MICROORGANISMS IN FUNAAB WELLS WATER
MICROORGANISMS IN UNAAB WELLS WATER
BY
MATRIC NO: 07/0617
A PROJECT REPORT SUBMITTED TO
DEPARTMENT OF MICROBIOLOGY
COLLEGE OF NATURAL SCIENCE
UNIVERSITY OF AGRICULTURE, ABEOKUTA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE IN MICROBIOLOGY
JUNE, 2011
CERTIFICATION
This is to certify that this work was carried out by Abolade Isaac Temitiope (Matric. No. 07/0617) in the Department of Microbiology, College of natural Science University of Agriculture, Abeokuta.
……………………………. ……………………………
Dr. (Mrs.) O.R. Afolabi Dr. (Mrs.) O.R. Afolabi
Project Supervisor Head of Department
DEDICATION
This project work is dedicated to Almighty God for giving me wisdom, knowledge and understanding throughout the year of my study and to my late father Mr. A.J. Samuel.
ACKNOWLEDGEMENTS
With all thanks to God Almighty, I herby express my heartfelt gratitude to my lecturers and supervisor Dr. (Mrs.) O.R. Afolabi whose accessibility and hard work give both the lead and inspiration for the timely and successful completion of this research work. I also appreciate the cooperation of member of staff of the Department of Microbiology. My parents of whom words alone cannot express how grateful I am for their love, support, prayer and encouragement. I thank you so much for being there for me all the way I am also grateful to all my brothers and sisters, friends, God bless you all.
ABSTRACT
Well water in the University of Agriculture UNAAB was microbiologically examined for isolation and identification of organism present in them. All the well water examined did not meet the required international standard of three coliform per 100ml and 60% did not meet the required international standard of zero E.coli per 100ml of drinking water. The total viable count of bacteria per ml ranges from 1.2x105to 9.8x107 and the total viable count of fungi ranged from 1.0x107and 1.9x107. All the well sampled had a pH range of 6.90 to 8.2 thus meeting the range of the pH of the world health organization standard.
The results are discussed with reference to the sanitary sitting and handling of the wells.
TABLE OF CONTENTS
CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Abstract v
Table of contents vi
List of tables viii
CHAPTER ONE
1.0 Introduction 1
CHAPTER TWO
2.0 Literature review 2
CHAPTER THREE
3.0 Materials and Methods 7
3.1 Collection of Samples 7
3.1.1 Laboratory Analysis 7
3.2 Preparation of Media 7
3.2.1 TCBS Agar 7
3.2.2 Salmonella ShIgella Agar (SSA) 7
3.2.3 Eosin Methylene Blue Agar (Emb agar) 8
3.2.4 pH 8
3.2.5 Total viable count of bacteria 8
3.2.6 Coliform test 9
3.2.7 Presumptive test 9
3.2.8 Confirmed test 9
3.2.9 Completed test 10
3.2.10 Identification of isolates 10
3.2.11 Biochemical test 10
3.2.12 Methyl red test 11
3.2.13 Glucose fermentation test 11
3.2.14 Nutrient agar 12
3.2.14.1Nutrient agar slant 12
3.2.15 Serial dilution 12
3.2.16 Gram staining 12
CHAPTER FOUR
4.0 Results and discussion 13
4.1 Results 13
4.2 Discussion 18
CHAPTER FIVE
5.0 Conclusion and Recommendation 20
5.1 Conclusion 20
5.2 Discussion 20
Appendix 23
LIST OF TABLES
The Description of wells in UNAAB
The pH of the most probable number (MPN) and the viable microbial count of microorganism in UNAAB well water.
The colonial, morphological and biochemical characteristics of bacterial isolated from UNAAB wells water.
The colonial morphological and biochemical characteristics of coliform obtained from UNAAB wells water.
CHAPTER ONE
1.0 INTRODUCTION
Water is a natural resources and has been a source of continuous preoccupations for humans since the beginning of mankind. Water consumption can be divided into the following categories, domestic, trade, Agricultural, Public, losses (consumer wastage, distribution losses, metering and other losses). (Degremont, 1991). Water is the most common mineral on the earth’s surface with an estimate volume of 1470 million cubic kilometres (Degremont, 1991).
Water can be obtained from a number of sources namely streams, lakes, rivers, ponds, rain water, springs, wells, all of which habour microorganisms.
