CA1037366A - Tissue staining method - Google Patents

Abstract

TISSUE STAINING METHOD

Abstract of the Disclosure A superior and extremely rapid histological stain-ing method is described in which frozen tissue is briefly immersed in an aqueous alcoholic solution comprsing trichloro-acetic acid, zinc chloride and formaldehyde and then in a second solution comprising hematoxylin. The tissue can be further counterstained if desired.

Description

~ 137361~;
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The pre~ent invention pertains to a his~ological ~tain- :~
ing method for tissue specimens prefatory to microscopic examina-tion. The selective staining o tissue cells both to identify various parts of the cell ~nd to distinguish different types of cells is a well kno~ diagnostic techni~ue. Suitable techniques yielding reproducible resul~s are however o~ten time consuming and cumbersome. In a typical cryogenic application, two major techni-ques are followed. The tissue is rapidly frozen and sectioned in part. These sections are fixed in, for example, formaldehyde or alcohol, stained most frequently with a single stain, coverslipped and examined This preparation is not permanent and has a limited ; llfe of only several hours. ~ile it perm~ts a more or less rapid examination of the tissue, the ~echnique has several disadvantages, most notably the diferent appearance, principally tinctorial, of the specimen from the usual microscopic preparations which thereby increases the difficulty of obtaining a corre~t interpretation.
In order to have a more definitive microscopic analysis and a permanent preparation of the original tissue a second techni-que is followed. Here, the froz~n tissue is thawed, fixed, dehy-drated, infiltrated with organic solvents, embedded in paraffin and sectioned. These sections are in turn deparaffinized9 hydrated, stained, dehydrated and roverslipped This provides a permanen~
microscopic preparation of the material originally studied in the ~emporary cryogenic method. As noted, this technique is extremely ,. -. , . ~ 1.'.. , . .. , , ~ .
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1~373~6 lengthy and c~bersome and moreover the specimens are not an exact replica of the origlnal tissue. In addition to the possible damage caused by ice crystal formation and tha~ing, the specimen may have differen~ orientation leading to a di~lerent plane o~ sectioning.
Tissue is also lost in the course of the chemical and physical pro-cessing.
The interplay of these two techniques, and their inherent disadvantages, can be most vividly seen in, for example, the case of a suspected malignancy~ Typically, a tissue sample removed from a patient in the course of exploratory surgery will be subjected to the first technique in order to permit the surgeon to make an imme-diate decision concerning the nature and extent o radical surgery.
Preparation of a permanent microscopic specimen to permit confirma-tion of the diagnosis can require as much as twenty-four hours or more which obviously limits the microscopic examination of tissues removed during surgery to the less definitive temporary prepara-~ions since the patient cannot be maintained in ~he operating room for the longer period required for a permanent preparation. Moreover, final results of tissue studies are not known for one or several days, thereby increasing or prolonging the natural apprehension o patient and family. Since the gross specimens upon which the diag-nosis were based are often not suitable for retention~ elaborate preservation steps must be taken to document the medical condition ~nd provide a long term reference. It occasionally happens that the tissue lost in processing~ although small, represents that por-tion of the tissue in the temporary preparation upon which the microscopic diagnosis was based.

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T~e e is described ln~coDendi~lg applicati~l Serial No.
/ ~ 3~ a novei histolo~ica1 fix~tive comprising a solution of trichloroacetic acid, zinc chloride and formaldehyde in an aqueous lower allcanol. Thls ~ixative causes minimal damage to the cells of the tissue, is convenien~ in actual use, and can be employed with a wide variety of tissue types. It has now been discovered that this ~ixative can be employed in a vastly superior method of staining tissue specimens. The entire stain procedure can be completed in a matter of minutes and the resulting stained specimens demo~strate suficient permanence to permit their reten~ion for months without special prec~utions.
The tissue to be stained is rapidly frozen ~y any of the well known techniques. The froze~ tissue is then sectioned with a conventional microtome and the sections then immersed in a fixing solution co~?rising an aqueous lower alkanolic solution o~ tri-chloroacetic acid, zinc chloride and formaldehyde. The section has of course already thawed as of this i~mersion but is fixed in ; a matter of seconds. A typical period of immersion is five seconds and ~hile this period may be extended, no advantages result from 20 a longer immersion.
The section is next immersed in an aqueous solution of hematoxylin. Hemato~ylin is a well kno~n stain ~?hich che~icallJ

is 7,11b-dihydrobenz[b]indenoEl,2-d]pyran-3,4,6a,~,10(6H)-pentol.
This step o~ the process can be conducted in one stage or in two stages. In one embodimen~, the section is immersed in the solu-tion of hematoxylic until staining is comple~e, generally from 10 to 30 seconds. In a second embodiment, the specimen is im-mersed fox a period in a first solutlon o hematoxylin, removed - ~ 4 ~

