WO2023221509A1 - 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法 - Google Patents

一种土壤/底泥活体藻细胞分离提取与快速分类计数方法 Download PDF

Info

Publication number
WO2023221509A1
WO2023221509A1 PCT/CN2022/141757 CN2022141757W WO2023221509A1 WO 2023221509 A1 WO2023221509 A1 WO 2023221509A1 CN 2022141757 W CN2022141757 W CN 2022141757W WO 2023221509 A1 WO2023221509 A1 WO 2023221509A1
Authority
WO
WIPO (PCT)
Prior art keywords
sediment
soil
centrifuge tube
extraction
sample
Prior art date
Application number
PCT/CN2022/141757
Other languages
English (en)
French (fr)
Inventor
林莉
贾迪
张雨婷
董磊
潘雄
Original Assignee
长江水利委员会长江科学院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 长江水利委员会长江科学院 filed Critical 长江水利委员会长江科学院
Publication of WO2023221509A1 publication Critical patent/WO2023221509A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1425—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements

Definitions

  • the present disclosure belongs to the field of microorganisms and relates to a method for separation, extraction and rapid classification and counting of living algae cells in soil/sediment.
  • Algal cells are widely distributed in nature. For example, in various water bodies, soils and other environments, sediment algae play an indispensable role in the material cycle and energy flow of ecosystems. Some algae species will concentrate in the bottom mud when they are dormant over the winter. The algae cells at this time will act as a "source”. When the temperature rises next year, the "source” algae in the bottom mud will continue to be released into the water body, resulting in the emergence of water bodies. phenomena such as bloom outbreaks, which directly or indirectly affect the survival of aquatic life and the safety of the ecosystem. Understanding the types and numbers of living algal cells in the sediment is the basis for conducting research on the mechanism of algal bloom formation and technical research on bloom prevention and control.
  • statistical methods for algal biomass include microscopic hemocytometer, photosynthetic pigment content determination, optical density value method and other innovative methods.
  • the microscopic hemocytometer method requires a large amount of sampling and transportation, and takes a long time to settle and concentrate. It requires scientific researchers to continuously observe multiple fields of view, so counting is easy to be repeated or missed, and the accuracy and efficiency are low. Moreover, the level and experience requirements for scientific researchers are relatively high. In addition, due to the presence of a large amount of impurities in the soil/sediment, it is difficult to observe and count algal cells under microscope.
  • imaging flow cytometry adhesion counting methods based on two-way background difference method Wang Junsheng, Zhang Hongyue: An imaging flow cytometry adhesion counting and activity detection method and device based on two-way background difference method [P] , CN113222969A, 2021-08-06
  • algae cell counting and algae species identification method based on line scanning Raman microscopy imaging He Shixuan, Xie Wanyi, Fang Shaoxi, Wang Deqiang: Algae cell counting and algae species identification based on line scanning Raman microscopy imaging Different identification methods, CN106442463B, 2019-03-08), etc., the counting of algal cells will be interfered by sediment.
  • the photosynthetic pigment content determination method requires steps such as suction filtration, grinding, extraction, centrifugation, and colorimetric determination.
  • the operation is complex, time-consuming, and low-efficiency.
  • the statistics of algal biomass by this method may be affected by inactive damaged or dead algal cells and residual photosynthetic pigments, so it is impossible to accurately count the number of active algal cells in the soil/sediment.
  • the solvents required for extraction operations are mostly organic solvents that are toxic to the human body, such as methanol, ethanol, and acetone.
  • the optical density value method (Su Wen, Kong Fanxiang, Zhao Xuhui: A rapid counting method for Microcystis aeruginosa [P], CN103276045A, 2013-09-04) is suitable for indoor simulation experiments of known algae and cannot identify algae. At the same time, when extracting algae from the soil, some sediment will be carried, which will also greatly interfere with the measurement data of the UV spectrophotometer. Therefore, this method can only be used to detect algae density in water bodies.
  • Zhang Chengxiang et al. proposed a method to separate algae cells from attached substrates such as submerged plants through the principle of ultrasonic cleaning, so that the algae cells enter distilled water to form algal liquid and collect statistics (Zhang Chengxiang, Lei Guangchun, Pei Hongcui: Attached algae separation sampling device, attached Algae content calculation system and method [P], CN111537678A, 2020-08-14).
  • This method and its device require the use of a suction filtration device.
  • this method is not applicable to soil/sediment, because the accumulation of large amounts of sediment not only easily blocks the filter membrane and device, but also causes the algae cells to be destroyed under high pressure.
