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Genet. Mol. Biol. vol.22 n.1 So Paulo Mar. 1999

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Hematoxylin: a simple, multiple-use

dye for chromosome analysis

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Marcelo Guerra
Departamento de Botnica, Centro de Cincias Biolgicas, Universidade
Federal de Pernambuco, Cidade Universitria, 50670-420 Recife, PE, Brasil.

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ABSTRACT

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A staining mixture of hematoxylin-iron alum combined with a

strong hydrochloric hydrolysis was successfully applied for
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chromosome observation of several kinds of plants and some
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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,
it is especially indicated for practical classes, due to its simplicity and high reproducibility
results. Moreover, the deep contrast observed makes this technique very useful for seque
staining of cells previously analyzed with other stains, as well as for materials with fixatio
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 mo

L. Taits, as early as 1875, drew attention to hematoxylin's capacity for preferentially staining the nucleus (
1929).

Wittmann (1962, 1965) demonstrated that an aceto-iron-hematoxylin solution produced excellent results f
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 chl
hydrate, made the technique seem more complicated and had less uniform results. Henderson and Lu (196
tried to simplify the technique, working with a stock solution of 2% hematoxylin and 0.5% iron alum, both
propionic acid. The results, however, vary depending on the ripening time of hematoxylin and the proportio
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 t
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 cytogenetic
mainly with plant species. Procedures for sequential staining of animal cells using other techniques are als
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 angiosperm
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 w
glass stick and left in a dark flask for one week. After that period, the mixture was filtered and kept in a da
glass. The solution could be used immediately or stored for an undeterminate period in the refrigerator. To
the best dye concentration, 2, 1, 0.5 and 0.2% solutions were prepared by direct dilution of the stock solu
(4%). The 1% hematoxylin was also prepared directly from 1 g hematoxylin plus 0.25 g iron alum in 100 m
45% acetic acid.
Methods

Two basic methods for slide preparation were tested in different organisms. In both cases, the tissues were
hydrolyzed in 5 N HCl and washed in distilled water. In the first method, the material was previously staine
with hematoxylin and then squashed under a coverslip. In the second method, the material was first squas
in 45% acetic acid and then stained with hematoxylin. The steps followed in the procedures, and described
detail below, were based on Guerra (1983) and Fujii and Guerra (1998). All photomicrographs were taken
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, followe
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 o
three drops of stain (polyethylene contact lens containers proved to be the most practical and safest tool).
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 term

meristem was dissected and fragmented into very small pieces. They were covered with a coverslip, tappe
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 th
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
possible in order to squeeze the meiocytes out. The anther wall debris were removed, the material was cov
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 tempera
- 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, squash
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
1B.
- The unstained material was macerated in a drop of 45% acetic acid and squashed. The coverslip was rem
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. Sta
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 Ente
or similar media.
- For meiotic analysis, the same procedure was followed, simply reducing the time of hydrolysis to between
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 success
washes in xylol.
- The slide was destained and air dried. Most dyes can be removed by washing the slide in 45% acetic acid
ethanol-acetic acid, or both alternately.
- The material was restained with hematoxylin as described in procedure 2A. If the cytoplasm becomes too
the slide can be simultaneously destained and hydrolyzed in 5 N HCl for 5 min and restained with hematox
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 ju
slightly stained cytoplasm. Tests with different hematoxylin concentrations showed that although the highe
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 in
the tissues slower, especially in anther tissues. Concentration of 0.5% was still efficient, while that of 0.2%
more heterogeneous, with many weakly stained areas. The ideal concentration was 1% (Figure 1b-g
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
represents 10 mm.

HCl hydrolysis greatly softens the cell wall (Fox, 1969), allowing a better spread of cells and chromosomes
result the cytoplasm becomes extremely transparent. On the other hand, it often hinders observation of th

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 me
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 presenc
HCl vestiges. However, carefully washing the tissues after hydrolysis overcomes this problem and guarante
very transparent cytoplasm. Henderson and Lu (1968) also verified the advantages of hydrochloric hydroly
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 HC
60oC due to its technical simplicity for routine work (see also Guerra, 1983). Moreover, optimal hydrolysis
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 softe
which makes anther dissection more difficult. Meiotic chromosomes can be clearly documented (
because the stain does not react with the microspore exine, pollen mitosis can also be observed (
In some cases, a very good contrast between cytoplasm and chromatin was obtained in meiocytes and pol
grains stained with hematoxylin without hydrolysis, but better results were obtained after hydrolysis. Kindi
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 laborato
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
homogeneous tissue staining and the distribution of chromosomes in different focal plains (Figure 1a,b
latter may hinder photographic documentation, except when slides are air dried and made permanent. A g
esthetical disadvantage of this staining is the brown color obtained with hematoxylin, which is not as appe
as the color obtained with most other dyes.

The sharp contrast observed among chromatin and cytoplasm makes hematoxylin staining especially indica
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, fixa
stored for 10 years or longer and field fixations maintained in inadequate temperature over a long time, w
tend to stain the cytoplasm darkly, exhibited surprisingly better results with hematoxylin than with other d

The technique was also useful for sequential staining. In prophase chromosomes of Citrus sinensis
it was possible to differentially stain the heterochromatin associated to NOR using the fluorochromes CMA
DAPI, destain the slide, restain it with hematoxylin and demonstrate the association of the nucleolus with t
NOR-bearing chromosome. Bat chromosomes sequentially stained with fluorochromes and hematoxylin als
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 poss
stain not only the nucleolus but also the pericentromeric heterochromatin of bivalents from Arabidopsis
Geniates borelli (Coleoptera), a similar heterochromatin differentiation was observed after conventional sta
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 facu
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 (
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,
Slides made permanent in this way have been maintained unaffected for at least two years.

ACKNOWLEDGMENTS

The author is very grateful to Dr. Maria Jos Lopes for supplying cytological material of b
and grasshoppers, Ana Emilia Barros and Silva for technical assistance and to his studen
Leonardo Flix, Gianna Carvalheira and Andr Vanzela for several tests and comments. T
work was supported by CNPq, BNB and FACEPE.

RESUMO

Uma mistura corante base de hematoxilina e almem frrico, combinada com uma hidr
clordrica forte, foi utilizada na observao de cromossomos de diversos tipos de vegetais
alguns animais, sempre com bons resultados. Procedimentos ligeiramente diferentes fora
desenvolvidos para diferentes finalidades e diferentes materiais. No caso das clulas veg
o aspecto tcnico mais importante foi o uso da hidrlise clordrica, que torna o citoplasm
muito transparente, combinado com a colorao intensa e muito especfica produzida pel
hematoxilina. A tcnica recomendada para anlise citogentica em geral, sendo
especialmente indicada para aulas prticas, devido simplicidade dos procedimentos e
repetibilidade dos resultados. Alm disso, devido ao maior contraste obtido entre citoplas
e cromatina, ela se mostrou muito til para a colorao seqencial de clulas previament
analisadas com outros corantes e para materiais com problemas de fixao.

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(Received February 13, 1998)

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