Sanaa Osman Yagoub Ahmed April - Neelain University

بسم الله الرحمن الرحيم

AL Neelain UniversityGraduate College

Effect of low Electrical voltages on the viable counting of

Escherechia coli and Staphylococcus aureus

A Complimentary Research Submitted inPartial Fulfillment of the Requirement for the

Degree of Master of Science (M.Sc.) in

Molecular Microbioloy

By

Muzdalifa Omer Adam Abd Arraheem

SupervisorProfessor: Sanaa Osman Yagoub Ahmed

April 2020

1

بسم الله الرحمن الرحيم

جامعة النيلين�كلية الدراسات العليا

أثر الجهد الكهربائي المنخفض على العد�

الحيوي لبكتيريا� اإلشريكية� القولونية�

والمكورة العنقودية� الذهبية�

بحث تكميلي لنيل درجه الماجستير في األحياء الدقيقة الجزيئية

:إعدادمزدلفة عمر آدم عبدS الرحيم

:إشرافأ.د سناء عثمان يعقوب

2020أبريل

2

اآلية الكريمة

قال تعالى:ذي تشربون ءأنتم68))أفرأيتم الماء ال

نشاء لو69نحن المنزلون أم المزن من أنزلتموه) (70تشكرون أجاجا فلوال جعلناه

صدق الله العظيمسورة الواقعة

Dedication

I

I dedicate this work to :

My mother

My aunt

My uncles

My grand mother

My brothers

My sisters

My friends

&

To all precious people with endless love

Acknowledgement

II

First of all I would like to express my deep gratitude to Allah for assisting me to complete this work.

Deep thanks to my supervisor Prof. Sanaa Osman Yagoub for her profound guidance.

I also would like to extend my thanks and appreciation for the people who helped me throughout the

research.

Particular thanks are also to Dr ibn aouf, Head of the Department of Microbiology and Molecular

Biology, Dr. Elfadil , Dr. Rabea, Miss. Amira, Mr. abdul hadi and Mr. abd assakhi who provided me

with whatever facilities needed during the course of study. My thanks also extend to my brighter

colleagues, and to everyone who participated in this research or helped me . Deep appreciations to the

faculty staff for the support provided throughout the study.

Last but not least I thank my great family for their aid and continuous support specially my uncles

who encouraged me during my study and making every thing in my life possible.

Abstract

III

Safe drinking water is necessary for human health all over the world. Being a universal solvent, water

is a major source of infection. According to world health organization (WHO) 80% of diseases are

water borne. 3.1% of deaths occur due to the unhygienic and poor quality of water. The direct current

(DC) effects on bacterial cells have been studied for several decades, viability studies have

concentrated on the use of pulsed high voltage for inactivation or moderate voltage for many hours or

many days. This study aimed to determine the effect of Alternating current on growth of E.coli and

Staphylococcus aureus at different voltages. Using a carbon wire, five, ten , fifteen, twenty , twenty-

five and fifty voltage alternating current (AC) current were adjusted in a voltmeter and electricity

was conducted for 10 minutes to each tube of 1×10 -1 to 10 -5 serially diluted E.coli and

Staphylococcus aureus and immediately cultured in plate count agar (PCA) medim; two plates for

each dilution, this dilution culture considered as after-electrical current treatment. The plates were

incubated at 37ºC for 24 hours, the bacterial colony were counted using colony counter. Statistical

analysis showed that both voltage and dilution of S. aureus and E. coli has clear effect on the

inhibition of the bacterial viable count. The difference in mean of data pre electrical current and after

electrical current treatment indicates that the electrical current has an effect on bacterial count. At 20

voltages above results showed that there was a complete inhibition of the growth of both tested

bacteria at 10 -4 and 10 -5 dilutions. This study encourage introduction of electric current for drinking

water treatment as alternatives for chemical disinfectants which has negative effect on human health.

IV

ملخ

ص البحث

تعتبر المياه الصالحة للشرب مهمة لص�حة االنس�ان في جمي�ع انح�اء الع�الم. ولم�ا ك�ان

% من االم��راض تنتق��ل80الماء مذيب عام فهو يعتبر مصدر رئيس للعدوى ل��ذلك ف��إن

% من الوفيات تحدث3.1عن طريق الماء وذلك حسب تقرير منظمة الصحة العالمية و

نتيجة تلوث المياه. لقد تمت دراسة اثر التيار المباش��ر على الخالي��ا البكتيري��ة من��ذ ع��دة

عقود ودرست فيها حيوية الخاليا بالتركيز على استخدام اما نبضات جهد كهرب��ائي ع��الي

لتثبيط النمو او جهد متوسط لعدة ساعات او ايام. هدفت هذه الدراس��ة الى تحدي��د أث��ر

التيار الكهربائي المتردد على نمو بكتريا االشريكية القولونية والمكورة العنقودية الذهبية

،15،� 10،� 5باستخدام عدة فروق جهد منخفضة . باستخدام سلك الكربون، و تم ضبط

10 فولت من التيار المتردد على جهاز الفولتميتر وتم توصيل التيار لمدة 50، و25،� 20

.10-� 5 الى 10-� 1دقائق النابيب اختبار تحتوي على البكتيريا مخففة تخفيفا عشريا من

بعد توصيل التيار مباشرة تم تزريع البكتيريا في بيئة اآلج��ار للع��د البكت��يري ؛ تم ال��تزريع

في طبقين لكل تخفيف ألخذ متوسط العد ليعتبر ذلك عدد البكتيريا بعد مرور التيار. وتم

ساعة واستخدم في الع��د جه��از ع��داد24م لمدة 37ºتحضين االطباق في درجة حرارة

المستعمرات. واثبتت التحاليل اإلحصائية ان هناك أثر إلختالف الجه��د و ترك��يز البكتيري��ا

على تثبيط العد الحيوي لبكتيريا االشريكية القولونية والمكورة العنقودية الذهبية ويت��بين

هذا األثر في الفرق بين متوسطات اعداد البكتيريا قبل م��رور التي��ار وبع��د م��رور التي��ار.

. 5- 10و 4- 10 فولت فاكثر حدث تثبيط تام للبكتيريا عند التخفيفين 20عند استخدام

هذه الدراسة تش��جع على اس��تخدام التي��ار الكهرب��ائي كطريق��ة لمعالج��ة مي��اه الش��رب

كبدائل للمعالجة الكيميائية والتي لها أثر واضح على صحة االنسان.

V

Table of Contents

Number Subject Page

Holy Quran I

Dedication II

Acknowledgement III

Abstract (English) IV

Abstract (Arabic) V

Table of contents VI

List of Tables X

List of Plates XI

List of Figures XII

List of Appendices XII

Chapter One : Introduction and Literature Review

1.1 Introduction 1

Problem statements 1

Objectives 2

1.2 Literature Review 2

1.2.1 Water pollution 2

1.2.1.1 Microbial pollution of water 3

VI

1.2.1.2 Chemical water pollution 3

1.2.2 Sources of water pollution 4

1.2.2.1 Point sources of pollution 4

1.2.2.2 Non-point sources of pollution 4

1.2.3 Water-borne diseases 5

1.2.3.1 Historical perspective of water-borne diseases

5

1.2.3.2 Bacterial water-borne diseases 5

1.2.3.3 Viral dater-borne diseases 6

1.2.4 Water Treatments 7

1.2.4.1 Traditional Treatment Methods 7

1.2.4.2 Advanced Methods of Treatments 8

1.2.5 Effect of Electrical Current on Bacteria 9

1.2.6 Test Organisms 9

1.2.6.1 Escherichia coli 9

1.2.6.2 Staphylococcus aureus 10

Chapter Two: Materials and Methods

2 Materials and Methods 13

2.1 Preparation of culture media 13

2.1.1 Nutrient agar 13

2.1.2 Eosine Methylene blue agar 13

2.1.3 MacConkey agar 13

2.1.4 Plate count agar 13

VII

2.1.5 Manitol salt agar 13

2.1.6 DNA agar 13

2.1.7 M R-Vp medium 14

2.1.8 Pepton water 14

2.2 Preparation of test organismos 14

2.3 Confirmation of bacterial isolates 14

2.3.1 Gram stain 14

2.3.2 Biochemical tests and culture 14

2.3.2.1 Culture in EMB and MacConkey agar media 14

2.3.2.2 Indol test 15

2.3.2.3 Methyl Red ( MR ) test 15

2.3.2.4 Voges Proskauer ( VP ) reaction 15

2.3.2.5 Motility test 15

2.3.2.6 Catalase test 15

2.3.2.7 Coagulase test 16

2.3.2.8 Oxidase test 16

2.3.2.9 Culture in manitol salt agar ( MSA ) 16

2.3.2.10 DNAse test 16

2.4 Preparation of water sample 17

VIII

2.5 Inoculation of water sample and bacterial count

17

2.6 Effect of AC current on S. aureus and E. coli

17

2.6.1 Conduction and application of current 17

2.6.2 Bacterial count 18

Chapter Three: Results

3 Results 20

3.1 Identification of bacterial isolates 20

3.2 Effect of 5 volt current on different dilutions of bacteria 20






3.8 Statistical analysis 33

3.8.1 Effect of electrical treatment on viable count of S. aureus 33

3.8.2 Effect of electrical current treatment on viable count of E. coli

35

Chapter Four: Discussion, Conclusion & Recommendations

4.1 Discussion 39

4.2 Conclusion 41

4.3 Recommendations 41

IX

References 43

Appendices 51

List of tables

Table 1 Characterization of S. aureus. 20Table 2 Characterization of E. coli. 20Table 3 S. aureus count before and after 5 volt treatment. 21Table 4 E. coli count before and after 5 volt treatment. 21Table 5 S. aureus count before and after 10 volt treatment. 25Table 6 E. coli count before and after 10 volt treatment. 25Table 7 S. aureus count before and after 15 volt treatment. 26Table 8 E. coli count before and after 15 volt treatment. 26Table 9 S. aureus count before and after 20 volt treatment. 29Table 10 E. coli count before and after 20 volt treatment. 29Table 11 S. aureus count before and after 25 volt treatment. 30Table 12 E. coli count before and after 25 volt treatment. 30

