Purple Sweet Potato (Ipomoea batatas L.) Peels Extract as an
Alternative Dye for Bacteria Gram Staining
Nastasya Nunki1, Diah Titik Mutiarawati2, Endah Prayekti1
1

Department of Medical Laboratory
Technology, Faculty of Health,
Universitas Nahdlatul Ulama
Surabaya, Surabaya, Indonesia
2

Department of Medical Laboratory
Technology, Poltekkes Surabaya,
Surabaya, Indonesia
Correspondence:
Nastasya Nunki,
Jl. Jemursari No. 51-57, Surabaya,
East Java, Indonesia
Zip Code : 60237
Email:
nastasya.nk14@student.unusa.ac.id
Received: July 12, 2020
Revised: July 22, 2020
Accepted: August 25, 2020

Abstract
Crystal violet and Safranin are dyes in Gram staining, which
are carcinogenic. Alternative safe materials are needed to
minimize the use of carcinogenic properties. Purple sweet
potato (Ipomoea batatas L.) peels were the candidate of the
alternative dye source because of its high anthocyanin
pigment. The purpose of this study was to determine purple
sweet potato (Ipomoea batatas L.) peels extract as an
alternative to Gentian violet in Gram staining of bacteria.
Extracts obtained from purple sweet potato peels studied
with varying concentrations of 50%, 60%, and 75% for 1, 3,
and 5 min as a substitute for Gentian violet on Bacillus sp.
The parameters observed from this study based on visual
field clarity, glass slide cleanliness, contrast, bacterial shape,
bacterial colour. Each extract concentration compared with
a control group using Gentian violet. The results showed
that optimum staining in 50% concentration for 5 min, 60%
concentration for 5 min, 75% concentration for 3 min, and 5
min respectively. The present study exhibited the potency
of Ipomoea batatas L. peels extract as an alternative
staining agent.
Keywords
Ipomoea batatas L., Gentian violet, bacteria Gram taining,
Bacillus sp.

INTRODUCTION
The microorganism is a living organism

intends to facilitate and assist in the
examination service in the laboratory (1).

that is very small in size, so microscopic

Bacteria have a strong cell wall as an

observation aids are needed. Microorganisms

outer part and maintain the shape of bacteria

studied in the laboratory for various

and protect against osmotic pressure. In

purposes. The study of these microorganisms

staining, there are variations in the structure
of cell walls. Where the complex structure,
semi-rigid with bacterial cell wall thickness
76

Nastasya Nunki, et al.

ranges from 10 to 35nm and surrounds the

reaction

is

related

to

morphological

cytoplasmic membrane, serves to give shape

characteristics in phylogenetic-related forms

to the cell and protect the contents of the cell

(4). Synthetic dyes are including Crystal

from outside cell influences (1).

violet, Safranin, Carbol

fuchsin, are

dyed

Bacterial cell walls are essentials for

commonly used in Gram staining. Crystal

growth and division. Based on the chemical

violet and Safranin are dyes that often used

composition of the cell wall, which causes

in Gram staining. Crystal violet is a human

cell wall rigidity is peptidoglycan. The

carcinogen (5).

mechanism of Gram staining based on the

To minimize the use of carcinogenic

structure and composition of the cell wall (2).

properties for gram staining, the research to

In the laboratory world, especially in the

find alternative materials are needed. To get

field of staining, microbiology is one of the

natural dyes that can be used as alternative

keys to helping to provide information about

dyes, extraction methods are needed to get

the diagnosis of a disease. The existence of

the best pigment (anthocyanin) from the

the development of staining procedures to

colour of these natural ingredients, thereby

assist in roughly observing the morphology

maximizing the quality and quantity. Purple

of microorganisms helps in identifying parts

sweet potato peel has an average anthocyanin

of the cell structure of microorganisms and

level of 521.84–729.74 mg / 100g (6). Thus,

helps differentiate similar microorganisms

in this research conducted further testing,

(1).

using peel extracts of purple sweet potatoes
The

importance

of

observing

the

(Ipomoea batatas L.).

morphology of microorganisms, where the

In the previous study (7), purple sweet

appearance of microorganisms in living

potato extract can be obtained manually and

conditions is quite complex, not only because

can produce reddish-purple colour. For this

of their size but also because they are

reason, it needs to develop research to

transparent and colourless when suspended in

maximize the content of dyes (anthocyanin)

a liquid medium. To study the properties and

in the purple sweet potatoes peels using

divide microorganisms into specific groups

extraction techniques MAE (microwave-

for diagnostic purposes, the biological dyes

assisted extraction) method. The previous

and staining procedures with a light

study only examined the coccus Gram-

microscope have become the main tools in

positive group without being compared with

microbiology (3).

