1

IV. RESEARCH PLAN

A. Statement of the Problem

This study will determine the antimicrobial assay of the combined Tinospora rumphii
Boerl (Makabuhay stem), Psidium guajava Linn.(Guava leaves), and Citrus microcarpa
(Calamansi) upon application on selected manure and stagnant water.
Specifically, it will seek to answer the following questions:
1) What is the effect of the combined Tinospora rumphii Boerl (Makabuhay stem), Psidium
guajava Linn. (Guava leaves), and Citrus microcarpa (Calamansi) on its application on selected
manure and stagnant water in terms of:
1.1) odor before and after application; and
1.2) zone of inhibition of bacteria?
2) Which among the three Treatments is the most effective in terms of:
2.1) odor before and after application; and
2.2) zone of inhibition of bacteria?
3) Is there a significant difference between the three setups in terms of:
3.1) odor before and after application?

B. Statement of Hypotheses
1) The combined Tinospora rumphii Boerl (Makabuhay stem), Psidium guajava Linn.(Guava
leaves), and Citrus microcarpa (Calamansi) has no effect on its application on selected
manure and stagnant water in terms of:
1.1) odor before and after application; and
1.2) zone of inhibition of bacteria.
2) The three setups are not effective in terms of:

2
1.1) odor before and after application; and
1.2) zone of inhibition of bacteria.
3) There is no significant difference between the three setups in terms of:
2.1) odor before and after application.

C. Methodology
The materials will be used in study are the following: one hundred grams of makabuhay
(Tinospora rumphii Boerl) stem, one hundred grams of guava (Psidium guajava Linn.) leaves,
one hundred grams of calamansi (Citrus microcarpa), water, five beakers, one sterilized bottle,
weighing scale, mortar and pestle, gauze, syringe, a stirring rod, three filter papers, three slides,
normal saline solution (NSS), gloves, masks, microscope, four glass container, casserole and
stove.

General Procedure
The calamansi (Citrus microcarpa), guava (Psidium guajava Linn.) leaves and
makabuhay (Tinospora rumphii Boerl) stem will be collected in the researchers back yard. One
hundred grams of guava leaves and one hundred grams of makabuhay stem will be pounded
using mortar and pestle. The solution will be prepared using the decoction process. The decocted
guava leaves and makabuhay stem will be filtered by using a filter paper. Fifty mL of decocted
guava leaves and makabuhay stem will be kept in a beaker. One hundred grams of calamansi will
be extracted using gauze and fifty mL of calamansi extract will be collected in a beaker. The
three solutions will be combined in a larger beaker and will be stirred in a clockwise direction.
Afterwards, the mixture will be transferred in a sterilized bottle. An actual test will be used to
obtain the results in terms of its odor by applying the three treatments in the piggeries, poultries

3
and stagnant water. Three samples from each area will be collected for the analysis of the
bacteria. An electronic microscope will be used in observing the bacteria present in the samples.
A Normal Saline Solution (NSS) will be needed in observing the specified materials under the
electronic microscope.

Testing Procedure

The following setups will be: Before application Setup 1 (Negative control), After
Application; Setup 2 (Positive Control) and Setup 3 (Experimental Control).
In gathering data, an actual test will be conducted on the piggeries, poultries and stagnant
areas in order to test the odor before and after the application. These will be rated by thirty
human respondents.
Three samples will be collected including pig manure, chicken manure and stagnant
water and will be placed in a clean container. A wet mount analysis will be used in testing the
anti-microbial activity of the treatments. Three samples will be prepared using slides with an
NSS. A small amount of waste material will be placed in a slide. Then this will be observed
under the microscope. Next, small amount of the treatments will be poured on the specimen.
Each of the treatments will be tested and analyzed in the laboratory to observe the presence of
the bacteria in the samples.

Risk and Safety

4
Gloves will be used while performing the experiment. Masks will be also used before
rating the odor of the selected manure and stagnant water in order to avoid disease-causing
bacteria that could be taken from the areas.

Data Analysis
The statistical tool that will be used in the study is Randomized Blocks ANOVA.

D. Bibliography
Website
Cabral, J.P.S. (2010). Water Microbiology. Bacterial Pathogens and Water. International Journal
of Environmental Research and Public Health, 7, 3657-3703.doi:10.3390/ijerph7103657
Encyclopedia
Grolier International (1995). Molasses. Grolier Encyclopedia of Knowledge (p.16). United States
of America: Grolier Incorporated.
Paton, J. (2000). Bacteria. Childrens Encyclopedia. (Vol.1, 61). Kingfisher Publications Plc.,
London.
Books
Joaquin, C., Lagunzad, C., & Rabago, L. (2003). Functional Biology. Quezon City: Vibal
Publishing House, Inc.
Kurian, J.C. (2010). Amazing Healing Plants. Manila: Philippine Publishing House.

