CN111238897A - Method and agent for treating biological specimen - Google Patents

Method and agent for treating biological specimen Download PDF

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Publication number
CN111238897A
CN111238897A CN202010098403.XA CN202010098403A CN111238897A CN 111238897 A CN111238897 A CN 111238897A CN 202010098403 A CN202010098403 A CN 202010098403A CN 111238897 A CN111238897 A CN 111238897A
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gelatin
biological specimen
specimen
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concentration
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CN111238897B (en
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许德田
王秀丽
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SHANGHAI DERMATOLOGY HOSPITAL
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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
    • G01N1/2806—Means for preparing replicas of specimens, e.g. for microscopal analysis
    • 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
    • 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
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • 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
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/2202—Preparing specimens therefor
    • 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
    • G01N2001/2873—Cutting or cleaving

Abstract

The invention provides a method and a treating agent for treating a biological specimen. The biological specimen processing method provided by the invention is to wrap gelatin on the surface of the biological specimen to form a gelatin wrapping layer before the biological specimen contacts a cross-linking agent. The processing method comprises the following main steps: step one, wrapping a biological specimen with heated gelatin hydrogel; and step two, cooling and solidifying the gelatin hydrogel, and then placing the biological specimen wrapped with the gelatin into a cross-linking agent for cross-linking treatment so as to enable the gelatin wrapping layer to form a fixed insoluble cross-linked substance. The biological specimen treating agent comprises gelatin, and the concentration range of the gelatin is 15-40%. The invention has the beneficial effects that: (1) reduce the pain and trauma of the patient; (2) ethical problems are avoided; (3) large-scale research is possible.

