Abstract：
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.
摘要：
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种群中的活嗜肺军团菌。而不是用InvitrogenBacLightLIVE/DEAD染色试剂盒进行连续的活力染色，然后用抗体-AlexaFluor（AF）647缀合物染色来鉴定活的肺炎支原体，开发了一种组合的单一混合溶液染色方案，以简化和加快在滤膜上检测混合物种种群中的活嗜肺乳杆菌血清群1（SG-1）的时间。染色鸡尾酒将有助于加速冷却塔的荧光显微镜分析，空气调节器和饮水机或其他液体样品的存在及其生存状态。明显红色染色的细胞被鉴定为死的非L。嗜肺SG-1细胞，而绿色荧光细胞代表有活力的非L。嗜肺SG-1细胞。由于也用抗体-AF647染色为红色,嗜肺乳杆菌SG-1细胞被假染色为蓝色以将它们与其他死细胞区分开。来自生存力染料(SYTO9和碘化丙啶)与抗体-AF647染色的嗜肺乳杆菌的荧光颜色发射混合导致其他荧光颜色。例如,绿色加假色蓝色AF647抗体标记的细胞被鉴定为活的青色嗜肺乳杆菌SG-1细胞。品红色细胞是由死的肺炎支原体细胞产生的，该细胞将红色碘化丙啶与蓝色假色AF647抗体发射相结合。测量RGB的分析(红色，绿色,蓝色）混合细菌种群的显微图像中的颜色值表明，可以轻松自动区分活的和死的肺炎克雷伯菌和非肺炎克雷伯菌的亚群。在组合的鸡尾酒中染色后，通过计算机在3维RGB颜色空间中检测嗜肺细菌，这将节省时间，以便更快速地显微检测军团病的潜在来源。
