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Course name: Principles in Microbiology

Course code: SBB3033


Report on mini project: Isolation and Identification of Fungi
Associated With Indoor Contamination
Lecturer’s name: Dr. Nurhaida Kamaruddin

Name Lecture Matric number


Group
NURNABILAH SYAHIRAH BINTI A D20151070964
JAFRI
NUR SHAHEEN BINTI RAMZI A D20151070998
Abstract

This project involves the isolation and identification of fungi associated with indoor
environment that pollute Faculty of Science and Mathematics buildings. This project was
conducted to know what type of antifungal agents that can be used to inhibit the growth of
the fungi by subculture few samples that had being taken from the source. Many parts of the
building of Faculty Science and Mathematics are contaminated with fungi. In this project, the
fungi that occupied the building was picked and being grew in the culture medium. They then
being isolated and identified the characteristics of the fungi. They also being tested with
antifungal agents to check the sensitivity of fungi towards the different types and
concentration of antifungal agents. It shows that fungus from lecturer’s room (Dr. Haida) is
sensitive to 20% tea tree oil, 50% white vinegar and biggest sensitivity in 80% of phenol
while fungi from toilet in block 3 FSMT building, the fungus is sensitive to 20% tea tree oil
and 80% of phenol. The characteristics of fungus observed in lecturer’s room (Dr. Haida)
showed it has septate hyphae, sporangiospore and the phylum is Ascomycota. Sample from
toilet in block 3, it has structure of coenocytic hyphae and it is from phylum Zygomycota.
Both samples have vegetative structure of moldlike growth. It is hoped that this project will
helps people to use antifungal agent that are suitable for the type of fungus formed to inhibit
its growth.

Introduction

The fungi, an often overlooked group of unicellular and multicellular organisms, have
a profound influence on ecology and human health. Along with bacteria, they are
decomposers and disease-causing organisms. Fungi made it possible for plants to colonize
land by associating with rootless stems and aiding in the uptake of nutrients and water.
Mushrooms for example are the multicellular spore-producing part of fungi that grow rapidly
under proper conditions. Yeast is used to make bread, but other cause diseases in plants
and animals. Yeasts are microscopic fungi consisting of solitary cells that reproduce by
budding. Fungi exist either in single-celled yeasts or multicellular form with several different
cell types. Their reproductions are in sexual or asexual and exhibit unusual form of mitosis.
They are specialized to extract and absorb nutrients from their surroundings through external
secretion of enzymes. (Mason et al, 2014)

Fungi takes important role in biological processes, including many processes that
support, and in other cases adversely affect, other forms of life. Fungi often interact with
other organisms forming beneficial or mutualistic associations. For example most terrestrial
plants form symbiotic relationships with fungi. Thus, the types of fungi in soil affect the types
of plants that thrive there, hence also the animals likely to make their home in that area. But
for certain time and place, fungal growth can give bad effect and disturbing our peace in a
particular area and place. When it comes to disturbing people health and cleanliness, it is
called as fungal contamination. Risk of health are the main point of us to investigate and find
a solution on how to inhibit the growth of fungal in certain area. (CR.1, 2011) state that the
microclimate of a room has an impact on human well-being, physical and mental health, on
work productivity and the preservation of good health. Several dozen species of bacteria can
live in buildings and more than 400 species of fungi (mainly Aspergillus, Cladosporium,
Penicillium, Fusarium genus).

An antimicrobial is an agent that kills microorganisms or inhibits their groth.


Antimicrobial medicines can be grouped according to the microorganisms they act primarily
against. For example, antibacterials are used against bacteria and antifungals are used to
against fungi. Antifungal agent is a drug that selectively eliminates fungal pathogens from a
host with minimal toxicity to the host (Dixon & Walsh). In medicine, they are used as a
treatment for infections such as athlete’s foot, ringworm and thrush and work by exploiting
differences between mammalian and fungal cells. As well as their use in medicine,
antifungals are frequently sought after to control mold growth in damp or wet home
materials. Some of antifungal agents are sodium bicarbonate and hydrogen peroxide.

Material and method

1. Inoculation of fungi from indoor environment.

a) The fungus that pollute the Faculty of Science and Mathematics buildings was
identified.
b) Sterilized wooden stick was used to picked the samples of fungi and inoculate on
SDA agar plates.
c) The plates were inverted and incubated in incubator at 30oC for 7 days.
d) Every 2-3 days checked if there was a growth on the plates, if no repeat the
samples.

