ANTIMICROBIAL ACTIVITY AND SYNERGISTIC EFFECTS OF PURE BEE

HONEY AND PROPOLIS PRODUCED BY Apis Mellifera (EUROPEAN

HONEYBEES)

____________________

An Undergraduate Thesis
Presented to the Faculty of the
COLLEGE OF NURSING
Mindanao State University - Iligan Institute of Technology

____________________

In Partial Fulfillment
Of the Requirements for the Degree
BACHELOR OF SCIENCE IN NURSING

____________________

MENDOZA, EUNICE C.
RESTOR, DENN ANTHONY CHRISTIAN A.
YAP, MARK ALLEN J.
ZAPANTA, TRIXIA JOY N.

____________________

March 2013

Chapter I
THE PROBLEM AND ITS SCOPE

Introduction
Honey and other bee products has been subjected to laboratory and clinical
investigations during the past few decades and the most remarkable discovery was
their antibacterial activity. Honey has been used since ancient times for the treatment
of some diseases and for the healing of wounds but its use as an anti-infective agent
was superseded by modern dressings and antibiotic therapy. However, the emergence
of antibiotic resistant strains of bacteria has confounded the current use of antibiotic
therapy leading to the re-examination of former remedies. Honey, bee pollen, bee
propolis, royal jelly, and bee venom have a strong antibacterial activity. Even
antibiotic-resistant strains such as epidemic strains of methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) have
been found to be as sensitive to honey as the antibiotic-sensitive strains of the same
species. Sensitivity of bacteria to bee products varies considerably within the product
and the varieties of the same product. Botanical origin plays a major role in its
antibacterial activity. Propolis has been found to have the strongest action against
bacteria. This is probably due to its richness in flavonoids. The most challenging
problems of using hive products for medical purposes are dosage and safety. Honey
and royal jelly produced as a food often are not well filtered, and may contain various
particles. Processed for use in wound care, they are passed through fine filters which
remove most of the pollen and other impurities to prevent allergies. Also, although
honey does not allow vegetative bacteria to survive, it does contain viable spores,

including clostridia. With the increased availability of licensed medical drugs

containing bee products, clinical use is expected to increase and further evidence will
become available. Their use in professional care centres should be limited to those
which are safe and with certified antibacterial activities. Antibiotic-resistant bacteria
have become a worrying global public health issue. Antimicrobial resistance not only
threatens to increase the cost of health care and jeopardise healthcare gains to society,
but it may even damage trade and impact the economy (WHO, 2012). Especially here
in the Philippines. We are a 3rd-world country and many people rather cling to the use
of herbal medicines and other therapeutic plant products, because of the lack of access
to treatment regimens and programs due to financial contraints. Moreover, because
our country is rich in folklore, especially with Mindanao and its diverse cultures. The
urgency for pursuing this research study is focused with the community and that the
results may be a promising form of awareness and could be a benchmark for future
studies concerning the use of herbal medicines for various health benefits, and other
plant products of the like.

Objectives of the Study

The researchers aim to determine the capacity of the antimicrobial activity of
pure bee honey and propolis produced by the genus Apis Mellifera (European
Honeybees), and whether it can perform an important role in fungicidal action and
bacteria-growth inhibition. Moreover, this study seeks to:
1. Determine if there is a significant difference between the zones of inhibition
produced by the extract with different concentrations and that of the negative and
positive controls at 0.05 significance level.

2. Examine whether the antibacterial aspect of the honey and propolis is

bacteriostatic or bactericidal in nature.
3. Hypothesize the possible biologically active components found in honey
and propolis that may be responsible for its antimicrobial activity.

Significance of the Study

To the Researchers: This study will enable the researchers to apply the
theories learned in nursing research and microbiology thus helping them hone their
skills. The findings of the study could be a means to improve the basic knowledge
concerning the use of bee products as an alternative and conventional treatment for
certain infections. Thus, providing awareness and recommendations to different
health care providers, as well as to those people who cant afford advanced treatment.

To the Nursing Profession: They will be given more knowledge on the proper
applications in using these bee products as an alternative and conventional treatment
of certain types of infection. They will be able to learn some techniques or actions on
how to at least alleviate the other symptoms associated by infections. Nursing
students will somehow be ready in using this honey with their health teaching if
someday these products will become licensed and indicated for treatment of certain
conditions.

To the Community: This study will give them knowledge in using honey as an
alternative treatment for certain infections and other ailments of the body. Moreover,
this could help them minimize their expenses with complying to their respective
treatment regimens.

To the Future Researchers: This study will serve as guide and source of
information to the future researchers thus making their paper more accurate and
efficient.