V. INTRODUCTION
A. Background of the Study

5
Doesnt curiosity bother you about the foul odor when you pass by a slaughter house?
Today, the most common problem in slaughter houses or pig farms is the increasing rate of
undesirable odor and diseases caused by rotting of organic matter of microorganisms. This
situation is provoked when the wastes are being pumped to the canals and other stagnant areas
which cause diseases to humans and animals.
Most of the farmers in rural areas have meager income. Money cannot be easily earn in
the farm because of some problems encountered. Most chickens and pigs tend to lay their
droppings everywhere and anywhere. Because of this, it gave way for pathogenic
microorganisms to thrive and multiply around the areas which caused diseases following an
undesirable odor to the pigs and poultry farms. It also results to pollution of water supplies which
is a major health problem today. Not only may sanitary sewage be discharge into water, but
complex wastes from agriculture may be also. In increasing volume such substances as animal
and vegetable matter, are released from poultry, processing plants, canals, oil fields and farms.
Bacterial diseases also affect animals, and they are responsible for various diseases of crop
plants.
The present condition in slaughter houses, poultry farm and stagnant water stress the need
for a fast and accurate solution for the problem. Thus, this study aimed to develop an effective
anti-microbial treatment from combined Tinospora rumphii Boerl (Makabuhay), Psidium
guajava Linn. (Guava), and Citrus microcarpa (Calamansi).

B. General Specific Objective

The specific objectives of this study were the following:

6
1) To determine the effect of the combined Tinospora rumphii Boerl (Makabuhay stem), Psidium
guajava Linn.(Guava leaves), and Citrus microcarpa (Calamansi) on its application on selected
manure and stagnant water in terms of:
1.1) odor before and after application; and
1.2) zone of inhibition of bacteria.
2) To distinguish which of the three setups is the most effective in terms of:
2.1) odor before and after application; and
2.2) zone of inhibition of bacteria.
3) To determine if there is a significant difference between the three setups in terms of:
3.1) odor before and after application.

C. Statement of the Problem

This study sought to determine the anti-microbial assay of combined Tinospora rumphii
Boerl (Makabuhay stem), Psidium guajava Linn.(Guava leaves), and Citrus microcarpa
(Calamansi) on selected manure and stagnant water.
Specifically, it aimed to answer the following question:
1) What is the effect of the combined Tinospora rumphii Boerl (Makabuhay stem), Psidium
guajava Linn.(Guava leaves), and Citrus microcarpa (Calamansi) on its application on selected
manure and stagnant water in terms of:
1.1) odor before and after application; and
1.2) zone of inhibition of bacteria?
2) Which among the three setups is the most effective in terms of:
2.1) before and after the application; and
2.2) zone of inhibition of bacteria?

7
3) Is there a significant difference between the three setups in terms of:
3.1) before and after the application?

D. Statement of Hypotheses
The following hypotheses will be tested:
1) The combined Tinospora rumphii Boerl (Makabuhay stem), Psidium guajava Linn.(Guava
leaves), and Citrus microcarpa (Calamansi) has no effect on its application on selected
manure and stagnant water in terms of:
1.1) odor before and after application; and
1.2) zone of Inhibition of bacteria.
2) The three setups are not effective in terms of:
2.1) odor before and after application; and
2.2) zone of Inhibition of bacteria.
3) There is no significant difference between the three setups in terms of:
3.1) odor before and after application.

E. Significance of the Study

This study aims to determine the anti-microbial assay of combined Tinospora rumphii
Boerl (Makabuhay stem), Psidium guajava Linn.(Guava leaves), and Citrus microcarpa
(Calamansi)on selected manure and stagnant water. Once this research study achieves positive
results, many will be benefiting from it:

Farmers and Local Units

This study can benefit people especially farmers and local units by reducing the
requirements of regular medication, antibiotics and disinfectants. Incidences of diseases like

8
diarrhea will also be reduced.

Community or Environment
This study will the community in eliminating foul odor of pig pens, poultry farms, and
stagnant water. In addition, it will help in reducing the incidence of flies and other undesirable
insect pests. Animals health will be significantly improved through reducing the stress factors
thereby strengthening their immunological system against diseases.

Future Researchers
This can benefit future researchers by giving them concepts and ideas on using
makabuhay, guava and calamansi as natural control for foul odor of pig pens, farms and stagnant
water and as an effective antimicrobial agent.

F. Scope and Limitations of the Study

This study was conducted to determine the anti-microbial assay of combined Tinospora
rumphii Boerl (Makabuhay stem), Psidium guajava Linn. (Guava leaves), and Citrus microcarpa
(Calamansi) on selected manure and stagnant water. The treatments were being applied on
selected manure and stagnant water. This will eliminate not only the odor but also the bacteria
present in those areas. The experiments were conducted at Maligaya, Gloria, Oriental Mindoro.
The time span of the experiments ranges from June 1 to July 26, 2013.
G. Definition of Terms
Alkaloids

Group of naturally occurring chemical compounds that contain mostly basic

nitrogen atoms. This group also includes some related compounds with neutral and even

9
weakly acidic properties. Alkaloids are highly reactive substances with biological activity
in low dose.
Bacteria

Bacteria are tiny living things- so tiny that they cannot be seen by your naked eye.

