REPORT ON ANTIMICROBIAL EFFECT OF METHYLATED SPIRIT ON ISOLATED

AND CHARACTERIZED BACTERIA FROM CURRENCY NOTES

BY:

BRIAN ORORI ONGOI

S131/0166/2016S

PRESENTED TO:

JARAMOGI OGINGA ODINGA UNIVERSITY OF SCIENCE AND TECHNOLOGY

SCHOOL OF BIOLOGICAL AND PHYSICAL SCIENCES

IN PARTIAL FULFILLMENT OF THE AWARD OF THE DEGREE THAT LEADS TO

BACHELOR OF SCIENCE IN BIOLOGICAL SCIENCES.

©2020
DECLARATION

I declare that this is my own duly done project and has not been presented in any university for
the award of any degree.

BRIAN ORORI ONGOI SIGN……………..

REG NO.: S131/0166/2016S DATE……………..

SUPERVISOR NAME:

Dr. BENSON ONYANGO

SIGN…………………………… DATE……………….
DEDICATION

First and foremost, I dedicate this project work to the all mighty God for strength and provision
of good health to carry out my project successful. I dedicate this too to my parents and also to the
school of biological and physical science for the award of the degree.
ABSTRACT

Paper currencies could be one of the most potential vehicles to transmit diseases amongst the
people. The daily transactions have made the paper currency to pass through many hands and
pathogens become imposed on them before they are finally deposited in banks. Modern scientific
studies have confirmed the presence of various pathogenic bacteria on paper currencies.
Amongst others, Staphylococcus aureus, Salmonella spps, Citrobacter spps, Mycobacterium
leprae, Shigella spps, Streptococcus spps, Pseudomonas aeroginosa, Klebsiella spps and
Escherichia coli were the dominant contaminants of paper currency samples.

Additionally, researches have also shown that paper currencies could be contaminated by several
fungal pathogens like Aspergillus niger, Aspergillus flavus, Rhizopus spps, Penicillium spps,
Candida spps Besides, several studies revealed that, paper moneys were also contaminated by
parasitic species of different helminthes that include parasitic nematodes and tapeworm like
Ascaris lumbricoides

Consequently, paper currency is largely contaminated with pathogenic microorganisms and this
contamination may play a significant role in the transmission of potentially harmful
microorganisms that are resistant to commonly used antibiotics and therefore represents risks and
public health hazards to the community and individuals handling paper currencies. So, there
needs frequent awareness development efforts to improve the poor hygienic practices being
exercised while handling paper currencies and this narrowed down to my study especially at our
school food Centre ,mess.
Table of Contents
DECLARATION………………………………………………………………………………………………………………………………2

