ABSTRACT
Bacteriological examination of well water samples
located near septic tanks in Abakaliki metropolis was investigated. 15 well
water samples from 15 different wells were used for the research. Measuring
tape was used to measure the distance, the colour and physical appearance of
the water was observed with the naked eyes, the temperature was measured with
mercury thermometer and its pH was measured with pH indicator. The pour plate
method was used to count cells.
In the bacteriological examination of the water
for coli- form, presumptive, confirmed and completed test were used while indole,
methyl red, vogues proskaeur and a citrate test where used for the
identification of E coli. The highest distance of 23.17 meters and the
shortest distance of 5 metres of the wells from the septic pit were the range
used. Physical and bacteriological characteristics of the well water samples
showed that 9 (60%) had colour ranging from slightly milky to light brown while
10 (66.67%) had presence of particles and cloudy appearance in it. The pH of
the well waters ranged from 6 to 8 with 10 (66.67) of samples having pH of 7. The
temperature range of the well water samples were between 31.80C to
370C. Three samples (20%) presented odour. The highest standard
plate counts of 2.9x105 cfu/ml were recorded while the lowest count
was 3.8x104. The highest most probable number (MPN) index per ml was
0.36 and the least 0.27. Thirteen samples (86.67%) showed the presence of coliforms
in them while five samples (33.33%) showed the presence of E coli. It
was observed that all the well water sample that give positive E coli
came from wells nearest to the septic tank. Well water samples from wells that
were 19 meter from the septic tank and beyond did not give positive coliform or
E coli. Recovering of E coli from the well water samples may
indicate contamination of the well waters from the septic tank. It is therefore
recommended that wells water should be sited at least 19 meters from septic
tank.
ENUMERATION OF COLIFORMS FROM WELL WATERS LOCATED NEAR
SEPTIC TANKS IN ABAKALIKI METROPOLIS, EBONYI STATE
A PROJECT SUBMITTED TO THE DEPARTMENT OF APPLIED
MICROBIOLOGY IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF
POSTGRADUATE DIPLOMA (PGD)
TABLE OF CONTENTS
Approval
Page ii
Certification iii
Dedication iv
Acknowledgements v
Abstract vi
Table
of contents vii
List
of Table xi
List
of Figures xii
CHAPTER
ONE:
1.0 Aim of the Research 1
CHAPTER
TWO:
2.0 Literature Review
2.1 Construction of Well 8
2.2.0 Microorganisms Associated with well 10
Water
Contamination
2.2.1 Coliform Bacteria 11
2.2.2 Gidrdia Lambia 13
2.2.3 Crptosporidium 14
2.2.4 Hepatitis 16
2.3 Sources of Contamination 16
2.4 Effect of Drinking Contamination Well Water 17
2.5 Classification of Well Water 18
2.6 The septic system 19
2.7 Major Chemical and Physical Properties of Water 21
2.8 Uses of Well Water 23
2.8.1 Agriculture 25
2.8.2 Drinking 25
2.8.3 Washing 26
2.8.4 Water Industry 27
2.8.5 Food Processing 27
CHAPTER THREE:
3.0 MATERIAL AND METHODS
3.1.0 Materials 29
3.1.1 Equipments 29
3.1.2 Media 29
3.1.3 Reagents 29
3.2.0 Study Area 29
3.3 Sample Collection 32
3.4 Analysis
of Physical Characteristics of the Well Water samples
3.4.1 Colour and Appearance 33
3.4.2 Determination of Temperature 33
3.4.3 Determination of pH 33
3.5 Microbiological Analysis 34
3.5.1 Standard Plate Count 34
3.5.2 Enumeration of Total Coliforms 35
3.5.2.1 Multiple Tube Method 35
3.5.2.2 Presumptive Test 35
3.5.2.3 Confirmed Test 36
3.5.2.4 Completed Test 38
3.5.2.5 Identification of Escherichia
coli Colonies on EMB
Agar plate 39
CHAPTER FOUR: RESULTS
4.1: Physical Characteristics of the Well Water Samples 41
4.2: Standard Plate Count of the Well Water Sample 44
4.3: Result
of the Presumptive Test and MPN of the Well Water
Samples 46
Colonial Characteristic of
the Organisms on Eosin Methylene
Blue (EMB) agar 48
Identification of E coli 50
CHAPTER FIVE
Discussion 52
Chapter SIX
Summary, Conclusion and Recommendation
CONCLUSION AND
RECOMMENDATION
Summary
Conclusion 55
Recommendation 56
Reference: 57
Appendix1: Equipments 62
Appendix 2: List of media, composition and their
preparation
List of media 63
Appendix 3: List of reagents 67
Appendix 4: Address and distances of well waters from
septic
Tank 68
Appendix 5: Picture showing well distance to septic Tank 69
Appendix 6: MPN table 70
CHAPTER ONE
1.0 INTRODUCTION
Water is a chemical substance with the formulae H2o (2
molecules of Hydrogen and I molecule of Oxygen) connected by covalent bonds.
Water is a liquid at ambient conditions, but it often co-exists on Earth with
its solid state as ice, and in gaseous state as water vapor or steam.
Water covers 70.9% of Earth surface
and is vital for all known forms of life (UN, 2008). On Earth, it is found mostly
in oceans, and other large bodies, with 1.6% of water below ground in aquifers
and 0.001% in the air as vapor, clouds (formed as solid and liquid water
particles suspended in air), and precipitation (UNEP, 2007). Oceans hold 97% of
surface water, glaciers and polar ice caps 2.4%, and other land surface water
such as rivers, lakes and ponds 0.6%. A very small amount of the Earth’s water
is contained within biological bodies and manufactured products (UN, 2008).
Ground water and freshwater are
useful or potentially useful to humans as water resources. Liquid water is
found in bodies of water, such as an Ocean, Lake, River, Stream, Canal, Pond,
or Puddle. The majority of water on Earth is Sea water.
It also exists as groundwater in aquifers. Groundwater
is present in most rocks, and the pressure of this groundwater affects pattern
of faulting (Ogan, 1992). Water in the mantle is responsible for the melt that
produces volcanoes at subduction zones. On the surface of the Earth, water is
important in both chemical and physical weathering processes (Campbell et
al., 2006). Groundwater is also extracted artificially in wells. This
water storage is important since clean, fresh water is essential to human and
other land-based life.
Hand-dug wells are circular holes
about one meter (m) in diameter and 10 to 30m in depth, dug with human labour.
The wells tap water from shallow aquifers for domestic water supply. The well is
for domestic uses like cooking, washing, drinking, in small-scale industries
and small irrigation schemes. Many hand dug wells exist in Nigeria and many
developing countries (Ogan, 1992).
This study is aimed at Enumeration
of coliforms from well waters in Abakaliki metropolis.
Fig 1: Model of
hydrogen bonds between molecules of Water
1.1 Aim of the Research
1.
