CHAPTER ONE

INTRODUCTION

1.1: BACKGROUND OF STUDY

Water plays a vital role in the productivity of aquatic habitats therefore in

understanding of the water physical and chemical properties serves as basis for

considering whether the water is rich or poor in biological production due to its

importance in the biotic community and significant role in the continuity of life

Olajuyigbe and Fasakin (2010). There are numerous scientific and economic

evidence such as industrial production, recreational activities that water pollution

can cause severe decrease in productivity and deaths of living species and

subsequent alteration of ecosystem services (Akpan et al, 2011). According to

Food and Agricultural Organization (FAO) African countries, particularly Nigeria,

water related diseases had been interfering with basic human development APHA

(2005), Improper management of huge amount of wastes generated by various

anthropogenic activities indiscriminate dumping of refuse (Bukar et al, 2015)

faecal, agricultural and industrial contamination or pollution which is continually

threatening aquatic ecosystem due to increasing exposure of untreated wastes and

urbanization in developing countries has gradually led to the deterioration of water

bodies in recent years (Akpan et al, 2011). Major agents of surface water pollution

include bacteria, viruses and other substances present in such concentration or

numbers to impair the quality of the water rendering it less suitable or unsuitable

for its intended use and presenting a hazard to man and other components of the

ecosystem. The increase in microorganisms and anthropogenic contaminants

enhances the risk of pathogen outbreaks, bacterial antibiotic resistance, and public

health costs Edungbola and Asaolu (2007). Incidence of diseases such as typhoid,

paratyphoid, giardiasis, infectious hepatitis, leptospiriosis, schistomiasis, Constant

monitoring of the quality of surface water cannot be overstressed especially now

that increase in population has resulted in generation of more waste thus exposing

the water to more pollutants (Egwuogu et al, 2016). According to Eja (2002), key

waste sources of rivers are dredging activities, markets, households, hotels,

hospitals but exclusive of toxic wastes. This problem has its roots to the

indiscriminate disposal of industrial waste, runoff of oil and grease from the

increasing filling stations, mechanic workshop and also the dumping of huge

amount of refuse in water bodies. Due to the increasing population in Imo state,

there has been an increase in waste generation and also more modern industries are

on the increase, but the fact still remains that the waste disposal system has not

grown as fast as the waste being generated (Akpan et al, 2011).

1.2: OBJECTIVES OF THE STUDY.

The general objective is to assess the physico-chemical and microbial load of

Oramiri-Ukwa river, Imo State.

The Specific Objectives are:

1. To determine the variation in the physico-chemical parameters

2. To compare the physico-chemical parameters with the WHO, NAFDAC
AND FAO standard
3. To determine the bacteria load of the water body
4. To evaluate the biochemical test of isolated organism

1.3: JUSTIFICATION OF STUDY

Water is a universal solvent as it tends to dissolve most substances. It is the natural

environment for aquatic life, these organisms a good carrier of micro-organisms

which could be transmitted to humans. Oramiri-Ukwa River along Ummahia-

Owerri road is surrounded by many inhabitants especially from Eyiogugu.

Sometimes, these villagers go to these water body to fetch drinking water of which

there is a nearby dredging activity going on. Also, similar research has been done

on Nworie river (Njoku et al, 2016), but no work has been done on Oramiri-Ukwa

river. This necessitates my interest to exploit and ascertain knowledge on this

natural water body which could be used for continuity of education and research.
 CHAPTER TWO

LITERATURE REVIEW

2.1 Review on Past Research by Several Authors

The various technical research paper on the assessment of water quality for lake,

river, sea and different areas have been presented at research level for the study.

Bhagat et al, 2013 has studied, in present investigation an attempt was made for

assessment of physical chemical parameter and quality of Sutlej River in Dogwe

area of Kaduna (Nigeria). The Physico-Chemical parameter were studied and

analyzed for the period of one year i.e. July 2010 to June 2011. Various physico-

chemical parameters such as water temperature, water colour, Turbidity, free

ammonia, Total dissolved solid, pH, Dissolved oxygen, Free CO 2, Total hardness,

total alkalinity, chlorides, BOD, Nitrates, Phosphates, Sulphates were studied. The

results revealed that there was significant seasonal variation in some

physicochemical parameters and River water was moderately polluted in Dogwa

area. On the basis of primarily study, it was apparent that water was not potable but

can be used for propagation of wildlife, fisheries and irrigation.

Manjusha et al. (2013), Has studied Water quality assessment of the River

Godavari, at Ramkunda Nashik. Three samples collected from three locations

along the area. Study area during the months

June, August, and October respectively. During present study some physical and

chemical properties were determined. The measurement of temperature, pH and

TDS were taken in the field. Immediately after the collection of samples, using a

portable water quality analyzer, Chlorides, Total hardness, Ca, Total alkalinity.

The result of the study shows that; the river is polluted at Ramkunda. It is believed

that continued pollution of the water sources by various human activities may lead

to any health problem to human. The values of correlation coefficients and their

significance levels will help in selecting the proper treatments to minimize the

contaminations of river water of Godavari. Mathuthu et al. (2016), have studied

physico-chemical parameter evaluation of water quality around Chandrapur

(Delimi). Two sampling stations were selected at the downstream of Chandrapur

town. The water sample was collected from the Wardha River at 3 different

selected stations. Over a period 12 months during the year. The various

physicochemical parameters were studied. In the present study it is our efforts to

evaluate many physico-chemical and its characteristic behavior of the river water

samples in different seasons and different sampling stations. Many values of

parameter crossed the maximum permissible limit. The study suggested immediate

need to take extensive water quality monitoring studies and to find the remedial

measures to perfect this important natural water source in the study area. Peter and

John (2011) has studied physico-chemical analysis water quality of Oramiri Ukwa.
We studied a four different stations for a period of one year during April-2011 to

March-2012 seasonal variation of different parameters investigated were as follows

pH (6.65 to 8.42), dissolved oxygen (3.25 to 11.78 mg/l), BOD (0 to 3.65 mg/l),

CO2 (0.55 to 2.3 mg/l), Electrical conductivity (0.36 to 29.1ms-1), Potassium (0.12

to 9.74 mg/l), Calcium (0.50 to 42.34 mg/l), Magnesium (0.25 to 109.5 mg/l),

Sodium (0.017 to 878.04 mg/l), bicarbonate (1.40 to 6.23 mg/l), Carbonate (nil),

Chloride (2.23 to 380.70 mg/l). The result of the study shows that as the season

changes there is fluctuation in the physic chemical characters of the water, this will

be due to ebb and flow flushing of rain water change in the temperature and

salinity as the season changes. In addition, intense pollution from both agricultural

input and shrimp culture ponds deteriorates the water quality of mangrove

ecosystems. J.G. Koliyar and N.S. Femi (207) have studied in order to understand

the water quality in Iholu Dam, Ibadan. The purpose of the survey was to collect

information concerning the use and value of water quality improvement at

Dorowa. The result is that the impurities are present in Dam. There are many

different parameters formed to be increased during summer season and got diluted

during rainy season. Lack of oxygen content can cause fish kills and lack of fish

enable malaria hosting mosquitoes as mosquitoes are natural food for fish.

Throwing waste material and garbage in the lake water should be strictly
prohibited proper bioremediation techniques should also use in order to improve

the water quality.

Abdul et al. (2014) has studied assessment of water quality in the Karanja

Reservoir. Monitoring of reservoir water quality on a regular basis plays an

important role in the detection and evaluation of water pollution. During the

present investigation different physical and chemical parameter were monitored

during dry season and rainy season for the period of 12 months (January -2014 to

December -2014). It is stated that at present the Karanja reservoir water is

relatively clean, the variation observed with respect to physical and chemical

variables of surface water are seasonal and are not significantly imparted by the

anthropogenic pressure. The load

of pollution in the Karanja reservoir is within the assimilate capacity of the body.

The data on nutrient level also indicate that sewage added into the water body,

either gets diluted or utilized as a source of nutrients for the photosynthesis.

2.2 Parameters Included in Water Quality Assessment

The following different physico-chemical parameter are tested regularly for

monitoring quality of water.

1) Temperature: Temperature is the most importance environment factor with

effect on plants and animals. Water has several unique thermal properties which
combine to minimize temperature change. The Water temperature depends on the

depth of the water column, climatic and topographic changes WQA 1992.