Clostridium perfringens

Spore-forming Gram-positive, non-motile, anaerobic, sulfite-

reducing rods. C. perfringens is present in higher numbers in the feces of some animals,
such as dogs, than in the feces of humans and less often in the feces of many other warmblooded animals. The numbers excreted in feces are normally substantially lower than
those of E. coli.
Cryptosporidium

A single-celled protozoan parasite commonly found in lakes and rivers,

especially when the water is contaminated with sewage and animal waste.
Cryptosporidium can cause gastrointestinal illness (e.g., diarrhea, vomiting, cramps).
Decoction

The process of boiling a substance in water to extract its essence.

Disinfectant Disinfectants are antimicrobial agents that are applied to non-living objects to
destroy microorganisms, the process of which is known as disinfection. It cleans an
article of some or all the pathogenic organisms which may cause infection.
Enterococci

Gram-positive, non-sporeforming, catalase-negative ovoid cells. Cells occur

singly, in pairs or short chains. Optimal growth for most species is 3537 C. Some will
grow at 4245 C and at 10 C. Growth requires complex nutrients but is usually
abundant on commonly used bacteriological media.
Enzymes

These are the biological catalyst supporting almost all chemical reactions that

maintain animal homeostasis. Because their role, the assay and pharmalogical regulation
of enzymes have become key elements in clinical diagnosis and therapeutics. The
macromolecular components of almost all enzymes are composed of protein, except for a
class RNA (ribonucleic acid) modifying catalyst known as rybozymes. Ribozymes are

10
molecules of ribonucleic acid that catalyze reactions on the phosphodiester bond of other
RNAs. Enzymes are found in all tissues and fluids of the body.
Escherichia coli

E. coli are bacteria whose presence indicates that water may be

contaminanted by human or animal wastes. Microbes in these wastes can cause short
term effects, such as diarrhea, cramps, nausea, headaches, or other symptoms. They may
pose a special health risk for infants, young children, and people with severely
compromised immune systems.
Flavonoids

Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms.

Flavonoids belong to the polyphenol family. Together with carotenes, flavonoids are also
responsible for the coloring of fruits, vegetables and herbs.
Gram Positive
for

the

Gram-positive organisms made up the phylum Firmicutes, a name now used

largest

group.

Staphylococcus,Streptococcus,

It

includes

many

Enterococcus,

well-known

(which

are

genera

cocci)

and

such

as

Bacillus,

Corynebacterium, Nocardia, Clostridium, Actinobacteria, and Listeria.

Gram Negative

Gram negative bacteria are the one who are mostly harmful therefore Gram

negative bacteria are pathogens (it includes bacteria like E-coli, Salmonella, seudomonas,
Moraxella, Helicobacter, Stenotrophomonas, Legionella, Acetic Acid Bacteria etc.).
Saponins

Saponins are glucosides with foaming characteristics. Saponins consist of a

polycyclic aglycones attached to one or more sugar side chains.

Staphylococcus aureus

Most common cause of staph infections. It is a spherical bacterium,

frequently part of the skin flora found in the nose and on skin.
H. Review of Related Literature and Studies

Related Literature

11
Makabuhay (Tinospora rumphii Boerl)
The Makabuhay plant belongs to the family of Menispermaceae, and can grow, or rather
climb, up to fifteen meters long. The plant is known for its bitter nature, and the fruits of this
plant are held in clusters, which can be as long as two centimeters. Only the leaves and the
stems of makabuhay have been found to cure particular health concerns. For the stems, both the
dried and the fresh stems are found to be beneficial. The stem of makabuhay plant is a primary
ingredient used to concoct preparations that would prevent spread of malaria, and may be used as
cleanser for skin ulcer and skin wounds. Clinical tests have shown that the decoction of this plant
cures diarrhea, indigestion and scabies.
The stem of the makabuhay plant contain alkaloids; berberine, palmatine, choline,
tinosporine, glycosides and steroid. Alkaloids have been found to have antimicrobial properties.
It also have been found to have microbiocidal effects (including against Giardia and Entamoeba
species), the major antidiarrheal effect is probably due to their effects on transit time in the small
intestine. Berberine is an important representative of the alkaloid group. It is potentially effective
against trypanosomes and plasmodia. The mechanism of action of highly aromatic planar
quaternary alkaloids such as berberine and harmane is attributed to their ability to intercalate
with DNA (Cowan, 2000; Stuart, 2012).

Guava (Psidium guajava Linn.)

Guava is a tropical plant, which is commonly seen and grown because of its many uses as
traditional remedy to treat various ailments. The use of the guava leaves is for cleaning and
disinfecting wounds by rinsing the afflicted area with a decoction of the leaves. The leaves are
also used as a mouth wash to treat gum infection and tooth decay.