other groups.
In this study, gram staining of Bacillus sp

methods used are Gram staining. The Gram

tested and also the optimum concentration

Ina J Med Lab Sci Tech 2020; 2(2): 76-84

77

The most basic and primary staining

Nastasya Nunki, et al.

and time of gram staining were studied. The

(anthocyanin) in the purple sweet potatoes

purpose of this study was to determine purple

peels.

sweet potato (Ipomoea batatas L.) peels

In sample preparation, the sample

extract as an alternative to Gentian violet in

extracted using the MAE. The extraction

Gram staining of bacteria.

process carried out with a concentration
variation of 50%, 60%, 75%, which approved
with a combination of staining time of 1

MATERIALS AND METHODS
The materials used in this study were the

minute, 3 minutes, and 5 minutes.
At

pure culture of Bacillus sp. obtained from

concentration

of Ipomoea

Central health laboratory (Balai Besar

batatas L. peel extract, a formulation was

Laboratorium

in

performed to obtain the appropriate color.

Surabaya, NaCl 0.85% (0,85 gr NaCl [SAP

50% concentration obtained by adding

chemicals]/100 mL distilled water), Gram

(extract 0.5 gr/10 mL ethanol 96%, 4mL HCl

stain

oil

2N, 3mL NH4OH 2 N), 60% concentration

from Ipomoea

obtained by adding (extract 0.6 gr/10 mL

batatas L. peel extract, ethanol 96% (SAP

ethanol 96%, 5 mL HCl 2N, 3.5 mL NH4OH

chemicals), HCl 1N (8.3 mL HCl 37% (SAP

2 N), a concentration of 75% was obtained by

chemicals)/100 mL distilled water), HCl 2N

adding (extract 0.75 gr/10 mL ethanol 96%,

(16.6 mL HCl 37% (SAP chemicals)/100 mL,

6 mL HCl 2N, 4.2 mL NH4OH 2N).

Kesehatan/BBLK)

hucker

method,

(Olympus),

staining

immersion

The

NH4OH 1N (16 mL HCl 37% (Merck)/100

smear

prepared

from

mL distilled water), NH4OH 2N (32 mL HCl

culture Bacillus sp. on a clean glass slide

37% (Merck) 100 mL distilled water).

allowed it to air-dried and fixed it by

The sample used was the peels of purple

flaming.
For the experimental group, at first, the

sweet potato (Ipomoea batatas L.) not other
types of sweet potato. This study consisted of

smear

several stages. The first stage was the process

with Ipomoea batatas L. extracts at each

of

on

a

glass

slide

was

added

using

MAE

concentration for 1 minute, 3 minutes and 5

extraction)

method

minutes. The Ipomoea batatas L. extract was

of

the

poured off and applied lugol for 1 minute and

concentration formula, the staining process,

washed with water, the smear decolourized

observation under a microscope, and lastly

with an organic solvent-alcohol for 30

data analysis. Extraction techniques MAE

seconds, or until the colour was oozed from

(microwave-assisted extraction) method was

the

sample

preparation

(microwave-assisted
extraction

78

each

(8),

determination

slide

and

washed

with

water.

selected to maximize the content of dyes
Ina J Med Lab Sci Tech 2020; 2(2): 76-84

Nastasya Nunki, et al.

The Safranin applied for 1-minute washed

RESULTS

with water and blot dried.

Ipomoea

The

slide

was

observed

under

batatas

L.

Peel

Extract

Concentration of 50%, 60%, 75%

magnification 100x after putting a drop of

Based on observations obtained by the

immersion oil then the results were compared

results of colouring where the extraction

with staining using Gentian violet for 1

concentration of 50% showed the best results

minute. For the control group, the first step

when staining for 5 minutes, the extract was

did not use extracts but Gentian violet and the

inadequate to penetrate bacteria, and some

next steps as in the experimental group until

bacteria were not completely stained. The

the observation was processed.

extraction concentration of 60% showed the

The results were compared directly

best results when staining for 5 minutes

between the Gram staining using Ipomoea

(Figure 1). The dye appears to penetrate the

batatas L. peel extract tested on Gram-

bacterial cell wall, but some bacteria were not

positive bacteria as an experimental group.

completely

Furthermore, in Gram staining using Gentian

concentration of 75% showed the best results

violet as a control group. The sampling

when staining for 3 minutes and 5 minutes,

technique in this study was a simple random

although some bacteria were not stained

sampling. The parameters observed in this

(Figure 2). In the Gentian violet stain, visible

study included visual field clarity, glass slide

dyes penetrate the bacterial cell wall and also

cleanliness, contrast, bacterial shape, and

the spores of Bacillus sp. (Figure 3).

bacterial colour.

stained.