Materials :

 Sabouraud Dextrose Agar (SDA) plates


 Sterile wooden stick
2. Isolation of pure colonies of fungus

a) 5 ml of sterile ddH2O was added on the cultured SDA agar plates.


b) Sterilized scalpel was used to scrape out the fungi mycelia and spores on agar
surface.
c) The spore suspension was sucked by using P1000 micropipette with 1-ml cut-end
tips and filter the spore via whatman filter paper (0.2μm pore size) into universal
bottle.
d) The spores count ( number of spores/ml) was determined using haemocytometer and
cell/number counter.
e) The filtered spore suspension to the concentration was diluted of 1.0×103/ml.
f) Used a hockey stick to spread the spore to get single colonies.
g) Appropriate amount of spore dilution was added onto a plate, managed to get 20
spores/plate. The spores was spreaded using a flamed spreader. Then the plate was
inverted in the incubator at 30oC for 1-2 days.
h) The single germinated spore was observed under light microscope.
i) Picked two most isolated germinated spores by excising the agar in cube shape and
transfer agar cube on to fresh SDA agar.
j) The plate was inverted and placed in incubator at 30oC for 7 days.
k) After 7 days, the spore was harvested ( steps 2a to 2d was repeated)
l) The fungal stock was prepared : in micro centri fudge tube 1.5ml, add 800 μl 80%
glycerol. Vortex before keep at-80oC freezer.

Materials:

 SDA agar plates


 Sterile scalpel
 Whatman flter paper (0.2 μm pore size)
 1 ml Tips
 Micropipette P1000
 Universal bottle
 Cell/number counter
 Haemocytometer
 Hockey stick
 Bunsen burner
 Micro centrifudge tube 1.5ml
 Autoclved Glycerol 80%
 Sterile ddH2O
3. Preparation of Methylene blue (LPCB) slide mounts

a) A microscope glass slide was cleaned with 70% alcohol.


b) A drop of LPCB stain was placed in the centre of a clean slide.
c) A fragment of fungus colony 2-3mm was removed from the colony edge using
flamed inoculating needle.
d) The fragment was placed in the drop of stain and tease gently.
e) A cover slip was applied on the slide. Do not tap the cover slip.
f) Observed the slide.

Materials :

 Microscope glass slide


 Cover slip
 Lactophenol cotton blue (LPCB)
 Inoculating needle
 Bunsen burner
 Light microscope

4. Observation of morphological characteristics of fungi

a) The slide was examined under 10×, 40× and 100× magnifications for the presence of
characteristics such as conidia, conidiophore, sporangium, hypha, septa et.
b) The observed fungus images was drew for each magnifications. Photos was snap for
record.
c) The morphological characteristics of fungus were described. The appropriate
references was consulted for diagnostic the features of fungi in clinical and non-
clinical specimens.

5. Evaluation of Efficacy of Antifungal Agents for the treatment of Fungal


Contamination in Indoor Environments.

a) The selected antifungal agents are tea tree oil, white vinegar, ethanol, hydrogen
peroxide and baking soda.
b) Each antifungal agent are prepared in different concentrations (70%, 50% and 20%)
c) 100μl (1×106) of spore suspension was spreaded on SDA plates by using flamed
hockey stick. The plate was let to dry at room temperature for 15 minutes.
d) A line was drew at the bottom pf the plate to divide the plate into to sections.
e) 20μl of each of the test antifungal agents was pipette onto sterilized Whatman filter
paper disc (0.2μm pore size) 9mm in diameter and placed them in the middle of each
section.
f) Include phenol (88%) as a positive control and sterilized distilled water as the
negative control.
g) The plate was sealed with parafilm and incubate at 30oC for 7 days.
h) After 7 days, the fungal growth and the formation of inhibition around disks was
observed.
i) The test was repeated for 3 times using different fungal cuture plates.
j) An agent is categorized as having antifungal activity when the diameter of the
inhibition is larger than 9.5mm, 0.5mm larger than the diameter of paper disc.

