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West Visayas State University

COLLEGE OF ARTS AND SCIENCES


Iloilo City

Chapter 1
Introduction to the Study
Chapter one, consists of five parts, namely: (1)
Background and Theoretical Framework of the Study, (2)
Statement of the Problem and Hypothesis, (3) Significance
of the Study, (4) Definition of Terms, and (5) Delimitation
of the Study.
Part One, Background and Theoretical Framework of the
study, gives the reasons for the choice of the problem and
the variables considered in the conduct of the study.
Part Two, statement of the Problem and Hypothesis,
describes the problem and its purpose in conducting the
study and the hypothesis to be tested.
Part Three, Significance of the Study, enumerates the
benefits that may be derived from the results of the study
and persons who would benefit from them.
Part Four, Definition of Terms, deals with the
conceptual and the operational definitions of important
terms used in the study.
Part Five, Delimitation of the Study, sets the limit
and scope of the study.

West Visayas State University


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Background and Theoretical Framework of the Study


Soil is a natural reservoir for microorganism and their
antimicrobial products (Alanis, 2005).
A significant number of these organisms are undocumented
and some of them may have an effect on pathogenic bacteria.
Barangay Sinapsapan is located at
on the lands

Jordan, Guimaras Island,

bordering to Panay Gulf with approximate coordinates

of N 010 35.00

E 122 30.47. The distance from the sea is about

270 meters at an elevation of about 30 meters above sea level. The


terrain is
rapidly
inland

a typical

to about

young coral

30-40

island

meters elevation

from the coastland some

100 meters.

with the land


when

rising

proceeding

Going further inland

elevation varies up and down forming valleys and ridges


(Eckerwall, Personal Communication, October 10, 2013).
Barangay Sinapsapan has many unexplored territories and which
is most likely to yield purposeful results towards isolation of
new antibiotics. There are areas that are not inhabited by people.
Most of the soil in that area is rich in organic matter since
leaves and twigs cover most of the land. The texture of the soil
is mostly coarse. With regards to its structure, the arrangement
of the soil particles is in different aggregates. Soil granules

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clump or bind together (Espaola, Personal Communication, October


10, 2013).
In a single gram of soil, there can be billions of bacteria
(Reid and Wong, 2005). There are estimated 60,000 different
bacteria species, most which have yet to be even named, and each
has its own particular roles and capabilities (Reid and Wong,
2005). Most live in the top 10 cm of soil where organic matter
is present (Reid and Wong, 2005). Soil bacteria perform
important services related to disease suppression (Martin,
1976).
Pathogenic bacteria are capable of causing disease. Humans
are generally most interested in the species of pathogenic
bacteria which can cause disease in humans, although these
bacteria can also infect other animals and plants.
Some notable pathogenic bacteria include Staphylococcus
aureus and Escherichia coli. Staphylococcus aureus, often
referred to simply as staph, are gram-positive spherical
bacteria that occur in microscopic clusters resembling grapes.
Bacteriological culture of the nose and skin of normal humans
invariably yields staphylococci (Todar, 2008). Escherichia coli
is the head of the large bacterial family, Enterobacteriaceae,

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the enteric bacteria, which are facultatively anaerobic gramnegative rods that live in the intestinal tracts of animals in
health and disease (Todar, 2008).
The science of antibiotics has remained and will
remain for many years, one of the most interesting natural
sciences, in both theoretical and practical aspects. Microbial
natural products still appear as the most promising source of
the future antibiotics that society is expecting (Pelaez,
2006).
For the past years, pathogens underwent mutation which
enabled them to resist antimicrobials, thereby threatening
millions of people worldwide. Thus, it is imperative to screen
more and more bacteria from different soil samples for
antimicrobial activity in hope of getting some bacterial strains
that produce antibiotics that have not been discovered yet and
active against drug resistant pathogens.

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The relationship between the variables of the study is


presented in Figure 1.
Independent Variables:

Dependent Variables:

Bacterial Isolates in
Brgy. Sinapsapan,
Jordan, Guimaras in
the high area
Bacterial Isolates in
Brgy. Sinapsapan,
Jordan, Guimaras in
the intermediate area
Bacterial Isolates in
Brgy. Sinapsapan,
Jordan, Guimaras in
the low area

Zone of inhibition
on Staphylococcus
aureus BIOTECH
1582

Zone of Inhibition
on Escherichia
coli BIOTECH 1634

Positive Control: 500


mg Ciprofloxacin / 10
ml distilled water

Negative Control:
distilled water

Figure 1. Paradigm showing the relationship between the


independent and dependent variables.

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Statement of the Problem and Hypotheses


This study was conducted to screen and determine the
antibacterial effects of soil bacterial isolates from three
sampling sites of Brgy. Sinapsapan, Jordan, Guimaras against S.
aureus and E. coli. Furthermore, soil bacterial isolates with
antibacterial properties against the test pathogenic bacteria
were characterized. Specifically, it sought to answer the
following questions:
1. How many bacterial isolates are present in the three
sampling sites of Brgy. Sinapsapan, Jordan, Guimaras?
2. What are the characteristics (colonial morphology and
gram staining reaction) of bacterial isolates that have
antibacterial properties against S. aureus and E. coli?
3. What are the effects of the bacterial isolates on S.
aureus and E. coli after 72 hours of incubation?
4. Are there significant differences on the zone of
inhibition of bacterial isolates from the three sampling
sites against S. aureus and E. coli?
Null Hypothesis:
1. There are no significant differences on the zone of
inhibition of bacterial isolates in the three sampling
sites against S. aureus and E. coli.

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Significance of the Study


The present study was undertaken to isolate bacteria from
the soil samples of Brgy. Sinapsapan, Jordan, Guimaras. Also, to
assess the soil bacteria for their anti-bacterial properties.
The resistance problem demands that to discover new
antibacterial agents effective against pathogenic bacteria
resistant to current antibiotics. Thus, it is imperative to
screen more and more bacteria from different soil samples for
antimicrobial activity in hope of getting some bacterial strains
that produce antibiotics that have not been discovered yet and
active against drug resistant pathogens.

Definition of Terms
The following terms are defined to ensure clarity and
understanding of the study:
Antibacterial effect- According to Mozo (2008), it is an
effect that is destructive to bacteria or suppressing their
reproduction or growth, effective against bacterial
infections.
In this study, antibacterial effect refers to the action
of screened bacterial isolates against S. aureus and E. coli.

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Barangay- is a village, especially when considered as the


smallest political unit (Department of Health, 2009).
In this study, barangay refers to Barangay Sinapsapan,
Jordan, Guimaras which is the sampling site of the experiment.
Escherichia coli- it is the head of the large bacterial
family, Enterobacteriaceae, the enteric bacteria, which are
facultatively anaerobic gram-negative rods that live in the
intestinal tracts of animals in health and disease (Todar,
2008).
In this study, Escherichia coli refers to one of the
bacterial pathogens tested.
Isolation - the process of separating, or the state of being
alone (OToole et al., 2003).
In this study, isolation refers to the process of
collecting the general bacterial populations in soil of Brgy.
Sinapsapan, Jordan, Guimaras.
High area sampling site- having an elevation of
approximately 21 meters- 30 meters above sea level (Eckerwall,
Personal Communication, October 10, 2013).

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In this study, high area refers to one of the sampling


areas of study from which the bacterial isolates were obtained.
Intermediate area sampling site- having an elevation of
approximately 11 meters- 20 meters above sea level (Eckerwall,
Personal Communication, October 10, 2013).
In this study, intermediate area refers to one of the
sampling areas of study from which the bacterial isolates were
obtained.
Low area sampling site- having an elevation of
approximately 0 meters- 10 meters above sea level (Eckerwall,
Personal Communication, October 10, 2013).
In this study, low area refers to one of the sampling
areas of study from which the bacterial isolates were obtained.
Soil- is a natural reservoir for microorganism and their
antimicrobial products (Alanis, 2005).
In this study, soil refers to the sample where the new
antibacterial agents are to be isolated.
Staphylococcus aureus- it is gram-positive spherical
bacteria that occur in microscopic clusters resembling grapes.

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Bacteriological culture of the nose and skin of normal humans


invariably yields staphylococci (Todar, 2008).
In this study, Staphylococcus aureus refers to one of
the bacterial pathogens tested.
Zone of inhibition- is the area of no bacterial growth
around an antimicrobial agent in the disk (Tortora, 2010).
In this study, zone of inhibition refers to the effect of
screened isolated antibacterial agents against S. aureus and E.
coli.

Scope and Limitation of the Study


This study aimed to determine the number of bacterial
isolates from the three sampling sites at Sinapsapan, Jordan,
Guimaras delineated by their elevation. The isolates are then
assessed for antibacterial effect using the agar disk diffusion
method against S. aureus and E. coli. All isolates with
antibacterial properties were then characterized based on their
colonial characteristics.

Aseptic technique was employed in the

experiment. This study used both the experimental and the


descriptive mode of investigation. The experimental mode
involved the bioassay of the bacteria and the descriptive mode
involved the isolation and characterization of the obtained

West Visayas State University


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bacterial isolates. It was conducted on May 5 to 24, 2014 at


Rizal Hall of West Visayas State University, La Paz, Iloilo
City.

11

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Chapter 2
Review of Related Literature
This chapter presents the conceptual literature regarding
this study to support the background for greater understanding
of the research outcome. This is consisted of the following:
(1)Staphylococcus aureus morphology, (2) its pathogenicity,
(3)Escherichia colis morphology, (4) colony characteristics,
(5) its pathogenicity, (6) soil, and Barangay Sinapsapan, Jordan
Guimaras.
Staphylococcus aureus
Morphology. According to Brooks, Carroll, Butel, Morse and
Mietzner (2010) staphylocci are gram positive spherical cells,
usually arranged in grapelike irregular clusters. They grow
readily on many types of media and are active metabolically,
fermenting carbohydrates and producing pigments that vary from
white to deep yellow.

Some are members of the normal flora of

the skin and mucous membranes of humans; others cause


suppuration, abcess formation, a variety of pyogenic infections
and even fatal septicemia. The pathogenic staphylococci often
hemolyze blood, coagulate plasma, and produce a variety of food

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poisoning is caused by a heat-stable staphylococcal enterotoxin.