The

extraction

At the percentage of the staining results,

In the data analysis, the observation was

at the extraction of 50% concentrations

results of each extract concentration of 50%,

obtained staining approaching control with

60%, and 75% with a staining time of 1

the results of the staining showed the

minute, 3 minutes, and 5 minutes compared

optimum results when staining for 5 minutes.

to the control, coding was carried out based

For extraction of a concentration of 60%, it

on 5 observation parameters to obtain

obtained staining that approached the control

quantitative data. Then, the calculation

with the results of the staining showed the

carried out with the formula for the total

optimum results when staining for 5 minutes.

number of coding values with 3 times

The extraction concentration of 75% attained

repetition / 15 (5 parameters repeated 3

a colour approaching the control with the

times) x 100%.

results of the staining showed the optimum

minutes (Figure 4).
Ina J Med Lab Sci Tech 2020; 2(2): 76-84

79

results when staining for 3 minutes and 5

Nastasya Nunki, et al.

a

b

Fig 1. Microscopic observation results of Bacillus sp. 100x magnification with Staining
of Ipomoea batatas L. for 5 minutes. (a) Extract Concentration of 50%, (b) Extract
Concentration of 60%

a

b

80

Fig 2. Microscopic observation results of Bacillus sp. 100x magnification with Staining
of Ipomoea batatas L. Extract Concentration of 75%. (a) Stainding for 3 minutes, (b)
Staining for 5 minutes

Fig 3. Microscopic Observation Results of Bacillus sp. 100x magnification with Gentian
violet for 1 minute

Ina J Med Lab Sci Tech 2020; 2(2): 76-84

Nastasya Nunki, et al.

Time of Staining

100%
80%
60%

80%

68%

68% 68%

50

60

80%

86%

68%

75

Control

Extract Concentration
Time of Staining 1 minute

Time of Staining 3 minute

Time of Staining 5 minute

Fig 4. Comparison Percentage of Staining Results of Ipomoea batatas L. Extract
Concentration and Time of Staining of Gentian violet
The results analyzed statistically using

no significant difference between the results

the one-way ANOVA test with SPSS

of staining using Ipomoea batatas L. extracts

instrument

and

version

16.0.

Where

the

control

(Gentian

violet).

The

concentration of 50% obtained α value of

determination of colour tendency was using

0.06, a concentration of 60% obtained α value

the colour matching with the RGB table, then

of 0.227, a concentration of 75% obtained α

compared with the control (Table 1).

value of 0.297, α value of >0.05. There was
Table 1.

RGB Code for Bacterial Gram Staining (RGB Color Chart.cdr - AWS)
RGB Code for Bacterial Gram Staining
Time of
Bacteria
Staining
Concentration of Staining
Control
(minute)
50%
60%
75%
Bacillus sp.
1
RGB: 180 RGB: 173 RGB: 180
RGB: 70
4 133
14 145
4 133
20 111

Color
Name
Purple

–
153 0 122
3

RGB: 173
14 145

RGB: 152
4 110

RGB: 153
0 122

-

5

RGB: 152
4 110

RGB: 180
4 133

RGB: 141
4 123

-

Ina J Med Lab Sci Tech 2020; 2(2): 76-84

81

–
153 0 122

Nastasya Nunki, et al.

Phenolic

DISCUSSION
The extraction concentration of 50%

batatas L. peel

which

of Ipomoea

included

in

the

showed the best results when staining for 5

flavonoid group and the content of phenol

minutes. The extract was inadequate to

compounds which is Ipomoea batatas L. peel

penetrate bacteria, and some bacteria were

is

not completely stained. The extraction

content. Gentian violet is a triaryl methane

concentration of 60% showed the best results

dye with three phenol groups arranged by

when staining for 5 minutes. The dye appears

methane groups. The composition contained

to penetrate the bacterial cell wall, but some

in Gentian violet is (2 gr crystal violet, 20 mL

bacteria were not completely stained. The

ethanol 95%, 0.8 gr ammonium oxalate, 100

extraction concentration of 75% showed the

mL distilled water).

