A kind of cytopathological slide fixative and preparation method thereof

Abstract

The present invention provides a cytopathology slide fixative, which comprises 800-900g of alcohols, 50-80g of polyethylene glycol, 30-50g of formaldehyde, and a phosphate buffer balance according to 1kg mass, wherein the alcohols comprise ethanol, n-butanol and tert-butanol, and the mass ratio of the ethanol, n-butanol and tert-butanol is 2-3:0.5-1:1-2. The present invention improves the fixative effect of the fixative by combining the three alcohol substances, and the specimen has good elasticity and a well-preserved structure. In the later coloring process, the coloring effect is excellent. By experimental comparison, the fixative effect of the fixative obtained by combining the three is better than that of the fixative obtained by simply using one alcohol.

Description

Technical Field

[0001] The invention relates to a cytopathological slide fixative and a preparation method thereof, belonging to the technical field of cytopathological fixatives. Background Art

[0002] Fixation is an indispensable basic technology in biomedical research. In the field of biology, the purpose of fixation is to inhibit the destruction and autolysis of organisms or their parts, and to preserve their appearance, internal structure, material composition, etc. in a state close to that of living organisms as much as possible, or to dissolve some substances and selectively preserve some internal structures and materials to observe the state of living organisms for the purpose of research, and to inhibit the growth of bacteria and molds. In order to facilitate observation with optical microscopes and electron microscopes, fixation can make embedding, sectioning, staining and other operation steps easier to carry out. Because the main components of cells are proteins and water, fixation is mainly carried out by coagulation and denaturation of proteins or freezing of water. The former is fixed by fixing solution or heating, and the latter is fixed by rapid cooling.

[0003] Fixatives are classified into aldehyde fixatives, mercury fixatives, alcohol fixatives, oxidant fixatives, picrate fixatives, etc. The more commonly used ones are formalin among aldehydes and ethanol among alcohols. However, formaldehyde has a pungent odor that can be smelled even at low concentrations. Its main hazard is irritation to the skin and mucous membranes.

[0004] The components of the current fixative are 160 mL of methanol, 30 mL of formaldehyde and 10 mL of glacial acetic acid. During the experiment, due to the high concentration of formaldehyde in the fixative, there is often a pungent smell, and the glacial acetic acid is volatile, which can cause irreversible damage such as corrosion to the respiratory tract of the experimenter. Summary of the invention

[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a cytopathology slide fixative.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned fixing liquid.

[0007] In order to achieve the first purpose, the present invention is implemented through the following technical scheme: a cytopathology slide fixative, based on 1kg mass, includes 800-900g of alcohol, 50-80g of polyethylene glycol, 10-50g of formaldehyde, and the remainder of phosphate buffer.

[0008] By adopting the above technical scheme, the fixing liquid does not contain glacial acetic acid, thereby avoiding the harm of glacial acetic acid volatilization to the human body, and the formaldehyde content added in the present application is low. After experimental verification, the formaldehyde volatility of the fixing liquid of the present invention is very low, and its volatilization concentration is much lower than that of the formalin fixing liquid available on the market.

[0009] Preferably, the alcohols include ethanol, n-butanol and tert-butanol.

[0010] Preferably, the mass ratio of the ethanol, n-butanol and tert-butanol is 2-3:0.5-1:1-2.

[0011] By adopting the above technical scheme, the fixation effect of the fixative is improved through the combination of the above three alcohol substances, the specimen has good elasticity, and the structure is well preserved. In the later coloring process, the coloring effect is excellent. Through experimental comparison, the fixation effect of the fixative obtained by the combination of the three is better than the fixation effect of the fixative obtained by using only one alcohol.

[0012] Preferably, the ethanol is 95% ethanol.

[0013] Preferably, the phosphate buffer is disodium hydrogen phosphate and sodium dihydrogen phosphate, and the mass ratio of the disodium hydrogen phosphate to the sodium dihydrogen phosphate is 1-2:2-3.

[0014] By adopting the above technical solution, a phosphate buffer is used as a buffer, which provides a good solution environment for the fixing solution.

[0015] Preferably, the pH of the fixing solution is 6.5-7.0.

[0016] In order to achieve the second object, the present invention is implemented by the following technical solution: a method for preparing a cytopathology slide fixative, comprising the following steps:

[0017] S1: 95% ethanol, n-butanol and tert-butanol are mixed according to a mass ratio to obtain an alcohol solution;

[0018] S2: adding polyethylene glycol and formaldehyde to the alcohol solution obtained in step S1 in sequence, heating and stirring;

[0019] S3: Add phosphate buffer to the solution obtained in step S2, heat, and stir to obtain a stationary solution.

[0020] Preferably, the heating temperature in step S2 and step S3 is 30-50° C., and the stirring conditions in step S2 and step S3 are: rotation speed 100-200 r / min, stirring 5-10 min.

[0021] Preferably, the fixative is used for fixation of animal, plant, bacterial, fungal and cell samples.

