CHAPTER 1

The Problem and It's Scope

Background of the Study

Molds are a natural part of the environment and can be found almost anywhere

that moisture and oxygen are present. They belong to the kingdom Fungi and live in

moist places such as soil, plants and dead or decaying matter. Outdoors, molds play a

part in nature by breaking down dead organic matter such as fallen leaves, dead trees

and other debris; however, indoors mold growth should be avoided.

Molds spread by producing tiny reproductive cells called spores that waft through

the air. Mold spores usually cannot be seen without magnification (ranging in size from

2-10 um) and are naturally present in both indoor and outdoor air. Some molds have

spores that are easily disturbed and settle repeatedly with each disturbance. Other

molds have sticky spores that will cling to surfaces and are dislodged by brushing

against them or by other direct contact.

Spores may remain able to grow for years after they are produced. In addition, whether

or not the spores are alive, the allergens in and on them may remain allergenic for years

(EPA.gov, 2017).

As said by Cook, M. (2018), black bread mold (Rhizopusstolonifer) is one of the

most common bread molds. It exists on every continent on Earth. In addition to bread,

black bread mold also appears on wild fruits and vegetables, especially if they are

growing in moist conditions. Its presence causes rotting in whatever organic material it

consumes, which means that black bread mold can kill plants.

1
 Black bread mold usually appears as fuzzy blue or green patches on the surface

of the bread. When left untouched, these patches develop black, splotchy centers,

which is how this bread mold got its name.It is never wise to eat bread molds or mold of

any kind. Certain molds can cause severe allergic reactions in some people. However,

for most people, eating black bread mold is not dangerous, though it can cause nausea,

indigestion and vomiting.

However, it was also stated by Tilden, E. (2018), molds causes the spores from

mold floating through the air land on bread and activate when moisture and temperature

conditions are right. Bread mold prefers warm, moist and dark environments. And its

types, although molds can be dry or slimy, the type of mold that afflicts bread is the dry,

cotton-textured mold, which grows in threads through the bread. The color of each

species of mold exhibits its own color. The Rhizopusstolonifer species appears black

and fuzzy, whereas the penicillium species appears blue-grayish-green with a white

border, and is also fuzzy. And it has also a benefits, the penicillium species of bread

mold is the same species from which scientists extract penicillin, which kills bacterial

infections within the body.

The scientific name for calamansi is Citrofortunellamicrocarpa. Belonging to the

Rutaceae family, calamansi is also commonly called kalamondin, kalamansi, Philippine

lemon and calamondin orange.Calamansi, which is native to Philippines, is a slightly

spiny citrus plant. Normally growing from 3 to 5 feet, it blooms throughout the year. Its

fruit turns from dark green to orange-yellow when ripe. Around 1.75 inches in diameter,

the fruit is acidic and juicy with a citrus flavor, similar to that of limes and lemons. It is

2
used in seafood dishes for improving iron absorption. Calamansi juice is applied to the

scalp after shampooing to improve hair growth or to get rid of scalps

itching.(Reference.com)

The Philippines calamansi peel contained the highest amount of total phenolic

acids. In addition, p-Coumaric acid was the dominant free phenolic acids, whereas

ferulic acid was the main bound phenolic acid (Elsevier, 2012).

The Researchers conducted this study because they want to help reduce molds

in a safe way without harming the environment.

3
Conceptual Framework

Independent Variable

 25 % Calamansi Extract

 50 % Calamanis Extract

 100% Calamansi Extract

Time of Exposure

36th hour Procedure

48th hour Adding Calamansi Extract to the Molds

90th hour ( RhizopusStolonifer)

Dependent Variable

Antifungal Activity of Calamansi Extract

against Black Bread Molds

The purpose of this paradigm is to show the relation between the various

concentrations against molds.

4
Statement of the Hypothesis

H0 = The different concentrations have no significant effect on the average zone of

inhibition of molds at different time of exposure.

H1 = The different concentrations have a significant effect on the average zone of

inhibition of molds at different time of exposure.