2) pH: pH, one of the most common analyses in soil and water testing, is the

standard measure of how acidic or alkaline a solution is. It is measured a scale

from 0 -14. pH of 7 is neutral, pH is less than 7 is acidic and pH greater than 7 is

basic. Aquatic organisms need the pH of their water body to be a certain range

optimal growth and survival. The presence of acid rain can lower the pH in lakes

making them more acidic (Gupta et al, 2009).

3) Electrical conductivity: Electrical conductivity (EC) a measure of the electric

current that solution carries. Electrical conductivity used to quickly estimate the

ionic or soluble salt concentration in soils, water supplies, fertilizer solution and

chemical solution APHA, 1985. It is measured with the help of EC meter which

measures the resistance offered by the water between two platinized electrodes.

The instrument is standardized with known values of conductance observed with a

standard KCl solution (Chavan et al, 2005).

4) Alkalinity: Alkalinity is a chemical measurement of water’s ability to neutralize

acid. Alkalinity is also a measure of a water buffering capacity or its ability to

resist changes in pH upon the addition of acids or bases (Devendra et al, 2014).

Alkalinity of natural water is due to primarily to the presence of weak acid salts,
although strong bases may also contribute (i.e. OH -) in the extreme environment.

Bicarbonate represents the major form of alkalinity in natural water, so its source

being the partitioning of CO2 from the atmosphere and the weathering of carbonate

minerals in rocks and soil (Patil et al, 2012). Other salts of weak acids, such as

borate, silicates, ammonia, phosphate, and organic bases from natural organic

matter may be present in small amounts (Patil et al, 2012).

5) Dissolved Oxygen: The amount of oxygen dissolved in water, such as a lake,

river or stream. Dissolved oxygen is the most important indicator of the health of

water bodies and its capacity to support a balanced aquatic ecosystem of plants and

animals (Patil et al, 2012). Warm water released from industrial outlets, flowages

or storm sewers can also reduce dissolved oxygen levels. Dissolved oxygen may

play a large role in the survival of aquatic life in temperature lakes and reservoirs

during summer months (Patil et al, 2012).

6) Total Hardness: Total hardness is defined as the sum of calcium and

magnesium hardness in mg/L as CaCO3.Total hardness in fresh water is usually in

the range of 15 to 375 mg/L as CaCO 3. Calcium hardness in freshwater is in the

range of 10 to 250 mg/L, often double that of magnesium hardness (5 to 125 mg/L)

and total hardness of 6630 mg/L as CaCO 3 APHA, 1985. A high concentration of

hardness may be due to leaching from of the soils or due to the high background

concentration of the waters. WHO permissible limit for total hardness of water is
150 mg L-1 and ISI desirable limit was 300 mg L -1. Suggested that the values

between 150 and 300 mg L-1 of TH means the water was hard, and TH greater than

300 mg L-1 means the water is very hard. High concentration of hardness may

cause the problem of heart disease and kidney stones (APHA, 1985).

7) Total dissolved solid: Total dissolved solids are the total amount of mobile

charged ions, including minerals, salts or metal dissolved in a given volume of

water in mg/L. TDS is directly related to the purity of water and the quality of

water purification system and affects everything that consumes, lives in, or uses

water, whether organic or inorganic, whether for better or for worse. Common

inorganic salts that can be found in water include calcium, magnesium, potassium

and sodium, which are cations and carbonates, nitrates, bicarbonates, chlorides and

sulphates which are anions (Gupta et al, 2009).

8) Chemical oxygen demand: The standard method for indirect measurement of

the amount of pollution in a sample of water Gupta et al, 2009. The chemical

oxygen demand test procedure is based on the chemical decomposition of organic

and inorganic contaminants, dissolved or suspended in water. In the present

investigation the recorded low value of DO and higher values of BOD and COD

can be described to the discharge of effluents and non-point source of pollution in

the coastal area and mangrove station (Mahesh and Prabhahar, 2012).
9) Biochemical oxygen demand: Biochemical oxygen measures the amount of

oxygen that microorganisms consume while decomposing organic matter, it also

measures the chemical oxidation of inorganic matter BOD is a measure of organic

material contamination in water, specified in mg/L. BOD is the amount of

dissolved oxygen required for the biochemical decomposition of organic

compounds and the oxidation of certain inorganic materials (e.g., iron, sulphites).

Typically, the test for BOD is conducted over a five-day period (Milacron

Marketing Co.).

10) Carbonates: Whenever the pH touches 8.3, the presence of carbonates is

indicated. It is measured by titration with standardized hydrochloric acid using

phenolphthalein as indicator. Below pH 8.3, the carbonates are converted into the

equivalent amount of bicarbonates. The titration can also be done pH metrically or

potentio-metrically (Devendra et al, 2014).

11) Bicarbonates: It is also measured by titration with standardized hydrochloric

acid using methyl orange as indicator. Methyl orange turns yellow below pH 4.0.

At this pH, the carbonic acid decomposes to give carbon dioxide and water.

12) Nitrate: Nitrate is naturally occurring inorganic ions present our environment.

The decomposition of organic materials in soils, releases ammonia. This ammonia

oxidizes to form nitrate. Drinking water containing nitrates. Wells with high levels
of nitrate can contribute to significant exposure. Eating foods containing nitrates

preservative, such as processed meats. Nitrates can change normal hemoglobin to

met hemoglobin Devendra et al, 2014. Nitrate test can be detected through urine

and blood test.

13) Chloride: Chloride, the ionized form of chlorine, is one of the most abundant

inorganic ions in natural water and wastewater (Tambekar, 2013) Though most

prevalent in sea water at concentration averaging 35000ppm, Chloride permits

rivers, lakes, and other freshwater systems. In normal fresh water, chloride

concentration is usually less than 10 ppm, but quite often less than 1 ppm

(Tambekar, 2013) The potentiometric method of chloride analysis by silver nitrate

titration is an effective technique of chloride level determination. The effect of

chloride on stomach discomfort, Eye/nose irritation (Devendra et al, 2014).

14) Sulphates: It is measured by the nephelometric method in which the

concentration of turbidity is measured against known concentration of synthetically

prepared sulphate solution (Tambekar, 2013) Barium chloride is used for

producing turbidity due to barium sulphate and mixture of organic substances

(Glycerol or Gum acetia) and sodium chloride is used to prevent the settling of

turbidity (Tambekar, 2013)

15) Calcium: It is measured by complexometric titration with a standard solution

of EDTA using patton’s and Reader indicator under the pH condition of more than

12.0

16) Iron: Iron is one of the most important constituents of blood in human and

another living organism. Iron is an essential element for human nutrition and

metabolism, but in excess quantities results in toxic effect like hemochromatosis in

tissues. The maximum permissible limit of iron in drinking water is 0.3 ppm.

(Tambekar, 2013).

2.3 MICRO-ORGANISM
exist as single-celled form or as a colony of cells. The possible existence of unseen

microbial life was suspected from ancient times, such as in Jain scriptures from

sixth century BC India (Tyrell and Kellyl , 2017).

The scientific study of microorganisms began with their observation under

the microscope in the 1670s by Anton van Leeuwenhoek. Louis Pasteur found that

microorganisms caused food spoilage, debunking the theory of spontaneous

generation. Robert Koch discovered that microorganisms caused the

diseases tuberculosis, cholera, diphtheria, and anthrax. Because microorganisms

include most unicellular organisms from all three domains of life they can be

extremely diverse (Schopf et al , 2017).

According to Gest (2005), Two of the three domains Archaea and Bacteria, only

contain microorganisms. The third domain Eukaryota includes all multicellular

organisms as well as many unicellular protists and protozoans that are microbes.

Some protists are related to animals and some to green plants. There are also many

multicellular organisms that are microscopic, namely micro-animals, some fungi,

and some algae, but these are generally not considered microorganisms.

Microorganisms can have very different habitats, and live everywhere from

the poles to the equator, deserts, geysers, rocks, and the deep sea (Schopf et al ,

2017). Some are adapted to extremes such as very hot or very cold conditions,

others to high pressure, and a few, such as Deinococcus radiodurans, to high

radiation environments. Microorganisms also make up the microbiota found in and

on all multicellular organisms (Tyrell and Kellyl , 2017). There is evidence that

3.45-billion-year-old Australian rocks once contained microorganisms, the earliest

direct evidence of life on Earth.

Microbes are important in human culture and health in many ways, serving

to ferment foods and treat sewage, and to produce fuel, enzymes, and

other bioactive compounds. Microbes are essential tools in biology as model

organisms and have been put to use in biological warfare and bioterrorism.