[0022] Preferably, the fixative is used for pathological tissue preparation and fixation of paraffin tissue section staining, and the fixative is used for conventional pathological detection, immunohistochemical detection, and molecular biology detection.

[0023] Beneficial effects of the present invention:

[0024] (1) Remove the glacial acetic acid from the original fixative to reduce the proportion of formaldehyde and reduce the damage caused by acidic gas to personnel during experimental work.

[0025] (2) The present application improves the fixation effect of the fixative by combining 95% ethanol, n-butanol and tert-butanol. DETAILED DESCRIPTION

[0026] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0027] Example 1

[0028] A cytopathology slide fixative comprises 800 g of alcohol, 80 g of polyethylene glycol, 50 g of formaldehyde, and a phosphate buffer added to 1 kg.

[0029] In this embodiment, the alcohols are 95% ethanol, n-butanol and tert-butanol in a mass ratio of 2:0.5:1.

[0030] In this embodiment, the phosphate buffer is disodium hydrogen phosphate and sodium dihydrogen phosphate in a mass ratio of 1:2.

[0031] In this embodiment, the pH of the stationary phase is 7.0.

[0032] A method for preparing a cytopathology slide fixative comprises the following steps:

[0033] S1: 95% ethanol, n-butanol and tert-butanol are mixed according to a mass ratio to obtain an alcohol solution;

[0034] S2: adding polyethylene glycol and formaldehyde to the alcohol solution obtained in step S1 in sequence, heating and stirring;

[0035] S3: Add phosphate buffer to the solution obtained in step S2, heat, and stir to obtain a stationary solution.

[0036] In this embodiment, the heating temperature in step S2 and step S3 is 30° C., and the stirring conditions in step S2 and step S3 are: rotation speed 200 r / min, stirring for 10 min.

[0037] Example 2

[0038] A cytopathology slide fixative, which is different from Example 1 in that: 900 g of alcohol, 50 g of polyethylene glycol, and 30 g of formaldehyde.

[0039] In this embodiment, the alcohols are 95% ethanol, n-butanol and tert-butanol in a mass ratio of 2:1:2.

[0040] In this embodiment, the phosphate buffer is disodium hydrogen phosphate and sodium dihydrogen phosphate in a mass ratio of 1:3.

[0041] In this embodiment, the pH of the stationary phase is 6.8.

[0042] A method for preparing a cytopathology slide fixative is different from that of Example 1 in that: the heating temperature in step S2 and step S3 is 40° C., and the stirring condition in step S2 and step S3 is: a rotation speed of 150 r / min.

[0043] Example 3

[0044] A cytopathology slide fixative, which is different from Example 1 in that: 850 g of alcohol and 30 g of formaldehyde.

[0045] In this embodiment, the alcohols are 95% ethanol, n-butanol and tert-butanol in a mass ratio of 3:1:1.

[0046] In this embodiment, the phosphate buffer is disodium hydrogen phosphate and sodium dihydrogen phosphate in a mass ratio of 1:1.

[0047] A method for preparing a cytopathology slide fixative is different from that of Example 1 in that the heating temperature in step S2 and step S3 is 50° C., and the stirring condition in step S2 and step S3 is a rotation speed of 100 r / min.

[0048] Example 4

[0049] A cytopathology slide fixative, which is different from Example 1 in that: 60 g of polyethylene glycol and 40 g of formaldehyde.

[0050] In this embodiment, the phosphate buffer is disodium hydrogen phosphate and sodium dihydrogen phosphate in a mass ratio of 2:3.

[0051] In this embodiment, the pH of the stationary phase is 6.5.

[0052] A method for preparing a cytopathology slide fixative is different from that of Example 1 in that the stirring condition in step S2 and step S3 is: stirring for 5 minutes.

[0053] Example 5

[0054] A cytopathology slide fixative, which is different from Example 1 in that it comprises 850 g of alcohol and 70 g of polyethylene glycol.

[0055] In this embodiment, the alcohols are 95% ethanol, n-butanol and tert-butanol in a mass ratio of 3:1:2.

[0056] A method for preparing a cytopathology slide fixative is the same as that in Example 1.

[0057] Test Example 1 Volatility Determination

[0058] Test groups: Comparative Example 1, Examples 1-5; wherein Comparative Example 1 uses commercially available 10% formalin fixative (formaldehyde content 3.6%).

[0059] Test method: Take 500ML of the products of Comparative Example 1 and Examples 1-5, respectively, and place them in wide-mouth bottles of the same caliber, open them and place them in a closed space (0.5m×0.5m×0.5m), let them stand for 3 minutes, and then conduct sampling and analysis according to GBZ 159-2004 and GBZ / T160.54-2004 "Determination of Aliphatic Aldehyde Compounds in Air Toxic Substances in the Workplace".

[0060] Test results: See Table 1 for details.

[0061] Table 1 Comparative Example 1, Example 1-5 Product Volatility Test Results

[0062]

[0063] As can be seen from Table 1, the product prepared by the present invention has very low volatility, and its volatility concentration is much lower than the commercially available formalin fixative, and the effect is very significant.

