Antibacterial Activity of Blumea balsamifera Leaf Extracts against Staphylococcus

epidermidis (ATCC 35983)

Mikaella Sofia E. Abraham
Patrick Eldwin F. Ana
Vince Russel E. Bernardo
Leslie Khryzel B. Daniel
Martin Ralph T. Presbitero
Runn Dominic L. Rebustillo
Aubrey Marie Roque

Senior High School Department, University Of Santo Tomas-Legazpi

ABSTRACT

Communicable disease are quite evident in our environment today. Staphylococcus epidermidis is a gram-
positive pathogen and is one of the bacteria which contribute to the proliferation of communicable diseases. At
present, antibiotic resistance is becoming a big threat to global health. Currently, researchers have focused on
plant extracts as an alternative remedy to these emerging illnesses. One of the herbal plants that is evident in
the Philippines is Blumea balsamifera, locally known as Sambong. Previous studies have proved the
antifungal, antibiotic, antioxidant and antidiarrheal activity of this herb. However, there is paucity in the
knowledge of the antibacterial aspect of this herb. A three-week experimentation was conducted to evaluate the
antibacterial activity of 100% Blumea balsamifera leaf ethanolic extracts against Staphylococcus epidermidis.
Furthermore, tests were made to compare and contrast the antibacterial susceptibility of the experimental
(Blumea balsamifera), negative control (tap water) and positive control groups (ceftriaxone). The Kirby-Bauer
disk diffusion susceptibility test and Pour plate method were performed to discern the differences between the
experimental and control groups. The widest zone of inhibition that was deduced from the experiment was 12
millimeters. Additionally, no colonies of bacteria emerged except on the negative control. One-way ANOVA
on the results of the said tests showed that there was a statistically significant difference between the control
and experimental groups. The results of the past investigation suggests that the leaf extracts of Blumea
balsamifera can be used as a potential antibacterial agent against Staphylococcus epidermidis. Thus, the
attained understanding could be pertinent in reducing the further proliferation of the said communicable
diseases.

Keywords: Blumea balsamifera, Staphylococcus epidermidis, zone of inhibition, colony count