Microbes are a vital component of fertile soil. In the human body, microorganisms

make up the human microbiota, including the essential gut flora.

The pathogens responsible for many infectious diseases are microbes and, as such,

are the target of hygiene measures (Tyrell and Kellyl , 2017).

2.3.1 BACTERIA

are ubiquitous, mostly free-living organisms often consisting of one biological cell.

They constitute a large domain of prokaryotic microorganisms (Norris et al , 2007)

Typically a few micrometres in length, bacteria were among the first life forms to

appear on Earth, and are present in most of its habitats. Bacteria inhabit soil,

water, acidic hot springs, radioactive waste, and the deep biosphere of Earth's

crust. Bacteria are vital in many stages of the nutrient cycle by recycling nutrients

such as the fixation of nitrogen from the atmosphere. The nutrient cycle includes

the decomposition of dead bodies; bacteria are responsible for

the putrefaction stage in this process (Norris et al, 2007). In the biological

communities surrounding hydrothermal vents and cold

seeps, extremophile bacteria provide the nutrients needed to sustain life by

converting dissolved compounds, such as hydrogen-sulphate and methane, to

energy. Bacteria also live in symbiotic and parasitic relationships with plants and

animals. Most bacteria have not been characterized and there are many species that

cannot be grown in the laboratory. The study of bacteria is known as bacteriology,

a branch of microbiology. Humans and most other animals carry millions of

bacteria. Most are in the gut, and there are many on the skin. Most of the bacteria

in and on the body are harmless or rendered so by the protective effects of

the immune system, though many are beneficial, particularly the ones in the gut

(Thanbichler et al , 2005)

However, several species of bacteria are pathogenic and cause infectious diseases,

including cholera, syphilis, anthrax, leprosy, tuberculosis, tetanus and bubonic

plague (Van, 2001). The most common fatal bacterial diseases are respiratory

infections. Antibiotics are used to treat bacterial infections and are also used in

farming, making antibiotic resistance a growing problem. Bacteria are important

in sewage treatment and the breakdown of oil spills, the production

of cheese and yogurt through fermentation, the recovery of gold, palladium, copper

and other metals in the mining sector, as well as in biotechnology, and the

manufacture of antibiotics and other chemicals (Thanbichler et al , 2005).

Once regarded as plants constituting the class Schizomycetes ("fission fungi"),

bacteria are now classified as prokaryotes. Unlike cells of animals and

other eukaryotes, bacterial cells do not contain a nucleus and rarely

harbour membrane-bound organelles (Schloss and Handelsman, 2004). Although

the term bacteria traditionally included all prokaryotes, the scientific

classification changed after the discovery in the 1990s that prokaryotes consist of

two very different groups of organisms that evolved from an ancient common
ancestor. These evolutionary domains are called Bacteria and Archaea (Schloss

and Handelsman, 2004).

Habitat

Bacteria are ubiquitous, living in every possible habitat on the planet including

soil, underwater, deep in Earth's crust and even such extreme environments as

acidic hot springs and radioactive waste (Schloss and Handelsman, 2004). There

are approximately 2×1030 bacteria on Earth (Euzéby, 2011) forming a biomass that

is only exceeded by plants. They are abundant in lakes and oceans, in arctic ice,

and geothermal springs (Schloss and Handelsman, 2004).

where they provide the nutrients needed to sustain life by converting dissolved

compounds, such as hydrogen sulphide and methane, to energy (Euzéby,

2011). They live on and in plants and animals. Most do not cause diseases, are

beneficial to their environments, and are essential for life. The soil is a rich source

of bacteria and a few grams contain around a thousand million of them. They are

all essential to soil ecology, breaking down toxic waste and recycling nutrients.

They are even found in the atmosphere and one cubic metre of air holds around

one hundred million bacterial cells. The oceans and seas harbour around 3 x
1026 bacteria which provide up to 50% of the oxygen humans breathe. Only around

2% of bacterial species have been fully studied.

Morphology

Bacteria display a wide diversity of shapes and sizes. Bacterial cells are about one-

tenth the size of eukaryotic cells and are typically 0.5–5.0 micrometres in length.

However, a few species are visible to the unaided eye—for

example, Thiomargarita namibiensis is up to half a millimetre long (Euzéby,

2011) and Epulopiscium fishelsoni reaches 0.7 mm. Among the smallest bacteria

are members of the genus Mycoplasma, which measure only 0.3 micrometres, as

small as the largest viruses. (Euzéby, 2011) Some bacteria may be even smaller,

but these ultramicrobacteria are not well-studied (Thanbichler et al , 2005).

Most bacterial species are either spherical, called cocci (singular coccus, from

Greek kókkos, grain, seed), or rod shaped, called bacilli (sing. bacillus,

from Latin baculus, stick) (Thanbichler et al , 2005). Some bacteria, called vibrio,

are shaped like slightly curved rods or comma shaped; others can be spiral shaped,

called spirilla, or tightly coiled, called spirochaetes. A small number of other

unusual shapes have been described, such as star-shaped bacteria (Louie,

2000). This wide variety of shapes is determined by the bacterial cell

wall and cytoskeleton, and is important because it can influence the ability of

bacteria to acquire nutrients, attach to surfaces, swim through liquids and

escape predators. Many bacterial species exist simply as single cells; others

associate in characteristic patterns: Neisseria forms diploids

(pairs), streptococci form chains, and staphylococci group together in "bunch of

grapes" clusters. Bacteria can also group to form larger multicellular structures,

such as the elongated filaments of Actinobacteria species, the aggregates

of Myxobacteria species, and the complex hyphae of Streptomyces species. These

multicellular structures are often only seen in certain conditions. For example,

when starved of amino acids, myxobacteria detect surrounding cells in a process

known as quorum sensing, migrate towards each other, and aggregate to form

fruiting bodies up to 500 micrometers long and containing approximately 100,000

bacterial cells. In these fruiting bodies, the bacteria perform separate tasks; for

example, about one in ten cells migrate to the top of a fruiting body and

differentiate into a specialized dormant state called a myxospore, which is more

resistant to drying and other adverse environmental conditions (Louie, 2000).

Bacteria often attach to surfaces and form dense aggregations called biofilms, and

larger formations known as microbial mats. These biofilms and mats can range

from a few micrometres in thickness to up to half a metre in depth, and may

contain multiple species of bacteria, protists and archaea. Bacteria living in

biofilms display a complex arrangement of cells and extracellular components,

forming secondary structures, such as microcolonies, through which there are

networks of channels to enable better diffusion of nutrients (Louie, 2000). In

natural environments, such as soil or the surfaces of plants, the majority of bacteria

are bound to surfaces in biofilms (Bryant et al, 2006). Biofilms are also important

in medicine, as these structures are often present during chronic bacterial infections

or in infections of implanted medical devices, and bacteria protected within

biofilms are much harder to kill than individual isolated bacteria (Bryant et al ,

2006).

2.4 MICROBIAL TESTING PROCEDURES

1. Surface Sampling

Although recovery of all microorganisms from a surface may not always be

possible, the consistent monitoring of specific areas in a food plant by surface

testing does provide valuable insight into the relative cleanliness of that area.

Swabbing

MO collected from a surface with sterile cotton or calcium alginate swabs (alginate

swabs are the best since the alginate can be readily dissolved in

hexametaphosphate), transferred to broth where they are dislodged, then diluted

and used with further tests to determine total numbers. Sponges can be used to

swab larger areas then placed in a buffer-filled bag (Bryant et al, 2006).

Contact plates

(Rodac plates) – raised agar plate that is pressed against a surface and then

incubated. A modified version of this technique is the agar syringe or

“sausage.” Tube full of agar, samples are pressed against a surface and then sliced

off into a Petri plate for incubation (Bryant et al, 2006).

Excision method

a plug of know surface area is taken from the food and then 1-3 mm is taken from

the surface end, homogenized and plated to determine total numbers. Commonly

used in whole meat cuts (Bryant et al, 2006).

