STUDIES ON THE AIR MICROFLORA OF FIVE EATRIES IN THE

UNIVERSITY OF ILORIN, ILORIN

BY

ABOYEJI, CHRISTIANAH ENIOLA

MATRIC NO: 18/55EJ011

A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF

MICROBIOLOGY, UNIVERSITY OF ILORIN, ILORIN, KWARA STATE,

NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

BACHELOR OF SCIENCE (B.Sc.) (HONS) DEGREE IN MICROBIOLOGY.

JULY, 2023.

i
 DECLARATION

I hereby declare that this project is my original work and is based on my research findings.

All errors of omission in this work are entirely mine and all materials consulted in the

course of this research work were duly acknowledged.

------------------------------- ---------------------------

ABOYEJI CHRISTIANAH ENIOLA DATE

ii
 CERTIFICATION

This is to certify that this project was carried out by Aboyeji Christianah Eniola with the

matric number 18/55EJ011 under my supervision and it is a fair reflection of the students

input.

………………………………… …………………………….

Dr. D. O. ADETITUN DATE

(SUPERVISOR)

………………………………… ………………………………

Dr. SALIU DATE

HEAD OF DEPARTMENT

………………………………….. ………………………………..

EXTERNAL EXAMINER DATE

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 DEDICATION

This project work is dedicated to God Almighty for seeing me through this far, my ever

loving Grandfather, HRH Oba G.K. Aboyeji for his undying love and support for me and

my Parents Dr. & Mrs. Aboyeji for their love support and understanding and for being a

great source of support in all my life endeavors.

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 ACKNOWLEDGEMENT

I would like to express my sincere gratitude to several individuals and organizations for

supporting me throughout my undergraduate study. First, I wish to express my sincere

gratitude to my supervisor, Dr. D.O. Adetitun, for his enthusiasm, patience, insightful

comments, helpful information, practical advice and unceasing ideas that have helped me

tremendously at all times in my research and writing of this project. His immense

knowledge, profound experience and professional expertise in Environmental

Microbiology has enabled me to complete this research successfully. Without his support

and guidance, this project would not have been possible. I could not have imagined having

a better supervisor in my study.

I am also grateful to my grandparents HRH & Olori Aboyeji and my parents, Mr. and Mrs.

Aboyeji for their consistent support, prayers and assistance and also to my big sis,

Oyeladun Aboyeji for being there anytime I needed her. Also, I’m sincerely grateful to

Omoyele Alex for his undying love, support and assistance over the years.

Finally, last but by no means least; also, to everyone in the Department of Microbiology,

it was great sharing premises with all of you during last four years.

Thanks for all your encouragement!

God bless us all!!!

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 TABLE OF CONTENT

Title page……………………………………………………………………………….i

Declaration……………………………………………………………………………...ii

Certification…………………………………………………………………………….iii

Dedication……………………………………………………………………………..iv

Acknowledgement……………………………………………………………………..v

Table of contents………………………………………………………………………vi

Abstract……………………………………………………………………………….x

CHAPTER ONE

1.0: Introduction…………………………………………………………………… 1

CHAPTER TWO

2.0: Sampling area…………………………………………………………........... 17

2.1: Sterilization of glassware and other materials……………………………….. 17

2.2: Preparation of media……………………………………………………….… 17

2.2.1: Preparation of nutrient agar………………………………………………… 18

2.2.2: Preparation of potato dextrose agar………………………………………… 18

2.3: Air sampling procedure……………………………………………………….. 19

2.4: Isolation and preservation of pure cultures……………………………………. 20

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2.4.1: Preparation of culture media for stock culture……………………………….20

2.4.2: Subculturing of bacterial colonies…………………………………………….20

2.4.3: Subculturing of fungal colonies………………………………………………..21

2.5.0: Characterization and identification of isolates……………………………........21

2.5.1: Characterization of bacterial isolates……………………………………….…..21

2.5.2: Gram staining………………………………………………………………...…22

2.5.3: Spore staining……………………………………………………………………23

2.5.4: Capsule test………………………………………………………………………24

2.5.5: Oxidase test…………………………………………………………….……..…24

2.5.6: Citrate test……………………………………………………………………… 25

2.5.7: Indole test………………………………………………………………………..25

2.5.8: Coagulase test……………………………………………………………………26

2..5.9: Triple Sugar Ion (TSI) test………………………………………………………26

2.6.0: Methyl Red and Vogues Proskauer test ………………………………………....27

2.6.1: Oxygen Relationship test...……………………………………..…………..…....28

2.6.2: Motility test ……………………………………………………………………..28

2.6.3: Catalase test……………………………………………………………….……..29

2.6.4: Sugar fermentation test……………………………………………………….…29

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2.6.5: Oil displacement test……………………………………………………………30

2.6.6: Haemolysis test…………………………………………………………………31

2.6.7: Drop collapse test………………………………………………………………31

2.7.0: Characterization and Identification of fungal isolates………………………….32

2.7.1: Characterization of fungal isolates.......................................................................32

2.7.2: Identification of fungal isolates………………………………………………….33

CHAPTER THREE

RESULTS

3.0: Bacterial isolates……………………………………………………………………34

3.1: Fungal Isolates………………………………………………………………………41

CHAPTER FOUR

Discussion………………………………………………………………………………53

CONCLUSION AND RECOMMENDATION

5.0: Conclusion…......................………………………………….................................70

5.1: Recommendation………………………………………………………………….70

REFERENCES………………………………………………………………………..72

APPENDIX…………………………………………………………………………....79

LIST OF TABLES

viii
Table 1………………………………………………………………………35

Table 2………………………………………………………………………36

Table 3……………………………………………………………………….37

Table 4………………………………………………………………………..38

Table 5………………………………………………………………………..39

Table 6…………………………………………………………………………43

Table 7………………………………………………………………………….44

Table 8………………………………………………………………………….45

LIST OF FIGURE

Figure 1………….……………………………………………………………..48

Figure 2………………………………………………………………………...49

Figure 3…………………………………………………………………………50

Figure 4…………………………………………………………………………51

Figure 5…………………………………………………………………………52

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 ABSTRACT

It is imperative to carry out microbiological analysis of indoor air in selected restaurants

located at University of Ilorin. Air samples were collected in duplicates using the open

plate method. Nutrient agar (NA) was used for the enumeration of total bacterial

concentrations while Potato dextrose agar (PDA) was used for the enumeration of total

fungal concentrations. The enumeration was been carried out for 5 days with the

concentration of bacteria in the selected locations ranging from 1667cfu/m³ to

107022cfu/m³ while the concentration of the fungi ranges from 4246cfu/m³ to

5819cfu/m³. The colony shape, surface edge, elevation, consistency, optical character and

cellular shape of the isolates was examined visibly. Stock culture was done for the

isolates for further biochemical test. The fungal isolates were identified based on their

morphological structure. A total of 10 bacteria and 5 fungal species were isolated at

varying frequency of distribution. Results revealed the presence of Bacillus,

Micrococcus, Staphylococcus, Streptococcus and Pseudomanas strains in the sampled

air, indicative of their prevalence in the eatery environment with Bacillus sp being the

highly occurring bacteria in all the sampling site being present for everyday of the

sampling period and Pseudomonas aeruginosa being the least. Presence of Rhizopus sp,

Aspergillus sp and Trichoderma sp were also sampled from the eateries air with

Trichoderma sp being the most prevalent and Rhizopus sp being the least occurred. This

x
study provides valuable insights into the airborne microbial community in eatery settings,

contributing to a better understanding of potential biosecurity and health implication.

xi
 CHAPTER ONE

INTRODUCTION

Nestled in the heart of Kwara State, Nigeria, the University of Ilorin stands as a towering

bastion of academic excellence and social vitality. Established in 1975, this institution has

burgeoned into a dynamic center for intellectual pursuits, cultural diversity, and vibrant

community interactions. Its sprawling campus, characterized by architectural splendors

and lush landscapes, serves as an epicenter where knowledge converges with camaraderie

(Ekanade et al., 2018). Eateries, in this academic microcosm, occupy a pivotal role. More

than just eateries, these are vibrant gathering places where students, faculty, staff,

and visitors not only dine but also foster connections and ideas.

Recognizing the prominence of these eateries unveils a profound truth—their significance

extends far beyond the culinary realm. These are spaces where academic discourse mingles

with laughter, where friendships are forged, and where the tapestry of campus life is woven.

Consequently, ensuring a healthy indoor environment within these eateries assumes

paramount importance, as it reverberates throughout the campus community, impacting the

well-being of its denizens (Zhang et al., 2016). This study embarks on an exploration of a

vital facet of these campus establishments—the quality of indoor air. Specifically, it

ventures into the intricate domain of air microflora, seeking to unravel its complex tapestry

and unveil its profound implications for indoor air quality.

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Air is an essential human need and contains many different particles and microorganisms,

so humans inhale many airborne particles when breathing in any environment. Of all the

environment, air is the simplest and it occurs in a single-phase gas. Unlike soil and water,

air does not contain the nutrients necessary for microorganisms to grow, it acts as a

medium to transfer microbial germs. Air quality is one of the most significant factor

affecting the health and wellbeing of people. It has been reported that a single person

inhales approximately 10m3 of air every day (Dacarro et.al, 2003). The atmosphere

consists of different components which enhances the survival of microorganisms in the air.

The relative quantities of various gases in air by volume percentage are:

i. Nitrogen – 78%

ii. Oxygen – 21%

iii. Argon – 0.9%

iv. Carbon dioxide – 0.03%

v. Hydrogen – 0.01%

vi. Other gases in trace amounts

Various layers can be recognized in the atmosphere up to a height of about 1000km. The

layer nearest to the earth is known as troposphere. In temperate regions, troposphere

extends up to about 11km where as in tropics up to 16km, troposphere is characterized by

a heavy load of microorganism

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In addition to gases, dust particles and water vapour. Air also contains microorganisms.

Microorganisms that make up the majority of atmospheric biomass have been found in

most environments, surviving and growing in extreme conditions such as heat, cold,

radiation, pressure, salinity, acidity and darkness. Air is not a natural environment for

microorganisms as it doesn’t contain enough moisture and nutrients to support their growth

and reproduction. Since air is often exposed to sunlight, it has a higher temperature and

lesser moisture. If not protected from desiccation, most of these microbial forms die.

