Huang C T, Yu F P, McFeters G A, Stewart P S
Department of Microbiology, Montana State University, Bozeman 59717, USA.
Appl Environ Microbiol. 1995 Jun;61(6):2252-6. doi: 10.1128/aem.61.6.2252-2256.1995.
Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4',6-diamidino-2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.
将荧光染色与冷冻包埋及图像分析相结合，以证明在一氯胺消毒过程中细菌生物膜内呼吸活性的空间梯度。在连续流动环形反应器中，肺炎克雷伯菌和铜绿假单胞菌在不锈钢表面共同生长形成的生物膜，用每升2毫克一氯胺（进水浓度）处理2小时。通过总细胞直接计数证明生物膜去除相对较少。平板计数（两种菌的总和）表明，暴露于每升2毫克一氯胺后平均减少1.3个对数级。荧光氧化还原指示剂5-氰基-2,3-二甲基氯化四氮唑（CTC）和DNA染色剂4',6-二脒基-2-苯基吲哚（DAPI）用于区分生物膜中进行呼吸和不进行呼吸的细胞。冷冻生物膜横截面的落射荧光显微照片清楚地显示了生物膜内响应一氯胺处理的呼吸活性梯度。通过计算图像分析测量的CTC和DAPI强度之比，对这些特定呼吸活性梯度进行了量化。生物膜-主体流体界面附近的细胞首先失去呼吸活性。经过2小时的杀菌剂处理后，生物膜深处比生物膜-主体流体界面附近保持着更高的呼吸活性。
