Life Science [SHS]

Team Category – District Level

Bacteriostatic Potential of Great Bougainvillea (Bougainvillea spectabilis) Leaf Extract

against Bacillus cereus
Erwin B. Mista1, Ariane Kate Z. Omnes2 & John Kerk B. Lizardo3
Research Advisers: Ms. Jereen A. Batomalaque and Mr. Rommel Gutoman
Batasan Hills National High School
IBP Road, Batasan Hills, Quezon City

ABSTRACT
Food contamination has become one of the severe problems of the world ever since the
world started to develop. Food contamination impedes socioeconomic development by harming
national economies and health care systems. In line with this, the problem of food poisoning
started to be one of the government’s problems since it affects the health of the people, economy
and industries. The research study was conducted to determine the potential of Great
Bougainvillea leaf extract in inhibiting the growth of bacteria that causes food poisioning. This
study aims to provide safe and useful bacteriostatic agent against Bacillus cereus using pure
extract from Great Bougainvillea leaves.
The researchers collected the materials around Quezon City. The specimen was plant
identified by the UP Institute of Biology. The 5 mL pure extract was collected from Great
Bougainvillea leaves using juice extractor and was put in a sterilized bottle afterwards. The
sample was then delivered to UP NSRI-MRSL for the proper antimicrobial assay against
Bacillus cereus and was compared to Chloramphenicol disca. The data was gathered through
measuring the clearing zones of each sample in millimeters.
The statistical treatment used in this study is Chi-Square Goodness of Fit Test to
determine the difference of samples and the effectiveness of the specimen as a bacteriostatic
agent. The experiment showed antimicrobial activity against B. cereus with an AI of 0.2.
The research study shows the potential of Great Bougainvillea as a bacteriostatic agent
against B. cereus. The researchers recommend trying using different specimen and methods in
testing Great Bougainvillea leaf efficiency.

Keywords: food contamination, bacteriostatic, antimicrobial assay, Great Bougainvillea

INTRODUCTION
Mild preservation technologies are becoming more important in modern food industries.
As a consequence, spore-forming microorganisms are likely to proliferate and hence become a
serious food safety risk. Food contamination is one of the most severe problems that our country
is experiencing right now. It greatly affects health care systems, industries and economy (World
Health Organization, 2017). A way to reduce the proliferation of microorganisms is the use of
essential oils and extracts. Bacillus cereus is a gram-positive aerobic or anaerobic spore-forming
rod. It is a cause of food poisoning, which is manifested by diarrhoeal or emetic syndromes. The
organism produces an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxin,
respectively (Helgason, Okstad & Caugant, 2015). Other toxins are produced during growth,
including phospholipases, proteases, and hemolysins, one of which, cereolysin, is a thiol-
activated hemolysin. These toxins may contribute to the pathogenicity of B. cereus in
nongastrointestinal disease (Ultee, Bennik & Moezelaar, 2016).
Great Bougainvillea Plants are abundant and commonly found here in our country. Its
hermaphroditic flowers, also referred as “paper flower”, come in different colors and are usually
use as home and garden decorations, while its leaves are usually kept aside (Great Bougainvillea,
2014).The study with regards to the use of its leaves for inhibiting the growth of this bacteria will
be a significant endeavor in promoting safeness in food owing to the fact that Great
Bougainvillea leaf plant are considered as non-edible plant and also, by preventing the toxins
production in food. According to Jin (2018), phytochemical analysis of Great Bougainvillea
leaves yielded the presence of flavonoids and tannins that which are commonly used components
against common bacteria. These also include the extraction processes that include ethanol and
methanolic extracts of the so called plant. It is suggested to use this leaves as an alternative
materials in creating antimicrobial products for the plant is common plant and have a fast growth
to achieve greater efficiency and lessen the production cost thus greatly reducing antimicrobial
products. On the other hand, commercialized antimicrobial products such as penicillin though the
price is relatively lower nowadays, the increase in resistance of pathogens specifically bacteria
against common antimicrobial products greatly enhanced because of repeated uses thus require
on the improvement products to eliminate harmful bacteria.
Through the evidences that were garnered by the researchers, assumptions were made.
The researcher assumed that Bougainvillea leaves have a significant effect in inhibiting the
growth of a gram-positive bacteria, bacillus cereus otherwise the leaves are not sufficient and
effective against the bacteria
Using the pure extract from Great Bougainvillea leaves will be more beneficial than
using any commercialized anti-bacterial agent. It will lessen the number of victims of food
poisoning in the country and the number of wastes specifically spoiled food that are inedible and
usually thrown anywhere. Since the growth of bacteria that cause this problem will be inhibited.
It will be cheaper and safer to use in inhibiting the growth of bacillus cereus. It also aims to
provide additional knowledge on people on the different benefits of using Great Bougainvillea
leaves and to provide safe production of food that can last longer.
This study will solely focus on the effect of great bougainvillea as a bacteriostatic agent
against Bacillus cereus. This study will include the growth rate of Bacillus cereus against the
amount of great bougainvillea and chloramphenicol disca as representative commercialized
antibacterial agent. Consequently, this study will produce appropriate information regarding
Bacillus cereus and great bougainvillea. Specifically, the method used for great bougainvillea
leaf extract. This study will be conducted in NSRI UP Diliman laboratory in 24 hours.

