Biotechnic & Histochemistry

ISSN: 1052-0295 (Print) 1473-7760 (Online) Journal homepage: http://www.tandfonline.com/loi/ibih20

How Romanowsky stains work and why they

remain valuable including a proposed universal
Romanowsky staining mechanism and a rational
troubleshooting scheme
RW Horobin
To cite this article: RW Horobin (2011) How Romanowsky stains work and why they remain
valuable including a proposed universal Romanowsky staining mechanism and a rational
troubleshooting scheme, Biotechnic & Histochemistry, 86:1, 36-51
To link to this article: http://dx.doi.org/10.3109/10520295.2010.515491

Published online: 14 Jan 2011.

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Date: 09 December 2015, At: 14:48

How Romanowsky stains work and why they

remain valuable including a proposed universal
Romanowsky staining mechanism and a rational
troubleshooting scheme
RW Horobin
School of Life Sciences, The University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK

Downloaded by [Universiteit Leiden / LUMC] at 14:48 09 December 2015

Abstract
An introduction to the nomenclature and concept of Romanowsky stains is followed by a
brief account of the dyes involved and especially the crucial role of azure B and of the impurity
of most commercial dye lots. Technical features of standardized and traditional Romanowsky
stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects,
staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted,
namely, the polychromasia achieved in a technically simple manner with the potential for stain
intensification of the color purple. Accounts are provided of a variety of physicochemically
relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures,
consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of the color purple, the substrate selectivity for purple
color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the
various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are
pulled together to generate a universal generic mechanism for these stains. Certain generic
problems of Romanowsky stains are discussed including the instability of solutions of acidic
dyebasic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.
Key words: AT-rich genome, bacteria, blood film, carmine, G-banded chromosome, Giemsa,
Leishman, malaria parasite, marrow smear, microorganism, paraffin section, parasite, resin section,
trouble-shooting, Wright

The term, Romanowsky stain, is a generic description of the azure B/polychromed methylene
blueeosin stains family, whose currently popular
variants include the Giemsa, Leishman and Wright
stains. Readers should see the historical account
by Dr. K. Krafts in this journal issue to appreciate how partial the term Romanowsky stain, or
Correspondence: Richard W Horobin, School of Life Sciences,
The University of Glasgow, University Avenue, Glasgow G12
8QQ, Scotland, UK. E-mail: RichardWHorobin@tomcroy.co.uk
2011 The Biological Stain Commission
Biotechnic & Histochemistry 2011, 86(1): 3651.

36

even its expansion, RomanowskyGiemsa stain,

really is.
The present article is a non-encyclopedic, nonhistorical review of the state of the art concerning staining mechanisms of Romanowsky stains.
Major current application areas of these stains are
addressed, including bacteriology, cytogenetics,
cytology, hematology and parasitology as well as
the staining of paraffin and resin histology sections. The relation of the staining mechanisms to
the peculiar advantages of Romanowsky staining is
discussed and a rational, i.e., staining mechanismbased, trouble-shooting guide is provided.
DOI:10.3109/10520295.2010.515491

So what are the characteristics of Romanowsky

staining? The most obvious is polychromy. Minimally, this requires just two dyes applied from
a single dye bath. In addition to red and blue
stained structures, a striking purple (Wittekind)
or magenta (Sumner) color also occurs in several structures (the Romanowsky-Giemsa effect).
The overall outcome is termed Romanowsky
staining.

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Nature of the dyes in Romanowsky

stains
Early investigators, using spectroscopic analyses, argued for the importance of azure B (Lillie
1977). This view was confirmed using dyes derived
from specific syntheses, followed by chromatographic purification; for a summary, see Wittekind
et al. (1978). These workers demonstrated that
the only dyes necessary for complete polychrome
Romanowsky staining were the basic (cationic)
dye azure B and the acid (anionic) dye, eosin Y. In
fact, the only basic dyes giving the color purple are
CH3
H3C

methyleneblue
S

azures A and B, and that occurring with azure A

is technically unsatisfactory owing to lack of color
contrast with basophilic cytoplasms as illustrated
by Wittekind (1986). The acidic dye, however, does
not have to be eosin Y and other halogenated fluoresceins such as eosin B perform satisfactorily (Wittekind 1983). The structures of the dyes named in
this review are shown in Fig. 1. Both azure B and
eosin Y currently are available commercially in a
satisfactorily pure state.
Since the earliest years (see Krafts, this issue),
and still today, however, nearly all laboratory workers have used polychromed methylene blue as a
source of azure B rather than the pure compound.
Polychrome methylene blue is a mixture of bluish
dyes, including azure B, produced from methylene
blue by oxidative treatments such as exposure to
atmospheric oxygen under alkaline conditions or
acidic dichromate solution as reviewed by Lillie
(1977). Exact compositions, and in particular the
amount of azure B present, varies among manufacturers and lots. This has been well and repeatedly
documented (e.g., Roe et al. 1940, Nerenberg and
Fischer 1963, Marshall et al. 1975).

CH3

CH3

CH 2

CH3

azureB
S

N
Cl

Cl
N
H
H3C

N
H

sym-dimethylthionine
N
S
Cl

H
CH 3

N
Cl

H
N

CH3

thionine
S

H3C

methylenevioletBernsthen
S
O

methythionoline
S

H3C

Br

Br
O

Br
eosinY

Cl

Cl
CH 3

CH 3

azure C
S

CH3

azureA
S

N
H

CH 3

COO
2Na

Br
O

Br

O2N

Br
O

O
NO2
COO

eosinB

2Na

Fig. 1. Structures of dyes discussed including azure B and eosin Y. The ionic species shown are those likely to predominate
under routine Romanowsky staining conditions. Only single resonance forms are shown.

How Romanowsky stains work 37

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The polychroming processes result in demethylation and eventually deamination of the tetramethylated methylene blue parent compound. Most
resulting dyes, including azure B, are expected to
be cationic under Romanowsky staining conditions
and all compounds retain the fused tricyclic heterocyclic ring structure. There is some variation of
hydrophilic-lipophilic character. These differences
are summarized numerically in Table 1 using the
parameters: electric charge (Z), largest conjugated
fragment (LCF), and log P, respectively; the latter
parameters were estimated as described by Horobin
(2001).
Halogenated-fluorescein acid dyes that have
been used or investigated for use in Romanowsky
stains typically are dianionic under Romanowsky
staining conditions. The sizes of the conjugated systems and lipophilicities vary slightly as summarized
in Table 1. Analytical studies have shown that many
commercial lots comprise mixtures of the nominal
dye with incompletely halogenated impurities that
are of lower lipophilicity (Marshall and Lewis 1974,
Marshall 1976).

