WO1996030738A1

WO1996030738A1 - Compositions and methods for permanently mounting biological specimens

Info

Publication number: WO1996030738A1
Application number: PCT/US1996/004274
Authority: WO
Inventors: Zahra P. Naser
Original assignee: Innovex Biosciences
Priority date: 1995-03-28
Filing date: 1996-03-28
Publication date: 1996-10-03

Abstract

The present invention provides compositions and methods for permanently mounting biological specimens. The mounting compositions of the present invention can be used for mounting stained tissue sections or cell smears for long-term storage. Such mounting compositions provide excellent optical clarity and resolution and, thus, the stained specimens mounted with the compositions of the present invention have greater transparency and visibility for microscopic examination. Preferably, the mounting compositions are aqueous based and, thus, slides do not require pre-mounting treatments such as dehydration in alcohol or clearing in xylene. The slides can be mounted directly from water. Moreover, the mounting compositions of the present invention are compatible with all aqueous and organic-soluble chromogens and dyes (e.g., AEC, Fast Red and Fast Garnet stains). In addition, because of their aqueous, non-toxic and non-hazardous nature, the mounting compositions of the present invention can be used in laboratory settings without a hood or other precautions.

Description

COMPOSITIONS AND METHODS FOR PERMANENTLY MOUNTING BIOLOGICAL SPECIMENS

BACKGROUND OF THE INVENTION The proper mounting and preservation of stained tissue sections and cell smears for microscopic examination is becoming increasingly important in view of the current requirements that pathological specimens be stored for twenty years. The process of mounting a biological specimen generally involves the application of a coverslip onto a microscope slide having a tissue section or cell smear thereon with the help of an adhesive agent known as the mounting medium. The mounting of a biological specimen in this manner protects the biological specimen and, in addition, lends the slide to microscopic examination with any objective lens of the microscope. The type of mounting medium used determines the permanency or non-permanency of the preservation as well as the longevity and optical clarity of the dyes and chromogens used in staining the biological specimen for microscopic examination. As such, the preservation of the biological specimen and the success or failure of the microscopic examination depends in large part on the type of mounting medium employed.

Currently, two types of mounting media are available for mounting a coverslip onto a microscope slide having thereon a tissue section or cell smear; they are the resinous type and the aqueous type. In the resinous type of mounting media, natural resins, such as Canada balsam and gum dammar, were initially used for mounting. Unfortunately, these natural resins were usually dissolved in xylene or toluene and, thus, they set and harden very slowly, often taking up to a few months for the stickiness to go away. In addition, such natural resins yellowed with time. Synthetic resins have now replaced the use of natural resins because they harden faster and do not yellow as much with age. However, such resinous type mounting media are not suitable for mounting biological specimens that have been stained with alcohol or organic soluble dyes and chromogens. For example, it has been determined that resinous mounting media cause gradual fading of the blue component of Romanowsky stains and in order to preserve this type of stain, the mounting process must be carried out with the use of heavy mineral oil which makes the process quite messy. In addition, it has been determined that resinous mounting media cannot be used for mounting biological specimens that are stained with alcohol soluble dyes and chromogens including, for example, immunoperoxidase chromogen, 9-amino-3-ethylcarbozole (AEC), immunoalkaline phosphatase chromogens, Fast Red, BCIPI/NBT, Oil Red O stain (Fat Stain used to demonstrate natural lipids in tissue sections), etc. As such, the use of the resinous type of mounting media is limited to mounting biological specimens that have been stained with dyes or chromogens that are insoluble in organic solvents.

Moreover, to mount a coverslip onto a microscope slide with the resinous type of mounting media, the microscope slide having the biological specimen thereon must be removed from the last rising fluid tank and then entered into an alcohol tank for the purpose of dehydrating the biological specimen. A complete dehydration of the biological specimen in alcohol must take place because failure to completely dehydrate the specimen can result in the appearance of cloudy areas and/or droplets of water in the specimen which may be visible macroscopically and/or microscopically. The dehydration step is then followed by a clearing step wherein the biological specimen is entered into a tank containing xylene or toluene. Slides are usually mounted from xylene and because the resins used are most often dissolved in toluene which is more volatile than xylene, air bubbles frequently form during the mounting process.

From the foregoing, it is readily apparent that mounting with the resinous type of mounting media is a laborious and time consuming process which requires that strict safety precautions (e.g. , working under a fume hood) be followed so as to prevent contact with or inhalation of xylene or toluene. In addition, slides mounted with this type of media are not reversible, i.e. , the coverslip cannot be removed without damage to the stain or tissue. Moreover, the resinous type of mounting media tend to evaporate quickly, thereby causing the media to thicken. As the mounting media thickens, the transparency of the tissue section decreases and, in turn, the refractive index is adversely affected.

