chronicles owa

Chapter I

Introduction

Background of the Study

Various researches regarding effectiveness of antibiotics are being held worldwide.

Innovations to existing antibiotics are being held to improve the performance of antibiotic drugs.

To test a certain antibiotic, a researcher must perform the method antibiotic sensitivity testing.

The aforementioned test requires the use of materials like Mueller-Hinton Agar. Any test held

requires the said medium. From this method of use, a problem has arisen.

Mueller-Hinton Agar is a microbiological growth medium that is commonly used

for antibiotic susceptibility testing. A subsequent international collaborative study confirmed the

value of Mueller-Hinton Agar for this purpose because of the relatively good reproducibility of

the medium, the simplicity of its formula, and the wealth of experimental data that had been

accumulated using this medium. Mueller Hinton Agar is recognized by all experts as being the

reference medium for the study of the susceptibility of bacteria to antibiotics and sulfamides. It is

an excellent base medium for the preparation of blood agars. This medium can be used with

complete confidence because it is rich in nutrients, able to grow fastidious organisms. The use of

a medium with suitable growth characteristics is essential to test the susceptibility of

microorganisms to antibiotics.

Mueller-Hinton Agar commonly have components like beef infusion, cantine, starch and

agar. Due to economic crises, the price of components like cantine and agar soars high because

our local suppliers experience shortage. Expensive and limited components are a great hindrance

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for a research study’s success. Finding an alternative for such components will be a great help

for researchers.

A potentially good alternative for cantine and beef infusion is the beef broth cube. Broth

cubes or soup base are derived from vegetable and animal protein. Bouillon cubes are convenient

and inexpensive but have essential nutritive value. Acid in broth cubes is being neutralized

which leaves behind salts that enhance the bouillon's flavor and extend shelf-life. Extended shelf

life and inexpensiveness gives great convenience for researchers.

Another potential alternative component is the “Sansaw” or much popularly known as

gelatin. It is a translucent, colorless and, flavorless solid substance derived from collagen

obtained from various animal by-products. It is commonly used as a gelling agent in food,

pharmaceuticals, photography, and cosmetic manufacturing. One characteristic of gelatin is its

soft texture. Sansaw and agar are both gelatinous in nature, which allows efficient growth of

bacteria. Experimentally proving that sansaw is a good alternative for base agar would be a great

significance to researchers.

This study was performed to determine if a modified media with beef cubes and gelatin

can be substituted to Mueller-Hinton agar in antibiotic sensitivity testing.

Objectives

This study generally aimed to determine potential use of modified culture media with

beef broth cubes as alternative to Mueller-Hinton agar for antibiotic sensitivity testing.

Specifically, this study aimed to determine the following:

1. To determine the zone of inhibition of the four test bacteria (Escherichia coli, Proteus

vulgaris, Streptococcus mutans, Staphylococcus epidermidis) grown in two types of

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modified media (modified Mueller-Hinton Agar with beef broth cubes and modified

Mueller-Hinton Agar with beef broth cubes and “Sansaw”) after treatment of five

selected standard antibiotic disks namely: cephazolin, ciprofloxacin, clindomycin,

doxycycline and erythromycin.

2. To determine the significant difference in terms of zone of inhibition of the four test

bacteria (Escherichia coli, Proteus vulgaris, Streptococcus mutans, Staphylococcus

epidermidis) grown in two types of modified media (modified Mueller-Hinton Agar with

beef broth cubes and modified Mueller-Hinton Agar with beef broth cubes and

“Sansaw”) after treatment of five selected standard antibiotic disks namely: cephazolin,

ciprofloxacin, clindomycin, doxycycline and erythromycin in comparison with the

control group (Mueller-Hinton agar).

Hypothesis

There is no significant difference in terms of zone of inhibition of the four test bacteria

(Escherichia coli, Proteus vulgaris, Streptococcus mutans, Staphylococcus epidermidis) grown

in two types of modified media (modified Mueller-Hinton Agar with beef broth cubes and

modified Mueller-Hinton Agar with beef broth cubes and “Sansaw”) after treatment of five

selected standard antibiotic disks namely: cephazolin, ciprofloxacin, clindomycin, doxycycline

and erythromycin in comparison with the control group ( Mueller-Hinton agar).

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Conceptual Framework

Independent Variable Dependent Variable

Types of Bacteria

1. Escherichia coli

2. Proteus vulgaris

3. Streptococcus mutans

4. Staphylococcus epidermidis

Zone of Inhibition (mm)

Types of Culture Media

1. Mueller-Hinton Agar

2. Modified Mueller-Hinton

Agar with Beef Broth Cubes

3. Modified Mueller-Hinton

Agar with Beef Broth Cubes

And “Sansaw”

Types of Antibiotics

1. Cephazolin

2. Ciprofloxacin

3. Clindamycin

4. Doxycycline

5. Erythromycin

Figure 1.1: Diagram showing the relationship between the variables.

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Scope and Limitations of the Study

This study focused only on the potential use of modified culture media with beef broth

cubes as alternative to Mueller-Hinton agar for antibiotic sensitivity testing.

The experiment was conducted at the Biochemistry Laboratory of the University of

Negros Occidental-Recoletos from August 4, 2012 to August 18, 2012 under the supervision of a

licensed microbiologist.