DEDICATION…………………………………………………………………………………………………………………………………3

CHAPTER 1 Background information……………………..………………………………………………………………………5

Abstract ……………………………………………………………………………………………………………………………………………5

Introduction………………………………………………………………………………………………………………………………….6

Objectives …………………………………………………………………………………………….6

Hypothesis…………………………………………………………………………………………6

Problem statement…………………………………………………………………………………6

Justification ……………………………………………………………………………………….7

CHAPTER 2 Literature review….………………………………………………………………….8

CHAPTER 3: Material and methods ……………………………………………………………….9

Study site…………………………………………………………………………………………9

Sampling design………………………………………………………………………………….9

Biochemical tests………………………………………………………………………………...10

Catalase test……………………………………………………………………………………10

KOH test………………………………………………………………………………………10

CHAPTER 4 Results and data analysis…………. ………………………………………………..12

CHAPTER 5: DISCUSSION……………………………………………………………………….16

Why 70% alcohol is preferred…………….…………………………………………………….16

Microbial contamination of currency note………………………………………………………16

Potential risk in handling currency notes………………………………………………………..17

CHAPTER 6…………………………………………………………………………………………17

Conclusion and Recommendations…………………………………………………………….17

Acknowledgement……………………………………………………………………………….18

References ……………………………………………………………………………………………19
CHAPTER ONE

BACKGROUND INFORMATION
We all understand that money is an essential tool for exchange of commodities and services. In
all our communities, regionals, at national level and worldwide at large. However, money
(currency notes and coins) also play a negative role and that is; acts as agents of transmission of
disease causing organisms. The currency is usually contaminated during transaction, handling,
storage and contact with dirty surfaces. There is a certain practice of wetting fingers before
counting money which introduces an array of bacteria to notes and this routes in conjunction
with other routes pose a significant impact on public health of a nation.
Many people do not care the level of cleanliness of their fingers when handling money and pick
paper currencies with contaminated hands, leading to the contamination of paper notes with
micro-organisms. Market men and women squeeze paper currencies and put them in their dirty
pockets. Meat sellers collect money with their hands contaminated with blood and animal
wastes. Such money handling habits introduces microbes to the notes.
Some of the bacteria previously isolated and identified to be found in bank notes include;
klebsiella spp staphylococcus aureus, Escherichia coli, Bacillus spp. The widespread occurrence
of these bacterial species could lead to outbreaks of infections that might result in high morbidity
and mortality with great implications. Paper currencies are found to be contaminated with fungi
including Aspergillus niger, A.flavus, candida spp, fusarium spp The study aims to determine the
prevalence of pathogenic bacteria in joust currency notes especially in the mess and investigating
their resistance profile.

OBJECTIVES:
i. Isolate bacteria found on currency notes
ii. Identify the kind of bacteria found on the money.
HYPOTHESIS
i. Food borne illness is not associated with microorganisms found on currency notes.
ii. Currency notes do not harbor microorganisms.

PROBLEM STATEMENT
Money provides a good transaction tool in the market industry where it is used as an exchange
tool. The problem comes in during handling of the money where there is microbial
contamination in a number of ways; wetting of fingers when counting, contamination by
counting machines, prolonged exposure to water, contamination by illicit drugs Abraham et al..,
(1994).
JUSTIFICATION
The use of contaminated currency notes has been on circulation for a long time and the effect is
becoming rampart causing a significant harmful effect to humans. More strains of bacteria are
emerging and an intervention has to be reached.
This study will be aiming to curb the problem of human related problems as a result of
circulation of contaminated currencies at our disposal by use of methylated spirit to test its
antimicrobial effect currency notes.
CHAPTER TWO
LITERATURE REVIEW
Money is an invention of the human mind. Before money was introduced in this world,
economic exchange was practiced by barter. The barter economy, which involved the direct
exchange of one good for certain amount of a different good, is a simple economy where people
produce goods either for self-consumption or for exchange with other goods which they want.
However, the barter system is inconvenient as it involves much effort on the part of people in
trying to exchange goods for services.
Paper currency refers to notes of different denominations made of paper and issued by the central
bank or the government of a country. Globally, paper currency is widely exchanged for goods
and services. However, the combination of its widespread use and its constant exchange make
paper currency a likely agent for disease transmission. The raw materials from which paper
currencies are made also play significant role in harboring high microbial load. As studies have
shown, those paper currencies that are made of mixture of cotton and linen usually offer surface
area for microorganisms to reside on both sides.
Nevertheless, according to Vriesekoop, F. et al., polymer-based paper currencies presented lower
bacterial counts than cotton-based paper currencies. This may be due to various physicochemical
parameters of polymers. It is likely that the fibrous surfaces of cotton-based paper currencies
provide a good surface for microbial attachment. They further showed that, the longer the paper
currencies remain in circulation, the more chance there is for them to become contaminated, and
lower-denomination notes receive the most handling because they are exchanged more
frequently. Furthermore, in poorer societies, low value denomination currency notes and
especially coins are regularly exchanged unlike in richer communities that use high value
denomination. Contamination of different objects by potential pathogenic microorganisms is the
serious concern of public health because items that passed from one to another hand could
generate a chance of contamination with wide range of pathogenic microorganisms.
Most of the things we use in our everyday life work as a potential carrier of pathogenic
microorganisms. Though we ignore, unknowingly we used to bear many pathogenic
microorganisms through some of the media which we use in our everyday life. One such media
is our currency, which is used by people of all categories. It is generally documented that
physical transfer of material from hands, surfaces, and the environment can contaminate paper
currencies since almost every socio-economic setting regularly hold and transfer paper
currencies
In summary, people living in unhygienic conditions with unhygienic practices will contaminate
the paper currencies with microorganisms in the course of improper hand washing after using the
toilet, counting paper currencies using saliva, coughing and sneezing on hands subsequently
exchanging currencies, and placement or storage of paper currencies on dirty surfaces leads to
the contamination and these currencies will act as a vehicle delivering microorganisms to
contaminate the hands of the next user. Consequently, paper currencies have significant role in
the transmission of pathogenic microorganisms and present sensible risk to public health.
CHAPTER THREE

