Preservative

Abstract

To provide a preservative solution that ensures biological safety, excels in specimen preservation, offers excellent workability, and allows for the specimen to be held in any position and shape, and to provide a method for preparing a specimen using the preservative solution.SOLUTION: A preservative solution comprises smectite dispersed in an aqueous medium. A method for preparing a specimen includes immersing the specimen in the preservative solution.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a preservation solution and a method for producing a specimen.

Background Art

[0002] When preparing a biological specimen, a 10-20% formalin solution is generally used as a fixative and / or preservation solution to fix the specimen target and prevent the decay of biological tissues. On the other hand, the formalin solution is a solution in which highly toxic formaldehyde and the like are dissolved in water, and the formalin solution itself is recognized as carcinogenic, so there is a demerit that it is very difficult to handle. Regarding disposal, after adding a large amount of water to the formalin solution to make a dilute aqueous solution, a treatment of adding an aqueous hypochlorite solution for decomposition treatment, or a treatment of making it alkaline with an aqueous sodium hydroxide solution or the like, adding hydrogen peroxide water for decomposition, and diluting with a large amount of water for treatment is required.

[0003] Furthermore, formaldehyde is highly volatile, and there is concern about the influence on the human body during specimen preparation by inhaling the volatilized formaldehyde even without direct contact with the formalin solution. As a method for reducing the amount of formaldehyde volatilized from the formalin solution used as a fixative during the preparation of resin-embedded specimens used for pathological specimens, for example, in Patent Document 1, a formaldehyde absorption sheet provided with a cloth containing highly absorbent fibers is used, and the formaldehyde absorption sheet is preliminarily laid on a cutting board, and a specimen immersed in the formalin solution is taken out and placed on the formaldehyde absorption sheet, and a small piece is cut out from the specimen on the absorption sheet. A method of using a formaldehyde absorption sheet is disclosed.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

[0005] In addition to the toxicity of formalin solution mentioned above, formalin solution has other disadvantages when used to prepare biological specimens. Formalin can fix specimens and prevent decay by forming cross-linked structures between proteins via aldehyde groups, but the strong cross-linking reaction can cause deformation of the specimen's appearance. Also, immersion in formalin causes the pigments of the specimen to fade, making it unsuitable for observing the specimen's colors over long periods. Furthermore, commonly used formalin solution is a solution containing 4-8% formaldehyde and methanol, resulting in low viscosity. When using formalin solution as a preservation solution to prepare liquid-immersed specimens, there is a problem that the specimen will sink, making it impossible to maintain the desired position and shape.

[0006] The present invention aims to provide a preservation solution that is highly biosafe, offers excellent preservation properties for specimens, and is highly workable, allowing specimens to be held in any position and shape, as well as a method for preparing specimens using the preservation solution. [Means for solving the problem]

[0007] In view of the above problems, the inventors conducted thorough research. As a result, they found that when they prepared a liquid-immersion specimen using a dispersion of smectite, which has a long track record of use in living organisms and is highly safe, in an aqueous medium, it exhibited transparency that allowed observation of the specimen inside, provided excellent preservation of the specimen, and allowed the specimen to be fixed in any position due to the thixotropy of the dispersion, and also provided excellent leakage prevention. In other words, they found that the above dispersion has suitable properties as a preservation solution to replace formalin. This invention was completed after further consideration based on these findings.

[0008] The above-mentioned problems of the present invention were solved by the following means. [1] A preservative solution comprising smectite dispersed in an aqueous medium. [2] The preservative solution according to [1], wherein the preservative solution contains alcohol. [3] The preservative solution according to [2], wherein the alcohol contains a polyhydric alcohol. [4] The preservative solution according to [3], wherein the polyhydric alcohol comprises at least one selected from glycerin, ethylene glycol, propylene glycol, polyethylene glycol, and polypropylene glycol. [5] The preservation solution according to [4], wherein the smectite is one or more selected from montmorillonite, byderite, nontronite, saponite, hectorite, soakonite, and stivunsite. [6] The preservation solution according to [5], wherein the particle size of the smectite is 10 to 500 nm. [7] The preservative solution according to any one of [3] to [6], wherein the content of the polyhydric alcohol in the preservative solution is 10 to 50% by mass. [8] The preservative solution according to any one of [3] to [6], wherein the smectite content in the preservative solution is 0.5 to 10% by mass. [9] A preservation solution for specimen preparation, as described in any of [3] to [6].

