CN106018027B - A kind of sample preparation methods of scanning electron microscope - Google Patents

Abstract

The invention discloses a sample preparation method for a scanning electron microscope. The steps of the method include the following steps: feces sample processing; nylon bag wrapping and washing; petri dishes standing and washing; high-speed water flow washing; water changing; water absorption and drying; sample platform preparation; Gold spray coating. Through drying, crushing, soaking, nylon bag wrapping and washing, high-speed water washing, water absorption, drying, gold plating and other processes, the scanning electron microscope sample prepared is clean and free of impurities, no gravel precipitation, small loss of residue, uniform distribution, and clear residue samples by visual observation , when observed by scanning electron microscope, the residue structure is clear and complete, the field of vision is clean, and the image has a strong three-dimensional effect, which is conducive to the observation and research of the ultrastructure of undigested forage residues in the feces of ruminants after digestion, and is very useful for intuitive understanding and evaluation of ruminant digestive tract microorganisms. The degradation of roughage fiber structure and the improvement of straw feed utilization are of great significance.

Description

A kind of sample preparation method of scanning electron microscope

technical field

The invention belongs to the technical field of microscopic observation samples, and relates to a scanning electron microscope sample preparation method, in particular to a scanning electron microscope (SEM) observation sample preparation method for forage grass residues in feces digested by ruminants.

Background technique

In recent years, with the rapid development of economy and society and the continuous improvement of people's living standards, my country's animal husbandry has developed rapidly. Therefore, the requirements for the quantity and quality of forage resources are constantly increasing, and relevant research is being carried out vigorously. Under this general development trend, the improvement of forage utilization rate is becoming a research hotspot, and various technologies to promote forage digestibility are emerging in an endless stream. At present, most of the research results on the degree of forage digestion are only reflected and compared with quantitative indicators, such as digestibility, rumen degradation rate, rumen disappearance rate, etc., and generally lack the fiber structure and cell wall changes that can directly reflect the forage passing through the digestive tract of ruminants visual image data. In order to more intuitively reveal the degree of forage fiber being utilized by animals after being digested by the digestive tract of ruminants, and to provide more accurate image data for the development of technologies and products that improve the digestibility of forage, the present invention proposes a ruminant-digested Sample preparation method for scanning electron microscopy (SEM) observation of forage residues in manure. The above-mentioned image data can be obtained through the method described in the present invention.

Contents of the invention

The purpose of the present invention is to overcome the defect that undigested forage grass residues in ruminant feces are difficult to separate and distinguish in the above-mentioned technology, and to provide a sample preparation method for scanning electron microscopy, through drying, crushing, soaking, nylon bag wrapping and washing, high-speed water flow Washing, water absorption, drying, gold plating and other processes, the prepared scanning electron microscope samples are clean and free of impurities, no gravel precipitation, the residue loss is small, the distribution is uniform, the residue samples are clear when observed with the naked eye, and the residue structure is clear and complete when observed by the scanning electron microscope, the field of vision is clean, and the image is three-dimensional It is conducive to the observation and research of the ultrastructure of undigested forage residues in the feces of ruminants after digestion, and is of great significance for intuitively understanding and evaluating the degradation of roughage fiber structure by microorganisms in the digestive tract of ruminants and improving the utilization rate of straw feed.

Its specific technical plan is:

A sample preparation method for a scanning electron microscope, comprising the following steps:

Step 1. Treatment of feces samples: Put 4 to 5 complete sheep feces samples collected with feces collection bags in a mortar, tap and crush them into granules, pay attention not to use too much force during the crushing process, and try to keep the animal The original shape and integrity of the fibrous structure of pasture residues after digestion;

Step 2. Nylon bag wrapping and washing: Wrap the crushed feces samples with 260-mesh nylon cloth prepared in advance, tie them tightly, put them under the tap, soak them with tap water, and then place them in a clean petri dish. Add water to make it completely submerged, and soak for about 10-15 minutes; during this period, use a glass rod to gently shake the nylon cloth wrapped in the feces to continuously dissolve the soluble matter in the feces and filter out the nylon cloth; change it every 2-3 minutes. once water;

