Technique FLAGELLAR STAINS
Principle  
Cautions Flagellar staining should not be undertaken lightly but with care, reasonably satisfactory results can be obtained

If care is taken in the preparation of the culture, the bacterial film and the reagents

Heat the ammoniacal silver nitrate solution nearly to boiling

Unless permanent preparations are made the stained films disintegrate after about a week on exposure to air
Method Preparation of bacterial suspension
1: Inoculate the organism on the surface of nutrient agar slopes
2: Incubate at 22-30 c for 18-24 hours.
3: Carefully pipette 2-3 ml of sterile distilled water into the tubes
4: Allow the bacteria to diffuse off the agar surface into the water.
5: When the suspension is turbid, transfer by pipette into a clean tube containing 1% formalin (0.4% formaldehyde ).The suspension should appear faintly opalescent. If it is turbid more formalin must be added.
6: The suspension may be washed by slow speed centrifugation and the deposit resuspended in 1% formalin solution.

Preparation of Films
1: Clean slides with hot dichromic acid,
2: Thoroughly wash to remove all traces of acid and
3; allow to drain, store in a closed container.
4: Place a loopful of the bacterial suspension near one end of the dry slide
5: Immediately tip the slide to a vertical position so that the drop runs down, leaving a thin film. The drop reaches the bottom in a few seconds if the slide is really clean
6: Dry the smear in the air at room temperature.
7: Keep the slide in the vertical position to prevent dust settling on the film

Specific Flagella Stains

See Cesares-Gill's Method

See Rhodes' Method

See Gray's Method
Results  
Positive control use peritrichous and polar flagellate organisms as controls
Negative control  
Reagents  
Reference  

 

Technique FLAGELLAR STAINS Cesares-Gill's Method
Principle  
Cautions Heat the ammoniacal silver nitrate solution nearly to boiling
Method Flagellar stain Cesares-Gill's Method
1: Treat with Kirkpatrick's fixative for 5 min.
2: Rinse thoroughly with water.
3: Filter the diluted Plimmer's mordant onto the slide
4: Leave for 5 min
5: Rinse with water.
6: Stain for 2 min with weak carbol-fuchsin.
7: Rinse with water
8: Dry in air.
Results  
Positive control use peritrichous and polar flagellate organisms as controls
Negative control  
Reagents Kirkpatrick’s fixative
Plimmer’s mordant
Carbol fuchsin
Reference H.G. Plimmer & S.. G. Paine 1921 J Path Bact 24 286)

 

Technique FLAGELLAR STAIN Rhodes' Method
Principle  
Cautions Heat the ammoniacal silver nitrate solution nearly to boiling
Method Rhodes' Method
1: Flood the smear with iron tannate mordant
2: Incubate at room temparature for 3-5 minutes.
3: Rinse thoroughly with water
4: Flood the slide with hot ammoniacal silver nitrate solution
5: Leave to act for 3-5 minutes.
6: Rinse thoroughly with water.
7: Drain or blot to dry.
Results  
Positive control use peritrichous and polar flagellate organisms as controls
Negative control Iron Tannate Mordant
Ammoniacal silver nitrate
Reagents  
Reference M.E. Rhodes 1958 J Gen Microbiol 18:639

 

Technique FLAGELLAR STAIN Gray's Method
Principle  
Cautions  
Method Gray's Method
1: Flood the smear with iron tannate mordant
2: Incubate at room temparature for 10 minutes.
3: Rinse thoroughly with water
4: Add Ziehl-Neelsen Carbol fuchsin
5: Leave on for 5-10 minutes.
6: Rinse with tap water,
7: Air dry
8: Examine under the oil immersion lenses
Results  
Positive control use peritrichous and polar flagellate organisms as controls
Negative control  
Reagents Iron Tannate Mordant
Carbol fuchsin
Reference Gray 1926 J.Bacteriol 12: 273