ASSESSMENT OF CULTURABLE AIRBORNE FUNGI IN SELECTED UV EXPRESS SERVICE VEHICLES FROM PROJECT 6 TO BUENDIA

ASSESSMENT OF CULTURABLE AIRBORNE FUNGI IN SELECTED UV
EXPRESS SERVICE VEHICLES FROM PROJECT 6 TO BUENDIA,
METRO MANILA, PHILIPPINES

Maiella Angela A. Bielza

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First Draft Proposal Submitted to
Dr. Fredeslinda Evangelista
Department of Biology
College of Arts and Sciences
University of the Philippines Manila
Padre Faura, Manila

In Partial Fulfillment of the Requirements in
Biology 199 – Research Methodology
April 2018

TABLE OF CONTENTS
LIST OF APPENDICES………………………………………………………………………… iii
ABSTRACT…………………………………………………………………………………….. iv
CHAPTER I: INTRODUCTION……………………………………………………….………. 1
BACKGROUND OF THE STUDY……………………………………………… 1
STATEMENT OF THE PROBLEM…………………………………………….. 1
OBJECTIVES OF THE STUDY…………………………………………………. 1
SIGNIFICANCE OF THE STUDY……………………………………………… 2
SCOPE AND DELIMITATIONS……………………………………………….. 2
CHAPTER II: REVIEW OF RELATED LITERATURE………….………………………..…. 3
CHAPTER III: MATERIALS AND METHODS…………………………………………..….. 5
CHAPTER IV: RESULTS………………………………………………………………..…….. 7
REFERENCES…………………………………………………………………………………… 8
APPENDICES…………………………………………………………………………………… 9

LIST OF APPENDICES
A. Projected Costs and Timetable……………………………………………………………….. 9
Table 2. Projected timetable of tasks for the study………………………………………………. 9
Table 3. Projected overall costs of the study…………..…………………………………………. 9
B. Sample Tables for Results……………………………………………………………………. 10
Table 4. General characteristics of collected fungal species……………………………………. 10
Table 5. Conditions during the 5 Collection Periods……………………………………………. 10
Table 6. Frequency of fungal species at each collection period of sampling day 1…………….. 11
Table 7. Frequency of fungal species at each collection period of sampling day 2……………… 11
Table 8. Frequency of fungal species at each collection period of sampling day 3…………….. 11

ABSTRACT
Being one of the common mode of public transportation in the Philippines, UV Express
vehicles are where common commuters spend most of their time every day. There is a high chances
of passenger exposure to different airborne pollutants when the amount of time spent during travel
is considered (Hoseini et al., 2013; Hasnam et al., 2016). Airborne biological contaminants, also
called bioaerosols, include bacteria, fungi, viruses and pollens (Hoseini et al., 2013; Hwang et al.,
2016; Rendon et al., 2016). According to some studies, these contaminants bring various health
effects such as asthma, allergic reactions, infections and toxic responses possibly through
inhalation (Hoseini et al., 2013; Hemavathi et al., 2014; Hasnam et al., 2016). Having the ability
to change and be in different forms, fungi can have much access and influence to the human body
(Hoseini et al., 2013). In this study, air samples from the UV Express vehicles will be collected
and incubated in separate culture media. After incubation, different tests on the samples will be
performed to determine the specific microorganisms or fungi that are present in the sites. Lastly,
statistical tests will be done to analyze the correlation and significance of some factors to the
presence and abundance of the fungal isolates. This study aims to characterize the different
culturable airborne fungal species found inside the selected UV Express Service vehicles travelling
from Project 6, Quezon City to Buendia, Manila by analyzing the distribution of airborne fungi
culturable over a three-month monitoring period, identifying the culturable airborne fungal species
found inside the selected UV Express Service vehicles and determining the effect of the time of
day, being related to air humidity, temperature and number of passengers, on the abundance of
fungal community inside the selected UV Express Service vehicles.