Technical features of Romanowsky

staining systems
Protocols for preparing and using Romanowsky
stains show considerable variation, even within a
single application area. These differences probably
arose from efforts to optimize staining when using
dye lots that varied greatly in composition and, in

Table 1. Some physicochemical features of dyes mentioned in this review

Dye properties1
Dye name (CI number)

Methylene blue (52015)

Azure B (52010)
Symmetrical dimethylthionine
Azure A (52005)
Azure C (52002)
Thionine (52000)
Methylene violet Bernsthen
(52041)
Methylthionoline
Eosin B (45400)
Eosin Y (45380)

LCF

1
1
1
1
1
1
0

18
18
18
18
18
18
18

0.9
1.2
1.6
1.5
1.9
2.2
2.8

18
18
18

1.9
0.6
0.3

0
2
2

log P

Dyes typically derived from methylene blue are listed in order of

decreasing methylation/amination. Acid (anionic) dyes are listed
in order of increasing halogenation.
1Z, the nominal electric charge; LCF, the largest conjugated
fragment; log P, the logarithm of the octanolwater partition
coefficient.

38

Biotechnic & Histochemistry 2011, 86(1): 3651

particular, azure B content. Despite such technical variation, it is striking how many laboratories
are not carrying out Romanowsky staining in the
precise sense, because their published works show
cells with blue, not purple, nuclei. Indeed, many
workers in reputable institutions are content to
use such non-Romanowsky Romanowsky staining as can be seen readily by keying Giemsa
into Google images. Having made these general
points, some comments on technical variations are
pertinent.
Dyes of standardized and traditional stains
Only azure B plus various halogenated fluoresceins
produce the Romanowsky polychromy including
the color purple. Other components of polychrome
methylene blue fail to either give the color purple
or provide contrast between nucleus and cytoplasm. Methylene blue itself does not give purple
nuclei, but does provide sky blue basophilic cytoplasms. Consequently, two types of standardized
Romanowsky stains have been developed.
The first are two-dye mixtures of azure B plus
eosin Y. One such example, using azure B isothiocyanate and eosinic acid, was proposed by an expert
panel (Wittekind et al. 1976) of the International
Council for Standardization in Hematology (ICSH).
Another two-dye stain used the more soluble azure
B chloride plus the more widely available sodium
eosinate (Marshall et al. 1981). The second type is
a three-dye mixture, again containing azure B and
eosin Y, but with methylene blue added (Marshall
et al. 1975). The rationale was that when azure B
is contaminated with other oxidation products, the
cytoplasmic staining becomes grayer and less contrasted with purple nuclei; however, this is compensated for by the blue staining dye. Note that
successful staining with many polychrome methylene blue-eosin mixtures, often with low azure B
content, can thus be understood as inadvertently
impure three-dye mixtures.
The ratio of the acid and basic dyes is significant.
Work with pure dyes showed that a wide range of
molar ratios, up to 16:1 azure B:eosin, gave satisfactory staining of blood and marrow films (Wittekind 1983). Use of azure eosinate precipitates,
however, with a molar ratio around 2:1, are not
satisfactory. Consequently, as use of polychrome
methylene blue makes precise control of the azure
B-eosin ratio unachievable, it is understandable that
staining recipes since Giemsas first Romanowsky
formulas have provided an excess of cationic
dye over anionic eosin. The pure dye experiments
also demonstrated that for successful histological

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application, the azure B-eosin molar ratio had to

be much lower, in the range of 63:1 (Wittekind
1983).

Although this does not preclude the Romanowsky

effect, such preparations require longer staining
times and/or higher dye concentrations.

Solvent effects

Exploitation of characteristic features

of Romanowsky staining

The Marshall standardized methods noted above

used azure B chloride and the traditional methanolic stock solutions for both two- and three-dye variants. The ICSH (or Wittekind) variant, however,
used the less soluble isocyanate, which required the
superior, albeit more potentially hazardous solvent,
dimethylsulfoxide. Both solvents, when diluted with
buffer in the working solutions, permit generation
of the color purple. Dimethylsulfoxide, however,
provides a longer working solution usable lifetime
than methanol. Also, the color purple is unstable
in the presence of high concentrations of the lower
alcohols, methanol and ethanol, as reported at least
as early as 1944 (Lillie 1944 ). This is one reason
why published histological applications of the
Romanowsky stain often failed to generate purple
cell nuclei; they still used the traditional alcoholic
dehydrating agents (e.g., Cramer et al. 1973). These
problems can be avoided by using isobutanol or isopropanol as dehydrating agents (Wittekind 1983).
The color purple also is removed quickly by dilute
acetic acid.
In fact, the effects of pH are marked regardless
of the nature of the acid involved. The relation of
pH to acidophilicbasophilic balance, and so to
overall color, are well known. Erythrocytes, for
example, become blue-green if the pH is too high.
The occurrence of the color purple, however, also
is pH sensitive. After methanolic fixation, the color
often most is intense at pH 7-8, diminishes at pH
6-5, and is completely inhibited at pH 3.5.

Romanowsky staining is characterized by polychromasia, particularly the formation of a distinctive

purple coloration in chromatin and the granules
of neutrophils and platelets and, in tissue sections,
collagenous connective tissue and mucin. This
polychromasia is used to distinguish populations
of morphologically related cells, especially for identification of the cells in blood and marrow smears
and cytological preparations.
Another key feature is that considerable intensification of this purple staining, without overstaining of blue and red colors, can be achieved
in a technically straightforward way, e.g., by
extending staining time (Boon and Drijver 1986;
Plate 6) or by increasing dye concentrations. Such
intensification facilitates detection of small cells
and cellular components near the limit of normal
optical resolution. Examples include chromosome
G-bands, neutrophil and platelet granules, and
bacterial spores.
Not only can such polychrome staining with
intensification be obtained in a technically simple
way (two dyes and a single staining solution), but
it can be achieved on films, smears, dabs etc., or
paraffin or resin sections. A variety of fixatives
can be used, although modifications of the staining protocol may be needed. For a dramatic visual
demonstration of the colored detail to be seen in
Romanowsky stained resin embedded histology
sections, see Wittekind (1991; Figs. 4 and 5).

