This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection of live Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify live L. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire’s disease.

该报告描述了一种组合的免疫荧光和荧光活力染色剂，用作一种染色溶液，用于快速检测混合细菌群体中的活嗜肺军团菌。开发了一种组合的单一鸡尾酒溶液染色方案来简化和加快检测时间，而不是先使用 Invitrogen BacLight LIVE/DEAD 染色试剂盒进行连续活力染色，然后再进行抗体-Alexa Fluor (AF) 647 缀合物染色来识别活的嗜肺军团菌。滤膜上混合物种种群中活的嗜肺军团菌血清群-1 (SG-1) 的数量。该染色混合物将有助于加速冷却塔、空调和饮水机或其他液体样品的荧光显微镜分析，以确定嗜肺军团菌的存在及其活力状态。明显红色染色的细胞被鉴定为死亡的非嗜肺军团菌SG-1细胞，而绿色荧光细胞代表活的非嗜肺军团菌SG-1细胞。由于还用抗体 AF 647 染色为红色，嗜肺军团菌SG-1 细胞被伪着色为蓝色，以将其与其他死亡细胞区分开。活力染料（SYTO 9 和碘化丙啶）与抗体 AF 647 染色的嗜肺军团菌混合发出的荧光颜色产生其他荧光颜色。例如，绿色加假彩色蓝色 AF 647 抗体标记的细胞被鉴定为活青色嗜肺军团菌SG-1 细胞。洋红色细胞是由死亡的嗜肺军团菌细胞产生的，该细胞将红色碘化丙啶与蓝色假色 AF 647 抗体发射物结合在一起。 对混合细菌群体显微图像中测得的 RGB（红、绿、蓝）颜色值的分析表明，计算机可以在 3 维 RGB 中轻松自动区分活的和死的嗜肺军团菌和非嗜肺军团菌亚群。在组合混合物中染色后的颜色空间，这将节省时间，以便更快速地显微镜检测军团病的潜在来源。