MICROBIOLOGICAL QUALITYOF INDOOR AIR IN VEHICLES PLYING EGERTON- NAKURU ROAD.

ELVIS MUTISYA NGILA

S12/21139/12

A Research Proposal Submitted to the Undergraduate School in Partial Fulfillment for the Requirement of
Degree in Biomedical Science and Technology of Egerton University.
 DECLARATION AND RECOMMENDATION

DECLARATION

This proposal is my original work and has not been submitted or presented for examination in any other institution.

Elvis Mutisya Ngila.

Signature:……………………………… Date:……………………………….

RECOMMENDATION

This proposal has been submitted for examination with my approval as a University supervisor.

Dr. Lelmen

Egerton University

Signature:……………………………. Date:………………………………
 ABSTRACT

The average human breathes nearly 500 million liters of air in lifetime. Much of the air inhaled contains microorganisms
in the dust, hence forming a potential source of inoculums for respiratory infections. Contamination can also result from
other people especially during talking, spitting, coughing or sneezing. Nowadays people travel from places to places by
vehicles. A good number of populations in towns like Nakuru spend some time travelling, to or from work, school or
hospitals. Passengers travelling are therefore exposed to polluted air inside vehicles. Among other pollutants are molds
and microorganisms some of which are pathogenic and therefore a problem to immunocompromised individuals as they
develop some conditions such as allergies and respiratory diseases.

Therefore there is a need to determine the concentration of molds and microbes in the air inside vehicles and
subsequently isolate and characterize them. The significance of this study will be to understand some of the common
microbes travelers are exposed to and means by which they can be reduced or completely eliminated. Settle plate method
will be used to collect molds and microbes in the air inside vehicles. After incubation, the collected microorganisms will
grow and be counted. Some of them will be identified using morphological structures and biochemical tests.
Table of Contents

DECLARATION AND RECOMMENDATION ....................................................................................................................................... ii

DECLARATION ..................................................................................................................................................................................... ii

RECOMMENDATION ....................................................................................................................................................................... ii

ABSTRACT ........................................................................................................................................................................................... iii

LIST OF ABBREVIATIONS ....................................................................................................................Error! Bookmark not defined.

CHAPTER ONE ...................................................................................................................................................................................... viii

1.0 INTRODUCTION ............................................................................................................................................................................. viii

1.1 BACKGROUND INFORMATION ............................................................................................................................................... viii

1.2 STATEMENT OF THE PROBLEM ................................................................................................................................................ ix

1.3 MAIN OBJECTIVE .......................................................................................................................................................................... x

1.4 SPECIFIC OBJECTIVES .............................................................................................................................................................. x

 1.5 JUSTIFICATION .............................................................................................................................................................................. x

CHAPTER TWO ...................................................................................................................................................................................... xii

2.0 LITERATURE REVIEW ................................................................................................................................................................ xii

2.1 INTRODUCTION TO AIR QUALITY ....................................................................................................................................... xii

2.2 AIRBORNE TRANSMISSION OF DISEASES ........................................................................................................................ xiii

2.3 CULTURING OF MICROBES .................................................................................................................................................. xvi

2.4 ENUMERATION OF MICROBES ............................................................................................................................................ xix

2.5 CONTROL OF AIRBORNE INFECTION ................................................................................................................................ xix

2.6 AIR SAMPLING TECHNIQUES ............................................................................................................................................... xx

2.7 STORAGE OF MICROBIAL STOCK SAMPLES COLLECTED IN AGAR PLATES .......................................................... xxi

CHAPTER THREE ............................................................................................................................................................................... xxiv

3.0 MATERIALS AND METHODS ................................................................................................................................................. xxiv

 3.1 STUDY AREA.......................................................................................................................................................................... xxiv

3.2 STUDY DESIGN ...................................................................................................................................................................... xxiv

3.2.1 Sample Collection .................................................................................................................................................................. xxiv

3.2.2 Sampling Procedure ................................................................................................................................................................ xxv

3.2.3 Statistical Analysis ................................................................................................................................................................. xxvi

CHAPTER FOUR ..................................................................................................................................Error! Bookmark not defined.

4.0 WORK PLAN ................................................................................................................................................................................. xxvii

CHAPTER FIVE .......................................................................................................................................Error! Bookmark not defined.

