Utilization of 1% of Methylene Blue in Staining Histopathological
Preparations at Anatomic Pathology Laboratory
Tri Rahmawati1, Yadi Apriyadi2, Mamay1
1

Department of Medical Laboratory
Technology, STIKes Karsa Husada,
Garut, Indonesia
2

Departement Pathology of
Anatomi, Regional public hospital
Dr. Slamet, Garut, Indonesia
Correspondence:
Mamay,
Jl. Nusa Indah No.1, Jayaraga, Kec.
Tarogong Kidul, Kabupaten Garut
Zip Code : 44151
Email: mamay.1745@gmail.com
Received: May 31, 2020
Revised: June 19, 2020
Accepted: August 25, 2020

Abstract
Tissue staining using hematoxylin-eosin (HE) is a standard
method of histopathological staining. The tissue staining
is hampered when there is no hematoxylin reagent in
laboratory. Therefore, other reagents are needed that can
replace the use of hematoxylin. Methylene blue is a basic
dyes that interact with cell nuclei which has a negative ionic
charge of the tissue. It can be used as an alternative nuclei
staining. This study aims to evaluate the use of 1% of
methylene blue in cell nuclei staining in histopathological
preparations. The research sample were 15 pathology
preparations which were randomly selected including breast
cancer, cervical cancer and ovarian cancer in the bank of
sampel at anatomical pathology laboratory of RSUD Dr.
Slamet Garut, Indonesia. The experiment showed that the
methylene blue dyes yielded “worth” result (40%) and
“poorly” result (60%). Further research can be carried out
by modifying the pH of 1% of methylene blue reagent so
that it can maximize the staining preparations results as good
as those using hematoxylin.
Keywords
Cancer, hematoxylin, histopathology, methylene blue

prevalence of tumors or cancer in Indonesia

INTRODUCTION
Cancer prevalence is increasing in the

shows an increase from 1.4 per thousand

the

populations in 2013 to 1.7 per thousand

International Agency for Research on Cancer

populations in 2018 (1). In Garut Regency,

(IARC), the number of cancer patients

especially

worldwide increases since 2012. More

laboratory of RSUD dr. Slamet Garut, there

than14 million new cases of cancer, of which

were 189 cancer patients (44.9% of breast

8 million or more cases, died from cancer.

cancer patients, 10.5% of cervical cancer,

Based on the Ministry of Health of the

8.9% of lymph node cancer, 7.4% of colon

Republic of Indonesia data in 2018, the

cancer, 5.8% of ovarian cancer, 4.7% of

world

every

year.

According

to

in

the

anatomic

pathology

93

Tri Rahmawati, et al.

thyroid cancer, 3.7% of bone cancer, 2.1% of

more easily colored into pink with an acidic

prostate cancer, 1.05% of parotid cancer

staining called acidophilic using a substance

(salivary glands), 1.05% of molar pregnancy,

eosin color (7).

1.05% of cancer of body fluids, and other
types of cancers which count up to 9.5%).

laboratory are often hampered when the

Precancerous and cancer is characterized

hematoxylin reagent runs out and it takes a

by excessive cell proliferation due to the

long time for the reagent to arrive since the

increase in mitotic cells (2). The increased

availability of hematoxylin is quite rare.

and abnormal mitosis indicates a damaged

Thus, the process of hematoxylin maturation

tissue of an important feature in precancerous

requires a long time (8). Nevertheless, the

and cancer tests. The identification and

examination

service

quantification of mitotic cells is used in

pathology

laboratory

histological examination to support the

continuously. Therefore, other reagents are

diagnosis (3). Histological staining has been

needed that

widely used in pathology (4). Examination of

hematoxylin

the development of cancer can be conducted

preparations staining.

microscopically

through

histopathology

examination.

94

The services in the anatomic pathology

in

the

must

can replace
in

anatomic
be

the

done

use

of

histopathological

The other reagents that can be applied for
staining are crystals violet (2, 3, 9), toluidine

Important stages of histopathological

blue, neutral red, methylene green, and

examination starts from fixation, processing,

methylene blue (4, 10, 11). Methylene blue is

embedding, cutting and staining (5). Tissue

a cationic dye that has a positive ionic charge

staining using the hematoxylin-eosin (HE) is

so that it will interact with cell nuclei that

a standard method of histopathological

have a negative ionic charge of the tissue (4)

staining. Hematoxylin is a basic dye that is

Löffler's alkaline of methylene blue is used in

commonly used for staining cell nuclei and

histopathologically conjunctival epithelial

provides a bluish tint while eosin yields a

cells and blue-colored neutrophils and more

pink dye (2). The principle of such coloration

intense colored nuclei (12). Previous stain

is chemical interactions binding between

with 1% of methylene blue detect oral

tissue and dyes (4). The tissues with negative

dysplasia and carcinoma resulted in good

or anionic charges are more easily stained

validity (11). However, the utilization of 1%

with a blackish-blue color with an alkaline

of methylene blue for histopathological

staining called basophilic using hematoxylin

staining cancer mitosis has not been reported.