Staphylococci rapidly develop resistance to many antimicrobial
agents and present difficult therapeutic problems. The genus
Staphylococcus has at least 40 species. The three most
frequently encountered species of clinical importance are
Staphylococcus aureus, Staphylococcus epidermis, and
Staphylococcus saprophyticus. Staphylococcus aureus is
coagulase-positive, which differentiates it from the other
species. Staphylococcus aureus is a major pathogen for humans.
Almost every person will have some type of Staphylococcus aureus
infection during a lifetime, ranging in severity from food
poisoning or minor skin infections to severe life-threatening
infections. Staphylococci are spherical cells about 1 um in
diameter arranged in irregular clusters. Single cocci, pairs,
tetrads, and chains are close also seen in liquid cultures.
Young cocci stain strongly gram-positive; on aging, many cells
become gram-negative. Staphylococci are non-motile and do not
form spores. Under the influence of drugs like penicillin,
staphylococci are lysed. The pathogenic capacity of a given
strain of a Staphylococcus aureus is the combined effect of
extracellular factors and toxins together with the invasive
properties of strain. At one end of the disease spectrum is

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staphylococcal food poisoning, attributable solely to the


ingestion of performed enterotoxin; at the other end are the
staphylococcal bacteremia and disseminated abscesses in all
organs. Pathogenic invasive of Staphylococcus aureus produces
coagulase and tend to produce a yellow pigment and to be
hemolytic. The prototype of Staphylococcal lesion is the
furuncle or other localized abscess. Group of Staphyloccus
aureus established in a hair follicle leads to tissue necrosis
(dermonecrotic factor). Coagulase is produce d and coagulates
fibrin around the lesion and within the lymphatics, resulting in
formation of a wall that limits the process and is reinforced by
the accumulation of inflammatory cells, and, later, fibrous
tissue.
Pathogenicity. According to Talaro (2008) it is surprising
that a bacterium with such great potential for virulence as
Staphylococcus aureus is a common, intimate human associate. The
microbe is present in most environments frequented by humans and
is readily isolated from fomites (Talaro, 2008).Colonization of
some infants begin within hours after birth and continues
throughout life. The carriage for normal healthy adults varies
anywhere from 20% to 60%, and the pathogen tends to be harbored
intermittently rather than chronically. Carriage occurs mostly

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in the anterior nares (nostrils) and, to a lesser extent, in the


skin, nasopharynx, and intestine. Usually this colonization is
not associated with symptoms, nor does it ordinarily lead to
disease in carriers or their contacts. Circumstances that
predispose an individual to infection include poor hygiene and
nutrition, tissue injury, preexisting primary infections,
diabetes mellitus, and immunodeficiency states. Staph infections
in the newborn nursery and surgical wards are the third most
common nosocomial infection. The so-called hospital strains
can readily spread in an epidemic pattern within and outside the
hospital. A serious concern has arisen from the increase in
community infections strains of Staphylococcus aureus called
MRSA (methicillin resistant S. aurues). Several outbreaks have
been reported in prison inmates, athletes, and schoolchildren.
The infections are spread by contact with skin lesions, and have
proved to be very difficult to treat or control (Talaro, 2008).
According to Roche (2003) Staphylococcus aureus is a
complex pathogen with numerous classes of virulence factors.
Protein secretion principally occurs via the Sec system and is
required to render many virulence factors functional. Compounds
which selectively block bacterial protein secretion while
leaving the host system unaffected may lead to novel

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antimicrobial therapies. Adherence to host tissues involves


MSCRAMMs (Microbial Surface Components Recognizing Adhesive
Matrix Molecules) which are redundant and overlapping. Microbial
Surface Components Recognizing Adhesive Matrix Molecules
(MSCRAMMs) continue to be targets of interest for vaccine
development, although no current immunization studies involve
children. The role of vaccines in combating S. aureus disease
and the identication of populations which should be targeted
for immunization strategies are questions yet to be resolved.
The importance S. aureus capability to invade and replicate in
nonprofessional phagocyte cells is unclear; intracellular
persistence with subsequent decreased antibiotic exposure could,
in the future, impact decisions of drug choice and therapy
duration for infections such as endocarditis and osteomyelitis.
An improved understanding of protein secretion, tissue adherence
and internalization in S. aureus pathogenesis carries the
promise of identication of new targets for novel therapies for
preventing and treating both acute and chronic S. aureus
infections.
According to Todar (2008) Staphylococcus aureus causes a
variety of suppurative (pus-forming) infections and toxinoses in
humans. It causes superficial skin lesions such as boils, styes

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and furuncles; more serious infections such


as pneumonia, mastitis, phlebitis, meningitis, and urinary tract
infections; and deep-seated infections, such
as osteomyelitis and endocarditis. Staphylococcus aureus is a
major cause of hospital acquired (nosocomial) infection of
surgical wounds and infections associated with indwelling
medical devices. S. aureus causes food poisoning by releasing
enterotoxins into food, and toxic shock syndrome by release of
superantigens into the blood stream.
According to Todar (2008) although methicillin-resistant
Staphylococcus aureus (MRSA) have been entrenched in hospital
settings for several decades, MRSA strains have recently emerged
outside the hospital becoming known as community associatedMRSA( (CA-MRSA) or superbug strains of the organism, which now
account for the majority of staphylococcal infections seen in
the ER or clinic. For the majority of diseases caused by S.
aureus, pathogenesis is multifactorial, so it is difficult to
determine precisely the role of any given factor. However, there
are correlations between strains isolated from particular
diseases and expression of particular virulence determinants,
which suggests their role in particular diseases. The
application of molecular biology has led to advances in

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unraveling the pathogenesis of staphylococcal diseases. Genes


encoding potential virulence factors have been cloned and
sequenced, and many protein toxins have been purified. With some
staphylococcal toxins, symptoms of human disease can be
reproduced in animals with the purified protein toxins, lending
an understanding of their mechanism of action. Human
staphylococcal infections are frequent, but usually remain
localized at the portal of entry by the normal host defenses
(Todar, 2008). The portal may be a hair follicle, but usually it
is a break in the skin which may be a minute needle-stick or a
surgical wound. Foreign bodies, including sutures, are readily
colonized by staphylococci, which may make infections difficult
to control. Another portal of entry is the respiratory tract.
Staphylococcal pneumonia is a frequent complication of
influenza. The localized host response to staphylococcal
infection is inflammation, characterized by an elevated
temperature at the site, swelling, the accumulation of pus, and
necrosis of tissue. Around the inflamed area, a fibrin clot may
form, walling off the bacteria and leukocytes as a
characteristic pus-filled boil or abscess. More serious
infections of the skin may occur, such as furuncles or impetigo.
Localized infection of the bone is called osteomyelitis. Serious

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consequences of staphylococcal infections occur when the


bacteria invade the blood stream. A resulting septicemia may be
rapidly fatal; a bacteremia may result in seeding other internal
abscesses, other skin lesions, or infections in the lung,
kidney, heart, skeletal muscle or meninges (Todar 2008)
Escherichia coli
Morphology. According to Brooks et al. (2010), The
Enterobacteriaceae are short gram-negative rods. Typically
morphology is seen in growth on solid media in vitro, but
morphology is highly variable in clinical specimens. Capsules
are large and regular in Klebsiella, less so in Enterobacter,
and uncommon in the other species. Escherichia coli and most
other enteric bacteria form circular, convex, smooth colonies
with distinct edges. Enterobacter colonies are similar but
somewhat more mucoid. Klebsiella are large and very mucoid and
tend to coalesce with prolonged incubation. The salmonellae and
shigellae produce colony similar to E. coli but do not ferment
lactose. Some strains of E. coli produce hemolysis on blood
agar. According to Todar (2008) E. coli is facultatively
anaerobic Gram-negative rod that live in the intestinal tracts
of animals in health and disease. The Enterobacteriaceae are

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among the most important bacteria medically. A number of genera


within the family are human intestinal pathogens
(e.g. Salmonella, Shigella, Yersinia). Several others are normal
colonists of the human gastrointestinal tract (e.g.
Escherichia, Enterobacter, Klebsiella), but these bacteria, as
well, may occasionally be associated with diseases of humans.
Colonial characteristics. According to Todar (2008) E.
coli is versatile and well-adapted to its characteristic
habitats. It can grow in media with glucose as the sole organic
constituent. Wild-type E. coli has no growth factor
requirements, and metabolically it can transform glucose into
all of the macromolecular components that make up the cell. The
bacterium can grow in the presence or absence of O2. Under
anaerobic conditions it will grow by means of fermentation,
producing characteristic "mixed acids and gas" as end products.
However, it can also grow by means of anaerobic respiration,
since it is able to utilize NO3, NO2 or fumigate as final
electron acceptors for respiratory electron transport processes.
In part, this adapts E. coli to its intestinal (anaerobic) and
its extra intestinal (aerobic or anaerobic) habitats (Todar,
2008).E. coli can respond to environmental signals such as

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chemicals, pH, temperature, osmolarity, etc., in a number of


very remarkable ways considering it is a unicellular organism.
For example, it can sense the presence or absence of chemicals
and gases in its environment and swim towards or away from them.
Or it can stop swimming and grow fimbriae that will specifically
attach it to a cell or surface receptor. In response to change
in temperature and osmolarity, it can vary the pore diameter of
its outer membrane poring to accommodate larger molecules
(nutrients) or to exclude inhibitory substances. With its
complex mechanisms for regulation of metabolism the bacterium
can survey the chemical contents in its environment in advance
of synthesizing any enzymes that metabolize these compounds. It
does not wastefully produce enzymes for degradation of carbon
sources unless they are available, and it does not produce
enzymes for synthesis of metabolites if they are available as
nutrients in the environment.
Pathogenecity. According to Brooks et al. (2010),
Escherichia coli is the most common cause of urinary tract
infection and accounts approximately 90% of first urinary tract
infection in young women. The symptoms and signs include urinary
frequency, dysuria, hematuria, and pyuria. Flank pains are

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associated with upper tract infection. None of these symptoms or


signs is specific for E. coli infection. Urinary tract infection
can result in bacteremia with clinical signs of sepsis. Most of
the urinary tract infections that is involve the bladder or
kidney in an otherwise healthy host are caused by a small number
of O antigen types that have specifically elaborated virulence
factors that facilitate colonization and subsequent clinical
infections. These organisms are designated as uropathogenic E.
coli. Typically these organisms produce hemolysin, which is
cytotoxic and facilitates tissue invasion. Those strains that
cause pyelonephritis express K antigen and elaborate a specific
type of pilus, P fimbriae, which binds to the P blood group
antigen. However, the regular presence of E. coli in the human
intestine and feces has led to tracking the bacterium in nature
as an indicator of fecal pollution and water contamination. As
such, it is taken to mean that, wherever E. coli is found, there
may be fecal contamination by intestinal parasites of humans.
The commensally E. coli strains that inhabit the large intestine
of all humans and warm-blooded animals comprise no more than 1%
of the total bacterial biomass. The E. coli flora is apparently
in constant flux. One study on the distribution of different E.
coli strains colonizing the large intestine of women during a

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one year period (in a hospital setting) showed that 52.1%


yielded one serotype, 34.9% yielded two, 4.4% yielded three, and
0.6% yielded four.

The most likely source of new serotypes

of E. coli is acquisition by the oral route. To study oral


acquisition, the carriage rate of E. coli carrying antibioticresistance plasmids (R factors) was examined among vegetarians,
babies, and non vegetarians. It was assumed that non vegetarians
might carry more E. coli with R factors due to their presumed
high incidence in animals treated with growth-promoting
antimicrobial agents. However, omnivores had no higher an
incidence of R-factor-containing E. coli than vegetarians, and
babies had more resistant E. coli in their feces than non
vegetarians (Todar,2008). No suitable explanation could be
offered for these findings. Besides, investigation of the
microbial flora of the uninhabited Krakatau archipelago has
shown the presence of antibiotic-resistant E. coli associated
with plants. The bottom line seems to be that most of us have
more than one strain of E. coli in our gut, and intestinal
strains tend to displace one another about three or four times a
year (Todar,2008).