The Randomized Complete Block Design (RCBD) was used in this study. All materials

and equipment were acquired from Biochemistry Laboratory of the University of Negros

Occidental-Recoletos. The four test bacteria used in this study are the following: Escherichia

coli, Proteus vulgaris, Streptococcus mutans, and Staphylococcus epidermidis. The three types

of media used in this study are the following: Mueller-Hinton Agar, Modified Mueller-Hinton

Agar with Knorr cubes and Modified Mueller-Hinton Agar with Knorr Cubes and “Sansaw”.

This study required the use of five antibiotics namely: Cephazolin, Ciprofloxacin, Clindomycin,

Doxycycline and Erythromycin.

The experimental groups are as follows: (Treatment A) Mueller-Hinton Agar with Broth

Cubes, (Treatment B) Modified Mueller-Hinton Agar with Broth Cubes and “Sansaw”. The

Control Group for this study is (Negative Control) Original Mueller-Hinton Agar. Each

treatment had three replicates for each type of bacteria with a total of thirty-six experimental

units.

The data and results were analyzed using the Multiple Analysis of Variance at α=0.05 to

determine the significant difference in terms of zone of inhibition of the four test bacteria grown

in two types of modified media after treatment of five selected standard antibiotic disks in

comparison with the control group.

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Definition of Terms

The following key terms were defined conceptually and operationally for better

understanding of the readers:

Antibiotic Sensitivity Testing

Conceptual- a laboratory test that determines how effective an antibiotic therapy is

against a bacterial infection (pharmagenerics.com, 2012).

Operational- The method used by the researcher to determine the zone of inhibition of

four test bacteria in three different media treated with five different antibiotics.

Broth Cubes (Beef)

Conceptual- Broth cubes or soup base are derived from vegetable protein (found in wheat

flour, maize meal, rice gluten etc.) and animal protein (Consumers Demand, 2012).

Operational- This served as substitute for cantine and beef infusion of Mueller-Hinton in

this study.

Cephazolin (CZ)

Conceptual- mainly used to treat bacterial infections of the skin. It can also be used to

treat moderately severe bacterial infections involving the lung, bone, joint, stomach,

blood, heart valve, and urinary tract (pharmagenerics.com, 2012).

Operational- One of the five antibiotics used in this study.

Ciprofloxacin (CIP)

Conceptual- is a synthetic antibiotic of the fluoroquinolone drug class. It is a second-

generation fluoroquinolone antibacterial (pharmagenerics.com, 2012). 6


Clindamycin (CD)

Conceptual- is a lincosamide antibiotic. It is usually used to

treat infections with anaerobic bacteria but can also be used to treat

some protozoa diseases, such as malaria (pharmagenerics.com, 2012).


Culture Media

Conceptual- a substance or solution used to cultivate microorganisms (Groiler.com,

2012).

Operational- the researcher used three types of media namely: Standard Mueller-Hinton

Media modified Mueller-Hinton Agar with beef broth cubes (MHB) and modified

Mueller-Hinton Agar with beef broth cubes and “Sansaw” (MHG).

Doxycycline (DXT)

Conceptual- is a member of the tetracycline antibiotics group, and is commonly used to

treat a variety of infections (pharmagenerics.com, 2012).


Erythromycin (ERY)

Conceptual- is a macrolide antibiotic that has an antimicrobial spectrum similar to or

slightly wider than that of penicillin, and is often used for people who have

an allergy to penicillin (pharmagenerics.com, 2012).

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Escherichia coli

Conceptual- are a Gram-negative, rod-shaped bacterium that is commonly found in the

lower intestine of warm-blooded organisms (Groiler.com, 2012).

Operational- One of the four different bacteria used in this study.

Mueller-Hinton Base Agar

Conceptual- is a microbiological growth medium that is commonly used for antibiotic

susceptibility testing (Groiler.com, 2012).

Operational- The standard media used by the researcher in this study.

Proteus vulgaris

Conceptual- is a rod-shaped, Gram negative bacterium that inhabits the intestinal tracts of

humans and animals. It can be found in soil, water and fecal matter (Groiler.com,

2012).


Staphylococcus epidermidis

Conceptual- is gram-positive and coagulase-negative staphylococci. Is one of 33 known

species belonging to the genus Staphylococcus (Groiler.com, 2012).


Streptococcus mutans

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Conceptual- is a Gram-positive bacterium that lives in the mouth. It can thrive in

temperature ranging from 18-40 degrees Celsius. It metabolizes different kinds of

carbohydrates, creating acidic environment in the mouth as a result of this process

(Groiler.com, 2012).


Zone of Inhibition

Conceptual -area without bacterial growth surrounding an antimicrobial-impregnated disk

in an antimicrobial sensitivity test (Groiler.com, 2012).

Operational- the parameter measured and compared by the researcher of the study.

Significance of the Study

Results and findings of this study would be beneficial to each of the following:

Department of Science and Technology (DOST): Results of this study would provide

information regarding the use of alternative materials in preparing a culture media for antibiotic

testing.

Medical Industries: The viability of the study could provide a basis for introducing new and

alternative type of Mueller Hinton Agar base.

