CN104075922A - Epoxy resin embedding method for preparing parasite sample for scanning electron microscope - Google Patents
Epoxy resin embedding method for preparing parasite sample for scanning electron microscope Download PDFInfo
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- CN104075922A CN104075922A CN201410316245.5A CN201410316245A CN104075922A CN 104075922 A CN104075922 A CN 104075922A CN 201410316245 A CN201410316245 A CN 201410316245A CN 104075922 A CN104075922 A CN 104075922A
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Abstract
The invention discloses an epoxy resin embedding method for preparing a parasite sample for a scanning electron microscope. The method specifically comprises the following steps: preliminarily treating the sample, fixing the sample with glutaraldehyde and osmic acid, washing the sample with PBS (phosphate buffered saline), performing gradient dehydration with acetone, dipping in an epoxy resin embedding medium, displacing, embedding, washing with acetone, polymerizing and coating a film on the sample, so as to complete preparation of the parasite sample for the scanning electron microscope. The method has the advantages that (1) the treatment of the parasite sample before fixing is favorable for keeping the normal morphology of a parasite, so that sample loss is reduced; and (2) 1% of osmic acid is used for fixing for the second time, so as to blacken the parasite, so that the fixing effect for the second time can be achieved, additionally, the parasite is fixed to be black, so as to be easily observed by naked eyes, and subsequent operation is facilitated. According to the method, a traditional drying step is replaced by gradient dipping, displacement and embedding in the epoxy resin embedding medium, because the rigidity of epoxy resin is large after polymerization, the integral structure of the parasite is stably supported inside, the parasite sample can be stored for a long time, and original morphological characteristics can be vividly displayed.
Description
Technical field
The invention belongs to parasite electron microscopic sample preparing technical field, particularly a kind of epoxy resin embedding method of preparing scanning electron microscope use parasite sample.
Background technology
Parasite is large numbers of bodies, parasitize in host's cell, tissue or cavity, cause a series of damage, and cause various diseases, can there is because of the impact of parasitic environment form structure and change in parasite, so morphological observation is the parasitic basis of research and focus.In early days, the traditional form methods such as applied optics microscope are identified parasite, along with scientific and technological progress and development, it is comparatively advanced and conventional means that sem observation has become the parasitic morphosis of research, the fine structure at each position is had to the very high sharpness of distinguishing, than carrying out Species identification more accurately and reliably with optical microscope." the simple and easy preparation method of parasite scanning electron microscopic observation sample " of Agricultural University Of Shenyang's journal publication in 1988, provide a kind of parasite scanning electron microscopic observation sample simple and easy preparation method, the method is applied in the deficiency existing in parasite scanning electron microscope to be had: 1) 70% ethanol is fixed, fixed effect is poor, and the fixing rear storage time of sample collection is short, can not preserve the long period; 2) not with filter paper parcel, easily loss; 3) direct 70% fixing dehydration, does not have gradient slowly to dewater, and the easy dehydration of parasite is out of shape too soon; 4) in 100 % ethanol, only have 10 minutes, can not ensure abundant dehydration, affect later stage displacement; 5) CO
2do critical point drying, equipment, isoamyl acetate purity requirement are high, parasite surface or can produce damage fold in various degree in dry run, and effect is not remarkable especially, easy slight deformation, and success ratio is not high; 6) vacuum plated aluminum film, electric conductivity is general.Because parasite is more, be that software is biological, form is less, is easily out of shape and loses in sample preparation process.In order to adapt to the requirement of parasite scanning electron microscope, better appear configuration of surface structure, in sample making course, must carry out special processing, could prepare the parasite scanning electron microscopic observation sample that keeps high integrity and good Morphologic Characteristics.The method of current this processing is perfect not enough, therefore needs further research.
Summary of the invention
The object of the invention is to provide in order to overcome above the deficiencies in the prior art a kind of epoxy resin embedding method of parasite sample for scanning electron microscope of preparing, we have explored a kind of epoxy resin embedding legal system of using for the method for parasite sample for this reason, we prepared a large amount of parasite samples to utilize the method, it is not yielding that result proof the method is prepared parasite, form is true to nature, success ratio is higher, and can longlyer naturally preserve.
