Indonesian Journal of Medical Laboratory Science and Technology

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p-ISSN 2684-6748 e-ISSN 2656-9825

Indonesian Journal of Medical Laboratoy Science and Technology (IJLMST) is a peer-reviewed journal published by Universitas Nahdlatul Ulama Surabaya (UNUSA). This journal is established by the lecturers of Medical Laboratory Technology Department, Faculty of Health, in 2018. IJLMST is registered to International Standard Serial Number (ISSN) with p- ISSN: 2684-6748 and e-ISSN: 2656-9825. This journal is also registered to CrossRef system with a DOI 10.33086/ijmlst. IJMLST is currently accredited on the Science and Technology Index with the predicate of Sinta 3.

FOCUS AND SCOPE

IJLMST focuses on the field of Medical Laboratory including hematology, clinical chemistry, immunoserology, microbiology, parasitology, cyto- histotechnology, clinical toxicology and molecular biology.

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CONTACT ADDRESS

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E-mail: [email protected] Website: http://journal2.unusa.ac.id/index.php/IJMLST

Indonesian Journal of

Medical Laboratory

Science and Technology

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p-ISSN 2684-6748 e-ISSN 2656-9825

EDITORIAL TEAM

Editor in Chief

Maharani Pertiwi K

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

Associate Editor

Samsul Bahari Shamsudin

Community and Family Medicine Department, Faculty of Medicine and Health Science, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia.

Journal Editor

Achmad Syafiuddin

Department of Public Health, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia.

Citrawati Dyah Kencono Wungu

Department of Medical Biochemistry, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia.

Prima Nanda Fauziah

Department of Medical Laboratory Technology, Faculty of Health, Universitas M. H. Thamrin, Jakarta, Indonesia.

F. X. Himawan Haryanto Jong

Department Anatomy Histology, Faculty of Medicine, Universitas Katolik Widya Mandala Surabaya, Surabaya, Indonesia.

Copyeditor

Fildzah Karunia Putri

Department of Nutrition, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia.

Proofreader

Agusniar Furkani Listyawati

Department of Medical Microbiology, Faculty of Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia.

Endry Nugroho Prasetyo

Departemen of Biology, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember, Surabaya Indonesia.

Layout Editor

Gilang Nugraha

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

Peer Reviewers

Win Darmanto

Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia.

Umi Hamidah

Research Unit for Clean Technology, Indonesian Institute of Sciences, Bandung, Indoneisa.

Dewi Noor Hidayati

Farma Veterinary Center, Surabaya, Indonesia.

Ummy Mardiana

Department of Medical Laboratory Technology, STIKes Bakti Tunas Husada, Tasikmalaya, Indonesia.

Muthi' Ikawati

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia.

Sri Agung Fitri Kusuma

Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Padjadjaran, Sumedang, Indonesia.

Yurnaliza

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia.

Esti Mumpuni

Department of Pharmacy, Universitas Pancasila, Jakarta, Indonesia.

Arli Aditya Parikesit

Department of Bioinformatics, School of Life Sciences, Indonesia International Institute for Life Sciences, Jakarta, Indonesia.

Megga Ratnasari Pikoli

Department of Biology, Faculty of Science and Technology, UIN Syarif Hidayatullah Jakarta, Indonesia.

Dwi Wahyu Indriarti

Indonesian-Japan Collaborative Research Center for Emerging and Re-emerging Infectious Diseases, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia.

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p-ISSN 2684-6748 e-ISSN 2656-9825

Content

Indonesian Journal of Medical Laboratory Science and Technology Volume 3 Number 1, April 2021

ARTICLES

Ability Of Ethanol Extract from Ajwa and Sukkari Dates (Phoenix dactylifera L.) in Inhibiting The Growth of Methicillin-Resistant Staphylococcus aureus (MRSA)

Putra Rahmadea Utami, Sri Indrayati, Nur Hayatang

1 – 8

Chemometric Analysis of Serum Magnesium Calculations Using Mg-Xylidyl Blue-I Method Based on Molar Absorptivity

Ally Kafesa, Nadira Nur Hajah Lutfi, Cep Wahyu

9 – 18

Comparation Between Mac conkey and Coconut Water Medium as a Growth Medium for Escherichia coli

Endah Prayekti, Suliati, Dwi Agustin Wulandari

19 – 25

In Silico Analysis of Antiviral Activity and Pharmacokinetic Prediction of Brazilein Sappan Wood (Caesalpinia sappan L.) Against SARS-CoV-2 Spike Glycoproteins

Dwi Krihariyani, Edy Haryanto, Retno Sasongkowati

26 – 37

Liver Histopathological Change and Malondialdehyde Level of Rattus Norvegicus on Administration of Curcuma Zedoaria and

Paracetamol Toxic Dose

Putu Oky Ari Tania, Puja Ayu Misuari, Satya Yudhayana, Ketut Ayesha Edelwise Prayoga

38 – 46

Relation of Parasites in Soil with The Existence of Parasites on Farmer's Nails

Edza Aria Wikurendra, Merry Crismiati, Globila Nurika

47 – 55

The Antibacterial Activity of Thermoactinomyces sp. (H24) Extract Against Escherichia coli and Staphylococcus aureus

Julia Nanda Puspita, Rikhsan Kurniatuhadi, Rahmawati

56 – 63

The Potential Use of EDTA as an Alternative to Defibrination in Preparing Blood Agar Plates with Human AB Blood on Staphylococcus aureus Culture

Dora Dayu Rahma Turista, Eka Puspitasari, Fanny Kurnanda

64 – 71

EDITORIAL

RETRACTION NOTICE TO ‘‘N-Terminal pro-Brain Natriuretic Peptide (NT-proBNP) in Stage 1 and Stage 2 Hypertension Patients” [Ina. J. Med.

