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On differential staining.

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Osborti Zodloyzcul Laborntory, Y a l e University
I n studying cytoplasmic inclusions in the cells of the digestive system of spiders T was confronted with the problem
of finding stains which would permit clear differentiation.
The inclusions are all granular or globular, of intergrading
sizes and of not less than six different organic substances,
exclusive of fats and lipoids. Several kinds occur jointly in
the same cells and cannot be easily separated either by their.
size or shape. I n may cases they all stain alike or else exhibit
a bewildering series of intergrading colors. This is especially
true in the case of triple and quadruple staining such a s
Xlillot 's ( '26) acid fuchsin, metanil yellow, light green,
toluidin blue method which, as I am 11om quite certain, led
him to wrong conclusions. To settle the question as to whether
the intergrading colors were clue to differences in the chemical
nature of the inclusions or to faults inherent in the staining methods themselves, an extensive analysis was found
to be necessary. A great many stains and staining methods
after difierent fixing fluids were tried with variable success.
It m7us at this stage of my work that I had R conference with
my friend Doctor Tolstoouhov, on the application of pH
control in staining. Unfortunately, neither his methods, nor
those of other investigators who used buffered solutions of
stains gave the desired results. However, they put me on the
track of certain phenomena involved in differential staining,
which finally gave me the clue to a proper and reliable method.
This method combining the principle of destaining with
control of PIT promises to have such wide application in
T H E A N 4 T 0 3 1 1 C A L RECOKI), \OT.. 6 8 , NO.
histology that it seertied desirable to publish it in siicli form
its to make it possible f o r othei. investigators to a p p l ~it to
ot h el* material.
Let us first coiisider the factors involved in differential
staining. In doing so we may disregard the question a s t o tlicl
physical or cheniical nature of staining and use the ex1)ression ‘staining affinity ’ for the reaction which takes place
wlieii a substance arid a stain a r e brought together. Diffcretitintion is a function of the staining affinity and may be due
t o one or several of the following factors: The chemical
c~mpositionand physical state of tlic substrata, their change
uiidel. the influence of various fixing fluids, the isoelectric
iwint, solubility in the staining and washing fluids, the
chemical composition, purity ant1 dissociation coilstant of the
stain, the pII of the stain, tlie concentration of the stain, cluration of staining, tho tclmpcrature at which the stain is used
a i i d the respective properties of the washing fluids and
niounting medium. 31aiiy of these factors are interrelated
and have been extensively studied hy various investigators,
making their further, detailed consideration liere unnecessary. A short list of references is given at the e i d of this
paper. Further references will be found in the papers listed.
F o r the better widerstanding of the method to which we
may now turn oiir attention, the folloming points must be
cmliliasi zed.
1. Fixation has a profound effect on staining. Thus the
so-called refractive bodies i n tlie interstitial tissue of the
diqestive orgaiis of spiders reniain colorless in acid fiichsin
a t all pH values at room temperature after fixation in almost
all fixing fluids geneidly ernployed, but stain a deep red after
fixation in absolute alcohol or paranitroformol (4 parts of
my cupric pai.ariitroy)lieiiol fixing- fluid with 1 part of 37 to
40r/c1reagent grade formalin, freshly added).
2. Duration of staining has an effect only when it is shorter
than the time required for the completion of the staining reaction. From that time on duration has iio effect whatsoever.
To achieve identical results one sliould, therefore, always
briiig staining t o completion.
3. The solvent used in the preparation of the staining fluid
has a distinct effect on staining even though not easily
noticeable at a glance. Alcoholic and aqueous solutions of
the same stain in equal eoncentration and adjusted to the same
pH produce a different degree of diff’erentiation, aqueous
solutions being distinctly more selective. Ruff er solutions
used a s solvents for the stains show the same effect, The
greatest differentiation is obtained by aqueous solutions
adjusted to a certain pH by the addition of HC1 or NaOH.
Nearest in effect come stains dissolved in acetate, phosphate
and borate buffers. Citrate buffers a r e less suitable because
they give a more diffuse staining. Stains dissolved in
phthalate buffers give very poor differentiation and should
be avoided.
