BACTERIAL ANALYSIS OF URINE POLLUTED

ENVIRONMENT IN FEDERAL POLYTECHNIC NEKEDE,

OWERRI

BY

--------------------------
---------------

SUBMITTED TO THE DEPARTMENT OF SCIENCE

TECHNOLOGY,

SCHOOL OF INDUSTRIAL AND APPLIED SCIENCE,

------------------------------

IN PARTIAL FUFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF -------------------------- IN SCIENCE TECHNOLOGY
(MICRO BIOLOGY OPTION)

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SUPERVISED BY: MR. ------------------

OCTOBER, ---------------

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 CERTIFICATION

This project was dully certified and approved for the award of
--------------------- in the Department of Science Technology,
-----------------

………………………… …………………………
Mr. ------------------ Date
(Project Supervisor)

………………………… …………………………
--------------- Date
(Head of Department)

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 DEDICATION

This research work is dedicated to the most High God for his knowledge
and inspiration towards me and my beloved parents, Chief and Chief Mrs.
--------------- for their assistance towards making my dreams come true.

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 ACKNOWLEDGEMENT

My hearty appreciation first of all goes to the Almighty God for his
guidance and sustenance throughout the period of this research work till
now.

My gratitude as well goes to my beloved parents, Chief and Chief Mrs.

------------- for their financial support all these years.

A special note of thanks go to my supervisor, Mr. ------------- for his close

supervision to make this work a successful one.

My appreciation also goes to my elder sister Mrs. -----------, my beloved

---------------, my friends -------,--------- for their installation during the
time of this work.

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 ABSTRACT

The bacteriological status of soil environment polluted with urine was

analyzed, using standard microbiological methods.
The identification test of bacteria revealed the isolation of proteus spp.
Pseudmonas spp, Escherichia coli, Enterobacteria and Staphylococcus
aureus from the contaminated soil. Bacillus spp, Pseudomonas spp and
Staphylococcus aureus were obtained from the polluted bathroom and
klebsiella spp, Pseudomonas spp, and Escherichia coli were isolated from
the cleaned bathroom. Pathogenicity test carried out revealed that while
all isolates from the polluted bathroom except E. coli to be pathogenic,
only proteus from contaminated soil was found to be pathogenic amongst
the isolates from contaminated and uncontaminated soil. Susceptibility
tests from contaminated and uncontaminated soil susceptibility test
revealed that among the disinfectants used for the study namely, Detol,
Izal, Lysol and jik, Lysol was found to be effective against all the test
pathogens. The study showed the diversity of pathogens in polluted
environment and the efficacy of Lysol in decontaminating the polluted
environment that harbour pathogenic organisms, but can be controlled
through disinfection.

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 TABLE OF CONTENT
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Abstract v
Table of content vi

CHAPTER ONE
1.1 Introduction 1
1.2 Aim of project 3

CHAPTER TWO
Literature review 4
MATERIAL AND METHODS
2.1 Sample collection 17
2.2.1 Nutrient agar 18
2.2.2 Macconkey agar 18
2.3.1 Isolation of total hetetrophic bacteria count 19
2.4 Identification of bacteria isolates 20
2.5 Gram reaction 20
2.6 Biochemical characterization of the isolates 21
2.6.2 Oxidase test 21
2.6.3 Indole test 22
2.6.4 Motility test 23
2.6.5 Citrate test 23
2.6.6 Spore stain test 24

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2.6.7 Mannitol fermentation (Sugar) 25
2.6.8 Coagulase 25
2.6.9 Lactose fermentation 26
2.6.10 Urease test 26
2.7.1 Pathogenicity test 27
2.7.1.1 Medium preparation 27
2.7.1.2 Method of culture 28
2.7.2 Antimirobial susceptibility testing 28
2.7.2.1 Prparation of antibaceterial disc 29
CHAPTER THREE
RESULT
3.1 Count of distinct colonies of the isolates 30
3.1.1 Microbial load of contaminated soil sample 30
3.1.2 Bacterial count for sample B 31
3.1.3 Microbial load for sample C (contaminated
Bathroom) 31
3.1.4 Microbial load for sample D (cleaned bathroom 32
3.3 Microbiological analysis 33
3.3.1 Result of Pathogenicity Tests 33
3.3.2 Result of Antimicrobial susceptibility 35
CHAPTER FOUR
4.0 DISCUSSION, CONCLUSION AND RECOMMENDATION
4.1 Discussion 38
4.2 Conclusion 43
4.3 Recommendation 44
References 45

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 CHAPTER ONE

1.1 INTRODUCTION
Urine is a liquid waste product from the kidney of both animals and
humans. It is collected in the bladder and excreted through the urethra. As
a waste liquid product, it contains some dissolved substances such as
ammonia, urea, uric acid, and creatinine. These constitute the organic
solids in the urine. Urine also contains inorganic dissolved substances
such as sodium chloride, calcium, potassium, phosphate and sulfates
(Cobire and Wewedo, 2002).

