US3546334A - Composition for fixing and protecting a smear of body cells and method of applying same - Google Patents

Composition for fixing and protecting a smear of body cells and method of applying same Download PDF

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Publication number
US3546334A
US3546334A US457842A US3546334DA US3546334A US 3546334 A US3546334 A US 3546334A US 457842 A US457842 A US 457842A US 3546334D A US3546334D A US 3546334DA US 3546334 A US3546334 A US 3546334A
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smear
composition
alcohol
slide
water
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US457842A
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Irwin S Lerner
Hugh J Davis
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LERNER LAB Inc
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LERNER LAB Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions
    • G01N2001/307—Fixative compositions non-toxic, no Hg, no formaldehyde

Definitions

  • This invention relates to a novel cytological fixative and protective composition, and to a method for applying such composition. More particularly, this invention relates to the treating of a slide having thereon a smear of body cells for microscopic examination, so as to fix and preserve the cells for such examination, and involves depositing on the slide a composition comprising an alcohol, water, a polyalkylene glycol, and a ketone.
  • Another object is to provide a method of simultaneously fixing the specimen on the slide and protecting it with a coating, the fixing and protecting being effected in a single step, whereby the slide may then be handled without damaging the specimen.
  • a cytological fixative and protective composition comprising an alcohol, water, a polyalkylene glycol, and a ketone.
  • the alcohol functions as a fixative and advantageously contains from about 1 to 10 carbon atoms, such alcohols as methanol, ethanol, and isopropanol being preferred. We have found that particularly desirable results are obtained using isopropanol.
  • the polyalkylene glycol serves as a fixative and also remains as a residual film on the surface of the slide, thus serving to protect the smear.
  • the polyalkylene glycol should have a molecular weight of from about 1000 to 4000.
  • polyethylene glycol we have found polyethylene glycol to be especially advantageous because it is non-toxic as well as water soluble. Accordingly, when the smear is to be subjected to microscopic examination, the protective polyethylene glycol film is easily removed by treatment with an aqueous liquid.
  • the ketone functions as a fixative and additionally enables the overall composition to rapidly penetrate the smear. This is highly advantageous, because the cells should be fixed while still fresh and moist, that is, before appreciable drying has occurred. Otherwise, changes in morphology invariably occur.
  • the ketone may be repersented by the formula 0:0 R2 wherein R and R may be alkyl containing from about 1 to 10 carbon atoms, aryl such as e.g. phenyl or naphthyl, alkaryl, or aralkyl.
  • R and R may be alkyl containing from about 1 to 10 carbon atoms, aryl such as e.g. phenyl or naphthyl, alkaryl, or aralkyl.
  • the preferred ketone is acetone.
  • the amount of water in the water-alcohol mixture may be from about 4 to cc., with the amount of alcohol being correspondingly from about 96 to 20 cc. If desired, a minute amount of a dye may also be incorporated.
  • the foregoing liquid composition is desirably applied in dropwise manner to the slide to thereby cover the smear, e.g., by means of a dropper applicator, by spraying, or the like.
  • the composition may be caused to flow over the slide surface, provided that care is taken to prevent any portion of the cells of the specimen from being washed off.
  • the slide Upon covering the smear, the slide is permitted to dry. This serves to fix the cells, while at the same time an appreciable proportion of the volatile constituents of the composition, e.g., the alcohol and ketone, will evaporate off, thereby leaving a protective film residue made up primarily of the polyethylene glycol.
  • the slide may now be handled without damage to the specimen.
  • the film is readily removed by washing with an aqueous liquid, e.g., water.
  • the polyethylene glycol readily dissolved so that the overall formulation was a liquid solution.
  • the foregoing solution was applied to a slide having thereon a freshly prepared smear of body cells.
  • the application was dropwise by spraying the solution onto the slide surface so as to completely cover the smear.
  • the slide was allowed to dry, thereby fixing the cells and leaving a protective film of polyethylene glycol covering the smear. It was found that the slide could then be handled and/or stored without damage to the smear.
  • the slide subesequently was prepared for microscopic examination by Washing with water to thereby dissolve and remove the polyethylene glycol protective film.
  • Microscopic examination showed the cells of body tissue to exhibit a high degree of transparency, with no change in morphology.
  • a cytological fixative and protective composition comprising a liquid solution of an alcohol containing from about 1 to 10 carbon atoms, water, a polyalkylene glycol having a molecular Weight of from about 1000 to 4000, and acetone, the proportions being, per 100 cc. of alcohol-water, from about 1 to 40 grams of said polyalkyl ene glycol and from about 5 to 50 cc. of acetone, the proportions of said alcohol-water being from about 20 to 96 cc. of alcohol and correspondingly from about 80 to 4 cc. of water.
  • composition of claim 1 wherein said alcohol is isopropyl alcohol, and said polyalkylene glycol is polyethylene glycol.
  • composition of claim 1 wherein there is additionally present a dye 3.
  • composition of claim 3 wherein said dye is toluidine blue.
  • a method of treating a slide having thereon a smear of body cells for microscopic examination comprising depositing dropwise on said slide a composition comprising a liquid solution of an alcohol containing from 1 to 10 carbon atoms, water, a polyalkylene glycol having a molecular weight of from about 1000 to 4000, and acetone, the proportions being, per 100 cc. of alcohol-water, from about 1 to 50 grams of said polyalkylene glycol and from about 5 to 50 cc. of acetone, the proportions of said alcohol-water being from about 20 to 96 cc. of alcohol and correspondingly from about 80 to 4 cc. of water.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)