4. The pI-1 value of the staining solution is of the highest
importance in itself. Moreover, il is intimately related to
the method of fixation. The same stain used after different
fixing fluids may have the same effect, but more often has a
different effect. I n the latter case, a t least after some fixing
fluids, it may or may not be possible to achieve the same result
by using the staining fluid at some other p H value. It is an
old experience that staining after fixation in picric acid mixtures is difficult and unsatisfactory. The reason for this lies
not in an inability of the tissues so fixed to become stained,
but in the fact that they stain diffusely at all values of pH.
5. The concentration of the stain, when the stain is allowed
to act to the completion of its reaction, has no noticeable effect
on the result within very wide limits. A 0.017. solution, let
us say, of toluitlin blue gives the same depth of staining a s
a 0.1% or a 1%solution when used at the same pH to the
completion of its reaction. When unbuffered aqueous solutions a r e used, the degree of concentlation may lead to a
considerable change of the ~ € 1 .Thus a 2yj aqueous solution
of acid fuchsiii showed a p H value of 2.85, while the same
solution further diluted with distilled water to 0.257. had a
p H 3.64. Griibler’s aurantia in a saturated aqueous solution
had a pII 7.8. A freshly prepared solution of 0.1% had a pH
THE A N A T O M I C 4 1 ~RKCOBII, VOI.. 6 8 , NO.
of ca. 6.4. It is clear that the superiority of staining in dilute
unbuffered aqueous solutions as often recommended in preference to more concentrated solutions, is due to the change in
the pH value and not to concentration as such.
6. The temperature at which a stain is used has little effect
on the result within a wide range, provided the pH is kept
constant. The latter, of course, changes with temperature.
If the same pH is to be used at considerably different
temperatures, the buffer solution has to be adjusted correspondingly. A t temperatures over 60°C. a profound change
i n staining properties may be observed. Thus the above
mentioned refractive bodies after fixation in Regaud remain
colorless in acid fuchsin a t all pH values within usual room
temperature limits, but stain a deep red at a temperature of
60°C. or more in the acid range up to about pH 4.
7. The pH of the washing fluid has a profound effect on
the result, an effect which is different from that of the pH
of the staining fluid. I n view of its importance in the application of the method proposed here, it will be discussed in
greater detail below.
To achieve uniform results the sources of error were
eliminated by the following methods :
a. Stock solutions of a certain concentration were made
f roin samples of certified stains further purified whenever
possible. Weighing was done on an analytical balance.
Water was twice distilled in a Pyrex distilling apparatus.
A volumetric flask was used for dilution.
b. Measurements of pH were made to the secoiid decimal
a t a temperature of 25°C. with the aid of a glass electrode
and a n amplifier with a pliotron tube.
c. Temperature was controlled by a n airbath of special
construction, having a tolerance of
O.O2"C., and a water
bath with a tolerance better than O.l°C.
d. Long series of 7 p sections were made from the same
block and mounted without albumen, by stretching over
warm distilled water in the usual way. The same species of
spider was used in all experiments, namely Phormictopus
cancerides (Latreille), a native of Haiti. I n addition, some
experiments were made with smears of human blood and
sections through the intestine of the leopard frog and the
newt. I t may be worth while mentioning that some of the cell
inclusions in the spider material a re proteins and others
e. The sections were kept a t constant temperature in the
staining solutions for 12 hours, although it was found that
normal staining was attained i n about 1hour.
f. F o r the study of the effect of p I I of the stain, the slides
were washed directly in pure dioxan. Most stains are not o r
very little soluble i n dioxan. It was found that even prolonged sojourn i n dioxan did not affect the staining density,
but for convenience of handling the slides were transferred
to xylene before mounting in dammar.
g. I n the procedure of the staining method finally adopted,
HC1 and distilled water mere used
buffer solutions,
for washing and destaining before dehydration with dioxan.
The first step consists in the preparation of a series of
staining fluids having the same concentration of stain at all
values of ~ € 1 . It was found desirable to begin at the lowest
pH a t which the stain does not precipitate and to make the
series a t 0.5 pH intervals all the way up to pH 1 2 or to
such a pH in the alkaline range at which the stain does not
precipitate or become decolorized. The range is, of course,
a different one for each stain. Some stains, such as eosin, are
not soluble i n water at lower values of pH and as they are
transformed into a corresponding acid, the latter was used
in preference to the salt. Eosinic acid of a high degree of
purity may be easily prepared from certified water soluble
eosin Y (di-sodium eosinate). Dried and weighed, it can then
be dissolved in absolute alcohol and used as a stock solution
f o r the preparation of 0.1% solutions in 50% alcohol by
adding the required volumes of HCl or NaOH and water.