Microbiologists: Results of this study would benefit microbiologists because they will use the

alternative medium as a substitute for the Original Mueller Hinton medium.

Researchers: Provide knowledge and alternative media for future studies regarding the use of

culture media and bacteria.

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Chapter II

Review of Related Literature

This chapter presents and describes the concepts related in the study. This includes the

information and recent studies relational to this study.

Conceptual Literature

Antibiotic Sensitivity Testing

Antibiotic sensitivity testing is a laboratory test that determines how effective an

antibiotic therapy is against a bacterial infection.

It aims to determine the susceptibility of an isolate to a range of potential therapeutic

agents. This can be with a view to individualizing the antibiotic to be administered or to monitor

resistance patterns developing in that environment, gathering this information is important for

revising and updating the standard antibiotic prescribing policy for a particular population or

institution.

Resistance to antibiotics can either be naturally occurring for a particular organism or

drug combination or acquired resistance, where mis-use of anti-microbial results in a population

being exposed to an environment in which organisms that have genes conferring resistance have

been able to flourish and spread. Identification of an organism normally goes hand in hand with

the AST test, knowing what organism you have isolated together with knowledge of the isolation

site, will give an indication of what type of antibiotics should be considered. The sensitivity of an

isolate to a particular antibiotic is measured by establishing the Minimum Inhibitory

Concentration (MIC) or breakpoint; this is the lowest concentration of antibiotic at which an

isolate cannot produce visible growth after overnight incubation.

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Broth Cubes (Beef)

Broth cubes or soup base are derived from vegetable protein (found in wheat flour, maize

meal, rice gluten etc.) and animal protein (found in tankage, fishmeal, etc.). Hydrochloric acid is

used to break down the protein into individual amino acids. Next, the acid is neutralised, leaving

behind the salts that enhance the bouillon's flavour and extend shelf-life. Today it is more

common for producers to use enzymes instead of acid to break down the protein into amino

acids.

Broth made from rehydrated cubes is different in taste from fresh broth because of its

higher salt content and flavours changed by the boiling process. Bouillon cubes are convenient

and inexpensive but have little nutritive value. The cubes are widely used in English cooking to

add flavour, particularly in soups, stews and casseroles. They often contain flavour enhancer–

either yeast extract or monosodium glutamate from other sources.

Knorr (a brand of Bouillon cubes) contains the following ingredients: Salt, Monosodium

Glutamate, Beef Fat, Cottonseed Oil Partially Hydrogenated, Yeast Extract, Caramel Colour,

Beef Stock Dehydrated, Vegetable Dried (Onion), Carrot, Parsley, Turmeric, Disodium

Inosinate, Disodium Guanylate, Spice. Based on chemical analysis of Bureau of Food and Drugs,

a 13.3 g of bouillon cube contain proteins such as Vitamin A, beta-carotene, Vitamin C, Calcium

Iron, Vitamin D, Vitamin E, Riboflavin, Niacin, Vitamin B6, Vitamin B12,Phosphorous,Iodine,

Zinc, Magnesium, Copper and Folic Acid. Based on lab results, there is indeed a great potential

for bouillon cubes as alternative in preparing culture media.

Cephazolin

Cephazolin is mainly used to treat bacterial infections of the skin. It can also be used to

treat moderately severe bacterial infections involving the lung, bone, joint, stomach, blood, heart

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valve, and urinary tract. It is clinically effective against infections caused

by staphylococci and streptococci of Gram-positive bacteria. These organisms are common on

normal human skin. Resistance to cephazolin is seen in several species of bacteria. Cephazolin is

extensively used as prophylaxis antibiotic before wide range of surgical operations.

It is also a first-generation cephalosporin antibiotic. Cephalosporin is derivatives of 7-

aminocephalosporic acid and is closely related to penicillin in structure. Cephazolin is effective

in a large variety of bacterial infections, such as respiratory tract, skin and urinary tract

infections. Antibiotics require constant drug level in body for therapeutic effect. This is achieved

by taking the medication at regular interval of time throughout the day and night as prescribed.

Ciprofloxacin

Ciprofloxacin is a synthetic antibiotic of the fluoroquinolone drug class. It is a second-

generation fluoroquinolone antibacterial. It kills bacteria by interfering with the enzymes that

cause DNA to rewind after being copied, which stops synthesis of DNA and of protein. It is used

to treat infections caused by certain bacteria. It is most commonly used to treat infections of the

skin, sinuses, bone, lung, ear, abdomen, kidney, prostate, and bladder.

Ciprofloxacin has 12 FDA-approved human uses and other veterinary uses, but it is often

used for unapproved uses. Ciprofloxacin interacts with other drugs, herbal and natural

supplements, a characteristic it shares with other widely used antibacterial drugs such as

amoxicillin, trimethoprim, azithromycin, cephalexin, and doxycycline.

Clindamycin

It is usually used to treat infections with anaerobic bacteria but can also be used to treat

some protozoa diseases, such as malaria. It is a common topical treatment for acne and can be

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useful against some methicillin-resistant Staphylococcus aureus (MRSA) infections. It is also

used to treat certain types of bacterial infections, including infections of the lungs, skin, blood,

female reproductive organs, and internal organs. Clindamycin is in a class of medications called

lincomycin antibiotics. It works by slowing or stopping the growth of bacteria. Antibiotics such

as clindamycin will not kill the viruses that cause colds, flu, and other infections.

