Chitarra L G, Breeuwer P, Van Den Bulk R W, Abee T
Department of Food Technology and Nutritional Sciences, Wageningen University and Research Centre, The Netherlands.
J Appl Microbiol. 2000 May;88(5):809-16. doi: 10.1046/j.1365-2672.2000.01014.x.
The viability of Clavibacter michiganensis subsp. michiganensis (Cmm) was determined by measuring the intracellular pH (pHin) as a viability parameter. This was based on the observation that growth of Cmm was inhibited at pH 5.5 and below. Therefore, viable cells should maintain their pHin above this pH value. The pHin of Cmm was determined using the fluorescent probe 5(and 6-)-carboxyfluorescein succinimidyl ester (cFSE). The pHin of Cmm cells exposed to acid treatments was determined using fluorescence spectrofluorometry, and for cells exposed to elevated temperatures, the pHin was determined using fluorescence spectrofluorometry and flow cytometry (FCM). A good correlation was found between the presence of a pH gradient and the number of colony-forming units (cfu) observed in plate counts. However, with the spectrofluorometry technique, the analysis is based on the whole cell population and the detection sensitivity of this technique is rather low, i.e., cell numbers of at least 107 cfu ml-1 are needed for the analysis. Using FCM, heat-treated and non-treated Cmm cells could be distinguished based on the absence and presence of a pH gradient, respectively. The major advantage of FCM is its high sensitivity, allowing analysis of microbial populations even at low numbers, i.e., 102-103 cfu ml-1.
通过测量细胞内pH值（pHin）作为活力参数来确定密执安棒杆菌密执安亚种（Cmm）的活力。这是基于观察到Cmm在pH 5.5及以下时生长受到抑制。因此，活细胞应将其pHin维持在该pH值以上。使用荧光探针5（和6-）-羧基荧光素琥珀酰亚胺酯（cFSE）来测定Cmm的pHin。对于经酸处理的Cmm细胞，使用荧光分光光度法测定其pHin；对于经高温处理的细胞，则使用荧光分光光度法和流式细胞术（FCM）测定其pHin。在平板计数中观察到的pH梯度的存在与菌落形成单位（cfu）数量之间发现了良好的相关性。然而，对于荧光分光光度法技术，分析基于整个细胞群体，且该技术的检测灵敏度相当低，即分析至少需要107 cfu ml-1的细胞数量。使用FCM，经热处理和未经处理的Cmm细胞可以分别基于pH梯度的不存在和存在来区分。FCM的主要优点是其高灵敏度，即使在细胞数量较少（即102 - 103 cfu ml-1）时也能对微生物群体进行分析。