Description

United States Patent 3,546,334 COMPOSITION FOR FIXING AND PROTECTING A SMEAR OF BODY CELLS AND METHOD OF APPLYING SAME Irwin S. Lerner, Greenwich, Conn., and Hugh J. Davis, Baltimore, Md., assignors to Lerner Laboratories, Inc., Greenwich, Conn., a corporation of Connecticut No Drawing. Filed May 21, 1965, Ser. No. 457,842 Int. Cl. G01n 1/00, 1/30 US. Cl. 4243 Claims ABSTRACT OF THE DISCLOSURE Cytological fixative and protective composition that includes a liquid solution of an alcohol, water, a polyalkylene glycol, and a ketone. The composition may also contain a dye.
This invention relates to a novel cytological fixative and protective composition, and to a method for applying such composition. More particularly, this invention relates to the treating of a slide having thereon a smear of body cells for microscopic examination, so as to fix and preserve the cells for such examination, and involves depositing on the slide a composition comprising an alcohol, water, a polyalkylene glycol, and a ketone.
It is conventional practice to place a smear on a slide and examine it under a microscope. If such a smear is permitted to dry in the air, the cells generally are not suitable for obtaining a clear picture under the microscope. To avoid drying of the smear, various fixative compositions have been applied. Thereafter, a protective coating has been applied in order to protect the surface of the smear.
It is an object of the present invention to provide a novel and improved cytological fixative and protective composition.
Another object is to provide a method of simultaneously fixing the specimen on the slide and protecting it with a coating, the fixing and protecting being effected in a single step, whereby the slide may then be handled without damaging the specimen.
Additional objects and advantages will become apparent from the following detailed description.
In accordance with one aspect of our invention, there is provided a cytological fixative and protective composition comprising an alcohol, water, a polyalkylene glycol, and a ketone.
The alcohol functions as a fixative and advantageously contains from about 1 to 10 carbon atoms, such alcohols as methanol, ethanol, and isopropanol being preferred. We have found that particularly desirable results are obtained using isopropanol.
The polyalkylene glycol serves as a fixative and also remains as a residual film on the surface of the slide, thus serving to protect the smear. The polyalkylene glycol should have a molecular weight of from about 1000 to 4000. We have found polyethylene glycol to be especially advantageous because it is non-toxic as well as water soluble. Accordingly, when the smear is to be subjected to microscopic examination, the protective polyethylene glycol film is easily removed by treatment with an aqueous liquid.
The ketone functions as a fixative and additionally enables the overall composition to rapidly penetrate the smear. This is highly advantageous, because the cells should be fixed while still fresh and moist, that is, before appreciable drying has occurred. Otherwise, changes in morphology invariably occur.
The ketone may be repersented by the formula 0:0 R2 wherein R and R may be alkyl containing from about 1 to 10 carbon atoms, aryl such as e.g. phenyl or naphthyl, alkaryl, or aralkyl. The preferred ketone is acetone.
The preferred proportions for our fixative-protective composition are as follows:
Water-alcohol-IOO cc. Polyalkylene glycoll-5O gm. Ketone-550 cc.
The amount of water in the water-alcohol mixture may be from about 4 to cc., with the amount of alcohol being correspondingly from about 96 to 20 cc. If desired, a minute amount of a dye may also be incorporated.
The foregoing liquid composition is desirably applied in dropwise manner to the slide to thereby cover the smear, e.g., by means of a dropper applicator, by spraying, or the like. Alternatively, the composition may be caused to flow over the slide surface, provided that care is taken to prevent any portion of the cells of the specimen from being washed off. Upon covering the smear, the slide is permitted to dry. This serves to fix the cells, while at the same time an appreciable proportion of the volatile constituents of the composition, e.g., the alcohol and ketone, will evaporate off, thereby leaving a protective film residue made up primarily of the polyethylene glycol. The slide may now be handled without damage to the specimen.
