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Pol yehromat i c Stai ni ng of Pl ant Cell Walls

by Tol ui di ne Blue O
By
T. P. O' Br i e n ~, N. F e d e r ~ and M. E. Mc Cu l l y ~
With 4 Figures
(Received July 22, 1964)
The val ue of basi c dyes as r out i ne st ai ns f or t he wal l s of p l a n t t i ssues
was r ecogni zed 34 ye a r s ago b y Cz a j a (1930):
, , Umgekehr t h a b e n wi r i n den s ubs t a nt i ve n Fa r bs t of f e n ei n sicheres Mi t -
t el i n der Ha n d , di e di f f er ent e St r u k t u r und Per meabi l i t ~i t sver h~i l t ni sse ver -
sehi edener, sogar d i r e k t be na e hba r t e r Zel l en oder aueh ver sehi edener Sehieh-
t en ei ner und der s el ben Z. el l wand mi t Lei eht i gkei t zu er mi t t el n. " 2
Al t hough t ol ui di ne bl ue O is used r out i ne l y as a s t ai n in a ni ma l cyt ol -
ogy, a nd .its me t a e hr oma t i c pr ope r t i e s .are wi de l y k n o wn (B e r g e r o n and
S i n g e r 1958), one fi nds but ca,suM ref. erenee to t he use of a n y of t he
t hi azi n dye s in p l a n t hi st ol ogy. I ndeed, J e n s e n (1962) r e c e nt l y r e ma r ke d,
"Thi s s t ai n ( Azur e B) has been l i t t l e used b y bot ani st s, but shows gr eat
pr omi s e bot h as a s t ai n f or t he nucl ei c aci ds a nd f or l i gni n. "
The p u r p o s e of t hi s not e i.s t wof ol d: to d r a w a t t e nt i on to t he we a l t h of
s t r uc t ur e r eveal ed b y t ol ui di ne bl ue O whe n it is used to st ai n f r es h or
f i xed p l a n t t i ssues and to out l i ne v e r y s i mpl e a nd r a p i d pr oc e dur e s f or
obt ai ni ng t e mp o r a r y or p e r ma n e n t mount s of s t ai ned sect i ons. The met hods
ar e so . simple t ha t one ma y pr oc e e d f r om t he i nt act p l a n t v i a t i ssue sec-
t i ons (10 50 #) cut f r e e - ha nd wi t h a r azor bl a de to an e x a mi n a t i o n of t hese
sect i ons i n t he mi cr os cope i n a ma t t e r of 10 mi nut es. The s t ai ni ng pr oce-
~lure ma y al so be a p p l i e d to sect i ons of f i xed and e mb e d d e d ma t e r i a l .
Magni f i cat i ons of up to X10O0 ma y be us e f ul l y e mpl oye d. The val ue of
such a t echni que f or t eachi ng a nd r esear ch is evi dent .
Authors' address: The Biological Laboratories, Harvard, Cambridge, Mass.,
U. S. A.
2 Conversely, in the substantive dyes we have available a sure means of easily
ascertaining the different st ruct ure and permeabi l i t y relations of different cells,
even of i mmedi at el y adj acent cells, or even of different layers of one and the
same cell wall.
l~rotoplasma, Bd. LIX, H. 2 O'Brien, Feder and Culley Table
Figs. I - 4. Al l phot omi crographs are of pea epicotyl, fixed in acrolein and
embedded in pol yest er wax and sectioned at 8#.
Fig. t. Di fferent i at i ng fibers in the "caps" of ~he vascul ar bundles, st ai ned with
t ol ui di ne bl ue O. The lignified compound mi ddl e l amel Ia (blue-green) is shar pl y
di fferent i at ed from the as yet unlignified secondary wal l (reddish purple). X860.
Fig. 2. Vascul ar bundl e st ai ned wi t h t ol ui di ne bl ue O. The legend is given in
Fig. 3 in which par t of the same field is reproduced. The lignified secondary wal l s
of the t racheary elements (te) st ai n blue-green, which cont rast s shar pl y wi t h the
reddi sh pur pl e of the unlignified mi ddl e l amel l a (ml) separat i ng two such lignified
elements. The walls of unlignified t racheary elements (ute) and those of xyl em
parenchyma stain vari ous shades and intensities of reddi sh purpl e. X860.