5.0 BUDGET ....................................................................................................................................................................................... xxviii

6.0 REFERENCES ................................................................................................................................................................................ xxix

LIST OF ABBREVIATIONS

IAQ- Indoor Air Quality

OAQ- Outdoor Air Quality

IEQ- Indoor Enviromental Quality

SBA- Sheep Blood Agar

SDA- Sabouraud Dextrose Agar

WHO- World Health Organisation

m/sec-metres per seconds

 CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND INFORMATION

How safe is the air inside public vehicles that you spend some your time in? Indoor environments are fundamental
environment factors capable of impacting negatively or positively on health. According to (Gupte, 2010) there has never
been pure air. Air quality of indoor environments is one of the main factors affecting health, well being and productivity
of people. Indoor air quality is affected by the presence of microorganisms which include bacteria, molds and viruses
[WHO, 2009], [Wamedo et al.,2012], and people spend 80%-90% of their time in indoor environments [Awad and
Farag,1999], by breathing on average 14m3of air per day [Brochu et al.,2006]. These make people highly exposed to
indoor environments. As of these, in recent years there has been a growing interest in indoor microbe studies -[Awad and
Farag,1999], -[Soto et al.,2009] [WHO, 2009] [Hospodosky et al.,2012]

The activity of people and equipment within the indoor environments is thought to be a principal factor contributing to
the build up and spread of airborne microbial contamination [Hospodosky et al.,2012], [Qian et al.,2012], [Taubel et
al.,2009]. Particular activities like eating, talking, sneezing, coughing, walking and washing can generate airborne
biological particulate matter. Food stuffs, house plants and flower pots, house dust, textiles, carpets, wood material and
furniture stuffing occasionally release various fungal spores into the air. Moreover the environmental factors mainly
include temperature, humidity, air exchange rate, air movement, building structures and location, poor design, ventilation
system as well as interior or redesign which enhance microorganisms growth and multiplication in the indoor atmosphere
[Wamedo et al.,2012], [Meadow et al.,2014], [Graudenz et al.,2005]
A review made by WHO on the number of epidemiological studies show that, there is sufficient evidence for an
association between indoor dampness-related factors and wide range of effects on respiratory health, including asthma
development, asthma exacerbation, current asthma respiratory tract symptoms, cough, wheeze and dyspnoea [WHO,
2009].

Thus microbiological air quality is an important criterion that must be taken into account when indoor places such as
inside public vehicles are designed to provide a safe environment. This research study will provide current concentration
of microorganisms and describe bacterial and fungal loads for different vehicles of Nakuru Town.

Moreover, co-existence of bacteria and fungi will be established to see the impact of environmental factors on their
multiplication and growth in the indoor air of vehicles. Therefore, my study aim will be to establish the number of
microorganisms in the randomly picked personal and public vehicles plying Egerton-Nakuru road.

1.2 STATEMENT OF THE PROBLEM

Air quality plays an important role in occupational and environmental medicine. The population of industrialized areas
like cities and towns spend more than one hour each day in vehicles. In this respect, numerous studies have so far
addressed indoor and outdoor air pollution that arises from traffic. By contrast, only little is known about indoor air
quality in vehicles and influences by non-vehicle sources, therefore my research study aims to investigate indoor air
quality in vehicles. In addition, many airborne factors negatively influence human health. Microbial air contamination
can lead to outbreak of travelling sick problems associated with clinical manifestations such as allergy, rhinitis, asthma
and conjunctivitis. Egerton University and her neighbors, is one of most places in Kenya with a large population that is
affected.
1.3 MAIN OBJECTIVE

To analyze the air quality environment in vehicles plying Egerton-Nakuru Town

1.4 SPECIFIC OBJECTIVES

i. To evaluate the concentration of bacteria and fungi in the vehicles plying Egerton-Nakuru Road
ii. To identify particular microbes available in the vehicles plying Egerton-Nakuru Road.

1.5 HYPOTHESIS

i. The level of concentration of microbial air contamination in public vehicles is high as compared to that of private
vehicles.
ii. Some of indoor microbes in vehicles are pathogenic.