The most severe common adverse effect of clindamycin is Clostridium difficile-

associated diarrhea. Although this side effect occurs with almost all antibiotics, including beta-

lactam antibiotics, it is classically linked to clindamycin use.

Doxycycline

Doxycycline is a member of the tetracycline antibiotics group, and is commonly used to

treat a variety of infections. Doxycycline is a semisynthetic tetracycline invented and clinically

developed in the early 1960s by Pfizer Inc. and marketed under the brand name Vibramycin.

Vibramycin received US Food and Drug Administration approval in 1967becoming Pfizer's first

once-a-day, broad-spectrum antibiotic. Other brand names include Monodox, Microdox,

Periostat, Vibra-Tabs, Oracea, Doryx, Vibrox, Adoxa, Doxyhexal, Doxylin, Doxoral, Doxy-1

and Atridox (topical doxycycline hyclate for periodontitis).

It is used in the treatment and prophylaxis of anthrax (caused by Bacillus anthracis). It is

also effective against Yersinia pestis (the infectious agent of bubonic plague), and is prescribed

for the treatment of Lyme disease, ehrlichiosis and Rocky Mountain spotted fever. In fact,

because doxycycline is one of the few medications shown to be effective in treating Rocky

Mountain spotted fever (with the next-best alternative being chloramphenicol), doxycycline is

indicated even for use in children for this illness. Otherwise, it is not indicated for use in children

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under the age of eight years. Doxycycline, like other antibiotics, will not work for colds,

influenza, or other viral infections.

Erythromycin

Erythromycin is a macrolide antibiotic that has an antimicrobial spectrum similar to or

slightly wider than that of penicillin, and is often used for people who have an allergy to

penicillin. Macrolide antibiotics slow the growth of, or sometimes kill, sensitive bacteria by

reducing the production of important proteins needed by the bacteria to survive. For respiratory

tract infections, it has better coverage of a typical organism,

including Mycoplasma and legionellosis. Erythromycin is an antibiotic used to treat certain

infections caused by bacteria, such as bronchitis; diphtheria; Legionnaires' disease; pertussis

(whooping cough); pneumonia; rheumatic fever; venereal disease (VD); and ear, intestine, lung,

urinary tract, and skin infections. It is also used before some surgery or dental work to prevent

infection. Antibiotics will not work for colds, flu, or other viral infections.

Escherichia coli

Escherichia coli are a Gram-negative, rod-shaped bacterium that is commonly found in

the lower intestine of warm-blooded organisms. Several types of E. coli exist as part of the

normal flora of the human gut and have several beneficial functions, such as the production

of vitamin K2. They also prevent harmful bacteria, known as pathogenic bacteria, from

establishing themselves in the intestine. Escherichia coli is one of the most frequent causes of

many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract

infection (UTI), and traveler's diarrhea, and other clinical infections such as neonatal

meningitis and pneumonia. The genus Escherichia is named after Theodor Escherich, who

isolated the type species of the genus. Escherichia organisms are gram-negative bacilli that exist

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singly or in pairs. E coli is facultatively anaerobic with a type of metabolism that is both

fermentative and respiratory. They are either nonmotile or motile by peritrichous flagella. E

coli is a major facultative inhabitant of the large intestine.

Figure 2.1: Photo of Escherichia coli under the microscope (photobucket.com, 2012)

Gelatin (Sansaw)

A translucent, colorless, brittle (when dry), flavorless solid substance, derived from

collagen obtained from various animal by-products. It is commonly used as a gelling agent in

food, pharmaceuticals, photography, and cosmetic manufacturing. Substances containing gelatin

or functioning in a similar way are called gelatinous. Gelatin is an irreversibly hydrolyzed form

of collagen, and is classified as a foodstuff. It is found in most gummy candies as well as other

products such as marshmallows, gelatin dessert, and some ice cream, dip and yogurt. Household

gelatin comes in the form of sheets, granules, or powder. Instant types can be added to the food

as they are; others need to be soaked in water beforehand. Gelatin forms a solution of high

viscosity in water, which sets to a gel on cooling, and its chemical composition is, in many

respects, closely similar to that of its parent collagen. Because of its viscosity, it has a high

potential for substituting agar in preparation of culture media.

Mueller-Hinton Base Agar

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Mueller-Hinton agar is a microbiological growth medium that is commonly used

for antibiotic susceptibility testing. Mueller Hinton Agar was originally developed for the

cultivation of pathogenic Neisseria. However, these organisms are now commonly isolated on

selective media. Because clinical microbiology laboratories in the early 1960s were using a wide

variety of procedures for determining the susceptibility of bacteria to antibiotic and

chemotherapeutic agents, Bauer, Kirby and others developed a standardized procedure in which

Mueller Hinton Agar was selected as the test medium.2,3. A subsequent international

collaborative study confirmed the value of Mueller Hinton Agar for this purpose because of the

relatively good reproducibility of the medium, the simplicity of its formula, and the wealth of

experimental data that had been accumulated using this medium.

