African pour plate technique for total viable

African Journal of Microbiology Research Vol. 6(10), pp. 2462-2468, 16 March, 2012
Available online at
DOI: 10.5897/AJMR11.1531
ISSN 1996-0808 ©2012 Academic Journals

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Bacteriological analysis of borehole water from
different towns in Ogun State, Nigeria

Aina, D. A.1*, Olawuyi, O. J.1, Coker, O. A.1, Ojelabi, D. O.2 and Alatise, F. A.1
1Department of Biosciences and Biotechnology, Babcock University, Ilishan-Remo, Ogun State, Nigeria. 2Department of Botany and Microbiology, University of Ibadan, Nigeria. Accepted 6 February, 2012
Samples of borehole water were collected from Iperu, Ilishan, Sagamu, Ogere, Ilara, Ikenne, Irolu, Ode-
remo and Babcock community, all in Ogun state. They were analyzed microbiologically using pour plate
technique for total viable counts and tube fermentation technique for Most Probable Number (MPN)
counts. Thirteen samples were positive for coliforms while remaining five were non-coliforms. Three
samples satisfied the W.H.O. standard requirements of coliform count between 1-3/100 ml. Four
samples were suspicious having coliform count ranging from 4-8/100 ml and eleven samples with too
numerous counts did not satisfy W.H.O. standard requirements. Six bacteria isolates were obtained
from the samples and identified as Escherichia coli, Klebsiella sp., Proteus sp., Enterobacter
aerogenes, Staphylococcus aureus and Streptococcus sp. Characterization of isolates was carried out
by Gram staining reaction and biochemical tests. Gram-negative bacilli (72.22%) were more prevalent in
the water samples compared to Gram Positive Bacilli (11.11%) and Gram Positive Cocci (16.67%). E. coli
gave the highest percentage of occurrence of isolates (33.33%) followed by Klebsiella sp. (27.78%)
while the percentage of occurrence of Proteus sp., S. aureus and E. aerogenes were the least (5.56%).
Two samples were confirmed for Streptococcus sp. (11.11%) and Clostridium sp. (11.11%). 50% of the
isolates tested positive to acid and gas.

Key words: Bacteriological analysis, water, bacteria.


The health of the people depends solely on the quality of
water available for consumption. The health aspects of
environmental quality were among the first to receive
scientific consideration through the recognition of water-
borne diseases (Olawuyi, 2006). W ater pollution as a
result of microbial contaminants and pollutants has
resulted in epidemics of water-borne diseases such as
typhoid fever, cholera and dysentery (Reeves et al.,
1989). Waterborne diseases are caused by pathogenic
microorganisms which are directly transmitted when
contaminated fresh water is consumed (Potter, 2006).
Contaminated fresh water, used in the preparation of
food, can be the source of food borne disease through

*Corresponding author. E-mail: [email protected] Tel:
consumption of the same microorganisms. According to
the W orld Health Organization (2008), diarrhea disease
accounts for an estimated 4.1% of the total global burden
of disease and is responsible for the deaths of 1.8 million
people every year. It was estimated that 88% of that
burden is attributable to unsafe water supply, sanitation
and hygiene, and is mostly concentrated in children in
developing countries (Bagley, 1985). Waterborne disease
can be caused by protozoa, viruses, or bacteria, many of
which are intestinal parasites (WHO, 2000).
Coliform bacteria are commonly-used bacterial
indicator of water pollution, which is present in the
environment particularly in the faeces of all warm-
blooded animals and humans (Howard et al., 2002). Their
presence in drinking water indicates that disease-causing
organisms could be in the water system and may pose an
immediate health risk the water (Tebutt, 2007).
The greatest risk from microbes in water is associated

