CN113237817A - Depolymerization observation method and application of benthic cyanobacteria algae pad - Google Patents
Depolymerization observation method and application of benthic cyanobacteria algae pad Download PDFInfo
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Abstract
The disclosure relates to the technical field of cell detection, and particularly provides a depolymerization observation method and application of a benthic cyanobacteria algae pad. The depolymerization observation method of the benthic cyanobacteria algae pad comprises the following steps: and (3) crushing the benthic cyanobacteria pad, and observing the form of the algae filaments under a high-power optical microscope. The method not only can be beneficial to the identification and counting of the subsequent optical microscope, but also can avoid the influence on the identification and counting accuracy of the optical microscope caused by the massive damage of the algae cells.
Description
Technical Field
The disclosure relates to the technical field of cell detection, and particularly provides a depolymerization observation method and application of a benthic cyanobacteria algae pad.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Along with global warming and water environment change, harmful benthic blue algae growing on the bottom matrix of rivers and lakes and reservoirs propagate in a large quantity, the own ecological structure and function of the water body can be damaged by the water bloom of the benthic blue algae, and a large amount of algal toxins and odor substances secreted by the benthic blue algae enter the water body to seriously influence the water quality of water supply. Bloom formation is a continuous, predictable process by which algal biomass gradually increases in a body of water. The monitoring and early warning work of the benthic cyanobacterial bloom of the water body in rivers and lakes is developed, which is helpful for water environment protection and related departments of drinking water supply to master the dynamic change of the benthic cyanobacterial population, actively remove the potential toxic cyanobacterial bloom in the water body, effectively prevent and control the risk of the outbreak of the surface cyanobacterial bloom, and has important significance for guaranteeing the water environment ecology of rivers and lakes and ensuring the water quality safety of drinking water.
Because the benthic cyanobacteria is usually filamentous cyanobacteria attached to bottom sediments or solid surfaces and intertwined with each other to form a cluster in the growth process, the benthic cyanobacteria sample can be identified and counted by an optical microscope after being pretreated and dissociated. The existing depolymerization method of the pad of the benthic cyanobacteria alga mainly comprises mechanical grinding and mild ultrasound. The inventor finds that because the filament-shaped benthic cyanobacteria is seriously wound in the growth process, the two methods can depolymerize the algae filaments, but cause serious breakage of the algae filaments and massive damage of algae cells, and seriously affect the accuracy of identification and counting of the benthic cyanobacteria.
Disclosure of Invention
In order to solve the defects of the prior art, the purpose of the present disclosure is to provide a method for effectively depolymerizing benthic cyanobacteria algae mats for observation, which not only can facilitate the subsequent identification and counting of an optical microscope, but also can avoid the damage of a large amount of algae cells so as to influence the identification and counting accuracy of the optical microscope.
In one or some embodiments of the present disclosure, a disaggregation observation method of a benthic cyanobacteria algae mat is provided, which includes the following steps: and (3) crushing the benthic cyanobacteria pad, and observing the form of the algae filaments under a high-power optical microscope.
In one or some embodiments of the disclosure, the application of the depolymerization observation method of the benthic cyanobacteria algae pad in algae removal and water purification is provided.
In one or some embodiments of the present disclosure, there is provided a use of the disaggregation observation method of the benthic cyanobacteria algae mat described above in biological environment simulation.
In one or some embodiments of the present disclosure, the application of the depolymerization observation method for the benthic cyanobacteria algae mat in algae cultivation is provided.
One or some of the above technical solutions have the following advantages or beneficial effects:
1) the benthic cyanobacteria algae pad is crushed, the long-length algae filaments in the cyanobacteria algae pad are reserved, fragmentation of the algae filaments is avoided, the visual field is clear under a high-power microscope, fragmentation algae filament interference is avoided, and the algae filament morphology and cell morphology can be observed conveniently.
2) The crushing mode of the method is mild, cell rupture is basically avoided, ruptured cell liquid is leaked out, visual field observation is influenced on the one hand, and cell counting accuracy is low on the other hand.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.