The present invention realizes by following technological means:
Prepare an epoxy resin embedding method for parasite sample for scanning electron microscope, comprise the following steps:
Step 1, sample is processed in earlier stage: after parasite live body is killed rapidly, be wrapped in qualitative filter paper;
Step 2, sample is fixed: sample is placed in to glutaraldehyde and fixes 2～4 hours, then use PBS damping fluid that sample surfaces is cleaned up, remove glutaraldehyde, then fix with osmic acid, parasite is smoked into black;
Step 3, sample cleans and gradient dehydration: use PBS damping fluid that sample surfaces is cleaned up, remove osmic acid, then with the acetone that volumetric concentration is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%, 100%, do gradient dehydration;
Step 4, displacement: flood embedding in the mixed liquor of employing epoxy resin embedding agent and acetone after parasite dehydration;
Step 5, clean: the parasite after epoxy resin embedding agent dipping is chosen and is put on the screen cloth that mesh is less than parasitic polypide, polyase 13～5 hour under 70 ℃ of conditions, after naturally cooling to room temperature, with acetone, clean, until nematode surface becomes tarnish, then polymerization 12～24 hours under 70 ℃ of conditions;
Step 6, sample coating: by the good ion sputtering instrument metal spraying 90 ~ 120s for sample stage of polymerization, evenly plate the golden film of one deck conduction at parasite sample surfaces, complete the preparation of parasite scanning electron microscope example.
The epoxy resin embedding method of parasite sample for described preparation scanning electron microscope, in step 1, live body parasite can kill rapidly under condition in 60 ℃, 3 ~ 5 minutes in test tube.
The epoxy resin embedding method of parasite sample for described preparation scanning electron microscope, in step 2, the mass concentration of glutaraldehyde can be 2%.
The epoxy resin embedding method of parasite sample for described preparation scanning electron microscope, in step 2, the mass concentration of osmic acid can be 1%.
The epoxy resin embedding method of parasite sample for described preparation scanning electron microscope, in step 4, replacing embedding can be to flood 2 hours in the epoxy resin embedding agent of 1:3 and the mixed liquor of acetone for proceeding to volume ratio after parasite is fully dewatered, then proceeding to volume ratio is to flood 4 hours in the epoxy resin embedding agent of 1:1 and the mixed liquor of acetone, proceeding to volume ratio is to flood 6 hours in the epoxy resin embedding agent of 3:1 and the mixed liquor of acetone again, finally proceeds in pure epoxy resin embedding medium and floods 8 ~ 12 hours.
The epoxy resin embedding method of parasite sample for above-described preparation scanning electron microscope, wherein said parasite is: sample is relatively little, form is variable, the Platyhelminthes or the protozoa that in body, without vertebral bones, support.
The present invention is to the fixedly pre-treatment of parasite sample, principle: 1) parasite vitality is indomitable, and live body is directly fixing in 2.5% glutaraldehyde, because struggling, roll up, parasite morphosis is had to considerable influence, and high temperature kills rapidly parasite, will be easy to keep parasite normal morphology; 2) parasite is little, with qualitative filter paper parcel, is easy to reduce the loss because of sample in operating process.Epoxy resin embedding agent dipping displacement embedding principle: epoxy resin embedding agent be easy to body in acetone displacement, be the conventional embedding medium of transmission electron microscope, hardness is high, soaks into easily.
The invention has the advantages that: 1) parasite sample fixedly pre-treatment be easy to keep parasite normal morphology, reduce the loss of sample; 2) again fixing with 1% osmic acid, parasite is smoked into black, bring into play again fixing effect, parasite is fixed into the black of easy observation simultaneously, be convenient to follow-up operation; 3) in the conventional method for making of parasite Sample Scan Electronic Speculum, need CO
2critical-point drying method or lyophilization, two kinds of seasonings all need special drying equipment, desiccant CO
2, the purity requirement of uncle-butanols is high, otherwise sample drying is insufficient, in dry run, parasite is prone to damage fold in various degree simultaneously, dried sample needs to observe in time, can not long-term outdoor preservation.Epoxy resin embedding agent gradient dipping displacement embedding substitutes drying steps, can overcome above shortcoming, can preserve for a long time, and displaying original form feature that can be more true to nature.