Lab. Sci. Tech. 2019;1(2): 64-67]

Supriati Wila Djami

72

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Ability of Ethanol Extract from Ajwa and Sukkari Dates (Phoenix dactylifera L.) in Inhibiting the Growth of Methicillin-Resistant Staphylococcus aureus (MRSA)

Putra Rahmadea Utami

¹

, Sri Indrayati

¹

, Nur Hayatang

¹

1Department of Medical Laboratory Technology, Faculty of Health Science, Universitas Perintis Indonesia, Padang, Indonesia Correspondence:

Putra Rahmadea Utami, Jl. Adinegoro KM 17, Simpang Kalumpang, Lubuk Buaya, Padang, West Sumatera, Indonesia

Zip Code: 25586 Email:

[email protected] Received: December 8^th, 2020 Revised: March 12^th, 2021 Accepted: March 15^th, 2021 Published: April 28^th, 2021 DOI: 10.33086/ijmlst.v3i1.1848

Abstract

Staphylococcus aureus is a pathogenic bacterium that spread throughout the world and still a problem that continues to increase both in hospitals and the community.

Infections due to S. aureus usually treated with antibiotics, but in some cases, several strains of S. aureus found to be resistant to antibiotics, such as Methicillin-Resistant Staphylococcus aureus (MRSA). Based on the previous research, the ethanol extract from Ajwa and Sukkari dates formed an inhibitory zone against the MRSA bacteria growth. This study aims to determine the inhibition of the ethanol extract from Ajwa and Sukkari variety of dates (Phoenix dactylifera L.) on the S. aureus growth. The ethanol extract from Ajwa and Sukkari dates with a concentration of 5 mg/mL, 10 mg/mL, 15 mg/mL, and 20 mg/mL resulted in the same inhibition zone with a diameter of ≤ 6 mm which categorized as weak (resistant), whereas the positive control ciprofloxacin had a resistance zone with a diameter of 9 mm. This study results concluded that the ethanol extract of Ajwa and Sukkari dates only has a maximum concentration of 20 mg/mL, which is still classified as a low concentration and has not been able to inhibit MRSA bacteria growth.

Keywords

Dates Ajwa, Dates Sukkari, Extract Ethanol, MRSA.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2021 by author.

INTRODUCTION

Staphylococcus aureus is treatable with antibiotics, but several strains of S.

aureus found to be resistant to antibiotics, such as Methicillin-Resistant Staphylococcus

aureus (MRSA). The spread of MRSA has been the subject of several studies because the danger of antibiotic resistance is a serious world health problem in both developed and developing countries. In 2010, the prevalence

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Putra Rahmadea Utami, et al.

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of MRSA is 28% (Hong Kong and Indonesia) and 70% (Korea) among all clinical isolates of S. aureus, while the S. aureus infections found in related communities in Asian countries varied widely, from 5–35% (1).

The presence of MRSA found in the hospital environment. The study of Vysakh &

Jeya (2) obtained 450 S. aureus isolates collected from patients from several hospitals in India, which 121 positives for MRSA (27%) and 329 as MSA (73%). The University of Alexandria, which collected 50 isolates of S. aureus strains from several hospitals, found 40% strains resistant to the antibiotics Oxacillin and cefoxitin, which indicates the presence of MRSA in these isolates (2). However, research conducted in Indonesia found that the prevalence of MRSA in RSUD Dr. Sutomo Surabaya, Indonesia was 8.2%. Despite the results, the study showed that with the low prevalence of MRSA in Indonesia, a coping strategy still needed to prevent further infection (1,3).

Dates are one of the plants in their role as medicine and food. Dates are a food ingredient that is rich in vitamins, minerals, fiber and sugar. In some varieties, the sugar content of dates can reach up to 88%. Many researchers researched twelve dates varieties to find out their chemical content, such as flavonoid compound (1,2). Aldaihan and Bhat examined the benefits of dates (Phoenix dactylifera L) in vitro and found that one of its benefits is that they have antibacterial

properties. This research showed that flavonoids are the active substances in dates.

Flavonoids can be used as immunomodulators to increase the work of macrophages on pathogenic microbial phagocytes that attack the body (4,5). Based on the previous research, the ethanol extract from Ajwa and Sukkari dates formed an inhibitory zone against the MRSA bacteria growth (2).

Extraction is a process of separating a substance based on differences in its solubility in two different insoluble liquids, usually water and other organic solvents such as nutritious substances or active substances from a part of medicinal plants, animals, and several types of fish, including marine biota (3). Due to the different thickness of plant and animal cells, an extraction method with a particular solvent needed. This extraction method is based on the principle of mass transfer agent component in a solvent, wherein the displacement began in the interface layer and then diffuses into the solvent (6). This study aims to determine the inhibition of the ethanol extract from Ajwa and Sukkari variety of dates (Phoenix dactylifera L.) on the S. aureus growth.

MATERIALS AND METHODS

This research used an experimental research design and conducted in July- August 2020 at the Microbiology Laboratory of Andalas University, Padang, Indonesia.

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Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 1–8 Putra Rahmadea Utami, et al.

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This study performed to determine the inhibition zone of the ethanol extract from Ajwa and Sukkari dates against the MRSA.

This study used the ethanol extract from Ajwa and Sukkari dates. Extraction was carried out using 96% ethanol solvent for 3x24 hours and evaporated using a rotary evaporator to obtain a thick extract of ± 150 g. The extract concentrations used were 5 mg/mL, 10 mg/mL, 15 mg/mL and 20 mg/mL. The study used MRSA bacteria, namely S. aureus. Bacterial isolates obtained from RSUP Dr. M. Djamil Padang, Indonesia. Bacteria isolates were resistant to penicillin-derived antibiotics. The positive control used ciprofloxacin, while the negative control used sterile aquadest for the treatment.

RESULTS

Characteristics of Ethanol Extract of Sukkari and Ajwa Dates

The pulp of Ajwa and Sukkari dates each

±500 g separated from the seeds.

Furthermore, from the ethanol extract, each obtained ±150 g of thick extract. The thick extracts from Ajwa dates and Sukkari dates shown in Figure 1. The color of Sukkari dates is darker than Ajwa dates.