Statement of the Problem

This study aims to investigate the effectiveness of Calamansi Extract against

molds.

This will specifically answer the ff. questions:

1.) Which of the different concentrations ( 10%, 50%, 100% ) of calamansi extract has

the highest zone of inhibition in ;

a. 48th hour

b. 72nd hour

c. 96th hour

2.) Which concentrations has the highest zone of inhibition?

a. 25 % Calamansi Extract

b. 50 % Calamansi Extract

c. 100 % Calamansi Extract

3.) Which of the different concentrations of calamansi extract is most comparable to the

commercial fungicide?

5
Significance of the Study

Bread is a staple food prepared by baking dough of flour and water. It is popular

around the world, in every household in Ireland and is one of the world’s oldest

foods.Bread may be served in different forms at any meal of the day, eaten as a snack

and is even used as an ingredient in other culinary preparations. As a basic food

worldwide, bread has come to take on significance beyond mere nutrition, evolving into

a fixture in religious rituals, secular cultural life and language. The researchers

conducted this study because they want to help reduce molds.

Environment - it does not damage the environment because it uses a pure

concentration in making.

Consumer - it really helps the consumer in terms of occasions or snacks.

Researchers - it helps to improve the study more and can help future researchers of

the researchers.

Vendors – they can sell good quality of bread and earn more capital.

Scope and Delimitations

This study will be conducted in the Department of Agriculture Regional Office 7.

Calamansi is indigenous in the Philippines. The fruit will be bought on October 19, 2018.

The study will start on October 19, 2018 and ends on October 31, 2018. The

researchers will only use calamansi extract, ethanol and commercial fungicide because

the researchers will only focus on the antifungal activity of calamansi extract and if it

6
could be comparable to the commercial fungicide . The researchers will not use other

citrus fruit extract in conducting the study.

This study

Definition of Terms

For the purpose of this study and to facilitate the comprehension of this work, the

terms below are hereby defined.

McFarland Standards- as used in the study the McFarland Equivalence Standards are

intended to be part of a quality control program for adjusting densities of fungal

suspensions that are used for identification and susceptibility testing. (J.Amer)

Positive control- refers to the set-ups that were treated with the commercial fungicide.

Negative control- refers to the setups that were treated with ethanol only.

PDA (Potato Dextrose Agar) - is a nonselective medium for the cultivation of yeasts

and molds.

7
 CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDY

Review of Related Literature

The researchers are looking for an eco friendly fungicide that are usually found

locally. Calamansi is an example of this local ingredient that is needed for the fungicide.

Citrofotunella microcarpa is commonly known as calamansi or orange calamondin.

Cirofortunella microcarpa is a small, bushy, evergreen tree or shrub which is abundantly

found in China or the Philippines. It is believed to be a cross or combination

between Citrus reticulata (mandarin) and Fortunella japonica (kumquat) It is

commercially grown in the Philippines, tropical Asia and parts of Latin America where

the edible fruit is commonly consumed as a food. Calamansi is a small tree or shrub to

6-20' tall (smaller when grown in containers as houseplants). The tree produces thin-

skinned, juicy, golf ball sized orange fruit which is edible but it's pulp and juice are

acidic. The fruit remains in the plant for a long time. Each fruit contains 6-9 fleshy

segments. Fragrant white 5-petaled flowers bloom primarily in spring, but basically may

produce 4-5 smaller flushes throughout the year. Seeing flowers and ripe fruit on the

plant at the same time is not unusual. Branches are clad with oval, rich, slightly glossy,

evergreen green leaves. ( Missouri Botanical Garden).

