ISOLATION AND IDENTIFICATION OF BIOFILM FORMING MICROORGANISMS FROM STORAGE WATER CANS IN HOSTELS

ABSTRACT

This study evaluated the isolation and identification of biofilm forming microorganism from storage water cans in MOUAU female hostel. From this study a total of 5 bacterial strains and 3 fungal stains were obtained and identified using morphological characteristics, pigmentation on media, microscopy, biochemical and sugar fermentation methods. This reveals the major bacterial isolates to belong to Escherichia coli, Staphylococcus aureus, Pseudomonas spp, Klebsiella spp and Bacillus species respectively, while the details of the fungal isolates include; Aspergillus spp, Rhizopus  spp and Penicillium spp. The total heterotrophic plate count (THPC) of the storage water can samples ranged from 2.8 × 105cfu/ml to 4.8 × 105cfu/ml The same sequence, the total coliform plate count (TCPC) of the storage water can samples ranged from 3.0 × 105cfu/ml to 4.2 × 105cfu/ml. The total fungal plate count (TFPC) of the storage water can samples ranged from 2.4 × 105cfu/ml to 3.9 × 105cfu/ml. It was observed that Escherichia coli is the most frequently occurring bacteria isolate from the storage water can samples with a percentage occurrence of (33.3%), followed by Staphylococcus aureus with a percentage occurrence of (28.2%), then, Bacillus spp with a percentage occurrence of (17.9%), Pseudomonas spp with a percentage occurrence of (12.8%), whereas Klebsiella spp had the least percentage occurrence of (7.7%). In the same sequence, Aspergillus spp is the most frequently occurring fungal isolate from the storage water can samples with a percentage occurrence of (42.9%), followed by Rhizopus  spp with a percentage occurrence of (33.3%) whereas Penicillium spp has the least percentage occurrence of (23.8%). The rate of Biofilm production by the isolates from the storage water can samples from MOUAU female hostels was found to be 9 representing (23.1%), with 2 representing (22.2%) from the Bacillus spp isolates, 4 representing (44.4%) from the Staphylococcus aureus isolates whereas Escherichia coli with 3 representing (33.3%). The general insanitary conditions at the various hostels, poor hygienic practices by the female students and poor quality of drinking water were the major contributors to the microbial contamination of storage water cans. The presence of these biofilm forming organisms in the storage water can samples poses potential health hazards to the general public because their low numbers do not prevent them from causing diseases.

TABLE OF CONTENTS

Title Page                                                                                                                                 i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgement                                                                                                                  v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          ix

Abstract                                                                                                                                  x

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aims and Objectives                                                                                                  3

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1       Waterborne Diseases                                                                                                  5

2.2       The Microbiological Quality of Water                                                                       6

2.2.1    Heterotrophic plate counts                                                                                          7

2.2.2    Total coliform bacteria                                                                                               8

2.2.3    Faecal coliform bacteria                                                                                             9

2.2.4    Escherichia coli bacteria                                                                                            10

2.2.5    Faecal enterococci bacteria                                                                                        10

2.2.6    Clostridium perfringens bacteria                                                                                11

2.2.7    Bacteriophages                                                                                                           12

2.3       Source Water Supplies                                                                                               12

2.3.1    Water collection from the source water supply                                                          14

2.3.2    Interventions to improve source water supplies                                                         15

2.4       Biofilms in Drinking Water Distribution Systems                                                     15

2.5       Biofilm Prevention and Control in Drinking Water Distribution Systems                       17

2.5.1    Pre-treatment                                                                                                              17

2.5.2    Material selection                                                                                                       18

2.5.3    Hydrodynamics                                                                                                          18

2.5.4    Chemical disinfection and alternative techniques                                                      19

2.5.5    Targeting key microbes                                                                                              20

CHAPTER THREE                                                                         

3.0       Materials and Methods                                                                                               21

3.1       Collection of Samples                                                                                                21

3.2       Sterilization of Materials                                                                                            21

3.3       Preparation of Culture Media                                                                                     21

3.4       Inoculation and Isolation                                                                                            22

3.5       Purification of Isolates                                                                                               22

3.6       Identification of the Isolates                                                                                       22

3.7       Gram Staining                                                                                                            22

3.8       Biochemical Test                                                                                                        23

3.8.1    Catalase Test                                                                                                               23

3.8.2    Indole Test                                                                                                                  23

3.8.3    Citrate Utilization Test                                                                                               23

3.8.4    Hydrogen Sulphide (H₂S) Production Test                                                                24

3.8.5    Starch Hydrolysis                                                                                                       24

3.8.6    Motility, Indole, Urease (MIU)                                                                                  24

3.8.7    Coagulase Test                                                                                                           25

3.8.8    Oxidase Test                                                                                                               25

3.9       Detection of Biofilm by Congo Red Agar (CRA) Method                                        25

CHAPTER FOUR

4.0       Results                                                                                                                        27

4.1       Total viable microbial count from storage water can samples                                 27

4.2       Morphological identification, Biochemical Identification, Gram Reaction and  

Sugar Utilization Profile of bacterial isolates from storage water can samples     27

4.3       Cultural Morphology and Microscopic Characteristics of the Fungal Isolates           

From storage water can samples                                                                                27

4.4       Percentage occurrence and distribution of the bacteria isolates from storage

Water can samples                                                                                                      28

4.5       Percentage occurrence of the fungal isolates from storage water can samples     28

4.6       Detection of Biofilm formation by the bacterial isolates from storage water  

Can samples by Conga red agar method                                                                    28