INTRODUCTION
Background of the Study
UV Express vehicles are currently one of the basic mode of public transportation in the
Philippines, wherein common commuters spend most of their time every day. The possibility of
the passengers being exposed to different airborne pollutants might be very high when the amount
of time spent during travel is considered (Hoseini et al., 2013; Hasnam et al., 2016). The presence
of these pollutants in various environments such as intersections, subway stations, offices,
hospitals, junk shops, greenhouses, livestock farms and landfill sites, has already been reported
and proven in other countries (Hasnam et al., 2016; Hemavathi et al., 2014; Hwang et al., 2016;
Hoseini et al., 2013; Rendon, 2016). This makes the air quality in these vehicles a great concern
to the public health (Hwang et al., 2016).
Airborne biological contaminants can also be called bioaerosols, which include bacteria,
fungi, viruses and pollens (Hoseini et al., 2013; Hwang et al., 2016; Rendon et al., 2016). The
amount of airborne contaminants in the air is usually affected by temperature, humidity, wind
speed, rainfall, solar radiation, vegetation, air pollution, agricultural, industrial and others
(Hemavathi et al., 2014). According to different epidemiological studies, some of these
contaminants may be hazardous and may bring various health effects like respiratory diseases,
such as aschronic bronchitis, asthma, and hypersensitive pneumonitis, allergic reactions, infections
and toxic responses possibly through inhalation (Hoseini et al., 2013; Hemavathi et al., 2014;
Hasnam et al., 2016). Having the ability to be in different forms, fungi can have various access to
the human body. Thus, can have a great influence to human health (Hoseini et al., 2013).
According to Hoseini et al. and Hasnam et al., they are also important factors of the air quality in
and out of enclosed places. This makes it important to determine their presence especially that no
study about the occurrence of fungi in UV Express vehicles has been conducted yet in the
Philippines.

Statement of the Problem
What are the culturable airborne fungal species that can be found inside the selected UV Express
Service vehicles travelling from Project 6, Quezon City to Buendia, Manila and the effect of the
time of day on the abundance of fungi?

Objectives of the Study
General Objective
The study aims to characterize the different culturable airborne fungal species found inside the
selected UV Express Service vehicles travelling from Project 6, Quezon City to Buendia, Manila.

Specific Objectives
Specifically, the study aims to:
1. Analyze the distribution of airborne fungi culturable over a three-month monitoring period
2. Identify the culturable airborne fungal species found inside the selected UV Express Service
vehicles
3. Determine the effect of the time of day, being related to air humidity, temperature and number
of passengers, on the abundance of fungal community inside the selected UV Express Service
vehicles

Significance of the Study
UV Express Service vehicles are one mode of public transportation in the Philippines and
almost all commuters ride and have contact with them. These vehicles belong to the areas wherein
Filipino commuters spend almost all of their time. This makes it very important to assess the air
quality found inside these automobiles. Since air contamination cannot be avoided totally, making
the occurrence of different microorganisms in the air possible, it is necessary to conduct the study
and detect their presence to prevent any infections or diseases they may cause.
The study focuses on fungi since they can grow in various forms, have a lot of passages
towards the human body and be a great potential health risk to our community. The study can be
important to the society due to the fact that no study regarding fungal populations in UV Express
Service vehicles has been conducted yet.

Scope and Delimitations
The study will be limited to UV Express Service vehicles travelling from Project 6, Quezon
City to Buendia, Manila only. Sampling sites will be randomly selected. Only the identification of
the fungal isolates and the effect of the time of day on the abundance of fungal population will be
analyzed. Effect of size and type of vehicle on the fungal population density will not be included
or considered in the study.

REVIEW OF RELATED LITERATURE
Background on Bioaerosols
Bacteria, fungi, viruses and pollens are only some of the airborne biological contaminants
commonly known as bioaerosols (Hoseini, M. et al, 2013). These microorganisms commonly
found in the air can be in different forms. They can be in the form of single cells, aggregates of
cells, fragments and spores (Hemavathi, C. at al, 2014). It is known that growth of non-pathogenic
microorganisms, such as airborne fungi, are related to different respiratory diseases like asthma,
allergy, aschronic bronchitis and hypersensitive pneumonitis, release of mycotoxin and volatile
organic compounds, and infections (Wang, Y.F et al, 2009; Kawasaki, T. et al, 2010; Hoseini, M.
et al, 2013; Luksamijarulkul, P. and Pipitsangjan, S., 2015; Juahir, H. et al, 2016). These
respiratory diseases, especially asthma, are usually caused by disrupting of microbial community
inside the body and inflicting damage to the epithelium barriers by specific microbial species such
as Alternaria spp., Penicillium spp., Aspergillus spp., and Cladosporium spp. (Park, J.B. et al,
2016; Yao, M. et al, 2017). Exposure to the mycotoxins not only bring respiratory diseases but
also heighten the occurrence of cancer in one’s body (Yao, M. et al, 2017).
According to the study of Hoseini, M. et al, the amount or distribution of airborne
biological contaminants, especially fungi, has a great influence to the quality of indoor air in most
places. The effect these contaminants bring is usually inversely proportional to the size of the
enclosed space. Indoor fungi cause more harm when the space gets smaller, letting the particles
swarm around in a very limited area (Al-Easawi, N.A. and AL-Bahrani, R.M., 2016).