If desired, one may use aqueous solutions from about p H 5 to
pH 12 by dissolving dried eosinic acid in graded solutions of
NaOH. I t is advisable to measure the pH of each stock solution, as this facilitates a subsequent adjustment of the stain. A
curve is made by plotting the volume of
HC’1 and
against the pH for a constant volume aiid concentration of
the resulting staining solution. Once the curves have been
prepared, staining solutions of any desired value of pH may
be easily aiid quickly made from the graph.
In cases of buffered stain solutions one should follow the
same procedure because the stain often causes considerable
change in the pH value of the buffer solution unless the concentration of the stain is very small.
The next step is the plotting of curves f o r the density of
staining of the various cell components a t all values of pH.
Color comparators, unless specially designed and constructed
for such work, a r e neither suitable, nor really necessary, because all that one wants t o know is the approximate re1n t’ive
density in terms of maximum, medium, slight or zero, when
staining is completely absent. Similar curves liave been published by other investigators and could be omitted here.
However, a n interesting feature manifested itself when
several curves were compared with each other. This is the
presence of an optimum or greatest density of staining a t a
certain pH, the curve droppiiig from this point in both directions (fig. 1). Another remarkable feature is that a curve
for the same stain used after another fixing fluid may not
only be different in shape, but show a constant rise up t o
pH 12 (fig. 2). Higher values of pH were not tried because
of the deleterious effeot on tissues.
An examination of the curves thus constructed, permits at
a glance the selection of the pH at which greatest differentiation takes place. Thus f o r methylene blue after fixation in
paranitroformol the differentiation is greatest between pH 2
and pH 3.3, because in this range neither the zymogen globules,
nor the refractive bodies are stained.
The last step is the selection of the proper pH value of the
buffer solution to be used in destaining. It may be accepted
P H 2
Fig. 2
Methylene blue after Regaud.
as a general rule that the capacity of retaining a stain in a
destaining fluid of a given pH is not of the same order a s the
staining affinity at that pH. A few examples may illustrate
this. Acid fuchsin, after fixation in paranitroformol, barely
stains at pI-1 8 and does not stain at all a t pH 10. Maximum
staining is at pI-1 2 when all cell organs and inclusions are
almost equally dark red. At pH 7 the refractive bodies and
zymogen globules are stained helow medium density, while
nuclei and cytoplasm are of a very faint pink color. But if
a section is stained at pH 2, or even a t pH 2.85, and then
destained for half an hour or more in a buffer solution a t pH 7,
all cell organs and inclusions loose their color completely,
except the zyrnogen globules, the refractive bodies and the
plasmasomes of interstitial nuclei. They retain their dark
red color. I n the case of light green the maximum density
of staining is between pH 2 and pH 2.5, while no staining
of any kind takes place at and above pH 8 after fixation in
Regaud. But if a section is stained a t pH 2.4 and destained
in a buffer solution at pH 8, the zymogen globules remain dark
green, while all other cell organs and inclusions become completely decolorized. The same holds true for orange G, only
that in this case the refractive bodies as well as the zymogen
globules remain colored after fixation in Regaud. After
fixation in paranitroformol, staining in orange G at pH 1.8
and washing in a buffer solution at pH 6 the refractive bodies
alone remain colored, the rest of the structures losing the
color completely.
The analysis of the curves and the capacity of stain retention by certain structures at a pH at which staining is
otherwise impossible open the way to correct double staining
without intergrading colors. If a staining solution is used,
containing a mixture of two stains, one is bound to get intergrading colors because both stains a r e of necessity used at
the same pIi no matter what the value of the latter may be.
Take for example Wright’s blood stain. If one adjusts i t to
a certain pH, one gets without fail a beautiful double staining
of human blood because of the great differences i n the isoelectric point and chemical composition of the various cellular
structures. Even so, some of them show intergrading colors
which do not appear after staining with separate solutions
of eosin a n d methylene blue. Tf Wright’s stain is used for
staining other material, such a s sections through digestive
organs of spiders or vertebrates, one gets besides pure blue
and pink all kinds of intermediate purplish colors. This is
always the case when the staining optima of both stains coincide or lie close together and the staining affinity of the substratum is more or less of the same order for both stains.
