ENUMERATION OF COLIFORMS FROM WELL WATERS LOCATED NEAR SEPTIC TANKS IN ABAKALIKI METROPOLIS, EBONYI STATE



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.                          
                
REFERENCES
Aghamelu, O.P, Nnabo, P.N, and Ezeh, H.N. (2011).Geometrical and            environmental problems related to shales in the Abakaliki area     South Eastern Nigeria African Journal of Environmental
            Science and Technology Vol. 5 (2),          Pp.       80-88. 
Alabi, S.A, Audo, R.A, Oyedeji K.S, Mafe I.G; Uhuanghi         J.E.(1998).     Viral, bacteria and parasitic agents associated with         infantile         diarrhea in Lagos. Nigeria Journal of Medical  Research ; 2: 29-32.
Babaniyi, O.A (1991). Oral rehydration of children with diarrhea in             Nigeria. 9-2 years review of impact on morbidity and     mortality from diarrhea disease and diarrhea treatment
            practices Journal of  Tropics 37:57-60. 
Baroni, L.; Cenci: L.; Tettamant; M.; Berati M. (2007) “Evaluating    the environmental impact of various dietary patterns        combined with different food production systems” European
            Journal of Climate Nutrition 61 (20): 279-286. 
 Braun, C. L.; Sergei N. S. (1993). Why is water             blue?
            “Journal of Chemical Education. 70 (8): 612.
Burkhard, M (2007). Prevention of Salmonella contamination. Seite 2.        Agent Detection. www.biotracer.org/assets/pdf-docume
Campbell, N.A.; Brad,W  Robin, J.H (2006). Biology: Exploring        Life.    Bosten, Massachusetts: Pearson Prentice Hall.Pp 499       
Cheesbrough M,(2005) District Laboratory Practice in Tropical        Countries       Part 1 2nd edition Cambridge University Press
            Pp.126.
Cheesbrough, M. (2004). District Laboratory practices in tropical     countries part 2. Cambridge University Press. Pp.225
Craun, G.F.(1996). Waterborne Disease in the United States. CRC     Press Inc. Boca Raton, FL.Pp 20-23
DeMan, John M. (1999). Principles of Food Chemistry. Springer.      http://books.google.com/
DHEC, S.C (2009). Effect on Health-fecal Coliform Bacteria in         Water. Journal of Environmental Health perspet. 102:854-856
Diehl, H.S (1960). Test book of Healthful Living 6th ed., McGraw Hill,                     York. Pp. 310
Dombek, D.E. and Sadowsky, M.J. (2004).Determining Sources of    faecal pollution. Applied Environmental microbiology
            70:4478-4485.
Egwari, L and Aboaba O.O. (2002). Environmental impact on the      bacteriological quality of domestic water supply in Lagos         Nigeria. Department of Botany and Microbiology, Faculty of
            Science University of Lagos. Akoka Lagos Nigeria  36:4.   