2.5 MOLECULAR METHODS TO DETECT BACTERIA

Oligonucleotide DNA Probes

This test is based on DNA or DNA-RNA complementary base pairing or

hybridization.Go through a hybridization procedure; include colony hyb (can run

up to 69 filter w/48 CFU per filter in one Reaction), dot blots or blots of restriction

digests. can use radioactive or non-isotopic labels on probes. Reactions can

require anywhere from 10 h - 2 days. this technique requires that the sequence of

the target nucleic acid (frequently a gene for toxin production or a species-specific

16S rRNA sequence) be determined and unique to the organisms sought. As we

discussed earlier in the lectures on taxonomy, 16S rRNA contains widely

conserved as well as species (and sometimes subspecies) -specific regions. Probes

to the species-specific regions are used to detect particular bacteria such

as Salmonella. One advantage to targeting the 16S rRNA versus a DNA sequence

in the chromosome is that there are several copies of the 16S rRNA per cell. The

same advantage applies to genes located on multicopy plasmids. Gene probes may

be based on the DNA sequence of particular toxins such as Clostridium

perfringens or staphylococcal enterotoxins. At least one test for Salmonella has

AOAC approval.

Polymerase Chain Reaction (PCR-DNA Amplification)

This technique is more useful than DNA probing when small numbers of a

particular microbe are present. It can amplify a single copy of any DNA sequence

107 times within a few hours. Amplified DNA can then be detected by agarose gel

electrophoresis or hybridization. PCR requires primers based on the known nucleic

acid sequence of the amplification target. PCR has been used to detect 1-5 CFU

of E. coli in 100 ml of water, useful for any organism where a unique nucleic acid

sequence is known, also for diagnosis by amplification of 16s rRNA (ASM

handout “new vistas for bacteriologists). Very powerful tool with many, many
applications; the latest clinical manual for diagnostic microbiology is almost

completely based on PCR assays.

Enzyme-Linked Immunosorbent Assay (ELISA)

Another very powerful technique that uses mono- or polyclonal antibodies to

detect specific antigens in a sample. Commercial ELISA kits are available for a

variety of applications (e.g. home pregnancy tests) including the detection of

various microbes including Salmonella, S. aureus and its enterotoxins, molds and

mycotoxins, botulinal toxins and E. coli strains and toxins.

Immunoprecipitation (Immunodiffusion)

Another immunological technique commonly used for toxin identification. Several

variations but the basic principle involves a reaction between antigen (toxin) and

antibody that forms a precipitate in an agar gel. Immunodiffusion methods can

detect as little as 0.1-0.01 µg of toxin. Incubation times increase with smaller

amounts of antigen (1-6 d range).

2.6 METHODS OF DETERMINING TOTAL MICROBIAL NUMBERS

Once the sample is prepared, there are several methods available to determine total

cell numbers in the food. Many of these methods also allow for the enumeration of

important groups of bacteria, such as coliforms. A few can allow you to count the

number of microbes from a specific genus like Salmonella (Thanbichler et al,

2005).
Standard Plate Count (SPC)

By far the most widely used method for determining the number of viable colonies

forming units (CFU) in a food. Use a spread or pour plate (psychrotrophs may not

survive as well in pour plates) that includes homogenized food sample (Davey and

O'toole, 2016). Incubation is aerobic at 35oC for 48± 2 h.

APC–aerobic plate count

cells spread over agar surface and incubated aerobically.

Spiral Plate Counter:

An automated version of SPC is the spiral plater, a device that distributes a

continuously decreasing volume of liquid over a single rotating agar plate (the

dispensing arm moves like a needle on a turntable, only backward). The agar is

then incubated and counts are made.

Dry Petri-films

Two plastic films held together on one side and coated like a sandwich with

culture media ingredients, tetrazolium dye (a reducing dye) and a water

soluble gelling agent (Davey and O'toole, 2016). 1 ml of sample in diluent is

placed between the films and gently spread around by pressing the 2 sides of

film. After incubation, cell growth reduces the dye and gives red colonies.

Most Probable Numbers

A method based upon statistical probability. Food samples are prepared like

SPC. Three serial dilutions are prepared and then transferred to 9 or 15 tubes (3-

or 5-tube method). The numbers of organisms/g are then estimated using standard

MPN tables.

Impedance

Impedance is a measurement based on the resistance in an electric circuit to the

flow of alternating current. Microorganisms metabolize substrates of low

conductivity into products of higher conductivity. As a result, resistance decreases

with growing cell numbers. With proper instruments, the change in impedance can

be followed and used to detect as few as 10 to 100 cells in a sample within about 6

h (impedance detection time or IDT). The technique is faster than SPC and gives

results that are within 90-95% agreement with the former technique but is not

AOAC approved (Davey and O'toole, 2016).

 CHAPTER THREE

MATERIALS AND METHODS

3.1: STUDY AREA

Oramiri-ukwa River is located at Azaraegbelu, Emekuku in Owerri North Local

Government Area of Imo State, Southeast Nigeria. It sits at approximately latitude

5O15` and 5O 35’ N and longitude 6O33` and 7O 15’ E. Oramiri-Ukwa is a typical

rain forest river. On both sides of the main river channel are large fridges of heavy

forested swamps dominated by the raffia palm. The river flows through a highland

in Okigwe and joins the Mbaa River, to flow through Okahia Ezihe in Isiala

Mbano Local Government Area, through Opara-nadim in Mbaise to Onu-ngara

Avuvu in Ikeduru Local Government Area of Imo State, Nigeria. Oramiri-ukwa

flows southward for about 5.8km before reaching Otamiri River and Nworie River

both are tributaries of the larger Imo River which drains into the Atlantic Ocean

south-east Nigeria. The climate of the area is characterized by (2) two district

seasons, the dry (November – March) and rain seasons (April – October). The

River is the main source of water supply to the towns and villages through which it

flows, especially during the dry seasons (Adaka, et. al, 2015).

Fig 1: Sketch Map of Oramiri-Ukwa River

Source: (Adaka, et al, 2015).

3.2: SAMPLE COLLECTION

Using sterile bottle containers, water samples was collected from three (3) different

points (A, B and C) of the river. The water samples were labelled thus: A, B, C

respectively indicating samples from the three (3) different sampling points. Water

samples from the River was collected monthly from April to June, 2023, using

clean polypropylene plastic container washed with 0.01% of HCl. The container

was washed again and rinsed several times with distilled water. Prior to collection

of water samples at the site, the containers were rinsed with the water samples.

Water samples was collected in different containers by dipping it into the River,

allowing it to fill to the brim and immediately the containers were corked and

transported to Federal Medical Center (FMC).

3.3: MATERIALS AND EQUIPMENT

Materials and equipment to be used in the analysis includes the following:

Weighing balance, Autoclave, Inoculation needles, Forceps, Wire loops, Bunsen

burner, Glass-wares (Beakers, Conical flask, Petri-dishes, Test-tubes, Measuring

cylinders etc.), Cotton wool, Whatman filter paper, Incubator. The reagents used

are distilled water, iodine, crystal violet, safranine, KOVAC’s reagent, ethanol etc.

Media to be used include: Nutrient agar, MacConkey agar, Eosine Methylene Blue

agar, Sabour and dextrose agar for microbiological studies. Diluents include

distilled water and some biochemical reagents.

3.4: STERILIZATION OF MATERIALS

Glass-wares was sterilized using hot air oven at 1700 C for two (2) hours. Moist

heat, dry heat, directs flaming and chemical methods of sterilization as will also be

adopted for the sterilization of materials.

3.5: Physicochemical Analysis

3.3.1 Temperature

The temperature of various samples was determined by dipping a thermometer

calibrated in degree centigrade (00C) into the water and reading the corresponding

temperature.

3.3.2. pH

the pH of the water samples was determined using Philips Model pw 9418 pH

meter. After allowing the pH meter to warm up for about 15 minutes and dully
calibrated with standard buffers pH 4.0, 7.0 and 9.0, the pH of the water was

determined by dipping the electrode into the water samples.

3.3.3. Conductivity

Conductivity was determined using Bridge type M.C. 3 conductivity meter

calibrated

3.3.4. Hardness

To 100 ml of the water sample was added 2 ml of NH4/NH4Cl buffer to bring the

pH value to 9.0. About 0.2 g of Erichrome black T indicator was added which

formed a wine red colour. The whole content was stirred and titrated against 0.01

M EDTA to blue end point.

(0.01M of EDTA x Titration value x molarmassofCaCO 3 x 1000) ÷ Sample (ml)

3.3.5 Chloride (Cl-)

Titri-metric method was employed in which 50 ml of the samples were titrated

against a standard solution of 0.0257M silver nitrate (AgN03) solution with 1 ml

solution of 5% K2Cr2O7 as indicator to a reddish-brown end point.

