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RESEARCH ARTICLE

Open Access Full Text Article

Bacteriological and physiochemical analysis of spring water:

Case study of Osan-Ekiti, Nigeria

Abdullahi Attah Alfa

1

and Abayomi Ajayi

1*

1Department of Anatomy, Faculty of Medicine, Kogi State University PMB 1008 Anyigba Kogi State, Nigeria.

*For correspondence: attahalfa@gmail.com

Article Info: Received 05 Sep 2014; Revised: 17 Nov 2014; Accepted 27 Nov 2014.

ABSTRACT

The bacteriological and ecological quality of major sources of drinking water was carried out using five samples of spring water collected from various sites in Osan-Ekiti, South West Nigeria. Enterococcus spp.,

Enterobacter spp., Klebsiella spp., Proteus spp., and Escherichia coli were isolated and antibiotics sensitivity test was carried out using disc diffusion method and Gram positive antibiotic disc. Enterobacter spp.,

Enterococcus spp., Escherichia coli and Proteus spp. showed resistance to Gentamycin showed high resistance. E. coli, Enterobacter spp., Enterococcus spp., Klebsiella spp., and Proteus showed high resistant to Tetracycline (25 µg/ml). Enterobacter spp., Enterococcus spp. and Klebsiella spp. show low resistance to Chloramphenicol (30 µg/ml). Klebsiella spp., Enterobacter spp., Enterococcus spp. and E. coli was resistant to Cloxacillin (5 µg/ml). Enterococcus spp.shows low resistance to Erythromycin (15 µg/ml). Escherichia coli, Enterobacter spp., Enterococcus spp., Klebsiella spp., and Proteus spp. were resistance to Augumentin (20

µg/ml). Enterobacter spp.,and Enterococcus spp. were resistance to Cotrimoxazole (10 µg/ml), Proteus spp. shows low resistance to Cotrimoxazole (10 µg/ml). Klebsiella spp., E. coli and Proteus spp. were resistance to a Gram negative antibiotic disc Ampicillin (10 µg/ml). E. coli and Proteus spp. show low resistance to Streptomycin (10 µg/ml) and Enterobacter spp., Enterococcus spp., E. coli and Proteus spp.show resistance to Gentamycin (25 µg/ml). E. coli, Enterobacter spp., Enterococcus spp., Klebsiella spp.,and Proteus spp. show high resistant to Tetracycline (25 µg/ml). Enterobacter spp., Enterococcus spp., and Klebsiella spp. show high resistance to Cotrimoxazole (10 µg/ml). from the sanitary point of view, it shows that almost all the spring water have the tendency of causing disease, which makes them unsafe for human consumption and most of the bacteria isolated were resistant to the antibiotics used one way or the other.

Keywords: Water, Enterococcus spp., antibiotics, resistance, physiochemical, mineral analysis

1.

INTRODUCTION

Water is a liquid compound which is chemically composed of hydrogen and oxygen in the ratio of 2:1 by volume or mass respectively. It is a universal solve

-nt and is essential for the growth and survival of many things on the planet, including microbes. Water serve as the medium in which all microbial and

Malaya

Journal of

Biosciences

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biochemical reactions occur and it also play a vital role in the evolution of biological system [1, 2]. Water resources are sources of water that are useful or potentially useful to humans. It is important because it is needed for life to exist. Uses of water

include; agricultural, industrial, household,

recreational and environmental activities. Virtually all of these human uses require fresh water, although only 2.7% of water on the earth is fresh water and over two third of this is frozen in glaciers and polar ice caps, leaving only 0.007% available for human use. Fresh water is a renewable resource, yet the world's supply of ocean, fresh water is steadily decreasing. Water demand already exceeds supply in many part of the world and as the population of many countries keep rising at an unprecedented rate, many more areas are expected to experience this imbalance in the near future [3].

Surface water is undoubtedly the most precious natural resources that exist without which life on earth will be non-existent. It is water in a river, lake or fresh water wetland. Surface water is naturally replenished by precipitation and mostly discharge to the oceans, through evaporation and sub-surface seepage. Although the only natural input to any surface water system is precipitation within it watershed, the total quantity of water in that system at any given time is also dependent on many other factors. These factor include storage capacity in lake, wetlands and artificial reservoirs, the permeability of the soil beneath these storage bodies, the runoff characteristics of the land in the watershed, the timing of the precipitation and local evaporation rates. All of these factors also affect the proportions of water loss through discharge to the ocean, evaporation and sub-surface seepage. Human activities can however have large input on these factors [4].