X

Table 13 S. aureus count before and after 50 volt treatment. 32Table 14 E. coli count before and after 50 volt treatment. 32Table 15 Statistical analysis for S. aureus numbers before and after treatment. 33Table 16 Paired sample t test of S. aureus before and after treatment. 33Table 17 S. aureus count (mean, medium, standard deviation) before and after current 34Table 18 Statistical analysis for E. coli numbers before and after treatment. 35Table 19 paired sample t test of E. coli before and after treatment. 36Table 20 E. coli viable count (mean, medium, STD) before and after current. 37

List of Plates

Plate 1 Steps of treatment using volts 18

Plate 1 S. aureus count before and after treatment with 5 volt electricity 22

Plate 3 S. aureus viable cell count of 10-4 dilution before treatment with 5 volt. 23

Plate 4S S. aureus viable cell count of 10-4 dilution after treatment with 5 volt. 23

Plate 5 S. aureus viable cell count of 10-5 dilution before treatment with 5 volt. 24

Plate 6 S aureus viable cell count of 10-5 dilution tube after treatment with 5 volt. 24

Plate 7 E. coli viable cell count of 10-1 dilution treated with 15 volt 27

Plate 8 E. coli cell count of 10-2dilution before and after treatment with 15v. 27

Plate 9 E. coli cell count of 10-3dilution before and after treatment with 15v. 28

Plate 10 E. coli 10-4 dilution before and after 15v treatment. 28



XI

List of Figures

Figure 1 Effect of voltage and dilution on S. aureus count. 34Figure 2 Effect of voltage and dilution on E. coli viable count 36

List of appendices

Appendix 1descriptive statistic of S. aureus count result pre- and after current passage. 51

Appendix 2descriptive statistic of E. coli count result pre- and after current passage. 56

Appendix 3Statistical analysis of S. aureus data shows dilution effect. 61

Appendix 4Statistical analysis of S. aureus data shows paired sample differences64

Appendix 5Statistical analysis of E. coli data shows paired sample differences 64

XII

CHAPTER ONE

INTRODUCTION AND LITRATURE REVIEW

XIII

CHAPTER ONE

INTRODUCTION AND LITRATURE REVIEW

1.1 Introduction:

Since along time, water has been recognized as important as shown “By means of water God gives life

to every living thing”, (Islam: Quran 21:30). “Whoever believes in me, stream of living water will pour

from within him” (Christianity: John 7:38), it is the main dietary components and drinking water has to

be visually acceptable, being clear, colorless and without disagreeable taste or odor. Many

pathogens can be transmitted through water and cause diseases to humans and animals. Possible

pathogens include viruses, bacteria, including Salmonella, Vebrio cholerae, Campylobacter and

Shigella, and protozoa, including, Giardia lamblia and other Cryptosporidia (Berrin, 2008).

Water treatment is any process that improve the quality of water to make it more acceptable for a

specific end-use. however the use of different forms of chlorine for drinking water disinfection was a

major public health breakthrough to avoid outbreaks of waterborne diseases, but their use can lead to the

formation of unwanted disinfection by-products (DBPs), of which some are bioactive and genotoxic

(Richardson et al., 2007; Li and Mitch, 2018). In epidemiological studies, exposure to DBPs in

chlorinated drinking water has been associated with various human health effects, such as bladder

cancer, miscarriage and birth defects (Villanueva et al., 2015; Bove et al., 2002). There are numerous

conventional water treatment technologies available in rural areas of developing countries. Traditional

water purification methods include boiling, filtration, sedimentation and solar radiation (Sadhana and

Ashok , 2016 ). an innovative water treatment process, including suspended ion exchange, ozonation, in-

line coagulation, ceramic microfiltration, and granular activated carbon were developed(Johan et

al.,2019).

Problem Statement:

Despite the vital importance of water, the citizen of the Khartoum state has been suffered from the

scarcity of safe and potable drinking water, lack of sufficient amount of water as well as Lack of

sanitary measures to protect water plants. Otherwise an epidemic water-borne diseases have been

reported in some areas in Khartoum state, this necessitate to conduct periodic water researches to solve

problems and provide sufficient amount of water to meet all needs of increasing population ( WHO/

UNICEF, 2017).

1

Objectives:

This work aimed to:

- Examine the effect of low voltages alternative current on growth of E.coli and Staphylococcus aureus .

- Examine the activity of different voltages toward different concentration of bacterial cells.

-Determine electric power efficiency in removing bacterial contamination of water and improve new

treatment technologies that are more safe and cost- effective to solve consumers complains towards

the quality of water and consumers acceptance.

1.2 Litrature review:

1.2.1 Water pollution

Water pollution occurs when unwanted materials enter into water (e.g. lakes, rivers, oceans, aquifers)

and contaminate the water. This form of degradation occurs when pollutants are directly discharged into

water bodies without adequate remove of harmful compounds. This is harmful to environment and

human health. Water pollution affects the biosphere of plants and organisms living in these as well as

organisms and plants that might be exposed to the water. In almost all cases the effect is damaging not

only to individual species and populations, but also to the natural biological communities (Wikipedia,

2018).

According to a 2007 World Health Organization (WHO) report, 1.1billion people lack access to an

improved drinking water supply; 88% of the 4 billion annual cases of diarrheal disease are attributed to

unsafe water and inadequate sanitation and hygiene, while 1.8 million people die from diarrheal disease

each year. The WHO estimates that 94% of these diarrheal disease cases are preventable through

modifications to the environment, including access to safe water.

The specific contaminants leading to pollution in water include a wide spectrum of chemicals,

pathogens, physical contaminants … etc. Discharge of domestic and industrial effluent wastes, leakage

from water tanks, marine dumping, radioactive waste and atmospheric deposition are major causes of

water pollution. Water pollutants are killing sea weeds, mollusks, marine birds, fishes, crustaceans and

other sea organisms that serve as food for human. Mishra (2010) points several reasons of water

pollution in Delhi such as sewage and waste water, dumping of solid wastes and litters in water bodies,

industrial waste, acid rain, global warming, eutrophication.

2

1.2.1.1 Microbial pollution of water:

The world health organization (2006) in its" guideline for drinking water quality" publication

highlighted at least seventeen different and major genus of bacteria that may be found in tap water

which are seriously affecting human health .

Biofilm in distribution system may provide a favorable condition for opportunistic pathogens (e.g.

Legionella spp., Pseudomonas aeruginosa and Mycobacterium avim ) to colonize and may harbor

pathogens such as Salmonella enterica serovar typhimurium, which can enter the distribution System

(Berry et al., 2006 ; Parsek and Singh , 2003). In general, the greatest microbial risks are associated

with ingestion of water that is contaminated with human or animal feaces. Wastewater discharges in

fresh waters and costal seawaters are the major source of fecal microorganisms, including

pathogens (Grabow, 1996 ; WHO, 2008). Polluted water causes infectious diseases, like cholera,

typhoid fever and other diseases like gastroenteritis, diarrhea, vomiting, skin and kidney problems (Owa,

2013). Wastewater discharges in fresh waters and costal seawaters are the major source of fecal

microorganisms, including pathogens (Grabow, 1996 and WHO, 2008).

1.2.1.2 Chemical water pollution:

Heavy metals, industrial waste and toxins in industrial waste are the major cause of immune

suppression, reproductive failure and acute poisoning. Dhote et al. (2001) argue that the toxic chemicals

used in making the idols tend to cause serious problems of water pollution and also pose a serious threat

to the underwater ecological system. When immersed, these colors and chemical dissolve slowly leading

to significant alteration in the water quality. Kaur et al. (2013) study on assessment of idol immersion on

physio-chemical characteristics of river Yamuna in Delhi stretch revealed that idol immersion activity

has negative impact on water quality of river Yamuna. The composed data was analyzed for the year

2011, to understand deterioration in the water quality of the river due to idol immersion practices.

According to the results, the value of DO, BOD, Total Solids and COD were found to vary from 6.0-7.5

mg/L; 3.3-38 mg/L; 430-1268 mg/L; 28- 136 mg/L respectively. The low levels of DO and high BOD

and Total solids levels at different sites indicate the poor water quality due to idol immersions. .

Insecticides like DDT concentration is increasing along the food chain, These insecticides are harmful

for humans (Owa, 2013).