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Soil
According to Alanis (2005) serious infections caused by
bacteria that have become resistant to commonly used antibiotics
have become a major global healthcare problem in the 21st
century. Staphylococcus aureus, for instance, a virulent
pathogen that is responsible for a wide range of infections
including pimples, pneumonia, osteomyelitis, endocarditis and
bacteremia, has developed resistance to most classes of
antibiotics. For more than two decades, clinicians and public
health officials have faced hospital acquired methicillinresistant Staphylococcus aureus (MRSA), which also bears
resistance too many antibiotics. During much of this time,
vancomycin has been the therapeutic answer to MRSA, but that
paradigm has changed. Vancomycin resistant strains have emerged
clinically. Vancomycin-resistant S. aureus (VRSA) challenges
clinicians, not only because of vancomycin and methicillin
resistance, but also because of resistance to many other
antibiotics, including aminoglycosides, macrolides, and
fluoroquinolones. Fortunately, newer therapeutic agents,
daptomycin, linezolid, and a streptogramin combination
(quinupristin/dalfopristin) have entered the clinical arena in
the past few years. However, certain undesirable side effects

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and the spread of pathogens with this new antimicrobial drug


resistance emphasize the need for the development of other newer
antimicrobial agents with activity against such Gram positive
bacteria. Another cause of great concern is the Gram negative
antibiotic-resistant opportunistic pathogens. Gram negative
environmental and enteric organisms currently threaten patients
in hospitals and communities with multi-drug resistance,
including broad resistance to first, second, and third
generations of penicillin's and cephalosporin's. These bacteria,
like Pseudomonas aeruginosa, are common environmental organisms,
which act as opportunistic pathogens in clinical cases where the
defense system of the patient is compromised (Lyczak, Cannon and
Pier, 2000). In addition, other intrinsically antibiotic
resistant organisms such as Stenotrophomonas maltophilia
(Saiman, Chen, Gabriel & Knirsch, 2002) are emerging as
opportunistic pathogens. The end result of this phenomenon is
that many strains of bacteria have become resistant, and in many
cases multi-resistant to these therapeutic agents, thus
rendering these drugs ineffective as treatments of choice for
severe infections caused by these pathogens (Saiman et. al.,
2002). Rising numbers of antibiotic unresponsive infectious
disease agents confront patients worldwide and consensus has

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emerged that it is essential that novel antibiotic classes be


developed as part of the strategy to control the emerging drugresistant pathogens. In response, there is a renewed interest in
discovering novel classes of antibiotics that have different
mechanisms of action. Search for new antibiotics effective
against multi-drug resistant pathogenic bacteria is presently an
important area of antibiotic research. Natural products having
novel structures have been observed to possess useful biological
activities. Soil is a natural reservoir for microorganisms and
their antimicrobial products. Filamentous soil bacteria
belonging to the genus Streptomyces are widely recognized as
industrially important microorganisms because of their ability
to produce many kinds of novel secondary metabolites including
antibiotics. Of all known drugs 70% have been isolated from
Actinomycetes bacteria of which 75% and 60% are used in medicine
and agriculture respectively. Indeed, different Streptomyces
species produce about 75% of commercially and medically useful
antibiotics (Saiman et. al., 2002). They have provided more than
half of the naturally occurring antibiotics discovered to date
and continue to be screened for useful compounds. In the course
of screening for new antibiotics, several studies are oriented
towards isolation of Streptomyces from different habitats.

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Presently, there is little documented information of the


occurrence of Streptomyces spp. in the soil of Turkey with a
potential to produce antimicrobial compounds. In the present
study, the isolation and characterization as well as the
inhibitory effects of local Streptomycete isolates tested
against various multiple antibiotic resistant bacteria and yeast
were reported, along with some chemical properties of secondary
metabolites with high biological activities.
According to Prescott, Harley and Klein (2008) soil
scientists would describe soil as weathered rock combined with
organic matter and nutrients. An agronomist would point out that
soil supports plant life. However, a microbial ecologists knows
that the formation of organic matter and the growth of plants
depend on the microbial community within the soil. Historically,
the complexity of the soil as a habitat has been a challenge to
understanding soil microbial ecology. Soil is very dynamic and
is formed in a wide variety of environments. These environments
range from Arctic tundra regions, where approximately 11% of the
Earths soil carbon pool is stored, to Antarctic dry valleys,
where there are no vascular plants. In addition, deeper
subsurface zones, where plant roots and their product cannot
penetrate, also have surprisingly large microbial communities

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(Prescott et al., 2008). Microbial activities in these


environments can lead to the formation of minerals such as
dolomite, microbial activity also occurs in deep continental oil
reservoirs, in stones, and even in rocky outcrops. These
microbes are dependent on energy sources from photosynthetic
protists and nutrients in rainfall and dust. Most soils are
dominated by inorganic geological materials, which modified by
the biotic community, including microorganisms and plants, to
form soils. The spaces between soil particles are critical for
movement of water and gases. Total pore space, and thus gas
diffusion, is determined by the texture of the soil (Prescott
et. al., 2008).
Barangay Sinapsapan, Jordan, Guimaras
According to Eckerwall (Personal Communication, October 10,
2013) barangay Sinapsapan is located at
on the lands

Jordan, Guimaras Island,

bordering to Panay Gulf with approximate coordinates

of N 010 35.00

E 122 30.47.

The distance from the sea is about 270 meters at an elevation


of about 30 meters above sea level.
The terrain is
land

rising rapidly

proceeding inland

a typical
to about

young coral
30-40

islands

meters elevation

from the coastland some

100 meters.

with the
when
Going

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COLLEGE OF ARTS AND SCIENCES
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further inland elevation varies up and down

29

forming valleys and

ridges.
According to Espaola (Personal Communication, October 10,
2013) many areas of the barangay are still untouched and
unexplored due to the accessibility. The barangay is composed of
different soil colors. It varies from dark brown and reddishyellowish brown. The texture of the soil is mostly coarse. With
regards to its structure, the arrangement of the soil particles is
in different aggregates. Soil granules clump or bind together.
According to Eckerwall (Personal Communication, October 10,
2013) in the valleys
found

but

the ridges

coral rocks

and

corn is planted.
larger trees.
Rocks
reach

some

The

10 cm

offers

little

or topsoil or even more

only barren land

soil between , where

can be

with sharp
mainly

fruit and

The spontaneous growth is bushes and a few


lots

give the roots

of

holes and canals

a possibility to

water, often salty.

inside the Coral

stretch very deep

and

The proof of this statement is that so

much growth still survives in long periods of drought. The soil


quality is influenced by the strong winds with high salt content
blowing in from SW during Habagat.

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30

Chapter 3
Research Design and Methodology
This chapter consists of Three (3) parts: Part One
Purpose of the Study and Research Design, Part Two Method, and
Part Three Data Analysis Procedure.
Part One, Purpose of the Study and Research Design,
elucidates on the purpose of the study and describes the
research design.
Part Two, Method, the instrument used, and the procedures
employed in gathering the data pertinent to the study.
Part Three, Statistical Data Analyses, explains the
statistical tools employed in the treatment of data and the
quantitative part which includes the measurement of the zone of
inhibition.

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Purpose of the Study and Research Design


The study employed both the experimental and descriptive
method of investigation. The main purpose of the study is to
screen and determine the antibacterial effects of soil bacterial
isolates from the three sampling sites of Brgy. Sinapsapan,
Jordan, Guimaras against S. aureus and E. coli. The independent
variables are the bacterial isolates from Brgy. Sinapsapan,
Guimaras in the high, intermediate, and lowareas. The dependent
variable is the zone of inhibitions exhibited by S. aureus and
E. Coli.
Materials
The research was conducted at the Central Science Laboratory
room at Rizal Hall of the Biological Science Department of West
Visayas State University, La Paz, Iloilo City.

Equipment and Apparatus


The following equipment and apparatuses were used: autoclave
machine, incubator, beakers, Erlenmeyer flask, culture tubes,
Pipette, test tubes, Petri dish and wire loop.

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Chemicals used
The following chemicals were prepared and used for this
study: Nutrient broth, Nutrient Agar, McFarland 0.5, Normal Saline
Solution, mineral oil, safranin, grams iodine, crystal violet,
Fluconazole and 70% Alcohol (as disinfectant).

Procedures
Sterilization of Materials. The glassware were washed,
dried and wrapped with a newspaper. They were placed inside the
autoclave at 15 psi for 15-20 minutes at 121C. After
sterilization, they were dried in a drying oven. The materials
were stored for later use.
Sampling Site Description. The sampling sites are located
in Barangay Sinapsapan,

Jordan, Guimaras Island,

on the lands

bordering to Panay Gulf with approximate coordinates of


35.00

E 122 30.47.

N 010

The distance from the sea is about 270

meters at an elevation of about 30 meters above sea level


(Eckerwall, Personal Communication, October 10, 2013).
Preparation of Nutrient Agar Plates. In one liter of
distilled water, 23 grams of nutrient agar was dissolved and
thoroughly mixed in an Erlenmeyer flask. After complete
dissolution of the media, it was autoclaved for 15 minutes at

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121

33

C at 15psi.The culture media was allowed to cool and

aseptically transferred to sterile petri dish containing 10 ml


each. All NA plates were allowed to solidify and was
refrigerated for future use.
Preparation of Diluents. In one liter of distilled water, 9
grams of sodium chloride were completely dissolved in an
Erlenmeyer flask. After complete dissolution of salt, it was
autoclaved for 15 minutes at 121

0C

at 15 psi. The solution was

allowed to cool and aseptically transferred to a 25 ml culture


tubes containing 9 ml each. All test tubes containing the
solution were tightly sealed and kept refrigerated for further
use.
Preparation of Nutrient Broth. In one liter of nutrient
broth solution, 13 grams of nutrient broth were dissolved and
thoroughly mixed in an Erlenmeyer flask. After complete
dissolution of the media, it was autoclaved for 15 minutes at
121

C at 15 psi. The culture media was allowed to cool and

aseptically transferred to sterile culture tubes containing 5 ml


each. All culture tubes were allowed to cool and kept
refrigerated for further use.
Soil Sample Collection. Twenty-seven soil samples were
collected from three sampling sites of Brgy. Sinapsapan, Jordan,

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34

Guimaras. The three sampling sites were based on there elevation


and labelled as high, intermediate and low. Each collection was
done from 10-15 cm depth of the soil. Approximately 10 g of soil
was scooped and placed into sterile plastic bags.
Isolation and culture condition. For each collected sample,
1g of the soil was suspended in 100 ml of normal saline
solution. It was incubated in an orbital shaker incubator at 280C
with shaking at 200 rpm for 30 min. Mixtures were allowed to
settle, and serial dilutions up to 10-6 were prepared using
sterile normal saline solution and were agitated normally. An
aliquot of 0.1 ml of each dilution specifically 10-2,10-4, and 106

were taken and spread evenly over the surface of nutrient agar

medium. The medium was encircled with Fluconazole (75 mg/ml) to


inhibit fungal contamination. Plates were incubated at 350C, and
were monitored after 24 hours. Repeated streaking on NA agar
plates of isolated colonies was done to purify bacterial
colonies and then incubated for 18-24 hours. The procedure was
repeated three times to ensure purification and obtain wellisolated bacterial colonies. The purified colonies were
described based on colony characteristics on agar media as seen
with the naked eye as described by Harley (2005). These were
described according to form, elevation, margin, pigmentation or

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35

color, appearance, optical property and texture. The isolated


strains were preserved at 40C overlaid with sterile mineral oil
for further use and were maintained for longer period by serial
subculture. Isolates with antibacterial properties in the
different sampling sites were provided with code as H1 to H6 for
high sampling site bacterial isolates, I1 to I6 for intermediate
sampling site bacterial isolates, and L1 to L6 for low sampling
site bacterial isolates.
Test organisms. The test organisms used in this study were
the gram-positive bacterium, Staphylococcus aureus BIOTECH 1582
and the gram-negative bacterium, Escherichia coli BIOTECH 1634.
All test bacteria were purchased from the Philippine National
Collection of Microorganisms (PNCM), University of the
Philippines Los Baos (UPLB) Biotech in Laguna. A letter for
requisition of purchase of the said bacteria was done ahead
prior to the microbial assay. Upon receiving the said culture of
bacteria, they were overlaid with sterile mineral oil and were
furthered sub-cultured for further use.
Broth Culture of Test Isolates and Test Pathogens. All subcultured test pathogens and characterized test isolates were
inoculated using a sterile wire loop (loopful of each bacterium)
in each prepared broth culture using aseptic technique.