To determine the total coliforms
from well waters near septic
Tanks.
2. To investigate for faecal
contamination of such well waters.
3. To
determine the shortest distance at which septic tank can contaminate well
waters.
CHAPTER TWO
2.0 LITERATURE
REVIEW
Water is universally consumed in large quantity and it
is important to know the types and number of microbes taken in by drinking
water (Geldreich, 1990). In developed and developing countries, well waters
supplies at least 100 million people with drinking water (WHO, 1998).Globally,
underground water provides 25% of drinking water (Encarta encyclopedia, 2004).
More than 100 million people in the United States use ground water as their
source of drinking water (Tuthill et al., 1998).
Septic tanks serve primarily as settling chambers
removing solids from the sewage. In sand and gravel aquifers characterized by
large pore sizes that allow for relatively easy and rapid transport of water
and contaminant, concentrated plumes of dissolved constituents from septic
systems can occur in the shallow part of the aquifer and can affect the quality
of drinking water withdrawn from domestic wells (Ingrid et al., 2004). In
Nebraska, a large number of shallow sand-point wells are used to obtain
drinking water in private households, even though their construction for
consumptive uses has been banned since 1987 (Nebraska Department of Health and
human Services, 2002.)
The quality of water from underground
contain higher concentration of dissolved chemicals which makes water treatment
and purification before drinking important (Nester et al., 2001).This
can also be further polluted and made unsafe for human consumption by man’s
activities like influx of sewage and industrial waste into them which may lead
to eutrophication of these water and subsequent increase of the microbial load
(Nester et al., 2001).
The great dependence on this resource has
not resulted in a corresponding understanding of microorganism and
microbiological process that occur in the well water environment (Prescott,
2002). Quality standards of purified water are graded according to the amount
of biological matter, dissolved organic and inorganic matter it contains
(Cheesbrough, 2005).
Maintenance of the microbiological quality
and safety of water system used for drinking, recreating and in harvesting of
seafood is imperative. Water contaminated with faeces is generally regarded as
a greater risk to human health, as they are more likely to contain human
specific enteric pathogens, including salmonella (Dombek and Sadowsky, 2004).
Understanding the origin of faecal
pollution is paramount in assessing associated health risk as well as the
actions necessary to remedy the problem while it still exists (Dombek and Sadowsky,
2004). Traditionally, an alternative indicator has been used for many years to
predict the presence of faecal pollution in water (Dumbek, and Sadowsky, 2004).
Clean drinking water is essential to human
and other life forms. Access to safe drinking water has improved steadily and
substantially over the last decades in almost every part of the world (Lomborg,
2001). There is a clear correlation between access to safe water and GDP per
capita. However, some observers have estimated that by 2025, more than half of
the world population will be facing water-based vulnerability (Kulshrestitha,
1998). A record report suggests that by 2030, in some developing regions of the
world, water demand will exceed supply by 50% (MDG, 2009). Water plays an
important role in the world economy, as it functions as a solvent for a wide
variety of chemical substances and facilitates industrial cooling and transportation.
Approximately 70% of freshwater is consumed by agriculture (Baroni et al.,
2007).
2.1
Construction
of Well
, small-scale industries and small irrigation schemes.
Many hand dug wells exist in Nigeria and in many developing countries. They are
important in both urban and rural communities. The wells are dug with hoes,
shovels, pickaxes and diggers. Water and cuttings are removed from the hole
using a human powered bucket-rope-pulley arrangement. Three to four men dig the
well in shifts.
In creating any new well, there are 4 important steps,
which require consideration:
i. Consultation
This is of particular importance
when sinking well that is to be used by a community, as issues of appropriate
access and well maintenance need community input for success.
ii. Siting
Other than the cultural
considerations affecting access, there are several physical requirements for
the sitting of a well. It must be preferably uphill and at least 30m from pit
latrines (Ingrid et al., 2004). The water table must be at least 3m
below ground level in all season, as above this the water quality is equal to
that of surface water (Ingrid et al., 2004). In balance with this, a
site needs to be selected that provides easy access to the water table, as the
deeper the well, the more sophisticated the equipment needed to dig it. The
presence of existing vegetative growth is an important clue as to the height of
the water table.
iii. Design and Construction
The choice of well design and
construction is largely depended on the resources and skills available to the
community, and also on geographical characteristics of the location.
iv. Protection
This is a very important aspect of
well management as a contaminated well has the capacity to affect the health of
a large number of people before detection of problem. There are several
protective components of the well structure that are evident from the design
given above. The apron of the well is a circular concrete plate that surrounds
the opening of the well. It functions as a barrier to contaminated water that leaches
down from around the entrance of the well. It must cover at least a 1.5m radius
extending from the well opening, and should include a channel that diverts the
waste water to a soak away pit that is at least 10m from the apron. The well
casing consists of concrete rings that line the well from top to bottom. Like
the apron, it functions to prevent contamination from leaching through surrounding
soils. A well cover and pomp can be added to stop any gross contamination from
above. Finally, if there is no pump, there should only be one rope and bucket
used to collect the water, suspended from the wellhead so that it cannot be
removed or touch the ground.
2.2.0 Classification
of Well Water
The development of unplanned houses
to accommodate rapidly growing population leads to the proliferation of refuse
(waste) dumps, which invariably pose disposal problems. This is usually a
common problem with many rapidly developing towns in Nigeria.
The high population growth, poor
developmental plan, chronic unhygienic habits and lack of enforcement or
regulations have served collectively as a recipe for environmental pollution.
The problem of acute water supply has resulted in widespread use of hand dug
wells among which some are located in unhygienic areas. Thus it is suspected
that water from wells in these areas could be contaminated, according to their
proximity to sources of pollutants (Eqwari and Abaoba, 2002). A well is
classified into two; deep and shallow. It is classified as deep if it is
difficult to draw water out of it with a cord 10 meters long even after heavy
rainfall and as shallow if there is considerable up-welling after heavy
rainfall.
Wells are also grouped into A, B,
and C based on the quality of the well and its location. Class A wells have
high concrete elevation (0.3 to 0.5m) above ground level with covers made of
corrugated iron sheets. The catchment’s area is cemented and the wells are not
located near a refuse dump or septic pit, class B are similar to class A in
design but the catchment’s have no cemented pavement. Class C wells have high
elevation concrete walls but without covering lid (Egwari and Abaoba, 2002).
2.3.0 Chemical and physical properties
of water
Water is a tasteless, odorless liquid at standard temperature and
pressure. The color of water and ice is, intrinsically, a very slight blue hue,
although water appears colorless in small quantities. Ice also appears colorless,
and water vapor is essentially invisible as a gas (Braun and Sergei, 1993).