  • the collected soil/sediment samples were air-dried and then ground. Add 90% acetone solution and Tris buffer to the samples respectively. Leave to stand for 8 to 10 hours and then centrifuge. Then place the filter membrane in a volumetric flask for filtration and repeat extraction for 3 to 10 hours. After 4 times, the volume was adjusted to a constant volume, and then the contents of chlorophyll and phycocyanin were measured through fluorescence analysis to obtain the algal community biomass (Chen, C., Yang, Z., Kong, F., Zhang, M., Yu, Y. ., & Shi, However, this method requires grinding the sediment sample and adding acetone and other solutions, which will cause the algae cells to lose activity, and the operation is complicated and takes a long time.
  • this disclosure proposes a simple, efficient and accurate method for separation, extraction and rapid classification and counting of living algal cells in soil/sediment, which is of great significance for research on soil/sediment algae biology and environmental ecology.
  • the present disclosure aims to provide a soil/sediment living algae cell separation, extraction and rapid classification and counting method, so as to be able to completely separate and extract algae cells from soil/sediment. And keep the activity of algae cells from being destroyed, while achieving the purpose of quickly and accurately counting the number, judging the structure and abundance of algae community.
  • the present disclosure provides a method for separation, extraction and rapid classification and counting of living algae cells in soil/sediment, which includes the following steps:
  • the amount of soil or sediment is 1-2 mL.
  • the small amount of sample required can significantly reduce the workload of sample collection and transportation, and ensure high extraction efficiency.
  • the dosage of the Percoll solution is 5-10 mL, and the concentration is 15%-55%.
  • Percoll separation solution is a silica gel particle coated with vinylpyrrolidone, which does not penetrate the cell membrane and has no toxic effect on cells. In this way, not only the intact living algae cells can be separated from the sediment, but also the living algae cells can be separated from the damaged algae cells and their fragments. In order to ensure that living algae cells can be fully separated into the solution, the amount of Percoll solution should not be too small. Through experimental analysis, it was found that 5 to 10 mL is appropriate.
  • the concentration of the Percoll solution should not be too low, preferably higher than 15%. At the same time, in order to reduce material consumption, the concentration should be as low as It is appropriate to set it at 55%.
  • the ultrasound time set by the ultrasonic cleaning instrument is 1 to 3 minutes, and the power is 20 to 360W.
  • Ultrasonic operation can promote the separation of algae cells from the sediment. However, if the power is too small and the time is too short, the algae cells cannot be completely separated. If the power is too high and the time is too long, the algae cells will be damaged, rupture, and die. According to the test The analysis shows that good separation effects can be achieved under the conditions of ultrasonic time of 1 to 3 minutes and power of 20 to 360W.
  • the centrifuge is set to a centrifugation time of 3 to 30 minutes and a rotation speed of 1000 to 6000 r/min. Centrifugal operation can make impurities such as sediment quickly settle to the bottom, while algae cells stay in the separation liquid. However, the centrifugation time and rotation speed should not be too large to prevent the algae cells from falling to the bottom again or being damaged due to excessive force. At the same time, the centrifugation time and rotation speed should not be too small, otherwise it will not be able to achieve a good separation effect and lead to sedimentation. A large amount of suspension affects the accurate operation of the instrument. The specific centrifugation time and speed can be selected within the above-mentioned centrifugation time and speed range according to the sedimentation conditions.
  • the amount of supernatant taken is 10 to 5000 ⁇ L
  • the flow cytometer is run at a slow speed (10 ⁇ L/min) for 1 to 5 minutes.
  • the flow cytometer can be run and counted for 1 minute to obtain the required data.
  • it can be run for 3 to 5 minutes and the same sample can be measured three times to reduce errors.
  • the filter uses a 200-400 mesh filter, which can not only ensure that the instrument is not damaged, but also retain the algal cells in the sample.
  • the Percoll separation solution is obtained by adding nine parts of Percoll stock solution to one part of 1.5M NaCl.
  • the soil or sediment sample before placing the soil or sediment sample into the centrifuge tube, the soil or sediment sample is placed in a beaker, and the beaker is placed in an autoclave for sterilization. For operation, set the working temperature to 120°C and sterilize for 30 minutes.
  • a certain amount of the sterilized soil or sediment is placed in a small beaker, a certain amount of algal liquid with a known concentration is added and mixed to obtain a known algae cell concentration of 80,000 cells/mL. Soil or sediment samples.
  • Figure 1 is a flow chart of the disclosed soil/sediment living algae cell separation, extraction and rapid classification and counting method
  • Figure 2 is a scatter plot of algae cell fluorescence signal distribution using a flow cytometer for extracting algal cell counting according to the present disclosure.
  • the present disclosure discloses a method for separation, extraction and rapid classification and counting of living algae cells in soil/sediment. Those skilled in the art can learn from the contents of this article and appropriately improve the process parameters.