Scope and Limitation of the Study

The study utilized pure bee honey and propolis that were used in the
antimicrobial activity testing and evaluation. The samples were acquired from the
local bee farm, Spiritans Apiary, situated at Pindugangan, Tipanoy, Iligan City and
produced by the genus Apis Mellifera (European Honeybees). Sterile technique was
used during the collection process, with the aid of the apiarist, and homogeneity of the
honey and propolis were ensured by thorough agitation at the time of extraction from
the combs. The honey and propolis samples were stored in the dark at 5C in
polyethylene buckets with tight-fitting lids during the transportation from the farm to
the school; and from there on, all the procedures were done only at the Microbiology
and Parasitology Laboratory (NR9), of the College of Nursing. The researchers made
sure that the samples were indeed handled aspetically from the collection process until
the testing and evaluation of antimicrobial activity; thus, preventing any errors or
alterations with the results. Test specimens were not acquired from a bodily wound;

but rather from fresh preserves in the culturing laboratory (GL2) at the Department of
Biological Sciences, College of Science and Mathematics. Crucial tests that were
done are the Ethanolic Extraction of Propolis (EEP), Mueller-Hinton (MH) and
Sabouraud Dextrose (SD) Agar Plating and the Kirby-Bauer Disk Diffusion Assay.
The emphasis on this experiment was to examine whether honey and propolis could
be a promising conventional and alternative medicine for antibiotics in the treatment
and management of infections caused by the bacteria: Escherichia coli,
Staphylococcus aureus, Bacillus subtilis, and the fungi: Apergillus niger,
Saccharomyces cerevisiae; and whether it offers other contributory benefits to the
field of medicine and to human health. This study is limited only to the testing and
evaluation of the antimicrobial capacity of honey with propolis and does not touch
humans as the subjects, only the five identified specimens. Also, it does not concern
the actual intake/application of the substance and also its positive and negative effects
on the human anatomy.

Definition of Terms
The following are operational terms defined by the researchers to ensure clearer
understanding and appreciation of the study:
Aerobic- A living organism or occurring only in the presence of oxygen.
Agar- A gelatinous material derived from certain marine algae and is used as a base
for bacterial culture media and as a stabilizer and thickener in many food products.
Anaerobic- An organism that can live in the absence of atmospheric oxygen.
Antimicrobial- is an agent that kills microorganisms or inhibits their growth.

Antibiotic- are used to treat infections caused by bacteria.

Antibacterial- is an agent that either kills bacteria or inhibits their growth.
Antifungal- the agent that inhibits or limits the growth of fungi.
Aseptic Technique- refers to a procedure that is performed under sterile conditions.
Bacteria- a very large group or domain of Prokaryotic microorganism.
E. coli- is a Gram-negative, facultative anaerobic, rod-shaped bacterium that is
commonly found in the Gastrointestinal tracts.
Facultative- Capable of functioning under varying environmental conditions. Used of
certain organisms, such as bacteria that can live with or without oxygen.
Fungi- a taxonomic kingdom, or in some classification schemes a division of the
kingdom Plantae, comprising all the fungus groups and sometimes also the slime
molds.
Germs- is the earliest form of an organism; a seed, bud, or spore.
Glucose Oxidase- A monosaccharide sugar, C6H12O6, occurring widely in most plant
and animal tissue. It is the principal circulating sugar in the blood and the major
energy source of the body.
Gram-Positive- designating bacteria that retain the violet stain in Gram's method
Gram-Negative- designating bacteria that fail to retain the violet stain in Gram's
method.
Honey- a sweet, sticky, yellowish-brown fluid made by bees out from collecting
nectars from flowers in their surroundings.

Hydrogen Peroxide- A colorless, strongly oxidizing liquid, used as a mild antiseptic,

an oxidizing agent, and a laboratory reagent.
Infection- invasion by and multiplication of pathogenic microorganisms in a bodily
part or tissue, that may produce subsequent tissue injury and progress to disease.
Inoculum- The material used in an inoculation. Also called inoculant.
in vitro- Outside the living organism
in vivo- Within a living organism
Microbe- is a minute life form, a single cell or a multicellular organism especially a
bacterium that causes disease.
Minimum inhibitory concentration (MIC)- is the lowest concentration of an
antimicrobial that will inhibit the visible growth of a microorganism after overnight
incubation
Obligate- Capable of functioning under varying environmental conditions. Used of
certain organisms, such as bacteria that can live with or without oxygen.
Photo-degradation- Capable of being chemically broken down by light.
Phytochemicals- A nonnutritive bioactive plant substance, such as a flavonoid or
carotenoid, considered to have a beneficial effect on human health. Also called
phytonutrient.
Zone of Inhibition (ZOI)- The clear region around the paper disc saturated with an
antimicrobial agent on the agar surface

