A technique to quantify the population size and composition of the biofilmcomponent in communities of bacteria in the phyllosphere

The presence of microbial biofilms in the phyllosphere of terrestrial plant s has recently been demonstrated, but few techniques to study biofilms asso ciated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is as sembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities . This technique is based on removal of bacteria from leaves by gentle wash ing, separation of biofilm and solitary bacteria by filtration, and disinte gration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solita ry bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here wer e also evaluated. Isolation and quantification of bacteria by this techniqu e is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this tech nique. Preliminary results from estimates of biofilm abundance in phyllosph ere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.