MATERIALS AND METHODS

Study site
The study was conducted around Jaramogi Oginga Odinga University. The University is located
in Bondo town in Siaya County. The university is located approximately 65km by road west of
Kisumu city. The Geographical coordinates are 0005’38.0”S,34015’31.0”E.

Sampling Design
A simple random sampling design will be used since every unit in the population has an equal
chance of being selected.
Sample collection
Samples were collected randomly from the cash drawer and kept in sterile carrier bags and taken
to the lab for analysis. Samples collected included 50, 100, 200, 500 and 1000 notes. An
additional samples of sterile notes of same denomination were be given in exchange with the
contaminated samples for use.
Media preparation
Nutrient Agar was used. The media was prepared according to manufactures instructions. The
required amount was measured into a reagent bottle and suspended into the required amount of
purified water. The bottle was heated with frequent agitation to boil until the media is completely
dissolved. The media was then autoclaved for 15 minutes at 1210C. The media was then be
maintained in a water bath at 500C.

Isolation of microbes from samples

Sterile cotton were dipped in the sterile distilled water and rubbed on both the surfaces of
currency notes, Serial dilution method was used where 9ml of sterilized distilled water was
added to falcon tubes labeled 10-1 to 10 -5 for each currency sample; the 50, 100, 200, 500 and
1000 notes. 1 ml of the initial suspension was transferred into the first tube 10-1. 1ml of this was
again transferred to the next tube labeled 10-2 up to the last tube.

Culturing
Pour plate method was used according to the method given by Maturin and peeler 2001.
1ml of each sample was transferred into a sterile labeled Petri dish. The nutrient agar media was
then poured into each Petri dish until the samples were fully covered then swirled left to right
then back and forth to ensure uniform spread. The Petri dishes were left to solidify in the
biosafety cabinet then incubated at 370C for 24 hours.

Microbial count and morphology Identification

The results were captured after 24 hours of incubation and colonies were counted by the use of
head colony counter. Counting of the colonies was done on two successive plates with the first
plate retained having countable number of colonies ranging 30-300.
Characterization and identification of the colony isolates was achieved by initial morphological
examination of the colonies in the plate for colonial appearance, size, elevation, color and
edge.