Characteristics of Methicillin-Resistant Staphylococcus aureus (MRSA)

The bacteria used were MRSA bacteria obtained from the Microbiology Laboratory of Andalas University, Padang. The MRSA bacterial suspension is already present in the media (Figure 2).

Inhibition Test of Ethanol Extract of Ajwa Dates and Sukkari on the Growth of Methicillin-Resistant Staphylococcus aureus

The test results of the inhibition of the ethanol extract of Ajwa and Sukkari dates with various concentrations of 5 mg/mL, 10 mg/mL, 15 mg/mL, and 20 mg/mL can be seen in Figure 3. This study result showed that the ethanol extract compounds from Sukkari and Ajwa dates could not inhibit MRSA bacteria growth. After that, it observed that there was no clear zone or zone of inhibition around the disc.

In this study, the positive control were used ciprofloxacin 50 mg, while the negative control were used sterile distilled water.

Table 1 showed that the extract from Ajwa and Sukkari dates could not inhibit MRSA bacteria growth. A seen in Figure 2, the zone of clearance appears from disk diameter paper discs. Repetition performed three times to show the inhibition zone diameter ≤ 6 mm (diameter paper discs).

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Figure 1. Dates extract using ethanol solvent. (a) Sukkari, (b) Ajwa.

Figure 2. Characteristics of MRSA. (a) MRSA turbidity compared to McFarland standard of 0.5 (left) and bacterial suspense (right), (b) The arrow in figure B shows the MRSA colony of

bacteria, (c) The arrow in Figure C shows cefoxitin resistance.

.

Figure 3. Inhibition test. (a) Sukkari dates ethanol extract concentrations of 5 mg/mL, 10 mg/mL, 15 mg/mL, and 20 mg/mL, (b) Ajwa date ethanol extract concentrations of 5 mg/mL, 10

mg/mL, 15 mg/mL, and 20 mg/mL, (c) and Ciprofloxacin.

a b

a b c

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Table 1. The results of the inhibition test of MRSA Ethanol Extract

Dates

Concentration (mg/mL)

Extract Repetition (mm)

X

1 2 3

Ajwa dates

5 ≤ 6 ≤ 6 ≤ 6 ≤ 6

10 ≤ 6 ≤ 6 ≤ 6 ≤ 6

15 ≤ 6 ≤ 6 ≤ 6 ≤ 6

20 ≤ 6 ≤ 6 ≤ 6 ≤ 6

Sukkari dates

5 ≤ 6 ≤ 6 ≤ 6 ≤ 6

10 ≤ 6 ≤ 6 ≤ 6 ≤ 6

15 ≤ 6 ≤ 6 ≤ 6 ≤ 6

20 ≤ 6 ≤ 6 ≤ 6 ≤ 6

Control positive Ciprofloxacin 50 mg

9

Negative control Aquadest ≤ 6

Note: Description of disc paper diameter = 6 mm

DISCUSSION

The study results showed no inhibition zone formed from the ethanol extract of Ajwa and Sukkari dates against the growth of MRSA bacteria. Ajwa and Sukkari dates ethanol extract with concentrations of 5 mg/mL, 10 mg/mL, 15 mg/mL and 20 mg/mL formed weak (resistant) inhibition zone diameter of ≤6 mm. Meanwhile, the positive control ciprofloxacin formed an inhibition zone with a diameter of 9 mm (7).

This condition happened because the bacteria used for this study was MRSA, where these bacteria are a group of bacteria that are already resistant to antibiotics.

MRSA is a S. aureus that immune to methicillin-type antibiotics. MRSA experiences resistance due to genetic changes caused by irrational exposure to antibiotic therapy (8). Besides, the research results also

prove that MRSA is also resistant to other antibiotics. This methicillin resistance will be followed by the simultaneous emergence of resistance to a large number of other antibiotic classes through the acquisition of additional receptor determinants, or as a result of mutations. All of which will cause the receptors at the target site to become resistant (9,10).

One of the factors of differences in sensitivity patterns or determinants of bacterial resistance to antimicrobials carries by genetic information outside the chromosomes, namely plasmid (11). S.

aureus is a bacterium that has small plasmids and large plasmids that have more than one resistance gene. Based on the study result, it can be assumed that there are large plasmids that carry more than one antibiotic-resistant gene in MRSA. Additionally, the methicillin-

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resistant gene may be linked to other antibiotic-resistant genes. MRSA bacteria, which are Gram-positive bacteria, have a peptidoglycan layer that is 20–80 nm thick.

Gram-positive bacteria also have a cell wall containing teichoic acid, which is a water- soluble polymer that functions as a transport for positive ions in and out (11,12,13).

Other studies conducted on the use of plant extracts to inhibit MRSA growth. One of the studies showed that the clove flower extract has an antimicrobial effect, which containing eugenol, flavonoids, tannins, saponins, alkaloids and phenols that can damage the structure of bacterial cells. The results showed that the Minimum Bactericidal Concentration (MBC) was 0.39%. One Way ANOVA test showed a significant difference (p < 0.05) between clove flower extract concentration and the number of MRSA colonies (1). Clove flower extract can kill MRSA by damaging the structure of bacterial cells. Another study was to test the inhibition of the ethanol extract of bitter melon (Momordica charantia) against the growth of MRSA. The ethanol extract of bitter melon fruit used as a test solution with a concentration (w/v) of 20%, 40%, 60%, and 80%. The results showed that the average diameter of the inhibition zone of the ethanol extract of bitter melon against the growth of MRSA with concentrations of 20%, 40%, 60%, and 80% was 6.16 mm, 9.5 mm, 10.83 mm, 12.3 mm respectively. The higher the

ethanol extract concentration of bitter melon fruit, the higher the inhibition power of the growth of MRSA (14,15,16).