The dissolved substances in the urine can be utilized by microorganisms

of various groups as nutrients whenever urine finds its way into the
environment. This is evidenced by the fact that urine polluted
environments usually have very strong odour, signifying that the
biological oxygen demand (BOD) is high. This phenomenon is observe in
toilets, bathrooms, street corners and fallow grounds. (Deni and Pennick,
1999).
The different groups of microorganisms can represent different microbial
functions and activities. Some can be harmful relating to public health
risk, or beneficial relating to positive economic value. Urine leach into
ground and surface waters often with much of the nitrogen intact. When
microorganisms in lakes and other surface waters consume the nitrogen. It
results into a great bloom of growth. When this dies and decomposes, it
pulls oxygen from the water or euthrophies, which can suffocate fish and
other aquatic life. Underground nitrogen can seep into drinking water,
posing a potential health hazard.

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Urine contains micro pollutants such as synthetic hormones,
pharmaceuticals and their metabolites, that is mainly excreted via urine
(Alder, 2002) and may be harmful to the ecosystems and human health
(Daughton and Ternes, 1999). Today, many micro pollutants reach the
aquatic environments because their degradation in waste water treatment
plant is poor (Barker and Jones, 2005).
More than just dirt hanging around the environment especially urine
polluted is unhealthy.
This work plans to asses the level of bacterial building in urine
contaminated campus environments and thus suggest control measures to
prevent the invasion of our environment by bacterial pathogens especially
the campus female hostel bathrooms which are usually polluted with
urine.
1.2 AIM OF PROJECT
The aim of this work is the bacteriological analysis of urine polluted soil.
1.3 STATEMENT OF THE STUDY
Urine generally is usually regarded as menace or nuisance in the environment
especially when poorly disposed. This is because of the inability to convert urine, an
organic waste to human use in urban and peri-urban centres. Improper urine disposal
constitute bad odour problems in the society. These problems come as a result of
accumulation of fresh urine at pH of 6.7. Excess urine in the soil can introduce
toxic levels of nutrient into the soil and thus kill the plant. Therefore, there the
should be a programme enlighting people about how urine can pollute the soil.
1.4 OBJECTIVES OF THE STUDY
The study of bacteriological analysis of urine polluted soil seek to:
1. Identify the bacteria found in the soil that lead to pollution in the
environment.

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 2. To examine the bacteriological analysis of urine polluted soil.
Justification
1.5 Justification of the Study
The aim of the present study is the bacteriological analysis of urine
polluted soil. The safety of soil raises the importance of efforts that should
be exerted towards evaluation and detection of bacterial hazard, which
represents a great risk to the soil. The results of this project may also be
beneficial to other soil scientist, by potentially heightening the awareness
for the control of bacteria through urine in a soil. This awareness may lead
to better prevention and control from urine contamination in the soil. This
project will address these gaps by discovering the bacteria contamination
and safety concerns of bacteriological analysis of urine in polluting the
soil, which will add to the existing theoretical knowledge regarding
bacterial contamination in the soil. The collected data can potentially be
used by soil scientist in preventing bacterial contamination of the soil
through urine. The project is also important to researchers and scholars, as
the collected data will potentially provide a foundation for further
research on the topic of bacteriological analysis of urine polluted soil.

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Soil microbiology is the study of microorganisms in soil, their functions,
and how they affect soil properties. This led to more advanced
microorganisms, which are important because they affect soil structure
and fertility

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CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 BACTERIOLOGICAL ANALYSIS OF URINE
A number of studies relating to the bacteriological analysis of many
environments and the sanitation of such environments like bathrooms
have already been executed. Sanitary conditions in public places have
always been a major problem, especially bathrooms. Health departments
are continually checking the cleanliness and safety of these bacteria
breeding grounds to prevent the spread of sickness and disease (Nester et,
al, 1995)

In a research experiment conducted in 2000, Barter and Bloomfield

studied the presence of Salmonella in domestic bathrooms, it was found
that in four out of six bathrooms tested, Salmonella bacteria existed in
close proximity to toilet. Despite the fact that Salmonella is usually
contacted by consumption of contaminated food, this study shows that the
bacteria was commonly spread by air and physical contact in four out of
six households. It is difficult to remove the bacteria with common
household cleaners. This is because it can get embedded into the biofilm
on the underside of the toilet bowl and just below the water level (Barter
and Bloomfield, 2000). This experiment was pursued after a report of an
attack of Salmonellosis in the home of one of the test bathrooms. In recent
studies done in England, it was found that infectious intestinal diseases,
such as the Salmonella found in Barker and Bloomfield’s research,
occured in one out of five people each year. Also in reports done by the
communicable disease surveillance center, (1999) it was calculated that
136 unreported cases in community caused considerable morbidity.

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According to the study by (Daharan, et al, 1999), the use ammonia-based
detergent promoted the growth of bacteria colonies.
This is because of the nitrogen contained in these products. In addition,
because the detergent being used becomes contaminated, it spreads the
bacteria to new surfaces as the individual continues to clean other areas.