The present invention is applicable to the processing of any parasite sample, principle: parasite comprises Platyhelminthes (be the most original acoelous Triploblastica, comprise most of parasite); (be the most original, the simplest, minimum etc. protistan, protozoic change of shape is very large for protozoa.There is arbitrarily mobile, the indefinite amoeba of shape of protoplasm, have delicate structure just like carving radiolitid and the foram of the handicraft of decorative pattern); Common feature is that sample is relatively little, and form is variable, in body, without vertebral bones, supports, and conventional sample preparation dehydrates and easily causes Folding Deformation, can not perfect displaying configuration true to nature.
Technical characterstic of the present invention is method innovation, simple and easy to operate, and usable range is wide, and parasite morphosis is complete, indeformable, and success ratio is higher, and technical costs is low, can be applicable to the preparation of parasite scanning electron microscope example.
Accompanying drawing explanation
Fig. 1 is that in embodiment, gained parasitic nematode scanning electron microscope example amplifies the head scanning Electronic Speculum figure of 2300 times;
Fig. 2 is that in embodiment, gained parasitic nematode scanning electron microscope example amplifies the middle part scanning electron microscope (SEM) photograph of 2000 times;
Fig. 3 is that in embodiment, gained parasitic nematode scanning electron microscope example amplifies the afterbody scanning electron microscope (SEM) photograph of 3000 times;
Fig. 4 is that the conventional vacuum freeze-drying method gained of scanning electron microscope microscope parasitic nematode amplifies the head scanning Electronic Speculum figure of 5000 times;
Fig. 5 is that the conventional vacuum freeze-drying method gained of scanning electron microscope microscope parasitic nematode amplifies the afterbody scanning electron microscope (SEM) photograph of 1600 times.
embodiment:
Embodiment 1
A parasite scanning electron microscope example epoxy resin embedding preparation method, realizes according to the following steps:
Step 1, sample is processed in earlier stage: live body Li Shi long-tail nematode kills rapidly in test tube in 60 ℃, 5 minutes, and parasite is wrapped in qualitative filter paper;
Step 2, sample is fixed: is placed in 2% glutaraldehyde and fixes 4 hours, PBS buffer solution for cleaning three times, each 15 minutes, then fix 2 hours with 1% osmic acid, parasite is smoked into black;
Step 3, sample cleans and gradient dehydration: PBS buffer solution for cleaning three times, each 15 minutes, with 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%, 100% acetone, do gradient dehydration, each 20 minutes;
Step 4, displacement: parasitic nematode proceeds to volume ratio after fully dewatering be to flood 2 hours in the epoxy resin embedding agent of 1:3 and the mixed liquor of acetone, proceeding to volume ratio is to flood 4 hours in the epoxy resin embedding agent of 1:1 and the mixed liquor of acetone, proceeding to volume ratio is to flood 6 hours in the epoxy resin embedding agent of 3:1 and the mixed liquor of acetone again, finally proceeds in pure embedding medium and floods 8 hours.
Step 5, cleans: the parasitic nematode after pure embedding medium dipping is picked out to be put into 300 order fine sieves online, under 70 ℃ of conditions, polymerization is 4 hours, and cooling clean dropwise with acetone afterwards, until nematode surface becomes tarnish.Under 70 ℃ of conditions, complete polymerization is 20 hours;
Step 6, sample coating: by the dried ion sputtering instrument metal spraying 100s for sample stage of polymerization, the golden film that evenly plates one deck conduction at parasitic nematode sample surfaces, completing gold-plated parasitic nematode sample can observe under scanning electron microscope, completes the preparation of parasitic nematode scanning electron microscope example.
The pattern of the parasitic nematode scanning electron microscopic observation of making by the inventive method is as shown in Fig. 1,2,3.The whole microscopic morphology that can observe intuitively parasitic nematode from 3 figure, under scanning electron microscope, parasitic nematode is indeformable, and form complete, the cuticula of nematode true to nature, ring grain, head, afterbody are high-visible.And Fig. 4 and Fig. 5 are for adopting conventional vacuum freeze drying
methodthe Electronic Speculum figure of the live body Li Shi long-tail nematode that method was processed, can find out, although parasite complete form, the cuticula of nematode, ring grain, head, afterbody are all unintelligible.
