CHAPTER I

INTRODUCTION

1.1 Background of the Study

Over time, the world has advanced in many aspects such as in the field of electronics,

business, and most especially healthcare and medicine. However, as new means of curing

diseases are made, the microbes responsible for its harmful effects to society also evolve and

adapt to these new developments. Staphylococcus aureus and Escherichia coli are two of the

most common disease-causing bacteria that can be found in the environment and even within

the body. Staphylococcus aureus can cause infections that are contagious and may even cause

pneumonia, skin disease, and inflammations in different parts of the body (Stoppler, 2018).

Escherichia coli, on the other hand, can cause intestinal sickness such as bloody diarrhea,

dehydration, or even kidney failure. Everyday, we come in contact with these microbes

unknowingly. They could be on the ground, water, animals, and even within us and with the

passing of time comes the development of their immune system, making it hard to combat their

destructive effects.

Herbal medicine is a common practice in rural areas in the Philippines. Practitioners

use a wide range of medicinal plants as they are easily found and are inexpensive especially

for those who cannot afford modern medicine and treatments. Even with the new medicinal

technology generated, plants contain organic antibacterial sources. Plants contain secondary

metabolites such as Tannins and Flavonoids that serve as defense mechanisms against

predation by microorganisms .
 2

The researchers chose the Acalypha indica linn (Kalipipi), which is commonly

mistaken as a weed despite its various medicinal properties. The different parts of the plant

have been used in a variety of ailments and diseases such as asthma, cough, dog bites, snake

bites, rheumatism, and many more.

The researchers want to investigate on the antimicrobial potential of the Acalypha

indica linn and provide alternative treatments and supply the demand of natural compounds

from medicinal plants.

1.2 Statement of the Problem

The main objective of this study is to investigate and determine the bioactivity of the

leaf extracts of Acalypha indica linn. Specifically, this study will aim to

1.2.1 Measure the zones of inhibition, which is parallel to the antimicrobial activity,

of the leaf extract of Acalypha indica linn against Echerichia coli and

Staphylococcus aureus using the Disk Diffusion Method.

1.2.2 Identify the effectivity of Acalypha indica linn leaf extracts against the E. coli

and S. Aureus through the interpration based on Quinto and Santos (2005): > 19

mm (very active), 13-19 mm (active), 10-12 mm (partially active), and < 10 mm

(inactive).

1.2.3 Determine if there is a significant difference between the zones of inhibiton

of the positive control, the negative control, and the extract.

 3

1.3 Significance of the Study

With the abundance of the Acalypha indica linn as a common weed in the Philippines,

this study will be beneficial to Filipinos everywhere if it exhibits antimicrobial properties.

Medicinal plants are usually used as they are readily available, cost-friendly, and they are more

effective compared to modern medicines. Although supplied with unapproved therapeutic

claims, this emphasizes the possibility of antimicrobial properties of Acalypha indica linn. The

study would also give future researchers a head start on other possible properties of the plant.

1.4 Scope and Limitations

This experiment was conducted to determine the presence of antimicrobial properties

in Acalypha indica linn leaf extracts. The leaves were gathered from Minglanilla, Cebu,

Philippines only. The experiment was limited to investigating the antibacterial scavenging

activity of pure compounds of the leaf extracts. For the leaf extracts, the inhibition zone of

bacteria was also identified. The study was conducted within the vicinity of Philippine Science

High School- Central Visayas Campus. Finally, the experiment was limited within the best

efforts and errors of the researchers in conducting the study.

1.5 Definition of Terms

Metabolites - It is the intermediates and products of metabolism. It is a substance essential to

the metabolism of a particular organism to a particular metabolic process.

 4

Tannins – It is any of the soluble astringent complex phenolic substances of plant origin

used especially in tanning leather, manufacturing inks and in medicine

Flavonoids – It is any of a large group of typically biologically active water- soluble

plant compounds that include pigments ranging in color from yellow to red to blue and

occur especially in fruits, vegetables, and herbs.

