HistochemicalJournal, 6 (I974), 679-684

The mechanism of the ferric ferricyanide

reduction reaction

R. D. L I L L I E and P. T. DONALDSON
Department of Pathology, Louisiana State University Medical Center,
New Orleans, Louisiana 70112, USA

Received I3 November 1973

Synopsis. The ferric ferricyanide reaction has been generally considered to depend on
the reduction of ferricyanide to ferrocyanide and, in the presence of the ferric ion, the
consequent production of Prussian Blue. In testing the ability of ferricyanide to prevent
the azo-coupling reactions ofenterochromaffin cells and of the noradrenaline islets of the
adrenal medulla, ferricyanide proved to be an effective oxidant in alkaline solution, but
failed to quinonize the two catechols below pH 6. Similar results are obtained in oxidative
blocldng of cutaneous sulphydryl sites against the ferric ferricyanide reaction and against
staining by the mercaptide acid azo-coupling reaction of Lillie & Glenner (1965).
Ferric ferricyanide solutions are used at pH 2-2.5 and the ferric ion is an effective
quinonizing and thiol-oxidizing agent at this pH level; the ferricyanide ion is not. The
ferric ferricyanide reaction depends on the reduction of ferric to ferrous ions in the
presence of ferricyanide, with the production of Turnbull's Blue.

Introduction
Theoretically, two mechanisms are possible for the ferric ferricyanide reaction. One,
that the ferricyanide ion is reduced to ferrocyanide and reacts with ferric ions to form
Prussian Blue; the other is that the ferric ions are reduced to ferrous and react with the
ferricyanide to form Turnbull's Blue.
Beginning with the introducers of the reaction, Golodetz & Unna (19o9), almost all
authors have adopted the ferricyanide reduction theory (Staemmler, I924; Ch~vremont
& Frederic, 1943; Massa, 1946; Gomori, 1948, 1952; Lillie & Burtner, I953; Lillie,
1954; Pearse, 1953, 1960 , 1968 , i972; Adams, 1956; Davenport, 196o; Lison, 196o;
Thompson, 1966; Ganter & Jolles, 197o).
Schmorl (1928, p. 205) gave the technique of the reaction and reported the colour
changes that occur, but gave no chemical explanation. Lillie (1948, p. I29) stated that the
ferric salt was reduced to ferrous and reacted with the ferricyanide to form Turnbull's
Blue. No source for this statement was given, and no experimental evidence was reported
at that time. Lillie & Burtner (1953) and Lillie (1954, p. 173) adopted the Golodetz-Unna
9 I974 Chapman and Hall Ltd 679
680 Lillie and Donaldson
dictum that ferricyanide is reduced to ferrocyanide and reacts with the ferric ion to form
Prussian Blue.
These elections were apparently based on the known reduction of ferricyanide by
thiols and other reagents in vitro. Leuckart (1890) used alkaline solutions of potassium
ferricyanide repeatedly for the conversion of aromatic mercaptans to disulphides. The
reaction does not appear to have been new to him. The oxidation ofdiphenols to quinones
by ferric salts stems from Vulpian's (x856) observations, as we have previously used it
(I96Ia,b, I973a,b).
Lillie & Burtner (I953) reported eighteen reagents which give direct colour reactions
with ferric chloride, and the same colour reactions with ferric ferricyanide. All of these
reagents, on mixing directly with ferricyanide solution and then adding a ferric salt,
produced Prussian Blue from the reduced ferricyanide.
In x96I Lillie (I96Ia) showed that when enterochromaffin cells were treated with
i mM solutions of FeC13 or FeSO4, washed and then reacted with ferricyanide, iron was
bound as Fe (II) in both cases and that the azo-coupling reaction was prevented by the
Fe (III) but not by the Fe (II) treatment. This raised a question as to the correctness of
the generally accepted theory of a ferricyanide reduction to ferrocyanide as the basis of
the ferric ferricyanide reduction test.
Lillie (i965, p. 2o8) wrote 'Ferric ions are reduced to ferrous and in the presence of
ferricyanides in the solution yield insoluble Turnbull's blue' and similarly (I969, p. 327)
in Conn's Biological Stains.
It is apparent that many reactive substances will reduce both Fe (III) and K~Fe(CN)6
to Fe (II) and K4Fe(CN)6 respectively. Generally the ferricyanide solutions so used are
neutral or even deliberately alkaline, as in Weigert's borax mixtures. No exploration
seems to have been made of the oxidative capacity of ferricyanides at low pH levels. The
pH of the Ch~vremont-Frederic mixture was 2. I4, the pH of 1% ferric chloride solutions
alone is 2.25. The Golodetz-Unna mixture is found to yield pH 2.3.
To help resolve the question of the actually operative mechanism, it was decided to
test the effect of FeCI~ and K3Fe(CN)6 at x% concentrations and at various pH levels
in prevention of the azo-coupling reactions of enterochromaffin and noradrenaline.