Conceptual Framework
This study aims to evaluate the efficiency of the antimicrobial activity of Great
Bougainvillea leaves extract against Bacillus cereus. The independent variable of the study is the
amount of pure leaves extract of Great Bougainvillea because it is the one that will be
manipulated. It will be measured in milliliters. The dependent variable of the study is the amount
of Bacillus cereus inhibited. The output or outcome of the study is that the pure leaves extract of
Great Bougainvillea has an antimicrobial activity against Bacillus cereus because the cleared
zone of B. cereus depends on the amount of pure leaves extract of Great Bougainvillea.
 INDEPENDENT VARIABLE DEPENDENT VARIABLE

Amount of pure leaves extract of Amount of Bacillus cereus

Great Bougainvillea leaves inhibited

OUTPUT

The pure leaves extract of Great

Bougainvillea as a bacteriostatic
agent against Bacillus cereus

Figure 1. Research Paradigm of the Study

METHODOLOGY
The research study was conducted from August 25, 2018 to September 08, 2018 at
Batasan Hills National High School Science Laboratory and Natural Science Research Institute
(NSRI) in U.P. Diliman for testing.

Phase I
The researchers collected the materials required to perform the preferred method in
conducting this research study. The main specimen which is the Great bougainvillea was
collected in Quezon City Memorial Circle and other materials such as juicer and sterilized glass
bottle were collected in the researcher’s house and UP Diliman, MRSL respectively.

Phase II
The researchers brought Great Bougainvillea plant to UP Diliman, Institute of Biology
for plant identification to assure if the plant garnered is truly great bougainvillea. After, it was
therefore identified that the plant is Bougainvillea spectabilis from Nyctaginaceae family.