Groundwater is a vital source of water supply in which its main source is precipitation, which may penetrate the soil directly to the groundwater or may enter surface stream or penetrate from these channels to the groundwater. (Linsley et al., 1999). Groundwater continues to be affected by faecal contamination originating from a variety of human associated sources in both rural and urban areas (Sundaresan, 1994). It is generally known that groundwater systems are prone to microbial infestations and colonized by higher plants (Crane and Moore, 1992). The potential for contaminating groundwater by pathogens can be raised based on increasing demands placed on currently available water resources. (Cubitt, 1991).
A well, is a shaft usually lined with brick or stone for obtaining water from an underground source or a device for extracting water from the ground. Well water undergo contamination from ground leakages and refuse disposal site and as a result there could be water related diseases. A well water could be clean, cloudy, dirty, brown that is, well water have different physical appearances.
UNAAB is an institution along Alabata road and located in Abeokuta South Local Government of Ogun State. In this area, well water supplies predominate the tap water supplies The well water are used for various domestic purposes including washing regardless of the microbial load present in the well water.
All these variously dug wells are not standardized and have been randomly placed since well water could serve as a source of epidemic of water borne diseases. This project was carried out to examine the water in then to ascertain the microorganism and the possible sources of contamination or pollution and possible control measure to make the water suitable for use.
CHAPTER TWO
2.0 LITERATURE REVIEW
Water is necessary for the existence of all living creatures. The human body needs about 2-10litres of water each day for normal physiological functions depending on climate and workload. About 1 litre of water is provided by daily food consumption (Sundaresan, 1994). The total water consumption per day is determined by a number of factors such as availability, quality cost, income and size of the family, cultural habits, standard of living, ways and means of water distribution and climate (Sundaresan, 1994).
The type of microorganism found in water reveal their possible sources of contamination and control (WHO, 1997). Water borne disease could be checked or controlled through three major ways depending on the source of water (WHO, 1997) which may be natural water, such as deep wells, boreholes, which could be protected, rainwater which could he hygienically collected to give clean and pure water, or unprotected water such as rivers, and lakes which could be treated to give tap water.
According to (Pean et al., 1990) the water resource survey state that water can be obtained from surface water, groundwater, water reclamation which is through desalination and blending, reuse of treated effluent, bulk supply from adjacent undertaking, In this study, an intensive bacteriological investigation was made of the incidence, numbers and bacterial species present in the drinking water of private wells and springs which serve a modern rural community. (Pean et al., 1990).
Groundwater is the subterrenial waters that occurs where all pores in the soil or rock containing materials are saturated. It is the water found under earth’s surface in rock formations and soil pores. The source of groundwater is precipitation and the infiltration of surface water from run-offs and streams. The deeper groundwaters show increasing temperature with increasing depth (Ayodele, 1988). The sequence of events when water drops from the atmosphere to the earth and hydrosphere, then goes back to the atmosphere is known as the hydrologic cycle (Awah et al., 1993).
A well, is a shaft usually lined with brick or stone for obtaining water from an underground source or a device for extracting water from the ground. At all times and all over the world, there is a movement of water in its various forms and this is called hydrological cycle.
Potable water is the water that is free from contaminant of any form and suitable for consumption. Microbiological monitoring of drinking water has been practiced in the United States and other countries since the beginning of this century. Well water can become polluted with coliforms arid pathogenic bacteria from normal, diseased, or carrier human and animal excrements. This can occur by cross connections between water main and a sewer, especially when the pressure in the water main becomes less than atmospheric pressure from the entry of sewage water through leaks in damaged pipes. Treatment deficiencies may allow the escape of either unharmed or reversibly inactivated organisms, especially when the source water contain high densities of coliforms and pathogenic bacteria.
Microorganisms can gain access to water from air-water interface in the distribution system such as in a storage tank or may remain unaffected by the chlorine treatment. For example Legionella species not only survive but multiply in storage tanks and other water systems (Ritonga, 1989). World wide, the most common bacteria diseases transmitted through water are caused by Shigella, Salmonella, Enterotoxigenic E coli, Camphylobacter jejuni and Vibrio cholerae (Ritonga, 1989). Transmission of Salmonella typhi through drinking water is existent and outbreaks of water borne cholera is common in Nigeria.
During the survey, 75% of the households were found to be using water contaminated with coliforms, faecal coliforms or Staphylococcus aureus or with standard plate counts exceeding 500/ml (Lechevalier et al., 1988).