  • the present disclosure will be described in detail below with reference to specific embodiments. The described embodiments are helpful for the understanding and implementation of the present disclosure and do not constitute a limitation of the present disclosure.
  • the implementation of this disclosure is not limited to the description of the following specific embodiments.
  • those skilled in the art can make equivalent or equivalent substitutions or changes based on the geographical location and natural conditions of different lakes. .
  • the protection scope of the present disclosure is not limited by the specific implementation, but is defined by the claims.
  • Example 1 Taking the classification, identification and counting of mud algae at the bottom of Houguan Lake in Wuhan City as an example
  • the area involved is Houguan Lake, a shallow lake in Wuhan City.
  • Houguan Lake a shallow lake in Wuhan City.
  • Example 2 Comparison of methods described in the present disclosure and conventional methods
  • the current conventional method for maintaining the activity of algae cells and counting them is to add distilled water to the sample, stir thoroughly, repeat the extraction three times, and use a microscope to count. Because microscopy has high requirements on operators and the accuracy is low when the concentration of algae cells is low, a flow cytometer was used instead of manual counting under a microscope during the experiment. After testing, it has been proven that the concentration of algae cells calculated by this conventional method is 55,140 cells/mL. According to the known algae cell concentration of 80,000 cells/mL, the extraction efficiency is only 69.30%, and this conventional method has the following shortcomings: 1. During the standing process, fine sediment is difficult to settle, the extraction of algal cells is incomplete, and centrifugation can easily destroy the integrity of the algal cells; 2.
  • the concentration of algae cells calculated using the extraction method of the disclosure is 72,900 cells/mL, and the extraction efficiency of the method of the disclosure can reach 91.12%.
  • the experimental group using this method was extracted again by the above conventional method, and it was found that only a very small number of algal cells remained.
  • the disclosed method can completely extract algal cells from the sediment and accurately count them according to categories. Compared with the current conventional methods of counting numbers, the disclosed method is simpler and faster to operate. , small error and other advantages, it is convenient for scientific researchers to analyze and study the structure and abundance of soil/sediment algae communities.
  • the above method is applied in the field of algae community cell detection, and is suitable for soil/sediment algae separation and extraction, classification and counting, and community characteristic analysis.
  • Experimental results show that compared with conventional technologies, the present disclosure has the following advantages.
  • This disclosure proposes for the first time a method for live separation, extraction, classification and counting of soil/sediment algae.
  • the method described only needs to take a small amount of sample, and by adding Percoll separation solution, performing ultrasound, centrifugation and other simple operations, the algae cells can be completely separated from the soil/sediment.
  • it can further achieve in vivo separation. Rapid differential counting of algal cells.
  • the optimal process parameters for separation and extraction, as well as the optimal conditions for rapid classification and counting were obtained. The two were organically combined to form a complete set of soil/sediment living algae cell separation, extraction and rapid classification and counting. method.
  • the method of the present disclosure directly measures the number of living algae cells and quickly counts them according to the reflected signal of the flow cytometer. It is simple to operate, complete separation, has a small sampling volume, and reduces the cost of manpower and material resources. consumption, the number of samples measured and the accuracy are greatly improved in the same time.
  • the method disclosed in the present disclosure can quickly count the biomass of living algae cells, identify the species of algae, does not damage the algae cells, is short in time, easy to operate, and highly efficient, and the reagents used are It is non-toxic to the human body.
  • the present disclosure has the advantages of low cost, simple operation, strong reproducibility, direct and rapid analysis, and the ability to keep cells intact for subsequent experiments.
  • This disclosure can obtain complete relevant information on the algae community in the native environment without using separation culture and molecular analysis techniques.
  • the above are only preferred embodiments of the present disclosure. It should be understood that, without departing from the principles of the present disclosure, skilled persons can also make several improvements and modifications to the methods of the present disclosure, and these improvements and modifications should also be regarded as the present disclosure. scope of protection.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Signal Processing (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本公开提供了一种土壤/底泥活体藻细胞分离提取与快速分类计数方法，包括：取土壤或底泥样品放入离心管中，向离心管中加入Percoll溶液，其中，在向所述离心管中放入所述样品之前和之后称量离心管的重量，以得到样品净重；轻微震荡离心管后，将离心管放入超声波清洗仪；待超声波清洗仪工作完成后取出离心管，将离心管放入离心机；待离心机运转完毕后取出离心管，轻微震荡离心管后用滤网过滤上清液，取部分上清液至流式细胞仪的流式细胞管，并运行流式细胞仪，利用流式细胞仪对上清液中的藻细胞进行藻种类和藻密度的检测与统计分析，得出所需藻细胞种类与数量。