Material and methods

For the demonstration of oxidative quinonization of catechols, formalin- and glutaralde-
hyde-fixed guinea-pig and hog duodenum and glutaraldehyde-fixed rat adrenals were
used. For demonstration of conversion ofthiols to disulphides, Carnoy-fixed guinea-pig
skin was used.
p-Nitrodiazobenzene at pH 8.5, 5 mM, 3 rain, was used to demonstrate the phenolic
phase of enterochromaffin (Lillie, I965). The diazosulphanilic acid, p H I Azure A
sequence as reported by Lillie et al. (I966, I973 a) was used for noradrenaline islet
demonstration. The ferric ferricyanide reaction itself under special conditions detailed
hereafter, and Lillie & Glenner's p-N,N-dimethylaminophenylmercuriacetate, acid azo-
coupling sequence (Lillie, I965, p. 215) were used to demonstrate sulphydryl groups.
Samples of Mercuroehrome proved inert.
The conditions of application of ferric chloride and potassium ferricyanide and con-
trol ferrocyanide treatments are presented directly with the experiments.
Ferric ferricyanide reaction mechanism 68 I

Experimental
In our first trial, sections of formalin- and glutaraldehyde-fixed hog and guinea-pig
duodenum were reacted 3 min in 5 mM p-nitrodiazobenzene at p H 8. 5 (Lillie, 1965,
p. 224) directly and after IO min baths in i % ferric chloride (FeC1 a" 6 H20 ) at p H 2.25,
in 1% KaFe(CN)6 in 2 N acetic acid (pH 2.35 ) and 1% KaFe(CN)6 in 0.2% barbitone
sodium (pH 9.5). T h e azo-coupling reaction of the enterochromaffin cells was positive
in the direct control and with the acid KaFe(CN)6, negative after FeC1 a at p H 2.25 and
KaFe(CN)6 at p H 9.5. A control 1% K4Fe(CN)6 at p H 9.5 also gave positive staining.
T h e experiment was repeated on the same material with 5 mM diazosafranin 3 min at
p H 8. An alkaline ferrocyanide control was included, to exclude air oxidation at alkaline
pH. T h e results conformed, though contrasts were less satisfactory. T h e acid ferri-
cyanide solution and the alkaline ferrocyanide failed to block, the alkaline ferricyanide
and the acid ferric chloride blocked the azo coupling reaction.
I n a further experiment I ~ KaFe(CN)6 IO min was used at p H 2.35 , 3.5, 4-5, 5.5, 6.5,
7.5, 8.5 and 9.5. Azo-coupling was then done, 2 min 5 mM p-nitrodiazobenzene at
p H 8.5, using formalin- and glutaraldehyde-fixed tissue. Positive azo-coupling reactions
were obtained in the direct control and in the ferricyanide tests up to p H 5-5 with
glutaraldehyde-fixed material and to p H 6.5 with formalin-fixed tissue. Complete
blockade occurred at the higher p H levels.
Similar tests were made on glutaraldehyde-fixed rat adrenal and the noradrenaline
islets were then stained by the diazosulphanilic acid, p H I Azure A sequence (DAS-
AzA).
Lillie et al. (1973) reported that a 2 hr 2.7% FeC1 a. 6H20 (o.I M) prevented the dark
blue D A S - A z A staining of noradrenaline islets. Results of experiments with 1% solu-
tions of ferric chloride and potassium ferricyanide are presented in Table I. An alkaline
ferrocyanide treatment is included as an additional control.
This Table again demonstrates that ferricyanide is active as a quinonizing reagent only
in alkaline solution, stopping abruptly at about p H 6. T h e alkaline ferrocyanide had no
quinonizing effect.

