Biochemical as well as molecular tools were used to characterize the cultured actinomycetes. Mucus of four healthy individuals of the coral A. digitifera were collected from Hare Island (9°12′N latitude and 79°5′E longitude), the largest island in the Gulf of Mannar, Tamil Nadu, India. Coral mucus was collected using sterile cotton swabs (Guppy & Bythell, 2006). The coral surface mucus layer was swabbed using sterile cotton swabs. Mucus samples of c. 1 cm2 coral surface area were taken with these swabs. After swabbing, the swabs were immediately placed in sterile polypropylene tubes. Seawater samples were collected with 50-mL sterile tubes that were opened underwater adjacent to the

same corals. Sediment samples were collected from right below the corals. All samples were transported to the laboratory (in about 4-h time) in ice-cold condition and were plated for isolation of bacteria. HSP inhibitor review The mucus swab samples were transferred to sterile

tubes with 1 mL of autoclave-sterilized seawater, in a sterile hood. The cotton swabs were vigorously vortexed to suspend the bacteria in seawater (Guppy & Bythell, 2006). Actinomycetes were isolated using standard serial dilution and plating techniques in BYL719 price triplicate on starch casein agar supplemented with actidione (40 μg mL−1) (Himedia Laboratories, Mumbai, India) found to inhibit the growth of fungi (Goodfellow & Williams, 1988) and nalidixic acid (10 μg mL−1) (Himedia Laboratories), which inhibits the bacteria capable of swarming without affecting the growth of actinomycetes (Nonomura & Hayakawa, 1988). Actinomycetes colonies were recognized these on the basis of morphological and physiological characteristics following directions given by the International Streptomyces Project (Shirling & Gottlieb, 1966). Morphological characteristics were studied under a light microscope after

15 days of growth on oatmeal agar (ISP3) (Shirling & Gottlieb, 1966). Actinomycetes counts were recorded as CFUs and expressed as CFU per 1 cm2 of coral surface area for mucus and tissue. Culturable actinomycetes from seawater and sediment were recorded as CFU mL−1 (of seawater) and CFU g−1 (of sediment), respectively. The isolated actinomycetes were identified by performing various biochemical tests according to the Bergey’s manual and Lampert et al. (2006). Carbohydrate tests were performed using the HiCarbohydrate kit (Himedia Laboratories). The sensitivity of the actinomycetes to various antibiotics was determined after incubation for 24–48 h at 30 °C on ISP2 (International Streptomyces Project) agar (Himedia Laboratories). Total genomic DNA was extracted using a modified cetyltrimethylammonium bromide–NaCl protocol. For each isolate, a loopful of mycelium and spores was scraped from colonies grown on Starch Casein Agar (SCA) and resuspended in TE buffer as described previously (Zin et al., 2007). As suggested by Stach et al.