The total quality of water available at any given time is an important consideration. Some human water users have an intermittent need for water, for example, many farms require large quantities of water in the spring and low quantities in the winter. To supply such a farm with water, a surface water system may require a large storage capacity to collect water throughout the year and release it in a short period of time. Other users have a continuous need for water, such as a power plant that requires water cooling. To supply such a power plant with water, a surface water system may require a large storage capacity to collect water throughout the year and release it in a short period of time. Nevertheless, over the long term the average rate of precipitation within watershed is the upper bound for average

consumption of natural surface water from that watershed. Natural surface water can be augmented by importing surface water from another watershed through a canal or pipeline. It can also be artificially augmented from any of the other source earlier mentioned. Man can however cause surface water to become unusable through pollution [5].

Sub-surface water or ground water located in the pore space of soil and rock. It is also the water that flows without aquifers below the water table. Sometime it is useful to make distinctions between sub-surface water that is closely associated with surface water and deep sub-surface water in aquifers (sometimes called "fossil water"). Nevertheless, over the long term the average rate of seepage above sub-surface water source is the upper bound for average consumption of water from the source. The natural input to sub-surface water is seepage from surface water. The natural output from surface is springs and seepage to oceans [6].

Enterococcus is a genus of lactic acid bacteria of the phylum Firmicutes. Member of this genus were classified as group D Streptococcus when genuine DNA analysis indicated that a separate genus classification was appropriate. Enterococci are gram positive cocci which often occur in pairs (diplococci) and are difficult to distinguish from streptococci on physical characteristics alone. Two species are commonly found as commensal organisms in intestine of humans; Enterococcus feacalis (90-95%) and Enterococcus faecium (10-15%). Enterococcus

are facultative organisms i.e. they prefer the use of oxygen but they can survive in the absence of oxygen. They typically exhibit gamma haemolysis on sheep blood agar. Other species in this genus are E. aviumos, and E. solitarium [7]. Clinical infections caused by Enterococcus include urinary tract infections, bacteraemia, bacterial endocarditis, diverticulitis and meningitis. Sensitive strains of these bacteria can be treated with Tetracyclin and Gentamycin.

From a medical stand point, the most important feature of the genus is their high level of endemic

antibiotic resistance. Some Enterococci are

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the strictest tolerances in the United States, the limit of water off its beaches is 7 colony forming unit per 100ml of water. Above which the state may post warning to stay out of the ocean. In 2004, Enterococcus spp. took the place of faecal coliform as the new federal standard for water quality at public beaches. It is believed to provide a higher correlation than coliform with many of the human pathogens often found in sewage.

Water can act as vehicle for the transmission of a number of serious infectious diseases, or as potential carrier of pathogenic microorganisms which can thus

endanger health and life [8]. Hence, the

bacteriological quality of water is of utmost importance and careful monitoring must be given the highest priority [9]. This research work was carried out to determine the bacteria count of outflow spring water samples from Osan-Ekiti, Southwest Nigeria. The antibacterial susceptibility of the isolated bacteria from the spring water as well as the physicochemical and mineral quality of the water samples from the outflow spring water was also determined.

2.

MATERIALS AND METHODS

2.1 Collection of samples

The water samples were collected with sterilized bottles pointed slightly upward and directed against the water current. The bottles were never allowed to brush anything before they were fixed in a cooler/warmer parked with iced, then closed and saved until when needed.

2.2 Sterilization

All glass wares were first washed with detergents and rinsed thoroughly with distilled water and air dried before sterilization by dry heat in an oven at a temperature of 1600C for 1 hour. Pipettes were

plugged with absorbent cotton wool and wrapped with aluminium foil paper after drying before sterilization to prevent external contamination. Work bench surfaces were cleaned with cotton wool soaked previously in disinfectant before and after any experiment was carried out to prevent contamination.