Description

一种土壤/底泥活体藻细胞分离提取与快速分类计数方法
相关申请的交叉引用
本申请要求于2022年05月18日提交中国专利局、申请号为202210550437.7、发明名称为“一种土壤/底泥活体藻细胞分离提取与快速分类计数方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开属于微生物领域，涉及一种土壤/底泥活体藻细胞分离提取与快速分类计数方法。
背景技术
藻细胞广泛分布于自然界，例如，在各种水体、土壤和其他环境中，底泥藻类扮演着生态系统的物质循环和能量流动不可或缺的角色。部分藻种类越冬休眠时会集中在底泥内，此时的藻细胞起到“种源”作用，待来年升温时底泥中的“种源”藻类会不断释放至水体中，从而导致出现水华爆发等现象，进而直接或间接地影响水生生物的生存及生态系统的安全。掌握底泥中活体藻细胞的种类和数量，是开展藻类水华形成的机理研究和水华防治的技术研究工作的基础。
通常，藻类生物量的统计方法有显微镜血球计数法、光合色素含量测定法、光密度值法及其他创新方法。
显微镜血球计数法采样及运输量大、沉降浓缩耗时长，需要科研人员连续观察多个视野，从而计数容易重复或遗漏，准确率、效率较低。并且，对科研人员的水平和经验要求较高。另外，由于土壤/底泥中存在大量杂质，因此存在藻细胞镜检观察困难、计数难度大的问题。除镜检计数法外，诸如基于双向背景差分法的成像流式细胞粘连计数方法(王俊生,张红月：一种基于双向背景差分法的成像流式细胞粘连计数与活性检测方法、装置[P]， CN113222969A,2021-08-06)、基于线扫描拉曼显微成像的藻细胞计数及藻种判别方法(何石轩,谢婉谊,方绍熙,王德强：基于线扫描拉曼显微成像的藻细胞计数及藻种判别方法，CN106442463B,2019-03-08)等，对藻细胞的计数都会受到泥沙干扰。
光合色素含量测定法需经过抽滤、研磨、萃取、离心、比色测定等步骤，操作复杂，耗时长、效率低。该方法对藻生物量的统计可能受无活性的被破损或死亡的藻细胞及残留光合色素影响，因而无法准确统计土壤/底泥中具有活性的藻细胞数量。同时，提取操作所需的溶剂多为甲醇、乙醇、丙酮等对人体有毒害的有机溶剂。
另外，光合色素在操作过程中易分解，容易产生误差，无法判定浮游植物的种类。光密度值法(苏文,孔繁翔,赵旭辉：一种铜绿微囊藻的快速计数方法[P]，CN103276045A,2013-09-04)适用于已知藻类的室内模拟实验，无法对藻类进行鉴定。同时，从泥土中提取藻类时会携带部分泥沙，也会对紫外分光光度计的测量数据有较大干扰。故，此方法只能运用于水体中藻密度的检测。
土壤/底泥中的活体藻细胞难以统计的重要原因之一是活体藻细胞较难从土壤/底泥中进行充分分离。张呈祥等提出了一种通过超声波清洗原理将藻细胞从沉水植物等附着基质上分离，使藻细胞进入蒸馏水形成藻液并统计的方法(张呈祥,雷光春,裴洪翠：附着藻类分离取样装置、附着藻类含量计算系统及方法[P]，CN111537678A,2020-08-14)。该方法及其装置需使用抽滤装置。然而，经室内实验尝试后发现此方法对土壤/底泥无法适用，因为大量泥沙堆积不仅容易堵塞滤膜与装置，更会使藻细胞在大压强作用下被破坏。
针对土壤/底泥中藻细胞的计数，目前使用的方法如下。
取一定量混匀的土壤/底泥样品，加入Percoll悬浮液与甲醛混合溶液，静置后用倒置荧光显微镜进行镜检分类并计数(邹万生,王智,刘良国,王文彬,石迎普：冲天湖底泥表层微囊藻休眠体复苏与菌群动态[J]，生态学报,2017,37(19):6597-6606.)。但是，该方法向样品中加入了甲醛溶液，使得藻 细胞丧失活性，无法对藻细胞开展与生理生态特征相关的后续研究。
将采集的土壤/底泥样品风干后研磨，向样品中分别加入90％丙酮溶液与Tris缓冲液，静止8～10h后离心，然后将滤膜置于容量瓶中进行过滤并在反复萃取3～4次后定容，接着通过荧光分析测定叶绿素与藻蓝素的含量，从而得出藻群落生物量(Chen,C.,Yang,Z.,Kong,F.,Zhang,M.,Yu,Y.,&Shi,X.(2016).Growth,physiochemical and antioxidant responses of overwintering benthic cyanobacteria to hydrogen peroxide.Environmental Pollution,219,649–655.https://doi.org/10.1016/j.envpol.2016.06.043)。但是，该方法需对底泥样品进行研磨并加入丙酮等溶液，将使得藻细胞丧失活性，且操作复杂，所需时间较长。
将土壤/底泥过0.2mm筛后再用25号浮游植物网过滤，向残留物样品中倒入蒸馏水，充分搅拌泥水混合物并待静置后取上清液，重复至少三次，提取藻细胞镜检(李阔宇,宋立荣,万能：底泥中微囊藻复苏和生长特性的研究[J]，水生生物学报，2004(02):113-118.)。由于，该方法仅通过向底泥中加入蒸馏水搅拌并混合，然后静置取上清液，难以保证藻细胞从底泥中得到充分的提取和分离，检测结果误差较大。
