Cappelier J M, Lazaro B, Rossero A, Fernandez-Astorga A, Federighi M
Ecole nationale vétérinaire de Nantes, unité associée Inra d'hygiène alimentaire, France.
Vet Res. 1997 Nov-Dec;28(6):547-55.
Direct microscopic enumeration of viable Campylobacter jejuni cells (ie, respiring bacteria) were performed in both culturable and non-culturable states. Five different C jejuni strains were used, including a reference strain, ATCC 33291. Cells from all five strains were incubated alone with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a redox dye. It was reduced by an electron transport chain to an insoluble red fluorescent CTC formazan salt, which accumulated intracellularly. The presence of these red CTC crystals in the bacteria cells was indicative of cellular respiratory activity. Counterstaining with 4'-6 diamino-2 phenylindole (DAPI), which fluoresces in blue, made a suitable contrast and allowed simultaneous enumeration of total and viable bacteria on a single filter. Four hours of incubation with 5 mM CTC under a microaerobic atmosphere was found to be the optimal condition yielding the maximum number of respiring cells (both culturable and non-culturable). When used in combination with standard culture techniques, double staining makes it possible to monitor the viable but non-culturable cells of jejuni obtained by starvation more easily than with the direct viable count procedure.
对空肠弯曲杆菌活菌（即进行呼吸作用的细菌）在可培养状态和不可培养状态下均进行了直接显微镜计数。使用了五种不同的空肠弯曲杆菌菌株，包括一株参考菌株ATCC 33291。将来自所有五种菌株的细胞单独与一种氧化还原染料5-氰基-2,3-二苯基四氮唑氯化物（CTC）一起孵育。它通过电子传递链被还原为不溶性的红色荧光CTC甲臜盐，该盐在细胞内积累。细菌细胞中这些红色CTC晶体的存在表明细胞具有呼吸活性。用发蓝色荧光的4'-6-二氨基-2-苯基吲哚（DAPI）进行复染形成了合适的对比，使得能够在单个滤膜上同时对总细菌数和活菌数进行计数。发现在微需氧气氛下用5 mM CTC孵育4小时是产生最多呼吸细胞数（包括可培养和不可培养的细胞）的最佳条件。当与标准培养技术结合使用时，双重染色比直接活菌计数法更易于监测通过饥饿获得的空肠弯曲杆菌的活但不可培养细胞。
