CN102818755B - Method for actual measurement of microcystis density and population size by using laser particle analyzer - Google Patents

Abstract

The invention relates to a method for actually measuring the density and population size of Microcystis by using a laser particle size analyzer. Through preprocessing on-site samples, the laser particle size analyzer can quickly and simultaneously measure the density of Microcystis and the particle size distribution of the population. The specific implementation steps are as follows: step 1, let the sample stand for 12 hours, collect the supernatant and supplement the same amount of distilled water as the bottom miscellaneous liquid; step 2, set the laser particle size analyzer absorption rate to 0.1, set the refractive index to 1.40, and cycle The pump speed is 1500rad/min; step 3, add V ₁ (600-700mL) volume of clear water to scan the background value, then gradually add the sample to be tested, when the shading reaches 10-20%, record the amount of sample added V ₂ and the corresponding The shading degree O _V , then measure the particle size distribution of Microcystis and calculate the density of Microcystis.

Description

Method for Measuring Microcystis Density and Colony Size Using Laser Particle Size Analyzer

technical field

The invention relates to a method for actually measuring the density and group size of Microcystis by using a laser particle size analyzer. Through the use of this method, the density and population size distribution of Microcystis can be determined rapidly and simultaneously. Unlike some previous methods for measuring Microcystis density and colony size, this method can simultaneously measure Microcystis density and colony size without breaking the Microcystis colony or spending a lot of time using a microscope to measure Microcystis Colony size, this method can not only quickly obtain the density and population size of Microcystis, but also obtain the particle size distribution of Microcystis population. The invention belongs to the field of resource environment.

Background technique

Cyanobacteria blooms are generally characterized by a large number of reproduction and enrichment of Microcystis. The microcystis cell density in lakes is the basic data for the research, prediction and control of water blooms. Generally, the research on the biomass of Microcystis is mostly based on the determination of cell density, such as microscope counting, spectrophotometry, and chlorophyll content, which are often used to evaluate the biomass of Microcystis. Microscopic observation will have certain limitations when studying the number of groups, group size, and number of cells in the group. Although the number of groups is easy to obtain by counting, the number of cells in each group is difficult to count due to the three-dimensional structure and mutual occlusion of the group cells. . When observing Microcystis colonies with a microscope, due to the small sampling volume (0.1μL-0.1mL) and the need for 1- to 10-fold dilution, the number and size of the colonies appearing in the field of view are quite random, which will cause resulting in poor representativeness of the evaluation results. At the same time, 400-600 algae units have to be counted for each sample when performing population evaluation, and the workload is also very huge. Microscopic observation plays an important role in the classification of phytoplankton and the measurement of the biomass of single-celled algae, but there are problems in operability and accuracy in the evaluation of the population characteristics of Microcystis that appear in colonies. Therefore, it is very necessary to seek a simple method that is applicable to both field and indoor samples and can not only evaluate the total cell density of Microcystis but also evaluate the population size and particle size distribution of the population.

Contents of the invention

The purpose of the present invention is to establish a method capable of rapidly and simultaneously measuring the density, population size and particle size distribution of Microcystis. By adopting the determination method of the patent of the present invention, the related data of Microcystis density and population size can be quickly determined.

The technical scheme of the present invention is characterized in that microcystis samples obtained in actual lakes are simply pretreated, and directly measured by a laser particle size analyzer, the data of microcystis density, population size and particle size distribution can be calculated and obtained.

Use a 65 μm zooplankton net to catch Microcystis floating on the surface of the water, fix it with formalin, and bring it back to the laboratory for storage.

The samples taken in the field need to be pretreated because there are many suspended sediments. Taking advantage of the fact that suspended sediment and other sundries settle faster than heavy sedimentation, the samples were placed in a 500mL water sample bottle for 12 hours. After the sediment and sundries settle to the lower part of the water sample bottle, extract the clean algae liquid from the upper part. At the same time, add the same amount of distilled water as the bottom miscellaneous liquid to ensure the original density of the algae liquid. Indoor purely cultured algal samples can be tested directly.

Laser particle size analyzer analysis: using the British Malvern company Mastersize2000 laser particle size analyzer. Through microscope observation and comparison with the reference absorption rate atlas, the absorption rate of the laser particle size analyzer is set to 0.1, the refractive index is set to 1.40 by trying, and the circulation pump speed is 1500rad/min to ensure sufficient mixing without obvious bubbles.

First, add V ₁ (600-700mL) volume of clear water to scan the background value, then gradually add the sample to be tested, and observe the change of shading degree at the same time, when the shading degree reaches 10-20%, record the amount V ₂ of the sample added and the corresponding The shading degree is O _V , and then the particle size distribution of the sample can be obtained by measuring, and the density of Microcystis can be calculated through V ₁ , V ₂ and O _V. The laser particle size analyzer obtains the total shading degree of suspended particles in the liquid while obtaining the microcystis Algal population size and particle size distribution.

Description of drawings

Figure 1 is the relationship between cell density and shading;

Figure 2 is a comparison of cell density and microscope counting results;

Figure 3 is the measured results of the total volume percentage of different particle sizes in the four test samples: (a, 11; b, 5; c, 4; d, 4).

