The Problem and Its Setting: Wesleyan University-Philippines

1
WESLEYAN UNIVERSITY-PHILIPPINES
College of Nursing and Allied Medical Science
Chapter 1
THE PROBLEM AND ITS SETTING
INTRODUCTION
Culture media contains nutrients and physical growth parameters
necessary for microbial growth. It could be a solid or liquid preparation for
the growth, transport and storage of microorganism. It is important for
most microbiological tests to obtain pure cultures, to grow and count
microbial cells, and to cultivate and select microorganisms. Differential
culture medium contain compounds that allow groups of microorganisms
to be visually distinguished by the appearance of the colony or the
surrounding media. Selective media contain ingredients that inhibit the
growth of some organisms but allow others to grow (Acharya, 2010).
Mannitol Salt Agar (MSA) as a selective, differential and
indicator medium used to isolate and identify Staphylococcus aureus from
the clinical specimen. Its purpose is to see if the microbe can ferment the
carbohydrate mannitol as a carbon source. This is a medium that is
composed of Peptone, as the source of Nitrogen, Vitamin and Carbon,
Phenol Red, as the indicator for organisms that can ferment Mannitol
which is the major and differential ingredients of MSA and lastly the
Sodium Chloride which is 7.5% concentration can inhibit the other
organism that cannot tolerate the high saline levels (Acharya, 2013).
2
Staphylococcus aureus is a gram-positive bacterium that is found on
the skin and in the nasal passages of about a quarter of humans. It is a
facultative anaerobe, meaning it can produce energy either in the presence
or absence of oxygen. The bacteria tend to infect the skin often causing
abscesses. However, the bacteria can travel through the bloodstream and
infect almost any site in the body, particularly heart valves and bones. As
a Laboratory Testing, this bacteria is isolated in the mannitol salt agar,
with a presence of Growth and Yellow Medium that indicates S. aureus a
Mannitol Fermenter (Al-Araji, 2015).
Brassica oleracea var. botrytis also known as Cauliflower is a flower
that is also a good source of protein and vitamins and mineral to humans.
Since Brassica oleracea var. botrytis is generally accepted that its extracts
contain a variety of components, such as polysaccharides, proteins, some
lectins and polyphenols like mannitol (Scott, 2016).
This research study aims to produce a differential culture medium from
the Brassica oleracea var. botrytis for the growth of Staphylococcus aureus.
3
STATEMENT OF THE PROBLEM
This study generally aimed to test or determine the effectiveness of
Cauliflower as substitute ingredient for Mannitol in Mannitol Salt Agar to
differentiate Staphylococcus aureus to other Gram Positive bacteria.
The specific problems were:
1. Does Staphylococcus aureus grows in the Cauliflower agar?
2. Does the use of Cauliflower as Mannitol in MSA provide a more
effective differential culture medium for growing Staphylococcus
aureus than Mannitol Salt Agar?
STATEMENT OF HYPHOTHESES
The following hypotheses were stated in null form
1. The Staphylococcus aureus did not grow on the Cauliflower agar.
2. The use of Cauliflower as a substitute mannitol in Mannitol Salt agar
does not provide a more effective differential culture medium for
growing Staphylococcus aureus than in Mannitol Salt Agar.
SIGNIFICANCE OF THE STUDY
In this study, the researcher will be able to produce a substitute
Mannitol for differentiating Staphylococcus aureus from other Gram
Positive bacteria.
The result will be focused on the following benefits:

4
The Researchers. They will be able to enhance their observing skills
in terms of experimental research design and in discovering new culture
medium that can provide nutrient and differentiate Staphylococcus aureus
from other Gram positive bacteria.
The Future Researchers. They can use this study as a guide in
pursuing a deeper study.
The Medical Technology Students. They will be knowledgeable
about the capability of fruits and vegetables to provide nutrient and
differentiate microorganisms.
Registered Medical Technologist. They will be able to increase
their knowledge in making a substitute culture medium that are used to
differentiate different types of microbes.
SCOPE AND LIMITATION OF THE STUDY
This study is focused in identifying the effectiveness of Cauliflower
as a substitute for Mannitol in Mannitol Salt Agar on isolating
Staphylococcus aureus only. This study was conducted at Wesleyan
University-Philippines Medical Technology Laboratories on month of
March and April 2018.
The researcher limited the study on observation and documentation
to obtain the desired result. Determination of the specific component
responsible for the bacterial growth were also not included in this study.
5
REVIEW OF RELATED LITERATURE AND STUDIES
Cauliflower
Cauliflower is a member of the cruciferous vegetable or
brassicaceae family-along with broccoli, cabbage, kale, brussels, sprouts
and some other less common varieties.
Recent studies suggest that cruciferous vegetables are an
excellent source of natural antioxidants due to their high levels of various
phytochemicals, as well as good suppliers of essential vitamins,
carotenoids, fiber, soluble sugars, minerals, and phrenolic compounds. In
fact, it is believed that brassica vegetables are the largest source of
phenolic compounds in the human diet (Axe, 2015).
Mannitol
Mannitol is added to a wide variety of processed products (simply
check the packaging of your shopping for ‘mannitol’ or food additive
number e421. On top of this, Mannitol is naturally occurring. Cauliflower,
for example, contains 3 grams of Mannitol per every 100 grams of weight.
We are all consuming it on a daily basis (Simon, 2016). Mannitol naturally
occurs in high levels in a range of fruit and vegetables like watermelon,
clingstone peaches, button mushrooms, cauliflower, celery, snow peas,
butternut squash and sweet potato (Muir et al., 2009). According to Smith,
L (2017) Mannitol has a long history of safe consumption in many