The refractive index can be defined as the ratio of the velocity of light in air to the velocity of light in liquid or solid medium. As the refractive index of the mounting medium gets closer to the refractive index of the tissue, the tissue becomes more transparent and, thus, unstained sections become indiscernible. Most biological tissues have an average refractive index of about 1.53 to 1.54 and in order for the tissue to be visible directly, the refractive index of the mounting medium must be slightly above or slightly below the refractive index of the tissue. Most synthetic resins in solution have a refractive index of about 1.51 to 1.55. As explained, however, such resinous mounting media tend to thicken over time due to the rapid evaporation of the organic solvent, and in order to prevent such media from thickening, they must be thinned down with xylene or toluene which adversely affect the refractive index.

Moreover, if such media are thinned with too much xylene, a retraction of the mounting medium from under the coverslip may result and air bubbles can be introduced between the tissue and the coverslip, thereby making microscopic examination extremely difficult. In addition to the resinous type mounting media, aqueous type mounting media are also commercially available. Aqueous type mounting media are generally used when the resinous type mounting media and the dehydration and clearing processes associated therewith adversely affect the staining results. Aqueous type mounting media are typically made of simple syrups, glycerol-gelatin, and gum Arabic media. Of these, glycerol-gelatin based aqueous type mounting media are the most frequently used. However, aqueous type mounting media made of glycerol-gelatin are not fluid at room temperature and, thus, such media require pre-warming to a liquid state before they can be applied for coverslipping purposes. In addition, aqueous type mounting media made of giycerol- gelatin remain wet and sticky for a long period of time. As with glycerol- gelatin based aqueous type mounting media, the aqueous type mounting media made from syrups do not dry thoroughly and, thus, they too remain wet regardless of the climate.

Moreover, when aqueous mounting media are used, the chromogens and dyes used to stain the biological specimen fade away over a period of time. As such, the aqueous type of mounting media do not lend themselves to long term storage. In fact, the stickiness of this type of mounting media causes microscope slides to stick to adjacent microscope slides when stored in batches, thereby making future retrieval of such slides from a storage container quite difficult. Moreover, aqueous type mounting media have index of refraction that differ greatly from that of most biological tissues and, as a result, the transparency of biological tissues is not as great as with synthetic resins and, thus, microscopic evaluation is difficult with a 45X objective lens or higher.

Aqua Mount™, which is obtainable from Learner Laboratories (Pittsburgh, Pennsylvania), is an example of a commercially available aqueous mounting medium. Aqua Mount™ contains polyvinyl alcohol and can be used with many chromogens and dyes that require the use of an aqueous mounting medium. It, however, is not suitable for use with immunoperoxidase stains, such as 9-amino-3-ethylcarbozole (AEC), which are alcohol soluble chromogens. In addition, Aqua Mount™ displays inward retraction of the mounting media from the edge of the slide, and this retraction continues expanding with time. Moreover, this media is also quite thick and preparations mounted with this medium are rarely ever air bubble or air pocket free.

Crystal Mount™ (Biomeda Corp., Foster City, California) and Super Mount™ (BioGenex, San Ramon, California) are two other aqueous based mounting media that are designed to protect the biological specimen without the need for a coverslip. Both of these mounting media are applied and allowed to set and harden at room temperature or with application of heat. The dried coating creates a clear barrier that protects the biological specimen and, thus, the microscope slides prepared with Crystal Mount™ or Super Mount™ can be examined without further treatment. If, however, the biological specimens are to be stored for a long period of time, or if the biological specimens are to be examined under a 40X or higher objective lens of a microscope, coverslips are necessary. Such coverslips are usually applied with synthetic resins because application with either Crystal Mount™ or Super Mount™ will create huge air pockets upon crystallization of the polymers under the coverslip. As such, Crystal Mount™ and Super Mount™ are not ideal mountants since the application of a coverslip onto a microscope slide still remains the method of choice for mounting biological specimens for the purpose of diagnostic microscopy since coverslips are not scratchable, they better lend themselves to high magnification microscopy and finger prints are removable therefrom.