Chapter II
REVIEW OF RELATED LITERATURE

A. THE SUBSTANCE
A.1 Honey
PNRI Develops Wound Dressing From Honey
The Philippine Nuclear Research Institute Department of Science and
Technology (PNRI-DOST) has developed an effective wound dressing from local
honey sources in the Philippines.
Science research specialists from PNRIs Biomedical Research Section are
taking advantage of the antimicrobial properties of these local products to produce a
cheaper and comparable alternative if not a better one already to antibiotics for
treating exudating wounds and burns.
Honey has, since ancient days, been used for medicinal purposes; its
composition makes it a very effective agent for healing wounds, said Biomedical
Research Section head Zenaida De Guzman.
According to Ms. De Guzman, honey is ideal as a wound dressing not only for
its antimicrobial and potentially anti-inflammatory composition, but also for its low
pH level that is suitable for fast healing.
Its sugar content helps in the granulation of wounds, while its low moisture
gives honey a longer shelf-life. Furthermore, honeys low water activity helps the
dressing draw out water and pus, thereby drying the wound and reducing the chances

of infection.
Among the samples obtained from the University of the Philippines Los
Baos, three indigenous types of honey stood out: the pineapple flower honey from
Bacolod which proved comparable to the average antibiotic, the scarce coconut honey
from Mindanao and the natural dark honey found in the highlands of Northern Luzon,
both of which matched and at times even bested antibiotics in dealing with pathogens
such as Staphylococus aureus.
As they are readily available, these honey samples provided the material for
the research sections honey dressings.
Results from initial testing in rabbits showed that the dressing healed the
wounds around the same time as the generic Neomycin; in some cases, the honey
treatment was a day ahead of that with the antibiotic.
Pre-clinical testing conducted in a government hospital showed that with the
Honey dressing, full treatment of a burn patient was achieved earlier by a month than
the usual healing time.
Sodium alginate made from brown algae, already used by hospitals for
dressings, serves as a base for the honey. They are mixed and molded into a gauze
before being sprayed with calcium chloride to bind them.
After being cured, dried and packaged in vacuum-packed aluminum foil, the
dressing is irradiated at 25 kilogray at PNRIs Multipurpose Irradiation Facility to
keep it microbe-free and longer-lasting.
The Biomedical Research Section applied for the honey dressings patent last
year and hopes to eventually finish the clinical tests. Ms. De Guzman expects the

products commercialization to begin by 2015.1

A.2 Propolis
Ilog Maria Bee Propolis
Ilog Maria Bee Propolis is nature's most powerful antibiotic. Propolis is an
essential part of the immune system of trees. It is a sticky resinous substance secreted
by flowering buds and under the bark of trees. Our honeybees collect propolis to seal,
varnish and, thus, disinfect their honeycombs and insides of their beehives. Ilog Maria
Bee Propolis possesses antiviral, antibacterial and fungicidal properties. It is even
capable of destroying some bacteria & viruses that have now become resistant to
modern synthetic antibiotics. Ilog Maria Bee Propolis is effective against infections of
the skin, mouth, throat, respiratory and digestive systems. Ilog Maria Bee Propolis
stimulates tissue regeneration, antibody formation and strengthens & stimulates the
immune system. It is also effective in treating hypertension, arteriosclerosis and
coronary disease. Dermatological disorders and rheumatism respond to propolis
therapy. Ilog Maria Bee Propolis is a potent anti-oxidant, which combats the ill effects
of eating processed and preserved foods.2

B. THE PHYTOCHEMICALS
1

http://www.pnri.dost.gov.ph/ by the Philippine Nuclear Research Institute

Department of Science and Technology (Wednesday, 19 February 2014)
2

http://www.ilogmaria.com/aboutus/benefits-of-bee-products/ by the Ilog Maria

Honeybee Farm (Page 3-4)

B.1 Flavonoids
B.2 Galangin
B.3 Hydroxycinnamic acids
B.4 Caffeic acid

C. THE TEST ORGANISMS

C.1 Escherichia coli
Enterohemorrhagic E. coli (EHEC) Diarrhea. This disease consists of a
hemorrhagic, watery diarrhea with abdominal cramping. Usually, patients have no
fever or only a slight fever. About 5% of infected people (especially children younger
than age 5 and the elderly) develop hemolytic-uremic syndrome (HUS), with anemia,
low platelet count, and kidney failure. The first recognized outbreak of diarrhea
caused by enetrohemorrhagic E. coli (0157:H7) occured in 1982, involving
contaminated hamburger meat - hamburger meat contaminated with cattle feces.
Since then, several well-publicized epidemics involving the same serotype have
occured. Not all of the outbreaks involved meat; some resulted from ingestion of
unpasteurized milk or apple juice, lettuce, or other raw vegetables. It has been
estimated that E. coli 0157:H7 infection accounts for as many as 73,000 cases of
illness and 60 deaths in the United States per year.
Pathogens. E. coli 0157:H7 (a serotype that possesses a cell wall antigen
designated 0157 and a flagerllar antigen designated H7) is the most commonly
involved EHEC serotype. Other EHEC serotypes include 026:H11, 0111:H8, and

0104:H21. These are all Gram-negative bacilli that produce potent cytotoxins called
Shiga-like toxins - named for their close resemblance to Shiga toxin, produced by
Shigella dysenteriae.
Reservoirs and Mode of Transmission. Reservoirs include cattle and
infected humans. Transmission occurs via the fecal-oral route; inadequately cooked,
fecally-contaminated beef; unpasteurized milk; person-to-person contact; or fecallycontaminated water.
Laboratory Diagnosis. E. coli 0157:H7 infection should be suspected in any
patient with bloody diarrhea. Stool specimens should be inoculated onto sorbitolMacConkey (SMAC) agar. Colorless, sorbitol-negative colonies, should then be
assayed for 0157 antigen using commercially available antiserum. Other
immunodiagnostic procedures are available.
Patient Care. Use Standard Precautions for hospitalized patients. Add
Contact Precaution for diapered or incontinent patients.