Step 3. Wash the petri dish standing still: take out the feces soaked in step 2, gently squeeze out the excess sewage; then unfold the nylon cloth, and use a syringe filled with water to wash the filter residue stuck on the nylon cloth to the petri dish Medium; continue to add water to the petri dish, stir, and continue to dissolve the impurities in the residue; at this time, fine sand, soil and other insoluble particles will gradually settle and the grass-like residue will float on it. Let it stand for 2 to 3 minutes without a needle. Gently suck the grass sample floating on the surface of the petri dish with the syringe, repeat 2-3 times;

Step 4. Rinse with high-speed water flow: Continue to use a syringe without a needle to inhale the pasture residue and water to remove fine sand and soil, then quickly push it back into the culture dish, repeat it several times, and use high-speed water to deeply clean the surface of the dung-like pasture residue ;

Step 5. Change the water: filter the washed pasture residue with nylon cloth, filter out the sewage, and add clean water to the culture dish; rinse the pasture residue stuck to the nylon cloth in the culture dish again with a syringe; repeat steps 4 and Step 5, until the washing water is clear and no longer slightly yellow; use distilled water for the last water change;

Step 6. Water absorption and drying: use filter paper to absorb excess water in the petri dish; place the petri dish containing pasture residue at room temperature for 48 hours to allow it to dry naturally, and ensure that it is completely dry when it is placed on the sample stage, and the residue is concentrated;

Step 7. Sample stage preparation: Paste the conductive double-sided tape on the sample stage, lightly stick the above-mentioned dried residue sample with fingertips, ensure that the residue on the pasted surface is evenly distributed, there is no overlap and damage residue, and stick it firmly on the tape in order superior;

Step 8. Samples are sprayed with gold coating: fresh samples themselves can conduct electricity, but dried biological samples cannot conduct electricity; when such non-conductive samples are observed under SEM, charge accumulation will occur and affect the stability of observation; dry samples can be used The ion spray gold instrument msp-1s needs to spray a layer of gold film on the surface of the sample;

Further, the scanning electron microscope sample prepared after gold spraying was observed with S3400N HITACHI scanning electron microscope, the image mode was SE, the accelerating voltage was 1.00KV-2.00KV, the inclination of the sample stage was 0, the brightness contrast was automatic plus manual, and the focus was manual. The magnification depends on the purpose of observation. The observed residue structure is clean, clear and complete, and has a strong three-dimensional effect.

Compared with prior art, the beneficial effect of the present invention:

The operation steps of the invention are simple and convenient, the cost is low, and the structure of the prepared scanning electron microscope sample is clear, which is conducive to the observation and research of the ultrastructure of the forage residue after digestion, which is useful for qualitatively and quantitatively understanding and evaluating the effect of rumen microorganisms on the roughage fiber structure of ruminants. Degradation and livestock are of great significance to the efficient utilization of crop straw in agricultural areas.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments.

Example 1

A sample preparation method for a scanning electron microscope, comprising the following steps:

Step 1. Treatment of manure samples: Put 4 complete sheep manure samples collected with manure collection bags into a mortar, tap and crush them into granules, pay attention not to use too much force during the crushing process, and try to keep the animals after digestion. The original shape and integrity of the fibrous structure of forage residues;

Step 2. Nylon bag wrapping and washing: Wrap the crushed feces samples with 260-mesh nylon cloth prepared in advance, tie them tightly, put them under the tap, soak them with tap water, and then place them in a clean petri dish. Add water to make it completely submerged, and soak for about 10 minutes; during this period, use a glass rod to gently shake the nylon cloth wrapped in the feces sample, so that the soluble matter in the feces sample is continuously dissolved and filtered out of the nylon cloth; change the water every 2 minutes;

Step 3. Wash the petri dish standing still: take out the feces soaked in step 2, gently squeeze out the excess sewage; then unfold the nylon cloth, and use a syringe filled with water to wash the filter residue stuck on the nylon cloth to the petri dish Continue to add water to the petri dish, stir, and continue to dissolve the impurities in the residue; at this time, fine sand, soil and other insoluble particles will gradually settle and the grass-like residue will float on it. Suction the grass sample floating on the surface of the petri dish, repeat 2 times;