Isolation characterization
The different identified colonies were isolated into different plates for characterization. Nutrient
agar media was used. The media was first prepared according to the manufacturer’s instructions,
autoclaved and maintained in a water bath at 550C. Streak plate method was used. The
inoculating loop was first sterilized in a Bunsen burner flame until it is red hot then allowed to
cool. One isolated colony was picked from each agar plate. A well was first made at a point near
the edge of the plate from where streaks were made in different patterns with the plate held at an
angle.
Gram staining
This was done using four stains; crystal violet, Iodine, Alcohol and Safranin. Slides of the cell
samples were made by picking bacterial colonies using a heated wire loop and smearing on
sterilized glass slide to a diameter of 5mm. The slides were heat fixed by carefully passing the
slide over the flame of a Bunsen burner three times. Primary stain; crystal violet was added to the
sample slides then incubated for 1 minute. The slides were then rinsed gently with water for 5
seconds to remove unbound crystal violet.
Grams iodine was added- this acts as a mordant or an agent that fixes the crystal violet into the
bacterial cells. The samples were then rinsed with alcohol for three seconds then rinsed with a
stream of water. The secondary stain Safranin was added to the slides and incubated for 2
minutes and washed gently with a stream of water

Biochemical tests
Catalase test
This was done by transferring a small amount of colony growth in the surface of clean, dry glass
slides using a sterile wire loop. A drop of 3% hydrogen peroxide was then placed on the sample
on the glass slide. The slides were then examined for the evolution of oxygen bubbles.

KOH test
This was done by first preparing 3% potassium hydroxide solution. 3g of potassium hydroxide
pellets were weighed and transferred to a clean reagent bottle. 50ml of distilled water was added
and mixed well until the chemical was completely dissolved. The remaining 50ml of water was
then added to make the volume 100ml. A drop of 3% KOH was then applied on a clean
sterilized glass slide. Using a wire loop a generous amount of bacteria was transferred to the drop
of KOH.
Control experiment with sterilized currency
A control experiment was conducted where the currency notes in question were sufficiently
sterilized by gently rubbing off the surface with 70% alcohol containing cotton, after which
swabs were carried out to collect bacterial isolate from the surface of the notes; 50, 100, 200, 500
and 1000 notes.

Media preparation
Nutrient Agar was used. The media was prepared according to manufactures instructions. The
required amount was measured into a reagent bottle and suspended into the required amount of
purified water. The bottle was heated with frequent agitation to boil until the media is completely
dissolved. The media was then autoclaved for 15 minutes at 1210C. The media was then be
maintained in a water bath at 500C.

Culturing
Pour plate method was used according to the method given by Maturin and peeler 2001.
1ml of each sample was transferred into a sterile labeled Petri dish. The nutrient agar media was
then poured into each Petri dish until the samples were fully covered then swirled left to right
then back and forth to ensure uniform spread. The Petri dishes were left to solidify in the
biosafety cabinet then incubated at 370C for 24 hours.

Microbial count and morphology Identification

The results were captured after 24 hours of incubation and were observed no colonies were
formed or had grown.
CHAPTER FOUR

RESULTS AND DATA ANALYSIS

The money currencies collected from the counter at the mess were analyzed for bacterial
contamination. A number of bacterial isolates from different genera including; Streptococcus
spp. Staphylococcus spp, Micrococcus spp, Escherichia spp. Bacillus Enterococcus were isolated
from the samples. This confirmed that there are indeed bacteria on currency surfaces and this
therefore led to the rejection of the second null hypothesis; currency note do not harbor
microorganisms.

Table 1
Objective 2: Bacterial counts before and after sterilization from the currency samples
Currency notes Before sterilization After sterilization
50 note (10-5) 160 0
100 note (10-5) 102 0
200 note (10-5) 64 0
500 note (10-5) 5 0
1000 note (10-5) 3 0

Four different bacterial colonies were isolated from the initial cultures. These were characterized
according to colony characteristics as shown in table 2 below;

Table 2

Objective 3: Isolation and characterization

Colony Characteristics

1 Cream-yellow, round, raised, smooth margin and opaque

2 Large white, serrated, round, flat and translucent

3 Large white, filamentous, flat and undulated

4 Small white, raised and pinctiform

Gram staining and microscopy

Was done to characterize the bacteria and the following results were obtained.

fig 1,Gram negative bacterial cells under a microscope fig 2; Gram positive bacterial cells under a microscope

Gram staining of bacterial colonies showing gram negative rods

image; colony forming units in a 50 note 10-1

 image displaying KOH test taking place

Table 3
Identification of possible bacteria from the study

No. Gram stain description Possible bacteria

1 Gram positive Cocci Bacillus spp, staphylococcus aureus
2 Gram negative Chains, rods Enterobacteriaceae spp, Escherichia coli,
Enterobacillus

3 Gram negative Cocci, singly, Neisseria meningitis, N. gonorrhoeae, Maraxella

chains catarrhalis, Haemophilus influenza.