The plant extracts used also contain content that is almost the same as other plants, namely eugenol, flavonoids, tannins, saponins, alkaloids and phenols, which can damage the structure of bacterial cells.

However, in this study, the ethanol extract of dates did not have a significant ability to inhibit the growth of MRSA (2). Whereas in research, a combination of extracts with different plants can inhibit the growth of other bacteria. In the research of Utami et al. (13), the results of testing the combination of Chinese petai extract and Aloe vera showed significant differences (p < 0.05) at the concentrations of 25 g, 50 g, 75 g, and 100 g. The ethanol extract of Chinese petai and Aloe vera can inhibit the growth of E. coli.

The results showed the most effective concentration of 100 g/mL resulted in an interaction between the ethanol extract of Aloe vera and china petai in inhibiting the growth of E. coli (12,17). In contrast, this Ajwa and Sukkari date research, only have 20 mg/mL as the maximum concentration, which still classified as a low concentration.

As a result, the active compound content is the only low level and unable to inhibit the growth of MRSA bacteria (4,18,19).

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7 CONCLUSIONS

The ethanol extract of Ajwa and Sukkari dates with a concentration of 5 mg/mL, 10 mg/mL, 15 mg/mL, and 20 mg/mL formed the same inhibition zone with a diameter of

≤6 mm. It conclusion, this study indicate that the ethanol extract of Ajwa and Sukkari dates only has a maximum concentration of 20 mg/mL, which is still classified as a low concentration and has not been able to inhibit MRSA bacteria growth by damaging the bacterial cell structure.

AUTHOR CONTRIBUTIONS

Putra Rahmadea Utami:

conceptualization, methodology, writing- reviewing and editing. Sri Indrayati: data curation, writing-original draft preparation, supervision. Nur Hayatang: visualization, investigation.

ACKNOWLADGEMENTS

This study was supported by the Universitas Perintis Indonesia.

CONFLICT OF INTEREST

There are no conflicts of interest.

REFERENCES

1. Setiawan HG, Kaligis AHM, Assa YA.

Description of serum apolipoprotein B (APO-B) levels in lacto-ovo vegetarians. Ebiomedik.

2017;5(1):2–5.

2. Kuntaman K, Hadi U, Setiawan F, Koendori EB, Rusli M, Santosaningsih D, et al. Prevalence of methicillin resistant Staphylococcus aureus from nose and throat of patients on admission to medical wards of Dr Soetomo hospital, Surabaya, Indonesia. Southeast Asian J Trop Med Public Health. 2016;47(1):66–70.

3. Kader OA, El-Batouti GA, Ghazal AA, Baraka KM. Hospital-acquired methicillin resistant Staphylococcus aureus: analysis of mec A gene and taphylococcal cassette chromosome. Int J Curr Microbiol Appl Sci. 2015;4(9):805–15.

4. Nismawati, Rizalinda S, Agus R. Deteksi Methicillin Resistant Staphylococcus aureus (MRSA) pada pasien Rumah Sakit Universitas Hasanuddin dengan metode kultur Detection of Methicillin Resistant Staphylococcus aureus (MRSA) in Hasanuddin University Hospital patients by culture method. Pros Semin Nas Biol.

2018;4(1):978–602.

5. Hamad I, Abdelgawad H, Al Jaouni S, Zinta G, Asard H, Hassan S, et al. Metabolic analysis of various date palm fruit (Phoenix dactylifera L.) cultivars from Saudi Arabia to assess their

nutritional quality. Molecules.

2015;20(8):13620–41.

6. Hariadi B, Widodo A. The Effect of Dates (Phoenix Dactylifera L.) Extract of Ajwa varieties on no levels in Balb/C mice infected with Salmonella typhimurium. Diponegoro Med J (Jurnal Kedokteran Diponegoro). 2018;7(2):751–

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7. Syarif RA, Sari F, Ahmad AR, Indonesia UM, Urip J, Km S. Rimpang kecombrang (Etlingera elator Jack.) as a phenolic source. J Feto Ina.

2000;2(2):102–6.

8. Fiţ NI, Chirilă F, Nadăş G, Pall E, Mureşan R.

Comparative testing of antimicrobial activity of aqueous extracts of Aloe vera and Lycium barbarium. Bull Univ Agric Sci Vet Med Cluj- Napoca - Vet Med. 2013;70(1):72–6.

9. Hu W, Li C, Dai J, Cui H, Lin L. Antibacterial activity and mechanism of Litsea cubeba essential oil against methicillin-resistant Staphylococcus aureus (MRSA). Ind Crops Prod.

2019;130(November 2018):34–41.

10. Bussmann RW, Glenn A, Sharon D. Antibacterial activity of medicinal plants of Northern Peru - can traditional applications provide leads for modern science?. Indian J Tradit Knowl. 2010;9(4):742–

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11. Craft KM, Nguyen JM, Berg LJ, Townsend SD.

Methicillin-resistant: Staphylococcus aureus (MRSA): Antibiotic-resistance and the biofilm

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phenotype. Medchemcomm. 2019;10(8):1231–

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12. Suryati N, Bahar E, Ilmiawati I. Antibacterial effectiveness test of Aloe vera extract against Escherichia coli growth in vitro. J Kesehat Andalas. 2018;6(3):518.

13. Rahmadea UP, Chairani, Yudha H. Combination test of chinese leaf extract (Leucaena leucocephala folium) and Aloe Vera inhibiting growth Escherichia coli. Indones J Med Lab Sci Technol. 2020;2(2):60–7.

14. Utami PR, Chairani C, Ilhamdi I. Interaksi ekstrak etanol daun petai cina (Leucaena leucocephala folium) dan lidah buaya (Aloe vera L.) menghambat pertumbuhan Staphylococus aureus secara invitro [The Interaction of ethanol extract of chinese petai leaves (Leucaena leucocephala folium) and Aloe vera (Aloe vera L.) inhibiting the growth of Staphylococus aureus by invitro]. J Kesehat PERINTIS (Perintis’s Heal Journal).