The present embodiment is prepared gained parasitic nematode scanning electron microscope example, and with S-3000N (HITACHI) scanning electron microscopic observation, Electronic Speculum parameter arranges: image model is SE, working distance 26.61-27.3mm, sample stage tilts 0, and accelerating potential is 15.00KV, enlargement factor 2.0K～3.0K.
Embodiment 2
A parasite scanning electron microscope example epoxy resin embedding preparation method, realizes according to the following steps:
Step 1, sample is processed in earlier stage: the different hook pincers of live body worm killed rapidly under condition in 60 ℃, 4 minutes in test tube, and parasite is wrapped in qualitative filter paper;
Step 2, sample is fixed: is placed in 2% glutaraldehyde and fixes 3 hours, PBS buffer solution for cleaning three times, each 15 minutes, then fix 2 hours with 1% osmic acid, parasite is smoked into black;
Step 3, sample cleans and gradient dehydration: PBS buffer solution for cleaning three times, each 15 minutes, with 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%, 100% acetone, do gradient dehydration, each 20 minutes;
Step 4, displacement: proceeding to volume ratio after different hook pincers worm fully dewaters is to flood 2 hours in the epoxy resin embedding agent of 1:3 and the mixed liquor of acetone, proceeding to volume ratio is to flood 4 hours in the epoxy resin embedding agent of 1:1 and the mixed liquor of acetone, proceeding to volume ratio is to flood 6 hours in the epoxy resin embedding agent of 3:1 and the mixed liquor of acetone again, finally proceeds in pure embedding medium and floods 10 hours.
Step 5, clean: the different hook pincers worm after pure embedding medium dipping is picked out to be put into 200 order fine sieves online, polyase 13 hour under 70 ℃ of conditions, cooling clean dropwise with acetone afterwards, until nematode surface becomes tarnish.Under 70 ℃ of conditions, complete polymerization is 24 hours;
Step 6, sample coating: by the dried ion sputtering instrument metal spraying 90s for sample stage of polymerization, the golden film that plates one deck conduction at different hook pincers worm sample product surface uniform, completing gold-plated different hook pincers worm sample product can observe under scanning electron microscope, completes the preparation of parasitic nematode scanning electron microscope example.
Embodiment 3
A parasite scanning electron microscope example epoxy resin embedding preparation method, realizes according to the following steps:
Step 1, sample is processed in earlier stage: live body Myxosporean killed rapidly under condition in 60 ℃, 3 minutes in test tube, and parasite is wrapped in qualitative filter paper;
Step 2, sample is fixed: is placed in 2% glutaraldehyde and fixes 2 hours, PBS buffer solution for cleaning three times, each 15 minutes, then fix 2 hours with 1% osmic acid, parasite is smoked into black;
Step 3, sample cleans and gradient dehydration: PBS buffer solution for cleaning three times, each 15 minutes, with 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%, 100% acetone, do gradient dehydration, each 20 minutes;
Step 4, displacement: Myxosporean proceeds to volume ratio after fully dewatering be to flood 2 hours in the epoxy resin embedding agent of 1:3 and the mixed liquor of acetone, proceeding to volume ratio is to flood 4 hours in the epoxy resin embedding agent of 1:1 and the mixed liquor of acetone, proceeding to volume ratio is to flood 6 hours in the epoxy resin embedding agent of 3:1 and the mixed liquor of acetone again, finally proceeds in pure embedding medium and floods 12 hours.
Step 5, cleans: the parasitic sporozoite after pure embedding medium dipping is picked out to be put into 400 order fine sieves online, under 70 ℃ of conditions, polymerization is 5 hours, and cooling clean dropwise with acetone afterwards, until nematode surface becomes tarnish.Under 70 ℃ of conditions, complete polymerization is 12 hours;
Step 6, sample coating: by the dried ion sputtering instrument metal spraying 120s for sample stage of polymerization, the golden film that evenly plates one deck conduction at sporozoite sample surfaces, completing gold-plated sample can observe under scanning electron microscope, completes the preparation of sporozoite scanning electron microscope example.
