荧光法测定水体叶绿素藻标准品制备技术研究

发布时间：2018-02-17 01:27

  本文关键词： 叶绿素a测定 荧光检测法 藻体标准品 悬浮稳定剂 真空冷冻干燥法　出处：《河北科技大学》2016年硕士论文　论文类型：学位论文

【摘要】：叶绿素a含量是水体富营养化指标,荧光法因测定时间短、可以实现现场在线测定等优势在水体叶绿a含量测定中得到广泛应用,但在测定过程,存在着藻细胞在水体分散不均匀、藻体悬浮体系不稳定、易沉降,无藻体标准品等问题,影响测量结果的准确度。论文研究了利用水体悬浮剂制备藻体标准品用于叶绿素荧光现场测定的制备技术,技术研究对水体叶绿素荧光法测定的方法标准化有重要意义。文章简述了荧光检测法对叶绿素a测定的国内外研究进展,微生物悬浮稳定技术、保存技术的研究进展。以叶绿素a的实验室荧光法测定方法为基础,得到如下结论:1)基于蛋白核小球藻的自身优势及实验优势,确定蛋白核小球藻作为目标藻体,建立了蛋白核小球藻的吸光度值与荧光强度值的标准曲线y=74.831x+16.94(R2=0.9945),最优D(663 nm)范围为0.219~0.777,对应的荧光强度值为31.606~74.722。2)藻细胞沉降,导致其荧光值受静置时间影响较大,静置1 min时,荧光值的衰减率达△F/F0=-9.96%,无法获得准确的藻浓度。3)藻体培养过程中向培养基中添加琼脂粉0.8~1.0 g·L-1培养蛋白核小球藻,藻液的悬浮性、均匀性、稳定性都明显增强。4)在荧光法测定水样叶绿素过程中,提出一种藻体悬浮稳定技术,在藻液荧光测定环节添加复合悬浮剂,即琼脂0.06%、黄原胶0.20%、卡拉胶0.40%,T=1h时荧光值的变化率为-1.16%,并对实际水样测定,T=1 h时荧光值的变化率小于±3.5%、精密度较高小于1.5%。5)对长势较好的蛋白核小球藻在最佳离心条件下,即离心转速为4 500 r.min-1,离心时间为为10min浓缩100倍;保护剂:15%脱脂奶粉,体积比1:1混合;预冻方式:4℃预冻12 h+液氮(-196℃)冷冻12 h;冻干保存温度:-20℃。保存时间:40 d(存活率高于40%);100 d(存活率高于30%)。
[Abstract]:Chlorophyll a content is a eutrophication index of water body. Because of the short measuring time, fluorescence method can be used widely in the determination of leaf green a content in water body, but it can be used in the determination process. There are some problems such as uneven dispersion of algal cells in the water body, unstable suspension system of algae body, easy settling, and no algal body standard, etc. In this paper, the preparation technology of algae standard for chlorophyll fluorescence in situ was studied. The technical research is of great significance to the standardization of the methods for the determination of chlorophyll a in water body. This paper briefly introduces the domestic and foreign research progress of fluorescence detection for the determination of chlorophyll a and the technique of microorganism suspension stabilization. Based on the fluorescence assay of chlorophyll a in laboratory, the following conclusions are obtained: 1) based on the advantages of Chlorella protein and its experimental advantages, Chlorella Proteinuca is identified as the target alga. The standard curve of absorbance value and fluorescence intensity value of Chlorella pyrenoidosa was established. The optimum range of DX 663nm was 0.2196nm and the corresponding fluorescence intensity value was 31.606n 74.722.2) the fluorescence value of Chlorella globosa was significantly influenced by standing time, and the fluorescence value was statically affected by 1 min. The attenuation rate of fluorescence value was as high as F / F _ 0 ~ (-9.96). It was impossible to obtain accurate algal concentration. 3) adding Agar powder (0.81.0 g 路L ~ (-1)) to the culture medium of Chlorella vulgaris, the suspension and homogeneity of algal fluid, were obtained by adding Agar powder (0.81.0 g 路L ~ (-1)) to the culture medium. In the process of determination of chlorophyll in water samples by fluorescence method, a suspension stabilization technique for algae was put forward, in which compound suspensions were added in algal liquid fluorescence determination. That is, Agar 0.06, Xanthan 0.20, carrageenan 0.40T + 1 h fluorescence change rate is -1.16, and the change rate of fluorescence value is less than 卤3.5 at 1 h and higher precision is less than 1.5. 5) under the best centrifugation conditions. The centrifugal speed is 4 500 r.min-1, the centrifugation time is 10 min, the concentration is 100 times, the protective agent is 1: 15% skim milk powder, the volume ratio is 1: 1; The pre-freezing method was pre-frozen at 4 鈩，

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