2019;6(2):186–92.

15. Azizah A, Suswati I, Agustin SM. Anti-microbial effect of clove flower extract (Syzygium Aromaticum) against Methicillin-Resistant Staphylococcus Aureus (MRSA) in vitro. Saintika Med. 2018;13(1):31.

16. Suhaimi S, Indrawati T, Kumala S. Combination test of crude fruit extracts (Aloe vera. (L) brum.

F.) and blue leaves (Piper crocatum ruiz & pav) extracts for purple leaf (Piper crocatum ruiz &

pav) antibacteries for acne. JIFFK J Ilmu Farm dan Farm Klin. 2018;15(01):12.

17. Cuny C, Friedrich A, Kozytska S, Layer F, Nübel U, Ohlsen K, et al. Emergence of Methicillin- Resistant Staphylococcus aureus (MRSA) in different animal species. Int J Med Microbiol.

2010;300(2–3):109–17.

18. Stefani S, Chung DR, Lindsay JA, Friedrich AW, Kearns AM, Westh H, et al. Meticillin-resistant Staphylococcus aureus (MRSA): Global epidemiology and harmonisation of typing methods. Int J Antimicrob Agents.

2012;39(4):273–82.

19. Pratama P, Purwanta M, Qurnianingsih E. The effectiveness of ethanol extract of Egyptian date palm seeds (Phoenix dactylifera L.) as an antibacterial agent against Streptococcus pyogenes in vitro. 2019;19(3):135–40.

20. Vysakh PR, Jeya M. A comparative analysis of community acquired and hospital acquired methicillin resistant Staphylococcus aureus. J Clin Diagnostic Res. 2013;7(7):1339–42.

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9

Chemometric Analysis of Serum Magnesium Calculations Using Mg-Xylidyl Blue-I Method Based on Molar Absorptivity

Ally Kafesa

¹

, Nadira Nur Hajah Lutfi

¹

, Cep Wahyu

¹

1Medical Laboratory of Technology Study Program, Rajawali Institute of Health, Bandung, Indonesia Correspondence:

Ally Kafesa,

Rajawali Barat No.38, Kota Bandung, West Java, Indonesia Zip Code: 40184

Email: [email protected] Received: January 7^th, 2021 Revised: March 27^th, 2021 Accepted: April 2^nd, 2021 Published: April 28^th, 2021 DOI: 10.33086/ijmlst.v3i1.1876

Abstract

The concentration of magnesium is determined based on the absorbance of the Mg-Xylydil Blue-I complex solution use spectrophotometer. Based on the Lambert-Beer rule, the calculation of sample concentration is based on the formula A = Ԑ. b. C. Generally, the thickness of the cuvette (b) and the molar absorptivity (Ԑ) factor will be ignored because it is considered to have a fixed value, therefore the sample concentration is measured based on the ratio of the absorbance of the sample against the standard solution.

However, the standard solution contains pure magnesium and has a different matrix than the sample matrix, so this condition can give analytical errors and lead to misinterpretation of the results. The purpose of this study was to determine the accuracy and the precision of serum magnesium calculation by the principle of the Mg- Xylydil Blue-I complex reaction based on molar absorptivity compared to the general method. This research uses comparative study design methods. The serum sample used was the patient's serum specimen who has a normal magnesium level. The results showed that the significance value of the paired t-test statistical was 0.000 (p < 0.05). The accuracy value (d%) of the calculation formula uses Ɛ is 0.00 and the precision value (CV%) is 0.53. While the accuracy value (d%) of the calculation formula without Ɛ is 0.00 and the precision value (CV%) is 0.38. Calculations based on molar absorptivity (Ɛ) can measure more significant serum magnesium than those calculated based on standard magnesium solutions.

Keywords

Epsilon, Magnesium, Mg-Xylydil Blue-I Complex Reaction, Serum, UV-Vis Spectrophotometry.

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Ally Kafesa, et al.

10 INTRODUCTION

Long-term deficiency of magnesium (Mg) can cause hypomagnesemia with clinical manifestations including numbness, tingling, muscle cramps, seizures, personality changes, and abnormal heart rhythm. On the other hand, excessive consumption of magnesium from drugs containing Mg (laxatives or antacids) led to hypermagnesemia, hyperthyroidism, kidney failure, and liver failure. Therefore, it is important to maintain the stability of the balanced micronutrient magnesium content in the body (1,2).

Magnesium is an essential element that composes the coenzyme for signal transfer in neurons and enzymes for cardiac contraction.

In assition, magnesium is required for the metabolism of carbohydrates, fats, and amino acids as micronutrients. Magnesium has medicinal value as a general laxative, antacid (e.g. milk of magnesia), and to stabilize abnormal nerve excitation or spasm of blood vessels in conditions such as eclampsia (2).

An analytical method (qualitative and quantitative analysis), e.g. chemometric analysis method, is needed to determine the content of magnesium. Chemometrics work by combining statistical values with chemistry, especially analytical chemistry.

The chemometric analysis uses statistical principles to design; select an optimal analytical procedure and experiment, and provide maximum and relevant chemical

information through chemical data analysis (4,5).

The spectrophotometer is widely used in quantitative measurements of magnesium because the amount of light absorbed by the particles in the solution depends on the type and number of particles (6,7). The photometer spectrum is based on the Lambert-Beer law. Lambert-Beer law states that the concentration of the standard solution is directly proportional to the value of light absorption (absorbance) (8). This law applies to monochromatic rays e.g. light with a single wavelength or that has an adjacent wavelength band. To calculate the absorbance of a sample, it is necessary to know the molar absorption denoted by Ɛ (epsilon), which is a molecule or ion that absorbs a solvent with a certain wavelength but does not depend on a particular concentration and wavelength or through radiation (9).

The Ɛ (epsilon) cannot be removed because it is affected by solvents. The solvent determines the addition of the electron transition energy in the compound, thus the wavelength that becomes the energy will be absorbed in a certain color. If the solvent has many mixtures, it will require a large amount of energy due to the interaction of the solvent (energy π-π* can be smaller or greater).