Table I. Effect of oxidation on diazosulphanilic acid-pH I Azure A reaction of adrenal medulla

Oxidizing Com. Time pH pH Adrenaline Noradrenaline

reagent (min) buffer recorded Nuclei cells islets

. . . . . G Gi B++
FeClz 1% IO -- 2.2 G G• GB
FeCla I~o 20 - - 2.1 GA: Y:V CG•
KaFe(CN)6 1% 20 -- 6.16 YG Y• B/B•
KaFe(CN)6 1% IO 2 2. 3 G G• B+
KaFe(CN)6 1% 20 2 1.85 G G:7 B++
KaFe(CN)~ o/
I/o 20 5 4-95 G y~ B+
KaFe(CN)o 1% IO 9-5 9.5 G G• G•
KaFe(CN)6 1% 2o 9.5 9.86 G fi: Y k_ YG •
K~Fe(CN)6 1% 20 9"5 9.96 G-- G:V B+
(control)

Abbreviations: C-gray, G-green, B-blue, Y-yellow, + + - dark, + - strong, ~ - pale, ~ - faint

colour.
682 Lillie and Donaldson
Since Ch~vremont & Frederic (1943), Adams (1956) and others have used the ferric
ferricyanide reaction for localization of thiols in tissue sections, it seemed proper to use
this tissue element also, for appraising the effect of ferricyanide and ferric ion as
oxidants.
Since the reagent itself contains ferricyanide ions, and ferric ion is known to be taken
up by tissue amino and guanidino groups (Lillie & Pizzolato, 1972) it was necessary after
ferric chloride oxidation to remove chelated iron from the tissue with 1 N HC1 (3 ~ min,
6o~ when using the ferric ferricyanide reaction itself as a thiol detection reagent.
Lillie (1961) used ferricyanide to localize Fe (II) taken up at reducing sites from Fe (III)
solutions. Pearse (1968) comments that ferricyanide ions are not so bound in sections
that a two phase ferric ferricyanide reaction can be performed in the reverse direction.
Carnoy-fixed guinea-pig skin showed a strong ferric ferricyanide reaction in the root
zones of many hairs and moderately deep blue in the stratum lucidum-granulosum of the
epidermis. After first treating I hr in o. I M ferric chloride, washing in distilled water and
then extracting 3~ min in I N HC1 at 6o~ to remove chelated iron, sections were washed
and reacted IO min in ferric ferricyanide according to Lillie (1965, p. 21 I). The reaction
in stratum lucidum was decreased to pale blue, that in hair root zones to pale blue-green.
A I hr treatment in 2/o~ 6 in lO% acetic acid (pH 2.3) left the reaction of
hair root zones and stratum lucidum almost as strong as in the untreated control. A I hr
treatment with 2% KaFe(CN)6 in 1% borax (pH 9.I) reduced the reaction of hair root
zones and stratum lucidnm-granulosum to pale yellow, without greenish tint.
To further confirm the necessity for an alkaline medium for ferricyanide blockade of
thiol groups, an organic mercurial reagent was used as demonstration reagent. Mercuro-
chrome, used by Cowden & Curtis (I97O), was chosen as the most readily available
reagent. Two samples of Mercurochrome, Chroma 71o5 and LaPine's 'Merbromin'
I8763 as well as a small sample from the Charity Hospital drug room were used. All
behaved simply as anionic dyes. The two larger samples failed to give a positive control
reaction by the Cowden & Curtis method, by an o.o1% isopropanol solution overnight
or by a 24 hr exposure to the colour acid prepared from aqueous Mercurochrome by
HCl-precipitation with a 24 hr exposure to I mg/ioo ml solution in n-propanol after the
Bennett mercury orange method;
The Lillie-Glenner mercaptide azo-coupling method (Lillie, 1965, p. 2I 5) gave usable
results. A freshly synthesized sample ofp-N,N-dimethylamino-phenylmercuriacetate
(0.7% in 99% isopropanol; 18 hr 25~ followed by acid azo-coupling with freshly
diazotized Safranin O (used in glacial acetic acid) gave deep red basal hair shaft staining
on a colourless background. Post-treatment with I mM 2-mercaptoethanol in isopropanol
decolourizes the stain; isopropanol alone does not, thus proving the specificity. Pre-
treatment I hr in o.I m ferric chloride (pH 2.25) or in o.I M KaFe(CN)6 in I~ borax
(pH 9.1) reduced the reaction to pale red grading to faint pink. A similar exposure to
o. I M K~Fe(CN)~ in IN acetic acid (pH about 2.4) yielded again the deep red reaction of
basal hair shafts.