1.6 JUSTIFICATION

Public vehicles are important indoor places where significant numbers of people spend their time, either daily for drivers
and conductors or even some time for most of the travelers. Therefore, indoor air quality of the vehicles should be
optimal in terms of number of microbial contamination. Especially presence of harmful microbes in the air should be
avoided or at least their presence should be below their pathogenic level. The quality of vehicle indoor air should be
improved by procedures such as window-opening or the correct use of fans or automated air conditioning system.
Moreover, in each sampling, the number of occupants in each vehicle will be recorded to evaluate a possible relationship
with the microbial pollution. Identification of microorganisms will also be done by morphological analysis (shape, size,
arrangement, motility, gram stain and spore stain) and biochemical tests (fermentation of glucose, sucrose, lactose and
mannitol among others) (Madigan et al.,2003)

1.6 EXPECTED OUTPUT

i. Different vehicles will have different numbers or concentrations of mold and microbial concentrations upon
counting.
ii. Personal vehicles will have less microbes than public vehicles. This is probably because air inside public
vehicles is more contaminated than in private vehicles.
 CHAPTER TWO

LITERATURE REVIEW

2.1 INTRODUCTION TO AIR QUALITY

Air quality can be defined as the value of air in terms of pollution either from industrial sources or microbial pollution. In
this project microbial air pollution will be of main interest. There are two types of air quality, namely IAQ and OAQ.
Indoor air quality is part of IEQ. Microbial air quality refers to the level of concentration of microbes in the air.
Microbial air quality can either be indoor or outdoor. Indoor air is inside the buildings, vehicles or any other enclosed
space. Outdoor air is simply the atmosphere. The microbial contents of outdoor air depends on: density of human and
animal population, nature of the soil, amount of vegetation, humidity of air, temperature of air, wind conditions, rainfall
and sunlight. Outdoor air contains usually non pathogenic microorganisms like micrococcus, fungi, spores and Bacillus
subtilis. The air in ocean is almost free from bacteria. The number of organisms present in outdoor air is less than in
indoor air. Air at a higher altitude contains much less bacteria (Gupte,2010)

Microbial pollution is a key element of indoor air pollution. Microbial indoor air pollution results in increased
prevalence of respiratory diseases, allergies, asthma and disturbance of the immune system. Some of the common indoor
microbial pollutants include; bacteria, mold spores, algae, microbial volatile organic compounds, mold and mildew
structures. (WHO, 2009)
2.2 AIRBORNE TRANSMISSION OF DISEASES

Air is not a suitable medium for the growth of microorganisms. Organisms found in air are derived from soil, water,
plants, animals, people or other sources (Frobisher et al., 1974). Microbial numbers indoors are considerably higher than
those outdoors and the organisms are the most commonly found in the human respiratory tract. Windblown dust carries
with it significant microbial populations that can travel long distances. Most microorganisms survive poorly in air and so
effective transmittal to another human occurs only over short distances (Madigan et al., 2003).

However, certain human pathogens such as staphylococcus and streptococcus survive under dry conditions fairly well
and remain in dust for long periods of time.

Gram positive bacteria are in general more resistant to drying than gram negative bacteria because of their thicker, more
rigid cell wall. Spore forming bacteria are extremely resistant to drying but are not generally passed from human to
human in the spore form. An enormous number of moisture droplets are expelled during sneezing and a considerable
number are expelled during coughing and talking. Each infectious droplet has a size of about 10 micrometers and
contains one to two bacteria. The speed of the droplet movement is about 100m/sec in a sneeze and ranges from 16-
48m/sec during coughing or shouting. The number of bacteria in a single sneeze varies from 10000 to 100000. Because
of the small size of the droplets, the moisture evaporates quickly in the air leaving behind a nucleus of organic matter and
mucus to which microbial cells are attached (Madigan et al., 2003).

