Mucus sampling: Mucus was obtained from the surfaces of replicate orange and red colonies of Montipora capitata collected in Kaneohe Bay, Oahu, using a sterile pipettor and frozen immediately.
Bacterial strains tested: The strains used for the antibacterial assays were selected as model systems to represent a range of potential bacterial pathogens from the marine environment, including known coral pathogens (Aurantimonas coralicida, Serratia marcescens, Vibrio coraliilyticus, Vibrio shiloi) and human enteric bacteria that have the potential to enter near-shore waters and can survive in the marine environment (Yersinia enterocolitica), and strains previously isolated from the surfaces of Hawaiian corals (Klebsiella pneumonia, Vibrio agarivorans) (Gochfeld & Aeby 2008). In addition, bacterial strains isolated from surfaces of Montipora capitata (OCN001, OCN002, OCN003, OCN008, O1-1, O2-8, R1-13, R5-5, R5-29) by Dr. Sean Callahan’s lab at University of Hawaii were also used. Two of these, OCN002 and OCN008, are pathogens associated with Montipora White Syndrome (Ushijima et al. 2012, 2014).
Growth inhibition assay (Gochfeld & Aeby 2008): Assays were run in triplicate in 96 well plates. Plates contained 3 wells of bacteria + each mucus sample (n = 5 replicate corals of 2 color morphs), along with the following controls: bacteria only, media only, and mucus solution to control for the coloration of extracts. Twenty-four hour bacterial cultures in exponential growth were diluted to optical density (OD) 600, and 100 μl were added to each well. 10 μl of mucus were added to the experimental wells. Wells were mixed by carefully pipetting their contents, and an initial reading (time 0) of OD was made on a BioTek Synergy™ HT Multi-Detection Microplate Reader. Plates were then covered with foil and incubated on a shaker for 24 h and a final reading was made. Prior to each reading, wells were mixed again to resuspend any settled material.
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