ABSTRACT Haematoxylin and eosin (H&amp;E) is the most popular routine stain used in pathology laboratories for highlighting cellular structures. To study the effect of tap water &apos;softening&apos; (i.e. calcium extraction) on H&amp;E stains, 5 sets of slides from 30 different paraffin-embedded human pathologic tissue blocks were prepared in the same way except for washing with 5 different types of water. Slides washed in untreated tap water showed the best results concerning differentiation and colour intensity, while slides washed with softened or other treated water showed poorer degrees of differentiation and colour intensity. The worst results were obtained from slides washed with water containing sodium bicarbonate. Low calcium and magnesium ions and high sodium ions in soft water adversely affect the results of routine H&amp;E stain

M O Easa

R M Abu-Hazeem

S R Malkawi

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Eastern Mediterranean Health Journal, Vol. 9, Nos 5/6, 2003 1109
Effect of water softening on the
colour intensity of routine
haematoxylin and eosin stain
S.R. Malkawi,1 R.M. Abu-Hazeem1 and M.O. Easa1
 1Princess Iman Research and Laboratory Sciences Centre, King Hussein Medical Centre, Amman,
Jordan.
Received: 27/05/02; accepted: 21/04/03
ABSTRACT Haematoxylin and eosin (H&E) is the most popular routine stain used in pathology laboratories
for highlighting cellular structures. To study the effect of tap water ‘softening’ (i.e. calcium extraction) on H&E
stains, 5 sets of slides from 30 different paraffin-embedded human pathologic tissue blocks were prepared
in the same way except for washing with 5 different types of water. Slides washed in untreated tap water
showed the best results concerning differentiation and colour intensity, while slides washed with softened or
other treated water showed poorer degrees of differentiation and colour intensity. The worst results were
obtained from slides washed with water containing sodium bicarbonate. Low calcium and magnesium ions
and high sodium ions in soft water adversely affect the results of routine H&E stain.
Effet de l’adoucissement de l’eau sur l’intensité de la couleur de la coloration de routine à
l’hématoxyline-éosine
RESUME L’hématoxyline et l’éosine sont les colorations les plus couramment utilisées dans les laboratoires
de pathologie pour la mise en évidence des structures cellulaires. Afin d’étudier l’effet de l’adoucissement de
l’eau du robinet (extraction du calcium) sur les colorations à l’hématoxyline et à l’éosine, cinq (5) séries de
lames issues de 30 blocs de tissus pathologiques humains différents inclus en paraffine ont été préparées
de la même manière, sauf pour le lavage qui a été effectué avec cinq types d’eau différents. Les lames lavées
à l’eau du robinet non traitée ont montré les meilleurs résultats pour ce qui concerne la différenciation et
l’intensité de la couleur, tandis que les lames lavées à l’eau adoucie ou avec une autre eau traitée affichaient
des degrés plus faibles de différenciation et d’intensité de la couleur. Les plus mauvais résultats ont été
obtenus avec les lames lavées à l’eau contenant du bicarbonate de sodium. La teneur faible en ions calcium
et magnésium et élevée en ions sodium de l’eau douce affecte négativement les résultats de la coloration de
routine à l’hématoxyline-éosine.
27 Effect of water softening.pmd 3/3/2005, 12:17 AM1109
1110 La Revue de Santé de la Méditerranée orientale, Vol. 9, No 5/6, 2003
 Introduction
Haematoxylin and eosin (H&E) is the most
widely applied routine stain in the histopa-
thology laboratory because of its ability to
highlight tissue structures and because it is
an inexpensive, quick method that is readily
available as powders or ready-to-use solu-
tions.
Haematoxylin stain is extracted from
the logwood of a tropical tree, Haematoxy-
lon campechianum [1]. The nuclei of cells
are stained by its active product, haematin,
which is produced when an oxidizing agent
is added. Haematin by itself has poor affin-
ity to stain nuclei without combining with a