3

1. 2.2 sources of water pollution:

The most significant sources of water pollution are Sewage ( Waste Water ), Agricultural

Pollution, Oil Pollution, Radioactive Substances, River dumping and Marine Dumping. Pollution is the

result of the cumulative effect over time. All plants and organisms living in or being exposed to

polluted water bodies can be impacted. The effects can damage individual species and impact the

natural biological communities (wikipedia, 2019).

The causes of water pollution include a wide range of chemicals and pathogens as well as physical

parameters. Contaminants may include organic and inorganic substances. Elevated temperatures can

also lead to polluted water. A common cause of thermal pollution is the use of water as a coolant by

power plants and industrial manufacturers. Elevated water temperatures decrease oxygen levels, which

can kill fish and alter food chain composition, reduce species biodiversity, and foster invasion by new

thermophilic species.( Goel, 2006; Edward, 2018). Sources of water pollution are either point sources or

non-point sources.

1.2.2.1 Point source of pollution

Point source of pollution refers to discharges that enter surface waters through a pipe, ditch or other well

defined point of discharge. The term applies to wastewater and storm water discharges from a variety of

sources. Point sources have one identifiable cause of the pollution, such as a storm drain, wastewater

treatment plant or stream ( Brian, 2008).

1.2.2.2 Non point source of pollution (NPS) :

NPS pollution generally results from land runoff, precipitation, atmospheric deposition, drainage,

seepage or hydrologic modification. NPS pollution, unlike pollution from industrial and sewage

treatment plants, comes from many diffuse sources. NPS pollution is caused by rainfall or snowmelt

moving over and through the ground. As the runoff moves, it picks up and carries away natural and

human-made pollutants, finally depositing them into lakes, rivers, wetlands, coastal waters and ground

waters (EPA, 2019).

4

https://en.wikipedia.org/wiki/Wastewater_treatment_plant

https://en.wikipedia.org/wiki/Wastewater_treatment_plant

1.2.3 Water-borne diseases:

1.2.3.1 Historical perspective of water-borne diseases

Safe drinking water is necessary for human health all over the world. Being a universal solvent, water is

a major source of infection. According to world health organization (WHO) 80% of the diseases are

water borne. 3.1% of deaths occur due to the unhygienic and poor quality of water (Pawari and

Gawande, 2015). The Pollution across rivers have been causing acute water-borne diseases and health

problems that are affecting the human population which needs to be treated and also poses an economic

cost on people (Shahid and Saba, 2018). The same authors added that in a study published by John

Hopkins states that Pneumonia and Diarrhoea claimed lives of 1.5 million child under the age of five.

This prevails disproportionately in a few countries as 72 percent of these two diseases among children

deaths occur in just 15 countries and that In India, 296,279 children under the age of five died due to

Pneumonia & Diarrhoea in 2016.

Diarrhoeal disease alone amounts to an estimated 3.6 % of the total DALY global burden of disease

and is responsible for the deaths of 1.5 million people every year. It estimated that 58% of that burden,

or 842 000 deaths per year, is attributable to unsafe water supply, sanitation and hygiene and includes

361 000 deaths of children under age of five, mostly in low-income countries(WHO, 2019).

Although water-associated diseases in developing countries are prevalent, they are also a serious

challenge in developed countries. A study by Arnone and Walling (2007), who compiled data of

outbreaks in the U.S. (1986 – 2000), reported 5,905 cases and 95 outbreaks associated with recreational

water. Drinking water supplies in United States are among the safest in the world. However, even in the

U.S., drinking water sources can become contaminated, causing sickness and disease from

waterborne germs, such as Cryptosporidium, E. coli, Hepatitis A , Giardia intestinalis , and other

pathogens.

1.2.3.2 Bacterial waterborne diseases :

Gastrointestinal Illness (GI) caused by variety of different microbes and germs, which causes

symptoms, such as diarrhea, nausea, vomiting, fever, abdominal pain, was responsible for about 29.53%

cases. More than 27% of cases were caused by Shigella spp. Nearly 21% of the outbreaks were caused

5

https://www.cdc.gov/healthywater/drinking/public/water_diseases.html

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077002/#B2

by Shigella spp, In addition, 12.63% of the outbreaks were caused by E. coli 0157:H7. Besides acute

gastroenteritis, major etiological agents such as E. coli 0157:H7, V. cholera, and Salmonella were the

agents responsible for many outbreaks (Craun et al., 2006).

Although cholera infections have not been reported in recent years in developed countries mainly due to

improved sanitation, millions of people each year continue to get infected by Vibrio cholera in

developing countries (Nelson et al., 2009). A work by Edge et al. (2010) detected water-borne E. coli in

80% of water samples with E. coli levels of less than 100 CFU/100 ml. Another study by Wade et al.

(2006) reported significant positive trends between increased GI illness and indicator organisms at the

Lake Michigan beach, and a positive trend with indicators such as E. coli at a Lake Erie beach. Recently,

the use of indicator organisms (e.g., fecal coliforms, E. coli) for assessing pathogen levels has been

debated more often than ever; however, the use of indicator organisms is likely to continue for assessing

pathogen levels in water resources potentially because of the lack of an alternative reliable solution.

1.2.3.3 Viral water-borne disease:

Water-transmitted viral pathogens that are classified as having a moderate to high health significance by

the World Health Organization (WHO) include adenovirus, astrovirus, hepatitis A and E viruses,

rotavirus, norovirus and other caliciviruses, and enteroviruses, including coxsackieviruses and

polioviruses (WHO, 2011). Also, viruses that are excreted through urine like polyomaviruses ( WHO,

2011) and cytomegalovirus (Cannon et al., 2011) can potentially be spread through water. Other

viruses, such as influenza and coronaviruses, have been suggested as organisms that can be transmitted

through drinking water, but evidence is inconclusive. Most of the above viruses are most commonly

associated with gastroenteritis, which can cause diarrhea as well as other symptoms including abdominal

cramping, vomiting, and fever. It should be noted that some of these viruses could also cause more

severe illnesses including encephalitis, meningitis, myocarditis (enteroviruses), cancer (polyomavirus),

and hepatitis (hepatitis A and E viruses). Hepatitis E virus can also cause a mortality rate of up to 25%

in pregnant women. Viral infections are usually self-limiting in healthy individuals. They can cause

greater morbidity in children under the age of five, the elderly, immune-compromised people, and

pregnant women. Waterborne virus-based diseases may be higher in developing regions, where there is

widespread malnutrition and large populations of HIV-positive people. Regardless, there are few broad

spectrum anti-viral drugs to treat these diseases ( WHO, 2011). Several of these viruses have extremely

low infectious doses; the probability of infection from exposure to one rotavirus particle is 31%

6





( Reynolds et al., 2008). Viruses are shed in faeces in very high numbers even asymptomatically. For

example, up to 1011 norovirus particles can be present per gram of stool (Hall, 2012). In addition, non-

enveloped viruses can persist in water for long periods of time. When considering these characteristics,

inadequate disinfection of feacally contaminated drinking water could easily lead to outbreaks of viral

gastroenteritis from ingestion. Notably, drinking water can also transmit viruses via inhalation (e.g.,

showering) or contact with skin and eyes (e.g., swimming) causing respiratory and ocular infections.

( Fong and Lipp , 2005).

1.2.4 Water Treatments:

Access to safe water is fundamental human need and a basic human right declared Kofi Annan former

United Nations Secretary. General and Economically a country is pulled back when drinking water

supply is not proper (WHO, 2003), hence, Recognizing an economic and easily accessible system for

improving water quality remains as essential for a community especially when it is isolated from

mainland.

1.2.4.1 traditional treatment methods:

UN and UNICEF (2005) promote household water treatment and safe storage. Some places community

may have their own simple purification method. If such traditional knowledge made available will be

useful to others in own country as well as in other countries. Babu and Chaudhry (2005) reported a

filtration method using natural coagulant. This method could be preferred by rural people. WHO (2003)

had elaborated simple filtration method by using fabric mesh of stainless steel or polyester (aperture 50-

45 um), which was sufficient for removing algae cells and large protozoa. Chlorine is commonly used as

a disinfectant. Probably these two measures would be easily adopted by villagers. This could be supplied

to public by government with minimum financial expenditure. However, the purity of drinking water

from this process alone is not sufficient (Lechevailler and Keung, 2004).

an Ayurveda classic had written water purification method for drinking purpose by using following

flowers ,Utpala (Nelumbo nucifera), Naga (Mesua ferrea), Champaka (Michelia champaca) and

Patala (Stereospermum suaveolens) . Among different communities this is stated as an accepted method

for purifying water. According to the method, keep one of these flowers in water to be purified and after

a period of time decant for portability. However, a standard measure for this is not known. The flowers

mentioned are being regularly used by people in different countries. However, it is not known whether

7

water is fully purified by this simple procedure ( Skandhan et al., 2011). Hackett and Kingrey (2006)

invented a treatment system for treating fresh water in a cost effective manner to mix the disinfectant

with water. The invention includes a water storage tank connected to a water source, a chlorination

device connected to the water storage tank, a media filter partially contained within the water storage

tank and a pump connecting the media filter vessel with the water storage tank. Flow through the

chlorination device is regulated to provide adequate amounts of disinfectant to be released in tablet

form.

