CN113075217A - Quick and efficient sporopollen extraction method - Google Patents
Quick and efficient sporopollen extraction method Download PDFInfo
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- CN113075217A CN113075217A CN202110267631.XA CN202110267631A CN113075217A CN 113075217 A CN113075217 A CN 113075217A CN 202110267631 A CN202110267631 A CN 202110267631A CN 113075217 A CN113075217 A CN 113075217A
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- G01N1/00—Sampling; Preparing specimens for investigation
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- G—PHYSICS
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Abstract
The invention discloses a quick and efficient sporopollen extraction method which comprises the steps of pretreatment, fine screening, temporary flaking, microscopic observation and sealing in sequence, and the impurities of the temporary flaking with more impurities which do not meet the requirements are removed in a mode of ring probe impurity removal in the microscopic observation process, so that the damage and loss of sporopollen are reduced as much as possible in the secondary impurity removal before flaking, the secondary impurity removal process is simplified, the experiment cost is further reduced, and the experiment period is shortened.
Description
Technical Field
The invention is mainly applied to the field of sediment sporopollen analysis, and particularly relates to a quick and efficient sporopollen extraction method.
Background
The quaternary sporopolleology is used as a substitute index for reconstructing ancient vegetation and ancient environment, plays an important role in reconstructing past earth change, researchers often need to carry out identification work on the morphology or microstructure of sporopollen, a pretreatment experiment needs to be carried out to remove impurities from collected soil samples before a microscope is used for identifying a slide, and then extracted sporopollen is collected by a small test tube and is dropwise added with glycerol. The traditional method is generally divided into the following steps in the experimental process: coarse screening, pretreatment, fine screening, temporary flaking, microscopic observation, sample loading, permanent flaking, identification, marking and the like. Optical identification generally requires that the slides be free of a large number of sight-obstructing impurities and that the spore powder density be relatively modest. The conventional method would be to perform a temporary slide after fine screening and to place it under a microscope for inspection of the previous step for impurities. If more impurities are observed, secondary treatment is needed to be carried out on the sample, silicate impurities are removed by hydrofluoric acid, carbonate impurities are removed by hydrochloric acid, organic matters and the like are removed by weak alkaline solution, and if more impurities are still observed and the impurities are insoluble in acid and alkali through multiple experiments, a heavy liquid flotation method is adopted. Although such an operation can further remove impurities, the probability of damage of the sporopouenin and the preparation period are increased, resulting in a decrease in experimental efficiency, and the consumption of an acid-base solution is increased, resulting in an increase in experimental cost.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects of the prior art, in the secondary impurity removal process of spore powder extraction, the traditional method uses an HF screening method and a heavy liquid flotation method, so that part of spore powder is easily lost, the experimental period and the cost are increased, the technology optimizes the manufacturing process aiming at the problems and uses a new tool, specifically, the invention is used under a microscope for selecting impurities in a slide, so that secondary impurity removal is realized, and the processed slide is sealed and stored. The fast and efficient sporopollen extraction method can effectively solve the problem of time and material consumption in the traditional scheme.
The technical scheme is as follows: in order to achieve the purpose of the invention, the invention adopts the technical scheme that:
a quick and efficient sporopollen extraction method comprises the following specific steps:
1) pretreatment
Taking 3-5 g of a soil sample, adding lycopodium spores before treatment, adding a 10% HCl solution, stirring, standing, placing the soil sample in a water bath pot for heating for 9-12min when no bubbles exist, removing carbonate impurities, pouring off a supernatant after centrifugation, adding water to wash the sample to be neutral, then performing water bath heat treatment by using a 10% KOH solution for 18-22mim, removing organic matters in the sample, pouring off the supernatant after centrifugation, adding water to wash the sample to be neutral, adding a 40% HF solution, standing for 1-2 days, removing silicate impurities, pouring off the supernatant after centrifugation, washing with water to be neutral, adding glacial acetic acid to remove moisture, treating the sample by using an acetolysis method, pouring off the supernatant after centrifugation, washing with glacial acetic acid, pouring off the supernatant after centrifugation, washing with water to be neutral, and preparing for a next step of fine screening;
2) fine screen
Sieving with 8-11 μm sieve cloth combined with ultrasonic oscillator, collecting the residual concentrate, adding water, centrifuging, and sucking the supernatant with a suction tube to obtain sample;
3) temporary tableting
Wiping the glass slide and the cover glass completely, dripping a drop of glycerol on the glass slide, uniformly stirring the sample prepared in the step 2) with water, sucking a drop of mixture by using a suction pipe, dripping the mixture on the glycerol, fully mixing the glycerol and the mixture by using the suction pipe, and covering the cover glass;
4) observation by microscope
Putting the temporary slide down and observing under a microscope, judging the amount of impurities in the temporary slide, directly loading samples with less impurities, and removing the impurities on the temporary slide by adopting a ring probe impurity removal method;
5) sealing sheet
And (4) performing mounting on the temporary slide sheet meeting the requirements in the step (4), when the spore powder can be clearly observed under a microscope, coating the brilliant nail oil on the periphery of a cover glass, and marking the mount to prepare a permanent slide sheet.