Staining times

Some physicochemical background

Crucially, formation of the color purple follows an

initial blue staining. Preparations do not merely
become more intensely stained, they also become
more purple as staining times are lengthened.
Optimal staining times are related to specimen
thickness, e.g., cell monolayers such as blood
smears show purple colors sooner than tissue
sections do.

The physicochemical phenomena underlying the

mechanisms of Romanowsky stains are considered
here in a general way. Not all phenomena discussed
apply to all Romanowsky staining methods and the
staining mechanisms of particular applications are
discussed later.
Acidic and basic dyeing mechanisms

Fixation
Fixation also influences uptake of acid and basic
dyes, and formation of the color purple. Aldehyde
fixatives, e.g., for histology, increase basophilia.

A simple way to conceptualize acid and basic dyeing is to view it as an ion-exchange process (Fig. 2).
Colored anions, e.g., those of eosin Y, exchange with
the mobile inorganic anions to neutralize the fixed
positive ions of proteins. Colored cations, e.g., those
How Romanowsky stains work 39

Basic dyeing of anionic biopolymer by azure B

Na+

Na+
Azure B
Na+

Cl
+

Na

Na+
Cl

Azure B

eg DNA or RNA
+

Na

Na+
Cl
Eosin Y

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Cl

2 Na+

Cl

2 Cl

Na+

Eosin Y

eg protein at low pH
Cl
Acid dyeing of cationic biopolymer by eosin Y

Fig. 2. Acid and basic dyeing seen as ion exchange processes. Mobile counterions, shown as Na and Cl, in actuality
depend on the species present in the staining solution.

of azure B, exchange with the mobile inorganic

cations to neutralize the fixed negative ions on such
biopolymers as DNA, RNA or acidic glycosaminoglycans (GAGs). Such a model accounts for both
affinity, which as described is entirely an entropy
driven process, and selectivity, which in this model
depends only on the fixed charges on the biopolymers, given that electrical neutrality of tissue and
dye bath must be maintained.
Such an ion exchange picture also can explain
the effects of pH on dye uptake. Consider cellular
biopolymers carrying acidic and basic substituents whose pKa values fall within the pH range of
Romanowsky stains. Their electrical charges vary
with pH, and as the charges vary, so will the ion
exchange possibilities as illustrated in Fig. 3.
But dyes are not merely ions. They have large
aromatic systems that provide regions of both
hydrophobicity (lipophilicity) and polarizable
electrons. So acid and basic dyeing is not just ion
exchange, but typically involves other phenomena that contribute to dyebiopolymer affinity.
Consider three possibilities. First, there may be
dyebiopolymer attractive forces such as those
occurring between the polarizable aromatic rings
of dyes and of the bases of nucleic acids. Second,
the hydrophobic effects that arise in aqueous milieu
when both dye and biopolymer have hydrophobic
regions can come into contact. Both of these processes are involved in dye binding that involves the
40

Biotechnic & Histochemistry 2011, 86(1): 3651

insertion of cationic dyes between the base pairs of

the double helix of DNA (intercalation). A third
contribution to dyebiopolymer affinity may be
formation of dyedye complexes such as occurs
with metachromasia, e.g., the staining of mast cell
granules by many basic dyes. The formation of the
color purple, as described below, probably results
from the formation of a dye complex of a somewhat
different type.
Acid dye-basic dye mixtures
After mixing an acid (anionic) and basic (cationic)
dye in a single solution, one possible outcome is
the formation of an insoluble salt. All of the cationic dyes present in polychrome methylene blue
do precipitate from aqueous solutions upon addition of eosin; the material precipitated contains
the basic dye and acid dye in approximately 2:1
molar ratio, which corresponds to neutral salts.
Water miscible organic solvents, such as methanol, dimethylsulfoxide or glycerol, reduce hydrophobic effects between the hydrophobic species
in aqueous solutions, such as the Romanowsky
staining solutions, which reduces precipitation.
The aqueous solutions mixed to produce such
azure eosinates contain dye at high concentrations
and precipitation is rapid. Dye concentrations
in staining solutions are lower and precipitation
is slower. Nevertheless, precipitation of azure

Protein in an
acidic dyebath

Cl+
H3N

Generic zwitterionic
protein in a neutral
dyebath

H3N

NH3+
Cl

HOOC

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H2N

OOC

HOOC

Exchangeable
anions, so
acidophilic

Protein in an
alkaline dyebath

OOC
Na+
NH3+

OOC

No
exchangeable
mobile ions

NH2

OOC
Na+

Exchangeable
cations, so
basophilic

Fig. 3. Effect of changes in pH on the ionic status of

biopolymers, thus on uptake of acid and basic dyes.

eosinates onto slides or within the pipework of

autostainers can be a significant technical problem
(Gregory and Maher 2009). The molar ratio of the
basic and acid dyes in the precipitate is approximately 2:1 and the presence of excess acid or,
especially, basic dye tends to reduce precipitation
(Lillie 1977).

Consequences of differential staining rates

Two dye binding sites with equal affinity for a dye
can nevertheless be colored differentially if they stain
at different rates. In progressive staining, the site
that binds dye fastest becomes selectively stained
after short periods of staining. Regressive staining,
which involves differentiation of over-stained
preparations, also fits this picture. If a preparation
is over-stained, then de-stained, sites slowest to destain retain dye longest and so become selectively
colored. Note that similar outcomes could follow if
some cellular targets have a higher density of binding sites than other targets with the same affinity for
dye. For reasons of simplicity, however, this is not
stated repeatedly.
Rate control of Romanowsky staining patterns
has diverse mechanisms, and several are discussed
below. For convenience, such effects are categorized as either of biological origin or technically
produced. This topic has been discussed at length
elsewhere, albeit without reference to Romanowsky
stains (Horobin 1982).

