Assessing diatom-associated bacteria by a specific CARD-FISH protocol

The cell surfaces of phytoplankton algae including diatoms are usually colonized by specific bacterial populations which interact with and affect growth of the host species. Catalyzed Reported Deposition Fluorescence in situ Hybridizazion (CARD-FISH) is a well-suited technique to visualize and identify algal-associated bacterial cells. Autofluorescence and the strongly structured cell surface of the algal cell make it difficult to quantify distinct populations of the colonizing bacterial communities. To overcome these limitations, we adopted a CARD-FISH method to this specific application by reducing the algal autofluorescence by an extra ethanol treatment and by stacking epifluorescence micrographs taken at different focal planes and merging them into a composite image. Cells of the diatom Thalassiosira rotula were used as host and incubated with a consortium of different bacterial strains and a natural bacterial community. Samples were concentrated either by filtration onto polycarbonate membranes or by centrifugation and analyzed with probes CF319a, GAM42a and ROS536. The results showed easily quantifiable bacterial cells and did not reveal any significant differences in the abundance of diatom-associated bacterial populations assessed by both methods. Our CARD-FISH protocol persuasively demonstrated that bacterial populations can be easily and reliably enumerated on diatom cells and presumably also on other algal cells and autotrophic biofilms.