Fig. 3. Par t of the same field as Fig. 2, but phot ogr aphed with light passi ng Corn-
ing yel l ow filter # 3484. X860.
Fig. 4. The same section as shown in Fig. 2 and 3 aft er t reat ment with phl oro-
gl uci nol -HCt to demonst rat e the Wi esner-posi t i ve lignin. The st ai ned section was
phot ogr aphed with the l i ght passi ng a Corni ng green filter # 1010 to enhance
contrast. There is an excel l ent correl at i on between wal l s or wal l l ayers which
are Wi esner positive, and the devel opment of a bl ue-green to green color wi t h
t ol ui di ne bl ue O. X860.
T. P. O' Brien, N. Feder a. M. E. McCully: Polyehromatie Staining of Pl ant . . . 369
Materials and Methods
S t a i n i n g s o 1 u t i o 11 : An aqueous sol ut i on cont ai ni ng 0.05% of
t ol ui di ne bl ue O (C. I. # 52040) s in 0.1 M p h o s p h a t e buf f er at p H 6.8.
P r o c e d u r e s : Fr e s h or f i xed t i ssues ma y be used ( but see c omme nt s
under F i x a t i v e s f or t he l i mi t at i ons i mpos e d b y f i xat i ves whi ch cont ai n
h e a v y met al s) . The t i ssues ma y be s ect i oned b y ha nd wi t h a r azor , or wi t h
a s l i d i n g - mi c r o t o me or c r yos t a t e d mi cr ot ome. Speci mens e mbe dde d in
pol yes t er wa x ma y be sect i oned i n t he us ual wa y (S i d m a n , M o t t I a,
a nd F e d e r 1961). The li.st of ma t e r i a l s whi ch we ha ve e xa mi ne d f r om
ha nd- c ut f r esh ma t e r i a l i ncl udes st or age t i ssues of c a r r ot a nd pot at o, epi -
cot yl , st em, r oot and cot yl edons of pea, pet i ol es of cel er y, s t e m of Elodea
der~sa (Planch. ) and Hippuris oulgaris L., l eaf of Zea mays L., st ems of
Coleus sp. , Thuja occidentalis L. and Pinus Strobus L., col eopt i l es of ADena
sativa L. a nd Zea mays L., and r hi z ome of Lycopodium sp.
1.
a)
s oak
b )
t hem
e)
For h a n d - c u t sect i ons of f r es h ma t e r i a l :
Cut sect i ons wi t h a r azor bl a de i nt o t a p wa t e r and al l ow t he m to
f or at l east 2- 3 mi nut es.
Tr a n s f e r sel ect ed sect i ons ( 10- 50#) to t he s t ai ni ng sol ut i on. I mme r s e
f or 1 mi nut e.
Wa s h t he s t ai ned sect i ons f or / - 2 mi nut es in t a p wat er .
d) Ex a mi n e t he s t ai ned sect i ons mount e d in t a p wa t e r unde r a cover -
sl i p, or p h o t o g r a p h at magni f i cat i ons up to X250 i n t he mi cr os cope.
e) For e x a mi n a t i o n wi t h h i g h - d r y and oil i mmer s i on obj ect i ves r ei nove
as much of t he wa s h wa t e r as possi bl e f r om a r ound t he sect i on on t he
sl i de and mount it unde r a cover s l i p i n a dr op of t he f ol l owi ng l i qui d:
Ca d mi u m i odi de 2 g.
Pot a s s i um t hi oc ya na t e 4 g.
Fr uc t os e 10 g.
Wa t e r 4 ml.
Thi s l i qui d is r e l a t i ve l y vi scous, has a r e f r a c t i ve i nde x of a p p r o x i ma t e l y
1.5, a nd sect i ons mount e d in it ma i n t a i n t hei r f ul l col or di f f er ent i at i on f or
at l east 2 days . The l i qui d does not set and t he mount s ar e t e mp o r a r y .