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Inoculation was done twice to ensure growth of bacterial culture


in broth solution. All bacterial broth cultures were incubated
at 370C for 24 hours.
Preparation of 0.5 McFarland Standard. The following
solutions were mixed, 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.036
N H2SO4.About 5 ml of the solution of the same proportions as
those used in preparing the culture suspension were transferred
in a 25 ml screw-cap tube. The culture tube were tightly sealed
and stored in the dark at room temperature. Prior to use, the
turbidity standard was shaken thoroughly. The Mc Farland
standard was used to adjust the turbidity of the inoculum prior
to microbial assay. The turbidity standard may contain
approximately 1.5 x 108 CFU/ml of the cultured bacteria (Quinto &
Santos, 2005).
Preparation of Antibiotic Solution. An antibiotic
ciprofloxacin was purchased from a local drug store. The
preparation of the solution was based on the indicated
concentration i.e. 50 mg/ml (Allen & Ansel, 2013), wherein 500
mg of the antibiotic tablet was pulverized and dissolved in 10
ml sterile distilled water.
In vitro screening of Soil Bacterial Isolates for Antibacterial Activity. Morphologically distinct colony of bacterial

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isolates was subjected for anti-bacterial activity screening


against the test organisms using the modified agar disk
diffusion method on agar medium. In this method, pure bacterial
isolate was inoculated in an agar disk with a thickness of 2mm
and a diameter of 10 mm on a plate of screening media spread
plated with the test pathogen. The plates were incubated for 24
h at 37

0C.

Antagonism was measured by the size of the inhibition

zone (Madigan et al., 1997). The presence of zone of inhibition


of the test bacteria were derived using a formula and evaluated
according to the observed and corresponding modified inferences
from Quinto and Santos (2005):
=

( )

Where
X = diameter of the petri dish used (90 mm)
Y = diameter of the punched hole on the agar plate (10 mm)
Z = number of descriptive scale used (4)

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Table 1. Descriptive Inferences of the Zone of Inhibition


Measurement

Description

0-20 mm

inactive;

21-40 mm

partially active

41-60 mm

Active

61-80 mm

very active

Each bacterial isolate was tested on the test organism in


three replicates and in three trials. Zone of inhibition was
recorded every after 24 hours for three days.
Gram staining of Bacterial Isolates with Antibacterial
Properties. A smear of culture was taken in a clean glass slide
and was heated gently over a flame. The smear was covered with a
thin film of crystal violet for 1 min and was washed gently in
slow running tap water. Grams iodine solution was flooded over
the smear for 1 min and was washed with tap water. Alcohol was
used to decolorize the smear until the violet color ceased to
flow away. The slide was washed with water and counter stained
with safranin. The stain was flooded over the smear for 2 min,
then the slide was washed, drained, air dried, and viewed under
microscope. The culture retaining the violet color indicates a

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gram-positive organism. However, the culture with pink color


indicates a gram-negative organism.
Data Collection Procedure. The data were collected based on the
presence of zone of inhibition of bacterial isolates on S.
aureus and E. coli. The zone of inhibition was measured with a
ruler in millimeter units. Each bacterial isolate was tested in
three replicates and in three trials. The measurement of the
zone of inhibition on the test bacteria was noted.
Descriptive Statistics. The mean zone of inhibition on each
test bacterium was tabulated. Furthermore, the zone of
inhibition of each bacterial isolate was evaluated with the aid
of a descriptive scale on the zone of inhibition modified from
Quinto and Santos (2005).
Inferential Statistics. Repeated measures of Analysis of
Variance (rANOVA) was used to compare the average zone of
inhibition of the different bacterial isolates against E. coli
BIOTECH 1634 and S. aureus BIOTECH 1582over a seventy-two hours
incubation period.

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Chapter 4
Results and Discussion

Chapter Four has three parts: (1) Descriptive Data


Analysis, describes the results shown in the experiment; (2)
Inferential Data Analysis, shows the level of significance based
on the result; and (3) Discussion, discusses the reason behind
the effectiveness of the different bacterial isolates against
the test bacteria.

Descriptive Data Analysis


Bacterial Isolates in the Three Sampling Sites of Barangay
Sinapsapan, Jordan, Guimaras. Table 2 shows that there were six
bacterial isolates obtained from the three sampling sites of
Barangay Sinapsapan, Jordan, Guimaras.

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Table 2
Bacterial isolates obtained in the three sampling sites of Brgy.
Sinapsapan, Jordan, Guimaras.
Sampling Site

Number of Isolates

High Area

Intermediate Area

Low Area

Characteristics of Bacterial Isolates in the Different


Sampling Sites in Barangay Sinapsapan, Jordan, Guimaras. Table 3
shows the characteristics of the six bacterial isolates in each
sampling site as characterized according to morphology which
include shape, elevation, margin, color, pigmentation, and
texture, cell arrangement, and Gram staining reaction. This is
further supported with the physical appearances of the colonies
and cell morphology in Appendices B, C, and D.

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42

Table 3
Characteristics of Bacterial Isolates in the High, Intermediate
and Low Area Sampling Sites of Brgy. Sinapsapan, Jordan,Guimaras
Isolate Number

Sampling site

H1

High

H2

High

H3

High

H4

High

H5

High

H6

High

I1

Intermediate

I2

Intermediate

I3

Intermediate

I4

Intermediate

I5

Intermediate

I6

Intermediate

L1

Low

L2

Low

L3

Low

L4

Low

L5

Low

L6

Low

Colony Characteristics
Irregular; Flat; Undulate; Dull;
Opaque; Non- pigmented; Smooth
Circular; Flat; Entire; Dull; Opaque;
Non- pigmented; Smooth
Punctiform; Flat; Entire; Dull;
Transluscent;
Nonpigmented;
Smooth
Rhizoid; Flat; Filamentous; Dull;
Transluscent;
Nonpigmented;
Smooth
Circular; Flat; Entire; Dull; Opaque;
Yellow; Smooth
Circular; Flat; Entire; Dull; Opaque,
Orange; Smooth
Rhizoid; Flat;Filamentous; Dull;
Transluscent;
Nonpigmented;
Smooth
Circular; Flat; Entire; Dull; Opaque;
Non- pigmented; Smooth
Punctiform; Flat; Entire; Dull;
Transluscent;
Nonpigmented;
Smooth
Irregular; Flat; Undulate; Dull;
Opaque; Non- pigmented; Smooth
Filamentous; Flat; Filamentous;
Dull; Opaque; Non- pigmented;
Smooth
Circular; Flat; Entire; Dull; Opaque;
Yellow; Smooth
Rhizoid;
Flat;Filamentous;
Dull;Opaque;
Non- pigmented;
Smooth
Filamentous; Flat; Filamentous;
Dull; Opaque; Non- pigmented;
Smooth
Punctiform; Flat; Entire; Dull;
Transluscent;
Nonpigmented;
Smooth
Irregular; Flat; Undulate; Dull;
Transluscent;
Nonpigmented;
Smooth
Circular; Flat; Entire; Dull; Opaque;
Yellow; Smooth
Circular; Flat; Entire; Dull; Opaque;
Orange; Smooth

Cell Shape & Cell


Arrangement

Gram
Reaction

Bacilli occur singly

Gram negative (-)

Cocci occur singly

Gram negative (-)

Bacilli in clusters

Gram positive (+)

Cocci in clusters

Gram positive (+)

Cocci in chain

Gram positive (+)

Cocci in clusters

Gram positive (+)

Diplobacilli

Gram positive (+)

Cocci occur singly

Staining

Gram negative (-)

Cocci in clusters

Gram negative (-)

Cocci in clusters

Gram positive (+)

Cocci in clusters

Gram positive (+)

Cocci occur singly

Gram negative (-)

Bacilli occur singly

Gram positive (+)

Cocci in clusters

Gram positive (+)

Cocci in clusters

Gram negative (-)

Bacilli occur singly

Gram positive (+)

Cocci occur singly

Gram negative (-)

Cocci occur singly

Gram negative (-)

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Zone of Inhibition of Bacterial Isolates in the Three


Sampling Sites Against Staphylococcus aureus BIOTECH 1582.
Figure 2 shows the mean zone of inhibition of highland soil
bacterial isolates against S. aureus BIOTECH 1582 after 24, 48
and 72 hours of incubation. H4 (M=25.89mm, SD=6.41)and H1
(M=34.22mm, SD=5.91; M=35.33mm, SD=4.97) have the highest zone
of inhibitions and described as partially active after 24, 48
and 72 hours of incubation among the isolates, respectively.
However the positive control antibiotic, ciprofloxacin has the
highest zone of inhibition after 24, 48 and 72 hours (M=28.55
mm, SD=10.33; M=49.67 mm, SD= 9.12; 54.67 mm, SD=10.78),
respectively.

43

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60

44

Active

50
isolate 1
40

Partially Active

isolate 2

isolate 3
30

isolate 4
isolate 5

Inactive

20

isolate 6
Antibiotic

10

Negative Control

0
24 hours

48 hours

72 hours

Figure 2. Zone of inhibition of highland soil bacterial isolates


against S. aureus BIOTECH 1582

Figure 3 shows the mean zone of inhibition of intermediate


land soil bacterial isolates against S. aureus BIOTECH 1582
after 24, 48 and 72 hours of incubation. I5 (M=28.67mm,
SD=10.31) and I2 (M=33.34mm, SD=4.21; M=29.44mm, SD=3.91) have
the highest zone of inhibitions and described as partially
active after 24, 48, and 72 hours of incubation among the
isolates, respectively. However, the positive control antibiotic
ciprofloxacin has the highest zone of inhibition after 24, 48

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and 72 hours (M=28.55 mm, SD=10.33; M=49.67 mm, SD=9.12; 54.67


mm, SD=10.78), respectively.
60

Active

50

isolate 1

Partially Active

40

isolate 2

isolate 3
30

isolate 4

Inactive

20

isolate 5
isolate 6

10

Antibiotic

0
24 hours

48 hours

72 hours

Negative
Control

Figure 3. Zone of inhibition of intermediate land soil bacterial


isolates against S. aureus BIOTECH 1582

Figure 4 shows the mean zone of inhibition of lowland soil


bacterial isolates against S. aureus BIOTECH 1582 after 24, 48
and 72 hours of incubation. L4 (M=25.33, SD=2.92) and L1 (M=29,
SD=3.04; M=38, SD=5.79) have the highest zone of inhibitions and
described as partially active after 24, 48, and 72 hours of
incubation among the bacterial isolates, respectively. However
the positive control antibiotic ciprofloxacin has the highest

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zone of inhibition after 24, 48 and 72 hours (M=28.55 mm,


SD=10.33 M=49.67 mm, SD=9.12; 54.67 mm, SD=10.78), respectively.
Active

60
50

isolate 1

Patially
Active

40

isolate 2
isolate 3

30

isolate 4

Inactive

20

isolate 5
isolate 6

10

Antibiotic

Negative
Control

24 hours

48 hours

72 hours

Figure 4. Zone of inhibition of low land soil bacterial


isolates against S. aureus BIOTECH 1582

Zone of Inhibition of Bacterial Isolates in the Three


Sampling Sites Against Escherichia coli BIOTECH 1634. Figure 5
shows the mean zone of inhibition of highland soil bacterial
isolates against E. coli BIOTECH 1634 after 24, 48 and 72 hours
of incubation. H2 (M=20.11mm, SD=9.37)and H4 (M=26.22mm,
SD=3.67; M=34.22mm, SD=5.29) have the highest zones of
inhibitions and described as partially active after 24, 48, and

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72 hours of incubation among the isolates, respectively.