Proteus vulgaris

Proteus vulgaris is a rod-shaped, Gram negative bacterium that inhabits the intestinal

tracts of humans and animals. It can be found in soil, water and fecal matter. It is grouped with

the Enterobacteriaceae and is an opportunistic pathogen of humans. It is known to cause urinary

tract infections and wound infections.

Figure 2.2: Photo of Proteus vulgaris under the microscope (photobucket.com).

Staphylococcus epidermidis

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Staphylococcus epidermidis is gram-positive and coagulase-negative staphylococci. It is

part of human skin flora (commensal), and consequently part of human flora. It can also be found

in the mucous membranes and in animals. Due to contamination, it is probably the most common

species found in laboratory tests. It typically lives on the human skin and mucosa and the most

common infections on catheters and implants. S. epidermidis is one of five most common

organisms that cause noscomial infections due to the increase in usage of biomaterials in the

clinical environment.

Figure 2.3: Photo of Staphylococcus epidermidis under the microscope (sciencephoto.com).

Streptococcus mutans

Streptococcus mutans is a Gram-positive bacterium that lives in the mouth. It can thrive

in temperature ranging from 18-40 degrees Celsius. It metabolizes different kinds of

carbohydrates, creating acidic environment in the mouth as a result of this process. This acidic

environment in the mouth is what causes the tooth decay. It is the leading cause of dental caries

(tooth decay) worldwide. S. mutans is considered to be the most cariogenic of all of the

oral Streptococci. Although S. mutans and S. sobrinus can be distinguished by appropriate

laboratory tests, these are expensive and time-consuming so it is not always practicable to

identify down to the species level in large-scale epidemiological studies.

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Figure 2.4: Photo of Streptococcus mutans under the microscope (sciencephoto.com).

Research Literature

A recently described medium (Haemophilus test medium [HTM]) for antimicrobial

susceptibility testing of Haemophilus influenzae was evaluated in this study for broth

microdilution testing of Streptococcus pneumoniae. All isolates of S. pneumoniae produced

acceptable growth in both media, and MICs determined in HTM agreed closely with those

determined in lysed horse blood. Drugs which provided a MIC within 1 log2 concentration

difference in both media included penicillin (100%), ampicillin (98.0%), amoxicillin-clavulanate

(100%), ampicillin-sulbactam (100%), cephalexin (98.9%), cefaclor (96.8%), cefuroxime

(99.0%), chloramphenicol (96.2%), tetracycline (96.2%), and erythromycin (100%). HTM MICs

with trimethoprim-sulfamethoxazole were 1 to 2 log2 concentration increments higher in 92.0%

of isolates than MICs determined in lysed horse blood. Based on the results of this study, HTM

appears to represent a promising alternative medium for broth microdilution susceptibility testing

of S. pneumoniae.

The researchers of the aforementioned study aimed to generate a new type of media by

replacing the type of media in their experiment. In the same manner, the researcher of this study

aims to formulate an alternative modified media to MH agar.

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Chapter III

Methodology

This chapter presents and describes the materials used during the experiment, the method

and design of the investigation and the procedure to obtain the desired data. The experiment

employed certain statistical tool for analyzing the result of the study.

Research Design

This study required the use of experimental design since the researcher aimed to compare

the results statistically in terms of variation in zone of inhibition. The Randomized Complete

Block Design was used in this study to allow the researcher to group the subjects accurately due

to the high number of treatments. The independent variables for this study were the types of

bacteria, types of media and the types of antibiotics. The said variables were compared in terms

of zone of inhibition.

The four test bacteria used in this study were the following: Escherichia coli, Proteus

vulgaris, Streptococcus mutans, and Staphylococcus epidermidis. The three types of media used

in this study were the following: Mueller-Hinton Agar, Modified Mueller-Hinton Agar with

broth cubes (Beef Flavored) and Modified Mueller-Hinton Agar with broth cubes and “Sansaw”.

This study used of five antibiotics namely: Cephazolin, Ciprofloxacin, Clindomycin,

Doxycycline and Erythromycin.

The experimental groups are as follows: (Treatment A) Modified Mueller-Hinton Agar

with Broth Cubes, (Treatment B) Modified Mueller-Hinton Agar with Broth Cubes and

“Sansaw”. The Control Group for this study is Original Mueller-Hinton Agar (Negative Control).

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Each treatment had three replicates for each type of bacteria treated with five different antibiotics

with a total of thirty-six experimental units.

Materials and Equipment

This study required the use of the following materials: Escherichia coli, Proteus vulgaris,

Streptococcus mutans, Staphylococcus epidermidis, Mueller-Hinton Agar, Modified Mueller-

Hinton Agar with broth cubes, Modified Mueller-Hinton Agar with broth cubes and “Sansaw”,

Cephazolin, Ciprofloxacin, Clindomycin, Doxycycline and Erythromycin.

This study required the use of the following equipment: Erlenmeyer flask, gas burner,

graduated cylinder, gyrotoric shaker, incubator, petri dishes, pippetor, test tube, test tube holder,

vortex mixer. All equipment required in this study were borrowed from the Biochemistry

Laboratory of University of Negros Occidental – Recoletos.

