Some bacteria such as Azotobacter sp. contains cysts which are visible with following reagent.
Glacial acetic acid 8.5 ml
Sodium sulphate (anhydrous) 3.25 g
Neutral red 200 mg Light green S.F. yellowish 200 mg
Ethanol (95%) 50 ml
Distilled water 100 ml
Add the chemicals and dyes in water with continuous stirring and filter (through 0.5 µm).
(i) Immerse the specimen in the reagent and examine the wet preparation.
(ii) The young vegetative azotobacters are yellowish green. Cyst forms of Azotobacter will appear with central body dark green and colourless intine and brownish red exine.