GB2161822A - Nontoxic composition for use in mounting microscope slides - Google Patents

Abstract

A composition for use in mounting specimens on microscope slides comprises, in the form of a homogenous solution: (a) an amorphous and essentially linear homopolymer of styrene; (b) a plasticizer, (c) an UV adsorber; and (d) a terpene which solubilizes the component (a). The composition may also include an antioxidant. The terpene component (d) has the advantage that it is non-toxic, unlike aromatic solvents which have been commonly used previously.

Description

SPECIFICATION Nontoxic composition for use in mounting microscope slides The present invention relates to a new nontoxic composition that can be used for mounting stained thin sections of biological tissue on microscope slides, for cytologic, hystologic and pathologic studies.

The invention also provides a method for mounting microscope slides by the use of said nontoxic composition as a mounting medium, for purposes of coating and causing preservation of the specimen, and adhesion of the cover glass to the slide.

Furthermore, the invention provides microscope slides mounted by the hereabove said method.

It is common practice in the medical field to provide thin sections of biological specimens on glass slides for microscopic studies, such sections having been stained, dehydrated and subjected to a mount ing process. Generally, such mounting process encloses each individual specimen section in a medium supported on a microscope slide, where it is preserved indefinitely.

According to the present general trend, a solution of a suitable mountant in an appropriate solvent is dispensed over the specimen, in a controlled volume, and there, to provide a planar, upper surface, nec essary for proper microscopic examination, it is common practice to apply a glass cover slip over the dispensed mountant, while still in liquid form. To remove the solvent the slides are subjected to a drying process which is essential to harden the mountant and to seal the cover slip.

Generally, the mounting and drying processes are carried out manually in a laboratory.

Commonly, after removing the slide from the clearing solution and quickly draining the same, one to several drops of mountant are deposited over the specimen and then, immediately, the cover slip is carefully positioned by the technician and gently pressed upon the mountant. Any surplus of mountant is usually quickly and carefully removed, for example with absorbent paper wet with the clearing agent.

The most common solvents constituting the commercially available clearing agents and mounting me dia are: xylol, benzene, toluene, dimethyl benzene, diethyl benzene and other aromatics. Many of these solvents are well known for their characteristics of toxicity, carcinogenicity, mutagenicity, teratogenicity.

The use of benzene, for example, is now forbidden by law in many countries because concerned with the development of leukaemia in people using it.

As all the operations hereabove described are usually performed, in a laboratory where the technicians come always into contact of the required solvents, breathing and touching them, it is evident the importance of finding a solvent which is equally useful but devoided of said toxicity problems.

According to the invention a nontoxic wholly satisfactory solvent is provided for use in preparing a composition suitable for mounting biological sections on microscope slides. The composition of the invention, besides showing absence of toxicity also possesses all the main properties recognized to be essential or desired for an appropriate mounting medium, such: do not cause stained specimens to fade; yellow minimally with age, and have optimum refractive index allowing to achieve an almost perfect transparency.

The invention provides a composition for use in mounting specimens, such as stained sections of biological tissue, on microscope slides, which composition comprises, in the form of a homogenous solution: (a) an amorphous and essentially linear homopolymer of styrene; (b) a plasticizer; (c) an UV adsorber; and (d) a terpene which solubilizes the component (a).

Though the relative proportions of the components (a), (b) and (c) in the above composition may vary within certain ranges, in a preferred embodiment of the invention the composition contains 20 to 35 per cent of the component (a), 0.5 to 10 per cent of the plasticizer component (b), 0.02 to 2.3 per cent of the UV adsorber component (c) and 55 to 79.5 per cent of the solvent component (d), the respective amounts of components (a), (b) and (d) being based on the total amount of these three components and the amount of component (c) being calculated with respect to the amount of component (a).

Percentages are by weight throughout except in the context of light transmission.

In addition to the above indicated essential components, the composition provided by the invention for use as mounting medium may include, as an optional component, also an antioxidant. Component (a) is an essentially linear (i.e. not branched) homopolymer of styrene, preferably having a molecular weight equal to or higher than 200.000.

