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. For this reason, a variety of specific microscopic stains have been developed and used with varying degrees of success. In this study we initially compared two stains: a hot malachite green stain (requires heating the slide) and a cold malachite green stain. Representative isolates from the genera Bacillus, Paenibacillus, and Clostridium were grown overnight in culture and then slides were prepared for staining. Each of the two spore stains was performed and then compared. The hot malachite green stain was found to be superior for all organisms evaluated. More spores appeared to take up the stain, and the contrast between spores and vegetative bacteria was greater. Although the hot malachite green staining procedure has superior staining properties and is more rapid than the cold malachite green procedure, aerosol formation during the staining procedure is a potential laboratory safety concern, particularly if the culture preparation is with a highly dangerous organism such as Bacillus anthracis. For this reason, we are exploring modifications of the cold malachite green stain to improve the staining characteristics. One such modification is substitution of malachite green with the fluorescent dye, auramine. A preliminary experiment demonstrated excellent staining with this fluorescent stain, offering the advantages of good contrast between spores and vegetative cells, as well as decreased preparation time (less than 5 minutes are required to prepare the stained slide). We will extend this experiment to compare the fluorescent stain with the malachite green stains.