INTRODUCTION

Currently, people all over the world have diseases. Communicable diseases are acquired
been encountering diverse infections and illnesses, most commonly through direct contact and
which have been emerging in our environment physical interaction with the infected individual
(Morse, 2009). The one-celled organisms (Tartt, 2016).
responsible for some of these unfolding sicknesses
are the billions of bacteria that are present in the Staphylococcus is a type of microscopic
air that people breathe. A person can already organism that has more than thirty (30) types.
acquire an illness just by breathing (Weatherspoon, (MedlinePlus, 2016) However, one type of which
2016). Not only that, physical interactions between is the Staphylococcus epidermidis (Nguyen, Park
people occur every single minute, not being aware & Otto, 2017). This bacterium is purple in color
of the fact that they could have already transferred under a gram stain because it is a gram-positive
the bacteria present in their hands to others or the pathogen (Bukhari, 2004). This bacterium is
other way around. These bacteria found in the proven to be present in the skin and in the hands
hands often lead to illnesses (BetterHealth, 2015). (Micropia, nd). It is also present in saliva and
But specifically, it results to communicable dental plaque and is thought to be related with
1
pericoronitis, dry socket, periodontitis, acute and 2017) and Urinary Tract Infection (UTI) (Gelano,
chronic pulpitis, and angular stomatitis (Zhou & 2014) are the other medicinal benefits that this
Li, 2015). Infections of large wounds are often plant offers. Phytochemical analysis determined
associated with this type of bacterium (Bukhari, that the leaves of Blumea balsamifera contained
2004). Moreover, this bacterium has the highest sufficient amount of volatile oil and flavonoids
percentage and is the leading among all coagulase- (Fan, et al., 2015). Moreover, its leaves have been
negative Staphylococci making it the primary recommended for its various healing properties to
cause of clinical infections (Namvar, et al., 2014). certain diseases (Chu, Du & Liu, 2013).
Additionally, septicemia and endocarditis are also
diseases, which are caused by this type of According to the Phytochemical Analysis
pathogen that have symptoms such as fever, conducted by Chu, Du and Liu (2013), the main
headache, and dyspnea (Bukhari, 2004). In components of the extracted oil from the leaves of
Tacloban City, the number of carabao deaths Blumea balsamifera are 1, 8-cineole (20.98%),
reached up to 192 due to a hemorrhage septicemia borneol (11.99%), β- caryophyllene (10.38%),
outbreak (Desacada, 2007). Infective endocarditis camphor (8.06%), 4-terpineol (6.49%), a-terpineol
(IE) at the Philippine Heart Center has an annual (5.91%), and caryophyllene oxide (5.35%).
incidence of 10 out of 10,000 admissions because Due to the recommendations of prior
of this, 239 individuals with infective endocarditis studies, the leaves extract of Blumea balsamifera,
from 1979 to 2001 were studied and found out that which contains bacteria-reducing components,
95% of the patients aged 18-60 were infected due were chosen as the most beneficial for the study
to bacteremia from skin lesions (Pasaporte & Pena, compared to its stem, roots and other parts. It was
nd). An important study regarding infections was observed that current studies and literature focused
conducted in Naples between the years 1996 to on varying concentration of Blumea balsamifera
1998, results showed that out of the total of 184 leaves on wound healing and were used for its
infections, 56 of those were directly attributed to many benefits; this study focused on the
Staphylococcus epidermidis (Bukhari, 2004). antibacterial activity of Blumea balsamifera leaf
In our modern times, technology has extracts against Staphylococcus epidermidis.
been our mainstay in terms of medicine and This study is the pioneering study in
healthcare (Hofmann, 2001). However, herbal terms of the assessment of the antibacterial activity
medicine has been never left out in the limelight, it of Blumea balsamifera leaf extracts on a gram-
is still being widely used all over the world. The positive pathogen, the Staphylococcus epidermidis.
World Health Organization has assessed that over Several studies have already studied its