The most common cause of water borne disease that results from the Consumption of untreated, inadequately treated or ineffectively filtered surface water is Giardiasis (Reynolds et al., 1996). Thus, the occurrence of disease outbreaks is frequent where heavy reliance is placed on the disinfection of surface water. The flagellated protozoan parasite Giardia lambia causes the most common parasite infection in the human population and causes a watery diarrhoea spread by the faecal oral route (Abbaszadegan et al., 1993). A more rapid method for detection of Gardia cysts in the environment would enhance our ability to evaluate the quality of water. Gene-specific probes have recently been shown to be very useful in the detection of a number of microorganisms.
Mycobacterium avium is often isolated from aquatic environment. There are four routes, in which water related infections are transmitted namely water borne, water-washed, waterbased and water related insect vector.
In water borne route, transmission, occurs when the pathogen is in water that is drinking by a person or animal that may then become infected. Potentially, water-borne diseases include the classified infections, notably cholera and typhoid and a wide range of other disease such as infectious hepatitis and some diarrhoea and dysentary. It is essentially transmitted by faecal oral route with water as the central vehicle (Feachem, 1984).
Water washed route involves a water washed disease whose transmission will be reduced following a decrease in the volume of water used for hygienic purposes irrespective of the quality of water. They include some of the faecal oral diseases, disease of the skin such as Rabies, Trichomonas, Fever. All related primarily to water quantity and not quality (Feachem, 1984).
Water based route is one in which a pathogen spends part of its life cycle in a water-snail or other intermediate aquatic host. All these diseases are due to infections by parasitic worms (helminths) which depend on aquatic host to complete their life cycles The larva of guinea worm escapes from the skin lesion and may be washed into the stream or well where they develop in the host called cyclops which infest man. Another example is the Schistosoma spp (Feachem, 1984).
Insect vector route is the mechanism in which water related microorganisms spread by insects that either breed in or bites near water. Malaria, yellow fever, dengue and onchocerciasis (river blindness) are transmitted by insects that breed in water, whereas West African trypanosomiasis (Gambian sleeping sickness) is transmitted by the riverine tsetsefly (Glosina spp) which bits near water (Feachem, 1984),
Hellyer et al., (1993) supported the hypothesis that the gastrointestinal tract is the portal of entry for environmental mycobacteria and confirmed that mycobacterium avium intracellular infection is a manifestation of late stage HIV disease.
Pasteur, specific media and reactions have been developed to determine the type and the virulence of all pathogenic bacteria. The main difficulty inherent in aquatic environment is the degree of dilution of the bacteria and the state of stress that their stay in water sometimes bring. Special concentration methods (filtering membranes) and renofication techniques (incubation at several temperature levels) are therefore used to isolate them.
Bacteria flora in water can be conveniently divided into three groups of natural water bacteria, soil bacteria and sewage bacteria depending on their sources of origin (Wilson and Pike, 1990). Natural water bacteria include organisms that are commonly found in water free from gross pollution. They consist of mainly saprophytic organism belonging to the genera Microccus, Pseudomonas, Serratia, Flavobacterium, Corynebacterium and Acinebacter and Alcaligenes (Wilson and Pike, 1990).
Soil bacteria though not normally inhabitants of water, are frequently washed into it during heavy rains. Most of them belong to the group of aerobic spore bearing bacilli such as Bacillus subtilis, Bacillus megaterium and Bacillus mycoides. By the use of special media, other organisms such as iron and manganese oxidizers and the autothrophtic nitrifying and photosynthetic sulphur bacteria may be isolated (Wilson and Pike, 1990).
Sewage bacteria include many organisms that are normally inhabitants of the intestine of man such as E coli, Streptococcus faecalis, Clostridium perfringes others live chiefly on decomposing organic matter of either animal or vegetable origin such as Proteus vulgaris (a sewage proper bacterium) Clostridium sporogenes and Zoogloea ramigera a floc forming bacteria while filamentous bacteria include Sphaerotilus natans, Heliscomenobacter hydrossis, Nostocoda limicola Microthrix, Parvicella, Flexibacter Microscilla, Norcardia (Eikelboom, 1975; Wilson and Pike 1990). Histoplasma capsulation is the only type or specie of fungi that infest main piping and causes histoplasmosis (Degremont, 1991). Amoebae are able to survive for over a month in water in the form of cysts. Two waterborne specie of amoebae which are pathogenic to man are Entamoeba histolytica which causes serious and sometimes fatal dysentery and Neagleria gruberi which causes meningitis transmitted through water, especially in swimming pool, This organism is resistance to disinfection which is greater than most bacteria (Degremont, 1991). Biological activity reaction test (BARTS) have been developed to monitor the presence and activity of three important groups of bacteria found in wells. These are the iron related bacteria (IRB), sulphur reducing bacteria (SRB) and slime forming bacteria, the three groups contribute to problems relating to quality (Mansuy et al., 1990).
