76 

Purple Sweet Potato (Ipomoea batatas L.) Peels Extract as an 

Alternative Dye for Bacteria Gram Staining 
 

Nastasya Nunki1, Diah Titik Mutiarawati2, Endah Prayekti1 
 

1Department of Medical Laboratory 

Technology, Faculty of Health, 

Universitas Nahdlatul Ulama 

Surabaya, Surabaya, Indonesia 

2Department 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 

that is very small in size, so microscopic 

observation aids are needed. Microorganisms 

studied in the laboratory for various 

purposes. The study of these microorganisms 

intends to facilitate and assist in the 

examination service in the laboratory (1).  

Bacteria have a strong cell wall as an 

outer part and maintain the shape of bacteria 

and protect against osmotic pressure. In 

staining, there are variations in the structure 

of cell walls. Where the complex structure, 

semi-rigid with bacterial cell wall thickness 



 

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

Nastasya Nunki, et al. 
7
7

 

ranges from 10 to 35nm and surrounds the 

cytoplasmic membrane, serves to give shape 

to the cell and protect the contents of the cell 

from outside cell influences (1). 

Bacterial cell walls are essentials for 

growth and division. Based on the chemical 

composition of the cell wall, which causes 

cell wall rigidity is peptidoglycan. The 

mechanism of Gram staining based on the 

structure and composition of the cell wall (2). 

In the laboratory world, especially in the 

field of staining, microbiology is one of the 

keys to helping to provide information about 

the diagnosis of a disease. The existence of 

the development of staining procedures to 

assist in roughly observing the morphology 

of microorganisms helps in identifying parts 

of the cell structure of microorganisms and 

helps differentiate similar microorganisms 

(1). 

The importance of observing the 

morphology of microorganisms, where the 

appearance of microorganisms in living 

conditions is quite complex, not only because 

of their size but also because they are 

transparent and colourless when suspended in 

a liquid medium. To study the properties and 

divide microorganisms into specific groups 

for diagnostic purposes, the biological dyes 

and staining procedures with a light 

microscope have become the main tools in 

microbiology (3). 

The most basic and primary staining 

methods used are Gram staining. The Gram 

reaction is related to morphological 

characteristics in phylogenetic-related forms 

(4). Synthetic dyes are including Crystal 

violet, Safranin, Carbol fuchsin, are dyed 

commonly used in Gram staining. Crystal 

violet and Safranin are dyes that often used 

in Gram staining. Crystal violet is a human 

carcinogen (5). 

To minimize the use of carcinogenic 

properties for gram staining, the research to 

find alternative materials are needed. To get 

natural dyes that can be used as alternative 

dyes, extraction methods are needed to get 

the best pigment (anthocyanin) from the 

colour of these natural ingredients, thereby 

maximizing the quality and quantity. Purple 

sweet potato peel has an average anthocyanin 

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

in this research conducted further testing, 

using peel extracts of purple sweet potatoes 

(Ipomoea batatas L.). 

In the previous study (7), purple sweet 

potato extract can be obtained manually and 

can produce reddish-purple colour. For this 

reason, it needs to develop research to 

maximize the content of dyes (anthocyanin) 

in the purple sweet potatoes peels using 

extraction techniques MAE (microwave-

assisted extraction) method. The previous 

study only examined the coccus Gram-

positive group without being compared with 

other groups.  

In this study, gram staining of Bacillus sp 

tested and also the optimum concentration 



 

 

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

Nastasya Nunki, et al. 

 
7
8

 

and time of gram staining were studied. 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. 

 

MATERIALS AND METHODS 

The materials used in this study were the 

pure culture of Bacillus sp. obtained from 

Central health laboratory (Balai Besar 

Laboratorium Kesehatan/BBLK) in 

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

chemicals]/100 mL distilled water), Gram 

stain hucker method, immersion oil 

(Olympus), staining from Ipomoea 

batatas L. peel extract, ethanol 96% (SAP 

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

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

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

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

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

37% (Merck) 100 mL distilled water). 

The sample used was the peels of purple 

sweet potato (Ipomoea batatas L.) not other 

types of sweet potato. This study consisted of 

several stages. The first stage was the process 

of sample preparation using MAE 

(microwave-assisted extraction) method 

extraction (8), determination of the 

concentration formula, the staining process, 

observation under a microscope, and lastly 

data analysis. Extraction techniques MAE 

(microwave-assisted extraction) method was 

selected to maximize the content of dyes 

(anthocyanin) in the purple sweet potatoes 

peels. 

In sample preparation, the sample 

extracted using the MAE. The extraction 

process carried out with a concentration 

variation of 50%, 60%, 75%, which approved 

with a combination of staining time of 1 

minute, 3 minutes, and 5 minutes. 

