Hematoxylin: a simple, multiple-use dye for

chromosome analysis

Marcelo Guerra
Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Cidade
Universitária, 50670-420 Recife, PE, Brasil.

ABSTRACT
A staining mixture of hematoxylin-iron alum combined with a strong hydrochloric
hydrolysis was successfully applied for chromosome observation of several kinds of
plants and some animals. Slightly different procedures were developed for different
materials and objectives. For plant cells, the most important technical aspect was
the use of 5 N HCl hydrolysis, which resulted in a very transparent cytoplasm,
combined with an intense, specific hematoxylin stain. This technique is
recommended for cytogenetical analysis in general, and it is especially indicated for
practical classes, due to its simplicity and high reproducibility of results. Moreover,
the deep contrast observed makes this technique very useful for sequential staining
of cells previously analyzed with other stains, as well as for materials with fixation
problems.

INTRODUCTION
Hematoxylin, combined in the proportion of 4:1 with alum,
was one of the first staining solutions used to analyze
chromosomes, and for a long time it was among the stains
preferred by classic cytogeneticists. Hematoxylin, itself, is
not a dye. First, it needs to be oxidized, either by alum or
by employing a tissue mordant. L. Taits, as early as 1875,
drew attention to hematoxylin's capacity for preferentially
staining the nucleus (Conn, 1929).

Wittmann (1962, 1965) demonstrated that an aceto-iron-

hematoxylin solution produced excellent results for
chromosome analysis of plants, animals and humans.
However, this technique had little acceptance among
cytogeneticists. Probably, the need for additional chemicals
to stain, such as mordants and the clarifier chloral-hydrate,
made the technique seem more complicated and had less
uniform results. Henderson and Lu (1968) tried to simplify
the technique, working with a stock solution of 2%
hematoxylin and 0.5% iron alum, both in propionic acid.
The results, however, vary depending on the ripening time
of hematoxylin and the proportions used in the stock
solution mixtures. With the development of simpler
techniques, this dye was almost universally substituted by
the similar carmin and orcein. Yabu and Tokida (1966),
however, demonstrated the superior quality of Wittmann's
staining for alga chromosomes. Fujii and Guerra (1998)
introduced some modifications in the technique for
chromosome staining of algae, with superior results to
those obtained previously.

In the present work some alternative procedures are

presented for hematoxylin use in general cytogenetics,
mainly with plant species. Procedures for sequential
staining of animal cells using other techniques are also
presented. In all cases, results were characterized by great
technical simplicity and higher contrast in chromosome
staining.

MATERIAL AND METHODS

Material

Mitotic and meiotic chromosomes from several species of

algae, bryophytes, pteridophytes and angiosperms were
examined. Insect and bat chromosomes were also tested.

Preparation of the staining (4% hematoxylin)

The traditional staining mixture was prepared by dissolving

4 g of hematoxylin and 1 g of iron alum
(FeNH4(SO4)2.12H2O) in 100 ml of 45% acetic acid at room
temperature. The mixture was homogenized with a glass
stick and left in a dark flask for one week. After that period,
the mixture was filtered and kept in a dark glass. The
solution could be used immediately or stored for an
undeterminate period in the refrigerator. To test the best
dye concentration, 2, 1, 0.5 and 0.2% solutions were
prepared by direct dilution of the stock solution (4%). The
1% hematoxylin was also prepared directly from 1 g
hematoxylin plus 0.25 g iron alum in 100 ml of 45% acetic
acid.

Methods

Two basic methods for slide preparation were tested in

different organisms. In both cases, the tissues were first
hydrolyzed in 5 N HCl and washed in distilled water. In the
first method, the material was previously stained with
hematoxylin and then squashed under a coverslip. In the
second method, the material was first squashed in 45%
acetic acid and then stained with hematoxylin. The steps
followed in the procedures, and described in detail below,
were based on Guerra (1983) and Fujii and Guerra (1998).
All photomicrographs were taken with Agfa Pan film and
Kodak Kodabromid F3 paper.

