Hatzinger Paul B, Palmer Patrick, Smith Richard L, Peñarrieta Cecilia T, Yoshinari Tadashi
Envirogen, Inc., 4100 Quakerbridge Road, Lawrenceville, NJ 08648, USA.
J Microbiol Methods. 2003 Jan;52(1):47-58. doi: 10.1016/s0167-7012(02)00132-x.
A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.
开展了一项研究，利用四氮唑盐测量马萨诸塞州科德角西部一个砂卵石含水层中本土细菌的有氧呼吸，并通过气相色谱法（GC）直接测量氧气消耗量。在地下水和含水层浆液中，通过直接GC测定法计算出的有氧呼吸速率比使用四氮唑盐2-(4-碘苯基)-3-(4-硝基苯基)-5-苯基氯化四氮唑（INT）的方法高出600多倍。为了解释这种差异，研究了INT以及另外两种四氮唑盐——3'-[1-(苯基氨基)-羰基]-3,4-四氮唑]-双(4-甲氧基-6-硝基)苯磺酸水合物（XTT）和5-氰基-2,3-二甲基氯化四氮唑（CTC）对含水层细菌分离株的毒性。观察到这三种四氮唑盐中的每一种在电子传递系统（ETS）和活力测定中通常使用的浓度下，对一些地下水分离株都有毒性。例如，用XTT（3 mM）孵育细胞导致所测试的五种地下水菌株中的四种密度下降超过四个数量级。在孵育4小时后，被CTC和INT杀死的细胞中有相当比例（>57%）含有可见的甲臜晶体（盐的不溶性还原形式）。因此，许多细胞在死亡前还原了足够的CTC或INT，以至于通过显微镜评估被认为是有活力的。然而，有一种细菌（荧光假单胞菌）在INT和CTC存在的情况下仍具有活力且可培养，即使在孵育24小时后，也只有不到百分之几的细胞中含有甲臜晶体。基于传统的四氮唑盐还原测定，该菌株会被认为是无活力的。数据表明，四氮唑盐测定可能会极大地低估地下水中总的ETS活性，并且尽管它们可能对地下水中细菌群落中有活力细胞的数量提供一个合理的总体估计，但由于盐的摄取或还原不佳，一些特定菌株可能会被该测定错误地认为是无活力的。