Enterotoxigenic E. coli (ETEC) Diarrhea (Travelers Diarrhea). This

disease consists of watery diarrhea with or without mucus or blood, vomiting, and
abdominal cramping. Dehydration and low-grade fever may occur. Enterotoxigenic
strains of E. coli are the most common cause of travelers diarrhea worldwide and a
common cause diarrheal disease in children in developing countries.
Pathogen. ETEC diarrhea is caused by many different serotypes of
enterotoxigenic E. Coli that produce a heat-labile toxin, a heat-stable toxin, or both.
Reservoir and Mode of Transmission. Infected humans serve as reservoirs.

Transmission occurs via the fecal-oral route; ingestion of fecally contaminated food
or water.
Laboratory Diagnosis. ETEC diarrhea is diagnosed by isolation of the
oragnism from stool specimens, followed by demonstration of enterotoxin production,
molecular diagnostic procedures, or immunodiagnostic procedures.
Patient Care. Use Standard Precautions for hospitalized patients. Add
Contact Precautions for diapered and incontinent patients.3

C.2 Staphylococcus aureus

Staphylococcal Skin Infections (Follicutlitis, Furuncles, Carbuncles,
Abscesses, Impetigo, Impetigo of the Newborn, Scalded Skin Syndrome).
Virtually all infected hair follicles, boils (furuncles), carbuncles, and stys involve
Staphylococcus aureus. The majority of common skin lesions are localized, discrete,
and uncomplicated. However, seeding of the bloodtsream may lead to pnewumonia,
lung abscess, osteomyelitis, sepsis, endocarditis, meningitis, or brain abscess With
impetigo, which occurs mainly in children, pus-filled blisters (pustules) may appear
anywhere on the body. Impetigo of the newborn (impetigo neonatorum) and
staphylococcal scalded skin syndrome (SSSS) may occur as epidemics in hospital
nurseries.
Pathogen. Most staphylococcal infections (staph infections) are caused by
S. aureus, a Gram-positive coccus. Impetigo may also be caused by Streptococcus

BURTONS MICROBIOLOGY FOR THE HEALTH SCIENCES 9th Edition; by Paul

G. Engelkirk; Section VIII: Major Infectious DIseases of Humans; Chapter 19:
Bacterial Infections; Table 19-8: Enterovirulent Escherichia coli (Page 338)

pyogenes, which is a nother Gram-positive coccus. S. Aureus spreads through skin by

producing hyaluronidase. SSSS is produced by strains of S. aureus that produce
exfoliative (or epidermolytic) toxin, which causes the top layer of skin (epidermis) o
split from the rest of the skin.
Reservoirs and Mode of Transmission. Infected humans serve as reservoirs.
Persons with draining lesion or any purulent discharge are the most common sources
of epidemic spread. Transmission occurs via direct contact with a person having a
purulent lesion or is an aysmptomatic carrier. In hospitals, staphylococcal infections
can be spread by the hands of healthcare workers.
Laboratory Diagnosis. The infecting strain must be isolated on culture media
and identified using a variety of phenotypic characteristics, including reactions in
biochemical- or enzyme-based tests. Susceptibility testing must be performed because
many strains of S. aureus are multidrug resistant.
Patient Care. Use Standard Precautions for skin, burn, and wound infections
if they are minor or limited, and Contact Precautions if they are major. Use Contact
Precautions for patients with SSSS. Use Standard Precautions for infections caused by
methicillin-resistant S. aureus (MRSA); add Contact Precautions if wounds cannot be
contained by dressings. Use Contact Precautions for diapered or incontinent children
with enterocolitis (staph food poisoning), for the duration of illness.4

Otitis Externa (External Otitis, Ear Canal Infection, Swimmers Ear).

Otitis externa is an infection of he outer ear canal with itching, pain, a malodorous
4

BURTONS MICROBIOLOGY FOR THE HEALTH SCIENCES 9th Edition; Section

VIII: Major Infectious DIseases of Humans; Chapter 19: Bacterial Infections; Table
19-2: Bacterial Infections of the Skin (continued) (Page 326)

discharge, tenderness, redness, swelling, and impaired hearing. Otitis externa is most
common during he summer swimming season; trapped water in the external ear canal
can lead to wet, softened skin, which is more easily infected by bacteria or fungi.
Otitis externa is referred to as swimmers ear because it often results from
swimming in water contaminated with Pseudomonas aeruginosa.
Pathogens. The usual causes of otitis externa are the bacteria Escheria coli, P.
aeruginosa, Proteus vulgaris, and Staphylococcus aureus. Fungi, such as Aspergillus
spp. Are less common causes of otitis externa.
Reservoirs and Mode of Transmission. Reservoirs include contaminated
swimming pool water, sometimes indigenous microflora, or articles inserted into the
ear canal for cleansing out debris and wax.
Laboratory Diagnosis. Material from the infected ear canal should be sent to
the microbiology laboratory for culture and susceptibility (C&S). Most strains of P.
aeruginosa are multidrug resistant.5