Preparation of Four Test Bacteria

The four test organisms namely: Escherichia coli, Proteus vulgaris, Streptococcus

mutans, and Staphylococcus epidermidis were acquired from Philippine National Collection of

Microbial Strains- Biotech (PNCM-Biotech). The organisms were cultured in University of

Negros Occidental – Recoletos (UNO-R) Biochemistry Laboratory.

Approximate amount of 5 ml of the four test bacteria were cultured in Luria Broth having

an approximate amount of 50 ml. Each of the bacteria was stored in approximate temperature of

15 C° for 72 hours culturing.

Preparation of the Three Culture Media

The ingredients required for the preparation of the three culture media were acquired

from the University of Negros Occidental – Recoletos (UNO-R) Biochemistry Laboratory. The

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components of Mueller-Hinton per liter were the following: beef infusion (150.00 g.), Cantine

(17.50 g.), starch (1.50 g.), and agar (17.00g.). For the MHB or modified Mueller-Hinton with

broth cubes, the beef infusion and cantine were substituted with 75 g beef broth cubes. For the

MHG or modified Mueller-Hinton with broth cubes and “Sansaw”, the beef infusion and cantine

were substituted with 75 g broth cubes while the agar was substituted with 17.00 g block of

“Sansaw”.

The agar for the MHB was first melted in a glass cylinder exposed to flame. Upon

boiling, the broth cubes and other components were added. The components were boiled for 10

minutes or until all components were liquefied. The MHG was prepared in the same procedure as

the MHB except for the gelatin which was substituted for agar. The media were cooled for 30

minutes for the inoculation.

Inoculation of Bacteria

Before the inoculation, 36 sterile petri dishes were prepared for the inoculation. Each of

the media was poured in 9 petri dishes. The media were then cooled for 30 minutes. The 4 test

bacteria were then inoculated in 3 petri dishes for each type of media. The petri dishes were then

stored in incubator to allow the test bacteria to grow in the three types of media. The subjects

were allowed to grow for 48 hours.

Bacteria Sensitivity Test

The five antibiotics required in the experiment were acquired from the Biochemistry

Laboratory. The Kirby-Bauer Method was used in this study. After the 48 hour incubation, each

of the petri dish was treated with 5 different kinds of antibiotics. The subjects were stored in

room temperature for 24 hours before analysis and data gathering. The zone of inhibition of the

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bacteria was measured for each antibiotic in every type of media. The zone diameters were

measured in mm scale. The setup for the petri dish is shown in figure 3.1:

Figure 3.1: Diagram showing the setup used by the researcher.

Disposal of Test Subjects

The materials used in the experiment were disposed and disinfected properly at the

University Negros Occidental Recoletos. The equipment used was sterile by the laboratory

personnel for future use.

Data Analysis and Treatment

The data and results were analyzed using the Multiple Analysis of Variance at α=0.05 to

determine the significant difference in terms of zone of inhibition of the four test bacteria grown

in two types of modified media after treatment of five selected standard antibiotic disks in

comparison with the control group. Using the formula: LB(-)/UB(+)=X ± tα/2(R/√n), the

researcher was able to get the two values under which the mean falls, the lower boundary and the

upper boundary, and the confidence interval which is always at 95%.

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Chapter IV

Results and Discussions

This chapter presents that data gathered in connection with the problems of the

investigation, the analyses of these data with the use of the identified appropriate statistical tools

and the interpretation of the results derived from the analyses.

Dependent Variable: Zone of Inhibition (mm)

BACTERIA Mean Std. Error

95% Confidence Interval

Lower Bound Upper BoundE. coli 17.911 .453 17.015 18.807S. epidermidis 21.956 .453 21.060 22.852

P. vulgaris 16.089 .453 15.193 16.985S. mutans 9.978 .453 9.082 10.874Table 4.1: Estimated Marginal Means of Different Types of Bacteria.

The above table showed that the mean scores of the four types of bacteria in terms of

zone of inhibition are not significantly different. The four types of bacteria were able to react to

the treatment of five different antibiotics since the results showed that the bacteria had a

significant zone of inhibition.


MEDIA Mean Std. Error


Lower Bound Upper BoundMueller Hinton Agar 16.467 .392 15.691 17.243Modified MH with Broth cubes 16.400 .392 15.624 17.176

Modified MH with Sansaw and Broth cubes

16.583 .392 15.807 17.359

Table 4.2: Estimated Marginal Means of Different Types of Media

Table 4.2 showed that the mean scores of the three different kinds of media are not

significantly different with each other since each value fell in the 95% confidence interval.

Results showed that the three types of media were able to cultivate the four types of bacteria.

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ANTIBIOTIC Mean Std. Error95% Confidence Interval

Lower Bound Upper BoundDoxycycline 21.611 .506 20.609 22.613

Erythromycin 9.639 .506 8.637 10.641

Ciprofloxacin 29.889 .506 28.887 30.891

Cephazolin 15.278 .506 14.276 16.280

Clindomycin 6.000 .506 4.998 7.002

Table 4.3: Estimated Marginal Means of Different Types of Antibiotic

Table 4.3 showed that the five types of antibiotics have no significant difference with

each other since each value fell in the 95% confidence interval. Results also showed that each

antibiotic had effect on the zone of inhibition of the four types of bacteria cultured in different

media.