Phase III
The researchers then used the collected Great Bougainvillea leaves to produce a leaf
extract. First, the researchers thoroughly washed each of the leaves until the dirt was removed
and the water that was retained on the surface of the leaves were removed using a clean and
sterilized cloth to make sure no other substance was mixed on the extract. Next, the leaves were
then placed on the juicer and the process was controlled by one of the researchers until the liquid
extract started to appear and leaves were completely used. Lastly, the 5mL liquid extract was
then put on a sterilized container to avoid any form of contamination and was properly labeled to
avoid any kind of confusion.
Phase IV
The sample was then brought to UP Diliman, Natural Sciences Research Institute
(NSRI)–Microbiological Research and Services Laboratory (MRSL) by the researchers for
proper anti-microbial analysis and anti-microbial assay that verified the legibility of the data
gathered in the experiment. Microbial suspension was prepared from 24-hour old culture of the
test organism. The suspending medium used was 0.1% peptone water.
Pre-poured Nutrient Agar (NA) plates about 3mm thick, were inoculated with the
respective microbial suspension by swabbing the agar surface. The cotton swab on an applicator
stick was dipped into the microbial suspension, rotated several times and pressed firmly on the
inside wall of the tube above the fluid level to remove excess inoculum from the swab. The swab
was streaked over the entire agar surface. This procedure was repeated two more times, rotating
the plate 60° each time to ensure even distribution of the inoculum. Three (3) equidistant wells
were made on the agar plate using cork borer (10mm diameter). Two hundred (200) µl portion of
the sample was placed in each of the wells.
The NA plates were incubated at 35°C and observed after 24 hours. The clearing zone
was measured in millimeters and the average diameter of the clearing zones was calculated. The
antimicrobial index (AI) was computed using the following formula:
Diameter of clearing zone − Diameter of well
AI =
Diameter of well
Phase V
The researchers used the MRSL laboratory safety guidelines as a base for the waste
disposal in the experiment. The researchers used washed hands using a germicidal soap to
eliminate possible bacteria on the skin and used disinfectant on things such as bench and ball pen
as safety precaution. The laboratory equipment used was sterilized before washed and disposable
things were disposed on the trash bin after use.

Phase VI
The results on showed in the antimicrobial index are used as variables that serve as data
used in the statistical tool appropriate for determining the conclusion. The researchers used chi-
square ‘’goodness of fit test’’ to find out how the observed data are significantly different for the
expected data. The results of the statistical test determine the stand that the researcher provide an
answer to the questions posed earlier before experimentation. To compute for the value of Chi-
Square goodness of fit, the following formula was use:
Σ(𝑂−𝐸)2
𝑋2 = .
E
where: O – the observed frequency in a given category
E – the expected frequency in a given category
𝑋 2 – Chi - square Goodness of Fit Test
 RESULTS
The experimentation was composed of two samples with three trials each. Same amount
of Great Bougainvillea Leaf Extract and Chloramphenicol disca were applied to three trials of
clearing zones on the wells of the agar plate. This was implied to further justify the potential of
Great Bougainvillea leaf extract to inhibit the growth of Bacillus cereus and to compare the
effectiveness of the sample to commercialized one.
The results obtained were recorded after 1 week. The table 1 below shows the clearing
zones in each sample. The clearing zones were measured in millimeters and the average diameter
of the clearing zones was calculated. Great Bougainvillea leaf extract has a clearing zone of 12
mm in trial 1, 12 mm in trial 2, and 12 mm in trial 3. While the commercialized antibacterial,
chloramphenicol disca, has a clearing zone of 20 mm in each trials.

Test organism Sample Clearing zone, mm

AI
1 2 3

Great Bougainvillea leaf 12 12 12 0.2

B. cereus extract

Chloramphenicol disca 20 2.3

a
6-mm diameter disc, contains 30 µg chloramphenicol
Table 1. Clearing zones (mm) in each sample and the antimicrobial index

25

20

15 Trial 1
Trial 2
10 Trial 3

0
Bougainvillea Leaf Extract Chloramphenicol disc

Figure 2. Data interpretation after 1 week of experimentation

 The bar graph on Figure 2 shows the results of the experimentation after 1 week. The
antimicrobial index (AI) was computed based on the results gathered. Great Bougainvillea leaf
extract showed antimicrobial activity against B. cereus with an AI of 0.2.

Statistical Treatment
𝐻𝑜 : There is no significant effect that shows the potential of Great Bougainvillea leaf extract as a
bacteriostatic agent against B. cereus.
𝐻𝑎 : There is a significant effect that shows the potential of Great Bougainvillea leaf extract as a
bacteriostatic agent against B. cereus.
The three trials each had the same amount of Great Bougainvillea leaf extract and
Chloramphenicol disca applied before the experimentation. After the observation of clearing
zones, the sample showed consistency of data in clearing zones in each trial.
Level of Significance = 0.05
Trial 1 Trial 2 Trial 3