Airborne bacterial diseases affect respiratory tract. These diseases are spread where people are crowded or conditions are
substandard (Frobisher et al., 1974). In an enclosed space like vehicles, occupants are most likely exposed to these
diseases but due to strong immune system they end up being not infected. For people whose body’s defenses are
compromised such as by viral infection, allergic reaction, extensive surgery, malnutrition, excessive smoking or
depression systems are infected in the long run. Such diseases include tuberculosis, meningitis, diphtheria, whooping
cough, Legionnaire’s disease, primary atypical pneumonia, bacterial conjunctivitis, strep throat and scarlet fever. Viral
diseases such as measles, rubella, colds, influenza, chicken pox and shingles are also spread through air. Some airborne
diseases transmitted by exhalation droplets include;

Tuberculosis: Causative agent is Mycobacterium tuberculosis. The disease is usually of crowded populations especially
lower socioeconomic groups; from respiratory droplets of victims. The small rod enters respiratory tract and grows on the
lung issue. Some of the signs and symptoms include; chronic cough, chest pains, high fever and flow of thick,
expectorated matter named sputum among others (Livingstone, 2007).

Meningococcal meningitis: Causative agent is Neisseria meningitidis, a small gram negative diplococcus commonly
called the meningococcus (Gilligan, 2003). The organism enters the body by droplets often from carriers. It may pass
rapidly through the mucous membranes of the upper respiratory tract into the blood stream. Here it multiplies quickly as
large amounts of endotoxins are released. An endotoxin shock called meningococcemia takes place. These events may
occur within several hours of infection and lead to substantial vascular damage or even death. Following blood phase,
they localize on the coverings of the spinal cord and brain known as meninges. Patient experiences violent headaches,
characteristic stiffening of the neck and hemorrhagic rash on the skin.

Haemophilus meningitis: Caused by Haemophilus influenzae, which was first identified by Richard Pfeiffer in
1892,naming it as Pfeiffer’s bacillus. This bacteria is an important cause of meningitis in children between six months
and two years of age. Pattern of infection is similar to that of Meningococcal meningitis (Livingstone, 2009). The
bacterium passes from the throat to the blood and then establishes itself on the meninges where it induces stiff neck,
severe headache and other neurological symptoms. The condition is named Haemophilus meningitis to distinguish it
from meningococcal, fungal and other types of meningitis. In older people Meningitis is caused by Listeria
monocytogenes (Ackerman, 1991).

Diphtheria: This condition is caused by Corynebacterium diphtheriae, a gram positive non-motile aerobic bacterium
whose rods are irregularly shaped or club-shaped. This bacterium causes acute diphtheria in children. It enters via
respiratory route with the bacterial cells lodging themselves onto the throat and tonsils.

Whooping cough: Causative agent is Bordetella pertussis. This bacterium is a small, gram-negative aerobic
coccobacillus bacteria. Whooping cough is a potentially serious childhood respiratory disease that affects children under
five years.

Legionnaire’s Disease: Caused by Legionella pneumophila, a gram negative rod. It is a fairly common form of
pneumonia in older or immunocompromised people. It is seldom transmitted directly from person to person. The
bacterium is an aquatic rod-shaped species with a temperature optimum of about 360C and is a common inhabitant of
warm-water systems in buildings. Infection occurs when people inhale aerosol droplets containing the bacteria.

Primary Atypical Pneumonia (PAP): This type of pneumonia is caused by an Eaton agent known as Mycoplasma
pneumoniae. This bacterium lacks a cell wall hence not susceptible to penicillin. However, erythromycin or tetracycline
have proved effective in treatment. The cell assumes variety of shapes hence said to be pleomorphic. They are passed by
respiratory droplets where crowded conditions exist. The bacterium is common in military installations, college
dormitories and prisons.

Bacterial conjunctivitis(pink eye): This condition is considered as an airborne disease because the causative organisms
are transmitted by respiratory droplets. They may also be passed by face to face contact, contaminated towels, eye gnats
or optometric instrument. The agent is a small gram negative rod named Haemophilus aegypticus. This bacterium causes
conjunctivitis, the disease of the conjunctiva (the thin mucous membrane that covers the cornea and forms the inner eye
lid. The membrane becomes pink with inflammation, hence the name pink eye. It results in copious discharge that runs
down the cheek in the day time and crusts the eyelids shut when person sleeps. The eye becomes swollen and one cannot
see clearly in a bright light, a condition called photophobia.