Water may serve as a vehicle for many parasitic worms affecting man and animals, these worms and eggs are usually large enough to ensure filtration which practically eliminate the risks of contamination, since they are not destroyed by infection. Examples of some of the worm are Taenia Bothriocephallis latus, Pistona spp, causing bilharziasis, Ascaris lumbricoides, Oxyuria vermicularia, Enstrongylus gigas, Ancloystoma duodenale, filaria worms and Anguillula intestinallis (Degremont, 1991).
Recently, regulatory agencies in the United State have proposed that the total bacteria counts in finished drinking water should not exceed 500cfu/ml in order to reduce interference with the detection of coliform bacteria and to reduce subsequent potential health risk (Payment, 1989). The search for an ideal indicator bacteria system to determine the microbiological water quality has been elusive. An ideal indicator should, always be present in the presence of pathogens, be specific for faecal contamination, be able to resist water treatment and disinfectious processes to the same or a slightly greater extent than the pathogens, and be detectable by simple and rapid methods convectional indicator bacteria were not just monitored but all coliforms presents were indentified to species (Lechevallier et al., 1988).
The detection of any enteric bacteria, even of low pathogenecity, in the aquatic environment may serve as a warning of unsafe recreational water or breach in the integrity of disinfection or distribution systems of potable water (Elkelboom, 1975)
The overall microbiological quality of water is determined on a routine basis by detecting the coliform group of bacteria that ferments lactose and constitutes a part of the normal mammalian intestinal flora. However, some members of genera such as klebsiella, Enterobacter and Citrobacter also have an extraintestinal prevalence and may gain entry into water from environmental sources (Jenneman et al., 1 998).
The faecal coliform bacteria, predominantly E coli, which originate primarily in the mammalian intestine, are detected by their ability to ferment lactose at 445°c. these bacteria are not usually long term occupants of aquatic ecosystems (Mansuy et al., 1998).
Thus, their presence in water serve as a useful indicator of recent faecal contamination, which is the major source of many enteropathogenic diseases transmitted through water, (Jenneman et al., 1998). These indicator bacteria which are relatively easy to detect and enumerate, suggest the potential existence of entropathogens in surface and potable water (Reynold et al., 1996).
The concept of a relationship between a colifom bacteria and enteropathogens Such as cholera and typhoid bacilli was introduced during the early part of this century and published in 1905 in the first edition of standard methods for the examination of water and wastewater. (Harvey, 1989). The methods described were widely used and were partially responsible for the subsequent reduction of water borne cases of typhoid.
To ensure safe recreational water and a continued supply of potable water, frequent monitoring of both raw water sources and finished for the presence of pathogens is very important.
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 COLLECTION OF SAMPLES
Well water was collected from six wells in UNAAB: COLNAS, COLANIM, S.U.B., Comfort Zone, Organic farm,female Hostel,male Hostel,COLANIM FARM,COLVET,Health centre. During the collection immediate surrounding of the well was observed for cleanliness. Six (10) sterile sampling bottles (100ml each) were used to take the sample water, the well water to be collected was first used to rinse the bottle then this was filed to the brim and covered by screwing the cap tightly and properly labeled, were taken to the laboratory for analysis.
3.1.1 LABORATORY ANALYSIS
Petridishes were washed, cleaned, drained to dry and put into the hot air oven for sterilization at 160°c for 1 hour. The petri dishes were allowed to cool to about 45°c and care was taken to avoid contamination by opening the head of the Petri dishes. These sterile petri dishes were used in the preparation of needed Agar media.
The pipettes were washed, rinsed, dried and cotton plugged, the pipettes were wrapped with thick foil paper and sterilized using the hot air oven at 160°c for 1 hour and allowed to cool to about 45° c ready for use. -
The medical bottles (100rnl) were washed properly with detergent, rinsed and dried, they were then sterilized using the hot air oven at 160° c for 1 hour and allowed to cool to about 45°c ready for use.
3.2 PREPARATION OF MEDIA
3.2.1 TCBS AGAR
88g of TCBS agar powder was weighed and dissolved into 500ml of distilled water in a conical flask. It was then brought to boil. After boiling it was allowed to cool and poured into a petridish with its replicate for cultivation of microorganism.
