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標題: 附著矽藻的分離、鑑定與種原保存方法之探討
Methods for the isolation, identification and preservation of benthic diatoms
作者: 張育堂
Chang, Yu-Tang
Contributors: 王敏盈
Min-Ying Wang
關鍵字: 矽藻;18S rDNA;型態學
Diatom;18S rDNA;Morphology
日期: 2013
Issue Date: 2013-11-18 10:44:35 (UTC+8)
Publisher: 生物科技學研究所
摘要: 矽藻是自然界種類最多的單細胞藻類,幾乎在任何水域都可發現。本研究目的為建立一個從野外採集、分離到矽藻種源鑑定與保存的平台。主要的樣本採集自墾丁,藻種的分離採用微量吸管在顯微鏡下吸取單一顆矽藻並移至培養盤內培養。依此方法共分離出13種矽藻,以其矽殼形態與18S rDNA做為鑑定依據,發現分離出的矽藻有6株屬於Nitzschia sp.,各有2株分屬於Psammoneis sp.和Hyalosynedra sp.,其他3株分屬於Diploneis sp.、Halamphora sp.和Cyclophora sp.。在保存方法上,我們以易生長且操作容易的Nitzschia sp. AQ 1為測試樣本。評估褐藻膠包埋與 -80 oC低溫保存兩種方法。結果顯示褐藻膠保存法在25 oC、1000 lux光照下優於4 oC、無光照環境,保存最久可達半年;在-80oC低溫保存方面,我們測試樣品中添加20 %、15 % 與10 % dimethyl sulfoxide(DMSO)對矽藻保存於 -80oC的影響。一週後解凍所測得之回復率顯示以上三種DMSO濃度間並無顯著差異,但不添加DMSO的樣品因冰晶造成機械性損傷而無法存活;將保存一個月後的藻種解凍,僅20 % DMSO的樣品仍可回復生長;第二個月後全數樣本皆無法回復生長。以上結果顯示褐藻膠包埋法在矽藻的保存上是一個操作簡單且符合成本考量的選擇。本研究所建立的矽藻分離、種源鑑定與保存之平台,可做為後續更進一步研究的基礎。
Diatoms are single-cell algae found in almost all types of waters. The goal of this study is to establish a system that includes efficient ways to collect diatoms from natural water sources, to isolate them into single species, to identify and classify them and to store and preserve them. Diatoms were collected from marine water sources collected from a coral rock found in Kenting Area, Pingtong County, Taiwan and separated into single-cell diatom cultures using a glass micropipette system to isolate and transfer them into culturing plates. This method was used to separate 13 kinds of diatoms. Frustule morphology and 18S rDNA analysis was used to identify and classify the diatoms species and as such, it was found that the isolated algae species belong to different genera, including Nitzschia (6 strains), Halamphora (1 strain), Diploneis (1 strains), Psammoneis (2 strains), Hyalosynedra (2 strains) and Cyclophora (1 strain). For the preservation studies, we chose one Nitzschia species (AQ 1) as the test sample because of its robustness and ease of growth. Two preservation methods were compared, one using a solution of sodium alginate and the other, a cryopreservation method that allows cells to be stored at -80 ℃ . The results of alginate immobilization showed that cells kept at 25 ℃ in weak light (1000 lux) were healthier after recovery than cells kept in the dark (at 4 ℃), and that at those conditions, they could be stored for about six months with a reasonably good survival rate. Tests for cryopreservation were done adding 20%, 15% and 10% dimethyl sulfoxide (DMSO) and stored at -80℃. After one week of storage, the recovery rates showed no significant difference between the three groups after re-culturing, and that diatoms can not regrow in DMSO-free culture. After one month of preservation, only the group to which 20% DMSO had been added could regrow, but all the tested groups died after two month of preservation. These results indicate that the alginate method is simpler and more cost-effective and should be considered the best choice for field preservation of isolated diatoms. This study established a system for collection, separation, identification and preservation, thus allowing isolated diatoms to be used as the basis for subsequent further study.
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