12
Guava plant contains alkaloids, flavonoids, glycosides, polyphenols, reducing
compounds, saponins and tannins. It also have active principles including tannins, vitamin C,
pectins, calcium oxalate terpenoids, essential oils, limonenes, methanol and guijavalic acid. Leaf
extract possessed trypanocidal properties attributed to broad anti-microbial and iron chelating
activity of flavonoids and tannins. Iron chelation was suggested as an effective way of killing
trypanosomes (Kurian, 2010; Uddin, 2013).
Guava possess alkaloid which are commonly found to have antimicrobial properties. It
also contain flavonoids which are hydroxylated phenolic substances but occur as a C6-C3 unit
linked to an aromatic ring. Since they are known to be synthesized by plants in response to
microbial infection, it should not be surprising that they have been found in vitro to be effective
antimicrobial substances against a wide array of microorganisms. Their activity is due to their
ability to complex with extracellular and soluble proteins and to complex with bacterial cell
walls. More lipophilic flavonoids may also disrupt microbial membranes. Catechins are the most
reduced form of C3 unit in flavonoid compound and this compounds inhibited bacteria and
microorganisms (Cowan, 2000).
Guava leaves have essential oils which are highly concentrated oils that often contain the
chemicals responsible for the characteristic scent of the plant. Essential oils have antiseptic and
antifungal properties. Combined with the pleasant scent of many essential oils, this makes them
excellent tools for removing odors (Jackson, 2013).

Calamansi (Citrus microcarpa)

13
The whole calamansi fruit contains a small level of carbohydrates (3%), minerals (1%),
ascorbic acid (0.1%), and citric acid (3%). The extract is rich in essential oils and ascorbic acid
(0.15%). The juice is very acidic and contains 5.5% citric acid. The acid in this fruit helps
suppress the odor-causing bacteria (Stuart, 2012).
The Calamansi scent has been proven to help alleviate depression and anxiety. Essential
oil found in calamansi extract is a great additive cleansing product since few aromatics of the
extract just not masks bad smell, but completely neutralizes them (Lorenzo, 2012; Jackson,
2013).
The juice was used to clear up acne vulgaris; and the essential oils from calamansi have
shown promising results against Staphylococcus aureus. It is often used in body deodorant
applications (King, 2008).

Related Studies
In the study of Vermani, Navneet and Gautam (2013), crude extracts of Tinospora
cordifolia was investigated for its ability to inhibit the growth of dental (bacterial) pathogens i.e.,
Staphylococcus aureus (MTCC 1144), Streptococcus mutans (MTCC 890), Streptococcus
salivarius (MTCC 1938), Lactobacillus acidophilus (MTCC 447), Streptococcus sanguinis
(ATCC 10556) and their isolates. Collected stems of T. cordifolia were properly washed and
shade dried at room temperature, crushed and extracted in petroleum ether (PET), chloroform
(CHCl3), methanol (MeOH) and aqueous (H2O) by using Soxhlet apparatus. The antimicrobial
activity of extracts was examined by agar well diffusion method at 200 mg mL -1 sample
concentration. Phytochemical analysis was done for plant extract. The result of antibacterial
activity was found that MeOH extract of T. cordifolia was most effective against all tested

14
bacterial pathogens. Maximum antibacterial activity was observed against S. sanguinis (23 mm)
and lowest activity against S. salivarius (17 mm). The phytochemical analysis revealed the
presence of alkaloids, flavonoids, tannins, phenols, saponins, glycosides, amino acids and
steroids which an accountable for its antimicrobial potential. The results validate the traditional
uses of T. cordifolia in treatment of dental diseases.
This study was related to present study because it focuses on the antibacterial activity of
Tinospora cordifolia (Makabuhay) extract against dental pathogens. This study concentrate on
T. cordifolia that possess alkaloids, flavonoids, tannins, phenols, saponins, glycosides, amino
acids and steroids which are known for its antimicrobial potential.

In the study of Mahalel (2012) entitled Antibacterial Sensitivity for some Chemically
Diverse Steroidal Glycosides In Vitro, susceptibilities (in vitro) of the steroidal glycosides were
tested against Bacillus cereus, Staphylococcus aureus, Serratia marcescens, Escherichia coli and
Pseudomonas putida using the disc diffusion method. These steroidal glycosides were identified
as alpinoside C (1) [kidjolanin3-O--D-glucopyranosyl-(14)--D-glucopyranosyl-(14)--Doleandropyranosyl-(14)--D-cymaropyranosyl-(14)--D-cymaro-pyranoside]; Ghalakoside
(2) [cardiac glycoside] and pentandroside A (3) [(22S,25S)-16,22,26- trihydroxycholest-4-en-3one-16-O--D-glucopyranosyl-(13)--D-xylopyranoside],

which

were

isolated

from

Oxystelma esculentum var. alpine; Pergularia tomentosa and Tribulus pentandrus, respectively.
Almost all of the selected compounds produced significant antibacterial potentialities against all
tested microorganisms and the data were compared to the standard antibiotics. All compounds
exhibited remarkable susceptibilities against Gram negative (S. marcescens) and Gram positive
(B. cereus). The minimum inhibitory concentrations (MIC) of compounds ranged from 10 to 50
g/disc.

