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Methods for observing microbial biofilms directly on leaf surfaces and recovering them for isolation of culturable microorganisms

Epifluorescence microscopy, scanning electron microscopy, and confocal laser scanning microscopy were used to observe microbial biofilms directly on leaf surfaces. Biofilms were observed on leaves of all species sampled (spinach, lettuce, Chinese cabbage, celery, leeks, basil, parsley, and broad-leaved endive), although the epifluorescent images were clearest when pale green tissue or cuticle pieces were used. With these techniques, biofilms were observed that were about 20 (mu)m in depth and up to 1 mm in length and that contained copious exopolymeric matrices, diverse morphotypes of microorganisms, and debris. The epifluorescence techniques described here can be used to rapidly determine the abundance and localization of biofilms on leaves. An additional technique was developed to recover individual biofilms or portions of single biofilms from leaves and to disintegrate them for isolation of the culturable microorganisms they contained. Nineteen biofilms from broad-leaved endive, spinach, parsley, and olive leaves were thus isolated and characterized to illustrate the applications of this technique.