 CHAPTER II

REVIEW OF RELATED LITERATURE

2.0 Introduction

Presented in Chapter 2 is a synthesis of researches that supports. Included in the

chapter are an overview of the Acalypha indica Linn, its habitat and multiple purposes

in medicine, an overview of the bacteria to be used, namely Staphylococcus aureus and

Escherichia coli; the background of the Disk Diffusion Method, and the preparation

and process to perform the Disk Diffusion Test.

2.1. Antimicrobial

An antimicrobial is an agent that kills or stops the growth of microorganisms

(Saga, 2009). The main classes of antimicrobial agents are disinfectants, antiseptics,

and antibiotics.

2.1.1. Classification

Disinfectants are antimicrobial agents that are applied to the surface of

objects to kill microorganisms dwelling on the objects. Disinfectants work by

interfering with the metabolism or destroying the cell wall of the microbes.

Disinfectants such as sanitizers, alcohols, and bleaches are commonly used in

hospitals, dental surgeries, kitchens and bathrooms to kill infectious organisms

(“Cleaning and Disinfecting”, 2011). Antiseptics are antimicrobial substances

that kill microorganisms on living tissue. Antiseptics such alcohols, hydrogen

peroxide, and iodine, are applied to living tissue or skin to reduce the risk of

infection, sepsis, and putrefaction (McDonnell, 1999). Antibiotics are a type of

 6

antimicrobial agent that is crucial for fighting bacterial infections. Antibiotic

medicine is also used to prevent such infections, by either killing the bacteria or

preventing its growth (“Antibiotics”, 2014).

2.1.2. Microorganisms

A microorganism, or microbe, is an organism that is invisible to the

naked eye and are often described as single-celled or unicellular, however, some

multicellular species are microscopic. Microorganisms can be fungi, bacteria,

archaea or protists, but not viruses and prions since the two are classified as

non-living. Microorganisms are present wherever there is liquid water or even

the slightest amount of moisture. Many microorganisms are crucial to nutrient

recycling in the ecosystem by acting as a decomposer while some also convert

nitrogen from the air to a form usable by plants. Microorganisms rapidly

multiply under good conditions, often contributing to its host organism and exist

in a healthy dynamic balance. However, in some cases, pathogenic microbes

invade larger organisms and cause disease (“Microorganism”, n.d.). Examples

of pathogenic microbes that cause infectious diseases are pathogenic bacteria,

which cause plague, tuberculosis and anthrax; protozoan parasites, which cause

diseases such as malaria, sleeping sickness, dysentery and toxoplasmosis; and

fungi, which cause diseases such as ringworm, candidiasis, and histoplasmosis.

2.2. Bacteria

Bacterium, or Bacteria in plural form, is a prokaryotic microorganism, with

shapes ranging from spheres to rods and spirals, and are typically a few micrometers in
 7

length. Bacteria consist of a single cell without a nucleus and form one of the three

domains of life; the others being archaea and eukaryotes (“Bacteria”, n.d.).

2.2.1 Staphylococcus aureus

The Staphylococcus aureus is a round-shaped bacterium commonly

found in the nose, respiratory tract, skin, and is a normal inhabitant of the lower

reproductive tract of women. It is not always pathogenic, however, it is

responsible for various skin infections including abscesses, respiratory

infections such as sinusitis, and food poisoning. The S. aureus can cause a wide

array of diseases, from minor skin infections such as pimples, impetigo, boils,

and cellulitis, to life threatening diseases such as pneumonia and meningitis. It

is one of the five leading causes of hospital-acquired infections and often the

cause of wound infections following surgery. Each year, around 500,000

patients in hospitals of the United States contract a staphylococcal infection,

chiefly by S. aureus (Bowersox,

1999). Additionally, there is no approved vaccine for the S. aureus.

It is a gram-positive bacterium, indicating that it takes up the crystal

violet stain in the Gram stain test developed by Danish bacteriologist Hans

Christian Gram, and appears to be purple-colored when viewed through a

microscope. When it does not take up the crystal violet stain in the Gram stain

test, it is classified as a gram-negative bacteria.