However, positive control antibiotic has the highest zone of
inhibition after 24, 48 and 72 hours of incubation (M=22.33 mm,
SD=3.39; 30.33 mm, SD=6.84; 36.65 mm, SD=5.66), respectively.
Partially Active

40
35

isolate 1

30

isolate 2

25

isolate 3

Inactive

20

isolate 4

15

isolate 5

10

isolate 6

Antibiotic

Negative
Control

24 hours

48 hours

72 hours

Figure 5. Zone of inhibition of highland soil bacterial isolates


against E. coli BIOTECH

1634

Figure 6 shows the mean zone of inhibition of intermediate


land soil bacterial isolates against E. coli BIOTECH 1634 after
24, 48 and 72 hours of incubation. I4 (M=19.44mm, SD=4.00), I1
(M=27mm, SD=3.50), and I6 (M=32.78mm, SD=6.65) have the highest
zones of inhibition and described as partially active after 24,

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48, and 72 hours of incubation, respectively. However, positive


control antibiotic has the highest zone of inhibition after 24,
48 and 72 hours of incubation (M=22.33 mm, SD=3.39; 30.33 mm,
SD=6.84; 36.67mm, SD=5.66), respectively.

40

Partially Active

35
30

isolate 1
isolate 2

25

isolate 3

Inactive

20

isolate 4
isolate 5

15

isolate 6
10

Antibiotic
Negative Control

5
0
24 hours

48 hours

72 hours

Figure 6. Zone of inhibition of intermediate land soil bacterial


isolates against E. coli BIOTECH 1634

Figure 7 shows the mean zone of inhibition of lowland soil


bacterial isolates against E. coli BIOTECH 1634 after 24, 48 and
72 hours of incubation. L1 (M=23mm, SD=10.77), L4 (M= 30.89mm,
SD=4.88), and L5 (M=34.78mm, SD=3.60) have the highest zones of

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inhibitions and described as partially active after 24, 48 and


72 hours of incubation, respectively. However, positive control
antibiotic has the highest zone of inhibition after 72 hours of
incubation (M=36.67 mm, SD=5.66).

40

Partially Active

35
isolate 1

30

isolate 2

25

isolate 3
20

Inactive
isolate 4

15
isolate 5
10

isolate 6

Antibiotic

0
24 hours

48 hours

72 hours

Negative Control

Figure 7. Zone of inhibition of lowland soil bacterial isolates


against E. coli BIOTECH 1634

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Inferential Data Analysis

Zone of Inhibition of Bacterial Isolates in the Different


Sampling Sites against Staphylococcus aureus BIOTECH 1582. Table
4 shows a no significant difference in the analysis of variance
with repeated measures for the zone of inhibition of bacterial
isolates in the different sampling sites against Staphylococcus
aureus BIOTECH 1582 after 72 hours of incubation, p < 0.05. This
may indicate that different soil bacterial isolates may have
comparable effects with the antibiotic ciprofloxacin.
Furthermore, this is supported by the Least Significant
Difference for pair wise comparison as indicated in Appendix F.
There were significant differences among the following: all
isolates and the antibiotic ciprofloxacin; and between isolate 6
and isolates 3, 4, and 5, p < 0.05.

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Table 4
Analysis of Variance with Repeated Measures for the Zone of
Inhibition of Bacterial Isolates in the Different Sampling Sites
against Staphylococcus aureus BIOTECH 1582.
Effect

Time*Isolate

Wilks
lambda

Value

Hypothesi
s df

Error
df

Signifi
cance

Partial
Eta
Squared

0.825

1.868

74

0.125

0.092

P > 0.05 is not significant.

Zone of Inhibition of Bacterial Isolates in the Different


Sampling Sites against Escherichia coli BIOTECH 1634. Table 5
shows a significant difference in the analysis of variance with
repeated measures for the zone of inhibition and time of
incubation of bacterial isolates in the different sampling sites
against Escherichia coli BIOTECH 1634 after 72 hours of
incubation, p < 0.05. This may indicate that soil bacterial
isolates have varying effects on E. coli as indicated in Figure
7. Furthermore, this is supported by the Least Significant
Difference for pair wise comparison as indicated in Appendix G.
There were significant differences among the following: all

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isolates and the antibiotic ciprofloxacin; isolates 3 and 6;


isolates 4 and 5, and isolates 4 and 6, p < 0.05.

Table 5
Analysis of Variance with Repeated Measures for the Zone of
Inhibition of Bacterial Isolates in the Different Sampling Sites
against Escherichia coli BIOTECH 1634.
Effect

Wilks
lambda

Value

Hypothes
is df

Error
df

Signifi
cance

Partial
Eta
Squared

Time*Isola
te

Wilks
lambda

0.681

3.915

74

0.006*

0.175

*P < 0.05 is significant.

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Discussion
A total of 18 bacterial isolates in the 3 sampling sites
with variable colony and cell characteristics in each sampling
site were collected. The bacterial isolates are all flat, dull,
non-pigmented and smooth. I1 is a diplobacilli while the rest
are

either

clustered

or

occur

singly.

Amongst

the

different

microorganisms inhabiting in the soil, bacteria are the most


abundant and predominant organisms (Sylvia, Fuhrmann, Hartel, &
Zuberer, 2005). Sylvia et al. (2005) added that morphologically,
soil

bacteria

are

divided

into

three

groups

viz

Cocci

(round/spherical), (rod-shaped) and Spirilla/ Spirillum (cells


with long wavy chains). Bacilli are most numerous followed by
Cocci

and

influence

Spirilla
the

in

soil.

microbial

The

major

population,

soil

factors

distribution

and

which
their

activity in the soil are soil fertility, cultural practices,


soil moisture, soil temperature, soil aeration, light, soil pH,
organic matter, food and energy supply, nature of soil, and
microbial associations (Stephanie, Breznak, & Schmidt, 2007).
All these factors play a great role in determining not only the
number

and

type

of

organism

but

also

their

activities.

Fertilizers and manures applied to the soil for increased crop


production, supply food and nutrition not only to the crops but

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also

to

microorganisms

in

soil

and

thereby

54

proliferate

the

activity of microbes. Optimum soil moisture (range 20 to 60 %)


must be there for better population and activity of microbes in
soil.

Seasonal

changes

in

soil

temperature

affect

microbial

population and their activity. The organic matter in soil being


the

chief

source

of

energy

and

food

for

most

of

the

soil

organisms, it has great influence on the microbial population.


Organic

matter

influence

directly

or

indirectly

on

the

population and activity of soil microorganisms. It influences


the structure and texture of soil and thereby activity of the
microorganisms.

Ecological

relationships

among

soil

organisms

are influenced by soil structure. Changes in resource locations


may allow for microorganisms to colonize another area (Sylvia,
Fuhrmann, Hartel, & Zuberer, 2005).
Bacteria are usually the most abundant group in soils in
terms of numbers. The genus Bacillus is very common in soil.
Bacillus,

Gram

positive,

aerobic

or

facultative

endospore

forming motile bacteria belongs to family Bacillaceae (Claus and


Berkeley 1986). Isolates H3, L1, and L4 reflect the cellular
characteristics of the Genus Bacillus. Thus, it may be implied
that these isolates belong to this genus. Bacillus can resist
and survive in a variety of environmental stresses and adverse

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55

conditions and considered as very important microbiota due to


its diverse ecophysiology, direct and indirect functions such as
N2

fixer

(Liu

et

al.,

2006),

denitrifiers

(Suharti

et

al.,

2004), antibiotic (Bacon and Hinton, 2002), and phytohormones


(Araujo et al., 2005) producers.
In

general,

the

soil

bacterial

isolates

exhibited

an

antimicrobial activity after 24 hours of incubation. Most of the


zone of inhibition became even larger after 48 and 72 hours of
incubation. It can then be said that this study is likewise
comparable to the study of Das et al. (2006) that the soil
bacterial

isolates

may

be

producers

of

novel

bioactive

compounds. Various antimicrobial substances from soil bacterial


isolates

have

been

aminoglycosides,
macrolides,

isolated

anthracyclines,

nucleosides,

polyketides,

and

characterized

glycopeptides,

peptides,

actionomycins,

and

betalactams,

polyenes,

tetracyclines

including

polyester,

(PrashithKekuda,

Shobha, Onkarappa, Goutham & Raghavendra, 2012). Most of the


antibiotics

are

extracellular

metabolites

which

are

normally

secreted in culture media and have been used as herbicides,


anticancer
drugs

agents,

(Kekuda,

gram-positive

drugs,

Shobha,
rods

&

grow

immunoregulators
Onkarappa,
as

2010).

filaments,

and

antiparasitic

Actinomycetes

branching

rods,

are
and

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56

diphtheroidal rods (Bowden, 1996). One of the soil bacterial


isolates,

I1

Actinomycetes

has
as

almost

parallel

gram-positive,

characteristics

filamentous

and

to

diplobacilli.

Thus, I1 could probably be an Actinomycete isolate.


Bacillus spp. are considered to be the safe microorganisms
that hold remarkable abilities for synthesizing a vast array of
beneficial
(Stein,

substances

2005). They

that
can

may

have

produce

IAA,

antibacterial

property

siderophore,

phytase,

organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate


oxidase,

and

solubilized

various

sources

of

organic

and

inorganic phosphates as well as potassium and zinc.


According

to

Sharma

et

al.

(2001),

the

ability

of

microorganisms to produce enzymes that may have antibacterial


properties

is

influenced

by

environmental

conditions

such

as

temperature, pH, and presence of inductors or repressors.


According to Hoagstron (2011), a common statement of the
competitive exclusion principle is as follows: species whose use
of resources is very similar cannot live in the same place for
an extended period of time. That is, if two or more species eat
the same things, use the same hiding places, occupy the same
habitats,

etc.

one

species

will

be

more

efficient

than

the

others and will fill the niche with its offspring leaving no

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57

resources for other species. In this way, the more efficient


species will competitively exclude the less efficient species.