1 - Staining-followed-by-squashing method

A - Mitotic plant chromosomes

- Fixation. Root tips in active growth were fixed in 3:1

acetic ethanol for 2-3 h.
- Washing. Root tips were transferred from the fixative to a
recipient with distilled water for 5 min, followed by a
second 5-min bath.
- Hydrolysis. Root tips were plunged in 5 N HCl for 20 min.
- Washing. Repeat item b with baths of 10 min.
- Staining. Root tips were slightly dried on filter paper and
transferred to a small recipient containing two or three
drops of stain (polyethylene contact lens containers proved
to be the most practical and safest tool). The recipient was
maintained closed at room temperature for at least 30
min.
- Slide preparation. A root tip was transferred to a small
drop of 45% acetic acid on a clean slide. The terminal
meristem was dissected and fragmented into very small
pieces. They were covered with a coverslip, tapped very
lightly with a needle and squashed between sheets of filter
paper.
- Preparation of permanent slide. The coverslip was
removed by freezing in dry ice or liquid nitrogen and the
slide was thoroughly air dried. The material was quickly
washed with a jet of distilled water to remove the excess
dye, air dried again and mounted in Canada balsam or
similar.

B - Meiotic plant chromosomes

Basically, the above procedure can be adapted as follows

for meiotic analysis of plants:

- Very young anthers or flower buds were fixed and washed

as described before.
- Since flower buds are more fragile than root tips, they
were hydrolyzed in 5 N HCl for 10 min or less. Excessively
soft material can be easily lost during handling.
- The material was washed and stained as described in
procedure 1A.
- A single anther was transferred to a drop of 45% acetic
acid on a clean slide and cut into pieces as small as possible
in order to squeeze the meiocytes out. The anther wall
debris were removed, the material was covered with a
coverslip, gently squashed and analyzed.

C - Meiotic grasshopper chromosomes

- Testes fixed in ethanol-acetic acid (3:1) were directly

transferred to the stain for 30 min at room temperature.
- The stained tissue was slightly washed and left in distilled
water.
- Two or three tubuli were transferred to a drop of 45%
acetic-acid on a slide, cut into small pieces, squashed under
a coverslip and analyzed.
- A permanent slide was prepared as in procedure 1A.

2 - Staining-after-squashing method
A - Plant chromosomes

- Root tips or flower buds were fixed, washed, hydrolyzed

and washed again as described in procedures 1A and 1B.
- The unstained material was macerated in a drop of 45%
acetic acid and squashed. The coverslip was removed, and
the slide air dried as in procedure 1A.
- A drop of hematoxylin solution was added to the dried
cells on the slide and covered with a coverslip. Staining
occurs within a few seconds, and the slide can be analyzed
immediately.
- To turn the slide permanent, the coverslip was removed
with a jet of water, air dried and mounted in Entellan or
similar media.
- For meiotic analysis, the same procedure was followed,
simply reducing the time of hydrolysis to between 5 and 10
min.

B - Animal chromosomes

The traditional techniques of slide preparation for mitotic

and meiotic cells of mammals and insects were followed,
substituting the usual dyes with hematoxylin.

3 - Sequential staining

Cells previously stained with orcein, Giemsa, fluorochromes

or C-banding treatment can be destained and restained
with hematoxylin. In these cases, the following procedure
was used:

- The coverslip was removed by freezing, and the mounting

medium was completely eliminated by successive washes in
xylol.
- The slide was destained and air dried. Most dyes can be
removed by washing the slide in 45% acetic acid, 3:1
ethanol-acetic acid, or both alternately.
- The material was restained with hematoxylin as described
in procedure 2A. If the cytoplasm becomes too dark, the
slide can be simultaneously destained and hydrolyzed in 5
N HCl for 5 min and restained with hematoxylin. When
necessary, permanent preparations were made as in
procedure 1A.