2.3 Culture media

Macconkey agar

Macconkey agar was prepared according to manufacturer's instructions. It was used for the enumeration of coliforms in the water samples. It is also used to obtain pure culture from Macconkey

broth and in the confirmation of Escherichia coli type 1 (Eijkman test).

Nutrient sensitivity test agar

About 28g of the nutrient agar powder was weighed and dissolved in 100ml of distilled water; this was boiled to dissolve completely and rendered sterile in an autoclave at 121°C for 15minutes. The

medium was allowed to cool before it was poured into sterile petri-dishes and then allowed to gel.

Eosin methylene blue agar (EMB)

Eosin Methylene Blue Agar was prepared according to manufacturer's instructions. It was used for the enumeration of coliforms in the water samples. It was also used to obtain pure culture from nutrient agar and in the confirmation of Escherichia coli type 1 (Eijkman test).

2.4 Preparation of stock culture

Microbiology analysis

Serial dilutions was done by setting up series of test tubes each containing 9 ml of sterile distilled water, then a sterile pipette was used to transfer 1ml from the stock samples into the first test tube containing 9 ml of distilled water and rub between palms to ensure even mixture of the sample with water, then another sterile pipette was used to transfer 1 ml from the first test tube into the next tube containing 9 ml of distilled water. This was repeated until the appropriate dilution was obtained at 10-6.

1ml of inoculums samples from 10-5 and 10-6 of each

sample were introduced with the aid of sterile pipette into a series of petri-dishes. Then, prepared Bile Esculin Agar was poured into it, swirled gently to mix the content of the petri-dishes. This was allowed to set and incubated at 37°C for 24 hours. After 24

hours of incubation, the plates were examined for growth of Enterococcus spp. and the colonies were transferred to a freshly prepared plate to obtain pure culture which was later isolated and kept on an agar slant with the aid of wire loop.

2.5 Culturing

The test was carried out using the pour plate method. This was carried out after serial of dilutions as mentioned above about and about 10-6 inoculums

were made for each sample. 1ml of the inoculums from 10-5 and 10-6 of each sample was introduced

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hours. After 24 hours of incubation, the colonies were counted.

2.6 Isolation of organism

After 24 hours of incubation, the plates were examined for the growth of Enterococcus spp. and a strain of the organism was transferred to a freshly prepared plate to obtain a pure culture which was later isolated and kept on agar slant with the aid of wire loop.

2.7 Antibiotic sensitivity

15 ml of sterilized sensitivity test agar was poured into sterile petri-dishes and was then allowed to gel. The organism was seeded on the surface of the agar. The antibiotic disc was carefully and firmly placed on the seeded plate using a sterile forceps. The plates were then incubated avertedly for 24 hours at a temperature of 37°C. The gram positive disc

constitutes Amoxilin (AMX, 10µg/ml),

Cotrimoxazole (COT, 10µg/ml), Chloramphenicol (CHL, 30µg/ml), Gentamycin (GEN, 25µg/ml), Augumentin (AUG, 20µg/ml), Cloxacilin (CXC, 15µg/ml) and Tetracyclin (TET, 25µg/ml). The gram negative disc constitutes Ampicillin (AMP, 10µg/ml), Streptomycin (STR, 10µg/ml), Gentamycin (GEN,

25µg/ml), Cotrimoxazole (COT, 10µg/ml),

Cloxacilin (CXC, 15µg/ml) and Tetracyclin (TET, 25µg/ml).

2.8 Physico-Chemical and Mineral Analysis

Determination of conductivity and pH

Conductivity and pH were measured with the use of conductivity meter pH meter respectively.

Determination of alkalinity

About 50ml of the water sample was pipette into a conical flask and 3 drops of phenolphthalein indicator was added and the solution turned colourless, which indicated that no carbonate was present in the solution and the process was processed further by adding 2 drops of methyl orange indicator was further added to the sample. The colour then changed to yellow and it was the titrated with 0.02N of sulphuric acid until a permanent reddish colour appears.

Alkalinity = Titre value × 0.05 × 100000 Volume of the sample

Determination of hardness

About 0.01ml of EDTA solution was put inside a burette and standardized against 0.1ml of Na2CO3.