向土壤/底泥样品倒入蒸馏水，充分搅拌泥水混合物后取上清液，对上清液进行稀释与平板涂布，培养至藻落长出后进行计数(吴生才,陈伟民,高光：太湖冬季底泥中活体藻类的检测[J]，湖泊科学，2003(04):339-344.)。该方法一方面仅通过向底泥中加入蒸馏水搅拌混合，然后静置取上清液，难以保证藻细胞从底泥中得到充分的提取和分离；另一方面，通过平板涂布法进行藻细胞的培养，耗时较长，培养操作复杂，而不同藻细胞生长速度与条件各不相同，培养后计数不能准确反映底泥中藻类实际的种群结构，只能证明底泥中存在活体藻细胞，具有较大局限性。
鉴于以上方法的不足，本公开提出一种简洁高效准确的土壤/底泥活体藻细胞分离提取与快速分类计数方法，对于土壤/底泥藻类生物学与环境生态学研究具有重要意义。
发明内容
为克服藻细胞提取与计数方法的不足，本公开旨在提供一种土壤/底泥活体藻细胞分离提取与快速分类计数方法，以能够从土壤/底泥中完整地分离和提取出藻细胞，并保持藻细胞活性不被破坏，同时达到快速精确统计数量、判断藻类群落结构与丰度等目的。
本公开提供一种土壤/底泥活体藻细胞分离提取与快速分类计数方法，包括以下步骤：
取混合均匀、无明显大型颗粒物及杂质的土壤或底泥样品放入离心管中，向离心管中加入Percoll溶液，其中，在向所述离心管中放入所述样品之前和之后称量离心管的重量，以得到样品净重；
轻微震荡离心管后，将离心管放入超声波清洗仪；
待超声波清洗仪工作完成后取出后离心管，将离心管放入离心机；
待离心机运转完毕后取出离心管，轻微震荡离心管后用滤网过滤上清液，取部分上清液至流式细胞仪的流式细胞管，并运行流式细胞仪，利用流式细胞仪对上清液进行统计与分析，得出所需藻细胞种类与数量。
在一实施例中，土壤或底泥的量为1～2mL。所需样品的量少，可以显著减少采集样品与运输的工作量，且可保证提取效率较高。
在一实施例中，所述Percoll溶液用量为5～10mL，浓度为15％～55％。Percoll分离液是一种包有乙烯吡咯烷酮的硅胶颗粒，其不会穿过细胞膜，对细胞无毒害作用。这样，不仅能够使完整的活体藻细胞与泥沙分离，同时还能使活体藻细胞与受损藻细胞及其碎片分离。为保证活体藻细胞能充分分离至溶液中，Percoll溶液用量不宜过少，通过试验分析研究发现以5～10mL为宜。为确保藻细胞经离心之后依旧悬浮于上清液中，防止藻细胞离心时再次沉降至底部，Percoll溶液浓度也不宜偏低，以高于15％为宜，同时为减少材料消耗，浓度以低于55％为宜。
在一实施例中，超声波清洗仪所设置的超声时间为1～3min，功率为20～360W。超声操作可促使藻细胞从底泥上较好地分离，但是功率过小、时间过短无法使藻细胞分离完全，而功率过大、时间过长又会导致藻细胞受损破裂死亡，通过试验分析得出在超声时间为1～3min，功率为20～360W 条件下可以达到良好的分离效果。
在一实施例中，离心机所设置的离心时间为3～30min，转速为1000～6000r/min。离心操作可使泥沙等杂质快速沉降至底部，而藻细胞停留于分离液中。但是，离心时间、转速不宜过大，以防藻细胞重新降至底部或受力较大被破坏，同时，离心时间、转速也不宜过小，否则无法起到很好的分离作用，导致泥沙大量悬浮而影响仪器的准确运行。具体离心时间及转速可根据泥沙沉降情况，在上述的离心时间和转速范围内进行选择。
在一实施例中，上清液的取用量为10～5000μL，流式细胞仪以慢速(10μL/min)运转1～5min。其中，流式细胞仪运行计数1min即可得到所需数据，为确保数据更准确，可运行3～5min，对同一样品测定3次以减小误差。
在一实施例中，滤网选用200～400目滤网，既可保证仪器不受损，也能保留样品中的藻细胞。
在一实施例中，所述Percoll分离液通过用一份1.5M的NaCl加九份Percoll原液而得到。
在一实施例中，在将所述土壤或底泥样品放入所述离心管之前，取所述土壤或底泥放于烧杯内，并将所述烧杯置于高压灭菌锅内进行灭菌操作，设置工作温度120℃，灭菌30min。
在一实施例中，取一定量的灭菌后的所述土壤或底泥于小烧杯内，加入一定量已知浓度的藻液进行混合，得到已知藻细胞浓度为80000个/mL的所述土壤或底泥样品。
附图说明
图1为本公开的土壤/底泥活体藻细胞分离提取与快速分类计数方法的流程图；
图2为本公开提取藻细胞计数的流式细胞仪的藻细胞荧光信号分布散点图。
具体实施方式
本公开公开了一种土壤/底泥活体藻细胞分离提取与快速分类计数的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数。下面结合具体实施方式对本公开进行详细描述。所述的实施方式有助于对本公开的理解和实施，并非构成对本公开的限制。实施本公开并不限于以下具体实施方式的记载，对于其中某些特定的技术参数和技术手段，本领域技术人员可以根据不同湖泊的地理位置和自然条件，而进行等同或等效的替换或变化。本公开的保护范围并不以具体实施方式为限，而是由权利要求加以限定。
为了使本领域的技术人员更好地理解本公开的技术方案，下面结合具体实施例对本公开作进一步的详细说明。
