CN111474021A - Method for rapidly fixing and identifying phytoplankton in water body - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 51
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- 238000000967 suction filtration Methods 0.000 claims abstract description 20
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- 238000003756 stirring Methods 0.000 claims abstract description 8
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- 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
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- 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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- 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
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- 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
- G01N2015/1006—Investigating individual particles for cytology
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- 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
- G01N2015/1024—Counting particles by non-optical means
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- 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
- G01N2015/1028—Sorting particles
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Abstract
The invention discloses a method for quickly fixing and identifying phytoplankton in a water body, which comprises the following steps: step one, sampling; taking a certain amount of water sample in a water body in which phytoplankton grows; step two, suction filtration; filtering the phytoplankton to be identified by a suction filtration device to be retained on a filter membrane; step three, stirring uniformly; putting the filter membrane into a container, adding distilled water, and stirring for multiple times until the mixture is uniform; step four, sampling and identifying; and taking a small amount of uniformly mixed sample to be tested from the container for identification. The method solves the problems of accuracy and safety of phytoplankton counting in the traditional method, ensures the accuracy of the identification result, does not need to add any medicine in the experimental process, is easy to realize the whole experimental process, needs simple instruments and equipment, and has utilizable value and important significance for identifying the phytoplankton.
Description
Technical Field
The invention belongs to the technical field of environmental monitoring, relates to the field of water environment, and particularly relates to a method for quickly fixing and identifying phytoplankton in a water body.
Background
Phytoplankton, namely planktonic algae, is a general term for a plurality of algae living in suspension in water, is widely existed in rivers, lakes and oceans, is influenced by natural conditions such as water flow, water temperature, nutritive salt, illumination and other biological factors such as death, sedimentation, aggregation, predation and the like of the algae, is very sensitive to environmental change, is an indicator organism for measuring water quality, and has an abundance degree and community composition which are important indexes for reflecting water eutrophication.
The main problems in the prior art include:
the phytoplankton individuals are tiny and can only be observed through a microscope, and the phytoplankton individuals are easily decomposed by microorganisms. And the added reagent has great harm to human body, and the experiment process may cause harm to experimenters.
The precipitation method in the existing fixing method is that a collected water sample is added with a Luge reagent after quantification to precipitate phytoplankton, then a siphon method is used to suck away supernatant fluid, the precipitated phytoplankton is transferred to a brown reagent bottle added with formaldehyde, the constant volume is stored, and then the identification is carried out by an optical microscope.
In the phytoplankton net method in the prior art, the phytoplankton net is a net with a certain pore size, so that only most of phytoplankton with the particle size larger than that of the phytoplankton net can be collected, and the part of phytoplankton with the particle size smaller than that of the phytoplankton net is not collected, so that the phytoplankton runs off, the total number of the phytoplankton is not counted, and like a precipitation method, formaldehyde is added during storage, so that the phytoplankton net method is harmful to human.
In the above two methods, if the storage time is too long, phytoplankton in the water may be decomposed by microorganisms, etc., and the amount of phytoplankton finally identified will also decrease, affecting the result. In addition, when the sample is collected, the space occupied by the water sample is large, and the transportation and the storage are inconvenient.
Therefore, if a method for rapidly fixing and identifying the field phytoplankton, which can not cause the loss of the phytoplankton during the previous treatment and can not decompose the phytoplankton during the preservation, can be found, the counting result is more accurate, and simultaneously, any reagent harmful to the health of human bodies does not need to be added, so as to ensure the health of experimenters, the effect of the phytoplankton on the water quality monitoring can be more exerted, the obtained result is more accurate on the monitoring of the phytoplankton of the eutrophic water body later, and the experimental process is more convenient.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for quickly fixing and identifying phytoplankton in a water body, which solves the problems of accuracy and safety of phytoplankton counting in the traditional method, ensures the accuracy of an identification result, does not need to add any medicine in an experimental process, is easy to realize the whole experimental process, needs simple instruments and equipment, and has a utilizable value and important significance for identifying the phytoplankton.
Therefore, the invention adopts the following technical scheme:
a method for quickly fixing and identifying phytoplankton in a water body comprises the following steps:
step one, sampling; taking a certain amount of water sample in a water body in which phytoplankton grows;
step two, suction filtration; filtering the phytoplankton to be identified by a suction filtration device to be retained on a filter membrane;
step three, stirring uniformly; putting the filter membrane into a container, adding distilled water, and stirring for multiple times until the mixture is uniform;
step four, sampling and identifying; and taking a small amount of uniformly mixed sample to be tested from the container for identification.