The calamansi is primarily valued for its acid juice. In the Philippines it is

commercially processed into bottled concentrate and juice. It is made into marmalade,

or preserved whole in sugar syrup. It is used in making chutneys and as a flavor

8
enhancer for dishes comprising seafood or meat. The juice can be used as a stain

remover, body deodorant, skin bleach and hair shampoo. It is also used to treat skin

irritation, as a cough remedy, an antiphlogistic ( is an agent that reduces inflammation )

laxative and, when combined with pepper, it is prescribed to expel phlegm. The roots

are used for a traditional treatment at childbirth, the distilled oil of the leaves to cure

flatulence. Bees gather the nectar to make honey. The calamansi also serve as

rootstock for lemons and the oval kumquat. It is popularly used as a potted ornamental

plant in many countries. The Philippines has produced over 51 291 t of calamansi. (

Plant Use ).

Calamansi grows best in lowland areas or places where it is warmer. It can also

grow on colder area but it must be frost free. As the temperature gradually lowers the

plant becomes dormant. This specie can tolerate short periods of low temperature.

Areas that have a long dry period are equally suitable in planting calamansi as long as it

has water availability. It requires a position that is fertile and well drained. It must require

sunlight. IT is better to plant it on a well drained land, like sandy or clay loam soil. The

tree grows throughout the year. (Tropical Plants Database, Ken Fern.

tropical.theferns.info. 2019-02-18 ).

Calamansi helps strengthen our immune system. It helps quicken the body's

ability to repair wounds. Calamansi juice serve as a liver cleanser and helps keep our

liver clean. It helps eliminate toxins in our bodies that results in a faster weight loss . It

helps reduce burning sensation. It helps control high blood pressure. ( Yayang, 2014 ).

9
 Molds are fungi that can be found both indoors and outdoors. Molds grow best in

warm, damp and humid conditions. Mold spores can survive in harsh environmental

conditions such as dry conditions that do not support mold growth. Symptoms that may

be caught from mold exposure are nasal, sinus congestion, eye irritation, blurred vision,

sore throat, chronic cough and skin rash. People who are mostly affected by molds are

people with a weak immune system, such as people receiving treatments for cancer,

people who had an organ or stem cell transplant and people taking medicines that

suppress the immune system are most likely to get mold infections. To clean up molds,

eliminate sources of moisture, reduce indoor humidity and clean any damp or wet

building materials and furnishing within 24-48 hours. (https://www.epa.gov/molds/ten-

things-you-should-know-about-molds).

Review of Related Studies

According to Anonymous, 2014, More calamansi fruits is grown mainly in

Vietnam and Brunei also in Philippines. Some of them was used as an ornamental

plant, it is frost sensitive and therefore limited to warm climates. If the plant is potted it

may be brought indoors when it is winter or a cold climate or areas.

The study that evaluated the antigiogenic and antioxidant properties of calamansi

(citrus microcarpa) ethanolic peel extract can lower the number blood vessels that can

trigger cancer progression and more powerful antioxidant property than the ascorbic

acid. (Anonymous, 2015)

10
 To clean up molds, eliminate sources of moisture, reduce indoor humidity and

clean any damp or wet building materials and furnishing within 24-48 hours. Molds can

survive harsh environmental conditions. ( Anonymous, 2003 )

To make the Calamansi Extract a volatile ethanol was mixed to complete the

required quality to be mixed in a certain molds. ( King, 2002 ).

Rhizopus stolonifer has the ability to transform steroids, such as progesterone, in

order to treat individuals who have hormonal deficiencies. Rhizopus stolonifer has a

steroid hydroxylating enzyme complex and binding sites on its plasma membrane,

which enables it to be so effective at producing the steroids. Birth control pills are multi-

purpose medications used by women to prevent pregnancy and to treat emotional

problems they may experience related to their monthly periods. Rhizopus

stolonifer provides the necessary component of steroids for birth control pills in order for

these emotional problems due to hormonal changes during a woman's life to be

controlled. hizopus stolonifer is a common member of the fungal phylum Zygomycota.

