CN101865915A - 2.5% glutaraldehyde fixative solution for electron microscope experiment and preparation method thereof - Google Patents

Abstract

The invention discloses a 2.5% glutaraldehyde stationary liquid for electron microscope experiment and a preparation method thereof. The method comprises the following steps: (1) preparing phosphate buffer: liquid A, adding distilled water to 71.64g of disodium hydrogen phosphate*12 crystal water to 1000ml; liquid B, adding distilled water to 31.21g of sodium dihydrogen phosphate*2 crystal water to 1000ml; preparing the 36ml of liquid A and the 14ml of liquid B into 0.2mol phosphate buffer with a pH value of 7.2; (2) preparing a fixing agent of glutaraldehyde: adding 5ml of 50% glutaraldehyde to the 50ml of prepared 0.2mol phosphate buffer with the pH value of 7.2, then adding the distilled water to the mixed solution to 100ml and adding 2g of active carbon, keeping the solution from the sun, closing, detecting and then packing. The invention has the advantages of high quality, experiment time and cost saving, less environment pollution due to the outdated reagents, reduced damage to the health of experimenters due to the preparation of the reagents, long quality guarantee period and the like.

Description

2.5% glutaraldehyde fixative solution for electron microscope experiment and preparation method thereof

technical field

The invention relates to a 2.5% glutaraldehyde fixative for electron microscope experiments and a preparation method thereof, more specifically a method for commercializing the 2.5% glutaraldehyde fixative for electron microscope experiments.

Background technique

At present, most of the 2.5% glutaraldehyde fixatives used in electron microscopy experiments are prepared by the laboratory, that is, the raw materials are purchased and configured according to the needs of the experiment. 2.5% glutaraldehyde, and the 2.5% glutaraldehyde fixative used in immunoelectron microscopy and cytochemical experiments needs to be further purified to meet the experimental requirements, and this process is very cumbersome and cumbersome in the laboratory. It is difficult to realize; in addition, because the amount of reagents used in electron microscopy experiments is generally small, 1mL of 2.5% glutaraldehyde fixative solution for a sample can meet the needs, and the minimum packaging of raw materials purchased by self-prepared reagents: 100mL of 25% glutaraldehyde, phosphoric acid Sodium dihydrogen 500g, disodium phosphine 500g, will cause self-prepared reagents like this, there is still a large amount of remaining reagents after the experiment, and these remaining reagents are due to very short shelf life, therefore, the reagents that can't be used up can only be used in the end. Abandoned, this not only wastes the experimental funds of the experimenters, but also wastes resources, and the abandoned reagents will also cause environmental pollution; secondly, in terms of labor, it takes about 2 days for the self-prepared reagents to clean the container, dry the container, weigh, and prepare. Time, this will affect the experimental process, and the toxicity of glutaraldehyde will also have a negative impact on the health of the preparer.

Based on this, the applicant has made the present invention.

Invention content:

In view of the above-mentioned problems existing in the prior art, the main purpose of the present invention is to provide a high-quality, low-cost, environmentally friendly, and long-lasting 2.5% glutaraldehyde fixative for electron microscopy experiments.

The technical scheme adopted in the present invention is as follows, a 2.5% glutaraldehyde fixative for electron microscopy experiments is characterized in that it is configured by using the following raw materials and methods: (1), using disodium hydrogen phosphate solution and sodium dihydrogen phosphate solution It is configured as a phosphate buffer solution, double-checked during the configuration, and the intermediate inspection is carried out after the configuration is completed; (2), the phosphate buffer solution configured in step 1 is added to glutaraldehyde and distilled water, and the pH value is checked after purification; (3) The solution prepared in step 2 is melt-sealed and sub-packaged in a brown ampoule, and the finished product is inspected after the packaging is completed and then released from the warehouse.

In the present invention: the pH value of the product during preparation can be any value within the range of 1-14.

The intermediate test in step 1 refers to the content and pH value test of the solution.

The purification method in step 2 can be selected from any one of purification methods such as decompression distillation, activated carbon purification, barium carbonate adsorption.