Therefore, mathematically, the Ɛ factor in the calculation equation cannot be ignored.

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Ina. J. Med. Lab. Sci. Tech. 2021; 3(1): 9–18 Ally Kafesa, et al.

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The use of different measuring formulas of magnesium content may provide different interpretations of the results. Comparative analysis of results is needed to compare the accuracy, precision, and statistical difference between the measurement results of the two formulas.

MATERIALS AND METHODS

This research used comparative study design methods. The sample used in this study was serum of healthy patients with normal magnesium levels in June 2020. The equipment used was a spectrophotometer (GenesysTM 10S). Reagents used in this study are Magnesium XL FS from DiaSys Diagnostic Systems (Germany) contains ethanolamine pH 11.0 750 mmol/L, Glycoletherdiamine-tetraacetic acid (GEDTA) 60 µmol/L, Xylydil Blue-I (248266 Sigma Aldrich, CAS Number 14936-97-1) 110 µmol/L and 2 mg/dL standard. The sample was measured by a photometer at a wavelength of 520 nm (11).

Samples that met the inclusion criteria were 3 samples. The inclusion criteria in this study were patients who were willing to become respondents by filling out an informed consent sheet and the sample volume had to reach 3 mL. The sampling technique used was random sampling with the criteria of non-lysed, non-icteric, and non-lipemic blood sample examination (11).

The samples obtained were centrifuged at 3.000 rpm for 15 minutes. Total samples were analyzed for Magnesium levels with the photometric test method using Xylydil Blue- I. The magnesium ion forms a purple complex with Xylydil Blue-I in an alkaline condition. In the presence of the calcium ion complex GEDTA, the reaction is specific.

The intensity of the purple color is proportional to the concentration of magnesium (11).

After the data on serum magnesium absorbance are collected, the magnesium content was calculated based on two formulas: Formula type A and Formula type B.

Calculation A Cspl =Aspl x Cstd

Astd ... 1 Cspl: Sample Concentration (mg/dL) Aspl: Sample Absorbance

Cstd: Standard Concentration (mg/dL) Astd: Standard Absorbance

Calculation B Cspl =Aspl x MW

Ɛ_1𝑐𝑚^1%. ... 2 Cspl: Sample Concentration (mg/dL) Aspl: Sample Absorbance

MW: Molecular Weigth of Xylydil Blue- I (513,5 g/mol)

Ɛ_1𝑐𝑚^1%.: Molar Absorptivity of Xylydil Blue-I (49.000 L M^-1cm^-1)

The calculation results of the two formulas were analyzed using the Paired T-

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test. The accuracy and precision analysis are calculated based on the deviation from repeated measurements of 3 times.

RESULTS

This research begins by creating a calibration curve to get the liner value.

Standard curve determination was performed with several concentrations (2, 4, 6, 8 mg/dL).

Figure 1. Curve calibration of magnesium standard solution

The calibration data shows that R = 0.9278, which means that the data shows a linear correlation between the concentration of magnesium and absorbance value.

The data collected was analyzed by statistical tests following certain conditions.

Data on 20 healthy patients were obtained with a mean serum magnesium level of 3.077 mg/dL, the lowest value was 1.387 mg/dL, and the highest value was 5.581 mg/dL. All patients had normal serum magnesium levels (one day before sampling) and did fasting for 10 – 12 hours to minimize the influence of food and activity. The accuracy and precision

of the Magnesium level of patients was determined by statistical analysis (Table 1).

Table 1. Quality test data

No Parameter Quality

1 Mean 3.077

2 Standard Deviation (SD) 1.712 3 Coefficient of Variation (CV) 0.556

4 Accuracy (D%) 0.057

5 Total Error (TE) 1.17 %

6 Total Error allowable (TEa) 4%

*based on magnesium test

Normal data distribution is obligatory before paired t-test conducted. The data normality test results show in Table 2. Result shows that all variable is normality distributed (p-value < 0.05).

y = 0,0174x + 0,6435 R² = 0,9278

0,66 0,68 0,7 0,72 0,74 0,76 0,78 0,8

0 1 2 3 4 5 6 7 8 9

Absorbance at 520 nm

Concentration (mg/dL)

Abs Linear (Abs)

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Figure 2. Serum magnesium level curve with 2 formulas: non (Ԑ) and (Ԑ)

Table 2. Variable test of normality test results for each sample

Kolmogorov-Smirnov Shapiro-Wilk

Statistic Df Sig. Statistic Df Sig.

Kit_Insert 0.198 16 0.093 0.898 16 0.076

Epsilon 0.116 16 0.200 0.925 16 0.205

The sample size is lower than 50, therefore the Shapiro-Wilk normality test was used. A significance value lower than 0.05 means the data is normally distributed.

The significance value of Epsilon is 0.205 (p

< 0.05) and the significance value of the Insert Kit is 0.076 (p < 0.05) so that the next test uses the paired T-test.

Table 3. Results of t-test statistics – paired one-sample test

Test Value = 0

t Df Sig.

(2-tailed)

Mean Difference

95% Confidence Interval of the Difference Lower Upper

Kit_Insert 10.40 15 0.000 2.42 1.92 2.91

Epsilon 7.52 15 0.000 2.88 2.06 3.69

Statistical test (Table 3) shows that the significance value is 0.000 (p < 0.05) for both the formula using the kit-insert and epsilon, therefore there is a significant difference

between the calculation formula for the results of the chemometric analysis with Ɛ and analysis without Ɛ.

0 1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Mg Serum Concentration mg/dL

Sample No.

non (Ԑ) (Ԑ)

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Ally Kafesa, et al.