But we have seen that staining affinity and capacityto retain
stain at certain values of pH a r e of different order. Herein
as in staining with single stain solutions, the way is open f o r
differentiation without intergrading colors. The stains must
be used separately, one after the other a t the pH of greatest
density and washing after each stain done in fluids having a
pH at which stain will be retained only in the desired cellular
component. Thus in the case of the digestive organs of the
spiders, if the sections are first stained in eosinic acid at
pH 2.1, washed in 6 HC1, stained in methylcne blue a t pH 3.6
to 4 and rinsed in distilled water, only the zymogen globules
in the enzyme cells are stained pink, refractive bodies remain
colorless, while all the other cell components are stained in
various degrees of density of pure blue color. Toluidin blue
may be substituted for methylene blue a t the same pH with
similar result. If it is desirable to retain blue color in nothing
but basophilic granules, the second washing solution should
have a lower pH. I n this case & HC1 or even 2 HC1 should
be used. If acid fuchsin is used instead of eosinic acid, the
sections should be stained in it at pH 2.8 to pH 3.5 and
washed in a buffer solution at pH 7 o r 8 depending upon the
fixing fluid, then counterstained with either mcthylene blue
or toluidin blue as before.
But this is not the only result which may be achieved by
the method here described. An examination of the curves of
some stains, such as eosin (fig.3), orange G (fig. 4) and aniline
blue shows that they cross each other at certain values of
pH. In the case of eosin all cell components are stained with
ail equal degree of density. S t p H 3 zymogen globules are
stained deepest, nuclei and cytoplasm less so and refractive
bodies least of all. At p H 10.5 refractive bodies are stained
deepest, zymogen globules less so and cytoplasm and nuclei
least of all. This permits the use of the same stain, only at
a different pH, f o r differentiation of other cell components.
F o r example, if we stain the sections in eosin at p H 9 (instead
of pH 2.15) wash in distilled water and counterstain with
methylene blue or toluidin blue as before, the refractive bodies
are stained pink, all the other small components presenting
different shades of blue, iiicluding the zymogen globules which
appear light blue.
At first sight, the method appears to be a complicated and
troublesome one. In reality its practical application is simple,
reliable arid f w e from errors inhereiit in other methods.
Moreover the results a r e strikingly clear aiid beautiful. One
must remember that each curve is good only for staining after
/ * I 2
Fig. 3
Ithsinic acid :iftt.r paranitroforniol.
Fig. 4
0r:rnge G a f t e r sul)liniatc forniol.
the particular fixing fluid for which it was drawn arld
possibly only for the material wliicli mas used. IIumaii or
amphibian tissues may require different values of pH of
!he staining fluids. But once a curve has been made it is
not necessary to prepare stains or buEer solutions of the
entire series of pH. The optimum value is all that is needed
for stains, while for washing
HCl,’ buffer solutions of pH 3, 6, 7 and 8 and distilled water will meet all requirements. Selection of a proper pfI is done from examination of the curve. Staining may be safely done at room
temperature for not less than 1hour, still better over night.
Rinsing in distilled water is not only permissible, but fully
warranted whenever the pH of the water (ca. 5.5) comes to
lie between that of the stain and the destaining buffer solution.
Moreover, such rinsing prevents rapid deterioration of buffer
solutions. After final rinsing in distilled water the stained
slides are dehydrated in pure dioxan and mountcd in dammar.
This prevents loss i n staining density and leaves the color a s
permanent a s the nature of the respective stain permits.
Xylene may be used between dioxaii and dammar, if one so
desires. Alcohol should be avoided under all circumstances
and is not only undesirable on account of its destaining effect
on many stains, but also, fortunately, quite unnecessary.
F o r the convenience of those who may wish to t r y the
method, but lack equipment for proper measurement of p H
of colored solutions the following instructions for the
preparation of staining fluids of certain pH values may be
of use. It innst be remernbcred, however, that samples of
even certified stains vary appreciably and that the resulting
values of pH may dif€er from those given here.
Acid fuchsin, 2% aqueous solution-pH
2.85. To make
50 cc. of a 0.2576 solution having a pH 2, take 25 cc. of a
0.5% solution, add 0.5 cc. of ,”, HC1 and dilute with water to
50 cc.
Anilivze hlice, make a 0.5% stock solution. To prepare a
0.1% aqueous solution having a pH of ca. 7.4 dilute the stock
solution with distilled water. To prepare a 0.1% solution
having a pH 2, add to 10 cc. of the stock solution 6 cc. of
HCl and dilute with water to 50 cc.