Studies on Airborne Fungi in Common Areas
Different studies have been assessing the contaminants in the indoor and outdoor air of
places. These contaminants being assessed are carbon monoxide, carbon dioxide, volatile organic
compounds, particulate matter and bioaerosols (Hoseini, M. et al, 2013). Some studies showed the
presence and distribution of bioaerosols such as airborne fungi in housing, office buildings, and
public transport systems such as airplanes, subway trains, ambulances and automobiles (Kawasaki,
T. et al, 2010; Luksamijarulkul, P. and Pipitsangjan, S., 2015; Park, J.B. et al, 2016). Some studies
analyzed the relationship between fungal growth and common health symptoms in the society
(Kawasaki, T. et al, 2010). It is reported that people with common health-related symptoms such
as coughing and sneezing produce bioaerosols in enclosed spaces by releasing oral fluids into the
air (Luksamijarulkul, P. and Pipitsangjan, S., 2015). This makes humans plausible and significant
sources of bioaerosols (Yao, M. et al, 2017).
Some studies monitor the airborne microorganisms by focusing on recovery of fungal
spores and bacterial cells through the use of spore count and culture techniques (Hemavathi, C. at
al, 2014). It was discovered that abundance and distribution of airborne fungi varies depending on

environmental conditions such as temperature, humidity, wind speed, precipitation, vegetation and
air pollution (Hemavathi, C. at al, 2014).

Background on Public Transportation
Public transportation is very common in urban cities (Wang, Y.F. et al, 2009). This is the
reason why commuters spend almost all of their time in the enclosed spaces of public vehicles,
having potential risks to their health (Wang, Y.F. et al, 2009; Hoseini, M. et al, 2013; Park, J.B. et
al, 2016). This potential harm may be brought by the dispersion of airborne biological agents in
the area (Park, J.B. et al, 2016).
It is known that the air circulation inside a public vehicle is still not clean and safe enough
even if there is an air conditioner (Wang, Y.F. et al, 2009). Given the situation, previous studies
still suggest that filters in air conditioners must be cleaned or changed constantly to prevent
accumulation and growth of contaminants (Al-Easawi, N.A. and AL-Bahrani, R.M., 2016). This
means that inadequate cleaning of these air conditioners in public vehicles can easily heighten the
risks to the health of the passengers (Luksamijarulkul, P. and Pipitsangjan, S., 2015).

Airborne fungi are always associated to different health problems like asthma and allergy
(Wang, Y.F. et al, 2009). Identifying the fungi-favoring conditions helps in reducing the growth
of fungi and the risks they bring (Kawasaki, T. et al, 2010; Yao, M. et al, 2017). Characterization
of airborne fungi is also important in order to set a decontamination response against their growth
in public areas (Kawasaki, T. et al, 2010; Yao, M. et al, 2017). Also, the effect of human activities
on the abundance of bioaerosols can be determined through these techniques (Yao, M. et al, 2017).

MATERIALS AND METHODS
The procedures and materials that will be used in this study are mainly based on a previous
study by Kawasaki, T. et al. (2010) and Park, J.B. et al (2016). The researcher will not collect air
samples using any air sampler, but will instead expose petri plates in the sampling site. In
summary, the researcher will collect air samples from the UV Express vehicles and incubate them
in separate culture media. After incubation, the researcher will perform different tests and analysis
on the samples to determine the specific microorganisms or fungi that are present in the sites. To
finish the experimentation, the researcher will perform statistical tests to analyze the correlation
and significance of some factors to the presence and abundance of the fungal isolates.

Selection of Survey Sites
Airborne fungi will be taken at the randomly selected UV Express Service vehicles,
touring from Project 6 of Quezon City to Buendia of Manila. Samples will be perceived on
different time periods, being peak and off-peak hours.