3.2.2 SALMONELLA SHEGELLA AGAR (SSA)
60g of Salmonella shegella powder was weighed and dissolved into 500ml of distilled water in a conical flask and was brought to boil. After boiling it was allowed to cool and poured into a sterile petridish with its replicate for cultivation of microorganism.
3.2.3 EOSIN METHYLENE BLUE AGAR (EMB AGAR)
Eosin methylene blue agar (EMB) agar weighing 18.0g was dissolved in 500ml of distilled water in a conical flask. It was then sterilized at 1210C for 15 minutes in an autoclave, it was brought out of the autoclave and allowed to cool before pouring into the sterile petridishes. The poured eosin methylene blue in the petri dishes were allowed to set before inoculating the organisms picked by a sterile inoculating loop in the positive test tubes meant for the presumptive test. This medium was used for the confirmed test.
3.2.4 pH
The pH meter was used to measure the pH of each well water. The electrode was standardized using buffer ‘7’. The electrode was then placed in the water sample and the pH value was read and recorded.
3.2.5 TOTAL VIABLE COUNT OF BACTERIA
One millilitre (lml) of dilution 10-1, 10 -2,10-3, 10-4 10-5 was pipetted into the plate depending on the appearance of the water though, three consecutive dilutions were used millilitre of each three consecutive dilutions was used pipetted aseptically into sterile petridishes. The inoculum was covered with sterile nutrient agar and the plates were immediately covered. The inoculum and the agar medium were thoroughly mixed together
The medium was allowed to set and incubated at 37° c for 24hour. The number of colonies per plate were counted and recorded. The different microorganisms observed on each culture plate after counting were subcultured into fresh sterile nutrient agar plated and incubated at 37° c for 24 hours. These isolates were purified. The pure culture streaked on the agar slant using sterile inoculating loop and incubated at 37oc for 24 hours and the number of colonies per plate were counted and recorded. The different microorganisms observed on each culture plate counting were subcultured into fresh sterile nutrient agar plate and incubated at 37°c for 24 hours. The pure culture was streaked on the agar slant using a sterile inoculating loop and incubated at 37° c for 24 hours and the slant bottles stored in the refridgerator.
The original culture per ml of the sample was determined by multiplying the number of colonies by dilution factor.
3.2.6 COLIFORM TEST
The coliform bacteria are members of the family Enterobacteriaceae. In analyzing water for coliform it is strictly Escherichia coli being tested for because while E. coli is a faecal coliform, one other common coliform, Enterobacter aerogenes, is non faecal (Fawole and Oso 1988).
This coliform test is used to determine thee presence or absence of coliform bacteria in water sample. The method used are:
3.2.7 PRESUMPTIVE TEST
This is used to enumerate and identify the presence of coliform organism in water sampled. The production of acid and gas in the Durham tube shows a positive presumptive test within 24-48 hours at 37oC. The first set of three test tubes contain Durham tube and 10ml of sterile distilled lactose broth was inoculated with 10ml of water sample. Each of the second set of three test tubes contain Durham tubes, 10ml of sterile lactose broth and 10ml of water sample. To the third set of three test tubes Durham tubes were put, 1.0ml of sterile lactose broth and 0.1ml of water. All the test tubes were incubated at 37oC for 24hours to 48hours and changing of the lactose broth colour from deep yellow and the presence of gas formation showed a positive presumptive test while no gas formation after 48hours of incubation constitute a negative test that is absence of coliform in water sample. Thee most probable number of the organism was recorded using the most probable number table.
3.2.8 CONFIRMED TEST
The inoculums from the presumptive test which is positive test were aseptically streaked on a sterile and solidified Eosin methylene blue agar (EMB) with sterile inoculating loop and incubated at 37oC for 24hours. Colonies of Enterobacter aerogenes were seen as pinkish mucoid dark colonies with dark centres while the colonies of E. coli were seen as greenish metallic sheen with dark colonies.
3.2.9 COMPLETED TEST
Typical colonies from Eosin methylene blue (Emb) plates were transferred to a nutrient agar slope and a lactose broth fermentation tubes and incubated at 37oC for 24hours. After the incubation, growth of colourless colonies was seen on the nutrient agar showing the presence of coliform while the presence of gas formation in the lactose broth tube also shows the presence of coliform.
3.2.10 IDENTIFICATION OF ISOLATES
Isolates were identified by gram staining, biochemical tests for coliform plates and bacteria while the isolates from fungi plates were identified with the use of cotton-blue lactose using a microscope.