RESULTS AND DISCUSSION

All the procedures described above provided well-stained
chromosomes and a completely transparent or just slightly
stained cytoplasm. Tests with different hematoxylin
concentrations showed that although the highest
concentrations (4% and 2%) resulted in intense chromatin
staining and high cytoplasm transparency, the texture of
chromosomes and interfase chromatin may be lost due to
overstaining (Figure 1a). One percent hematoxylin allowed
better visualization of nuclear structure chromocenters and
late condensed regions of prophase chromosomes. On the
other hand, the higher viscosity of 4% hematoxylin turns
its penetration into the tissues slower, especially in anther
tissues. Concentration of 0.5% was still efficient, while that
of 0.2% was more heterogeneous, with many weakly
stained areas. The ideal concentration was 1% (Figure 1b-
g), although in some special cases, as in the sequential
staining of old slides, a 4% concentration is recommended.
Figure 1 - Plant and animal chromosomes stained with hematoxylin. a, b - Root tip mitotic cells
of Allium cepa stained according to procedure 1A with 4% hematoxylin (a) and procedure 2A with 1%
hematoxylin (b). Note the excessive chromatin staining in a and the chromosome arms in different focal
plains (arrowheads). c - Meiotic chromosomes of Nothoscordum sp. showing five bivalents (three
metacentrics and two acrocentrics) and a slightly stained nucleolus (arrowhead). Arrow points to the
centromere of one of the metacentrics. d, e - Pollen mitoses in Nothoscordum sp. showing the five
metaphase chromosomes (d) and a binucleated pollen grain (e). f - Interphase nucleus and metaphase
chromosomes of Artibeus jamaicenses (male: 2n = 31). g - Meiosis of Xyleus angulatus, showing
heteropycnosis of X chromosome during zygotene (left) but not in metaphase I (right). All photographs
were taken from permanent slides stained with 1% hematoxylin (except a). Bar in g represents 10 mm.

HCl hydrolysis greatly softens the cell wall (Fox, 1969),

allowing a better spread of cells and chromosomes; as a
result the cytoplasm becomes extremely transparent. On
the other hand, it often hinders observation of the
nucleolus. When the nucleolus must be observed, the
hydrolysis step should be tentatively suppressed or reduced
to as short a time as possible (see also Henderson and Lu,
1968). Nevertheless, in mitosis and meiosis of some plant
specimens fixed at room temperature for 20 h or longer,
the nucleolus was often well stained, even after 20 min of
hydrochloric hydrolysis (Figure 1c).
Wittmann (1965) observed that chromatin staining with
hematoxylin can be greatly harmed in the presence of HCl
vestiges. However, carefully washing the tissues after
hydrolysis overcomes this problem and guarantees a very
transparent cytoplasm. Henderson and Lu (1968) also
verified the advantages of hydrochloric hydrolysis (1 N at
60oC, 5 min) in place of the clearing agent chloral-hydrate
used in the technique of Wittmann. In the present work,
hydrolysis with 5 N HCl at room temperature was preferred
to the more conventional 1 N HCl at 60oC due to its
technical simplicity for routine work (see also Guerra,
1983). Moreover, optimal hydrolysis is not so readily
obtained using this latter hydrolysis as it is with 5 N HCl
(Fox, 1969).

For meiotic analysis of plants, floral buds must be

hydrolyzed for a very short time to avoid excessive
softening which makes anther dissection more difficult.
Meiotic chromosomes can be clearly documented (Figure
1c) and, because the stain does not react with the
microspore exine, pollen mitosis can also be observed
(Figure 1d,e). In some cases, a very good contrast between
cytoplasm and chromatin was obtained in meiocytes and
pollen grains stained with hematoxylin without hydrolysis,
but better results were obtained after hydrolysis. Kindiger
and Beckett (1985) suggested successively heating slides
to reduce the darkening of pollen grain cytoplasm stained
with 2% hematoxylin. Although heat is effective in reducing
cytoplasm staining, the final contrast remains poor, when
compared with hydrolyzed pollen.

