CN101545876B - Method for observing collagenous fibre of fresh and alive trepang with electron microscope - Google Patents
Method for observing collagenous fibre of fresh and alive trepang with electron microscope Download PDFInfo
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- CN101545876B CN101545876B CN2009100149585A CN200910014958A CN101545876B CN 101545876 B CN101545876 B CN 101545876B CN 2009100149585 A CN2009100149585 A CN 2009100149585A CN 200910014958 A CN200910014958 A CN 200910014958A CN 101545876 B CN101545876 B CN 101545876B
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Abstract
The invention relates to a method for observing the collagenous fibre of fresh and alive trepang with an electron microscope, which is characterized by comprising the steps: firstly, the body wall muscles of the trepang are cut into pieces, the trepang pieces are cleanly washed by phosphoric acid buffer solution, the cleanly washed trepang pieces are fixed by glutaraldehyde phosphoric acid buffer solution for 0.5-5 hours under the temperature of -5-10 DEG C, and a trepang sample which is soaked in NaOH aqueous solution for 1-10 hours under the temperature of 10-50 DEG C is washed by normal saline solution until the PH value reaches 6.0-8.0; and secondly, the trepang sample is dyed by tannin, fixed by the glutaraldehyde phosphoric acid buffer solution under the temperature of 0-10 DEG C, sequentially dehydrated by alcohol with concentration ranging from low to high, dried at critical point, processed by gilded film and observed by a scanning electron microscope. Because trepang sample has the characteristics of easy putrescence, easy degenerescence of the marine product and isophagy per se, the two steps of collagenous fibre fixation and conductive dying are added to the preparation process of the electron microscope sample thereof, and the tissue constitution of the collagenous fibre of the fresh and alive trepang can be observed, thereby achieving the ideal effect.
Description
Technical field
The present invention relates to a kind of sea life food sea cucumber collagenous fibres, particularly relate to a kind of electron microscopic observation method of collagenous fibre of fresh and alive trepang.
Background technology
Sea cucumber is listed in one of " marine products eight delicacies " with its higher nutrition and pharmacy value, be nourishing, body-building, cure the disease, anti-old good merchantable brand.Sea cucumber body wall muscle accounts for more than 85% (M/M) of sea cucumber general assembly (TW), constitute by the corium connective tissue, and the principal ingredient of corium connective tissue is collagenous fibres more than 70%, and texture characteristic that we can say sea cucumber is that the histological structure's characteristic by collagenous fibres is determined to a great extent.So observe the histological structure of collagenous fibre of fresh and alive trepang and at the intramuscular existence of body wall, significant to edible, nutritive peculiarity and the fabricated product quality quality thereof of understanding sea cucumber.
Observational technique at sea cucumber collagenous fibres structure has two kinds at present, and a kind of is by decoration methods such as Van-Gieson, Mallory three looks each composition in the sea cucumber muscle to be dyeed, and distinguishes according to each difference that becomes branch to dye color.Though this method can be judged the existence of collagen in the musculature qualitatively, can not effectively observe in the institutional framework of giving birth under the body state it; Another kind method is to extract by the collagen in the muscle being carried out target, observes its corresponding microtexture and composition then, and extracting method mainly contains acid system, alkaline process, enzyme process, salt method and hot water extraction etc.Yet no matter the sort of extracting method all can cause the collagen sex change in various degree, and the single collagen that extracts can not reflect its real existence in musculature.
Summary of the invention
Purpose of the present invention provides a kind of electron microscopic observation method of collagenous fibre of fresh and alive trepang, and it can overcome the above-mentioned shortcoming of prior art.
A kind of electron microscopic observation method of collagenous fibre of fresh and alive trepang, it is characterized in that earlier sea cucumber body wall muscle being cut into bulk, clean with phosphate buffer, use glutaraldehyde phosphate buffer fixedly 0.5-5 hour under the-5-10 ℃ of condition, with the sea cucumber sample under 10-50 ℃ of condition, with the NaOH aqueous solution soaking after 1-10 hour, be washed till pH6.0-8.0 with normal saline solution, then with tannic acid dyeing 0.5-5 hour, use glutaraldehyde phosphate buffer fixedly 10-60 minute under the 0-10 ℃ of condition, after dewatering one by one with concentration alcohol from low to high, carry out critical point drying, critical point temperature 25-40 ℃, pressure 30-50kg/cm
2, gold-plated film is used scanning electron microscopic observation after handling.
Advantage of the present invention is to have easily corruption of aquatic products at the sea cucumber sample, perishable, self has the characteristics of autolysis again, fixing and two steps of conduction dyeing of collagenous fibres in its preparing electron microscopy specimen process, have been increased, can observe collagenous fibre of fresh and alive trepang histological structure, obtain even more ideal effect.