Strep throat and scarlet fever: This is a common bacterial infection caused by Streptococcus pyogenes. The organism
reaches upper respiratory tract by airborne droplets. It results in high fever and a sore throat. Pharyngeal muscles become
red and a ‘beefy’ appearance about the tonsils occurs with swollen lymph glands in the neck. Many strains of
Streptococcus pyogenes produce toxin called erythrogenic toxins. The toxin damages capillaries and increases their
permeability, allowing blood to leak into the skin tissues. The blush like rash characteristic of scarlet fever soon appears.
Cases of scarlet fever or Streptococcal disease may be followed by rheumatic heart disease or rheumatic fever.

Aspergillosis, Histoplasmosis and Coccidioidomycosis are examples of serious fungal infections of humans iniatiated
by spores deposited in the alveoli. They can be life threatening diseases of immunocompromised people, when the fungi
disseminates from the lungs to the major organs of the body. However, in all cases the infection of the humans is
incidental to the fungus, playing no part in its normal biology. These are fungi that grow naturally as decomposes
organisms in soil, bird faeces or other organic substrates

2.3 CULTURING OF MICROBES

The five I’s according to (Talaro, 2005) are basic techniques used by microbiologists to manipulate, grow, examine and
characterize microorganisms in the laboratory. The procedures make it possible to handle and maintain microorganisms
as discrete entities whose detailed biology can be studied and recorded. Specimen is collected from any object, material
or air spaces which serve as sources of microbes. The common sources are foods, water, air, soil, body fluids and tissues
(Mueller et al., 2011). Specimen are removed by some form of a sampling device which may be a swab, syringes or a
special transport system that holds, maintains and preserves the microbes in the sample. The five I’s include;
Inoculation, Incubation, Isolation, Inspection and Identification (Talaro, 2005).

Inoculation: The sample is placed into a container of sterile medium that provides microbes with appropriate nutrients to
sustain growth. One can use a sterile tool such as a flamed inoculating wire to spread the sample onto the surface of the
solid medium or introduce the sample into a flask or a tube. For air sampling, inoculation involves opening the petri
dishes to let the microbes saturated in the air settle (Fekadu and Melaku, 2014). Selection of media with specialized
functions can improve later steps of isolation and identification. Some microbes may require a live organism (animal,
egg) as the inoculation medium.

Incubation: An incubator is adjusted for proper growth conditions of a sample. Optimum temperature and gas content
should be set properly to promote multiplication of the microbe over the period of predetermined period/time. After
incubation a visible culture growth of the microbe in the medium is produced.

Isolation: The microbe is isolated in macroscopic form. They take the form of separate colonies (discrete mounds of
cells) on solid media or turbidity in broths. The isolated culture can be further be isolated in a process known as
subculturing or passaging. This process involves taking a tiny bit of growth from an isolated colony and inoculating a
separate medium. It is actually one of the ways to make a pure culture that contains only a single species of microbe.

Inspection: The growth cultures are examined or observed macroscopically for obvious growth characteristics such as
colour, texture, arrangement, shape and size that could be useful in analyzing the specimen contents. Slide is also made
to assess the microscopic details such as the cell shape, size and motility. Staining techniques may be used to gather
specific information on the microscopic morphology (Madigan et al, 2003).

Identification: This is the major purpose of a microbiologist as it encompasses determining the type of microbe usually to
the level of species. Characteristics are developed to identify and characterize the microbes isolated. Information can
include relevant data already taken during inspection and additional tests that further describe and differentiate the
microbes. Specialized tests include biochemical tests to determine metabolic activities specific to the microbe,
immunologic test and genetic analysis.

Pure cultures refer to the growth of a single species of microbe (Talaro, 2005). After obtaining a mixture of cultures in a
plate, pure cultures need to be isolated for further studies, identification or storage. There are two main methods of
obtaining pure cultures; Streak plate method and Pour plate method (Black J. G. and Black L. J., 2013).

Streak plate method: Uses agar plates. Microbes are picked up on a sterile wire loop and the wire is moved lightly along
the agar surface depositing streaks of microorganisms on the surface. The inoculating loop is flamed and a few microbes
picked from the region already deposited and streaked onto a new region. Fewer and fewer microbes are deposited as
streaking continues, and the loop flamed after each streaking. Individual organisms are deposited in the region streaked
last. After the plate is incubated, at a suitable growth temperature for the organism, small colonies-each derived from a
single microbial cell appear. A wire loop is used to pick up a portion of an isolated colony and transfer it to any
appropriate sterile medium for further study. The use of aseptic technique ensures that the new medium will contain
organisms of only a single species.