In view of the foregoing, there exists a need in the art for compositions and methods for the permanent mounting of biological specimens which avoid the limitations of the currently available mounting media. SUMMARY OF THE INVENTION The present invention provides compositions and methods for permanently mounting biological specimens. The mounting compositions of the present invention can be used for mounting stained tissue sections or cell smears for long-term storage. Such mounting compositions provide excellent optical clarity and resolution and, thus, the stained specimens mounted with the compositions of the present invention have greater transparency and visibility for microscopic examination. Preferably, the mounting compositions are aqueous based and, thus, slides do not require pre-mounting treatments such as dehydration in alcohol or clearing in xylene. The slides can be mounted directly from water. Moreover, the mounting compositions of the present invention are compatible with all aqueous and organic-soluble chromogens and dyes (e.g. , AEC, Fast Red and Fast Garnet stains). In addition, because of their aqueous, non-toxic and non- hazardous nature, the mounting compositions of the present invention can be used in laboratory settings without a hood or other precautions. As such, in one aspect, the present invention provides a composition for mounting a coverslip onto a microscope slide having thereon a biological specimen, the composition comprising: a water-soluble polymer having a weight average molecular weight in the range of about 1,000 to about 250,000, a polyether having a weight average molecular weight in the range of about 200 to about 15,000, and a polyhydric alcohol, wherein the composition has a pH of about 6.0 to about 11.0. In another aspect, the present invention provides a method for mounting a coverslip onto a microscope slide having thereon a biological specimen, the method comprising: (a) providing a microscope slide having thereon a biological specimen; (b) applying one to three drops of a mounting composition onto the biological specimen, the mounting composition comprising a solution of: a water-soluble polymer having a weight average molecular weight in the range of about 1,000 to about 250,000; a polyether having a weight average molecular weight in the range of about 200 to about 15,000; and a polyhydric alcohol; the mounting composition having a pH of about 6.0 to about 11.0; (c) applying a coverslip onto the biological specimen; and (d) drying. Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In one aspect of the present invention, a composition is provided for mounting a coverslip onto a microscope slide having thereon a biological specimen, the composition comprising: a water-soluble polymer having a weight average molecular weight in the range of about 1,000 to about 250,000, a polyether having a weight average molecular weight in the range of about 200 to about 15,000, and a polyhydric alcohol, wherein the composition has a pH of about 6.0 to about 11.0.

As used herein, "water-soluble polymer" refers to a polymer which exhibits solubility in an aqueous solution. Suitable water-soluble polymers include, but are not limited to, sythetic resins which have adesive properties and which form transparent, stable solutions when dissolved in water. Examples of such water-soluble polymers include, but are not limited to, polyvinylpyrroiidone (PVP) and polyvinyl alcohol (PVA). Typically, the water-soluble polymer will have a weight average molecular weight in the range of about 1,000 to about 250,000. More preferably, the water-soluble polymer will have a weight average moleculare weight in the range of about 5,000 to about 160,000. Even more preferably, the water-soluble polymer will have a weight average molecular weight in the range of about 10,000 to about 80,000. In a presently preferred embodiment, polyvinylpyrroiidone is the water-soluble polymer used in the compositions of the present invention. In an equally preferred embodiment, polyvinyl alcohol (PVA) is the water-soluble polymer used in the compositions of the present invention.

"Polyvinylpyrroiidone, " as used herein, refers to a polymer having the formula (CβHjNO),,. The polyvinylpyrroiidone used in the compositions of the present invention has a weight average molecular weight in the range of about 1 ,000 to about 250,000. In a presently preferred embodiment, the polyvinylpyrroiidone has a weight average molecular weight in the range of about 5,000 to about 160,000. In an even more preferred embodiment, the polyvinylpyrroiidone has a weight average molecular weight in the range of about 10,000 to about 80,000. In an even further preferred embodiment, the polyvinylpyrroiidone has a weight average molecular weight of about 10,000. In an equally preferred embodiment, the polyvinylpyrroiidone has a weight average molecular weight of or of about 40,000. In addition to the foregoing, it will be understood by those of skill in the art that blends of polyvinylpyrrolidones having various molecular weights can be used in the compositions of the present invention. For example, polyvinylpyrroiidone having a molecular weight of 10,000 can be blended or mixed with polyvinylpyrroiidone having a molecular weight of 40,000 and the resulting polyvinylpyrroiidone mixture used in the compositions of the present invention. Polyvinylpyrrolidones of various molecular weights are commercially available from a number of different chemical companies. For example, polyvinylpyrrolidones having weight average molecular weights of 10,000, 40,000 and 160,000 are commercially available from ISP Technologies, Inc. (Bound Brook, New Jersey). In addition, polyvinylpyrrolidones having weight average molecular weights of 10,000, 28,000 and 40,000 are commercially available from Aldrich Chemical Co. (Milwaukee, Wisconsin). It will be readily apparent to those of skill in the art that polyvinylpyrrolidones having weight average molecular weights in the range of 1,000 to 250,000 can be prepared from vinyl (-CR:CH_) monomers and pyrrolidone (e.g. , 2- pyrrolidone) monomers using standard polymerization reactions known to those of skill in the art (See, e.g. , U.S. Patent No. 2,317,804 (April 27, 1943); U.S. Patent No.