Not only is it the most common, it is the fastest growing of the Zygomycota. Rhizopus

stolonifer is also commonly known as a bread mold that is filamentous and prefers moist

environments. In order to learn more about the areas in which Rhizopus stolonifer lives,

please proceed to habitat. Also, please visit classification to learn what this mold

upholds. Specifically, if you would like to learn about another type of fungus that is the

most common of the fungi family Agaricaceae, then proceed to learn about the Giant

Puffball. Also, you can learn about a different type of filamentous organism

named Spirogyra longata. ( Christina Olbrantz, 2011).

11
 CHAPTER 3

Research Methodology

Experimental Design

The researchers will not use Randomize Complete Block Design in conducting

the study. There will be ( 5 ) five treatments namely ; T 1 = commercial fungicide, T2 =

25% Calamansi Extract , T3 = 50% Calamansi Extract, T4 = 100 % Calamansi Extract

and T5 = Negative Control.

Treatments Commercial 25% 50% 100% Negative

Fungicide Calamansi Calamansi Calamansi Control
Extract Extract Extract

Name of
Molds Molds Molds Molds Molds
fungus used

Incubator

Temperature 35 oC 35 oC 35 oC 35 oC 35 oC

used

Storage Properly Properly Properly Properly Properly

Sealed Sealed Sealed Sealed Sealed

Filter Paper
Puncher Puncher Puncher Puncher Puncher
Disk ( size ) Size Size Size Size Size

12
 FLOW OF THE STUDY

Isolation of Pathogen

Extraction of Calamansi Fruit

Preparation of Treatments

Preparation of Culture Media

Preparation of the Fungal Standard Solution

Impregnation of Filter Paper Disk

Application of Discs on PDA

Incubation of Agar Plates

Disposal of PDA with Rhizopus stolonifer

Data Gathering Technique

Statistical Treatment

13
Research Locale

The study will be conducted in the Department of Agriculture, Regional Field,

Office 7, Mandaue City. This research is a qualitative research as it will observe the

effects of calamansi extract against molds in the given time of inhibition ( 36th hour,

48th hour and 90th hour ). Calamansi will be bought in local markets. The fruit will be

washed and extracted in Department of Agriculture.

Bread will be obtained in local bakeries near the area. The researchers will need

3 breads of different kinds to see which of the breads will grow more black bread molds

and which breads will grow the said molds.

The concentrations will be 10% calamansi extract, 50 % calamansi extract and

100% calamansi extract with the negative control ethanol.

14
Materials Needed

For the extract, calamansi will be used.

For the negative control, ethanol will be used.

For the medium, Bread of any different kinds will be used.

For storing the molds after isolation, disks will be used.

For storing the bread which has molds, zip lock will be used.

For safety so that the researchers will not get any disease, masks and lab gowns will be

used.

For the lesser possibility of not contaminating the microorganism, rubbing alcohol will be

used.

15
Research Procedure

A. Isolation of Pathogen

The researchers will collect the molds in the bread. They were placed together in

a zip lock plastic bag to ensure that no other pathogen could contaminate. The molds

were brought to the Department of Agriculture- in Maguikay, Mandaue City. The bread

molds were examinedthrough microscopes. The molds were transferred through a petri

dish. They were set aside to allow the growth of the fungi. The fungal growths were

transferred to an agar slant for pure culture. Samples were taken from the agar slant

were dyed with methylene blue. Once dyed, each of them was mounted on a compound

light microscope. Pure cultures were then placed into previously plated PDA and were

allowed to grow for about two weeks. The said petri dishes were sealed again with

parafilm to prevent contamination.

B. Extraction of Calamansi Fruit

The extraction will be done at Department of Agriculture- in Maguikay, Mandaue

City. The researchers brought Calamansi in the laboratory and cut it all. After cutting it,

it was placed inside a 500 ml beaker with ethanol. It was set aside for 24 hours. After 24

hours the set aside beaker was prepared and cheese cloth also was prepared for

filtration. When the filtration was done the extract was put into a rotary evaporator for

the ethanol to be separated from the calamansi extract.

16
C. Preparation of Treatments

100 ml beakers were prepared for the calamansi extract, positive control and

negative control.