Another object of the present invention is to provide a kind of preparation method of 2.5% glutaraldehyde fixative for electron microscope experiment, comprising the following steps:

1. Inspection of raw materials and auxiliary materials: the raw materials, auxiliary materials and equipment used for configuration shall be inspected according to the standards and then reserved;

2. Phosphate buffer preparation:

Liquid A: disodium hydrogen phosphate * 12 crystal water 71.64g, add distilled water to 1000ml;

Liquid B: Sodium dihydrogen phosphate * 2 crystal water 31.21g, add distilled water to 1000ml;

Prepare 36ml of solution A + 14ml of solution B into 0.2mol pH7.2 phosphate buffer;

3. Glutaraldehyde fixative preparation:

Add 50ml of 0.2molpH7.2 phosphate buffer prepared in step 1, add 5ml of 50% glutaraldehyde, then add distilled water to 100ml, add 2g of activated carbon, keep away from light, and stir for 1h; then carry out pH test: pH7. 2±0.1, filter ball filter, filter membrane filter;

4. Packaging and inspection:

After cleaning and drying the brown ampoules, they are packaged and sealed in a fusion sealing machine, and packaged after light inspection.

The beneficial effects of the present invention are as follows:

1. The product of the present invention is checked by two people during the preparation process. The key items (such as: content, pH, etc.) intermediate inspection are carried out before packaging, and the finished product inspection is carried out after packaging. From the perspective of process control, self-preparation of reagents is avoided to the greatest extent. Possible mistakes ensure the improvement of experimental quality.

2. The product of the present invention is produced professionally in batches, has the ability to use professional equipment, can achieve conditions such as light protection and sterility, and ensures that the quality of the product glutaraldehyde is higher than that of self-prepared reagents.

3. The product of the present invention has formulated a quality standard that can fully meet or be higher than its experimental requirements. In the production process, an activated carbon purification step has been added, and the quality standard of the product must be formulated to meet the immune electron microscope experiment. The commercialized reagent can guarantee to provide high High-quality reagents can also greatly reduce the cost of labor and equipment for self-preparation reagents.

4. Inspection of validity period: It is impossible to use up the self-prepared reagents at one time. During the placement period, due to different storage conditions, the reagents vary greatly. When they are used next time, it cannot be guaranteed whether they can meet the experimental requirements. Through process control, the shelf life is effectively extended. Practice has proved that the brown glass ampoule fusion seal can completely isolate the contents from external oxygen, carbon dioxide, etc., and effectively avoid light. In addition, process control such as nitrogen filling and aseptic operation can make the product The shelf life of the contents is greatly extended, and the shelf life can reach two years; this technically lays a technical foundation for extending the validity period of reagents and ensuring the production of commercialized reagents.

5. In terms of preparation safety: since most reagents for electron microscopy experiments are toxic or radioactive, it is impossible for laboratories to equip operators with expensive labor protection for infrequently prepared reagents; and specialized production enterprises must consider safe production.

6. Packaging specification: Product reagents can prepare 1ml, 2ml, 10ml, and different specifications of products according to user needs, and can be combined and purchased according to experimental needs, so that users can obtain economical and high-quality products; self-prepared reagents are prepared each time The amount is far higher than the product specification, resulting in waste of reagents and environmental pollution.

The present invention will be further described below in combination with specific embodiments.

Detailed ways:

A preparation method of 2.5% glutaraldehyde fixative for electron microscopy experiments of the present invention, the specific process is as follows: raw material, auxiliary material inspection-professional preparation-intermediate inspection-filtering-subpackage, fusion sealing-lamp inspection-packaging-finished product test.

1. Inspection of raw and auxiliary materials: The raw and auxiliary materials and equipment involved in this embodiment are shown in Table 1.

Table 1: Inspection list of raw and auxiliary materials and equipment

Raw materials, auxiliary materials, equipment Quality standard, model 50% glutaraldehyde (imported) chemically pure

Raw materials, auxiliary materials, equipment Quality standard, model Sodium dihydrogen phosphate chemically pure Disodium phosphate chemically pure activated carbon Medical standard Process water Double distilled water   Dosing container Made of stainless steel, with stirring and heating devices PH meter PHS-3C (Shanghai Precision Science Instruments) Filter Ball, Membrane Filter Medical standard brown ampoule 1ml, 2ml, 10ml, medical standard Melting sealing machine 1ml, 2ml, 10ml bottle washing machine Self-made, with spraying and drying functions Drying box DGG-9140B (Shanghai Senxin) printing machine 1ml, 2ml, 10ml

2. Preparation process:

1), Phosphate buffer preparation: double check during preparation, intermediate inspection after preparation.