实施例1：以武汉市后官湖湖底的泥藻类的分类鉴定与计数为例
所涉及区域为武汉市浅水湖泊后官湖。采用本公开的方法，对后官湖水域越冬水华蓝藻种源进行分离提取，并进行数量、门类统计。
后官湖水域越冬水华蓝藻种源的分离提取统计方法流程参见图1，包括以下步骤：
1)从采样点分别采集三份平行样品，切取每份样品最上层2cm左右厚度的表层底泥，并将所切取的表层底泥移至密封袋中，将每份样品混合均匀。
2)按百分比两步稀释法配制40％Percoll分离液，先用一份1.5M的NaCl加九份Percoll原液得到100％Percoll分离液，再使用0.15M的NaCl稀释所述100％Percoll分离液进而得到40％Percoll分离液。
3)对流式细胞仪进行开机预热、质检，用已知门类的纯种藻液划定荧光信号的范围。
4)用电子天平称量10mL的空的离心管质量，取1mL混匀的底泥样品放入离心管中(注意避开石块、杂草等杂物)，再次称量放入样品后的离心管的重量，得到样品净重并记录。继续向放入样品的离心管中加入9mL的40％Percoll分离液，轻微震荡，使泥样分散在分离液中并与之充分接触。
5)将离心管盖紧后放入超声波清洗仪，设置超声波清洗仪的频率为40kHz，功率为80W，工作时间2min。
6)将超声后的样品放入离心机，设置相对离心力为3000r/min，离心时间8min，以使泥沙沉至底部，藻细胞分离至分离液当中。
7)轻微震荡离心管，混匀离心管中的上清液，将上清液过300目滤网，以防止流式细胞仪堵塞。取1mL上清液于流式细胞管中，调取设置完毕参数的模板，运行流式细胞仪2min，运行完毕后即可得到所需藻细胞的数量，同一样品检测三次求平均值，测得底泥中蓝藻数量为5500个/mL，绿藻为26500个/mL。
实施例2：本公开所述方法与常规方法的对比
于实验室内进行模拟实验，取一定量湖泊底泥放入烧杯中，用铝箔纸密封。将该烧杯及玻璃棒、小烧杯、移液枪头等其余实验所需用品置于高压灭菌锅，设置工作温度120℃，灭菌30min，确保底泥与实验用品中无活体藻细胞。待上述物品冷却至室温后，取一定量的灭菌后的底泥于小烧杯内，加入一定量已知浓度的藻液进行混合，得到已知藻细胞浓度为80000个/mL的泥样。考虑超声的功率、时间；分离液种类、浓度等因素设置正交实验，每个实验组取1mL上述泥样，5mL分离液于离心管内，进行相应处理，并对各实验组进行计数。
目前保持藻细胞活性并计数的常规方法为：向样品中加入蒸馏水，充分搅拌，反复提取三次，使用显微镜进行计数。因镜检对操作者要求较高，藻细胞浓度低时准确度低，故实验时使用流式细胞仪代替显微镜人工计数。经试验后证明该常规方法提取计算获得的藻细胞浓度为55140个/mL，根据已知藻细胞浓度为80000个/mL可得提取效率仅为69.30％，并且该常规方法有以下缺点：1.静置过程中细泥沙难以沉降，藻细胞提取不彻底，离心容易破坏藻细胞完整性；2.静置过程中藻细胞的种群结构易产生变化，如出现藻的繁殖、种间竞争等状况；3.泥沙较多的情况下使用流式细胞仪计数，不仅误差较大，同时也容易导致仪器堵塞；4.泥沙较多的情况下使用血球计数法观察难度较大，耗时较长，准确度低。
使用本公开提取计算得藻细胞浓度为72900个/mL，本公开的方法提取效率可达91.12％。为证明本公开方法的优越性，将使用该方法的实验组再次通过上述常规方法进行提取，发现仅有极少数藻细胞残留。
对比本公开的方法与常规方法结果，本公开能将藻细胞完整地从底泥中提取出来并依据门类准确计数，与目前统计数量的常规方法相比，本公开的方法具有操作更简便、快速、误差小等优点，便于科研人员进行土壤/底泥藻类群落结构与丰度的分析研究。
上述方法应用于藻类群落细胞检测领域，适用于土壤/底泥藻类分离提取、分类计数、群落特征分析，实验结果表明，较常规技术相比，本公开具有以下优点。
本公开首次提出一种对土壤/底泥藻类进行活体分离提取、分类计数的方法。所述方法仅需取少量样品，并通过加入Percoll分离液、执行超声、离心等简单操作，即可实现从土壤/底泥中完整地分离藻细胞，同时结合流式细胞仪，可进一步实现活体藻细胞的快速分类计数。通过大量的对比试验，得出了分离提取的最优工艺参数，以及快速分类计数的最优条件，并将二者有机结合，形成了一整套土壤/底泥活体藻细胞分离提取与快速分类计数方法。
与传统镜检计数法相比，本公开的所述方法直接测定活体藻细胞的数量，根据流式细胞仪的反映信号对其进行快速计数，操作简单，分离彻底，采样量少，减少人力物力的消耗，相同时间内测定样品数量及准确度大幅提升。
与光合色素含量测定法相比，本公开的所述方法可快速统计活体藻细胞生物量、鉴定藻的种类、对藻细胞无损伤，耗时短、操作简便、效率高，并且所使用的试剂对人体无毒害。
与其他技术相比，本公开具有成本低廉、操作简便、可重复性强、可直接快速分析、可保持细胞完整以进行后续实验等优点。本公开无需采用分离培养与分子分析技术，即可获得原生环境藻类群落完整的相关信息。以上所述仅是本公开的优选实施方式，应当理解，在不脱离本公开原理的 前提下，技术人员还可以对本公开的方法做出若干改进和润饰，这些改进和润饰也应视为本公开的保护范围。