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~73~;6 and rinsed with water and then immersed in a second solution of hematoxylin. In a preferred aspect of this second embodiment, the immersion of the specimen in the second solution of hema-toxylin is preceded by a brief immersion in a solution of lithium carbonate which acts in the capaci~y of an accelerator. Thus.for example, the section emerging from the fixi~g solution i5 immersed in a first hematoxylin solution for a period of about 15 seconds, rinsed by dipping into water, briefly immersed in a solution of lithium carbonate, say for example for a period of 4 seconds,and again immersed in a solution of hematoxylin for about 5 seconds.
At this point, the specimen ha~ fixed and the cell nuclei stained with a bluish-purple hue. ~he specimen can then be prepared for mounting by washing and drying, the latter being performed in the conventional manner of sequential immersion in progressively more hydrophobic organic media, e.g. ~5% ethanol, absolute ethanol and xylol.
. I desired, ~e specimen can also be counterstained using any of the well known differentiating stains, as for example eosin, a fuchsin such as rosaniline, pararosaniline, magenta II, magenta III or acid fu hsin; picric acid or the like.
As noted above, the fixative comprises a solution of trichloroacetic acid, zinc chloride and formaldehyde in an aqueous lower alkanol. The lower alkanol can be any of the well known alcohols having from 1 to 4 carbon atoms as for example methanol, e~hanol, n-propanol~ isopropanol9 n~butanol and the various branched butanols. Methanol is highly satisfactory aDd preferred ..

3i73 ~torn ttll~ .q~:nr~ olllt ol~ c3coll0mlcrl nnd .~olvent pow~r. Thc ~queou~
lo~r nll~fln(~l. wlll ll~v~ ~ voltlme rr~tio o~ nlknnol to total watcr o~ f.~om nl~ut l~ o rlho~ 3. ~ p~f~c~rred rn~l.o 1~ ~bf~u~ 1:1.1 ~:o nl~o~t 1: l . 5 . r)i.~q~olved in ttl~3 .EIqt~eo~l.s a lknnol i~ ~ mlxture oJ~ tr.l.c~llo~nc(3t:1.c nci.d, 7.inc chlor:Lcl(3 ~ncl ~ormaldehydc. 1~ r~tio ol~ lnc clll~rl.lla to ~:rl.cl~loronca~ acid, on .~ wai~ht b~si~, Is ~rom ~botlt: 2:1 t:o about 4:1. ~ rr~t:lo o 3:1 i8 highly 9~tl~nctory.
Th~ rn~.ic oE orrn~lll(~hy(l~ o tho trichloroncctic acicl i8 r~rom nbclut: 8:1 to ~Ibou~ 10:1, n typic~ll rnt:io bc~n~ 9:1.
. ~t prcEQtr~cl flnfll concentrnl~ion i~ nbou~ 107. w~igh~/volt!rn~
o~ tll~ trlchloronc~tlc nclcl, zinc chlorid~ ~Ind Eormaldehyd~ in th~
nqu~ou~ low~ nl.lc~n~l~
Th~ hemn oxylln cnn be irl nny o~ th~ u~utll Porrn~. Thcse i.nc~Id~ IInrri~ lIamntoxylisl, M~y~r~ Hn~mn1um, Erlich~ Hematoxyl~n, 1~ ~h~ nlum h~m~toxylln d~scrib~d by Li~ ot al., ~fayo Cllnlc Proc~ed-in~,~; 4~ 31~ (~fay 1971), ~nd the lik~
On~ p~r~icularly u~fu~ prep~r~ion i~ that containlng hema~ox~r1in, y~l1O~ merc-Ir1c oxid~ and alumlnum Rmm~nium 8ul~te in Llqueou~ ~lyc~ln, tl~i~ r~nE~nt ~ener~11y being known a~ al~n hematoxylln.