 8

2. Escherichia coli

The Escherichia coli, abbreviated as E. coli, are rod-shaped bacteria

found in the environment, foods, and intestines of humans and animals. Most E.

coli are harmless, and are actually an important component of a healthy human

intestinal tract. However, some E. coli are pathogenic, indicating that it can

cause illness, either diarrhea or illness outside of the intestinal tract.

E. coli is a Gram-negative bacterium, meaning it does not retain the

crystal violet stain used in the gram-staining method of bacterial differentiation

(E. coli, n.d.). The bacteria that are identified as gram-negative are an important

medical challenge, as their outer membrane protects them from many antibiotics

(including penicillin). Additionally, the outer leaflet of this membrane contains

a complex lipopolysaccharide (LPS) whose lipid A component can cause a toxic

reaction when the bacteria are lysed by immune cells, which can cause a life-

threatening condition known as septic shock characterized by fever, an

increased respiratory rate, and low blood pressure.

2.3. Acalypha indica linn

The Acalypha Indica linn, coined as kalipipi in the Philippines, is an annual

herbaceous shrub that is common in the Tropics, such as Southeast Asia, India,

Oceania and parts of Africa. It grows in moist and shaded places and disturbed places

such as wastelands, wall crevices, roadsides, rocky hillsides, riverbanks and forest

edges (“Acalypha indica L.”, n.d). The Acalypha indica Linn is considered by most

people as a weed in the areas it is found. However, locals have acknowledged it to

 9

possess medicinal properties that are useful for therapeutic purposes such as an

anthelmintic, anti-ulcer, bronchitis, asthma, wound healing, anti-bacterial, etc.

In traditional medicine, the kalipipi provides various uses. It is used as a

diuretic, anthelmintic and for respiratory complications such as asthma, pneumonia,

and bronchitis (Varier, 1996). In the Seychelles, a decoction or infusion of the root is

taken to treat asthma and to cleanse the liver and kidneys, and to treat stomachaches

and purge intestinal worms. A leaf decoction, on the other hand, is used as a massage

cream to treat joint pain in Comoros. In East Africa, the leaf sap is used as eye drops to

treat eye infections. In Namibia however, ground leaves infused in water serve as an

alternative for leaf sap in curing eye infections. The Acalypha indica Linn’s leaf powder

is also applied to maggot-infested wounds (“Acalypha indica L.”, n.d).

The kalipipi is an erect annual herb that belongs to the Euphorbiaceae family

and is is distinguishable by the cup-shaped involucre surrounding the small flowers in

the catkin-like inflorescence. It can grow up to a height of 30 to 80 centimeters. Its

leaves are ovate and 3 to 6 centimeters long, shorter than the long stalks, and has toothed

margins. Its flowers are greenish and sessile, born on numerous lax, erect axillary

spikes. The male flowers are very small and clustered

near the summit, while the female flowers are solitary and scattered, each with a large

and leafy bract that is about 5 to 6 millimeters long (Stewart, 2019).

 10

Table 1. Classification of the Acalypha indica L.

KINGDOM Plantae

SUBKINGDOM Tracheobionta

SUPERDIVISION Spermatophyta

DIVISION Magnoliophyta

CLASS Magnoliosipda

SUBCLASS Rosidae

ORDER Euphorbiales

FAMILY Euphorbiaceae

GENUS Acalypha L.

SPECIES Acalypha indica L.

2.4. Disk Diffusion Method

The disk diffusion test, or also coined as the agar diffusion test, or Kirby-Bauer

test is a test of the antibiotic sensitivity of bacteria. It uses antibiotic discs to test the

extent of the how far the antibiotics affect the bacteria. In this test, wafers containing

antibiotics are placed on an agar plate where bacteria have been placed, and the plate is

left to incubate. If an antibiotic stops the bacteria from growing or kills the bacteria,

there will be an area around the wafer where the bacteria have not grown enough to be

visible, which is called the zone of inhibition (Brown, 1975).

