Evaluation Of Microscopic Stains For Spore-forming Bacte

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Identification of aerobic and anaerobic gram-positive rod-shaped bacteria is frequently difficult. One important differential test is the detection of endospores in organisms such as Bacillus and Clostridium. Whereas some organisms sporulate freely and most organisms form endospores when the culture is old or maintained in unfavorable growth conditions, the detection of endospores may be difficult in relatively young cultures. We previously compared two staining methods: a hot malachite green stain (requires heating the slide) and a cold malachite green stain. Representative isolates from the spore-forming genera Bacillus, Paenibacillus, and Clostridium were grown overnight in culture and then slides were prepared for staining. We found that the hot malachite green stain was superior to the cold stain; however, concern with aerosol formation during the hot staining procedure led us to explore alternative cold stains. We evaluated using two fluorescent stains: auramine O and acridine orange. Both fluorescent stains were superior to either the hot or cold malachite green stains. The contrast between spores and vegetative cells (i.e., bacterial cells without spores) was excellent, and the microscopic slide could be examined rapidly using low power magnification. Because acridine orange stains can be purchased commercially, we found using this dye more convenient than auramine O.