with consumption of drinking-water that is contaminated
with human and animal excreta (Ito et al., 1991). Also,
infectious diseases caused by pathogenic bacteria,
viruses and parasites (e.g., protozoa and helminths) are
the most common and widespread health risk associated
with drinking-water (De Zuane, 2009). Other
microorganisms sometimes found in surface waters
which have caused human health problems include;
Burkholderia pseudomallei, Cryptosporidium parvum,
Giardia lamblia, Salmonella, Clostridium, Streptococcus ,
Parasitic worms (helminths), Novovirus and other viruses
(Olawuyi, 2006; Schleifer and Kilpper-Balz, 2008).
The immunity of individuals also varies considerably,
whether acquired by contact with a pathogen or
influenced by such factors as age, sex, state of health
and living conditions. Part of the demonstration of
pathogenicity involves reproducing the disease in suitable
hosts. For pathogens transmitted by the faecal–oral
route, drinking-water is only one vehicle of transmission.
Contamination of food, hands, utensils and clothing can
also play a role, particularly when domestic sanitation
and hygiene are poor. Improvements in the quality and
availability of water, in excreta disposal and in general
hygiene are all important in reducing faecal–oral disease
transmission (Heidelberg, 2000; Amyes, 2007; Genthe
and Strauss, 2007).
Borehole operators are required to deliver safe and
reliable drinking water to their consumers 24 h a day, 365
days a year. If the water supply becomes contaminated,
consumers can become seriously ill (Howard et al.,
2002). The underground water supplies are usually
considered safe provided they are properly located,
constructed and operated according to the W orld Health
Organization Guidelines for Drinking Water (WHO, 1976).
Boreholes as a low-cost technology option for domestic
water supply in developing countries are generally
considered as ‘safe sources’ of drinking water. However,
it is the collection, transportation, storage and decanting
of water that can lead to subsequent contamination. Most
pathogens that can contaminate water supplies come
from the feces of humans or animals (Edema and
Omemu, 2001).
When coliforms and other bacteria are found there is
the need to investigate and find out the sources of
contamination in the water. Conformation with
microbiological standards is of special interest because of
the capacity of water borne disease within a large
population (Edema and Omemu, 2001). Therefore, this
study was aimed at characterizing bacteria isolates in
borehole water of some towns in Ogun state and
assessing the quality of water and potential sources of
water contamination in such areas. MATERIALS AND METHODS Collection of samples Eighteen borehole water samples were collected from nine different
Aina et al. 2463

towns in Ogun state for analysis. The tap faucets were surfaced sterilized with cotton wool soaked in 75% ethanol, and then flamed. The tap was allowed to run for a 60 s before collecting in a 500 ml sterile bottle which was carefully covered with its screw cap. All samples collected from all sources were taken to the laboratory for analysis within six hours after collection. Each of the sample bottles was labeled with sample code number and they were thoroughly mixed, before testing.

Preparation of medium Seventy-three grams of lactose broth were weighed using an analytical weighing balance and dissolved in 1000 ml of sterile distilled water inside a conical flask. 52 g of MacConkey agar powder were dissolved in 1000 ml of sterile distilled water. 36 g of Eosin Methylene Blue powder was dissolved in 1000 ml of distilled water; 28 g of nutrient agar powder was also dissolved in 1000ml of distilled water and 32 g of Mueller Hinton agar powder was dissolved in 1000 ml of distilled water. For proper dissolution and homogenization, the media were shaken vigorously and melted using a water bath at the temperature at 45°C for 40 min before sterilizing in an autoclave at 121°C for 15 min. Media were aseptically dispensed into oven-sterilized Petri-dishes and allowed to solidify under laminar air-flow. Isolation of bacteria from water samples Water samples were subjected to colony count (using MPN standard table procedure) and multiple tube fermentation technique (using MPN procedure). Screening for acid and gas producing coliform bacteria Presumptive test 1 ml of the water sample was inoculated into test tubes containing 9 ml of Lactose broth with Durham tubes which was incubated at 37°C±0.5°C for 24 to 48 h. Gas production within 24 to 48 h indicated a positive presumptive test while absence of gas production indicates negative presumptive test. The appearance of air bubbles in Durham tubes before incubation was not confused with the actual gas production. The presence of acid production within 48 h of incubation is indicated by the change in color, showing a positive presumptive test.

Confirmed test A loopful each of the water sample in the positive presumptive test tubes was streaked on Eosin Methylene Blue and incubated at 37°C ± 0.5°C for 24 h. Growth of bacterial colonies indicated that the confirmed test is positive while absence of growth was considered as negative.

Completed test Melted MacConkey agar was distributed into plates using pour plates technique and allowed to solidify. A loopful of broth from each of the positive tubes was streaked on each of the MacConkey agar plates. The plates were incubated at 37°C for 24 h. Gram stained preparation from slant cultures corresponding to the positive tubes that showed acid and gas formation were thoroughly examined.

