0022-1554/78/2602-0138$02.O0/0 THE JOURNAL OF Hi

Copyright

ocllzrbfls’rity

Vol. 26, No. 2, pp.

CYTOCHEMISTRY Society, Inc.

AND

Histochemical

© 1978 by The

Brief

TISSUE

FIXATION

AND ROLE

Department

of Chemistry,

Imperial

NIELSON

College

WILLIAM

AND

of Science

Received

in U.S.A.

Reports

STAINING WITH OSMIUM OF PHENOLIC COMPOUNDS

ALASTAIRJ.

1978

138-140,

Printed

for publication

THE

P. GRIFFITH’

Technology,

and

TETROXIDE:

October

London

SW7

2AY,

United

Kingdom

24, 1977

It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic. Recently,

cytochemical

centration cells

evidence

of phenolic

and

features fixation

tissues

has

observed

with osmium

compounds been

in electron tetroxide

higher

(2, 10) and

micrographs (0s04)

(6,7)

include dense globular in vacuoles or vesicles

characteristically plasm with The

by lead features regions

plants

lower

some

inference

increased degradation

has

been

with

typical after usu-

and

or uranyl thus reof cells in and

animals

or other

depos-

and in areas

of

density in the cytoof normal details. drawn

that

phenolic

materials are osmiophilic (2), and it has been shown that 0s04 alone, without post-staining by other metal salts, will impart electron-density to phenolic-containung areas (3). In this communication, the chemistry 5), we report

as part of our continuing study of of biological fixation by 0s04 (4, experiments which show that 0804

reacts stable

o-dihydroxy complexes.

with chelate

chelates are areas hitherto nolics, mary

the sites

responsible inferentially o-dihydroxy of attachment

phenols to give We believe that for units for

MATERIALS

con-

in particular

correlated

ally with additional staining salts. Typical ultrastructural vealed in phenolic-containing (16) may its, often

for high

RESULTS

In water idly

very these

AND

or aqueous

with

lutions These

o-dihydroxy

from solids

DISCUSSION

acetone,

0804

phenols

to

which deep are chemically

reacts give

rap-

blue

so-

blue solids precipitate. very stable and have

the basic polymeric structure (I). In phosphate or cacodylate buffers at pH 6.8, o-dihydroxy phenols form red solutions with 0804 containing the

as pri-

red

cationic

species

(II).

In

non-polar

sol-

vents such as chloroform the few phenols soluble in such media give tris-chelates of type (III)

osmium.

We thank the Science Research Council doctoral fellowship to one of us (A.J.N).

METHODS

Pure phenolic compounds and 0504 were mixed in: a, water b, aqueous acetone (1:1);c, 0.025 M sodium phosphate buffer, pH 6.8, in 1:1 aqueous acetone; d, 0.025 M sodium cacodylate buffer, pH 6.8, in 1:1 aqueous acetone or e, chloroform, according to the solubility of the phenol, and allowed to react for up to 2 hr. The osmium:phenol mole ratio was varied from 1:2 to 1:5. The resulting products were separated from the reaction mixture by filtration (a, b), as insoluble tetraphenylphosphonium salts (c, d), or by crystallization (e). Their stoichiometries were established by elemental analyses and the complexes were structurally characterized where possible by infrared, Raman and ‘H resonance spectroscopy, mass spectroscopy (and in one case by a full single-crystal x-ray study).

the electron-dense associated with phefunctioning

AND

(one been

for a post-

ray

of these, with 3,5,-di-tert-butylcatechol, fully characterized by a single analysis).

In

all

cases,

138

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reaction

crystal was

has x-

essen-

BRIEF

139

REPORTS

.nHO

(II)

(I)

R-Os

(III)

tially

complete

normally permit tion

within

allowed penetration

with

0804.

is simulated use for

The

acid,

three with but

reaction 0804

groups.

aqueous

by the solvents (II) are

species which

two

media

within

containing guiacol,

droxyl and hydroxybenzoic

the

(a) only

o-dihydroxy

ultrastructure five-membered

be especially

period

group (b)

period

(d) those

groups within

is carried chelate stable

out. rings, metal

in

fixation

be

with

an

o-dihyfound in reaction (5), and inter-

of 0804 with lipids or fixation (12). We have

(VI) phenol less reactive

from olefins. Their due to conjugation

species (I) and than the esters

enhanced with

is permitted groups

by

The

to (I) by acid,

stability aromatic

the

coordination

to osmium.

if they formation

plain the of certain with

the

is

of the

red

species

or by the

polymer (I). that, in those plant

in which phenolics 0804 will react

(II)

concentra-

reach rapidly

contain

o-dihydroxy

of such

extra contrast animal tissues

o-dihydroxy

and

complexes induced when

phenolic

animal

a high concenwith such comgroupings. may

also

ex-

in the cytoplasm they are pre-fixed

species

such

as

tannic

acid (1, 15) or D-catechin (8) before conventional fixation with 0804. We note that the comparable images of cytoplasmic microtubules previously

