Factors affecting quantitative assessment of Pseudomonas aeruginosa adherence to buccal epithelial cells

The influence of experimental protocol on estimation of adherence of P. aeruginosa to buccal epithelial cells is highlighted. Use of membrane filtration to remove non-adherent bacteria was significantly affected by membrane pore size and rinse volume, but was not affected by inoculum size or test isolate. Even with optimum filtration conditions, over 58% of the non-adherent bacterial population were retained on the filters. Separation of non-adherent bacteria was greatly improved when density centrifugation was employed, with less than 30% of non-adherent bacteria present in the buccal epithelial suspension. Adherence values measured by a radiological and fluorometric method were significantly higher than those determined by microscopic counting. Radiological assays had the lowest variance. Gradient centrifugation was used to prepare a standard suspension of buccal epithelial cells. However, cells collected on different days showed significantly different adherence measurements. Adherence of bacteria to trypsinized buccal epithelial cells and to buccal epithelial cells from cystic fibrosis patients was similar, whereas adherence to normal buccal epithelial cells was significantly lower.