2464 Afr. J. Microbiol. Res.

Characterization of coliform isolates Morphological Growth and Identification of Isolates on media The cultural characteristics of the isolates on solid MacConkey agar were examined. The growth patterns, colony size, edge, elevation on the plates were recorded after 48 h of incubation at 37°C. Gram staining technique was carried out for the identification and differentiation of each isolated bacteria. The size and arrangement of colonies into shapes (rods or round and chains) were also recorded (Ryan and Ray, 2008). Biochemical tests for identification of bacteria isolates Some tests were carried out namely: Catalase, Urease, Oxidase, Indole and Citrate following standard procedures with reference to Bergey’s Manual of Systematic Bacteriology (Sneath, 1986).


In this study, eighteen samples of borehole water
collected from different towns in Ogun state, Nigeria were
tested for the presence of bacteria. Screening for acid
and gas production was carried out at 37°C (Figures 1, 2
and 3). Fifty percent (50%) of the isolates produced acid
and gas with colour changes on MacConkey Lactose
Broth medium and gas production was shown in Durham
tubes. The isolates that produced acid only accounted for
33.33% while 16.67% isolates did not produce acid and
gas (Figure 2).
From the morphological characterization of the isolates
(Table 1), E. coli isolated were rod shaped, soft, and
smooth and 2-4 mm in size, while Klebsiella spp. were
rod shaped, mucoid, rough, heaped or doomed, opaque
and pinkish colonies with sizes ranging from 2-4 mm.
Isolated Streptococcus spp. were cocci in chains, soft,
raised opaque and pink colonies with sizes ranging from
0.2-0.6 mm. S. aureus isolated were cocci in clusters,
rough, raised, mucoid, opaque, creamy with sizes
ranging from 1-1.8 mm, while Clostridium spp. showed
rod shape, serrated flat, soft, opaque, and white colonies
with sizes ranging from 4-5 mm. E. aerogenes showed
rod shaped, rough, slightly raised, mucoid, opaque, and
pink colonies with sizes from 4-5 mm, while Proteus also
showed rod –shaped, smooth, flat, mucoid, opaque and
pink colonies with sizes ranging from 2-4 mm.
In isolation and characterization of bacteria, E. coli
gave the highest percentage of occurrence (33.3%)
followed by Klebsiella sp. (27.78%), while percentage of
occurrence of Proteus sp. and E. aerogenes were the
least (5.56%). Three samples were confirmed to contain
Streptococcus sp. (11.11%) and Clostridium sp. (11.11%)
and 5.56% of S. aureus (5.56%) (Figure 2).
Table 2 showed that E. coli was indole and methyl red
positive while, Voges prokauer, citrate, litmus milk,
catalase, oxidase, coagulase, oxygen relationship,
DNAse and urease tests were negative for the organism.
Streptococcus sp. produced haemolysis on blood agar

but negative to Voges prokauer, citrate, litmus milk,
catalase, oxidase, coagulase, oxygen relationship tests,
methyl red, urease, DNAse and spore staining tests.
Clostridium sp. was positive for litmus milk, spore
staining, oxygen relationship and haemolysis tests but
negative to Voges prokauer, methyl red, citrate, oxidase
and coagulase tests. S. aureus was positive for catalase,
coagulase and DNase tests. Acid producing Klebsiella
sp. was Voges proskauer positive but negative to other
biochemical tests, while acid and gas producing
Klebsiella sp. was positive methyl red and citrate tests
but negative to other biochemical tests. E. aerogenes
was positive to indole, Voges proskauer, and citrate but
negative to other biochemical tests while Proteus was
positive to Voges proskauer and urease but negative to
other biochemical tests. High proportion of the isolated
bacteria (72.22%) were Gram negative bacilli, 16.67%
were Gram positive cocci and 11.11% were Gram
positive bacilli (Figure 3). The Most Probable Number
(MPN) of coliform bacteria was estimated from Monica C
(2006) MPN standard Table (Table 3). Three water
samples satisfied the WHO standard recommending a
coliform count between 1-3/100 ml, three were
suspicious, while eleven samples with too numerous to
count did not satisfy the WHO standard requirements.


Water must meet internationally acceptable standards
and be in line with guidelines stipulated by the World
Health Organization to be fit for drinking. Water quality
criteria are a function of several parameters. Appropriate
parametric limits for water quality standards have been
established (W HO, 1976). The bacteriological analysis of
the water samples are shown in Tables 2 and 3. The total
viable count (TVC) indicates that water samples from
Ilishan, Ogere, Sagamu, Ikene, Irolu, and Ode, had high
coliform counts far above the recommended value after
incubation for 24 h. The result of the bacteriological
examination of the water samples obtained also showed
that three of the samples (IPR1, SG2, OGR2) are within
the range of satisfactory values of 1-3/100 ml of coliform
count (but the most acceptable limit is 0 coliform count
which was stamped as Excellent), four samples (ILH1,
ODE2, BAB1 and BAB2) were suspicious, while eleven
samples (IPR2, ILH2, SG1, OGR1, IKN1, IKN2, ILA1,
ILA2, IRO1, IRO2, ODE1) were unsatisfactory. This
implies that they do not satisfy the World health
Organization standard requirements.
Faecal contamination appears to be the most serious
form of water contamination obtained from various
sources. Poor methods of faecal waste management like
refuse disposal, shallow depth of well and uncontrollable
use of inorganic fertilizers are possible source of
However, illegal dumping of domestic wastes, livestock