80% of the total population in developing antibacterial activity on other types of bacteria.
countries depend and utilize primarily herbal Also, this study used an ethanolic type of
medicine for healthcare uses (Tilburt & Kaptchuk, extraction on the leaves of the said herb.
2002). Blumea balsamifera or lakad-bulan as its
local name is a green endemic plant which is a Since medicinal herbs are widely known
well-known herbal medicine in the Southeast Asia, and being used globally, the results of this study
specifically in China, Thailand, Malaysia, Vietnam will redound to the benefit of the medical
and the Philippines (Fan, et al., 2015). This plant community. By understanding the needs of people
herb is often provided by traditional healers to convenient and inexpensive medicine, these
(albularyo) for its healing effects (Atrillano & people will surely be provided with alternative
Cipriano, 2014). This plant belongs to the Blumea medicine. The future researchers will be also given
genus (Alonzo, 2016). At present, this plant herb a brighter understanding of the different
can be utilized as an antibacterial (Sakee, possibilities of herbal plants to the field of
Maneerat, Cushnie & De-eknamkul, nd), medicine and this can serve as a basis among
antifungal (Fan et al, 2007), expectorant (Tropical researchers undertaking related field.
the ferns, 2014), and an antibiotic (Fujita, 2005)
and an antioxidant (Haider, 2014). The leaves are The study mainly extracted the leaf
applied in the forehead to relieve headache (Co, extracts of Blumea balsamifera through an
Ragasa & Rideout, 2003), leaf decoctions are used ethanolic extraction. Additionally, the research
as an antidiarrheal (Ahsan, 2016), antidiabetic only focused on one bacterium, specifically the
(Roy, Saha, Biswas, Ahmed & Mariappan, 2013) Staphylococcus epidermidis. Moreover, the study
and antigastralgic (Truon, 2012), for stomach only concentrated on the antibacterial activity of
pains and for aromatic baths in rheumatism (Co, et Blumea balsamifera on the said pathogen. Thus,
al., 2003). the research did not anymore include other types
of Staphylococcus bacteria or other pathogens as
Additionally, treating wounds (Fan, et al., well as any other plant leaves.
2015), coughs and colds (Gelano, 2014), lowering
blood pressure (Oyson, 2013), and curing kidney The general objective of this study is to
stones (Montealegre & De Leon, 2016), liver extract the leaves of Blumea balsamifera.
disease (Haider, 2014), abdominal pain (DOST,
2
The specific objectives of this research are to the subsequent procedure: (1) sterilize the agar by
discern parameters set for the study specifically the autoclaving it at 121 degrees Celsius for fifteen
zone of inhibition and number of colonies, minutes; (2) when the agar has cooled down
determine the effectiveness of 100% concentration around 50-55 degrees Celsius, mix it well and
of Blumea balsamifera leaf extracts against distribute it to sterile petri dishes and (3) store the
Staphylococcus epidermidis and to ascertain if petri dishes in plastic bags at 2-8 degrees Celsius
there is a significant difference on the possible to avoid loss of moisture.
antibacterial effects of Blumea balsamifera leaf
extracts from ceftriaxone (positive control) and tap Incubation of Staphylococcus epidermidis
water (negative control). The purchased Staphylococcus
epidermidis from Biowell Medical Enterprise in
Rizal Street, Legazpi City was swabbed in a
METHODS
Mannitol Salt Agar plate and was incubated at the
The study used a quantitative research Science Laboratory of University of Santo Tomas-
approach, since the study modified data to Legazpi while the actual experiment has not yet
numerical and experimental findings that are been conducted. The temperature was maintained
expressed in tables. Under the quantitative at thirty-seven (37) degrees Celsius. (Graw, 2018)
research approach, an experimental research
The Extraction Process of Blumea balsamifera
design was used since the dependent variable such
as the antibacterial activity of the Staphylococcus
epidermidis was manipulated by the independent The freshly picked leaves of Blumea
variable, which was the Blumea balsamifera leaf balsamifera were air dried for three (3)
extracts. consecutive days.