Aurnizfia, 0.1% aqueous solution-pH ca. 6.4. To make a
0.1% solution having a pH 2.4, take 25 cc. of a 0.2% solution
“Fixanal’ rengcntv of tlic E. de Haen Company, Germany, Pfaltz and Bsuer
sole agents for U.S.A., were found t o be quite satisfactory.
of aurantia, add 3 cc. of $ HC1 and dilute with distilled water
to 50 cc.
Benxonmriizc, after paranitroformol fixation gives double
staining a t pH 5.5 to 6.0. To get a solution having a p H 5.5
make a 0.1% solution of benzoazurine powder in pH 5.5 acetate
Eosin yellowish in a 0.5% aqueous solution has a pH of
ca. 9.5.
Eosiizic acid (tetrabromfluorescic acid), stock solution 0.2%
in absolute alcohol. To make a 0.1% solution i n 50% alcohol
having a pH 3.3 take 25 cc. of the stock solution and dilute
with distilled water to 50 cc. To make a similar solution
having a pI3 2.6 take 25 cc. of stock solution, add 2 cc. of
HC1 and dilute with water to 50 cc. To make a solution
having a pH 2.1 take 25 cc. of stock solution, add 7 cc. of
HC1 and dilute with water to 50 cc. F o r pH 2.25 use only
5 cc. of & HCl.
Light green. To make a 0.1% solution i n 50% alcohol
having a pH 2.4 take 10 cc. of 0.5% aqueous solution, add
25 cc. of absolute alcohol and 4 cc. of ,”, HC1 and dilute with
water to 50 cc.
Metaail yellow, 1%aqueous solution-ca. pH 8.0. To make
a 0.1% solution having pH 2.4 take 25 cc. of a 0.2% aqueous
HC1 and dilute with water to 50 cc.
solution, add 3 cc. of This dye is soluble in dioxan in place of which chloroform
should be used.
Methylew blue, U.8.P.-aqueous
stock solution 0.5%. To
make a 0.25% solution having a pH 4.0 take 25 cc. of stock
solution and dilute with water t o 50 cc. To make a solution
having a pfI 3.0 take 25 cc. of stock solution, add 0.5 cc. of
,”, HC1 and dilute to 50 cc. To make a solution having a
pH 2.0 take 25 cc. of stock solution, 5 cc. of -Z HCl and dilute
to 50 cc. with water.
Ora?zge G , 0.1% aqueous solution has a pH of ca. 8.4. To
make a 0.1% solution having a pH 2.0 take 25 cc. of a 0.2%
soliition, add 5 cc. of ,”, HC1 and dilute with water to 50 cc.
Toluidim blue, a 0.1% aqueous solution has a pH ca. 4.3.
h 0.1% solution i n 50% alcohol has a pH ca. 4.6. To make a
0.1% aqueous solution having a pH 2.1 take 10 cc. of a 0.5%
aqueous solution, add 4 cc. of ,”, HC1 and dilute with water
to 50 cc.
W r i g h t ’ s blood stain, to adjust the pH to 7.36 which gives
the greatest differentiation with human blood smears make
a 1%stock solution by dissolving the powder in pure methyl
alcohol and add an equal volume of a phosphate buffer solution pH 6.3.
Double s t a i n k g with eosin-methyle9ze blue in separate solutions to obtain greatest differentiation without intergrading
colors. Pass the blood smear over a flame and fix it for 1 or
2 minutes in pure methyl alcohol. Stain in 0.1% eosinic acid
in 50% alcohol, rinse in distilled water, stain in 0.1% solution
of methylene blue, U.S.P. in acetate buffer pH 5.5, rinse in
water and dry.
For double staining with eosin-methylene blue of oxyntic
cells in the stomach of vertebrates, fixed in Zenker’s fluid,
sublimate formol or paranitroformol, stain the sections in
0.1% eosinic acid in 50% alcohol adjusted to p H 2.2 to 2.3
wash in approximately 0.01 normal hydrochloric acid, rinse
in distilled water, stain in 0.1% solution of methylene blue,
U.S.P. in acetate buffer pH 4, rinse in distilled water and
dehydrate directly in dioxan. Do not expect to obtain the
same result a t other values of pH.
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