FIG. 1 is a histogram of the relationship between different pulverization times and the content of chlorophyll a in benthic cyanobacteria when the mild pulverization power is 150W in example 1 of the present disclosure;
FIG. 2 is a histogram of the relationship between different pulverization times and the content of chlorophyll a in benthic cyanobacteria when the mild pulverization power is 200W in example 2 of the present disclosure;
FIG. 3 is a histogram of the relationship between different pulverization times and the content of chlorophyll a in benthic cyanobacteria when the mild pulverization power is 250W in example 3 of the present disclosure;
FIG. 4 is a photomicrograph of the algal filament morphology at different pulverization times ((a)0, (b)1s, (c)2s, (d)3s, (e)4s and (f)5s) for a mild pulverization power of 250W according to example 3 of the present disclosure;
FIG. 5 is a bar graph of the relationship between different ultrasonic times and the content of chlorophyll a in benthic cyanobacteria when the mild ultrasonic power of comparative example 1 of the present disclosure is 20W;
FIG. 6 is a photomicrograph of the filament morphology of comparative example 1 of the present disclosure at 20W mild sonication power for varying sonication times ((a)0, (b)2s, (c)5s, (d)10s, (e)15s, and (f)20 s);
FIG. 7 is a bar graph of the relationship between the number of grinding times and the content of chlorophyll a in the benthic cyanobacteria of comparative example 2 in the present disclosure;
fig. 8 is a microscope photograph of algal filament morphology at various grinding times ((a)0, (b)5, (c)10, (d)20, (e)40, and (f) 100) of comparative example 2 of the present disclosure.
Detailed Description
The technical solutions in the embodiments of the present disclosure will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present disclosure, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the disclosure without making any creative effort, shall fall within the protection scope of the disclosure.
In order to solve the defects of the prior art, the purpose of the present disclosure is to provide a method for effectively depolymerizing benthic cyanobacteria algae mats for observation, which not only can facilitate the subsequent identification and counting of an optical microscope, but also can avoid the damage of a large amount of algae cells so as to influence the identification and counting accuracy of the optical microscope.
Algae broadly refers to plants that grow in water, and also includes certain aquatic higher plants. Algae is a large group of cryptophyte, has no difference of roots, stems, leaves and other parts, can produce nutrients by oneself with chlorophyll, has a plurality of varieties, is available in seawater and fresh water, and can live in the shady and humid places of land for a very few times. Common algae such as seaweed and chlorella.
Benthic algae mat (periphyton) is a general name of algae growing on a substrate immersed in water, and is generally formed by winding a plurality of algae in the growing process.
Generally speaking, there are many different cells in the same alga to achieve different physiological functions, for example, a houttuynia cordata is disclosed in "an anabaena ultrastructure" (wakayama, beijing university student system, 1981), and the authors of the document observed that tens of cells on each algal filament of the anabaena are differentiated into three types, namely, a vegetative cell with photosynthetic capacity, a heterocyte and a chlamydospore, and further judged physiological activities such as an energy storage mode, a photosynthesis mode and the like of the houttuynia cordata, which is very meaningful research for biology.
However, in the description of the article, "Tilletia foetida is collected in depression of Yuanmingyuan in Beijing Yuanmingyuan", the sample collected by the author of the article is very convenient to observe and contains complete algae filaments, so that the valuable research results can be obtained.
However, for the benthic algae pad, the benthic algae pad is formed by winding a plurality of algae filaments, and usually depolymerizes by mechanical grinding and mild ultrasound in the prior art, but the inventor finds that, in the experimental process, the benthic algae pad subjected to mechanical grinding and mild ultrasound post-treatment has more fragments and is difficult to obtain a complete algae filament, and in the observation process, a plurality of fragmented algae filaments and damaged cells are observed, and it is difficult to judge which specific algae filament cell is and the specific distribution mode of each functional cell, i.e. it is difficult to judge the real structure of one algae filament, and it is also difficult to accurately identify the species of the algae filament.
In addition, in the process of growth of algae, the algae filaments also change along with the growth of the algae, for example, the process of degradation of the algae filaments into nutrient algae filaments (figure 8) is found by examining shell filaments of porphyra yezoensis shells in microscopic observation of shell filaments before picking seedlings of porphyra yezoensis shells (Zhujiayi, Luassian and the like, aquaculture [ J ], 2003), which is very important for researching the growth process of the algae and further realizing the culture of the algae.
For the benthic algae pad, the benthic algae pad may be formed by winding various different types of algae filaments, and if there is no one or more complete algae filaments, a large amount of algae filament fragments in various forms appear in the visual field of an observer, and it is difficult to really distinguish the specific structure of the specific benthic algae pad according to the algae filaments, which causes great difficulty in research.