Step 4. Rinse with high-speed water flow: Continue to use a syringe without a needle to inhale the pasture residue and water to remove fine sand and soil, then quickly push it back into the culture dish, repeat it several times, and use high-speed water to deeply clean the surface of the dung-like pasture residue ;

Step 5. Change the water: filter the washed pasture residue with nylon cloth, filter out the sewage, and add clean water to the culture dish; rinse the pasture residue culture dish stuck to the nylon cloth with a syringe again; repeat steps 4 and 1 for several times 5. Until the washing water is clear and no longer slightly yellow; use distilled water when changing the water for the last time;

Step 6. Water absorption and drying: use filter paper to absorb excess water in the petri dish; place the petri dish containing pasture residue at room temperature for 48 hours to allow it to dry naturally, and ensure that it is completely dry when it is placed on the sample stage, and the residue is concentrated;

Step 7. Sample stage preparation: Paste the conductive double-sided tape on the sample stage, lightly stick the above-mentioned dried residue sample with fingertips, ensure that the residue on the pasted surface is evenly distributed, there is no overlap and damage residue, and stick it firmly on the tape in order superior;

Step 8. Samples are sprayed with gold coating: fresh samples themselves can conduct electricity, but dried biological samples cannot conduct electricity; when such non-conductive samples are observed under SEM, charge accumulation will occur and affect the stability of observation; dry samples can be used The ion spray gold instrument msp-1s needs to spray a layer of gold film on the surface of the sample;

Observe the scanning electron microscope sample prepared after gold spraying with S3400N HITACHI scanning electron microscope, the image mode is SE, the accelerating voltage is 1.00KV-2.00KV, the inclination of the sample stage is 0, the brightness contrast is automatic plus manual, focus manual, magnification Depends on what you want to observe. The observed residue structure is clean, clear and complete, and has a strong three-dimensional effect.

Example 2

A sample preparation method for a scanning electron microscope, comprising the following steps:

Step 1. Feces sample treatment: Put 5 complete sheep feces samples collected with feces collection bags into a mortar, tap and crush them into granules, pay attention not to use too much force during the crushing process, and try to keep the animals after digestion. The original shape and integrity of the fibrous structure of forage residues;

Step 2. Nylon bag wrapping and washing: Wrap the crushed feces samples with 260-mesh nylon cloth prepared in advance, tie them tightly, put them under the tap, soak them with tap water, and then place them in a clean petri dish. Add water to make it completely submerged, and soak for about 15 minutes; during this period, use a glass rod to gently shake the nylon cloth wrapped in the feces sample, so that the soluble matter in the feces sample is continuously dissolved and filtered out of the nylon cloth; change the water every 3 minutes;

Step 3. Wash the petri dish standing still: take out the feces soaked in step 2, gently squeeze out the excess sewage; then unfold the nylon cloth, and use a syringe filled with water to wash the filter residue stuck on the nylon cloth to the petri dish Medium; continue to add water to the culture dish, stir, and continue to dissolve impurities in the residue; at this time, fine sand, soil and other insoluble particles will gradually settle and the grass-like residue will float on it. Suction the grass sample floating on the surface of the petri dish, repeat 3 times;

Step 4. Rinse with high-speed water flow: Continue to use a syringe without a needle to inhale the pasture residue and water to remove fine sand and soil, then quickly push it back into the culture dish, repeat it several times, and use high-speed water to deeply clean the surface of the dung-like pasture residue ;

Step 5. Change the water: filter the washed pasture residue with nylon cloth, filter out the sewage, and add clean water to the culture dish; rinse the pasture residue culture dish stuck to the nylon cloth with a syringe again; repeat steps 4 and 1 for several times 5. Until the washing water is clear and no longer slightly yellow; use distilled water when changing the water for the last time;

Step 6. Water absorption and drying: use filter paper to absorb excess water in the petri dish; place the petri dish containing pasture residue at room temperature for 48 hours to allow it to dry naturally, and ensure that it is completely dry when it is placed on the sample stage, and the residue is concentrated;

Step 7. Sample stage preparation: Paste the conductive double-sided tape on the sample stage, lightly stick the above-mentioned dried residue sample with fingertips, ensure that the residue on the pasted surface is evenly distributed, there is no overlap and damage residue, and stick it firmly on the tape in order superior;

