In situ identification of bacterial species in marine microfouling films by using an immunofluorescence technique

An immunofluorescence technique was developed for the in situ identification of specific bacteria in marine microfouling films. Microorganisms adherent to glass plates after 30 days of immersion in a synthetic seawater system were cultured and classified by biochemical tests, flagellar arrangement, and the API 20E system. All isolates were gram-negative aerobic or facultative motile rods, predominantly Pseudomonas spp. Rabbit antisera to the five dominant organisms including Achromobacter spp., Comamonas terrigena, P. putrefaciens, a yellow-pigmented Pseudomonas sp., and Vibrio alginolyticus were prepared. These antisera were shown to be species specific in indirect immunofluorescence assays against a battery of 26 marine isolates from 14 bacterial species, with the exception of antisera to the Pseudomonas spp, which cross-reacted with each other but not with test bacteria of other genera. These immunofluorescent reagents enabled the in situ identification of all five bacterial species in microfouling films. Low-surface-energy test plates had smaller numbers of adherent bacteria in microfouling films than medium-surface-energy test plates, suggesting that the degree of microfouling may be influenced by the surface energy. In addition, the reagents could identify up to 39% of the attached bacteria in microfouling films spontaneously formed on steel plates in flow cells deployed in different areas of the Atlantic Ocean. The microbial composition of the ocean-formed films varied with the geographical area of their formation. The present results indicate that immunofluorescence techniques may provide a rapid and reliable means to identify, in situ, specific bacteria in marine microfouling films.