Comparison of a culture‐based and a PCR‐based methods for estimating bacterial abundance on eggshells,with comments on statistical analyses

Field ornithologists have used traditional culture‐based techniques to determine the presence and abundance of microbes on surfaces such as eggshells, but culture‐independent PCR‐based methods have recently been introduced. We compared the traditional culture‐based and the real‐time PCR‐based methods for detecting and quantifying Escherichia coli on the eggshells of Eurasian Magpies (Pica pica). PCR estimates of bacterial abundance were ～10 times higher than culture‐based estimates, and the culture‐based technique failed to detect bacteria at lower densities. When both methods detected bacteria, bacterial densities determined by the two methods were positively correlated, indicating that both methods can be used to study factors affecting bacterial densities. The difference between the two methods is consistent with generally acknowledged higher sensitivity of the PCR method, but the extent of the difference in our study (10×) may have been influenced by both a PCR‐based overestimation and culture‐based underestimation of bacterial densities. Our results also illustrate that bacterial counts may sometimes produce left‐censored data (i.e., we did not detect E. coli in 62% of our samples using the culture‐based method). Specific statistical methods have been developed for analyzed left‐censored data, but, to our knowledge, have not been used by ornithologists. In future studies, investigators studying bacterial loads should provide information about the possible degree of left censoring and should justify their choice of statistical methods from the broad set of available methods, including those explicitly designed for censored data.