Bacteria in the air often come from the activity of soil, water and living organisms. The

air micro biome is dynamic and is affected by temperature, wind speed, humidity,

pollution, and other human and animal activities. They are of great importance because

their presence in the air can have adverse effects on the health of people and cause

degradation, promote composting, biodegradation, etc. Air is mainly a transport medium

or dispersal medium for microorganisms. They occur in relatively small numbers in air

when compared with soil or water. Invisible to the naked eye, these microorganisms

include bacteria, viruses, fungi, and even some micro-animals. They are transported

through the atmosphere by wind currents, dust particles, and aerosols. Understanding their

presence and activity is essential as they can impact climate, agriculture, human health,

and ecosystems. Air movement aids in the dispersal and transport of particles and

microorganisms. The airborne microorganisms that raise public health hazards are those

that are causative agents of infectious disease and allergies (Burge and Hoyer, 1990).

Microorganisms abound in the earth’s atmosphere as a particle or bacteria, fungi, lichen

3
and algal cell. The composition and concentration of these particles are generally related

to man activities (Lacey and West, 2006). Airborne microorganisms typically originate

from a variety of natural sources, including soil, animals, human activities such

as wastewater treatment, plant and animal utilization, fermentation processes, and

agricultural activities, all of which release microorganisms into the air. . Several

studies have identified human activities such as talking, sneezing, and coughing as sources

of microorganisms in the air, whereas other human activities such as vehicular

transportation and human movements, washing in homes and business centers, flushing of

toilets and sewages, and sweeping of floors and road sides can indirectly generate bio

aerosols. Since microorganisms can lodge in or on dust particles, dust therefore is a

potential source of bioaerosols (Chen and Hildermann, 2009).

Bio aerosol is a colloidal suspension, formed by droplets and particles of solid matter in

the air, whose components can contain or have attached to them viruses, fungal spores and

conidia, bacterial endospores, plant pollen and fragments of plant tissues (Karwowska,

2005). Biological contamination of air is mostly caused by bacteria, moulds and yeasts

(Flannigan, 2001; Daisey et al., 2003; Pieckova and Kunova, 2002). Bioaerosols enter the

human body by a variety of routes (inhalation, ingestion, or skin absorption) and cause a

variety of health impacts such as communicable infections, acute toxic effects, allergies,

and cancer. Inhalation is the most common way for these germs to enter the body.

Respiratory infection and reduced lung function are created by health effects caused by bio

aerosols (Haliki-Uztan et. al., 2010). Unlike chemicals, exposure to bio aerosols does not

4
have any health threshold due to the type of microorganisms, entrance way, and difference

in individual immune response (Mandal and Brandl. 2011). Although several studies have

been performed to determine the air born bacteria of indoor and outdoor (Balasubramanian

et. al., 2012, Faridi et. al., 2015), few of those have evaluated indoor and outdoor Air born

bacteria in the kitchen of eateries. Nowadays, food preparation is occasional in our lifetime

and people have to pass a significant part of their time in eateries. Therefore, good air

quality in restaurants not only protects people's health, but also enhances their enjoyment

of food.

Indoor Air Quality (IAQ) stands as a fundamental determinant of human health,

influencing our well-being in profound ways within the confines of indoor environments.

The spaces where we live, work, and spend much of our lives are not just physical

constructs but dynamic ecosystems where the air we breathe carries an array of factors that

can significantly affect our health and overall quality of life (Mendell et al., 2011).

IAQ doesn't concern itself with the mere presence of breathable air; rather, it delves into

the intricate interplay of elements that collectively determine the quality of air within

enclosed spaces. In this context, indoor air quality has never been more important

to human health, from chemicals such as volatile organic compounds (VOCs) emitted

by furniture and cleaning products to mold spores, bacteria, and more. It contains a

wide range of contaminants, ranging from, to biological substances such as

viruses.

5
Bacterial communities within indoor air are particularly noteworthy due to their direct

relevance to human health. Indoor air can host diverse bacterial species, and their presence

can exert a profound impact on the well-being of occupants. Certain bacteria are known to

be pathogenic and can cause respiratory infections, making them a notable health risk,

particularly in enclosed spaces where people congregate (Adams & Pasut, 2019).

The implications of poor IAQ extend beyond the realm of bacteria to encompass fungi,

including molds. Mold spores, ubiquitous in the environment, can proliferate within indoor

spaces under favorable conditions. As molds grow, they release allergenic particles and

mycotoxins, which, when inhaled or come into contact with occupants, can lead to a

spectrum of health issues, ranging from allergies and asthma exacerbation to more severe

respiratory conditions (Adams & Pasut, 2019).

The health risks associated with subpar IAQ are both insidious and multifaceted.

Individuals exposed to indoor air pollutants may experience immediate discomfort,

including symptoms such as headaches, fatigue, respiratory irritation, and skin problems

(Sundell et al., 2011). However, the long-term consequences are equally concerning.

Chronic exposure to poor IAQ can contribute to the development and exacerbation of

various health conditions, including chronic respiratory diseases, cardiovascular problems,

and even cognitive decline (Mendell et al., 2011).

Furthermore, the ramifications of IAQ extend beyond individual health to encompass

broader public health concerns. In densely populated indoor spaces such as schools, offices,

6
and healthcare facilities, the risk of disease transmission increases when IAQ is

compromised. Airborne diseases can spread quickly in closed environments, affecting not

only those nearby but also the surrounding community.

Addressing the complex relationship between IAQ and health necessitates a multifaceted

approach. Comprehensive IAQ assessment involves not only identifying and quantifying

pollutants but also considering factors such as ventilation, humidity, and temperature

(Mendell et al., 2011). Adequate ventilation, for instance, helps dilute indoor air pollutants,

reducing the risk of exposure and mitigating health risks.

The inextricable bond between indoor air quality and human health is a cardinal truth

(Mendell et al., 2011). The enclosed indoor environment, where individuals spend most

of their lives, has an enormous influence on their physical health and mental harmony.

These enclosed spaces can harbor an arsenal of pollutants, ranging from insidious chemical

contaminants to the subtle machinations of biological agents (Adams & Pasut, 2019).

Among these biological agents, microbial entities take center stage. These microscopic life

forms, including bacteria, fungi, and viruses, exert a formidable influence on indoor air

quality (Jayaprakash et al., 2017).

The repercussions of subpar indoor air quality are multifold and insidious. They encompass

a spectrum of health risks, from the stealthy propagation of respiratory infections to the

exacerbation of allergies and the aggravation of pre-existing chronic conditions (Sundell

7
et al., 2011). The implications, however, transcend the individual, extending to encompass

broader concerns of public health within the context of a bustling university campus.

Within this ecosystem, eateries serve as crucibles where students, faculty, and visitors

converge in close proximity. Therefore, an urgent need to ensure optimal indoor air

quality in these facilities is emerging, potentially shaping collective well-being.

Addressing the complex relationship between IAQ and health necessitates a multifaceted

approach. Comprehensive IAQ assessment involves not only identifying and quantifying

pollutants but also considering factors such as ventilation, humidity, and temperature

(Mendell et al., 2011). Adequate ventilation, for instance, helps dilute indoor air pollutants,

reducing the risk of exposure and mitigating health risks.

In conclusion, IAQ is far from a passive component of indoor environments; it is a dynamic

and influential factor that significantly shapes human health. Recognizing the intricate

relationship between IAQ and health is imperative for creating healthier indoor spaces,

safeguarding individual well-being, and addressing broader public health concerns. By

understanding the complex interplay of pollutants and environmental factors within indoor

environments, we embark on a journey toward healthier living and working spaces for all.

Within the intricate mosaic of indoor environments, microbial contaminants assume a

prominent role, wielding a profound influence on indoor air quality (Adams & Pasut,

2019). These microorganisms, representing many life forms of bacteria, fungi and

viruses, reside in the air we breathe and exist in a state of dynamic equilibrium. Their

8
presence is not static; rather, it is formed by a complex interaction of factors, including

human activity, environmental conditions, and ventilation system performance.

Eateries, characterized by diverse activities including food preparation, diverse human

interactions, and complex HVAC systems, serve as hubs of microbial dynamics. The

patrons, as they come together for meals and conversations, unwittingly engage with an

invisible realm of microflora. Understanding the origins, dynamics, and impacts of

microbial contaminants in these environments becomes imperative as they provide the

foundation upon which campus hygiene, public health, and health eating experience.

Indoor air, often taken for granted, is a complex ecosystem that houses a diverse

community of microorganisms. These microscopic inhabitants, both benign and potentially

harmful, subtly influence the quality of the air we breathe within enclosed spaces. This

exploration dives into the world of microbial contaminants in indoor air, revealing the

hidden influences that shape our living and working environments. Microbial contaminants

within indoor air encompass an array of microorganisms, including bacteria, fungi, viruses,

and allergenic mites (Adams & Pasut, 2019). These entities exist ubiquitously, forming an

intricate ecosystem within the very air that fills our homes, offices, and public

buildings.The sources of these microorganisms are diverse, from the intrusion of

outside air to humans and their daily activities.

Bacteria, though often overlooked, are prominent members of this microbial community,

silently shaping the indoor air quality (Jayaprakash et al., 2017). They are introduced

9
through various pathways, including human occupants who shed skin-associated bacteria

and outdoor air that carries environmental bacteria (Adams & Pasut, 2019). These bacteria

may not always be pathogenic, but their presence can influence the microbial balance

within indoor spaces.

Fungi, including molds, represent another facet of indoor air microflora. Mold spores, often

invisible to the naked eye, can proliferate in environments with favorable conditions. When

mold growth occurs, it can release allergenic particles and mycotoxins, potentially leading

to health issues ranging from allergies to respiratory problems (Adams & Pasut, 2019).

Viruses, although less frequently considered in discussions of indoor air quality, can also

play a role in indoor environments. The transmission of respiratory viruses, such as

influenza and COVID-19, can occur in enclosed spaces, especially when the space is

overcrowded. Proper ventilation and air filtration are crucial in mitigating the risk of

airborne viral transmission.

The consequences of inadequate indoor air quality, influenced by microbial contaminants,

are far from trivial. Subpar indoor air can contribute to health issues, including respiratory

irritation, allergies, and discomfort (Sundell et al., 2011). Chronic exposure to poor indoor

air quality may lead to more severe conditions, such as chronic respiratory diseases,

cardiovascular problems, and cognitive decline (Mendell et al., 2011).

Beyond individual health, the implications of microbial contaminants extend to broader

public health concerns. In crowded indoor spaces like schools, offices, and healthcare

10
facilities, the risk of disease transmission escalates when indoor air quality is compromised

. Controlling the spread of airborne diseases within such environments becomes

paramount.

while microbial contaminants within indoor air often go unnoticed, their influence on

indoor air quality and human health is undeniable. Bacteria, fungi, viruses, and other

microorganisms coexist within the air we breathe, shaping our indoor environments in

subtle yet impactful ways. Recognizing the importance of maintaining a healthy indoor

microbial balance is essential for safeguarding both individual well-being and public

health. By understanding the complex interplay of microorganisms within enclosed spaces,

we take a step toward creating healthier and safer indoor environments for everyone.