Claims (6)
1. prepare an epoxy resin embedding method for parasite sample for scanning electron microscope, it is characterized in that, comprise the following steps:
Step 1, sample is processed in earlier stage: after parasite live body is killed rapidly, be wrapped in qualitative filter paper;
Step 2, sample is fixed: sample is placed in to glutaraldehyde and fixes 2～4 hours, then use PBS damping fluid that sample surfaces is cleaned up, remove glutaraldehyde, then fix with osmic acid, parasite is smoked into black;
Step 3, sample cleans and gradient dehydration: use PBS damping fluid that sample surfaces is cleaned up, remove osmic acid, then with the acetone that volumetric concentration is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%, 100%, do gradient dehydration;
Step 4, displacement: flood embedding in the mixed liquor of employing epoxy resin embedding agent and acetone after parasite dehydration;
Step 5, clean: the parasite after epoxy resin embedding agent dipping is chosen and is put on the screen cloth that mesh is less than parasitic polypide, polyase 13～5 hour under 70 ℃ of conditions, after naturally cooling to room temperature, with acetone, clean, until nematode surface becomes tarnish, then polymerization 12～24 hours under 70 ℃ of conditions;
Step 6, sample coating: by the good ion sputtering instrument metal spraying 90 ~ 120s for sample stage of polymerization, evenly plate the golden film of one deck conduction at parasite sample surfaces, complete the preparation of parasite scanning electron microscope example.
2. the epoxy resin embedding method of parasite sample for preparation scanning electron microscope according to claim 1, is characterized in that, in step 1, live body parasite killed rapidly under condition in 60 ℃, 3 ~ 5 minutes in test tube.
3. the epoxy resin embedding method of parasite sample for preparation scanning electron microscope according to claim 1, is characterized in that, in step 2, the mass concentration of glutaraldehyde is 2%.
4. the epoxy resin embedding method of parasite sample for preparation scanning electron microscope according to claim 1, is characterized in that, in step 2, the mass concentration of osmic acid is 1%.
5. the epoxy resin embedding method of parasite sample for preparation scanning electron microscope according to claim 1, it is characterized in that, in step 4, replacing embedding is to flood 2 hours in the epoxy resin embedding agent of 1:3 and the mixed liquor of acetone for proceeding to volume ratio after parasite is fully dewatered, then proceeding to volume ratio is to flood 4 hours in the epoxy resin embedding agent of 1:1 and the mixed liquor of acetone, proceeding to volume ratio is to flood 6 hours in the epoxy resin embedding agent of 3:1 and the mixed liquor of acetone again, finally proceeds in pure epoxy resin embedding medium and floods 8 ~ 12 hours.
6. the epoxy resin embedding method of parasite sample for preparation scanning electron microscope according to claim 1, is characterized in that, described parasite is: sample is relatively little, and form is variable, the Platyhelminthes or the protozoa that in body, without vertebral bones, support.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104977431A (en) * | 2014-04-02 | 2015-10-14 | 中国科学院植物研究所 | Young plant sample critical point drying method for scanning electron microscope |
| CN105158518A (en) * | 2015-09-24 | 2015-12-16 | 中国科学院西北高原生物研究所 | Method for preparing trichophyton scanning electron microscope sample |
| CN117593452A (en) * | 2023-11-21 | 2024-02-23 | 中国科学院南京地质古生物研究所 | Three-dimensional reconstruction method of radioworm micro-body fossil |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104977431A (en) * | 2014-04-02 | 2015-10-14 | 中国科学院植物研究所 | Young plant sample critical point drying method for scanning electron microscope |
| CN105158518A (en) * | 2015-09-24 | 2015-12-16 | 中国科学院西北高原生物研究所 | Method for preparing trichophyton scanning electron microscope sample |
| CN117593452A (en) * | 2023-11-21 | 2024-02-23 | 中国科学院南京地质古生物研究所 | Three-dimensional reconstruction method of radioworm micro-body fossil |
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Application publication date: 20141001 |