Great Bougainvillea 12 mm 12 mm 12 mm
leaf extract

Chloramphenicol 20 mm 20 mm 20 mm
disca

(𝑂 − 𝐸) -8 -8 -8

(𝑂 − 𝐸)2 64 64 64

𝑋2 3.2 3.2 3.2

Σ𝑥 2 = 9.6
Table 2. Statistical Analysis

DISCUSSION
To test both null and alternative hypothesis, the Chi-square goodness of fit was used. The
Chi-square goodness of fit is used to compare the difference between the samples tested in
finding out the potential of Great Bougainvillea leaf extract in inhibiting the growth of B. cereus.
Σ(𝑂−𝐸)2
The formula of 𝑋 2 is 𝑋 2 = .
E
Having 0.05 level of significance, the Chi-square goodness of fit suggested that there is a
significant effect that shows the potential of Great Bougainvillea leaf extract as a bacteriostatic
agent against Bacillus cereus.
The chi-square suggested that the null hypothesis of this study was rejected which means
that the research study showed a bacteriostatic potential against Bacillus cereus when Great
Bougainvillea leaf extract was used.
Conclusion
After the samples were tested, the clearing zones were constant in each sample. The
researchers have done analyzing and interpretation of results. The results showed that the Great
Bougainvillea leaf extract showed bacteriostatic potential in against Bacillus cereus with an AI
of 0.2. Therefore, it is concluded that the Great Bougainvillea (Bougainvillea spectabilis) leaf
extract has a significant effect in inhibiting the growth of Bacillus cereus.

Recommendation
It is therefore recommended to future researchers to try using other leaf extracts as an
antibacterial agent against Bacillus cereus. It is also advised to future researchers to try other
concentration of Great Bougainvillea leaf extract and use more effective methods and techniques
including the ethanolic and methanolic extraction of the extract, in harnessing Great Bougainvillea leaf
extract and retain the antibacterial property within Great Bougainvillea leaf. A different technique may
yield better results or concentrations, so it is recommended to test other techniques as it may
increase the efficiency of the results. It is also recommended to conduct a research that will have
a study and gather data on different active components that may have been overlooked and could
inhibit the growth of certain bacteria.
 REFERENCES

1. Great Bougainvillea. Bougainvillea spectabilis - Overview. (2014). Retrieved from

http://eol.org/pages/2899871/overview
2. Helgason, E., Økstad, O. A., Caugant, D. A., (2015, June 01). Bacillus anthracis, Bacillus
cereus, and Bacillus thuringiensis-One Species on the Basis of Genetic Evidence.
Retrieved from https://aem.asm.org/content/66/6/2627.short
3. Mao Bao Jin. (2018). Bogambilya. Retrieved from
http://www.stuartxchange.org/Bogambilya
4. World Health Organization. (2017, October 31). Food safety. Retrieved from
http://www.who.int/news-room/fact-sheets/detail/food-safety
5. Ultee, A., Bennik, M. H., & Moezelaar, R. (2016, April 01). The Phenolic Hydroxyl Group
of Carvacrol Is Essential for Action against the Food-Borne Pathogen Bacillus cereus.
Retrieved from https://aem.asm.org/content/68/4/1561.short
APPENDICES
Experimentation

Figure 3. Inquiry of bacteria

Figure 4. Inquiry of the

requirements for plant identification

Figure 5. Collection of Great

Bougainvillea plant in Quezon
City Memorial Circle
Figure 6. Plant identification at UP Diliman, Institute of Biology

Figure 7. The researchers gathered the materials

 Figure 8. The researcher washed Great Bougainvillea leaves

Figure 9. The researcher used a Figure 10. The researcher went

juicer to extract from Great to MRSL laboratory for testing
Bougainvillea leaves
 Figure 11. The researchers went
to MRSL laboratory to claim the
experiment results

Figure 12. Front view of the Figure 13. Back view of the
Nutrient Agar plate used for Nutrient Agar plate used for
experiment experiment
Figure 14. Certificate of Authentication for Plant Identification
Figure 15. Service report for Antimicrobial Assay (page 1)
Figure 16. Service report for Antimicrobial Assay (page 2)
Hypothesis Testing

