New Method for Counting Bacteria Associated with Coral Mucus

Abstract

ABSTRACT 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 × 10 5 ± 1.0 × 10 5 cells ml −1 to 1.8 × 10 6 ± 0.2 × 10 6 cells ml −1 ) than in the surrounding seawater (1.9 × 10 5 ± 0.1 × 10 5 cells ml −1 to 4.2 × 10 5 ± 0.2 × 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 × 10 5 ± 0.1 × 10 5 cells ml −1 to 3.4 × 10 5 ± 0.3 × 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.