Materials The air dried leaves were grinded through

the use of an electric blender and underwent the
Five hundred (500) grams of Blumea percolation process which followed the subsequent
balsamifera from Barangay Budiao, Daraga, Albay procedure: (1) fill the bottom part of the percolator
was used for the study which made an extract of with a handful of cotton; (2) place a filter paper on
17.8 grams of Blumea balsamifera leaf extracts. top of the cotton and make sure that the filter paper
Blumea balsamifera’s availability is all year round perfectly fits the percolator; (3) add the cheese
but it is most abundant from February to April. cloth with the grinded Blumea balsamifera leaves;
(4) add filter paper; (5) pour six hundred (600)
The test subjects were divided into three milliliters of 100% ethanol into the percolator; (6)
(3) groups. The first group was labelled as the add marbles; and (7) cover the percolator with
experimental group named specifically as the E three layers of aluminum foil and let the setup stay
groups. The second group was the negative control for about a week.
group, labelled specifically as the N groups.
Lastly, the third group was the positive control The leaf extracts of Blumea balsamifera
group, which was labelled as the P groups. was extracted through the use of Steam Distillation
Process (Nichols, 2018) which followed the
Petri dishes, inoculation loops, medicine subsequent procedure: (1) pour the percolated
droppers, Erlenmeyer flasks, beaker, graduated mixture into a large distillation flask, not more
cylinder, spatula, thermometer, iron clamp, iron than half full; (2) use an iron clamp to secure the
stand, rubber stopper, hot plates, distillation flasks, large distillation flask to the iron stand; (3) place a
marbles, percolator, filter papers, alcohol burner, hole at the center of the rubber stopper and affix a
100% ethanol, cotton, aluminum foil, condenser, thermometer into the hole. Make sure that the
rubber tubing, mannitol salt agar, nutrient agar, thermometer perfectly fits the hole. Thus, it must
automated digital colony counter, Mueller-Hinton not slide downwards. Attach a rubber stopper on
agar and stirring rods were used in the study. the distillation flask to close the system; (4) attach
Samples a rubber tubing on one of the mouths of the
condenser that is connected to a water source on
A Staphylococcus epidermidis (ATCC the other end. Attach another rubber tubing on
35983) bacteria sample was used in the study. The another mouth that will serve as the passageway of
said sample was bought from Biowell Medical water to be released; (5) heat the distillation flask
Enterprise, Legazpi City. using a Bunsen burner or a hotplate; (6) attach a
separatory funnel or glass tubing if it is anticipated
Procedure that the substance mixed with the plant material
such as ethanol or water will need to be
Preparing the Mannitol Salt Agar
replenished during the distillation; (7) the
receiving flask can be a graduated cylinder,
According to MicrobeOnline (2013), the Erlenmeyer flask, or round bottomed flask; (8)
preparation of the mannitol salt agar will follow collect distillate at a rate of 1 drop every second;
3
and (9) the sticky solid that is left on the distilled water, the prepared nutrient agar was
distillation flask is now your pure plant extract. already considered as the negative control.
Preparing the Mueller-Hinton Agar The experimental and control groups had
three (3) petri dishes each which contained ten
According to Aryal (2015), the (10) milliliters of nutrient agar mixed with Blumea
preparation of the Mueller-Hinton agar follows the balsamifera leaf extracts, ten (10) milliliters of
following procedure: (1) suspend 28 grams in 1000 nutrient agar mixed with ceftriaxone powder and
milliliters distilled water; (2) heat to boiling to ten (10) milliliters of plain nutrient agar.
completely dissolve the medium; (3) autoclave at
121°C for 15 minutes for sterilization and cool to Adding the Staphylococcus epidermidis
room temperature; and (4) pour the cooled MHA
(Mueller-Hinton agar) into sterile petri dishes and A small amount of bacteria strain from
make sure to pour on a uniform depth. the incubated mannitol salt agar plate was mixed
with distilled water in a test tube. A sterile swab
Adding the Staphylococcus epidermidis
was used in swabbing the strain into the nine (9)
petri dishes that contained the nutrient agar.
An inoculation loop was used in getting a
small amount of bacteria strain from the incubated Incubation of the Nutrient Agar plates
plate and in mixing the strain with distilled water
in a test tube. A sterile swab was used in swabbing The agar plates were incubated for 24
the strain into three (3) petri dishes that contained hours.
Mueller-Hinton Agar. Observation of the Three (3) Experimental
Adding the Blumea balsamifera leaf extracts, Setups
ceftriaxone and tap water discs An observation took place at the Science
The sterile individual discs were loaded Laboratory of University of Sto. Tomas- Legazpi
with Blumea balsamifera leaf extracts, ceftriaxone in order to record colony count in each of the
and tap water. The experimental and control experimental setups.
groups each had three (3) disks. The disks were RESULTS AND DISCUSSION
added to the Mueller-Hinton Agar plates with
streaked Staphylococcus epidermidis. Table 1 shows the comparison of the zone
of inhibition of Staphylococcus epidermidis.
Incubation of the Mueller-Hinton Agar plates
Table 1. Comparison of zone of inhibition of
The agar plates were incubated for 24 Staphylococcus epidermidis among the three (3)
hours. setups
Observation of the Three (3) Experimental
Setups
An observation took place at the Science Zone of Inhibition
Laboratory of University of Santo Tomas- Legazpi Setup Trials of Staphylococcus
in order to record the zone of inhibition that epidermidis
occurred in the experimental setups.
E Groups
Preparing the Nutrient Agar (with 1st Trial 10.9 mm
Blumea
According to MicrobeOnline (2015), the balsamifera 2nd Trial 12 mm
preparation of the nutrient agar follows the leaf
following procedure: (1) dissolve 14 grams of the extracts) 3rd Trial 8.8 mm
medium in 500 milliliters of distilled water; (2)
heat to boiling to completely dissolve the powder; P Groups 1st Trial 38 mm
(3) autoclave at 121°C for 15 minutes for (with
2nd Trial 39 mm
sterilization and cool to room temperature; and (4) ceftriaxone)
3rd Trial 42 mm
pour the cooled nutrient agar into sterile petri
N Groups 1st Trial 0 mm
dishes and make sure to pour on a uniform depth.
(with tap nd
Adding the Blumea balsamifera leaf extracts, water) 2 Trial 0 mm
ceftriaxone and tap water to the Nutrient Agar 3rd Trial 0 mm
8.9 grams of Blumea balsamifera leaf
extracts and 8.9 grams of ceftriaxone powder were
mixed with thirty (30) milliliters of nutrient agar
separately. Since the agar was already mixed with