Rate effects of biological origin

Some basophilic molecules such as acid GAGs
found in the granules (lysosomes) of neutrophils are
highly swollen in aqueous media, which permits
rapid dye diffusion. Other basophilic biopolymers
are intimately associated with proteins and it is the
overall permeability of the nucleoprotein complex
that controls dye entry. Thus the RNA in the dense
nucleolar or cytoplasmic ribosomes stains slowly
with a basic dye, whereas the DNA in chromatin
stains faster (Goldstein 1963a and b).
Even within cell monolayers, the thickness of a
cell influences stain penetration; thus, in general,
parasites require longer staining than blood elements (Clark 1973) and the cells of parasites
are in general larger than those of human blood.
In particular, the morphology can influence rate of
formation of the color purple. This occurs first in the
thinner portions of the preparation (Wittekind 1979,
Horobin et al. 1989). It is intriguing that similar rate
effects also have been reported in cell-free DNA
films of varying thickness (Friedrich et al. 1990).
When slabs of coherent biological material are
used, i.e., tissue sections, the situation becomes
more complex. The porosity of some acidophilic
entities, typically protein or protein-rich, also has
been assessed using the refractive index as a measure. Relative porosity fell in the sequence: collagen fibers smooth muscle cytoplasm pancreatic
exocrine secretion granules red blood cells (Goldstein 1965). In keeping with a diffusion rate control
model, staining rates also fall into this sequence
(Horobin 1974, cited in Horobin 1982).
Rate effects technically produced
Morphology is influential in this case also. Thus
marrow smears stain faster, both overall and in
terms of how soon the color purple is seen, than do
sections of bone marrow (Wittekind 1983, Wittekind
et al. 1984). Moreover, as considered in more detail
later, resin sections stain more slowly than paraffin
sections. By analogy, in the thicker portions of cell
smears, the nuclei stain blue, not purple (Boon and
Drijver 1986). Formaldehyde fixation also influences
(retards) the rate of formation of the color purple
compared to the rate following methanolic fixation
(e.g., Wittekind 1983). As described in more detail
in the Fixation section below, this is probably due
to the loss of free amino groups in proteins, which
reduces the rate of uptake of eosin.
Finally, note that dye molecules of different
sizes tend to diffuse through specimens at different rates as demonstrated dramatically a century

How Romanowsky stains work 41

ago by Mann (1902). Large dyes typically stain

slower, perhaps a direct effect of particle size (Seki
1932), and perhaps also because tissue affinity of
large dyes is often greater due to their larger conjugated systems, which favor dye-tissue van der
Waals attractions (Horobin 1982). Taking the relative molecular masses of the dyes as an indication
of size, eosin Y is almost three times the size of
azure B, so the latter may be expected to stain considerably faster.

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The chemical meaning of the color purple

The color purple produced within biological specimens following azure Beosin staining has an
absorption maximum near 550 nm. This usually is
regarded as resulting from formation of an azure
Beosin or azure Beosinbiological substrate complex (Wittekind 1985, Friedrich et al. 1990, respectively). An alternative view, which regarded this
absorption as overlapping of absorption peaks of
constituent dyes (Galbraith et al. 1980), cannot be
correct for many tissue sites, e.g., chromatin, mucus
and neutrophil granules, which have no significant affinity for eosin in the absence of azure B as
pointed out for chromosomes by Sumner (1990). This
absorption does not arise in solution, rather only in
some biological matrix or at an interface such as
the surface of a solution (Sumner and Evans 1973,
Wittekind 1983). Examples of biological sites where
the color purple arises include chromatin and granules of neutrophils and platelets in blood smears
and tissue sections, in the bands of G-banded chromosomes, and in collagenous connective tissue and
mucus granules of tissue sections.
Although most investigators have regarded formation of the color purple as resulting from complex formation, the specific interactions between
the dyes, and perhaps also the biosubstrate, still are
debated. An early suggestion proposed formation
of an acceptor/donor azure B-eosin charge transfer
complex (Zanker 1981). This concept was adopted
by Wittekind (1985) who noted the possible role
of hydrogen bonding between the NH groups of
azure B and eosin for facilitating electron transfer.
The latter feature would account for lack of purple
staining when methylene blue is used with eosin
Y and its occurrence when azure A, with two NH
groups, is used; however, the question of why less
methylated dyes, e.g., thionine, do not show the
Romanowsky effect remains unresolved.
Wittekind (1985) also considered that azure B
and eosin are associated in part due to hydrophobic
effects, which would explain the lability of the complex when exposed to solvents that disrupt water
42

Biotechnic & Histochemistry 2011, 86(1): 3651

structure or remove the water completely such

as when lower alcohols are used for dehydration.
Zimmermanns group carried out a detailed spectroscopic study of a DNA, azure B, eosin Y model
system, and they also favored hydrophobic effects
as a major contributor to the dye-dye binding
involved in formation of the color purple, although
in their picture the DNA formed part of the complex (Friedrich et al. 1990). Because the conditions of
the latter study were markedly different from those
of practical Romanowsky staining and because the
color purple arises at sites not containing DNA, the
significance of the study is not clear.
Finally, note that additional azure B-eosin
complexes may exist. For example, in strongly acidophilic substrates, such as eosinophil granules,
an initial uptake of eosin is followed by binding
of azure B, which gives a red-brown coloration
that might be due to a different complex (Wittekind
1991). Yet another possible case arises with the carmine or red nuclear coloration of Romanowsky
stained malarial parasites (for an historical account,
see Baker 1958). Regarding the latter, it is worth noting that Plasmodium falciparum DNA contains the
unusual feature of super AT-rich regions in its
genome (Woynarowski et al. 2007), and, of course,
AT-rich DNA is significant in Romanowsky staining
of banded chromosomes.
Substrate selectivity for formation of the
color purple
Whatever the origin of the color purple, whether it is
a complex or merely an azure eosinate salt, its substrate-selective formation constitutes a distinct problem. Why, for example, is DNA-rich chromatin purple,
but not the adjacent RNA-rich nucleoli? Although
such questions cannot yet be answered definitively,
contributing possibilities can be proposed.
The color purple requires the presence of
both azure B and eosin in the same region of the
specimen. Therefore, both affinities for and rates
of uptake into a substrate could control selectivity. Rate effects certainly play a part. Chromatin,
for example, first stains blue, then purple. At the
stage of staining when chromatin becomes purple,
basophilic cytoplasms still are blue, but if staining
times are prolonged sufficiently, these cytoplasms
also become purple. With extremely extended
times, many other substrates become purple as well
(Horobin and Walter 1987). The causes of such rate
effects are varied and are considered as appropriate for each of the staining applications, e.g., blood
smears, G-banding, resin section staining etc., discussed below.

