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The Dorner method of endospore staining is a differential technique used to selectively stain bacterial endospores. Published by Dorner in 1922, this method uses carbol fuchsin as the primary stain, acid alcohol as the decolorizer, and nigrosin as the counterstain. It employs a lengthy heating step but results in differential staining of endospores and vegetative cells in the same sample.
In 1933, Shaeffer and Fulton devised ‘Schaeffer Fulton Stain’ a modification of Dorner’s method to make the process faster.
Endospores are highly resistant structures some bacteria produce as a defensive strategy against unfavorable environmental conditions. Special techniques are required for staining because of their resistance to heat and chemicals.
This article will explore the principle, procedure, and results of the Dorner method of endospore staining and its importance in microbiology.
Also read: Endospore staining techniques
Dorner Method of Endospore Staining
History of the Dorner method
In 1922, Dorner published a method for staining endospores. It employed a lengthy heating step but resulted in differential staining of endospores and vegetative cells in the same sample. Endospores and free spores appeared green or blue in contrast to the red dye taken up by the vegetative cells.
Principle of the Dorner method
The Dorner method of endospore staining is based on the principle of using carbol fuchsin as the primary stain. When applied to a heat-fixed slide and heated, the carbol fuchsin softens the structure of the bacterial spores, allowing the basic fuchsin to penetrate the spores. The slide is then decolorized with acid alcohol, which removes the color from the vegetative cells, leaving them colorless. Nigrosin is used as a counterstain.
This method involves a lengthy heating step but results in the differential staining of endospores and vegetative cells within the same sample. Endospores and free spores appear green or blue, in contrast to the red dye taken up by the vegetative cells.
Dorner method stain procedure
- Make a smear of the test organism on a clean glass slide.
- Allow it to air dry/ heat fix.
- Cover the slide with blotting paper.
- Saturate the blotting paper with carbol fuschin dye and steam for 5 to 10 minutes.
- Add more dye as required to prevent it from drying out.
- Remove the blotting paper.
- Decolorize the stain with acid alcohol for 1 minute.
- Rinse with running water and air dry.
- Place a drop of nigrosin dye on one end of the slide and make a thin smear over the slide.
- Examine under oil immersion on a microscope.
Dorner method stain procedure variation
- Mix equal volumes of carbol fuschin dye and an aqueous suspension of bacteria in a test tube.
- Immerse the test tube in a boiling water bath for 10 minutes.
- Place a loopful of 7% nigrosin on a glass slide.
- Mix a loopful of the boiled carbol fuchsin-bacteria suspension with the nigrosin on the slide.
- Allow the preparation to air dry to a thin film.
- Examine under oil immersion on a microscope.
Results
The Dorner method of endospore staining produces differential staining of endospores and vegetative cells within the same sample. Endospores and free spores will appear green or blue, while vegetative cells will take up the red dye.
This result allows for the identification of bacterial spores and the differentiation of spore-forming bacteria from non-spore-forming bacteria. The location and shape of the spores can also provide useful diagnostic information. Spores may be in the middle of the cell, at the end, or between the end and middle of the cell. The shape of the spores may be spherical or elliptical.
Examples of spore-forming bacteria include Bacillus subtilis, Clostridium sporogenes, Clostridium tetani, Bacillus anthracis, Sporolactobacillus spp, Bacillus cereus, Clostridium perfringens, C. botulinum, Desulfotomaculum spp, and Sporosarcina spp.
Examples of non-spore-forming bacteria include Salmonella spp and E. Coli.
Conclusion
The Dorner method of endospore staining is an important technique in microbiology for the identification and differentiation of bacterial spores. Published by Dorner in 1922, this method uses carbol fuchsin as the primary stain, acid alcohol as the decolorizer, and nigrosin as the counterstain. It employs a lengthy heating step but results in differential staining of endospores and vegetative cells in the same sample.
The expected result of this method is differential staining of endospores and vegetative cells, with endospores and free spores appearing green or blue in contrast to the red dye taken up by the vegetative cells. This allows for the identification of bacterial spores and the differentiation of spore-forming bacteria from non-spore-forming bacteria.
