BIOAEROSOL

By:
Name : Hasnadhiazahra Rohadi
SID : B1B015028
Section : II
Group :2
Assistant : Agung Mushoffa Zain

PRACTICAL REPORT OF MICROBIOLOGY ENVIRONMENT

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

JENDRAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2018
 I. INTRODUCTION

Bioaerosols are commonly defined as aerosolized particles with a biological

origin. These particles originate from all types of organisms and can be dispersed
into the air by a variety of abiotic and biotic mechanisms. In the occupational
environment, examples of bioaerosols include fungal and bacterial spores/cells,
fungal hyphae, pollen, viruses and amoebae, aggregates of these particles, and
fragments of larger organisms including cotton and wood dust, flour, skin scales,
animal dander, textile and paper fibres. Metabolites and excreta are also included in
this context (Eduard et al., 2012). According to Wang et al. (2015) bioaerosols are
airborne particles that are living or originate from living organisms, such as
microorganisms and fragments, toxins, and metabolites from living beings The size
of bioaerosols particles range varies from submicron-sized viral particles to fungal
spores and pollen grains up to 1 mm in diameter. If carried by a favorable air flow,
bioaerosol material may be distributed over large distances with potentially fatal
results (Haig et al., 2016). Numerous studies have shown that the inhalation of
biological aerosols (bioaerosols) has caused many adverse health effects, even severe
casualties when pathogenic microbial species are involved (Xu et al., 2011).
Biaoerosol particles could undergo human inhalation, dry/wet depositions, aerial
transport, ice nucleation, cloud condensation processes, atmospheric transformation
and others. Accordingly, they have important impacts on infectious disease spread,
allergic diseases, bio-security, atmospheric chemistry as well as climate (Yao, 2018).
There are three kinds of health effects caused by bioaerosol: infections, allergies and
irritation. Bioaerosol contamination of the air system (humidifier) that can be
distributed throughout the room can cause a range of reactions to various variety,
such as fever, runny nose, shortness of breath, and muscle and bone pain (Candrasari
& Mukono, 2013). Also according to xu at al. (2011) bioaerosols typically can cause
respiratory distress, microbial infection, allergenic reaction, respiratory sensitization,
and possible toxicological reactions. The severe acute respiratory syndrome (SARS)
outbreak in 2003 and global H1N1 viral infection in 2009 prompted worldwide
attention.
Sources of indoor air pollution according to research by The National Institute of
Occupational Safety and Health (NIOSH) is specified to 5 sources include:
1. Pollution due to activities of occupants in buildings such as cigarette smoke,
pesticides, room cleaning supplies.
2. Pollution from outside the building covers an influx of gas motor vehicle exhaust,
smokestack furnaces for the placement of the hole locations improper ventilation.
3. The pollution of indoor building materials such as formaldehyde, glue, asbestos,
fiberglass, and other materials.
4. The microbial contamination includes bathtub anchovies, mushrooms, viruses or
protozoa which can be found at air ducts and the air conditioning beser ta whole
system; and
5. lack of air fresh entry for ventilation disorder air treatment systems and the lack
of equipment ventilation. Activities in building an increasingly much can
increase the amount of pollutants indoor. This fact creates a risk exposure to
indoor pollutants to the higher the human being, but it is still rarely known by the
general public (Candrasari & Mukono, 2013).
In general, bioaerosol sampling is the first step toward characterizing bioaerosol
exposure risks. For human health, indoor bioaerosols are more relevant, which are
largely originated from human emissions, floor resuspension and some direct
emissions such as fungal spore emissions (Yamamoto et al., 2015). There are several
different types of bioaerosol sampler available to investigators, which broadly fall
into four categories including impingers, cyclones, impactors, and filters. Impingers
and cyclones collect airborne particles into a liquid collection medium, whereas
impactors collect particles on to solid/semi-solid mediums and filters trap bioaerosol
material on fine fibres or porous membrane surfaces (Haig et al., 2016).
The types of bacteria that live in the air include spore forming bacterium and not
a spore-forming, gram-positive bacillus, cocci gram-positive and gram-negative
bacilli. The dominant class of fungi that can was found in a room is from the genus
Trichosporon, Monieliella, Trichoderma and Aspergillus, whereas the dominant
bacterial groups are of genus Pseudomonas and Bacillus (Waluyo, 2008). According
to Lisyastuti (2010), environmental factors that affect air microbial is atmospheric
temperature, humidity, wind, altitude, and others. Temperature and relative humidity
are two important factors that determine the viability of microorganisms in aerosols.
Studies with Serratia marcesens and E. coli showed that survival is closely related to
air temperature. Increasing the temperature causes a decrease in survival time. There
is a progressive increase in mortality with an increase in temperature from -18 ° C to
49 C. The virus in aerosols exhibit the same behavior. Particle influenza, polio and
vaccinia virus is able to survive at low temperature, 7-24 ° C. The relative humidity
level (RH) optimum for the survival of the microorganisms is between 40 and 80%.
The relative humidity is higher or lower causes the death of microorganisms. The
influence of the wind also determine the presence of microorganisms in the air. In
still air, the particles tend to fall by gravity.
According to Volk & Wheeler (1989), to calculate a volume of organisms in the
air used qualitative techniques as simple as exposing petridish which composed of
nutrient medium or in the air for some time. During this exposure time, some of the
bacteria in the air will settle on the exposed plate. The more bacteria, bacteria that
settle in the petridish more. The petridish then incubated for 24 hours to 48 hours it
will appear colonies of bacteria, yeasts and molds that can grow on the medium used.