Further, the method for removing impurities by the ring probe is specifically that the titanium alloy ring probe is used and extends into a gap between the glass slide and the cover glass, and under the observation of a microscope, the ring probe is used for hooking out large organic matters and mineral matters in the glass slide, so that spore powder is not lost as far as possible.
Further, in the step 2), in order to not disturb the spore powder at the bottom of the test tube and reduce the loss caused by sucking away the spore powder, 1-3 ml of water is reserved at the bottom of the test tube.
Further, in the step 3), for the convenience of observation, it should be ensured that there is no bubble between the slide and the cover glass as much as possible.
Further, in said step 5), it must be ensured that the small amount of impurities remaining in the slide before the mounting is not sufficient to interfere with the observation of the morphology and structure of the sporopollen.
Furthermore, the sporopollen is different in size, most of the sporopollen is 10-200 mu m in diameter, and a ring probing needle with the diameter of about 18-22 mu m is adopted.
Has the advantages that: compared with the prior art, the invention has the advantages that the classification management is clear at a glance,
1) the mode of impurity removal by a probing needle is adopted, so that the damage and loss of sporopollen are reduced as much as possible in secondary impurity removal before tabletting, the secondary impurity removal process is simplified, the experiment cost is further reduced, and the experiment period is shortened;
2) as the spore powder has different sizes and most diameters fall between 10 and 200 mu m, the spore powder loss can be avoided to the maximum extent on the premise of ensuring that the probe with the diameter of about 20 mu m is not easy to damage by adopting the probe with the diameter of about 20 mu m;
3) the material of the ring probe is titanium alloy which is found to be an alloy material with higher hardness at present, and the ring probe can be conveniently operated and stored when being manufactured into a fine ring probe.
Drawings
FIG. 1 is a flow chart of the method of the present invention;
fig. 2 is a structural schematic diagram of the probe ring needle.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to be limiting.
The traditional sporopollen extraction method comprises the steps of taking 3-5 g of soil sample, adding lycopodium clavatum spores before treatment, adding 10% HCl solution for stirring, standing, putting the mixture into a water bath pot for heating for 10min when no bubbles exist, removing carbonate impurities, pouring out supernate after centrifugation, adding water to wash the sample to be neutral, then, performing water bath heat treatment on the sample by using 10% KOH solution for 20mim to remove organic matters in the sample, then adding 40% HF solution, standing for 1-2 days to remove silicate impurities, after HF treatment, washing with water to neutrality, treating the sample by acetic acid hydrolysis method, washing with water to neutrality, sieving with 10 μm sieve cloth combined with ultrasonic oscillator, collecting the residual concentrate, tabletting, observing the impurity removal condition under microscope, generally, a large amount of silicate impurities appear, 40% HF solution is required to be added and placed for 1-2 days, the solution is poured out after centrifugation, and a sample is washed to be neutral. If more impurities still exist, the heavy liquid flotation method is adopted, and the heavy liquid using process of the heavy liquid flotation method, including the processes of heavy liquid preparation, heavy liquid recovery, sample dewatering, heavy liquid flotation centrifugation, dilution and the like, is time-consuming and labor-consuming.