Aina et al. 2465

Figure 1. Percentage of isolates in water sample.

Figure 2. Percentage of isolates screened for Acid and Gas production in water samples. A: Acid Only; AG: Acid and Gas; G: Gas Only; NAG: No Acid, No Gas.
management, faecal deposit and waste dumps also affect
bacterial concentration in run-off water. Faecal
contamination caused by E. coli was dominant. The
pollution caused by human activities includes the
indiscriminate habit of the people in the use of latrines
and siting wells close to the toilets. Bagley and Seidler

Escherichia coli





Enterobacter aerogenes

Staphylococcus aureus

2466 Afr. J. Microbiol. Res.

Table 1. Morphological Characteristics of isolated bacteria.
Colour Size (mm) Surface Shape Elevation Consistency Odour Opacity
IPR1 Pink 3-4 Rough Rod Heaped Mucoid No odour Opaque
IPR2 Pink 3-4 Rough Rod Heaped Mucoid No odour Opaque
ILH1 Pink 2-4 Smooth Rod Slightly Raised Soft Flowery Opaque
ILH2 Pink 2-4 Rough Rod Heaped Mucoid No odour Opaque
SG1 Pink 2-4 Smooth Rod Slightly raised Soft Flowery Opaque
SG2 Pink 2-4 Smooth Rod Slightly raised Soft Flowery Opaque
OGR1 Pink 2-4 Smooth Rod slightly raised Soft Flowery Opaque
OGR2 White 0.2-0.6 Smooth Cocci Raised Soft Flowery Opaque
IKN1 Pink 3-4 Rough Rod Heaped Mucoid Flowery Opaque
IKN2 Pink 2-4 Smooth Rod Slightly raised Soft Flowery Opaque
ILA1 White 4-5 Serrated Rod Flat Soft Flowery Opaque
ILA2 White 4-5 Serrated Rod Flat Soft No odour Opaque
IRO1 Pink 2-4 Smooth Rod Slightly raised Soft Flowery Opaque
IRO2 Pink 4-5 Rough Rod Slightly raised Mucoid Flowery Opaque
ODE1 PINK 3-4 Rough Rod Heaped Mucoid No odour Opaque
ODE2 Creamy 1-1.8 Rough Cocci Raised Mucoid Flowery Opaque
BAB1 Pink 2-4 Smooth Rod Flat Mucoid Flowery Opaque
BAB2 Creamy 0.2-0.6 Smooth Cocci Raised Soft Flowery Opaque

Table 2. Gram reaction and Biochemical tests of Bacteria isolates.

Gram reaction


Methyl red

Voges proskauer







Dnase test

Acid and gas production

IPR 1 GNB – – + – – – – – – – A – Klebsiella sp.
IPR 2 GNB – + – + – – – – – – AG + Klebsiella sp.
ILH 1 GNB + + – – – – – – – – AG + E. coli
ILH 2 GNB – + – + – – – – – – AG + Klebsiella sp.
SG 1 GNB + + – – – – – – – – AG + E. coli
SG2 GNB + + – – – – – – – AG + E. coli
OGR 1 GNB + + – – – – – – – – AG + E. coli
OGR 2 GPC – – – – – – – – – – A – Streptococcus sp.
IKN 1 GNB – + – + – – – – – – AG + Klebsiella
IKN 2 GNB + + + + – – – – – – AG + E. coli
ILA1 GPB – – – – – – – + – + NAG – Clostridium
ILA2 GPB – – – – – – – + – + NAG – Clostridium
IRO1 GNB + + – – – – – – – – AG + E. coli
IRO2 GNB – – + + – – – – – – A + Enterobacter aerogenes
ODE1 GNB – – + – – – – – – – A – Klebsiella sp.
ODE2 GPC – – + – + – – – + – NAG – Staphylococcus aureus
BAB1 GNB – – + + – – – – – + A – Proteus sp.
BAB2 GPC – – – – – – – + – – A – Streptococcus sp. +: Positive; GNB: Gram Negative bacilli; -: Negative; A: Acid only; GPC: Gram Positive cocci; AG: Acid and Gas; GPB: Gram Positive bacilli; NAG: No Acid and Gas.