[10] A preservation solution according to any one of [3] to [6], which is used by immersing the object to be preserved in the aforementioned preservation solution.

[11] A method for producing a specimen, comprising immersing the specimen in any of the preservation solutions described in [3] to [6]. [Effects of the Invention]

[0009] The preservation solution of the present invention offers excellent biosafety and can preserve the object to be preserved. Furthermore, when the preservation solution of the present invention is used for specimen preparation, it has transparency that allows observation of the specimen placed inside the preservation container, and also offers excellent leakage prevention and position retention of the specimen. Furthermore, according to the specimen manufacturing method of the present invention, specimens possessing the above-mentioned characteristics can be obtained. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a photograph used as a substitute for a drawing, showing a specimen prepared using the preservation solution of the present invention. [Modes for carrying out the invention]

[0011] The following describes preferred embodiments of the present invention in detail, but the present invention is not limited to these forms other than those specified herein.

[0012] [Preservation solution] The preservation solution of the present invention is a dispersion obtained by dispersing smectite in an aqueous medium. By using the preservation solution of the present invention, objects to be preserved can be effectively preserved. In this invention and specification, "preservation" by the preservation solution is used in a broader sense than usual. That is, in addition to maintaining the appearance of the object to be preserved before preservation, even if changes in the appearance of the object to be preserved (spoilage, deterioration, discoloration, etc.) occur, if the preservation solution has an inhibitory effect on such changes, it is included in the concept of "preservation" in this invention and specification.

[0013] The preservation solution of the present invention can be used, for example, as a preservation solution for specimen preparation. Figure 1 is a photographic representation of an example of a specimen prepared using the preservation solution of the present invention. The preservation solution used for the specimen on the left side of FIG. 1 is a preservation solution composed of 2.6% by mass of smecton - ST (synthetic stibnite site), 43.9% by mass of glycerin, and 53.5% by mass of water. This is the specimen after 97 days at 25°C after immersing Aomemezo in the preservation solution. Also, the preservation solution used for the specimen on the right side of FIG. 1 is a preservation solution composed of 1.7% by mass of smecton - SWF (synthetic hectorite), 44.2% by mass of ethylene glycol, and 54.1% by mass of water. This is the specimen after 145 days at 25°C after immersing Aomemezo in the preservation solution. In both specimens, the appearance of Aomemezo, which is the specimen target, has been preserved over a long period. Note that the composition of the preservation solution of the present invention is not limited to the composition of the preservation solution used for the specimen in FIG. 1, except as defined in the present invention. In addition to the above smectite, the preservation solution of the present invention may contain various compounds within a range that does not prevent the effects of the present invention. Each component that constitutes or can constitute the preservation solution of the present invention will be described below.

[0014] (Smectite) In the preservation solution of the present invention, the smectite is considered to exhibit an action of suppressing changes in the appearance of the preservation target (such as changes in appearance that occur over time with preservation). Also, since the dispersion has thixotropy, the viscosity can be reduced by applying shear stress, resulting in excellent workability. Moreover, when left standing, the viscosity can be increased to make it substantially solid, and the position of the specimen target can be fixed at any desired location.

[0015] The type of the smectite contained in the preservation solution of the present invention is not particularly limited and can be appropriately set according to the purpose. Smectite itself is known and is also commercially available. The smectite contained in the preservation solution of the present invention may be natural smectite or synthetic smectite. From the perspective of ensuring the transparency of the preservation solution, it is preferable that the smectite is synthetic smectite. Furthermore, the smectite is preferably one or more selected from montmorillonite, beiderite, nontronite, saponite, hectorite, soakonite, and stivunsite, and more preferably one or more selected from montmorillonite, beiderite, nontronite, saponite, hectorite, soakonite, and stivunsite.

