CN106018027B - A kind of sample preparation methods of scanning electron microscope - Google Patents
A kind of sample preparation methods of scanning electron microscope Download PDFInfo
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- CN106018027B CN106018027B CN201610365082.9A CN201610365082A CN106018027B CN 106018027 B CN106018027 B CN 106018027B CN 201610365082 A CN201610365082 A CN 201610365082A CN 106018027 B CN106018027 B CN 106018027B
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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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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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
- G01N1/34—Purifying; Cleaning
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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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2866—Grinding or homogeneising
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Abstract
The invention discloses a kind of sample preparation methods of scanning electron microscope, this method step includes the following steps：The processing of excrement sample；Nylon Bag package washing；Culture dish stands washing；High-velocity flow rinses；Change water；Absorbent drying；Sample stage prepares；Sample metal spraying plated film.Pass through drying, grind broken, it impregnates, Nylon Bag package washing, high-velocity flow rinses, water suction, it is dry, the techniques such as gold-plated, the clean free from admixture of the scanning electron microscope example of preparation, no gravel precipitating, residue loss is small, it is evenly distributed, it is clear to visually observe residue sample, residue is clear in structure complete when scanning electron microscopic observation, the visual field is clean, image three-dimensional sense is strong, be conducive to carry out the observation and research for not digesting herbage residue ultra microstructure after ruminant digestion in excrement, it is significant to the degradation of roughage fibre structure and raising straw feed utilization rate for intuitively understanding and evaluating ruminant digestion road microorganism.
Description
Technical field
The invention belongs to microexamination sample technical fields, are related to a kind of sample preparation methods of scanning electron microscope, specifically
Say, be related to it is a kind of after ruminant digestion in excrement herbage residue scanning electron microscope (SEM) observation sample preparation method.
Background technique
In recent years, with the fast development of economic society and the continuous improvement of living standards of the people, China's animal husbandry is obtained
Quick development is arrived.Therefore, the quantity of Forage Germplasm Resources and quality requirements are continuously improved, related research is just in mushroom development.
Under this big development trend, the raising of forage grass utilization rate just constantly becomes research hotspot, various promotion herbage digestibilities
Technology emerges one after another.The result of study of herbage digestible degree is only embodied with quantitative index and is mutually compared mostly at present
Compared with, such as digestibility, rumen digestibility, cud disappearance rate etc., general lack of can intuitively reflect herbage by ruminant digestion road
Fibre structure and its cell wall variation intuitive image data.Disappear to more intuitively disclose herbage fiber through ruminant
Change after road digests by the degree of animal use, also improves the technology of herbage digestibility for research and development and product provides more exact figure
As data, the invention proposes a kind of, and the scanning electron microscope (SEM) of herbage residue observes sample in excrement after ruminant digestion
Preparation method.The available above-mentioned image document of the method illustrated through the invention.
Summary of the invention
It is an object of the invention to overcome not digesting herbage residue in ruminant excrement existing for above-mentioned technology and be difficult to point
From the defect with resolution, a kind of sample preparation methods of scanning electron microscope are provided, by drying, stone roller is broken, impregnate, Nylon Bag package is washed
It washs, high-velocity flow flushing, water suction, dry, the techniques such as gold-plated, the clean free from admixture of the scanning electron microscope example of preparation, no gravel precipitating,
Residue loss is small, is evenly distributed, and it is clear to visually observe residue sample, and residue is clear in structure complete when scanning electron microscopic observation, the visual field
Completely, image three-dimensional sense is strong, is conducive to carry out the observation for not digesting herbage residue ultra microstructure after ruminant digestion in excrement
And research, for intuitively understanding and evaluating ruminant digestion road microorganism to the degradation of roughage fibre structure and improve stalk
Efficiency of feed utilization is significant.