Cl- = ((A-B) x 35400) ÷Sample

A=Volume of AgNO3 used for sample titration M=Molarity of AgNO3 used

B= Volume of AgNO3 used for blank titration

M = Volume of AgNO3 used for titration

3.3.6 Sulphate

To 10 ml samples in 25 ml Erlenmeyer flask was added 5 ml of gelatin barium

chloride. Stirring continued for 1 minute and stand for 30 minutes. A cloudy colour

was observed. The absorbance was then determined at 420 nm by corning

colorimeter 253. Standards ranging from 2ppm-10ppm SO42- were prepared from

sodium sulphate stock solution and treated as above. Sulphate concentration was

read from the standard curve and results was calculated from the equation given as

SO42- = Mass ((curve) x 1000) ÷ Sample

3.3.7. Phosphate

4 ml of prepared ascorbic acid were pipette and 1.06 g of ascorbic acid was

weighed and made up to 200 ml. The sample was added to make the required

volume 50 ml. The solution was thoroughly mixed and allowed to stand for 30

minutes. A cloudy colour was observed. The absorbance was then determined at

420 nm using corning colorimeter 253. Readings of unknown were read from the

standard curve and the result was calculated as follow:

PO43- = (Curver reading x 1000 x D) ÷ Sample

Where D: Is the dilution factor.

3.3.8. Nitrate

10 ml of each sample were poured into an evaporating dish and evaporated to

dryness using a hot plate at 1050C and allowed to cool after which 2 ml of phenol-

2,4 di-sulphuric acid reagent was added to each evaporating dish. After 10 mins,

10 ml of distilled water were added to dilute each solution which was then

transferred to 100 ml volumetric flask. This was followed by the addition of 200

ml of NH4OH to make it alkaline and a yellow colour was developed. The content

was finally made up to mark with distilled water. Then the absorbance of the

yellow solution was measured at 460 nm using corning colorimeter 253. Standard

were prepared from KNO3, with 10 ml diluted with 90 ml of distilled water to

make 100 ppm.

3.3.9. Nitrite

The sample prepared was left overnight to oxidize and then the absorbance was

read from the corning colorimeter and the concentration of the unknown was

determined from the calibrating graph.

3.3.9 Chemical Oxygen Demand (COD)

100 ml of the prepared sample was measured and titrated against sodium

thiosulphate. The colour was observed from deep and the readings taken.
Biological Oxygen Demand (BOD)

This was determined according to APHA (American Public Health Association)

(2014) method. At the field, the reagent bottles set aside for BOD was filled with

water samples and wrapped with black polythene bags to avoid any form of light

penetration. The samples were then transported to laboratory and kept in a dark

cupboard. After five (5) days, the procedure for carrying out dissolved oxygen was

repeated to check the amount of oxygen that has been used up by microorganisms.

Calculation: BOD = DO1 – DO5 (mg/l)

DO1 = Initial dissolve oxygen at the first day

DO5 = Dissolved oxygen reading after 5 – days

Results are expressed in milligrams per litre

Total Alkalinity

This was determined by titration methods following the method of APHA

(American Public Health Association) (2014). The collected water was measured

to 50 ml and poured into a clean 150 ml conical flask, and then 3 drops of

phenolphthalein indicator was added. The sample was titrated with 0.05ml of

H2SO4, until the colour disappeared. To the colorless solution, 3 drops of methyl

orange indicator was added and titrated further until the colour change from yellow
to permanent reddish or orange red colour and the titrated values were recorded

and used to compute the alkalinity.

Mercury, Iron, Copper and Lead

Other parameters (Mercury, Iron, Copper, and Lead), were analyzed using Atomic

Absorption spectrophotometer (AAS) (FAO 2004). (Buck scientific 200A).

3.5.6: MICROBIOLOGICAL ANALYSIS

Media prepared included Nutrient agar, MacConkey agar, Eosine Methylene Blue

agar and Sabourand dextrose agar for the study and the preparations was done in

accordance with the manufacturer’s directives.

3.4 IDENTIFICATION AND CHARACTERIZATION OF BACTERIA

ISOLATES

3.4.1 GRAM STAINING TECHNIQUE OF THE BACTERIA ISOLATES

Working solution of reagents used for the Gram staining technique was prepared

according to manufacturer’s instruction. Staining was carried out by emulsifying

approximately one isolated 18- 24hours old colony in a drop of water placed at the

center of a clean grease free slide until a thin smear was made. The smear was air
heat fixed by passing the slide through a Bunsen burner flame and then air dried.

The heat fixed smear was flooded with a basic aniline dye (crystal violet) for 60

seconds. This was flooded with Lugol’s iodine and allowed to remain for 60

seconds. This was then rinsed off with running tap water. The smear was

decolorized with 70% ethanol which was immediately washed out to avoid total

decolorization. The smear was counter stained with safranin for 60seconds, washed

off with running tap water and blot-dried. The slide was then examined under oil

immersion objective microscope. Organisms that retained the purple colour of

crystal violet- iodine complex (CV-1 complex) were recorded as Gram- positive,

while those that appeared pink were Gram- negative.

3.5 BIOCHEMICAL TESTS OF THE ISOLATED ORGANISMS

3.5.1 CATALASE TEST

This test detects the presence of catalase enzyme when present in a bacterium, it

catalyzes the breaking down of hydrogen peroxide with the release of oxygen as

bubble. With a wire loop, a colony was picked from the pure culture and was

transferred to the Centre of a glass slide. 1- 2 drops of 3% hydrogen peroxide was

added to the bacterial isolates. Immediate production of bubbles indicated positive

result and if no bubble indicated negative (Cheesebrough, 2006)

2H2O2→2H2O + O2

3.5.2 OXIDASE TEST

The isolated organisms were inoculated and grown in Nutrient broth for 24 hrs at

37°C. After 24 hrs Oxidase strip was dipped into the broth and colour change was

observed. Bacteria Isolates were oxidase positive when the colour changes to

purple within 15 seconds to 30 seconds and oxidase negative when the colour did

not change at al. (Cheesebrough, 2006).

3.5.3 INDOLE TEST

This test demonstrates the ability of certain bacteria to decompose the amino acid

tryptophan to indole which then accumulates in the medium for indole production.

Bacterial isolates were inoculated not peptone water medium contained in a sterile

test tubes then incubated at 37°C for 48 hours. After the incubation period about 3

drops of kovac’s indole reagent was added to the peptone water culture. The

bottles were shaken thoroughly and allowed to stand and observed for colour

development. A red colour ring at the interface of the medium denotes a positive

result. And if the isolate is negative, the reagent layer will remain yellow or

slightly cloud (Cheesebrough, 2006).

3.5.4 UREASE TEST

The Urease test is used to identify those organisms that are capable of hydrolyzing

urea to produce ammonia and carbon dioxide. In this test each isolate was

inoculated into test tubes containing sterilized urea agar medium and incubated at

37°C. The medium was observed for a colour change at 24hrs and everyday up to 6
days. Urease production was indicated by a bright pink colour throughout the

medium (Cheesebrough, 2006).

3.5.5 SIMON’S CITRATE TEST

The citrate test screens bacterial isolates for the ability to utilize citrate as its

carbon and energy source. Citrate agar was prepared and homogenized on a

magnetic stirrer after which it was dispensed into test tubes and sterilized in the

autoclave and slants were prepared. The slants were inoculated with the test

organisms and incubated at 37°C for 24hrs. Slant culture was observed for the

growth and coloration of the medium, positive with blue colour and negative with

green colour (Willey et al , 2008).

3.6 SUGAR FERMENTATION OF THE BACTERIA

Isolates: This test shows the ability of microorganisms to ferment certain sugars.

Five sugars were used; mannitol, sucrose, maltose, galactose and fructose using

(Hoffman-Soommer et al, 2020).

Mannitol: 3g of peptone powder was dissolved in 180 ml of distilled water in

appropriately labeled conical flask and 0.5g of phenol red was added. 1g of

Mannitol sugar was added into the conical flask and shaken thoroughly. The

solution was dispensed in 5ml amounts into test tubes with inverted Durham’s

tubes and autoclaved for 15 minutes. The test tubes were then inoculated with loop

full of test organisms and incubated at 37°C for maximum of 48 hours. The test
was observed for acid production leading to colour change (red to yellow) as well

as gas production that causes the displacement of the liquid in the inverted

Durham’s tubes which indicates a positive test (Hoffman-Soommer et al, 2020).