dyes. Meanwhile, tissue components with a

Therefore,

positive or cationic charge (cytoplasm) are

methylene blue compound in the process of

the

author

aims

to

apply

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Tri Rahmawati, et al.

histopathological preparations as a substitute

70oC for 45 minutes, starting from 70%, 80%

for hematoxylin in the hematoxylin-eosin

and 95%. The clearing step used xylol I and

staining.

xylol II, respectively, for 45 minutes in the
oven with a temperature of 65oC–70oC. The

MATERIALS AND METHODS
The tools used in this study were
microtome knife, pencil, camera, tissue, label
paper, oven, cover glass, object-glass, beaker
glass, microscope, microtome, water bath,
cassette, tweezers, mold, refrigerator, pipette,
and staining jar. The materials used include
tissue cuts, 10% of buffered formalin,
absolute alcohol, 96% of alcohol, 80% of
alcohol, 70% of alcohol, xylol, paraffin,
lithium carbonate, Mayer hematoxylin, eosin,
blue methylene, tap water, distilled water,

This research was conducted in the
sitohistotechnology laboratory from July to
August 2019. Sample were obtained from 15
pathology preparations and were randomly
selected, including breast cancer, cervical
cancer and ovarian cancer in the bank of
sampel at anatomical pathology laboratory of
RSUD Dr. Slamet Garut, Indonesia. The
quality assessment of the preparation is
carried out by an anatomist pathologist.
The sample was fixed in the second stage
with a 10% of formalin buffer solution for 24
hours. The tissue preparation consisted of
stages,

minutes in an oven with a temperature of
65oC–70oC. Furthermore, the embedding step
was done to plant the tissue into paraffin
molds then it was stored in the refrigerator for
2 hours until it was solidified completely.
Lastly, a gross cut with a thickness of 4
microns was done to obtain a network band.
The tissue tape was affixed to the glass object
and was followed by mounting in a water
bath with a temperature of 40oC to obtain
histopathological preparations that were
ready to be coloured.

and entelan.

several

impregnation step used liquid paraffin for 45

including

dehydration,

clearing, impregnation, embedding and tissue
alcohol in an oven at a temperature of 65 oC–
Ina J Med Lab Sci Tech 2020; 2(1): 93-100

control group used the Mayer hematoxylineosin staining method while the experimental
group used methylene blue-eosin staining.
The blue methylene used 1% of distilled
water. The results of staining was analysed
under a microscope with a magnification of
400x. An optimal-quality staining as nuclei
exhibiting

‘blue

hematoxylin’

with

chromatin patterns, permit the differentiation
of distinct cell type (13). A sub-optimal
staining, the nuclei were weakly stained and
appear pale (14). The data were categorized
based on the quality of the stained nuclei
(Chapman category modified), it is classified
as “good” if the nucleus was clearly stained
and the chromatin was clearly visible;

95

cutting. The dehydration process used

Histopathological preparations for the

Tri Rahmawati, et al.

“worth” if the nucleus was stained but less

were three lobes in one cell), as shown in

clear and the chromatin was lacking clearly

figure 1 (a). The results of staining the cell

visible, and it was classified as “poorly” if the

nucleus with methylene blue-eosin in a breast

cell nucleus is not colored.

cancer preparation showed poor staining
quality that was characterized by colorless
cell nuclei, mitotic cell nuclei and chromatin

RESULTS
The quality of staining cell nuclei using
hematoxylin-eosin in five breast cancer
preparations has good quality (the cell nuclei
were visible with coarse chromatin). The
results of the staining showed the presence of
mitotic cell nuclei (observed cell nuclei that

a

b

were not observed, as shown in Figure 1 (b).
Meanwhile,

the

four

breast

cancer

preparations have a pretty good staining
quality, the cell nucleus was colored but it
was not seen and the chromatin was less
clear, as shown in figure 1 (c).

c

Fig 1. Staining cell nuclei in breast cancer preparations with 400x magnification. (a)
Hematoxylin, (b–c) methylene blue

good quality. The cell nucleus was stained

hematoxylin-eosin in four cervical cancer

but it was not seen and chromatin was less

preparations showed good staining quality

clear so that there should be a cell nucleus, as

with clear observations of cell nuclei. The

in figure 2 (b). Meanwhile, the four cervical

appearance of mitotic cell nuclei is shown in

cancer preparations showed poor quality

figure 2 (a). The results of cell nucleus

colour which means that the cell nucleus was

staining with methylene blue-eosin in a

not stained and chromatin was not observed,

cervical cancer preparation showed a quite

as shown in Figure 2 (c).