Its specific technical solution is：
A kind of sample preparation methods of scanning electron microscope, include the following steps：
Step 1, the processing of excrement sample：By using collection colostomy bag collect come 4~5 complete sheep dung samples be placed in mortar, tapping
Stone roller is broken into granular, and paying attention to should not be firmly excessive during grinding broken, the original of herbage residue fibre structure as far as possible after holding animal digestion
Shape and integrality；
Step 2, Nylon Bag package washing：The excrement sample for grinding broken is wrapped up with 260 mesh nylon cloth of preprepared, is pricked
Tightly, it is placed under tap, with originally water-soaked, then places it in clean culture dish, water is added to be allowed to flood completely, soak
Bubble about 10~15 minutes；Period constantly gently stirs the nylon cloth of package excrement sample with glass bar, makes soluble matters in excrement sample not
Disconnected dissolution filters out nylon cloth；Change a water within every 2~3 minutes；
Step 3, culture dish stand washing：Excrement sample soaked in step 2 is taken out, extra sewage is gently squeezed out；Then
Nylon cloth is unfolded, rinses the filter residue being sticked on nylon cloth into culture dish with the syringe water flow for filling water；Continue to culture dish
In plus water, agitation, continue dissolved residue in impurity；Fine sand at this time, the refractory particulate matters such as soil can be precipitated gradually and careless sample residue
It then floats on above, stands 2~3 minutes, with the careless sample for floating on culture dish surface is gently inhaled without needle applicator, repeat 2~3
It is secondary；
Step 4, high-velocity flow rinse：Continue with sucked without needle applicator the herbage residue for removing fine sand and soil and
Then it is pushed into culture dish rapidly by water again, repeated multiple times, cleans excrement sample herbage residue table using high-velocity flow depth
Face；
Step 5 changes water：The herbage residue of washing is filtered with nylon cloth, filters off sewage, culture dish rejoins completely
Water；Again the herbage residue on nylon cloth is sticked in culture dish with irrigation with syringe；Step 4 and step 5 is repeated several times, until
Washing clarification of water no longer have it is yellowish until；Last time uses distilled water when changing water；
Step 6, absorbent drying：Excessive moisture in culture dish is blotted with filter paper；Fill the culture dish of herbage residue at room temperature
It places 48 hours, it is made to dry out naturally, and guarantee to be completely dried when upper sample stage, concentrate residue；
Step 7, sample stage prepare：Conductive double-sided tape is pasted on sample stage, be gently stained with finger tip state it is dried
Residue sample, guarantee are pasted face residue and are uniformly distributed, and residue is not overlapped and damages, and patch jail is on adhesive tape in order；
Step 8, sample metal spraying plated film：Fresh sample itself is with regard to electrically conductive, and passing through dry biological sample cannot be conductive；
When this nonconducting sample is observed in the secure execution mode (sem, charge accumulated can be generated and influence to observe stability；Dry sample ion
Gold spraying instrument msp-1s need to spray one layer of golden film in sample surfaces；
Further, scanning electron microscope example obtained after metal spraying is observed with S3400N HITACHI scanning electron microscope, is schemed
Picture mode is SE, and acceleration voltage 1.00KV-2.00KV, sample stage gradient is 0, and luminance contrast is to add automatically manually, is focused
Manually, amplification factor be depending on it oneself will observe purpose,.The residue structure observed is clean, complete display, and three-dimensional sense is strong.
Compared with prior art, beneficial effects of the present invention：
Operation of the present invention step is simple and convenient, at low cost, and the scanning electron microscope example of preparation is clear in structure, is conducive to development and disappears
The observation and research of herbage residue ultra microstructure after change, this is for qualitative and quantitative understanding and evaluation ruminant tumor gastric microorganism
Degradation and domestic animal to roughage fibre structure efficiently utilize agriculture district crop material significant.
Specific embodiment
Technical solution of the present invention is described in more detail below with reference to specific embodiment.
Embodiment 1
A kind of sample preparation methods of scanning electron microscope, include the following steps：
Step 1, the processing of excrement sample：By using collection colostomy bag collect come 4 complete sheep dung samples be placed in mortar, tapping stone roller break
Granulate, paying attention to should not be firmly excessive during grinding broken, as far as possible after holding animal digestion the original state of herbage residue fibre structure and
Integrality；
Step 2, Nylon Bag package washing：The excrement sample for grinding broken is wrapped up with 260 mesh nylon cloth of preprepared, is pricked
Tightly, it is placed under tap, with originally water-soaked, then places it in clean culture dish, water is added to be allowed to flood completely, soak
Bubble about 10 minutes；Period constantly gently stirs the nylon cloth of package excrement sample with glass bar, keeps the soluble matters in excrement sample constantly molten
Solution filters out nylon cloth；Change a water within every 2 minutes；
Step 3, culture dish stand washing：Excrement sample soaked in step 2 is taken out, extra sewage is gently squeezed out；Then