* Since
the water molecule is not linear and the oxygen atom has a higher electronegativity
than hydrogen atoms. It carries a slight negative charge, whereas the hydrogen
atoms are slightly positive. As a result, water is a polar molecule with an
electrical dipole moment. The net interactions between the dipoles on each
molecule cause an effective skin effect at the interface of water with other
substances or air at the surface, the latter given rise to water’s high surface
tension. This dipolar nature contributes to water molecules tendency to form
hydrogen bonds which cause water’s many special properties (Campbell et al.,
2006).
* Water
is a good solvent and is often referred to as the universal solvent. Substances
that dissolve in water, e.g., salts, sugars, acids, alkalis and some gases –
especially, oxygen, carbon -dioxide (carbonation) are known as hydrophilic
(water loving) substances, while those that do not mix well with water e.g.
fats and oils, are known as hydrophobic (water-fearing) substances.
* Pure
water has a low electrical conductivity but this increases significantly with
the dissolution of a small amount of ionic material such as sodium chloride.
* The
boiling point of water is dependent on the barometric pressure. For example, on
the top of Mount Everest, water boils at 680C (154 0f),
compared to 1000C (2120f) at sea level. Conversely water
deep in the ocean near geothermal vents can reach temperatures of hundreds of
degrees and remain liquid.
* Water
has the second highest molar specific heat capacity of any known substance,
after ammonia, as well as a high heat of vaporization (40.65KJ mol-1),
both of which are as a result of the extensive hydrogen bonding between its
molecules. These two unusual properties allow water to moderate Earth’s climate
by buffering large fluctuation in temperature.
* The
maximum density of water occurs at 3.980C (39.16F) (Kotz et al.,
2005). It has the anomalous property of becoming less dense, not more, when it
is cooled down to its solid form, ice. It expands to occupy 9% greater volume
in this solid state, which accounts for the fact of ice floating on liquid
water.
* Water
is miscible with many liquids such as ethanol, in all proportion, forming a
simple homogeneous liquid. On the other hand, water and most oils are
immiscible usually forming layer according to increasing density from the top.
As a gas, water vapor is completely miscible with air.
2.4.0 Uses
of Well Water
Water fit for human consumption is
called drinking water or potable water. Water that is not potable may be made
potable by filtration or distillation, or by a range of other methods (UNEP,
2002).
Water that is not fit for drinking
but is not harmful for human when used for swimming or bathing is called by
various names other than portable or drinking water, and is sometimes called
safe water, or “safe for bathing” (UNEP, 2002). Chlorine is a skin and mucous
membrane irritant that is used to make water safe for bathing or dinking. Its
use is highly technical and is usually monitored by government regulations
(typically 1 part per million (ppm) for drinking water, and 1 - 2 ppm of
chlorine not yet reacted with impurities for bathing water) (UNEP, 2002). Water
for bathing may be maintained in satisfactory microbiological condition using
chemical disinfectants such as chlorine or ozone or by the use of ultraviolet
light.
In the USA, non-potable forms of
waste water generated by humans may be referred to a grey water, which is
treatable and thus easily able to be made potable again, and black water, which
generally contains sewage and other forms of waste which require further
treatment in order to be made reusable (UNEP, 2002).
Grey water is 50-80% of residential
wastewater generated by a household’s sanitation equipment (sinks, showers, and
kitchen run off, but not toilets, which generate blackwater).
This natural resource is becoming
scarcer in certain places, and its availability is a major social and economic
concern. Currently, about a billion people around the world routinely drink
unhealthy water (WHO 2008).
Poor water quality and bad
sanitation are deadly; some five million deaths a year are caused by polluted
drinking water. The World Health Organization estimates that safe water could
prevent 1.4 million child deaths from diarrhea each year (WHO, 2008). Water,
however is not a finite resource, but rather re-circulated as potable water.
Therefore, it is the relatively
small quantity of water in reserve in the earth (about 1% of over drinking
water supply, which is replenished in aquifers around every 1 to 10 years),
that is a non-renewable resource, and it is, rather than the actual amount of
it that exist on the earth. Water-poor countries use importation of goods as
the primary method of importing water (to leave enough for local human
consumption), since the manufacturing process uses around 10 to 100 times
products masses in water (Baroni et al., 2007).
2.4.1 Agriculture
The most important use of water in
agriculture is for irrigation, which is a key component to produce enough food.
Irrigation takes up to 90% of water withdrawn in some developing countries
(National Atlas 2001) and significant properties in more economically developed
countries (in United States, 30% of freshwater usages is for irrigation)
(National Atlas, 2001).
2.4.2 Drinking
The human body is anywhere from 55%
to 78% water depending on body size (Jeffrey, 2007). To function properly, the
body requires between one and seven liters of water per day to avoid
dehydration; the precise amount depends on the level of activity, temperature,
humidity and other factors. An original
recommendation for water intake in 1945 by the Food and Nutrition Board of the
National research council read; “an ordinary standard for diverse person is 1
milliliter for each calorie of food. Most of this quantity is contained in
prepared foods. The latest dietary reference intake report by the united
National Research council in general recommended (including food source): 2.7
liters of water total for women and 3.7 liters for men. Specifically, pregnant
and breast feeding women need additional fluids to stay hydrated.
Humans require water that does not
contain too many impurities. Common impurities include metal slats and oxides
(including copper, iron, calcium and lead) and/or harmful bacteria, such as
Vibrio, E coli and others. Some solutes are acceptable and even
desirable for taste enhancement and to provide needed electrolytes (Maton, et
al.,1993).
2.4.3 Washing
The propensity of water to form
solutions and emulsions is useful in various washing processes. Many industrial
processes rely on reactions using chemicals dissolved in water, suspension of
solid in water (slurries) or using water to dissolve and extract substances.
Washing is also an important component of several aspect of personal body
hygiene.
2.4.4 Water Industry
Drinking water is often collected at
springs extracted from artificial boring (wells) in the ground, or pumped from
lakes and rivers. Building more wells in adequate places is thus a possible way
to produce more water, assuming the aquifers can supply an adequate flow. The
distribution of drinking water is done through municipal water systems, tankers
delivery or as bottled water.
2.4.5 Food Processing
Water plays many critical roles
within the field of food science. It is important for a food scientist to
understand the roles that water plays within food processing to ensure the
success of their products. Solutes such as salts and sugars found in water
affect the physical properties of water. The boiling and freezing points of
water are affected by solutes, as well as air pressure, which is in turn
affected by altitude (Deman, 1999). Water boils at lower temperatures with the
lower air pressure which occurs at higher elevation. Solutes in water also
affect water activity which affects many chemical reactions and the growth of
microbes in food (Deman, 1999). Water activity can be described as a ratio of
the vapor-pressure of water in a solution to the vapor-pressure of pure water.
2.5.0 Microorganisms Associated with well water
Contamination.