Materials:

 Antifungal agents:- 1) tea tree oil 2) white vinegar 3) ethanol 4) hydrogen peroxide 5)
baking soda( each in concentrations of 70%, 50%, 20%.
 SDA plates
 Spore suspension (1×106spore/ml)
 Hockey stick
 Bunsen burner
 Sterile Whatman filter paper disc (0.2μm pore size) 9mm I diameter
 Phenol (88%)
 Sterile ddH2O
 Parafilm
Result

The location of samples taken Picture of the sample taken

On the surface of the inner


cupboard

On the mirror of the cupboard

On the surface of the outer


cupboard
In 1 week of incubation

The location of samples Picture of the sample after Observation


taken 1 week

On the surface of mirror Fungus growth


cupboard

On the surface of the inner


cupboard No fungus growth

On the surface of the outer No fungus growth


cupboard
Isolation of pure colonies of fungus

Sample of the surface on mirror Sample of the surface on mirror

Sample of the surface on mirror


Sample of the surface on mirror
Diagram 1.0 Isolation of pure colony part 1

Sample of the surface on mirror Sample of the surface on mirror

Sample of the surface on mirror Sample of the surface on mirror

Diagram 1.0 Isolation of pure colony part 2


Spore count

Sample Shaheen Nabilah


Cermin 1 42 15
Cermin 2 15 39
Number of spore count under haemocytometer

Sample Spore count (Total Number of


Conidia/ml)
Nabilah – cermin (1) 6.00 × 108
Nabilah – cermin (2) 1.56 × 1010
Shaheen – cermin (1) 1.68 × 1010
Shaheen – cermin (2) 6.00 × 109

Example of calculation spore count:

Total number of conidia/ml = (# conidia in the 4 squares / 4) x 16 x dilution x 104)

Shaheen – cermin (1)

42
Total number of conidia/ml = ( )×16 × 104 × 104 = 1.68 × 1010
4

Shaheen – cermin (2)


15
Total number of conidia/ml = ( ) × 16 × 104 × 104 = 6.0 × 109
4

Nabilah – cermin (1)


15
Total number of conidia /ml =( ) × 16 × 103 × 104 = 6.0 × 108
4

Nabilah- cermin (2)


39
Total number of conidia/ml = ( ) × 16 × 104 × 104 = 1.56 × 1010
4
Amount of spore dilution onto a plate

Calculation:

𝑚1 𝑣1 = 𝑚2 𝑣2

Shaheen – cermin (1)

Step 1
𝑚1 𝑣1 = 𝑚2 𝑣2
1.68 × 1010 𝑣1 = 1 × 109 × 1000𝜇𝐿
𝑣1 = 59.5 𝜇𝑙
Step 2
𝑚1 𝑣1 = 𝑚2 𝑣2
1 × 109 𝑣1 = 1 × 107 × 1000𝜇𝐿
𝑣1 = 10 𝜇𝐿 (spore suspension) + 990 𝜇𝐿
Shaheen – cermin (2)
Step 1

𝑚1 𝑣1 = 𝑚2 𝑣2
6.0 × 1010 𝑣1 = 1 × 108 × 1000𝜇𝐿
𝑣1 = 16.67 𝜇𝑙
Step 2
𝑚1 𝑣1 = 𝑚2 𝑣2
1 × 108 𝑣1 = 1 × 106 × 1000𝜇𝐿
𝑣1 = 10 𝜇𝐿 (spore suspension) + 990 𝜇𝐿
Nabilah – cermin (1)
Step 1
𝑚1 𝑣1 = 𝑚2 𝑣2
6.0 × 108 𝑣1 = 1 × 107 × 1000𝜇𝐿
𝑣1 = 166.7 𝜇𝑙
Step 2
𝑚1 𝑣1 = 𝑚2 𝑣2
1 × 107 𝑣1 = 1 × 105 × 1000𝜇𝐿
𝑣1 = 10 𝜇𝐿 (spore suspension) + 990 𝜇𝐿
Nabilah-cermin (2)
Step 1

𝑚1 𝑣1 = 𝑚2 𝑣2
1.56 × 1010 𝑣1 = 1 × 109 × 1000𝜇𝐿
𝑣1 = 641.03 𝜇𝑙
Step 2
𝑚1 𝑣1 = 𝑚2 𝑣2
1 × 109 𝑣1 = 1 × 107 × 1000𝜇𝐿
𝑣1 = 10 𝜇𝐿 (spore suspension) + 990 𝜇𝐿

Spore formation (first trial)

Shaheen- cermin (1) Shaheen – cermin(2)

Nabilah –cermin(1)
Nabilah- cermin(2)
(no isolated colony)- rejected
Observation of morphology characteristic of fungi

First observation (first trial)

Shaheen – cermin (1)

400x total magnification 1000x total magnification

Shaheen - cermin (2)

400x total magnification 1000x total magnification

Nabilah - cermin (2)

400x total magnification 1000x total magnification


Second observation (second trial)

Type of sample LPCB stain Methylene blue stain


Lecturer’s room (Dr. Haida)

400x total magnification 400x total magnification

1000x total magnification


Toilet in Block 3 FSMT
buildings

400x total magnification


400x total magnification

1000x total magnification


Evaluation of efficacy of antifungal agents for the treatment of fungal contamination
in indoor environments