D. Preparation of Culture Media

The Potato Dextrose Agar (PDA) was used as the culture medium for growth of

the pathogen. It will be prepared by dissolving 7.8 grams of PDA in 200ml distilled water

and was sterilized. Autoclave for 15 minutes at 121°C. Sterile PDA media was distribute

in a petri dish inside the biosafety cabinet and was set aside to solidify. The bread was

used as the culture medium for the pathogen. The researchers will wait for the bread to

have molds.

E. Preparation of the Fungal Standard Solution

Exactly 0.5 McFarland Standards equivalent turbidity was prepared for

comparison. Suspension of Rhizopus stolonifer was prepared by aseptically inoculating

in a sterile saline solution. The inoculated tube was compared to 0.5 McFarland

Standards against black and white line. The sample, Rhizopus stolonifer, was

transferred to the test tube containing 10ml of distilled water. The 30ml PDA in

Erlenmeyer flask was inoculated with sample of Rhizopus stolonifer. It will be incubated

for 24 hours to produce the active strain.

17
F.Impregnation of Filter Paper Disk

Calamansi extracts, the positive (commercial fungicide) and negative control will

be prepared. Then, fifty pieces of punched filter paper was produced. It will serve as the

discs. Ten (10) discs were soaked into the desired treatments for 12 hours. After the

12th hour of soaking, they were taken and placed in a previously prepared in the fungal

culture.

G. Application of Discs on PDA

Using a pen, the dishes was divided into 5 labels. 10% Calamansi Extract, 50%

CalamansiExtract , 100% Calamansi Extract, Commercial fungicide and Negative

Control. The pointed forceps was soak first in an 80% ethanol solution and was passed

over the flame to kill microorganisms. They were leave to cool for 5 seconds. The discs

were placed with the sterilized forceps into its designated areas in the previously

inoculated plates. Each of the discs was pressed gently into the PDA until it was in full

contact with the PDA surface. Each were place equidistant from each other for at least

10mm.

H. Incubation of Agar Plates

After the application of discs, plates were incubated for 42 hours at 35°C.

18
I. Disposal of PDA with Rhizopus stolonifer

After measurements and observations were done, the petri dishes was secured

in plastic autoclave bag and were sterilized for15 minutes in the autoclave for121°C

prior to disposal to kill the Rhizopus stolonifer. After sterilizing, the agar in each dish

was threw in a laboratory wastebasket, and then the dishes were washed with water

and dishwashing liquid. Water was allowed to drip before wiping them with a clean

cloth.

J. Data Gathering Technique

After the different discs was placed unto the fungal colony, the change of

diameter was determined by measuring the zone of inhibition using a ruler after 48th

hour, 72nd hour and 96th hour using this process. The change of diameter of the fungal

colony was measured by subtracting the diameter in the previous time to the current

time during the time of observations.

K. Statistical Treatment

In getting the average change of colonies per set up per trial, it was done by

adding all the zone of inhibition and divides them by the number of the addends. The

mean was used to determine the statistical significance through the use of one way

ANOVA

19
 CHAPTER IV

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter presents the findings of the investigation in the different aspects of

the study. It aims to answer the problems stated in the previous chapters.

To achieve the goal of the study, the researchers would need a laboratory that

has the materials needed in the study. The researchers went to the Department of

Agriculture Regional Field Office 7, Mandaue City. The succeeding findings were

presented under headings that coincide to the various points of the problem.

Table 2. The average zone of inhibition of rhizopus stolonifer in each different

timezones.

Average Zone of Inhibition

Treatments 48th hour 72nd hour 96th hour

T1 T2 T3 T1 T2 T3 T1 T2 T3

25% 0.03 0.4 0.7 0.23 0.5 0.6 10.8 0.86 0.87

Calamansi

Extract

50% 0.75 0.89 0.97 1.1 1.3 0.53 1.7 0.79 1.8

Calamansi

Extract

100% 0.9 1.1 0.8 0.33 0.5 0.45 0.7 1.4 0.96

Calamansi

Extract

20
Positive 3.0 4.1 4.2 5.32 4.35 6.36 4.54 4.4 4.8

Control (

Commercial

Fungicide )

Negative 0 0 0 0 0 0 0 0 0

Control

(Ethanol)

The table shows the result by using the Randomized Complete Block Design.