Detailed ways

Implementation case:

(1) Instruments and samples

The test adopts the Mastersize2000 laser particle size analyzer of British Malvern Company.

The microcystis species were collected from the Institute of Hydrobiology, Chinese Academy of Sciences, and cultured in a 250ml Erlenmeyer flask using BG-11 medium, with a light intensity of 5-50μE·m ^-2 ·s-1, a culture temperature of 25°C, and a light-to-dark ratio of 12:12 Hour. Under different light intensities and perturbation conditions, three types of samples with different population characteristics were obtained: the naked eye and microscope observation can clearly distinguish: type a samples are mainly single cells, a total of 24, and b samples are group-based. Main, a total of 13 samples, c samples of single cells, small populations and larger populations mixed, a total of 9 samples.

In June 2010, the sampling location was Gonghu Bay of Taihu Lake. The microcystis floating in the surface of the water where the microcystis was more obvious was fished with a 63 μm zooplankton net, fixed in formalin and brought back to the laboratory for storage. Both naked eye and microscope observation can clearly distinguish Microcystis in the form of large groups, and occasionally single cells and small groups (d, a total of 13 samples).

(2) Standard curve drawing

Microscopic observation and laser particle size analyzer analysis were carried out on the four samples respectively.

Microscopic observation: sample a was counted directly through a microscope to obtain the algae density D _m , samples b, c, and d were boiled with alkali to disperse the Microcystis population and then counted the cells to obtain the algae density D _m .

Laser particle size analyzer analysis: using the British Malvern company Mastersize2000 laser particle size analyzer. Through microscope observation and comparison with the reference absorption rate atlas, the absorption rate of the laser particle size analyzer was set to 0.01, and the refractive index was set to 1.50 by trying. The circulating pump speed was 1500rad/min to ensure sufficient mixing without obvious bubbles.

First, add V ₁ (600-700mL) volume of clear water to scan the background value, then gradually add the sample to be tested, and observe the change of shading degree at the same time, when the shading degree reaches 10-20%, record the amount V ₂ of the sample added and the corresponding Shading O _V , and then measure the particle size distribution of the sample.

Using the principle of shading degree of suspended particles, the cell number of Microcystis was measured by laser particle size analyzer.

The shading degree O _M of the sample with a volume of V ₂ can be calculated according to the formula (2) according to the measured shading degree O _V after dilution and the volume V ₁ of the diluent

O _M =O _V (V ₁ +V ₂ )/V ₂ (2)

Using this method, the shading degree of 4 kinds of 35 samples (a, 13; b8; c, 5; d, 9) was measured, and the cell density was measured by microscope at the same time. The relationship between density and shading is plotted in Figure 1. From the measured data, it can be seen that there is a very obvious linear correlation between the two, and the conversion formula between cell density and shading is obtained (3)

D _c = kO _M (3)

In the formula: D _c is the cell density indirectly measured by the laser particle size analyzer, and k is the shading coefficient: through this experiment, the indoor culture sample k=22.98; the field sample k=9.03.

(3) Determination of the algae density of the sample

In order to prove the accuracy of this method, 24 unused samples (a, 11; b, 5; c, 4; d, 4) of the 4 samples were measured using a laser particle size analyzer to obtain The cell density was compared with the microscope counting results, and the results are shown in Figure 2. It can be seen from Figure 2 that the results obtained by the laser particle size analyzer are basically consistent with the results obtained by the microscope.

(4) Microcystis population size and particle size distribution

In addition to obtaining the total shading degree of suspended particles in the liquid, the laser particle size analyzer can obtain the particle size and particle size distribution curve of the sample. The measured results of the total volume percentages of different particle sizes in the four test samples are shown in Figure 3.

Claims (1)

1. A method for using a laser particle size analyzer to actually measure Microcystis density and population size, characterized in that it is realized in the following steps: A. The method of on-site sample pretreatment is: put the sample in a 500mL water sample bottle for 12 hours; wait for the sediment and sundries to settle to the lower part of the water sample bottle, extract the clean algae liquid from the upper part; at the same time replenish the miscellaneous liquid at the bottom The same amount of distilled water to ensure the original density of the algae liquid; B. Using a laser particle size analyzer: set the absorption rate of the laser particle size analyzer to 0.1, set the refractive index to 1.40, and the circulating pump speed to 1500rad/min; C. The basic steps of laser particle size analyzer measurement are: add _V1 water with a volume of 600-700mL to scan the background value, then gradually add the sample to be tested, and observe the change of shading degree at the same time. When the shading degree reaches 10-20%, record the addition The amount V ₂ of the sample and the corresponding shading degree O _V , and then the particle size distribution of the sample can be obtained by measuring, and the density of Microcystis can be calculated through V ₁ , V ₂ and O _V ; D. The conversion formula between cell density and shading is: D _c = _kOM , where D _c is the cell density indirectly measured by the laser particle size analyzer, k is the shading coefficient, and _OM is the sample with a volume of _V2 Shading degree; among the parameters mentioned above, k=22.98 for indoor samples; k=9.03 for outdoor samples; The shading degree O _M of the sample whose volume is V ₂ can be calculated by the formula according to the measured shading degree O _V after dilution and the volume V ₁ of the diluent, and its characteristic is O _M =O _V (V ₁ +V ₂ )/V ₂ .