US2645647A - Clearing agents for histological tissue - Google Patents
Clearing agents for histological tissue Download PDFInfo
- Publication number
- US2645647A US2645647A US182145A US18214550A US2645647A US 2645647 A US2645647 A US 2645647A US 182145 A US182145 A US 182145A US 18214550 A US18214550 A US 18214550A US 2645647 A US2645647 A US 2645647A
- Authority
- US
- United States
- Prior art keywords
- tissue
- clearing agent
- butyl acetate
- histological tissue
- clearing
- 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.)
- Expired - Lifetime
Links
- 239000003795 chemical substances by application Substances 0.000 claims description 24
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 14
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 10
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 6
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims 1
- 230000008595 infiltration Effects 0.000 description 5
- 238000001764 infiltration Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000002380 cytological effect Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- KPZGRMZPZLOPBS-UHFFFAOYSA-N 1,3-dichloro-2,2-bis(chloromethyl)propane Chemical compound ClCC(CCl)(CCl)CCl KPZGRMZPZLOPBS-UHFFFAOYSA-N 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000010186 staining Methods 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
Definitions
- This invention relates to the preparation of histological tissue specimens for microscope examination and, more particularly to clearing agents employed in treating the tissue in the course of such preparation.
- the preparation of tissue to enable the microscopic examination thereof involves a series of treatments of the tissue prior to the cutting of the sections from the tissue specimens for the staining and mounting of the sections on the microscope slides. More particularly, in the preparation of the tissue it is necessary to immerse the tissue successively in a series of liquid agents for certain lengths of time, first to fix the tissue, then to wash the same for removing the fixative, then to dehydrate the tissue, usually by immersion of the tissue successively in alcohols or other dehydration agents, then to immerse the tissue in a clearing agent, and thereafter to infiltrate the tissue with an infiltration agent such as, for example, parafiin, celloidin, etc. After the tissue is thus treated, it is cut into sections of the desired thickness; then the parafiin or other infiltration medium is removed from said sections usually by a solvent, after which the sections are stained and mounted on the slides.
- an infiltration agent such as, for example, parafiin, celloidin, etc.
- tissue-clearing agent be capable of removing all such liquid from the tissue and have a high degree of solvency for, or miscibility with, parafiin so as to facilitate the infiltration of the paraflin into the tissue. It is also very important that the clearing agent be of such character that it does not harden the tissue and does not impair the cytological structures of the tissue.
- the clearing agent be non-inflammable.
- the clearing agent of the present invention possesses these and other desirable characteristics and advantages. as will hereinafter more fully appear, and is moreover less expensive than the clearing agent heretofore preferred by the pathologist.
- the clearing agent usually preferred by the pathologist consisted of butyl acetate.
- This substance although fairly satisfactory in certain respects, especially in that it does not harden and distort the tissue, is not satisfactory in other respects of considerable importance, more especially in that butyl acetate is not as good a solvent for paraffin as maybe desired, has a pungent odor, and is rather expensive.
- the primary object of the present invention is the provision of an improved clearing agent in the respect that it possesses the desirable properties of butyl acetate but has in addition a consider ably greater solvent action on fats and waxes and has a much more pleasant odor than butyl acetate.
- the clearing agent comprises N. butyl acetate, ethylene dichloride and carbon tetrachloride.
- the function of the ethylene dichloride in the composition of the present clearing agent is to impart to the latter the desired degree of solvency for fats and waxes, which the composition would otherwise not possess in view of the considerably lower degree of solvency of butyl acetate.
- the function of the carbon tetrachloride is to raise the flash point of the composition so that the clearing agent is non-inflammable.
- Tissue blocks comprising tissue which has been treated with the clearing agent of this invention have greatly improved cutting qualities, the cytological structures of the tissues are eifectively preserved, and the appearance of the tissue sections is considerably improved.
- a clearing agent for use in the preparation consisting essentially of N. butyl acetate; ethylene? dichloride and carbon tetrachloride, in which the amount by volume of N. butyl acetatezisapproxiea mately equal to the amountstofcbothithe:ethylenet dichloride and carbon tetrachloride, the amount Number of ethylene dichloride exceeds thelamountioflcare bon tetrachloride and the amount of carbon-i.tet+r--- rachloride is sufficient to render the clearing agent substantially non-inflammable.
Description
Patented July 14, 1953 ICE CLEARING AGENTS FOR ,VHISITOLOGICAL TISSUE Andrs Ferrari, Jr., Uniondale, N.'Y.,.assignor to Technicon Chemical Company, Inc., N. Y., a corporation of New York; I
New York,
No Drawing Application August 29,1950,
Serial No. 182,145
2 Claims.
This invention relates to the preparation of histological tissue specimens for microscope examination and, more particularly to clearing agents employed in treating the tissue in the course of such preparation.
