Evaluation Of Microscopic Stains For Spore-forming Bacte

产芽孢细菌显微染色的评价

• 批准号: 6825567
• 负责人: Patrick R Murray
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6825567
• 关键词: Bacillus; Clostridium; aniline dye; bacterial capsules; cold temperature; fluorescent dye /probe; gram positive bacteria; heat; laboratory accident /infection; method development; microorganism classification; stainings; tissue /cell culture

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.

鉴定需氧和厌氧革兰氏阳性杆状细菌常常是困难的。一种重要的鉴别试验是检测芽孢杆菌和梭状芽孢杆菌等生物体中的内孢子。当培养物陈旧或保持在不利的生长条件下时，一些生物可以自由产生孢子，大多数生物体形成内孢子，但在相对年轻的培养物中可能很难检测到内孢子。我们之前比较了两种染色方法：热孔雀绿染色(需要加热载玻片)和冷孔雀绿染色。芽胞形成属芽孢杆菌、拟青霉属和梭状芽孢杆菌的代表性菌株在培养中过夜培养，然后制备玻片进行染色。我们发现热孔雀绿染色优于冷染色；然而，对热染色过程中气溶胶形成的担忧促使我们探索替代冷染色。我们用两种荧光染料：金胺O和吖啶橙进行了评估。这两种荧光染料均优于孔雀石绿的冷染剂和热染剂。孢子和营养细胞(即没有孢子的细菌细胞)之间的对比度很好，使用低倍放大可以快速检查显微切片。因为吖啶橙染料可以在商业上买到，我们发现使用这种染料比使用金胺O更方便。