• Home
  • »
  • Micro & Molecular Biology

Micro & Molecular Biology

Image

Standard microbiological techniques will be used to identify the microbial populations associated with lesion and healthy coral samples. Briefly, once a lesion is observed during the survey, a water sample is collected from above the sampling site as a reference and then a small portion of the coral lesion is removed and placed in a plastic bag at depth. Control samples are taken from healthy looking coral on the same head and from nearby coral (same species) that has no apparent lesions. All samples are placed in a cooler for transport to the laboratory at HIMB. Immediately upon return to the lab, coral mucus and water samples (50 ml) are used to inoculate growth media and for isolation of genomic DNA. Culture medium used most routinely include marine agar and TCBS (selective media for Vibrio spp.). Culture plates are incubated at ambient conditions and monitored on a daily basis. As individual bacterial colonies develop they are examined under a dissecting microscope for distinguishing morphologies and routinely stained for identification (Gram stain, acid-fast stain, etc.). Isolates are maintained in the lab and also in glycerol stocks (20% glycerol at -80° C).

DNA is extracted from mucus and water samples as well as culture isolates and amplified by polymerase chain reaction (PCR) to identify bacteria by ribotyping (16S rDNA). PCR amplicons are run on a temperature gel gradient electrophoresis apparatus (BioRad) to identify the ribotype fingerprint. These will be used to compare similarities/differences in samples based on differences in DNA bands amplified by PCR and selected bands are sequenced to identify bacteria present in each sample. This method complements the culture work described above to identify bacterial genera/species from the samples but also allows us to examine samples for the presence of unculturable bacteria.


Navigation :