Claims (12)

  1. 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述方法包括：
    取混合均匀、无明显大型颗粒物及杂质的土壤或底泥样品放入离心管中，向离心管中加入Percoll溶液，其中，在向所述离心管中放入所述样品之前和之后称量所述离心管的重量，以得到样品净重；
    轻微震荡所述离心管后，将所述离心管放入超声波清洗仪；
    待所述超声波清洗仪工作完成后取出后所述离心管，将所述离心管放入离心机；
    待所述离心机运转完毕后取出所述离心管，轻微震荡所述离心管后用滤网过滤上清液，取部分所述上清液至流式细胞仪的流式细胞管，并运行所述流式细胞仪，利用所述流式细胞仪对所述上清液进行统计与分析，得出所需藻细胞种类与数量。
  2. 如权利要求1所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述土壤或底泥的量为1～2mL，所述Percoll溶液的用量为5～10mL，浓度为15％～55％。
  3. 如权利要求1或2所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述超声波清洗仪所设置的超声时间为1～3min，功率为20～360W。
  4. 如权利要求1-3中任一项权利要求所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述离心机所设置的离心时间为3～30min，转速为1000～6000r/min。
  5. 如权利要求1-4中任一项权利要求所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述上清液的取用量为10～5000μL，所述流式细胞仪的运转时间为1～5min。
  6. 如权利要求5所述的土壤/底泥活体藻细胞分离提取与快速分 类计数方法，其中，所述流式细胞仪的运转时间为3～5min，对同一样品测定3次。
  7. 如权利要求1-6中任一项权利要求所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述滤网选用200～400目滤网。
  8. 如权利要求1-7中任一项权利要求所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述Percoll分离液通过用一份1.5M的NaCl加九份Percoll原液而得到。
  9. 如权利要求1-8中任一项权利要求所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述Percoll分离液是一种包有乙烯吡咯烷酮的硅胶颗粒。
  10. 如权利要求1-9中任一项权利要求所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，其中，所述流式细胞仪的运转速度为10μL/min。
  11. 如权利要求1-10中任一项权利要求所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，在将所述土壤或底泥样品放入所述离心管之前，取所述土壤或底泥放于烧杯内，并将所述烧杯置于高压灭菌锅内进行灭菌操作，设置工作温度120℃，灭菌30min。
  12. 如权利要求11所述的土壤/底泥活体藻细胞分离提取与快速分类计数方法，取一定量的灭菌后的所述土壤或底泥于小烧杯内，加入一定量已知浓度的藻液进行混合，得到已知藻细胞浓度为80000个/mL的所述土壤或底泥样品。
PCT/CN2022/141757 2022-05-18 2022-12-26 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法 WO2023221509A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210550437.7 2022-05-18
CN202210550437.7A CN115046819A (zh) 2022-05-18 2022-05-18 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法

Publications (1)

Publication Number Publication Date
WO2023221509A1 true WO2023221509A1 (zh) 2023-11-23

Family

ID=83160347

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/141757 WO2023221509A1 (zh) 2022-05-18 2022-12-26 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法

Country Status (2)

