GB2192712A - Fixative - Google Patents

Fixative Download PDF

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Publication number
GB2192712A
GB2192712A GB08614120A GB8614120A GB2192712A GB 2192712 A GB2192712 A GB 2192712A GB 08614120 A GB08614120 A GB 08614120A GB 8614120 A GB8614120 A GB 8614120A GB 2192712 A GB2192712 A GB 2192712A
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United Kingdom
Prior art keywords
fixative
anhyd
fixing
acid
pictric
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GB08614120A
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GB8614120D0 (en
GB2192712B (en
Inventor
Joseph Mcdermott
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LAB SUPPLIES AND INSTR
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LAB SUPPLIES AND INSTR
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Priority to GB8614120A priority Critical patent/GB2192712B/en
Publication of GB8614120D0 publication Critical patent/GB8614120D0/en
Publication of GB2192712A publication Critical patent/GB2192712A/en
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Publication of GB2192712B publication Critical patent/GB2192712B/en
Anticipated expiration legal-status Critical
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305Fixative compositions

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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)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

A histology fixative comprises from 2.75-3.25% w/v of potassium acetate (anhyd.) and from 2.75-3.25% (w/v) of potassium nitrate (anhyd.) in an aqueous solution comprising a quantity of formaldeyde or glutaraldehyde and from 0.25-0.30% (w/v) of picric acid. A cytology fixative can be prepared from the histology fixative by mixing it with 50% v/v absolute alcohol.