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                                        35

5.1       Discussion                                                                                                                   35

5.2       Conclusion                                                                                                                  37

5.3       Recommendation                                                                                                       38

References                                                                                                                  39

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

Water is a transparent, colourles, odourless and tasteless liquid that makes up the sea, lakes, rivers, rainfall as well as the liquid that makes up living organisms (Michael, 2000). Water is a compound of two elements; hydrogen and oxygen atoms with a chemical formula H₂O and it is known to make up about 70 percent of the earth surface. Rivers, streams, wells and more recently boreholes, serve as the main source of drinking water and domestic use in developing countries like Nigeria, where most of the people reside in rural areas (Ibe and Okplenya, 2005). According to the World Health Organisation guidelines for drinking water underground water supplies are usually considered safe provided they are properly located, constructed and operated to WHO regulatory standards. Boreholes with hand pumps are commonly used by poor rural communities and this amounts to approximately 250,000 hand pumps in Africa. Studies have shown that water may become contaminated at any point between collection, storage and usage (Tambekar et al., 2006). Also, storing water in plastic containers and handling procedures of water at homes, hotels or restaurants causes’ water quality deterioration to such extent that it becomes potential risk of infection to consumers. Microorganisms associated with contaminated water includes; Salmonella sp, Escherichia coli and Vibro cholera. Water borne diseases often arises when pathogenic microorganisms associated with contaminated water is consumed. Boreholes and wells are polluted industrially, domestically or agriculturally. Industrial pollution may involve seepages of used water containing chemicals such as metals and radioactive compounds while domestic pollution may involve seepage from broken septic tanks, pit latrines and privies. Runoff water after rainfall carrying pesticides, fertilizers, herbicides and faecal matter may contribute to agricultural pollution. However the pollution sources, the quality of packaging plastic bottle cannot guarantee safety from contamination. In the natural environment there are compound that have the potential to disturb equilibrium in living organisms and are mistakenly recognized by oestrogen receptors, treated the same as those naturally present in the organism. Substances of this type are known as Endocrine Disrupting chemicals. Bisphenol A is one of the highest volume chemicals produced worldwide with more than 6million pounds produced each year (Burridge, 2003). It serves as a base line in the manufacturing of plastics and a major compound in the production of epoxy resins, printers ink, powdered paints, dental sealants and composites (Vandenberg et al ., 2009; Markey et al., 2003). Bisphenol A can be released and leached into water from packaging materials. Hence, through consumption due to it use in packaging and storage containers, consumers are directly or chronically exposed to BPA. Several health cases have been attributed to bisphenol A and studies have been carried out on the chemical component at different scales. Some researchers have focused on the detection and measurement of bisphenol A whiles others on the effects on humans and laboratory animals (Chang et al., 2009).

Furthermore, biofilm formation on the inner surface of storage vessels has been reported to offer a suitable medium for the growth of microorganisms and consequently to contribute to the deterioration of drinking water quality in homes (Vander-Merwe et al., 2013). Studies have shown high counts of heterotrophic bacteria and faecal coliforms in stored drinking water, which by far exceeded the limits set for human consumption. Given the right conditions, a small number of microorganisms in water may also provide a seed, which will allow them to multiply in the storage containers. A study by Momba and Kaleni (2002) has shown that microorganisms attached on the surface wall of such containers during storage multiplied at the expense of low concentrations of carbon in water. These authors found a direct relationship between the degree of bacterial re-growth on drinking water storage vessels and the storage materials and the quality of the intake water [(such as temperature, turbidity and concentrations of organic nutrient (dissolved organic carbon–DOC)]. A comparison of the quality of drinking water stored in polyethylene (PE) and galvanized steel (GS) containers by the rural communities of South Africa revealed a higher re-growth of total coliforms on PE than on GS containers as the former contained higher DOC concentrations than the latter (Momba and Kaleni, 2002). However, plastic-based materials are the most widely used for water storage containers in the developing world as rural communities can afford these products. There is therefore a need to improve the quality of drinking water stored in these plastic-based vessels.

Deterioration of drinking water quality during storage is one of the major difficulties not only experienced in decentralised systems, but also by potable water suppliers in centralised systems. The most alarming situation is the occurrence of pathogenic and opportunistic bacteria such as Legionella spp., Pseudomonas, Mycobacterium, Campylobacter, Klebsiella, Aeromonas, Helicobacter pylori, Salmonella and E. coli within biofilms. It is therefore important for suppliers of home drinking water treatment technologies to make sure that the technologies provide safe drinking water after treatment and during storage in homes. Although various systems and devices have been extensively reported to improve the quality of water and the health of the population, scant attention has been given to the potential sustainability of these devices and very little guidance is available in terms of maintenance to prevent the development of biofilms during storage. Taking into consideration the deterioration of the stored water and the health risk associated with pathogenic microorganisms within this type of water, this study investigated the efficiency of the household drinking water treatment technologies developed by the Tshwane University of Technology (TUT) in inhibiting the growth of biofilms on the most widely used water storage container prior to their deployment in South African rural communities. We were also able to provide information regarding their potential sustainability as well as guidance in terms of their maintenance.

1.1       AIM AND OBJECTIVES

The aim of this study is to isolate and identify biofilm forming microorganisms from storage water cans in MOUAU female hostel

The objectives are;

1.     To isolate and characterize the isolates from the storage water cans in MOUAU female hostel

2.     To determine the percentage occurrence of the isolates from the storage water cans

3.     To determine biofilm formation among the isolates from the storage water cans samples

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