In this intricate milieu, this study embarks on a mission of paramount significance—to

investigate the air microflora in five carefully selected eateries nestled within the

University of Ilorin campus. This endeavor holds immense relevance in the context of

campus hygiene, public health, and the holistic well-being of the campus community

(Sundell et al., 2011). Beyond its immediate impact, it has the potential to ripple across

academic halls and have broader implications in the field of indoor air quality research.

In the search for energy efficiency, more self-contained or airtight, energy-efficient

homes that reduce cooling and heating costs are built. However, there is reason to be

concerned about the indoor air quality of this self-contained building with adequate

ventilation. Fresh air is an essential condition for healthy living and lifestyle. The

11
quality of air in homes, offices, eateries, schools, day care centres, public buildings, health

care facilities and other private and public buildings where people spend over 80-90% of

their time daily is crucial for healthy living and people’s well-being (Quarcoo et.al., 2012).

The National Health and Medical Research Council (NHMRC) defines indoor air as air

within a building occupied by people of varying states for a period of at least one hour

(NHMRC, 1996). Buildings covered by this definition include homes, schools, eateries,

public buildings, residential institutions, offices, etc. Eateries have extensively evolved to

become controlled environments with sophisticated ventilating and airconditioning

systems. However, little is known about IAQ of these modern eateries. In recent years,

eateries have not been extensively studied compared to other equally important indoor

environments in terms of time spent by the population, such as dwellings, office and

schools (Gobo et.al., 2009). Humans need a regular supply of food, water and essentially

a constant supply of air because they spend most of their time breathing air in an

enclosed space where there are many sources of pollution. This is an important risk

factor for the health of the general population.To avoid poisoning, illness and even death,

good hygiene practices such as hand washing, maintaining general cleanliness and being

aware of the dangers of crosscontamination between raw and cooked foods are essential

for food preparation, not only in the food industry but also the domestic setting (Johnson,

2016).

The University of Ilorin campus is a microcosm of academic excellence and cultural

diversity. Established in 1975, it has evolved into not just an academic institution but a

12
dynamic community where ideas flourish, friendships are nurtured, and Nigerian culture is

celebrated (Ekanade et al., 2018). This unique blend of intellectual vitality and cultural

richness makes the campus an ideal setting for exploring the hidden world of air microflora

within its eateries. These eateries are more than places to grab a meal; they serve as vibrant

social spaces where academic and social worlds intersect. Students gather for nourishment,

discussions, and collaborative study sessions. Professors engage in informal conversations

with their students, fostering mentorship and camaraderie. Visitors to the campus

experience the warmth of Nigerian hospitality through the diverse culinary offerings

available in these eateries.

However, amid the liveliness of these spaces lies an invisible world - the air microflora.

Comprising bacteria, fungi, viruses, and other microorganisms, this microbial community

inhabits the air within eateries, often unnoticed by those who frequent them. These

microorganisms, while unseen, have the potential to significantly impact the health and

well-being of individuals within these spaces. Understanding the composition and

dynamics of the air microflora in eateries is of paramount importance. One key aspect

pertains to hygiene and health. Microbes present in the air can influence food safety,

potentially impacting both the staff who prepare meals and the patrons who consume them.

Bacterial contamination, for instance, can pose risks to food quality and safety (Sapkota et

al., 2020). Fungal spores, on the other hand, may contribute to allergenic reactions when

inhaled, potentially affecting both employees and diners (Adams & Pasut, 2019).

13
Moreover, in the context of the ongoing COVID-19 pandemic and concerns over airborne

transmission of respiratory diseases, understanding the microbial landscape of indoor

spaces like eateries gains heightened relevance (Morawska & Cao, 2020). The presence of

respiratory viruses in indoor air can have direct implications for public health and safety.

This study's significance transcends the academic realm, touching upon the daily lives of

students, staff, and visitors to the University of Ilorin campus. By shedding light on the

hidden microbial world within eateries, it contributes to the enhancement of campus

hygiene, food safety, and public health. The findings from this research may not only

benefit the campus community but also provide insights and guidelines applicable to a

broader context, particularly in the context of indoor air quality and microbial

contamination in public spaces.

To contextualize the findings, the study aims to compare the data collected in the eateries

to established indoor air quality standards or guidelines. Such comparisons help assess

whether the microbial concentrations and overall air quality meet recognized health and

safety standards. Any deviations from these standards may indicate areas that require

attention and improvement.

The salience of this study lies in its capacity to unravel the enigma of air microflora within

eateries. By peering into this microbial tapestry, we not only gain insights into the

intricacies of indoor air quality within a bustling campus but also pave the way for

interventions and recommendations that can reverberate throughout the domain of public

14
spaces and indoor environments. Ultimately, based on the research findings, this study aims

to provide practical recommendations for enhancing indoor air quality and mitigating

microbial contamination within the selected eateries. These recommendations may

encompass strategies related to ventilation, hygiene practices, and facility management.

The goal is to contribute valuable insights into the microbial realities of campus eateries

and promote improvements in campus hygiene, public health, and the well-being of the

campus community.

These objectives collectively form a comprehensive approach to understanding and

addressing the microbial landscape within eateries on the University of Ilorin campus. The

study seeks to unravel the hidden world of air microflora and translate its findings into

actionable recommendations for a healthier dining environment. Achieving these

ambitious objectives necessitates a robust research methodology. The study employs a

multifaceted approach, encompassing air sampling, microbial identification through state-

of-the-art techniques, and a rigorous data analysis protocol (Meadow et al., 2015). The

foundation of the study's validity lies in the selection of five representative eateries,

meticulously chosen on the basis of their diversity and relevance within the University of

Ilorin campus. Studies for assessing the indoor air quality of eateries are required for

evaluating the potential health risks, establishing guidelines, and advocating possible

control measures for ensuring the healthy workplace environment (Zhao i 2010) Although

studies dealing with eateries’ air quality are scarce in the literature, few of such studies

have been carried out in different parts of the world which indicated a serious indoor air

15
quality problem, particularly in developing countries ( Zhao et.al., 2014). In conclusion,

this introduction serves as the vanguard of a journey into the concealed world of indoor air

quality within eateries. It has laid the foundation upon which the subsequent chapters are

constructed, and it has illuminated the path toward a holistic comprehension of air

microflora in a campus setting.

In the chapters that ensue, we traverse the realms of microbiology, environmental science,

and public health, seeking to unearth the nuances that govern the air we breathe as we dine

and converse within the eateries of the University of Ilorin.

16
 CHAPTER TWO

MATERIALS AND METHODS

2.0: Description of Sampling site.

Five eateries were chosen to be researched in various regions of University of Ilorin

Campus (permanent site). Eateries were chosen because they are public spaces where

various individuals are served.

2.1: Sterilization of glass wares and other materials.

Conical flasks, flavor bottles, beakers and other glass wares used were thoroughly washed,

air dried, wrapped with aluminum foil and sterilized in the hot air oven at 1600c for 1hour.

The petri dishes used were disposable plastic types which had already been sterilized from

the manufacturing industry. The culture media used were sterilized in the autoclave at

1210c for 15 minutes. The sterilization of inoculating needles and inoculating loops was

done by flaming till they are red hot. Disinfection of the work bench was done by swabbing

with cotton wool soaked in 70% ethanol. All sub culturing and inoculation were done in

the inoculating chamber to avoid contamination. The inoculating chamber was sterilized

with ultra violet lamp.

2.2: Preparation of Culture media.

Two different culture media were used for the isolation of the air micro flora from the study

sites. Nutrient agar and Potato dextrose agar were used for the isolation of bacteria and

17
fungi respectively. The preparation of the media was done according to the manufacturer’s

instruction.

2.2.1: Preparation of Nutrient agar.

According to the manufacturer’s instruction, 28g of dehydrated nutrient agar should be

dissolved in 1000mls of distilled water. Aluminum foil was used to measure 7g of

dehydrated nutrient agar on a weighing balance and dissolved in 250mls of distilled water

in a 250mls conical flask, the medium was stirred to dissolve the agar and was allowed to

properly homogenize by heating. After heating, the conical flask was corked with a cotton

wool and wrapped with aluminum foil and ready for sterilization.

The medium was sterilized in an autoclave at a temperature of 1210c for 15 minutes after

which the medium was brought out of the autoclave and allowed to cool down to about

450c before pouring into the sterile petri dishes aseptically. After pouring into the petri

dishes, the plates were allowed to cool and solidify before labelling the plates, the needed

plates for sampling were stacked and wrapped with an aluminum foil to be taken to the

sampling site.

2.2.2: Preparation of Potato dextrose agar.

According to the manufacturer’s instruction, 39g of commercial potato dextrose agar

(PDA) should be dissolved in 1000mls of distilled water. Aluminum foil was used to

measure 9.75g of commercial potato dextrose agar on a weighing balance and dissolved in

18
250mls of distilled water in a 250mls conical flask, the medium was stirred to dissolve the

agar and was allowed to properly homogenize by heating. After heating, the conical flask

was corked with a cotton wool and wrapped with aluminum foil and ready for sterilization.

The medium was sterilized in an autoclave at a temperature of 1210c for 15 minutes after

which the medium was brought out of the autoclave and allowed to cool down to about

450c before pouring into the sterile petri dishes aseptically. After pouring into the petri

dishes, the plates were allowed to cool and solidify before labelling the plates, the needed

plates for sampling were stacked and wrapped with an aluminum foil to be taken to the

sampling site.

2.3: Air Sampling Procedure.

For this research study, the settling plate technique was used to sample the air in five (5)

restaurants in the University of Ilorin. The sampling was carried out for 5 consecutive days.

Samples were collected by exposing 2 petri dishes containing solidified nutrient agar and

2 petri dishes containing solidified potato dextrose agar in selected parts of the sampling

site (i.e. one nutrient agar plate and one potato dextrose agar each in different parts of the

restaurant) for 10 minutes. After the sampling the air in the sites, the petri dishes containing

nutrient agar were incubated inversely at 370c for 24 hours, while the plates containing

potato dextrose agar were incubated at 250c (room temperature) for 72 hours. After

incubation, colonies developed were enumerated and characterized.

19
2.4: Isolation and Preservation of Pure culture.