For this reason, the inventors have recognized that it would be greatly convenient for the observer to observe if there could be a way to maintain a large number of long, highly intact algal filaments during the disaggregation of the benthic algal mat.
In one or some embodiments of the present disclosure, a disaggregation observation method of a benthic cyanobacteria algae mat is provided, which includes the following steps: the benthic cyanobacteria mat is appropriately crushed, and then the shape of the algal filaments is observed under a high-power optical microscope.
The crushing special refers to crushing by using a blade, and because in the cutting process of the blade, a part of algae filaments in the benthic algae pad can pass through a gap between the blade and the blade, a large amount of algae filaments with complete lengths are reserved, and compared with the fragmentation treatment modes of the whole algae pad, such as ultrasonic and grinding, the crushing mode is more suitable for observation under a high-power optical microscope.
Of course, it should be understood that in the prior art, one algae filament can be easily obtained for free algae, and then the cells can be directly observed by using an optical microscope, but the prior art is not suitable for the benthic cyanobacteria mat, because the foregoing has been fully analyzed, and the detailed description is omitted.
The method for depolymerizing the pad of the benthic cyanobacteria can be helpful for subsequent optical microscope identification and counting, can avoid a large amount of broken algae filaments and a large amount of damaged algae cells, and ensures the accuracy of subsequent identification and counting of the benthic cyanobacteria.
The benthic cyanobacteria algae pad sample is an algae pad which is collected on the bottom matrix of water bodies in the nature, including rivers, lakes, reservoirs and the like and contains cyanobacteria.
Preferably, the pulverization is carried out by using a pulverizer;
preferably, the crushing apparatus comprises a crushing chamber, and crushing blades transversely mounted are fixed in the crushing chamber.
The pulverizer disclosed by the present disclosure is composed of a stainless steel upper cover and a lower body pulverizing chamber, is closed by a screw fastener, and drives a transversely installed pulverizing blade by the high-speed operation of a vertical motor to perform impact and shear type pulverization on a sample, such as a small pulverizer, specifically, a Target, a hand-held mixer, and a TARHB40S are used in the embodiment of the present disclosure.
Preferably, the pulverizer is a small pulverizer;
preferably, the crushing power is 150-250W;
or pulverizing for 1-5 s.
Preferably, the magnification of the high power optical microscope is 100-400,
preferably, the eyepiece is 10 x and the objective lens is 10 x to 40 x.
Preferably, the method also comprises a blue algae identification process, and comprises the following steps: judging the blue algae species according to the observed algae filament morphology and cell morphology.
Preferably, the method also comprises an algae cell counting process, which comprises the following steps: selecting the algae filament in a certain visual field, classifying different cells in the algae filament, and counting the same kind of cells.
Preferably, the method also comprises a slide preparation process, which comprises the following steps: and fully shaking the crushed algae samples uniformly, taking 0.1mL of the sample into a counting frame by using a dropper, covering a cover glass, standing for 3-5 min after the sample is prepared to enable the algae bodies to sink to the bottom of the frame, and performing microscopic examination.
In one or some embodiments of the disclosure, the application of the depolymerization observation method of the benthic cyanobacteria algae pad in algae removal and water purification is provided. The algae species and cell characteristics in the algae pad are researched, and then a reasonable algae cleaning mode is formulated.
In one or more embodiments of the present disclosure, there is provided a use of the disaggregation observation method for the benthic cyanobacteria algae mat in an algae observation experiment. That is, the observer can select the benthic cyanobacteria algae pad at different periods to perform physiological observation, including but not limited to, predominant species of cyanobacteria, morphological characteristics of cyanobacteria cells.
In one or some embodiments of the present disclosure, the application of the depolymerization observation method for the benthic cyanobacteria algae mat in algae cultivation is provided. The characteristics of cells and algae filaments of algae in different growth periods are researched, nutrients are correspondingly supplemented according to the characteristics of the cells and the algae filaments, and a proper growth environment is created.
Example 1
The embodiment provides a depolymerization observation method of a benthic cyanobacteria algae pad, which comprises the following steps:
collecting benthic cyanobacteria and algae pads from a certain reservoir in Shandong, wherein the main harmful cyanobacteria in the benthic cyanobacteria and algae pads is basket algae, adding 200mL of the cyanobacteria pad samples into a small-sized pulverizer, wherein the power of the pulverizer is 150W, pulverizing for different times (1, 2, 3, 4 and 5s), taking out the samples, and then measuring the chlorophyll a content change of the samples and observing the morphological change condition of the algae filaments under a high-power optical microscope.
