Preliminary Study Of Contamination And Its Control In Cell Culture Of Marine Sponge Hymeniacidon Perleve

The contamination in sponge cell culture is one of the major obstacles to establish a long-term cell culture system. The characterization of various types of contamination and their control techniques were studied in the cell culture of marine sponge, Hymeniacidon perleve collected at the China Yellow Sea. The effects of these control techniques on sponge cell culture were also investigated.Based on the establishment of cultivation of sponge primmorphs from H. perleve, microbes and protists were detected as the two major sources of contamination and their damaging effects on sponge cell culture were also investigated. Bacterial contamination was the predominant type, and the culture was completely contaminated within 3 days without using suitable antibiotics as the control agents. Protist contamination occurred more frequently in the cell cultures using natural seawater media, and the addition of suitable antibiotics may inhibit the protist growth to certain extent.Different methods of controlling the microbial contamination were studied. Soaking sponge tissues in nature seawater containing antibiotics before preparing dissociated cells proved to be efficient in removing microbial contamination. Ficoll density gradient centrifugation that was used to enrich archaeocytes eliminated a large portion of bacterial contamination. During the cell cultue process, gentamycin was found to be the most efficient antibiotic in controlling the microbial growth among six antibiotics tested, and no apparent negative effect on sponge cell attachment, aggregation and the formation of primmorphs was observed upon the addition of gentamycin.The protist contamination and its controlling techniques were also investigated. Two types of protists Bodo and Gymnodinium were isolated, purified, and structurally confirmed. Ecological characteristic including the effects of pH, salinity and competition on population growth were studied. It was found that Bodo was the food source of Gymnodinium and high salinity inhibited the growth of Bodo. Increasing the salinity of the cell culture media was an effective method to control the contamination of Bodo, but it affected the growth of sponge cell and the formation of primmorphs. Treatment with 4 to 10ppm CuSO4 within less than 5min, proved effective to kill Gymnodinium. No apparent effects on the metabolizing activity of sponge cells were observed, while the formation of prommorphs was slightly affected.