Typically, the said homopolymer consists of an amorphous polymeric species, without crystallites, having a Vicat softening point (determined according to ASTM D 1525-load 1 Kg/mm2) ranging from 99"C to 102"C, an Izod impact strength (determined according to ASTM D 256-sample 1/2" x 1/8") ranging from 1.5 to 2 Kg cm/cm about at 23"C), and a specific gravity (determined according to ASTM D 792) of 1.05 g/ cm3. It looks as a crystal clear resin with an average per cent of light transmission (determined according to ASTM D 1003 on a crystal 1.3mm thick) ranging from 88 to 90% in the wavelength range from 420 nm, to 700 mm, and a high refractive index of 1.59, as n (D,20) value measured according to ASTM D 542.

As far as the refractive index is concerned, it is presently considered desirable to have a refractive index far from the critical range comprised between n(D,20) =1.530 and n(D,20) = 1.540, which is characteristic for refractive index of tissues. The best optical resolution was obtained using media having refractive indices ranging from 1.44 to 1.50 or above 1.58. In fact, as the refractive index of the mounting medium approaches that of the tissue, the latter becomes more and more transparent, so that unstained objects may be difficult to discern. The refractive index of the mounting composition of the invention in its whole, can be easily brought to a desired value by simply adjusting the relative proportions of components (a), (b) and (d), preferably within the above indicated ranges.The plasticizer component (b) has the specific purpose of further enhancing the performances of the component (a).

More particularly, it reduces the hardness of the resins, which ranges from 78 to 82 if determined according to ASTM D 785 (Rockwell hardness - M scale), and contributes to optimize the refractive index value.

The kind of plasticizer component (b) and its relative proportion with respect to component (a) are chosen so as to obtain the desired softness in the resulting resin, and the desired refractive index value for the composition. A suitable plasticizer may be, for instance, a phthalic acid di -C1 -ClO -alkylester, for example phthalic acid diethyl-ester, phthalic acid di-n-butylester or phthalic acid di -n-octylester, more preferably phthalic acid di -n-butylester.The adsorber of UV radiations component (c), which may be any known commercially available product having the said function, may be, for example, benzophenone or a derivative thereof such as, e.g., 2-hydroxy -4-methoxy -4'-methyl-benzophenone and 2- -hydroxy -4-methoxy-benzophenone, or other compounds such as, for instance, 2,2'-di-phenoxy -4,4' -di-phenyl - biphenyl or a benzotriazole derivative such as, e.g.,2-(2-hydroxy-t-octylphenyl) benzotriazo le.

Preferably the U.V. adsorber is a benzotriazole derivative of the kind hereabove specified, which may be added to the composition in an amount of, e.g., from 0.05 to 2 per cent by weight with respect to component (a). The solvent component (d) may be any terpene derivative able to easily and completely dissolve component (a) and to give a homogenous solution when admixed with the plasticizer (b), the UV adsorber (c) and the possibly present antioxidant. The solvent is, preferably, a monoterpene such as, for instance, 2-methyl-6-methylene 2,7 -octadiene, 2-methyl methylene -1,7 -octadiene, 1-methyl -4-(1methylethenyl)-cyclohexene, or 4-isopropyl methylene cyclohexene, in racemic (dl) or optically active form. The racemic or optically active forms of 1-methyl-4-(1- -methylethenyl) cyclohexene are particularly preferred solvents.

As already said, the mounting medium composition subject of the present invention may also contain, as an additional component, an antioxidant, which may be any known commercially available product having the said function; it may be, for example, 2,6 -bis (1,1-dimethylethyl)-4-methylphenol. Although the presence of an antioxidant is normally not necessary, nevertheless, if desired, an amount of, e.g., from 0.02 to 2 per cent by weight of antioxidant with respect to component (a) may be added to the composition.

A preferred composition provided by the invention is a composition comprising (a) 23 to 33 per cent of an amorphous and essentially linear homopolymer of styrene having a molecular weight equal to or higher than 200.000; (b) 0.8 to 8 per cent of a plasticizer chosen from the group of the C,-C10 alkyl esters of the phthalic acid; (c) 0.05 to 2 per cent of a UV adsorber, and (d) 59 to 76.2 per cent of a monoterpene as solvent; the respective amounts of components (a), (b) and (d) being based on the total amount of these three components and the amount of component (c) being caluclated with respect to the amount of component (a).