[0016] In this invention, "smectite" refers to smectite in particulate form. More specifically, the smectite preferably has an average particle diameter (average primary particle diameter) of 10 to 500 nm, more preferably 30 to 400 nm, even more preferably 40 to 380 nm, and particularly preferably 50 to 370 nm. By setting the average particle diameter of the smectite within the above preferred range, the preservation solution of this invention can be made substantially transparent, and an appropriate viscosity or thixotropy can be imparted to the preservation solution. In this invention, the "average particle diameter" of the smectite in the aqueous dispersion refers to the volume-based median diameter. This average particle diameter can be determined, for example, by a laser diffraction / scattering particle size distribution analyzer.

[0017] The intercalated cation species of smectite used in this invention are not particularly limited. From the viewpoint of easy water swelling when used as an aqueous dispersion or aqueous paste, monovalent metal ions are preferred, and lithium ions and / or sodium ions are more preferred. Furthermore, the cation exchange capacity (CEC) of the smectite is preferably 20 meq (milliequivalent) / 100g or more, more preferably 25 meq / 100g or more, and even more preferably 30 meq / 100g or more, from the viewpoint of improving swelling properties during water dispersion. In addition, the cation exchange capacity of the smectite used in the present invention is usually 250 meq / 100g or less.

[0018] There are no particular restrictions on the smectite content in the preservation solution of the present invention, but from the viewpoint of preservation properties (preservation properties, color preservation properties, etc.), viscosity, positional fixation of the specimen, and maintaining transparency that allows observation of the interior, the content is preferably 0.5 to 10% by mass. By setting the smectite content to 0.5% by mass or more, the preservation effect of smectite and the positional fixation due to the thickening effect can be fully exhibited. Furthermore, by setting the smectite content to 10% by mass or less, the workability of the preservation solution can be improved, and transparency can be maintained so that the inside of the preservation container can be observed. From the same viewpoint as above, it is more preferable that the content be 1 to 7% by mass, and even more preferable that be 1.5 to 5% by mass.

[0019] (aqueous medium) In the preservation solution of the present invention, the smectite is dispersed in an aqueous medium. The aqueous medium is preferably water or an aqueous solution. In the case of an aqueous solution, examples of water-soluble components other than water that constitute the aqueous medium include various organic solvents such as alcohols, ester compounds, ketone compounds, aromatic compounds, and carbonate compounds. There are no particular restrictions on the water used in the aqueous medium described above. In addition to ordinary tap water, purified water such as distilled water or ion-exchanged water, from which ionic components have been removed, can also be used. The water is preferably water from which ions have been removed, in order to rapidly exfoliate and swell the smectite and to increase viscosity and impart antiseptic properties. Specifically, the ionic conductivity of the water is preferably 10 μS / m or less, more preferably 5 μS / m or less, and even more preferably 2 μS / m or less. Furthermore, dissolved oxygen may be removed from the water in the aqueous medium by subjecting it to reduced pressure treatment or inert gas bubbling.

[0020] The preservation solution of the present invention preferably contains alcohol along with water in its aqueous medium. The inclusion of alcohol in the preservation solution of the present invention further improves its preservative properties and allows it to maintain transparency for a longer period. When the preservation solution of the present invention contains alcohol, the smectite is dispersed in the alcohol-containing aqueous medium to form the preservation solution. There are no particular restrictions on the molecular weight of the alcohol; lower alcohols with 5 or fewer carbon atoms and higher alcohols with 6 or more carbon atoms can be used. Furthermore, the alcohol may have other functional groups besides hydroxyl groups, such as amino groups, carboxyl groups, or ketone groups, and may also have ester bonds, urethane bonds, urea bonds, imide bonds, acid anhydride bonds, etc.