14 DISCUSSION

Spectrophotometric analysis work by white light or radiation passed through a colored solution, then radiation with a certain wavelength will be absorbed and other will be transmitted. The absorbance value depends on the content of the substances, the more molecules absorb light, the greater the absorption value. Therefore, the absorbance value will be directly proportional to the concentration of the substance contained in a sample (9). Curve calibration of magnesium standard solution have slope 0.0174 and intercept 0.6435 with equation y = 0.0174x + 0.6435. The correlation between analyte level (x) and instrument response (y) is expressed as the correlation coefficient (r = 0.9278) (Figure 1). Ideally, the intercept is zero. It is expected that no instrument response will occur when analyte free water or blanks are measured. But in this research, we find instrument response occurs due to small interaction, interference, noise, contamination or other sources of bias.

Therefore, the intercept (a) in this calibration curve can be considered as the signal from the blank. While the slope (b) is a measure of the sensitivity of a test method. We have greater the value of b, so this method provides a higher sensitivity or the instrument's response is strong enough to change the magnesium existing levels. Based on the correlation coefficient obtained, it shows a linear relationship between magnesium

concentration and absorbance. The linear relationship that occurs is positive and strong.

In this study, curves of serum magnesium levels in 20 serum samples with 2 formulas (non (Ԑ) and (Ԑ)) showed an average difference in yield of 15.27%, where serum magnesium levels calculated using Ԑ had higher levels (Figure 2). At magnesium levels

<2.5 mg/dL, the difference in calculations is not too far away, but at magnesium levels

>2.5 mg/dL, there is a very large difference.

This shows that the calculation of serum magnesium using the molar absorption of Xylydil Blue-I can bind magnesium ions more than calculated compared to the absorbance of standard magnesium solutions.

The molecule that receives visible light at the appropriate frequency will experience a transfer of energy to a higher level (transfer of electrons from the ground state to the excited state). This electron transition (µ-µ*) absorbs specific energy and can be detected at certain wavelengths. The specificity and quantity of absorbed light energy are determined based on solubility. The more dissolved a compound, the more energy it will absorb. Conversely, the harder it is to dissolve, the lighter energy is transmitted and this will give false results of the measured number of molecules (8).

The calculation formula used to measure magnesium in serum is as follows formula 3:

Cspl =Aspl x Cstd

Astd ... 3

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This calculation formula comes from:

1. 𝐴𝑠𝑝𝑙1 = 𝑏1 𝑥 Ԑ1𝑥 𝐶𝑠𝑝𝑙1 2. 𝐴𝑠𝑝𝑙2 = 𝑏2 𝑥 Ԑ2 𝑥 𝐶𝑠𝑝𝑙2

If there are two similar solutions measured, the fixed factor in the formula can be removed and combine:

1. 𝐴𝑠𝑝𝑙1 = 𝑏 𝑥 Ԑ 𝑥 𝐶𝑠𝑝𝑙1 2. 𝐴𝑠𝑝𝑙2 = 𝑏 𝑥 Ԑ 𝑥 𝐶𝑠𝑝𝑙2

Cspl1 = Aspl x Cspl2 Aspl2

Cspl1: Sample Concentration

Aspl1: Sample absorbance (sample 1) Cspl2: Standard Concentration

Aspl2: Standard absorbance (sample 2)

In this formula, the concentration of the magnesium in the standard solution (pure solvent) is used as a reference for calculating the level of magnesium in serum. The serum is a matrix containing solutes (enzyme, protein, clotting agents, immune system);

body essentials such as vitamins and hormone; and dispersed cell components with a pH between 7.35 – 7.45. The solubility of magnesium ions in the serum matrix differs from the solubility of magnesium in pure solvents which do not contain other solutes. A standard solution of magnesium dissolved in water at a pH of 9.0. The difference in pH of this solution reduces the reaction of magnesium with Xylydil Blue-I

the magnesium ion forms a purple complex with Xylydil Blue-I in an alkaline condition.

In the presence of the calcium ion complex GEDTA, the reaction is specific. The intensity of the purple color is proportional to the magnesium concentration (8).

Figure 3. Chemical Structure of Xylidyl blue-I (C25H20N3NaO6S) (7).

Figure 3 show the structure of Xylydil Blue-I. IUPAC name of Xylidyl Blue-I is sodium; 3-[[3-[(2,4-dimethylphenyl) carbamoyl] -2-hydroxynaphthalen-1-yl]

diazenyl]-4-hydroxy benzene sulfonate.

Xylydil Blue-I is a synthetic colorimetric reagent for Mg detection with a molecular weight of 513.5. The aqueous solution of XB- 1 is red and turns reddish-violet in the presence of Mg at pH 9 (maximum wavelength: 510 nm, molar absorptivity:

49,000, detection range 0.02-0.4 ppm.

Xylydil Blue-I can react specifically with magnesium ions in the presence of a glycoletherdiamine tetraacetic acid

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Ally Kafesa, et al.

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(GEDTA). GEDTA is a substance to bind and control metal ions because it can remove water hardness (chelating agent). That is the greater affinity of chelating ligands for a magnesium ion than that of similar nonchelating (monodentate) ligands for the same metal (6).

Xylydil Blue-I has a very good molar absorption (Ɛ = 49,000) value in polar solvents therefore it can be used for the detection of compounds in polar matrices.

When Xylydil Blue-I reacts with magnesium ions at pH < 9, the stoichiometry of the reaction will shift to the left and the complex mg-Xylydil Blue-I products formed are getting less. Polar sulfonyl groups (SO3-) can interact favorably with similar water molecules. Therefore, the short-chain hydroxyl-benzene is soluble in water.

However, since the organic portion (more C atoms) gets bigger (the longer chain in this case), this interaction is less effective and water solubility decreases (8,6).

Serum pH does not support the Mg- Xylydil Blue-I reaction because the electrons in the sulfonyl group do not have enough energy to bind protons to the magnesium ion at pH 7. On the other hand, the standard solution of magnesium has a pH of 9. If this difference is calculated using formula type A, the magnesium test results will be inconsistent, thus the calculation of serum magnesium levels must be divided by the molar absorptivity of xylidyl blue-I.