In the said preferred composition the plasticizer component (b) is, preferably, phthalic acid di -n-butyl ester, the UV absorber component (c) is, preferably, 2-(2-hydroxy-5-t-octyl- -phenyl)-benzotriazole, and the solvent component (d) is, preferably, the dl-form of 1-methyl-4-(1-methylethenyl)- cyclohexene or an optically active form thereof, most preferably the d form.

Such a preferred composition may also contain an antioxidant, e.g. of the kind previously specified, as additional component. The invention provides also a method for mounting a specimen on a microscope slide, which method comprises mounting the specimen on the slide using a composition according to the invention as the mounting medium. The specimen placed on a supporting slide is completely immersed in the clearing agent which can be, and preferably is, the same solvent component (d) indicated in the above described composition.

After removing the slide from the clearing solution, the specimen may be coated with the composition of the invention and with a covering slide, and then the solvent of the composition removed.

To remove the solvent, the so-mounted slides are subjected to a drying process which is usually and preferably carried out at room temperature, for several hours.

The method of the invention is particularly applicable to mounting biological tissue, for example sections of tissue. The tissue may be stained. The mounting medium of the invention coats and preserves the tissue.

Few drops of the mounting composition are normally sufficient for the coating process and care must be taken that air bubbles are avoided between the supporting and the covering slide. Though glass slides are preferred, slides made of any other suitable material can also be used according to the invention. The major advantage and improvement obtained with the composition and method of the invention resides in the fact that they involve the use of a solvent, namely the terpene component (d) previously specified, which is devoided of any toxicity and, at the same time, possesses all requisites recognized to be essential or desired for a solvent to be used as a component of a mounting medium.

As already said, absence of toxicity is a very important factor in the laboratory.

In particular, for example, a comparison between d-1-methyl-4(1-methyl ethenyl)-cyclohexene, which is a particularly preferred solvent according to the invention, and xylene and toluene, which are solvents currently used in histology laboratories, indicates that the solvent of the invention allows superior results. The vapors of the latter solvent are nontoxic and are classfied as generally regarded as safe by the Food and Drug Administration and, furthermore, the flammability aspect of xylene and toluene usage is eliminated because the said solvent is rated only as combustible material (flash point 136"F).

The potential health hazards associated with xylene and toluene are therefore eliminated with the solvent of the invention.

As already said, microscope slides mounted with the above described composition and mounting method, are a further object of the present invention.

The use of the styrene homopolymer component (a) combined with the terpene solvent component (d) allows to finally obtain mounted slides having improved characteristics with respect to the slides mounted with the currently used or commercially available mounting media.

Indeed, the light transparency degree and the optimal refractive index of said polymeric material allow a clear, detailed and almost perfect view of the biological specimen at the microscope.

The use of a mounting medium which is already in polymerized form avoids damages due to possible occurence in situ of polymerization processes, e.g. by action of light or U.V. radiations or chemical catalysts.

The microscope slides obtained according to the invention were tested for their stability to light, U.V.

radiations and temperature.

Exposure to light and U.V. radiations, even for periods of 5-6 months, did not reveal any change with respect to the freshly mounted slides, and the same was observed after slides were kept at temperatures from -30 C to 40"C for similar periods. Also the adhesion between the supporting and the covering slides remained unchanged.

Thus, according to the invention, microscope slides are provided showing a high degree of light and UV radiations stability, as well as temperature and weathering-resistance.

Furthermore, if necessary, the slides of the invention can be easily demounted, e.g., by simple immersion in the same terpene solvent used as clearing agent and as solvent component (d) in the mounting procedure, for instance the dl-form of 1-methyl -4-(1-methylethenyl)cyclohexene or an optically active form thereof.

The abbreviation ASTM used in the text of the present invention stands for "American Society for Testing Materials". The following examples illustrate but do not limit in any way the invention.

Ratios are by weight.