At each concentration of Ipomoea 

batatas L. peel extract, a formulation was 

performed to obtain the appropriate color. 

50% concentration obtained by adding 

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

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

obtained by adding (extract 0.6 gr/10 mL 

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

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

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

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

The smear prepared from 

culture Bacillus sp. on a clean glass slide 

allowed it to air-dried and fixed it by 

flaming.  

For the experimental group, at first, the 

smear on a glass slide was added 

with Ipomoea batatas L. extracts at each 

concentration for 1 minute, 3 minutes and 5 

minutes. The Ipomoea batatas L. extract was 

poured off and applied lugol for 1 minute and 

washed with water, the smear decolourized 

with an organic solvent-alcohol for 30 

seconds, or until the colour was oozed from 

the slide and washed with water. 



 

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

Nastasya Nunki, et al. 
7
9

 

The Safranin applied for 1-minute washed 

with water and blot dried. 

The slide was observed under 

magnification 100x after putting a drop of 

immersion oil then the results were compared 

with staining using Gentian violet for 1 

minute. For the control group, the first step 

did not use extracts but Gentian violet and the 

next steps as in the experimental group until 

the observation was processed. 

The results were compared directly 

between the Gram staining using Ipomoea 

batatas L. peel extract tested on Gram-

positive bacteria as an experimental group. 

Furthermore, in Gram staining using Gentian 

violet as a control group. The sampling 

technique in this study was a simple random 

sampling. The parameters observed in this 

study included visual field clarity, glass slide 

cleanliness, contrast, bacterial shape, and 

bacterial colour. 

In the data analysis, the observation was 

results of each extract concentration of 50%, 

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

minute, 3 minutes, and 5 minutes compared 

to the control, coding was carried out based 

on 5 observation parameters to obtain 

quantitative data. Then, the calculation 

carried out with the formula for the total 

number of coding values with 3 times 

repetition / 15 (5 parameters repeated 3 

times) x 100%. 

 

 

RESULTS 

Ipomoea batatas L. Peel Extract 

Concentration of 50%, 60%, 75% 

Based on observations obtained by the 

results of colouring where the extraction 

concentration of 50% showed the best results 

when staining for 5 minutes, the extract was 

inadequate to penetrate bacteria, and some 

bacteria were not completely stained. The 

extraction concentration of 60% showed the 

best results when staining for 5 minutes 

(Figure 1). The dye appears to penetrate the 

bacterial cell wall, but some bacteria were not 

completely stained. The extraction 

concentration of 75% showed the best results 

when staining for 3 minutes and 5 minutes, 

although some bacteria were not stained 

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

dyes penetrate the bacterial cell wall and also 

the spores of Bacillus sp. (Figure 3). 

At the percentage of the staining results, 

at the extraction of 50% concentrations 

obtained staining approaching control with 

the results of the staining showed the 

optimum results when staining for 5 minutes. 

For extraction of a concentration of 60%, it 

obtained staining that approached the control 

with the results of the staining showed the 

optimum results when staining for 5 minutes. 

The extraction concentration of 75% attained 

a colour approaching the control with the 

results of the staining showed the optimum 

results when staining for 3 minutes and 5 

minutes (Figure 4). 



 

 

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

Nastasya Nunki, et al. 

 
8
0

 

  

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% 

 

 

  

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 

 

 

a b 

a b 



 

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

Nastasya Nunki, et al. 
8
1

 

 

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 

the one-way ANOVA test with SPSS 

instrument version 16.0. Where the 

concentration of 50% obtained α value of 

0.06, a concentration of 60% obtained α value 

of 0.227, a concentration of 75% obtained α 

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

no significant difference between the results 

of staining using Ipomoea batatas L. extracts 

and control (Gentian violet). The 

determination of colour tendency was using 

the colour matching with the RGB table, then 

compared with the control (Table 1). 

 

 

60%
68% 68%

100%

68% 68%

80%80% 80%
86%

50 60 75 Control

T
im

e
 o

f 
S

ta
in

in
g

Extract Concentration

Time of Staining 1 minute Time of Staining 3 minute Time of Staining 5 minute

Table 1. RGB Code for Bacterial Gram Staining (RGB Color Chart.cdr - AWS) 

Bacteria 

Time of  

Staining 

(minute) 

RGB Code for Bacterial Gram Staining 
Color 

Name 
Concentration of Staining 

Control 
50% 60% 75% 

Bacillus sp. 1 RGB: 180 

4 133 

  

RGB: 173 

14 145 

 

RGB: 180 

4 133 

 
– 

153 0 122 

 

RGB: 70 

20 111 

 

Purple 

  

3  RGB: 173 

14 145 

  

RGB: 152 

4 110 

 

RGB: 153 

0 122 

 

- 

5  RGB: 152 

4 110 

 

RGB: 180 

4 133 

 
– 

153 0 122 

 

RGB: 141 

4 123 

 

- 



 

 

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

Nastasya Nunki, et al. 

