Evaluation Of Microscopic Stains For Spore-forming Bacte

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。为此，各种特殊的显微染色已经被开发和使用，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：热孔雀绿染色(需要加热载玻片)和冷孔雀绿染色。芽孢杆菌属、芽孢杆菌属和梭状芽孢杆菌属的代表性菌株在培养上过夜培养，然后制备玻片进行染色。对这两种孢子进行染色，然后进行比较。热孔雀绿染色被发现对所有被评估的生物都是更好的。更多的孢子似乎吸收了污渍，孢子和营养细菌之间的反差更大。尽管热孔雀绿染色程序具有更好的染色性能，并且比冷孔雀绿程序更快，但在染色过程中形成气溶胶是一个潜在的实验室安全问题，特别是当培养准备含有高度危险的有机体，如炭疽芽孢杆菌。为此，我们正在探索对冷孔雀绿染色进行修改，以改善染色特性。一种这样的修饰是用荧光染料极胺取代孔雀石绿。初步实验表明，这种荧光染料染色效果很好，提供了孢子和营养细胞之间良好的对比度，并减少了准备时间(制备染色玻片所需时间不到5分钟)。我们将扩展这个实验来比较荧光染色和孔雀石绿染色。

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.