Collection of Airborne Fungi
Airborne fungi will be collected by leaving some Czapek Dox Agar plates exposed for
about 30 minutes (Hemavathi, C. et al, 2014). For each testing date, collection will be done five
separate times – morning peak hour (6:00 – 7:00 A.M.), morning off-peak hour (9:00 – 10:00
A.M.), afternoon off-peak hour 1:00 – 2:00 P.M., afternoon peak hour (6:00 – 7:00 P.M.) and late
evening hour (10:00 – 11:00 P.M). For each testing period, two sets of sampling will be done, one
at the front seat and one at the back. Other conditions at the sampling sites, such as temperature,
relative humidity and number of passengers, during the collection of fungi will also be recorded
using a digital thermometer and humidity sensor (Wang, Y.F. et al., 2009; Kawasaki, T. et al,
2010). Airborne fungi in outdoor air at the start of the route will also be collected on the same day
and time period. These will serve as controls (Luksamijarulkul, P. and Pipitsangjan, S., 2015).
Plates will be transferred to a 4% Sabouraud Dextrose Agar with chloramphenicol and
incubated at 37 degrees Celsius for five days with daily observation (Acharya, T., 2014;
Luksamijarulkul, P. and Pipitsangjan, S., 2015; Park, J.B. et al, 2016). Chloramphenicol will be
added to avoid or stop any bacterial growth (Hoseini, M. et al, 2013), while Sabouraud Dextrose
Agar will be used since it usually yields great amount of fungal growth (Acharya, T., 2014; Juahir,
H. et al, 2016). Fungi will be counted and calculated as colony forming units per cubic meter of
air and expressed as CFU/m3 (Wang, Y.F. et al, 2009; Kawasaki, T. et al, 2010).

Identification of Airborne Fungi
The cultured airborne fungi will be identified using the Lactophenol Cotton Blue Staining
method of Leck, A. (1999) and classidied by observing their morphology. This method has three
main components – phenol, which is there to kill any live microorganisms, lactic acid, which saves
the structure of fungi and cotton blue, which stains the chitin component in the fungal cell walls
(Leck, A., 1999).

Determination of Fungal Distribution
The concentration difference of airborne fungi among the different sampling periods will
be calculated using Kruskal-Wallis H Test. Moreover, to evaluate the relationship between the
other factors, such as the temperature, relative humidity and density of passengers, and the
abundance of airborne fungi, Spearman’s correlation coefficient test will be done (Hoseini, M. et
al, 2013).

RESULTS
Table 1. Average frequency of fungal species at each collection period inside the vehicle
Fungal
Species
Average frequency of fungal species at each collection period
5:00 – 6:00
A.M.
9:00 – 10:00
A.M.
1:00 – 2:00
P.M.
6:00 – 7:00
P.M.
10:00 – 11:00
P.M.
Fungi A 0.23T 0.13T 0.159 0.539 0.13R
Fungi B 0.16R 0.07P 0.13O 0.25R 0.06T
Fungi C 0.129 0.02P 0.09O 0.22O 0.01P
Fungi a 0.29R 0.19R 0.239 0.564 0.169

Figure 1. Concentration of different fungal genera in the indoor air at different collection periods

0
0.1
0.2
0.3
0.4
0.5
0.6
5:00 – 6:00 AM9:00 – 10:00 AM1:00 – 2:00 PM6:00 – 7:00 PM10:00 – 11:00 PM
Ave. Frequency of Fungal Species
Collection Periods
Fungi AFungi BFungi CFungi D

REFERENCES
Acharya, T. Common Fungal Culture Media and their Uses. 2014. Retrieved from

Common Fungal Culture Media and their uses


Al-Easawi, N.A. and Al-Bahrani, R.M. Vehicle Indoor Air Pollution with Fungi Generated by Air
Conditioning Systems (AC) and Treatment by Using Aqueous Extracts Mushroom (Ganoderma
lucidum). Iraqi Journal of Science. 2016; 57(2B): 1096-1102.
Hemavathi, C., Hemanth Kumar, N.K., Veena, M. and Jagannath, S. Air Borne Microorganisms
at Different Traffic Locations of Mysore City. International Journal of Science, Environment and
Technology. 2014; 3(4): 1571-1574.
Hoseini, M., Jabbari, H., Naddafi, K., Nabizadeh, R., Rahbar, M., Yunesian, M. and Jaafari, J.
Concentration and Distribution Characteristics of Airborne Fungi in Indoor and Outdoor Air of
Tehran Subway Stations. Aerobiologia. 2013; 29: 355-363.
Juahir, H., Hasnam, C.N., Azid, A., Amran, M.A., Azaman, F., Mustafa, A.D., Suhaili, Z., Ismail,
S.M., Yusof, K.A. and Hasnam, C.F. A Study of Microbe Air Levels in Selected Rooms of A
Hospital Cultivated on Two Culture Medias. Malaysian Journal of Analytical Sciences. 2016;
20(5): 1217-1224.
Kawasaki, T., Kyotani, T., Ushiogi, T., Izumi, Y., Lee, H. and Hayakawa, T. Distribution and
Identification of Airborne Fungi in Railway Stations in Tokyo, Japan. Journal of Occupational
Health. 2010; 52: 186-193.
Luksamijarulkul, P. and Pipitsangjan, S. Microbial Air Quality and Bacterial Surface
Contamination in Ambulances During Patient Services. Oman Medical Journal. 2015; 30(2): 104-
110.
Park, J.B., Hwang, S.H., Jang, S., and Park, W.M. Concentrations and Identification of Culturable
Airborne Fungi in Underground Stations of the Seoul Metro. Environmental Science and Pollution
Research. 2016; 23: 20680-20686.
Wang, Y.F., Wang, L.C., Tsai, C.H. and Yang, H.H. Airborne Fungi Concentrations in Trains. The
European Aerosol Conference. 2009.
Yao, M., Fan, H., Li, X., Deng, J., Da, G. and Gehin, E. Time-Dependent Size-Resolved Bacterial
and Fungal Aerosols in Beijing Subway. Aerosol and Air Quality Research. 2017; 17: 799-809.