6
products commonly used and consumed by pregnant women, including
folic acid supplements, vitamins, candy and baked goods. Mannitol also
occurs naturally in many foods, including cauliflower, mushrooms, snow
peas, and peaches. The amount of mannitol used as a non-medicinal
ingredient in medications is extremely small—usually less than 0.25
grams. The amounts of mannitol found naturally in foods are much
higher. For example: cauliflower has about 2.6 grams of mannitol per 100
grams; mushrooms have about 2.6 grams of mannitol per 100 grams;
snow peas have about 1.2 grams of mannitol per 100 grams; peaches have
about 0.5 grams of mannitol per 100 grams.
Cauliflower contains a type of sugar-alcohol, or polyol, called
mannitol. The mannitol is poorly absorbed in many people diagnosed with
IBS and avoiding foods containing mannitol can help alleviate the
symptoms (Jacob, 2017).
Mannitol Salt Agar
Differential media contain compounds that allow groups of
microorganisms to be usually distinguished by the appearance of the
colony or the surrounding media, usually on the basis of some biochemical
difference between the two groups (Arvidson, 2010).
Mannitol Salt is a selective bacterial growth medium because it has
a very high concentration of NaCl (7.5%). Most bacteria cannot survive in

7
this highly saline, hypertonic environment. But the
genus Staphylococcus has a protective slime layer that protects it in a
harsh, salty environment. So Staph grow well in this media. This growing
medium is also differential because it contains a dye that identifies types
of Staphylococcus that produce an organic acids from
mannitol fermentation (eating mannitol, a type of alcohol). The bacterial
waste products generated, organic acid metabolites, change
the pH indicator in MSA from red to bright yellow. Pathogenic Staph, such
as Staphylococcus aureus, are mannitol fermenters, and when growing on
Mannitol Salt Agar, their wastes turn the MSA a bright yellow color. By
contrast, nonpathogenic Staph such as Staphylococcus
epidermidis (a.k.a. Staph epi), the normal flora that grows on human skin,
does not ferment mannitol. When Staph epi grows on Mannitol Salt, the
naturally orange-pink color of the agar doesn’t change, since S.
epidermidis doesn’t eat mannitol or produce the resulting organic acid
wastes. Mannitol salt agar (MSA), a medium generally used in human
medicine for differentiating Staphylococcus aureus from coagulase-
negative staphylococci (CNS), for culturing bovine-associated CNS species.
Based on the research done by Bereda et.al., (2016) Staphylocccus
spp. are incubated and observed at 12 and 48 hours with a temperature
of 32°C.
8
According to the United States Pharmacopeial Convention, Inc., 2008
Plates are incubated at 35 ± 2°C in an aerobic atmosphere. Examine plates
after 18 to 24 and 48 h for growth, colony size, pigmentation and
selectivity. Typical reactions are as follows: Strains Growth results
Staphylococcus aureus Medium-sized yellow colonies, Staphylococcus
epidermidis ATCC 12228 Small to medium-sized white colonies, medium
red.
Staphylococcus aureus
Staphylococcus aureus is a catalase positive, gram positive coccus
usually arranged in clusters. In laboratory, Staphylococcus can be
differentiated with other species by coagulase test: S. aureus is coagulate
– positive. It is a major cause of skin, soft tissue, respiratory bone, joint,
endovascular and wound infection. Growth of bacterial cultures is an
increase in number of bacteria in a population occurs in a geometric or
exponential manner: with each division cycle (generation), one cell gives
rise to 2 cells, 4 cells then 8 cells, then 16 and 32 and so forth. There is a
required time for the incubation (Cauz, 2014). Staphylococcus aureus,
sometimes called golden staph is a common bacterium that lives on skin
or in the nose. It can cause a range of mild to severe infections and may
cause death. Some strains are resistant to antibiotics. Hospital patients
are more likely to be infected by Staphylococcus aureus because of surgical
or by wounds. In most situations, Staphylococcus aureus is harmless.