The Pour plate method: Makes use of serial dilutions. A series of dilutions are made such that the final dilution contains
about 1000 microorganisms. Then 1 ml of liquid medium from the final dilution is placed in 9 ml of melted agar medium
(450C) and the medium is quickly poured into a sterile plate. The resulting pour plate will contain a small number of
bacteria, some of which will form isolated colonies on the agar. Because this method embeds some organisms in the
medium, it is particularly useful for growing microaerophiles that cannot tolerate exposure to oxygen in the air at the
surface of the media.

2.4 ENUMERATION OF MICROBES

The number of microbes is established to determine the concentration of microorganisms in a certain specimen collected.
Turbidity readings are useful to evaluate relative amounts of growth. Viable colony count can also be done. Other
methods include; direct or total cell count which involves counting the number of cells in a sample microscopically using
a microscope slide cytometer calibrated to accept a tiny sample that is spread over a premeasured grid. Counting can be
automated by sensitive devices such as the Coulter counter which electronically scans a culture as it passes through a tiny
pipette. As each cell flows by, it is detected and registered on an electronic sensor. A flow cytometer works on a similar
principle, but in addition to counting, it can measure cell size and even differentiate between live and dead cells. When
used with fluorescent dyes and antibodies to tag cells, it has been used to differentiate between gram positive and gram
negative bacteria.

2.5 CONTROL OF AIRBORNE INFECTION

Most airborne diseases are transmitted by either infectious dust or droplet nuclei via the respiratory route. Therefore there
is a great need to control airborne infection through the following methods;
 a) Dilution of contaminated air by ventilation: It is a very effective method especially indoors; however, it is
expensive because of cost of heating and installation of air ducts and blowers. This method only reduces the
number of microorganisms but not necessarily disinfection or sterilization.
b) Irradiation with ultraviolet light: UV light is lethal to numerous microorganisms. A radiation wavelength 254 nm
is generally used as this is sufficiently microbicidal and not excessively irritating. (Frobisher et al.,1974 ).
Ultraviolet producing electric lamps may be attached to walls, overhead or at other strategic points. Deflectors are
used to prevent direct exposure of persons to the rays, which can cause serious burns and protect the eyes which
may be seriously and permanently injured by more than very limited direct observation of an ultraviolet source.
c) Application of Bactericidal vapors and aerosols: Many substances are lethal to microorganisms in the vapor form.
Such substances include; formaldehyde, propylene glycol, triethylene glycol, ethylene oxide and beta-
propiolactone. (Frobisher et al.,1974 ). Propylene glycol and triethylene glycol are the most effective for
disinfection of air. They are odorless, tasteless, non-irritating, non-toxic and not corrosive or explosive.
Furthermore, they are highly effective in killing bacteria in the air in vapor form. Other agents such as ortho-
phenyl phenol and related compounds are recommended for surface applications to supplement aerosols as phenyl
phenol clings to the surfaces on which dust settles rendering them bactericidal for prolonged periods under
ordinary conditions.

2.6 AIR SAMPLING TECHNIQUES

A number of methods can be used to test air for mold and microbial contamination including:

 The settle plate method: This method is also called sedimentation method. It is based on the principle of
measuring the rate at which microbial carrying particles settle down by gravity (Gupte,2010). Open petri
dishes or plates containing different culture media are exposed to air for a certain predetermined period and
 incubated at 370C for 24 hours. The number of colonies are then counted. The method gives an impression
about the numbers of microorganisms and species present in air. Blood agar plates can be used to detect both
pathogenic and non-pathogenic organisms and are especially used for testing air in surgical theatres and
hospital wards. This method is simple but capable of measuring only the rate of deposition of large particles
from air. For mold testing, agar plates containing malt extract agar (MEA) supplemented with antibiotics to
suppress bacterial growth would be used. Alternatively, Sabouraud Dextrose Agar can be used for
determination of fungal contamination. Tryptone soy Agar can also be used for the determination of bacterial
contamination. Counting of colonies can be achieved by hemacytometer or direct plate counting in terms of
colony forming units [CFU’s] per unit time. The counted colonies can then be further characterized to genera
or species. Higher numbers of CFU’s and/or presence of pathogenic or toxigenic molds such as Aspergillus
fumigatus and Stachybotyrs chartarum are indicators of a problem (Fekadu and Melaku, 2014)
 Silt sampler method: It gives count bacteria carrying particles containing a given volume of air. This
method is better than settle plate technique as efficiency of detection of even small particles containing
microorganisms is quite high. A known volume of air is directed onto a plate of culture medium through a slit
of 0.25 mm width, the plate being mechanically rotated in order to ensure uniform distribution of organisms
all over the plate containing the medium (Gupte, 2010). One cubic foot of air is allowed to pass through the
slit and likewise ten cubic feet of air is tested. The culture media are incubated and colonies counted which
gives the bacterial number present in the air.