Encarta encyclopedia for windows, (2004). Compendium of methods for the Microbiological Examination of Foods         Microsoft Corporation Seattle Washington USA, 1-2.
Federal Ministry of Health of Nigeria.
            WHO/UNICEF/USAID/CCCD.(1995). Lagos; Nigerian
            Control of      Diarrhea Disease Programme         1991-1995.
            Lagos: Federal          Ministry of Health.
Freedman, B.(1997). Quality of Drinking Water in Sanitarians           handbook theory and Administrative Practice for Environmental            Health. Peerless Publishing USA  Pp.194.
Geldreich, E.E (1990). Microbial indicators of pollutions. Journal of           Water Pollution Control Federation 52 (3): 1774-1783.
Gerba, C.P, and Bitton, Gabriel, (1984). Microbial pollutants-their    survival and transport pattern to ground water, in Bitton,      Gabriel, and Gerba, C.P.; eds.; ground water Pollution Microbiology: New York, John Willey and Sons, Pp.65-88.
Harold J. B. (1989). Microbiological applications. A laboratpry         manual in general microbiology 5th ed WCB Brown publishers,     U.S.A. Pp78-79.      
Ingrid M. Verstracten, Greg S. Fetterman, Sonja K: Sebree, M.T         Meyer, and T.D Bullen (2004). Is septic waste affecting drinking      water from shallow domestic wells along the platte river in
            Eastern Nebraska, Prentice Hall Pp1-4. 
Jeffrey Utz, M.D. (2007). What percentage of the human body is       composed of water? Madsci Network. www.madsci.org
John Dezyane, P.E (1990). Handbook of drinking water quality,        Van     Nostrand Reinhold, N.v. Pp.53   
Juranek, D.D.(1995). Cryptosporidiosis: sources of infection and     guidelines for prevention. Centers for Disease Control and            Prevention, Atlanta GA. www.ncbi.nlm.nih.gov/pubmed/8547513
Kotz, J.C.; Treichel, P; and Weaver, G.C (2005). Chemistry and         Chemical Reactivity. Thomson Brooks/Cole pressPp76 
Kulshrestitha, S. N.(1998). “A global outlook for water resources to             the year2025” Water resources management.12(3):67-84
Lomborg, Bjorn (2001). The Skeptical Environmentalist.        Cambridge     University Press Pp. 22.
Malalcoff, D. (2002). Water quality. Microbiologists on the Trail of             Pollution Bacteria. New Focus Science 295:2352-2353.   
Maton, Anthea; Jean Hopkins, Charles William McLaughlin, Susan Johnson, Maryanna Quon Warner, David Lahtart, Jill D.           Wright (1993). Human Biology and Health. Englewood Cliffs,           New Jersey, USA; Prentice Hall.  Pp70-72
Millennium development goal(MDG) (2009) “Charting our water     future: Economic Frameworks to             inform decision-making.
MDG Report, United Nations. Pp16-20
National Atlas Gov (2001).estimated Water use in the United States.             www.nationalatlas.gov/mld/wu2ooot.html
National Groundwater Association (1998), 601 Dempsey Road,        Westervlle, Ohio 614-898-7791,
http://www.h20-       ngwa.org/pubaff/bac9-a.html.
Nebraska Department of Health and Human Services,(2002).             Regulations governing water well construction, pump installation and water well decommissioning standards:          Department of Health and Human Services Regulation and        Licensure. Pp 33-45
Nester, W.E, Anderson, D.G, Robert Jr, C.E Pearsall, N.N and             Nester, M.T (2001). Microbiology, a human perspective, 3rd        ed. The McGram Hill Companies Inc. New York 604-636.
Ogan, M.T. (1992). Magnitude of federal contamination of rural        community well waters in Nigeria and its relationship to well         and      water properties. Environmental health journal 73 (2): 168-174.
PEACH inspections(1996), Water quality facts. The national             association of certified home inspectors.www.callpeachstate.com/default.asp 
Prescott, L.M., Harley, J.P, Klein, D.A, (2002). Microbiology 5th ed. McGraw Hill Company Inc, New York 650-661,987-994.
Rand, M.C., Greenberge, A.E and Taras, M.J (1975).     Microbiological examination of water in standard method, for    the examination of water and waste water, 14 ed., P 875          New    York: American Public health Association, American Water Works Association and Water Control.  Pp875
Trivedi, R.K and Goel, P.K. (1986). Chemical and Biological Methods for Water Pollution Studies. Environmental          Publication Karad 415110.India. Pp70
Tuthill, M.S, Meikle, D.B; and Alavanja, M.C.R, (1998), Coliform     bacteria and nitrate contamination: Environmental Health,1:16-          20.  
U.S. Environmental Protection Agency, (2000), Current drinking      water standards-national primary and secondary drinking water regulations: office of Ground water and Drinking water,          accessed             URL:   http://www.epa.gov/safewater/standards.html.
U.S. Geological survey (2005). Microbiological contamination of     water U.S Department of the interior
            http://pubs.water.usgs.gov/fs07203  
Ugwuja, E.I. and Ugwu, N.C. (2007). Helicobacter pylori         uninvestigated          dyspepsia in primary cares in Abakaliki,   Nigeria. Online journal of   Health Allied Sciences. 1:4.

UNEP International Environment (2007). Water vapour in the Climate         System, Special Report (AGU). IWA publishing Pp78
UNEP International Environment (2002). Environmentally Sound     Technology for Wastewater and Storm water Management: An international source Book. IWA Publishing. Pp25-33  

UNEP International Environment (2002). Environmentally sound     technology for wastewater and storm water management: An        International Source book. IWA Publishing.
United Nations (2008). International Decade for Action water for life
2005-2015 www.un.org/u/quality.shtanl
W.H.O (1996). Guidelines for drinking water quality:  Health criteria          and other supporting information. World health
            Organizations, Geneva.  2nd edition volume 2:7-10
W.H.O (2008), “Safe water as the key to Global
            Health.www.inweh.unu.edu/documents/safewat
W.H.O Geneva (1998). Guidelines for drinking water quality             volumes 122 Surveillance and Control of Community         Supplies 2nd   ed.      
Wootlen Home Inspection (2004).Free Encyclopedia of building and           environmental inspection, Testing, Diagnosis and repair. Salisbury press Pp32
                           
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.

Share on Google Plus

Declaimer - MARTINS LIBRARY

NB: Join our Social Media Network on Google Plus | Facebook | Twitter | Linkedin