1.2.4.2 Advanced methods of treatment:

New biotechnological tools helps environmental sustainability and Innovative drinking water treatment

techniques reduce the disinfection-induced oxidative stress and genotoxic activity (Johan et al., 2019).

Enhanced biological phosphorus removal (EBPR) is an important biological process in wastewater

treatment where P can be removed without addition of chemicals ( Blackall et al., 2002 ; Melia et al.,

2017). EBPR exploits the capability of certain microorganisms, termed polyphosphate (poly-P)

accumulating organisms (PAO), to store large quantities of orthophosphate (ortho-P) intracellularly

as poly-P. This P-enriched biomass can be removed from the treated wastewater as surplus sludge

and used directly as fertilizer or for recovery of P. A new water purification techniques including

Boiling, Distillation, Reverse osmosis, Desalination, and In Situ Chemical Oxidation are invented and

used a form of advanced water treatment in addition to Bioremediation which is a technique that uses

microorganisms in order to remove or extract certain waste products from a contaminated area. About

filtration systems, Baird (2007) disclosed a water purification system consisting of a two-stages reverse-

osmosis (RO) filtration process utilizing a carbon block pre-filter and RO filter using an annular design.

Yoon et al. ( 2005) developed nanofiltration membrane based water purifier without the need for a

storage tank. The water purifier uses a nanofiltration membrane filter as the main filtering section and

does not require a storage tank for containment of the purified water. Tonelli et al. (2001) disclosed a

method for producing high purity water using dealkalization and a double pass reverse osmosis

membrane system with enhanced membrane life. The treatment method involves coagulant addition,

membrane filtration, ion exchange for dealkalization, decarbontation, and pH adjustment. Archer (2007)

developed a drinking water filter which can remove major contaminates from tap water and other

drinking water sources, and adjust pH. The water filter contains a cylindrical cartridge with sponge

filters used as dividers between different layers of filtration material and along a length of the cartridge.

8

https://www.ncbi.nlm.nih.gov/pubmed/?term=Skandhan%20KP%5BAuthor%5D&cauthor=true&cauthor_uid=22661834

The same author added that beside those there are other techniques as ion exchange systems, in which

Ion exchange resins used for water softening divided into two categories as cationic and anionic resins.

1.2.5 Effect of electrical current on bacteria:

The direct current (DC) effects on bacterial cells have been studied for several decades (Pareilleux and

Sicard,1970 ; Rosenberg et al., 1965) and the studies have focused mainly on the viability, metabolism,

and transport of the cells. In particular, viability studies have concentrated on the use of pulsed high

voltage for inactivation ( Dreesa et al., 2003) or moderate voltage for many hours or many days in some

cases (Valle et al., 2007). Even in bacterial biofilms; surface-adhering bacteria that form colonies

characterized by the production of an exopolysaccharide matrix in which they reside ( Hall-Stoodley et

al., 2004), the bioelectric phenomenon is a synergy between a relatively weak DC and the antibiotic

used to eradicate the biofilm bacteria ( Sandvik et al., 2013). There are limitations for the use of DC

currents, including weak currents, for the killing of vegetative bacteria Alternatively, new trials using

AC instead of DC have been conducted. Very high electric field strength ( Smith et al., 2009) or

moderate electric field strength for longer time was used to exert heating effect ( Lee et al., 2012). The

electrochemical method for the oxidation of organic pollutants for waste water treatment has attracted a

great deal of attention recently, mainly due to the development of new effective anode material (Mericas

and Wagoner, 1994) .In fact it has been found that the oxidation of organics takes place always with

simultaneous oxygen evolution, this has allow to search new anode material with high oxygen evolution

over potential in order to favor the reaction of organics oxidation over the side reaction of oxygen

evolution ( Errami et al., 2011 ; Bouya et al., 2012 ; Id El Mouden et al., 2012).

1.2.6 Test organisms:

1.2.6.1 Escherichia coli:

E. coli is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus

Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms)

(Tenaillon, 2010). Most of the E. coli strains are harmless, but some serotypes can cause serious food

poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination

(Vogt and Dippold, 2005 ; CDC, 2012). The harmless strains are part of the normal microbiota of the

9

https://en.wikipedia.org/wiki/Escherichia

https://en.wikipedia.org/wiki/Escherichia

https://en.wikipedia.org/wiki/Warm-blooded

https://en.wikipedia.org/wiki/Intestine

https://en.wikipedia.org/wiki/Genus

https://en.wikipedia.org/wiki/Coliform_bacteria

https://en.wikipedia.org/wiki/Bacillus_(shape)

https://en.wikipedia.org/wiki/Facultative_anaerobic_organism

https://en.wikipedia.org/wiki/Gram-negative_bacteria

gut, and can benefit their hosts by producing vitamin K2, and preventing colonization of the intestine

with pathogenic bacteria, having a symbiotic relationship (Wikipedia, 2019).

Escherichia coli remain one of the most frequent causes of several common bacterial infections in

humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and

other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with

diarrhoea in pet and farm animals ( Nerino et al., 2013). As a commensal it lives in a mutually

beneficial association with hosts, and rarely causes disease. It is, however, also one of the most common

human and animal pathogens as it is responsible for a broad spectrum of diseases. The peculiar

characteristics of the E. coli such as ease of handling, availability of the complete genome sequence and

its ability to grow under both aerobic and anaerobic condition, makes it an important host organism in

biotechnology. E. coli is used in a wide variety of applications both in the industrial and medical areas

and it is the most used microorganism in the field of recombinant DNA technology (Yoo et al., 2009).

Antimicrobial resistance is a major and increasing global healthcare problem (WHO, 2012). It was also

observed in animals, where the antimicrobials are used for therapy and prophylaxis of infectious

diseases (Szmolka and Nagy, 2013). As it present in the intestine of men and animals and are released

into the environment in faecal material. faecal matter is the main source for disease causing agents in

water and faecal bacteria are widely used as indicators of contamination which can affect rivers, sea

beaches, lakes, ground water, surface water, recreational water and the many diverse activities

associated with these (Ishi and Sadowsky, 2008).

1.2.6.2 Staphylococcus aureus:

Staphylococcus aureus is Gram-positive bacteria that are cocci-shaped and tend to be arranged in

clusters that are described as “grape-like.” On media, these organisms can grow in up to 10% salt,

and colonies are often golden or yellow. These organisms can grow aerobically or anaerobically

(facultative) and at temperatures between 18° C and 40° C. Typical biochemical identification

tests include catalase positive (all pathogenic Staphylococcus species) , coagulase positive (to

distinguish Staphylococcus aureus from other Staphylococcus species), novobiocin sensitive (to

distinguish from Staphylococcus saprophyticus), and mannitol fermentation positive (to distinguish

from Staphylococcus epidermidis) ( Rasigade and Vandenesch, 2014).

10

https://www.ncbi.nlm.nih.gov/pubmed/?term=Allocati%20N%5BAuthor%5D&cauthor=true&cauthor_uid=24287850

Infections are common both in community-acquired as well as hospital-acquired settings and treatment

remains challenging to manage due to the emergence of multi-drug resistant strains such as Methicillin-

Resistant Staphylococcus aureus ( Boucher and Corey, 2008).

Transmission is typically from direct contact. However, some infections involve other transmission

methods ( Rasigade and Vandenesch , 2014). Health-care-associated infections represent a major health

concern with a substantial impact on morbidity and mortality. The prevalence of nosocomial infections

is especially high in intensive care units (ICUs), where the occurrence of multidrug-resistant pathogens

is highest in the hospital ( Vincent et al., 2009). Staphylococcus aureus is a major agent of health-care-

associated infections that causes a wide range of diseases from mild to life-threatening conditions. It is

one of the most prevalent causes of nosocomial bacteraemia, hospital-acquired pneumonia, and surgical

site infections ( Vincent et al., 2009; Weiner et al., 2016 ).

11

CHAPTER TWO

MATERIALS AND METHODS

12

CHAPTER TWO

MATERIALS AND METHODS

2.1 Preparation of culture media:

All used media were prepared according to the manufacture instructions

2.1.1 Nutrient agar:

Twenty-eight grams of powder weighted using sensitive balance, added to flask of 1L of D.W. , mixed

and heated in water bath, sterilized by autoclave at 121°C for 15 minutes, and then poured (15ml /plates)

into Petri dishes that pre-sterilized in an oven and allowed to solidify .

2.1.2 EMB :

Prepared by added 35.9 gm of powder to 1L of D.W., mixed , heated in water bath, autoclaved at 121°C

for 15 minutes, then poured into sterile plates and allowed to solidify.

2.1.3 Macconkey agar:

Prepared by suspended 51.53 grams of powder in one liter of D.W. , boiled untill agar is dissolved

completely. Sterilized by autoclave at 15 Ibs pressure 121°C for 15 minutes, cooled to 45°C , poured

aseptically in sterile plates.

2.1.4 Plate count agar:

Prepared by dissolved 17.5 grams of powder in 1000ml of D.W., heated to be dissolved, autoclaved at

121°C for 15 minutes, poured aseptically in sterile plates.