15
This study was related to present study because Steroidal Glycosides was proven to have
remarkable susceptibilities against Gram negative and Gram positive.
Disinfectant properties of essential oils from Salvia officinalis L. cultivated in Tunisia
were evaluated. Essential oils were obtained by hydro-distillation of the aerial part of Salvia
officinalis L. cultivated in Sfax gardens, Tunisia. The obtained oils were analyzed by gas
chromatographymass spectrometry (GCMS) and 44 compounds were identified. Strong
bactericidal and fungicidal effects were shown using the NCCLS broth dilution, EN 1275 and
EN 1276 st]andard methods. The minimal cidal concentrations (MCCs) values ranged from
0.031 to 0.25 L mL1. The essential oils concentrations of 0.5% and 1% (v/v) resulted in a
reduction in viability higher than 5 and 4 log units per mL for the standard bacteria and fungi,
respectively, within a contact time of 5 min. Using an air sampler and an aroma dispenser,
vaporisation of 0.25 mL m3 of S. officinalis essential oils resulted in (72%, 73% and 70%) and
(54%, 55% and 55%) reduction of the total microbial count and the total count of yeasts and
moulds, after a residence time of 1 h, 6 h and 24 h in a selected testing room, respectively. S.
officinalis essential oils showed a potent vapour activity against a panel of bacteria, yeasts and
fungi. This supported their use as a natural eco-friendly disinfectant to manage airborne microbes
(Bouaziz, 2009).
This study was related to the present study because the essential oils were proven as an
effective disinfectant against a panel of bacteria, yeast and fungi.
In the study of Yu (2005) entitled Antimicrobial activity of berberine alone and in
combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus,
the antimicrobial activity of berberine was investigated. The main antibacterial substance of
Coptidis rhizoma (Coptis chinensis Franch) and Phellodendri cortex (Phellodendron amurense
Ruprecht), against clinical isolates of MRSA, and the effects of berberine on the adhesion to

16
MRSA and intracellular invasion into human gingival fibroblasts (HGFs). Berberine showed
antimicrobial activity against all tested strains of MRSA. Minimum inhibition concentrations
(MICs) of berberine against MRSA ranged from 32 to 128 microg/mL. Ninety percent inhibition
of MRSA was obtained with 64 microg/mL or less of berberine. In the checkerboard dilution test,
berberine markedly lowered the MICs of ampicillin and oxacillin against MRSA. An additive
effect was found between berberine and ampicillin, and a synergistic effect was found between
berberine and oxacillin against MRSA. In the presence of 1-50 microg/mL berberine, MRSA
adhesion and intracellular invasion were notably decreased compared with the vehicle-treated
control group. These results suggest that berberine have antimicrobial activity and the potential
to restore the effectiveness of beta-lactam antibiotics against MRSA, and inhibit the MRSA
adhesion and intracellular invasion in HGFs.
This study was related to present study because berberine was proven to have
antimicrobial activity against against methicillin-resistant Staphylococcus aureus (MRSA).

VI. METHODOLOGY
A. Materials and Equipment
The main materials and equipment used for the preparation of the product were one
hundred grams of makabuhay stem, one hundred grams of guava leaves, one hundred grams of
calamansi, water, five beakers, one sterilized bottle, weighing scale, mortar and pestle, gauze,

17
syringe, a stirring rod, three filter papers, three slides, normal saline solution (NSS), gloves,
masks, microscope, four glass container, casserole and stove.

B. General Procedure
The calamansi (Citrus microcarpa), guava (Psidium guajava Linn.) leaves and
makabuhay (Tinospora rumphii Boerl) stem were collected, washed and weighed. One hundred
(100) grams of guava leaves and one hundred (100) grams of makabuhay stem were pounded
and boiled to obtain a decoction. The decocted guava leaves and makabuhay stem were filtered
by using a filter paper. Fifty (50) mL of decocted guava leaves and makabuhay stem were kept in
a beaker. One hundred grams of calamansi were extracted using gauze and fifty mL of calamansi
extract were collected in a beaker. The three solutions were combined and stirred in a larger
beaker. The mixture was then transferred in a sterilized and labeled bottle. Other set ups like
positive and negative control were also prepared. An actual test was used to obtain the results in
terms of eliminating foul odor in the pig pens, poultries and stagnant water. Three samples from
each area were collected for the analysis of the presence of bacteria. An electronic microscope
was used in observing bacterial presence in the samples. A Normal Saline Solution (NSS) was
used in observing the specified materials under the electronic microscope.