 
8
2

 

DISCUSSION 

The extraction concentration of 50% 

showed the best results when staining for 5 

minutes. The extract was inadequate to 

penetrate bacteria, and some bacteria were 

not completely stained. The extraction 

concentration of 60% showed the best results 

when staining for 5 minutes. The dye appears 

to penetrate the bacterial cell wall, but some 

bacteria were not completely stained. The 

extraction concentration of 75% showed the 

best results when staining for 3 minutes and 

5 minutes, although some bacteria were not 

stained. 

The colour produced by the Ipomoea 

batatas L. peel is purple. The colour 

produced by Ipomoea batatas L. peel is more 

stable under acidic conditions and the 

increase in pH affects the resulting colour. 

Besides the use of polar solvents makes it 

easier to dissolve the peel content of Ipomoea 

batatas L. (6).  

Based on the study results of Yuniarti 

and Misbach (7) that Ipomoea batatas L. can 

give colour to bacteria. On microscopic 

observations, Staphylococcus aureus shaped 

coccus with reddish-purple. In this study, the 

formulation was needed as an anthocyanin 

proof test that was appropriate in 

processing Ipomoea batatas L., so that the 

colour produced under the microscope can 

resemble the purple colour produced 

by Gentian violet. 

Phenolic compounds of Ipomoea 

batatas L. peel which included in the 

flavonoid group and the content of phenol 

compounds which is Ipomoea batatas L. peel 

is the same as Gentian vielot 

content. Gentian violet is a triaryl methane 

dye with three phenol groups arranged by 

methane groups. The composition contained 

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

ethanol 95%, 0.8 gr ammonium oxalate, 100 

mL distilled water). 

Based on a study conducted Virgianti and 

Lucyana (9), KMnO4 used as an oxidizer 

when added to the solution of Angkak and 

teak leaves as a cover dye in Gram staining. 

The alleged positive charge of potassium and 

dye complexes forming dissociation, so that 

it can be bound to bacterial cell components 

which have a negative charge. For this 

reason, the process of extraction results 

requires the addition of ethanol to match the 

composition of Gentian violet. Besides, the 

addition of acid-base was useful to get the 

appropriate violet colour. 

Violet colour obtained when it was at 

anthocyanin equilibrium, which was a 

quinoidal base for the addition of NH4OH 

and HCl serves as a counterweight when trial 

and error. Bacillus sp. bind dyes 

from Ipomoea batatas L., after the 

formulation and was obtained purple colour, 

which comes from the reaction between HCl 

and NH4OH, forming compounds NH4Cl. 



 

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

Nastasya Nunki, et al. 
8
3

 

In this study, the dyes formed tend to be 

alkaline dyes, to maintain the purple colour 

of the extraction of the peel of Ipomoea 

batatas L. Alkaline dyes are substances that 

produce cations. The ionization of the 

chromogen component shows a positive 

charge that can bind to the cell component, 

which is a negative charge. The nucleic acid 

is a negatively charged cell component (9, it 

also can accept positively charged dyes such 

as the Gentian violet, or in this study, the 

dyes derived from the peel extract 

of Ipomoea batatas L. 

Assumes that the positive charge of NH4 

ions dissociates with anthocyanin dyes in the 

form of cyanide which has a negatively 

charged OH group from the results 

of Ipomoea batatas L. peel extracts. Positive 

charge ions resulting from dissociation with 

anthocyanin dyes bind with protoplasm ions 

of bacterial cells that have negatively charged 

nucleic acids in the results of the Ipomoea 

batatas L. peel extract form of phosphate 

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

group and an experimental group. The 

control group Bacillus sp. were stained 

with  Gentian violet, lugol, alcohol, 

and Safranin. 

This study has limitations. The further 

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 

of Ipomoea batatas L. has not maximally 

penetrated the bacterial wall so that the 

colour of bacteria did not entirely derive from 

the extract. The author assumes this was 

coming from the residual colouring 

of Safranin. To minimize information bias, 

the authors recommend that further study on 

this topic, especially regarding the 

mechanism of alternative dyes in penetrating 

bacterial cell walls, so that we can find out 

more about the ability of alternative dyes to 

stain bacterial cell walls. 

 

CONCLUSIONS 

The present study demonstrated that 

Ipomoea batatas L. peel extract can be used 

as an alternative staining agent, but to 

substitute Gentian violet in Gram staining of 

bacteria, need conducting further studies on 

this topic and observe to several aspects. 