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Chapter 5
Summary, Conclusions, Implications, and Recommendations
Chapter Five consists of four parts: (1) Summary of the
Findings, (2) Conclusions, (3) Implications, and (4)
Recommendations.
Part One, Summary of the findings; state the salient points
of the study.
Part Two, Conclusions, presents the interferences drawn
from the results of the study.
Part Three, Implications, clarifies the relationships
between the present investigation and the theories presented in
relation to the antibacterial screening of soil bacterial
isolates from Sinapsapan, Jordan, Guimaras against Escherichia
coli and Staphylococcus aureus.
Part Four, Recommendations, offer some suggestions in terms
of the findings, conclusions, and recommendations.

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Summary of the Problem


This study was designed to screen and determine the
antibacterial effects of soil bacterial isolates in three
sampling sites of Brgy. Sinapsapan, Jordan, Guimaras against S.
aureus and E. coli.
This study sought to answer the following questions:
1.

How many bacterial isolates are obtained in the three

sampling sites of Brgy. Sinapsapan, Jordan, Guimaras?


2.

What are the characteristics (colonial morphology and

gram staining reaction) of bacterial isolates that have


antibacterial properties against S. aureus and E. coli?
3.

What are the effects of the bacterial isolates on S.

aureus and E. coli after 72 hours of incubation?


4.

Are there significant differences on the zone of

inhibition of bacterial isolates in the three sampling sites


against S. aureus and E. coli?
Unexplored and untouched site in Brgy. Sinapsapan, Jordan,
Guimaras was selected and the soil sampling was done in three
different areas namely: high, intermediate and low. Experimental
method of investigation was employed to screen the antibacterial
properties of the different bacterial isolates from the sampling
site. Isolation and purification of the bacterial isolates were

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60

done using serial dilution, spread plate method, and streak


plate method. Characterization was done based on colonial
morphology and Gram staining for cellular morphology and
staining properties. Agar disc diffusion method was used to
assess the antibacterial properties of the isolates against S.
aureus and E. coli after 24 to 72 hours of incubation. Each was
done in three replicates and three trials.
For the antibacterial activity, the zone of inhibition was
measured in millimeter using a 30cm ruler. The zone of
inhibition was analyzed using ANOVA with repeated measures
together with the positive and the negative controls.

Conclusions
Based on the experimental study, the results showed the
following:
1. There were 6 soil bacterial isolates obtained in the
high, intermediate, and low sampling sites in Brgy. Sinapsapan,
Jordan, Guimaras.
2. Based on the characterization of the bacterial isolates
in Brgy. Sinapsapan, Jordan, Guimaras, the shared

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characteristics of the isolates in the three sampling sites are


elevation, appearance, pigmentation, and texture wherein they
are all flat, dull, no pigmentation and smooth, respectively.
The only characteristic that differs is the arrangement of
isolate 1 in the intermediate area which is diplobacilli while
the rest are either clustered or occur singly. Isolates 1 and 5
were the most effective soil bacterial isolate against S. aureus
and E. coli, respectively. This is may be due to the fact that
they can degrade and inhibit bacterial growth due to their
secretion of digestive enzymes.
3. After 72 hours of incubation, all bacterial isolates
exhibited variations on their antibacterial effect against S.
aureus and E. coli. For S. aureus, the following isolates had
the widest zone of inhibitions after 72 hours of incubation: in
the highland sampling site, isolate 4; in the intermediate
sampling site, isolate 2; and lowland sampling site, isolate 1.
For E. coli, the following isolates had the widest zone of
inhibitions after 72 hours of incubation: in the highland
sampling site, isolate 4; in the intermediate sampling site,
isolate 6; and lowland sampling site, isolate 5.
4. There was a significant difference on the zone of
inhibition of the different bacterial isolates in the three

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sampling sites due to varying antibacterial effects of the


isolates against S. aureus and E. coli. This may be due to
differences on the amount of substrates produced by the
different bacterial isolates which inhibit the growth of the
test pathogens, S. aureus and E. coli.

Implications
This study was conducted to screen the antibacterial
properties of soil bacterial isolates from Sinapsapan, Jordan,
Guimaras against S. aureus and E. coli. The findings have shown
that different isolates are more effective on S. aureus than on
E. coli. It is therefore possible for the isolates to be
considered as a prospective source of antibiotic against on S.
aureus than E. coli. Searching for new possible source of
antibiotics is necessary because it becomes a manner for
bacteria to be resistant to certain antibiotics. Antibiotic
resistant bacteria are increasing nowadays in number that makes
the treatment for some diseases to be difficult.
Bacteria become resistant more quickly when antibiotics are
used improperly. Since there are kinds of bacteria that are

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resistant to antibiotics and are untreatable, there is a greater


must to search for more possible sources of antibiotics like
from unexplored soil sources in the nearby locality.
As the number of antibiotic resistant bacteria increases,
the quantity of antibiotics they are resistant to also rises.
According to Collignon (2000), these bacteria are known as
superbugs. They have adapted to resist multiple antibiotics
including the last-line drugs. Different strains of bacteria
have become superbugs, even the bodies normal micro flora such
as S. aureus and E. coli are on the list. Due to resistance of
these bacteria to combinations of antibiotics, infection caused
by these multi-resistant bacteria becomes unstoppable. Thus, it
is imperative to screen more and more bacteria from different
soil samples for antimicrobial activity in hope of getting some
bacterial strains that produce antibiotics that have not been
discovered yet and active against drug resistant pathogens.
Since there were six isolates that were obtained from the
sampling sites, it could therefore be implied that there are
more isolates that could be obtained from the area that have
antimicrobial properties since the area is vast and due to

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limited sampling sites and time of sampling, the researchers


were not able to maximize the soil sample collection.
Since most of the characteristics shared by the bacterial
isolates were almost all the same but one isolate (Isolate 1)
from the intermediate site which is diplobacilli, it can be
implied that there might be more other isolates that have
different characteristics which might be a more effective
isolate against E. coli and S. aureus. This may also implied
that further molecular characterization of the different
bacterial isolates to differentiate the properties among the
isolate.

Recommendations
Based on the conclusions of the study, the following are
recommended:
1. The study was limited only on the characterization of
the colonies and gram staining technique, it is further
recommended to do special staining methods such as flagellar
staining to determine the arrangement of the flagella, endospore
staining to determine whether the isolates are spore-former and

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the position of their spores, capsule staining to determine


whether the isolates are virulent or not, and negative staining
to reveal the morphology of the cell.
2. The study did not determine the genera or the species of
the isolates with antibacterial properties, it is recommended to
do biochemical tests to identify the genus of each isolate and
confirm using Bergeys Manual of Systematic Bacteriology for the
identification of the isolate. Furthermore, molecular
characterization of the isolates to reveal the identity of each
species of the isolate is also suggested. Phylogenetic analysis
should be done to determine the group of each isolate. This will
also help to determine if the bacterial isolates that were used
in this study are newly discovered or not.
3. The study was limited only on the antibacterial
screening of soil bacterial isolates on S. aureus and E. coli.
It is recommended that other pathogenic bacterial strains and
fungi be tested against this bacterial isolates to determine
their range of effectivity.
4. For further study, other microbiologist are advised to
explore more on the site for more bacterial isolates screening

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that have potential antibacterial properties on pathogenic


bacteria.
5. Microbiologists whose interest is on antibioticproducing bacteria are suggested to search further for isolates
in the same location but in different area as the unexplored
area is still vast.
6. The study was limited only to the use of modified agar
disc diffusion method to test the antibacterial potential of the
different isolates. It is recommended to use other antimicrobial
assays or techniques in the confirmation of the antibacterial
properties of the said isolates.

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References
Alanis A.J. (2005), Resistance to Antibiotics: Are We in the
Post-Antibiotic Era? Archives Med Research ,36, 697-705.
Araujo, F.F., Henning, A.A. and Hungria, M. (2005).
Phytohormones and antibiotics produced by Bacillus
subtilis and their effects on seed pathogenic fungi and on
soybean root development. World J. Microb. Biot., 21:
1639-1645.
Bacon, C.W. and Hinton, D.M. (2002). Endophytic and biological
control potential of Bacillus mojavensis and related
species. Biol. Control., 23: 274-284.
Boirivant, M. and Strober, W. (2007). The mechanism of action of
probiotics.

Retrieved

from

http://journals.1ww.com/co-

gastroenterology/Abstract/2007/11000/The_mechanism_of_actio
n_of_probiotics.16.aspx
Brooks, G., Carroll, K.C., Butel , J., Morse, S. and Mietzner,
T. (2010). Jawetz, Melnick & Adelbergs Medical
Microbiology, 25th Edition, USA: McGraw Hill Companies.
Bowden, G.H.W. (1996). Actinomyces, Propionibacterium
propionicus, and Streptomyces. In: Baron S, editor. Medical
Microbiology. 4th edition. Galveston (TX): University of
Texas Medical Branch at Galveston; Chapter 34. Retrieved

West Visayas State University


COLLEGE OF ARTS AND SCIENCES
Iloilo City

68

from: http://www.ncbi.nlm.nih.gov/books/NBK8385
Claus, D. and Berkeley, R.C.W. (1986). Genus Bacillus Cohn 1872,
174AL. In: Bergeys Manual of Systematic Bacteriology, 2,
1105-1139.
Collignon, P. (2000). Millenium Bugs. Retrieved from:
http://www. abc.net.au.com.htm
Das, S., Lyla, P. S. and Ajmal Khan, S. (2006). Marine microbial
diversity and ecology: present status and future
perspectives. Current Science,90(10): 1325-1335.
Department of Health (2009). National health data dictionary
Retrieved from:http://uhmis1.doh.gov.ph/standards/sdetails.
php?data_element=BARANGAY
Dong H., Gao, S., Han, S. & Cao, S. (1999). Purification and
characterization

of

a Pseudomonas sp.

lipase

and

its

properties in non-aqueous media, Biotechnology and Applied


Biochemistry, vol. 30, no. 3, pp. 251256.
Dotan,

I.

and

Rachmilewitz,

D.

(2005).

Probiotics

in

inflammatory bowel disease: possible mechanisms of action.


Retrieved

from:http://journals.lww.com/co-gastroenterology/

Abstract/2005/07000/Probiotics_in_inflammatory_bowel_diseas
e__possible.7.aspx.

West Visayas State University


COLLEGE OF ARTS AND SCIENCES
Iloilo City

Enright,

M.C.

(2003).

Staphylococcus

Structure

aureus in

of

the

complex

MecI

with

69

repressor

the

from

cognate

DNA

operator of mec Curr Opinion Pharmacol, 3, 5, 474- 479.


Greg R. and Percy W. (2005). Soil Bacteria. State of New
South Wales Department of Primary Industries.
Harley, J.P. (2005). Laboratory exercises in Microbiology,
(6thed.) Boston: McGraw Hill Higher Education.
Hoagstrom, C. (2011). Competitive exclusion principle. Retrieved
from: http://www.eoearth.org/view/article/151404
Ingham,

E.R.

(2011).

Soil

Biology.

Retrieved

from:

http://soils.usda.gov/sqi/concepts/soil_biology/soil_food_w
eb.html
Kekuda, T.R.P., Shobha, K.S., & Onkarappa, R.(2010). Fascinating
diversity and potent biological activities of Actinomycete
metabolites. Journal of Pharmacy Research 3(2): 250-

256.