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Intensification of the color purple

by a template effect
Almost a century after the introduction of the polychrome methylene blue-eosin staining system, an
explanation was offered for the stains characteristic of intense purple staining of extremely small
objects such as the submicrometer lysosomes of
neutrophils or the tiny G-bands of chromosomes.
Sumner (1980), who was concerned with the latter structures, proposed that because the levels of
staining of nuclei and chromosomes with Giemsa
and similar mixtures are far greater than those normally attained with methylene blue perhaps
Giemsa is not dyeing chromosomes in the normal
sense, but is in fact being precipitated preferentially
in them the initial stages of Giemsa staining
involve ionic binding of thiazines alone A second
process in staining would then be the binding of
thiazines to eosin to form a precipitate, thereby freeing the reactive groups on the DNA to bind more
thiazine. Therefore, this involves a DNA-induced
co-precipitation, which will be termed here a template effect and which proceeds without reaching
equilibrium. In the context of staining blood smears
and other complex tissues, one can, in fact, replace
Sumner s term DNA with other anionic biopolymers, e.g., hyaluronic acid in the lysosomes of neutrophils and platelets. Such a generalized template
effect mechanism is illustrated in Fig. 4, where the
precise chemical nature of the azure B-eosin complex is not specified.
To appreciate the shift in mechanistic perspective
represented by the template intensification effect,
consider the views of two well known figures in histochemistry and biotechnique. John R. Baker, in his
classic monograph, considered that an azureeosin
complex was unlikely to transfer from solution onto
DNA; however, he had no suggestions regarding
what did happen (Baker 1958). Moreover, Ralph D.
Lillie, an investigator with considerable experimental experience in the field, argued as late as 1977 that
staining of the characteristic granules of neutrophils
reflected their affinity for an azure Beosin compound
(Lillie 1977).

sections than in smear preparations, however, but

that certain regions stain very much slower.
This markedly non-uniform staining inhibition
is due to non-uniform resin infiltration. This in turn
is due to the viscous and, even when water miscible,
slightly hydrophobic character of the infiltrating
resin monomers. These properties cause resin to be
largely absent from dense structures, e.g., chromosomes, elastic fibers, nucleoli, red blood cells, and
from hydrophilic structures, e.g., collagen fibers,
GAG-rich lysosomes and mucus granules. Other
regions, such as nuclear chromatin and the cytoplasmic matrix, though not the small, dense ribosomes, are infiltrated well by the resin (Horobin
1983, Gerrits et al. 1990).
Staining of resin sections consequently displays
evidence of site-selective staining inhibition by resin
superimposed upon the rate and affinity effects
owing to the biological substrates. It may be noted
that resin inhibition can arise from both a reduction
of free volume available for dye diffusion and dyepolymer affinity as when a lipophilic dye stains the
resin (Horobin et al. 1992).
What this implies for Romanowsky staining is
straightforward. A small, weakly hydrophilic dye
such as azure B can access resin sections with only
slight inhibition of entry. Access by the three times
larger and weakly lipophilic eosin Y, however, is
inhibited significantly in resin-containing structures
such as chromatin and the cytoplasmic matrix. As
an example of the resulting staining outcomes, consider cytoplasmic basophilia, namely, the staining
of ribosomal RNA. This staining is blue In smears,
because the dense ribosomes impede entry of the
eosin Y. In resin sections, however, because ribosomes are dense, they are, unlike the cytoplasmic
matrix, poorly infiltrated. Because routine staining
times for resin sections are necessarily longer or
staining temperatures higher than for smears, eosin
has relatively better access to ribosomes than to
matrix. Consequently, ribosomes that are exposed
on the surface of the section stain purple and this
reversal of the normal staining pattern has been
observed in glycol methacrylate sections of bone
marrow (Horobin and Boon 1988).

Effects of resin embedding media

Chemical and physical effects of fixation

Resin (plastic) embedding media have an inhibitory

influence on dye uptake when sections are stained
with the resin still present. For example, staining
rates of resin sections of bone marrow are slower
than those of marrow smears unless the stain is
heated (Horobin and Boon 1988). It is not merely
that many structures stain somewhat slower in resin

Changing the fixative used could influence

Romanowsky staining in two ways. First, chemically,
e.g., aldehyde fixatives such as formaldehyde react
with tissue amino groups, which results in decreased
acidophilia (Horobin 1982). This has been observed
to lead to preparations with an overall bluer coloration (Wittekind 1983). Different fixatives, however,
How Romanowsky stains work 43

small,

Cl

fast diffusing
Azure B
Na+
Azure B

Na

Na+

polyanion

2 Na

2 Cl

Azure B

Na+

Na+

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Azure B
Eosin Y
Eosin Y

Azure B

Na+

Large,
slow diffusing

insoluble azure-eosin
complex deposited
nearpolyanion

Fig. 4. Intensification of the Romanowsky effect with continued staining seen as a template effect. Note that the precise
relation of azure B to eosin is not defined.

also alter the native morphology in different ways.

Thus the classic alcohol and acidic alcohol fixations
give rise to shattered preparations at a microscopic level, whereas aldehyde fixation produces a
more coherent structure (Horobin 1982).
Empirically, alcoholic fixatives produce specimens that tend to stain faster than formalin fixed
preparations. Where two dyes of different sizes are
used simultaneously, faster staining typically favors
entry of the larger dye. Either of these mechanisms
apparently could underlie the observed effect of formalin fixation, namely, that the color purple fails
to occur in nuclear chromatin, which instead stains
blue. If staining times are lengthened or eosin binding is enhanced by lowering the pH of the dye bath
(Lillie 1977), the purple staining is restored. It has
been established, however, by comparing preparations fixed in alcohol followed by formaldehyde
to those fixed in formaldehyde followed by alcohol that the effect is due to the chemical changes
(Horobin and Walter 1987).