When the smear is to be subjected to microscopic examination, the film is readily removed by washing with an aqueous liquid, e.g., water.
The following example will further illustrate our invention.
EXAMPLE The following formulation was prepared:
Ingredient: Amount Isopropyl alcohol cc Water cc l0 Polyethylene glycol (Molecular weight of about 1540) g 5 Acetone cc 20 Dye (toluidine blue) cc 0.01
The polyethylene glycol readily dissolved so that the overall formulation was a liquid solution.
The foregoing solution was applied to a slide having thereon a freshly prepared smear of body cells. The application was dropwise by spraying the solution onto the slide surface so as to completely cover the smear. The slide was allowed to dry, thereby fixing the cells and leaving a protective film of polyethylene glycol covering the smear. It was found that the slide could then be handled and/or stored without damage to the smear.
The slide subesequently was prepared for microscopic examination by Washing with water to thereby dissolve and remove the polyethylene glycol protective film. Microscopic examination showed the cells of body tissue to exhibit a high degree of transparency, with no change in morphology.
Variations can of course be made Without departing from the spirit of the invention.
Having thus described our invention, what we desire to secure and claim by Letters Patent is:
1. A cytological fixative and protective composition comprising a liquid solution of an alcohol containing from about 1 to 10 carbon atoms, water, a polyalkylene glycol having a molecular Weight of from about 1000 to 4000, and acetone, the proportions being, per 100 cc. of alcohol-water, from about 1 to 40 grams of said polyalkyl ene glycol and from about 5 to 50 cc. of acetone, the proportions of said alcohol-water being from about 20 to 96 cc. of alcohol and correspondingly from about 80 to 4 cc. of water.
2. The composition of claim 1 wherein said alcohol is isopropyl alcohol, and said polyalkylene glycol is polyethylene glycol.
3. The composition of claim 1 wherein there is additionally present a dye.
4. The composition of claim 3 wherein said dye is toluidine blue.
5. A method of treating a slide having thereon a smear of body cells for microscopic examination, comprising depositing dropwise on said slide a composition comprising a liquid solution of an alcohol containing from 1 to 10 carbon atoms, water, a polyalkylene glycol having a molecular weight of from about 1000 to 4000, and acetone, the proportions being, per 100 cc. of alcohol-water, from about 1 to 50 grams of said polyalkylene glycol and from about 5 to 50 cc. of acetone, the proportions of said alcohol-water being from about 20 to 96 cc. of alcohol and correspondingly from about 80 to 4 cc. of water.
6. The method of claim 5 wherein said alcohol is isopropyl alcohol, and said polyalkylene glycol is polyethylene glycol.
7. The method of claim 5 wherein there is additionally present a dye.
8. The method of claim 7 wherein said dye is toluidine blue.
9. The method of claim 5 wherein said dropwise deposition is by spraying.
10. The method of claim 5 wherein after said dropwise application said slide is permitted to dry whereby the volatile constituents of said composition evaporate to thereby leave a residual protective film of said polyalkylene glycol and treating said slide with a aqueous liquid prior to microscopic examination to thereby dissolve said film.
References Cited UNITED STATES PATENTS 3,389,052 6/1968 Ehrenreich 4243 FOREIGN PATENTS 799,458 8/1958 Great Britain 167-88 OTHER REFERENCES ALBERT T. MEYERS, Primary Examiner A. P. FAGELSON, Assistant Examiner U.S. Cl. X.R. 424-7, 32
US457842A 1965-05-21 1965-05-21 Composition for fixing and protecting a smear of body cells and method of applying same Expired - Lifetime US3546334A (en)