Diameter of clear
Zone (cm) Lecturer’s room (Dr. Toilet in Block 3 FSMT
Haida) buildings
Type of antifungal
70% No clear zone formed No clear zone formed
Tea tree oil 50% No clear zone formed No clear zone formed
20% 1.1 cm 1.2 cm
70% No clear zone formed No clear zone formed
White vinegar 50% 1.5 cm No clear zone formed
20% No clear zone formed No clear zone formed
70% No clear zone formed No clear zone formed
Ethanol 50% No clear zone formed No clear zone formed
20% No clear zone formed No clear zone formed
Hydrogen 70% No clear zone formed No clear zone formed
peroxide 35% No clear zone formed No clear zone formed
25% No clear zone formed No clear zone formed
Baking soda 70% No clear zone formed No clear zone formed
50% No clear zone formed No clear zone formed
20% No clear zone formed No clear zone formed
Phenol 80% 3.7 cm 1.5 cm

Dh2o No clear zone formed No clear zone formed


Diameter of clear
Zone (cm) Lecturer’s room (Dr. Toilet in Block 3 FSMT
Haida) buildings
Type of antifungal

Tea tree oil

White vinegar

Ethanol

Hydrogen peroxide

Baking soda

Phenol 80%
Discussion

In this project, we had searched and examined the fungi that were pollutes Faculty of
Science and Mathematics buildings. For our group, we had identified the fungus that was in
the lecturer’s room (Dr. Nurhaida).The fungus that had identified was collected using a
sterile wooden stick and then being brought to the laboratory to culture the fungus in SDA
agar plates. The samples were taken from a cupboard, at mirror, inner side of cupboard and
outer side of cupboard. The plates were incubated in the incubator for grow. The results
obtained after left for 7 days, only one sample that had grown fungi which is from the mirror,
and the other plates did not showed any forms of growth. We then took another samples
from mirror in the Dr Nurhaida’s room and the fungi grew after few days. The other samples
that were not grow may not favorable with the temperature of the incubator as it is hot
temperature compared in the cold room.

For the next step, the isolation of pure colonies of fungus was carried out. The original
samples were then being isolates and placed on a new agar plates to get single colony of
fungus. The plates then incubated for another 6 days. After 6 days, the cultured medium was
added with sterile distilled water and the colony of fungus on the medium was scraped and
being filtered using Whatman filter paper to get spores and removed the mycelium in the
spore suspension. The spore suspension was then being diluted until dilution factor of 104.
The spores count was determined using haemocytometer under light microscope. The
spores counted were then calculated using a formula. The spores formed were not too many
and we proceed with calculation of amount of spore dilution that need to place in a plate.
The value that we had got were too small to be suck by the micropipette. We then, calculate
again by using the same formula and get volume that were reasonable. Then, the spore
suspension was sucked and placed on the SDA agar plate and incubated for 4 days long as
there was holiday during the period. The samples observed were in yeast-like form. We then
proceed with another subculture of isolated spores from the samples and incubated for
another 6 days.

Next, we had prepared slides to observe the structure of fungus under light microscope.
A drop of Lactophenol cotton blue (LPCB) stain is placed at centre of slide and a fragment of
fungus was placed in the stain. The slide was observed under light microscope starts with
total magnification of 40× until 1000×. The slides of methylene blue stain also being made as
the structure in slide of LPCB stain cannot be observed clearly compared to methylene blue
stain.
By our first observation on the morphological characteristic of fungi was different with our
expectations, on our first observation from the single spore isolation under the microscope,
we are unable to found the morphological characteristic of our fungi because the
characteristic shown are such as bacteria, not a fungi with its conidia, sporangium, hyphae,
septa and many more. We thought that our spore isolation was contaminated with bacteria
therefore we unable identify the morphological characteristic of fungi in our experiment.
Because we observe our sample that the characteristics was rod shape and does not shows
the characteristics of fungi. But we try to make second times of isolation of fungi based on
our first sample on the early experiment to identify the morphological characteristic of fungi.

By our observation of sample, we prefer to use methylene blue to stain our slide because
its show the good picture to determine the characteristics of our sample fungus. In our
second observation, we just back to our original sample in the early step, we isolate for 3
times isolation, the spore suspension also are made but the serial dilution was not done. So
that in our second observation, it is not a single colony of fungus because the isolation of
fungus was not successful show the positive result on the morphological characteristics of
our fungi sample. For our second observation, not all of our sample fungus grow within
7days, only one of our sample the fungus grow. Therefore, another sample we took from
other group to proceed with observation of the morphological characteristics of fungi. So
that, in our final result in this experiment is only 2 sample. One sample are from lecture’s
room, and the other one sample was taken in the toilet at Block 3 FSMT buildings.

