Quantification of relative proportions of intact cells in microbiological samples using the example of Cryptosporidium parvum oocysts

Abstract

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.