2,665,271 (January 5, 1954); Copenhaver and Bigelow, Acetylene and Carbonmonoxide Chemistry (Reinhold Publishing, New York, 1949, p. 68); and Breitenbach and Schmidt, Monatsh. Chem. , 83:833 (1952)). Moreover, it will be readily apparent to those of skill in the art that a polyvinylpyrrolidone-derivative can be used in place of polyvinylpyrroiidone in the aqueous mounting compositions of the present invention. Such pyrrolidone-derivatives include, but are not limited to, the following: _v-ethyl-2- pyrrolidone, _v-cyclohexyl-2-pyrrolidone, _v-dodecyl-2-pyrrolidone, _v-methyl-2- pyrrolidone, yv-hydroxyethyl-2-pyrrolidone and iV-(4-hydroxybenzyl)-pyrrolidone.

As used herein, "polyvinyl alcohol" refers to a polymer having the general formula [-CH₂CH(OH)-]_B. The polyvinyl alcohol used in the compositions of the present invention has a weight average molecular weight in the range of about 1,000 to about 250,000. In a presently preferred embodiment, the polyvinyl alcohol has a weight average molecular weight in the range of about 5,000 to about 150,000. In an even more preferred embodiment, the polyvinyl alcohol has a weight average molecular weight in the range of about 10,000 to about 85,000. In addition to the foregoing, it will be understood by those of skill in the art that blends of polyvinyl alcohols having various molecular weights can be used in the compositions of the present invention. For example, polyvinyl alcohol having a molecular weight of 10,000 can be blended or mixed with polyvinyl alcohol having a molecular weight of 40,000 and the resulting polyvinyl alcohol mixture used in the compositions of the present invention.

Polyvinyl alcohols of various molecular weights are commercially available from a number of different chemical companies. For example, polyvinyl alcohols having weight average molecular weights of 9,000-10,000, 13,000-23,000,

31,000-50,000, 85,000-146,000, etc. are commercially available from Aldrich Chemical Co. (Milwaukee, Wisconsin). In addition, it will be readily apparent to those of skill in the art that polyvinyl alcohols having weight average molecular weights in the range of 1,000 to 250,000 can be prepared by hydrolysis of a polyvinyl ester (e.g. , polyvinyl acetate) using standard reactions known to those of skill in the art (See, e.g. , U.S. Patent No. 2,317,804 (April 27, 1943); U.S. Patent No. 2,665,271 (January 5, 1954)).

As used herein, "polyether" refers to a polymer containing the -(CH₂-CHR-O-)_B linkage in the main chain or side chain. The polyether used in the aqueous mounting compositions of the present invention has a weight average molecular weight in the range of about 200 to about 15,000. Suitable polyethers for use in the mounting compositions of the present invention include, but are not limited to, polyethylene glycol (PEG) and its derivatives. In a presently preferred embodiment, polyethylene glycol, a polymer of ethylene glycol with the general formula HOCH₂(CH₂OCH₂)„CH₂OH or H(OCH₂CH_)_BOH, is the polyether used in the mounting compositions of the present invention. Other polyethylene glycol derivatives suitable for use in accordance with the present invention include, but are not limited to, methoxypolyethylene glycol, methoxypolyethylene glycol derivatives and polyoxyethylene. Polyethylene glycol, methoxypolyethylene glycol, its derivatives and polyoxyethylene of varying molecular weights are commercially available from a number of different chemical companies including, for example, Sigma Chemical Co. (St. Louis, Missouri). Alternatively, such polyethers can be prepared from the appropriate monomer(s) using standard polymerization reactions known to those of skill in the art.

"Polyhydric alcohol" (or, alternatively, "polyol" or "polyalcohol"), as used herein, refers to an alcohol with two or more hydroxyl (-OH) radicals. Suitable polyhydric alcohols for use in the mounting compositions of the present invention include, but are not limited to, glycerol and sucrose. In a presently preferred embodiment, glycerol, a compound having the formula (CH₂OH)₂CHOH, is the polyhydric alcohol used in the mounting compositions of the present invention. As with polyvinylpyrroiidone and the polyethers set forth above, polyhydric alcohols are commercially available from a number of different chemical companies including, for example, Aldrich Chemical Co. (Milwaukee, Wisconsin). In addition to the foregoing, the mounting compositions of the present invention can further include a preservative. Preservatives suitable for use in accordance with the compositions of the present invention include, but are not limited to, sodium azide and sodium benzoate. In a presently preferred embodiment, sodium azide is the preservative used in the compositions of the present invention.

