proteins comprise the outer rings
-Flagellar antigens – “H Antigens”

10. L & P rings (Outer rings)
• L ring is associated with LPS
• P ring is associated with peptidoglycan layer
• Function as bearings
• Minimize friction & leakage of materials from the cell at the
points of flagellar insertion
S ring (Inner ring)
• Associated with periplasmic space

11. M ring (Inner ring)
• Associated with plasma membrane
• Rotates as a part of energy dependent reaction
Causes rigid flagellar helix to turn like a propeller

12. In GP bacteria,
2 rings
-Inner ring associated with plasma membrane
-Outer ring associated with peptidoglycan layer

14. Phase variation
 Ability of organisms to express 2 types of flagella
alternately
 Occurs by differential expression of Chromosomal
genes that code for 2 variously structured flagellin
proteins
 1st recognized in enteric GN bacteria (Salmonella spp)
 Also seen in N. gonorrhoeae

15. Organisms with modified flagellar
structures
Vibrio
• Flagellum is encased in a sheath derived from outer
membrane of cell wall
Spirochetes
• Flagellum lies within a sheath exterior to protoplasmic
cylinder of the cell body
• Endoflagellum/axial filament arises from 1 pole of the cell
& wraps around the cell body internal to the sheath

16. Type of motility Bacteria
Tumbling Listeria
Gliding Mycoplasma
Stately Clostridium
Darting V.cholerae
Campylobacter
Swarming on agar plate Proteus
C.tetani
Corkscrew/lashing/flexion-extension Spirochete

19. Demonstration of flagella
Direct demonstration of flagella
 Tannic acid staining(Leifson’s method & Ryu’s method)
 Electron microscopy
Indirect means by demonstrating the motility
 Cragie tube method
 Hanging drop method
 Semisolid medium
 Dark ground/Phase contrast microscopy

20. Ryu’s method
Mordant solution
 10 ml 5% Phenol
 2g Tannic acid
 10 ml saturated aqueous solution of Aluminium potassium
sulphate
Ryu’s stain
 10 ml Crystal Violet, saturated ethanolic solution (12g/100ml of
95% ethanol)
 100 ml Mordant solution

21. Method
 Grow bacteria for 16-24 hours on a a non inhibitory medium
 Touch a a loopful of water onto the edge of a colony & let
motile bacteria swim into it
 Transfer this into a loopful of water on a slide to get a turbid
suspension
 Cover with coverslip
 After 5-10 mins, add 2 drops of Ryu’s stain to the edge of
coverslip
 Leave for 5-15 mins at ambient temperature
 Examine under oil immersion
 Flagella stains violet

23. Leifson’s method
Leifson’s stain
 1.2% Basic fuschin 95% alcohol
 3% Tannic acid in water
 1.5% NaCl in water
 Final stain,
-Combine 3 stock solutions in equal volumes
-Use immediately after preparation
-Store in tightly stoppered bottle
-Precipitate formed during storage should not be disturbed
-Remove staining solution from the top with a pipette

24. Method
 Place 2 drops of bacterial suspension towards 1 end of acid
cleaned microscope slide & air dry
 Draw a perpendicular line on the glass surface with a wax
pencil towards opposite end from dried suspension
 Place the slide on a a tilted rack
 Overlay with thin film of stain
 After 5-15 mins, as alcohol evaporates, precipitate forms
 Rinse the slide with running water & air dry
 Flagella stains red to blue black

26. Hanging drop preparation
 Use a hollow ground slide (cavity slide)
 Encircle the concavity with a line streak of soft petroleum jelly
applied with a glass rod to the surface of the slide just outside
the concavity
 Draw a V shaped line on the surface of coverslip
 Place the suspension on the same side of the coverslip within
the angle of the “V”
 Invert the slide over the coverslip, then quickly turn around
the slide so that the coverslip is uppermost
 The drop will be hanging from the coverslip in the centre of
the concavity

28. Craigie tube method

29. Detection of motility using semi solid agar
 0.4% Japanese agar/0.2% New Zealand agar dissolved in
nutrient broth or peptone water
 Final medium – Clear & transparent
 Pour 10 ml amounts in test tubes & leave to set
 Inoculate with a straight wire ,making a single stab down
the centre of the tube to half the depth of the medium
 Incubate under conditions favouring motility

30. Motilityusingsemisolidagar
Non Motile Bacteria
-Growth confined to stab line
-Sharply defined margins
-Surrounding medium clearly
transparent
Motile Bacteria
-Diffuse,hazy growth/ swarming
extending as a zone of turbidity
from the stab line
-Medium becomes opaque