Purpose

The objectives of this practical work is to know the effect of activity in a room
on microbial population density and its diversity.
 II. MATERIAL AND METHOD

A. Materials

Materials used in this practical were petri dishes, wrapper, pippete, ose needle,
bunsen, tissue and labelled.
Tools used in this practical were Nutrient agar medium (NA), Potato Dextrose
Agar medium (PDA), safranin solution, crystal violet, lugols iodine, alcohol, and
distilled water.
B. Method

2. 1 Isolation
Practicans received 3 Natrium Agar (NA) and 3 Potato Dextrose Agar (PDA)
medium to be isolated for 15 minutes in several spots that not in near distance. Each
practican held 2 medium consists of 1 NA medium an 1 of PDA medium to be
isolated, then placed the medium in the spots were chosen. After that, let the medium
opened and wait for 15 minutes. Finally, after reached 15 minutes the medium was
closed also being wrapped again and the medium being incubated for 2 x 24 hours
for bacteria observation and 5 x 24 hours for fungi observation.

2.2 Observation

The NA and PDA medium that already incubated for 2 x 24 and 5 x 24 hours
being observed by it’s density and the diversities in bacteria and fungi that has found.
For the observation of diversity can observed by see the morphology of air microbial
colonies (colony shape, edge, elevation, color, diameter and amount).

2.3 Gram Staining

Each colony of bacteria, then swab in object glass added by aquadest. Do
fixation two or three times in bunsen burner, drop crystal violet solution and wait for
one minute. After that wash, dry, and aired the object glass therefore drop lugol’s
iodine solution and wait for another one minute. Wash, dry, and aired again, then
drop ethanol and wait for only fifteen seconds. Before and after we drop safranin as
the last solution for 45 seconds, we must wash, dry, and aired the object glass.

2.4 Counting
 The density of bacteria and fungi that has been known is calculated by the
5𝑎 𝑥 104
formula of CFU’s = .
𝑏𝑥𝑡
 III. RESULT AND DISCUSSION

Table 3.1 The Bio-Aerosol Observation Results

Section Group Time & Density Diversity
Sampling
Bacteria Fungi Bacteria Fungi
location
I 1 Morning (15 0,94×103 1,756×103 6 7
minutes),
Micology lab.
2 Evening (15 2,096×10 1,519×103 3 11
3
minutes),
Micology lab.
3 Morning (15 0,435×10 2,34×103 13 27
3
minutes), Old
musholla
II 1 Morning (15 1,96×103 9,905×103 7 7
minutes),
Central Park
Fabio
2 Evening (15 4,45×103 0,436×103 6 6
minutes),
Central Park
Fabio
III 1 Morning (15 2,56×103 6,34×103 7 8
Minutes),
Fabio cafeteria
2 Evening (15 2,16×103 0,54×103 6 6
minutes),
Fabio Cafetaria