The preparation of tissue to enable the microscopic examination thereof involves a series of treatments of the tissue prior to the cutting of the sections from the tissue specimens for the staining and mounting of the sections on the microscope slides. More particularly, in the preparation of the tissue it is necessary to immerse the tissue successively in a series of liquid agents for certain lengths of time, first to fix the tissue, then to wash the same for removing the fixative, then to dehydrate the tissue, usually by immersion of the tissue successively in alcohols or other dehydration agents, then to immerse the tissue in a clearing agent, and thereafter to infiltrate the tissue with an infiltration agent such as, for example, parafiin, celloidin, etc. After the tissue is thus treated, it is cut into sections of the desired thickness; then the parafiin or other infiltration medium is removed from said sections usually by a solvent, after which the sections are stained and mounted on the slides.
As pathologists are well aware, the purpose of treating tissue with a clearing agent before infiltrating the tissue with parafiin is to eliminate from the tissue all liquid which, if not removed, would decrease the refractive index of the tissue and would interfere with the thorough infiltration of the paraffin into the tissue resulting in poorly cut tissue sections. It is of the utmost importance, therefore, that a tissue-clearing agent be capable of removing all such liquid from the tissue and have a high degree of solvency for, or miscibility with, parafiin so as to facilitate the infiltration of the paraflin into the tissue. It is also very important that the clearing agent be of such character that it does not harden the tissue and does not impair the cytological structures of the tissue. Further, among other things, it is desirable that the clearing agent be non-inflammable. The clearing agent of the present invention possesses these and other desirable characteristics and advantages. as will hereinafter more fully appear, and is moreover less expensive than the clearing agent heretofore preferred by the pathologist.
Heretofore, the clearing agent usually preferred by the pathologist consisted of butyl acetate. This substance, although fairly satisfactory in certain respects, especially in that it does not harden and distort the tissue, is not satisfactory in other respects of considerable importance, more especially in that butyl acetate is not as good a solvent for paraffin as maybe desired, has a pungent odor, and is rather expensive.
The primary object of the present invention is the provision of an improved clearing agent in the respect that it possesses the desirable properties of butyl acetate but has in addition a consider ably greater solvent action on fats and waxes and has a much more pleasant odor than butyl acetate.
Pursuant to the present invention, the clearing agent comprises N. butyl acetate, ethylene dichloride and carbon tetrachloride. The function of the ethylene dichloride in the composition of the present clearing agent is to impart to the latter the desired degree of solvency for fats and waxes, which the composition would otherwise not possess in view of the considerably lower degree of solvency of butyl acetate. The function of the carbon tetrachloride is to raise the flash point of the composition so that the clearing agent is non-inflammable.
The following is an example of the presently preferred composition of the clearing agent of the present invention:
N. butyl acetate percent 50.0 Ethylene dichloride do 37.5= Carbon tetrachloride do 12.5
properties of the composition pursuant to the primary object of the invention, as will be apparent to those skilled in the art and in the light of the presence disclosure.
It will be readily understood, of course, that in using the clearing agent of the present invention, the tissue is immersed therein after the dehydration treatment and before the paraflin infiltration process. Tissue blocks comprising tissue which has been treated with the clearing agent of this invention have greatly improved cutting qualities, the cytological structures of the tissues are eifectively preserved, and the appearance of the tissue sections is considerably improved.
It will be understood that various changes may be made in the clearing agent of the'present invention within the skill of the art, without departing from the underlying idea or principles of nliy invention within the scope of the appended c aims.
about theaproportions, by. volume of r.5( )%,.-0f; N.
butyl acetate, ,37.5%"ofethylene dichloride,.,and 12.5% of carbon tetrachloride.
2. A clearing agent for use in the preparation"? of histological tissue for microscopicexamination consisting essentially of N. butyl acetate; ethylene? dichloride and carbon tetrachloride, in which the amount by volume of N. butyl acetatezisapproxiea mately equal to the amountstofcbothithe:ethylenet dichloride and carbon tetrachloride, the amount Number of ethylene dichloride exceeds thelamountioflcare bon tetrachloride and the amount of carbon-i.tet+r-- rachloride is sufficient to render the clearing agent substantially non-inflammable.
v ANDRES FERRARI, JR.
References Cited in the file of this patent UNITED-. STATES PATENTS Number Name Date 1,307,562 Metz et a1 June 24, 1919 1,434,465 Webb Nov. 7, 1922 1,464,170 Carroll Aug. 7, 1923 1,915,163 Morrison et al June 20, 1933 2,393,580 Weiskopf Jan. 22, 1946 FOREIGN PATENTS Country Date 46.1,034L. France Dec. 1'7, 1913 390,867 Great Britain Apr. 20, 1933
Claims (1)
1. A CLEARING AGENT FOR USE IN THE PREPARATION OF HISTOLOGICAL TISSUE FOR MICROSCOPIC EXAMINATION, COMPRISING THE FOLLOWING INGREDIENTS IN ABOUT THE PROPORTIONS, BY VOLUME, OF 50% OF N. BUTYL ACETATE, 37.5% OF ETHYLENE DICHLORIDE, AND 12.5% OF CARBON TETRACHLORIDE.