Liu, X., Zhao, H. and Chen, S. (2006). Colonization of maize and


rice plants by strain Bacillus megaterium C4. Curr.
Microbio., 52: 186-190.
Lyczak, J.B., Cannon, C.L. & Pier G.B. (2000). Establishment of
Pseudomonas aeruginosa infection: lessons from a versatile
opportunist, Microbes and Infection 2, 9, 1051-1060.

West Visayas State University


COLLEGE OF ARTS AND SCIENCES
Iloilo City

70

Madigan, M. T., J.M., Stahl, D.A. & Clark, D.P. (2012). Block
biology of microorganism, (13thed.) Boston: Benjamin
Cummings.
Martin, J.P., (1976). Soil Microbiology and Biochemistry.
SSSA; Madison, WI.
Mozo, M.V. (2008). Bacteria. Retrieved from: http://www. biology
-online.org/dictionary/Bacteria
Oskay, M., Tamer, A.U. & Azeri, C. (2004). Isolation of soil
Streptomyces as source antibiotics active against
Antibiotic-resistant bacteria. African J Biotechnol, 3
(9), 441-446.
OToole, G.H., Kaplan, H.B., & Kolter, R. (2000).
Biofilm formation as microbial development. Retrieved
from: http://medicalmycology.org/biofilms.htm.
Pela ez, F. (2006). The historical delivery of
antibiotics from microbial natural productsCan
history repeat?. Biochemical Pharmacology, 71, 981990.
Pommier,

Y.,

Leo,

topoisomerases

E.,
and

Zhang,
their

H.,

Marchand,

poisoning

by

antibacterial drugs" Chem. Biol 17: 421-433.

C.

(2010).

DNA

anticancer

and

West Visayas State University


COLLEGE OF ARTS AND SCIENCES
Iloilo City

Prashith

Kekuda,

S.A.,

T.R.,Shobha,

Raghavendra,

H.L.

K.S.,

Onkarappa,

(2012).

71

R.,

Screening

Goutham,
biological

activities of a Streptomyces species isolated from soil of


Agumbe, Karnataka, India. Int. J. Drug Dev. & Res. 4 (3):
104-114.
Prescott, L.M., Harley, J.P. & Klein, D.A.(1993). Microbiology.
2nd ed. Wm. C. Brown Communications, Inc. USA.
Quinto, E. A. & Santos, M. A. G., (2005). A guidebook to
plant screening : Phytochemical and biological,
Microbiology section. Espaa, Manila; University of
Santo Tomas Publishing House.
Reid, G. & Wong, P. (2005). Soil bacteria, State of New South
Wales Department of Primary Industries.
Roche F. (2003), The Staphylococcus aureus surface protein
SasG. Microbiology ,149, 27592767.
Saiman L., Chen, Y., Gabriel P.S., & Knirsch C. (2002).
Synergistic activities of macrolide antibiotic against
Pseudomonas aeruginosa, Burkholderia cepacia,
Stenotrophomonas maltophilia, and Alcaligenes
xylosoxidans isolated from patients with cystic
fibrosis. Antimicrobial Agents and Chemotherapy 46, 4,
1105-1107.

West Visayas State University


COLLEGE OF ARTS AND SCIENCES
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72

Sharma, R., Chisti, Y., & Banerjee, U.C. (2001). Production,


purification,

characterization,

and

applications

of

lipases, Biotechnology Advances, vol. 19, no. 8, pp. 627


662.
Stein,

T.

(2005).

syntheses

Bacillus

and

subtilis

specific

antibiotics:

functions,

structures,

MolMicrobiol,

56

(4)(2005), pp 1669-1679.
Stephanie,

A.,

Breznak,

E.J.

A.

&

Schmidt,

Isolation and characterization of soil


define

'

Terriglobis

'

gen.

T.

M.

(2007).

bacteria

nov.,

in

the

that
Phyllum

'Acidobacteria' Appl. And Env. Microbiology Vol. 73, No. 8,


p.2708-2717.
Suharti, Heering, H.A. and de Vries, S. (2004). NO Reductase
from Bacillus azotoformans is a bifunctional enzyme
accepting electrons from menaquinol and a specific
endogenous membrane-bound cytochrome c551. Biochem.,
43: 13487-13495.
Sylvia, D. M., Fuhrmann, J. F., Hartel, P. G., & D.A Zuberer.
2005). Principles and applications of Soil Microbiology.
New Jersey, Pearson Education Inc.

West Visayas State University


COLLEGE OF ARTS AND SCIENCES
Iloilo City

Talaro,

K.P.

(2008).

Foundations

in

73

Microbiology,

(7thed.)

Boston: Mc Graw Hill Co.


Todar, K. (2008). An Introduction to Bacteriology:

University

of Wisconsin Inc.
Tortora, G.J., Funke, B.R., & Case, C.L. (2010). Microbiology:
an introduction, 10th ed. Pearson Education, Inc. USA.
Williams S.T., Sharmeemullah, M., Watson E.T., & Mayfield
C.I. (1972). Screening of Actinomycete Isolates from Niche
Habitats in Manipur for Antibiotic Activity. Retrieved
from: thescipub.com/pdf/10.3844/ajbbsp.2009.221.225.

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Appendix A
Sampling Site Map

74

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Appendix B
Characteristics of Bacterial Isolates in the High Area Sampling
Site of Brgy. Sinapsapan, Jordan Guimaras.

Figure 8.1. Colonial (left) and cell morphology


(right;600X magnification) of isolate 1.

Figure 8.2. Colonial (left) and cell morphology (right;


600X magnification) of isolate 2.

Figure 8.3. Colonial (left) and cell morphology (right;


600X magnification) of isolate 3.

Figure 8.4. Colonial (left) and cell morphology (right;


600X magnification) of isolate 4.

Figure 8.5. Colonial (left) and cell morphology (right;


600X magnification) of isolate 5.

Figure 8.6. Colonial (left) and cell morphology (right;


600X magnification) of isolate 6.

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Appendix C
Characteristics of Bacterial Isolates in the Intermediate Area
Sampling Site of Brgy. Sinapsapan, Jordan Guimaras.

Figure 9.1. Colonial (left) and cell morphology (right;


600X magnification) of isolate 1.

Figure 9.2. Colonial(left) and cell morphology (right;


600X magnification) of isolate 2.

Figure 9.3. Colonial (left) and cell morphology (right;


600X magnification) of isolate 3.

Figure 9.4. Colonial (left) and cell morphology (right;


600X magnification) of isolate 4.

Figure 9.5. Colonial (left) and cell morphology (right;


600X magnification) of isolate 5.

Figure 9.6. Colonial (left) and cell morphology (right;


600X magnification) of isolate 6.

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Appendix D
Characteristics of Bacterial Isolates in the Low Area Sampling
Site of Brgy. Sinapsapan, Jordan Guimaras.

Figure 10.1. Colonial (left) and cell morphology (right;


600X magnification) of isolate 1.

Figure 10.2. Colonial (left) and cell morphology (right;


600X magnification) of isolate 2.

Figure 10.3. Colonial (left) and cell morphology (right;


600X magnification) of isolate 3.

Figure 10.4. Colonial (left) and cell morphology (right;


600X magnification) of isolate 4.

Figure 10.5. Colonial (left) and cell morphology (right;


600X magnification) of isolate 5.

Figure 10.6. Colonial (left) and cell morphology (right;


600X magnification) of isolate 6.

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Appendix E
Antibacterial Data Sheet
Sampling Site: ____HIGH______ Test Pathogen:____E. coli______

78

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Antibacterial Data Sheet


Sampling Site: ___INTERMEDIATE____ Test Pathogen:___E.coli______

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Antibacterial Data Sheet


Sampling Site: __LOW______ Test Pathogen: ____E.coli____________

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Antibacterial Data Sheet


Sampling Site: ___HIGH___ Test Pathogen: _____S. aureus________

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Antibacterial Data Sheet


Sampling Site: _____INTERMEDIATE___ Test Pathogen: ___S. aureus_

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Antibacterial Data Sheet


Sampling Site: _____LOW____ Test Pathogen: ___S. aureus_

83

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Appendix F
Least Significant Difference for Staphylococcus aureus Multiple
Comparisons
95% Confidence Interval
(I) Group
Isolate 1

(J) Group
Isolate 2
Isolate 3

Mean Difference
(I-J)
-.2067

Std. Error
.91919

Sig.
.823

Lower
Bound
-2.0675

Upper
Bound
1.6541

-1.2574

.91919

.179

-3.1182

.6034

Isolate 4

-1.3422

.91919

.152

-3.2030

.5186

Isolate 5

-.5404

.91919

.560

-2.4012

1.3204

Isolate 6

1.6200

.91919

.086

-.2408

3.4808

-18.4530(*)

1.29993

.000

-21.0845

-15.8214

.2067

.91919

.823

-1.6541

2.0675

Isolate 4
Isolate 5

-1.0507
-1.1356
-.3337

.91919
.91919
.91919

.260
.224
.719

-2.9115
-2.9964
-2.1945

.8101
.7252
1.5271

Isolate 6

1.8267

.91919

.054

-.0341

3.6875

-18.2463(*)

1.29993

.000

-20.8779

-15.6147

Isolate 1

1.2574

.91919

.179

-.6034

Isolate 2

1.0507

.91919

.260

-.8101

2.9115

Isolate 4

-.0848

.91919

.927

-1.9456

1.7760

Isolate 5

.7170
2.8774(*)

.91919
.91919

.440
.003

-1.1438
1.0166

2.5778
4.7382

-17.1956(*)

1.29993

.000

-19.8271

-14.5640

Isolate 1

1.3422

.91919

.152

-.5186

Isolate 2

1.1356

.91919

.224

-.7252

2.9964

Isolate 3

.0848

.91919

.927

-1.7760

1.9456

Isolate 5

.8019

.91919

.388

-1.0589

2.6627

Isolate 6

2.9622(*)

.91919

.003

1.1014

4.8230

-17.1107(*)

1.29993

.000

-19.7423

-14.4792

Isolate 2
Isolate 3

Positive
Control
Isolate 3

Isolate 6
Positive
Control
Isolate 4

Positive
Control
Isolate 5

Positive
Control
Isolate 1

Isolate 1

.5404

.91919

.560

-1.3204

Isolate 2

.3337

.91919

.719

-1.5271

2.1945

Isolate 3

-.7170

.91919

.440

-2.5778

1.1438

Isolate 4

-.8019

.91919

.388

-2.6627

1.0589

Isolate 6

2.1604(*)

.91919

.024

.2996

4.0212

-17.9126(*)

1.29993

.000

-20.5442

-15.2810

Isolate 1

-1.6200

.91919

.086

-3.4808

-1.8267

.91919

.054

-3.6875

.0341

Positive
Control
Isolate 6
Isolate 2
Isolate 3

-2.8774(*)

.91919

.003

-4.7382

-1.0166

Isolate 4

-2.9622(*)

.91919

.003

-4.8230

-1.1014

Isolate 5

-2.1604(*)

.91919

.024

-4.0212

-.2996

-20.0730(*)

1.29993

.000

-22.7045

-17.4414

Positive
Control
Positive
Control
Isolate 2

Isolate 1

18.4530(*)

1.29993

.000

15.8214

18.2463(*)

1.29993

.000

15.6147

20.8779

Isolate 3

17.1956(*)

1.29993

.000

14.5640

19.8271

Isolate 4

17.1107(*)

1.29993

.000

14.4792

19.7423

17.9126(*)

1.29993

.000

15.2810

20.5442

20.0730(*)

1.29993

.000

17.4414

22.7045

Isolate 5
Isolate 6

3.1182

3.2030

2.4012

.2408

21.0845

Based on observed means.