Based on the results showed that the bacteria density showed the higher
results in the central park of biology faculty with the time was in the evening and the
density amount of 4,45×103 CFU’s/ ml. and also the results showed that the highest
diversity of bacteria is in the micology laboratorium with the amount of 13. For the
indoor there are micology laboratory, the old musholla building, and cafeteria of
biology faculty. The bio-aerosol test results showed that in the micology laboratory
the higher bacterial density occurs in the evening with the amount of 2,096×103
CFU’s/ ml and the diversity of each bacteria and fungi were 3 and 11, while in the
morning the density was 0,94×103 CFU’s/ ml and the diversity amount of each
bacterial and fungi were 6 and 7. The density of fungi in the micology laboratory
were 1,756×103 CFU’s/ ml in the morning and 1,519×103 in the evening. The least
amount of bacteria density in indoor sampling location was showed in the old
musholla building with the amount of 0,435×103 CFU’s/ ml and the fungi density
was 2,34×103. The diversity amount of each bacterial and fungi in the old musholla
building were 13 and 27. By the results we could see that the higher density of
bacterial is in the central park of biology faculty and the higher density of fungi was
also in the central park of biology faculty but in the morning, according to Hussin et
al. (2011), The outdoor air bioaerosol concentrations were strongly affected by the
nearby regional vegetation activities and by the presence of sources from outdoor
soil, plants and other factors such as humidity and etc. Also according to Hussin et al.
(2011) the temperature and relative humidity were higher in comparison with those
in cold countries due to less variation in the temperature and climate that prevail
throughout the year. Studies in cold countries found that the outdoor temperatures
and relative humidity were lower than corresponding indoor levels. Because of the
higher temperature and humidity, the bio-aerosols in here are more favorable for the
microorganisms to growth than in an indoor location. Also the highest amount of
bacterial occurs in the evening, this is because from the morning to the evening there
has been a lot of activity going on so that a lot of factors which was mentioned
before might be affected and it may increase the density of the bacterial. While the
highest amount of fungi occurs in the morning, this might happened because the
humidity were higher and the temperature was colder in the morning, as mentioned
before it could be a favorable condition for fungi to grow.
The highest density of the bacterial bio aerosol located in the indoor area
were in cafeteria of biology faculty which occur in the morning and in the micology
laboratory that occur in the evening and the highest density of fungi were located in
cafeteria biology faculty which occur in the morning with the diversity amount of 8.
According to Lisyastuti (2010), stated that the concentration of microbes in the room
will be larger/ higher depend on a conducive growth in the rooms, for example of
humidity, temperature and human activity. Biological material flowing in the air and
piled up in the room and cover the interior surface will lead to changes in indoor air
quality. Few sources of carbon and water in the room will be a microorganism
growth medium. According to Adams et al. (2015) Human occupants are an
important source of microbes in indoor environments. On indoor surfaces, direct
contact leads to a rapidly generated signature of the occupants, one that is predictable
based on the nature of the human contact. Airborne microbial levels increase when
rooms are occupied compared to unoccupied conditions, and humans have been
reported to be a source of bacteria and fungi in settled dust samples. And also
according to Hussin et al. (2011) the indoor air could be contaminated with airborne
microorganisms from outdoor airborne microorganisms by natural ventilation or due
to inside generative sources. According to Pudjiastuti et al. (1998) stated that the
factor that influenced the quality of bio-aerosol in room are environment condition
inside of the room, humidity and air flow. The three factors caused the absorption of
chemical pollutant inside of the room increase, growth of microorganism in air.
Others factor is construction of building or furniture and bad ventilation will caused
circulation of fresh air decrease.
The microorganisms in air consists of a complex between the bio-aerosol
compositions such as fungi, bacteria and allergens and non-biological particles such
as smoke, burning particle generator and others. More than 80 genera of fungi
associated with the incidence of allergy symptoms (Horner, 1995). According to
Hamdi (2013) the level of relative humidity (RH) optimum for the survival of the
microorganisms is between 40 and 80%. The relative humidity is higher or lower
causes the death of microorganisms. The influence of the wind also determine the
presence of microorganisms in the air. In still air, the particles tend to fall by gravity.
According to Hussin et al. (2011) the most common indoor airborne fungal genera
isolated were Aspergillus, Penicillium, Fusarium, Rhizopus and Zygomycetes while
the most common air borne bacterial genera were Straphylococcus, Acinetobacter,
Bacillus, Corynebacterium, Enterobacteriaceae, and etc.