[0021] The alcohol may be a monohydric alcohol or a dihydric or more hydric alcohol (polyhydric alcohol), and it is preferable that it contains a polyhydric alcohol. Examples of monohydric alcohols that can be used in the preservation solution of the present invention include water-soluble alcohols such as methanol, ethanol, 1-propanol, and 2-propanol, and amino alcohols such as ethanolamine. The inclusion of a polyhydric alcohol in the alcohol makes it less likely to cause denaturation of the proteins constituting the object to be preserved compared to the case where a polyhydric alcohol is not included, and the appearance of the object to be preserved can be maintained for a long period of time. It is also preferable that all of the alcohol is a polyhydric alcohol. The polyhydric alcohol is not particularly limited as long as it is a compound that has two or more hydroxyl groups in one molecule and can form an aqueous solution, and examples include glycerin, ethylene glycol, propylene glycol, polyethylene glycol, and polypropylene glycol, sugars, polysaccharides, sugar alcohols, polyvinyl alcohol and its derivatives. In particular, from the viewpoint of having a high preservative effect and being less susceptible to changes in the object to be preserved due to long-term storage, it is preferable that the polyhydric alcohol contains at least one selected from glycerin, ethylene glycol, propylene glycol, polyethylene glycol, and polypropylene glycol.

[0022] In the preservation solution of the present invention, there are no particular restrictions on the content of the alcohol (preferably the polyhydric alcohol), but from the viewpoint of preservation and maintaining the transparency of the preservation solution, it is preferably 10 to 50% by mass. By setting the content to 10% by mass or more, the preservation properties of the preservation solution can be further improved, and the transparency of the preservation solution can be maintained for a longer period of time. Furthermore, by setting the content to 50% by mass or less, sufficient preservation and thickening properties can be achieved without impairing the dispersibility of the smectite in aqueous media, and the fading of the pigment of the specimen caused by the alcohol can be reduced. From the same viewpoint as above, it is more preferable that the content be 15 to 47% by mass.

[0023] (Other ingredients) The preservative solution of the present invention may contain other components as long as they do not impair the effects of the present invention. For example, known and commonly used additives such as dispersants, surfactants, defoamers, wetting agents, polymer materials, organic solvents, thickeners, and inorganic salts may be used. Furthermore, various water-soluble or water-dispersible preservatives, fungicides, etc. may be included for the purpose of further improving preservation.

[0024] [Method for producing preservative solution] The preservation solution of the present invention can be obtained by mixing smectite in an aqueous medium while simultaneously detaching and dispersing it. Known and conventional methods can be used to detach and disperse the smectite. If the preservation solution of the present invention contains alcohol, the alcohol may be mixed with the smectite dispersion (dispersion), or the alcohol may be mixed with water or the like beforehand, and the preservation solution of the present invention may be obtained by dispersing the smectite in this mixture.

[0025] [Method of producing specimens] A specimen (liquid-immersed specimen) can be produced by immersing the specimen (object to be preserved) in the preservation solution of the present invention. The specimen may be immersed in the preservation solution as is after collection, or it may be fixed with formalin or the like before being immersed in the preservation solution. It is preferable that the entire specimen is immersed in the preservation solution of the present invention.

[0026] The preservation solution of the present invention exhibits thixotropy. Therefore, from the viewpoint of workability, such as filling a preservation container with the preservation solution, it is preferable to reduce the viscosity of the preservation solution by applying shear stress, such as shaking or stirring, to the preservation solution when immersing the specimen object in the preservation solution of the present invention. It is also preferable to immerse the specimen object in the preservation solution after the viscosity of the preservation solution has been reduced by the above process. By allowing the specimen object to stand after immersion, the viscosity of the preservation solution increases, so that the position of the specimen object in the preservation solution and the shape of the specimen object can be appropriately adjusted and fixed.

[0027] (Sampling target / Preservation target) When preparing specimens using the preservation solution of the present invention, there are no particular restrictions on the specimen subject (the preservation subject whose appearance is to be suppressed by the preservation solution of the present invention), and animals and plants can be used as specimen subjects and preservation subjects. If the subject is an animal, it may be the whole animal, or it may be a part of the animal such as living tissue or organs. For example, a specimen subject that is conventionally preserved by formalin immersion can be used as a specimen subject for specimens using the preservation solution of the present invention. From the viewpoint of liquid-immersed specimens, the preservation solution of the present invention can be suitably used for animals with a high water content in their bodies, and can be suitably used for vertebrates such as mammals, birds, reptiles, amphibians, and fish, as well as animals other than vertebrates that do not have an exoskeleton (insect larvae, parasites, etc.). [Examples]