C.3 Bacillus subtilis

C.4 Aspergillus niger
C.5 Sacchaomyces cerevisiae

D. THE DRUGS
5

BURTONS MICROBIOLOGY FOR THE HEALTH SCIENCES 9th Edition; Section

VIII: Major Infectious DIseases of Humans; Chapter 19: Bacterial Infections; Table
19-3: Viral and Bacterial Ear Infections (Page 328)

D.1 Antibiotic
Antibiotics are drugs used to treat bacterial infections. Unfortunately, an
increasing number of bacteria are developing resistance to currently available
antibiotics. The resistance develops in part because of the overuse of the antibiotics.
Therefore, new antibiotics are constantly being developed to combat increasingly
resistant bacteria. Eventually though, the bacteria will also become resistant to the
newer antibiotics.
Antibiotics are classified on the basis of their strength. Bactericidal antibiotics
actually kill bacteria; bacteriostatic antibiotics merely prevent them from multiplying,
allowing the body to eliminate the remaining bacteria. For most infections, the two
types of antibiotic seem equally effective, but if the immune system is impaired or the
person has a severe infection, such as a bacterial endocarditis or meningitis, a
bactericidal anibiotic usually is more effective.6

D.2 Anti-fungal
Antifungal drugs may be applied directly to a fungal infecion of the skin or
other surface, such as the vagina or the inside of the mouth. Antifungal drugs may
also be taken orally or injected.
Generally, antifungal drugs cause more side effects than do antibiotics.
Antifungal drugs also are generally less effective, so fungal infections are difficult to
treat and often become long-lasting (chronic). Antifungal therapy often lasts for

THE MERCK MANUAL OF MEDICAL INFORMATION Home Edition; Chapter 173:

Anti-infective Drugs (Page 926)

weeks and must be repeated.7

Chapter III
METHODOLOGY

Research Design
The study entitled Antimicrobial Activity and Syngergistic Effects of Honey
and Propolis produced by Apis Mellifera (European Honeybees) is an experimental
research that aims to determine the capacity of the antimicrobial activity of honey and
whether it can perform an important role in fungicidal action and bacteria-growth
inhibition. Special procedures that are crucial in the study are the Ethanolic Extract of
Propolis (EEP), Mueller-Hinton (MH) and Sabouraud-Dextrose (SD) Agar Plating,
and the Kirby-Bauer Disk Diffusion Susceptibility Test, which is known best for
determining the sensitivity or resistance of pathogenic aerobic and facultative
7

THE MERCK MANUAL OF MEDICAL INFORMATION Home Edition; Chapter 173:

Anti-infective Drugs (Page 931)

anaerobic

bacteria,

to

various

antimicrobial

compounds

used

by

many

pharmaceuticals and laboratories in order to assist physicians in selecting treatment

options for their patients.

This study is a true experimental research because the researchers consider the
four fundamental properties of an experimental research and the study is to be
conducted in a laboratory. Also, because the researchers follow a standard protocol to
prevent bias in any factor and ensure accuracy and consistensy with the results. Pure
bee honey and propolis were decided as the subject to be proven as claims about these
substances for treatment of different kinds of ailments and infection has been long
known, but up until now there are minimal laboratory testings or evidences for its
medicinal properties. Quantitative methods were employed to determine specific
results necessary for generating the desired research outputs.

Locale of the Study

Figure 1. Location of MSU-IIT, CSM

First, the collection of the honey and propolis samples were done at the local
bee farm Spiritans Apiary, situated at Pindugangan, Tipanoy, Iligan City. Next, the
conditioning of the extract was done at the Microbiology and Parasitology Laboratory
(NR9), in the College of Nursing of Mindanao State University - Iligan Institute of
Technology. Lastly, the antimicrobial activity testing was done at the GL2 of the
College of Science and Mathematics, with the supervision of Prof. Mark Anthony
Jose, a microbiologist and faculty member of the Department of Biological Sciences.

Preparation of the Extract

A. Collection of Honey and Propolis samples

The samples were acquired from the local bee farm, Spiritans
Apiary, situated at Pindugangan, Tipanoy, Iligan City and produced by the genus
Apis Mellifera (European Honeybees). Sterile technique was used during the
collection process, with the aid of the apiarist, and homogeneity of the honey and
propolis were ensured by thorough agitation at the time of extraction from the combs.
The honey and propolis samples were stored in the dark at 5C in polyethylene
buckets with tight-fitting lids during the transportation from the farm to the school.
Samples of honey and propolis for testing were handled aseptically to avoid any
occurence of contamination, and protected from bright light to prevent
photodegradation of the glucose oxidase that gives rise to hydrogen peroxide in
honey.