The levels of bacteria in cold and hot water were compared in another
study by Bloomfield and Scott, (2001). They concluded that cold water
contained a higher amount of mesophilic bacteria than other parts of the
bathroom. However, the decrease in the hot water temperatures coming
from a shower-head and the cooling of the standing water promotes
increase in microbial growth.
The potential spread of infection caused by aerosol contamination
surfaces after flushing a domestic toilet was studied. It is pointed out that
although a single flush deduced the level of microorganisms in the toilet
bowl, water when contaminated at concentrations reflecting pathogens
shedding, larger numbers of microorganisms on the toilet the bowl surface
and in the boil water which were disseminated into the air by further
flushes.
Large numbers of bacteria which seeded into household toilets were
shown to remain in the bowl after flushing, and even continual flushing
could not remove a persistent fraction. This is found to be due to the
adsorption of the organisms to the porcelain surfaces of the bowl, with
gradual elution occurring after each flush. Droplets produced by flushing
toilets were found to harbour bacteria which had been seeded, the
detection of bacteria falling out onto surfaces in bathrooms after flushing

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indicated that they remain airborne long enough to settle on surface
throughout the bathroom. Thus, there is a possibility that a person may
acquire an infection from an aerosol produced by toilet (Gerba, et al,
1975).

A bacteriological investigation of the effectiveness of cleaning and

disinfection procedures for toilet hygiene by Bloomfield and Scott, (1985)
showed that the effect of daily disinfection with hypochlorine or a
quaternary ammonium product, or with a continuous release of
hypochlorite disinfectant system, based on the chlorine-releasing agent
tricholorosocyanuric acid, produced some reduction in contamination
contaminations compared with daily cleaning, the reductions were less
than that associated with the continuous release system and indicated the
inadequacy of daily disinfection and / or cleaning of toilets where more
effective procedures are required.

Although detergent –based cleaning using a typical bowl –wash routine

without rinsing produced some risk reduction (from 100 to 61.4% of
contaminated surfaces). It was insufficient to consistently restore surfaces
to a hygienic state. By combing detergent-based cleaning with a rinsing
step or with hypochlorine at 500 ppm (or available chlorine) some further
reduction in microbial risk was achieved, but was not considered
satisfactory for food hygiene purposes. By contrast the risk reduction
produced by hypochlorite at 500 ppm was highly significant and was
sufficient to reduce the number of contaminated surfaces to 2.9%, (Boln,
1995).

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Everyone expects that there will be bacteria growth on bathroom surfaces.
However, the magnitude pf bacteria found in bathrooms, used so
frequently and by many people that live in a community will be alarming.
Even though our immune systems can resist these common bacteria, such
high exposure could pose a throat to the health of the users. One common
bacterium is streptococcus pyognes and is found in the throats of healthy
people. This bacterium can be transmitted by air or saliva. However, it
can cause diseases such as pharyngnitis, impetigo, meningitis, and toxic
shock syndrome (Geldreich, et al 1976). Staphylococcus, E. Coil and
many other normal microfolra can be spread among the students
potentially causing major problems.
In a research work to compare the bacteria growth in male and female
bathrooms, it was reported that female bathrooms contained more bacteria
than the male bathroom. Between the two bathrooms they found seven
different types of bacteria including: E.Coil, Streptococcus,
Streptococcus, Azobacter, proteus, Candida. In order to keep these
bacteria quantities at a safe level the bathrooms must be cleaned daily or
even twice a day. This cleaning process will eliminate large quantities or a
continual build-up of bacteria. This will make for a healthier, safer, and
happier bathroom experience (Davis, et al, 2002).
The most common soil bacterial are rod-shaped, a microorganism
(1/25,000 of an inch) or less in diameter and up to a few microns long
(Grey, 1986).
2.2 SOIL BACTERIA
Soil bacteria may be divided into two large groups based on their energy
requirements. The two group are:

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1. The heterotrophic bacteria, which obtain their energy and carbon
source from complex organic substances.
2. The autotrophic bacteria, which can obtain their energy from the
oxidation of inorganic elements or compounds, obtaining their
carbon from carbon dioxide, and their nitrogen and other minerals
from inorganic compounds (Alexander, 1977). In the autotrophic
group are found such organisms as the nitrate formers, the sulfur
oxidizing bacteria, the iron oxidizers, and those that act on
hydrogen and its compounds. Most of the soil bacteria require
oxygen from the soil air and are classified as aerobes. Some aerobic
bacteria can adapt to living where the soil is devoid of oxygen, they
are facultative aerobes. Other cannot live in the presence of oxygen
and are anaerobes. The soil bacteria also differ considerably in their
nutrition and in their response to environmental conditions.
Consequently, the kinds and abundance of bacteria depend both on
the available nutrients present and on the soil environmental
condition.
It has been established that the genetic diversity of soil bacteria is high
and that soils contain many bacterial specie or lineage for which no
known cultivated isolates are available. Many soil bacteria are referred to
as uncultured or even nonculterable. Many of these bacteria are in fact
culturable using relatively simple techniques (Jansen, et al 2002).
All natural water contains bacteria. The aerobic gram negative rods of the
genera Pseudomonas, Alcalignes and Flavobacterium as well as others are
common in water. Water has special unique properties that help make it a
necessary part of the environment for many bacteria and contributes to its
essential function within the living cell (Nester et al, 1995).

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Bacteria in particular form quite an essential part of stream water. Some
bacteria such as animal pathogens and other soil species are carried into
rives and lakes by run off water. Other species of bacteria grow in nature
only in such environments, and are called “indigenous population”
Species of representative bacteria in stream water varies considerably in
their relative numbers and kinds from one stream to another depending on
the source, kinds, activities and population source and types (Nester et al,
1995).