For the morphology of our fungi sample, we observed that our sample in lecturer’s room
(Dr. Haida) showed a septate hyphae which is contain cross-walls and we found that the
spore was a sporangiospore which is enclosed in a sac when we observed under light
microscope in 400x total magnification. It is show that this fungi reproduce asexually by
sporangiospore while, sexually with zygospore. For the vegetative structure we observed
that our sample was in moldlike growth. From the characteristics showed from the sample,
we identified that sample in lecturer’s room (Dr. Haida) was from phylum Ascomycota fungi.

Sporangiophore

Sporangiospores
Septate
hyphae
In sample of the toilet at Block 3 FSMT buildings we found that the morphology
characteristics of the sample was coenocytic hyphae which is non septate. Since we are
unable to observed the type of spore of the fungi, we assume that it is from phylum
zygomycota because it is the only phylum that was non septate. The vegetative structure we
observed that our sample was in moldlike growth in the nutrient plate.

Non septate
hyphae

After we observed and identified our fungi sample, we had done an evaluation of
Efficacy of antifungal agents for the treatment of fungal contamination in indoor
environments. In this evaluation involve 5 types of antifungal agents with 3 differents
concentration to determine the suitable concentration of certain antifungal for inhibit the
growth of fungi. The antifungal used in this experiment was tea tree oil, white vinegar,
ethanol, hydrogen peroxide and baking soda with different concentrations 70%, 50%, and
20%. But the concentration of hydrogen peroxide was prepared by the lab assistant are
70%, 35% and 25%. Phenol 88% concentration was include as a positive control and sterile
distilled water as a negative control.

The result after 6 day incubation with the antifungal agents was found that, only tea
tree oil and phenol show a diameter of the inhibition zone. The others antifungal agents was
not show a positive result with a diameter of the inhibition zone. The fungal was grow
heavyily in the plate that contain white vinegar, ethanol, hydrogen peroxide and baking soda
antifungal agents. In our observation, tea tree oil with concentration of 20% which is the
diameter of the inhibition zone is 11mm for lecturer’s room and 12mm for the sample in toilet
at Block 3 FSMT buildings. Based on our reading found that tea tree oil has a properties as
an antibacterial, antimicrobial, antiseptic, antiviral, balsamic, cicatrisant, expectorant,
fungicide, insecticide, stimulant and sudorific substance. Besides, the fact of tea tree oil is
that it is extracted from the seed of the Tea plant. Thus, the Tea Tree Essential Oil is as
effective against fungal infections as it is against any bacterial or microbial infections. It
inhibits fungal growth and cures diseases like dermatitis and Athlete’s Foot. Although
internal fungal infections can be very dangerous, and even deadly, never ingest tea tree oil,
even in extremely diluted forms, as it is toxic.
In other hand, we also found that phenol with 88% concentration show a positive result,
which is the diameter of the inhibition zone is larger than tea tree oil. The diameter of the
inhibition zone of phenol with 88% concentration is 37mm for lecturer’s room and 15mm for
the sample in toilet at Block 3 FSMT buildings. Based on our literature review, phenolic
compound always used against fungal disease and their antifungal mechanism of action was
observed. There are various phenolic compounds which affect this pathogen by affecting its
dimorphic transition. For instance three new phenolic compounds isolated from the leaves of
Baseonema acuminatum P. Choux (Asclepiadaceae) has been known to show antifungal
activity against two different strains of C. albicans. The structures of the phenolic compounds
is such that they can diffuse through the microbial membrane and can penetrate into the cell,
where they can interfere in the metabolic pathways by interfering with the synthesis of
ergosterol, glucan, chitin, proteins and glucosamine in fungi.

Conclusion

The objectives of this project have achieved. We was done the isolation of fungi that
contaminating buildings of Faculty of Science and Mathematics which is from Lecturer’s
room (Dr. Haida) and toilet in Block 3 FSMT buildings. Thus, the fungi that contaminating
FSMT building was been identified based on morphological characteristics, which is from
zygomycota and ascomycota phylum of fungi. By this project also, we have done a
evaluation of efficacy of antifungal agents towards the fungi for a treatment of fungal
contamination in indoor environments, that is from our experiment found that tea tree oil and
phenol was effective to inhibit the growth of fungal.
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