Optimal staining and sample storage time for direct microscopic enumeration of total and active bacteria in soil with two fluorescent dyes
Optimal staining and sample storage time for direct microscopic enumeration of total and active bacteria in soil with two fluorescent dyes
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使用两种荧光染料直接显微计数土壤中总细菌和活性细菌的最佳染色和样品储存时间
DOI：
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发表时间：
1995
影响因子：
4.4
通讯作者：
Eric A. Strauss
中科院分区：
文献类型：
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作者：
Wei Yu;Walter K. Dodds;M. K. Banks;J. Skalsky;Eric A. Strauss
直接计数技术，首先开发的水生样品，可用于计算土壤和地下水沉积物中的细菌。用5-氰基-2，3-二甲苯基四唑氯化物（CTC）和4（prm 1），6-二脒基-2-苯基吲哚（DAPI）两种荧光染料对土壤中呼吸活跃的细菌和细菌总数进行了荧光直接计数。两种染料可用于同一土壤样品，而不影响计数结果。用CTC染色8小时和用DAPI染色40分钟产生最大数量的染色细胞。最佳DAPI染色浓度为10 mg/L（sup-1）。制备后，载玻片应在4 ℃下储存，CTC在2天内计数，DAPI在24小时内计数。在计数之前，使用PP钠（infi）或氯化钠溶液从土壤中解吸细菌。当使用氯化钠时，计数显着更高。
Direct counting techniques, first developed for aquatic samples, can be used to enumerate bacteria in soil and groundwater sediments. Two fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for actively respiring bacteria and 4(prm1),6-diamidino-2-phenylindole (DAPI) for total bacteria, were tested for their usefulness in epifluorescent direct bacterial enumeration in soil. Both dyes can be used for the same soil sample without affecting enumeration results. Staining for 8 h with CTC and for 40 min with DAPI resulted in maximum numbers of stained cells. The optimal DAPI staining concentration is 10 mg liter(sup-1). After preparation, slides should be stored at 4(deg)C and counted within 2 days for CTC and within 24 h for DAPI. Sodium PP(infi) or sodium chloride solutions were used to desorb bacteria from soil prior to counting. Counts were significantly higher when sodium chloride was used.