B. Conditioning of the extract

Thereafter, the propolis samples were cut into small pieces, transferred
into a beaker, mixed with 50ml of pure honey, heated in low heat, stirred constantly
and allowed to meld. Next, the now liquid mixture of the sample was transferred into
a new beaker after being cooled down. It was then added with a technical grade
ethanol about 150ml, covered with foil to avoid evaporation, and soaked at a dark
room for 48 hours. After that, the mixture was then poured and filtered using a filter
paper into an erlenmeyer flask. This step was repeated again and again until the fluid
becomes clear and all residue be filtered to prepare the sample for Rotary
Evaporation. And then, the now clear fluid had undergone the process of Rotary
Evaporation (ROTAVAP) until only the substance remained. Afterwards, the extract

was poured in petri plates on minute amounts, just to fill up the base of the plates and
were set out to be air-dried for about 48 hours. The now air-dried extracts were then
scraped, weighed using an analytical electronic balance, and put into vials with
labeled concentrations marked 1,000 ml, 750ml, 500ml, and 250ml, respectively.
Each of the vials were then mixed with the technical grade ethanol for dilutions,
measured an amount of 10ml using a graduated cylinder. Constant shaking of the vials
for a couple of minutes was done to ensure that the extract blended with the ethanol.
After that, concentrations of 100mg/ml, 75mg/ml, 50mg/ml, and 25mg/ml were now
obtained.

Kirby-Bauer Disk Diffusion Susceptibility Test Protocol

A. Preparation of research instruments
A.1 Materials
Antibiotic disk
18- to 24-hour old pure culture of the organism to be tested
Wickerham card
Mueller-Hinton agar plates, 100 mm or 150 mm
Caliper or ruler
Forceps
Sterile swabs
Inoculating loop or needle

Bact-cinerator or Bunsen burner

Alcohol pads or isopropyl alcohol in a tube
A.2 Equipments
Vortex mixer
35C to 37C non-CO2 incubator
A.3 Chemicals
Barium sulfate standard
Sterile saline in 2-ml tubes
0.5 McFarland standard

B. Use of the McFarland Standard

Add a 0.5-ml aliquot of a 0.048 mol/liter BaCl2 (1.175% wt/vol BaCl2
2H20) to 99.5 ml of 0.18 mol/liter H2SO4 (1% vol/vol) with constant stirring to
maintain a suspension. Verify the correct density of the turbidity standard by
measuring absorbance using a spectrophotometer with a 1-cm light path and matched
cuvette. The absorbance at 625 nm should be 0.08 to 0.13 for the 0.5 McFarland
standard. Transfer the barium sulfate suspension in 4- to 6-ml aliquots into screw-cap
tubes of the same size as those used in standardizing the bacterial inoculums. Tightly
seal the tubes and store in the dark at room temperature. Prior to use, vigorously
agitate the barium sulfate standard on a mechanical vortex mixer and inspect for a
uniformly turbid appearance. Replace the standard if large particles appear. If using

a standard composed of latex particles, mix by inverting gently, not on a vortex mixer.
As the student adds bacterial colonies to the saline in the preparation of the
inoculum step of the procedure, he or she should compare the resulting suspension to
the McFarland standard. This is done by holding both the standard and the inoculum
tube side by side and no more than 1 inch from the face of the Wickerham card (with
adequate light present) and comparing the appearance of the lines through both
suspensions. Do not hold the tubes flush against the card. If the bacterial suspension
appears lighter than the 0.5 McFarland standard, more organisms should be added to
the tube from the culture plate.

If the suspension appears denser than the 0.5

McFarland standard, additional saline should be added to the inoculum tube in order
to dilute the suspension to the appropriate density. In some cases it may be easier to
start over rather than to continue to dilute a bacterial suspension that is too dense for
use.

C. Preparation of Mueller-Hinton Agar plate

Allow a MH agar plate (one for each organism to be tested) to come to
room temperature. It is preferable to allow the plates to remain in the plastic sleeve
while they warm to minimize condensation. If the surface of the agar has visible
liquid present, set the plate inverted, ajar on its lid to allow the excess liquid to drain
from the agar surface and evaporate. Plates may be placed in a 35C incubator or in a
laminar flow hood at room temperature until dry (usually 10 to 30 minutes).
Appropriately label each MH agar plate for each organism to be tested.

D. Preparation of inoculums
Use a sterile inoculating loop or needle to touch four or five isolated
colonies of the organism to be tested. Suspend the organism in 2 ml of sterile saline.
Vortex the saline tube to create a smooth suspension. Adjust the turbidity of this
suspension to a 0.5 McFarland standard by adding more organism if the suspension is
too light or diluting with sterile saline if the suspension is too heavy. Use this
suspension within 15 minutes of preparation.