Participants/Sampling
Thirty (30) individuals, of varying age (18-55) and gender (male/female) from Maligaya
Gloria, Oriental Mindoro, were first taken as respondents for the study.
Non-probability, volunteer sampling was the method used since the study called for a
predefined group or groups of people as subjects of the investigation. Taking into consideration

18
that this study focused on odor before and after application on selected manure and stagnant
water, non-probability, purposive sampling was used as the sampling procedure. To address the
sensitivity of the topic for some people, only those (30 persons) who volunteered to participate in
the study were taken as respondents. This was to ensure that information was gained from key
informants alone as well as upholding the ethics of research. The participants were instructed that
they can decide not to answer any specific question as mentioned in the Informed Consent Form.
Upon deciding not to participate, it was made sure that there would not be any negative
consequences. In addition, the information they provided would be treated with utmost
confidentiality.
Research Instrument
A questionnaire containing 3 questions on perceived elimination of foul odor of the
different areas tested was distributed among the participants. Answers were measured using a 4point Likert scale with 1 as the least effective (Odorous) and 4 as the most effective (Odorless).
The parameters used in rating the odor were: 4- Odorless, 3-Moderately Odorous, 2-Slightly
Odorous and 1- Strongly Odorous

C. Data Gathering Procedure

The following setups were used to gather data: before application Setup 1 (Negative
control), After Application; Setup 2 (Positive Control) and Setup 3 (Experimental Control) the
experimental product. An actual test was conducted on the pig pens, poultries and stagnant areas

19
in order to test the odor before and after the application of the products. These were rated by
thirty respondents.

Three samples were collected including pig manure, chicken manure and stagnant water
and placed in a clean container. A wet mount analysis was used in testing the anti-microbial
activity of the treatments. Three samples were prepared using slides with a Normal Saline
Solution. A small amount of waste material was placed in a slide. Then, this was observed under
the microscope. Next, small amount of the treatments were poured on the specimen. Each of the
treatments were tested and analyzed in the laboratory to observe the presence of bacteria in the
samples. To ensure safety during experimentation, gloves were used while performing the
experiment. Masks were also used before rating the odor of the selected manure and stagnant
water in order to avoid diseases-causing bacteria that could be taken from the areas.

VII. RESULTS AND DISCUSSION

Table 1. Ratings of the 30 Respondents Before and After the Application
of the Setups in Pig Manure in terms of odor

20

Respondents
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total
Mean

Before Application
Setup 1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
30
1.00

After Application
Setup 2
Setup 3
4
4
3
4
3
3
4
4
4
4
3
4
3
4
4
4
3
4
4
4
4
3
4
3
4
3
4
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
4
3
3
3
3
4
3
4
3
4
3
3
4
3
4
4
4
110
110
3.67
3.67

Based on table 1, Setup 1 got a mean of 1.00 while Setup 2 and 3 had the same mean of
3.67. Randomized Block ANOVA was used to determine if there are significant differences
among the three treatments. Based on the statistical tool used, the computed p-value was 4.07 x
10 36 which is lower than the 0.05 level of significance. This means that the null hypothesis was
rejected. Therefore, there is a significant difference in terms of its odor before and after the
application.

21
Post hoc analysis was used to support this result. Setup 2 and 3 has no significant
difference since the p-value was 1.0 which was higher than the 0.05 level of significance. It
shows that setup 2 and 3 are better than setup1 in terms of the odor.
The results of the study was supported by Jackson (2013) which stated that guava
(Psidium guajava Linn.) leaves extract and calamansi (Citrus microcarpa) extract have essential
oils which are highly concentrated oils that often contain the chemicals responsible for the
characteristic scent of the plant. It was proven that these have antiseptic and antifungal
properties. Combined with the pleasant scent of many essential oils, make these as excellent
tools for removing odors.

Table 2. Ratings of the 30 Respondents Before and After the Application

of the Setups in Chicken Manure in terms of odor
Respondents
1
2

Before Application
Setup 1
1
1

After Application
Setup 2
Setup 3
4
3
4
3

22
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total
Mean

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
30
1.00

4
4
4
3
3
3
4
3
4
4
3
4
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
114
3.80

4
4
4
4
4
4
4
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
4
4
4
4
115
3.83

Based on table 2, Setup 1 got a mean of 1.00 while Setup 2 had a mean of 3.80. It was
shown that Setup 3 is the best Setup in terms of eliminating the foul odor in poultry farms since
it got the highest mean which was 3.83. Randomized Block ANOVA was used to determine if
there are significant differences among the three treatments. Based on the statistical tool used,
the computed p-value was 1.73 x 10

42

which is lower than the 0.05 level of significance. This

means that the null hypothesis was rejected. Therefore, there is a significant difference among
the three setups in terms of odor.

23
Post hoc analysis was used to support this result. Setup 2 and 3 has no significant
difference since the p-value was 0.6891 which was higher than the 0.05 level of significance. It
implies that Setup 2 and 3 are better than Setup 1 in terms of the odor.

The results of the study was supported by Lorenzo (2012) which stated that calamansi
(Citrus microcarpa) extract have essential oils which contain the chemicals responsible for the
characteristic scent of the plant. It was proven that these have antiseptic and antifungal
properties. Combined with the pleasant scent of many essential oils, make these an excellent tool
for removing odors.