Staining mechanisms of Romanowsky

and related stains
While all methods use the typical Romanowsky
staining system based on azure B, usually in the
form of polychrome methylene blue, and eosin Y,
44

Biotechnic & Histochemistry 2011, 86(1): 3651

the procedures used for preparing specimens, the

morphology of the material so prepared, and the
staining procedures vary markedly. These differences are outlined below.
Specimen pretreatment
Fixation of cell smears for hematology or cytology routinely use methanol. Chromosome spreads
usually are prepared using methanol-acetic acid.
Tissue prepared for sectioning, whether in paraffin or resin, usually has been fixed in formalin,
although other fixatives occasionally have been used.
Chromosome banding uses various pretreatments
of the fixed chromosome spreads prior to staining
in addition, e.g., with a solution of a hot neutral
aqueous buffer, a surfactant, or a protease.

Morphology of the prepared specimen

Hematological or cytological preparations are cell
monolayers produced as brushings, dabs, smears,
spins etc. For diagnosis of protozoal parasitic
infections, thick, i.e., many cells thick, films sometimes are used. Cytogenetic chromosome spreads
are monolayers of chromosomes. Paraffin sections
and sections cut from methylmethacrylate resin
blocks are stained after removing the embedding

media using suitable solvents. Glycol methacrylate

sections are stained with the resin in situ; these are
the eponymous resin sections. Sections are typically 0.55.0 m thick with the resin sections falling
at the thin end of this range.

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Staining conditions
Blood and marrow monolayers, e.g., smears, are
stained for up to 30 min at room temperature. Staining times for chromosome spreads are much shorter,
while those for sections are longer. The staining of
smears and sections, and in particular resin sections,
has on occasion been accelerated considerably by
heating the solutions, sometimes with a microwave
oven (Boon and Kok 1987). Section staining sometimes uses regressive procedures with differentiation in aqueous acetic acid or more exotic mixtures
(Wittekind et al. 1991). To retain the color purple,
however, contact with acetic acid differentiation
solutions must be minimized, and dehydration must
avoid use of the lower alcohols and instead use air
drying or solvents such as butanol or isopropanol.
Mechanistic summaries of each general methodology are given below.

Blood, bone marrow and cytological smears

Explanations for development of the full
Romanowsky polychromasia will be considered
below. The specific issues are: why do any dyes
bind, and in particular, why do some substrates
stain red, some blue and some purple? In addition,
why are certain tiny granules in neutrophils and
platelets stained so strongly as to be readily noticeable? To deal with these questions three aspects of
mechanism are relevant as follows.
1. Acid and basic dyeing occurs in sites rich in
polycations and polyanions, respectively. This,
in the final stage of the staining, gives rise to
blue staining of ribosome-rich (basophilic)
cytoplasms and nucleoli and red staining of
eosinophil granules and red blood cells.
2. An additional color, purple, is produced in a
limited number of relatively fast staining sites,
notably chromatin and certain granules in
neutrophils and platelets. Such rate-controlled
selectivity involves transformation of initial
azure B staining into an azure B-eosin complex
as illustrated in the color plates in Boon and
Drijver (1986). The faster staining is due to
DNA-rich chromatin, which has a more porous
structure than RNA-rich ribosomes, and to the

swollen character of GAG-rich lysosomes in

partially aqueous media. Failure to stain purple, as with the lysosomes of monocytes, also
is explained by this model, because monocyte
lysosomes lack significant GAG content
(Fedorko and Morse 1965).
3. The easy visibility of the purple staining of
tiny granules, which occurs only if the batch
of Romanowsky stain used contains sufficient
azure B, is due to intensification owing to a
template effect.
Banding of chromosome spreads
Several distinct phenomena need explanation. Why
do the chromosomes stain, and in particular, why
do they stain purple? Why is it that only certain
bands stain strongly? And how is it that these tiny
structures are stained so intensely as to be noticeable? To address these questions, four aspects of
mechanism are discussed below.
1. The first step involves basic dyeing of DNA by
azure B.
2. The blue staining subsequently is converted to
purple as an azure Beosin complex is formed.
Note that if the chromosomes are covered with
incompletely dispersed cytoplasmic material,
the entry of the larger eosin ions is retarded
and this step is inhibited (Sumner 1990).
3. The question of selectivity is more contentious. Various suggestions have been made to
explain why dye binding favors certain limited regions of the chromosomes, the G-bands.
For example, it is known that G-bands correspond to AT-rich regions. Because AT base
pairs interact less strongly than GC base pairs,
this location would be expected to favor insertion of an intercalating dye such as azure B,
and indeed in an azure Beosin YDNA
model system, the azure B was observed to be
intercalated (Friedrich et al. 1990). This obviously is not the only, or perhaps not even the
overriding, phenomenon, however, because
strongly stained G-bands occur only following
various pretreatments. These are varied, e.g.,
proteases, detergents, hot buffer, and it is not
apparent what the common features are. The
subject of selectivity is still a topic of lively
debate; for a recent contribution see Ushiki
and Hoshi (2008). Unfortunately, the view
Sumner espoused in 1990, we still lack a satisfactory hypothesis, remains the case today,
as he confirmed to the author recently (Sumner 2008, personal communication).
How Romanowsky stains work 45

4. The visibility of the tiny purple stained

G-bands is due to a template intensification
effect.