2.1. 5 Manitol salt agar:

Prepared by dissolving 111.2 grams of media in 1000 ml of D.W., Boiled in water bath, sterilized in

autoclave at 121°C for 15 minutes, allowed to be cool and poured aseptically in sterile plates.

2.1.6 DNA agar medium:

Prepared by dissolving 42 grams of the medium in 1000 ml of D.W. , boiled in water bath, autoclaved

13

at 121°C for 15 minutes, allowed to cool to 45- 50 °C and then poured into plates.

2.1.7 MR –VP medium:

Prepared by adding 17.0 gms of powder to1liter of D.W, mixed well, distributed into test tubes and

Autoclaved at 121°C for 15 minutes.

2:1:8 Peptone water:

Prepared by adding 15 gms of powder to one liter of D.W , well-shacked , and distributed in test tubes

as 2 ml in each tube and sterilized by autoclaving at 121°C for 15 minutes .

2.2 preparation of test organisms:

E. coli and S. aureus isolates were obtained from National Center For Research, Khartoum, Sudan.

Single colony of each of E.coli and S. aureus was inoculated into nutrient agar medium to be confirmed.

2. 3 Confirmations of bacterial isolates:

2. 3. 1 Gram stain:

A drop of D.W was taken on a clean dry glass slide, a part of colony was taken using a sterile loop and

smear was made, allowed to dry in air, fixed using a flame, crystal violet was flooded on smear, allowed

to stay for 1 minute, washed by D.w, then the smears were flooded by logols iodine for 1 minute,

washed by D.W., de-stained by applying alcohol for 10 seconds, at last saffranin was applied for 2

minutes as counter stain, washed, allowed to dry, and then a drop of immersing oil was added and

examined in an oil immersion microscope lens. Gram positive organisms were purple, while gram

negative ones appeared as red- colored organisms.

.

2.3.2. Biochemical tests and culture:

Identification tests performed according to Monica Cheesbrough, (2008).

2.3.2.1 Culture in EMB and MacConkey agar media :

Using a loop, under aseptic condition, a part of single colony of bacterial culture was taken and

14

streaked in EMB medium, from the same colony a part was streaked into MacConkey agar medium,

cultured plates were incubated overnight at 37°C.

2.3.2.2 Indol test:

The test organisms were cultured in peptone water medium which contained tryptophan amino acid, the

inoculated cultures were incubated at 37 C° for 24 hours. Indol production was detected by kovac's

reagent, reddening of strip indicated positive test.

2.3.2.3 Methyl red (MR) Test

Glucose phosphate medium was inoculated with test organisms and incubated for 48 hours at 37 C°, two

drops of methyl red solution were added and the tube was shaken. Red color indicated positive reaction

and an orange color indicated negative reaction.

2.3.2.4 Voges Proskauer (V/P) Reaction:

The tested organism was inoculated in glucose phosphate medium and incubated for 48 hours at 37° C.

0.6 mL of 5% α-naphthol and 0.2 mL of 40% aqueous solution of potassium hydroxide were added, the

tube was well shaken and examined after 15 minutes and after one hour. A positive reaction was

indicated by appearance of strong red color.

2.3.2.5 Motility test:

A drop of the bacterial suspension was placed in the center of slides and covered with a cover glass

avoiding the trapping of air bubbles, using the corner of the heated slide, a drop of molten Vaseline was

placed on each corner of the cover glass. The slide was examined for motile bacteria using the 40X

objective lens.

2.3.2.6 Catalase Test:

A drop of 3% aqueous solution of hydrogen peroxide was placed on a clean glass slide, a small part of

the bacterial colonies was then placed in the hydrogen peroxide drop using a glass rod. Production of gas

bubbles indicated catalase expression.

15

2.3.2.7 Coagulase Test:

To detect bound Coagulase (clumping factor) a drop of physiological saline was placed on each two

slides. A small part of the bacterial colonies was emulsified in each slide to make two thick suspensions.

A drop of undiluted human’s plasma was added to one of the suspensions and mixed gently. Clumping

of the organisms within 10 seconds was considered as a positive reaction compared with the other slide

that was control.

2.3.2.8 Oxidase Test:

The organism was grown on nutrient agar, A piece of filter paper- approximately 2 cm, in diameter was

placed in Petri dish and drops of Tetramethyle 1-p Phenylene Diamine Di-hydrochloride were added,

small part of the colonies of the test organisms were streaked in the filter paper using non steel loop and

rubbed on the filter paper, dark purple color that developed within 5 to10 seconds was considered

positive result.

2.3.2.9 Culture in Manitol salt agar:

a single colony from N.A culture was taken and streaked into manitol salts agar ( MSA) medium,

Cultured plate was incubated overnight at 37c. Yellow colored colonies were noticed and confirmed as

S. aureus

2.3.2.10 DNAse test:

This was done for identification of Staphylococcus aureus , which hydrolyzed the DNA, a colony of

Staphylococcus aureus under test was inoculated onto small area in the middle of the DNA medium,

then the plates were incubated aerobically at 37°C for 18 to 24 hours and flooded with a few milliliters

of 1 mol/litter (3.6%) hydrochloric acid to be examined against a dark background, unhydrolyzed DNA

was precipitated and produced a white opacity or cloudiness in the Agar DNA , positive cultures were

surrounded by clear, unclouded zones.

16

2.4 preparation of water sample:

Nine ml of a tap water was collected in 5 clean, dry test tubes of 10 ml capacity and covered well using

a cotton and aluminum foil. Another water sample was taken in a clean, dry bottle of 250 capacities with

screw cap. All water samples were autoclaved at 121 ºC, 15psi for 15 minutes.

2. 5 Inoculation of water sample and bacterial count:

Under aseptic condition , from 18 hours E. coli culture plate, a loopfull was taken and diluted into a 250

ml capacity bottle containg sterile tap water to achieve a turbidity equivalent to 0.5 McFarland standard

(approximately 1.5 × 108CFU/ml). One ml of bacterial diluents (0.5 McFarland standard) was taken into

a tube of 9 ml sterile tap water and serial 10 fold dilutions was performed until 10−5. Then 100µ of each

dilution was taken into plates of plate count agar ( PCA) medium and speeded (two plates for each

dilution) , the plates were then incubated at 37ºC for 24 hours and this culture is considered as pre-

electrical current application culture.

2.6 Effect of AC current on S. aureus and E. coli:

Electrical current thoroughly affected bacterial cells resulted on cell death due to electrical shock. This

identified by decrease in numbers of bacteria (CFU) which demonstrated by viable cell count.

Electrical current applied for 10 minutes and cells was counted before and after

current application. Different electrical voltages used )5v, 10v, 15v, 20v, 25v and

50v( with different dilutions )10^-1 to 10^-5( to determine the effect of voltage

and if the density of cells can negatively or positively affect treatment process.

Normal drinking water used with its natural salts to identify true result regarding

natural conductivity of water.

2.6.1 Conduction and application of current:

Using carbon wire, 5volt current was adjusted in voltmeter and electricity was conducted for 10 minutes

to each tube of serial dilution immediately after culturing in PCA media. After this, another 100 µ was

taken immediately and distributed in PCA agar, two plates for each dilution was cultured. This culture

considered as after-electrical current culture. The plates were incubated overnight at 37ºC. This

procedure was applied using 10, 15, 20, 25 and 50 voltage electrical current for E. coli and S. aureus

bacteria separately.

17

2. 6.2 Bacterial count:

After incubation, the bacterial colonies were counted using colony counter, the mean of the two plates

for each dilution is counted and result was recorded.

18

Plate 1: Steps of water treatment using volts

CHAPTER THREE

RESULTS

19

CHAPTER THREE

RESULTS

3:1 Identification of bacterial isolates:

Bacteria were identified according to their morphological appearance, cultural characteristics and

biochemical reactions as S. aureus ( Table 1) and E. coli (Table 2 ).

Table 1: Characterization of S. aureus

Test ResultGram stain +ve cocciCulture in MSA agar +ve (manitol fermenter)

DNAse test +veCatalase test +veCoagulase test +veMotility test -ve

+ve: positive result, -ve: negative result

Table 2: Characterization of E. coli

Test ResultGram stain -ve rodsCulture in EMB agar Metalic green colonies ( metachromatic propertyCulture in MaCconkey agar Pink colonies (lactose fermenter)MR test +veVP test -veIndol test +veMotility test +ve

3.2 Effect of 5 volt current on different dilutions of bacteria:

It was clear that the activity of current was strong with the higher dilutions of tested bacteria. At 103,

104 and 105 , the numbers of S. aureus were dramatically reduced ( Table 3). At 103 and 104 , the numbers

of Staphylococcus aureus reduced from uncountable to 2120 and 350 CFU/100µl , respectively ,

while at dilution of 105 the number was reduced from47900 to only 480 CFU/ml ( Plates 2,3,4,5 and6).

Similarly in the same table the numbers of E. coli was reduced, at dilutions 103, 104 and 105 , E coli

numbers was reduced from uncountable numbers to 20000 and 5000 at dilutions of 103and 104

respectively. At dilutions of 105 E. coli number was reduced from 64000 CFU/ml to only 420 CFU/ml

(Table 4).