After the incubation period, the number and types of colonies on both the nutrient agar and

potato dextrose agar plates were observed and recorded. Sub culturing was done from

mixed cultures to obtain pure cultures. The pure cultures were then sub cultured into agar

slants in McCartney bottles and kept as stock culture.

2.4.1: Preparation of Culture Media for Stock culture.

For the nutrient agar, 7g of nutrient agar was dissolved in 250mls of distilled water in a

conical flask and then heated to homogenize. The properly homogenized nutrient agar was

the transferred into McCartney bottles, sterilized by autoclaving at 1210c for 15 minutes, it

was the left to cool in a slant state.

2.4.2: Sub culturing of Bacterial Colonies

The sub culturing of bacterial colonies was carried out in the inoculating chamber

aseptically by picking the intended colony to be sub cultured from the mixed culture using

a sterile inoculating loop and streaking on a new solidified nutrient agar plate. The plates

were labelled properly and incubated inversely at 370c for 24 hours. After the incubation

period, the bacterial pure culture were sub cultured by inoculating each bacterium from

pure cultures onto sterile nutrient agar slant in McCartney bottles. The bottles were labelled

properly and incubated at 370c for 24 hours. The stock cultures obtained were preserved

by refrigeration.

20
2.4.3: Sub culturing of Fungal Colonies.

The sub culturing of fungal colonies was carried out in the inoculating chamber aseptically

by cutting out the intended colony to be sub cultured from the mixed culture using a sterile

inoculating needle and transferred onto a new solidified potato dextrose agar plate. The

plates were labelled properly and incubated inversely at 250c for 72 hours. After the

incubation period, the fungal pure culture were sub cultured by inoculating each fungus

from pure cultures onto sterile potato dextrose agar slant in McCartney bottles. The bottles

were labelled properly and incubated at 250c for 72 hours. The stock cultures obtained were

preserved by refrigeration.

2.5: Characterization and Identification of Bacterial Isolates.

Bacterial isolates will be characterized and identified based on their cellular morphology,

colonial morphology, gram staining, spore staining and biochemical tests. The biochemical

tests includes catalase, oxidase and oxidase, also the ability of the bacterial isolates to make

use of biological molecules such as starch, sucrose, maltose etc. will also be tested.

For the Characterization of bacterial isolates, the colonial morphology was based on:

• The shape of the colony e.g. circular, rhizoid etc.

• The edge of the colony e.g. entire, lobate, dentate etc.

• The elevation of the colony e.g. flat, convex, raised etc.

• The optical characteristics of the colony e.g. transparent, translucent or opaque.

21
 • The surface of the colony e.g. dull, smooth, rough etc.

• The consistency of the colony e.g. viscid, butyrous, granular etc.

• The pigmentation i.e. the color of the colony which may be white, red, pink,

light yellow, deep yellow, orange, cream etc. (Fawole & Oso, 2004)

The cellular characteristics were detected by gram staining, spores staining, capsule

staining and motility test. Biochemical tests such as catalase test, citrate test, starch

hydrolysis, coagulase test, oxygen relationship tests etc. serves as a supplement colonial

and cellular morphology in order to fully characterize and identify the isolates.

Identification of bacterial isolates was based on the colonial morphology, cellular

characteristics and morphology and biochemical characteristics. References were made to

a standard microbiology text book Badgis Manual of Determinative Microbiology to get

the appropriate names for the bacterial Isolates.

2.5.1: Gram Staining

Smears of bacterial isolates were prepared on glass slides (an isolate per slide). In preparing

the smear, a drop of sterile distilled water will be placed in the middle of the slide. A

sterilized inoculating loop will be used to pick from the bacteria colony and rubbed on the

slide containing a drop of sterile water. The bacteria cells will be spread into a thin smear,

air dried and heat fixed. The slides were flooded with crystal violet for 60 seconds and

22
drained off, the slide were rinsed off and then flooded with iodine solution for 60 second

and drained off, rinsed with water, 95% ethanol was used to wash the slides for about 10 –

15 seconds and then rinsed with water. The slides were then flooded with safranin for 30

seconds, drained off, rinsed with water, blotted dry and examined under the microscope

with oil immersion X100 objective. Gram positive cells are expected to appear purple while

gram negative cells are expected to appear pink. The cellular shape and arrangement were

also observed. Some of the organisms appeared to be cocci shaped and others rod shaped.

Base on cell arrangement some appeared to be in clusters, some arranged in a single manner

and others in chains (Fawole and Oso, 2004)

2.5.2: Spore Staining

Smears of bacterial isolates were prepared on glass slides (an isolate per slide). In preparing

the smear, a drop of sterile distilled water will be placed in the middle of the slide. A

sterilized inoculating loop will be used to pick from the bacteria colony and rubbed on the

slide containing a drop of sterile water. The bacteria cells will be spread into a thin smear,

air dried and heat fixed. The slides were flooded with malachite green and steamed for 5 -

7 minutes (the malachite green should not dry out during the steam), the malachite green

was drained off and the slides were left to cool for 1 - 2 minutes before washing off with

water. The slides were then flooded with safranin for 30 seconds, rinsed off, blotted dry

and viewed under the microscope with oil immersion at X100 objective. While the

23
vegetative cell of the bacteria stained pink, the spores appeared to be green colored (Willey

et al., 2009)

2.5.3: Capsule Staining

Smears of bacterial isolates were prepared on glass slides (an isolate per slide). In preparing

the smear, a drop of crystal violet will be placed in the middle of the slide. A sterilized

inoculating loop will be used to pick from the bacteria colony and rubbed on the slide

containing a drop of crystal violet water. The bacteria cells will be spread into a thin smear,

air dried. The slides were then washed with aqueous cupric sulphate and then blotted dry.

The slides were then viewed under the microscope with oil immersion at X100 objective.

Capsules will appear as faint blue-violet zones around dark blue bacterial cells.

2.5.4: Oxidase test

This is a test carried out to determine if an organism possesses the Cytochrome C oxidase

enzyme. The reagent Tetra-methyl-P-phenylenediamine dihydrochloride is used as an

artificial electron donor for cytochrome C. when the reagent is oxidized by Cytochrome C,

it changes from colorless to a dark blue or purple color. Oxidase Reagent was prepared

with the measurement 0.1 in 10mls of distilled, filter paper was then soaked in the prepared

in the prepared reagent and allowed to dry. Swab of each isolated were picked and placed

on different spots on the filter paper. A blue colour change within 10seconds will indicate

the production of the enzyme oxidase.

24
2.5.5: Citrate test

6.07g of Simmons citrate agar was dissolved in 250mls of distilled water in a 250mls

conical flask, heated to homogenize and poured into McCartney bottles and sterilized in

the autoclave at 1210c for 15minutes and the left to cool and solidified in a slant state. Each

bottle was then inoculated with different bacterial isolates by streaking on the slant surface

using a sterile inoculating loop, labelled properly and incubated at 350c for 18-48hours.

The observation of a color change from green to blue indicated the ability of the bacterium

to utilize citrate as sole source of carbon (McWilliams, 2009)

2.5.6: Indole test

This test demonstrates the ability of a certain bacteria to decompose the amino acid

tryptophane to indole which accumulates in the medium, this test is carried out by a chain

of a number of different intracellular enzymes, generally known as Tryptophanes. A

bacteria isolate can be tested positive for indole if the bacteria possessing the enzyme

tryptophanase can hydrolyze with amino acid tryptophane to form indole, ammonia and

pyruvic acid. Tryptone Soy A broth was prepared with the measurement 30g to 1litre od

distilled water and poured in to McCartney bottles and sterilized in the autoclave at 1210c

for 15minutes and allowed to cool to avoid death of inoculum. Each bottle containing the

broth was inoculated with different bacterial isolates. Kovac’s reagent was the added to the

25
culture after 48hours and shaken gently. Red color at the reagent layer indicates indole

production while green color indicates no indole production.

2.5.7: Coagulase test

This is a biochemical test that is carried out to differentiate Staphylococcus aureus from

other Staphylococci species. Some bacteria produces enzymes known as Coagulase that

converts fibrinogen to fibrin, which means they can coagulate plasma. Drops of deionized

water was placed on glass slides, isolates from pure culture were picked and placed on the

slides containing deionized water using a sterile inoculating loop and emulsified to form a

smooth-milky suspension. A drop of human plasma was then added to the suspension on

each slides and clumping is observed within 10 seconds. Agglutination of the bacterial cells

after the plasma has been added indicates a positive result while lack of agglutination

indicates negativity.

2.5.8: Triple Sugar Ion (TSI) test

Triple sugar ion agar was prepared by weighing 16.13g of Triple sugar iron powder and

transfer into a conical flask, dissolved with 250ml of water and boiled to homogenize. The

medium was then poured into test tubes, autoclaved and slanted to solidified. The bacteria

isolates were picked with an inoculating needle and stabbed into the Agar slants three times

and then streaked the surface of the slant after stabbing. The isolates were incubated at

37°C overnight and for 48hours for the production of H2S. Bubbles in the agar indicates

gas gormation, yellow slant and butt indicate lactose or sucrose fermentation, yellow butt

26
and red slant indicates glucose fermentation, not lactose nor sucrose, red butt and slant

indicates no utilization of sugar a black colour indicates H2S production.

2.5.9: Methyl Red and Voges-Proskauer test

Methyl red test is carried out to detect if an organism has the ability to produce and maintain

acid end products from glucose fermentation. Voges proskauer test is used to determine if

an organism produces acetymethyl carbinol from glucose fermentation, it is used to detect

acetoin in a bacterial broth culture. MR-VP broth was prepared and transferred into test

tubes, the test tubes were covered and labelled properly and sterilized in the autoclave at

1210C for 15 minutes. After sterilization, the test tubes were allowed to cool, bacterial

isolates were inoculated in the broth using a sterile inoculating loop, the test tube were

incubated at 350C for 48 hours. After 48 hours the bacteria inoculated MR-VP broth was

divided into two test tubes. For the methyl red test, 5 drop of methyl red indicator was

added to one of the test tubes while for Voges Proskauer test, 15 drops of reagent A and 5

drops of reagent B was added to the second test tube, the test tubes were shaken thoroughly

and left undisturbed for 20 minutes.The methyl red test is done alongside Voges proskauer

test since they are both performed on cultures grown in MR-VP broth. Red colouration

indicates a positive methyl red test while yellow or orange colouration indicates negativity.

For Voges Proskauer test, A pink to red coloration shows a positive result which indicate

the presence of acetymethylcarbinol while a negative result was shown by a reddish-brown

coloration (Winn et al., 2006).