The results shows that the positive control ( commercial fungicide ) has the highest

zone of inhibition, followed by the experimental ( 100% Calamansi Extract, 50%

Calamansi Extract, 25% Calamansi Extract ) and lastly the negative control ( ethanol )

which did not show any zone of inhibition

21
Table 3. Results on the zone of inhibition of Rhizopus stolonifer during 48th hour

Trial 1 Trial 2 Trial 3

Treatments R1 R2 R3 R1 R2 R3 R1 R2 R3

Positive 1.1 0.9 1.0 2.1 1.3 0.7 0.8 1.4 2.0

Control

Negative 0 0 0 0 0 0 0 0 0

Control

25% 0 0.2 0.1 0.1 0.1 0.2 0.4 0.1 0.2

Calamansi

Extract

50% 0.26 0.27 0.22 0.4 0.3 0.19 0.32 0.32 0.33

Calamansi

Extract

100% 0.4 0.2 0.3 0.4 0.5 0.2 0.3 0.3 0.2

Calamansi

Extract

This table shows the results on the zone of inhibition in the 48th hour. The results

shows that the zone of inhibition is higher on the positive control and that it is not in

increasing order. There is a change of zone of inhibition.

22
Table 4. Results on the zone of inhibition of Rhizopus stolonifer during 72nd hour

Trial 1 Trial 2 Trial 3

Treatments R1 R2 R3 R1 R2 R3 R1 R2 R3

Positive 2.2 2.0 1.12 1.14 2.1 1.11 1.16 2.9 3.1

Control

Negative 0 0 0 0 0 0 0 0 0

Control

25% 0.13 0.9 0.1 0.2 0.1 0.2 0.1 0.3 0.2

Calamansi

Extract

50% 0.5 0.4 0.2 0.4 0.2 0.7 0.16 0.19 0.18

Calamansi

Extract

100% 0.1 0.1 0.13 0.2 0.1 0.2 0.13 0.22 0.1

Calamansi

Extract

This table shows the results on the zone of inhibition in the 72nd hour. The

results show that the zone of inhibition is higher in the positive control and lower in the

negative control. The results are not in increasing order which proves the change of

zone of inhibition.

23
Table 5. Results on the zone of inhibition of Rhizopus stolonifer during 96th hour

Trial 1 Trial 2 Trial 3

Treatments R1 R2 R3 R1 R2 R3 R1 R2 R3

Positive 2.13 1.21 1.2 1.3 1.9 1.2 1.4 1.6 1.8

Control

Negative 0 0 0 0 0 0 0 0 0

Control

25% 0.1 0.2 0.1 0 0.1 0 0.1 0.1 0

Calamansi

Extract

50% 1.0 0.3 0.4 0.31 0.25 0.23 0.5 0.4 0.9

Calamansi

Extract

100% 0.2 0.3 0.2 0.3 0.7 0.4 0.33 0.32 0.31

Calamansi

Extract

This table shows the results on the zone of inhibition in the 96th hour. The results

shows that the zone of inhibition is higher in the positive control and lower in the

negative control. This proves the change in the zone of inhibition as there is some

Molds growing while the others are getting removed or reduced.