Cspl =Aspl x MW

Ɛ_1𝑐𝑚^1%. ... 4 Type B formula (Lambert-Beer) involves the molecular weight and molar absorptivity of Xylydil Blue-I. Consequently, the measured Mg-Xylydil Blue-I complex reaction corresponds to the actual reaction product. The pH changes and the number of magnesium ions in the serum matrix will not affect the detected reaction product suitability. Calculation of serum magnesium using this formula has units of mole/Liter, therefore it must be converted to mg/dL. This research was done in the patient who had normal magnesium levels. More comprehensive studies must be conducted in patients with pathological conditions that allow the maximum amount of magnesium ions that can form bonds with Xylydil Blue- I.

CONCLUSIONS

The serum magnesium level formula that include molar absorptivity factor have a significant difference result (α<0.05) compared to formula without molar absorptivity factor. There is a significant difference between serum magnesium levels using the absorbance calculation formula with formula A (without Ɛ factor) and formula B (with Ɛ factor). Calculations based on molar absorptivity can measure more significant serum magnesium than those

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calculated based on standard magnesium solutions.

AUTHOR CONTRIBUTIONS

Ally Kafesa: conceptualization, methodology, writing-original draft, visualization, supervision, funding acquisition. Cep wahyu: methodology, supervison. Nadira Nur Hajah Lutfi: formal analysis, investigation, resources.

ACKNOWLADGEMENTS

The authors express their sincere gratitude to the Rajawali Institute of Health, Bandung, Indonesia.

CONFLICT OF INTEREST

We declare that we do not have any commercial or associative in-terest that represents a conflict of interest in connection with the worksubmitted.

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Comparation Between Mac conkey and Coconut Water Medium as a Growth Medium for Escherichia coli

Endah Prayekti

¹

, Suliati

²

, Dwi Agustin Wulandari

¹

1Department of Medical Laboratory Technology, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia

2Department of Medical Laboratory Technology, Poltekkes Surabaya, Surabaya, Indonesia

Correspondence:

Endah Prayekti,

Jl. Jemursari No. 51-57, Surabaya, East Java, Indonesia

Zip Code: 60237

Email: [email protected] Received: January 28^th, 2021 Revised: March 15^th, 2021 Accepted: March 15^th, 2021 Published: April 28^th, 2021 DOI: 10.33086/ijmlst.v3i1.1906

Abstract

Escherichia coli is the bacteria that can cause diarrhea in humans and often used as a parameter of stool environmental pollution. Culture of E. coli from the sample often requires Mac Conkey as commercial media which is able to distinguish it from other bacteria in the Enterobacteriaceae group. Commercial media such as Mac Conkey certainly has a price that is quite expensive because of its ability as a growth medium for Enterobacteriaceae.

Therefore, in the study tested natural ingredients that can be used for growth media, such as coconut water. The purpose of this study was to compare the ability of Mac Conkey media and coconut water to support the growth of E. coli.

This research is an experimental study with a completely randomized design. The concentration of coconut water tested was 0%, 20%, 40%, 60%, 80%, and 100%. The results showed that at the concentration of coconut water 20% to 60% the number of E. coli colonies on coconut water media was slightly below the Mac Conkey Agar media, while in coconut water a concentration of 80% showed a greater number of colonies than Mac Conkey. The Mann Whitney test showed a significant difference between the number of colonies on 80% coconut water media and Mac Conkey Agar, which was equal to 0.004 (p < 0.05). Based on these results, coconut water has the potential to be used as a growth medium for E. coli.

Keywords

Coconut Water, Escherichia coli, Mac Conkey.

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Indonesia is an archipelago which produces coconut fruit in most of provinces (1). Indonesia is one of country in the world which produces a high number of coconut fruit per year. Coconut were being consumed for its water and flesh, and or its product as kopra and coconut oil (1). In coconut water contain carbohydrate, protein, fat, vitamin, and minerals (2,3). Coconut water can be used for a body electrolytes subtitution, antidotes, antioxidants, to antibacterial beside for being consumed (4,5,6). Coconut water can also be used as a medium for making food products such as nata de coco (7, 8). Some traditional markets in Indonesia sell coconut meat and often not use the water.

Abundance and ease of the access to obtain coconuts can be used as opportunities for wider use of coconut water. Utilization of coconut water for bacterial growth media in the laboratory is one of the ways to access it.

The examination parameter which often carried out in the laboratory is the examination of Escherichia coli.

E. coli is the bacteria that can cause diarrhea in humans (9, 10, 11) and often used as a parameter of stool environmental pollution (12, 13, 14). Culture of E. coli from the sample often requires Mac Conkey as commercial media which is able to distinguish it from other bacteria in the Enterobacteriaceae group. Commercial media such as Mac Conkey certainly has a

quite expensive price because of its ability as a growth medium for Enterobacteriaceae.

Therefore, in this study were tried to compare viable count of E. coli recovered from Coconut water medium and Mac Conkey as gold standard for growing E. coli.

MATERIALS AND METHODS

This study was an experimental study using complete randomized design. Variable of this study were coconut water concentration (0%, 20%, 40%, 60%, 80%, 100%) and numbers of E. coli colony. This study conducted in Microbiology Laboratorium at Universitas Nahdlatul Ulama Surabaya, Indonesia.

This study used E. coli ATCC 25922 that were obtained from BBLK (Balai Laboratorium Kesehatan) Surabaya. E. coli need to subculture in nutrient agar slant before used. Twenty-four bacterial culture in nutrient agar slant then suspend in sterilize 0.85% natrium chloride until it reaches Mac Farland 0.5 turbidity.

Coconut water between 6 and 8 months of age were obtained from traditional market.

Coconut water was extracted by breaking coconut endocarp.

Coconut water obtained then analyse for chemical characters, namely carbohydrate, protein and fat component in Balai Riset dan Standarisasi Industri Surabaya (Baristand Industri Surabaya) Indonesia. Composition of the coconut water media per 100mL were