4 Gram positive Cocci, rods Bacillus spp, staphylococcus aureus, streptococcus

pyogenes, Staphylococcus epidermidis.

Biochemical tests were also performed using Catalase and KOH tests and the following results
were obtained.

Table 4; Biochemical Tests

Colony Biochemical test

Catalase test KOH test Coagulase test

1 Negative Positive Negative

2 Negative Positive Positive

3 Positive Negative Negative

4 Negative Positive Negative

DISCUSSION

Why 70% Alcohol is preferred and not 100%

70% of alcohol is ideal to a stronger solution. If this concentration of alcohol is poured to an

organism or microbe, the alcohol coagulates the protein, but at a slower rate, so that it penetrates
all the way through the cell before coagulation can block it. Then the entire cell is coagulated and
the organism dies. Ethanol percentage of 50-80% destroys the cell wall/membrane of bacteria by
denaturing their proteins and dissolving their lipids. It is effective against most bacteria, fungi
and some viruses.

Therefore, the ethanol has to pass through the bacterial membrane/wall to get into the bacteria- if
100% alcohol concentration is used the bacteria get sealed and they will survive…another
mechanism is the high osmotic pressure of ethanol/water- mixture, and the 70% has the highest
one. 90% ethanol concentration will evaporates fast.

70% alcohol has been found to be effective in sterilizing lab bench work area, killing the
microbes and other contaminants. Its mode of operation is by cell protein denaturation.

Microbial Contamination of Paper Money and Sources of Contamination

Some epidemiological studies have shown that contamination of different objects by potential
pathogenic microorganisms is of public health importance as contaminated materials can be
possible sources of transmission of such pathogens. Accordingly, several studies revealed that
paper currency have been identified as one of the Vehicle through which pathogens could be
transmitted and could be a particular risk to public health. Many people do not care how dirty
their fingers are when handling money: the butcher with the bloody fingers, the street-food
vendor with the wetly-oily fingers and others receive or pick the paper currency with
contaminated hands and therefore, leading to the contamination of the notes with
microorganisms.

Microbial contamination of paper currency could be from several sources, it could be from the
counting machine, atmosphere, during storage, usage, or handling. Daily transactions have made
the paper currency to pass through many hands and pathogens become imposed on them before
they are finally deposited in banks, Ogo, et al. also reported that the source of contamination
could be as a result of poor money handling practices like spraying during ceremonies where
such notes may be trampled upon when they fall on the ground.

The presence of various pathogenic microorganisms such as E. coli, Pseudomonas sps,

Klebsiella sps, Streptococcus sps and Staphylococcus sps, which are known to be responsible for
watery diarrhea, mouth skin diseases, pneumonia, respiratory track diseases, gastro- intestinal
diseases etc.
Potential Health Risk of Handling Contaminated Paper Currency Notes

Paper currency is commonly handled by various categories of people during transaction. Paper
money, therefore presents a particular risk to public health, since communicable diseases can
spread through contact with fomites and harbor various deadly pathogenic microorganisms.

Pathogenic microorganisms that may survive on paper currency may serve as a potential source
of enteropathogens causing food poisoning because food vendors may serve food with the hands
and at the same time handle paper currency as they sell. Such practices transfer bacteria from
paper currency to humans through food. This is enough to reject the first null hypothesis;
foodborne illness is not associated with microorganisms present on the currency notes.