24
 CHAPTER V

SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary

The researchers conducted an experimental study about the Antifungal Activity of

Calamansi Extract ( citrofortunella microcarpa ) against Black Bread Molds ( rhizopus

stolonifer ). The study used randomize complete block design in the study. The study

was conducted in the Department of Agriculture Regional Office 7, Mandaue City where

in the researchers used their materials and assistance in conducting the study. The

independent variables in the study is the positive control-commercial fungicide, 25%

Calamansi Extract, 50% Calamansi Extract, 100% Calamansi Extract and negative

control-ethanol. The highest zone of inhibition is commercial fungicide and followed by

the 100% Calamansi Extract. The lowest zone of inhibition is the negative control which

is the ethanol. The Calamansi Fruit was extracted and the molds was isolated in the

laboratory. The molds grew for 2 weeks in the laboratory and after the weeks, the

extract was applied in the molds. The area and diameter of the zone of inhibition was

noted by the researcher, as well as the certain time zones ( 48th hour, 72nd hour and

96th hour ). In finding the statistical treatment, One way ANOVA was used.

Conclusion

The study entitled Antifungal Activity of Calamansi Extract ( citrofortunella

microcarpa ) against Black Bread Molds ( rhizopus stolonifer ) found out that calamansi

25
extract has the potential to remove and reduce molds. Based on the tests conducted in

the study, the positive control ( commecial fungicide ) has the highest zone of inhibition

followed by the 100% Calamansi Extract , 50% Calamansi Extract and 25% Calamansi

Extract. The lowest zone of inhibition has the lowest zone of inhibition with no results.

The results on the test statistics was that there is no significant effect on the

average zone of inhibition..

Recommendations

For the development of the experimental study, the researchers would like to

recommend a few advices in future researchers conducting the study. It is much better if

the researchers would use a different citrus fruit and compare its ability to remove

molds, if it is comparable to the calamansi fruit and find out which fruit is much more

affordable and money saving .

Future researchers should have a field test after the experimental test to check

the bakeries on which bread grows mold the fastest and the slowest. There should be a

longer time in doing the study.

26
REFERENCES

27
http://patthebaker.com/home/the-importance-of-bread/

www.britannica.com/science/plant-disease

http://bioweb.uwlax.edu/bio203/2011/olbrantz_chri/

http://bioweb.uwlax.edu/bio203/2011/olbrantz_chri/classification.htm

http://www.missouribotanicalgarden.org/PlantFinder/PlantFinderDetails.aspx?taxonid=2
91787

pfaf.org/User/Plant.aspx?LatinName=Citrofortunella+microcarpa

https://uses.plantnet-project.org/en/Citrofortunella_microcarpa_(PROSEA)

https://www.britannica.com/science/Rhizopus-stolonifer

Skinner, C. (1930). Molds, Yeasts and Actinomycetes. John Wiley and Sons.

28
APPENDICES

29
 APPENDIX A

March 11,2018

Mrs. Maricor Mandawe

Research Adviser

Talamban National High School

Talamban, Cebu City

Dear Madame,

Greetings!

We the students of Grade 9 St Luke will be having an investigatory project as a

requirement for our Research II. We have decided to conduct our study entitled " The
Antifungal Activity of Calamansi Extract ( citrofortunella microcarpa ) against Black
Bread Molds ( rhizopus stolonifer )".

In this connection, we would like to ask for your permission for us to be able to
conduct at the Department of Agriculture Regional Office 7 due to the fact that their
laboratory holds the material for our experimentation.

We are hoping for your approval regarding the matter. Thank You and God
Bless!!

Sincerely,

JAYLA MAE MONTEBON

JODI GABRIANNE T. SORIANO

NEIL IVAN ARDIENTE

Noted by: Approved by:

FARAH CENIZA MARICOR MANDAWE

Research II Teacher Research Adviser

30
 APPENDIX B

STEP 1: State the Hypotheses.

H0 = The different concentrations have no significant effect on the average zone of

inhibition of molds at different time of exposure.

H1 = The different concentrations have a significant effect on the average zone of

inhibition of molds at different time of exposure.

STEP 2: State the Alpha.