20

Table 3: S aureus count before and after 5 volt treatment for ten minutes:

Dilution Bacterial count pre-electrical current ) CFU/100 µl )

Bacterial count after-electricalcurrent treatment ) CFU/100 µl )

10-1 Uncountable Uncountable 10-2 Uncountable Uncountable10-3 Uncountable 2120

10-4 Uncountable 350

10-5 4790 48

Table 4: E coli count before and after 5 volt treatment for ten minutes:

Dilution Bacterial count pre-electricalcurrent ) CFU/100 µl )

Bacterial count after-electricalcurrent ) CFU/100 µl )

10-1 Uncountable Uncountable 10-2 Uncountable Uncountable10-3 Uncountable 200010-4 Uncountable 50010-5 6400 42

Generally , 5 volt current is very low voltage and it was found to affect the numbers of Staphylococcus

aureus and E. coli moderately when compared to high voltages . the results showed that the higher

dilutions (10-3 , 10-4 and 10-5) which contained low numbers of bacterial cells were affected more than

low dilutions ( 10-1 and 10-2) as it contains uncountable numbers )1×108 CFU/ml and 1×107 CFU/ml),

respectively which is considered as a very high bacterial cell numbers.

21

Plate 2: S. aureus count befor and after treatment with 5 volt electricity, Up: before treatment; down:

after treatment starting from low dilution leftmost and abstractly to high dilution rightmost.

22

Plate 3 : S. aureus viable cell count of 10-4 dilution before treatment with 5 volt.

Plate 4 : S. aureus viable cell count of 10-4 dilution after treatment with 5 volt.

23

Plate 5: S. aureus viable cell count of 10-5 dilution before treatment with 5 volt.

Plate 6: S. aureus viable cell count of 10-5 dilution tube after treatment with 5 volt.

24

3.3. Effect of 10 volt current on different dilutions of bacteria:

On the different dilutions of the bacteria at ten volt current treatment the activity was more strong than 5

volt current against the same bacteria. S. aureus numbers at 10-3 and 10-4 dilutions is reduced from

uncountable numbers to 15600 and 2800 CFU/ml respectively, while dilution of 10 -5 showed reduction

of bacterial number from 50000 to 300 ( Table 5 ). On the other hand E. coli numbers was not affected

at low dilutions ( 10-1 and 10-2 ) while at higher dilutions ( 10-3 and 10-4) that contained uncountable

numbers, they reduced respectively to 3120 and 1000 CFU/ml. At 10-5 dilution E. coli was reduced

sharply from 59000 to only 100 CFU/ml (Table 6). From the two tables (Table 5 and 6) It appeared that

the ten voltage of current was more active against E. coli than Staphylococcus aureus

Table 5: S. aureus count before and after 10 volt treatment for ten minutes:

Dilution Bacterial count pre-electricalcurrent (CFU/100µl)

Bacterial count after-electrical currenttreatment (CFU/100µl)

10-1 Uncountable Uncountable10-2 Uncountable Uncountable10-3 Uncountable 156010-4 Uncountable 280 10-5 5000 30

Table 6: E. coli count before and after 10 volt treatment for ten minutes:



10-1 Uncountable Uncountable10-2 Uncountable Uncountable10-3 Uncountable 31210-4 Uncountable 100 10-5 5900 10


25

Similar to 5v and 10v, when 15 volt electrical current used, Staphylococcus aureus numbers showed no

apparent effect at 10-1 and 10-2 dilutions. At 10-3 and 10-4 dilutions the numbers of Staphylococcus

aureus was reduced from uncountable numbers to 7480 and 1000 CFU/ml respectively whereas at 10 -5

the count was reduced from 49500 to only 100 CFU/ml ( Table 7). At same voltage of current at 10 -3 and

10-4 dilutions the numbers of E. coli were reduced from uncountable to 5000 and 800 CFU/ml,

respectively. At 10-5 dilution the numbers were reduced from 65000 to 40 CFU/ml ( Table 8, Plates 7,8

and 9).

Table 7: S. aureus count before and after 15 volt treatment for ten minutes:



10-1 Uncountable Uncountable10-2 Uncountable Uncountable10-3 Uncountable 74810-4 Uncountable 10010-5 4950 10




10-1 Uncountable Uncountable10-2 Uncountable Uncountable10-3 Uncountable 50010-4 Uncountable 8010-5 6500 4

26

Plate 7: E. coli viable cell count of 10-1 dilution treated with 15 volt, left: before treatment ; right:

after treatment.

Plate 8: E. coli cell count of 10-2dilution before and after treatment with 15v. left: before treatment;

right: after treatment

27

Plate 9: E. coli cell count of 10-3dilution before and after treatment with 15v. left plate: before

treatment; right 2 plates: after treatment

Plate 10: E. coli 10-4 dilution before and after 15v treatment, left: before treatment; right: after

treatment

28


As shown in Table 9 the 20 volt current has strong activity against all dilutions, the numbrs of

Staphylococcus aureus were reduced from uncountable to 860 , 170, 40 and zero CFU/ml at

dilution of 10-1, 10-2, 10-3 and 10-4 respectively. At 10 -5 dilution the numbr of Staphylococcus aureus

was reduced from 40000 to zero CFU/ml . Similar results were obtained with E coli bacteria

where it reduced from uncountable numbers to 500, 30, 40 and 10 CFU/ml at dilutions of 10 - 1,

10-2,10-3 and 10-4 respectively, and at dilution of 10-5 the E. coli count was reduced from 48800

to zero CFU/ml (Table 10) .

Table 9: Staphylococcus aureus count before and after 20 volt treatment for ten minutes:



10-1 Uncountable 8610-2 Uncountable 1710-3 Uncountable 410-4 Uncountable 010-5 4000 0






It is clear that the high voltage current has stronger activity against the bacteria. The number of

Staphylococcus aureus ( Table 11) were reduced from uncountable to 330 , 10 , 10 and zero

CFU/ml at dilutions of 10-1 ,10-.2, 10-3 and 10-4 , respectively, while E. coli numbers were reduced

29

to 300 , 10 , 20 and zero CFU/ml at the same last dilutions ( Table 12, Plate 11 and 12). At 10 -5

dilution the numbers of Staphylococcus aureus were reduced from 51000 to zero while the numbers

of E. coli were reduced from 58000 to zero in the same manner.





Table 12: E coli count before and after 25 volt treatment for ten minutes:




30

Plate 11: E. coli 10-4 dilution before and after 25v treatment, left: before treatment; right: after

treatment

Plate 12 : E. coli 10-2 dilution before and after 25v treatment, down: before treatment; up: after

treatment

31


Staphylococcus aureus was reduced from uncountable numbers to 20 , 30 , 20 and 10 at dilutions 10-1 ,

10-2, 10-3 and 10-4 respectively. At 10-5 the numbers of Staphylococcus aureus was reduced from

48000 to zero CFU/ml ( Table 13) . Similarly E coli showed reduction of the count from uncountable

numbers to 20, 10 , 10 and zero at dilutions of 10-1 ,10-.2, 10-3 and 10-4 respectively, while dilution of

10-5 showed reduction of E. coli number from 50000 to zero CFU/ml ( Table 14).







Bacterial count after-electrical current(CFU/100µl)

10-1 Uncountable 2

10-2 Uncountable 110-3 Uncountable 110-4 Uncountable 010-5 5000 1

3.8 Statistical analysis

SPSS v21 is used for analysis

32

Table 15: Statistical analysis for S. aureus numbers before and after electrical current treatment.

Mean N Std. Deviation Std. Error Mean

Pair 1

pre elect 2230954.60 30 3968672.078 724577.074

after elec

1479.87 30 2606.427 475.866

3.8.1 Effect of electrical current treatment on viable count of S. aureus :

Table 15 showed that there was difference in mean of data which was changed from 2230954.60

before electrical current treatment which revealed 1479.87 after electrical current treatment, this high

change indicate that the electrical current has an effect on bacterial count. Statistical result were

illustrated and explained at tables 15, 16 and 17 and Fig 13

Table 16 : paired sample t test of S. aureus before and after current treatment.

Paired Samples Test

Paired Differences t df Sig. (2-tailed)

95% Confidence Intervalof the Difference

Upper

Pair 1pre elect - after elec

3710977.565 3.078 29 .005

The sig.(2-tailed) column displays the probability of obtaining a t statistic whose absolute value is equal

to or greater than the obtained statistic. Since the significance value for change in 0.05, we concluded

that the average loss of 2229474.733 ( see appendix no4) is not due to chance variation, and can be

attributed to the passage of voltage and change in dilution.

33

Figure 1: Effect of both voltage and dilution on S aureus count.

Vertical axis represent numbers of bacterial cells.