the

same

as Gentian

vielot

best results when staining for 3 minutes and

Based on a study conducted Virgianti and

5 minutes, although some bacteria were not

Lucyana (9), KMnO4 used as an oxidizer

stained.

when added to the solution of Angkak and

The colour produced by the Ipomoea

teak leaves as a cover dye in Gram staining.

batatas L. peel is purple. The colour

The alleged positive charge of potassium and

produced by Ipomoea batatas L. peel is more

dye complexes forming dissociation, so that

stable under acidic conditions and the

it can be bound to bacterial cell components

increase in pH affects the resulting colour.

which have a negative charge. For this

Besides the use of polar solvents makes it

reason, the process of extraction results

easier to dissolve the peel content of Ipomoea

requires the addition of ethanol to match the

batatas L. (6).

composition of Gentian violet. Besides, the

Based on the study results of Yuniarti
and Misbach (7) that Ipomoea batatas L. can

82

compounds

addition of acid-base was useful to get the
appropriate violet colour.

give colour to bacteria. On microscopic

Violet colour obtained when it was at

observations, Staphylococcus aureus shaped

anthocyanin equilibrium, which was a

coccus with reddish-purple. In this study, the

quinoidal base for the addition of NH4OH

formulation was needed as an anthocyanin

and HCl serves as a counterweight when trial

proof

and

test

that

was

appropriate

in

error. Bacillus

sp. bind

processing Ipomoea batatas L., so that the

from Ipomoea

colour produced under the microscope can

formulation and was obtained purple colour,

resemble

which comes from the reaction between HCl

the

purple

by Gentian violet.

colour

produced

batatas L.,

dyes

after

the

and NH4OH, forming compounds NH4Cl.

Ina J Med Lab Sci Tech 2020; 2(2): 76-84

Nastasya Nunki, et al.

In this study, the dyes formed tend to be

penetrated the bacterial wall so that the

alkaline dyes, to maintain the purple colour

colour of bacteria did not entirely derive from

of the extraction of the peel of Ipomoea

the extract. The author assumes this was

batatas L. Alkaline dyes are substances that

coming

produce cations. The ionization of the

of Safranin. To minimize information bias,

chromogen component shows a positive

the authors recommend that further study on

charge that can bind to the cell component,

this

which is a negative charge. The nucleic acid

mechanism of alternative dyes in penetrating

is a negatively charged cell component (9, it

bacterial cell walls, so that we can find out

also can accept positively charged dyes such

more about the ability of alternative dyes to

as the Gentian violet, or in this study, the

stain bacterial cell walls.

dyes

derived

from

the

peel

from

topic,

the

residual

especially

colouring

regarding

the

extract

of Ipomoea batatas L.

CONCLUSIONS

Assumes that the positive charge of NH4

The present study demonstrated that

ions dissociates with anthocyanin dyes in the

Ipomoea batatas L. peel extract can be used

form of cyanide which has a negatively

as an alternative staining agent, but to

charged

results

substitute Gentian violet in Gram staining of

of Ipomoea batatas L. peel extracts. Positive

bacteria, need conducting further studies on

charge ions resulting from dissociation with

this topic and observe to several aspects.

anthocyanin dyes bind with protoplasm ions

Besides, the matter that needs to be

of bacterial cells that have negatively charged

considered is the storage method and storage

nucleic acids in the results of the Ipomoea

time for the extraction of Ipomoea batatas L.

batatas L. peel extract form of phosphate

peel. The author recommended conducting

groups (9). In this study, there was a control

further studies use the extract 100%

group and an experimental group. The

concentration and the staining time used for

control

5 minutes.

with

OH

group

from

the

group Bacillus sp. were
Gentian

violet,

lugol,

stained
alcohol,

and Safranin.
This study has limitations. The further

CONFLICT OF INTEREST
There are no conflicts of interest.

research is needed to use Ipomoea batatas L.
peel extract as a substitute for Gentian
violet in Gram stain. The author assumes that
the dye produced from the peel extract

Ina J Med Lab Sci Tech 2020; 2(2): 76-84

The author would like to thank the
laboratory assistants and several related
parties who have provided direction and

83

of Ipomoea batatas L. has not maximally

ACKNOWLEDGMENT(S)

Nastasya Nunki, et al.

support in this study. The author also would

laboratory at the Universitas Hang Tuah

like to thank BBLK Surabaya, Microbiology

Surabaya

laboratory faculty of health at the Universitas

laboratory technology faculty of health at the

Nahdlatul

Universitas Nahdlatul Ulama Surabaya.

Ulama

Surabaya,

Chemistry

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