Besides, the matter that needs to be 

considered is the storage method and storage 

time for the extraction of Ipomoea batatas L. 

peel. The author recommended conducting 

further studies use the extract 100% 

concentration and the staining time used for 

5 minutes. 

 

CONFLICT OF INTEREST 

There are no conflicts of interest. 

 

ACKNOWLEDGMENT(S) 

The author would like to thank the 

laboratory assistants and several related 

parties who have provided direction and 



 

 

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

Nastasya Nunki, et al. 

 
8
4

 

support in this study. The author also would 

like to thank BBLK Surabaya, Microbiology 

laboratory faculty of health at the Universitas 

Nahdlatul Ulama Surabaya, Chemistry 

laboratory at the Universitas Hang Tuah 

Surabaya and department of Medical 

laboratory technology faculty of health at the 

Universitas Nahdlatul Ulama Surabaya.

REFERENCES 

1. Pelczar, M J, Chan E C S. Fundamentals of 

Microbiology Volume 1. UI Press; 1988. 

2. Sri Harti A. Health Microbiology. Andi Offset; 

2015. 

3. Cappuccino J G, Sherman N. Microbiology 

Laboratory Manual edition 8. EGC; 2014. 

4. Geo F B, Karen C C, Janet S B, Stephen A M, 

Timothy A M. Jawetz, Melnick, Adelberg. 

Medical Microbiology edition 25. EGC; 2012 

5. Sun M, Ricker K, Osborne G, Marder M E, 

Schmitz R. Evidence on the arcinogenicity of 

Gentian violet. OEHHA; 2018. 

6. Andarwulan, Nuri, Faradilla R H F. Natural 

Dyes for Food. SEAFAST center IPB; 2012. 

7. Yuniarty T, Misbach S R. Utilization of Purple 

Sweet Potato Extract (Ipomoea batatas L.) as a 

Staining Substance in Staphylococcus aureus 

Staining. TEKNOLAB. 2016; 5(2): 59-63. 

8. Agung L, Yunianta. Anthocyanin Extraction 

from Purple Sweet Potato Skin Waste (Ipomoea 

batatas L.) Microwave-Assisted Extraction 

Method (Study of extraction time and solvent). 

Artikel Ilmiah Malang: Universitas Brawijaya. 

9. Virgianti D P, Lucyana C. Use of Combination 

of Angkak Extract and Teak Leaves as Closing 

Dyes in Gram Staining. Jurnal Kesehatan Bakti 

Tunas Husada. 2017;1 7(1): 66-72. 

10. Subandi. Microbiology. Development of Studies 

and Observations in Islamic Perspectives: 

Remaja Rosdakarya; 2010. 

11. Aballe, L M, Questo I A R, Dimacuta S S. 

Basella alba (Alugbati) Berries and Hibiscus 

Rosa Sinensis (Gumamela) flower crude 

Extracts as Alternative Dyes for Gram Staining. 

Medical Laboratory Science of San Pedro 

College; 2016. 

12. Aishah B, Nursabrina M, Noriham A, Norzizah 

A R, Shahrimi M. Anthocyanins from Hibiscus 

sabdariffa, Melastoma malabathricum, and 

Ipomoea batatas and its color properties. 

International Food Research Journal. 2013; 

20(2): 827-834. 

13. Jiwintarum Y, Rohmi, Prayuda I D P M. Dragon 

Fruit (Hylocereus Polyrhiuz) as a Natural Color 

For Bacterial Staining. Jurnal Kesehatan Prima. 

2016; 10 (2): 1726-1734. 

14. Kamel F H, Najmaddin C. Use of Some Plants 

Color as Alternative Stain in Staining of 

Bacteria. Kirkuk University Journal Scientific 

Studies (KUISS). 2016; 11(3): 248-253. 

15. Azmi, A N, Yunianta. Extraction of 

Anthocyanin from Mulberry Fruit (Morus alba 

L.) Method of Microwave-Assisted Extraction 

(Study of extraction time and the ratio of 

ingredients: solvent). Jurnal Pangan dan 

Agroindustri. 2015; 3 (3): 835-846. 

16. Martha E, Kresno S. Qualitative Research 

Methodology for Health. Raja Grafindo Persada; 

2016. 

17. Notoatmodjo. Health Research Methodology. 

Rineka Cipta; 2012. 

18. Misnadiarly, Djajaningrat H. Microbiology for 

Clinic and Health. Rineka Cipta; 2014. 

19. Radji M. Microbiology Textbook for Pharmacy 

and Medical Students. EGC; 2011.