About 50ml of water sample was pipette into a

conical flask and 2 ml of buffer solution was added followed by a pinch of (KCN) potassium cyanide, which was added as a masking agent, about 3 drops of indicator Erichrome Black T was added and the solution was titrated against about 0.01 ml of EDTA standard until the colour changed from wine red to blue.

Hardness = Titre value × 100 Volume of the sample

Determination of turbidity

The turbidity of the water sample was determined using spectrophotometer at wavelength of 520nm. The sample was poured into the cell of the spectrophotometer after being standardized. Turbidity value was read off spectrophotometer read out device. The unit is NTU (Normal Turbidity Unit).

Determination of dissolved solid

About 50ml of water sample was placed in a weighing crucible and kept in an oven to evaporate into dryness at 1100C, the crucible was allowed to

cool in desiccators and weighed to a constant weight. The total solid was calculated using the formula:

Determination of Dissolve Solid = W2 − W1 Volume of used sample

Where, W1 = initial of crucible and W2 = weight of crucible + residue.

Determination of suspended solid

Filter paper was first weighed and about 50ml of the water sample was weighed and poured into the filter paper and filtered. The filter paper was then dried inside the oven at a temperature of 1050C. The

filter paper was allowed to cool in the desiccators before it was weighed again. This was done for a period until a constant weight was observed. The differences in weight represent the total suspended solid.

Determination of acidity

About 50ml of the water sample was pipette into a conical flask and 3 drops of phenolphthalein was added and the solution turns pink, the solution was then titrated with 0.05 NaOH and pink colour changed to orange at the end point. Acidity (A) was measured using the following formula, A = M1V1 = M2V2.

3.

RESULTS

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Nigeria were investigated for their microbial qualities, their significance to human health and their antibiotic resistance bacteria. The constituent of the contaminants of ground surface water consist largely of soluble particles, microorganisms and chemicals as observed in this work and this agrees with the microbial contamination of spring water especially by Enterococcus spp. which is viewed with serious concern because of the potential health risk that could result thereof. This is because the rural populaces do not treat the spring water before use. Table 1 shows the antibiotic sensitivity test using an antibiotic sensitivity discs (Gram positive). Gentamycin, Tetracyclin, Chloramphenicol and Erythromycin were detected among the antibiotic resistant to which

Enterococcus spp. was at zero degree of inhibition.

Enterococcus infection showing pink colonies were observed on selective agar (EMB) plate.

Table 1. Shows the antibiotic sensitivity test using gram positive antibiotic sensitivity test disc.

S.No TET CXC GEN COT CHL AUG AMX ERY

1. R R R R R R R R

2. R R I R I R R R

3. R R S R I R R I

4. R R S R S R R R

5. R R R R R R R R

6. R R I R R R R R

7. R R I R S R R R

8. R R S R I R R R

9. R R S R R R R R

10. R R R R S R R R

11. R R I R S R R R

12. R R R R I R R R

13. R R R R I R R R

14. R R R R I R R R

I = Inhibition; R = Resistance; S = Sensitivity

Table 2 shows the antibiotic sensitivity test using an antibiotic sensitivity test (Gram negative) discs. Table 3 shows the biological and ecological qualities of major sources of drinking water in the samples.

S.No GEN CLT AMP CXC COT TET STR ERY

1. R R R R R I R R

2. R R R R R R R R

3. R R R R R R R R

4. R R R S R I R R

5. I R R R R R I R

6. R R R R R I R R

I = Inhibition; R = Resistance; S = Sensitivity

4.

DISCUSSION

Water is indispensable to man and other living organisms as its essential for health and sanitation. Being the cheapest and principal raw material used in all industries and being the major component of every living organism, it can act as an avenue for the transmission of a number of serious infectious diseases [10].

The general view of spring water for domestic use of the general public in Osan-Ekiti appears to almost be in the negative, in terms of microbiological and or bacteriological quality. Consequently, a greater amount of organic and inorganic wastes spread back into the neater sources so that less portable water eventually becomes available [11]. Based on the drinking water quality standard of the Environmental

Protection Agency (EPA) for "potable

uncontaminated water", as reported by Drezeen (1996) [12], water with counts under 100 cfu/ml should be considered "potable" and values between 100-500 cfu/ml as "questionable". One can safely conclude that the quality of several water samples examined is bacteriologically poor, since there is the presence of bacteria, which are even antibiotic resistant in the water samples. Therefore, based on this, failure of all the water samples to meet the standard requirement may be as a result of contamination either from the source of the environment or possibly from defecation.

