Evaluation Of Microscopic Stains For Spore-forming Bacte

产芽孢细菌显微染色的评价

• 批准号: 6675252
• 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/6675252
• 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. 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.

需氧和厌氧革兰氏阳性杆状菌的鉴定通常是困难的。一个重要的鉴别试验是检测生物体中的内生孢子，如芽孢杆菌和梭菌。然而，当培养物老化或维持在不利的生长条件下时，一些生物体自由地产生孢子，并且大多数生物体形成内生孢子，在相对年轻的培养物中检测内生孢子可能是困难的。出于这个原因，已经开发了各种特定的显微镜染色剂，并取得了不同程度的成功。在这项研究中，我们最初比较了两种染色：热孔雀石绿色染色（需要加热载玻片）和冷孔雀石绿色染色。将芽孢杆菌属、类芽孢杆菌属和梭菌属的代表性分离株在培养物中生长过夜，然后制备载玻片用于染色。分别进行两种孢子染色，然后进行比较。发现热孔雀石绿绿色染色对所有评价的微生物均具有上级效果。更多的孢子似乎占据了染色，孢子和营养细菌之间的对比更大。尽管热孔雀石绿色染色程序具有上级的染色性能，并且比冷孔雀石绿色染色程序更快，但染色程序期间气溶胶的形成是一个潜在的实验室安全问题，特别是如果培养物制备物是高度危险的生物体，例如炭疽杆菌。为此，我们正在探索改良的冷孔雀石绿色染色剂，以改善染色特性。一种这样的修饰是用荧光染料金胺取代孔雀石绿色。初步实验表明，这种荧光染料染色效果极佳，孢子和营养细胞之间的对比度良好，制备时间缩短（制备染色载玻片所需时间不到5分钟）。我们将扩大这个实验，以比较荧光染色和孔雀石绿色染色。