Table 17: S aureus data (mean, medium, standard deviation) before and after electrical powerapplication .

voltag

e

Mean)befo

re

current(

Mean)aft

er

current(

median)befo

re(

Median-

after

STD)befor

e (

STD.after

5 volt 22311449.20

3108.40 100000.00 2000.00 4362407.5 3199.25

10 volt 22311449.20

2684.40 100000.00 312.00 4362407.5 3502

15 volt 22311449.20

2716.8 100000.00 500 4362407.5 3476.7

20 volt 22311449.20

11.6 100000.00 3 4362407.5 21

25 volt 22311449.20

6.6 100000.00 1 4362407.5 13.107

50 volt 22311449 1.8 100000.00 1 4362407.5 .707

34

.20

Constant value of 22311449.20 obtained as a mean for all five dilutions before electrical current

application while when numbers of viable cell count analyzed after electrical treatment they were

3108.40 , 2684.40, 2716.8, 11.6, 6.6 and 1.8 for 10 -1 , 10-2 , 10-3 ,10-4 and 10-5 respectively with also

fixed value of mediums that was 100000.00 for all five dilutions before electrical current versus

2000.00 , 312.00 , 500 , 3 , 1 and 1 for 10-1 , 10-2 , 10-3 ,10-4 and 10-5 after current treatment

respectively . This constant values of means obtained before current indicate that no significant effect on

data. The mean value decreased gradually from 3108.40 to 2684.40, 2716.8, 11.6, 6.6 and 1.8 and the

numbers are inversely proportion to the voltage used. This proportioning appeared in median and STD

values too. All changes occurred indicate how voltage and dilutions affected living S aureus cells.

3.8.2 Effect of electrical treatment on viable count of E. coli:

Statistical results of the effect of the electrical current treatment were illustrated and explained at tables

18, 19 and 20 and Fig 14.

Taple 18: Statistical analysis of E coli shows the difference in mean before and after electrical

current.

Paired Samples Statistics

Mean N Std. Deviation Std. Error Mean

Pair 1

pre elect 2231149.20 30 3968559.174 724556.460

after_elect

1421.47 30 2619.569 478.266

The mean was changed from 2231149.20 to 1421.47 and the standard deviation was changed from

3968559.174 also, The difference in mean and STD of data pre electrical current and after electrical

current treatment indicate that the electrical current has an effect on bacterial count.

35

Figure 2: Effect of both voltage and dilution on E. coli viable count.

Table 19: paired sample t test of E coli before and after current treatment.

Paired Samples Test

Paired Differences t df Sig. (2-tailed)95% Confidence

Interval of theDifference

Upper

36

Pair 1pre elect - after_elect

3711177.589 3.078 29 .005

The sig.(2-tailed) column displays the probability of obtaining a t statistic whose absolute value is equal

to or greater than the obtained statistic. Since the significance value for change in 0.05, we concluded

that the average loss of 2229727.733 ( see appendix no 5) is not due to chance variation, and can be

attributed the passage of voltage and change in dilution

table 20: E coli viable count (mean, medium, standard deviation) before and after electrical power

application .

voltage Mean

)before

current(

Mean

)after

current(

Median

)before(

Median-

after

STD

)before (

STD. after

5 volt 22311449.2

0

3108.40 100000.00 2000.00 436207.57 3199.25

10 volt 22311449.2

0

2684.40 100000.00 312.00 436207.57 3502.7

15 volt 22311449.2

0

2716.8 100000.00 500.00 436207.57 3476.7

20 volt 22311449.2

0

11.60 100000.00 3.00 436207.57 21

25 volt 22311449.2

0

6.60 100000.00 1.00 436207.57 13.107

50 volt 22311449.2

0

1.00 100000.00 1.00 436207.57 0.707

E coli also showed constant value of 22311449.20 obtained as a mean for all five dilutions before

electrical current application while when numbers of viable cell count analyzed after electrical treatment

they were 3108.40 , 2684.40, 2716.8, 11.60, 6.60 and 1.00 for 10 -1 , 10-2 , 10-3 ,10-4 and 10-5

respectively. A fixed value of mediums that was 100000.00 for all five dilutions also was obtained

for data before electrical current versus 2000.00 , 312.00 , 500 , 3.00 , 1.00 and 1.00 for 10-1 ,

10-2 , 10-3 ,10-4 and 10-5 after current treatment respectively. These constant values of means obtained

before current indicate that there were no effect on data. The mean value decreased gradually from

37

3108.40 to 2684.40, 2716.8, 11.60, 6.60 and 1.00 and the numbers are inversely proportion to the

voltage used. This proportioning appeared in median and STD values that were also decreased

gradually. All changes occurred indicate effect of voltage and dilutions in E coli counts.

CHAPTER FOUR

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

38

CHAPTER FOUR

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

4.1 Discussion

This study considers Gram negative and Gram positive bacteria in an effort to contribute to scarce

knowledge available concerning the different effects of low intensity alternating currents on bacterial

cell counting, it focused on the effects of low electric current on the growth of E. coli and

Staphylococcus aureus. The effect of the current treatment with 5, 10 and 15 volt on 10-1 and 10-2

dilution culture of both E. coli and Staphylococcus aureus revealed no effect, whereas notable

inhibition was observed using 20, 25 and 50 volt. At dilution of 10-5 both tested bacteria showed clear

reduction of the total viable count at all tested current volt, this confirm the finding of Kekez et al.

( 1996) who reported that treatment of bacteria with high intensity current causes an irreversible loss of

membrane function. The enumeration of both bacteria was decrease with the increase of the current

strength and increase of the bacterial dilution , these might be explained by findings of Valle et al.(2007)

who found that when low voltage was used membrane damage causing permeability alteration and the

leakage of cellular contents; according to authors microbial death may be caused by the influx of toxic

substances through the disrupted cell membrane, whereas under much stronger field current, permanent

membrane disruption occurs and the death of cells occurs by mechanical causes. The lethal AC high

electric field effect mentioned in many researches is due to either the direct energy effect on high

voltage pulse on the cell membranes causing electroporation or to the release of toxic ions from the

used electrodes due to oxidation of the metal ions of the anode resulting in the dissolution of the

anode (Giladi et al, 2008).

39

In this study, E.coli seems to be more sensitive to increasing current intensity than Staphylococcus

aureus. The high sensitivity of Gram negative bacteria towards electric current was confirmed by Davis

et al. (1989) who found that both E. coli and Salmonella typhimurium were inhibited and killed by low

microamperage. The authors evidenced that the effectiveness of electric current in inhibition of growth

and mortality is directly related to increasing microamperage and inversely related to the bacterial

concentration. Bayer and Sloyer (1990) concluded that different parameters could affect behavior, like

the presence of lipopolysaccharides (in the Gram negative bacteria), capsule and surface proteins. Some

studies carried out with low voltage current investigated the effects of microamperage on the viability of

bacteria such as E. coli and Proteus sp. The results showed that even low microamperage can be

effective in reducing the number of microorganisms and inhibiting bacterial growth (Davis et al., 1989) ,

Palaniappan et al.( 1992) related the reduction of the Yeast and bacteria to the changes in the cell

morphology, with the formation of nonhomogeneous area in the cytoplasm and large concentrations of

debris in the culture depending on the voltage intensity and electric discharge frequency. Our results are

in agreement with literature data, in fact Valle et al.(2007) studied the effect of low electric current

(LEC) treatment on pure culture of E. coli and Bacillus cereus , the authors concluded that the

bacteriocidal effect depended on the current passing through the cell suspension and the bacterial death

are extremely complex and involve a number of interactions between microorganism medium and

electrode materials.

The DC effects on the bacterial cells have been studied for several decades (Pareilleux and Sicard, 1970)

and the studies have focused mainly on the viability, metabolism and transport of cells. In particular,

Viability studies have concentrated on the use of pulse high voltage for inactivation (Dreesa et al., 2003)

or moderate voltage for many hours or many days in some cases (Valle et al., 2007). Alternatively, new

trials using Ac instead of DC have been conducted. Very high electric field strength (Smith et al., 2009)

or moderate electric field strength for longer time were used to exert heating effect (Lee et al. ,2012)

both condition are still not optimum for in vivo application. Some studies have reported the effect of

the low electrical current (LEC) on yeast cells , Ranalli et al. (2002) demonstrated that LEC

intensities (10, 30 and 100 mA) could reduce the ATP content and viability of both S. cerevisae and H.

guilliermondii culture. In addition LEC treatment on S. cerevisae resulted in the loss of integrity of the

cytoplasmic membranes.

40

Many experiments in this field have been described, but the standardizing of the processes has incurred

difficulties related to the nonhomogeneous experimental conditions and the numerous parameters that

must simultaneously be taken into consideration ( voltage, current intensity, possible electrode use and

duration of the treatment).

As many industries produce wastewater containing toxic organic pollutants, there has been a notable

increase in both research and the number of businesses concerned with the treatment of such industrial

effluents, including biological, physical and chemical processes. Disinfection of drinking water using

chlorine can lead to formation of genotoxic by-products when chlorine reacts with natural organic

matter (NOM). Electric current has recently been targeted for the development and optimization of

innovative techniques to disinfect water and food, but its application is still far from being well-

established.

Studies have reported successful electric current applications on complex matrices of continuous low

intensity electrical current in the biomedical field (Stoodley et al., 1997), animal manure treatment

(Ranalli et al., 1996) and in food sector (Lustrato et al., 2006). There were some researches for using

AC on bacteria to avoid drawback of using DC (Del Pozo et al., 2009; Dzidic et al., 2008) but most of

them by using high pulsed electric fields as in case of food preservation (Lee et al., 2013).