Level of Significance = 0.05

STEP 3: Calculate the Degrees of Freedom

N= 45 n= 5

Dfbetween = a-1

= 3-1

=2

Dfwithin =N-a

=15-3

=12

Dftotal =N-1

31
 =15 - 1

=14

STEP 4: State the Decision Rule

If F is greater than 3.21, reject the null hypotheses

STEP 5: Calculate Test Statistics

Source of Sum of Degrees of Mean of F

Variation Squares Freedom Squares

Between 3.02 2 1.51 0.21

Treatment
Within 87.3677 12 7.28
Treatment
14
Total 47.84

  a  T Zone of Inhibition:
2
2
i
SSBT =
n N

48th Hour: 0.38 + 0.87 + 0.93 + 3.77 + 0

=5.952 + 7.192 + 11.212 -24.352 = 5.95
5 15

72nd Hour: 0.44 + 0.98 + 0.43 + 5.34 + 0

= 42.5525 - 39.928 = 7.19

= 3.02
96th Hour: 4.18 + 1.43 + 1.02 + 4.58 + 0
= 11.21

32
   a 
2

SSWT =  Y  2 i

= 87.3677 - 5.952 + 7.192 +

11.212

= 87.3677 - 42.55254

= 44.82

0.382 + 0.872 + 0.932 + 3.772 + 02 +

0.442 + 0.982 + 0.432+ 5.342 + 02 + 4.182

+ 1.432 + 1.022 + 4.582 + 0 2= 87.3677

33
 =44.82

T2
SSTT = Y 2  N

= 87.3677 - 24.352

15

= 47.84

SS BT
MSBT =
Df BT

= 3.02
2

=1.51

SSW T
MSWT =
Df W T

= 87.3677
12

= 7.28

MS BT
F=
MSW T

= MS BT
MS WT

= 1.51
7.28

= 0.21

34
STEP 6: State Results

If F is greater than 3.89, reject the null hypothesIs.

F= 0.21

STEP 7: State conclusion

Therefore, The different concentrations have no significant effect on the average

zone of inhibition of molds at different time of exposure.

35
 APPENDIX C

Documentation

Isolation of Pathogen Extraction of Calamansi Fruit

Preparation of Treatments Preparation of Culture Media

36
Preparation of the Fungal Standard Impregnation of Filter Paper Disk

Solution

Application of Discs on PDA Incubation of Agar Plates

Disposal of PDA with Rhizopus stolonifer

37
 APPENDIX D

Expenses

Travel Expenses 208

Calamansi Fruit 30

Bread 15

TOTAL P 253.00

38
 CURRICULUM VITAE

Personal Data

Name : Jodi Gabrianne T. Soriano

Home Address : Villa Leyson, Bacayan, Cebu City

Age : 14

Date of Birth : October 6, 2004

Place of Birth : Chong Hua Hospital, Cebu City

Parents

Father : Dexter O. Soriano

Mother : Josephine T. Soriano

Educational Background

Elementary : Bacayan Elementary School

Bacayan, Cebu City

S.Y. 2010 - 2016

Secondary : Talamban National HIgh School

Talamban, Cebu City

S.Y. 2016 - Present

39
 CURRICULUM VITAE

Personal Data

Name : Neil Ivan Ardiente

Home Address : Baugo, Budla-an, Cebu City

Age : 15

Date of Birth : February 13, 2004

Place of Birth : Cebu City

Parents

Father : Arnil Ardient

Mother : Cherry Ardiente

Educational Background

Elementary : Talamban Elementary School

Talamban, Cebu City

S.Y. 2010 - 2016

Secondary : Talamban National HIgh School

Talamban, Cebu City

S.Y. 2016 - Present

40
 CURRICULUM VITAE

Personal Data

Name : Jayla Mae Montebon

Home Address : Cabancalan, Talamban, Cebu City

Age : 15

Date of Birth : January 24, 2004

Place of Birth : Cebu City

Parents

Father : Edito Montebon

Mother : Concepcion Montebon

Educational Background

Elementary : Talamban Elementary School

Talamban, Cebu City

S.Y. 2010 - 2016

Secondary : Talamban National HIgh School

Talamban, Cebu City

S.Y. 2016 - Present

41