2.7 STORAGE OF MICROBIAL STOCK SAMPLES COLLECTED IN AGAR PLATES

Microbial samples collected can be stored for future research, diagnostic or teaching purpose. Certain factors need to be
considered though. Some important considerations are microbial compatibility, experimental purpose and cell viability.
The specific length of time a culture will remain viable in a given storage condition depends upon the microbial strain.
Although cell death during storage is inevitable, it should be minimized as much as possible. For the microbial cultures
that are used regularly, either daily or weekly, they can be stored on agar plates or in stab cultures in a refrigerator at 40C.
But if cultures will not be used for long, more long term storage method should be considered for maximum bacterial
viability. Therefore, storage of microbial cultures can be categorized as short-term or long-term.

Short-term storage: This can either be on agar plates or agar stab cultures. Working bacterial stocks can be streaked
onto agar plates and stored at 40C for daily or weekly use. The culture petri dishes should then be wrapped with a
laboratory sealing film (plastic or paraffin) and stored upside down (agar side up) to minimize contamination and keep
both the culture and agar properly hydrated. Microbial strains in agar stab cultures can be transported to other research
facilities. Stab cultures are prepared by first sterilizing the agar and then transferring the warm liquid agar to screw cap
vials using the appropriate aseptic technique. After the agar has solidified, a single colony is picked from an actively
growing culture using a sterile, straight wire. The wire with the bacteria is then plunged deep into the soft agar several
times, and the vial is incubated at 370C (ideal temperature conditions) for a specified period of time with the cap slightly
loose. The vial is then sealed tightly and stored in the dark at 40C (Perry, et al., 2002)

Long term storage: Decreasing temperature can maximize microbial cells viability. Freezing and thawing cells at an
appropriate rate and maintaining the frozen stocks at the proper storage temperature help to minimize damage from the
freezing process. The cells should also be in greater density for better recovery after thawing of cells especially a density
of 107 cell/mL results in adequate recovery if all conditions are properly maintained. Use of cryoprotectants such as
dimethylsulfoxide and glycerol in the right percentages volume is also necessary (5-15% v/v). During freezing water in
cells is converted to ice, solutes accumulate in the residual free water. This localized increase in salt concentration can
denature biomolecules in microbial cultures. In addition ice crystal formation can damage cell membranes. Therefore,
cryoprotectants additives are mixed with the microbial suspension before freezing to lower the freezing point and protect
the cells during freezing to minimize the detrimental effects of increased solute concentration and ice crystal formation.
Non permeable additives used as cryopreservants such as polysaccharides, proteins and dextrans, adsorb to the surface of
microorganisms and form a viscous layer that protects membranes, making these agents particularly useful for
cryopreservation. Other commonly used additives include blood serum, ethylene glycol, methanol, skim milk, yeast
extracts and tripticase soy

Freezing samples: To prepare glycerol stocks, the glycerol is first autoclaved and allowed to cool. The appropriate
volume of the glycerol is added to a suspension of log phase bacteria and vortexed to dissociate the cells and ensure even
mixing of the bacteria with the glycerol. After aliquoting the suspension into cryogenic screwcap vials, the cells are snap-
frozen by immersing the tubes either in ethanol-dry ice or liquid nitrogen and then stored in freezers (-200C to -800C) or
liquid nitrogen (-1500C). Repeated thawing and freezing of the microbial stocks reduces cell viability and therefore
should be avoided. When recovering strains with antibiotic selection markers, culturing them on selective media will
ensure that the bacterial stocks were not contaminated (Brown, 2000)

