This study focuses on isolating and identifying bacteria and fungi contaminants from bottled waters sold within the University’s main campus. A sample size of 6bottled water of 6different brands was collected at random and was analyzed microbiologically. The study utilizedthe spread plate technique using commercially prepared Sabouraud’s Dextrose Agar (PDA) forthe isolation of fungi, and the multiple tubefermentation technique with MacConkeybroth and Nutrient agar used as reagents for bacteria isolation. This study revealed that, out of the 6 bottled water samples, fungalspecies were isolated in 5 and bacterial species were isolated in all while 1 was devoid of fungal contaminants. Out of the 6 selected brands, fungi wereisolated in five brands, while bacteria were isolated in all the six brands. Among themicrobes isolatedareAspergillus species and Alternaria alternata. Staphylococcus aureus, Escherichia coli and Bacillus cereus were the predominant isolates. The findings revealed the presenceof coliform and other pathogens in the samples which indicate risk involved inconsumption of such products and therefore could be hazardous to human health.Tosafeguard the health of the consumers, there is need for regular monitoring of theproduction and quality by National Agency for Food and Drug Administration andControl.


Title page

Certification                                                                                                                i

Dedication                                                                                                                  ii

Acknowledgment                                                                                                       iii

Abstract                                                                                                                      iv

Table of contentS                                                                                                        v


Introduction                                                                                                                1

Literature review                                                                                                         4

  • Drinking Water and its Sources             4
  • Accessibility and availability of safe drinking water             6
  • Packaged drinking water                                                                         9


Materials and methods                                                                                                14

  • Study Area                                                                                     14
  • Samples used for the analysis             14
  • Sample Collection 14
  • Experimental Design                                                                                     14
  • Laboratory used for the analysis 14
  • Media used                                                                                                 14
  • Culture Media Preparation for Isolation of Fungi and Bacteria 14
  • Methods for Isolation of Fungi                                                             15
  • Methods for isolation of bacteria 15
  • Incubation of water sample                                     15
  • Subculturing of the colonies             16
  • Preservation of pure cultures 16
  • Identification of Fungi             17
  • Identification of Bacteria 17
  • Coliform identification 18
  • Biochemical tests                                                                         18
  • Sugar fermentation                                                                         21


Results                                                                                                                                    25

Discussion                                                                                                                   38


Conclusion                                                                                                                  40

Recommendation                                                                                                        41

References                                                                                                                  42


  1. Distribution of the fungi in the bottled water 27
  2. Macroscopic identification of fungi on Sabouraud’s Dextrose agar (SDA) 28
  3. Distribution of the bacteria in the bottled water 29
  4. Colour of the isolated organisms on the different agar medium used

for their culture                                                                                                           30

  1. Sugar fermentation of the bacterial isolates 31
  2. Microbial examination of the ICP 32
  3. Microbial examination of the EVR 33
  4. Microbial examination of the EVT                                                                         34
  5. Microbial examination of the IV                                                             35
  6. Microbial examination of the AYK                                                             36
  7. Microbial examination of the RCT 37
  8. Microscopic and biochemical tests results for the identification of the

isolated bacteria                                                                                                          38






Water is one of the most essential commodities for the survival of all lives. It is abundantin nature and occupies about 70% of the earth’s crust (Kalpana et al., 2011). Water is thebiological medium on earth and the only common substance that exists in nature in allthree physical states of matter: solid, liquid and gas. It is the most universally usedsolvent and common route of transmission of diseases.According to World Health Organization statistics,only 36% of Nigerians have access to potable water and 6% have access to improved sanitation (WHO, 2014). An estimated 748 million people all over the world lack access to potable water and close to 2.5 billion persons are not provided with adequate sanitation (WHO, 2014). Water is said to be potable when the physical, chemical and microbiological qualities conform to specified standard. To achieve this, such raw water is subjected to purification processes that range from simple long-term storage to enable sedimentation of some suspended solid to aeration, coagulation, flocculation and disinfection among other treatments (Ajewole, 2005).Water is indispensible to life as it is required for allphysiological processes which demand that all livingorganisms have ready access to water. This need is noless important to human beings who have to drink plenty of water every day (Nester et al., 2004). Apart from drinking,man uses water for many domestic, industrial andrecreational purposes which include washing,bathing, cooking, food processing, brewing andbeverage bottling as well as sporting activities. Thismeans that there is a need for the constant supply ofpotable water to all human communities and in areaswhere such supplies are lacking, a great deal of timeand effort are devoted to finding a suitable source ofsupply. Unfortunately, such water sources even whenthey are available, are seldom safe or reliable andwaters obtained there from need to be treatedappropriately in order to make them potable.

