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Endospores are surrounded by a highly resistant spore coat, The spore coat is highly resistant to excessive heat, freezing and desiccation as well as chemical agents. More importantly, for identification, spores are resistant to commonly employed staining techniques; therefore alternative staining methods are required. Moeller staining involves the use of a steamed dye reagent in order to increase the stainability of the spores; Carbol fuchsin is the primary stain used in this method. Endospores are stained red, while the counterstain, Methylene blue stains the vegetative bacteria blue.
Carbol fuchsin is applied to a heat-fixed slide. The slide is then heated over a bunsen burner, or suspended over a hot water bath, covered with a paper towel, and steamed for 3 minutes. The slide is rinsed with acidified ethanol, and counter-stained with Methylene blue. An improved method involves the addition of the surfactant Tergitol 7 to the carbol fuchsin stain, and the omission of the steaming step.
- Hayama, Masayoshi; Oana, Kozue; Kozakai, Tomonori; Umeda, Shuhei; Fujimoto, Junkoh; Ota, Hiroyoshi; Kawakami, Yoshiyuki (2007). "Proposal of a simplified technique for staining bacterial spores without applying heat - Successful modification of Moeller's method". European Journal of Medical Research. 12 (8): 356–9. PMID 17933713. hdl:10091/10132.
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