Freeze-drying: Microbes can be freeze-dried by suspending log-phase cells in a lyophilization medium and then freeze-
drying the suspension. Not all microbes can be successfully freeze-dried. Certain strains might not survive the process or
die rapidly once freeze-dried. Once freeze dried, it is best to store the bacteria at or below 40C.
 CHAPTER THREE

MATERIALS AND METHODS

3.1 STUDY AREA

Egerton University is a public institution in Kenya with its main campus located at Njoro, near the town of Nakuru,
Kenya. The institution was founded in 1939 as a school to prepare White European youth for Careers in Agriculture. In
1987 it was recognized as a Chartered Public University. The University currently holds a great population. It is located
about 20 km from Nakuru.

The study will involve vehicles travelling from Egerton, Njoro to Nakuru Town. The vehicles will be randomly recruited
ranging from Egerton Welfare Buses, Nissan Matatus and Personal Cars. The study will be conducted between January
to May, 2016.

3.2 STUDY DESIGN

3.2.1 Sample Collection

Bacteria and fungi present inside vehicles will be collected by passive air sampling procedure. The settle plate method
using 9cm diameter Petri dishes will be particularly used. The sampling height will be approximated to human breathing
zone that is one meter above their feet when seated and at the centre of the vehicle. Two of the plates in each vehicle will
contain 2% Nutrient Agar and the other two 4% SDA. The sampling times will be set at 30 minutes for twice a day. The
morning sample will be done from 9.30 am while the evening sample will be done from 4.00 pm EAT.

3.2.2 Sampling Procedure

After exposure the samples will be taken to the laboratory{Department of Biochemistry and Molecular Biology, Egerton
University} and incubated at 370C for 24 hours for bacteria and at 250C for 3 days for fungi. Colony forming units (CFU)
will be enumerated. In addition CFU/m3 will be determined according to (Omeliansky, 1940);

N= 5a×104 (bt)-1

Where: N=Microbial CFU/m3 of indoor air.

a=Number of colonies per petri dish

b=Dish surface area in cm2

t=Exposure time in minutes

Thereafter identification of isolates according to standard methods will be done.

3.2.3 Statistical Analysis

Statistical difference between the concentration of bacteria and fungi will be measured at different sampling splices as
well as linearity between the concentration of bacteria and fungi. The indoor air microbial loads of nine vehicles plying
Egerton-Nakuru Road will be analyzed into the concentration, concentration range, arithmetic mean and standard
deviation. The type of microorganisms will also be indicated.

3.3 Data analysis

Data obtained will be recorded in tables. This will help in comparing the concentration of microbes in different vehicles
and draw conclusions from the same. Tabulation of the results will also compare the sampling times of day at different
times of exposure. The type of microorganisms isolated from each vehicles plying Egerton-Nakuru Road will be
tabulated. Line of best fit will also be drawn.
 WORK PLAN
TIME ACTIVITIES

SEPT OCT NOV DEC JAN-16 FEB-16 MAR APR MAY

15TH 15TH 15TH 15TH 16TH 16TH 16TH
Identifying a Supervisor

Writing a concept note

Writing Project Proposal

Submission to the Supervisor

Collection of Samples

Carrying out Practical

Report Writing

Report Presentation and

Defending of the Hypothesis
ITEM PARTICULARS QUANTITY UNIT COST TOTAL COST
A. LABORATORY CHEMICALS
Nutrient Agar 500g 3500 3500
Sabouraud Dextrose Agar 500g 5000 5000
Alcohol 1l 2500 2500
SUBTOTAL 11000

B. MATERIALS AND APPARATUS

Aluminium foil 2 rolls 400 800
Disposable Surgical Rubber Gloves 1 pkt 300 300
Stationery materials, Printing, Photocopy and 1000
Internet
Microscope Slides 2 pkts 1000 2000
Petri Dishes 100 pieces 40 4000
SUBTOTAL 8100

C. PERSONAL AND LOCAL

TRAVEL
Communication 1000
Transport 2000

CONTINGENCY ( 10% OF SUBTOTAL 3430

A+B+C)
GRAND TOTALS 35310
SOURCE OF FUNDING: UNIVERSITY AND SELF
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