27
2.6.0: Oxygen Relationship

Nutrient agar using 4.5g/250mls of distilled water was prepared and dispensed into test

tubes, the test tubes were cocked with cotton wool and sterilized in the autoclave at 1210c

for 15minutes, the test tubes were allowed to cool to solidify after sterilization. Bacterial

isolates were inoculated by stabbing the isolates into the solidified agar in the test tubes

using a sterile needle, the test tubes were incubated at 350c for 24 hours.The pattern of

growth of the organisms along the length of the stab was observed. Obligate aerobes were

observed to grow on the surface of the media, growth through out the length of the stab

indicated facultative anaerobes while growth at the bottom of the bottle indicated anaerobes

(Fawole and Oso, 2004)

2.6.1: Motility test

Nutrient agar using 4.5g/250mls of distilled water was prepared and dispensed into test

tubes, the test tubes were corked with cotton wool and sterilized in the autoclave at 1210c

for 15minutes, the test tubes were allowed to cool to solidify after sterilization. Bacterial

isolates were inoculated by stabbing the isolates into the solidified agar in the test tubes

using a sterile needle, the test tubes were incubated at 350c for 24 hours. Growths were

observed after 24 hours and the test tubes were incubated for another 6days. Movement of

organism from the initial growth position indicates a motile organism.

28
2.6.2: Catalase test

This test is carried out to detect the presence of catalase enzyme is a bacterial isolate .

Catalase is an iron containing enzymes which catalyzes the of release of oxygen from

hydrogen peroxide, it is formed by most aerobic bacteria. 2-3 drops of hydrogen peroxide

was placed on a clean glass side, a sterile inoculating loop was used to pick the isolates and

place on the slide containing 3 drops of hydrogen peroxide. Evolution of a gas as a white

froth indicated a catalase positive reaction while absence of gas indicated a catalase

negative reaction (Barrow and Feltham, 2003).

2.6.3: Sugar Fermentation test.

This test is carried out to test the ability of the bacterial isolates to make use of biological

molecules such as starch, sucrose, maltose etc. Nutrient broth was prepared by dissolving

7.0g of nutrient agar in 250mls of distilled water in a 250mls conical flask, shake gently to

properly dissolve, the mixture was allowed to settle and decanted, the sediment was

discarded, after then the liquid was allowed to settle again and decanted again to obtain

nutrient broth. 1.25g of starch was dissolved in the broth, then 0.03g of phenol red indicator

was added too and shaken gently. The broth was then transferred into test tubes, Durham's

tube were placed inside each test tube inversely, the test tubes were covered and labelled

properly with cotton wool and sterilized in the autoclave at 1210C for 15 minutes. After

sterilization, the broth was allowed to cool before inoculating a bacterial isolate in it to

prevent death of isolate, bacterial isolates were inoculated in to the broth using a sterile

29
inoculating loop. The test tubes were incubated at 370C for 24-48 hours. The above

procedure was repeated for other sugar tests, 1.25g of the sugar(sucrose, inositol, maltose)

was added to 250ml of nutrient broth. Colour change from red to yellow indicates acid

production and bubbles in the Durham's tube indicates gas production.

2.6.4: Oil displacement test

Bacterial Isolates were inoculated in McCartney bottles containing nutrient broth, the

bottles were labelled properly (an isolate per bottle) and incubated at 37oc for 48 hours.

After which the bacteria inoculated nutrient broth was poured into plain sample bottles and

centrifuged at 3500rpm for 10minutes to obtain a cell free supernatant. Petri dish of 90mm

base diameter was filled with 30ml of distilled water, and 50ul of crude oil was carefully

placed on the water surface uniformly, after which 30ul of the Centrifuged supernatant was

added to the center of the hydrocarbon film which is coated on the surface. This process

was repeated for kerosene and engine oil respectively. Clear zone occurrence indicated

biosurfactant production. The binomial diameter of the clear zone was measured to

calculate the area of clearance. The mathematical representation of oil displacement test is

calculated as : (mm)= diameter of the clear zone before-diameter of the clear zone after.

The activity of the collected supernatant was compared with the control (water) as

described by Seema and Nakuleshwar (2012).

30
2.6.5: Haemolysis test

Nutrient agar was prepared and placed in water bath, transfer the nutrient agar in water

bath at 50°C. Bring blood at room temperature before adding to NA. Add sterile blood (50

ml for 1000 ml NA) aseptically and mix well to gently avoiding bubble formation.

Dispense the blood agar aseptically in sterile petri dishes. The plates were left to solidify

and bacterial isolates were inoculated aseptically by streaking. Alpha haemolysis is shown

by a clear zone around bacterial growth, beta haemolysis is shown by partially clear zone

around the bacterial growth while gamma haemolysis is shown by the absence of a clear

zone around the bacterial growth.

2.6.6: Drop collapse test

Bacterial Isolates were inoculated in McCartney bottles containing nutrient broth, the

bottles were labelled properly (an isolate per bottle) and incubated at 37oc for 48 hours.

After which the bacteria inoculated nutrient broth was poured into plain sample bottles and

centrifuged at 3500rpm for 10 minutes, to obtain a cell free supernatant. 10ul of petrol was

used to thinly coat the surface of a glass slide. The 25ul of the CFS was added to the center

of the coated slides. The presence of biosurfactant was detected from the collapsing of the

CFS on the slides with 10 seconds, the test was negative when the drop remained. A 20 𝜇L

distilled water was used as control (Hasham et al., 2012).

31
2.7: Characterization and Identification of Fungal Isolates

Fungal isolates will be characterized and identified using microscopic and macroscopic

characteristics i.e. the cultural and morphological features which includes colony growth

pattern, conidial morphology and pigmentation (color).

2.7.1: Characterization of Fungal Isolates

After establishing a pure culture of each of the fungal isolates, the pure culture was

macroscopically and microscopically examined. The colonial and morphological

characteristics of the isolates were used to characterize them. The colonial morphology of

each isolate was detected macroscopically, i.e. with the naked eye, and characteristics such

as the color of the colony and the color of the reverse side of the colony, the form of fungus

growth, and the rate of colony growth were recorded. To establish the morphological traits,

a drop of distilled water was placed in the center of a glass slide, and a portion of the fungus

was cut out and placed on the drop of distilled water on the glass slide. The specimen was

teased with two sterile needles before it was covered with a cover slip. The slide was placed

on the stage of the microscope and examined at x100 objective. The morphological

characteristics includes the characteristics of the vegetative and reproductive structure of

the fungus. The nature of the hyphae was observed whether septate or aseptate, colour or

colourless hyphae, presence of rhizoids, sporophore type which can be branched or

unbranched, attatchment of sporophore to the vegetative hyphae, type and colour of spores

produced were all recorded.

32
2.7.2: Identification of Fungal Isolates

After the characteristics of the fungal isolates were determined, the fungi isolates were

identified according to microbiology textbook (Nester et al., 2007).

33
 CHAPTER THREE

RESULTS

3.0: Bacterial Isolates

A total of ten bacterial isolates were obtained after a sampling period of five days. The

bacterial isolates obtained from the sampling site were identified as: Bacillus cereus,

Micrococcus luteus, Bacillus Subtilis, Staphylococcus epidermis, Pseudomonas

aeruginosa, Staphylococcus haemolyticus, Streptococcus pyogenes, Staphylococcus

hominis, Micrococcus lylae and Streptococcus viridans. The colonial morphology, cellular

and biochemical characteristics of the ten isolates are presented in Table 1. A total of nine

Gram positive and one Gram negative isolates were obtained. The Gram-positive

organisms obtained include; Bacillus subtilis, Bacillus cereus, Micrococcus luteus,

Micrococcus lylae, Staphylococcus epidermis, Staphylococcus haemolyticus,

Staphylococcus hominis, Streptococcus pyogenes and Streptococcus viridans while

Pseudomonas aeruginosa was Gram negative. The cellular arrangement of the isolates

varied from single to clusters and to chains.

Table 1 shows the number of colonies of the bacterial isolates obtained during the five days

of sampling. Table 2 shows the cellular and morphological characteristics and biochemical

test results of isolates Table 3 shows Biosurfactant test results of each isolate. The

occurrences of the bacterial isolates are presented in Table 4

34
 Table 1: Daily counts of Bacteria growths from each sampling sites.

SAMPLING SS A SS B SS C SS D SS E

PERIOD

DAY 1 326 426 8 64 537

DAY 2 116 61 6 43 122

DAY 3 47 42 6 39 78

DAY 4 81 51 5 39 100

DAY 5 57 45 6 35 88

Key: SS- Sampling Site

35
 TABLE 2: COLONIAL MORPHOLOGY, CELLULAR CHARACTERISTICS, AND BIOCHEMICAL CHARACTERISTICS OF BACTERIAL ISOLATES

MORPHOLOGY CELLULAR CHARACTERISTICS BIOCHEMICAL TESTS -

SURFACE TEXTURE

VOGES PROSKEUR
ARRANGEMENT
CONSISTENCY

OXYGEN REL.

METHYL RED
SPORE STAIN
GRAM STAIN

COAGULASE

CELL SHAPE

ORGANISMS
ELEVATION

CATALASE
MOTILITY
ISOLATES

MALTOSE

INOSITOL
SUCROSE
CAPSULE

CITRATE

OXIDASE
OPTICAL

STARCH
INDOLE
COLOR
SHAPE

EDGE

TSI
A C CW E CX R G O CH + + + + + - FA - - + + - RR + + + - RO Bacillus cereus

B C Y E CX S V O TE + - - - + - AE - - - + + RY - + - - CO Micrococcus luteus

C C W L FL R G T CH + + + - + - FA - + + + + YY + + + + RO Bacillus subtilis

D C CR E R S V O CL + - - + + - FA - - + - - YY - + + - CO Staphylococcus epidermidis

E I BG U R S B T CH - - + - + - FA - - - + + RR - - - - RO Pseudomonas aeruginosa

F C CW E R S V O CL + - - - + - FA - - - - - RY - + + + CO Staphylococcus

haemolyticus

G R CR R F S V T CH + - - + - - FA - + - - - RY - + + - CO Streptococcus pyogenes

H C CW E R S V O CL + - - - + - FA - - - - - RR - + + - CO Staphylococcus hominis

I C CW E CX S V O TE + - - - + - AE - - - + + RY - + + - CO Micrococcus lylae

J C GW E R S V T CH + - - + - - FA - - - - - RY - - + - CO Streptococcus viridans

