Lim E L, Caron D A, Delong E F
Biology Department, Woods Hole Oceanographic Institution, Massachusetts 02543, USA.
Appl Environ Microbiol. 1996 Apr;62(4):1416-23. doi: 10.1128/aem.62.4.1416-1423.1996.
A fluorescent in situ hybridization method that uses rRNA-targeted oligonucleotide probes for counting protists in cultures and environmental water samples is described. Filtration, hybridization, and enumeration of fixed cells with biotinylated eukaryote-specific probes and fluorescein isothiocyanate-conjugated avidin were performed directly on 0.4-microns-pore-size polycarbonate filters of Transwell cell culture inserts (Costar Corp., Cambridge, Mass.). Counts of various species of cultured protists by this probe hybridization method were not significantly different from counts obtained by the 4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods. However, counts of total nanoplankton (TNAN) based on probe hybridizations in several field samples and in samples collected from a mesocosm experiment were frequently higher than TNAN counts obtained by staining with DAPI or AO. On the basis of these results, 25 to 70% of the TNAN determined with probes were not detectable by DAPI or AO staining. The underestimation of TNAN abundances in samples stained with DAPI or AO was attributed to the existence of small nanoplanktonic cells which could be detected with probes but not DAPI or AO and the difficulty associated with distinguishing DAPI- or AO-stained protists attached to or embedded in aggregates. We conclude from samples examined in this study that enumeration of TNAN with oligonucleotide probes provides estimates of natural TNAN abundances that are at least as high as (and in some cases higher than) counts obtained with commonly employed fluorochrome stains. The quantitative in situ hybridization method we have described here enables the direct enumeration of free-living protists in water samples with oligonucleotide probes. When combined with species-specific probes, this method will enable quantitative studies of the abundance and distribution of specific protistan taxa.
本文描述了一种荧光原位杂交方法，该方法使用针对核糖体RNA的寡核苷酸探针来计数培养物和环境水样中的原生生物。使用生物素化的真核生物特异性探针和异硫氰酸荧光素偶联的抗生物素蛋白对固定细胞进行过滤、杂交和计数，直接在Transwell细胞培养插入物（美国马萨诸塞州剑桥市康宁公司）的孔径为0.4微米的聚碳酸酯滤膜上进行。通过这种探针杂交方法对各种培养原生生物的计数与通过4',6-二脒基-2-苯基吲哚（DAPI）和吖啶橙（AO）染色方法获得的计数没有显著差异。然而，在几个野外样本和从中尺度生态实验收集的样本中，基于探针杂交的总微型浮游生物（TNAN）计数通常高于通过DAPI或AO染色获得的TNAN计数。基于这些结果，用探针测定的TNAN中有25%至70%无法通过DAPI或AO染色检测到。用DAPI或AO染色的样本中TNAN丰度被低估，这归因于存在小型微型浮游生物细胞，这些细胞可以用探针检测到，但不能用DAPI或AO检测到，以及区分附着在聚集体上或嵌入聚集体中的DAPI或AO染色的原生生物存在困难。我们从本研究中检查的样本得出结论，用寡核苷酸探针计数TNAN可提供自然TNAN丰度的估计值，这些估计值至少与（在某些情况下高于）常用荧光染料染色获得的计数一样高。我们在此描述的定量原位杂交方法能够用寡核苷酸探针直接计数水样中自由生活的原生生物。当与物种特异性探针结合使用时，该方法将能够对特定原生生物类群的丰度和分布进行定量研究。