Example 1 A composition for use as mounting medium of microscope slides was prepared by the following general procedure.

To a 300 ml round bottom flask, fitted with a water condenser and equipped with a magnetic stirrer, the solvent (a monoterpene chosen from dl 1-methyl-4-(1-methylethenyl)cyclohexene or an optically active form thereof; dl -4-isopropyl-1-methylene-2-cyclohexene or an optically active form thereof; and 2-methyl-6-methylene-2,7-octadiene) and then the polystyrene resin [EDISTIR SNI 410 (Registered Trademark)j added under stirring at room temperature.

The mixture was heated gently at about 40-45"C and then stirring was continued at this temperature for 15-20 hours to get complete dissolution of the polymeric material. The plasticizer (phthalic acid di-ethyl ester, di n-butyl ester or di n-octyl ester), the UV adsorber (benzophenone or benzotriazole derivative) and, if desired, the antioxidant [2,6-bis(1 ,1 -dimethyl-ethyl)-4-(methylphenol)] were added, always under stirring.

By the said procedure the following compositions (A) to (E) were prepared with the indicated relative proportions of the various components: (A) (a) polystyrene resin -na (D,20) = 1.59 50 g.

(b) phthalic acid di n-butyl ester -nb (D,20) = 1,49 2 g.

(c) 2-(2-hydroxy-5-t-octylphenyl)-benzotriazole 0.1 g.

(d) d-form of 1-methyl-4(1-methylethenyl)cyclohexene-nc (D,20) 126 g.

1,47 (e) 2,6-bis (1,1-dimethyl-ethyl)-4-methyl phenol 0.05 g.

Considering only the major components of the mixture, namely (a), (b) and (d), it is possible to determinate their respective ratios by weight in the resulting solution, namely: 0.28 for component (a), 0.01 for component (b) and 0.71 for component (d). Determining the respective values of the refractive indices for these three components, it is possible to make theoretical evaluation of the refractive index of the resulting mixture nM (D,20), using the relation: Ratio by weight Ratio by weight Component (a) .na (D,20) + Component(b) .nb(D,20)+ + Ratio by weight nd(D,20) = nM(D,20) Component (d) So, for the particular case of composition (A) it was precalculated: 0.28 . 1,59 + 0.01 . 1,49 + 0.71 . 1,47 = 1.5038, and this value was found to be in perfect agreement with the experimental one which was: 1.503.

(B) (a) polystyrene resin -nn(D,20) 1.591 45.g.- (b) phthalic acid di n-diethylester -nb(D,20) = 1,505 2 g.

(c) 2-(2-hydroxy-5-t-octytphenyl)-benzotriazole 0.5 g; (d) dl form of 1-methyl-4(1-methytethenyl)cyclohexene - n4(D,20) =-1.47 125 g.

nM (D,20) = 1.487 (theoretical value); n(D,20) =1-.489 (experi mental value) (C) (a) polystyrene resin -na(D,20) = 1.59 509.

(b) phthalic acid di n-dibutylester -nb(D,20) = 1.49 10g.

(c) 2-(2-hydroxy-5-methylphenyl)-benzotriazole 1 g.

(d) 2-methyl-6-methylene-2,7-octadiene-nd(D,20) = 1.471 125 g.

nM (D,20) = 1.489 (theoretical value); n(D,20) = 1.49 (experi mental value) (A) (D) (a) polystyrene resin in (D,20)-- 1.59 -53 g, (b) phthalic acid di n-dibutylester -n,(D,20) = 1.49 . 6 g.

(c) benzpphenone 0.3 g.-.

(d) deform of 1-methyl-4-(1-methylethenyl) cyclohexene nd (D,20) = 1.47 127 g.

nM (D,20) = 1.490 (theoretical value); n(D20) = 1.49-(experi mental value) (E) (E) (a) polystyrene resin -n (D,20) = 1.59 55 g.

(b) phthalic acid di n-octylester -n,(D,20) = 1.485 4 g.

(c) 2-hydroxy-4-methoxybenzophenone 0.11 g.

(d) d-form of 4-isopropyl-1 -methylene-2-cyclohexene- nd (D,20) = 1.479 120 g.

