US3737335A - Method for preparing a specimen - Google Patents

Abstract

A PROCESS FOR PREPARING A SPECIMEN TO BE VIEWED IN A MIROSCOPE, IN WHICH A POLYVINYL ALCOHOL (PVA)-FIXATIVE-SPECIMEN MIXTURE IS PROTECTED AGAINST THE DELETERIOUS EFFECTS OF ALCOHOL DURING THE STAINING PROCEDURE. THIS PROCESS MAY BE ACCOMPLISHED BY SURROUNDING THE PVAFIXATIVE-SPECIMEN MIXTURE WITH A BUFFER ZONE. THE BUFFER ZONE CAN INCLUDE ANY SUBSTANCE OR SOLUTION THAT BLOCKS THE WETTING ACTION OF ALCOHOL, FOR EXAMPLE, WATER, SALINE OR CLYCERINE TO NAME A FEW.

Description

United States Patent O 3,737,335 METHOD FOR PREPARING A SPECIMEN Sheldon N. F einberg, 98 Broadway, Hillsdale, NJ. 07642 No Drawing. Filed July 17, 1970, Ser. No. 55,982

Int. Cl. G02b 21/34 US. Cl. 117-72 8 Claims ABSTRACT OF THE DISCLOSURE Alternatively, the PVA-fixative-specimen mixture may be protected against separation by promoting a better PVA bonding to the slide or coverslip by the use of an insolubilizing agent (e.g. di-methylolurea), a coupling agent (e.g. silane), a first paint substance (e.g. neoprene), a Wetting agent or by using specimen slides with a roughened surface or granular coating.

BACKGROUND OF THE INVENTION It is Well known in the prior art that polyvinyl alcohol (PVA) is used with specimens as an adhesive solution to enable the specimen to adhere to a slide or a coverslip and to prevent the specimen, especially protein in the specimen, from breaking down. Generally the specimen to be examined is mixed with the polyvinyl alcohol-fixative solution and placed on a slide or coverslip. The mixture is permitted to dry for twelve to twenty-four hours and then can be passed through a series of staining solutions which, in a known manner, brings out the details of the cells. During the twelve to twenty-four hour drying period, hours of what can be precious time are lost. An additional problem relates to the use of alcohol in the staining process. It has been found that the wetting action of the alcohol deleteriously affects the polyvinylalcohol specimen mixture by loosening the adhesion between the polyvinyl alc'ohol-specimen mixture and the slide or coverslip. When this occurs, a new specimen must be prepared and more time is lost.

An example of a type of specimen which can be prepared is a stool specimen. When a patient who has diarrhea is examined at the physicians office, a stool specimen can be rapidly stained in the new method described below. The stained specimen is examined under the microscope and serves as a valuable diagnostic aid to the physician.

Diarrhea is most usually caused by infection. Infection is of three types: (1) viral, (2) bacterial or (3) protozoan. Viral diarrheas subside naturally and no treatment is known; however, bacterial and protozoan caused diarrheas are amenable to treatment. But, the treatments are different and it is potentially harmful to treat diarrhea without knowledge of the exact cause. Properly selected drugs can be life saving but areoften characterized by hazardous side effects Emetine hydrochloride, for example, is very effective in severe protozoan diarrhea but can cause carditis (infiamation of the heart). iEmetine, therefore, should never be used in diarrhea of bacterial etiology. Thus the exact cause of diarrhea must be determined before a drug is selected. It is important also to identify the cause of diarrhea rapidly so that treatment may be started as soon as possible. However, prior 3,737,335 Patented June 5, 1973 methods are not satisfactory mainly because of time delays.

A 5-7 day delay occurs when the diarrhea stool is sent to the laboratory for growth and identification of pathogenic bacteria. Meanwhile the patient can become severely ill and even die.

Though it is possible to examine the patients stool under the microscope, the method as previously practiced has several drawbacks. When the stool is not preserved and stained, cells are difiicult to differentiate and what is worse, they deteriorate within 30 minutes. The specimen must then be discarded. When the stool is preserved and stained by the prior art method, the process is so complicated and time consuming that few hospitals are prepared to use the test.

lIn the prior art, a 12 to 24 hour drying period was necessary before the PVA-specimen mixture could be stained. 'If stained without this long delay for drying, the alcohol used in the stain prevented the specimen from adhering to the glass surface of the slide or coverslip. It is felt that the wetting action of the alcohol used in the staining process produces the poor adhesion of the PVA- specimen mixture.