Microbiological contamination of water has long been a
concern to the public, from the 1920’s to1960’s, the bacteria which causes typhoid
fever was considered a major problem in the water supply (Craun, 1996). Once it
was eradicated, new microbes were present to take its place. In parts of the
United States, concern is increasing due to outbreaks of Coliform bacteria,
Giardiasis, Cryptosporidiosis and Hepatitis A (Craun, 1996). Some of these are
bacteria, while others are viruses or protozoa. The presence of fecal Coliform
in drinking or bathing water proves that human or animal wastes have been or is
present.
Two water borne disease are Giardiasis
and Cryptosporidiosis; both cause stomach illness. E coli 0157.H7 has
also been associated with drinking contaminated water. It can cause intestinal
illness and in very rare cases, a serious kidney condition called haemolytic
uremic syndrome.
Coliform bacteria can multiply
rapidly or die off quickly depending upon water temperature and other Variable
(Juranek, 1995).
2.5.1 Coliform Bacteria
Coliform bacteria live in soil or
vegetation and in the gastrointestinal tract of animals. Coliforms enter water
supplies from the direct disposal of waste into streams or lakes, or from
runoff, from wooded areas, pastures, feedlots, septic tanks, and sewage plants
into streams or groundwater. In addition, Coliforms can enter an individual
house via back flow of water from a contaminated source, carbon filters or
leaking well caps that allow dirt and dead orgasms to fall into the water
(National ground water association, 1998). Coliforms are not a single type of
bacteria, but a grouping of bacteria that includes many strains, such as E
coli. They are ubiquitous in nature, and many types are harmless. Therefore
it is not definitive that Coliform bacteria will cause sickness. Many variables
such as the specific type of bacteria present, and your immune systems effectiveness
will determine if you will get sick.
Total Coliform is the standard by
which microbial contamination is measured. Coliforms will be one of the first
bacteria to present in the water should contamination occur, and they will be
in much larger quantities than some pathogenic microbes that may be present.
Therefore, coliform act as indicators of possible contamination. The presence
of coliforms does not necessarily mean that pathogenic members are also present
rather it suggests the presence of sewage (WHO, 1996). Indicator organisms are
used because even when a person is infected with more pathogenic bacteria, he
will still be excreting many millions time more indicators organisms than
pathogens. It is therefore reasonable to surmise that if indicator organism levels
are low, then pathogen level will be very much lower or absent (Gerba and
Bitton, 1984). Coliform bacteria can multiply rapidly or die off quickly
depending upon water temperature and other variables (EPA, 2000).
2.5.2 Giardia Lambia
Giardia has become more prevalent in
the past few years as a water borne disease, and a few large outbreaks have
occurred in the U.S (Juranek, 1995) Giardia are flagellated protozoa that are
parasitic in the intestines of humans and animals (WHO, 1996). They have two
stages, one of which is a cyst form that can be ingested from contaminated
water. Once the cyst enters the stomach, the organism is released into the
gastrointestinal tract where it will adhere to the intestinal wall. Eventually
the protozoa will move into the large intestine where they encyst again and are
excreted in the faeces and back into the environment (WHO, 1996).
Once in the body, the Giardia causes
giardiasis, a disease characterized by symptoms such as diarrhea, abdominal
cramps,, nausea, weight loss and general gastrointestinal distress. These
symptoms last for about a week; however some people can undergo a more chronic
infection with similar symptoms and an even greater degree of weight loss
(Juranek, 1995).
Giardiasis
is rarely fatal (National Academy of Sciences, 1997).
Giardia enters the water supply via
contamination by faecal material. The faecal material can enter the water from:
* Sewage
discharged into the water via cross-contamination of sewage and water lines.
* Sewage
directly discharged from small sewage plants into lakes or streams.
* Sewage discharged into lakes or
streams from cabin toilets.
* Rainfall
moving the cyst deposited from animals on the soil into a body of water.
2.5.3 Cryptosporidium
Cryptosporidium parvum
is a protozoan parasite that causes cryptosporidiosis which has gained
notoriety in the past five years (USGS, 2005). In 1993, over 400,000 people in
Milwaukee, Wisconsin became ill with it after drinking contaminated water (EPA
1997). Since this outbreak, there has been a greater impetus to remove the cryptosporidium
from municipal water supplies. Cryptosporidium is spread by the transmission of
Oocysts via drinking water which has been contaminated with infected faecal
material. Oocysts from humans are infective to humans and many other mammals,
and many animals act as reservoir of Oocysts which can infect humans. Once
inside its host, the Oocyst breaks, releasing four movable spores that attach
to the walls of the gastrointestinal tract and eventually form Oocysts again
that can be excreted (WHO, 1996). Symptoms occur 2 to 10 days after infection
(EPA, 1997). These symptoms include diarrhea, headache, abdominal cramps,
nausea, vomiting and a low fever. There is no treatment against the protozoa,
although it is possible to treat the symptoms. After about 1-2 weeks, the
symptoms subside as the immune system stops the infection. However, for persons
with a compromised immune system such as infants, aged, those with AIDS, or transplantees,
Cryptosporidisis may become life threatening (WHO, 1996). Cryptosporidium
infected faecal material enters the water supply either from cross
contamination of sewage lines with water lines, or surface water infected with
contaminated animal waste. Water treatment processes that utilize coagulation,
sedimentation, filtration and chlorination may remove it (EPA, 1997) However, contamination
of a well may occur from a leaking or improperly placed septic tanks, or animal
waste, so it may be a good idea to test for total Coliforms. If the amount of Coliforms
are low, then more than likely cryptosporidium is not a problem and if it is
high, boiling of water is necessary (National Ground Water Association, 1998).
2.5.4
Hepatitis
Hepatitis
A is an enteric Virus that is very small. It can be transferred through
contaminated water, causing outbreaks (John, 1990). The virus is excreted by a
person carrying it, and if the sewage contaminates the water supply, then the
virus is carried in the water until it is consumed by a host. Symptoms such as
an inflamed liver, accompanied by lassitude, anorexia, weakness, nausea, fever
and jaundice are common. A mild case may only require a week or two of rest,
while a severe case can result in liver damage and possible death (WHO, 1996).
Generally, water systems utilize chlorination, preceded by coagulation,
flocculation settling and filtration to remove the virus (John, 1990)
2.6.0 Sources
of Contamination
* Contaminated groundwater from a nearby
failing septic tank.
* A
non-air tight well cover or improperly welded or grouted well pipe, which can
allow Coliform bacteria to enter the water supply from runoff.
* Insects,
snakes, mice or other creative in the well (DHEC, 2009).
