Department of Microbiology, Faculty of Biology, Universitat de Barcelona, Barcelona, Spain.
Lett Appl Microbiol. 2014 Jan;58(1):70-8. doi: 10.1111/lam.12157. Epub 2013 Sep 30.
The fast analysis of relative proportions of live and dead cells can be of great value whether for comparing inactivation efficiencies of different biocidal treatments or for monitoring organisms of interest in environmental samples. We introduce here a straightforward method to determine the percentage of intact cells based on treatment of samples with the viability dye propidium monoazide (PMA). PMA selectively enters membrane-damaged cells and suppresses their PCR detection through modification of their DNA. The study was performed using Cryptosporidium parvum oocysts as a model although the principle should be applicable to other organisms. Validation was performed with defined mixtures of live and heat-killed oocysts and by exposing oocysts to a heat stress gradient. The method correctly indicated increasingly lower proportions of intact cells with increasing temperatures. When comparing the loss of membrane integrity of UV-killed (40 mJ cm(-2) ) oocysts during storage in nonsterile tap water, results suggested that integrity declines slowly (over weeks) and at a rate comparable to non-UV-exposed oocysts. For all experiments, the amplification of longer DNA sequences was found beneficial. In the UV experiment, longer amplicons revealed not only higher sensitivity in excluding membrane-damaged oocysts, but also in excluding DNA with UV-induced damage.
Whether in the context of microbial ecology or in an industrial context, many questions in microbiology are linked to microbial viability. As cultivation of micro-organisms can be long or may not be possible, fast methods to assess the numbers of live cells are in great demand. We present here a straightforward strategy to determine the relative proportions of intact cells. The PCR-based rapid method is expected to be useful where relative information is sufficient (e.g. for comparing the effect of different antimicrobial treatments on known numbers of micro-organisms) or when the presence of PCR inhibitors does not allow absolute quantification.
快速分析活细胞和死细胞的相对比例无论对于比较不同杀菌处理的灭活效率，还是对于监测环境样本中感兴趣的生物，都具有重要价值。我们在此引入一种简单的方法，该方法基于用荧光染料碘化丙啶单（PMA）处理样品来确定完整细胞的百分比。PMA 选择性地进入受损细胞膜，并通过修饰其 DNA 来抑制其 PCR 检测。该研究使用微小隐孢子虫卵囊作为模型，但该原理应该适用于其他生物。通过使用活细胞和热灭活卵囊的定义混合物以及使卵囊暴露于热应激梯度来进行验证。该方法随着温度的升高正确地指示出完整细胞的比例越来越低。当比较在非无菌自来水储存过程中 UV 杀死（40 mJ cm(-2) ）卵囊的膜完整性丧失时，结果表明完整性缓慢下降（数周），并且与未暴露于 UV 的卵囊的下降速度相当。对于所有实验，较长 DNA 序列的扩增被发现是有益的。在 UV 实验中，较长的扩增子不仅显示出更高的灵敏度，可以排除受损的卵囊，而且可以排除具有 UV 诱导损伤的 DNA。
无论是在微生物生态学背景下还是在工业背景下，微生物学中的许多问题都与微生物的生存能力有关。由于微生物的培养可能需要很长时间或者可能不可行，因此需要快速评估活细胞数量的方法。我们在此提出了一种简单的策略来确定完整细胞的相对比例。基于 PCR 的快速方法有望在相对信息足够的情况下（例如，用于比较不同抗菌处理对已知数量的微生物的影响）或当 PCR 抑制剂的存在不允许绝对定量时，会非常有用。