The size of the zone of inhibition is influenced by multiple factors, one being

the effectiveness of the antibiotic in stopping the growth of the bacteria. Another factor

is the diffusion of the antibiotic with the agar medium and can vary based on the

molecular configuration of the antibiotic. Once the zone diameter is measured it must
 11

be compared to a database of zone standards to determine if the bacterium being studied

is susceptible, moderately susceptible or resistant to the antibiotic in question.

Table 2. Factors that influence zones of inhibition and the diffusion of the antibiotic

FACTORS THAT INFLUENCE FACTORS THAT INFLUENCE THE

ZONES OF INHIBITION
Concentration of bacteria spread onto
agar plate
Pathogen susceptibility
Antibiotic diffusion effects
Agar depth
Growth temperature
Nutrient availability
Drug antagonists
DIFFUSION OF THE ANTIBIOTIC
Concentration of antibiotic
Molecular weight of antibioticA
Water solubility of antibiotic
pH and ionization
Binding to agar

2.4.1. Preparation

The preparation of the test is standardized in order to ensure consistent

and accurate results. The media used in Kirby–Bauer testing must be Mueller-

Hinton agar at 4 mm deep and poured into either 100 mm or 150 mm Petri

dishes. The culture must be the Mueller-Hinton agar because it is an agar that

has been thoroughly tested for its pH level and composition. Using this agar

ensures that zones of inhibition can be produced from the same organism. The

agar’s pH level must be between 7.2 and 7.4. This ensures reproducibility and

standardization.

The size of the inoculated organism must also be standardized, because

if the size of the inoculum will be too small, the zone of inhibition will be larger
 12

than supposed to. Alternatively, if the size of the inoculum is too large, the zone

of inhibition will be smaller (“Kirby-Bauer Method”, 2013).

2.4.2. Procedure

Using an aseptic technique, place a sterile swab into the broth culture

of a specific organism and remove the excess liquid by gently rotating or

pressing the swab against the inside of the tube.

Streak the Mueller-Hinton agar plate to form a bacterial lawn using the

swab. To obtain uniform growth, streak the plate with the swab in one direction,

rotate the plate 90° and streak the plate again in that direction, and repeat this

rotation 3 times. Allow the plate to dry for approximately 5 minutes.

Use an antibiotic disc dispenser to dispense discs containing specific

antibiotics onto the plate.

Using flame-sterilized forceps, gently press each disc to the agar to

ensure that the disc is attached to the agar.

Plates should be incubated overnight at an incubation temperature of

37 °C (98.6 °F).

2.4.3. Interpretation of Results

A noticeable “clearing” zone should appear around each of the antibiotic

discs after the plates have been incubated. The diameter of each zone should be

measured and recorded in millimeters (mm).

Each measurement is compared to a zone-size interpretive chart, and the

organism can be characterized as being resistant, intermediate, or susceptible to

 13

the specific antibiotic. Intermediate susceptibility means that some inhibition

from the antibiotic occurred but not sufficiently enough to inhibit the growth of

the organism (“Kirby-Bauer Method”, 2013).

Table 3. Zone size Interpretive Chart for the Kirby-Bauer Test

ANTIMICROBIAL DISC R = mm or I = mm MS = S = mm or
AGENT CODE less range more
Amoxicillin (Staph) AMC 19 20
Amoxicillin (other AMC 13 14-17 18
bacteria)
Ampicillin (Staph) AM 28 29
Ampicillin (other AM 11 12-13 14
bacteria)
Carbenicillin CB 13 14-16 17
(Pseudomonas)
Carbenicillin (other CB 17 18-22 23
bacteria)
Cefotaxime CTX 14 15-22 23
Cephalothin CF 14 15-17 18
Chloromaphenicol C 12 13-17 18
Erythromycin E 13 14-22 23
Gentamycin GM 12 13-14 15
Methicillin (for M or 9 10-13 14
Staph only) DP

Penicillin P 28 29
Streptomycin S 11 12-14 15
Sulfamethoxazole- SXT- 10 11-15 16
trimethoprim TMP
Tetracycline 14 15-18 19

R = resistant I = intermediate S = sensitive MS = moderate sensitive

 14

2.5. Related Studies

A previous study on the antimicrobial activity of extracts of the Acalypha indica Linn

utilized the dilution method to determine the effect of petroleum ether extract (40-60 Degree)

chloroform and methanolic extract of dried leaves of Acalypha indica Linn (Euphorbiaceae)

against fungi (Candida albicans) and bacteria (Escherichia coli, Staphylococcus aureus,

Pseudomonas aeruginosa, Salmonella typhosa, Bacillus substilis, Klebsiella pneumoniae).