[0064] Test Example 2 Fixation Effect and Stability Test

[0065] Test groups: Comparative Example 1, Examples 1-3

[0066] Test method:

[0067] (1) Fixation effect test: Take fresh pig liver tissue, cut an area of ​​1 cm×1 cm and a thickness of 0.4 cm; soak the cut tissue in the fixative of Comparative Example 1 and Examples 1-3, respectively, with the amount of fixative being 8 times the volume of the tissue, fix and stain for 5 hours, perform routine sectioning according to pathological examination, and observe under a microscope.

[0068] (2) Stability test: After the fixatives of Comparative Example 1 and Examples 1-3 were placed at 37° C. for 10 days, the pig liver tissue was fixed according to the above-mentioned fixation effect test method to further verify the stability of the fixative.

[0069] Test results: See Table 2 for details.

[0070] Table 2 Results of fixation effect and stability test of fixatives of Comparative Example 1 and Examples 1-3

[0071]

[0072] With reference to Table 2, the specimen after the fixed staining of the tissue fixative of 1-3 in the embodiment, observed under a microscope after sectioning, its result is suitable with commercially available formalin fixation effect, has improved the accuracy of the test result judgment. And after fixing 5h, the effect is still good, showing that the tissue fixative of the present invention is evenly fixed, and painted lasting. Meanwhile, by stability test, after the fixative was placed for 10 days, its fixative performance was still stable, and the fixing effect did not deteriorate.

[0073] Experimental Example 3 Effect of the Combination of Ethanol, N-Butanol and T-Butanol on the Fixation Effect

[0074] Test groups: Example 1, Comparative Examples 2-4, wherein the alcohol in Comparative Example 2 is 95% ethanol, the alcohol in Comparative Example 3 is n-butanol, the alcohol in Comparative Example 4 is tert-butanol, and the others are the same as in Example 1.

[0075] Test method: Take fresh pig liver tissue, cut an area of ​​1cm×1cm and a thickness of 0.4cm; soak the cut tissue in the fixative of Example 1 and Comparative Examples 2-4 respectively, the amount of fixative is 8 times the volume of the tissue, fix and stain for 5 hours, perform routine sectioning according to pathological examination and observe under a microscope.

[0076] Test results: See Table 3 for details.

[0077] Table 3 Effect of the combination of ethanol, n-butanol and tert-butanol on the fixation effect

[0078] Fixed effects Comparative Example 2 Pig liver tissue is elastic, well-preserved in structure, slightly contracted, and clearly colored, allowing for identification of small tissues. Comparative Example 3 Pig liver tissue has poor elasticity, well-preserved structure, slight shrinkage, and poor coloration Comparative Example 4 The liver tissue has poor elasticity, well-preserved structure, no shrinkage, obvious coloration, and small tissues can be identified. Example 1 The liver tissue is elastic, well-preserved, has no shrinkage, is clearly colored, and small tissues can be identified.

[0079] Referring to Table 3, through the combination of the above three alcohol substances, the fixation effect of the fixative is improved, the specimen has good elasticity, and the structure is well preserved. In the later coloring process, the coloring effect is excellent. Through experimental comparison, the fixation effect of the fixative obtained by the combination of the three is better than the fixation effect of the fixative obtained by using only one alcohol.

[0080] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0081] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A cytopathology slide fixative, characterized in that: According to 1kg mass, it includes 800-900g of alcohol, 50-80g of polyethylene glycol, 30-50g of formaldehyde, and the balance of phosphate buffer, wherein the alcohol includes ethanol, n-butanol and tert-butanol; The mass ratio of the ethanol, n-butanol and tert-butanol is 2-3:0.5-1:1-2, the ethanol is 95% ethanol, the phosphate buffer is disodium hydrogen phosphate and sodium dihydrogen phosphate, the mass ratio of disodium hydrogen phosphate and sodium dihydrogen phosphate is 1-2:2-3, and the pH of the fixing solution is 6.5-7.

0.

2. A method for preparing the fixing liquid as claimed in claim 1, characterized in that: The steps include: S1: 95% ethanol, n-butanol and tert-butanol are mixed according to a mass ratio to obtain an alcohol solution; S2: adding polyethylene glycol and formaldehyde to the alcohol solution obtained in step S1 in sequence, heating and stirring; S3: Add phosphate buffer to the solution obtained in step S2, heat, and stir to obtain a stationary solution.

3. The method for preparing a cytopathological slide fixative as claimed in claim 2, characterized in that: The heating temperature in step S2 and step S3 is 30-50° C., and the stirring conditions in step S2 and step S3 are: rotation speed 100-200 r / min, stirring 5-10 min.

4. The fixing liquid according to claim 1, characterized in that The fixative is used for fixing animal, plant, bacterial, fungal and cell samples.

5. The fixing liquid according to claim 1, characterized in that The fixing solution is used for pathological tissue preparation and fixation of paraffin tissue section staining. The fixing solution is used for conventional pathological detection, immunohistochemical detection, and molecular biological detection.

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