The presence of some bacteria in stream water invariably are essential for
the growth of other organisms, for instance, bacteria produce vitamin
(B12) that are essential for the growth of many algae. Most bacteria are
Gram negative, motile aerobes or facultative anaerobes while strict
anaerobes are found only in aquatic animals and sediments where
anaerobic conditions exist (Nester et. Al 1995).
Bacteriology of urine from patients with long term urinary catheters by
(Hayson and Sharp, 2005) was reviewed. The study pointed out that
bacteria associated with long term urinary catheters (those in place for
greater than or equal to 30 days) appears to be the most common source of
nosocomial infection in US. Medical care facilities. The bacteriuria is
polymicrobial and dynamic and accompanied by fevers, catheter
obstructions, bacterimias, and deaths.

2.3 PATHOGENIC BACTERIA IN URINE

In a healthy individual the urine is sterile in the bladder. When the
transported out of the body, different types of dermal bacteria are picked

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up and freshly excreted urine normally contains up to 10,000 bacteria per
ml. In urinary tract infections, which in more than 50% of cases are
caused by E. Coli (Ivanov et. al, 2006), significantly higher amounts of
bacteria are excreted. However, these have not been reported to be
transmitted to other individuals through environments. Pathogens causing
veneral diseases may occasionally be excreted in urine but there is no
evidence that their potential survival outside the body would be of health
significance (Ivanov et. Al, 2006). The pathogens traditionally known to
be excreted in urine are Leptospira interogans, Salmonella typhl,
Salmonella paratyphi and Schistosoma harmatobium (Ivanov et. al,
2006).
2.4 PATHOGENS IN URINE AND THEIR DISEASE
Pathogens that may be excreted in urine and the diseases they cause.
Bacteria Diseases
Salmonella Typhi Typhoid fever
Salmonella paraty[hl Paratyphoid fever
Leptospora Leprospirosis
Yersinia Yersinisiosis
Escherichia coli Diarrhea
Schistosoma harmatobium Schistosomiosis

2.5 ORGANIC SUBSTANCES IN URINE

The concentration of organic substances in urine is high, about
10,000COD/M3. On a COD basis, organic acids, creatinine, amino acids
and carbohydrates are the main organic urine compounds. Nitrification
and autotrophic gentrification of source separated urine by pointed out
that on a molar basis, urea, which has no COD, is the most important of
the organic substances in urine. Biological degradation of the organic
compounds will occur, if urine gets in contact with anaerobic

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microorganisms which can use the organic compounds as electron
acceptors. Examples are sulphate reducers. Fermenters may also growin
urine.
Hogland et al (1998) on evaluation of facial contamination and microbial
die-off in urine separating systems noticed that bacteria of the fermenting
gernus Clostridium is persistent in urine. Methane production would be
critical, but is unlikely because of the high ammonia concentration. Not
only anaerobic, but also aerobic degradation can occurm if substantial
amount of oxygen diffuse through the tank walls (Grillies and Dodds,
1976). The organic substances in urine also include micro pollutants.

2.6 EFFECTS OF URINE ON THE CONTAMINTED

ENVIRONMENTS
Urine on a clay loam soil increases soil microbial biomass carbon and
nitrogen contents by about 20% but there was no specific effect of urine.
Urine however, caused an increased in soil respiration of greater than 50%
and the average increase greater for cow’s urine that for artificial urine.
Urine disposition on grassland causes significant N2O losses, which in
some cases may result from increased identification stimulated by labile
compounds released from scorched plants. In the soil contaminated with
urine, the ammonium is nitrite, releasing two protons. The nutrient
balance and content of the urine will reflect what the crops have removed
from the fields and thus the average need of fertilization. In other words,
urine supplies nutrient to the soil, which serves as a fertilizer in the soil.

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 CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 SAMPLE COLLECTION
Urine polluted soil sample was collected from a urine polluted area
behind biology lab in the campus, of Federal Polytechnic, Nekede.
Unpolluted soil sample was collected from a garden located within the
polytechnic premises as the soil samples were transferred into sterile
comical flasks and wrapped with aluminum foil.

Samples were also collected from the Polytechnic’s female hostel

bathroom using cotton wool swab. This was done by swabbing the floor
of the hostel bathroom after cleaning it with diluted disinfectant (Izal)
(2000ml of water and 20ml of Izla) and detergent (Omo).
All the samples were immediately sent to the laboratory for analysis.

3.2 PREPARATION OF MEDIA FOR BACTERIAL ISOLATION

3.2.1 NUTRIENT AGAR
Two point eight grams 92.8g) of nutrient agar was dissolved in 100ml of
distilled water shaken to dissolve completely, it was then autoclaved at
121oC for 15 minutes at 15psl and allowed to cool. On cooling, 20 ml was
aseptically dispensed into sterile Petri dishes, and allowed to solidify.
Nutrient agar is a general purpose medium suitable for cultivation of non-
fastidious organisms. In this worth the medium was used for isolation of
pure cultures and as slants for preparing isolates.