In the secondary impurity removal process in the spore powder extraction in the traditional method, a part of spore powder is easily lost by using an HF screening method and a heavy liquid flotation method, and the experimental period and the cost are increased, so that the manufacturing process is optimized aiming at the problems, a new tool is used, specifically, impurities in a glass slide are selected under a microscope, secondary impurity removal is realized, and the treated glass slide is sealed and stored, so that the problem of time consumption and material consumption in the traditional scheme can be effectively solved by providing a quick and efficient spore powder extraction method; the specific method comprises the following steps: as shown in fig. 1
1) Pretreatment
Taking 3-5 g of a soil sample, adding lycopodium clavatum spores before treatment, adding a 10% HCl solution, stirring, standing, putting the soil sample into a water bath pot for heating for 10min when no bubbles exist, removing carbonate impurities, pouring off a supernatant after centrifugation, adding water to wash the sample to be neutral, then carrying out water bath heat treatment by using a 10% KOH solution for 20 mm, removing organic matters in the sample, pouring off the supernatant after centrifugation, adding water to wash the sample to be neutral, adding a 40% HF solution for standing for 1-2 days, removing silicate impurities, pouring off the supernatant after centrifugation, washing to be neutral, adding glacial acetic acid to remove water, treating the sample by using an acetification method, pouring off the supernatant after centrifugation, washing by using glacial acetic acid, pouring off the supernatant after centrifugation, and washing to be neutral so as to prepare for a next fine sieve;
2) fine screen
Sieving with 8-11 μm sieve cloth combined with ultrasonic oscillator, collecting the residual concentrate, adding water, centrifuging, and sucking the supernatant with a suction tube to obtain sample;
3) temporary tableting
Wiping the glass slide and the cover glass completely, dripping a drop of glycerol on the glass slide, uniformly stirring the sample prepared in the step 2) with water, sucking a drop of mixture by using a suction pipe, dripping the mixture on the glycerol, fully mixing the glycerol and the mixture by using the suction pipe, and covering the cover glass;
4) observation by microscope
Putting the temporary slide down and observing under a microscope, judging the amount of impurities in the temporary slide, directly loading samples with less impurities, and removing the impurities on the temporary slide by adopting a ring probe impurity removal method;
5) sealing sheet
And (4) sealing the temporary slide sheet meeting the requirements in the step (4), coating the brilliant nail oil on the periphery of a cover glass when the spore powder can be clearly observed under a microscope to achieve the effect of isolating the cover glass from the outside air, and marking the seal sheet to prepare a permanent slide sheet.
The spore powder identification can be carried out after the preparation of the permanent tablet.
In this example, the method for removing impurities by using the probe ring needle is to use the titanium alloy probe ring needle to extend into a gap between the glass slide and the cover glass, and hook out large organic matters and minerals in the glass slide by using the probe ring needle under observation of a microscope, so that spore powder is not lost as much as possible.
In this example, further, in the step 2), in order to not disturb the spore powder at the bottom of the test tube and reduce the loss caused by the spore powder being sucked away, 1-3 ml of water is retained at the bottom of the test tube.
In this example, further, in the step 3), for the convenience of observation, it should be ensured that there is no air bubble between the slide glass and the cover glass as much as possible.
In this example, further, in said step 5), it must be ensured that the small amount of impurities remaining in the slide before the coverslipping is not sufficient to interfere with the observation of the spore pollen morphology and structure.
In this example, further, since the sporopous have different sizes and most diameters fall between 10 μm and 200 μm, as shown in fig. 2, the probe ring needle structure is schematically illustrated, and the probe ring needle with a diameter of about 20 μm is used in this example, so that sporopou loss can be avoided to the maximum extent on the premise that the probe ring needle is not easily damaged, wherein the probe ring needle is made of a titanium alloy material.
Compared with the traditional method, the method comprises the following steps:
1) after the sample prepared in the step 2) is screened, impurities are removed by adopting a mode of impurity removal by a probe ring needle, so that the loss of spore powder caused by secondary impurity removal by an HF fine screening method and a heavy liquid flotation method is avoided;
2) the method for removing impurities by using the probe ring can reduce the time and cost of the experiment.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. A quick and efficient sporopollen extraction method is characterized in that: the method comprises the following specific steps:
1) pretreatment
Taking 3-5 g of a soil sample, adding lycopodium spores before treatment, adding a 10% HCl solution, stirring, standing, placing the soil sample in a water bath pot for heating for 9-12min when no bubbles exist, removing carbonate impurities, pouring off a supernatant after centrifugation, adding water to wash the sample to be neutral, then performing water bath heat treatment by using a 10% KOH solution for 18-22mim, removing organic matters in the sample, pouring off the supernatant after centrifugation, adding water to wash the sample to be neutral, adding a 40% HF solution, standing for 1-2 days, removing silicate impurities, pouring off the supernatant after centrifugation, washing with water to be neutral, adding glacial acetic acid to remove moisture, treating the sample by using an acetolysis method, pouring off the supernatant after centrifugation, washing with glacial acetic acid, pouring off the supernatant after centrifugation, washing with water to be neutral, and preparing for a next step of fine screening;
2) fine screen
Sieving with 8-11 μm sieve cloth combined with ultrasonic oscillator, collecting the residual concentrate, adding water, centrifuging, and sucking the supernatant with a suction tube to obtain sample;
3) temporary tableting
Wiping the glass slide and the cover glass completely, dripping a drop of glycerol on the glass slide, uniformly stirring the sample prepared in the step 2) with water, sucking a drop of mixture by using a suction pipe, dripping the mixture on the glycerol, fully mixing the glycerol and the mixture by using the suction pipe, and covering the cover glass;
4) observation by microscope
Putting the temporary slide down and observing under a microscope, judging the amount of impurities in the temporary slide, directly loading samples with less impurities, and removing the impurities on the temporary slide by adopting a ring probe impurity removal method;
5) sealing sheet
And (4) performing mounting on the temporary slide sheet meeting the requirements in the step (4), when the spore powder can be clearly observed under a microscope, coating the brilliant nail oil on the periphery of a cover glass, and marking the mount to prepare a permanent slide sheet.