2.7.0 Effect of Drinking Contaminated
Well Water
Water remains the major source of
transmission of enteric pathogens in developing countries. Notified cases are
mostly in children especially those under 5 years of age (Federal Ministry of
Health of Nigeria, 1995). In whom gastroenteritis usually manifest as acute
diarrhea and often many require hospitalization. The conditions are usually
less severe in adults and may resolve without serious medical attention (Egwari
and Aboaba, 2002). In Nigeria, there is a high incidence of childhood diarrhea
despite the intensive activities of the national diarrhea control program
(Egwari and Aboaba, 2002). This is due to the unavailability of potable water especially
in rural communities, and mothers usually obtain water from unhygienic sources
for preparing weaning foods (Babaniyi, 1991). In reviewing the prevalence of diarrhea
in Nigerian children over a period of 9 to 12 years, it was observed that
315,000 children under the age of 5 years died annually from this disease
condition. Also children within this age range (0-5 years) are reported to
experience an average of 4.3 episodes of diarrhea annually (Federal ministry of
health of Nigeria, 1995), Studies carried out across the country have shown
that viruses, bacteria, protozoa, and helminthes are variously responsible for
diarrhea disease in children (Alabi et al., 1998). In one study, bacteria
cause 48.6% of the diarrhea cases, 30.6% were caused by viruses, 8.2% by
enteric parasite (protozoa and helminthes) and 6.9% were of dual etiology
(Alabi et al., 1998).
During period of water shortage,
people buy water of doubtful source from vendors; others resort to borehole or
well for water.
2.8.0 The septic system
The septic system consists of the
sewer line, septic tank, distribution tank and drain field. The system works on
gravity. The main sewer line running to
the tank is generally a three or four inch line that slopes, allowing a flow by
gravity of the waste from the house to the septic tank smaller lines, typically
one and one half inch for sinks, two inch for showers and three inch for
toilets would feed into the main line. The pipe, on systems installed within
the past thirty years, is polyvinyl chloride commonly called PVC. This pipe
typically requires little or no maintenance.
Modern septic tanks are made of
concrete, there are two hatches, square opening, on the top of the tank used
for cleanout. The size of the tank required is determined by the number of
bedrooms in the house. Biodegradable waste breaks down to mostly liquid and sludge
in the septic tank. The liquid rises to the top and overflows through a pipe
near the top of the septic tank and flows to the distribution tank. The
distribution tank has several ports which the drain field pipes are connected.
The liquid waste water then flows, again by gravity, out into the drain field
pipes.
The drain field consists of a
number, generally two to four pipes three inches in diameter. These pipes have
holes in the side along their length to allow the waste liquid to escape. The
size location, length and depth of the system determine the soils ability to
absorb the waste water. The waste water must be absorbed and go down into the
ground. Being allowed to come to the surface of the ground would result in a
health hazard (Wootlen home inspection, 2004).
CHAPTER THREE
MATERIALS AND METHODS
3.1.0 Materials
3.1.1
Equipment
For the list of equipment used, see Appendix 1
3.1.2 Media
List of media used and their preparation are given in Appendix
2
3.1.3 Reagents
List of reagents used are given in Appendix
3
3.2.0 Study Area
Abakaliki is the administrative
capital of Ebonyi state. It is located at the intersection of the Enugu, Afikpo
and Ogoja roads. Abakaliki was formerly known for its Guinea worm pandemic;
however, years of sustained provision of potable water program for the public
have eradicated Guinea worm cases from the state. Abakaliki is on latitude
6.28N and longitude 8.08E elevated at 380ft above sea level. Two main seasons
exist in the Abakaliki area; the dry season which lasts from November to March
and the rainy season which begins in April and ends in October with a short
period of reduced rains in August commonly referred to as “August break”. Temperature in the dry season ranges from 20
to 30OC and result in high evapotranspiration while during the rainy
season temperature ranges from 16 to 280C, with generally lower evapotranspiration.
The main occupation of the people is subsistence farming (mainly yam and
cassava) with some animal husbandry and other professions and/or activities
such as civil service, trading, artisans, and stone quarrying.
The sources of potable water to this
area are harvested rain water during the raining season, pipe borne water that
runs occasionally bore holes and hand dug wells which is the major source of
portable water especially during the dry season. (Aghamelu et al., 2011,
Ugwuja and Ugwu, 2007).
3.3 Sample Collection
15 samples of well water were
collected from wells located at different areas within Abakaliki metropolis (see
Appendix 4 for location of wells). The distances of the well water from the
septic tanks were measured with measuring tape.
The samples were collected with a
clean sterile 500ml plastic bottles tied to a weighted length of a rope.
Sterilization of the plastic bottles was done by rinsing with 1ml of ethanol
and finally rinsed again with water boiled at 100oC. The boiled
water was removed at the point of sample collection.
A heavy piece of metal of about 1kg
was attached to the bottle as supporting weight to enable it immerse deeply
into the well.
The cap of the bottle was
aseptically removed and the hot water poured out and the bottle lowered into
the well to a depth of about 1 metre from the surface water. When the air
bubbles stop to rise to the surface, the bottle was raised out of the well and
carefully the cap was replaced. The bottle was labeled, put into an insulated
cold box and transported to the laboratory and examined within 1 hour of
collection.
3.4
Analysis
of physical characteristics of the well water samples
3.4.1 Colour and Appearance
The colour and the physical
appearance of the water was observed with the naked eye and observation
recorded.
3.4.2 Determination of Temperature
The temperatures of the different
well water samples were determined using a standard thermometer Brannan
(100mm). This was done by immersing the thermometer into the water samples and
the value recorded.
3.4.3 Determination of pH
The pH of the well water samples
were determined using an UNIVERSAL pH indicator paper Q/GHSCI544-2006 shanghai
sss Regent. This was done by immersing 1/4 of the test strip in the well water
sample for 45seconds. Colour developed was matched with that of the standard
value and the value recorded.
3.5.0 Microbiological Analysis
3.5.1 Standard Plate Count
Standard plate count was done using the pour plate
method according to the method of Harold (1989).
The
pour plate method is used for counting those Cells in the inoculum which are
capable of growing on the medium. Each colony is assumed to have risen from a
single cell.
Procedure
The bottom of the sterile plates were labeled, 1ml
each of the sample were inoculated into
one sterile Petri dish for each sample, The sterilized molten agar was mixed by
gentle movement, allowed to cool and aseptically 15ml of the molten agar was
dispensed into each of the Petri dishes. The Petri dishes were rotated (rocked)
to distribute the bacterial cells evenly in the agar and then allowed to
solidify (gel). They were incubated at 350C for 48 hours. The plates
were observed for growth and number of colonies counted and recorded.
3.5.2 Serial
Dilutions
A serial dilution is any dilution
where the concentration decreases by the same quantity in each successive step.
Ten
fold logarithmic serial dilutions was used in which each test tubes were filled
with 9ml of peptone water and 1ml of sample was introduce to the first tube and
thoroughly mixed and then serially transferred to the rest
3.5.3 Enumeration Total Coliforms
3.5.3.1
Multiple Tube Method
In the examination of water for coliforms, the procedure is carried out
as the presumptive test, the confirmed test and the completed test. This method
measures an estimate of the number of viable cells that are capable of
multiplying in a specified liquid medium.