In this and the following examples, the chlorophyll a measurement procedure was as follows:
1. after the sample is filtered, the filter membrane is placed into a refrigerator for refrigeration for 24h (the water content of the filter membrane is evaporated), the filter membrane is taken out and placed into a centrifuge tube, a small amount of magnesium carbonate powder and 5mL of 90% acetone are added, the mixture is ground by a glass rod, the mixture is stood for 5min and then centrifuged for 10min by a centrifuge (4000r/min), and the supernatant is sucked into a 10mL volumetric flask.
2. Then adding 3mL of 90% acetone, continuing milling and extracting, centrifuging for 10min, sucking the supernatant into a 10mL volumetric flask, and fixing the volume.
3. And (3) putting the supernatant on a spectrophotometer, and respectively reading the absorbances of 750nm, 663nm, 645nm and 630nm wavelengths by using a cuvette with a 1cm optical path, and measuring the blank absorbances by using 90% acetone to correct the absorbance of the sample.
4. Calculation method
Chl-a(mg/m3)
V-volume of water sample: 0.5L
V1 volume after volume fixing (mL) of extract: 10mL
δ — cell optical distance (cm): 1cm
Example 2
The embodiment provides a depolymerization observation method of a benthic cyanobacteria algae pad, which comprises the following steps:
collecting benthic cyanobacteria and algae pads from a certain reservoir in Shandong, wherein the main harmful cyanobacteria in the benthic cyanobacteria and algae pads is basket algae, adding 200mL of algae pad samples into a small-sized pulverizer, wherein the power of the pulverizer is 200W, pulverizing for different time (1, 2, 3, 4 and 5s), taking out the samples, and then measuring the chlorophyll a content change of the samples and observing the morphological change condition of algae filaments under a high-power optical microscope.
Example 3
The embodiment provides a depolymerization observation method of a benthic cyanobacteria algae pad, which comprises the following steps:
collecting benthic cyanobacteria and algae pads from a certain reservoir in Shandong, wherein the main harmful cyanobacteria in the benthic cyanobacteria and algae pads is basket algae, adding 200mL of the cyanobacteria pad samples into a small-sized pulverizer, wherein the power of the pulverizer is 250W, pulverizing for different times (1, 2, 3, 4 and 5s), taking out the samples, and then measuring the chlorophyll a content change of the samples and observing the morphological change condition of the algae filaments under a high-power optical microscope.
The depolymerization methods described in examples 1-3 are different in power, as shown in FIGS. 1-3, the content of chlorophyll a is similar at different powers, and it is clear that the influence of power on cyanobacteria chlorophyll a is not great at powers between 100 and 250W.
As shown in figure 4, as the crushing time is prolonged, the benthic cyanobacteria algae pad is depolymerized into algae filaments, the length of each algae filament is moderate, the algae filaments are relatively complete, the number of crushed algae cells is less, the visual field is clean, fragments of the crushed algae filaments do not interfere with the visual field, and the observation and counting of the shapes and the cell shapes of the algae filaments can be realized.
Comparative example 1
The embodiment provides a depolymerization observation method of a benthic cyanobacteria algae pad, which comprises the following steps:
collecting benthic cyanobacteria algae pads from a certain reservoir in Shandong, wherein the main harmful cyanobacteria in the benthic cyanobacteria algae pads is basket algae, putting 20mL of algae pad samples into an ultrasonic instrument, carrying out ultrasonic power of 20W, taking out the samples after carrying out ultrasonic treatment for different time (2, 5, 10, 15 and 20s), and then measuring the chlorophyll a content change of the samples and observing the morphological change condition of algae filaments under a high-power optical microscope.
As shown in fig. 5, the depolymerization method described in comparative example 1 has a larger chlorophyll-a loss rate than in examples 1 to 3, but the chlorophyll-a loss rate is not high as a whole, and it is presumed that the ultrasonic treatment causes the cyanobacterial cells to be ruptured to some extent. As shown in fig. 6, the benthic cyanobacteria are decomposed into cyanobacteria fragments, and many cyanobacteria fragments have a disturbed visual field and few long-length algal filaments, and it is difficult to observe the form of algal filaments, and it is also difficult to observe the cell form due to the overlapping effect of the fragmented algal filaments.