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3.2.2 MACCONKEY AGAR
This medium was prepared by dissolving 4.85g of powdered macconkey
in 100ml of distilled water, shaken to dissolve properly and sterilized at
121oC for 15 minutes at 15psi, it was then poured into Petri dishes and
differentiates between the enterabacteriaceae based on the ability to
ferment lactose pinte colonies indicted lactose fermentation while
colourless and indicates non-lactose fermenters.

3.3.1 ISOLATION OF TOTAL HETROTROPHIC BACTERIA

COUNT
One gram each of the soil samples (contaminated and uncontaminated)
was weighed out using weighing balance and aseptically added into 9ml
of sterile physiological saline contained in the test tube. Using ten –fold
serial dilution technique, the soil suspension was serially diluted to 10- 5
dilution. Aliquots (0.1ml) of appropriate dilutions were inoculated into
nutrient agar and into Macconkey agar plates in triplicate (one served as
control, and three were triplicate counts) using the spread plate technique.
The plates were inculcated at 370C for 24h.
Ten-fold serial dilutions of the bathroom samples were also made,
inoculated into nutrient agar and macconkey agar plates in duplicate (one
served as control) using the spread plate technique. The plates were
incubated at 370C for 24h.
3.4 IDENTIFICATION OF BACTERIA ISOLATES
This was done by the morphological appearance, Gram reaction and
biochemical characteristics of the isolates. For biochemical tests, standard
inocula were prepared in stocks used when needed and this was done by

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aseptically subculturing from the stock culture with freshly prepared
nutrient agar and incubated at 370C for 18-24th.

3.5 GRAM REACTION

Discrete colonies which developed after 24th picked with a sterile wire
loop onto a clean sterile glass slide, with a loopful of physiological saline
added to the slide to make a thin film of smear. The smear was allowed to
dry and then heat fixed by passing it thrice over the Bunsen blue flame.
The fixed smear was then stained for 60 seconds with crystal violet
solution. The stain was washed off by gently running tap water and then
flooding with Lugol’s iodine solution (a mordant) for 60 seconds. The
iodine was drained and the slide rinsed in running top water. The stained
film was then decolourized with 95% (v/v) ethanol until the entire bolet
colour disappeared. The smear was counterstained with safrainn solution
for 60 seconds. After the slide washed, blotted dry and observed under oil
immersion (x100) objective of the light microscope.

3.6 BIOCHEMICAL CHARACTERIZATION OF THE

ISOLATES
This test demonstrates the presence of catalase, an enzymes that catalyzes
the release of oxygen from hydrogen peroxide. The glass slide technique
was used. Drop of hydrogen peroxide was placed on a clean grease from
glass slide and smeared with a loopful of isolate collected from a 24h
fresh culture. The production of gas bubbles was an indication of a
positive test.

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3.6.2 OXIDASE TEST
This test was adopted from cheesbrough (2000) to differentiate between
bacterial groups through the production of oxidase. A few drops of freshly
prepared oxidase reagent (1% solution of tetramethylp-phenylene diamine
dihydrocholride) were placed on a piece of filter paper and allowed to dry.
A small quantity of growth from a fresh culture was smeared across the
filter paper. A positive result was indicated by a purple-blue coloration on
the filter paper within 10 seconds.

3.6.3 INDOLE TEST

Testing for indole production is important in the identification of
enterobacteria. Most strain of E. coli P, Viligaris, P. Rettgeri, M.
Morganii and Providencia species breakdown the amino acid trypiophan
with the release of indole.

PROCEDURE
The test organism was cultured in a medium which contains tryptophan.
Indole production was detected by kovac’s reagent which contained 4-(P)
dimethy laminobenzldehdyde. Production red colour indicated positive
result while no red colour was negative result.
3.6.4 MOTILITY TEST
The medium sued here was a semi-solid agar medium. The medium was
prepared by adding 4.0g of bacteriological agar to 15g of nutrient broth
and 1 litre and of deionized water. This was then heated to dissolve the
agar and 10ml amounts were dispensed into the test tubes and sterilized.
The test tubes were allowed to set in a vertical position. Inoculation was
done with a sterile straight inoculation wire making a single stab down the

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centre of the tubes to about half the depth of the medium. The tubes were
then incubated and growth examined after 24h. Diffused growth through
the medium from the line of inoculation undirected a positive result.

3.5.6 CITRATE TEST

This test was one of several techniques used to assist in the identification
of Enterobacteria. The test is based on the ability of an organism to sue
citrate as its only source of carbon and NH3 as its only source of nitrogen.

PROCEDURE
The test organism was cultured in a medium which contains sodium
citrate, an ammonium salt with the indicator bromothymol blue
(simmon’s citrate agar).
Growth in the medium was shown by turbidity and a change in colour of
the indicator from light green to blue, due to the alkaline reaction
following citrate utilization.

3.6.6 SPORE STAIN TEST

Smears from cultures were prepared, ail –dried and heat fixed. The slide
with smear was placed over beaker of boiling water. A small quantity of
malachite green (5% v/v) was dropped to cover the smear on the slide.
The sildes were washed with water and counter stained with safranin for
30 seconds. The slides were washed with water, allowed to dry and
examined under (x100) objective of the microscope. The spore stained
green and the vegetative cells red.