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4120991A (en) * 1976-12-10 1978-10-17 Technicon Instruments Corporation Process for mounting tissue sections with an U.V. light curable mounting medium
US4284495A (en) * 1979-12-10 1981-08-18 Newton William A Coating apparatus and method
US4595524A (en) * 1984-03-28 1986-06-17 Miles Laboratories, Inc. Two component stain composition for producing a Giemsa blood stain effect
EP0241025A1 (en) * 1986-04-08 1987-10-14 Whittaker Bioproducts, Inc. Method and substrate for immunofluorescent microscopy
EP0311035A2 (en) * 1987-10-09 1989-04-12 Dr. Barry A. Siegfried Histological fixative
US5401625A (en) * 1993-06-24 1995-03-28 E. K. Industries, Inc. Histological composition for light microscopy
US5453381A (en) * 1993-12-10 1995-09-26 Lipton; Stewart Method for adhering fecal samples to slides
US5540892A (en) * 1993-04-22 1996-07-30 Kidd; Marvin L. Urinalysis collection and testing kit and method
WO1999004240A1 (en) * 1997-07-15 1999-01-28 Bernard Pajak Method for fixing and embedding tissues for histological preparations
EP1005633A1 (en) * 1997-08-20 2000-06-07 The University Of Miami A high quality, continuous throughput, tissue fixation-dehydration-fat removal-impregnation method
US6337189B1 (en) 2000-02-08 2002-01-08 Streck Laboratories, Inc. Fixative system, method and composition for biological testing
DE10040448A1 (en) * 2000-08-18 2002-03-07 Osram Opto Semiconductors Gmbh Semiconductor chip and method for its production
US20030211452A1 (en) * 2002-05-10 2003-11-13 Vladimir Vincek Preservation of RNA and morphology in cells and tissues
US6793890B2 (en) 1997-08-20 2004-09-21 The University Of Miami Rapid tissue processor
US20070172911A1 (en) * 2006-01-13 2007-07-26 Michael Farrell Biological sample processing composition and method
US20070292899A1 (en) * 2006-05-11 2007-12-20 Stephen Lovell Method Of Protein Extraction From Cells
US20080261266A1 (en) * 2004-12-17 2008-10-23 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US7470401B2 (en) 2003-10-24 2008-12-30 The University Of Miami Simplified tissue processing
US20090123910A1 (en) * 2007-11-14 2009-05-14 Malick Adrien P Method of efficient extraction of protein from cells
US20090298172A1 (en) * 2008-05-28 2009-12-03 Steven Paul Wheeler Histological specimen treatment apparatus and method
US20110236895A1 (en) * 2008-12-05 2011-09-29 Olympus Corporation Method for preparing sample, solution for preparing sample and stool collection kit method for analyzing a nucleic acid
US10365189B2 (en) 2015-05-07 2019-07-30 Steven Wheeler Histological specimen treatment

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB799458A (en) * 1956-08-07 1958-08-06 Ernest Norland Improved means for dyeing the hair
US3389052A (en) * 1965-11-02 1968-06-18 Ehrenreich Theodore Fixing and drying cytological smears

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB799458A (en) * 1956-08-07 1958-08-06 Ernest Norland Improved means for dyeing the hair
US3389052A (en) * 1965-11-02 1968-06-18 Ehrenreich Theodore Fixing and drying cytological smears

