Design and validation of a novel quantitative method for rapid bacterial enumeration using programmed stage movement scanning electron microscopy

The adhesion of bacteria to surgical implants is the first stage of implant infection. The method for detecting bound bacteria is an important consideration in the study of bacterial adherence and colonisation. Enumeration of bacteria by direct visualisation techniques is labour intensive and time consuming. We have developed and validated a method for enumerating bacteria on porous material surfaces using programmed stage movement scanning electron microscopy and compared cumulative counts after 1-10 stage movements with absolute bacterial counts. We describe this method with three commercially sourced meshes used for abdominal wall hernia repair and with three different inoculums of Staphylococcus epidermidis. The results demonstrate significant correlation to the absolute count after five cumulative counts for all meshes analysed. The mean time saved by the cumulative counting method was 1h and 9 min per mesh. We conclude that advances in scanning electron microscopy and the advent of precise automated stage control have facilitated rapid data acquisition for bacterial counting purposes and that five cumulative counts at 1000× or 2500× magnification are a valid quantitative method for enumerating S. epidermidis bacteria on porous surfaces (with a pore size of up to 1.3 mm).