GB2192712A - Fixative - Google Patents
Fixative Download PDFInfo
- 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
- Authority
- GB
- United Kingdom
- Prior art keywords
- fixative
- anhyd
- fixing
- acid
- pictric
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000834 fixative Substances 0.000 title claims abstract description 47
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 58
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims abstract description 22
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims abstract description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 235000011056 potassium acetate Nutrition 0.000 claims abstract description 11
- 239000004323 potassium nitrate Substances 0.000 claims abstract description 11
- 235000010333 potassium nitrate Nutrition 0.000 claims abstract description 11
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 229960004279 formaldehyde Drugs 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 14
- 235000019256 formaldehyde Nutrition 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims 6
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 abstract description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 229960000583 acetic acid Drugs 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 239000012362 glacial acetic acid Substances 0.000 description 5
- 238000001574 biopsy Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229910001385 heavy metal Inorganic materials 0.000 description 3
- 229960002523 mercuric chloride Drugs 0.000 description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 210000003850 cellular structure Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 150000002731 mercury compounds Chemical class 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000012758 nuclear staining Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000012285 osmium tetroxide Substances 0.000 description 2
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 2
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000007982 barbital buffer Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 231100001223 noncarcinogenic Toxicity 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
Classifications
-
- 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
Landscapes
- 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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8614120A GB2192712B (en) | 1986-06-10 | 1986-06-10 | Fixative |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8614120A GB2192712B (en) | 1986-06-10 | 1986-06-10 | Fixative |
Publications (3)
Publication Number | Publication Date |
---|---|
GB8614120D0 GB8614120D0 (en) | 1986-07-16 |
GB2192712A true GB2192712A (en) | 1988-01-20 |
GB2192712B GB2192712B (en) | 1990-02-14 |
Family
ID=10599246
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB8614120A Expired - Fee Related GB2192712B (en) | 1986-06-10 | 1986-06-10 | Fixative |
Country Status (1)
Country | Link |
---|---|
GB (1) | GB2192712B (en) |
Cited By (3)
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)
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 |
-
1986
- 1986-06-10 GB GB8614120A patent/GB2192712B/en not_active Expired - Fee Related
Cited By (6)
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 |
Also Published As
Publication number | Publication date |
---|---|
GB8614120D0 (en) | 1986-07-16 |
GB2192712B (en) | 1990-02-14 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19940610 |