Sucrose: 3g of peptone powder was dissolved in 180ml of distilled water in

appropriately labeled conical flask and 0.5g of phenol red was added. 1g of

Sucrose sugar was added into the conical flask and shaken thoroughly. The

solution was dispensed in 5ml amounts into test tubes with inverted Durham’s

tubes and autoclaved for 15 minutes. The test tubes were then inoculated with loop

full of test organisms and incubated at 37°C for maximum of 48 hours. The test

was observed for acid production leading to colour change (red to yellow) as well

as gas production that causes the displacement of the liquid in the inverted

Durham’s tubes which indicates a positive test (Hoffman-Soommer et al, 2020).

Maltose: 3g of peptone powder was dissolved in 180ml of distilled water in

appropriately labelled conical flask and 0.5 g of phenol red was added. 1g of

Maltose sugar was added into the conical flask and shaken thoroughly. The

solution was dispensed in 5ml amounts into test tubes with inverted Durham’s

tubes and autoclaved for 15 minutes. The test tubes were then inoculated with loop

full of test organisms and incubated at 37°C for maximum of 48 hours. The test

was observed for acid production leading to colour change (red to yellow) as well
as gas production that causes the displacement of the liquid in the inverted

Durham’s tubes which indicates a positive test (Hoffman-Soommer et al, 2020).

Galactose: 3g of peptone powder was dissolved in 180ml of distilled water in

appropriately labeled conical flask and 0.5g of phenol red was added. 1g of

Galactose sugar was added into the conical flask and shaken thoroughly. The

solution was dispensed in 5ml amounts into test tubes with inverted Durham’s

tubes and autoclaved for 15minutes. The test tubes were then inoculated with loop

full of test organisms and incubated at 37°C for maximum of 48hours. The test was

observed for acid production leading to colour change (red to yellow) as well as

gas production that causes the displacement of the liquid in the inverted Durham’s

tubes which indicates a positive test (Oludare et al, 2020).

Fructose: 3g of peptone powder was dissolved in 180ml of distilled water in

appropriately labeled conical flask and 0.5 g of phenol red was added. 1g of

Fructose sugar was added into the conical flask and shaken thoroughly. The

solution was dispensed in 5ml amounts into test tubes with inverted Durham’s

tubes and autoclaved for 15 minutes. The test tubes were then inoculated with loop

full of test organisms and incubated at 37°C for maximum of 48 hours. The test

was observed for acid production leading to colour change (red to yellow) as well

as gas production that causes the displacement of the liquid in the inverted

Durham’s tubes which indicates a positive test (Fawole and Oso, 2004).
3.7 STARCH HYDROLYSIS TEST OF BACTERIA ISOLATES

Nutrient agar was prepared and the isolates were inoculated onto the plates with

sterile inoculating loop using streak method. The plates were incubated at 37°C for

24hrs, after incubation the plates were flooded with Gram’s iodine. Plates were

observing for clear zone around the test organisms (Oludare et al, 2020).

3.6: STATISTICAL ANALYSIS

The data was subjected to simple percentages, frequency and bar charts. The

physico-chemical parameters were analyzed using Analysis of Variance (ANOVA)

IBM SPSS version 14. Further test such as Tukey was carried out to ascertain the

significant difference among the parameters.

 CHAPTER FOUR

RESULTS AND DISCUSSION

Table 1. showed that The temperature ranged from 25.200C to 260C, D.O ranged

from 3.84 to 6.90 (Mg/L), phosphate ranged from 1.50-3.00 (Mg/L), sulphate

ranged from 200-550 (Mg/L), BOD ranged from 2.20-7.90, COD ranged from 3.30-

11.80, Mg hardness range from 0.50-4.00 (Mg/L), Ca hardness ranged from 4.80-

16.00 (Mg/L), nitrite ranged from 4.40-6.30 (Mg/L), Nitrate ranged from 2.20-

5.80(Mg/L), pH was 6.20 to 7.40 and the conductivity was 21.70-39.40 S/cm.

In Table 2, Station A (before the main road) has the lowest total viable count

(1.1X103), followed by station C (2.5X104), while station B has the highest value

(9.0X104). as for the Total coliform count, station B has the highest value (1.3X
105), followed by station C (along Agbara) (6.4.0X104) and station A has the

lowest (7.0X103)

The Biochemical and bacterial isolates in table 3; were Staphylococcus aureus

(Gram -, Non motile and cocci structure), Escherichia Coli (Gram +, Non motile and

rod structure), Klebsiella Sp (Gram +, Non motile and rod structure), Enterobacter

Sp (Gram -, motile and rod structure), Bacillus Sp (Gram -, motile and rod

structure), Pseudomonas sp (Gram +, Non motile and rod structure), Proteus SP

(Gram -, Non motile and rod structure), and Salmonella sp (Gram +, Non motile

and rod structure).

In Table 4, E. coli was smooth, milky, ternate transparent and forms a colony

shape. Proteus sp was rough, greenish, lobate, filamentous and transparent.

Pseudomonas sp. was dull, creamy, fimbriate, irregular and transparent.

Staphylococcus sp. was smooth, milky, round, form colony and was transparent.

Bacilus sp, was rough, creamy, entire, circular and opaque. Klebsiella sp. was

smooth, creamy, round, irregular and transparent. Salmonella sp. was smooth,

blue-black, lobate, forms colony and opaque. Enterobacter spp was dull, creamy,

round, irregular and transparent.

Table 5 showed that, station B recorded the highest number of organisms (52),

followed by station C and then station A. Salmonella spp has the highest
occurrence (27%) in station B, while Proteus spp and Klebsiella spp has the lowest

occurrence (8%). In station C, Klebsiella spp, has the highest occurrence (21%) in

station B, while Proteus spp has the lowest occurrence (7%). In station A, Bacillus

spp and Enterobacter spp, has the highest occurrence (21%), while Pseudomonas

spp has the no occurrence (0%). In all the overall occurrence of these organisms,

Salmonella spp had the highest (20%), followed by Bacillus spp (19%), while the

lowest was Pseudomonas spp (4%)

Table 1: Physico -Chemical Properties of Oramiri-Ukwa River in Comparison

WHO, NAFDAC and FEPA

Properties A B C WHO NAFDAC FAO

Temperature (0C) 25.20 26.50 25.00 <29 24 -29.70 26
Dissolved Oxygen 6.90 3.84b 6.37 >6 >5.50 >4
Phosphate (Mg/L) 1.50 b 5.30 3.00 5.0 - -
Sulphate (Mg/L) 200 b 550 300 <450 - -
Conductivity (S/cm) 21.70 39.40 b 26.10 100 > 70 50
pH 7.40 6.20 7.10 6.5 - 8.5 6.9 -7.3 6.2-7.1
Nitrate (Mg/L) 3.00 b 5.80 2.20 b <50 10 20
Nitrite (Mg/L) 6.30 6.60 4.40 <50 - -
Chloride (Mg/L) 4.10 10.20 b 7.40 <250 - -
Mg Hardness (Mg/L) 0.50 4.00 b 1.50 <500 100 500
Ca Hardness (Mg/L) 7.30 16.00 b 4.80 <500 500 500
C.O.D 3.30 11.80 b 6.10 - 10 -
B.O.D 2.20 b 7.90 4.70 b 10 - 10.5
 Mercury 0.000 0.002 0.001 0.005 - <0.005
Copper 0.10 0.17 0.12 1.0 <1.5 -
Lead 0.026 0.318 0.184 <0.5 - -
Iron 1.00 2.71 b 0.20 0.3 - <0.5

Values with the letter(b) are significantly different within rows

Table 2: Microbial Load of the Bacterial Isolates from Oramiri-Ukwa River

STATION TVCC (CFU/ml) TCC (CFU/ml)

A 1.1X103 7.0X103

B 9.0X105 1.3X 105

C 2.5X104 6.4X104

KEYS: Tvc = total viable count, Tcc = Total coliform count, CFU/ml = colony forming unit per milliliter

Station A (before the main road) has the lowest total viable count (1.1X10 3), followed by station
C (2.5X104), while station B has the highest value (9.0X104). as for the Total coliform count,
station B has the highest value (1.3X 10 5), followed by station C (along Agbara) (6.4.0X10 4) and
station A has the lowest (7.0X103)
 Table 3. Biochemical characteristics and sugar fermentation of
bacteria isolates in water Samples
Isolate M CAT IND OXI URE MOT SH CIT MAN SUC FRU MAL GAL Organism