It is basically the dilution Count
which is made more accurate by the use of several tubes. In ordinary dilution
count, a serial 10-fold dilution of the liquid whose bacterial load is to be
counted is made. The combination of positive and negative tubes is read off the
table against the sample sizes to determine the MPN.
Procedures:
3.5.3.2
Presumptive Test
9 bottles of bijoux bottle were used
in all, 6 bottles contain single strength lactose broth while 3 bottles contain
double strength lactose broth. To 3 tubes of the Bijoux bottle, that contained
single strength lactose peptone broth were added 1ml of the water sample, and
to the remaining 3 tubes of single-strength lactose peptone broth were added
0.1ml. To the 3 tubes of double-strength lactose peptone broth were added 10ml
of the same well water sample. This was done for the 15 samples. The bottles
were incubated at 350C for 24hrs and observed for evidence of acid
and gas production. It was then re-incubated for another 24 hours. Gas positive
tubes were separated from negative tubes.
3.5.3.3
Confirmed Test
From gas positive tubes of lactose
peptone broth, a loopfull were streaked on Eosine-methylene blue (EMB) agar
plate and slant and incubated at 350C for 48 hours. The growth
characteristic and colour in EMB were observed and recorded.
Fig
3: Picture showing the growth
characteristic and colour of coliforms on Eosin Methylene Blue (EMB) agar slant,
inoculated from EMB plates,
3.5.2.4 Completed Test
A typical coliform colony on the EMB
plate (coionies with dark or black colour with reflective green metallic sheen)
was inoculated into a nutrient agar slant and incubated at 350C for
24hours. Growth from nutrient agar slant was used for Gram staining
Gram
Staining
Procedure
A thin smear was made on a slide and
allowed to air dry. The smear was heat fixed by passing the bottom of the slide
over the blue flame of a Bunsen burner for 30 seconds. The smear was covered with
crystal violet stain for 60seconds after 60seconds it was washed off with clean
water and covered with lugol’s iodine for 40 seconds and washed off with clean
water. The smear was decolorized rapidly with acetone alcohol and washed
immediately with clean water. The smear was then counterstained with neutral
red stain for 1 minute. It was then washed off with clean water, and the slide
then placed in a draining rack for the smear to air dry.
It was then examined microscopically
first with 40 x objective and then with the oil Immersion objective. (Cheesbrough,
2004).
3.5.2.5 Identification of Escherichia coli Colonies
on EMB Agar Plate.
The Identification of Escherichia coli colonies
of EMB agar was done using the IMVIC test. IMVIC represent
I: indole
M: Methylred
V: Voges proskaeur and
C: Citrate Test
Colonies that produced the characteristic
E coli colonies (dark centered and flat with or without metallic sheen)
were transferred into nutrient agar slants and incubated at 350C for
24 hours
Indole
Test
The suspected E coli colonies
from the nutrient agar slant were inoculated into tubes containing indole
medium (tryptone broth) and incubated at 350C for 48 hours. 0.5 ml
of indole (kovac’s reagent) were added to the tube and the formation of a red
ring in the upper layer indicated indole production (Taras et al., 1986)
Methyl
Red Reaction
Suspected E. coli cells were
inoculates into MR-VP medium and incubated at 35oC for 2
days. Drops of methyl red solution were added, shaken and examined. Red colouration
of the mediums gave a positive test while yellow colour was negative (Taras et
al., 1986)
Voges-Proskaeur
(VP) Test
After the methyl red (MR) test was
completed, 0.5ml 5% alpha-naphthol solution and 0.5ml of 40% potassium
hydroxide aqueous solution were added to the methyl red medium. The tubes were
shaken vigorously, sloped and examined after 15min. a positive reaction was
indicated by pink or red colour (Taras et al., 1986)
Citrate
Utilization
Colonies from the nutrient agar
slant were inoculated into Simmon’s citrate agar and incubated at 350C
for 2 days. The presence of blue colour/colonies in the medium indicated a positive
result (Harold, 1989).
CHAPTER FOUR
RESULTS
4.1: Physical Characteristics of the Well Water
Samples.
Samples 1 and 5 had milky colour, while sample 7, 8, 9 and 13 had
slightly milky colour. Also samples 2, 3, and 6 had light brown colour while
samples 4, 10, 11, 12 and 15 were colourless. (Table 1).
The appearance of the samples as was
observed showed that samples 1, 4, 5, 7, 8, 10, and 13 had particles while
samples 9, 11, 12, 14 and 15 had no particles. Samples 2, 3 and 6 were observed
to be cloudy, (Table 1).
The result of the pH test showed
that samples 2 and 3 had the highest pH value of 8 while the lowest pH of 6 was
recorded from samples 1, 5 and 7. Also samples 4,6,8,9,10,11,12,13,14, and 15 had pH value of 7, (Table 1).
With respect to odour, samples 1,
3and 14 had odour while samples 2,4,5,6,7,8,9,10,11,12,13, and 15 had no odour
(table 1).
The result of the temperature test
showed that sample 3 had the highest value with370C followed by
sample 12 with 36.00C while sample 10 had the least value of 31.80C.
It was also observed that the temperature of 350C occurred in
samples 14, 8, 5 and 4, followed by 34.0 0C that occurred in samples
13, 9 and 7, (Table 1).
TABLE
1: Physical Characteristics of the Well Water Samples
|
Sample
|
Colour
|
Appearance
|
pH
|
Temperature
0c
|
Odour
|
|
1
|
M
|
PP
|
6
|
33.0
|
OP
|
|
2
|
LB
|
C
|
8
|
36.0
|
OA
|
|
3
|
LB
|
C
|
8
|
37.0
|
OP
|
|
4
|
CL
|
PP
|
7
|
35.0
|
OA
|
|
5
|
M
|
PP
|
6
|
35.0
|
OA
|
|
6
|
LB
|
C
|
7
|
34.5
|
OA
|
|
7
|
SM
|
PP
|
6
|
34.0
|
OA
|
|
8
|
SM
|
PP
|
7
|
35.0
|
OA
|
|
9
|
SM
|
PA
|
7
|
34.0
|
OA
|
|
10
|
CL
|
PP
|
7
|
31.8
|
OA
|
|
11
|
CL
|
PA
|
7
|
33.0
|
OA
|
|
12
|
CL
|
PA
|
7
|
36.0
|
OA
|
|
13
|
SM
|
PP
|
7
|
34.0
|
OA
|
|
14
|
CL
|
PA
|
7
|
35.0
|
OP
|
|
15
|
CL
|
PA
|
7
|
34.5
|
OA
|
KEY
M - Milky
LB
- Light
Brown
CL - Colourless
PA - Particles
absent
OA - Odour
absent
SM - Slightly
Milky
PP - Particles present
C - Cloudy
OP - Odour
present
4.2: Standard Plate Count of the Well Water
Sample
The standard plate count of the well water samples showed that sample 6
had the highest count of 2.96 X 105 cfu/ml followed by sample 3 with
count of 2.90 x 105 cfu/ml. The least count of 3.8 x 104
cfu/ml was obtained with sample 2 followed by sample 15 with 5.0x104
cfu/ml (Table 2).