[0028] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0029] [Preparation of preservation solution] (Example 1) In a sealed container, smecton-ST (product name: synthetic stibnsite, manufactured by Kunimine Industries Co., Ltd.) and distilled water were added in the proportions (composition, mass%) shown in Table 1 to a total of 250g. This was stirred for 5 minutes at a rotation speed of 2000 rpm using a rotation-revolution type mixer (product name: Awatori Rentaro, model number: ARE-310, manufactured by Shinky Co., Ltd.), and then the stirred mixture was allowed to stand for 24 hours to obtain the preservation solution of Example 1 containing 0.83% by mass of smectite.

[0030] (Examples 2-10) The preservatives of Examples 2 to 10 were obtained in the same manner as in Example 1, except that the formulations were as shown in Table 1 below.

[0031] (Comparative Example 1) Distilled water was used as the solution in Comparative Example 1. (Comparative Examples 2 and 3) Distilled water and water-soluble components were mixed in the ratios shown in Table 2, totaling 100 g, in a 200 mL beaker and stirred for 5 minutes using a stirrer. The resulting homogeneous aqueous solution was used as the solution for Comparative Examples 2 and 3. (Comparative Example 4) Isopropyl alcohol was used as the solution in Comparative Example 4.

[0032] Details of the solutions used in Examples 1-10 and Comparative Examples 1-4 are shown below. (Smectite) • Smecton-ST (Product name: Synthetic stivunsite, manufactured by Kunimine Industries Co., Ltd., particle size: 35 nm) • Smecton-SWN (Product name: Synthetic hectorite, manufactured by Kunimine Industries Co., Ltd., particle size: 70 nm) • Smecton-SWF (Product name: Synthetic hectorite, manufactured by Kunimine Industries Co., Ltd., particle size: 80 nm) • Smecton-SA (product name, synthetic saponite, manufactured by Kunimine Industries Co., Ltd., particle size: 100 nm) In all smectites, the interlayer cation is a sodium ion. (water) • Distilled water: Ionic conductivity 0.5 μS / cm (Water soluble component) -Polyhydric alcohols- • Ethylene glycol (99.5%, special grade reagent, manufactured by Kanto Chemical Co., Ltd.) Glycerin (99.5%, special grade reagent, manufactured by Kanto Chemical Co., Ltd.) • Polyethylene glycol 200 (99.5%, reagent grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) -Monohydric alcohol- • Isopropyl alcohol (99.5%, special grade reagent, manufactured by Kanto Chemical Co., Ltd.) Furthermore, in Table 1 below, if the above-mentioned water-soluble component includes water, that water shall be considered to constitute the water in Table 1 below.

[0033] [Physical property testing of preservative solution] (Evaluation of light transmittance) Each solution from Examples 1-10 and Comparative Examples 1-4 was filled into a 1 cm wide transparent plastic cell to prepare light transmittance evaluation samples. The light transmittance of each light transmittance evaluation sample at a wavelength of 500 nm was measured using a spectrophotometer ASV11D-H (manufactured by AS ONE Corporation). The results are shown in Table 1 below.

[0034] (Evaluation of leak prevention when the container is tilted) For each of the solutions in Examples 1-10 and Comparative Examples 1-4, 85g was transferred to a 100ml screw-cap vial (AS ONE NO.8, NO.9-852-10, container liquid-filled section height 10.1cm, container material glass). The vial was then tilted to 45° to check for leakage of the liquid inside. A "○" was given if the liquid inside the container was retained and did not leak, and a "×" was given if the liquid inside the container flowed and leaked. The results are shown in Table 1 below.

[0035] [Performance testing of specimens] Each of the aforementioned containers (screw-cap bottles) containing the liquids of Examples 1-10 and Comparative Examples 1-4 was sealed with a blue-eyed fish (commonly known as mehikari) from Fukushima Prefecture, measuring 9 cm or less in length, for the purpose of preparing fresh fish specimens. At that time, the blue-eyed fish were positioned in the center of the container so that they did not come into contact with the outer perimeter (walls) of the container. The obtained specimens were left to stand in a room air-conditioned at 25°C and the following evaluation tests were performed.