Nylon cloth is unfolded, rinses the filter residue being sticked on nylon cloth into culture dish with the syringe water flow for filling water；Continue to culture dish
In plus water, agitation, continue dissolved residue in impurity；Fine sand at this time, the refractory particulate matters such as soil can be precipitated gradually and careless sample residue
It then floats on above, stands 2 minutes, with the careless sample for floating on culture dish surface is gently inhaled without needle applicator, be repeated 2 times；
Step 4, high-velocity flow rinse：Continue with sucked without needle applicator the herbage residue for removing fine sand and soil and
Then it is pushed into culture dish rapidly by water again, repeated multiple times, cleans excrement sample herbage residue table using high-velocity flow depth
Face；
Step 5 changes water：The herbage residue of washing is filtered with nylon cloth, filters off sewage, culture dish rejoins completely
Water；Again it is sticked in the herbage residue culture dish on nylon cloth with irrigation with syringe；Step 4 and step 5 is repeated several times, until washing
Wash clarification of water no longer have it is yellowish until；Last time uses distilled water when changing water；
Step 6, absorbent drying：Excessive moisture in culture dish is blotted with filter paper；Fill the culture dish of herbage residue at room temperature
It places 48 hours, it is made to dry out naturally, and guarantee to be completely dried when upper sample stage, concentrate residue；
Step 7, sample stage prepare：Conductive double-sided tape is pasted on sample stage, be gently stained with finger tip state it is dried
Residue sample, guarantee are pasted face residue and are uniformly distributed, and residue is not overlapped and damages, and patch jail is on adhesive tape in order；
Step 8, sample metal spraying plated film：Fresh sample itself is with regard to electrically conductive, and passing through dry biological sample cannot be conductive；
When this nonconducting sample is observed in the secure execution mode (sem, charge accumulated can be generated and influence to observe stability；Dry sample ion
Gold spraying instrument msp-1s need to spray one layer of golden film in sample surfaces；
Scanning electron microscope example obtained after metal spraying is observed with S3400N HITACHI scanning electron microscope, image model is
SE, acceleration voltage 1.00KV-2.00KV, sample stage gradient are 0, and luminance contrast is to add automatically manually, focus manually, amplify
Multiple is depending on it oneself will observe purpose.The residue structure observed is clean, complete display, and three-dimensional sense is strong.
Embodiment 2
A kind of sample preparation methods of scanning electron microscope, include the following steps：
Step 1, the processing of excrement sample：By using collection colostomy bag collect come 5 complete sheep dung samples be placed in mortar, tapping stone roller break
Granulate, paying attention to should not be firmly excessive during grinding broken, as far as possible after holding animal digestion the original state of herbage residue fibre structure and
Integrality；
Step 2, Nylon Bag package washing：The excrement sample for grinding broken is wrapped up with 260 mesh nylon cloth of preprepared, is pricked
Tightly, it is placed under tap, with originally water-soaked, then places it in clean culture dish, water is added to be allowed to flood completely, soak
Bubble about 15 minutes；Period constantly gently stirs the nylon cloth of package excrement sample with glass bar, keeps the soluble matters in excrement sample constantly molten
Solution filters out nylon cloth；Change a water within every 3 minutes；
Step 3, culture dish stand washing：Excrement sample soaked in step 2 is taken out, extra sewage is gently squeezed out；Then
Nylon cloth is unfolded, rinses the filter residue being sticked on nylon cloth into culture dish with the syringe water flow for filling water；Continue to culture dish
In plus water, agitation, continue dissolved residue in impurity；Fine sand at this time, the refractory particulate matters such as soil can be precipitated gradually and careless sample residue
It then floats on above, stands 3 minutes, with the careless sample for floating on culture dish surface is gently inhaled without needle applicator, be repeated 3 times；
Step 4, high-velocity flow rinse：Continue with sucked without needle applicator the herbage residue for removing fine sand and soil and
Then it is pushed into culture dish rapidly by water again, repeated multiple times, cleans excrement sample herbage residue table using high-velocity flow depth
Face；
Step 5 changes water：The herbage residue of washing is filtered with nylon cloth, filters off sewage, culture dish rejoins completely
Water；Again it is sticked in the herbage residue culture dish on nylon cloth with irrigation with syringe；Step 4 and step 5 is repeated several times, until washing
Wash clarification of water no longer have it is yellowish until；Last time uses distilled water when changing water；
Step 6, absorbent drying：Excessive moisture in culture dish is blotted with filter paper；Fill the culture dish of herbage residue at room temperature
It places 48 hours, it is made to dry out naturally, and guarantee to be completely dried when upper sample stage, concentrate residue；
Step 7, sample stage prepare：Conductive double-sided tape is pasted on sample stage, be gently stained with finger tip state it is dried
Residue sample, guarantee are pasted face residue and are uniformly distributed, and residue is not overlapped and damages, and patch jail is on adhesive tape in order；
Step 8, sample metal spraying plated film：Fresh sample itself is with regard to electrically conductive, and passing through dry biological sample cannot be conductive；
