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Separation of the acidophilic elements of the tissues into two groups.

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The prol'ouncliy differential staining reactions obtained by
the use of phosphotu~~gsticand phorphomolyhdic acid i n
corinectioii with certain acid dyes have long been knoma. AS
applied for example i n tlie well-known Xallory 's connective
tissue stain, the method sen'es to divide the cytoplasmic elements of mammalian tissue into t w o major groups according t o
their staining reactions. It consists in staining Z e n k e r - h c d
material in acid fuclisin, which confers a more o r less uniform
color upon the tissue. Sections a r e transferred t o pliosphomolylsclic or phosphotungstic acid f o r selective differentiation.
(lertain tissue elements, relatively acidophilic, including
general cytoplasm, muscle, erythrocytes, acidophil cells of the
pituitary, epmogen (~7hichwe may call claw 1) retain tlie acid
fuchsin ; others, iiicludiiig collagen, reticulum, cartilage, niucin,
basophil cells of tlie pituitary (class a), a r e completely decolorized. Since it is desirahle that these elements 1iax.e color,
a secondary dye, anilin blue, is used f o r iliat purpose. Various
modifications of Mallory's original stain employing other
fixatives and dyes, when intelligeiitly performed and microscopically controlled, >-ield similar results with respect t o the
staining reactions briefly described above,
Orange G may be incorporated with the secondary dye,
milin blue (Mallorp, '00) or with the primary dye, acid
fuchsin (Crossmon, '37). If material is fixed in Zenker's
or a bichromute fixative, erythrocytes possess a greater
affinity for orange G than acid fuchsiii and consequently a r c
stained orange. The application of orange G has h o n w e r
no bearing 011 the following discussion, and no further 1-cfereiice need be niade to this component of RLallory's stain.
The specific action of pliospliotungstic acid npoii certain
elements of the tissues is well illustratecl by the use of Congo
red. The dye acid is blue, but its sodium salt is red. The
red color of the salt is readily changed by weak acids into
blue. If sections a r e stained with a saturated solution of this
dye containing R small amount of sodium chloride, the tissue
is quite homogeneously stained red. Sections a r e transferred
to 2% phosphotungstic acid f o r a few seconds, qriicldy rinsed
in distilled water, and examined under the microscope.
Tissue elements of class 1 a r e red, those of class 2 a r e blue.
Phosphotungstic acid in aqueous solutioii is unable to remove
this dye selectirely from the secondary elements pet expresses
its specificity by changing their color to blue. The result is
a Mallory stain with the employmelit of one dye. Permaiient
preparations i n xylol balsam a r e however not possible brcause the blue coloration of the secondary clemcnts is
gradually discharged in ethyl alcohol.
Examination of sections stained 1)p Shllory 's original
method sliows that certaiii components forming class 1 are
more intensely stained than others, because these more
acidophil elements have a greater affinit~7for acid fuelisin.
This is also true when certain other dyes a r e iisecl as the
primary stain, e.g., poncenu tlc xylicliiie (Masson, '29).
Preparations stained with hematoxyliii and eosiii show this
same tinetorial differentiation. A few of these elements
long since observed to have greater affiriitv f o r eosin a n d
consequently t o be more intensely stained by it have been
termed eosinophilic, o r more generallj-, acidophilic or osyphilic.
It occurred to the Tvriter that with appropriate technique
and choice of dyes these moi*e intensely staining acidophil
elements of class 1 might be niorc sharply iliffereiitiated.
Experiment with rarious acid clyeq showed that they may
be divided illto three groups accordiiiq t o their reactions wit11
phosphotungstic acid.
1. Dyes that show no reaction to phosphotungstic acid.
Mercurochrome is the oiilj7 example arailable at this time.
2. Dyes such as acid fuchsin or ponceau de xylidine that
may be used for. the division of liistological elenleiits into two
major classes.
3. Dyes that hare affinity f o r only the strongly acidophil
elements of protoplasm. The fluorescein dyes eosin Y,
erythrosin, phloxiiie and rose beiigal are i n this category.
Chromotrope 2R, an azo dye closely relatcd to orange G,
is also in this group, hut its usc has not yet been fully
With this information, ihe following modification of Xallory’s stain, cmploying the iluoresceiii dyes in idace of acitl
fuchsin, has been devised.