SUMMARY OF THE INVENTION It has been discovered that by using a buffer zone surrounding the adhesive solution such as PVA mixed with a specimen, that the deleterious effects of the wetting action of alcohol can be successfully repulsed. With the use of such buffer substances the specimen may be passed through the alcohol staining solutions after only 1 hour of drying time. In addition, it has been found that the actual staining time can be reduced from 35 minutes to 15 minutes with the use of these buffer substances. An aqueous or non-aqueous solution or any substance or solution that blocks the wetting action of alcohol can be used to create a buffer zone. Examples of some materials which have been successfully used are Water, saline or glycerine.

It has also been discovered that the adhesive bond between the polyvinyl alcohol-specimen mixture and the slide or coverslip can be greatly improved by the use of 1) an insolubilizing agent such as dimethylolurea; (2) a coupling agent such as silane; (3) a first paint substance such as neoprene; (4) a wetting agent; or (5) the slides can have a roughened surface or a granular coating to improve adhesion.

It is therefore one of the principal objects of this invention to provide a method which will enable a. specimen to be preserved and stained in approximately one hour and fifteen minutes so that the diagnosis and treatment of diarrhea may be substantially hastened.

It is another object of the invention to improve the adhesion between a polyvinyl alcohol-specimen mixture and a slide or coverslip.

It is still another object of the invention to improve and greatly decrease the amount of time required to stain a specimen.

It is yet another object of the invention to improve the adhesion of a polymer to another member.

Further objects and advantages of the present invention will become apparent as the following description proceeds and the features of novelty which characterize the invention will be pointed out with particularity in the claims annexed to and forming part of this specification.

For the purpose of illustrating the present invention, a specific type of specimen and substances to be used therewith are disclosed. It should be understood, however, that the aspects of the present invention are applicable to the many varied types of specimens and numerous equivalent substances can be used where it is desired to secure a specimen to a slide or coverslip and then stain the specimen. The teachings of this invention can be applied in any environment where it is desired to promote polymer adhesion; the invention is not limited to use with a specimen but is intended to cover the field of polymer use. It is necessary to initially prepare a specimen so that it can be stained with various colored dyes and viewed in a microscope. The purpose for staining a specimen is to bring out the detail of the cells so that the practitioner can differentiate between the cells. For example, in examining a stool specimen, it is necessary to distinguish between a pus cell, a macrophage or a protozoan. By staining the specimen the differences are apparent to the skilled eye of the practitioner. Generally, a trichrome staining procedure is used and further detail concerning this procedure can be obtained in an article entitled A Rapid One-Step Trichrome Stain by George Gomori, M.D., published in the American Journal of Clinical Pathology, 1950, Volume 20 at page 661.

The specimen, which can be a stool, for example, is initially thoroughly mixed with a solution of polyvinyl alcohol (PVA) to which mercury bichloride has been added. PVA is readily obtainable and is marketed under the trade name Elvanol by E. I. du Pont de Nemours and Co., Wilmington, Del. The PVA is a polymer which acts as a preservative because it kills bacteria which attack and destroy the structure of cellular matter (pus cells, protozoans, etc.) in the stool specimen. The PVA also functions as an adhesive because it causes a wet stool specimen to adhere to a glass slide or coverslip.

The mercury bichloride which is added to the PVA solution functions as a fixative, i .e. it fixes or retards structural changes during the staining process. A drop of the PVA-specimen mixture is then placed on a slide or coverslip and is allowed to dry at room temperature for approximately one hour.

The dried PVA-specimen mixture is then pre-rinsed in a solution of normal saline for approximately thirty seconds. After the pre-rinse, the PVA-specimen mixture is ready to be stained and it is thereafter placed in the following solutions for the time period noted:

Solution: Time Iodine alcohol (70%) 5 minutes. Alcohol (70%) 1 minute. Alcohol (70%) '1 minute. Trichrome stain 5 minutes. Acid alcohol (90%) 3 minutes. Alcohol (100%) 3 seconds. Alcohol (100%) 30 seconds. Xylol '1 minute.