Preferably, in the first step, the sampled water body comprises a river channel, a lake, a reservoir and the like, 1.2L water is taken, and 1L water is measured by a measuring cylinder after shaking up to be used as a water sample.
Preferably, in the second step, a 0.45 μm mixed fiber membrane is used for suction filtration, the phytoplankton to be identified is retained on the filter membrane, the phytoplankton is preserved and the integrity of the phytoplankton is protected by means of cold storage of the filter membrane, and when the phytoplankton is required to be identified, a sample is directly taken from the filter membrane.
Preferably, in the third step, the filter membrane is placed in a 50m L beaker, distilled water is added, the mixture is stirred for 200 times by a glass rod, all phytoplankton on the filter membrane is put into the water, the water is moved to a 50m L brown reagent bottle, the volume is fixed to 50m L, a mixed water sample containing phytoplankton species consistent with the sampling water body is obtained, and the sample is uniformly mixed.
Preferably, in the fourth step, during identification, 0.1m L uniformly mixed sample to be detected is directly taken from a brown reagent bottle, the sample is placed on a counting area of a biological counting frame of a glass slide, then the cover glass is lightly covered, bubbles cannot be generated under the cover glass, an objective lens, an eyepiece, a reflector and the like of an optical microscope are adjusted to enable a visual field to be clear, a visual field counting method is selected, 100 visual fields are selected under the optical microscope for photographing and recording, and the final type and number are obtained through identification.
Preferably, the two slices are counted and averaged, and if the difference between the value of two slices and the average value is greater than ± 15%, the third slice is counted.
Preferably, the brown reagent bottle is fixed to 50m L for storage, so that a mixed water sample containing phytoplankton species consistent with the sampling water body is obtained, and the obtained sample to be detected has a certain amount and consistent concentration and contained species.
Preferably, the phytoplankton number per liter of water body is calculated by adopting the following formula
Wherein N is the number of phytoplankton per liter of water body in unit cfu/L, A is the area of counting frame in unit mm2；ACTo countNumber area, unit mm2I.e. the viewing area × number of views, VWThe volume of a sample obtained by washing a 1L water sample from a filter membrane after concentration and constant volume is m L, V is the volume of a counting frame and m L, n is the number of phytoplankton obtained by counting, and d is the dilution multiple.
Preferably, the equipment to be used comprises a plastic square barrel, a measuring cylinder, a suction filtration device, a filter membrane, a beaker, a glass rod, a brown reagent bottle and an optical microscope;
the plastic square barrel is a 1.2L white semitransparent water barrel, and is 142mm long, 74mm wide and 178mm high;
the measuring cylinder is a 1000m L transparent glass measuring cylinder, the height is 415mm, and the diameter of the tube is 66 mm;
the suction filtration device is a common glass suction filtration device and comprises a 300m L cylindrical glass funnel, an aluminum alloy clamp, a standard-mouth sand core filter and a standard-plug triangular flask;
the filter membrane is a white mixed fiber membrane with the diameter of 50mm and the pore diameter of 0.45 mu m;
the beaker is a 50m L transparent glass beaker and a common transparent glass rod;
the brown reagent bottle is a plastic bottle with the diameter of 3.8cm and the height of 8.8cm, and is a 50m L plastic bottle;
the optical microscope is a common optical microscope comprising an ocular lens, an objective lens, a glass slide of a biological counting frame, a cover glass and the like.
Preferably, tweezers for taking the filter membrane and the cover slip and a pipette for taking a sample and dripping the sample on the glass slide are also included.
Compared with the prior art, the invention has the beneficial effects that:
(1) the phytoplankton in the water body to be identified is fixed on the filter membrane in a suction filtration mode, and the phytoplankton is stored and the integrity of the phytoplankton is protected in a cold storage filter membrane storage mode, so that the problems of sample storage and transportation are solved; meanwhile, the sample is not transferred for many times in the process, and in the process of preserving the filter membrane, the environment on the filter membrane is not beneficial to the growth and the reproduction of microorganisms, so that phytoplankton on the filter membrane is not easily decomposed by the microorganisms, and the accuracy of the identification result is ensured.
(2) The method has the advantages that no medicine is added in the experimental process, the whole experimental process is easy to realize, required instruments and equipment are simple, the defects of the existing method in the aspects are overcome, and the method has a utilizable value and an important significance for identifying phytoplankton.
(3) The method can fully play the role of the phytoplankton in water quality monitoring, and enables the obtained result to be more accurate and the experimental process to be more convenient in the later monitoring of the phytoplankton in the eutrophic water body.
Drawings
FIG. 1 is a flow chart of a method for rapidly fixing and identifying phytoplankton in a water body provided by the invention.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments, which are provided for illustration only and are not to be construed as limiting the invention.