1. Fixation. The usual methods of fixation may he einployed. Bichromate-f orrnaliii (or Zenkcr-formaliii) is essential f o r the preservation of zj-mogen granules or other
histological elemeiits soluble in acid fixativcs. Bouin’s fluid
is useful for the demonstration of erythrocytes and othw
aciclopliilic clcrnents riot sohnbl e in acid fixatives, a i d when
used particularly f o r erythrocytes has the adrantage that
maiiy other strongly stainable siriall elements (e.g., zymogen
granules) are not pi~escrvccl. Neutral 10% formalin containing 0.85 gni. of sodium cliloride f o r each 100 cc. is also
a suitable general fixative.
The fixing finids should be prepared and used exactly a s
Rnclwoniate formolui (proportions as used by Masson, ’29)
3 % potassium bichromate in distilled water
Comincrcial f orinalin (neutralized)
90 cc.
1 0 cc.
Commercial formalin map be neutralized by the addition of
niarble chips a fern days prior to fixation. The mixture is
relatively unstable and should be p~epareclimmediately before use,
The duration of fixation is 18 to 24 houi*s,according to the
size of material. Pieces should not h s greater than 8 mm. in
thickness j their other dimensions are of no importance. The
quantity of fixing fluid should be a t least fifty times the volume
of the tissue. Precipitation gradually occiirs in the bichromate formalin mixture after a few hours. Pieces should
be transferred to freshly prepared fixative. One or two
changes are usually sufficient.
Following fixation, material is washed in running tap water
for 18 t o 24 hours and transferred to 70% ethyl alcohol,
preliminary to further dehydration.
Houin’s fluid
Picric acid, saturated aqueous solution
Commercial formalin
Glacial acetic acid
7 I ,> c r .
20 cc.
5 cc.
This mixture is stable and may be prepared in large
quantities. The size of pieces and time of fixation are as f o r
bichromate formalin.
Following fixation, material is washed in 70% ethyl alcohol.
Several changes should be used to remove excess picric acid.
2. Dehydrate completely, embed in paraffin, cut and mount
as usual.
3. Stain in Mayer’s acid liemalniil (standard formula as
given in Lee’s Vade-Xecum). The nuclei should be slightly
overs t ained.
4. Wash sections in running tap water €or at least 15
minutes followed by a change to distilled water.
5. Stain in thymol erythrosin.
Erythrosin, bluish (National A oiline Co.)
Thymol ergatal
Distilled water
0.5 gm.
0.1 gm.
Heat gently until thyniol and erythrosin are dissolved. Tlic
solution should be filtered, as a slight precipitate usually
results. The addition of thynzol is not necessary but of value
because it increases the intensity of staining. In this laboratory it is always used with eosin P wlienever that dye is used
as an acid counterstain, f o r example with hematoxylin.
Other fluorescein dyes iiiclnding eosiii 37, phlosine a n d rose
bengal may be substituted for erythrosin in the formula
above. As stated above, niercurochromc is an exception j
phosphotuiigstic acid shows no selective action with reference
t o this fluorcsceiii dye.
A suggested staining time is 20 minutes. Somewhat morc‘
intense results may be obtained by staining for a longer
6. Rinse i n distilled watcr. This sicp is sometimes omitted.
It is nsually necessary if tlie scctioiis contain striated muscle.
7. Transfer to 2% phosphotungstic acid i n distilled water.
Sections a r e left in this iaeagent 15 to 20 minutes or until
the elements of class 2 a r e microscopically orange, components in class 1remaining red. Like Congo red, erytlirosin
is an indicator. Phosphotungstic acid in aqueous solution is
unable to remove the dye from cornpoiients of class 2 yet
expresses its specificity hy changing their color.
F o r best results fresh phosphotungstic acid should he used
f o r every second rack of slides.
8. Rinse in distilled water. This step is sometimes omitted.
It is usually necessary if sections contain striated muscle.
9. Transfer t o absolute ethyl alcohol. Seetions shoulcl be
gently agitated to allow for imiform and rapid decolorization.
Elements of class 2, colored orange in step 7, a r e immediately
decolorized. Smooth rnuscle, skeletal muscle and general
cytoplasm, colored red in step 7, require a longer time. As
a final result, erythrosin is removed from all tissue elements
with the exception of thosc histological structurcs that arc
strongly acidophil.