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3.6.7 MANNITOL FERMETNATION (SUGAR)
This test is based on the fact that some microbes can ferment mannitol to
produce either acid or gas or both. One percent weight per volume of
sugar solution was prepared and sterilized by autoclaving for 6mins. One
milliliter of mannitol sugar solution was then added to the test tube,
containing 9ml of tryptone, water and inoculated with a 100pful of the test
organism incubation was for 24- 48hrs at 370C. Tubes were examined to
indicate change in colour of the medium from blue to yellowish green.

3.6.8 COAGULASE
The test was used to differentiate S. auerus which produces the enzyme
coagulase, a drop of saline water was placed on a clean slide with a sterile
loop, a trace of undiluted plasma was stirred with the bacteria suspension
on the slide coarse clumping within 5-10 seconds indicated a positive
result.

3.6.9 LACTOSE FERMENTATION

The ability to utilize lactose was determine by observing the growth of
organisms on Macconkey agar medium. The specimens were aseptically
plated on both media. Lactose fermenters were pink on Macconkey agar
and those of non-lactose fermenters were pale on both media.

3.6.10 UREASE TEST

Testing for urease enzyme activity was important in differentiating
enterobacteriaecae, proteus strains are urease producers. V. entrodica
also shows arease activity (weakly at 35-37 0C). shigella and Salmonella
do not produce urease.

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PROCEDURE
The test organism was cultured in medium which contained urea and the
indicator, pheonol red. Production of red-pink colour in the medium
indicated a positive urease result while no pink colour indicated negative
urease result.
MICROBIOLOGICAL ANALYSIS
The microbiological assays carried out in this study were pathogenicity
test and antimicrobial susceptibility tests.

3.7.1 PATHOGENICITY TEST

This experiment was carried out to determine whether the test organisms
are pathogenic to humans. This was done routinely using human serum by
determining the degree of virulence (haemolysis) by the test organisms.
The medium used was blood agar medium. A control was also provided
which did not contain the test organism.
3.7.1.1 MEDIUM PREPARATION
The medium was prepared by placing 9.6g of nutrient agar into a sterile
500ml conical flask 200ml of distilled was water was poured into it and
shaken thoroughly to dissolve the agar and to mix well. The medium was
sterilized by autoclaving at 1210C for 15min at 15psi on cooling at
temperature of 450C, 4ml of serum was added and shaken properly to mix
well with the nutrient. The prepared medium was then poured into Petri
dishes and allowed to solidity.

3.7.1.2 METHOD OF CULTURE

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The test organisms were streaked unto the solidified plate and incubated
at the temperature of 370C for 24h. The result was then recorded after 24h
incubation.

2.7.2 ANTIMICROBIAL SUSCEPTIBILITY TESTING

(USING DISINFECTANT)
This test assesses the ability of the test organism to be susceptible or
resistant to antimicrobial agents. The anitimicrobial agents used were four
different types of disinfectants, namely; Detol, Izal, Lysol, and Jik. The
test was carried out using agar disc diffusion method (the use of filter
paper method). The result of this test was determined by measuring the
zones of inhibition of the agents on the test organism.
3.7.2.1 PREPARATION OF ANTIBACERIAL DISC
Discs for sensitivity test were prepared using whatman filter paper No1
Method of preparation was the same was antibiotic disc preparation in the
laboratory as stated by Ogbulie et al (1998).

PROCEDURE
The absorbent whatman filter paper No 1 was perforated with the standard
office paper perforator, producing discs of 6mm I diameter. The discs
were put in one Petri dish and covered very well. Then, it was sterilized
using hot air oven at 1060C for 1 hour.
Serial dilution of each of the disinfectants in this work was made by
dissolving 1ml of each of the disinfectants in 9ml of sterile water
respectively. Then 1ml of this 100 concentration was transferred into 9ml
of sterile water again to obtain 10-1 concentration each the sterile
perforated disc measuring 6mm was held with sterile forceps and

28
dippened into the dilutions (10-1 of each of the disinfectants used). The
dipped disc was placed on cultured plates containing the test organism.
The se sensitivity plates were then incubated at 37 0C for 24h. The zones
of inhibition were measured using meter rule.

29
 CHAPTER FOUR
4.0 RESULTS
4.1 COUNT OF DISTINCT COLONIES OF THE ISOLATES
The result of the total bacterial load per ml of the samples are shown in
the tables below.