Examples
A method for quickly fixing and identifying phytoplankton in water body is disclosed, the flow is shown in figure 1, and the required equipment mainly comprises a plastic square barrel, a measuring cylinder, a suction filtration device, a filter membrane, a beaker, a glass rod, a plastic bottle and a complete set of device of an optical microscope.
The plastic square barrel is a 1.2L white semitransparent water barrel, and is 142mm long, 74mm wide and 178mm high;
the measuring cylinder is a 1000m L transparent glass measuring cylinder, the height is 415mm, and the diameter of the tube is 66 mm;
the suction filtration device is a common glass suction filtration device and comprises a 300m L cylindrical glass funnel, an aluminum alloy clamp, a standard-mouth sand core filter and a standard-plug triangular flask;
the filter membrane is a white mixed fiber membrane with the diameter of 50mm and the pore diameter of 0.45 mu m;
the beaker is a 50m L transparent glass beaker and a common transparent glass rod;
the plastic bottle is a 50m L brown reagent bottle, the diameter is 3.8cm, and the height is 8.8 cm;
the optical microscope is a common optical microscope comprising an ocular lens, an objective lens, a glass slide of a biological counting frame, a cover glass and the like.
Forceps are also required for taking the filter and cover slips, and a pipette is used for taking the sample and dripping it onto the slide.
The specific process is as follows:
sampling 1.2L water sample at a sampling point of a water body in which phytoplankton grows in a river, a lake, a reservoir and the like are put into a plastic square barrel, weighing 1L water sample by using a measuring cylinder after shaking uniformly, performing suction filtration by using a mixed fiber membrane with the diameter of 50mm and the aperture of 0.45 mu m through a suction filtration device, retaining the phytoplankton to be identified on a filter membrane, storing the phytoplankton by using a cold storage filter membrane, wherein the phytoplankton on the filter membrane is not easily decomposed by microorganisms and the like, so that the integrity of the phytoplankton is protected, the filter membrane is more convenient to store and carry when needing identification, directly sampling on the filter membrane when needing identification, directly taking the filter membrane when sampling, putting the filter membrane into a 50m L beaker, adding distilled water and stirring 200 times by using a glass rod, so that the phytoplankton the filter membrane is all in the water, so that the phytoplankton is not lost, enabling the result to be more accurate, transferring the water sample uniformly stirred in the glass rod into a 50m L reagent bottle, storing the sample in the 50m L, obtaining a sample containing the same type of the sample, obtaining a mixed optical microscope, and obtaining a final identifying result, and a final identifying result, wherein the sample, the sample is obtained by taking a sample, and a sample, wherein the sample is obtained by taking a sample, and a microscope, and a sample, the sample is not needed microscope, and a sample, wherein the sample is not needed by taking a sample, the sample is obtained by taking a sample, the sample is not needed.
The final phytoplankton number per liter of water can be obtained by the following formula
N-number of phytoplankton per liter of water, cfu/L;
a-area of counting frame, mm2；
AC-area of counting, mm2I.e., field of view area × field of view number;
VWthe 1L water sample is concentrated and washed from the filter membrane to obtain a constant volume sample volume m L;
v-count box volume, m L;
n-the number of phytoplankton obtained by counting;
d is dilution factor.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and scope of the present invention are intended to be covered thereby.
Claims (10)
1. A method for rapidly fixing and identifying phytoplankton in a water body is characterized by comprising the following steps:
step one, sampling; taking a certain amount of water sample in a water body in which phytoplankton grows;
step two, suction filtration; filtering the phytoplankton to be identified by a suction filtration device to be retained on a filter membrane;
step three, stirring uniformly; putting the filter membrane into a container, adding distilled water, and stirring for multiple times until the mixture is uniform;
step four, sampling and identifying; and taking a small amount of uniformly mixed sample to be tested from the container for identification.
2. The method for rapidly fixing and identifying the phytoplankton in the water body according to claim 1, wherein in the step one, the water body to be sampled comprises a river channel, a lake, a reservoir and the like, 1.2L is taken, and 1L is measured by a measuring cylinder after shaking up to be used as a water sample.
3. The method for rapidly fixing and identifying phytoplankton in water body according to claim 1, wherein the method comprises the following steps: and in the second step, a 0.45-micron mixed fiber membrane is used for suction filtration, phytoplankton to be identified is retained on the filter membrane, the phytoplankton is stored and the integrity of the phytoplankton is protected in a cold storage filter membrane storage mode, and when identification is needed, a sample is directly taken from the filter membrane.