It is necessary to remove the water from the PVA specimen because it can create a cloudy effect and render the specimen non-discernible under a microscope. By passing the specimen through increasingly greater concentrations of alcohol, the water content can be removed. The alcohol must be added progressively to prevent distortion and undue shrinkage of the specimen.

It has been discovered that the alcohol used in the staining procedure does not permeate between the polyvinyl alcohol specimen mixture and the glass interface providing that the PVA specimen mixture has been prerinsed in normal saline. Thus, the pre-treatment of the specimen with the normal saline apparently forms a buffer zone around the PVA-specimen that leads to increased resistance to reagent permeation of the interface between the glass and the PVA-specimen mixture.

EXAMPLE -I A drop of polyvinyl alcohol fixative solution mixed with a protozoan specimen was permitted to dry on a coverslip at room temperature for 1 hour. The coverslip was then dipped in a solution of normal saline (0.9 gm. NaCl per 100 cc. water) followed by a second saline rinse of one half normal saline (0.45 gm. per 100 cc. of water). Immediately thereafter, the PVA-specimen was immersed consecutively in the eight staining solutions outlined above. The PVA-specimen mixture was viewed in a microscope and the cellular detail was excellent. The specimen had excellent adherence to the coverslip and there was no significant loss of specimen material.

As a control, two coverslips having PVA specimen mixtures thereon were used which were not pre-rinsed in the saline solutions. The specimen was otherwise prepared identically to the other specimens in which the coverslips were pre-rinsed. The result was a complete wash away of the specimen otf the coverslip when the coverslips were immersed in the first alcohol staining solution indicating that the saline pre-rinse created a buffer zone through which the alcohol in the staining solutions could not penetrate.

Other substances which have been successfully used as pre-soak liquids are water and glycerol. All of the latter liquids blocked the Wetting action of alcohol on glass. Similar substances as for example, ethylene glycol, and certain PVA solvents, e.g. diethylene triarnine, would be expected to have a similar protective action if used as a pre-rinse.

In order to prove that a 'pre-rinse with normal saline prevents wash away of the specimens on a coverslip by some action other than normotonicity, the following experiments were conducted.

EXAMPLE II Sucrose was added to each staining solution until the concentration reached 10% (normotonicity). No saline prerinse was used. Two specimens were mixed in a polyvinyl alcohol fixative solution and were allowed to dry for 1 hour on a coverslip.

The specimens on the coverslips were passed through the staining solutions and the specimens were completely washed away thereby indicating that normotonicity of the staining solution does not prevent major wash away of the specimen. I

EXAMPLE 111 Two specimens were mixed with a polyvinyl alcohol fixative solution and allowed to dry for 1 hour on a coverslip. The specimens were passed through a pre-rinse of distilled water (no salt content) and thence through the 8 staining solutions.

The specimens exhibited no signs of being washed away and had good adherence to the coverslip. Thus, it is clear that nomotonicity of the pre-rinse solution is not necessary to prevent major wash away.

Another experiment was conducted to ascertain if glycerine added to the first alcohol rinse would prevent the wash away of the polyvinyl alcohol specimen. Two specimens were mixed with poly-vinyl alcohol and allowed to dry for 1 hour on a coverslip. The specimens were then passed through a first alcohol solution (8 cc.) to which 2 cc. of glycerine was added. Each specimen was then passed through the remaining 8 staining solutions. The major portion of each specimen remained adherent to the coverslip thereby proving that glycerine when added to the first alcohol solution prevents significant wash away of the specimen.

The agents described above block the deleterious wetting action of alcohol by forming a buffer zone that protectively surrounds the PVA-specimen drop. Fermentation of alcohol between the PVA specimen drop and glass can also be prevented by using methods that create a tighter bond between the PVA and the underlying glass.

A tighter bond prevents alcohol from seeping under the PVA. Substances which have worked satisfactorily by producing a better bond include a coupling agent, an insolubilizing agent and a paint coating.