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Staining of microorganisms and other

single cell entities
Microorganisms, whether bacterial, protozoal or fungal, sometimes are stained as monolayers (smears,
films or dabs) and sometimes within tissue sections.
We are concerned here only with the mechanisms
underlying selective visualization of the pathogens;
the staining of other tissue elements in sections is
discussed in a later section. The problems requiring
mechanistic explanation are as follows. Why do the
pathogens stain, and in particular, why stain purple? How is it that these often tiny cells are stained
sufficiently strongly to be noticed? These questions
and their implied extensions, are addressed below.
1. All the target organisms contain DNA, either
in nucleoids of bacteria or in chromatin of
eukaryotic cells. The first step is basic dyeing
of DNA by azure B.
2. This dye then interacts with eosin to yield the
purple azure Beosin complex. Note that preparations discussed in this section are, for various reasons, often slow staining. Thus, although
bacterial cells are tiny, they usually are very
dense; within a smear, the cells of parasites
often are larger and thicker than white blood
cells. In any event, it is common to use thick
films of blood for diagnosis of protozoal parasites in parasitological samples. The effects of
such rate limiting factors were discussed above.
The technical consequence of this is shown by
the commonplace use of diffusion-enhancing
procedures such as extended staining times
(even overnight for parasitic cells within paraffin sections; Bartlett 2008) or heated staining
solutions (for an illustration of the benefits of
which, see Boon and Kok 1987).
3. The visibility of tiny purple stained microorganisms is due to the template intensification
effect described above.
4. Selectivity of staining calls for comment. While
intense staining of morphologically discordant organisms is the primary feature for identification, the mechanisms of two additional
tactics should be noted. The first is differentiation with solvents, such as dilute acetic acid
or isopropanol, of slow staining specimens
subjected to extended or heated staining. The
mechanism of such destaining has been
discussed above. An additional source of
46

Biotechnic & Histochemistry 2011, 86(1): 3651

selectivity specific to the demonstration of

microorganisms is the use of various pre-treatments prior to staining. Thus, if the parasite
load is low, the sample size can be increased
by using thick blood films. In this case, however, the parasites may be obscured by the
mass of red blood cells, which are therefore
lysed either osmotically or enzymatically.

Three additional topics requiring

brief mention
1. Romanowsky stains also are widely used with
additional types of cellular monolayers, notably cytological specimens, e.g., pleural fluid
smears or lymph node aspirates. Another
example is the staining of spermatozoa, both
of humans and many other mammals, to assess
male reproductive status. Mechanistically, however, such cases are analogous to the blood
smears discussed above.
2. Multicellular parasites, e.g., tapeworms, also
may be Romanowsky stained. As this is done
within tissue sections, however, the process is
mechanistically unremarkable; see below.
3. The phenomenon of the carmine or red purple nuclear coloration of some Romanowsky
stained parasites was discussed above.
Tissue sections: paraffin
The full range of Romanowsky staining polychromasia, including the color purple, can be achieved
with paraffin sections. The technical requirements
are that sufficient azure B is present in the stain, that
the staining time is long enough, and that excessive
exposure to solvents such as acetic acid during differentiation or lower alcohols during dehydration
be avoided. Given these circumstances, the staining
is not particularly fixative-dependent.
Some of the questions requiring mechanistic
explanation are by now familiar. Why do the acidic
and basic dyes bind, and why is the color purple
generated? In particular, why do tissue elements
such as collagen fibers, cartilage matrix and some
mucins stain purple? The longer staining times and
use of formaldehyde as a fixative also call for discussion. The mechanistic factors needed to explain
these are listed below.
1. Acid and basic dyeing occurs in sites rich in
polycations and polyanions, respectively. This
is the cause of the red staining of muscle cytoplasm and of eosinophil granules and red
blood cells; this accounts also for the blue

2.

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3.

4.

5.

staining of ribosome-rich (basophilic) cytoplasms, e.g., of secretory cells.

The color purple is seen in chromatin and the
granules of neutrophils. This rate-controlled
selectivity is described above and involves
transformation of initial azure B staining into
an azure B-eosin complex. Because the color
purple is unstable in the presence of lower
alcohols, dehydration through ethanol must
be brief or be replaced by air drying or use of
agents such as butanol.
Rate effects also are seen. Paraffin sections are
thicker and more coherent, especially if formaldehyde fixed, than methanol fixed cell
monolayers. Consequently, such sections
require extended staining times: 90 min are
recommended for obtaining the full polychromatic staining pattern (Wittekind 1991). Such
staining can be usefully differentiated, traditionally with aqueous dilute acetic acid, but
also with media such as isopropanol containing tannic acid.
The occurrence of the color purple in tissue
elements such as collagen fibers, cartilage
matrix and mucus granules also can be
explained by a rate control mechanism. In paraffin sections, probably due to the swollen
character of the plentiful GAGs found at all
these sites, these are relatively rapidly staining
structures.
Inhibition, although not inevitable elimination, of formation of the azure B-eosin complex by formaldehyde fixation is probably a
chemical effect.

Romanowsky staining: is there a

universal mechanism?
The mechanisms of the major applications of
Romanowsky stains in modern biomedicine have
been sketched. It is obvious that some factors such
as specimen thickness, proportion of AT-bases in
DNA, and occlusion by resin embedding media,
have influence only in particular staining systems.
There is no single, all-inclusive mechanism. Nevertheless, there are features that are important for
all the staining systems described, namely, initial
acidic and basic dyeing, formation of a purple azure
Beosin complex in a rate-controlled manner, and
intensification of the purple staining due to a template effect. These factors do constitute a universal
mechanism of sorts, common to all methods as set
out diagrammatically in Fig. 5.

Romanowsky staining problems and

a staining mechanism-based
trouble-shooting scheme
Certain problems with Romanowsky stains, namely,
the unstable nature of a working Romanowsky
staining solution and the heterogeneous nature of
polychrome methylene blue, are inherent to the
routine Romanowsky procedures and so are discussed first, followed by comments on the problems
of stain standardization. Once again, the account
given here refers back to the discussion of general
physicochemical principles.
Inherent instability of Romanowsky stains

Tissue sections: resin

The full range of Romanowsky staining polychromasia including the color purple occurs; long
staining times again are required and over-staining
followed by differentiation can be used; fixation in
formaldehyde is possible if suitable modifications
to the protocols are made. So far, the phenomena
and the mechanistic explanations are the same as
for paraffin sections and the details do not call for
repetition.
An apparently novel phenomenon, however,
the effective reverse color coding of nuclear chromatin and basophilic cytoplasms has been reported
(Horobin and Boon 1988). The appearance of blue
nuclei and purple cytoplasms in lymphocytes, for
example, is due to a combination of differential
resin occlusion effects plus use of formaldehyde
fixation. Taken together, these phenomena reverse
some of the rate effects seen in smears.

