Methods for the isolation, identification and preservation of benthic diatoms

• Main Authors: Yu-Tang Chang, 張育堂
• Other Authors: Min-Ying Wang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/32432017050086458954

碩士 === 國立中興大學 === 生物科技學研究所 === 101 === 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.