(e) 2,6-bis (1,1-dimethyl-ethyl)-4-methyl phenol 0.06 g nM(D,20) = 1.514 (theoretical value); n(D,20) = 1.515 (experi mental value) Example 2 Tissue sections from different human and animal samples, were conventionally fixed and embedded, and conventionally supported on a glass slide.

The slide was previously immersed in a clearing agent, which was the same solvent component (d) of the mounting composition to be used in the subsequent step, then it was removed from the clearing solution and mounted as follows.

Few drops of a composition as obtained in example 1 were placed over the tissue section-supporting slide and then a covering glass slide was coated thereon having care to eliminate air bubbles between the slides.

The slides were then kept at room temperature for 4-5 hours in a horizontal position, and then examined at the microscope. The view of the histological preparate resulted perfectly detailed and clear.

Some slides were kept for six months at a temperature of 35"C in a thermostatic chamber.

Others were kept for the same period at a temperature of -20 C. Others were exposed to light for six months at about 25"C, and others again were exposed to U.V. radiation for two days at about 25"C.

After any of such treatment periods the slides resulted identical to the freshly mounted slides, and the view of the histological preparates was equally perfect.

Claims (17)

1. A composition for use in mounting specimens on microscope slides, which composition comprises, in the form of a homogenous solution: (a) an amorphous and essentially linear homopolymer of styrene; (b) a plasticizer; (c) an UV adsorber; and (d) a terpene which solubilizes the component (a).

2. A composition according to claim 1 containing 20 to 35 per cent of the component (a), 0.5 to 10 per cent of the plasticizer component (b), 0.02 to 2.3 per cent of the UV adsorber component (c), and 55 to 79.5 per cent of the solvent component (d) the respective amounts of component (a), (b) and (d) being based on the total amount of these three components and the amount of component (c) being calculated with respect to the amount of component (a).

3. A composition according to anyone of the preceding claims comprising (a) 23 to 33 per cent of an amorphous and essentially linear homopolymer of styrene having a molecular weight equal to or higher than 200,000; (b) 0.8 to 8 per cent of a plasticizer chosen from the group of the C,-C,0- alkyl esters of the phthalic acid; (c) 0.05 to 2 per cent of UV adsorber; and (d) 59 to 76.2 per cent of a monoterpene as solvent; the respective amounts of components (a), (b) and (d) being based on the total amount of these three components and the amount of component (c) being calculated with respect to the amount of component (a).

4. A composition according to anyone of the preceding claims wherein the plasticizer component (b) is phthalic acid di-n-butyl ester.

5. A composition according to anyone of the preceding claims wherein the UV adsorber component (c) is 2-(2-hydroxy- 5-t-octyl-phenyl)-benzotriazole.

6. A composition according to anyone of the preceding claims wherein the component (d) is the dlform of 1-methyl- 4-(1-methylethenyl)-cyclohexene or an optically active form thereof.

7. A composition according to claim 6 wherein the optically active form of 1-methyl-4-(1-methylethenyl)-cyclohexene is the d-form.

8. A composition according to anyone of the preceding claims containing, as additional component, an antioxidant.

9. A composition according to claim 8 wherein the antioxidant is 2,6-bis-(1,1-dimethylethyl)-4-methylphenol.

10. A method for mounting a specimen on a microscope slide which method comprises mounting the specimen on the slide using a composition as claimed in any one of the preceding claims as the mounting medium.

11. A method according to claim 10 in which a specimen of biological tissue is mounted on the slide.

12. A method according to claim 11 in which the specimen is a section of biological tissue.

13. A method according to claim 11 or 12 in which the biological tissue is stained.

14. A method according to any one of claims 10 to 13 in which the specimen, placed on a supporting slide, is coated with the mounting medium composition and with a covering slide and then the composition's solvent is removed.

15. A microscope slide having a specimen mounted thereon according to a method as claimed in any one of claims 10 to 14.

16. A composition for use in mounting specimens on microscope slides, said composition being any one of compositions (A) to (E) hereinbefore described.

17. A method of mounting a specimen on a microscope slide substantially as hereinbefore described in Example 2.