E. Inoculation of MHA plate

Dip a sterile swab into the inoculum tube. Rotate the swab against the
side of the tube (above the fluid level) using firm pressure, to remove excess fluid.
The swab should not be dripping wet. Inoculate the dried surface of a MH agar plate
by streaking the swab three times over the entire agar surface; rotate the plate
approximately 60 degrees each time to ensure an even distribution of the inoculum.
Rim the plate with the swab to pick up any excess liquid. Discard the swab into an
appropriate container. Leaving the lid slightly ajar, allow the plate to sit at room
temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the
agar plate to dry before proceeding to the next step.

F. Placement of the antibiotic disks

Place the appropriate antimicrobial-impregnated disks on the surface of
the agar, using either forceps to dispense each antimicrobial disk one at a time, or a
multidisk dispenser to dispense multiple disks at one time. To use a multidisk

dispenser, place the inoculated MH agar plate on a flat surface and remove the lid.
Place the dispenser over the agar plate and firmly press the plunger once to dispense
the disks onto the surface of the plate. Lift the dispenser off the plate and using
forceps sterilized by either cleaning them with an alcohol pad or flaming them with
isopropyl alcohol, touch each disk on the plate to ensure complete contact with the
agar surface.

This should be done before replacing the Petri dish lid as static

electricity may cause the disks to relocate themselves on the agar surface or adhere to
the lid. Do not move a disk once it has contacted the agar surface even if the disk is
not in the proper location, because some of the drug begins to diffuse immediately
upon contact with the agar. To add disks one at a time to the agar plate using forceps,
place the MH plate on the template provided in this procedure. Sterilize the forceps by
cleaning them with a sterile alcohol pad and allowing them to air dry or immersing
the forceps in alcohol then igniting. Using the forceps carefully remove one disk from
the cartridge. Partially remove the lid of the Petri dish. Place the disk on the plate
over one of the dark spots on the template and gently press the disk with the forceps
to ensure complete contact with the agar surface. Replace the lid to minimize
exposure of the agar surface to room air. Continue to place one disk at a time onto the
agar surface until all disks have been placed as directed in steps f. and g. above. Once
all disks are in place, replace the lid, invert the plates, and place them in a 35C air
incubator for 16 to 18 hours. When testing Staphylococcus against oxacillin or
vancomycin, or Enterococcus against vancomycin, incubate for a full 24 hours before
reading.
G. Incubation of the plates
A temperature range of 35C 2C is required. Do not incubate plates
in CO2 as this will decrease the pH of the agar and result in errors due to incorrect pH

of the media. Results can be read after 18 hours of incubation.

H. Measuring of the zone sizes

Following incubation, measure the zone sizes to the nearest millimeter
using a ruler or caliper; include the diameter of the disk in the measurement. When
measuring zone diameters, always round up to the next millimeter. All measurements
are made with the unaided eye while viewing the back of the Petri dish. Hold the
plate a few inches above a black, nonreflecting surface illuminated with reflected
light. View the plate using a direct, vertical line of sight to avoid any parallax that
may result in misreading. Record the zone size on the recording sheet. If the
placement of the disk or the size of the zone does not allow you to read the diameter
of the zone, measure from the center of the disk to a point on the circumference of the
zone where a distinct edge is present (the radius) and multiply the measurement by 2
to determine the diameter. Growth up to the edge of the disk can be reported as a zone
of 0 mm. Organisms such as Proteus mirabilis, which swarm, must be measured
differently than non-swarming organisms. Ignore the thin veil of swarming and
measure the outer margin in an otherwise obvious zone of inhibition. Distinct, discrete
colonies within an obvious zone of inhibition should not be considered swarming.
These colonies are either mutant organisms that are more resistant to the drug being
tested, or the culture was not pure and they are a different organism.

If it is

determined by repeat testing that the phenomenon repeats itself, the organism must be
considered resistant to that drug.

Chapter IV
RESULTS AND DISCUSSION

The researchers have prepared antimicrobial assays for bacteria (both Grampositive and Gram-negative) and fungi. For the bacteria, the researchers represented

the Gram-positives with Staphylococcus aureus and Bacillus subtilis, while

Escherichia coli for the Gram-negatives. For the fungi, the researchers used
Aspergillus niger and Saccharomyces cerevisiae. The bacteria were cultured for 24
hours, while the fungi for 36 hours, and then applied to the sterile petri plates for the
inoculation and antimicrobial testing. The zones of inhibition were then measured by
a vernier caliper after 18 hours from the application of the impregnated Whatmann
paper discs. As you can see from Figure 1 it shows the zones of inhibition on the three
trials of the Gram-positive bacteria Staphylococcus aureus. During the measuring of
zone sizes, the researchers observed that the Whatmann disc having the greatest zone
of inhibition is the positive control which is the Streptomycin having an average zone
size of 38mm, followed by the 100% concentration with 10mm, then 75% having
8mm, then 50% having 7mm, then 25% having 6mm, and lastly the negative control
which is the distilled water that made no effect with the bacteria.

Figure 1. Zones of inhibition for the Gram-positive bacteria Staphylococcus aureus

For Figure 2, it shows the zones of inhibition on the Gram-positive Bacillus
subtilis. The results show that the positive control yielded the greatest average zone

size which is 40 mm, followed by the 100% concentration having 14 mm, then 75%
having 9 mm, then 50% having 7 mm, then 25% having 7 mm, and lastly the negative
control which is the distilled water having a zone size of 6 mm.