EXAMPLE USING A COUPLING AGENT Two coverslips were coated (by brush) with a known coupling agent (10% silane) and were permitted to air dry at room temperature. A drop of polyvinyl alcohol was then placed on each coverslip and dried at room temperature for one hour. The coverslips were then passed through the aforementioned 8 staining solutions. Two control coverslips were also used. The controls were not coated with silane but were otherwise treated identically as the other coverslips.

Satisfactory adhesion of the polyvinyl alcohol to the coverslip was obtained. A 5% silane solution was also tried as above and similar results were produced. In the controls used in all cases, the polyvinyl alcohol washed away.

EXAMPLE USING AN INSOLUBILIZING AGENT Two coverslips were coted with 10% dimethylolurea and were permitted to air dry at room temperature. An insolubilizing agent such as dimethylolurea reacts with PVA, the adhesive solution, in a known manner to form an insoluble product which is diflicult to dissolve or to lift 011 a slide. Two controls were not coated with anything. Polyvinyl alcohol, mixed with a protozoan specimen, drops were applied to all four coverslips and allowed to dry for one hour. The coverslips were then passed through the aforementioned eight trichrome staining solutions.

It was then noted that the polyvinyl alcohol specimen mixture adhered better to the coverslips pre-ccoated with dimethylolurea than when not so coated as in the controls.

EXAMPLE USING A COATING OF PAINT Two coverslips were coated with neoprene G590 manufactured by Pittsburgh Plate Glass Industries, Inc., Bloomfield, N.I., and were allowed to air dry at room temperature. A drop of PVA-specimen mixture was applied to each coverslip and allowed to dry for one hour. The coverlips were passed through the eight trichrome staining solutions and satisfactory adhesion of the PVA specimen mixture occurred.

It should also be noted that the adhesion of a polymer such as PVA upon a slide or coverslip can be increased by making the surface of the slide, which is normally smooth, rough by scraping the surface or by etching the surface or by using any other known roughening technique. The surface of the slide or coverslip can also be lightly burred. When the polymer is placed on the slide or coverlip it become wedged in the minute interstices of the roughened or etched surface of the slide or coverslip and the alcohol used in the trichrome stain cannot permeate the interface between the polymer and the slide.

It will be obvious to those skilled in the art that various changes may be made without departing from the spirit of the invention, for example, polymers other than PVA can be used, such as ethyl cellulose or Kelmar made by Kelco Company to name two or any solution having adhesive properties can be used; therefore, the invention is not limited to what is described in the specification but only as defined within the true spirit and scope of the appended claims.

I claim:

1. A method for preparing a specimen to be viewed in a microscope comprising the steps of,

(a) mixing said specimen with a specimen adhesive,

(b) applying said specimen and specimen adhesive to a means facilitating the viewing of said specimen in a microscope,

(c) encircling said specimen adhesive with a liquid buffer which blocks the wetting action of alcohol used in a subsequent staining process.

2. A method for preparing a specimen as defined in claim 1 in which said means facilitating the viewing of the specimen comprises a slide.

3. A method for preparing a specimen as defined in claim 1 in which said liquid comprises water.

4. A method for preparing a specimen a defined in claim 1 in which said liquid comprises a saline solution.

5. A method for preparing a specimen as defined in claim 1 in which said liquid comprises glycerol.

6. A method for preparing a specimen as defined in claim 1 in which said liquid comprises diethylene glycol.

7. A method for preparing a specimen as defined in claim 1 in which said liquid comprises an insolubilizing agent which serves to render said adhesive solution insoluble.

8. A method for preparing a specimen as defined in claim 1 in which said adhesive solution comprises polyvinyl alcohol.

References Cited UNITED STATES PATENTS 3,341,399 9/1967 Hazdra et al. 117-124 FX 3,619,256 11/1971 Pepe et al 117-124 FX 3,498,860 3/1970 Pickett 424-3 OTHER REFERENCES Lillie, Histopathologic Technic and Practical Histochemistry, McGraw-Hill Book Co., New York, 1965, third ed., pp. 64, 77, 105, 159.

RALPH HU SACK, Primary Examiner US. Cl. X.R.