1 Rod + + - - NM - + + GP +GP + GP + GP + GP Escherichi

a coli

2 Rod + + + + NM + + + GP +NG +NG + GP - NG Proteus spp

3 Rod + + - + NM + + + GP +GP + GP + GP + GP Pseudomonas

spp

4 Cocci + - - - NM - + + GP +GP + GP + GP +NG Staphilococc

usspp

5 Rod + + - - NM + + + GP +GP + GP + GP + GP Klebsiellaspp

6 Rod + + - - M + + + GP +GP + GP + GP - NG Bacillus spp

7 Rod - + - - NM + + + GP +GP + GP + GP + GP Salmonella
spp

8 Rod - - - - M - + + GP +NG + GP + GP - NG Enterobact

er spp

Keys: CAT= Catalase test URE= Urease test SH= Starch hydrolysis MAN= Mannitol test SUC= Sucrose test IND=
Indole test MOT= Motility test OXI= Oxidase test CIT= Citrate test MAL= Maltose GAL= Galactose NM= Non
Motile FRU= Fructose GP= Gas production NG= No gas

Table 4: Morphological, Macroscopic and Cultural Characteristics of

bacterial isolates in Water Samples
Isolates Surface Colour Edge Shape Appearance in light

Escherichia coli Smooth Milk Ternate Colony Transparent

Proteus spp Rough Greenish Lobate Filamentous Transparent

Pseudomonas spp Dull Cream Fimbriate Irregular Transparent

Staphilococcusspp Smooth Milk Round Colony Transparent

Bacillus spp Rough Cream Entire Circular Opaque

Klebsiellaspp Smooth Cream Round Irregular Transparent

Salmonella spp Smooth Blue-black Lobate Colony Opaque

Enterobacter spp Dull Cream Round Irregular Transparent

Table 5: Frequency, Percentage and Incidence of Bacterial Pathogen Isolated
from Water Samples of Oramiri-Ukwa River
ORGANISMS A B C TOTAL(%)

Escherichia coli 2 (14%) 5(10%) 3(11%) 10 (11%)

Proteus spp 1(7%) 4(8%) 2(7%) 7 (7%)

Pseudomonas spp 0(0%) 3(6%) 1(4%) 4 (4%)

Staphylococcus spp 2(14%) 7(13%) 4(11%) 13 (14%)

Klebsiella spp 2(14%) 4(8%) 3(21%) 9 (10%)

Bacillus spp 3(21%) 9(17%) 6(11%) 18 (19%)

Salmonella spp 1(7%) 14(27%) 4(14%) 19 (20%)

Enterobacter spp 3(21%) 6(12%) 5(18%) 15 (15%)

 14 (100) 52 (100) 28 (100) 94 (100%)

From the table, station B recorded the highest number of organisms (52), followed by station C
and then station A. Salmonella spp has the highest occurrence (27%) in station B, while Proteus
spp and Klebsiella spp has the lowest occurrence (8%). In station C, Klebsiella spp, has the
highest occurrence (21%) in station B, while Proteus spp has the lowest occurrence (7%). In
station A, Bacillus spp and Enterobacter spp, has the highest occurrence (21%), while
Pseudomonas spp has the no occurrence (0%). In all the overall occurrence of these organisms,
Salmonella spp had the highest (20%), followed by Bacillus spp (19%), while the lowest was
Pseudomonas spp (4%)

CHAPTER FIVE

DISCUSSION

5.1 DISCUSSION

From table 1, temperature falls within the normal range while the pH ranged from

6.20 – 740 for all the stations. However, the pH of station B slightly acidic as it

was moving towards the acidic region of the pH scale. This could be attributed to

the fact that an increase in bacteria activities lead to break down of complex

organic matter of which the bi-product is acidic in nature. They were also

increasing in some heavy metals such as iron, copper etc. reason been that some of

the effect of dredging activities is the discharge of metals either through seepage

from the engines or from excess turnover of soil particles. Metals become
detrimental to both fish and humans when they become more in the body system.

Metals could affect the normal physiological activities of living things.

The high value of BOD (7.90) and COD (11.80) for station B shows that there are

more bacteria present in the water, probably due to the fact that dredging, farming

activities results to the deposition of more nutrients for rapid bacteria growth.

Respective salts, metals and nitrates seem to be high and either greater than WHO,

NAFDAC and FEPA standards or closer to their values, though other Physico-

chemical parameters were increasing as Oramiri-Ukwa flows and empties itself to

Otamiri River. Dredging activities tend to increase the metals of a water body due

to seepage (Yilmaz et al, 2007). In table 2, the total mean viable count ranged

from 1.2x103 to 9.0 x 105 CFU/ml, while the total mean coliform count was from

7.0x103 to 5.0x105 CFU/ml. Station B was high for TVCC (9.0 x 10 5) and TCC

(5.0x105), this may be as a result of so many activities going on there such as

washing if clothes by villagers, dredging and farming. The microbial load of both

the TVC and TCC were high which is a reflection of the input of microorganism

from external sources and availability of growth supporting organic matter (Sayler

et al, 1975). It also shows the level of water pollution as an indication of organic

matter present. The mean total bacterial counts obtained in this study especially

station B, followed by station C were high but it is not surprising because Oramiri-

Uwka River serves the population within Azara Egbelu and its environs for
domestic purposes. Table 3 reveals the biochemical characteristics and sugar

fermentation bacterial isolates from Oramiri-Ukwa River. Their presence in water

indicates faecal contamination of the water.

The coliform isolated is an indication of gross contamination of water.

Staphylococcus aurues, bacillus sp. Pseudomonas sp. and proteus sp may have also

come from current contamination of the river. Bacillus sp is a spore former and can

survive in harsh environmental condition, staphylococcus aureus may have come

from contamination before or during this study. Staphylococcus sp and bacillus sp

are pathogenic and non-pathogenic bacteria respectively. In water, bacillus sp is

harmless, their presence signifying waste materials decomposition in water body.

(Ogubile, 1999). The high value of Salmonella sp for station B (27%) is an

indication that the water should not be drank, as diseases such as typhoid fever

may occur According to Arvanitidov (2005), Salmonella typhyis responsible for

salmonellosis. This implies that controlled sewage water systems and personal

hygiene will help reduce the incidence of gastroenteritis Le Minor (2003). Also,

streptococci observed in this study is a health concern because their presence in

Oramiri-Ukwa water has made it prone to diseases such as cholera and Skin

infection on consumption as confirmed by the study of Nwanebu et al (2011). As

Escherichia coli was isolated from the water samples, it indicated recent faecal

contamination of the water sources. This result is supported by the works of

(Ogbulie et al, 2009) and (Cabral, 2010). While most strains of E. coli are non-

pathogenic, some can cause serious diarrheal infections in human (Ihejirika et al,

2011). The presence of Salmonella typhyin the water samples could be traced to

contamination from domestic sewage, agricultural waste and storm water runoffs.

This is in line with the report of Le Minor (2003)

and (WHO, 1998). Klebsiela spp can cause human diseases ranging from

asymptomatic colonization of the intestine, urinary or respiratory tract to fatal

septicemia (Popoff MY, 2005). Total coliform bacteria include the faecal coliforms

associated with faecal materials, species of Escherichia, Klebsiella, Enterobacter

and Citrobacter are important members of the total Coliform group (Grimont et al,

2003)
 CHAPTER SIX
CONCLUSION AND RECOMMENDATION
6.1 CONCLUSION

The evaluation of the physico-chemical and bacterial load of Oramiri-Ukwa

River quality has confirmed the major contaminant as faecal. Contaminated

water is an established source of most enteric disorders which in most cases

manifest as diarrhea, typhoid and skin infections. The implication of these

findings may be that people depended on this river water for domestic use

including cooking, bathing, washing and even drinking or for agricultural

uses like fishing and farming may be exposed to public health risks. We

therefore suggest that to maintain healthy living, health education, proper

waste disposal system and environmental sanitation remain key factors to

eradicating this menace of water pollution

6.2 RECOMMENDATION

Based on this study; it is recommended that Government should enact regulations

(monitoring agency) that will make sure people do not defecate in the river, do not

dump refuse and waste materials from both domestic and industrial activities and

this can be achieved through the organization of seminars/workshop from time to

time to sanitize the populace on the need for personal hygiene the populace within

Owerri municipal that make use of the water for domestic activities should

endeavor to subject the water through purification processes before usage.