Figure 2. Zones of inhibition for the Gram-positive bacteria Bacillus subtilis

For Figure 3, it shows the zones of inhibition on the Gram-negative bacteria

Escherichia coli. The results show that the 100% concentration yielded the greatest
average zone size which is 14 mm, followed by the 75% concentration having 11 mm,
then 50% having 8 mm as well the positive control which is Chloramphenicol, then
25% having 7 mm, and lastly the negative control which is the distilled water having a
zone size of 6 mm.

Figure 3. Zones of inhibition for the Gram-negative bacteria Escherichia coli

For Figure 4, it shows the zones of inhibition on the fungi Aspergillus niger.
The results show that the 100% concentration yielded the greatest average zone size
which is 11 mm, followed by the positive control which is Nystatin having 9 mm,
then 75% and the 50% having 8 mm, then 25% having 7 mm, and lastly the negative
control which is the distilled water having a zone size of 6 mm.

Figure 4. Zones of inhibition for the fungi Aspergillus niger

For Figure 5, it shows the zones of inhibition on the fungi Saccharomyces

cerevisiae. The results show that the 100% concentration yielded the greatest average
zone size which is 10 mm, followed by the 75% concnetration having 8 mm, then the
50%, 25% and positive control having 7 mm and lastly the negative control which is
the distilled water having a zone size of 6 mm.

Figure 5. Zones of inhibition for the fungi Saccharomyces cerevisiae

To determine the degree of differences among the zones of inhibition of the

four concentrations along with the positive and negative controls, the researchers
made use of the analysis of variance (ANOVA). Table 2 shows the ANOVA results on
the differences of the zones of inhibitions in different concentrations of honey and
propolis extract produced by the genus Apis mellifera. Significant relationship can be
found among various concentrations in terms of their zone sizes. Table 2 includes
the sum of squares between groups (between concentration samples) and that of
within groups (within a concentration sample). The obtained p-value is 0.007 at 0.05
significance level.
Table 1. One way ANOVA results of the zone of inhibition on the diffirent
concentrations of honey and propolis extract of Apis mellifera as well as the negative

and positive controls.

Source of
Variation
Between
Groups
Within
Groups

0.2776
0.3179
2

24

Total

0.5955
2

29

SS

df

MS
5

0.05552
0.01324
7

F
4.19124
3

P-value
0.00703
5

F crit
2.62065
4

Chapter V
SUMMARY OF FINDINGS, CONCLUSION, AND RECOMMENDATIONS
Conclusion

The use of honey in modern wound care is still met with some scepticism.
Since the advent of evidence-based medicine, changing clinical practice depends on
providing clinicians with appropriate levels of evidence of clinical efficacy. Although
honey has become a first-line intervention in some wound care clinics, larger and
better designed randomized controlled trials (RCT) are needed to cement the role of
honey in modern wound care. Medical devices (such as wound dressings) are not
required to demonstrate the same level of evidence in order to become licensed for
use, but high levels of evidence should be aimed for, and will widen use. However,
carrying out meaningful RCTs is difficult in complex and chronic wounds.
In the context of the continued emergence of antibiotic-resistant pathogens,
some alternative or ''traditional" topical antimicrobials have been reintroduced into
modern wound care, one such example being honey. While a range evidence is
available for the use of honey in wound management, definitive RCTs remain to be
undertaken.

Based on the obtained results, the following conclusions were made:

1. Propolis prevents the growth of the microorganisms in single and mixed
microbial cultures, and has synergistic effect when used with honey and/or ethyl
alcohol;
2. The antimicrobial property of propolis varies with geographical origin; and
3. This study will pave the way to isolate bioactive ingredients from honey
and propolis to be further tested individually or in combination against human
resistant infections.

Recommendations
For further advancement of the study, the researchers would recommend the
following:
1. Compare and contrast the antimicrobial activity of honey and propolis
produced by Apis Mellifera (European Honeybees) from other different kinds of bee
genus.
2. Include the use of Phytochemical Screening to examine various biologically
active compounds and chemicals found in the honey and propolis extract.
3. Include the use of Pollen Analysis for examination of pollen samples in the
honey to determine its vegetation history.
4. Include the use of Thin-Layer Chromatography (TLC) to utilize the
capillary action of a solvent for separation and determination of the various
compounds found in the extract.
5. Include the beeswax and bee pollen as samples.
6. Separate the antimicrobial testing of the honey from the propolis; but with
the same amount and number of concentrations.
7. Determine the Minimum Inhibitory Concentration (MIC) of the extract with
different levels of concentration, as well as both the positive and negative controls.
8. Increase the number of plates per test organism.
9. Measure the Zone of Inhibition (ZOI) after 24 hours.

10. Determine the toxicity and therapeutic levels of the extract through
Cytotoxic and Genotoxic assay.