The compos i t i on of t hi s mo u n t a n t is modi f i ed f r om t ha t pr opos e d b y
S p u r r (1954).
2. For sect i ons cut on a sl i di ng or cr-yost at ed mi cr ot ome:
i) Cut sect i ons at 1 0 - 1 6 # a nd al l ow t he m t o d r y on ge l a t i n- c oa t e d
sli.des in t he us ual wa y.
it) St ai n f or 1 mi nut e in t he s t ai ni ng sol ut i on, wa s h f or 1 mi nut e in t a p
wat er , a nd mount a nd e x a mi n e as f or 1. d) or e) above.
3 Available from National Aniline Division, Allied Chemical Corporation.
370 T. P. O'Brien, N. Feder and M. E. McCully
3. For sect i ons embedded in pol yes t er wa x:
S i d m a n, M o t t l a , and F e. d e r (1961) g.ive i nst r uct i ons f or pr e pa r i ng
sect i ons s t ai ned wi t h t ol ui di ne bl ue O whi l e st i l l in t he r i bbon of wax.
Fr om such sect i ons, p e r ma n e n t mount s can be . obt ai ned b y de wa xi ng t he
sect i ons i n xyl e ne and mount i ng in Di a pha ne . 4 Thi.s t r e a t me nt r esul t s in
some change f r ont t he colors devel oped by t he same mat er i al st ai ned whi l e
fresh. The col or change can be mi n i mi z e d b y a sl i ght r e hydr a t i on of t he
mat er i al bef or e mount i ng. Thi s is most s i mpl y done by br eat hi ng on t he
dr i ed wa x sections j ust bef or e i mmer si ng t hem in xyl ene.
Resul t s
Tol ui di ne bl ue O resol ves t i ssue sect i ons i nt o t hei r component cell t ypes
by col or i ng var i ous t ypes of wal l s t r i ki ngl y di f f er ent colors (Figs. 1 and 2).
I n some i nst ances the col or r esol ut i on ext ends to di f f er ent l ayer s of t he
Tabl e i .
Tissue Element Color Deveioped by Toluidine Blue 0
Tracheary elements (lignified wMls)
Lignified selerenehyma
Collenehyma
P a r e n c h y m a
Sieve tubes and companion cells
Unlignified compound middle lamellac
CMlose, starch
Green, or bluish green
Blue-green, but occasionally green
l{eddish purple
Reddish purple
Red
Reddish p~rple or red
Unstained
wal l of one cell. For exampl e, tile l i gni fi ed s econdar y wal l of t he t r aehear y
el ement s of pea epi eot yl ar e st ai ned a bl ui sh gr een whi l e t he compound
mi ddl e l amet l a s e pa r a t i ng t wo such adj acent el ement s i:s ,stained a br i l l i ant
r eddi s h pur pl e (F,ig. 2). The "caps " of t he vas cul ar bundl es of t he same
t i ssue ar e composed of fibers in whi ch l i gni f i eat i on commences i n t he
p r i ma r y wal l adj acent to t he i nt er cel l ul ar ai r spaces. A sect i on of such
young fibers st ai ned wi t h t ol ui di ne bl ue O shows t he l i gni f i yi ng p r i ma r y
wal l st ai ned a cl ear bl ue- gr een, whi l e t he mi ddl e l anrel l a in t he r egi on
adj acent to t he ai r spaces, and t he unl i gni f i ed s econdar y wal l s, ar e st ai ned
i nt ense r eddi s h pur pl e. As t he l i gni f i cat i on of t he fi bers pr oceeds, t he
bl ue- gr een st ai ni ng pr ogr esses i nt o t he mi ddl e l amel l a (Fig. 1) and f i nal l y
i nt o t he s econdar y wal l . Ti l e colors devel oped t ypi c a l l y by tolui.dine bl ue O
are shown i n Tabl e ~. I n al l cases "l i gni f i ed" means t hat t he pa r t i c ul a r
wal l or wal l - l ayer was posi t i ve in t he Wi es ner t est ( phl or oghmi nol - HC1,
J o h a n s e n 1940).