3.2.11 BIOCHEMICAL TEST
This test is basically for the identification of organisms present using various biochemical tests.
(a) Coagulase test
On a clean slide, a suspension of the organism was made ands a drop of fresh plasma was added and mixed properly. Coagulase production shows the immediate clumping of the suspension. This shows, the ability of the isolates to produce the enzyme coagulase plasma.
(b) catalase test
A loopful of the bacteria was placed on the sterile slide. A drop of 3% hydrogen peroxide (H2O2) was added to the isolate (bacteria isolate) production of gas bubbles or form showed catalase was present that is the bacteria isolate produced an enzyme called catalase.
(c) Citrate test
The inoculating wire loop was aseptically used to pick the isolate into solidified Simmon’s citrate agar plate incubated at 37oC for 48hours and observed for colour change. Colour change from green to blue indicate positive result, the negative result indicate the original green colour.
3.2.12 METHYL RED TEST
Five milliliter (5.0ml) of the sterile MR-VP medium was aseptically pipetted into a sterile test tube, organism was inoculated into this tube and incubated at 37oC for 48hours. Five drops of methyl red indicator was added and the medium examined for colour change. Yellow colour indicates a negative result while the red colour indicate a positive result.
3.2.13 GLUCOSE FERMENTATION TEST
Nutrient broth powder oxoid (8.0g), glucose (5g) and phenol red (0.1g) were weighed and dissolved in 100ml of distilled water. Ten milliliter (10ml) of this solution was pipetted into test tubes with inverted Durham tubes and autoclaved at 121oC for 15minute. To each of the test tube each isolates was inoculated and one tube was left uninoculated, which was the control. The tubes incubated at 37oC for 48hours.
Colour changes from red to yellow indicate the production of acid while the presence of gas at the Durham tube showed the production of gas.
Gelatin liquefaction test
Nutrient broth powder (oxoid) 4.0g
Distilled water 500ml
Gelatin powder 50g
500ml of distilled water was added to the nutrient broth powder and gelatin powder and autoclaved for 121oC for 15minutes. 5mls was pipetted into each test tube, an organism was introduced while one test tube was left uninoculated which was the control. The test tubes were incubated at 37oC for 5days and after incubation the tubes were refrigerated for 15 minutes and observed for gelatinase production.
The positive test is known when the broth remain liquid meaning that gelatinage was produced by the organism but negatively showed solidification in the test tubes.
3.2.14 NUTRIENT AGAR
Nutrient agar powder weighing 28g was dissolved in 1000ml of distilled water in the conical flask and brought to boil using the water both to enable the nutrient agar to dissolve. The nutrient agar was autoclaved at 1210C, 15 atmospheric pressure for 15 minutes and allowed to cool to about 450C. It was dispensed into petri-dishes and allowed to set and stored in the refridgerator after undergoing sterility test.
3.2.14.1 NUTRIENT AGAR SLANT
The molten nutrient agar which has been sterilized was put into the macCartney bottle and the macCartney bottle was placed in a slanting position to solidify. The nutrient agar slant was stored in the refridgerator and was used when required for the storage of isolates.
3.2.15 SERIAL DILUTION
One milliliter (1ml) of the original stock culture which was properly shaken, was pipette into the first test tube containing 9ml sterile distilled water making 10-1 dilution. To the other five test tube, 1ml of the distilled water was pipette into each of the test tubes and labeled 10-2, 10-3, 10-4, 10-5. From the 10-1 test tube, 1 ml is aseptically transferred to the 10-2 test tube and 1 ml is subsequently transferred aseptically.
3.2.16 GRAM STAINING
A thin smear of each isolate was made on a slide and air dried. The thin smear was stained with crystal violent for 60 seconds and washed with water. It was then stained with Gram’s iodine for 60 seconds, water was ran on it. The smear was decolorized with 75% alcohol for 15 to 30 seconds and water was ran on it. The smear was stained with safranine for 30 seconds, water was ran on it and air dried. This was observed under a microscope with the aid of an oil immersion on the slides.
The gram color and morphological shape were observed. Red color shows gram negative while the purple color shows the presence of gram positive bacteria.
CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
4.1 RESULTS
Well water in UNAAB were sampled for microbiological analysis for isolation and identification of microorganism present in them.
Table I gives the description of wells in UNAAB the immediate environment, mode of drawing, purpose it serves, colour and the water taste.
Table II shows the pH, the most potable number (MPN) and the viable microbial count of microorganisms in UNAAB well water.
Table III shows the colonial morphological and biochemical characteristics of bacteria isolated from the sampled water.