Discussion
The results demonstrate that potassium ferricyanide does not act effectively to quinonize
such catechols as that of the enterochromaffin cells or the noradrenaline cells of the
Ferric ferricyanide reaction mechanism 683

adrenal m e d u l l a below p H 6. Similar results were o b t a i n e d in blocking cutaneous

s u l p h y d r y l sites against reaction to ferric ferricyanide and to the L i l l i e - G l e n n e r m e r -
captide azo reaction d e m o n s t r a t e d the failure o f acid ferricyanide to oxidize hair base
thiols to disulphides, while acid ferric chloride and alkaline ferricyanide b o t h s u c c e e d e d
in p r e v e n t i n g b o t h thiol reactions.
T h e p H o f ferric ferricyanide m i x t u r e s is about that o f simple ferric chloride or ferric
sulphate solution, that is b e t w e e n p H 2.o and 2.5. F e r r i c chloride is an effective q u i n o n i z -
ing reagent at this p H level.
It is concluded, therefore, that in the ferric ferricyanide reaction as u s e d h i s t o c h e m i -
cally by all authors, the oxidizing agent is the ferric ion, w h i c h on r e d u c t i o n to the ferrous
state reacts w i t h the ferricyanide to f o r m T u r n b u l l ' s Blue.

Acknowledgement
A c k n o w l e d g e m e n t is due to D. D a r b y for preparation o f t h e manuscript.