O t h e r d y e s t u f f s : Sever al ot her member s of t he t hi azi n gr oup of
dyes wer e t est ed i n pr e l i mi na r y e xpe r i me nt s on f r esh sect i ons of pea stem.
Of t hose t est ed ( azur e A, azur e B, t ol ui di ne bl ue O, t hi oni n, me t hyl e ne
bl ue, new me t hyl e ne ]31ue, and me t hyl e ne vi ol et ) onl y t ol ui di ne bl ue O and
The Will Corporation, l/ochester 5, N.Y.
Polychromatie Staining of Plant Cell Walls by Toluidine Blue O 371
azure A v-ere satisfactory in the range and stability of the colors developed.
Thionin was also satisfactory, but the colors were shifted towards the red.
Although we selected toluidine blue O for our furt her tests, azure A should
be equally sat.isfactory and other members of this group of dyes might be
useful in certain circumstances.
Ef f e c t of pH: 0.1M phosphate buffer at pH6. S is recommended
because it is easy to prepare. However, tests on sections of pea epicotyls
cut on a cryostated microtome showed that the same colors were developed
by toluidine blue O dissolved in water or in buffers in the pH range 5 to 9.
At pH less than 4 the metaehromatic red colors are steadily- reduced
in intensity, but lignified walls will stain either blue or green even at
pH 1.o.
E f f e c t s o f F i x a t i v e s : Air-dried sections of pea stem.s were
fixed for 10 minutes, 1 bout or overnight in the following fixatives: 10%
acrolein, 10% formalin, 5% glutaraldehyde, FAA, Bouin's, CRAF, Zenker's,
and 1% osmium tetroxide. The fixed sections were washed for 1 hour in
distilled water, stained and exami ned in tap water. The colors developed
by sections fixed .in acrolein, formalin, glutaraldehyde, FAA, and Bouin's
were indistinguishable from those seen in sections soaked in water for the
same period. The same was true of sections fixed for 10 minutes in Zenker's,
CRAF, or 1% osmium tetroxide; however, aft er fixation for 1 hour or
overnight .in these fixatives a pronounced blue shift in the characteristic
colors developed, and t hey began to fade within a few minutes of staining.
This change could be prevented in material fixed in CRAF if the stained
sections were mounted in the liquid mountant described above.
Di s cus s i on
The fact that toluidine blue O would stain pl ant cell walls polyehro-
mafically has been known for many years ( Cz a j a 1934), but this dye
does not seem to have gained the widespread use it deserves in pl ant cy-tol-
ogy. Shortly after we began to use toluidine blue O for the st udy' of fresh
plant tissues, our attention was drawn to the extensive unpublished work
of E. Pt a s c h and H. H. S wi f t who have attempted to analyze the histo-
chemical basis of the polychromatic staining .developed by- cell walls of
different types in pl ant tissue embedded in paraffin. These workers are
the first in recent years to recognize the value of polychromafic staining
with toluidine blue O for the investigation of plant cell walls, and we are
pleased to t hank them for allowing us to see a prel i mi nary description of
their results, which are in good agreement with ours.
In the range of herbaceous materials examined here, there is an excel-
lent but not invariable correlation between the color developed by toluidinc
blue O a~d the presence of lignin. All walls which give the Wiesner reaction
(phloroglucinol-HCl) stain distinctly green, or blue-green. In the dif-
ferent.taring cortical fibers of pea epicotyl, it is very clear that the .intensely
blue-green, compound middle-lamella of these ceils (Fig. 1) is the only
part of the wall which gives the Wiesner reaction. Conversely, some of the
372 T.P. O'Brien, N. Feder and M. E. MeCully
traeheary elements in the xylem of this tissue have Wiesner-po.sitive second-
ary walls; t he compound midd!e-lamella is negative (Fig. 4). When stained
with toluidine blue O, the secondary wall stained green, the compound
middle-lamella a .distinct reddish purple (Fig. 2). However, in spit.e of
these results, it is not certain thai the green color necessarily indicates the
presence of lignin..Sections of pea epieotyl were "delignified" for various
periods of time by treatment with 2% sodium chlorite and ammoniaeial
70 % alcohol (B a r g h o o r n 1948). After treatment for 1 hour, the sections
appeared to be delignified as judged by the Wiesner test, but the colors
developed by toluidine blue O were identical to those of controls. Prolonged
delignification (overnight)did destroy all green colors when the delignified
sections were stained with toluidine blue O. Those areas which previously
stained green now stained a deep blue. However, different results were
obtained with Lycopodium rhizome, in which some of the heavily lignified
walls are Wiesner positive and also stain intensely green with toluidine
blue O. In thi,s t.issue treatment of sections with sodium chlorite for 8 hours
abolishes the Wiesner reaction but even treatment for two days has no
effect on the green staining of cell walls with toluidine blue O.