Description

Method and agent for treating biological specimen
Technical Field
The invention relates to a processing method and a processing agent for a biological specimen, in particular to a method for coating a tiny or extremely thin biological specimen by gelatin so as to keep the tissue structure or the internal components of the specimen and the integrity of microorganisms, belonging to the technical field of biology.
Background
When a specimen slice is required for observation with a microscope, it is difficult to produce a slice having a uniform thickness because the tissue is soft or the local hardness is uneven when producing a specimen slice or an ultrathin slice. Therefore, it is necessary to impregnate the inside of the tissue with a substance called an embedding medium, so that the whole tissue is uniformly hardened to facilitate slicing with a sharp knife.
The paraffin flaking method is a method of embedding a material in paraffin for slicing after fixing, dehydrating and waxing, and the paraffin is an embedding agent with moderate hardness. The method has wide application range and complete flaking means, can cut materials into extremely thin slices and can make continuous slices, which is difficult to achieve by other flaking methods, so the method is the most commonly used method in flaking technology of an optical microscope.
Since 1948, researchers have reported that, in the case of microscopic or extremely thin tissue specimens, the smallest specimens were unicellular trichomonas by a double coating method in which collodion (nitrocellulose), agar, gelatin, agarose, and the like are embedded in advance, and then paraffin embedding and sectioning are performed. The main purpose of these previous studies is to orient and position the specimen to obtain slices with a certain direction of section for easy observation.
In the process of researching the structure and microorganisms of the acne and comedo plug, researchers use the traditional paraffin embedding and slicing method to manufacture the sample, and the problems of structural damage and microorganism loss of the angle plug sample are encountered, so that the structure and the integrity of the microorganisms of the acne and comedo plug cannot be maintained, and follow-up research is difficult.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: how to maintain the integrity of the tissue structure and the microorganisms contained therein when making small or extremely thin biological specimens.
In order to solve the above technical problems, a first aspect of the present invention provides a method for processing a biological specimen, wherein a gelatin coating layer is formed by coating the surface of the biological specimen with gelatin before the biological specimen contacts a cross-linking agent.
In some embodiments, the concentration of gelatin ranges from 15% to 40%. Preferably, the concentration of gelatin is 25%.
In some embodiments, the gelatin coating has a thickness of 0.5 to 2 mm.
In some embodiments, the biological specimen is less than 5 millimeters in size.
In some embodiments, the method of processing a biological specimen includes the steps of:
step one, wrapping a biological specimen with heated gelatin hydrogel;
and step two, cooling and solidifying the gelatin hydrogel, and then placing the biological specimen wrapped with the gelatin into a cross-linking agent for cross-linking treatment so as to enable the gelatin wrapping layer to form a fixed insoluble cross-linked substance.
In some embodiments, formaldehyde or glutaraldehyde is selected as the crosslinking agent.
In some embodiments, the concentration of the cross-linking agent ranges from 3% to 15%.
In some embodiments, the crosslinking treatment time is 12 to 24 hours.
In a second aspect of the invention, a biological specimen processing agent is provided, which comprises gelatin, wherein the concentration of the gelatin ranges from 15% to 40%.
The invention has the beneficial effects that:
(1) reduce the pain and trauma of the patient;
(2) ethical problems are avoided;
(3) large-scale research is possible.
Drawings
FIG. 1 is a photomicrograph of a 25% gelatin coated acne keratoplug specimen coated with other materials, respectively, in example 1 of the present invention;
FIG. 2 is a photomicrograph of a sample of pilosebaceous follicles coated with 25% gelatin in example 2 of the present invention.
Detailed Description
Unless otherwise defined, technical or scientific terms used in the claims and the specification of this patent shall have the ordinary meaning as understood by those of ordinary skill in the art to which this patent belongs.
The preparation sequence of the common biological specimen is as follows: soaking a specimen in a formaldehyde solution, and denaturing protein in the specimen to fix the shape of the specimen; embedding the specimen with agar, gelatin, agarose and other material; then, replacing water in the specimen with alcohol with different concentrations to completely dehydrate the specimen; then, the specimen is soaked in the melted wax, the inner pore of the specimen is filled with the wax, and the outer surface of the specimen is wrapped by the wax; and finally, slicing the paraffin-embedded specimen to obtain a specimen slice for microscopic observation.
For a tiny or extremely thin biological specimen, if the specimen is directly soaked in a formaldehyde solution, a tiny tissue structure in the specimen or microorganisms in the specimen are damaged by the formaldehyde solution, the shape is difficult to maintain, and subsequent observation and research are prevented. If the specimen is wrapped with a substance to form a wrapping layer before the biological specimen is exposed to the formalin solution, it is possible to protect the micro-tissue structures inside the specimen or the shape of microorganisms therein. The above is the inventive concept of this patent.
According to the invention, various materials are tried, and the gelatin can play a good protection effect on the micro specimen. Collagen is a biopolymer, and is a major component in animal connective tissue. Gelatin is an irreversibly hydrolyzed form of collagen, and is a jelly with some viscosity.
In formaldehyde or glutaraldehyde solution, gelatin undergoes a cross-linking reaction and becomes denatured and hardened into a solid. Experiments show that the concentration range of the gelatin is 15% -40%, and the gelatin is suitable for preparing micro specimens. If the concentration of gelatin is too small, the crosslinked product formed in the formaldehyde solution is too soft, resulting in poor shaping. If the concentration of the gelatin is too high, the cross-linked substance is too hard to facilitate slicing of the specimen, and the wrapping and infiltrating effects are poor. The preferred gelatin concentration is 25%.
If the sample is a micro sample, the size of the sample is less than 5 mm. If the specimen is an extremely thin specimen, the thickness of the specimen is less than 2 mm. Gelatin is wrapped on the surface of the specimen, and the thickness of the formed wrapping layer is 0.5-2 mm.
The method for processing the biological sample comprises the following steps:
step one, wrapping the biological specimen with gelatin. The gelatin concentration ranges from 15% to 40%, preferably 25%. The gelatin hydrogel with the concentration is heated to be more than 40 ℃ and kept at the temperature, so that the gelatin hydrogel has fluidity. If the tiny specimen is adopted to make a specimen slice, the specimen can be placed still, and gelatin is dripped on the specimen by using a dripping tool, so that the tiny specimen is automatically and completely wrapped due to the fluidity and the permeability of the gelatin.
And step two, after the gelatin hydrogel is cooled and solidified, trimming the biological specimen wrapped with the gelatin, removing excessive gelatin, and then placing the biological specimen into a cross-linking agent for cross-linking treatment so as to enable the gelatin wrapping layer to form a fixed insoluble cross-linked substance. The cross-linking agent is formaldehyde or glutaraldehyde. The concentration of the cross-linking agent is in the range of 3% to 15%, preferably 4%. The specific operation is as follows: and immersing the biological specimen wrapped with the gelatin in the cross-linking agent and standing for 12-24 hours.
The first step and the second step are key steps of the invention, and in order to obtain a finished product of the specimen, the method also comprises the following conventional steps:
and step three, completely removing free moisture in the specimen, wherein the commonly used dehydrating agent is alcohol. The dehydration should be performed slowly, not too rapidly or for too long a period of time to avoid hardening and embrittlement of the tissue or shrinkage. The dehydration adopts gradient dehydration, namely, the dehydration is started from low-concentration alcohol and gradually replaced to high-concentration alcohol. The general formula is as follows: 70% alcohol → 80% alcohol → 95% alcohol → anhydrous alcohol (twice), each stage of the residence time is 2-4 hours or more, and finally the anhydrous alcohol treatment time is preferably short.
And step four, embedding paraffin and slicing. The melted paraffin was allowed to fill the inner space of the specimen, and then cut into thin sections required for microscopic observation according to a conventional procedure.
Example 1
The keratoplug of acne is a characteristic structure of common acne, contains a large amount of microbial information, and has important significance for exploring the etiology and mechanism of acne occurrence. The traditional sampling method relates to histology, and trephine biopsy sampling is carried out by punching holes in skin by using a trephine, so that the samples are very difficult to accept in the face, the appearance of a patient is damaged, the skin of the patient is damaged and the patient feels painful, and therefore, the traditional sampling method has ethical problems; even with the ability to take biopsy samples with patient consent, a large sample size is not possible, thus hampering the study of large samples.
If the acne plug is separated by a non-invasive extrusion method and is not processed by a proper coating method, the integrity of the structure and the microorganism contained in the acne plug cannot be guaranteed by slicing. Because the angle bolt has loose structure and lacks connexin, in the traditional embedding method treatment process, the angle bolt is loosened and broken due to the use of a large amount of multi-step organic solvents, and microorganisms in the angle bolt are lost, so that the research value is lost. Traditionally, pre-embedding methods using agar, agarose and the like mainly focus on the orientation and positioning of a specimen and do not relate to the integrity of the structure of the specimen and microorganisms therein; the method comprises the steps of fixing a specimen by using an organic solvent, pre-coating, dehydrating, waxing and slicing.
No one has previously treated free acne keratoplugs with double coating. The method provided by the invention comprises the steps of pre-coating, fixing, dehydrating, waxing and slicing. The invention utilizes the characteristic that gelatin is taken as a protein hydrolysate to form a fixed insoluble cross-linked substance in formaldehyde, so that the gelatin forms a coat on the outer surface of the specimen, and the damage and the loss of the structure and microorganisms of the gelatin are avoided. The method provided by the invention can bring great convenience for acne research.
The experiment was carried out in this example, and the overall method of the experiment was as follows: seeking 4 acne patients, taking 5 keratotic plugs from each patient by a non-invasive method, coating by different materials, setting a negative control group without pre-coating, staining and observing the keratotic plugs after slicing, and observing by a scanning electron microscope.
The specific steps of the experiment are as follows:
the study was determined to use 4 different materials as pre-encapsulation agents, including agar, agarose, gelatin, agar + low concentration gelatin, see table 1. In the experimental protocol using agar, the agar concentration was 2%. In the agarose protocol, the agarose concentration was 2%. In the experimental protocol with gelatin, different concentrations of gelatin were tried, with the best results when the gelatin concentration was 25%. In the agar + low gelatin protocol, the agar concentration was 2% and the gelatin concentration was 2.5%.
Taking the acne keratome of the same patient, placing the acne keratome on a warm glass plate, dripping the pre-wrapping material, putting the acne keratome into 4% formaldehyde solution for overnight after the material is gelled, taking out the acne keratome and placing the acne keratome in 75% alcohol for storage, and then carrying out conventional paraffin embedding and slicing on the specimen.
The results of comparing the properties of several pre-wrapped materials are shown in table 1 below:
TABLE 1
Figure BDA0002386028820000041
Note in table 1:
a thermal stability after fixation: the material was heated to 70 degrees infusible after overnight in 4% formaldehyde solution.
b, weldability: for a specimen with a slightly larger volume, if the material on one drop cannot completely coat the specimen, more material needs to be dropped. If the weldability is poor, the materials dripped twice cannot be well welded, and after the materials dripped twice are soaked in a 4% formaldehyde solution overnight, the materials dripped twice can crack from the junction, so that the coating fails.
c, convenience: refers to the reusability of the material which can be conveniently and easily heated and melted again after being used for one time, and the convenience which can be adjusted in the using process.
d, colorability: staining after sectioning, staining ability to dye.
The results of the microscopic observation are shown in FIG. 1. Different materials and different material concentrations are tested by the invention, and finally 25% of gelatin is found to have the most remarkable advantage. Therefore, 25% gelatin is the best pre-wrap material for a comedone keratoplug. The method can maintain the structural integrity of the angle plug during the treatment process, and can make the microorganism in the angle plug in situ, and can be used for the histopathology and microorganism space distribution research of acne. Agarose can retain the structure of the angle pin to some extent, but the angle pin structure can still become loose. Agar, agar + low concentration gelatin do not protect the structural integrity of the keratoplug.
With the method provided by the invention, biopsy sampling of acne patients is not required. Conventionally, a tissue of 0.5 cm diameter may be taken; using the method of the invention, only 1mm is required, which reduces the trauma to the patient by a factor of 24, i.e. 96% (note: areas of 5mm and 1mm in diameter differ by a factor of 25). Or, according to the traditional method, only 1 site can be taken at 0.5 cm; by using the method of the invention, 25 sites can be taken at the same 0.5 cm, thus more comprehensive detection, diagnosis and research can be carried out on the disease condition, the tissue and the pathology. The method can solve the ethical problem of research and sampling of the acne, simultaneously ensures the integrity of the structure and the microorganism in the sample, enables the research of the large sample of the acne to be possible, and is expected to become an important tool for researching the etiology of the acne.
In summary, the present example uses gelatin to coat the acne keratoplug specimen, which effectively maintains the structure and microbial integrity of the acne keratoplug. Reduce the trauma to the patient, reduce the ethical problem, and the precious small specimens can be well protected and researched.
Example 2
The embedding technology provided by the invention can be used for preparing samples of acne and comedo plugs and can also be widely used for preparing other tiny biological tissue samples. And a smaller sample is used to obtain more information, thereby bringing convenience to clinical research and diagnosis work.
In this example, a single pilosebaceous gland having a diameter of only about several hundred micrometers was successfully coated and sectioned using a double coating method similar to that of example 1, and the results are shown in fig. 2. The pilosebaceous structure and morphology in fig. 2 are very complete and can achieve the image quality obtained by the traditional slicing technology. This work is impossible without using the present method.
The invention is used for coating and processing the tiny and precious skin and whole body tissue samples, including samples with the size of hundreds of microns such as hair follicles, and has great significance for effectively utilizing the samples and reducing the pain and the wound of patients.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (10)