All of the extracts except for the petroleum ether extract showed prominent antimicrobial

activity. The methanolic extract was then further fractioned into acetone soluble and

insoluble parts, which both also showed prominent antimicrobial activity (Walter, 2007)
 CHAPTER III

METHODOLOGY

3.1 Research Processes

The experiment will be following the procedure as shown below.

Obtaining of Washing and Extraction from

Acalypha indica Drying of leaf
samples
the leaf samples
linn leaf samples

Statistical Disk Diffusion Obtaining of

Analysis method
bacterial samples

Figure 1. Research Design

3.2 Procurement of Materials

A sack of Acalypha indica Linn leaf samples were collected from parts of Minglanilla

and Argao, Cebu. The agar plates were procured from the CVisC Laboratory (Figures 2, 3, 4).

The bacteria S.aureus and E.coli were taken from the already present samples in the CVisC

Laboratory. The sample was verified in Cebu Technological University, Argao last December

05, 2018.

3.3 Acalypha indica Linn Leaf Extract Preparation

The Acalypha indica Linn leaf samples were washed with distilled water, were air dried

for 24 hours, and then homogenized into fine particles. Then, the samples were oven dried for

48 hours at 70 degrees Celsius.The dried samples were weighed. One hundred (100) grams of

the dried sample were soaked in 900mL 95% ethanol for 48 hours at room temperature. Filtrates

were collected using filter paper. The ethanol filtrates were put in a rotary evaporator (Figure
 16

5.) at 40 degrees Celsius to separate the ethanol and plant extracts. The extracts were collected

and were stored in a refrigerator until further use.

3.4 Bacterial Culture

The microorganisms that were used in this study are under the Biosafety-Level 1 Group,

meaning the agents used were highly unlikely to cause disease in healthy laboratory workers,

animals and plants, based on laboratory policy. The work was done in a laminar flow hood.

Standard microbiological process in the laboratory were used when working in the laboratory.

Decontamination were performed by autoclaving at 121° C for 30 minutes, and laboratory

safety equipment such as lab coats and gloves were required. An individual with knowledge

and training in this field will supervise the laboratory work, and the researchers were only

allowed to observe. The researchers will do the diameter zone of inhibition measurements.

A total of two microorganisms were used in the testing of antimicrobial activity:

Staphylococcus aureus as the Gram-positive bacteria and Escherichia coli as the Gram-

negative bacteria. The group were using the existent bacterial samples in the laboratory. The

bacterial cultures were maintained in nutrient agar (NA) medium, which were prepared by

heating the mixture of twenty-three (23) grams of NA powder in one thousand (1000) mL

distilled water and autoclaved at 121° C Celsius under 15 psi pressures for 20 minutes.

3.5 Antimicrobial Activity Testing

The Disk Diffusion method was used to test for the leaf extract’s antimicrobial

susceptibility. The test was carried out according to the standard method by Guevarra (2005)
 17

and by Clinical and Laboratory Standard Institute (2012) to assess the presence of antibacterial

activities of the plant ethanol extract. The entire procedure was conducted in a well-disinfected

area inside a laminar flow hood using 70% ethyl alcohol as general disinfectant. Standard size

blank Whatman paper discs, 6.00 mm in diameter, were sterilized in the autoclave for 20

minutes at 121° C Celsius at 15 psi. It was impregnated with the ethanol extract, and the known

standard reference antibiotics separately. Each test plate will comprise of three discs. The

standard antibiotic discs for Amoxicillin will serve as a positive control. The negative control

was 95% ethanol. The discs were placed on the surface of the sterilized medium inoculated

with the test organism by using a sterile swab and allowed to stand for five minutes. Samples

were incubated for 37 degrees Celsius for 24 hours, after which the zones of inhibition of

desired growth were collected and measured. The tests were repeated three times to ensure

reliability and the mean values were computed. The antibacterial activity was interpreted from

the size of the diameter of the zone of inhibition measured, it was observed as the clear zones

surrounding the hole. The zone of inhibition was qualitatively interpreted based on Quinto and