TABLE
2: Standard Plate Count of the Well Water Samples
|
Sample
|
Colony forming units (Cfu/ml) (x105)
|
|
1
|
1.10
|
|
2
|
1.70
|
|
3
|
2.90
|
|
4
|
0.86
|
|
5
|
0.47
|
|
6
|
2.96
|
|
7
|
2.70
|
|
8
|
2.40
|
|
9
|
2.45
|
|
10
|
0.60
|
|
11
|
1.20
|
|
12
|
0.38
|
|
13
|
1.10
|
|
14
|
0.80
|
|
15
|
0.50
|
4.3: Result of the Presumptive Test and MPN of
the Well Water Samples
The result of the most probable number of the well water samples showed
that samples 3 and 9 had the highest MPN index of 0.36 MPN/ml followed by
samples 6, and 7 with MPN index of 0.35 MPN/ml respectively. The lowest MPN
value of 0.27 MPN/ml were obtained with samples 1, 2, 4, 5, 8, 10, 11, 12, 13, 14
and 15 respectively (Table 3).
TABLE
3: Result of the Presumptive Test and
MPN of the Well Water
Samples
|
Sample
|
MPN
index/ml
|
|
1
|
0.27
|
|
2
|
0.27
|
|
3
|
0.36
|
|
4
|
0.27
|
|
5
|
0.27
|
|
6
|
0.35
|
|
7
|
0.35
|
|
8
|
0.27
|
|
9
|
0.36
|
|
10
|
0.27
|
|
11
|
0.27
|
|
12
|
0.27
|
|
13
|
0.27
|
|
14
|
0.27
|
|
15
|
0.27
|
4.4: Colonial Characteristics of the Organisms
on Eosin Methylene Blue (EMB)
Agar
The result showed that sample 1 had dark nucleated colonies, while
samples 2 and 4 had pink colonies with dark nucleated colonies too. Also
samples 3, 6 and 9 had only green metallic sheen while sample 7 and 8 had both
green metallic sheen and pink colonies. The result of samples 5, 10,
11,12,13,14 and 15 showed pink colonies only (Table 4).
TABLE
4: Colonial Characteristics of the Organisms on Eosin Methylene Blue (EMB) Agar
|
Sample
|
Characteristics
|
Organisms
|
|
1
|
Dark
nucleated colonies
|
other
Coliforms
|
|
2
|
Pink
colonies, dark nucleated
|
other
Coliforms
|
|
3
|
Green
metallic sheen
|
E
coli
|
|
4
|
Pink colonies,
|
other Coliforms
|
|
5
|
Pink colonies
|
other Coliforms
|
|
6
|
Green metallic sheen
|
E coli
|
|
7
|
Green metallic sheen, pink
colonies
|
E coli, other Coliforms
|
|
8
|
Green metallic sheen, pink
colonies
|
E coli, other Coliforms
|
|
9
|
Green metallic sheen
|
E coli
|
|
10
|
Pink colonies
|
other Coliforms
|
|
11
|
Colourless whitish colonies
|
Non coliforms
|
|
12
|
Colourless whitish and
light yellow colonies
|
Non coliforms
|
|
13
|
Pink colonies
|
other Coliforms
|
|
14
|
Pink colonies
|
other Coliforms
|
|
15
|
Pink colonies
|
other Coliforms
|
4.5: Identification of E coli
The result showed that samples
3,6,7,8 and 9, are gram negative, it also shows that the IMVIC test on samples
3,6,7,8 and 9 are indole positive, methyl red positive, citrate negative and Voges-Proskauer
negative (Table 5).
TABLE
5: Identification of E. coli
|
Sample
|
Gram
stain
|
Cell
morphology
|
Indole
|
MR
|
VP
|
Citrate
|
Organism
|
|
3
|
-ve
|
Rod shaped, slightly raised elevation with
smooth surface that appears in clusters.
|
+ve
|
+ve
|
-
ve
|
-ve
|
E
coli
|
|
6
|
-ve
|
Rod
shaped, slightly raised elevation with smooth surface that appears in
clusters.
|
+ve
|
+ve
|
-ve
|
-ve
|
E coli
|
|
7
|
-ve
|
Rod
shaped, slightly raised elevation with smooth surface that appears in
clusters.
|
+ve
|
+ve
|
-ve
|
-ve
|
E coli
|
|
8
|
-ve
|
Rod
shaped, slightly raised elevation with smooth surface that appears in
clusters.
|
+ve
|
+ve
|
ve
|
-ve
|
E coli
|
|
9
|
-ve
|
Rod
shaped, slightly raised elevation with smooth surface that appears in
clusters.
|
+ve
|
+ve
|
-ve
|
-ve
|
E coli
|
KEY
-ve = Negative
+ve = Positive
CHAPTER FIVE
DISCUSSION
Drinking water is expected to be
colourless as one of the characteristic of portability but the case is
different in some of the well water samples analysed (Table 1). The presence of
colour could be attributed to dissolved organic material from decaying
vegetation, certain inorganic matter and suspension of sediments. Although
colour itself is not usually objectionable from health standpoint, their
presences is aesthetically objectionable and suggest that the water needs appropriate
treatment, this is in line with the work of Thai industry standard, (2006).
Also
the presence of particles as shown in Table 1 may be due to the presence of
suspended material such as finely divided organic material, clay, silt and
other inorganic material in water otherwise known as turbidity in accordance
with the work of PEACH Inspections 1996. Turbidity acts as food source for
microorganisms allowing them to survive and multiply, and then triggering
possible threat to human health in line with the work of Burkhard, (2007). This
is also applicable to the presence of odour.
The pH value is a good indication of
whether water is hard or soft. The pH of pure water is 7, water with a pH lower
than 7 as seen in samples 1, 5, and 7 (Table 1) is considered acid and with pH
greater than 7 as seen in samples 2 and 3 (Table 1) as basic. Water samples
with a pH greater than 8 could be said to be hard. Hard waters does not pose a health risk but can also cause aesthetic problem like formation of scale
deposit on dishes, utensils and laundry, difficulty in getting soaps and
detergents to form lather and formation of precipitates on clothing while water
with pH less than 6 is acidic, soft and corrosive. Acidic water could contain
metal ions such as iron, copper, manganese, lead and zinc. In other words,
acidic water contains elevated levels of toxic metals this is in agreement with
the work of Burkhard, (2007).
The result of the standard plate
count on Table 2 shows that the least count is 3.8x104 cfu/ml against
the most commonly accepted maximum number of 0.27x102 cfu/ml and the
least MPN value of 27 MPN 100/ml as shown in Table 3 against the maximum
standard of 2.2 MPN/100ml as reported by Thai Industry Standard 2006. The
presence of coliforms as shown in Table 3 is an indication that the water is
unsafe and may cause illness.