4.1.1 MICROBIAL LOAD OF CONTAMINATED SOIL

SAMPLE
Table 2 Total bacterial count for sample A (contaminated soil)
Plate No of colonies on No of colonies on
nutrient agar macconeky agar
-5
10 6.0x104 5.2x104
10-4 5.6X105 5.4X105
10-5 5.4x106 5.0x105
Average No of 5.6x105 5.2x105
colonies (CFLMI)

Total bacterial count for sample B (uncontaminated soil)

4.1.2 table 3: Total bacterial count for sample B

(uncontaminated soil)
Plate No of colonies on No of colonies on
nutrient agar macconkey agar
-3
10 5.4x104 4.8x104
10-4 4.9x105 4.0x105
10-5 4.5x104 4.8x105
Average No of 4.9x105 4.5x105
colonies (CFLMI)

4.1.3 Microbial load for sample C (contaminated Bathroom)

30
Table 4 Bacterial count for uncleaned bathroom
Plate No of colonies on No of colonies on
nutrient agar macconkey agar
-3
10 4.7x104 4.8x104
10-4 4.4x105 8.0x105
Average No of 4.5x105 6.4x105
colonies (CFLMI)

4.1.4 Microbial load for sample D (Cleaned bathroom)

Table 5 table Bacterial count for cleaned bathroom
Plate No of colonies on Not of colonies on
nutrient agar macconeky agar
-3
10 4.2x104 5.9x104
10-4 4.0x105 3.9x105

Average No of 4.1x105 4.9x105

colonies (CFLMI)

31
4.2 MICROBIOLOGICAL ANALYSIS
4.2.1 Result of Pathogenicity Tests
In the contaminated soil, zones of haemolysis were exhibited by only the
proteus spp among the five different bacteria isolated. All the three
isolates (Proteus, websialla, and staphylecocus aureas) from polluted
bathroom showed under zones of haemolysis than the isolates the
contaminated soil in the cleaned bathroom only the websiella spp
exhibited zones of haemolysis while in the uncontaminated soil no zone
of harmolysis was obtained.
Table 8 result of pathogenicity tests of the all the isolates
Sample Isolates Location Diameter Result
A Proteus spp Contaminated 8mm +
soil
Pseudomona Contaminated
s soil
E. coli Contaminated - -
soil
Enterrobacter Contaminated - -
soil
S. aureus Contaminated - -
soil
B Bacillus Uncontaminated - -
soil
Pseudomona Uncontaminated - -
s soil
C Proteus Polluted 15mm ++
bathroom
S. Aureus Polluted 12mm ++
bathroom

32
D Websiella Cleaned 9mm +
bathroom
Pseudomona Cleaned 9mm +
s bathroom
E. coli Cleaned - -
bathroom

Key + = Positive (haemolysis); Negative (no haemolysis) ++ = Positive

haemolysis (Highly virulence).

4.2.2 Result of Antimicrobial susceptibility Test

The results of antimicrobial susceptibility test using four different
disinfectants (Detol, Izal, Lysol and Jik) on the isolates are as follows:

The isolates from contaminated soil were tested using these disinfectants
and it showed that proteus species was susceptible to Detol, Izal, Lysol
and Jik resistant to Jik, Lysol having the largest diameter of inhibition
zone pseudomonas was resistant to Detol, Izal and Jik but susceptible to
Lysol. E. Coli was resistant to all the four disinfectants. Enrerobacter was
susceptible to Izal, Lysol, and jik but resistant to Detol while
staphylococcus aureas was susceptible to Detol, Izal and Lysol but
resistant to Jik.
Proteus species from polluted bathroom was susceptible to Detolm Izal
and Lysol but resistant to Jik, S. aureaus was susceptible to Detol, Izal
and Lysol but resistant to Jik, Lysol and resistant to Izal and Jik.
Websiella from the cleansed bathroom floor was susceptible to Detol and
Lysol and resistant to Izal and Jik. Pseudomonas was resistant to Detol,
Izal and Jik but susceptible to Lysol, while E. Coli was resistant to all the

33
four disinfectants used table 9 below shows the results with the diameter
measurements (in millimeter) of each of the reagents on the isolates from
(contaminated soil, contaminated bathroom and cleansed bathroom). The
original diameter of the disc used was 6mm.

Table 9 Antimicrobial Susceptibility Results (0riginal)

Organism Site Diameter (mm) On the Agents
Proteus spp Detol Izal Lysol Jik
Contaminate 8.0 7.0 9.0 -
d soil
Pseudomona Contaminate - - 8.0 -
s spp d
E. coil Contaminate - - - -
d
Enterobacter Contaminate - 8.0 10.0 7.0
spp d
Staph aureus Contaminate 7.0 7.0 10.0 -
d soil
Proteus spp Polluted 8.0 9.0 9.0 -
bathroom
Staph aureus Polluted 7.0 6.5 10.0 -
bathroom
Websiella Polluted 7.0 - 9.0 -
spp bathroom
Websiella Cleaned 7.0 - 10.0 -
spp bathroom
Pseudomona Cleaned - - 7.0 -
s spp bathroom
E. coil Cleaned - - - -
bathroom

34
 Key: = No zone of inhibition

4.3 DISCUSSION
The bacteria isolated from soil contaminated with urine were Proteus spp,
Pseudomonas spp, Escherichia coil, Enterobacter, and Staphylococcus
aureus. Bacillus spp and pseudomonas spp were isolated from
uncontaminated soil.
In the urine polluted bathroom, proteus spp, Klebsiella spp and
Staphyloccus aureus were isolated while in the cleaned bathroom
Klebsilla spp, Pseudomonas spp and E. Coil were isolated.
The incidence of a close similarity in the genera of bacteria isolated from
the soil contaminated with bathroom contaminated with urine suggests the
fact that those organism are indeed associated with urine contaminated
environment. Though the biomass of the contaminated soil is higher than
the contaminated bathroom analogy indicates that the contaminated soil.
This is because soil ordinarily is the home of microorganisms. However,
urine availability is an additional nutrient, hence the cluster of these
species. The bathroom, being a constructed area specific for human use, is
amazing to harbour such a load of pathogenic biomass. Therefore, the
urine contaminated bathroom is a threat to health. The bacteria isolated
from it were Proteus spp, Klebsiella spp and Staphyloccus aureus. These
are well established causative agents of human diseases. Proteus spp
could be, Proteus mirabills and Proteus vulgaris. They cause urinary and
bloodstream infections. Staphyloccous aureaus cases wound infections,
food poisoning, abscesses, toxic shock syndrome and even infect the
intestine as well as urinary tract, which is most likely where the