BACTERIA MEDIA MeanStd.

Error


Lower Bound

Upper Bound

E. coli Mueller Hinton Agar 18.333 .784 16.781 19.885Modified MH with Broth cubes 18.000 .784 16.448 19.552Modified MH with Sansaw and Beef Broth cubes

17.400 .784 15.848 18.952

S. epidermidis Mueller Hinton Agar 20.400 .784 18.848 21.952

Modified MH with Broth cubes 22.200 .784 20.648 23.752

Modified MH with Sansaw and Beef Broth cubes

23.267 .784 21.715 24.819

P. vulgaris Mueller Hinton Agar 17.333 .784 15.781 18.885



15.800 .784 14.248 17.352

S. mutans Mueller Hinton Agar 9.800 .784 8.248 11.352



9.867 .784 8.315 11.419

Table 4.4: Estimated Marginal Means of Different Types of Bacteria and Media

24

Table 4.4 showed that at α=0.05 the zone of inhibition four types of bacteria each grown

in three different types of media is not significantly different. The mean scores of S. epidermidis

showed the highest mean scores followed by E. coli, P. vulgaris and lastly S. mutans. Growth of

S. epidermidis and S. mutans in modified media MHB was higher compared to the control while

E. coli and P. vulgaris had a lower mean score when compared to the standard MH. The growth

of S. epidermidis and S. mutans in MHB was higher compared to the control while E. coli and P.

vulgaris had a lower mean score when compared to the standard MH.


ANTIBIOTIC MEDIA MeanStd.

Error


Lower BoundUpper Bound

DXT Mueller Hinton Agar 19.667 .876 17.931 21.402Modified MH with Broth cubes 22.250 .876 20.515 23.985Modified MH with Sansaw and Broth cubes

22.917 .876 21.181 24.652

ERY Mueller Hinton Agar 8.833 .876 7.098 10.569

Modified MH with Broth cubes 10.167 .876 8.431 11.902Modified MH with Sansaw and Broth cubes

9.917 .876 8.181 11.652

CIP Mueller Hinton Agar 31.917 .876 30.181 33.652

Modified MH with Broth cubes 28.417 .876 26.681 30.152Modified MH with Sansaw and Broth cubes

29.333 .876 27.598 31.069

CZ Mueller Hinton Agar 15.917 .876 14.181 17.652



14.750 .876 13.015 16.485

CD Mueller Hinton Agar 6.000 .876 4.265 7.735



6.000 .876 4.265 7.735

Table 4.5 Estimated Marginal Means of Different Types of Antibiotic and Media

Table 4.5 showed that results for analysis at α=0.05 between antibiotics and media are

not significantly different. CIP got the highest mean scores followed by DXT, CZ, ERY and

lastly CD. DXT and ERY are more effective when treated to the two modified media compared

25

to the standard MH. CIP and CZ yielded higher results when treated to the standard MH

compared to the two types of modified media. CD had same effects on the three types of media.


ANTIBIOTIC BACTERIA Mean Std. Error


Lower Bound Upper BoundDXT E. coli 23.444 1.012 21.441 25.448

S. epidermidis 33.889 1.012 31.885 35.893

P. vulgaris 13.778 1.012 11.774 15.781

S. mutans 15.333 1.012 13.330 17.337

ERY E. coli 8.000 1.012 5.996 10.004

S. epidermidis 18.000 1.012 15.996 20.004

P. vulgaris 6.000 1.012 3.996 8.004

S. mutans 6.556 1.012 4.552 8.559

CIP E. coli 35.222 1.012 33.219 37.226

S. epidermidis 28.889 1.012 26.885 30.893

P. vulgaris 40.556 1.012 38.552 42.559

S. mutans 14.889 1.012 12.885 16.893

CZ E. coli 16.889 1.012 14.885 18.893

S. epidermidis 23.000 1.012 20.996 25.004

P. vulgaris 14.111 1.012 12.107 16.115

S. mutans 7.111 1.012 5.107 9.115

CD E. coli 6.000 1.012 3.996 8.004

S. epidermidis 6.000 1.012 3.996 8.004

P. vulgaris 6.000 1.012 3.996 8.004

S. mutans 6.000 1.012 3.996 8.004

Table 4.6: Estimated Marginal Means of Different Types of Antibiotic and Bacteria

Table 4.6 showed that there was no significant difference on the zone of inhibition of the

four types of bacteria treated with five different antibiotic at α=0.05. DXT showed its highest

results when treated to S. epidermidis followed by E. coli, S. mutans and P. vulgaris. ERY

showed its highest results when treated to S. epidermidis followed by E. coli, S. mutans and P.

vulgaris. Among all antibiotics, CIP showed the highest results. CIP yielded its highest result

when treated to P. vulgaris followed by E. coli, S. epidermidis and S. mutans. CZ is most

effective when treated to S. epidermidis followed by E. coli, P. vulgaris and S. mutans. CD

yielded the least results and its means scores were equal to all types of bacteria.