Table 3. Ratings of the 30 Respondents Before and After the Application

of the Setups in Stagnant Water in terms of odor

Respondents
1

Before Application
Setup 1
1

After Application
Setup 2
Setup 3
4
4

24
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Total
Mean

1
1
1
1
1
2
2
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
33
1.10

4
4
3
4
4
4
3
3
3
4
4
4
4
4
4
4
4
3
4
3
3
4
4
4
4
4
4
4
3
112
3.73

4
4
3
4
4
4
3
4
4
4
4
4
4
4
4
4
3
4
3
3
3
4
4
4
3
4
4
4
3
112
3.73

As can be gleaned from Table 3 above, Setup 1 got a mean of 1.10 while Setup 2 and 3
had the same mean of 3.73. Randomized Block ANOVA was used to determine if there are
significant differences among the three setups. Based on the statistical tool used, the computed pvalue was 2.14 x 1037 which is lower than the 0.05 level of significance. This means that the null
hypothesis was rejected. Therefore, there is a significant difference among the three treatments in
terms of odor.

25

Post hoc analysis was used to support the result and it shows that Setup 2 and 3 has no
significant difference since the p-value was 1.0 which was higher than the 0.05 level of
significance. It means that Setup 2 and 3 are better than Setup 1 in terms of the odor.

The results of the study was supported by Jackson (2013) which stated that guava
(Psidium guajava Linn.) leaves extract and calamansi extract have essential oils which are highly
concentrated oils that often contain the chemicals responsible for the characteristic scent of the
plant. It was proven that these have antiseptic and antifungal properties. Combined with the
pleasant scent of many essential oils, make these as excellent tools for removing odors.
Table 4. Summary on the different test areas
Location
Pig Manure
Chicken Manure
Stagnant Water
Average

Before Application
Setup 1
1.00
1.00
1.10
1.03

After Application
Setup 2
Setup 3
3.67
3.67
3.80
3.83
3.73
3.73
3.73
3.74

Based on table 4, Setup 1 got a mean of 1.03 while Setup 2 had a mean of 3.73. It was
shown that Setup 3 is the best Setup as an odor eliminator having an average of 3.74.
Randomized Block ANOVA was used to determine if there is a significant difference among the
three setups. Based on the statistical tool used, the computed p-value was 7.80 x 10 7 which is
lower than the 0.05 level of significance. This means that the null hypothesis was rejected.
Therefore, there is a significant difference among the three setups in terms of odor.

26
Post hoc analysis was used to support this result. Setup 2 and 3 has no significant
difference since the p-value was 0.84 which was higher than the 0.05 level of significance. It
implies that Setup 2 and 3 are better than Setup 1 in terms of the odor.

The results of the study was supported by Jackson (2013) and Lorenzo (2012) which
stated that guava (Psidium guajava Linn.) leaves extract and calamansi extract have essential oils
which are highly concentrated oils that often contain the chemicals responsible for the
characteristic scent of the plant. It was proven that these have antiseptic and antifungal
properties. Combined with the pleasant scent of many essential oils, make these as excellent
tools for removing odors.

To further validate the claim that the elimination of foul odor of the tested areas is
positively correlated to the elimination of disease-causing bacteria in the area, a gross
examination was conducted. In the gross examination, the following parameters used in the study
were:
+ 1 0-5 M present bacteria

+2 6-10 M present bacteria

+3 11-15 M present bacteria

Table. 5 Analysis on Zone of Inhibition of Bacteria before and after application

Location
Pig Manure
Chicken Manure
Stagnant Water
Average

Before Application
Setup 1
+3
+3
+3
+3

After Application
Setup 2
Setup 3
+1
+1
+1
+1
+1
+1
+1
+1

Based on the Gross Examination for the zone inhibition of bacteria, +3 with 11- 15 M
bacteria were obtained in Setup 1 (before the application). After application of the two setups,
each of the setup obtained +1 with 0-5 M present bacteria. It showed that Setup 2 and 3 were

27
both effective anti-microbial treatment in pig manure, chicken manure and stagnant water in
terms of zone inhibition of bacteria with the same average of +1.

The results of the study was supported by the study of Vermani, Navneet and Gautam
(2013) wherein the phytochemical analysis on crude extracts of Tinospora cordifolia revealed the
presence of alkaloids, flavonoids, tannins, phenols, saponins, glycosides, amino acids and
steroids are accountable for having anti-microbial potential.