Liquid A: disodium hydrogen phosphate * 12 crystal water 71.64g, add distilled water to 1000ml;

Liquid B: Sodium dihydrogen phosphate * 2 crystal water 31.21g, add distilled water to 1000ml;

0.2molpH7.2 buffer solution: A solution 36ml+B solution 14ml.

2) Preparation of glutaraldehyde fixative solution: 50ml of 0.2molpH7.2 buffer solution + 50% glutaraldehyde 5ml + distilled water to 100ml + activated carbon 2g;

3), avoid light, and stir in airtight for 1h;

4), pH test: pH7.2±0.1;

5), filter ball filter, filter membrane filter;

6), the ampoule is cleaned and dried;

7), the fusion sealing machine packs and seals;

8) Light inspection: under the light with a black background, observe the melted and sealed products, and remove unqualified products such as bubble heads, deflated heads, and carbonization points;

9), printing packaging;

10) Finished product inspection: The finished product inspection standard is shown in Table 2.

Table 2. List of inspection standards for finished products

Test items Quality Standard Visual inspection The printing is clear and the outside of the bottle is clean Glutaraldehyde content 2.5%±0.1 pH pH7.2±0.1

The products produced by the present invention have been used by users at present: Huajiachi Electron Microscope Laboratory of Zhejiang University, Zhejiang Kidney Disease Research Center, Zijingang Electron Microscope Laboratory of Zhejiang University, Electron Microscope Laboratory of Shaoxing College of Arts and Sciences, etc., after reflection: high product quality, low experimental cost and low cost. Programs are greatly reduced.

Claims (5)

1. a kind of 2.5% glutaraldehyde fixative for electron microscope experiment is characterized in that, adopts following raw material and method to configure: (1), adopt disodium hydrogen phosphate solution and sodium dihydrogen phosphate solution to be configured into phosphate buffer saline Preparation, two-person check during configuration, and intermediate inspection after configuration is completed; (2), after adding glutaraldehyde and distilled water to the phosphate buffer solution configured in step 1, purify, and check the pH value; (3), the step 2. The prepared solution is melt-sealed and sub-packaged in a brown ampoule, and the finished product is inspected after the packaging is completed and released from the warehouse. 2. A 2.5% glutaraldehyde fixative for electron microscopy experiments according to claim 1, characterized in that: the pH value in the preparation process is any value within the range of 1 to 14. 3. a kind of electron microscope experiment according to claim 1 uses 2.5% glutaraldehyde fixative, it is characterized in that: the intermediate inspection of step 1 refers to the content of solution, pH value inspection. 4. a kind of electron microscopy experiment according to claim 1 uses 2.5% glutaraldehyde fixative, it is characterized in that: the purification method in the step 2 is selected from the arbitrary method of decompression distillation method, gac purification method, barium carbonate adsorption method A sort of. 5. A preparation method for electron microscope experiment with 2.5% glutaraldehyde fixative, comprising the following steps: (1) Inspection of raw materials and auxiliary materials: the raw materials, auxiliary materials and equipment used for configuration shall be inspected according to the standards and then reserved; (2), phosphate buffer preparation: Liquid A: disodium hydrogen phosphate * 12 crystal water 71.64g, add distilled water to 1000ml; Liquid B: Sodium dihydrogen phosphate * 2 crystal water 31.21g, add distilled water to 1000ml; Prepare 36ml of solution A + 14ml of solution B into 0.2mol pH7.2 phosphate buffer; (3), preparation of glutaraldehyde fixative: 50ml of 0.2molpH7.2 phosphate buffer prepared in step 1, add 50% glutaraldehyde 5ml, then add distilled water to 100ml, add 2g of activated carbon, avoid light, Airtight stirring 1h; Then carry out pH test: pH7.2 ± 0.1, filter ball filter, filter membrane filter; (4) Packaging and inspection: After the brown ampoules are cleaned and dried, they are packaged and sealed in a fusion sealing machine, and then packed after light inspection.