“ANTIMICROBIAL ACTIVITY AND SYNERGISTIC EFFECTS OF PURE BEE HONEY AND PROPOLIS PRODUCED BY Apis Mellifera (EUROPEAN

HONEYBEES)”

____________________

An Undergraduate ThesisPresented to the Faculty of the

COLLEGE OF NURSINGMindanao State University - Iligan Institute of Technology

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In Partial FulfillmentOf the Requirements for the Degree

BACHELOR OF SCIENCE IN NURSING

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MENDOZA, EUNICE C.RESTOR, DENN ANTHONY CHRISTIAN A.

YAP, MARK ALLEN J.ZAPANTA, TRIXIA JOY N.

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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was superseded by modern dressings and antibiotic therapy.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 can’t 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 5°C 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 PNRI’s 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, honey’s 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

Baños, 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 section’s 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 PNRI’s Multipurpose Irradiation Facility to

keep it microbe-free and longer-lasting.

The Biomedical Research Section applied for the honey dressing’s patent last

year and hopes to eventually finish the clinical tests. Ms. De Guzman expects the

product’s 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

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. THE PHYTOCHEMICALS

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 fecally-

contaminated water.

Laboratory Diagnosis. E. coli 0157:H7 infection should be suspected in any

patient with bloody diarrhea. Stool specimens should be inoculated onto sorbitol-

MacConkey (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 (Traveler’s 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 traveler’s 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

3 BURTON’S 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)

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

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

4 BURTON’S MICROBIOLOGY FOR THE HEALTH SCIENCES 9th Edition; Section VIII: Major Infectious DIseases of Humans; Chapter 19: Bacterial Infections; Table 19-2: Bacterial

Otitis Externa (External Otitis, Ear Canal Infection, Swimmer’s Ear).

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

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 “swimmer’s 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

Infections of the Skin (continued) (Page 326)

5 BURTON’S 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)

C.5 Sacchaomyces cerevisiae

D. THE DRUGS

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.

6 THE MERCK MANUAL OF MEDICAL INFORMATION Home Edition; Chapter 173: Anti-infective Drugs (Page 926)

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

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

7 THE MERCK MANUAL OF MEDICAL INFORMATION Home Edition; Chapter 173: Anti-infective Drugs (Page 931)

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

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 5°C 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

35°C to 37°C 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 35°C 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 35°C 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 35°C ± 2°C 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 Gram-

positive 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.

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

Source of Variation SS df MS F P-value F crit

Between Groups 0.2776 5 0.05552 4.191243 0.007035 2.620654Within Groups 0.31792 24 0.013247

Total 0.59552 29

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.