As can be gleaned from Table 5 below, before the application, setup 1 obtained 0% zone
of inhibition of bacteria in the selected manure and stagnant water. 85% of the tested bacteria in
Pig manure were inhibited for the application of Setup 3 while in Setup 2, the zone of inhibition
of bacteria was 100%. In chicken manure, Setup 3 had inhibited 80% of the tested bacteria
while Setup 2 had inhibited 100% of the tested bacteria. In terms of the stagnant water, the zone
of inhibition of bacteria of Setup 3 was 90%. In Setup 2, 100% of the tested bacteria were
inhibited. Setup 3 had an average of 85% while setup 2 obtained an average of 100% for the
zone of inhibition of bacteria on selected manure and stagnant water.
Table 5. Percentage of Zone Inhibition of Bacteria Before and After Application
Location
Before Application
After Application
Setup 1
Setup 2
Setup 3
Pig Manure
0%
100%
85%
Chicken Manure
0%
100%
80%
Stagnant Water
0%
100%
90%
Average
0%
100%
85%

28
1.2
1
0.8
0.6
Before Application
0.4

Setup 1
Setup 2

0.2
0

Figure 1. Zone of Inhibition of bacteria before and after application

The results of the present study was supported by Cowan (2000) which stated that guava
(Psidium guajava Linn.) posses alkaloid and flavonoids which are phytochemical compounds

29
that have been found in vitro to be an effective anti-microbial treatment against a wide array of
microorganisms.

Table 6 below shows the bacteria found in the selected manure and stagnant water after
the tests conducted.
Table 6. Bacteria Found in the selected manure and stagnant water
Location

Bacteria

1. Pig Manure

Cryptosporidium, Clostridium Perfringens,

Escherichia coli (Fecal Coliform), Fecal streptococci

2. Chicken Manure

Clostridium Perfringens, Escherichia coli (Fecal

Coliform), Fecal streptococci, Cryptosporidium
Cryptosporidium, Escherichia coli (Fecal coliform),
Enterococci

3. Stagnant Water

VIII. CONCLUSION
Based on the foregoing results and analysis of data, this study found out that the
combined Tinospora rumphii Boerl (Makabuhay), Psidium guajava Linn.(Guava), and Citrus
microcarpa (Calamansi) had a positive effect on its application on selected manure and stagnant
water in terms of 1) Odor before and after application; and 2) Zone of Inhibition of bacteria.
The combined Tinospora rumphii Boerl (Makabuhay), Psidium guajava Linn.(Guava),
and Citrus microcarpa (Calamansi) was found to be the most effective treatment in terms of odor
of test samples before and after application. The Positive Control (Domex) is the most effective
in terms of zone of inhibition of bacteria.
In addition, there is a significant difference between the three setups in terms of odor
before and after application.

30

IX. RECOMMENDATIONS
With the results and conclusion presented, this study forwards the following
recommendations for further studies. First, it is highly recommended to use the combined extract
of makabuhay stem, guava leaves and calamansi as disinfectant on areas with foul odor like pig
pens, poultry farms and stagnant water. Further studies can also be made to test the effectiveness
of makabuhay stem, guava leaves and calamansi as an anti-microbial treatment. Additional
studies should also be conducted to determine other methods of applying the combined extract of
makabuhay stem, guava leaves and calamansi. When a wet mount analysis was used for the test,
gross examination should be used in interpreting the data being gathered after the test.

X. BIBLIOGRAPHY
Books
Joaquin, C., Lagunzad, C., & Rabago, L. (2003). Functional Biology. Quezon City: Vibal
Publishing House, Inc.
Kurian, J.C. (2010). Amazing Healing Plants. Manila: Philippine Publishing House.

Encyclopedia
Grolier International (1995). Grolier Encyclopedia of Knowledge (p.16). United States of
America: Grolier Incorporated.
Paton, J. (2000). Bacteria. Childrens Encyclopedia. (Vol.1, 61). Kingfisher Publications Plc.,
London.

31
Websites
Anwar et al. (2003). Young leaf extract of the plant possesses antibacterial and antifungal
properties, Retrieved August 9, 2013, from http://www.mpbd.info/plants/psidiumguajava.php.
Archa V., Navneet & Gautam, S. (2013). Screening of Antibacterial Activity of Tinospora
cordifolia Miers. Extracts Against Dental Pathogens, Retrieved August 1, 2013, from
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Jackson, N. (2013). Essential Oil Odor Removal. Retrieved August 1, 2013 from
http://www.ehow.com/info_8688040_essential-oil-odor-removal.html.
King,

M.

(2008).

Kalamansi.

Retrieved

August

1,

2013

from

http://www.in-

cosmetics.com/__novadocuments/2520.
Lorenzo,

T.

(2009).

Calamansi

Essential

Oil.

Retrieved

August

1,

2013

from

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from

http://www.thecasadelorenzo.com/2012/04/calamansi-essential-oil.html.
Stuart,

S.

(2012).

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http://www.stuartxchange.com.html.

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Bouaziz, M. (2009). Disinfectant properties of essential oils from Salvia officinalis L. cultivated
in Tunisia. Food and Chemical Toxicology, 47, 27552760.
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Cowan, M. (2000). Plant Products as Antimicrobial Agents. Clinical Microbiology Reviews, 12,
564582.

32
Mahalel, U. (2012). Antibacterial Sensitivity for some Chemically Diverse Steroidal
Glycosides in Vitro. Journal of Agriculture & Social Sciences, 25-28.
Yu, H. et al. (2005). Antimicrobial activity of berberine alone and in combination with ampicillin
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