4
 Table 2. Comparison of the colony count of
Staphylococcus epidermidis among the three (3)
setups

Colony Count of
Staphylococcus
Setup Trials
epidermidis
(After)

E Groups
(with 1st Trial 0 colony
Figure 1. Zone of inhibition of the control groups Blumea
balsami-
Figure 1 shows the zone of inhibition that fera leaf 2nd Trial 0 colony
occurred in the positive (ceftriaxone) and negative extracts)
(tap water) control groups.
3rd Trial 0 colony

P Groups
1st Trial 0 colony
(with
ceftriaxone)
2nd Trial 0 colony

3rd Trial 0 colony

N Groups
1st Trial 1922 colonies
(with tap
water)
2nd Trial 3670 colonies

3rd Trial 3670 colonies

Figure 2. Zone of inhibition of the experimental
group
Figure 2 shows the zone of inhibition that
occurred in the experimental group.

An ANOVA
Table 2 showsbetween the control
the number groups
of colonies
of Staphylococcus epidermidis that occurredthein F-
and the experimental group showed that
value
the (544.3665)and
experimental was greater
control than the F-critical
groups.
value (5.143253). A greater F-value shows that
there was a statistically significant difference
between the control and experimental groups in
terms of its zone of inhibition. Thus, the null
hypothesis was rejected.

Figure 3. Pour Plate Method of the Experimental

Group
Figure 3 shows that no colonies of
bacteria occurred in the experimental group.

5
 Table 3. An ANOVA for Kirby-Bauer disk
diffusion susceptibility test

Figure 4. Pour plate method of the positive control

group
Figure 4 shows that no colonies of
bacteria occurred in the positive control group.
An ANOVA between the control groups
and the experimental group showed that the
computed P-value (0.000255) was lesser than the
alpha value 0.05. A lesser P-value shows that there
was a statistically significant difference between
the control and experimental groups in terms of
colony count. Thus, the null hypothesis was
rejected

Table 4. An ANOVA for the pour plate method

Figure 5. Pour plate method of the negative

control group
Figure 5 shows the colonies of bacteria
that occurred in the negative control group.

The P-value indicates the level of

marginal significance within a statistical x
An ANOVA between the control groups
and the experimental group showed that the F-
value (544.3665) was greater than the F-critical
value (5.143253). A greater F-value shows that
there was a statistically significant difference
between the control and experimental groups in
terms of its zone of inhibition. Thus, the null
hypothesis was rejected.