36
Table 3: BIOSURFACTANT AND HAEMOLYSIS TEST
OIL DISPLACEMENT DROP COLLAPSE

Bacter CRUD KEROSEN ENGINE CRUD KEROSEN ENGIN HAEM

ial E OIL E E E E OLYSIS

OIL
Isolate
OIL OIL

A 4.3 3.5 0.5 - + + 𝜷

B 0.4 1.2 2.5 - + + 𝜷

C 5.8 2.8 0.7 - + + 𝜷

D 1.4 1.9 3.5 - + + 𝜸

E 7.8 4.2 1.3 - + + 𝜷

F 0.5 1.0 1.9 - + + 𝜷

G 0.3 0.7 1.5 - + + 𝜷

H 0 0 0.6 - + + 𝜸

I 0.2 1.3 2.2 - + + 𝜷

J 0 1.9 1.2 - + + 𝜶

KEY: C= Circular I= Irregular R= Rhizoid CR= Cream Y= Yellow E= Entire R= Raised S=Smooth WR= Wrinkled B=

Butyrous G= Granular O= Opaque CL= Cluster CH= Chain CO= Cocci RO= Rod AE= Aerobic FA= Facultative anaerobic

MA= Microaerobic CX =ConvexCW= Creamy white GW= Grey white TE= Tetrad RR= Red slant/Red butt RY= Red

slant/Yellow butt Y/Y= Yellow slant/yellow butt T= Translucent V= Viscious G= Granular

37
Table 4: Occurrence of bacterial isolates during the five days sampling period.

DESIGNATION BACTERIA ISOLATE 1 2 3 4 5

A Bacillus cereus + + + + +

B Micrococcus luteus + + + + +

C Bacillus subtilis + + + + +

D Staphylococcus epidermis + + + + +

E Pseudomonas aeruginosa + + - + +

F Staphylococcus aureus + + + + +

G Streptococcus pyogenes + - + - +

H Staphylococcus hominis + + + + +

I Micrococcus lylae + + + + +

J Streptococcus viridans + + + - +

KEY: + = PRESENT

38
 - = ABSENT

TABLE 5: Numbers of colonies of bacterial isolates obtained during the five days of sampling.
BACTERIAL ISOLATES SAMPLING PERIOD IN DAYS TOTAL

1st 2nd 3rd 4th 5th

Bacillus cereus 160 62 39 44 36 341

Micrococcus luteus 142 45 27 33 22 269

Bacillus subtilis 149 66 42 47 25 329

Staphylococcus epidermidis 151 47 08 16 28 250

Pseudomonas aeruginosa 70 12 0 12 07 101

Staphylococcus haemolyticus 128 34 35 39 32 268

Streptococcus pyogenes 125 0 07 12 08 152

Staphylococcus hominis 158 21 14 19 34 246

Micrococcus lylae 131 47 18 25 16 237

Streptococcus viridans 147 14 22 29 23 235

TOTAL 1361 348 212 276 231 2428

39
The bacterial load is thus calculated as N=5a×104(bt)-1

N= microbial cfu/m3, A= number of colonies per petri dish, B= dish surface, t= exposure

time, T=Total number of colonies

Day 1= 1361, Day 2= 348, Day 3= 212, Day 4= 276, Day 5= 231

Day 1 has the highest number of colonies with 1361n colonies while day 3 has the lowest

with 212n colonies.

B= 9cm diameter which makes an area of 63.585cm²

T= 10 minutes

Highest = 5×1361×104(63.585×10)-1

68050000×0.0015727

107022 cfu/m³

Lowest = 5×212×104(63.585×10)-1

10600000×0.0015727

16670 cfu/m³

The bacterial load ranged from 16670cfu/m³ to 107022cfu/m³

Figure 2-11 shows the microscopic view of the Gram staining results of the bacteria isolates

40
3.1: Fungal Isolates

Five fungal isolates were obtained after a sampling period of five weeks. The fungal

isolates obtained from the sampling site were identified as: Rhizopus stolonifer, Aspergillus

flavus, Aspergillus niger, Rhizopus oryzae and Trichoderma viride

3.1.1 Description of fungal isolates

Isolate A: It is macroscopically identified as white hairy spores and black edges.

Microscopically observed to have rapid growth and produces sporangia that bear

sporangiospores. Commonly known as black bread molds, identified as Rhizopus stolonifer

Isolate B: Macroscopically identified as powdery lemon green color which appears creamy

from the reverse side of the plate, tiny spores on top of the mycelia. Identified as

Aspergillus flavus.

Isolate C: Macroscopically, it has white edges with powdery black at the center, white to

creamy colour on the reverse side of the plate. Microscopically observed connection of

sporangia with tiny sporangiosphores on the top. Mostly round like structure (spores).

Identified as Aspergillus niger.

Isolate D: Macroscopically observed to be wooly with brownish-grey or black coloration.

Microscopically, it is characterized by presence of stolons and pigmented rhizoids, the

formation of sporangiophores, singly or in groups from nodes directly above the rhizoids,

41
and apophysate, columellate, multispored, generally globose sporangia. Identified as

Rhizopus oryzae

Isolate E: It is macroscopically identified with yellow-green pigmentation, wooly with

white edges. Microscopically observed to have rapid growth, dense conidia, branched

conidiophores, ampuliform phialides and slightly globose conidia. Identified as

Trichoderma viride

The average number of fungi counts from the sampling sites are presented in Table 6

The occurrence of each fungal Isolates during the 5day sampling period is presented in

Table 7.

The average distribution of fungal isolates is presented in Table 8

42
 Table 6: The average number of fungi counts from the sampling sites.

SAMPLING SS A SS B SS C SS D SS E

PERIOD

DAY 1 13 14 5 19 23

DAY 2 9 11 2 13 19

DAY 3 15 12 4 21 19

DAY 4 12 12 5 16 22

DAY 5 17 14 4 19 20

Key: SS- Sampling Site

43
Table 7: The occurrence of each fungal Isolates during the 5 days sampling period.

DESIGNATION FUNGAL ISOLATE 1 2 3 4 5

A Rhizopus stolonifera + - + - +

B Aspergillus flavus + + - + +

C Aspergillus niger + - + + +

D Rhizopus oryzae + + + - -

E Trichoderma viride + - + - +

KEY: + = PRESENT

- = ABSENT

44
Table 8: The average distribution of fungal isolates.

FUNGAL SAMPLING PERIOD IN DAYS TOTAL

ISOLATES
1st 2nd 3rd 4th 5th

Rhizopus stolonifer 04 0 04 0 07 15

Aspergillus niger 03 08 0 07 0 18

Aspergillus flavus 08 07 05 0 06 26

Rhizopus oryzae 02 04 03 06 0 15

Trichoderma viride 06 0 07 09 07 29

Total 23 19 19 22 20 103

45
The fungal load is thus calculated as N=5a×104(bt)-1

N= microbial cfu/m3 , NA= number of colonies per petri dish, B= dish surface, t=

exposure time, T=Total number of colonies

Day 1= 74, Day 2= 54, Day 3= 64, Day 4= 67, Day 5= 74

Day 1 and Day 5 has the highest number of colonies with 74n colonies each while day 2

has the lowest with 54n colonies.

B= 9cm diameter which makes an area of 63.585cm²

T= 10 minutes

Highest = 5×74×104(63.585×10)-1

3700000×0.0015727

5819cfu/m³

Lowest = 5×54×104(63.585×10)-1

2700000×0.0015727

4246 cfu/m³

The fungal load ranged from 4246cfu/m³ to 5819cfu/m³

46
47
Fig 1: Aspergillus niger under microscope X40 objective lens

48
Fig 2: Aspergillus flavus under microscope X40 objective lens

49
Fig 3: Rhizopus stolonifera under microscope X40 objective lens

50
Fig 4: Rhizopus oryzae under microscope X40 objective lens

51
Fig 5: Trichoderma viride under microscope X40 objective lens

52
 CHAPTER FOUR

DISCUSSION

Although air is not a suitable medium for microbial growth, the differences in the types

of microorganisms isolated demonstrate the general fact that the air micro biome does

not contain unique microorganisms. What is the only living thing in the air? The

surrounding air has many microorganisms carried in dust particles. Air disturbances

such as fans, sweeping, dancing, human and hand movements can carry microorganisms

and spores from surfaces into the air. It can be concluded that the number of

microorganisms in the air depends on the activities taking place in the environment that

stir up dust particles. The number and type of microorganisms falling on the contact

plates depends on the time of exposure, the movement of the individual and the air

content passing through at that time as well as the sampling method used. The

survival of airborne organisms depends greatly on the type of organism and atmospheric

conditions such as relative humidity, light exposure, and temperature. Although the air

inside restaurants is generally dry and free of microorganisms other than Gram-positive

and Gram-negative bacteria, this study found that bacteria are hardier and survive longer

in the air, as do many types of spores. The concentrations of bacteria and fungi observed

in this study can be linked to a number of sources of microbial contamination such

as restaurant staff, customers, clothing, poor ventilation, poor sanitation and food,

among other sources. The human body, like clothing, is a breeding ground for

53
microbial growth. A strong relationship between occupant density, sanitation, ventilation

system, human activity and microorganisms’ concentration in the indoor air has been

reported in past studies (Yoon et.al., 2011; Udochukwu et.al., 2016). The microorganisms

found present in the air of this eateries can also be as result of dust during cleaning

operations, cooking, rate at which customers come in and go out, human activities like

breathing, sneezing, coughing, talking, and movement, have been linked to the high

concentration of indoor air bioaerosols (Chen and Hildemann, 2009; Castillo et.al., 2012;

Bhangar et.al., 2014, 2015; Adams et.al., 2015; Meadow et.al., 2015; Nazaroff, 2015). Air-

borne Bacteria may be distributed in different forms:

• Attached to dust particles

• Contained in gross droplets released from nose and mouth

• Presence in droplets nuclei.

The study of the air microflora of five selected eateries in the University of Ilorin Campus

led to the isolation of a number of bacteria and fungi. The bacteria isolates were Bacillus

cereus, Micrococcus luteus, Bacillus Subtilis, Staphylococcus epidermidis, Pseudomonas

aeruginosa, Staphylococcus heamolyticus, Streptococcus pyogenes, Staphylococcus

hominis, Micrococcus lylae and Streptococcus viridans with Bacillus spp being the most

and the fungal isolates were Rhizopus stolonifer, Aspergillus flavus, Aspergillus niger,

54
Rhizopus oryzae, Trichoderma viride and Penicillium chrysogenum. Results revealed the

presence of Bacillus, Micrococcus, Staphylococcus, Streptococcus and Pseudomanas

strains in the sampled air, indicative of their prevalence in the eatery environment with

Bacillus spp being the highly occurring bacteria in all the sampling site being present for

everyday of the sampling period and Pseudomonas aeruginosa being the least. Presence of

Rhizopus sp, Aspergillus sp and Trichoderma sp were also sampled from the eateries air

with Trichoderma sp being the most prevalent and Rhizopus sp being the least occurred.