When this nonconducting sample is observed in the secure execution mode (sem, charge accumulated can be generated and influence to observe stability；Dry sample ion
Gold spraying instrument msp-1s need to spray one layer of golden film in sample surfaces；
Scanning electron microscope example obtained after metal spraying is observed with S3400N HITACHI scanning electron microscope, image model is
SE, acceleration voltage 1.00KV-2.00KV, sample stage gradient are 0, and luminance contrast is to add automatically manually, focus manually, amplify
For multiple depending on oneself observing purpose, minimum can see 50 times, even more small.The residue structure observed is clean, clear complete
Whole, three-dimensional sense is strong.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Claims (2)
1. a kind of sample preparation methods of scanning electron microscope, which is characterized in that include the following steps：
Step 1, the processing of excrement sample：By using collection colostomy bag collect come 4~5 complete sheep dung samples be placed in mortar, tapping stone roller break
Granulate, paying attention to should not be firmly excessive during grinding broken, as far as possible after holding animal digestion the original state of herbage residue fibre structure and
Integrality；
Step 2, Nylon Bag package washing：The excrement sample for grinding broken is wrapped up with 260 mesh nylon cloth of preprepared, is tightened,
It is placed under tap, with originally water-soaked, then places it in clean culture dish, water is added to be allowed to flood completely, impregnate
10~15 minutes；Period constantly gently stirs the nylon cloth of package excrement sample with glass bar, keeps the soluble matters in excrement sample constantly molten
Solution filters out nylon cloth；Change a water within every 2~3 minutes；
Step 3, culture dish stand washing：Excrement sample soaked in step 2 is taken out, extra sewage is gently squeezed out；Then it is unfolded
Nylon cloth rinses the filter residue being sticked on nylon cloth into culture dish with the syringe water flow for filling water；Continue to add in culture dish
Water, agitation continue impurity in dissolved residue；Fine sand at this time, soil refractory particulate matter can be precipitated gradually and careless sample residue then floats
In above, 2~3 minutes are stood, with the careless sample for floating on culture dish surface is gently inhaled without needle applicator, is repeated 2~3 times；
Step 4, high-velocity flow rinse：Continue with without needle applicator suck remove fine sand and soil herbage residue and water,
Then it is pushed into culture dish again rapidly, it is repeated multiple times, excrement sample herbage residue surface is cleaned using high-velocity flow depth；
Step 5 changes water：The herbage residue of washing is filtered with nylon cloth, filters off sewage, culture dish rejoins clean water；Weight
Newly the herbage residue on nylon cloth is sticked into culture dish with irrigation with syringe；Step 4 and step 5 is repeated several times, until washing
Clarification of water no longer have it is yellowish until；Last time uses distilled water when changing water；
Step 6, absorbent drying：Excessive moisture in culture dish is blotted with filter paper；The culture dish for filling herbage residue is placed at room temperature
48 hours, it is made to dry out naturally, and guarantee to be completely dried when upper sample stage, concentrates residue；
Step 7, sample stage prepare：Conductive double-sided tape is pasted on sample stage, is gently stained with finger tip and states dried residue
Sample, guarantee are pasted face residue and are uniformly distributed, and residue is not overlapped and damages, and patch jail is on adhesive tape in order；
Step 8, sample metal spraying plated film：Fresh sample itself is with regard to electrically conductive, and passing through dry biological sample cannot be conductive；It is this
When nonconducting sample is observed in the secure execution mode (sem, charge accumulated can be generated and influence to observe stability；Dry sample ion metal spraying
Instrument msp-1s need to spray one layer of golden film in sample surfaces.
2. a kind of sample preparation methods of scanning electron microscope according to claim 1, which is characterized in that obtained after metal spraying
Scanning electron microscope example is observed with S3400N HITACHI scanning electron microscope, image model SE, acceleration voltage 1.00KV-
2.00KV, sample stage gradient are 0, and luminance contrast is to add automatically manually, are focused manually, amplification factor will be observed according to oneself
Depending on purpose, minimum can see 50 times.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1161060A (en) * | 1994-09-12 | 1997-10-01 | M·G·伯杰龙 | Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis Lab. |
| CN105053595A (en) * | 2015-07-15 | 2015-11-18 | 甘肃农业大学 | Emergency and supplementary feeding complete compound pellet feed using maize straws for shepherding sheep and preparation method thereof |
-
2016
- 2016-05-27 CN CN201610365082.9A patent/CN106018027B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1161060A (en) * | 1994-09-12 | 1997-10-01 | M·G·伯杰龙 | Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis Lab. |
| CN105053595A (en) * | 2015-07-15 | 2015-11-18 | 甘肃农业大学 | Emergency and supplementary feeding complete compound pellet feed using maize straws for shepherding sheep and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 南方丘陵地区人工草地和旱作农田能量物质利用率;欧阳克蕙等;《草地学报》;20070131;第15卷(第1期);第35-39页 * |
| 根管内粪肠球菌生物膜的形成研究;张静兰等;《白求恩军医学院学报》;20131231;第513-514页 * |
| 陕北白绒山羊常用饲料原料小肠消化率研究;李占臻;《中国优秀硕士学位论文全文数据库 农业科技辑 》;20140515;第D050-100页 * |
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