Limnological Institute, Vijverhof Laboratory, 3631 AC Nieuwersluis, The Netherlands.
Appl Environ Microbiol. 1986 Dec;52(6):1266-72. doi: 10.1128/aem.52.6.1266-1272.1986.
Quantitative effects of several fixatives on heterotrophic nanoflagellates (HNAN) and phototrophic nanoflagellates (PNAN) were investigated by hemacytometer and epifluorescence counting techniques. Counts of Monas sp. cultures before and after fixation with unbuffered 0.3% glutaraldehyde and 5% formaldehyde showed no loss of cells during fixation, and cell concentrations remained constant for several weeks after fixation. Buffering of fixatives with borax caused severe losses, up to 100% within 2 h. Field samples from Lake Vechten showed no decline of HNAN and total nanoflagellate concentrations for at least 1 week after fixation with 5% formaldehyde and with 1% glutaraldehyde. With 1% glutaraldehyde, the chlorophyll autofluorescence of PNAN was much brighter than with 5% formaldehyde, although it was lost after a few days and thus limited the storage time of samples. However, when primulin-stained slides were prepared soon after fixation and stored at -30 degrees C, the loss of autofluorescence was prevented and PNAN and HNAN concentrations were stable for at least 16 weeks. Effects of filtration and centrifugation on HNAN were also studied. Filtration vacuum could not exceed 3 kPa since 10 kPa already caused losses of 15 to 20%. Similar losses were caused by centrifugation, even at low speed (500 x g).
通过血球计数器和荧光显微镜计数技术研究了几种固定剂对异养微型鞭毛虫（HNAN）和自养微型鞭毛虫（PNAN）的定量影响。用未缓冲的 0.3%戊二醛和 5%甲醛固定之前和之后的 Monas sp.培养物的计数显示，固定过程中没有细胞损失，并且固定后数周内细胞浓度保持不变。用硼砂缓冲固定剂会导致严重的损失，在 2 小时内高达 100%。Vechten 湖的现场样本表明，用 5%甲醛和 1%戊二醛固定后，至少在 1 周内 HNAN 和总微型鞭毛虫浓度没有下降。用 1%戊二醛固定时，PNAN 的叶绿素自发荧光比用 5%甲醛时亮得多，尽管几天后就会消失，因此限制了样品的储存时间。然而，当固定后立即制备并储存在-30 摄氏度的Primulin 染色载玻片时，可以防止自发荧光的损失，并且 PNAN 和 HNAN 浓度至少稳定 16 周。还研究了过滤和离心对 HNAN 的影响。过滤真空不能超过 3 kPa，因为 10 kPa 已经导致 15%至 20%的损失。即使在低速（500 x g）下，离心也会造成类似的损失。