* The mean difference is significant at the .05 level

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85

Appendix G
Least Significant Difference for Escherichia coli Multiple
Comparisons
95% Confidence Interval

(I) Group
Isolate 1

(J) Group
Isolate 2
isolate 3

Mean
Difference
(I-J)
.0863

Std.
Error
.80336

Sig.
.915

Lower Bound
-1.5400

Upper Bound
1.7126

-.7004

.80336

.389

-2.3267

.9260

isolate 4

-.7833

.80336

.336

-2.4097

.8430

isolate 5

.8626

.80336

.290

-.7637

2.4889

isolate 6

.9619

.80336

.239

-.6645

2.5882

Antibiotic

-4.3559(*)

1.13613

.000

-6.6559

-2.0560

Isolate 2

Isolate 1

-.0863

.80336

.915

-1.7126

1.5400

isolate 3

-.7867
-.8696

.80336
.80336

.334
.286

-2.4130
-2.4960

.8397
.7567

isolate 4
isolate 5

.7763

.80336

.340

-.8500

2.4026

isolate 6

.8756

.80336

.283

-.7508

2.5019

Antibiotic

1.13613

.000

-6.7422

-2.1423

isolate 3

-4.4422(*)
Isolate 1

.7004

.80336

.389

-.9260

Isolate 2

.7867

.80336

.334

-.8397

2.4130

isolate 4

-.0830

.80336

.918

-1.7093

1.5434

isolate 5

.80336
.80336
1.13613

.059
.045
.003

-.0634
.0359
-5.9555

3.1893
3.2885
-1.3556

isolate 4

1.5630
1.6622(*)
-3.6556(*)
Isolate 1

.7833

.80336

.336

-.8430

Isolate 2

.8696

.80336

.286

-.7567

2.4960

isolate 3

.0830

.80336

.918

-1.5434

1.7093

isolate 5

1.6459(*)

.80336

.047

.0196

3.2723

isolate 6

1.7452(*)

.80336

.036

.1189

3.3715

Antibiotic

-3.5726(*)

1.13613

.003

-5.8726

-1.2726

isolate 5
Isolate 2

Isolate 1
-.7763

-.8626
.80336

.80336
.340

.290
-2.4026

-2.4889
.8500

isolate 3

-1.5630

.80336

.059

-3.1893

.0634

isolate 4

-1.6459(*)

.80336

.047

-3.2723

-.0196

isolate 6

.0993

.80336

.902

-1.5271

1.7256

Antibiotic

-5.2185(*)
Isolate 1

1.13613

.000

-7.5185

-2.9185

-.9619

.80336

.239

-2.5882

isolate 3
isolate 4

-.8756
-1.6622(*)
-1.7452(*)

.80336
.80336
.80336

.283
.045
.036

-2.5019
-3.2885
-3.3715

.7508
-.0359
-.1189

isolate 5

-.0993

.80336

.902

-1.7256

1.5271

Antibiotic

-5.3178(*)

1.13613

.000

-7.6177

-3.0178

Antibiotic

Isolate 1

4.3559(*)

1.13613

.000

2.0560

Isolate 2

4.4422(*)

1.13613

.000

2.1423

6.7422

isolate 3

3.6556(*)

1.13613

.003

1.3556

5.9555

isolate 4

3.5726(*)
5.2185(*)
5.3178(*)

1.13613
1.13613
1.13613

.003
.000
.000

1.2726
2.9185
3.0178

5.8726
7.5185
7.6177

isolate 6
Antibiotic

isolate 6
Isolate 2

isolate 5
isolate 6

2.3267

2.4097

.7637

.6645

6.6559

Based on observed means.


* The mean difference is significant at the .05 level.

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86

Appendix H
Zone of inhibition data of S. aureus
Number of
trials and its

24 HOURS

48 HOURS

72 HOURS

corresponding

(mm)

(mm)

(mm)

replicates
1-1

52

55

59

1-2

31

38

38

1-3

20

61

74

2-1

37

50

55

2-2

22

48

51

2-3

20

48

53

3-1

25

62

65

3-2

25

35

43

3-3

25

50

54

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87

Appendix I
Zone of Inhibition data of E.coli
Number of
trials and its

24 HOURS

48 HOURS

72 HOURS

corresponding

(mm)

(mm)

(mm)

replicates
1-1

25

32

38

1-2

20

26

33

1-3

27

35

46

2-1

20

22

35

2-2

19

24

36

2-3

26

31

41

3-1

21

24

29

3-2

18

42

42

3-3

24

37

30

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Appendix J
Descriptive Statistics for Staphylococcus aureus
Zone of Inhibition: High Land

88

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Zone of Inhibition: Intermediate

89

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Zone of Inhibition: lowland

90

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Appendix K
Descriptive Statistics for Escherichia coli
Zone of Inhibition: High Land

91

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Zone of Inhibition: Intermediate

92

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Zone of Inhibition: lowland

93

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Appendix L
Picture of Zone of inhibition

94

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95

Appendix M
Letter to UPLB Biotech
April 8, 2014
DR. REYNALDO V. EBORA
BIOTECH Director
PNCM-BIOTECH, UPLB
Los Banos, Laguna 4031
Dr. Ebora:
Good day Sir!
I would like to request for the purchase of activated cultures
of Staphylococcus aureus and Escherichia coli that will be used
by my undergraduate advisees, Ma. Sophia Estella C. Tajolosa and
Kristine Joy R. Estrella for their antibacterial research study.
Aside from my supervision, our Biology department has also a
registered microbiologist to guide and assist them in handling
these bacteria. Our microbiology lab is also provided with the
necessary equipment and gadgets in order to qualify and fit for
a Biosafety level 2 organisms manipulation.
Attached herewith are the accomplished forms needed for our
request and for your perusal. I look forward for your favorable
acceptance of my request for the success of my students
undergraduate research study as part of their chosen career
path. Thank you and more power.
Very yours truly,
REY G. TANTIADO
Biological Science Instructor and Research Adviser
Bio. Sci., CAS, West Visayas State University

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96

Appendix N
Letter of Permission
April 29, 2014
MR. AUDIE A. SULADAY
Head
Central Laboratory Section
West Visayas State University
Dear Mr. Suladay:
Greetings!
We, Ma. Sophia Estella C. Tajolosa and Kristine Joy R. Estrella,
incoming fourth year students of BS Biology will be conducting a research
experiment as a partial fulfillment of the requirement in our course subject,
Thesis Writing in Biological Sciences (BIO 230A).
In line with this, we would like to request the following chemicals and
laboratory apparatus for our thesis entitled Antibacterial Screening of Soil
Bacterial Isolates from Sinapsapan, Jordan Guimaras against Escherichia coli
and Staphyloccocus aureus this May 2014 at RH 104.
Attached hereof is the list of the following apparatus and chemicals
that we are to use in the said study.
We are looking forward for the granting of our request.
Thank you very much.
Respectfully Yours,
______________________________
MA. SOPHIA ESTELLA C. TAJOLOSA
Researcher
_____________________________
KRISTINE JOY R. ESTRELLA
Researcher
Noted by:
______________________
PROF. REY G. TANTIADO
Thesis Adviser
_____________________
DR. NANCY S. SURMIEDA
Dean, College of Arts and Sciences
_______________________
MR. AUDIE A. SULADAY
In Charge, Central Science Laboratory

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97

April 29, 2014


MR. RUBEN C. ITABAG
Chief Administrative Officer
West Visayas State University
Sir:
Greetings!
We, Ma. Sophia Estella C. Tajolosa and Kristine Joy R. Estrella,
incoming fourth year students of BS Biology will be conducting a research
experiment entitled Antibacterial Screening of Soil Bacterial Isolates from
Sinapsapan, Jordan Guimaras against Escherichia coli and Staphyloccocus
aureus.
In view of this, we are asking permission to use the facilities of the
the Rizal Hall Room 104 from May 2014 October 2014 from 7:30 am to 5:00 pm.
We are hoping for your kind approval regarding our request.
Thank you very much.
Respectfully Yours,
______________________________
MA. SOPHIA ESTELLA C. TAJOLOSA
Researcher
_____________________________
KRISTINE JOY R. ESTRELLA
Researcher
Noted by:
_______________________
PROF. REY G. TANTIADO
Thesis Adviser
Endorsed By:
_______________________
DR. NANCY S. SURMIEDA
Dean, College of Arts and Sciences
_______________________
Romeo Y. Solano
GSO Supervisor
Approved by:
____________________
MR. RUBEN C. ITABAG
Chief Administrative Officer

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Apparatus and Materials:


(10) 20 ml Test tube
(1) Test tube rack
(2) 500 ml Erlenmeyer flask
(2) 250 ml Erlenmeyer flask
(15) Petri dish
(3) Spatula
(5) Stirring rod
(1) Electric Weighing balance
(1) Autoclave Machine
(1) Incubator
(3) Alcohol Lamp
(3) 100 ml Beaker
(3) 250 ml Beaker
(1) Colony Counter
(1) 100 ml Graduated Cylinder
(1) 25 ml Graduated Cylinder
(15) Pipette
Chemicals:
Nutrient agar
Nutrient broth
Mc.Farland 0.5
Normal Saline Solution
Mineral oil
Safranin
Grams iodine
70% Alcohol

98

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Appendix O: Soil Sample Collected

Appendix P: Pure Culture of Escherichia coli Biotech 1634

99

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Appendix Q: Pure Culture of Staphylococcus aureus Biotech 1582

Appendix R: Prepared Nutrient Agar and NSS

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Appendix S: Preparation of Nutrient Agar Plates

Appendix T: Labelling of Agar Plates

101

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Appendix U: Agar Slant Preparation

Appendix V: Serial Dilution of Soil Isolates

102

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Appendix W: Soil Bacterial Isolates in Preparation for


Incubation

Appendix X: Incubation

103

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Appendix Y: Recorded Characteristics of Soil Bacterial Isolates

Appendix Z: Stains used in Grams Staining

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Appendix A1: Gram staining of Bacterial Isolates with


Antibacterial Properties

Appendix A2: Stained Slides for Microscopic Observation

105

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RESEARCH CERTIFICATION
This is to certify that KRISTINE JOY R. ESTRELLA and MA.
SOPHIA ESTELLA C. TAJOLOSA have submitted to me their manuscript
entitiled ANTIBACTERIAL SCREENING OF SOIL BACTERIAL ISOLATES
FROM SINAPSAPAN, JORDAN, GUIMARAS AGAINST Escherichia coli
AND Staphylococcus aureus.
I have examined the same and found it in order.

REY G. TANTIADO Ph. D.


Research Adviser

JEANNEMAR GENEVIEVE YAP-FIGUERAS M. Sci.


Department Editor

PRENCY C. YERRO M. A.
Student Research Coordinator

GERARD L. PENECILLA Ed. D.


Department Chair

NANCY S. SURMIEDA Ph. D.


Dean

________________
Date

________________
Date

________________
Date

________________
Date

________________
Date