Santos (2005): > 19 mm (very active), 13-19 mm (active), 10-12 mm (partially active), and <

10 mm (inactive).

3.6 Statistical Analysis

The antimicrobial activity was expressed as mean or average of its inhibition zones.

One Way- Analysis of Variance (ANOVA) at 0.5 level of significance was used to determine

if the zones of inhibition have significant difference and compare the zones of inhibition that

have a significant difference. The program Minitab 18 was used for ANOVA and Tukey test.
 18

The leaf extract was then identified as very active, active, partially active, and inactive based

the Quinto and Santos interpretation.

 CHAPTER IV

RESULTS AND DISCUSSIONS

4.1 Antibacterial assay of the leaf extracts

About 5 ml of the extracts were taken from the rotary evaporation process. These

extracts were then used as treatments against S. aureus and E. coli. The experiment showed

that the Acalypha indica linn leaf exhibits antibacterial properties. As shown in Table 1, the

average zone of inhibition of the leaves against S. aureus is 15.54 mm, which is less compared

to that of the positive control, amoxicillin, which is 21. 09 mm. The leaf extracts show an

average zone of inhibition against E. coli of 9.31, which is also less than that of the positive

control which is 13.33 mm. The leaf extract is found to have better inhibited the growth of S.

aureus compared to E. coli. The same can also be said for the positive control, amoxicillin.

Table 4. Average Zone of Inhibition by Acalypha indica linn leaf extracts, negative control
Ethanol and positive control Amoxicillin against S. aureus and E. coli

TREATMENTS AVERAGE ZONES OF INHIBITION

S. aureus E. coli

LEAF ETHANOLIC EXTRACTS 15.54 9.31

POSITIVE CONTROL 21.09 13.33

(AMOXICILLIN)
NEGATIVE CONTROL (ETHANOL) 0.00 0.00
 20

4.2 Statistical Analysis

Using One-Way Analysis of Variance (ANOVA), it was found out that the p- values

for all treatments against S. aureus and E. coli are both 0.001and 0.004 respectively, which

are both less than 0.05 (Table 2 and 3). Thus, the difference among treatments is significant.

Tukey Test was used to compare the mean values of the zones of inhibition of the

treatments against the organisms (Appendix B). A value of 0.05 or less signifies a significant

difference, while a value greater than 0.05 means there is an insignificant difference. The mean

difference of the leaf extracts against S. aureus shows a significant difference from the positive

control and negative control, which means that it is not as effective as Amoxicillin, but has the

capacity to inhibit S. aureus.

The mean difference of the leaf extracts against E. coli is significant in comparison to

the positive and negative controls, thus it is similarly not as effective as Amoxicillin in S.

aureus. However, it still has the strength to inhibit E. coli compared.

Based on the results, the leaf extracts have more potential against S. aureus. On the

interpretation of the leaf extract’s inhibition zones on its effectivity, predicated from Quintos

and Santos (2005), the leaf extract against S. aureus and E. coli are active (13-19 mm) and

inactive ( < 10 mm) respectively.

 CHAPTER V

CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

The ethanolic extracts of Acalypha indica linn from its leaves manifested zones of

inhibition against both test organisms E. coli and S.aureus. This suggests that Acalypha indica

linn has antimicrobial activity against these two organisms.

According to the Tukey test result (Appendix), the leaf extracts are more effective against

S. aureus. The leaf extracts are active against S.aureus at (13-19 mm) and inactive at (<10 mm)

against E.coli. The antimicrobial activity of the ethanolic extracts and the positive control has

a significant difference which means that the extracts possess antimicrobial properties but they

cannot surpass the action of commercially available antimicrobial drugs.