66
 A one-way between groups analysis of in mean scores between groups were quite small.
variance was conducted to test the antibacterial Post-Hoc comparisons using the Games-Howell
effects of the experimental and the control groups test indicated the mean score for the positive
against Staphylococcus epidermidis. Subjects were control (M=39.6667, SD=2.08167) was
divided into three (3) groups namely the positive statistically significant from the negative control
control (ciprofloxacin), negative control (tap (M=.0000, SD=.0000). Experimental group
water) and the experimental group (Blumea (M=10.5667, SD=1.62583) also differed
balsamifera leaf extracts). There was a statistically significantly from the negative control. However,
significant difference at the p<.05 level for the it did not differ significantly from the positive
three (3) groups [F(2,6)-4.6,p=0.1]. Despite control.
reaching statistical difference, the actual difference

Table 5. Post-hoc analysis for zone of inhibition

Zone of Inhibition

A one-way between groups analysis of Despite reaching statistical difference, the

variance was conducted to test the antibacterial actual difference in mean scores between groups
effects of the experimental and the control groups were quite small. Post-Hoc comparisons using the
against Staphylococcus epidermidis. Subjects were Tukey test indicated the mean score for the
divided into three (3) groups namely the positive positive control (M=1.5000, SD=.54772) was
control (ciprofloxacin), negative control (tap statistically significant from the negative control
water) and the experimental group (Blumea (M=6.0000, SD=0000). Experimental group
balsamifera leaf extracts). There was a statistically (M=1.5000, SD=.54772) also differed significantly
significant difference at the p<.05 level for the from the negative control and the positive control
three (3) groups [F(2, 6)-9, p=0.16].

Table 6. Post-hoc analysis for colony count

Presence of Extract

7
 Resistance signifies that Staphylococcus inhibitory concentration (MIC) of 150 µg mL−1
epidermidis can penetrate the effects of a medium. against Bacillus cereu sand an MIC of
Therefore, colonies of the said pathogen are 1.2 mg mL−1 against Staphylococcus aureus and
present. On the other hand, susceptibility refers to Candida albicans. In addition, activity was also
the sensitivity of the bacteria when streaked on a discerned hexane extract against Enterobacter
medium. Thus, no colonies of Staphylococcus cloacae and S. aureus. On the other hand, this
epidermidis are present. study also focused on the utilization of Pour Plate
Method and Kirby-Bauer Disk Diffusion
Table 7 shows that the result displays a Susceptibility Test in evaluating the antibacterial
zone of inhibition for Staphylococcus epidermidis activity of Blumea balsamifera against
on both experimental and positive control thus, Staphylococccus epidermidis (Sakee, Maneerat,
graded as susceptible. Cushnie & De-Eknamkul, 2011).
Table 7. Results of first, second and third trial of In summarization, the results of this
the experiment research were supported by the study conducted by
Sakee, et al. in 2011 regarding the beneficial
Nega- properties of Blumea balsamifera on the
Experi-
Positive tive emergence of microbial diseases. Moreover, this
Bacteria mental
Control Con- research contradicted the study of Barraquia et al.
Group
trol in 2017. Thus, Blumea balsamifera leaf extracts is
a possible effective antibacterial agent compared
Staphylococcus
S S R to Hibiscus Rosa-Sinensis Flower extract.
epidermidis
CONCLUSIONS AND RECOMMENDATIONS