Historically, water has played a significant role in the transmission of human disease, such as, typhoid fever, cholera, infectious hepatitis, bacillary and amoebic dysentery and many varieties [13]. The

bacterial isolated from this study include

Enterococcus spp. This organism may be present in water as contaminant and may be of faecal origin. This is in agreement with (Drezeen, 1996) [12] who reported that spring water may have a wide range of organisms which includes indigenous species, saprophytic species as well as human pathogenic contaminant, indicating that Enterococcus can be ingested. From this investigation, the result shows that almost all the samples examined were polluted with microorganisms. It can thus be concluded that some of them do not meet the W.H.O. standard and this may be due to the source of water used.

The antibiotic resistivity pattern of this work has further thrown more light on the findings of (Marrit

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Table 3. Biological and ecological qualities of major sources of drinking water in Osan-Ekiti, Ekiti State

PARAMETER KAJOLA IGBOLOKE IGBOAPO OROKI OMI-ISUN

WATER CONDITION

Width (m) 3-5 1-2 3-5 6-1O >20

Depth (m) 0.7-1.0 0.1-0.3 0.4-0.6 >1.0 >0.1

Current Still Still Still Still Still

Velocity estimated <0.2 <0.2 <0.2 <0.2 <0.2

SUBSTRATE Boulders Nil Nil Nil Nil Nil

Large stone Nil Nil Nil Nil Nil

Small stone Nil Nil Nil Nil Nil

Silt <25% <25% <25% 25%-50% >50%

Filamentous algae <25% <25% <25% >50% >50%

Mosses <25% <25% <25% >50% >50%

Detritus <25% 25%-50% >50% 25%-50% 25%-50%

Submerged plants 25%-50% <25 25%-50% 25%-50% 25%-50%

BIOTIC QUALITY

Emerged plants >50% <25% 25%-50% >50% >50%

Marginal plants >50% <25% >50% >50% >50%

Amphibians <25% <25% <25% <25% <25%

Insects >50% 25%-50% 25%-50% 25%-50% 25%-50%

HABITAT % still water >50% >50% <25% >50% >50%

% flowing water <25% <25% 25%-50% <25% <25%

Shade Heavy Moderate Heavy Heavy Heavy

Protection measures Concrete banks Dammed with trees of raffia palm

Concrete banks Concrete banks Concrete banks

POLLUTION Accessibility of domestic animals

Rare Remote Remote No Common

Distance from the main road

=200m >500m >500m >300m <10m

Influx of effluent from town

Remote Remote Remote Remote Possible

Key: <25% = Rare; 25-50% = Intermediate; >50% = Dominant

Table 4. Physico-chemical and mineral analysis of major sources of drinking water

PARAMETER KAJOLA IGBOLOKE IGBOAPO OROKI OMI-ISUN

Sodium (Na) 50.1 50.1 49.9 50.0 65

Potassium (K) 111.0 111.5 88.5 85.5 120.1

Magnesium (Mg) 70.5 71.5 70.0 71.0 80.1

Calcium (Ca) 40.1 39.7 38.0 42.1 45.2

Iron (Fe) 0.8 0.5 0.4 0.7 1.0

Lead (Pb) Not found Not found Not found Not found 0.09

Zinc (Zn) 2.1 1.9 2.0 1.5 2.2

Copper (Cu) 0.09 0.09 0.05 0.08 0.1

Manganese (Mn) 0.04 Not found Not found 0.02 0.2

pH 7.0 7.0 7.0 6.9 7.0

Alkalinity 7.0 7.0 7.0 7.0 7.0

Dissolve oxygen mg/l 0.5 0.7 0.5 0.4 1.1

Dissolve solid mg/l 0.0123 0.0111 0.0145 0.0243 0.1042

Temperature 0C 28.5 35.0 29.1 27.8 29.2

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that contains a high frequency of antibiotics resistant bacteria. This resistance could greatly be attributed to drug abuse among people.