Aina et al. 2467

Figure 3. Percentage Gram reaction of isolates in water samples. GNB: Gram Negative Bacilli; GPC: Gram Positive Cocci; GPB: Gram Positive Bacilli. Table 3. Estimation of number of coliforms using MPN standard (Monica Cheesbrough, 2006).
Sample code (Volume of Sample in each bottle) Number of bottle used Coliform count using
MPN coliform/100 ml Remark 1 (50 ml) 5 (10 ml)
IPR 1 1 1 3 Satisfactory
IPR2 1 5 TNTC Unsatisfactory
ILH1 0 3 4 Suspicious
ILH2 1 5 TNTC Unsatisfactory
SG1 1 5 TNTC Unsatisfactory
SG2 1 1 3 Satisfactory
OGR1 1 5 TNTC Unsatisfactory
OGR2 1 0 3 Satisfactory
IKN1 1 5 TNTC Unsatisfactory
IKN2 1 5 TNTC Unsatisfactory
ILA1 1 5 11 Unsatisfactory
ILA2 1 3 TNTC Unsatisfactory
IRO1 1 5 103 Unsatisfactory
IRO2 1 5 TNTC Unsatisfactory
ODE1 1 5 TNTC Unsatisfactory
ODE2 1 0 4 Suspicious
BAB1 1 2 6 Suspicious
BAB2 1 3 8 Suspicious

(1976) and Olawuyi (2006), observed that irrespective of
other organisms present in water, only the isolation of
faecal indicator bacteria coupled with appreciable levels
of coliform MPN signifies faecal pollution as an
observation. In the tropics, a fairly high proportion of
coliform organisms in water are often found to be of
faecal origin. Since careful sanitary surveys have been
shown that such water may be free from exposure to
excretal contamination, it is clear that reliance on the
presumptive coliform count will result in unnecessary

Figure 3- Percentage Gram reaction of isolates in water samples

2468 Afr. J. Microbiol. Res.

condemnation of a number of unpolluted water.
Differentiation will generally be necessary and attention
should be mainly to the numbers of faecal coliform by
carrying out confirmed and completed tests. The absence
of evidence indicating faecal contamination does not
necessarily indicate that contamination has not taken
place. There might be no detectable evidence of
contamination as at the time the sample was examined.
Due to this reason, thorough and frequent bacteriological
examination of water samples is desirable with the
inclusion of tests for faecal Streptococcal and some
Clostridium sp. since growth on selective media such as
Blood agar and various biochemical tests carried out had
revealed the presence of Streptococcus sp. and
Clostridium sp. (Table 2).


The need for controls over the quality of water meant for
drinking purposes has been recognized by public health
and environmental officials for many years. The results of
this study demonstrate clearly the presence of
microorganisms in borehole water. Contamination from
faecal origin is the major and prominent source of
contaminant in water samples due to poor method of
faecal waste disposal (Olawuyi, 2006).
The presence of pathogenic organisms and indicator
organisms in some of the water samples renders it unfit
for drinking due to contamination. Water should meet
different quality specifications depending on the particular
use. Potable and domestic water should be harmless to
man. Water quality should be controlled in order to
minimize acute problems of water related diseases,
which are endemic to the health of man. Faecal waste
disposal along the major surface water should be
discouraged. However, the Federal Government of
Nigeria should educate people through the sanitary
control inspectors on the health hazards posed by
indiscriminate faecal waste disposal and they should be
educated on proper waste disposal management. Also,
more borehole systems should be constructed to replace
the shallow hand-dug wells. The result of their industrial
and domestic use after treatment and purification makes
borehole water free from pathogenic organisms. Coliform
bacteria, and in particular Enterobacter, Citrobacter,
Klebsiella and Serratia species, can bring on, as agents
with facultative pathogenicity, a large number of
infections in medical areas with predisposed or
immunodeficient patients. The present state of
knowledge concerning facultative pathogenic significance
is subject to a considerable process of change, with
microbiological research, in particular, resulting in new
insights and assignments of individual species. The
spectrum of nosocomial infections that they bring on
covers, among others: wound infections, catheter-related
infections, pneumonia and septicemia.

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