Obviously, the ultrasonic pulverization can realize the depolymerization of the algae filaments, but the pulverization degree of the degraded algae filaments is higher, the visual field is disordered under a microscope, and the detection of the algae filaments and cells is difficult to realize, namely, in the embodiment 1-3, the pulverization of the pulverizer is matched with a high-power optical microscope, so that a large amount of algae filaments with longer length are retained in the depolymerization process of the benthic algae, and the observation under the high-power optical microscope is convenient.
Comparative example 2
The embodiment provides a depolymerization observation method of a benthic cyanobacteria algae pad, which comprises the following steps:
collecting benthic cyanobacteria algae pads from a certain reservoir in Shandong, wherein the main harmful cyanobacteria in the benthic cyanobacteria algae pads is basket algae, putting 20mL of algae pad samples into a glass grinding tube, grinding for different times (5, 10, 20, 40 and 100 times), taking out the samples, measuring the chlorophyll a content change of the samples and observing the algae filament form change condition under a high-power optical microscope.
As shown in FIG. 7, the depolymerization method according to comparative example 2 showed a large amount of chlorophyll-a loss and apparently a high degree of cell disruption as compared with examples 1 to 3, and as shown in FIG. 8, a large number of blurred shadow portions appeared in a high magnification optical microscope field corresponding to an increase in the number of cell disruptions as the magnification was increased.
In addition, as seen from fig. 7, when the number of grinding times was 5 times and 10 times, the amount of chlorophyll a lost in the depolymerization method according to comparative example 2 was small, that is, the cell rupture was small, but as seen from fig. 8, although the number of grinding times was 5 times and 10 times, the cell rupture was small, the algal filaments were broken into pieces severely, the visual field was very disturbed, and the observation was very difficult.
That is, as can be seen from the data results of 5 times and 10 times in comparative examples 1 and 2 and the visual field under a high power optical microscope, the degree of fragmentation of the algae filaments is high even if the cell is not broken by the ultrasonic treatment or the grinding treatment, the visual field for observation is poor, and the morphological observation and counting requirements are difficult to realize. Thus, it will be appreciated that the relatively aggressive disaggregation approach, which reduces the number of treatments or time, is not suitable for disaggregation observations of the algal mat.
The disclosure of the present invention is not limited to the specific embodiments, but rather to the specific embodiments, the disclosure is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A depolymerization observation method of a benthic cyanobacteria algae pad is characterized by comprising the following steps: and (3) crushing the benthic cyanobacteria pad, and observing the form of the algae filaments under a high-power optical microscope.
2. The method for observing depolymerization of a pad of benthic cyanobacteria as claimed in claim 1, wherein the pulverization is carried out using a pulverizer;
preferably, the crushing apparatus comprises a crushing chamber, and crushing blades transversely mounted are fixed in the crushing chamber.
3. The method for observing depolymerization of a mat of benthic cyanobacteria of claim 1, wherein the pulverizer is a small pulverizer;
preferably, the crushing power is 150-250W;
or pulverizing for 1-5 s.
4. The method for disaggregating and observing a pad of cyanobacteria of claim 3, wherein the magnification of the high power optical microscope is 100 ×,
preferably, the eyepiece is 10 x and the objective lens is 10 x to 40 x.
5. The disaggregation observation method of the benthic cyanobacteria mat of claim 1, further comprising a cyanobacteria identification process comprising the steps of: and judging the type of the blue algae according to the observed algae filament morphology and cell morphology.
6. The disaggregation observation method of the cushion of benthic cyanobacteria as claimed in claim 1, further comprising an algal cell counting process comprising the steps of: and selecting a complete algae filament in the visual field, classifying different cells in the algae filament, and counting the cells of the same kind.
7. The disaggregation observation method of the benthic cyanobacteria algae mat of claim 1, further comprising a slide preparation process comprising the steps of: the crushed algae samples are fully shaken up, 0.1mL of the algae samples are taken by a dropper to a counting frame, and a cover glass is covered. And (5) standing for 3-5 min after the specimen is prepared to enable the algae to sink to the bottom of the frame, and performing microscopic examination.
8. Use of the depolymerization observation method for the pad of benthic cyanobacteria algae of any one of claims 1 to 7 for removing algae and purifying water source.
9. Use of the method for observing depolymerization of a pad of a benthic cyanobacteria alga of any one of claims 1 to 7 in an alga observation experiment.
10. Use of the depolymerization observation method for the cushion of benthic cyanobacteria algae according to any one of claims 1 to 7 in algae cultivation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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