From the investigation, Gentamycin has proved very potent as well as Streptomycin, Chloramphenic--ol, and Tetracyclin. This can be regarded as the best antibiotics to combat diseases caused by bacteria. Conversely, the highest incidence of resistance by bacteria isolates were recorded with Augumentin, Erythromycin, Amoxicillin, Contrimoxazole and Cloxacillin. The clinical management of such infections then becomes a major challenge [15]. With the advent of antibiotics, it was thought by many that as microorganisms are exposed, they would disappear, and bacteriology would as much as became a thing of the past. However, due to myriad of reasons, resistant organisms do develop and even pose more problems after antibiotics treatment. Resistance is therefore a major problem in chemotherapy and it’s mainly confined to

Escherichia coli and also to a lesser extent, the

Enterobacter spp [11]. The rate of emergence of resistant organisms varies with different antibiotics. For example, Escherichia coli resistant organisms vary with different antibiotics. Klebsiella strains are more resistant because the organisms undergo mutation. As more antibiotics is given, the sensitive organism will be killed and the resistant strains multiply under favourable conditions and the resistant strains thus emerge and adapting themselves to any increase in antibiotics dosage. So by sensitivity test and assays, the clinicians can be kept informed of the behaviour of the effective organism and the patient.

5. CONCLUSION

In conclusion, the present study has provided a clue as to the bacteriological properties of spring water in Osan-Ekiti, Southwest Nigeria. These findings are important not only for the assessment of water quality in this particular area but also for policy formulation and planning by the government.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Acknowledgement

Authors wish to thank Dr. Ajayi, Dept. of Microbiology, Ekiti State University and members of Dept. of Anatomy, Kogi State University, Nigeria

References

1. Akinyanju JA (1990). Bacteriological

Analysis of Drinking Water from Different

Sources in Ilorin: An Urban Center In Nigeria. Nigerian Journal of Pure and Applied Sciences. 2:1-5.

2. Nikkado H and Vaara N (1994). Molecular

Basis of Bacteria Outer Membrane

Permeability. Microbiological Review. 49: 1-32.

3. Stickler DC (1992). The Microbiology of Treated Natural Mineral Water. Journal of Social Health. 109: 118-124.

4. Gleick PH (1996). Water Resources: In Encyclopedia of Climate and Weather. New York; 2: 817-823.

5. Olutola PO, Famurewa O, Sountag MG

(1999). An Introduction to General

Microbiology: A Practical Approach.

Heidelberg, 146-148.

6. Famurewa and Sountag, M.G. (1999). An Introduction to General Microbiology: A Practical Approach. Heidelberg. Pg. 146-148.

7. Marrrit N, Mervi S, Seppo W (1983).

Antibiotic Resistance Among Different Species of Faecal Coliform Isolated From

Water Samples. Applied Environmental

Microbiology. 4873-4877.

8. Pelczar MJ, Chan ECS, Krieg NR (1999). Introduction to water analysis and the associated microbes. 5th Edition. Tata MC Graw Hill Publishing Company LTD. New York. 510-527, 570-571, 774.

9. Pipa E. (2000). Environmental Health

Perspective. Manitoba Canadian Survey. 108: 863-867.

10. Baker, F.J. and Breach, M. (1989).

Handbook of Bacteriological Technical. (2nd Edition). Butter Worth (Pub) LTD. 171-173.

11. Shuval H.T., 1992 Advances in water pollution Research, Oxford Pergamon press.

12. Drezeen, P.H. (1996). Biofilms: Key to Understanding and Controlling Bacteria Growth in Automated Watering Systems.

Published by Endostrom Industries Inc.

13. Gerger P.S. and Y. Argaman (Eds) (1984).

Assessment of Microbiology and Turbidity Standards for Drinking Water.

14. Walter, M.V. and Vennes, J.W. (1985). Occurrence of Multiple Antibiotic Resistant Enteric Bacteria in Domestic Sewage

Oxidation Lagoons. Applied and

Environmental Microbiology. 50: 930-933. 15. Odeyemi, A.T., Dada, A.C., Akinjogunla,

Figure

Table 2 shows the antibiotic sensitivity test using an antibiotic sensitivity test (Gram negative) discs
Table 3. Biological and ecological qualities of major sources of drinking water in Osan-Ekiti, Ekiti State

References

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