4.2 Conclusions

Electric current can be used as a physical method to kill E. coli and S aureus . This study concluded that

the electrical current has a clear effect on both E. coli and S aureus , The difference in mean and STD of

data pre electrical current and after electrical current treatment indicate that the electrical current has an

effect on bacterial count. It was clear that the best results were obtained with 10 -4, and 10-5 dilutions

and when the treatment was carried out at 20, 25 and 50 voltage, as both bacteria showed absence of the

growth in the plates indicating that the bacteria had been inhibited , this mean that the removal rate of

bacterial cell increases with increase of applied current voltage and increasing of dilution. Hence, this

study concluded that we can use electricity power for water treatment in order to improve water quality

and make it fit for drinking and of good quality in a bacteriological parameter. It was clear that electrical

power can be used successfully with low voltage as a physical method to remove or to kill two types of

41

bacteria E. coli and S. aureus present in water. Our results contribute to a better understanding of the

effects of applying LEC to bacteria, and enhance the potential for future applications in different fields.

4.3 Recommendations

This study recommended that:

1- More studies are needed in order to check if viable E. coli and S. aureus after electrical shock

are changed their characteristics or not , aware must be taken not to provide new strains of

bacteria

which may have new dangerous characters.

2- More efforts should be devoted to a better understanding of this new technology and more

researches should be complemented with the quantification of the amount of voltage taking in

consideration the current intensity and power of electricity in order to determine the exact price of

treatment process.

3- Study the effect of the electrical current on other water borne pathogens such as Shigella,

Salmonella , Vibrio …etc.

4- Study the effect of the electrical current using waste water and sewage.

5- More studies are needed to evaluate effect of the different duration of the treatment.

6- Further research on the feasibility and cost-benefit of scaling up of this process is needed.

42

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Epidemiol. 37: 1288-1301.

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https://en.wikipedia.org/wiki/PubMed_Central

https://doi.org/10.1177%2F003335490512000211




World Health Organization, 2006. Guidelines for Drinking water quality,

Volume 1: 3rd edition, WHO Press, Switzerland.

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Part 1:9-13; ISBN 978-92-4-159522-3. WHO Press, World Health Organization, 20 Avenue Appia,

1211 Geneva 27, Switzerland.

WHO (World Health Organization) 2008. Guidelines for Drinking-water Quality.

Incorporating 1st and 2nd Addenda, Volume 1, Recommendations. 3rd ed. WHO; Geneva, Switzerland.

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Press .

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sanitation-health/disease-risks/en/. [Date of access 15 august 2019].

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2003. WHO.

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Biotechnological Applications of Escherichia coli B: Is B for better. Springer; Berlin, Germany .

Yoon S.R., Kim S.S., Hyung H., Kim Y.H., 2005. Domestic nanofiltration membrane

based water purifier without storage tank. Publication of US6841068B1. Google patents.


Appendices:

Appendix 1: descriptive statistic of S aureus count result pre- and after current passage:

Descriptives

voltage Statistic Std. Error

pre elect 5 volt Mean 2231149.20 1950927.975

95% Confidence Interval for Mean

Lower Bound

-3185495.23

Upper Bound

7647793.63

51


https://en.wikipedia.org/wiki/Special:BookSources/978-92-4-159522-3


https://en.wikipedia.org/wiki/World_Health_Organization

5% Trimmed Mean 1923179.89

Median 100000.00

Variance1903059981330

3.203

Std. Deviation 4362407.571

Minimum 5746

Maximum 1E+007

Range 9994254

Interquartile Range 5472127

Skewness 2.188 .913

Kurtosis 4.815 2.000

10 volt

Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981330

3.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


15 volt Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance 19030599813303.203

52


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


20 volt

Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981330

3.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


25 volt Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981330

3.203


Minimum 5746

Maximum 1E+007

Range 9994254

53


Skewness 2.188 .913


50 volt

Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981330

3.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913

Kurtosis 4.815 2.000after_elect

5 volt

Mean 3108.40 1430.752


Lower Bound

-864.00

Upper Bound

7080.80


Median 2000.00

Variance 10235252.800


Minimum 42

Maximum 7000

Range 6958


Skewness .448 .913

Kurtosis -2.804 2.000

10 volt Mean 2684.40 1566.484

54


Lower Bound

-1664.86

Upper Bound

7033.66


Median 312.00

Variance 12269356.800


Minimum 10

Maximum 7000

Range 6990


Skewness .650 .913

Kurtosis -3.058 2.000

15 volt

Mean 2716.80 1554.854


Lower Bound

-1600.17

Upper Bound

7033.77


Median 500.00

Variance 12087851.200


Minimum 4

Maximum 7000

Range 6996


Skewness .642 .913

Kurtosis -3.043 2.000

20 volt Mean 11.60 9.626


Lower Bound

-15.13

Upper Bound

38.33


Median 3.00

55

Variance 463.300


Minimum 0

Maximum 50

Range 50


Skewness 2.206 .913


25 volt

Mean 6.60 5.862


Lower Bound

-9.67

Upper Bound

22.87


Median 1.00

Variance 171.800


Minimum 0

Maximum 30

Range 30


Skewness 2.214 .913


50 volt Mean 1.00 .316


Lower Bound

.12

Upper Bound

1.88


Median 1.00

Variance .500

Std. Deviation .707

Minimum 0

Maximum 2

Range 2


56

Skewness .000 .913


Appendix 2: descriptive statistic of E coli count result pre- and after current passage:

Descriptives

voltage Statistic Std. Error

pre elect5 volt

Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981

3303.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


10 volt Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981

3303.203


Minimum 5746

57

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


15 volt

Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981

3303.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


20 volt Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981

3303.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913

58


25 volt

Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981

3303.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


50 volt

Mean 2231149.20 1950927.975


Lower Bound

-3185495.23

Upper Bound

7647793.63


Median 100000.00

Variance1903059981

3303.203


Minimum 5746

Maximum 1E+007

Range 9994254


Skewness 2.188 .913


aft5 volt

Mean 3108.40 1430.752


Lower Bound

-864.00

59

er_elect

Upper Bound

7080.80


Median 2000.00

Variance10235252.80

0


Minimum 42

Maximum 7000

Range 6958


Skewness .448 .913

Kurtosis -2.804 2.000

10 volt

Mean 2684.40 1566.484


Lower Bound

-1664.86

Upper Bound

7033.66


Median 312.00

Variance12269356.80

0


Minimum 10

Maximum 7000

Range 6990


Skewness .650 .913

Kurtosis -3.058 2.000

15 volt Mean 2716.80 1554.854


Lower Bound

-1600.17

Upper Bound

7033.77


Median 500.00

60

Variance12087851.20

0


Minimum 4

Maximum 7000

Range 6996


Skewness .642 .913

Kurtosis -3.043 2.000

20 volt

Mean 11.60 9.626


Lower Bound

-15.13

Upper Bound

38.33


Median 3.00

Variance 463.300


Minimum 0

Maximum 50

Range 50


Skewness 2.206 .913


25 volt Mean 6.60 5.862


Lower Bound

-9.67

Upper Bound

22.87


Median 1.00

Variance 171.800


Minimum 0

Maximum 30

Range 30

61


Skewness 2.214 .913


50 volt

Mean 1.00 .316


Lower Bound

.12

Upper Bound

1.88


Median 1.00

Variance .500

Std. Deviation .707

Minimum 0

Maximum 2

Range 2


Skewness .000 .913


Appendix 3: Statistical analysis of S aureus data shows dilution effect.

Descriptivesa,b,c,d,e

dilution Statistic Std. Error

after_elect 10^-1 Mean 3513.67 1559.148


Lower Bound

-494.25

Upper Bound

7521.58


Median 3525.00

Variance14585656.66

7


62

Minimum 2

Maximum 7000

Range 6998


Skewness .000 .845

Kurtosis -3.333 1.741

10^-2

Mean 3000.83 1341.268


Lower Bound

-447.01

Upper Bound

6448.67


Median 3001.50

Variance10794001.36

7


Minimum 1

Maximum 6000

Range 5999


Skewness .000 .845

Kurtosis -3.333 1.741

10^-3 Mean 469.83 317.433


Lower Bound

-346.15

Upper Bound

1285.82


Median 158.00

Variance 604580.967


Minimum 1

Maximum 2000

Range 1999


Skewness 2.088 .845

63


10^-4

Mean 113.50 79.395


Lower Bound

-90.59

Upper Bound

317.59


Median 40.50

Variance 37821.500


Minimum 0

Maximum 500

Range 500


Skewness 2.176 .845


Mean 9.50 6.682


Lower Bound -7.68

Upper Bound 26.68


Median 2.50

Variance 267.900


Minimum 0

Maximum 42

Range 42


Skewness 2.178 .845


Appendix 4: statistical analysis of S aureus data shows paired sample differences

Paired Samples Test

Paired Differences

Mean Std.Deviation

Std. ErrorMean

95% ConfidenceInterval of the

Difference

64

Lower

Pair 1pre elect - after elec

2229474.733

3967537.449 724369.920 747971.902

Appendix 5: statistical analysis of E coli data shows paired sample differences

Paired Samples Test

Paired Differences

Mean Std.Deviation

Std. ErrorMean

95% ConfidenceInterval of the

Difference

Lower

Pair 1pre elect - after_elect

2229727.733

3967395.577 724344.017 748277.878

65

Sanaa Osman Yagoub Ahmed April - Neelain University

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