The mounting composition of the present invention can be either an aqueous solution or an organic solution. In a presently preferred embodiment, the mounting composition is an aqueous composition. If, however, the mounting composition is an organic solution, the solvent is an alcohol including, but not limited to, methanol, ethanol and isopropanol. As previously mentioned, the mounting composition of the present invention has a pH of about 6.0 to about 11.0. In a presently preferred embodiment, the mounting composition has a pH of about 7.0 to about 10.5.

In the mounting composition of the present invention, the water-soluble polymer is present at a concentration ranging from about 2% to about 41 % and, more preferably, at a concentration ranging from about 10% to about 30%. If the water- soluble polymer is polyvinylpyrroiidone, the polyvinylpyrroiidone is present at a concentration ranging from about 10% (w/v) to about 41 % (w/v). In a presently preferred embodiment, polyvinylpyrroiidone is present at a concentration ranging from about 15% (w/v) to about 35% (w/v). In an even further preferred embodiment, polyvinylpyrroiidone is present at a concentration ranging from about 17% (w/v) to about 33% (w/v). The foregoing concentration ranges are fully applicable to polyvinylpyrrolidone-derivatives. If the water-soluble polymer is polyvinyl alcohol, the polyvinyl alcohol is present at a concentration ranging from about 2% (w/v) to about 25% (w/v). In a presently preferred embodiment, the polyvinyl alcohol is present at a concentration ranging from about 5% (w/v) to about 15% (w/v).

In the mounting composition of the present invention, the polyether, e.g., polyethylene glycol, is present at a concentration ranging from about 0.01 % to about 5%. In a presently preferred embodiment, the polyether is present at a concentration ranging from about 0.03% to about 3%. In addition, the polyhydric alcohol, e.g. , glycerol, is present in the mounting composition of the present invention at a concentration ranging from about 0.01 % to about 10%. In a presently preferred embodiment, the polyhydric alcohol is present at a concentration ranging from about 0.04% to about 5%. If a preservative (e.g. , sodium azide or sodium benzoate) is included in the compositions of the present invention, it is generally present at a concentration ranging from about 0.01% (w/v) to about 3% (w/v).

It will be readily apparent to those of skill in the art that the concentration ranges of the various components, as set forth above, remain the same regardless of whether the mounting composition is an aqueous solution or an organic solution. In another aspect of the present invention, a method is provided for mounting a coverslip onto a microscope slide having thereon a biological specimen, the method comprising: (a) providing a microscope slide having thereon a biological specimen; (b) applying one to three drops of a mounting composition onto the biological specimen, the mounting composition comprising a solution of: a water-soluble polymer having a weight average molecular weight in the range of about 1 ,000 to about 250,000; a polyether having a weight average molecular weight in the range of about 200 to about 15,000; and a polyhydric alcohol; the mounting composition having a pH of about 6.0 to about 11.0; (c) applying a coverslip onto the biological specimen; and (d) drying.

"Coverslip," as used herein, refers to a thin slip of glass, plastic or other transparent, polymeric material used for covering a biological specimen on a microscope slide that to be observed under a microscope. The coverslip should be of a sufficient length and width to cover the biological specimen in its entirety. "Mountant" or "mounting composition" is used herein to refer the mounting medium of the present invention which can be used in the method set forth above to mount a coverslip onto a microscope slide having a biological specimen thereon. It should be understood that the prior discussion pertaining to the mounting composition of the present invention, its components and preferred embodiments is fully applicable to the mounting composition used in the method of the present invention and, thus, it will not be repeated again.

"Biological specimen" is used herein to refer to a tissue section or cell smear. Prior to mounting the coverslip, the biological specimen is generally subjected to a series of physical and chemical manipulations that include, for example, fixation, embedding, sectioning, mounting on the microscope slide and staining. Such physical and chemical manipulations are known to those of skill in the art and, thus, they will not be described only briefly hereinbelow. Once subjected to the foregoing physical and chemical manipulations, the biological specimen may be referred to as a "histochemical section" or "cytochemical smear." As such, "histochemical section" refers to a solid sample of biological tissue which has been frozen or chemically fixed and hardened by embedding in wax or plastic, sliced into a thin sheet, generally several microns thick, and attached to a microscope slide. Moreover, "cytochemical smear" refers to a suspension of cells, such as blood cells, which has been chemically fixed and attached to a microscope slide.