4. The method for rapidly fixing and identifying the phytoplankton in the water body according to the claim 1, characterized in that in the third step, the filter membrane is put into a 50m L beaker, distilled water is added, the glass rod is used for stirring for 200 times, so that all the phytoplankton on the filter membrane is put into the water, the water is transferred to a 50m L brown reagent bottle, the volume is fixed to 50m L, a mixed water sample containing phytoplankton species consistent with the sampled water body is obtained, and the sample is uniformly mixed.
5. The method for rapidly fixing and identifying the phytoplankton in the water body according to claim 4, wherein in the fourth step, when identifying, 0.1m L uniformly mixed samples to be detected are directly taken from a brown reagent bottle, placed on a counting area of a biological counting frame of a glass slide, then the glass slide is covered lightly, bubbles cannot be generated under the glass slide, an objective lens, an eyepiece, a reflector and the like of an optical microscope are adjusted to enable the visual field to be clear, a visual field counting method is selected, 100 visual fields are selected under the optical microscope for photographing and recording, and the final type and number are identified.
6. The method for rapidly fixing and identifying phytoplankton in water body according to claim 5, wherein the method comprises the following steps: counting two slices to obtain an average value, and counting the third slice if the difference between the values of the two slices and the average value is more than +/-15%.
7. The method for rapidly fixing and identifying the phytoplankton in the water body according to claim 4, wherein a brown reagent bottle is subjected to constant volume of 50m L for storage to obtain a mixed water sample containing phytoplankton species consistent with the sampled water body, and any one of the obtained samples to be detected is in a certain amount, and the concentration of the sample is consistent with the species contained in the sample.
8. The method for rapidly fixing and identifying phytoplankton in a water body according to any one of claims 1 to 7, wherein: calculating the phytoplankton number in each liter of water body by adopting the following formula
Wherein N is the number of phytoplankton per liter of water body in unit cfu/L, A is the area of counting frame in unit mm2；ACIs a counting area in mm2I.e. the viewing area × number of views, VWThe volume of a sample obtained by washing a 1L water sample from a filter membrane after concentration and constant volume is m L, V is the volume of a counting frame and m L, n is the number of phytoplankton obtained by counting, and d is the dilution multiple.
9. The method for rapidly fixing and identifying phytoplankton in water body according to claim 8, wherein the method comprises the following steps: the equipment to be used comprises a plastic square barrel, a measuring cylinder, a suction filtration device, a filter membrane, a beaker, a glass rod, a brown reagent bottle and an optical microscope;
the plastic square barrel is a 1.2L white semitransparent water barrel, and is 142mm long, 74mm wide and 178mm high;
the measuring cylinder is a 1000m L transparent glass measuring cylinder, the height is 415mm, and the diameter of the tube is 66 mm;
the suction filtration device is a common glass suction filtration device and comprises a 300m L cylindrical glass funnel, an aluminum alloy clamp, a standard-mouth sand core filter and a standard-plug triangular flask;
the filter membrane is a white mixed fiber membrane with the diameter of 50mm and the pore diameter of 0.45 mu m;
the beaker is a 50m L transparent glass beaker and a common transparent glass rod;
the brown reagent bottle is a plastic bottle with the diameter of 3.8cm and the height of 8.8cm, and is a 50m L plastic bottle;
the optical microscope is a common optical microscope comprising an ocular lens, an objective lens, a glass slide of a biological counting frame, a cover glass and the like.
10. The method for rapidly fixing and identifying phytoplankton in a water body according to claim 9, wherein the method comprises the following steps: the device also comprises tweezers and a pipette, wherein the tweezers are used for taking the filter membrane and the cover glass, and the pipette is used for taking a sample and dripping the sample on the glass slide.
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| CN113528503A (en) * | 2021-06-16 | 2021-10-22 | 中国科学院南京地理与湖泊研究所 | Fixing method for researching structural diversity of freshwater ultramicro eukaryotic algae community |
| CN113528503B (en) * | 2021-06-16 | 2023-03-24 | 中国科学院南京地理与湖泊研究所 | Fixing method for researching structural diversity of freshwater ultramicro eukaryotic algae community |
| CN113604339A (en) * | 2021-08-16 | 2021-11-05 | 黑龙江省庄禹检测科技有限公司 | Microbial sampler for environment detection |
| CN113604339B (en) * | 2021-08-16 | 2024-01-26 | 广东宏科检测技术有限公司 | Microorganism sampler for environment detection |
| CN114115221A (en) * | 2021-09-28 | 2022-03-01 | 中国科学院水生生物研究所 | Water ecological factor in-situ monitoring device and method based on unmanned ship |
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