mordant, such as aluminium, iron or other
metallic salts. Ripened haematoxylin can
stain nuclei by electrostatic attraction be-
tween its positive-charged ions with the
negative-charged contents in the nucleus
such as phosphate groups in DNA and RNA
and carboxylated or sulfated mucosub-
stances [2,3].
Eosin is an anionic dye that is usually
used as a counterstain to haematoxylin. It
can stain cytoplasmic organelles by elec-
trostatic attraction with positive-charged
ions in tissues such as collagen or muscle.
Many factors affect the colour intensity
of a stain, including the type of mordant,
the duration of each step in the staining
process, tissue fixation methods, pH and
many others. One of these factors may be
the tap water used for washing slides dur-
ing processing. At the laboratories of the
King Hussein Medical Centre in Jordan, it
was noticed that H&E stained slides
washed with ‘softened’ (treated) water be-
came faint compared with those previously
washed with ‘hard’ (untreated) tap water.
This led us to make a systematic compari-
son of different types of treated tap water
on the colour intensity of slides and to in-
vestigate changes in the chemical content
of tap water before and after treatment.
Methods
A total of 30 different human pathology tis-
sue specimens embedded in paraffin
blocks, previously fixed in 10% buffered
formalin, were processed routinely for his-
topathology. The tissue specimens included
samples from the endometrium, appendix,
gall bladder, spleen and other gastric and
pleural tissues obtained by surgery, curet-
tage and biopsy.  Five sections (5 µm thick)
were prepared from each paraffin block
and placed on different slides. The slides
were divided into 5 groups. Each group
was stained routinely with H&E stain under
the same conditions except that different
waters were used for the washing steps:
• tap water (untreated),
• soft water (treated tap water),
• 1% magnesium sulfate (MgSO4) dis-
solved in soft water,
• 1% calcium carbonate (CaCO3) dis-
solved in soft water, or
• 1% sodium bicarbonate (NaHCO3) dis-
solved in soft water.
The following H&E staining procedure
was applied:
1. De-wax slides in xylene.
2. Hydrate sections by applying different
concentrates of ethyl alcohol (70%,
85%, 95% up to absolute alcohol).
3. Wash in water.
4. Stain in Coles haematoxylin stain for 8
min.
5. Wash in water for 20 s.
6. Differentiate in 1% HCl dissolved in
70% alcohol for 10 s.
7. Wash in water for 20 s.
27 Effect of water softening.pmd 3/3/2005, 12:17 AM1110
Eastern Mediterranean Health Journal, Vol. 9, Nos 5/6, 2003 1111
8. Blue in Scott’s tap water solution for 1
min.
9. Wash in water for 1 min.
10.Stain in 0.5% eosin yellowish for 3 min.
11. Wash in water for 30 s.
12.Dehydrate through different concen-
trates of alcohol, clear in xylene, and
permanent mount with distrene-plasti-
cizer-xylene (DPX) synthetic resin.
All stained slides were reviewed micro-
scopically and evaluated for adequacy of
H&E stain, taking into consideration the
degree of differentiation, background stain
and the overall intensity of colour in com-
parison with slides washed in untreated tap
water.
Finally, water samples were taken from
the Kind Hussein Medical Centre before
and after water softening and the chemical
composition was analysed by the Water
Authority of Jordan to measure specific
conductance, pH and the concentrations of
various ions and heavy metals.
Results
Slides washed in raw untreated tap water
showed good differentiation and good co-
lour intensity for both the blue colour of
haematoxylin and red colour of eosin. This
group of slides washed in tap water was
used as a reference of colour intensity and
differentiation.
Slides washed in soft (treated) tap wa-
ter, showed faint blue colour and very faint
to almost absence of reddish colour of
eosin with poor differentiation (Figure 1).
Slides washed in 1% MgSO4 dissolved in
soft water showed slightly faint blue co-
lour, faint red colour of eosin with moder-
ate differentiation (Figure 2). Slides
washed in 1% CaCO3 dissolved in soft wa-
ter showed slightly faint blue colour, and