The quality of drinking water is of great concern tomankind, but drinking water supplies have a longhistory of being contaminated by a wide spectrum ofmicrobes including the fecal coliforms (Sadeghi et al., 2007).Contaminated water can cause a spectrum of diseasesranging from self-limiting gastrointestinaldisturbances to severe life-threatening infections (Akoto and Adiyiah, 2007).According to World Health Organization (WHO), 80 %of the diseases in developing countries are either water or sanitation related (Prasanna and Reddy, 2009).Recently, there has been a considerable worldwideincrease in the consumption of bottled water due toconsumer’s awareness regarding bottled water as ahealthy alternative to tap water. However, bottledwater is not necessarily safer than tap water. Manystudies have reported the presence of heterotrophicbacteria along with coliforms in bottled water incounts, exceeding national and international standards (Semerjian, 2011).Over one billion people out of seven do not have access to safedrinking water. Reasonable access to safe drinking water isdefined as the availability of at least 20 litres per person per dayfrom an improved source within 1 kilometre of the user’s dwelling.Approximately 40,000 humans die daily from diseases directlyrelated to consumption of contaminated water. Four thousandchildhood deaths every year worldwide are caused by the use ofcontaminated water (WHO, 2013).Safe and wholesome water has been defined as “water that is freefrom pathogenic agents, free from harmful chemical substances,pleasant to taste and smell”(Maxcy-Rosenau, 1998). In developing countries, such asPakistan, 60% of the population has no access to pure drinkingwater (Khan et al., 2000). Eighty-eight percent of the functional water supplyschemes in Pakistan provide water that is unsafe for drinkingbecause of microbiological contamination (PCRWR, 2012).

The demand for safe drinking water in Nigeria cannot be overemphasized, considering the inability of government to provide adequate pipe borne water to its citizens. Moreover, treated pipe borne water may be contaminated due to poor maintenance of broken and leaking pipes, especially those close to gutter and drainages. Consequently, anumber of small scale water producing industries are packaging and marketing factory filled sachet drinking water popularly called “pure water” that are considered as a safe source of potable water (Dodoo et al., 2006). In all urban areas in the developed countries,reliance is placed on the supply of adequately treatedwater by municipal authorities. In developingcountries however there is little or no access to suchtreated water and so, potable water is usuallydifficult or even impossible to get. This is becausevery little money has been made available for theappropriate municipal infrastructure and this beingso, a large percentage of people in these countrieshave to depend on their own individual efforts to getthe water which they need. One of the means ofsatisfying the need for potable water especially inurban communities is to consume packaged waterwhich in Nigeria is sold in plastic sachets called purewater or in plastic bottles (Omoniyi and Abu, 2012). Bottled water isdrinking water which has been packaged in plasticbottles ranging in size from small single servingpolyethylene terephthalate bottles of 500 ml −1.5 Lcapacity to large carboys (20 L) for water coolers (Reynolds, 2005).In Nigeria, bottled water is regarded as being saferthan water dispensed and sold in sachets, but it isalso about ten times more expensive which is why itis patronized mainly by people with a relatively largedisposable income.A large number of infectious diseases are transmitted primarilythrough water supplies, contaminated with human and animalexcreta (Ilyas et al., 2000). Outbreaks of water borne diseases occur throughoutthe world but are especially common in developing countries (WHO, 1993; Reynolds et al., 2007 and Jones et al., 2007).

The human pathogens, that present serious risk of diseaseswhenever present in the drinking water, include Salmonellaspecies, Shigella species, Yersinia enterocolitica, Campylobacterspecies, various viruses such as Hepatitis A virus, Hepatitis Evirus, Rota virus and parasites like Entamoeba histolytica, Giardialamblia and others (Geldreich, 1992 and Joklik et al., 1992).Apart from microbiological considerations, theupsurge in the demand for bottled water hasprompted the interest of many manufacturers in theproduction of bottled water. Close to two decadesago, bottled water was a product of a fewmultinational and large scale food processing andbeverage producing companies in Nigeria. Presentlyhowever, there is the involvement of very manywater bottling companies ranging from large scalemultinational companies to medium scale businessenterprises, institutional and government businessinvestment companies as well as small scaleentrepreneurs (Babatunde and Biala, 2010). These water bottling companiesuse various water purification methods which may beone of or a combination of two of filtration,ozonisation, ultra violet irradiation and chlorination.Bottled water has been reported to be associated withoutbreaks of infections in the last few years. In 2006,Salmonella enterica serovar Kottbus from bottled waterwas significantly associated with 41 cases in anoutbreak in infants in Gran Canaria.

Aim and Objectives

The aim of this study is to isolate fungi and bacteria contaminants and assess the drinking quality of different types of bottled water marketed within Nnamdi Azikiwe University, Awka.

The specific objectives were to;

  1. Isolate fungi and bacteria from different types of bottled water marketed within Nnamdi Azikiwe University, Awka.
  2. Identify the fungi and bacteria associated with the bottled water marketed within Nnamdi Azikiwe University, Awka.