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 APPENDICES
Appendix I: Descriptive Statistics of the Physico-Chemical Parameters of Oramiri-Ukwa River
Mean Standar Media Standard Sample Range Minimu Maximu Coun Confidence
d Error n Deviatio Varianc m m t Level(95.0%
n e )

Temperature 25.75 0.75 25.75 1.06 1.13 1.50 25.00 26.50 2.00 9.53
(0C)
Dissolved 5.11 1.27 5.11 1.79 3.20 2.53 3.84 6.37 2.00 16.07
Oxygen
Transparenc 0.65 0.15 0.65 0.21 0.05 0.30 0.50 0.80 2.00 1.91
y
Phosphate 4.15 1.15 4.15 1.63 2.65 2.30 3.00 5.30 2.00 14.61
(Mg/L)
Sulphate 425.0 125.00 425.00 176.78 31250.0 250.0 300.00 550.00 2.00 1588.28
(Mg/L) 0 0 0

Conductivit 32.75 6.65 32.75 9.40 88.45 13.30 26.10 39.40 2.00 84.50
y (S/cm)
pH 6.65 0.45 6.65 0.64 0.41 0.90 6.20 7.10 2.00 5.72

Nitrate 4.00 1.80 4.00 2.55 6.48 3.60 2.20 5.80 2.00 22.87
(Mg/L)
Nitrite 5.50 1.10 5.50 1.56 2.42 2.20 4.40 6.60 2.00 13.98
(Mg/L)
Chloride 8.80 1.40 8.80 1.98 3.92 2.80 7.40 10.20 2.00 17.79
(Mg/L)
Mg 2.75 1.25 2.75 1.77 3.13 2.50 1.50 4.00 2.00 15.88
Hardness
(Mg/L)
Ca Hardness 10.40 5.60 10.40 7.92 62.72 11.20 4.80 16.00 2.00 71.15
(Mg/L)
C.O.D 8.95 2.85 8.95 4.03 16.25 5.70 6.10 11.80 2.00 36.21

B.O.D 6.30 1.60 6.30 2.26 5.12 3.20 4.70 7.90 2.00 20.33

Mercury 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.00 0.01

Copper 0.15 0.03 0.15 0.04 0.00 0.05 0.12 0.17 2.00 0.32

Lead 0.25 0.07 0.25 0.09 0.01 0.13 0.18 0.32 2.00 0.85

Iron 1.46 1.26 1.46 1.77 3.15 2.51 0.20 2.71 2.00 15.95

ANOVA

Null hypothesis All means are equal

Alternative hypothesis Not all means are equal

Source of
Variation SS df MS F P-value F crit

Rows 332178.8 17 19539.9286 11.4566409 0001435 2.271893

Columns 2454.624 1 2454.62445 1.43919414 0.01263 4.451322

Error 28994.43 17 1705.55478

Total 363627.8 35
According to the ANOVA performed by IBM SPSS, 2018. Showed that the P-value (0.0014)

was less than alpha = 0.05, so we reject the null hypothesis and accept the alternative hypothesis

that there is a difference between and among means between rows. While between columns P-

value (0.012) is less than alpha = 0.05, so we reject the null hypothesis and accept the alternate

hypothesis

Model Summary
S R-sq R-sq(adj) R-sq(pred)
0.63 73.26% 76.51% 66.71%

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

Factor N Mean Grouping

Temperature (0C) 2.00 25.75

Dissolved Oxygen 2.00 5.11

b
Transparency 2.00 0.65
b
Phosphate (Mg/L) 2.00 4.15
b
 Sulphate (Mg/L) 2.00 425.00
b
Conductivity (S/cm) 2.00 32.75
b
pH 2.00 6.65

Nitrate (Mg/L) 2.00 4.00

b b
Nitrite (Mg/L) 2.00 5.50

Chloride (Mg/L) 2.00 8.80

b
Mg Hardness (Mg/L) 2.00 2.75
b
Ca Hardness (Mg/L) 2.00 10.40
b
C.O.D 2.00 8.95
b
B.O.D 2.00 6.30
b b
Mercury 2.00 0.00

Copper 2.00 0.15

Lead 2.00 0.25

Iron 2.00 1.46

b
Means that do not share a letter are significantly different.

Turkey pairwise comparison test showed that the means with the letter (b) are
different from other means. An adjusted R-Squared of 76.51% showed that about
66.71% of all the variations in the data are explained by the model.
Appendix II

Fig 1: Microbial Load of the Bacterial Isolates from Oramiri-Ukwa River

 140000

120000

100000

80000

60000

40000

20000

0
Station A Station B Station C
TVCC TCC Column1

Appendix III
Fig 1: Frequency and Incidence of Bacterial Pathogen Isolated from Water

Samples of Oramiri-Ukwa River

16

14

12

10

0
li
co s pp sp
p
s pp sp
p pp sp
p
hi
a us as us la ss la
ric e on c el ci llu el
e ot co
c
bs
i
on
ch Pr do
m lo e Ba lm
Es y Kl Sa
eu ap
h
Ps St

Station A Station B Station C

Appendix IV

Fig 4: Overall Frequency and Incidence of Bacterial Pathogen Isolated from

sampling stations of Oramiri-Ukwa River

13%

24%

9%

5%

16%
23%

11%

Escherichia coli Proteus spp Pseudomonas spp Staphylococcus spp

Klebsiella spp Bacillus spp Salmonella spp

ABSTRACT
Water bodies in Nigeria, faces significant level of physico-chemical parameters and bacterial
load fluctuations. In this research work, the physico-chemical and bacterial load of water from
Oramiri-Ukwa River was investigated. About three sampling stations of the Oramiri-Ukwa river
were chosen that is Station A (before the main road, Azara Egbelu), Station B (across the main
road Azara Egbelu) and Station C (along Agbara). These stations were selected for water
collection points from the river based on anthropogenic activities. Analyzed water samples
collected from the river showed that station A of the river was not contaminated, station B was
highly contaminated and station C partially contaminated. Also, the were increases in some
heavy metals such as iron and copper, which is a product of dredging activities. The temperature
ranged from 25.200C to 260C, D.O ranged from 3.84 to 6.90 (Mg/L), phosphate ranged from
1.50-3.00 (Mg/L), sulphate ranged from 200-550 (Mg/L), BOD ranged from 2.20-7.90, COD
ranged from 3.30-11.80, Mg hardness range from 0.50-4.00 (Mg/L), Ca hardness ranged from
4.80-16.00 (Mg/L), nitrite ranged from 4.40-6.30 (Mg/L), Nitrate ranged from 2.20-5.80(Mg/L),
pH was 6.20 to 7.40 and the conductivity was 21.70-39.40 S/cm. The total visible count ranged
from 1.1x103 to 9.0x105 CFU/ml while the total coliform count was from 7.0x10 3 to 1.3x105
CFU/ml which revealed increasing level of contamination. The biochemical characteristics and
bacterial isolates were Staphylococcus aureus (Gram -, Non motile and cocci structure),
Escherichia Coli (Gram +, Non motile and rod structure), Klebsiella Sp (Gram +, Non motile
and rod structure), Enterobacter Sp (Gram -, motile and rod structure), Bacillus Sp (Gram -,
motile and rod structure), Pseudomonas sp (Gram +, Non motile and rod structure), Proteus SP
(Gram -, Non motile and rod structure), and Salmonella sp (Gram +, Non motile and rod
structure). The morphological and cultural characteristics were; E. coli was smooth, milky,
ternate transparent and forms a colony shape. Proteus sp was rough, greenish, lobate, filamentous
and transparent. Pseudomonas sp. was dull, creamy, fimbriate, irregular and transparent.
Staphylococcus sp. was smooth, milky, round, form colony and was transparent. Bacilus sp, was
rough, creamy, entire, circular and opaque. Klebsiella sp. was smooth, creamy, round, irregular
and transparent. Salmonella sp. was smooth, blue-black, lobate, forms colony and opaque.
Enterobacter spp was dull, creamy, round, irregular and transparent. The coliforms were dominant occurring
in all the water samples and are indicators of faecal and dredging contamination. It is
recommended that people using the water especially for drinking and other domestic activities
should purify it properly to avoid possible health hazards associated with the identified microbes.

Keywords: Bacterial load, Oramiri-Ukwa River, total visible count total coliform count WHO,
NAFDAC, FAO