Diversity of bacteria in coastal seawater as investigated by cultivation on TCBS agar medium

• Main Authors: Huang-Ming Li, 李晃銘
• Other Authors: 謝文陽
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/43306781861510439896

碩士 === 國立臺灣大學 === 海洋研究所 === 92 === Seawater samples were collected five times from the coastal regions of Badouzih and Shenao, northern Taiwan between years 2002 and 2003 in the present study. Thiosulfate-citrate-bile salt-sucrose（TCBS）were used principally as the selective medium and Vibrio parahaemolyticus-sucrose-I（VPS-I）, Vibrio parahaemolyticus-sucrose-II（VPS-II）and tryptose-yeast-glucose（TYG） were also utilized as auxiliary media for enumeration of bacteria in seawater samples. The plate counting values of the bacteria were estimated to be in the range of 102-104 CFU/ml on TYG agar medium, and the values on the other three kinds of selective media were generally lower than half of the former. According to the differences of colony shapes, 213 bacterial strains were isolated from TCBS, VPS-I and VPS-II agar media, respectively. These 213 strains were divided into 15 groups on the basis of variations of phenotypic characterizations including Gram reaction, glucose fermentation, sucrose fermentation, Na+ requirement, swarming and agarase. Excluding the 2nd, 9th, 12th and 13th groups, the 16S rDNA sequence similarities of the representative strains of other 11 groups compared to the known bacteria in GenBank were higher than 97﹪. The 1st, 2nd, and 3rd groups containing 18 strains were not marine vibrio obviously. The remainder strains which belonged to the other twelve groups accounted for more than 90% of total amount of bacteria. Except the representative strains of the 12th group still needed to be further confirmed, these strains possessed the characteristics which were consistent with marine vibrios. This result proved that TCBS, VPS-I and VPS-II mediums containing cholate and sodium ion were effective to sieve vibrio out from marine water and repressed commonly the growth of bacteria belonging to non-marine vibrios and land-originated species, especially Gram-positive bacteria. Two strains of every group were selected for 16S rDNA/ RFLP analysis using three endonucleases including DdeI, RsaI and ScaI. The results showed that 13 genotypes were categorized from the multiple restriction patterns. The 16S rDNA sequences similarities among representative strains were higher than 97﹪, the genotypes of 16S rDNA/ RFLP were almost same from each other. But, the rule was not suitable for some strains. The representative strains of 9th and 13th groups with incomplete 16S rDNA sequence data had unique genotype of RFLP differentiated from other representative strains. Genomic DNA hybridization showed that all of strains with 16S rDNA sequence similarity high than 97﹪belonged different species. Based on 16S rDNA/ RFLP analysis, 16S rDNA-based phylogenetic analysis and DNA hybridization, the 15 representative strains were different from one another, and the representatives of the 2nd and 12th groups may belong to new species.