Remove the blotting paper and decolorize the film with acid-alcohol for 1 minute rinse with tap water and blot dry.
Alternatively, the slides may be steamed over a container of boiling water.
Saturate the blotting paper with carbolfuchsin and steam for 5 to 10 minutes, keeping the paper moist and adding more dye as required.
Endospore Staining by Dorner’s Methodĭissolve the basic fuchsin in the ethanol then add the phenol dissolved in the water. Spore shape may also be of diagnostic use. Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Vegetative Cells: Vegetative cells are brownish red to pink. Endospores are bright green and vegetative cells are brownish red to pink.
Examine the slide under microscope for the presence of endospores.
Counterstain with 0.5% safranin for 30 seconds.
Alternatively, the slide may be steamed over a container of boiling water.
Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required.
Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide.
Take a clean grease free slide and make smear using sterile technique.
Stock solution (2.5% (wt/vol) alcoholic solution)ġ00 ml of 95% ethanol Procedure of Endospore Staining Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution) At the end of the staining process, vegetative cells will be pink, and endospores will be dark green. Safranin is then applied to counterstain any cells which have been decolorized. Malachite green is water soluble and has a low affinity for cellular material, so vegetative cells may be decolourized with water. In the Schaeffer-Fulton`s method, a primary stain- malachite green is forced into the spore by steaming the bacterial emulsion. The bacteria can remain in this suspended state until conditions become favorable and they can germinate and return to their vegetative state. Principle of Endospore Stainingīacterial endospores are metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions. The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers. Shaeffer and Fulton modified Dorner’s method in 1933 to make the process faster The endospore stain is a differential stain which selectively stains bacterial endospores. In 1922, Dorner published a method for staining endospores.
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