A s,ample of Brauns' isolated native spruce lignin (kiudly given to us
by Dr. I. A. P e a r 1 of the Institute of Paper Chemistry) does stain an
intens,e bluish-green. The refractory nature of native lignin in situ raises
doubts ,about the validity of any histoehemieal procedure that claims to
identify lignin. Until more is known of the chemistry underlying the Wies-
net reaction and the blue-green colors developed by toluidine blue O in
lignified walls, it is impossible to decide with certainty which is more
reliable for the identification of lignin.
It is disappointing not to be able to attach histoehemieal significance in
some simple way to the polyehromatie staining observed. Nonetheless, the
convenience of the sectioning, staining and mounting procedures and the
quality of the polyehromatie staining achieved lead us to believe that the
method may find widespread application in the teaching of plant anatom}"
and in research into plant histology.
This work has been supported in part by Grant No. G 21.799 NSt;' to
Dr. Thi ma nn and USPH GM-08139 to Dr. Fe d er.
Summary
1. The polychromatic stain,ing of plant cell walls by toh, idine blue O is
described and illustrated.
2. The eff:eets .of various conmlon fixatives and the effects of the pH o[
tlre staining solution are evaluated.
5. Simple and rapid procedures are described for preparing stained
temporary mounts of fresh material, or permanent mounts of embedded and
sectioned material.
4. The relationship between the polyehromatie staining observed and
tl~e lignifieation of the walls is discussed.
Pol ydar omat i c St ai ni ng of Pl a nt Cel l Wal l s by Toluid, i ne Bl ue O 373
Ref erences
B a r g h o o r n, E. S., 1948: Sodi um chl ori t e as an ai d ill pa l e obot a ni c a l and
a na t omi c a l s t udy of pl a nt tissues. Sci ence 107, 480--481.
B e r g e r o n , J. A., and M. S i n g e r , 1958: Met achr omas y: an e xpe r i me nt a l and
t heor et i cal r eeval uat i on. J. Bi ophys. Biochem. Cyt oI . 4, 433--457.
C z a j a , A. Th. , 1930: Unt er s uchungen f i ber met achr omat i s dl e F~i rbungen yon
Pf l anzengeweben. 1. Subs t ant i ve Far bst of f e. Pl a nt a 11, 582--626.
- - 1934: Unt er s uchungen fi ber met adl r omat i s ehe F~i rbungen yon Pf l anzengeweben.
II. Basisd~e Far bst of f e. Pl a nt a 21, 531--601.
J e n s e n, W. A., 1962: Bot ani cal hi s t odl emi s t r y, p. 197. W. H. Fr e e ma n & Co.
J o h a n s e n, D. A., 1940: Pl a nt mi cr ot echni que, p. 194. Mc Gr a w- Hi l l Book Co.
S i d m a n , R. L., P. A. M o t t t a, a nd N. F e d e r , 1961: I mpr ove d pol yes t er wa x
e mbe ddi ng f or hi st ol ogy. St ai n Tech. 76, 279--284.
S p u r r, A. R., 1954: Pol yvi nyl al cohol wi t h c a dmi um i odi de a nd f r uct ose as an
aqueous mount i ng medi um. St ai n Tech. 29, 301--313.