The leaf extracts of 500 grams of Blumea

R= Resistant S= Susceptible
balsamifera were extracted using the Steam
Distillation method, which produced an extract of
According to a related journal from the
17.8 grams.
Lyceum of the Philippines entitled as Antibacterial
Activity of Ethanolic Extract of Hibiscus Rosa- Based on Kirby-Bauer disk diffusion
Sinensis Flower against Staphylococcus susceptibility test, the result of the zone of
Epidermidis and Staphylococcus Saprophyticus inhibition of the Staphylococcus epidermidis on
which also used the same ethanolic method of Blumea balsamifera leaf extract disks were half of
extraction, the extract of Hibiscus Rosa-Sinensis the outcome of the ceftriaxone disks.
Flower or locally known as gumamela is not
potent as treatment for the two bacteria. The Moreover, according to the Pour plate
results of the journal showed no visible zone of method, the number of colonies on the negative
inhibition in different concentrations (Barraquia, control, which was the plain nutrient agar summed
Cabuso & Narvacan, 2017). up to 7, 514 colonies. On the other hand, the
positive control and the experimental group both
Moreover, according to another research contained zero colonies.
journal entitled as Inhibition of Staphylococcus
epidermidis Biofilm Formation by Traditional Thai Due to the stated data above, the
Herbal Recipes Used for Wound Treatment which researchers concluded that Blumea balsamifera
used the same bacteria, the journal’s results clearly leaf extracts is a possible antibacterial agent
showed that THR-SK004E could prevent the against Staphylococcus epidermidis.
staphylococcal biofilm development, whereas both In conclusion, the results of the Kirby-
THR-SK004E and THR-SK010E contained Bauer disk diffusion susceptibility test and Pour
remarkable eradication ability on the mature plate method generally showed that there is a
staphylococcal biofilm (Chusri, et al., 2012). significant difference between the control and the
Additionally, another research journal experimental groups.
entitled as Antimicrobial activity of Blumea
A post-hoc analyses were also conducted
balsamifera (Lin.) DC Extracts and Essential Oil to evaluate the significant difference between the
used the same plant herb. The previous study also control groups and the experimental group which
used the disc diffusion assay and agar resulted to significant differences between the
microdilution method in evaluating the three (3) groups.
antibacterial and antifungal activities of Blumea
balsamifera leaves essential oil with a minimum

8
 The researchers emphasized several Preparation. Retrieved February 27, 2019
recommendations in order to furnish significant from microbiologyinfo.com/mueller-
ideas to future related researchers. The researchers hinton-agar-mha-composition-principle-
recommend the inclusion of the assessment of the uses-and-preparation/
25%, 50%, 75% and 100% oncentrations of
Blumea balsamifera leaf extracts against Atrillano, N. & Cipriano, M. C. (2014). Medicinal
Staphylococcus epidermidis to test the most plants and other forms of traditional
medicine used by the residents of dasma
effective antibacterial concentration. 3, golden city, barangay scalawag,
Additionally, the usage of other herbal dasmarinas cavite. Retrieved October 16,
plants in examining their antibacterial activity 2018 from
www.slideshare.net/emsicipriano/medicin
against Staphylococcus epidermidis is also highly
al-plants-and-other-forms-of-traditional-
recommended. medicine-used-by-the-residents-of-
Moreover, this study only used two (2) dasma-3-golden-city-barangay-salawag-
parameters. Thus, the researchers also recommend dasmarias-city-cavite
a much wider set of parameters that would test the
efficacy of Blumea balsamifera’s antibacterial
Barraquia, A., Cabuso, A. & Narvacan, C. (2017)
activity against Staphylococcus epidermidis and antibacterial activity of ethanolic extract
support its results such as the McFarland of hibiscus rosa-sinensis flower against
Standard. staphylococcus epidermidis and
staphylococcus saprophyticus. 2.2.
Also, the usage of other parts of the Retrieved March 16, 2019.
Blumea balsamifera plant herb such as the stem
and roots are recommended.
Blumea balsamifera. (n.d.) Retrieved October 17,
Another recommendation is to look for
2018 from tropical.theferns.info.
other methods of extraction such as the water bath
that would lead to a more abundant yield of leaf
extracts. Bukhari, M. (2004) Student presentation on
Staphylococcus Epidermidis. Retrieved
Furthermore, since the researchers used a
August 22, 2018 from
gram-positive bacterium, it is much recommended web.uconn.edu/mcbstaff/graf/Student%20
to use a gram-negative bacterium to test the presentations/S%20epidermidis/sepidermi
antibacterial activity of Blumea balsamifera. dis.html

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Bukhari, M. (2004) Student presentation on
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APPENDIX A

Preparing the mannitol salt agar

Incubating the Staphylococcus epidermidis

13
APPENDIX B

The extraction process of Blumea balsamifera

Preparing the Mueller-Hinton agar

14
APPENDIX C

Adding the Staphylococcus epidermidis to the three set-ups

Preparing the nutrient agar and adding the experimental and control groups

15
APPENDIX D

Petri dish streaking of Staphylococcus epidermidis

16