Feacal contamination is evidence by
the presence of coliforms and E coli as shown in Table 3, 4 and
its confirmation in Table 5. Their presence in water possibly indicates the
presence of enteric pathogens; this is in accordance with the work of Thai
Industry Standard (2006).
Among all the samples tested,
presence of E coli were observed only on those well waters at a distance
below 50 feet with the septic tanks though all the samples showed the presence
of coliforms except samples 11 and 12
CHAPTER SIX
6.0SUMMARY,
CONCLUSION, AND RECOMMENDATION
6.1 SUMMARY
A total number of 15 well water samples were analysed, the physical
characteristics of these samples were determined, these include the colour,
appearance, pH and temperature. Also the microbiological analysis were carried
out, these include standard plate count, enumeration of total coliform by multiple tube method; presumptive test, confirmed
test, and completed test. Out of the 15 samples analysed, 13 samples had
coliforms while 5 samples showed the prensence of E coli
6.2 Conclusion
The result of the analysis has shown that most of the well waters
analyzed were contaminated with coliforms and E coli and can cause
serious human illness if consumed. These bacterial contaminants can be
controlled by chlorination, proper septic system, well maintenance and good
sanitation practices. The physical appearances of water in terms of odourless,
colourless, and tasteless, are not the only conditions for the determination of
water purity. The microbiological analysis is very important and should not be
neglected because of the adverse health effects that arise from drinking water
contaminated by microbes.
6.3
Recommendation
We
are recommending that the wells should not be used for drinking and that the
wells used for domestic purposes be tested for the presence of coliform every
one to two years. There is therefore need for public enlightment on boiling of
well water prior to use and for health agencies to ensure regular chlorination
of such wells. In addition, efforts need to be made to establish and enforce a
safe well-to-latrine distance standard especially for new buildings. In the
construction of new wells, it is preferably to be sited uphill and at least 50
feet from septic tank or 100 feet from leach field. The well should have an
apron that will serve as a barrier to contaminated water that leaches down from
around the entrance of the well. It must cover at least a 1.5m radius extending
from the well opening, and should include a channel that diverts the waste
water to a soak away tank that is 15m away from the apron. The well should have
a casing consisting of concrete ring that line the well from top to bottom. A
well cover and pump can be added to stop any gross contamination from above. If
there is no pump, there should only be one rope and bucket for the use of
collecting the water, suspended from the well head so that it can not be
removed or touch the ground.
It is also
recommended that wells should be sited at least 19 metres from the septic tank.
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APPENDIX 1
Equipment
The following laboratory materials were used; sterile
Petri dishes, pipettes, droppers, bijoux bottles, slide, sterile inoculation
loop, Durham tubes, autoclave, incubator, weighing balance, masking tape, cotton
wool, aluminum foil, graduated cylinder,
spatula, glass rod, thermometer, pH scale, microscope, test tubes and
measuring tape.
pH
scale
pH 1-14 Q/GH ScCI544-2006 Universal Indicator Paper.
Shanghai SSS Regard Co. Ltd
APPENDIX 2: List of Media,
Composition and Their Preparation
List of Media
Glucose phosphate broth
Lactose peptone broth
Nutrient
agar
Eosine
methylene blue (EMB) agar
Simmon’s
citrate agar
Voges-proskauer (VP) mediun
Indole medium
COMPOSITION
OF MEDIA
MR-VP MEDIUM
Peptone - 7.0g
Glucose - 5.0g
Dipotassium
hydrogen phosphate - 5.0g
Distilled
water
- 1000ml
INDOLE
MEDIUM.
Tryptone 10.0g
Sodium chloride 5.0g
DL
tryptophan 1.0g
Distilled
water 1000ml
SIMMONS
CITRATE AGAR
Sodium
ammonium hydrogen sulphate 1.5g
Dipotassium
hydrogen phosphate 1.0g
Magnessium
sulphate 0.2g
Sodium
citrate 3.0g
Distilled
water 1000ml.
Agar 15.0g
EOSIN
METHYLENE BLUE AGAR Gram/Liter
Balanced
peptone no 1 10.0
Lactose
10.0
Dipotassium
hydrogen phosphate 0.7
Potassium
dihydrogn phosphate 1.3
Eosin
yellow 0.4
Methylene
blue 0.0065
Agar
No 2 15.0
NUTRIENT
AGAR Gram/Liter
Peptone 5.0
Beef
Extract 3.0
Sodium
Chloride 8.0
Agar
no 2 12.0
LACTOSE
PEPTONE BROTH Gram/Liter
Peptone
from casein 17. 0
Peptone
from soymeal 3.0
Lactose 10. 0
Sodium
chloride
5. 5
Bromocresol purple 0. 02
PREPARATIONS
Eosin Methyline Blue Agar
Weigh 37.5 grams of powder and disperse in 1 liter of
deionized water, Allow to soak for 16 minutes, swirl to mix and sterilize by
autoclaving at 1210C for 15 minutes. Cool to 470C and
gently agitate to ensure even distribution of the precipitate, before pouring
into sterile Petri dishes.
Nutrient Agar
Weigh 28 grams of powder, disperse in 1 litre of deionized water, allow
soaking for 10 minutes, then sterilizing by autoclaving for 15 minutes at 1210C.cool
to 470C, mix will then pour plate.
Lactose
Peptone Broth
Suspend 35g or 70g in 1 litre of deionized water;
disperse into reagent tubes fitted with fermentation tubes, autoclave for 15
minutes at 1210C.
APPENDIX 3: LIST OF
REAGENTS
Methyl
red
95%
ethanol
Alpha
naphthol
40%
potassium hydroxide
Crystal
violet
Lugol’s
iodine
Acetone
Neutral
red
Distilled
water
:
Address and Distances of Well Waters from Septic Tank
|
|
Address
|
Distance
(metre)
|
|
1
|
33
Chukwu Street
|
10
|
|
2
|
5
Chukwu Street
|
8
|
|
3
|
13
Chukwu Street
|
5
|
|
4
|
Ugwuachara
|
19.20
|
|
5
|
1
Akaeze Street
|
8
|
|
6
|
3
Aniekwena Street
|
5
|
|
7
|
Em
Chuks Villa Ishieke
|
8.22
|
|
8
|
Agility
lodge Ishieke
|
15.24
|
|
9
|
Aguluzigbo
Street
|
6.40
|
|
10
|
Saint
Luke’s lodge Ishieke
|
12.19
|
|
11
|
5
Nachi street Azuiyiokwu
|
29.87
|
|
12
|
Edeukwu
Road
|
23.17
|
|
13
|
13
igbokwe Street
|
7m
|
|
14
|
30B
Emefor Street
|
8.84
|
|
15
|
21B
Udude Street
|
9.30
|
Fig 4:
Picture showing one of the well distances from septic tank.