Step 8. Samples are sprayed with gold coating: fresh samples themselves can conduct electricity, but dried biological samples cannot conduct electricity; when such non-conductive samples are observed under SEM, charge accumulation will occur and affect the stability of observation; dry samples can be used The ion spray gold instrument msp-1s needs to spray a layer of gold film on the surface of the sample;

Observe the scanning electron microscope sample prepared after gold spraying with S3400N HITACHI scanning electron microscope, the image mode is SE, the accelerating voltage is 1.00KV-2.00KV, the inclination of the sample stage is 0, the brightness contrast is automatic plus manual, focus manual, magnification Depending on the purpose of your observation, you can see at least 50 times, or even smaller. The observed residue structure is clean, clear and complete, and has a strong three-dimensional effect.

The above is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed in the present invention can obviously obtain the simplicity of the technical solution. Changes or equivalent replacements all fall within the protection scope of the present invention.

Claims (2)

1. a sample preparation method for scanning electron microscope, is characterized in that, comprises the following steps: Step 1. Treatment of feces samples: Put 4 to 5 complete sheep feces samples collected with feces collection bags in a mortar, tap and crush them into granules, pay attention not to use too much force during the crushing process, and try to keep the animal The original shape and integrity of the fibrous structure of pasture residues after digestion; Step 2. Nylon bag wrapping and washing: Wrap the crushed feces samples with 260-mesh nylon cloth prepared in advance, tie them tightly, put them under the tap, soak them with tap water, and then place them in a clean petri dish. Add water to make it completely submerged, and soak for 10-15 minutes; during this period, use a glass rod to gently shake the nylon cloth wrapped in the feces to continuously dissolve the soluble matter in the feces and filter out the nylon cloth; change it every 2-3 minutes water; Step 3. Wash the petri dish standing still: take out the feces soaked in step 2, gently squeeze out the excess sewage; then unfold the nylon cloth, and use a syringe filled with water to wash the filter residue stuck on the nylon cloth to the petri dish Medium; continue to add water to the petri dish, stir, and continue to dissolve impurities in the residue; at this time, the fine sand and insoluble particles in the soil will gradually precipitate and the grass-like residue will float on it, let it stand for 2 to 3 minutes, and use a syringe without a needle Gently suck the grass sample floating on the surface of the petri dish, repeat 2-3 times; Step 4. Rinse with high-speed water flow: Continue to use a syringe without a needle to inhale the pasture residue and water to remove fine sand and soil, then quickly push it back into the culture dish, repeat it several times, and use high-speed water to deeply clean the surface of the dung-like pasture residue ; Step 5. Change the water: filter the washed pasture residue with nylon cloth, filter out the sewage, and add clean water to the culture dish; rinse the pasture residue stuck to the nylon cloth into the culture dish again with a syringe; repeat steps 4 and Step 5, until the washing water is clear and no longer slightly yellow; use distilled water for the last water change; Step 6. Water absorption and drying: use filter paper to absorb excess water in the petri dish; place the petri dish containing pasture residue at room temperature for 48 hours to allow it to dry naturally, and ensure that it is completely dry when it is placed on the sample stage, and the residue is concentrated; Step 7. Sample stage preparation: Paste the conductive double-sided tape on the sample stage, lightly stick the above-mentioned dried residue sample with fingertips, ensure that the residue on the pasted surface is evenly distributed, there is no overlap and damage residue, and stick it firmly on the tape in order superior; Step 8. Samples are sprayed with gold coating: fresh samples themselves can conduct electricity, but dried biological samples cannot conduct electricity; when such non-conductive samples are observed under SEM, charge accumulation will occur and affect the stability of observation; dry samples can be used The ion spray gold instrument msp-1s needs to spray a layer of gold film on the surface of the sample. 2. the sample preparation method of a kind of scanning electron microscope according to claim 1, it is characterized in that, the scanning electron microscope sample that makes after spraying gold is observed with S3400N HITACHI scanning electron microscope, image mode is SE, accelerating voltage 1.00KV- 2.00KV, the inclination of the sample stage is 0, the brightness contrast is automatic plus manual, and the focus is manual. The magnification depends on the purpose of observation, and the minimum can be seen at 50 times.