0804

observed

of cell

ascribed

can

reaction tissue

osmium much

which

tissues tration, pounds

The formation of which are known complexes,

the

to precipitate We conclude

The

hy-

systems

tion of phenol being high enough to overcome the effect of the buffer. We have observed that aqueous uranyl acetate is acidic enough (pH 2.0)

phe-

which with

in the in

to

formed probably

(e.g.,

m-dihydroxy

react

in phenolic

is converted

m- or p(c) o-di-

phenols

will which

for

(e.g., acid);

one

mediates lipoproteins

hydroxy

reaction react in with

hydroxyl

carbonyl group acid, salicycic

methoxy groups (veratrole); groups (resorcinol). It is evident that only the

2-hr

groups the to

occur

droxy unit. Five-membered rings are the osmium (VI) esters formed in the between 0804 and olefinic double bonds such esters are thought to be important

ring

resonance

o-hydroxy

one

by

of these phenols attacks the

vanillaldehyde);

one

and

also contain 0804 would

‘H magnetic

only

found (III)

phenols D-cateand o-dihydroxy

mixtures preferentially

That

acids,

ring are necessary for by the failure of 0804

p-cresol,

to

of tissues

closely

digallic plant acid

latter bonds

to react,

an aromatic was shown

during

most

and

occurring caffeic

of the that

hydroxy

contain

time

than non-polar species (I) and

gallic

These double

be expected

nols

fixation

studies

rather The

the naturally chin, quercetin,

spectra suggests

maximum

in such solutions by the o-dihydroxy catechol, methyl catechol, tert-butyl catechols, pyrogallol, 3,4-dihydrox-

ybenzoic

coumarin. olefinic

the

chemical

in these

of aqueous the reactions.

formed phenols substituted

2 hr,

in cytobiological fixation to and tissue component reac-

in certain (11)

and polyphenols.

plant

to the

tissues

natural

Finally,

have

been

of tannins

it is of historical

est to add that the discoverer son Tennant, noted the deep

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(9)

presence

of osmium, blue color

interSmithformed

BRIEF

140 by reaction and Schultze

of 0804 with and Rudneff

to suggest ing

that

areas

0804

in plant

“infusion in 1865

reacted

tissue

of galls” were the

with

(14), first

phenol-bear-

(13).

ACKNOWLEDGMENTS We thank

Mr.

W. C. Mueller the

course

A. D. Greenwood

for very of this

work,

Council to one

for a postdoctoral of us (A.J.N.)

Limited

for

the

helpful

loan

and

Professor

discussions

the

Science

during Research

research assistantship and Johnson, Matthey of 0804.

LITERATURE

CITED

1. Anderson WA, Trantalis J, Kang YH: Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat. Results after tannic acid-formaldehyde-glutaraldehyde fixation. J Histochem Cytochem 23:295, 1974 2. Baur PS, Walkinshaw CH: Fine structure of tannin accumulations in callus cultures of Pinus Elliotii (slash pine). Can J Bot 52:615, 1974 3. Beckman CH, Mueller WC: Distribution of phenols in specialised cells of banana roots. Phytopathology 1970 4. Cohn RJ, Griffith WP: Mechanisms of tissue component staining by osmium tetroxide. J Histochem Cytochem 22:992, 1974 5. Cohn RJ, Griffith WP, Phillips FL, Skapski AC:

60:79,

Staining

and

fixation

of

unsaturated

lipids by osmium tetroxide-crystal model osmium (VI) intermediate. phys Acta 320:745, 1973

membrane

structure Biochim

of a Bio-

REPORTS 6. Evans LV, Holligan MS: Correlated light and electron microscopic studies on brown algae. II. Physical production in Dictyota. New Phytol 71:1173, 1972 7. Fulcher RG, McCully ME: Histological studies on the genus Fucus. V. An autoradiographic and electron microscopic study of the early stages of regeneration. Can J Bot 49:161, 1971 8. Futaesaku Y, Mizuhira U: Fine structure of the microtubules by means of the tannic acid fixation. Proc VIII Int Congr Electron Microsc. 11:340, 1974 9. Ledbetter MC, Porter KR: Morphology of microtubules of plant cells. Science 144:872, 1964 10. Mueller WC, Beckman CH: Ultrastructure and development of phenolic-storing cells in cotton roots. Can J Bot 54:2074, 1976 11. Porter KR: Principles of Biomolecular Organisation. Edited by GEW Wolstenholme and M Connor. Churchill, London, 1966, p 308 12. Riemersma JV: Chemical effects of fixation in biological systems, Some Biological Techniques in Electron Microscopy. Edited by DF Parsons. Academic Press, New York. 1970, p 69 13. Schultze M, Rudneff M: Weitere mittheilungen #{252}berdie einwirkung der ueberosmiumsilure auf thierische gewebe. Arch Mikrbiol Anat 1:299, 1865 14. Smithson Tennant: On the discovery of two new metals in crude platina. Nicholson’s Journal of the Arts and Natural Philosophy 10:24, 1805 15. Tilney LG, Bryan J, Bush DJ, Fujiwara K, Mooseker MS, Murphy DB, Snyder DH: Microtubules-evidence for 13 protofilaments. J Cell Biol 59:267, 1973 16. Tranzer JP, da Prada M, Pletacher A: Storage of 5-hydroxytryptamine in megakaryocytes. J Cell Biol 52:191, 1972

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