In the method of the present invention, the biological specimen may or may not be fixed prior to mounting the coverslip. In a presently preferred embodiment, the biological specimen is fixed. A "fixed biological specimen, " as used herein, refers to a sample of biological cells which has been chemically treated to stabilize protems and to strengthen cellular structures, particularly membranes, against disruption by solvent changes, temperature changes, mechanical stresses, and drying, Cells may be fixed in suspension or contained in a sample of tissue, such as might be obtained during autopsy, biopsy or surgery. Cell fixatives generally are chemicals which cross-link the protein constituents of cellular structure, most commonly by reacting with protein amino groups. Preferred fixatives include, but are not limited to, buffered formalin, 95% ethanol, formaldehyde and glutaraldehyde.

Moreover, cells or tissues to be examined are usually embedded in warm, liquid paraffin wax. The wax, which both surrounds the tissue and infiltrates it, hardens on cooling, thereby supporting the tissue externally and internally. The resulting solid paraffin block is then trimmed to the appropriate shape before being sectioned. If ultrathin sections are required, the use of harder embedding and infiltrating materials, such as epoxy plastics, may be required. Such materials are initially in liquid form and are poured into small molds containing pieces of fixed tissues; on heating, the liquid undergoes polymerization to form a hard plastic.

The trimmed blocks containing the embedded samples are sectioned using a microtome. In this instrument, the block is sequentially swept over the blade of a knife that cuts the block into a series of thin sections. Such sections are then mounted on, i.e. , deposited on or attached to, a microscope slide and stained with dyes or chromogens of various colors that specifically attach to different molecular constituents of the cells. At this point, the coverslip can be mounted on the tissue or cell specimen in accordance with the method of the present invention. Using the method of the present invention, a tissue section or cell smear mounted microscope slide is removed from the last rinsing vessel containing water or water-based buffers (e.g. , phosphate buffered saline (PBS) or the like), transported to a bench top or a table and mounted, without any prior blotting or wiping off of any excess liquid, using the method and mounting composition of the present invention. More specifically, one to three drops (depending on the size of the tissue section or cell smear and the length of the coverslip used) of the mounting medium of the present invention are applied to the center of the tissue section or cell smear and a coverslip of an appropriate length and width is applied over die slide-mounted tissue section or cell smear so that the entire length of the tissue section or cell smear is covered. If any air bubbles are visible, they can be removed by applying slight pressure to the coverslip and moving the bubbles outward to the edges of the microscope slide.

Once the coverslip has been mounted using the mounting media of the present invention, the microscope slide can be heated in an oven or on a hot-plate at a temperature of about 37°C to about 80°C for about 10 to about 20 minutes. The heating process provides for a faster immobilization of the coverslip and, in addition, securely adheres the coverslip to the microscope slide for microscopic examination. Alternatively, the coverslip can be effectively immobilized by allowing the microscope slide to stand at room temperature with no heating for about 40 to about 60 minutes. One of the advantages of the mounting medium of the present invention is that it is reversible such that the coverslip can be readily removed, if needed, by immersing the coverslip mounted microscope slide in water. The time required to carry out the dismounting process will varying depending on the amount of time the coverslip has been mounted, but will generally be in the range of about 10 minutes to about 24 hours.

The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are intended neither to limit nor define the invention in any manner.

EXAMPLE I

This example illustrates the general protocols used to prepare the mounting compositions of the present invention. In accordance with the present invention, an aqueous mounting composition was prepared by dissolving 15 grams of polyvinylpyrroiidone (PVP) having a weight average molecular weight of 10,000 (obtained from Aldrich Chemical Company, Milwaukee, Wisconsin) in 90 ml of deionized water. The pH of the solution was adjusted to about 9.0 by dropwise addition of Tris base or a sodium hydroxide solution. 0.3 ml of polyethylene glycol and 0.3 ml of glycerol were added and the final volume was adjusted to 100 ml.

In accordance with the present invention, an organic mounting composition was prepared by dissolving 15 grams of PVP having a weight average molecular weight of 10,000 in 90 ml of methanol or reagent alcohol (mixture of methanol, ethanol and isopropanol). The pH of the solution was adjusted to about 9.0 by die dropwise addition of sodium hydroxide or Tris base. 0.3 ml of polyethylene glycol and 0.3 ml of glycerol were added and the volume was adjusted to 100 ml.

EXAMPLE II

Stained biological specimen mounted slides coverslipped with the mountant of the present invention were tested for performance criteria that are highly desirable in an optimally performing mounting media. Such criteria include optical clarity, general ease of use, stability, lack of air pocket/bubble formation, durability, lack of fading, lack of drying and/or cracking and lack of stickiness or messiness which is commonly associated with the use of gelatin-glycerol based aqueous type mounting media.