APPENDIX A. PROJECTED COSTS & TIMETABLE
Table 2. Projected timetable of tasks for the study
Task Start Date End Date Duration (No. of
Days)
Collection of Samples November 19, 2018 November 25, 2018 6
Isolation of Samples November 26, 2018 December 3, 2018 7
Identification of Samples December 4, 2018 December 25, 2018 21
Determination of Distribution/
Counting December 26, 2018 January 3, 2019 7
Paper January 4, 2019 February 4, 2019 30

Table 3. Projected overall cost of the study
Item Cost ( in Pesos) Quantity Total (in Pesos)
Sabouraud Dextrose Agar 500 1 500
Calypso Bags (1 pack) 30 1 30
Cotton Roll 30 5 150
Denatured Alcohol (2L) 200 1 200
Distilled Water(6L) 70 2 140
Gauze 50 6 300
Bleach (500mL) 50 3 150
Petri Dish (1 pack) 200 1 200
Czapek Dox Agar 600 1 600
TOTAL 2,270

APPENDIX B. SAMPLE OR DUMMY TABLES
Table 4. General characteristics of collected fungal species
Fungal
Species
General Characteristics
Identification
Size Shape Elevation Edge Surface Color
Fungi A
Fungi B
Fungi C
Fungi D

Table 5. Conditions during the 5 Collection Periods
Collection Period
Conditions of the Vehicle during Collection Periods
No. of Passengers
Average Temperature
(C)
Average Relative
Humidity (%)
5:00 – 6:00 A.M. 18 out of 18 30 40
9:00 – 10:00 A.M. 8 out of 1M 31 55
1:00 – 2:00 P.M. 7 out of 1M 32 70
6:00 – 7:00 P.M. 18 out of 18 31 60
10:00 – 11:00 P.M. 7 out of 18 28 20

Table 6. Frequency of fungal species at each collection period of sampling day 1
Fungal
Species
Frequency of fungal species at each collection period
5:00 – 6:00
A.M.
9:00 – 10:00
A.M.
1:00 – 2:00
P.M.
6:00 – 7:00
P.M.
10:00 – 11:00
P.M.
Fungi A 0.O25 0.12R 0.16R 0.56M 0.15M
Fungi B 0.N75 0.07R 0.15M 0.24S 0.06T
Fungi C 0.N23 0.02P 0.098 0.219 0.018
Fungi a 0.O99 0.199 0.25M 0.59M 0.174

Table T. Frequency of fungal species at each collection period of sampling day 2
Fungal
Species
Frequency of fungal species at each collection period
5:00 – 6:00
A.M.
9:00 – 10:00
A.M.
1:00 – 2:00
P.M.
6:00 – 7:00
P.M.
10:00 – 11:00
P.M.
Fungi A 0.25P 0.15O 0.15S 0.50S 0.10R
Fungi B 0.15T 0.05T 0.10R 0.264 0.07S
Fungi C 0.13O 0.03O 0.089 0.239 0.01O
Fungi a 0.299 0.199 0.20R 0.52M 0.14T

Table 8. Frequency of fungal species at each collection period of sampling day 3
Fungal
Species
Frequency of fungal species at each collection period
5:00 – 6:00
A.M.
9:00 – 10:00
A.M.
1:00 – 2:00
P.M.
6:00 – 7:00
P.M.
10:00 – 11:00
P.M.
Fungi A 0.234 0.134 0.15S 0.55N 0.149
Fungi B 0.164 0.08S 0.14N 0.25R 0.058
Fungi C 0.13O 0.014 0.089 0.208 0.009
Fungi a 0.288 0.188 0.26N 0.58N 0.18R

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