2. The method for quickly and efficiently extracting sporopollen as claimed in claim, wherein the sporopollen is prepared by the following steps: the method for removing impurities by the ring probe comprises the steps of extending the titanium alloy ring probe into a gap between a glass slide and a cover glass, and hooking out large organic matters and mineral matters in the glass slide by the ring probe under the observation of a microscope, so that sporopollen is not lost as far as possible.
3. The method for quickly and efficiently extracting sporopollen as claimed in claim, wherein the sporopollen is prepared by the following steps: in the step 2), in order to not disturb the sporopollen at the bottom of the test tube and reduce the loss caused by absorbing the sporopollen, 1-3 ml of water is reserved at the bottom of the test tube.
4. The method for quickly and efficiently extracting sporopollen as claimed in claim, wherein the sporopollen is prepared by the following steps: in the step 3), for the convenience of observation, it should be ensured that no air bubbles exist between the glass slide and the cover glass as much as possible.
5. The method for quickly and efficiently extracting sporopollen as claimed in claim, wherein the sporopollen is prepared by the following steps: in said step 5), it must be ensured that the small amount of impurities remaining in the slide before the mounting is not sufficient to interfere with the observation of the morphology and structure of the sporopollen.
6. The method for quickly and efficiently extracting sporopollen as claimed in claim, wherein the sporopollen is prepared by the following steps: the sporopollen is different in size, the diameter of most sporopollen falls to 10-200 mu m, and a ring probing needle with the diameter of about 18-22 mu m is adopted.
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| CN202110267631.XA CN113075217A (en) | 2021-03-12 | 2021-03-12 | Quick and efficient sporopollen extraction method |
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| CN202110267631.XA CN113075217A (en) | 2021-03-12 | 2021-03-12 | Quick and efficient sporopollen extraction method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103535835A (en) * | 2012-07-13 | 2014-01-29 | 北京市蜂业公司 | Solid beverage prepared from pollen soluble extracts |
| CN104941544A (en) * | 2015-05-29 | 2015-09-30 | 蔡文 | Sporopollen microcapsule, and preparation method and application thereof |
| CN108548704A (en) * | 2018-04-18 | 2018-09-18 | 中国科学院东北地理与农业生态研究所 | A kind of peat cryptogam rapid extracting method |
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2021
- 2021-03-12 CN CN202110267631.XA patent/CN113075217A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103535835A (en) * | 2012-07-13 | 2014-01-29 | 北京市蜂业公司 | Solid beverage prepared from pollen soluble extracts |
| CN104941544A (en) * | 2015-05-29 | 2015-09-30 | 蔡文 | Sporopollen microcapsule, and preparation method and application thereof |
| CN108548704A (en) * | 2018-04-18 | 2018-09-18 | 中国科学院东北地理与农业生态研究所 | A kind of peat cryptogam rapid extracting method |
Non-Patent Citations (4)
| Title |
|---|
| 刘斌生: "《食品微生物检验》", 31 January 2013, 中国轻工业出版社 * |
| 李宜垠 等: ""泥炭样品的AMS14C年龄测定：全样、植物残体和孢粉浓缩物"", 《第四纪研究》 * |
| 李育 等: "中国北方第四纪孢粉提取方法研究", 《沉积学报》 * |
| 果德安: "《中药鉴定学基础》", 31 March 2001, 中国医药科技出版社 * |
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