Trial 1 Trial 2 Trial 3

Great Bougainvillea 12 mm 12 mm 12 mm
leaf extract

Chloramphenicol 20 mm 20 mm 20 mm
disca

(𝑂 − 𝐸) -8 -8 -8

(𝑂 − 𝐸)2 64 64 64

𝑋2 3.2 3.2 3.2

Σ𝑥 2 = 9.6
Table 2. Statistical Analysis
Critical Value: 𝑋 2 𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 = 5.991
Σ(𝑂−𝐸)2
𝑋2 = . where: O – amount of observed diesel before experimentation
E
E – amount of observed diesel after experimentation
𝑋 2 - chi – square

(12 − 20)2 (12 − 20)2 (12 − 20)2

𝑋2 = + +
20 20 20

= 3.2 + 3.2 + 3.2

2
𝑋 = 9.6

Degrees of freedom: df = k – 1
=3–1
=2
The tabular value of 𝑋 2 at 0.05 level of significance with degrees of freedom of 2 is
5.991.

Acceptance
Region

𝑋 2 = 5.991 𝑋 2 = 9.6
Decision
Figure 17. Normal curve Since 𝑋 2 > 𝑋 2 , reject null
hypothesis.
 RESEARCH PLAN/PROJECT SUMMARY

Bacteriostatic Potential of Great Bougainvillea (Bougainvillea spectabilis) Leaf Extract

against Bacillus cereus
Erwin B. Mista1, Ariane Kate Z. Omnes2 & John Kerk B. Lizardo3
Research Adviser: Ms. Jereen A. Batomalaque
Batasan Hills National High School
IBP Road, Batasan Hills, Quezon City

Rationale
Food contamination has become one of the severe problems of the world ever since the
world started to develop. Food contamination impedes socioeconomic development by harming
national economies and health care systems. In line with this, the problem of food poisoning
started to be one of the government’s problems since it affects the health of the people, economy
and industries. Mild preservation technologies are becoming more important in modern food
industries. As a consequence, spore-forming microorganisms are likely to proliferate and hence
become a serious food safety risk. A way to reduce the proliferation of microorganisms is the use
of essential oils and extracts. Bacillus cereus is a gram-positive aerobic or anaerobic spore-
forming rod. It is a cause of food poisoning, which is manifested by diarrhoeal or emetic
syndromes. The organism produces an emetic or diarrheal syndrome induced by an emetic toxin
and enterotoxin, respectively (Helgason, Okstad & Caugant, 2015). Based on the issues
regarding health and economic concerns caused by food contamination, the researchers were
encouraged to conduct the study. The investigatory project with regards to the use of its leaves
for inhibiting the growth of this bacteria will be a significant endeavor in promoting safeness in
food owing to the fact that Great Bougainvillea leaf plant are considered as non-edible plant and
also, by preventing the toxins production in food. The research study was conducted to determine
the potential of Great Bougainvillea leaf in inhibiting the growth of Bacillus cereus. This study
aims to provide safe and useful agent for antimicrobial against Bacillus cereus using pure extract
from Great Bougainvillea.

Hypothesis
The role of the Great Bougainvillea leaves gives entitlement for it to inhibit the growth of
Bacillus cereus which can causehis investigatory project aims to determine the effect of Great
Bougainvillea leaves extract as a bacteriostatic agent against Bacillus cereus. It also aims to
provide additional knowledge on people about the various effects which are considered common
plant found in Philippines to provide safety in food comsumption.Specifically, the researchers
formulated this nill hypothesis “There is no significant effect in the potential of Great
Bougainvillea leaves as a bacteriostatic agent against Bacillus cereus” and alternative hypothesis
“There is a significant effect in the potential of Great Bougainvillea leaves as a bacteriostatic
agent against Bacillus cereus”.
Procedures
The research study was conducted from August 25, 2018 to September 08, 2018 at
Batasan Hills National High School Science Laboratory and Natural Science Research Institute
(NSRI) in U.P. Diliman for testing.