Description

SPECIFICATION Fixative This invention relates to a fixative which is particularly suitable for use in histology, but is not restricted to such use and may, for example, be used in cytology.
Histology is the study of the structure and chemical composition of tissues of animals and plants to determine their function, the primary aim being to understand how tissues are organized at all structural levels. Thus, interest lies in both the molecular and macromolecular, the entire cell and intercellular substances as well as the tissues and organs. The study of such biological material involves the use of microscopic, X-ray diffraction and radioactive tracer techniques and for preservation and examination of samples by such techniques fixation has proved invaluable.
Fixation is the process of killing cells while preserving their structure and internal organization in as life-like a condition as possible. Such a process involves the inactivation of enzyme systems, particularly those responsible for autolysis, and also ideally hardens cellular structures, rendering the cell components compatible with particular stains. However, most if not all methods of fixation produce artefacts to a lesser or greater degree.
Such partial denaturing of the cell's components may be seen in heat fixation which, although acceptable for bacterial smears, is unsuitable if fine cellular details are to be examined. Furthermore, in practice it cannot be relied on to kill all species of bacteria.
By the use of some chemical fixatives, a more accurate morphology may be obtained. Chemical fixatives permeate cells and stabilize their components by binding to them or denaturing then.
Although most fixatives react with proteins or lipids, some react with both, some fixatives such as ethanol, mercuric chloride, picric acid precipitate proteins thereby introducing a considerable disturbance in cellular organization: others such as formaldehyde, glutaraldehyde, potassium dichromate and osmium tetroxide do not cause precipitation and tend to preserve proteins in situ, the last two mentioned also being important lipid fixatives. A fixative comprising formaldehyde in the form of Formalin (40 /O solution), although not precipitating proteins has a slow rate of fixation, and is therefore not entirely satisfactory. One approach to provide more effective fixatives involves the addition of heavy metals to such reagents as Formalin.
Although trace amounts of certain heavy metals are essential for the growth of most microorganisms, higher concentrations may exhibit antimicrobial activity.
Several mercury compounds have been tried to date such as inorganic mercurials, eg, mercuric chloride and organic mercurials, which are generally less toxic and more effective antimicrobial agents. Unfortunately, most mercury compounds have no antisporidal activity.
Although the toxicity of mercuric fixatives such as mercuric formol has improved fixation, these mercuric salts have introduced new problems: their corrosive nature makes it difficult to use the fixative with automatic processing equipment, while their carcinogenic properties make them a general hazard.
Examination of specimens by light microscopy typically involves fixation thereof in, for example, Bouin's Fluid (a mixture of picric acid, formalim and glacial acetic acid), Carnoy's Fluid (either ethanol/glacial acetic acid in a 3:1 ratio, or ethanol/chloroform/glacial acetic acid in a 6:3:1 ratio), polyvinyl alcohol, Schaudinn's Fluid (mercuric chloride/ethanol/glacial acetic acid) or Zenker's Fluid (merduric chloride/potassium dichromate/ glacial acetic acid).
Fixation of specimens for electron microscopy is more critical since structural disturbances must be minimal. For such work, glutaraldehyde and osmium tetroxide are commonly used, usually in an alkaline phosphate buffer, or an acetate barbital buffer.
Specimens for Auto-radiography should be fixed in solutions which do not contain heavy metals since these may block or divert radiation from point sources in the specimen.
According to this invention there is provided a fixative comprising from about 2.75-3.25% w/v of potassium acetate (anhyd.) and from about 2.75-3.25% (w/v) of potassium nitrate (anhyd.) in an aqueous solution comprising a quantity of formaldehyde or glutaraldehyde and from about 0.25-0.30% (v/v) of picric acid.
Preferably the fixative comprises from 3.0-3.2% (w/v) potassium acetate (anhyd.), from 2.53.0% (w/v) potassium nitrate (anhyd.), from 18-19% (v/v) formaldehyde (as 36% w/v formalin solution) and 0.25-0.30% (v/v)- picric acid (aq.) sat (approx. 1.2%), and water.
An advantage of this fixative is that a pH of'7.4 is obtained, which is critical for the intended purpose.
The invention will now be described by way of the following example.
Brunnell's histology fixative 260 g Potassium acetate, anhyd.
250 g Potassium nitrate, anhyd.
1.6 -L Formalin(36%) 25 ml Picric acid, sat.
7 1 Water pH 7.4 In an alternative formulation glutaraldehyde is substituted for formalin. Equally from 240-280 g of potassium acetate (anhyd.) with from 240-280 gof potassium nitrate may be used to provide an acceptable fixative. The fixative of this invention (Brunnell's fixative) represents a single fixing material with a wide range of excellent properties which can meet the demands of almost any sample or processing requirement.
This new fixative is a modified buffered formalin solution to be used as a general primary histology fixative. Buffered to pH 7.4 it has several important advantages over standard buffered formalin.
Brunnell's fixative is extremely fast. The optimum length of time in solution is twelve hours, although- excellent results have been obtained with small blocks after three hours. This rate of fixation would be particularly useful in labaratories with a high throughput of blocks.
The fixative can be used satisfactorily with automatic processing equipment because it contains no mercuric salts.
Brunnell's fixative gives even penetration and can safely be used for a wide variety of tissue samples including brain. Nuclear staining is much improved compared with normal buffered formalin and, after processing, tissues section more readily. By maintaining enzyme activity samples processed in Brunnell's fixative for light microscopy can subsequently be used in electron microscopy procedures.
This new development in histology combines the advantages of mercuric formol without the use of hazardous mercuric salts.
Use as a Cytology Fixative Brunnell's fixative has been tested with sputum and smear samples and excellent results were obtained. The cytology fixative is prepared by mixing equal portions of Brunnell's and absolute alcohol.
Nuclear staining is enhanced using this mixture.
Technical Report on Brunnell's Fixative A variety of both surgical and post mortem tissues were sampled and secondary fixed in Brunnell's fixative. The biopsy tissues were fixed for six hours and the post mortem tissues fixed for 24 hours. The large biopsy specimens and post mortem organs were primarily fixed in 10% buffered formalin. The blocks were routinely processed on a 14 hour schedule.
The sections were all stained with the Haematoxylin and Eosin method. The blocks sectioned fairly easily and the staining result were good.
BIOPSY SPECIMENS POST-MORTE-M SPECIMENS Tissue Type Tissue Type Liver As for biopsy Kidney plus Spleen Brain Lung H e a r t Uterus Prostrate Brunnell's fixative gives a clearer stain than with mercuric formol and cell morphology is more definite.
Brunnell's is preferred to mercuric formol because it is non-carcinogenic and has no corrosive properties which are attributable to the use of mercuric salts.

Claims (13)