96

The quality of staining of cell nuclei with

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Tri Rahmawati, et al.

a

b

c

Fig 2. Staining cell nuclei in cervical cancer preparations with 400x magnification. (a)
Hematoxylin, (b–c) methylene blue
The results of cell nucleus staining with

quality of staining. It means that the cell

hematoxylin-eosin in five ovarian cancer

nucleus was colored but it was not seen, it

preparations showed good staining quality,

only looked like a small dot from the

dye the cell nucleus was stained and observed

cytoplasmic outline so that it could not be

with coarse chromatin as shown in Figure 3

seen, as well as the cell nucleus and

(a). The results of nucleus cell staining with

chromatin nuclei were also not observed, as

methylene blue-eosin in four ovarian cancer

shown in Figure 3 (c).

preparations showed poor quality meaning

The results of the breast cancer, cervical

that the cell nucleus was not stained with

and ovarian cancer staining preparations

chromatin which was not observed, as shown

using

in Figure 3 (b). Meanwhile, the four ovarian

methylene blue-eosin in the control group can

cancer preparations showed quite good

be seen in Table 2.

a

b

hematoxylin-eosin

method

c

97

Fig 3. Staining cell nuclei in ovarian cancer preparations with 400x magnification. (a)
Hematoxylin, (b-c) methylene blue

Ina J Med Lab Sci Tech 2020; 2(1): 93-100

and

Tri Rahmawati, et al.

Table 2. Results of staining cell nuclei of breast, cervical and ovarian cancer
Cancer
Quantity (Percentage%)
Dye
preparation
good
worth
Breast
5 (100%)
0
Hematoxyilin
Servical
4 (80%)
1 (20%)
Ovarian
5 (20%)
0
Total
14 (93%)
1(7%)
Breast
0
4 (80%)
Methylen blue
Servical
0
1 (20%)
Ovarian
0
1 (20%)
Total
0
6 (40%)

98

DISCUSSION

poorly
0
0
0
0
1 (20%)
4 (80%)
4 (80%)
9 (60%)

histopathological preparations resulted in

Methylene blue is a cationic dye which is

poor quality cell nuclei with as much as 60%

also known as basic dyes. It has a positive

of the cell nucleus was not successfully

ionic charge so it will interact with cell nuclei

stained. While, the other experiment has a

that have a negative ionic charge of the tissue

pretty good staining quality (40%) where the

(4). This dye can react with anionic groups as

nucleus cells were colored but the mitosis

phosphate groups of nucleic acids (DNA and

could not be seen from the cell nucleus itself.

RNA) which is commonly used as nuclear

Meanwhile, by using hematoxylin-eosin

stains. It is a basophilic dye which will bind

staining, 93% of the preparations were well

to acidic tissue. This relates to the concept of

colored and 7% were quite well colored. The

tissue staining, where acidic tissue is more

nucleus staining in the control group

easily stained with base or basic dyes (7).

underwent mitosis with three lobes, whereas

Bonding staining to tissue is not different

in the cell nucleus experiment, the shape was

from chemical bonds and the mechanism is

visible from the methylene blue staining. It

similar in other organic bonding components.

was also visible from outside the cytoplasm

The bonding of tissue coloring involves ion

so that there should be a cell nucleus division

interactions. It is a combined form and it can

with three lobes yet it was only visible in one

bind to tissues and dyes as long as two

lobe. The extracellular pH environment has

different ion poles interact properly (4).

more acidic pH than normal cells (the pH of

1% of methylene blue dye in previous

normal cells was 7.4 and pH of of tumor cells

research can detect the dysplasia and oral

was 6.5) (15). The charge variation of the

carcinoma. The validity of methylene blue to

breast cancer cells and fibroblasts was good

detect the dysplasia and carcinoma was

at pH of 2.5–9 (16). Therefore, the interaction

increase (11). In this study, 1% of methylene

between methylene blue dye is greater with

blue dyes in coloring the cell nucleus from

breast cancer tissue, so that the quality of the
Ina J Med Lab Sci Tech 2020; 2(1): 93-100

Tri Rahmawati, et al.

staining is better compared to cervical cancer

which used potassium hydroxide to increase

and ovarian cancer.

the pH to color all protein components and

The lack of staining quality by using
methylene

blue

is

affected

by

the

nucleic acids. Ultimately, the tissue will
appear blue (12).

composition of the dye and the acidity or pH
which

is

different

from

CONCLUSIONS

hematoxylin. Hematoxylin is mixed with

Staining

preparation

with

1%

of

potassium or ammonium alum, sodium

methylene blue can color the cell nucleus

iodate, and chloral hydrate (8). Meanwhile,

quite good. From the results of this study, it

methylene blue was not added with other

is necessary to modify the pH of the 1% of

strong alkaline materials to increase the pH.

methylene blue reagent in order to maximize

In acidic condition, the pH of methylene blue

the staining preparations to get good results

will produce a different color that will color

as well as those using hematoxylin.

the cell nuclei but the protein remains
colorless. Cell nuclei that are not successfully

ACKNOWLADGEMENTS

stained by methylene blue are also caused by
changing in pH because the ionic bonds in

The source of funding for this research
came from researchers' personal funds.

dyes are very sensitive to pH. To be conclude,
there was no interaction between tissue ions

CONFLICT OF INTEREST

and dye ions. Based on previous research,

There are no conflicts of interest.

maximum coloration obtained by methylene
blue with loffer formula produced best result,

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