EXAMPLE IIA

Tissue specimens stained with 3-amino-9-ethylcarbazole (AEC), 3,3'- diaminobenzidine (DAB) and 2-methoxy-4-nitrobenzenediazonium salt (Fast Red) and coverslipped with (1) the mounting medium of the present invention, (2) an aqueous- based, gelatin-glycerol mounting media and (3) Aqua Mount™ were evaluated for physical performance and stability by placing a set of each of such slides (1) at room temperature, (2) in an oven at 37°C for four weeks and (3) in an oven at 65 °C for four weeks. Following these incubation periods, each set of slides was examined for signs of physical deformation including, for example, the formation of air pockets, the cracking and/or drying of the mounted tissue and the mountant, and me inward retraction of the mountant from the edges of the slide. No air pockets or inward retraction of the mounting media were visible in the slides mounted with the mounting medium of the present invention. However, slides mounted with the aqueous-based gelatin-glycerol media and incubated at room temperature displayed approximately 10 small to medium-sized air bubbles, whereas the similarly mounted slides incubated at 37°C displayed approximately 15 medium-sized air bubbles. Moreover, the slides mounted with the aqueous-based gelatin-glycerol media and incubated at 65 °C displayed the same number of air bubbles as those incubated at 37°C, but, in addition, displayed drying and cracking of the mounting medium. The slides mounted with Aqua Mount™ did not display excessive air bubble formation under any of the three incubation conditions. However, at all temperatures tested, the slides mounted with Aqua Mount™ displayed inward retraction of the mounting media, leaving the long edges of die slides dry and unprotected by 2 mm. This inward retraction was increased by 2 mm for the slides incubated at 37°C, and by an additional 1 to 2 mm for the slides incubated at 65 °C. Cracking was not observed for slides mounted with Aqua Mount™. Similarly, no cracking of the mounting media was observed for slides mounted with the mounting media of the present invention regardless of d e incubations conditions used.

EXAMPLE IIB The slides, which were previously stained, mounted and incubated as described in Example II A, were examined for fading of the various stains following a four week incubation period. In doing so, it was found Uiat when examined microscopically or macroscopically, the stained specimens mounted with the mounting composition of the present invention did not display fading of the stains under any of the three incubation conditions described above. The stained specimens mounted with the aqueous-based gelatin-glycerol media and incubated at room temperature and at 37°C did not display fading of important measures, but similarly mounted specimens incubated at 65 °C displayed moderate fading. Moreover, the stained specimens mounted witii Aqua Mount™ displayed moderate to severe fading, especially for the slides incubated at 37 °C and at 65°C.

The permanency, i.e. , chemical stability, of the chromogen stains was further explored by storing in a slide storage cabinet a set of tissue specimen stained with light-sensitive chromogens AEC and DAB and with Fast Red, and mounted with (1) the mounting medium of the present invention, (2) an aqueous-based, gelatin-glycerol mounting media and (3) Aqua Mount™. Sets of similarly stained and mounted specimen were also placed in a well-lighted room for a period of 6 montfis. The light-exposed, stained tissue specimen coverslipped using the mounting medium of the present invention did not display fading of the AEC, DAB or Fast Red stains when examined microscopically and macroscopically. However, AEC, DAB and Fast Red stained specimen mounted with the aqueous-based, gelatin-glycerol media displayed moderate fading. Moreover, AEC, DAB and Fast Red stained specimen mounted with Aqua Mount™ displayed the highest degree of stain fading, especially with specimen stained with AEC and DAB.

EXAMPLE IIC

The quick drying property of the mounting media of the present invention was evaluated by measuring the time required for the slides to dry and for the coverslips to securely adhere to the slides. It is extremely important that the mounting medium used to mount the coverslip to the microscope slide dry quickly when the slides are to be examined microscopically. The mountant of the present invention dried in about 10 minutes when placed in an oven at a temperature of about 65 °C. When placed in an oven at a temperature of about 37^βC, the mountant of die present invention dried in about 20 minutes. When left to stand at room temperature, the mountant of the present invention dried in about 45 minutes. The quick drying property of the mounting medium of the present invention is a unique feature of this formulation and one not previously observed with other aqueous based mounting media. The drying time for other aqueous based mounting media took anywhere from three to four weeks to achieve.

EXAMPLE IID The optical clarity associated with the mountant media of the present invention was examined for tissue visibility and tissue transparency under all objective lenses of the microscope. Tissue specimen stained with DAB, a non-alcohol soluble chromogen, and mounted with the mounting media of the present invention equaled d e optical clarity of a similarly stained slide mounted with a resinous type mounting media which required pretreatment with both alcohol and xylene. The same comparison was not possible for AEC or Fast Red because they are alcohol soluble chromogens and, thus, such stains will not survive the dehydration step in alcohol and the clearing step in xylene required when mounting with a resinous type mounting media. It should be noted, however, that tissue sections stained with AEC and Fast Red and mounted with the mounting medium of the present invention were compared with similarly stained slides mounted with other gelatin-glycerol based mounting media under 10X, 40X and lOOX objective lenses and, in doing so, it was determined that d e optical clarity, tissue visibility and tissue transparency were superior in the slides mounted witii the mounting media of me present invention tiian with the gelatin-glycerol based mounting media.