Cited By (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4120991A (en) * 1976-12-10 1978-10-17 Technicon Instruments Corporation Process for mounting tissue sections with an U.V. light curable mounting medium
US4284495A (en) * 1979-12-10 1981-08-18 Newton William A Coating apparatus and method
US4595524A (en) * 1984-03-28 1986-06-17 Miles Laboratories, Inc. Two component stain composition for producing a Giemsa blood stain effect
EP0241025A1 (en) * 1986-04-08 1987-10-14 Whittaker Bioproducts, Inc. Method and substrate for immunofluorescent microscopy
EP0311035A2 (en) * 1987-10-09 1989-04-12 Dr. Barry A. Siegfried Histological fixative
EP0311035A3 (en) * 1987-10-09 1990-01-10 Xenetics Biomedical, Inc. Histological fixative
US5540892A (en) * 1993-04-22 1996-07-30 Kidd; Marvin L. Urinalysis collection and testing kit and method
US5401625A (en) * 1993-06-24 1995-03-28 E. K. Industries, Inc. Histological composition for light microscopy
US5453381A (en) * 1993-12-10 1995-09-26 Lipton; Stewart Method for adhering fecal samples to slides
US5607870A (en) * 1993-12-10 1997-03-04 Lipton; Stewart Parasitological preservative for fecal samples
WO1999004240A1 (en) * 1997-07-15 1999-01-28 Bernard Pajak Method for fixing and embedding tissues for histological preparations
US6793890B2 (en) 1997-08-20 2004-09-21 The University Of Miami Rapid tissue processor
EP1005633A1 (en) * 1997-08-20 2000-06-07 The University Of Miami A high quality, continuous throughput, tissue fixation-dehydration-fat removal-impregnation method
US7547538B2 (en) 1997-08-20 2009-06-16 The University Of Miami High quality, continuous throughput, tissue processing
US8221996B2 (en) 1997-08-20 2012-07-17 The University Of Miami High quality, continuous throughput, tissue processing
EP1005633A4 (en) * 1997-08-20 2003-01-22 Univ Miami A high quality, continuous throughput, tissue fixation-dehydration-fat removal-impregnation method
US6586713B2 (en) 1997-08-20 2003-07-01 The University Of Miami Apparatus for high quality, continuous throughput, tissue fixation-dehydration-fat removal-impregnation
US6337189B1 (en) 2000-02-08 2002-01-08 Streck Laboratories, Inc. Fixative system, method and composition for biological testing
DE10040448A1 (en) * 2000-08-18 2002-03-07 Osram Opto Semiconductors Gmbh Semiconductor chip and method for its production
US7138226B2 (en) 2002-05-10 2006-11-21 The University Of Miami Preservation of RNA and morphology in cells and tissues
US20030211452A1 (en) * 2002-05-10 2003-11-13 Vladimir Vincek Preservation of RNA and morphology in cells and tissues
US8288168B2 (en) 2003-10-24 2012-10-16 The University Of Miami Simplified tissue processing
US7470401B2 (en) 2003-10-24 2008-12-30 The University Of Miami Simplified tissue processing
US20090136992A1 (en) * 2003-10-24 2009-05-28 The University Of Miami Simplified tissue processing
US8288121B2 (en) 2004-12-17 2012-10-16 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8652803B2 (en) 2004-12-17 2014-02-18 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US8512978B2 (en) 2004-12-17 2013-08-20 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US20080261266A1 (en) * 2004-12-17 2008-10-23 Ventana Medical Systems, Inc. Methods and compositions for a microemulsion-based tissue treatment
US20070172911A1 (en) * 2006-01-13 2007-07-26 Michael Farrell Biological sample processing composition and method
US8962262B2 (en) 2006-05-11 2015-02-24 Arbor Vita Corporation Method of protein extraction from cells
US20070292899A1 (en) * 2006-05-11 2007-12-20 Stephen Lovell Method Of Protein Extraction From Cells
US9771398B2 (en) 2006-05-11 2017-09-26 Arbor Vita Corporation Method of protein extraction from cells
US20090123910A1 (en) * 2007-11-14 2009-05-14 Malick Adrien P Method of efficient extraction of protein from cells
US9207240B2 (en) 2007-11-14 2015-12-08 Arbor Vita Corporation Method of efficient extraction of protein from cells
US9995745B2 (en) 2007-11-14 2018-06-12 Arbor Vita Corporation Method of efficient extraction of protein from cells
US20090298172A1 (en) * 2008-05-28 2009-12-03 Steven Paul Wheeler Histological specimen treatment apparatus and method
US9366605B2 (en) 2008-05-28 2016-06-14 Steven Paul Wheeler Histological specimen treatment apparatus and method
US20110236895A1 (en) * 2008-12-05 2011-09-29 Olympus Corporation Method for preparing sample, solution for preparing sample and stool collection kit method for analyzing a nucleic acid
US10365189B2 (en) 2015-05-07 2019-07-30 Steven Wheeler Histological specimen treatment
US10641688B2 (en) 2015-05-07 2020-05-05 Steven Wheeler Histological specimen treatment
US11885723B2 (en) 2015-05-07 2024-01-30 Steven Wheeler Histological specimen treatment

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