Table IV shows the colonial morphological and biochemical characteristics of coliform obtained from the sampled well water.
Table 1: The description of wells in UNAAB
Well sample Mode of drawing Purpose it serves Colour Taste Immediate environment
COLNAS Drawing bucket Washing Very clean Tasteless About 35metre away from chemistry laboratory, situated on hilltop with a path leading to it.
COLANIM Drawing bucket Washing Very clean Tasteless Is located at the back of APH laboratory, beside generator house
Comfort zone Drawing bucket
Pumping machine Washing and soak away Slightly clean Sour About 40 metre away from multipurpose laboratory along comfort zone
SUB Drawing bucket Washing Dirty Tasteless At SUB block
Male hostel Drawing bucket Washing Slightly dirty Tasteless Very close to the male hostel
Organic farm Drawing bucket Irrigation Very dirty Located at the centre of the organic farm
COLANIM farm Drawing bucket Washing Very clean Sour very close to aquaculture building
COLVET Drawing bucket pumping machine Washing clean Tasteless At the COLVET laboratory
Female hostel Drawing bucket Washing Slightly dirty Tasteless At the back of female old hostel
Health centre Drawing bucket
Pumping machine Washing and cleaning Very clean Tasteless Beside old building at the health centre.
Table 2: The pH, the most probable number (MPN) and the viable microbial count of microorganism in UNAAB well water
Well samples pH MPN/100ml Total bacteria count/ml on Nutrient agar Total viable fungi count/ml on potato dextrose agar Specific bacteria present
COLNAS 7.10 6.1 1.5 x 105 2.4 x 107 Enterobacter aerogenes
COLANIM 6.91 9.0 2.3 x 105 - E. coli
Comfort zone 7.13 14 2.2 x 105 - E. coli
SUB 7.12 43 7.5 x 105 - Klebsiella pneumonia
Male hostel 8.0 9.1 6.3 x 105 1.1 x 105 Enterobacter aerogenes
Organic farm 7.6 22 4.4 x 105 1.5 x 105 E. coli
COLANIM farm 6.93 34 2.8 x 105 1.75 x 105 E. coli
COLVET 7.2 44 9.6 x 105 1.9 x 107 Enterobacter aerogenes
Klebsiella pneumonia
Female hostel 8.0 53 8.3 x 105 1.2 x 107 Enterobacter aerogenes
Klebsiella pneumonia
Health centre 7.10 29 9.6 x 105 1.9 x 107 Enterobacter aerogenes
Table 3: The colonial, morphological and biochemical characteristics of bacteria isolated from UNAAB well water
Well samples COLNAS COLANIM Comfort zone SUB Male hostel Organic farm COLANIM farm COLVET Female hostel Health centre
Cultural characteristics (morphology) Circular raised transparent smooth creamy Circular raised transparent smooth creamy Irregular raised transparent entire smooth creamy Circular flat transparent smooth creamy Circular raised entire transparent smooth creamy Circular flat entire transparent smooth creamy Irregular flat rhizoid translucent smooth creamy Circular flat entire transparent smooth creamy Irregular flat rhizoid translucent smooth creamy Irregular flat rhizoid translucent smooth creamy
Cell shape Short rods in singles Short rods in singles Short rods in singles Short rods in singles Short rods in singles Short rods in singles Short rods in singles Short rods in singles Short rods in singles Short rods in singles
Gram staining - - + + - + + + + +
Catalase + + + + + + + + - -
Coagulase - - - - - - + - + +
Citrate + + + + + + + + + +
Methyl red test + + + + + + + + + +
Glucose fermentation + + + + + + + + + +
Gelatin liquefaction test + + + + + + + + + +
identified organism Pseudomonas sp. Pseudomonas sp. Klebsiella pneumoniae Micrococcus sp. Pseudomonas sp. Micrococcus sp. Klebsiella pneumoniae Micrococcus sp. Klebsiella pneumoniae Klebsiella pneumonia
Table 4: The colonial morphological and biochemical characteristics of coliform obtained from UNAAB wells water
Well samples COLNAS COLANIM Comfort zone SUB Male hostel Organic farm COLANIM farm COLVET Female hostel Health centre
Appearance on EMB Agar Large pinkish mucoid with dark colonies Dark colonies with greenish metallic sheen Dark colonies with greenish metallic sheen Large pinkish mucoid with dark colonies Dark colonies with greenish metallic sheen Dark colonies with greenish metallic sheen Dark colonies with greenish metallic sheen Large pinkish mucoid with dark colonies Large pinkish mucoid with dark colonies Large pinkish mucoid with dark colonies
Methyl red test - + - - + + + - - +
Citrate test + - + + - - - + + +
Catalase test + + + + + + + + + -
Identified organisms Enterobacteer aerogenes E. coli E. coli Enterobacter aerogenes
Klebsiella pneumoniae E. coli E. coli Klebsiella pneumoniae Klebsiella pneumoniae Enterobacter aerogenes
Klebsiella pneumoniae Enterobacter aerogenes
Klebsiella pneumoniae
4.2 DISCUSSION
The Construction materials for these wells vary but included rings, concrete bricks. A well constructed with appropriate materials is protected from surface sipping in the well. Well heads varies from 0.45 metres to 0.95 metres. Hence 60% of the well sampled met the internationally accepted well head of 0.6 metres some of the well tops were completely covered with metals or planks and some were partially covered while two of them were not covered at all. The covering prevents atmospheric air contamination of water.
