Assessment of Bacterial Survival in the Presence of Nanomaterials: Is Colony Forming Unit Count Sufficient?

Assessment of bacterial survival is a common task in evaluating environmental impacts of nanomaterials. In such studies, the colony forming unit (CFU) method, that is, the enumeration of viable cells by counting colonies formed by individual cells is commonly used. This method is simple and straightforward and does not require much specialized expertise. However, the CFU method may generate misleading results for the biological systems in the presence of nanomaterials.

One basic assumption for the CFU method is that, when the cultures are diluted to a certain extent, each bacterial cell will develop into one single colony. This assumption is not valid in many studies in the presence of nanomaterials. In bacterial cultures under environmental stresses such as exposure to toxic nanomaterials, cells often form aggregates (Chua et al., 2015). For example, when Pseudomonas aeruginosa cells were exposed to a subinhibitory concentration of tellurite (∼1/10th of minimal inhibitory concentration [MIC]), about 80% cells were found to be associated with aggregates (∼1–3 mm in diameter) (Chua et al., 2015). In addition, cells may also coaggregate with nanomaterials (Chowdhury et al., 2012). Each aggregate often contains hundreds or thousands of individual cells that are usually tightly packed in a sticky matrix of self-produced extracellular polymeric substances. As a result, each aggregate instead of an individual cell would develop into one single colony in CFU-based viability assessment, resulting in highly underestimated cell viability.

Another assumption of the CFU method is that each bacterial cell is equally culturable under tested conditions. This assumption may be questionable for biological systems exposed to nanomaterials. Many bacteria are capable of transiting into the viable but nonculturable (VBNC) state, that is, a viable state with a very low metabolic activity and division rate, to protect themselves from environmental stresses. For example, recent studies showed that exposure to nanosilver induced a VBNC state in model environmental bacteria such as P. aeruginosa, in which nanosilver caused about 7-log reduction of culturable cells, although almost all the cells survived with an intact cell membrane (Königs et al., 2015). In addition, the VBNC state may also be induced by cell aggregation. When aggregates are large and/or tight enough to cause diffusion limitation, cells inside the aggregates may experience nutrient starvation, which may induce a VBNC state. Hence, when conducting a CFU-based comparison of cell viability between control and nanomaterial-treated experiments, we need to rule out the possibility of the presence of a significant amount of VBNC cells in the treated conditions.

In cases where the CFU method is not applicable to or insufficient for our studies, molecular biology techniques provide a good alternative or complementary approach. For example, reverse-transcription polymerase chain reaction (RT-PCR) is a promising analytic tool to evaluate bacterial viability. The mRNA is a highly labile molecule with a very short half-life (in minutes), which correlates with metabolic activity and resuscitation capability. Quantification of mRNA by RT-PCR gives a better indication of the viable cell population that includes cells in aggregates and the VBNC cells. There has been an increasing number of environmental toxicity studies which have adopted a combined method of CFU count and molecular biology techniques, although in many recent studies, assessment of bacterial survival still solely relies on the CFU method.

There is no doubt that the CFU method is a very useful tool and most likely will remain as a mainstream method in the environmental engineering research community. However, because of the above-discussed limitations of the CFU method, we urge the environmental science and engineering community to exercise caution when applying the CFU method for the assessment of cell survival in the presence of nanomaterials and, if possible, to use appropriate molecular biology techniques as an alternative or complementary approach.