1. The method for treating the biological specimen is characterized in that the surface of the biological specimen is coated with gelatin to form a gelatin coating before the biological specimen is contacted with the cross-linking agent.
2. The method of claim 1, wherein the gelatin concentration is in the range of 15% to 40%.
3. The method of claim 2, wherein the gelatin is present at a concentration of 25%.
4. The method as claimed in claim 1, wherein the gelatin coating layer has a thickness of 0.5-2 mm.
5. The method as claimed in claim 1, wherein the dimension of the biological specimen is less than 5 mm.
6. The method of claim 1, comprising the steps of:
step one, wrapping a biological specimen with heated gelatin hydrogel;
and step two, cooling and solidifying the gelatin hydrogel, and then placing the biological specimen wrapped with the gelatin in a cross-linking agent for cross-linking treatment so as to enable the gelatin wrapping layer to form a fixed insoluble cross-linking object.
7. The method as claimed in claim 6, wherein the cross-linking agent is formaldehyde or glutaraldehyde.
8. The method of claim 7, wherein the concentration of the cross-linking agent is in the range of 3% to 15%.
9. The method according to claim 6, wherein the cross-linking treatment time is 12 to 24 hours.
10. The biological specimen treating agent is characterized by comprising gelatin, wherein the concentration range of the gelatin is 15-40%.
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