New Method for Counting Bacteria Associated with Coral Mucus

The ability to count bacteria associated with reef-building corals in a rapid, reliable, and cost-effective manner has been hindered by the viscous and highly autofluorescent nature of the coral mucus layer (CML) in which they live. We present a new method that disperses bacterial cells by trypsinization prior to 4',6-diamidino-2-phenylindole (DAPI) staining and quantification by epifluorescence microscopy. We sampled seawater and coral mucus from Porites lobata from 6 reef sites influenced by wastewater intrusion and 2 reef sites unaffected by wastewater in Hawaii. Bacterial and zooxanthella abundances and cell sizes were quantified for each sample. Bacteria were more abundant in coral mucus (ranging from 5.3 x 10(5) +/- 1.0 x 10(5) cells ml(-1) to 1.8 x 10(6) +/- 0.2 x 10(6) cells ml(-1)) than in the surrounding seawater (1.9 x 10(5) +/- 0.1 x 10(5) cells ml(-1) to 4.2 x 10(5) +/- 0.2 x 10(5) cells ml(-1)), and the mucus-associated cells were significantly smaller than their seawater counterparts at all sites (P < 0.0001). The difference in cell size between mucus- and seawater-associated bacteria decreased at wastewater-influenced sites, where simultaneously mucus bacteria were larger and seawater bacteria were smaller than those at uninfluenced sites. The abundance of zooxanthellae in mucus ranged from 1.1 x 10(5) +/- 0.1 x 10(5) cells ml(-1) to 3.4 x 10(5) +/- 0.3 x 10(5) cells ml(-1). The frequency of dividing cells (FDC) was higher in the surrounding seawater than in mucus, despite finding that a 1,000-fold-higher zooxanthella biovolume than bacterial biovolume existed in the CML. Establishment of a standardized protocol for enumeration will provide the field of coral microbial ecology with the urgently needed ability to compare observations across studies and regions.