Description of drawings
Accompanying drawing is the figure of collagenous fibre of fresh and alive trepang histological structure.Collagenous fibre of fresh and alive trepang roughly is divided into crin and filament two classes, and the crin collagenous fibres present directivity longitudinally, and the filament collagenous fibres then are network structure.
Embodiment
Sea cucumber body wall muscle is cut into 5 * 5 * 3mm
3Bulk is cleaned with 0.1M pH7.4 phosphate buffer, is that 0.5M, pH are that 7.2 glutaraldehyde phosphate buffer is fixed 2 hours with concentration under 4 ℃ of conditions.Under 20 ℃ of conditions, with 2M NaOH aqueous solution soaking after 4 hours, is that 5% normal saline solution is washed till pH7.2 with mass percentage concentration with the sea cucumber sample, is that 2% tannic acid dyeed 1 hour with mass percentage concentration then.Be that 0.5M, pH are that 7.2 glutaraldehyde phosphate buffer is fixed 30 minutes again with concentration under 4 ℃ of conditions, after dewatering one by one with concentration alcohol 50%, 60%, 70%, 80%, 90%, 100% (V/V) from low to high, carry out critical point drying, critical point temperature is that 31.4 ℃, pressure are 39.5kg/cm
2, gold-plated film is used scanning electron microscopic observation after handling.
The concentration of the phosphate buffer described in the present invention is that 0.1M-1M, pH are 6.0-8.0; The concentration of described glutaraldehyde phosphate buffer is that 0.1M-1M, pH are 6.0-8.0; The concentration of described NaOH aqueous solution is 0.5M-5M; The mass percentage concentration of described normal saline solution is 1%-10%; The mass percentage concentration of described tannic acid is 0.5%-5%; The concentration expressed in percentage by volume of described alcohol is 10%-100% (V/V).
Claims (1)
1. the electron microscopic observation method of a collagenous fibre of fresh and alive trepang, it is characterized in that earlier sea cucumber body wall muscle being cut into bulk, with concentration is 0.1M-1M, pH cleans for the 6.0-8.0 phosphate buffer, be 0.1M-1M with concentration under the-5-10 ℃ of condition, pH is 6.0-8.0 glutaraldehyde phosphate buffer fixedly 0.5-5 hour, to be cut into block sea cucumber body wall muscle under 10-50 ℃ of condition, with concentration is that 0.5M-5M NaOH aqueous solution soaking is after 1-10 hour, is 6.0-8.0 with mass percentage concentration for the 1%-10% normal saline solution is washed till pH, be 0.5%-5% tannic acid dyeing 0.5-5 hour with mass percentage concentration then, use glutaraldehyde phosphate buffer fixedly 10-60 minute under the 0-10 ℃ of condition, with concentration expressed in percentage by volume is after 10%-100% alcohol from low to high dewaters one by one, carry out critical point drying, critical point temperature 25-40 ℃, pressure 30-50kg/cm
2, gold-plated film is used scanning electron microscopic observation after handling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100149585A CN101545876B (en) | 2009-04-29 | 2009-04-29 | Method for observing collagenous fibre of fresh and alive trepang with electron microscope |
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| CN2009100149585A CN101545876B (en) | 2009-04-29 | 2009-04-29 | Method for observing collagenous fibre of fresh and alive trepang with electron microscope |
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| CN101545876A CN101545876A (en) | 2009-09-30 |
| CN101545876B true CN101545876B (en) | 2011-02-16 |
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| CN2009100149585A Expired - Fee Related CN101545876B (en) | 2009-04-29 | 2009-04-29 | Method for observing collagenous fibre of fresh and alive trepang with electron microscope |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103175723B (en) * | 2011-12-22 | 2015-11-18 | 上海纳米技术及应用国家工程研究中心有限公司 | The visual preparation method of laser confocal scanning microscope macromolecular fibre |
| CN104198767A (en) * | 2014-08-04 | 2014-12-10 | 浙江大学 | Method for examining fresh cocoon raw silk through 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 |
| CN106918609A (en) * | 2017-03-24 | 2017-07-04 | 青岛农业大学 | It is a kind of distinguish different sea cucumber kinds analyze and identify method |
| CN110604126A (en) * | 2018-06-14 | 2019-12-24 | 宁波化奇工程技术有限公司 | Non-formaldehyde environment-friendly pathological tissue fixing agent |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1912567A (en) * | 2006-07-10 | 2007-02-14 | 中国水产科学研究院黄海水产研究所 | Paraffin section method for seawater fish egg |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1912567A (en) * | 2006-07-10 | 2007-02-14 | 中国水产科学研究院黄海水产研究所 | Paraffin section method for seawater fish egg |
Non-Patent Citations (2)
| Title |
|---|
| Xin GAO等.Rheological properties and structural changes in raw and cooked abalone meat.《Fisheries Science》.2001,第67卷第314-320页. * |
| 汤志旭等.即食海参质构及流变学特征的研究.《食品工业科技》.2007,第28卷(第10期),第57-60页. * |
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