35
Staphylococcus isolated came from Klebsiella spp causes chest and
urinary tract infections.

The pathogenicity test showed a positive haemolysis by the Proteus spp

isolated from contaminated soil after 24hr of incubation, Proteus spp,
Klebsiella spp, isolated from contaminated bathroom also exhibited
positive haemolysis on blood agar within the same period of incubation.
This proves these organisms are pathogenic on humans. The diameter of
the haemolytic zones produced by the isolates from contaminated
bathroom was wider than that produced by the Proteus spp from the
contaminated soil on blood agar. This suggests that the bacteria isolated
from contaminated bathroom have higher degree of virulence than that
found in the contaminated soil. The Klebsiella spp and Pseudomonas spp
isolated from cleansed bathroom exhibited positive haemolysis on blood
agar. This bathroom was cleaned with Izal diluted in water and detergent
(omo) and yet pathogenic organisms were isolated from it. This shows the
ineffectiveness of that disinfectant (Izal) and suggests that Klebsiella and
Pseudomonas are resistant to that disinfection. The antimicrobial
susceptibility tests of all the isolates to four different types of disinfectants
(Detol, Izal, Lysolm and Jik) were al surveyed.
In the contaminated soil Proteus was susceptible to Detol, Izal Lysol and
resistant to Jik, pseudomonas was resistant three disinfectant and
susceptible to Lysol, E. coli was resistant to susceptible to three
disinfections and staphylococcus aureus was susceptible to three
disinfectants and resistant to Jik. From the result, Lysol was showed to
have the highest susceptible reaction on the isolates therefore proved to be
the best disinfectant for exhibition of bacteria in the contaminated soil.

36
Result showed that Proteus from polluted bathroom was susceptible to
three disinfectants (Detol, Izal and Lysol) and resistant to Jik showing
Lysol and Izal to have the highest exhibition zones on the proteus, thereby
proving to be better disinfectant on Proteus from urine polluted
bathrooms. Staphylococcus aureus showed susceptibility reactions on
Detol, Izal and Lysol. Lysol had the highest inhibition zone.
Klebsiella spp was susceptible on Detol and Detol and Lysol and resistant
on Izal and Jik. Lysol had the highest inhibition reaction. This suggests
that using Lysol on the urine polluted bathroom can eliminate Klebsiella
spp, which was resistant to Izal, Pseudomonas pp was resistant to Detol,
Izal and Jik but susceptible to Lysol. This suggests none of the
disinfectants used in this study can inhibit the E.Coli strain. These results
on the susceptible tests suggest that the organisms that were resistant to
some of the disinfectants on them but can be eliminated or inhibited
through the use of the disinfectants in which they were susceptible to.
This work therefore proves the use of disinfection in the prevention of
infectious diseases. This agrees with the work of Cozad and Jones (2003)
which pointed out that one of the means of prevention of diseases is
through proper disinfection, even though the presence of pathogenic
bacteria cannot be eradiated disinfectants like Lysol has been proved to be
good disinfectant by this work. This is in agreement with the works of
Davis et al, (2002) which pointed out that disinfectants containing a
variety of active ingredients demonstrated efficacy against a broad
spectrum of pathogens and interrupted microbial transmission (Lysol
being the good disinfectant) and the use of disinfectant results in public
health benefit.

37
 CHAPTER FIVE
5.0 CONCLUSION AND RECOMMENDATION
5.1 CONCLUSION
This study reveals that the urine contaminated environments harbour
pathogenic organisms. It is extremely difficult to create an environment
completely hostile to all bacteria in order to prevent infection.
Nevertheless, it is important to eliminate as much of the pathogenic
bacteria as possible by using careful cleaning procedures. When a certain
bacteria evolves into an extremely dangerous form or there is an infection

38
that spreads among students grouped in such close quarters-sharing the
same fountains, the same toilets, and the same showers the entire
community can be infected.
5.3 RECOMMENDATION
This work will make people aware of the real possible threat of urinating
in bathroom and using public bathrooms and therefore recommended the
use of disinfectants in prevention of infections diseases in polluted
environment such as bathrooms. Even though the presence of pathogenic
bacteria cannot be eradicated but the use of disinfectant like Lysol can
inhibit the pathogenic organisms to lower numbers. This work also
recommends adequate and thorough cleaning of bathrooms as one of the
preventive measures. Even if the presence of harmful bacteria cannot be
eradicated, students can take precautions to protect themselves from
infections on their own.

39
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