Sometimes a little difficulty niay be experienced i n removing all dye from muscle substance, especially with thick o r
torn scctions. If this occurs, slides a r e transferred from the
absolute alcohol back to phosphotnngstic acid for a n additional time interval.
A t this stage the strongly acidopliilic elements are red, tlic
less strongly aeidophilic and the basophilic elements are colorless, and the nuclei have been stained by the hematoxylin.
The procedure limy be terminated by coinpleting dehydration
in one or more clianges of absolute alcohol, follomd by
clearing in xylol and mounting in xylol balsam.
It will however usually be desirable t o introduce color into
the elements left colorless by the preceding steps. Proceed
as follows.
10. Reniordaiit in 2% phospliotungstic acid (repeating
step 7) f o r 2 to 3 minutes.
11. Transfer to light green (or aiiilin blue).
liiglrt green (Iira11 Microcolor ; Eiiiicr :tncl L l ~ l ~ ( ~ n d )
Distillcd water
Glacial acetic acid
1 gm.
100 w.
1 cc.
Since it is dificnlt t o examine the preparation in this stain,
the snggestccl time is 2 to 4 minntm. Light green and aiiiliii
blue are mordanteil to the elements of class 2. With additional staining time the decolorized elements of class 1 map
be lightly stained.
The w e of' light green is suggcstecl for most preparations.
F o r the demonstration of the basophil cells of the pituitaq-,
iiiucin and certain other basophil structures, anilin blue is
superior. The formula is :
Aiiilin blue (Xational -\iuline C'o.)
I)istilld water
Glacial avrtic acid
2 gm.
100 ec.
2 cc.
The procedure is practically the same as with light green,
except that sections are left comparatively longer in the
glacial acetic acid (step 13) t o obtain optimum dilferentiation.
12. Rinse in clistilled water, renioring the sectioris inimecliat ely.
13. Traiisl'cr to 1% glacial acetic acid to remove the light
green or aiiiliii blue looscly bouiid to other than elements of
class 2. Sections shoiild be left in the acid until that result
is attained. Iiispection of a trial slide with the microscope
will determine optimum dillerentiation. Thirty to 60 seconds
is usually sufficient for light green.
The final result should he as indicated in the table at the
end of this paper. Those elements listed in class 1A still
retain the erythrosiri or other substituted fluorescein dye.
Those listed as class IB, having little aEnity for either
primary or secondary dye are colorless or very lightly stained
by the light green or aiiilin blue.
14. Rinse in distilled water removing the sections immediately.
15. Pass througli two or three changes of absolute ethyl
alcohol followed by three changes of sylol. Mount in balsam.
Results obtained, at this time of writing, with respect to
color are listed in the following table.
Class 9
Glusa 1
Colorless o r vei11 rightly
ntauied , q > c e n or blii?
( i r e m or blur
General cytoplasm
Smooth muscle
Collagenous fibers
Eosinophil granules of
Certain zyniogen granules
Russell bodies of carci
noma, sarcoma
Acidophil cells of the
Xeratinized epithelium
Skrletal musclr
Basophil cells of
The staining reactions of the components of class 2 mill be
well known to the reader through the use of the original
Xallory’s stain. The separation into two classes, of the
elements staining with the acid fuchsin in the original
Jlallory’s stain, which is achieved by the new technique herewith described, will require further study. Alan)- questions
suggest themselves as to chemical reasons f o r the high affinity
for acid dyes shown by the elenieiits of class l A , and also as
l o possible relationship (or lack of relation) kd,weeri these
elements, seemingly so diverse.
Specific applications are also a question for additional consideration. Other workers may firid that the Substitution of
other fluorescein dyes o r other methods of fixation serve better
a particiilav purpose. The time of staining or ileeolorization
should be considered as open to modification to suit specific
G. c. 1937 A modification of iVallorp’s connective tissue stain with
a discussion of the principles involved. Anat. Ree., vol. 69, pp. 33-38.
MALLORY,F. B. 1900 A contribution t o staining methods. J. Esp. hled.,
rol. 5, pp. 15-20.
MASSON, P. 1939 Some histological methods. Triclirome stainings and their
preliminary technique.
. I . Tech. Methods, >ol. 12, pp. 75-90.
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two, elements, separating, group, tissue, acidophilus
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