Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter
Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter
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DOI：
10.1023/a:1026579426265
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发表时间：
2000-08-01
影响因子：
2.6
通讯作者：
Iimura, Y
中科院分区：
文献类型：
--
作者：
Hayakawa, M;Otoguro, M;Iimura, Y
本文介绍了一种简单的富集技术，使不同的游动孢子放线菌属的快速和选择性的分离直接从土壤和植物凋落物。该技术称为再水化和离心（RC）法，包括将空气干燥的源材料浸入含有10%土壤提取物的10 mM磷酸盐缓冲液中，使制备物在30 ℃下静置90分钟，然后在1 ℃下离心流体，将含有放线菌游动孢子的部分上清液涂布在腐殖酸-琼脂培养基上。维生素琼脂，其补充有萘啶酸和甲氧苄啶作为革兰氏阴性菌和杆菌的选择性抑制剂。磷酸盐缓冲液-土壤提取液显著促进游动孢子从源材料中的释放。离心阶段大大消除了链霉菌和其他非运动放线菌从液相中，从而促进稀有，运动放线菌的选择性生长的隔离板上接种后。十个不同的土壤和落叶样品，从田野，森林和河岸，进行了检查。RC方法在所有样品中一致地实现了运动放线菌的优先分离，其占回收的总微生物种群的37-86%。最常见的运动放线菌是游动放线菌和Daclampussporangium。根据检查样品的性质，还回收了放线菌属、链板菌属和孢子虫属菌株。其他偶尔分离出的少量活动放线菌包括放线菌、嗜地皮菌和孢子鱼属。
The present paper describes a simple enrichment technique which enables rapid and selective isolation of diverse zoosporic actinomycete genera directly from soil and plant litter. This technique, designated the rehydration and centrifugation (RC) method, consists of immersing the air-dried source material in 10 mM phosphate buffer containing 10% soil extract, letting the preparation stand at 30 degreesC for 90 min, followed by centrifugation of the fluid at 1,500xg for 20 min. Portions of the supernatant containing actinomycete zoospores are plated on the humic acid-vitamin agar which is supplemented with nalidixic acid and trimethoprim as the selective inhibitors for Gram-negative bacteria and bacilli. The phosphate buffer-soil extract solution significantly promoted liberation of motile zoospores from the source material. The centrifugation stage greatly eliminated streptomycetes and other non-motile actinomycetes from the liquid phase, thereby facilitating selective growth of rare, motile actinomycetes on the isolation plates subsequent to inoculation. Ten different soil and leaf-litter samples, taken from fields, forests, and stream banks, were examined. The RC method consistently achieved preferential isolation of motile actinomycetes in all samples, which accounted for 37-86% of the total microbial population recovered. The most frequently isolated motile actinomycetes were Actinoplanes and Dactylosporangium. Strains of Actinokineospora, Catenuloplanes and Kineosporia were also recovered, depending on the nature of the samples examined. Other motile actinomycetes that were occasionally isolated in small numbers included Actinosynnema, Geodermatophilus and Sporichthya.