Bacillus cereus

Bacillus cereus is a gram-positive or gram-variable, rod-shaped, aerobic-to-

facultative, spore-forming bacterium. Its bacterial spores do not swell the sporangium and

sporulate only under aerobic conditions (Bottone, 2010). Bacillus cereus (B. cereus) is a

spore-forming microorganism commonly found in the environment. B. cereus is generally

considered to be a mesophilic microorganism with a growth temperature range of 10–

50 °C (optimum temperature 28–37 °C). Additionally, only a few strains can thrive

at temperatures below 7 °C and above 45 °C. This pathogen can grow at a pH of 4.3

to 9.3 (optimum is 6.0 to 7.0) and a minimum water activity (aw) of 0.92. B. cereus

spores are moderately heat resistant and tolerate freezing and drying. This bacteria

produces harmful substances (toxins) that can cause disease. It is found in soil, raw plant

foods such as rice, potatoes, peas, beans and spices are common sources of B. cereus. B.

cereus produces harmful substances (toxins), these toxins can cause food poisoning

55
(intestinal Bacillus cereus) or more serious health problems (non-intestinal Bacillus

cereus). Generally, infections caused by B. cereus have a short duration and are self-

limiting without recognized post-illness (Schmid et al., 2016).

Most people who get food poisoning recover within 24 hours, however, if your immune

system is weakened you are at higher risk for complications. Intestinal Bacillus

cereus which causes is a gastrointestinal disease that causes food poisoning.This disease

usually clears up on its own quickly, but if you have a weakened or weakened

immune system, you are at risk for more severe symptoms. Bacillus cereus food

poisoning is a common description of illness associated with this microorganism, but

the two recognized types of illness are caused by two different metabolites (toxins).

Diarrheal disease is caused by high molecular weight proteins, and emetic disease

(vomiting) is associated with cereulide, an acid- and heat-stable polypeptide. Vomiting

syndrome (Emetic disease) is mainly associated with starchy foods such as fried rice and

noodles. The disease is caused by the ingestion (i.e., poisoning) of preformed toxins

contained in food. On the other hand, diarrhea syndrome is associated with other foods

such as milk, salads, and meat. Unlike emetic syndromes, diarrheal diseases are caused

by the ingestion of large numbers of bacterial cells (i.e., virulent infections). To

control this bacterium in food, proper cooking and rapid cooling are required to prevent

spores from germinating. Identifying Bacillus cereus in restaurant air suggests that it may

have multiple sources of introduction such as contaminated food, water, air conditioning

systems, human activities or even contact surfaces within the restaurant.

56
Micrococcus luteus

Micrococcus luteus is a type of Gram-positive, round cocci (spherical) bacterium

that occur in clusters or tetrads. They are often yellow or golden in color, which is why

they are referred to as "luteus," meaning yellow in Latin. bacterium that is part of the

Micrococcus genus that is found in various environments, including soil, dust, water, and

air. It is considered a part of the normal microbial flora on human skin and mucous

membranes. It is part of the normal microbial flora and can be present in indoor spaces,

including eateries. However, the presence of Micrococcus luteus in the air of eateries is

generally not a cause for concern, as it is typically considered harmless to humans and not

associated with foodborne illnesses. Known for its ability to produce the enzyme catalase,

which helps it break down hydrogen peroxide, Micrococcus luteus is generally non-

pathogenic and not associated with causing diseases in healthy individuals. It is considered

to be a relatively harmless bacterium. It is also positive for the coagulase test, which helps

differentiate it from some other bacteria.

Eateries, like any indoor spaces, can have a variety of microorganisms in the air due to

human activity, food preparation, and ventilation systems. Maintaining proper hygiene and

ventilation in eateries is important to ensure the overall cleanliness and safety of the

environment, but the presence of Micrococcus luteus itself is not a specific health risk in

this context. Micrococcus luteus is a bacterium of scientific interest due to its

characteristics and applications in research and industry. It is generally considered

57
harmless to humans and is more often associated with its environmental and

biotechnological roles. Health and safety regulations in eateries typically focus on more

concerning pathogens, such as bacteria like Escherichia coli or Salmonella, which can

cause foodborne illnesses. Monitoring and control measures are in place to minimize the

risk of such pathogens in food service establishments.

Bacillus subtilis

Bacillus subtilis, a Gram-positive bacterium with exceptional versatility, recognized for its

rod-shaped (bacilli) morphology, is a member of the Bacillus genus with an impressive

range of physiological capabilities. Bacillus subtilis, also called hay bacillus or grass

bacillus, it was originally named Vibrio subtilis by Christian Gottfried Ehrenberg

(Ehrenberg, 1836) and renamed Bacillus subtilis by Ferdinand Cohn in 1872 (Cohn, 1872),

it is a Gram-positive, catalase-positive bacterium found in the soil and gastrointestinal

tract of ruminants, humans, and sponges. Bacillus subtilis is a spore former and normal

Flora of the soil, its presence in the air may be due to its ability to form endospores that are

resistant to heat, drying, disinfectants and other adverse conditions. Bacillus subtilis is of

great economic importance in food processing industries and it is also known to cause

contamination in dairy products. As with other members of the genus Bacillus, it can form

an endospore, to survive extreme environmental conditions of temperature and desiccation

(Madigan and Martinko, 2005). B. subtilis is a facultative anaerobe (Nakano and Zuber,

58
1999) and had been considered as an obligate aerobe until 1998. B. subtilis is heavily

flagellated, which gives it the ability to move quickly in liquids.

Staphylococcus epidermidis

Staphylococcus epidermidis is a type of bacterium commonly found on the skin and

mucous membranes of humans. It is part of the normal microbiota and is generally

considered to be harmless in healthy individuals. However, in certain situations,

Staphylococcus epidermidis can cause opportunistic infections, particularly in individuals

with weakened immune systems or when it enters the bloodstream through medical devices

like catheters or prosthetic implants. Proper hygiene and infection control measures are

important in healthcare settings to prevent the spread of Staphylococcus epidermidis and

related infections.

Staphylococcus epidermidis is not typically associated with the air in eateries. It is a

bacterium commonly found on the skin and mucous membranes of humans, and its

presence in the air of a restaurant or eatery would be unusual. However, the air in eateries

can contain various microorganisms, including bacteria, which can come from sources such

as food preparation, cooking, or the environment. Maintaining good hygiene and

cleanliness practices in eateries is essential to minimize the presence of harmful bacteria

and ensure food safety.

59
Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium notorious for its ability to cause

disease in individuals with compromised immune systems i.e. opportunistic pathogen.The

isolation of Pseudomonas aeruginosa from restaurant air samples raises red flags as it can

cause a range of infections, including respiratory tract infections, skin infections, and even

life-threatening systemic infections in susceptible individuals. Its presence in restaurant air

underscores the importance of maintaining stringent hygiene practices to protect both

customers and restaurant staff. Identifying Pseudomonas aeruginosa in restaurant air

suggests that it may have multiple sources of introduction. These could include

contaminated food, water, air conditioning systems, or even contact surfaces within the

restaurant.

The detection of Pseudomonas aeruginosa in restaurant air highlights the role of air as a

vector for the spread of pathogens. Airborne transmission can occur through coughing,

sneezing, or simply as a result of normal human activities within the restaurant. While

Pseudomonas aeruginosa is primarily associated with respiratory and wound infections,

its presence in restaurant air still has significant food safety implications. Airborne bacteria

can settle on food surfaces and equipment, potentially contaminating food items if hygiene

protocols are not rigorously maintained. Therefore, detecting Pseudomonas aeruginosa

should trigger heightened awareness of food safety practices and sanitation measures in

restaurants.

60
Staphylococcus haemolyticus

Staphylococcus haemolyticus is a member of the coagulase-negative staphylococci (CoNS)

family. It is part of the human skin flora and its largest populations are commonly found

in the axillary, perineal and groin areas. S. haemolyticus also colonizes primates and

livestock. Its presence in the eateries can be as result of a carrier such as the cook,

staff/workers and customers. It is a well-known opportunistic pathogen and the second

most frequently isolated CoNS (S. epidermis is first). It lives on a variety of substrates,

including glucose, glycerol, maltose, sucrose and trehalose. It is also catalase positive

and coagulase negative. Optimal growth occurs at temperatures between 30 and 40°C

in the presence of oxygen and 10% NaCl. However, some varieties can grow at

temperatures between 18 and 45°C. Growth at 15°C or 15% NaCl is poor or absent. S.

haemolyticus infection particularly affects immunocompromised patients and presents

primarily as blood- and device-related infections. Hospital isolates of S. haemolyticus

are classified as among the most antibiotic resistant among CoNS, and therefore antibiotic

treatment options are very limited. Staphylococcus haemolyticus can cause catheter-

associated urinary tract infections, wound infections, and conjunctivitis. The main route of

transmission is direct or indirect contact with contaminated people or objects.

Streptococcus pyogenes

61
Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is a bacterium that

can cause a range of infections in humans. It is responsible for conditions like strep throat,

skin infections, and in severe cases, invasive diseases like necrotizing fasciitis and toxic

shock syndrome. It is primarily transmitted through respiratory droplets from an infected

person or by direct contact with infected wounds or sores. Streptococcus pyogenes (Group

A Streptococcus) is not typically associated with the air in eateries. It is primarily a human

pathogen that is transmitted through respiratory droplets or direct contact with infected

individuals. In eateries, the main concerns are typically related to foodborne pathogens or

environmental factors that can impact food safety and quality. Maintaining proper hygiene,

food handling practices, and cleanliness in eateries are crucial for preventing the spread of

foodborne illnesses, but Streptococcus pyogenes is not a common concern in this context

Staphylococcus hominis

Staphylococcus hominis is a bacterium that is part of the normal microbiota found on

human skin and mucous membranes. Like other Staphylococcus species, it is generally

considered to be part of the natural microbial flora of the human body and is typically

harmless. However, in certain situations, Staphylococcus hominis can become an

opportunistic pathogen and cause infections, especially in individuals with weakened

immune systems or in healthcare settings.