Country Link
CN (1) CN115046819A (zh)
WO (1) WO2023221509A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115046819A (zh) * 2022-05-18 2022-09-13 长江水利委员会长江科学院 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009192450A (ja) * 2008-02-18 2009-08-27 Public Works Research Institute 植物プランクトンの計測法
CN102735671A (zh) * 2012-07-02 2012-10-17 中国科学院南京地理与湖泊研究所 大型浅水湖泊越冬蓝藻种源疏浚位点的确定方法
CN112921066A (zh) * 2021-04-25 2021-06-08 上海交通大学 基于流式细胞仪和高通量测序技术联用的超微藻检测方法
CN115046819A (zh) * 2022-05-18 2022-09-13 长江水利委员会长江科学院 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009192450A (ja) * 2008-02-18 2009-08-27 Public Works Research Institute 植物プランクトンの計測法
CN102735671A (zh) * 2012-07-02 2012-10-17 中国科学院南京地理与湖泊研究所 大型浅水湖泊越冬蓝藻种源疏浚位点的确定方法
CN112921066A (zh) * 2021-04-25 2021-06-08 上海交通大学 基于流式细胞仪和高通量测序技术联用的超微藻检测方法
CN115046819A (zh) * 2022-05-18 2022-09-13 长江水利委员会长江科学院 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOLANDA M. H. VERSPAGEN; EVELINE O. F. M. SNELDER; PETRA M. VISSER; KLAUS D. JÖHNK; BAS W. IBELINGS; LUUC R. MUR; JEF HUISMAN: "Benthic–pelagic coupling in the population dynamics of the harmful cyanobacterium Microcystis", FRESHWATER BIOLOGY, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 50, no. 5, 28 April 2005 (2005-04-28), GB , pages 854 - 867, XP071384860, ISSN: 0046-5070, DOI: 10.1111/j.1365-2427.2005.01368.x *
WANG CHUNBO; WU XINGQIANG; TIAN CUICUI; LI QIAN; TIAN YINGYING; FENG BING; XIAO BANGDING: "A quantitative protocol for rapid analysis of cell density and size distribution of pelagic and benthicMicrocystiscolonies by FlowCAM", JOURNAL OF APPLIED PHYCOLOGY., KLUWER, DORDRECHT., NL, vol. 27, no. 2, 9 June 2014 (2014-06-09), NL , pages 711 - 720, XP035477869, ISSN: 0921-8971, DOI: 10.1007/s10811-014-0352-0 *
YU XIAO-HAN; ZENG SONG-FU; CAO YU-FENG; CHEN YAO; XIE JIE-ZHEN; ZHENG SHAO-PING: "Identification of Red Tide Algae Based on FlowCAM", HAIYANG KEXUE JINZHAN - ADVANCES IN MARINE SCIENCE, GUOJIA HAIYANG-JU DI-1 HAIYANG YANJIUSUO, CN, vol. 31, no. 4, 31 October 2013 (2013-10-31), CN , pages 515 - 526, XP009550477, ISSN: 1671-6647 *
ZHOU, QUAN ET AL.: "A New Approach to The Ecological Study of Microcystis Aeruginosa in Lake Sediments", ABSTRACTS OF THE 30TH ANNIVERSARY AND 15TH SYMPOSIUM OF THE CHINESE ALGAE ACADEMIC RESEARCH ASSOCIATION, 9 January 2017 (2017-01-09) *

Also Published As

Publication number Publication date
CN115046819A (zh) 2022-09-13

Similar Documents

Publication Publication Date Title
Jin-Feng et al. Separation and identification of microplastics in digestive system of bivalves
Chioccioli et al. Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris
Buskey et al. Use of the FlowCAM for semi-automated recognition and enumeration of red tide cells (Karenia brevis) in natural plankton samples
CA2402538C (en) Process for preparing control samples of particles such as microorganisms and cells
CN112903349A (zh) 城市河道沉积物中微塑料提取、检测方法
WO2023221509A1 (zh) 一种土壤/底泥活体藻细胞分离提取与快速分类计数方法
CN110646334A (zh) 一种水样中小尺寸微塑料的快速分析方法
CN102321729B (zh) 一种检测土壤和沉积物中细菌数量的荧光显微计数方法
Günerken et al. Flow cytometry to estimate the cell disruption yield and biomass release of Chlorella sp. during bead milling
Alam et al. Standard techniques and methods for isolating, selecting and monitoring the growth of microalgal strain
CN113552244A (zh) 一种基于ASE-Py-GCMS测定纳米微塑料定性定量的方法
CN103555587A (zh) 一种从自然水体中筛选高油脂藻类的方法
CN117554152A (zh) 一种紫菜中微塑料的提取与检测方法
CN106018688B (zh) 一种金属纳米颗粒离子和纳米效应毒性贡献率的估算方法
CN108982192A (zh) 一种适于枣染色体倍性测定的细胞核悬浮液快速制备方法
CN115015061A (zh) 基于固体悬浮物浓度测定水环境微塑料含量的方法
CN207649987U (zh) 适用于水体中悬浮颗粒物和浮游生物的分离系统
Prata et al. Do's and don'ts of microplastic research: a comprehensive guide
CN217639050U (zh) 一种用于快速检测土壤中大丽轮枝菌微菌核的采样装置
CN116106092A (zh) 基于流式细胞仪鉴定马铃薯倍性的高通量样品制备方法
DeLeo et al. Enumeration and biomass estimation of bacteria in aquifer microcosm studies by flow cytometry
CN1244808C (zh) 一种适用于流式细胞术检测倍性的贝类样品制备方法
CN105112497A (zh) 一种河口及近岸海洋环境中大肠杆菌、金黄色葡萄球菌分离筛选及抗生素抗性评估方法
Dyachenko et al. Method development for microplastic analysis in wastewater
Goss et al. Quantifying particulate matter deposition in Niwot Ridge, Colorado: Collection of dry deposition using marble inserts and particle imaging using the FlowCAM

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 202327005971

Country of ref document: IN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22942529

Country of ref document: EP

Kind code of ref document: A1