1. Collection of Materials. The researchers collected the materials required to perform the
preferred method in conducting this research study. The main specimen which is the
Great bougainvillea was collected in Quezon City Memorial Circle and other materials
such as juicer and sterilized glass bottle were collected in the researcher’s house and UP
Diliman, MRSL respectively.
2. Plant Identification. The researchers brought Great Bougainvillea plant to UP Diliman,
Institute of Biology for plant identification to assure if the plant garnered is truly great
bougainvillea. After, it was therefore identified that the plant is Bougainvillea spectabilis
from Nyctaginaceae family.
3. Preparation of Great Bougainvillea leaf extract. The researchers then used the collected
Great Bougainvillea leaves to produce a leaf extract. First, the researchers thoroughly
washed each of the leaves until the dirt was removed and the water that was retained on
the surface of the leaves were removed using a clean and sterilized cloth to make sure no
other substance was mixed on the extract. Next, the leaves were then placed on the juicer
and the process was controlled by one of the researchers until the liquid extract started to
appear and leaves were completely used. Lastly, the 5mL liquid extract was then put on a
sterilized container to avoid any form of contamination and was properly labeled to avoid
any kind of confusion.
4. Antimicrobial Assay. The sample was then brought to UP Diliman, Natural Sciences
Research Institute (NSRI)–Microbiological Research and Services Laboratory (MRSL)
by the researchers for proper anti-microbial analysis and anti-microbial assay that
verified the legibility of the data gathered in the experiment. Microbial suspension was
prepared from 24-hour old culture of the test organism. The suspending medium used
was 0.1% peptone water. Pre-poured Nutrient Agar (NA) plates about 3mm thick, were
inoculated with the respective microbial suspension by swabbing the agar surface. The
cotton swab on an applicator stick was dipped into the microbial suspension, rotated
several times and pressed firmly on the inside wall of the tube above the fluid level to
remove excess inoculum from the swab. The swab was streaked over the entire agar
surface. This procedure was repeated two more times, rotating the plate 60° each time to
ensure even distribution of the inoculum. Three (3) equidistant wells were made on the
agar plate using cork borer (10mm diameter). Two hundred (200) µl portion of the
sample was placed in each of the wells. The NA plates were incubated at 35°C and
observed after 24 hours. The clearing zone was measured in millimeters and the average
diameter of the clearing zones was calculated.
5. The researchers used the MRSL laboratory safety guidelines as a base for the waste
disposal in the experiment. The researchers used washed hands using a germicidal soap to
eliminate possible bacteria on the skin and used disinfectant on things such as bench and
ball pen as safety precaution. The laboratory equipment used was sterilized before
washed and disposable things were disposed on the trash bin after use.
Data Analysis
A chi-square test for independence compares two variables in a contingency table to see if they
are related. The researcher used this for the antimicrobial activity of Great Bougainvillea leaves
extract as compared to Chloramphenicol against Bacillus cereus.

Bibliography
1. Great Bougainvillea. Bougainvillea spectabilis - Overview. (2014). Retrieved from
http://eol.org/pages/2899871/overview
2. Helgason, E., Økstad, O. A., Caugant, D. A., (2015, June 01). Bacillus anthracis,
Bacillus cereus, and Bacillus thuringiensis-One Species on the Basis of Genetic
Evidence. Retrieved from https://aem.asm.org/content/66/6/2627.short
3. Mao Bao Jin. (2018). Bogambilya. Retrieved from
http://www.stuartxchange.org/Bogambilya
4. World Health Organization. (2017, October 31). Food safety. Retrieved from
http://www.who.int/news-room/fact-sheets/detail/food-safety
5. Ultee, A., Bennik, M. H., & Moezelaar, R. (2016, April 01). The Phenolic Hydroxyl
Group of Carvacrol Is Essential for Action against the Food-Borne Pathogen Bacillus
cereus. Retrieved from https://aem.asm.org/content/68/4/1561.short

Checked by:

Ms. Jereen A. Batomalaque & Mr. Rommel Gutoman

Research Advisers