Incubation in NA medium Incubation in PDA medium

Figure 3.1. Observation result of incubation medium NA and PDA has

exposurred in the central park FABIO Air for 15 minutes.
Figure 3.1 showed that in NA medium after incubation for 2x24 hours in
room temperature there are various bacteria grow in it. After observed the diversity
and density, the amount of density and diversity calculated were relatively huge.
Also the amount of microorganism is different its depend on place and human
activity, ventilation and when the time of exposure occurs. Its in accordance to
Dwidjoseputro (2005), the level of air pollution in the room by microbes is affected
by several factors such as ventilation rate, the density of people, and the nature and
level of activities of persons occupying in the room. The group of microorganisms
most widely roam free air is a bacterial, fungal (including yeast) and microalgae. The
presence of the living bodies in the air, there are in the vegetative form or in the form
of generative (usually spores). Most of the fungal body consisting of top yarns -
threads called hyphae, which are interconnected to establish a sort of mesh that is
mycelium. The mycelium can be distinguished by a vegetative mycelium which
serves to absorb nutrients seep from the environment, and the mycelium fertile that
function in reproduction (Gandjar. 1999). Also based on picture 3.1 it show bacterial
and fungi was growth in medium that has been incubated and it can be use as the
parameter of air quality its accordance with The air quality is reviewed from
biological parameters are influenced by the presence of microbes and other
organisms that cause disease. Microbes originating from indoors such as bacteria,
mold (fungi), protozoa, algae, viruses, as well as small animals such as insects or
other animals 2 carrier microbes (Darkuni, 2001).

Figure 3.2 The gram staining results for bacteria microscopic observation
Based on the 3.2 showed kind of bacteria, and the result was found is the
bacteria are gram negative bacteria because the color become red while for the
bacteria gram positive the bacteria color become blue. According to Hadioetomo
(1993), gram positive bacteria are bacteria that retain methyl violet dye during the
Gram stain process. The bacteria of this type will be blue or purple under the
microscope, whereas gram negative bacteria will be red or pink. According to
Suriawiria (1999), the differences between the two types of bacteria classification is
mainly caused by the differences in the structure of their cell walls. Gram Positive
wall contains a lot of peptidoglycan, whereas Gram Negative bacterial wall contains
a lot of lipopolysaccharide.
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on this lab activity can be concluded that in the indoor room has lower
density and diversity, it is because the concentration of microbes in the room will be
larger/ higher depend on a conducive growth in the rooms, for example of humidity,
temperature and human activity. The effect of activity in a room on the population
and diversity of bacteria are in the central park of biology which the exposure occur
in the evening and the higher density of fungi was in the central park of biology
faculty which the exposure occur in the morning. The outdoor air bioaerosol
concentrations were strongly affected by the nearby regional vegetation activities and
by the presence of sources from outdoor soil, plants and other factors such as
humidity, temperature, the people activity etc.

B. Suggestion

Practican must be careful when working in lab or take the isolate or sample so
reduced the posibillity of contamination and the result of them will be observed
clearly.
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 ATTACHMENT

Attachment 1. The Calculation of Bacteria and Fungi Density

1.1 The density calculation of bacteria
5𝑎 ×104
CFU’s = 𝑏.𝑡
5(85)×104
= 63,6×15

425 × 104
= 954

= 0,445 × 104
= 4,45 × 103
1.2 The density calculation of fungi
5𝑎 ×104
CFU’s = 𝑏.𝑡

5(8,3)×104
= 63,6×15

= 0,436×103