1. A fixative comprising from 2.75-3.25% w/v of potassium acetate (anhyd.) and from 2.753.25% (w/v) of potassium nitrate (anhyd.) in an aqueous solution comprising a quantity of formal-dehyde or glutaraldehyde and from 0.25-0.30% (v/v) of pictric acid.
2. A fixative according to claim 1 comprising from 3.0-3.2% (w/v) potassium acetate (anhyd.), from 2.8 -3.0% (w/v) potassium nitrate (anhyd.), from 18-19% (v/v) formal-dehyde (as 36% w/v formalin solution) and 0.25 -0.30% (v/v) pictric acid (aq.) sat (approx. 1.2%), and water.
3. A fixative according to claims 1 or 2 adapted for use as a histology fixative.
4. A fixative according to claims 1 or2 adapted for use as a cytology fixative.
5. A fixative according to claim 4 comprising 50% (v/v) absolute alcohol.
6. A method of fixing a sample comprising preparing a fixative including from 2.75-3.25% w/v of potassium acetate (anhyd.) and from 2.75-3.25% (w/v) of potassium nitrate (anhyd.) in an aqueous solution comprising a quantity of formal-dehyde or glutaraldehyde and from 0.25-0.30% (v/v) of pictric acid, the pH being buffered to pH7.4 thereafter fixing.
7. A method according to claim 6 wherein the fixative comprises from 3.0-3.2% (w/v) potassium acetate (anhyd.), from 2.8-3.0% (w/v) potassium nitrate (anhyd.), from 18-19% (v/v) formal-dehyde (as 36% w/v formalin solution) and 0.25-0.30% (v/v) pictric acid (aq.) sat (approx. 1.2%), and water.
8. A method according to claim 6 or 7 wherein the sample is fixed with the fixative for twelve hours.
9. A method according to claims 6 to 8 wherein the fixative is adapted for use as a histologyfixative.
10. A method of fixing a tissue sample comprising primarily fixing said sample in 10% buffered formalin and thereafter further fixing the said sample in a fixative comprising 50% v/v absolute alcohol with 50% v/v of mixture including from 2.75-3.25% w/v of potassium acetate (anhyd.) and from 2.75-3.25% (w/v) of potassium nitrate (anhyd.) in an aqueous solution comprising a quantity of formal-dehyde or glutaraldehyde and f rom 0.25-0.30% (v/v) of pictric acid.
11. A method of fixing a tissue sample according to claim 10 wherein said mixture comprises from 3.0-3.2% (w/v) potassium acetate (anhyd.), from 2.8-3.0% (w/v) potassium nitrate (anhyd.), from 18-19% (v/v) formaldehyde (as 36% w/v formalin solution) and 0.25-0.30% (v/v) pictric acid (aq.) sat (approx. 1.2%), and water.
12. A method of fixing a sample as described with reference to the examples.
13. A fixative as described with reference to the examples.
GB8614120A 1986-06-10 1986-06-10 Fixative Expired - Fee Related GB2192712B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB8614120A GB2192712B (en) 1986-06-10 1986-06-10 Fixative

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Application Number Priority Date Filing Date Title
GB8614120A GB2192712B (en) 1986-06-10 1986-06-10 Fixative

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GB8614120D0 GB8614120D0 (en) 1986-07-16
GB2192712A true GB2192712A (en) 1988-01-20
GB2192712B GB2192712B (en) 1990-02-14

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0562877A2 (en) * 1992-03-27 1993-09-29 ORTHO DIAGNOSTIC SYSTEMS INC. (a New Jersey corp.) Cell fixative and method of staining cells without destroying the cell surface
WO2016041890A3 (en) * 2014-09-17 2016-06-09 Ventana Medical Systems, Inc. Compositions, methods, and systems for tissue fixation
US10126216B2 (en) 2011-02-17 2018-11-13 Ventana Medical Systems, Inc. Method for tissue sample fixation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114279801B (en) * 2021-12-24 2024-03-29 哈尔滨东尧科技有限公司 Formaldehyde-free fixing liquid and fixing method for fixing various fresh tissues

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0562877A2 (en) * 1992-03-27 1993-09-29 ORTHO DIAGNOSTIC SYSTEMS INC. (a New Jersey corp.) Cell fixative and method of staining cells without destroying the cell surface
EP0562877A3 (en) * 1992-03-27 1993-12-29 Ortho Diagnostic Systems Inc Cell fixative and method of staining cells without destroying the cell surface
US5597688A (en) * 1992-03-27 1997-01-28 Ortho Diagnostic Systems Inc. Cell fixative and method of analyzing virally infected cells
US10126216B2 (en) 2011-02-17 2018-11-13 Ventana Medical Systems, Inc. Method for tissue sample fixation
US11624684B2 (en) 2011-02-17 2023-04-11 Ventana Medical Systems, Inc. Method for tissue sample fixation
WO2016041890A3 (en) * 2014-09-17 2016-06-09 Ventana Medical Systems, Inc. Compositions, methods, and systems for tissue fixation

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Publication number Publication date
GB8614120D0 (en) 1986-07-16
GB2192712B (en) 1990-02-14

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PCNP Patent ceased through non-payment of renewal fee

Effective date: 19940610