A comparative analysis of in vitro assays of bacterial adherence

In recent years there has been a sharp increase in publications concerned with adherence of bacteria to animal cells. Surprisingly, the three types of assays used to measure such adherence — light microscopy, quantitative subculture and radiolabel assays — had not previously been compared in a systematic fashion to determine their reliability or relative merits. In the present report we undertake such a comparison. Our findings indicate that although each assay has unique advantages and limitations, the double radiolabel assay has the fewest major limitations of the three assays examined. Unfortunately, this assay can quantitative the number of bacteria adhering to a given number of cells, but not the actual percent of cells colonized. The microscopic assay, because of its subjectivity and inability to detect small or poorly staining microorganisms, or to differentiate morphologically similar bacteria in mixed populations, is the least useful of the adherence assays. Our data suggest that when this assay is used, no fewer than 50 cells should be examined. The quantitative subculture assay had the advantage of detecting living bacterial forms, but suffered from difficulty obtaining a homogeneous suspension of colonized cells. Our data suggest that when using this assay or the radiolabel assay, quantitation of both bacterial and animal cells is necessary even when using only confluent monolayers. To obtain a comprehensive picture of bacterial adherence, data obtained with one assay should be confirmed in at least one of the other two assay systems.