Priority Applications (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BE504617D BE504617A (en) | 1950-08-29 | ||
| NL73299D NL73299C (en) | 1950-08-29 | ||
| NL76786D NL76786C (en) | 1950-08-29 | ||
| US182145A US2645647A (en) | 1950-08-29 | 1950-08-29 | Clearing agents for histological tissue |
| US199561A US2645618A (en) | 1950-08-29 | 1950-12-06 | Clearing agents for histological tissue |
| GB14738/51A GB699776A (en) | 1950-08-29 | 1951-06-21 | Clearing agents for histological tissue |
| FR1064674D FR1064674A (en) | 1950-08-29 | 1951-07-02 | Thinning agents to increase the transparency of bisological tissues |
| CH303333D CH303333A (en) | 1950-08-29 | 1951-07-31 | Whitening preparations for histological preparations. |
| CH303335D CH303335A (en) | 1950-08-29 | 1951-07-31 | Whitening preparations for histological preparations. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US182145A US2645647A (en) | 1950-08-29 | 1950-08-29 | Clearing agents for histological tissue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US2645647A true US2645647A (en) | 1953-07-14 |
Family
ID=22667242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US182145A Expired - Lifetime US2645647A (en) | 1950-08-29 | 1950-08-29 | Clearing agents for histological tissue |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US2645647A (en) |
| BE (1) | BE504617A (en) |
| CH (2) | CH303335A (en) |
| FR (1) | FR1064674A (en) |
| GB (1) | GB699776A (en) |
| NL (2) | NL76786C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1115388B (en) * | 1976-08-26 | 1986-02-03 | Gen Electric | PRE-COLORED MICROSCOPE SLIDE COVER |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR461034A (en) * | 1912-11-15 | 1913-12-17 | Anilin Fabrikation Ag | Non-flammable solutions of cellulose nitrates, preferably usable as lacquers, and process for their manufacture |
| US1307562A (en) * | 1919-06-24 | Carbon-remover | ||
| US1434465A (en) * | 1922-02-23 | 1922-11-07 | Eastman Kodak Co | Cellulose-ether solvent and composition |
| US1464170A (en) * | 1921-04-05 | 1923-08-07 | Eastman Kodak Co | Cellulose-ether solvent and composition |
| GB390867A (en) * | 1930-10-20 | 1933-04-20 | Arthur Eichengruen | Process for the production of an uninflammable or difficultly inflammable solution of nitrocellulose |
| US1915163A (en) * | 1931-04-06 | 1933-06-20 | Du Pont | Cellulose nitrate composition and solvent mixture therefor |
| US2393580A (en) * | 1942-03-31 | 1946-01-22 | Edwin C Weiskopf | Method of preparing tissue |
-
0
- NL NL73299D patent/NL73299C/xx active
- NL NL76786D patent/NL76786C/xx active
- BE BE504617D patent/BE504617A/xx unknown
-
1950
- 1950-08-29 US US182145A patent/US2645647A/en not_active Expired - Lifetime
-
1951
- 1951-06-21 GB GB14738/51A patent/GB699776A/en not_active Expired
- 1951-07-02 FR FR1064674D patent/FR1064674A/en not_active Expired
- 1951-07-31 CH CH303335D patent/CH303335A/en unknown
- 1951-07-31 CH CH303333D patent/CH303333A/en unknown
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US1307562A (en) * | 1919-06-24 | Carbon-remover | ||
| FR461034A (en) * | 1912-11-15 | 1913-12-17 | Anilin Fabrikation Ag | Non-flammable solutions of cellulose nitrates, preferably usable as lacquers, and process for their manufacture |
| US1464170A (en) * | 1921-04-05 | 1923-08-07 | Eastman Kodak Co | Cellulose-ether solvent and composition |
| US1434465A (en) * | 1922-02-23 | 1922-11-07 | Eastman Kodak Co | Cellulose-ether solvent and composition |
| GB390867A (en) * | 1930-10-20 | 1933-04-20 | Arthur Eichengruen | Process for the production of an uninflammable or difficultly inflammable solution of nitrocellulose |
| US1915163A (en) * | 1931-04-06 | 1933-06-20 | Du Pont | Cellulose nitrate composition and solvent mixture therefor |
| US2393580A (en) * | 1942-03-31 | 1946-01-22 | Edwin C Weiskopf | Method of preparing tissue |
Also Published As
| Publication number | Publication date |
|---|---|
| BE504617A (en) | |
| FR1064674A (en) | 1954-05-17 |
| CH303333A (en) | 1954-11-30 |
| CH303335A (en) | 1954-11-30 |
| NL73299C (en) | |
| GB699776A (en) | 1953-11-18 |
| NL76786C (en) |
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