江苏沿海滩涂土壤中细菌培养方法的优选
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江苏省滩涂生物资源与环境保护重点建设实验和国家自然科学基金(40871117和40471072)


Culturability of bacteria in mudflat soils and optimization of culture schemes
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    摘要:

    研究选择江苏省盐城市王港附近一垂直于海岸线的断面(土壤含盐量近似递减),分别采集自海水淹没线至内陆农田相隔一定距离的土样(编号为Soil-1、Soil-2、Soil-3、Soil-4和Soil-5),代表海水淹没、裸滩、盐蒿、芦苇/白茅和农田植被环境。设计9种培养方案,包括不同浓度的NB培养基,配以不同浓度的海水,添加和不添加环腺苷(cAMP),平板和液体两种培养方式,用变性凝胶电泳检测可培养菌的多样性,并评价和优选培养方案。结果表明,经过自然脱盐的耕种土壤,培养基中配入10%的海水可培养菌多样性指数最高;其余4种土壤配入100%海水多样性指数最高。添加cAMP不能明显提高微生物可培养性。低浓度培养基较过去惯用浓度的培养基更有利于提高可培养微生物的多样性。液体培养基明显优于固体培养基。在10-8或更高稀释度下,液体培养的试管底部有粘稠状沉淀物为微生物细胞,但培养基清澈。将有沉淀物的试管视为阳性试管,用最或然数方法计的土壤中细菌数量可达1012 g-1。提出培养分离滩涂土壤培养方案为:用1/10浓度的NB配以100%海水的液体培养基培养,用相同浓度和成分的固体培养基分离,培养25d左右。

    Abstract:

    Mudflat soils different in salt concentration were collected from Dafeng, Jiangsu Province, China. The soils were tested for culturability of bacteria, using different culture schemes and the technology of DGGE (denaturing gradient gel electrophoresis). Results show that natural sea water was necessary as a media supplement in culturing bacteria in all soils. For cAMP did not show much improvement in bacterial culturability. Concentration of the medium was a key factor affecting the culturability. Prolonged incubation up to 25 days increased the number of colony-forming units on the plate. Liquid media were better than solid ones in terms of culturing maximal number of bacteria. By the most probable number method, it was noted that tubes with limpid solution and some mucous precipitation, which used to be overlooked as “negative”, should be taken as “positive”. Microscopic and sequence examination revealed that the mucous precipitation consisted mainly of small short rodshaped bacteria (0.6×1.2~2.6 μm), highly similar to Bacillus sp. An optimal culture scheme for the mudflat soils is proposed to have liquid culture medium consisting of 1/10 nutrient broth and 100% natural sea water, and 25-day incubation. Further study should be conducted to optimize carbon compounds in the media.

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张雯雯,陈新兵,赵秀锦,朴哲,周春霖,殷士学.江苏沿海滩涂土壤中细菌培养方法的优选[J].土壤学报,2010,47(3):529-536. DOI:10.11766/trxb200901220036 Zhang Wenwen, Chen Xingbing, Zhao Xiujing, Piao Zhe, Zhou Chunling, Yin Shixue. Culturability of bacteria in mudflat soils and optimization of culture schemes[J]. Acta Pedologica Sinica,2010,47(3):529-536.

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  • 收稿日期:2009-01-22
  • 最后修改日期:2009-11-04
  • 录用日期:2009-11-10
  • 在线发布日期: 2010-06-13
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