very faint red colour of eosin with poor dif-
ferentiation. The worst results were ob-
tained from slides washed in sodium bicar-
bonate (1% NaHCO3 dissolved in soft
water). They showed very faint blue co-
lour, and very faint to almost absence of
red colour of eosin with poor differentia-
tion.
Figure 1 Stained slide from colon specimen
treated with soft water
Figure 2 Stained slide from appendix
specimen treated with magnesium sulfate in
soft water
27 Effect of water softening.pmd 3/3/2005, 12:17 AM1111
1112 La Revue de Santé de la Méditerranée orientale, Vol. 9, No 5/6, 2003
Table 1 shows the chemical composi-
tion of water samples taken from the King
Hussein Medical Centre before and after
water softening. The amount of sodium
ions increased after water softening from
1.65 mEq/L in tap water to 7.98 mEq/L in
soft water, and the pH of soft water be-
came slightly more alkaline (from 7.60 to
8.02).
Discussion
Tap water contains large amounts of ions
such as calcium, magnesium and many
other minerals that make it ‘hard’ [4]. As
calcium and magnesium cations are rela-
Table 1 Chemical composition of water samples taken from King
Hussein Medical Centre before and after water softening
Chemical analysed Before After
softening softening
Specific conductance (µS/cm) 814 850
pH (units) 7.60 8.02
Ions
Calcium by titration (mEq/L) 4.40 0.00
Magnesium by calculation (mEq/L) 2.18 0.02
Sodium by flame photometry (mEq/L) 1.65 7.98
Potassium by flame photometry (mEq/L) 0.07 0.01
Chloride by titration (mEq/L) 3.01 2.88
Sulfate by titration (mEq/L) 0.75 0.75
Carbonate by titration (mEq/L) 0.00 0.00
Bicarbonate by H2SO4 titration (mEq/L) 4.18 4.09
Nitrate by spectrophotometry (mg/L as NO3) 30.71 30.61
Heavy metals
Iron (mg/L) 0.05 0.03
Manganese (mg/L) 0.015 0.013
Copper (mg/L) < 0.01 < 0.01
Lead (mg/L) < 0.01 < 0.01
Chromium (mg/L) < 0.01 < 0.01
Cadmium (mg/L) < 0.003 < 0.01
Zinc (mg/L) 0.17 0.01
Nickel (mg/L) < 0.01 < 0.01
tively insoluble in water, they tend to pre-
cipitate out as calcium carbonate, causing
scale and deposit problems in heat ex-
change equipment, boilers and pipelines.
‘Hardness’ of water is the concentration of
calcium and magnesium ions expressed in
terms of calcium carbonate. The amount of
hardness in natural waters may vary from
60 mg/L to over 180 mg/L calcium carbon-
ate. The main purpose of water softening in
big centres, hospitals and modern houses is
to eliminate calcium and magnesium ions,
which are replaced by sodium ions.
The increase in sodium ions as well as
the slightly more alkaline pH of soft water
shown here may be responsible for the de-
27 Effect of water softening.pmd 3/3/2005, 12:17 AM1112
Eastern Mediterranean Health Journal, Vol. 9, Nos 5/6, 2003 1113
terioration in the colour intensity of H&E
stain, mainly in the eosin red stain which
became faint or almost absent. It is highly
recommended to use raw untreated tap
water for washing steps in the routine H&E
stain and consideration should be given to
the effect of soft water on other stains.
Acknowledgements
We would like to thank Miss N. Abu-
Hazeem and engineer A-A. Al-Ajarmeh for
their support. The technical support of the
Water Authority of Jordan is highly appre-
ciated.
References
1. Stevens A. The haematoxylins. In:
Bancroft JD, Stevens A, eds. Theory and
practice of histological techniques.
Edinburgh, Churchill Livingstone, 1982:
109–21.
2. Baker JR, ed. Principles of biological mi-
crotechnique. London, Methuen, 1958:
238–41.
3. Singer M. Staining of tissue sections with
acid and basic dyes. International re-
views of cytology, 1952, 1:211–55.
4. Water sources and impurities. In: Hand-
book of industrial water conditioning, 8th
ed. Trevose, Pennsylvania, USA, Betz
Laboratories, 1980:10–3.
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Effect of water softening on the colour intensity of routine haematoxylin and eosin stain

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Effect of water softening on the colour intensity of routine haematoxylin and eosin stain

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