26

Dependent Variable: Zone of Inhibition(mm)

ANTIBIOTIC BACTERIA MEDIA Mean Std. Error 95% Confidence IntervalLower Bound Upper Bound

DXT E. coli Mueller Hinton Agar 19.000 1.753 15.530 22.470Modified MH with Broth cubes 25.333 1.753 21.863 28.804Modified MH with Sansaw and Broth cubes 26.000 1.753 22.530 29.470

S. epidermidis Mueller Hinton Agar 31.667 1.753 28.196 35.137Modified MH with Broth cubes 34.667 1.753 31.196 38.137Modified MH with Sansaw and Broth cubes 35.333 1.753 31.863 38.804

P. vulgaris Mueller Hinton Agar 13.333 1.753 9.863 16.804Modified MH with Broth cubes 14.000 1.753 10.530 17.470Modified MH with Sansaw and Broth cubes 14.000 1.753 10.530 17.470

S. mutans Mueller Hinton Agar 14.667 1.753 11.196 18.137Modified MH with Broth cubes 15.000 1.753 11.530 18.470Modified MH with Sansaw and Broth cubes 16.333 1.753 12.863 19.804

ERY E. coli Mueller Hinton Agar 10.667 1.753 7.196 14.137Modified MH with Broth cubes 7.333 1.753 3.863 10.804Modified MH with Sansaw and Broth cubes 6.000 1.753 2.530 9.470




CIP E. coli Mueller Hinton Agar 39.000 1.753 35.530 42.470Modified MH with Broth cubes 33.667 1.753 30.196 37.137Modified MH with Sansaw and Broth cubes 33.000 1.753 29.530 36.470




CZ E. coli Mueller Hinton Agar 17.000 1.753 13.530 20.470Modified MH with Broth cubes 17.667 1.753 14.196 21.137Modified MH with Sansaw and Broth cubes 16.000 1.753 12.530 19.470




CD E. coli Mueller Hinton Agar 6.000 1.753 2.530 9.470Modified MH with Broth cubes 6.000 1.753 2.530 9.470Modified MH with Sansaw and Broth cubes 6.000 1.753 2.530 9.470



S. mutans Mueller Hinton Agar 6.000 1.753 2.530 9.470Modified MH with Broth cubes 6.000 1.753 2.530 9.470

27

Table 4.7: Estimated Marginal Means of Different Types of Antibiotic, Bacteria and Media.

Results on table 4.7 showed that there is no significant difference in terms of zone of

inhibition of the four test bacteria (Escherichia coli, Proteus vulgaris, Streptococcus mutans,

Staphylococcus epidermidis) grown in two types of modified media (modified Mueller-Hinton

Agar with beef broth cubes and modified Mueller-Hinton Agar with beef broth cubes and

“Sansaw”) after treatment of five selected standard antibiotic disks namely: cephazolin,

ciprofloxacin, clindomycin, doxycycline and erythromycin in comparison with the control group

( Mueller-Hinton agar). DXT showed its highest mean score in S. epidermidis cultivated in MHG

while its least result was in P. vulgaris cultivated in the standard MH. ERY showed its highest

results in S. epidermidis cultivated in MHG while its lowest score was from E. coli cultivated in

MHG and P. vulgaris cultivated in three types of media which all showed equal mean scores.

CIP yielded its highest results from P. vulgaris cultivated in standard MH while its least score

was from S. mutans cultivated in MHB. CZ showed its best effect in S. epidermidis cultured in

MHG while its least score was from S. mutans cultured in MHG. CD showed the least score

among the five antibiotics and it showed same results for the four types of bacteria cultured in

three types of media.

28

Chapter V

Conclusions and Recommendation

Summary of Findings

Results showed that the three types of media were able to culture the four types of

bacteria which in turn allowed the bacteria to react to the five types of antibiotics. Results also

showed that there is no significant difference between the zone of inhibition of bacteria cultured

in the two modified media in comparison with the control group. Results also showed that

effectiveness of an antibiotic is not affected by the modification of bacteria since there is no

significant difference on the mean scores of the antibiotics treated to different bacteria cultured

in different media.

Conclusions

1. The two modified media can serve as alternative to the standard MH in antibiotic

sensitivity testing.

2. There is no significant difference in terms of zone of inhibition of the four test bacteria

(Escherichia coli, Proteus vulgaris, Streptococcus mutans, Staphylococcus epidermidis)

grown in two types of modified media (modified Mueller-Hinton Agar with beef broth

cubes and modified Mueller-Hinton Agar with beef broth cubes and “Sansaw”) after

treatment of five selected standard antibiotic disks namely: cephazolin, ciprofloxacin,

clindomycin, doxycycline and erythromycin in comparison with the control group

( Mueller-Hinton agar).

29

Recommendations

It has been proved that Modified MH with Broth Cubes, and modified MH with Broth

Cubes and Sansaw is as good as the Mueller-Hinton Agar in performing an antibiotic sensitivity

test. Broth Cubes and Sansaw may be used in the future and preset study that requires the use

culturing media for growth of microorganisms.

The modification of MH agar may also be used for further and deeper knowledge. Some

microbiologists may also use information provided for his or her inline studies. Moreover,

student researchers or researcher may use modifications as their treatment for the enhancement

research and innovation. Beef broth cubes and Sansaw may also be used for continuous studies

for further information. The modifications may also be used by researchers for their current

research projects.

30

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