In eateries or food-related environments, Staphylococcus hominis is not typically a major

concern, is not typically associated with eateries or foodborne illnesses. It's a bacterium

62
commonly found on human skin and mucous membranes and is generally not a major

concern in food safety. . Food safety practices mainly focus on preventing contamination

by more common foodborne pathogens like Salmonella, E. coli, or Listeria. To maintain

food safety in eateries, it's crucial to follow proper hygiene, sanitation, and food handling

practices. These measures help prevent the growth and spread of harmful bacteria and

ensure the safety of the food served to customers.

Micrococcus lylae

Micrococcus lylae is a gram-positive bacterium known for its widespread distribution in

various environmental niches, including soil, water, dust, and air. Its presence in eateries

air samples is not unexpected, as eateries are exposed to constant environmental factors

such as foot traffic, outdoor air, and food preparation activities. While Micrococcus lylae

is not typically associated with foodborne illnesses, its presence in indoor air could serve

as an indicator of overall indoor air quality. It is important to consider that microbial

contaminants in the air may originate from human skin, clothing, and respiratory emissions.

Therefore, the detection of Micrococcus lylae may not necessarily indicate unsanitary food

practices but rather the presence of humans in the restaurant environment. While

Micrococcus lylae itself is not a direct foodborne pathogen, it is crucial to note that the

presence of various microorganisms in restaurant air samples can indirectly impact food

63
safety. Airborne microorganisms can settle on food surfaces and equipment, potentially

leading to cross-contamination if proper sanitation practices are not maintained.

Streptococcus viridans

Streptococcus viridans is a group of bacteria that are part of the normal flora in the human

oral cavity, gastrointestinal tract, and other mucous membranes. While they are typically

harmless and commensal (meaning they coexist without causing harm) in healthy

individuals, they can occasionally cause infections, especially in individuals with

weakened immune systems or those undergoing certain medical procedures.

Streptococcus viridans is not typically associated with eateries or food safety concerns.

These bacteria are commonly found in the human oral cavity and other mucous membranes

and are usually considered harmless commensals in healthy individuals. In eateries or food-

related settings, Streptococcus viridans is not a typical concern for food safety. The primary

focus is on preventing contamination by foodborne pathogens that can cause foodborne

illnesses. Maintaining proper food handling practices, hygiene, and sanitation measures are

essential to ensure the safety of the food served to customers

Rhizopus stolonifer

64
Rhizopus stolonifer, commonly known as black bread mold, is a filamentous fungus that

can grow on various food items, especially those with high moisture content. It's often

associated with bread, fruits, and vegetables. This mold can cause food spoilage and is

known for its rapid growth and ability to spread quickly on the surface of foods. The

presence of Rhizopus stolonifer (black bread mold) in an eatery or food establishment is

generally considered undesirable. This mold can grow on various food items, particularly

those with high moisture content, and it can lead to food spoilage and a decline in food

quality.

In eateries and food-related environments, the presence of Rhizopus stolonifer on food

items is a concern because it can lead to the deterioration and spoilage of these items. To

prevent the growth of molds like Rhizopus stolonifer, it's essential to store food properly,

regularly inspecting and discarding moldy or spoiled food items is crucial to maintain food

safety and quality. Mold-contaminated food should not be served to customers to prevent

potential health hazards and maintain the reputation of the eatery.maintain cleanliness in

food preparation areas, and follow food safety guidelines to minimize the risk of foodborne

illnesses.

Aspergillus flavus

Aspergillus flavus is a type of fungus commonly found in the environment, especially in

soil and on plant material. It is known for producing a toxin called aflatoxin, which can

65
contaminate crops such as peanuts, corn, and various other nuts and grains. Aflatoxin is a

potent carcinogen and can pose serious health risks if consumed in contaminated food.

The presence of Aspergillus flavus in eateries or food establishments is a concern,

particularly if it contaminates food ingredients. Aspergillus flavus is known for producing

aflatoxins, which are potent carcinogenic toxins that can be harmful when consumed. To

prevent the growth and spread of Aspergillus flavus and the associated aflatoxin

contamination in eateries, it's essential to follow strict food safety and quality control

measures. This includes proper food storage, handling, and inspection of ingredients for

signs of mold or contamination. Food safety regulations and guidelines are in place to

minimize the risk of aflatoxin exposure to consumers, and eateries should adhere to these

standards to ensure the safety of the food they serve.

Aspergillus niger

Aspergillus niger is a common filamentous fungus belonging to the genus Aspergillus. It

is widely distributed in the environment and can be found in soil, decaying organic matter,

and indoor environments. Aspergillus niger has several industrial and scientific

applications. Aspergillus niger is a common fungus found in the environment, including

indoor spaces like eateries. It can be present in the air, especially in areas with high

humidity or poor ventilation. Aspergillus niger is a common fungus found in the

environment, including indoor spaces like eateries. It can be present in the air, especially

in areas with high humidity or poor ventilation. While Aspergillus niger itself is not

66
harmful, some strains can produce mycotoxins under certain conditions, which can be a

concern in food safety. However, it is also used in the production of various food and

beverage products, such as soy sauce and certain cheeses. It is used in the production of

citric acid, an important ingredient in the food and beverage industry. Aspergillus niger is

known for its ability to efficiently convert sugars into citric acid through fermentation.

However, it's important to note that the presence of Aspergillus niger in the air of eateries

is generally not a cause for concern unless it reaches elevated levels or if individuals have

specific health conditions that make them more susceptible to fungal infections. Eateries

should maintain proper hygiene and ventilation to minimize the growth of such fungi and

ensure the safety of their patrons. If you suspect an issue with indoor air quality, it's

advisable to consult with experts or relevant authorities for a proper assessment and

recommendations. Overall, while Aspergillus niger has both beneficial and potentially

harmful aspects, its significance lies in its diverse applications in various fields, including

biotechnology, research, and food production. Proper handling and monitoring are

essential in contexts where it may pose health or safety concerns. Eateries should maintain

proper hygiene and ventilation to minimize the growth of such fungi and ensure the safety

of their patrons. If you suspect an issue with indoor air quality, it's advisable to consult

with experts or relevant authorities for a proper assessment and recommendations.

67
Rhizopus oryzae

Rhizopus oryzae is a filamentous fungus commonly known as "rice mold" or "bread mold."

It is used in various food fermentation processes, particularly in the production of certain

Asian food products like tempeh and some types of rice wines. In these processes, Rhizopus

oryzae plays a beneficial role in breaking down starches and proteins, contributing to the

flavor and texture of the final product. However, outside of controlled food fermentation

processes, the presence of Rhizopus oryzae on food items is generally considered

undesirable. It can lead to food spoilage and deterioration.

The presence of Rhizopus oryzae in an eatery or food establishment is generally not

desirable, as it can lead to food spoilage and a decline in food quality. Rhizopus oryzae is

a type of mold commonly associated with bread mold and other food items with high

moisture content. To prevent the growth of molds like Rhizopus oryzae in eateries, it's

crucial to follow proper food storage, handling, and hygiene practices. Regularly inspecting

and discarding moldy or spoiled food items is important to maintain food safety and

quality. Mold-contaminated food should not be served to customers to prevent potential

health hazards and to uphold the reputation of the eatery.

Trichoderma viride

Trichoderma viride is a type of fungus commonly found in the environment, particularly

in soil and decaying organic matter. It is well-known for its biocontrol properties, as it can

act as a natural antagonist to certain plant pathogens, making it useful in agriculture.

68
Trichoderma viride is not typically associated with eateries or food establishments. This

fungus is commonly found in the natural environment, particularly in soil and decaying

organic matter. It is not considered a foodborne pathogen, and it is not typically associated

with food safety concerns in eateries.

In eateries or food-related settings, the presence of Trichoderma viride is generally not a

concern because it is not typically associated with food or food safety issues. Food

establishments primarily focus on preventing contamination by foodborne pathogens and

molds that are more commonly associated with food spoilage. Nevertheless, maintaining

good hygiene and cleanliness in food preparation and storage areas remains essential to

ensure the safety and quality of the food served to customers.

69
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

5.0: Conclusion

This research has successfully identified a diverse population of non-pathogenic

microorganisms in air of selected eateries in University of Ilorin campus. This finding

underscores the complexity of microbial communities present in such settings, many of

which are harmless and even beneficial. Although some of these microorganisms do not

cause disease in healthy people, they may become virulent with immunocompromised and

unhealthy individuals i.e. opportunistic pathogen.

Through rigorous sampling and analysis, we have established that the majority of

microorganisms in these restaurants pose no immediate health risks to patrons or staff. This

indicates that the restaurant maintains a reasonably hygienic environment with effective

sanitation practices in place.

5.1: Recommendations

❖ Sustain Sanitation Practices: Maintain and reinforce the current sanitation practices

within the restaurant. Ensure that cleaning schedules and protocols are consistently

followed to control microbial populations.

70
❖ Staff Training: Continue to educate restaurant staff on proper food handling,

hygiene, and cross-contamination prevention. Regular training sessions can help

uphold food safety standards.

❖ Regular Monitoring: Establish a routine microbial monitoring program to assess

the ongoing cleanliness of surfaces, equipment, and food products. Periodic checks

can identify any deviations from the norm and allow for prompt corrective actions.

❖ Proactive Maintenance: Implement a proactive maintenance plan for kitchen

equipment to prevent microbial buildup. Regular servicing and cleaning of

equipment can reduce the risk of contamination.

❖ Temperature Control: Emphasize the importance of maintaining proper

temperature control for food storage to prevent spoilage and the growth of

undesirable microorganisms.

❖ Customer Education: Develop informational materials or posters for customers that

highlight the restaurant's commitment to cleanliness and food safety, promoting

transparency and trust.

71
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 APPENDIX

Plate 1: Student carrying out the Oil displacement test.

79
Plate 2 : Bacillus cereus gram staining viewed under microscope X100 objective lens

80
Plate 3: Micrococcus luteus gram staining viewed under microscope X100 objective

lens

81
 .

Plate 4 : Bacillus subtilis gram staining viewed under microscope X100 objective

lens

82
 .

Plate 5 : Staphylococcus epidermidis gram staining viewed under microscope X100

objective lens

83
 .

Plate 6 : Pseudomonas aeruginosa gram staining viewed under microscope X100

objective lens

84
 .

Plate 7: Staphylococcus haemolyticus gram staining viewed under microscope X100

objective lens

85
 .

Plate 8 : Streptococcus pyogenes gram staining viewed under microscope X100

objective lens

86
 .

Plate 9 : Staphylococcus hominis gram staining viewed under microscope X100

objective lens

87
 .

Plate 10 : Micrococcus lylae gram staining viewed under microscope X100 objective

lens

88
 .

Plate 11: Streptococcus viridans gram staining viewed under microscope X100

objective lens

89