EXAMPLE HE

The general ease of use and die handling of the mounting media of die present invention was compared with the use of other aqueous-based and resinous-based types of mounting media. The mounting media of the present invention lacked die general tackiness and stickiness that is observed with most aqueous-based mountants. Slides mounted with the mountant of the present invention did not stick together upon archiving and, thus, they were easily retrieved. Moreover, it was found that unlike the commonly used gelatin-glycerol based aqueous mounting media which solidify at room temperature, the mounting media of die present invention is liquid at room temperature and, thus, no pre-warming of the mounting media is necessary prior to use. In addition, unlike the resinous types of mounting media, the mountant of the present invention does not require dehydration with alcohol or clearing with xylene before mounting. As such, contact with these undesirable solvents are avoided and. in addition, since the slides can be mounted directly from the bench top using the mounting media of the present invention, there is no need to work under a certified fume hood. The foregoing is offered for purposes of illustration. It will be readily apparent to tiiose skilled in die art that die operating conditions, materials, procedural steps and otiier parameters of die metiiods and compositions described herein may be further modified or substituted in ways without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. An aqueous composition for mounting a coverslip onto a microscope slide having thereon a biological specimen, said composition comprising: a water-soluble polymer having a weight average molecular weight in the range of about 1,000 to about 250,000; a polyether having a weight average molecular weight in the range of about 200 to about 15,000; and a polyhydric alcohol; wherein said composition has a pH of about 6.0 to about 11.0.

2. The composition of claim 1 wherein said water-soluble polymer has a weight average molecular weight of about 5,000 to about 160,000.

3. The composition of claim 1 wherein said polyvinylpyrroiidone has a weight average molecular weight of about 10,000 to about 80,000.

4. The composition of claim 1 wherein said water-soluble polymer is selected from me group consisting of polyvinylpyrroiidone and polyvinyl alcohol.

5. The composition of claim 1 wherein said water-soluble polymer is polyvinylpyrroiidone.

6. The composition of claim 1 wherein said water-soluble polymer is present at a concentration ranging from about 2% (w/v) to about 41 % (w/v).

7. The composition of claim 1 wherein said polyvinylpyrroiidone is present at a concentration ranging from about 10% (w/v) to about 30% (w/v).

8. The composition of claim 1 wherein said polyetiier is a member selected from the group consisting of polyethylene glycol and its derivatives.

9. The composition of claim 1 wherein said polyhydric alcohol is a member selected from the group consisting of glycerol and sucrose.

10. The composition of claim 1 wherein said polyether is present at a concentration ranging from about 0.01 % to about 5%.

11. The composition of claim 1 wherein said polyhydric alcohol is present at a concentration ranging from about 0.01% to about 10%.

12. The composition of claim 1 further comprising a preservative.

13. The composition of claim 12 wherein said preservative is present at a concentration ranging from about 0.01 % (w/v) to about 3% (w/v).

14. The composition of claim 12 wherein said preservative is a member selected from the group consisting of sodium benzoate and sodium azide.

15. The composition of claim 14 wherein said preservative is sodium azide.

16. The composition of claim 1 wherein said composition has a pH of about 6.5 to about 10.5.

17. A method for permanentiy mounting a coverslip onto a microscope slide having thereon a biological specimen, said method comprising:

(a) providing a microscope slide having thereon a biological specimen;

(b) applying one to three drops of an aqueous mounting composition onto said biological specimen, said aqueous mounting composition comprising: a water-soluble polymer having a weight average molecular weight in the range of about 1,000 to about 250,000; a polyether having a weight average molecular weight in the range of about 200 to about 15,000; and a polyhydric alcohol; said aqueous mounting composition having a pH of about 6.0 to about 11.0; (c) applying a coverslip onto said biological specimen; and

(d) drying.

18. The method of claim 17 wherein said biological specimen is fixed prior to step (b).

19. The method of claim 17 wherein said aqueous mounting composition further comprises a preservative selected from the group consisting of sodium benzoate and sodium azide.

20. The method of claim 17 wherein step (d) is carried out at a temperature ranging from about 35 °C to about 80°C.

21. A coverslipped, biological specimen mounted microscope slide produced in accordance widi die method of claim 17.