CONCLUSION AND RECOMMENDATIONS

Paper currencies are one of the important fomite for infectious diseases in various countries due
to their wide circulation. The common infectious microorganisms include Citrobacter spps,
Mycobacterium leprae, Salmonella spps, Shigella spps, Escherichia coli, Staphylococcus aureus,
Pseudomonas aeroginosa, Klebsiella spps, Streptococcus spps, Micrococcus spps, Acinetobacter
spps, Aspergillus niger

From this study it can be concluded that currency notes in Jooust student eatery center (mess) is
commonly contaminated with pathogenic bacteria and this contamination may play a significant
role in transmission of infectious diseases. It is therefore recommended that:

 Currency notes must be handled with caution and with great care especially during the
preparation and handling of food to avoid contamination.
 Personal hygiene to reduce risk of infection is recommended especially for those who
simultaneously handle food and money.
 Food sellers should be educated to avoid possible cross contamination between currency
notes and food.
 There should be public awareness of the fact that currency notes could be a source of
infection and could be dangerous to health.
 Regular microbial testing of currency notes and establishment of method for large scale
replacement of contaminated currency should be employed.
 Introduction of plastic currency notes which can be washed easily as in Australia can
serve as an alternative.
ACKNOWLEDGEMENT

I would like to send my gratitude to all who enabled me successfully carry out my
project work s. First I thank the almighty God. It will be unfair not recognizing the
effort of Mr. William Emitaro for consistently guiding me to do the right thing as
far as my project work is concerned. My gratitude also goes to my fellow
colleagues during consultations. Not forgetting my supervisor Dr. Benson
Onyango.
REFERENCES
1. Ogo, N. I., Ajayi, J. A., Madukeke, A. Eggs and Cysts of parasites
Girma G. Health Risk with Contaminated Paper Currencies. 6 Int J Food Nutr Sci. Volume 2:
contaminating Nigerian currency notes. (2004) Afr J Nat Sci 7: 40-42.

2. Ogbonda K H., Oku, I. Y., Okwelle, A. A., et al. The incidence of human disease-causing
fungi on Nigerian paper money. (2012) Int J Microbial Immmunol Res 2(1): 6-10.

3. Podhajny, M. R. How dirty is your money? Paper, Film and Foil Converter (PFFC). (2004)
Penton Media Inc. 2300: 60611-3698.

4. Uneke, C. J., Ogbu, O. Potential for parasite and bacterial transmission by paper currency in
Nigeria. (2007) J Environ Health 69(9): 54-60.

5. Alwakeel, S.S., Nasser, L. Bacterial and fungal contamination of Saudi Arabian paper
currency and cell phones. (2011) Asian J Biological Sci 4: 556-562.

6. El-Dars, F. M., Hassan, W. H. A preliminary bacterial study of Egyptian paper money. (2005)
Int J Environ Health Res 15(3): 235-239.

7. Vriesekoop, F., Russell, C., Alvarez-Mayorga, B., et al. Dirty money: an investigation into the
hygiene status of some of the world’s currencies as obtained from food outlets. (2010)
Foodborne Pathog Dis 7(12): 1497–1502.

8. Prasai, T., Yami, K. D., Joshi, D. R. Microbial load on paper/polymer currency and coins.
(2008) Nepal J Sci Tech 9: 105-109.

9. Hosen, J.M., Sarif, D.I., Pahman, M., et al. Contamination of coliforms in different paper
currency notes of Bangladesh. (2006) Pak J Biol Sci 9(5): 868-870.

10. Xu, J., Moore, J. E., Millar, B. C. Ribosomal DNA identification of the culturable bacterial
flora on monetary coinage from 17 currencies. (2005) J Environ Health 67(7): 51-55.

11. Ogbu. O., Uneke, C. J. Potential for parasite and bacterial transmission by paper currency in
Nigeria. (2007) J Environ Health 69