For demonstration of mitotic and meiotic cycles in practical

classes, the procedures 1A, B and C are much simpler than
traditional techniques. They combine perfect reproducibility
of results with minimum laboratory conditions, dispensing
the use of liquid nitrogen or dry ice for coverslip removal
(procedure 2A) as well as heating the slide for contrast
enhancement. The disadvantage of procedure 1, in relation
to procedure 2, is less homogeneous tissue staining and the
distribution of chromosomes in different focal plains (Figure
1a,b). The latter may hinder photographic documentation,
except when slides are air dried and made permanent. A
general esthetical disadvantage of this staining is the brown
color obtained with hematoxylin, which is not as appealing
as the color obtained with most other dyes.

The sharp contrast observed among chromatin and

cytoplasm makes hematoxylin staining especially indicated
in certain special cases. In algae and bryophyte tissues,
hematoxylin stains the chromatin, especially the interphase
nuclei, more intensely than other dyes (see also Fujii and
Guerra, 1998). In the same way, fixations stored for 10
years or longer and field fixations maintained in inadequate
temperature over a long time, which tend to stain the
cytoplasm darkly, exhibited surprisingly better results with
hematoxylin than with other dyes.

The technique was also useful for sequential staining. In

prophase chromosomes of Citrus sinensis, for example, it
was possible to differentially stain the heterochromatin
associated to NOR using the fluorochromes CMA and DAPI,
destain the slide, restain it with hematoxylin and
demonstrate the association of the nucleolus with the NOR-
bearing chromosome. Bat chromosomes sequentially
stained with fluorochromes and hematoxylin also exhibited
quite good staining, even on old slides (Figure 1f).

Ross et al. (1996), using a similar staining solution, found

that by reducing the hydrolysis time it was possible to stain
not only the nucleolus but also the pericentromeric
heterochromatin of bivalents from Arabidopsis. In Geniates
borelli (Coleoptera), a similar heterochromatin
differentiation was observed after conventional staining
with hematoxylin (Edgar Bione, personal communication).
However, conventional stainings may sometimes produce
false heterochromatin patterns since it cannot distinguish
condensed euchromatin from heterochromatin (Guerra,
1988). Heterochromatin always seems to be better
differentiated by C-banding procedure plus Giemsa than by
any other method (see Guerra, 1983). In grasshopper
meiocytes, the facultative heterochromatin of X
chromosome, classically observed after aceto-carmin or
aceto-orcein staining, was generally not identifiable in
metaphase I after utilizing the present hematoxylin staining
methods (Figure 1g).

To make the slide permanent, simply washing it in distilled

water conserves the chromosome staining and contrast far
better than the alcohol series recommended in traditional
techniques (Darlington and LaCour, 1976). Slides made
permanent in this way have been maintained unaffected for
at least two years.

ACKNOWLEDGMENTS
The author is very grateful to Dr. Maria José Lopes for supplying cytological
material of bats and grasshoppers, Ana Emilia Barros and Silva for technical
assistance and to his students Leonardo Félix, Gianna Carvalheira and André
Vanzela for several tests and comments. This work was supported by CNPq, BNB
and FACEPE.

RESUMO
Uma mistura corante à base de hematoxilina e alúmem férrico, combinada com
uma hidrólise clorídrica forte, foi utilizada na observação de cromossomos de
diversos tipos de vegetais e alguns animais, sempre com bons resultados.
Procedimentos ligeiramente diferentes foram desenvolvidos para diferentes
finalidades e diferentes materiais. No caso das células vegetais, o aspecto técnico
mais importante foi o uso da hidrólise clorídrica, que torna o citoplasma muito
transparente, combinado com a coloração intensa e muito específica produzida pela
hematoxilina. A técnica é recomendada para análise citogenética em geral, sendo
especialmente indicada para aulas práticas, devido à simplicidade dos
procedimentos e à alta repetibilidade dos resultados. Além disso, devido ao maior
contraste obtido entre citoplasma e cromatina, ela se mostrou muito útil para a
coloração seqüencial de células previamente analisadas com outros corantes e para
materiais com problemas de fixação.

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(Received February 13, 1998)