A. FI~ nr S~1U~i_n Four hundr~d ~ram~ cP ~inc chlorlde ~r~ c~mbined w~t~
120 ~ram~ of ~ric~ roac~tic acld and ~.64 li~ers of 37% form-aldehyd~ ~coxre~pondi~ o lQ55 ~rams of lOOZ~ for~ldeh~d~ in ^~

7.3 liters o~ 95~/~ methanol (corresponding to 7 l~ters of 100%
m~thanol) and 7 liters of water. A slight exothermic reaction occurs and ater the ~olution has reattained room tempera~ure, it is filtered and containerizèd. This solution, which is re~dy for use as a tissue fixatlve, corresponds to a 9.8% wt/vol. solu-tlon of trichloro2cetic acid, zlnc chlor~de and formaldehyde wherein the weight ratio of zinc chloride to trichloroacetic acid i~ about 3.33 :1 and the wei~ht ratio of formaldehyde to trichloro-acetic acid is a~out 9:1, in a~ueous metha~ol wherein the volume ratlo of alkanol to total water (including th~t added separately, ~hat contained in the 95% methanol and that cont&ined in the 37%
formaldehyde) ls about 1:1.3.

B. (1) Stainin~ Solution To a 801ution o~ 0.67 g of hematoxylin in 33 ml of absolute ethanol sre added 33 ml of distilled water, 33 ml o~
glycerol, 3.3 ml of glacial acetic acld and an excesg of potas~ium . aluminum sulfate. The mixture is shaken well and allowed to stand - for several weeks after which it i8 reAdy for ~se.

B. (2) Sr~ A ~

One grsm of hematoxylin, 0.5 g of yellow mercuric oxide and 12 g of alumi~um ammonium sulf~te are mixed with 140 ml of di8t~.11ed wa~er. Thi~ mi~ture is boiled for 10 minutes, cool~.d : and adiusted to its origin~l volume with di~illed water. Six~y m~lliliters of glycerin ~nd 8 ~1 of glacial acetic acid are added ~5 arld the mixture is then filtered.

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~ ~ 7 3 6 6 C, Gounters tainin~Solution These can be purchased as such and include aqueou3 solutions of eosin, basic fuchsim, plcric acid and the like.

Example 1 Frozen tlssu~ specimens are sectioned on a micro~ome . to a thiclless of about 5~G~. The individual sections are im-mersed in the fixing solution for approximately 5 seconds and then into staining solution B (2) for approximately 15 seconds, Af~er dipping inko distilled water or about 3 seconds, the 0 8ectio~ iS ~mmersed in ~ ~aturated solution of lithium carbonate for approximately 4 seconds and then im~ediately reimmersed in 8tainlng solution B(2) for ~bout 5 ~econds. The ixed and stained section.is then ri~sed in distilled water or 3 seconds and sequentially immersed in 957. ethanol, absolute ethanol, xylol ~o. 1 and xylol No. 2, each im~ersion being about 2 seconds, and i mounted with a coverslip.

A counterstained sectlon is obtained according to ~he - procedure of Example 1 by ri~sing the specimen wi~h distilled ~ater for 3 seconds followlng its removal from the second im- :
~ersio~ in the B(2) stsining solu~io~, ~mmersing it in 95%
ethanol for 2 seconds and ~hen ~mmersing ~he same in a standard eosln solu~ion for appro~imately 15 seconds. The ~hus di~feren~
~, ; tiated section is then sequentially immersed in 95~ ethanol 7 .~5 absolu~e ethanol and xylol as described ln Exampl~ 1.

Claims (7)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of staining tissue for microscopic exami-nation which comprises (a) immersing frozen tissue in an 8 to 20% wt/vol solution of a mixture of trichloroacetic acid, zinc chloride and formaldehyde wherein the weight ratio of zinc chloride to trichloroacetic acid is from about 2:1 to about 4:1 and the weight ratio of formaldehyde to trichloroacetic acid is from about 8:1 to about 10:1, in an aqueous lower alkanol wherein the volume ratio of alkanol to total water is from about 1:1 to about 1:3, and (b) thereafter immersing the tissue at least once in an aqueous solution comprising 0.1 to 2% hematoxylin.

2. The method according to claim 1 wherein solution (a) is a 9 to 11% solution wt/vol of a mixture of trichloro-acetic acid, zinc chloride and formaldehyde wherein the weight ratio of zinc chloride to trichloroacetic acid is from about 3:1 to about 3.5:1 and the weight ratio of formaldehyde to tri-chloroacetic acid is from about 8:1 to about 10:1, in aqueous methanol wherein the volume ratio of methanol to total water is from about 1:1.1 to about 1:1.5.

3. The method according to claim 1 wherein said hematoxylin is complexed with alum.

4. The method according to claim 1 wherein at least one of the immersion of said tissue in said solution of hematoxylin is preceded by immersion of said tissue in a solution of lithium carbonate.

5. The method according to claim 1 wherein said tissue is counterstained at least once after immersion in said solution of hematoxylin.

6. The method according to claim 5 wherein said counterstain is eosin.

7. The method according to claim 5 wherein said counterstain is a fuchsin.