References
ADAMS, C. W. M. (I956). A stricter interpretation of the ferric ferrieyanide reaction with particular
reference to the demonstration of protein-bound sulphydryl and disulphide groups. J. Histo-
chem Cytochem. 4, 23-35.
CHI~VREMONT, M. & FREDERIC, J. (I943). Une nouvelle mdthode histochimique de raise en
evidence des substances fi fonction sulfhydrile. Arch. Biol. (Paris) 54, 589-6o5 9
c o w DE~, R. R. & CURT I S, S. K. (I970). Demonstration of protein-bound sulfhydryl and disulfide
groups with fluorescent mercurials. Histochemie zz, z47-55.
DAVENPORT, H. A. (I960). Histological andHistoehemical Technics. Philadelphia: Saunders.
GANTER, P. & JOLLES, G. (I969--70). Histochimie Normale et Pathologique. Paris: Gauthier-
Villars.
GOLODETZ, L. & UNNA, e. G. (I909). Zur Chemic der Haut, I I I Das Reduktions-verm6gen der
histologischen Elemente der Haut. Monatsh. Prakt. Dermatol. 48, I49-66.
Go/vi o g I, G. (I948). Chemical character of the enterochromaffin cells. Arch. Path. 45, 48-55.
GOMORI, G. (I952). Microscopic Histochemistry, Principles and Practice. University of Chicago
Press, Chicago.
LEUCKART~ R. (I890). Eine neue Methode zur Darstellung aromatischer Merkaptane. J. prakt.
Chem. 4 I, I79-224.
LILLIE, R. D. (I948). Histopathologic Technic. Philadelphia: Blakiston.
LIZZIE, R. D. & BURTNER, H. J. (I953). The ferric ferricyanide reduction test in histochemistry.
J. Histochem. Gytochem. I, 87-92.
LILLIE, R. D. (I954). Histopathologic Technic and Practical Histoehemistry, 2nd Edn. New York:
Blakiston.
LILLIE, R. D. (I96Ia). Metal chelate reaction of enterochromaffin. J. Histochem. Cytochem. 9,
44-8.
L ILL I E, g. D. (1961 b). Investigations on the structure of the enterochromaffin substance.57. Histo-
chem. Cytochem. 9, 184- 9.
L ILLIE, R. D. (I965). Histopathologic Technic and Practical Histoehemistry, 3rd Edn. New York :
Blakiston Div., McGraw-Hill.
LILLIE, R. D. (ed). (I969). H. 57. Conn's Biological Stains. 8th Edn. Baltimore: Williams and
Wilkins.
LILLIE, R. D. & PIZZOLATO, r. (I972). Mechanisms of iron II and iron I I I sequence hema-
toxylin stains. J. Histochem. Cytochern. zo~ I I6-z 9.
684 Lillie and Donaldson
L I L L I E , R. D., PIZZOLATO, P., VACCA, L. L., CATALANO, R. A. & DONALDSON, P.T. (I973a).
U s e o f t h e diazosulfanilic acid, p H I azure A s e q u e n c e on adrenal m e d u l l a a n d t h e effect o f
prior oxidation a n d r e d u c t i o n on t h e reaction of several species. J . Histochem. Cytochem. 21,
448-54.
L I L L I E , R. D., PIZZOLATO~ P.~ VACCA, L. L.~ CATALANO~ R. A. & DONALDSON, P. T. (I973b).
H i s t o c h e m i c a l azo coupling reactions : A catecholamine in enterochromaffin cells in place o f or
in addition to 5 - h y d r o x y t r y p t a m i n e . J . Histochem. Cytochem. 2 1 , 4 5 5 - 6 3 .
L I S o N, L. (1960). Histochimie et Cytochimie Animales, 3rd Edn. Prais: Gauthier-Villars.
MA SSA, V. (1946). U n r6actif au ferricyanure ferrique p o u r la localisation de l'acide ascorbique dans
la tissues v6g&aux. Tray. Soc. Pharm. Montpetier 5, 14-16.
I'EARSE, A. G. E. (1953). Histochemistry, TheoreticalandApplied. Boston: Little, Brown.
P EAR SE, A. G. E. (I960). Histochemistry, Theortetical and Applied, 2 n d Edn. Boston: Little, Brown.
PEARSE, A. G. E. (1968). Histochemistry, Theoretical and Applied, 3rd E d n . Vol. I. L o n d o n :
Churchill.
PEARSE, A. G. E. (1972). Histochemistry, Theoretical and Applied, 3rd E d n . Vol. 2. E d i n b u r g h a n d
L o n d o n : Churchill, L i v i n g s t o n .
SCHMORL, G. (I928). Die pathologisch--histologischen Untersuchungs-Methoden. Leipzig: Vogel.
STAEMMLER, M. (1924). U n t e r s u c h u n g e n fiber a u t o g e n e P i g m e n t e . Virchows Arch. Path. Anat.
253,459-71.
VtlLI'IAN, F.. F. A. (1856). N o t e s u r q u e l q u e s r6actions p r o p r e s fi la s u b s t a n c e des capsules s u r r &
nales. G.r. Acad. Sci. (Paris) 96, 663-5.