Statistical assessment of the accuracy and precision of bacteria- and virus-sized microsphere enumerations by epifluorescence microscopy

Fluorescent microspheres are increasingly used in environmental studies to evaluate threats of viral and bacterial pathogens in drinking water and to investigate colloid-facilitated contaminant transport. A commonly accepted technique for the enumeration of viruses, bacteria, and virus- and bacteria-sized particles by microscopy involves a field-of-view (field) approach to estimate concentration. Few studies have focused on those factors that are most important in ensuring precise and accurate measures of concentration. Microsphere counts in suspensions of artificial groundwater and deionized water were contrasted in this study to gain a greater understanding of the effect of ionic strength and the presence of precipitates in groundwater matrices that can bias microsphere enumerations. To investigate microsphere enumeration with minimal bias from other factors, a commonly used standard method was used to prepare slides and enumerate microspheres, with particular care to randomly select fields for counting. A factorial experiment evaluated two factors, (1) the density of microspheres in each field and (2) the number of counts in an enumeration. Two parameters, relative standard deviation and percent error, were used to assess methodological precision and accuracy. Visual observations of the slides indicated that some biases, such as undulation in the filter membrane or bubble entrained in the mounting medium, create biases in microsphere enumeration. Additional biases were introduced by the presence of precipitates that form in artificial groundwater saturated with calcite. Microsphere density was found to be critical for ensuring methodological precision, whereas the total number of microspheres counted was essential to ensuring methodological accuracy. The results suggested that to minimize variability using the field approach, the enumeration of at least 350 microspheres and 25–40 microspheres field−1 is necessary.