As discussed earlier, as soon as the substantially

aqueous staining solutions are prepared, precipitation of material approximating azure- eosinates
commences. The amount of precipitation tends to
be minimized by factors such as minimal standing time before staining, low dye concentration
and low temperature, presence of excess acidic or
especially basic dye, presence of larger proportions
of organic solvent; dimethylsulfoxide and glycerol
are particularly effective inhibitors of precipitation.
Precipitation onto slides or within the pipework of
autostainers can constitute a significant technical
problem.
Inherent dye impurity
Dye lots labeled azure B, even when purchased
from reputable vendors, may contain some uncertain proportion of the named dye, but essentially
How Romanowsky stains work 47

Uptake of dye anions and

cations by simple acid and
basic dyeing

Application of Romanowsky stain,

containing the
red acid (anionic) dye eosin Y, plus the
blue basic (cationic) dye azure B

RED
stained
acidophilic
sites

BLUE
stained
basophilic
sites

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Uptake of second dye by faster

staining sites only, yielding an
azure Beosin Y complex

deposit of
PURPLE
complex
Continuous deposition
of the azure B-eosin Y
complex due to a
template effect giving
a stain intensification

No short-term changes of
color in slower staining sites

BLUE
stained
lymphocyte
cytoplasms,
& nucleoli

PURPLE
chromatin,
neutrophil
granules, &
platelets

RED stained
eosinophil
granules &
erythrocytes

Fig. 5. The Romanowsky staining mechanism: the universal core processes. For illustrative purposes, the diagram
assumes that a blood smear is being stained.

are relabeled polychrome methylene blue as

discussed earlier. A few vendors have supplied
azure B in a fairly pure state, but because this is considerably more expensive than azure B-containing
polychrome methylene blue, such material is not
likely to be used for the preparation of stains purchased by routine diagnostic laboratories and even
pure lots can be less pure than claimed (Marshall
1979). Because the concentration of the key ingredient, azure B, consequently varies markedly among
lots, the intensity of the Romanowsky effect also is
variable.

Standardization of Romanowsky stains

Presumably because of these phenomena, the color
balance and appearance of Romanowsky stained
preparations acceptable to different laboratories
also is varied. This may be appreciated most simply by inspecting the images of blood cells in different hematology atlases. Preferences also have
been investigated systematically by comparing the
opinions of experienced working hematologists
concerning the acceptability of various standardized formulations (Marshall et al. 1978).
48

Biotechnic & Histochemistry 2011, 86(1): 3651

Despite these problems, Romanowsky staining

can be standardized to varying degrees. If using
polychrome methylene blue, stains based on dye
lots certified by the Biological Stain Commission
are recommended. Such reagents are on average
no more expensive and when used with appropriate protocols, are more reliable. If more rigorous
standardization is desired, various standard staining procedures based on pure dyes have been
devised, tested and the protocols published (e.g.,
Marshall et al. 1975, 1981, Wittekind et al. 1976).

A trouble-shooting scheme for Romanowsky

staining
In the past, laboratory workers have tended to view
Romanowsky staining as capricious and difficult to
get right. A trouble-shooting scheme can be based
on the account offered above, however, and one is
provided in Table 2.

Conclusions
There is a universal staining mechanism for
Romanowsky stains that involves:

Table 2. Trouble-shooting Romanowsky staining of blood smears and related specimens.

Problem

A. Stain precipitates on slides or within pipework

of autostainer

B. Overall staining too pale, but color balance

satisfactory

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C. Cell nuclei blue, not purple

D. Neutrophils appear agranular in blood smears,

collagen fibers pink, not purple in histological
sections
E. Neutrophils appear toxic, i.e., grossly enlarged
granules; general purple tinge to basophilic
cytoplasms
F. Erythrocytes and eosinophil granules too blue

G. Erythrocytes and eosinophil granules too

brownish orange, not pink
H. Basophil granules in blood smears fail to stain

Possible causes

1. Buffer concentration too high; reduce

2. Methanol (glycerol, dimethylsulfoxide) content too low; increase
3. Staining solution too old; replace
4. Temperature too high; keep cooler
1. If pure azure B, dye content too low; due to: Impure lot of dye
or stock solution; replace Error in dilution or weighing; check
and correct Precipitation of stain; see A
1. If pure azure B, dye content much too low, see B.
2. If polychrome methylene blue, azure B content of lot is low; replace
3. If either dye type:
Methanol (etc.) content too high; reduce
pH of staining solution too low; check and correct
Staining time too short; lengthen
Staining temperature too low; check
Eosin concentration in stock solution too low; make fresh
Specimen too thick; lengthen staining time.
If histological section, formalin fixation too extended; lengthen
staining time or increase dye concentration
If histological section, acetic acid differentiation or alcoholic
dehydration too long; reduce or change solvents
Occlusion of specimen; check under the microscope for
presence of any overlying material.
Any of factors listed in C

pH too high; check

Staining time too long; reduce
Azure B concentration too high; check
pH too high; check
Wrong buffer used; check
Staining time too long; reduce
Some standardized stains give this appearance; rinse briefly in
distilled water
pH too high; check and adjust
Fix with azure dye dissolved in methanol

1. Acidic and basic dyeing to give a polychrome

effect.
2. Formation of an azure B-eosin Y complex,
which provides further polychromasia, the
Romanowsky-Giemsa effect.
3. Formation of this complex is rate-controlled, and exhibits a template intensification
effect.
4. The latter phenomenon provides the potential
for stain intensification.
Romanowsky stains have two advantageous
features:
1. They can provide a technically straightforward polychrome staining.
2. They can visualize entities at or below the
limits of optical resolution under a standard
microscope.
When technical problems arise with Romanowsky
stains, rational trouble-shooting is possible.

Acknowledgments and dedications

I wish to acknowledge Prof. I. McGrath, Division
of Integrated Biology, FBLS, University of Glasgow,
for providing facilities, and P.N. Marshall, A.T. Sumner and Richard Hartley (Department of Chemistry,
University of Glasgow) for helpful discussion and
comment. I dedicate this paper to R.D. Lillie and
D.W. Wittekind, two colorful giants of histotechnology. I had the benefit and, sometimes, pleasure of
meeting both men. The former told me long ago
Young men like theories, but I think you should go
back to the lab and do more experiments, Horobin,
while the latter said to me many times But
Dick Perhaps I should have listened to them
more attentively.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible
for the content and writing of the paper.
How Romanowsky stains work 49

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