[0036] (Evaluation of position retention) The positional stability of the specimens was visually observed 14 days after preparation. Specifically, a "○" indicated that the specimen was not touching the container wall 14 days after preparation, while a "×" indicated that the specimen was touching the container wall 14 days after preparation. The results are shown in Table 1 below.

[0037] (Evaluation of storage properties) After preparing the specimens, the condition of the blue-eyed fish inside the containers was observed over time. If decay, deterioration, and / or significant discoloration of the blue-eyed fish were observed within one week of specimen preparation, it was judged as "×". If decay, deterioration, and / or significant discoloration were observed between one week and four weeks after specimen preparation, it was judged as "△". If decay, deterioration, and / or significant discoloration were observed between four weeks and eight weeks after specimen preparation, it was judged as "〇". If no decay, deterioration, and / or significant discoloration were observed after eight weeks after specimen preparation, it was judged as "◎". The results are shown in Table 1 below.

[0038] [Table 1]

[0039] In Table 1, "◎※" indicates that the eyeball became cloudy within four weeks after one week had passed since the specimen was prepared, but no decay, deterioration, or significant discoloration was observed in the rest of the specimen eight weeks after the specimen was prepared.

[0040] In Comparative Examples 1-4, which did not contain smectite, the light transmittance was higher than that of Examples 1-10, but the leakage prevention performance was inferior. Furthermore, even when specimens were prepared using the solutions of Comparative Examples 1-4, the position retention and preservation performance were all inferior. Furthermore, the blue-eyed fish immersed in the solution of Comparative Example 1 completely decomposed in a short period of time, and its tissues disintegrated within one week of specimen preparation. The blue-eyed fish immersed in the solutions of Comparative Examples 2 and 4 contracted in volume immediately after specimen preparation, as if dehydrated, and a cloudy solid substance formed and settled on the surface of the specimen. The eyes of the specimen also turned white. The blue-eyed fish immersed in the solution of Comparative Example 3 underwent volume swelling within one week of specimen preparation, and its appearance deteriorated significantly.

[0041] In contrast, the preservation solutions of each of Examples 1 to 10, which contained smectite, were transparent enough to allow observation of the inside of the preservation container, and also had good leakage prevention properties. Furthermore, when specimens were prepared using the preservation solutions of Examples 1 to 10, all specimens exhibited excellent positional retention, and no decay, deterioration, or significant discoloration was observed for at least one week after preparation, demonstrating their preservation properties. Moreover, when specimens were prepared using the preservation solutions of Examples 6 to 10, which contain alcohol, the preservation properties were improved compared to specimens prepared using the preservation solutions of Examples 1 to 5.

Claims

1. A preservative solution comprising smectite dispersed in an aqueous medium, A preservative solution containing alcohol.

2. The preservative solution according to claim 1, wherein the alcohol contains a polyhydric alcohol.

3. The preservative solution according to claim 2, wherein the polyhydric alcohol comprises at least one selected from glycerin, ethylene glycol, propylene glycol, polyethylene glycol, and polypropylene glycol.

4. The preservative solution according to claim 3, wherein the smectite is one or more selected from montmorillonite, byderite, nontronite, saponite, hectorite, soakonite, and stivunsite.

5. The preservative solution according to claim 4, wherein the particle size of the smectite is 10 to 500 nm.

6. The preservative solution according to any one of claims 2 to 5, wherein the content of the polyhydric alcohol in the preservative solution is 10 to 50% by mass.

7. The preservative solution according to any one of claims 2 to 5, wherein the content of the smectite in the preservative solution is 0.5 to 10% by mass.

8. A preservation solution for specimen preparation, as described in any one of claims 2 to 5.

9. The preservation solution according to any one of claims 2 to 5, which is used by immersing the object to be preserved in the preservation solution.

10. A method for producing a specimen, comprising immersing the specimen in a preservation solution described in any one of claims 2 to 5.

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