5.2 Recommendations

The researchers recommend using more test organisms such as fungi and other

microorganisms as test organisms. Different methods of submereging in other substances such

as ethanol, methanol, and others to bring out the potential of the leaves against S.aureus are

also recommended. To obtain more accurate results, the researchers recommend conducting

more trials.
 22

BIBLIOGRAPHY

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 25

APPENDIX A

PHOTOS

Figure 2. The Acalypha indica Linn plant

Figure 3. The nutrient agar

 26

Figure 4. Nutrient agar diluted in distilled water

Figure 5. Distribution of diluted agar solution

 27

Figure 6. Rotary Evaporation of extracts

Figure 7. Escherichia coli

 28

Figure 8. Staphylococcus aureus

Figure 9. Placement of treatments

 29

Figure 10. Measurement of inhibition zones

 30

APPENDIX B

RESULTS OF ANOVA AND TUKEY TEST

Table 5. Results of One-Way ANOVA for the leaf extract against S. aureus

Sum of Squares DF Mean Square F Sig.

Between 2142.8 2 1071.40 194.9 0.000

Groups

Within 131.9 24 5.50

Groups

Total 2774.7 26

Table 6. Results of One-Way ANOVA for the leaf extract against E. coli

Sum of Squares DF Mean Square F Sig.

Between 842.0 2 420.979 95.16 0.000

Groups

Within 106.2 24 4.424

Groups

Total 948.1 26
 31

Table 7. Multiple Comparisons for the leaf extract against S. aureus

Mean Std. Error Sig. Lower Upper

Difference Bound Bound

Leaf Amox -5.69 1.11 0.000 -8.45 -2.93

Negative 15.40 1.11 0.000 12.64 18.16

Amox Leaf 5.69 1.11 0.000 2.93 8.45

Negative 21.09 1.11 0.000 18.33 23.85

Negative Leaf -15.40 1.11 0.000 -18.16 -12.64

Amox -21.09 1.11 0.000 -23.85 -18.33

Table 8. Multiple Comparisons for the leaf extract against E. coli

Mean Std. Error Sig. Lower Upper

Difference Bound Bound

Leaf Amox -4.02 0.991 0.001 -6.497 -1.547

Negative 9.311 0.991 0.000 6.836 11.786

Amox Leaf 4.02 0.991 0.001 -15.808 -10.858

Negative 13.333 0.991 0.000 10.858 15.808

Negative Leaf -9.311 0.991 0.000 -11.786 -6.836

Control Amox -13.333 0.991 0.000 -15.808 -10.858

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APPENDIX C

RAW DATA

Table 9. Zones of inhibition by leaf extracts in three trials with three tests each.

Zone of Inhibition

S. aureus E. coli

TEST 1 TEST 2 TEST 3 TEST 1 TEST 2 TEST 3

TRIAL 1 16.3 19.2 18 13 8 7
TRIAL 2 12 13.5 10.5 11 6 5.9
TRIAL 3 19 19.1 11 16 9.1 7.8

Table 10. Zones of inhibition by positive control, amoxicillin, in three trials with three tests
each.

Zone of inhibition

S. aureus E. coli

TEST 1 TEST 2 TEST 3 TEST 1 TEST 2 TEST 3

TRIAL 1 22 20.6 20 12.5 14.5 12.5
TRIAL 2 21.9 20.6 25.1 13.2 12 12.1
TRIAL 3 20 20.5 19.1 13.4 15.9 13.9
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Table 11. Zones of inhibition by negative control, ethanol, in three trials with three testseach.

Zone of inhibition

S. aureus E. coli

TEST 1 TEST 2 TEST 3 TEST 1 TEST 2 TEST 3

TRIAL 1 0 0 0 0 0 0
TRIAL 2 0 0 0 0 0 0
TRIAL 3 0 0 0 0 0 0
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APPENDIX D

VERIFICATION