The immediate environment of the location of the well were not quit favorable some of thee environment were bush with grasses growing in an on top of the well, drainage systems and bathroom waste water running around the well. Non of the well sampled met the UNESCO/UMO standard of 1991 of a well being sited at a distance of not les than 30 metres from the bathroom, toilet and other pollutant (UNESCO/UMO1991).
The physical properties of the water of these wells samples in terms of colour, odour, taste, presence or absence of precipitate 40% of them are unacceptable in terms of aesthetics as the contained precipitate. 60% of the wells tasted sour will 40% had offensive odour.
The pH range between 6.90 to 8.20 while the water that is potable should be neutral (pH7). The slight variation of above or below pH 7 maybe due to the type of soil around the well or the activities of micro organism in the well. The viable microbial counts were high for all the well waters while the MPN counts though low make the waters unsuitable for consumption (Jay 1991). How ever, only five of the well waters harbored E. coli confirming the possibility of their facal contamination.
CHAPTER FIVE
5.0 CONCLUSION AND RECOMMENDATION
5.1 CONCLUSION
All the well water sample in UNAAB consist of high microbial load majorly the entero pathogenic bacterial which are capable of causing gastrointestinal infection such as diarrhea, typhoid etc. Moreso, the water sample does not confirm with the standard of the World Health Organization (WHO) guideline for drinking water. If followed the result of the microbial analysis. The study has confirmed the effect of the environmental hazards from series of contaminants in ground water arising from the waste portability can not be only determined just by the physical appearance of water but the microbiological analysis has to be put into consideration. Efforts should be made in the provision of potable water for human consumption.
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APPENDIX
Preparation of media used
The media used are double strength and single strength Macconkey Agar, Nutrient Agar, Simon atrate Agar, Salmenella Shegella Agar, TCBs, and Eosin methylene Blue (EMB).
Nutrient Agar
Beef extract 5.0g
Agar powder 15.0g
Peptone 5.0g
Nacl 8.0g
Distilled water 1 litre
pH 7.1 after sterilization
Simmon citrate Agar
Sodium citrate 2.0g
Dipotassium phosphate 1.0g
Ammonium Dihydrogen phosphate 1.0g
Magnesium sulphate 0.2g
Nacl 5.0g
Bromothymol Blue 0.08g
Agar powder 20.0g
Distilled water 1 litre
pH 6.8
Eosin methylene Blue Agar (EMB)
Lactose 10g
Peptone 10g
Dipotasium hydrogen phosphate 2g
Methylene Blue 0.065g
Eosin Y 0.4g
MacConkey Agar (MCA)
Peptone 17.0g
Protosepeptone 3.0g
Lactose 10.0g
Bile salt 1.5g
Nacl 5.0g
Neutral red 0.001g
Agar powder 13.5g
Distilled water 1 litre
Samnella shigella agar (ssa)
Beef extract 5.0g
Peptone 5.og
Lactose 10.0g
Bile salt 8.5g
Sodium citrate 8.5g
Sodium thiosulphate 8.5g
Ferric citrate 1.0g
Brilliant green 0.00033g
Neutral red 0.25g
Agar no2 13.5g
T.C.B.S Cholera medium
Yeast extract 5.5g
Peptone mixture 10.0g
Sodium citrate 10.0g
Bile salt 9.0g
Sucrose 17.0g
Nacl 10.0g
Ferric citrate 1.0g
Bromothymol blue 0.04g
Thymol blue 0.04g
Agar no1 15.0g
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