9
However if it enters the body through a cut in skin, it may cause death
(Briones et.al., 2015).
According to the CDC, Food safety, Staphylococcus aureus is a type of
bacteria commonly found on the skin and hair as well as in the noses and
throats of people and animals. These bacteria are present in up to 25
percent of healthy people and are even more common among those with
skin, eye, nose, or throat infections. Staphylococcus can cause food
poisoning when a food handler contaminates food and then the food is not
properly refrigerated. Other sources of food contamination include the
equipment and surfaces on which food is prepared. These bacteria
multiply quickly at room temperature to produce a toxin that causes
illness. Staphylococcus is killed by cooking and pasteurization.
Staphylococcus aureus infections are more frequent than those by other
bacteria, particularly in setting with high HIV/AIDS prevalence. This
warrants correct identification of the isolates to achieve better treatment
outcomes (Katabazi et.al.,2010). Toxin-mediated diseases caused by
Staphylococcus aureus include scalded skin syndrome, food poisoning,
and toxic shock syndrome (McPherson & Pincus, 2011).
Staphylococcus aureus (S. aureus) causes a variety of infections,
ranging from a mild skin infection to blood stream infections and deep
seated infections. As Staphylococcus aureus bacteremia (SAB) has the

10
tendency to cause endovascular and metastatic infections, complications
can occur at almost all sites of the body (Abriouel el.al.,2010).
In another past study, it is also stated that Staphylococcus aureus
is a major cause of bacteremia, and S. aureus bacteremia is associated
with higher morbidity and mortality, compared with bacteremia caused by
other pathogens. The burden of S. aureus bacteremia, particularly
methicillin-resistant S. aureus bacteremia, in terms of cost and resource
use is high. The risk of infective endocarditis and of seeding to other
metastatic foci increases the risk of mortality and raises the stakes for
early, appropriate treatment. The incidence of S. aureus bacteremia and
its complications has increased sharply in recent years because of the
increased frequency of invasive procedures, increased numbers of
immunocompromised patients, and increased resistance of S. aureus
strains to available antibiotics. This changing epidemiology of S. aureus
bacteremia, in combination with the inherent virulence of the pathogen, is
driving an urgent need for improved strategies and better antibiotics to
prevent and treat S. aureus bacteremia and its complications (Naber,
2009).
Staphylococcus epidermidis
During the last decade, S. epidermidis has emerged as an important
opportunistic pathogen frequently causing infections linked to medical
devices such as intravascular catheters, ventricular shunts, prosthetic

11
heart valves, artificial lenses and orthopedic implants. The infection starts
with adherence of S. epidermidis to the biomaterial, followed by
colonization of the device surface and formation of a biofilm. Thus, the
ability of S. epidermidis to switch from a harmless commensal to an
opportunistic pathogen, which is closely linked to its capacity to form a
biofilm, increases its clinical importance (Expert Rev Vaccines, 2012).
Staphylococcus epidermidis is nowadays regarded as the most frequent
cause of nosocomial infections and indwelling medical device-associated
infections. One of the features that contributes to the success of this
microorganism and which is elemental to the onset of pathogenesis is its
ability to form biofilms. Cells in this mode of growth are inherently more
resistant to antimicrobials. Seeking to treat staphylococcal-related
infections and to prevent their side effects, such as the significant
morbidity and health care costs, many efforts are being made to develop
of new and effective antistaphylococcal drugs (Gomes et.al., 2013).
Coagulase-negative staphylococci (CoNS) constitute an indigenous part
of the microbiota of human and animal skin and mucosa. Over a period of
several decades, CoNS and particularly Staphylococcus epidermidis have
evolved as important opportunistic pathogens, primarily causing health
care-associated infections in patients with indwelling medical devices.
These infections were previously predominantly regarded as being of
endogenous origin, but considerable evidence has been accumulated

12
confirming that nosocomial genotypes of S. epidermidis colonize patients
and health care personnel and cause a substantial proportion of health
care-associated infections. S. epidermidis is currently the main pathogen
in catheter-related bloodstream infections and early-onset neonatal sepsis
and is also a frequent cause of prosthetic joint infections, prosthetic valve
endocarditis, and other biomedical device-related infections (Caroll, 2016).
Plants as Alternative Medium
According to a research, a vegetable waste was used to
formulate a cost effective media. Media with minimal ingredient was
formulated. The growth of different microorganism was comparable with
regular media (Dr. C. Berde and Dr. V. Berde,2015).
Cassava starch has been used successfully in plant tissue
culture media, and its potential and culturing fungi was evaluated for cost
reduction since agar is expensive. Cassava starch showed some level of
gelling ability. The cassava starch alone cannot be use for culture media
unless blended with some amount of agar (C.K. Kwoseh,2012).
A past study of Ravathie Arulanantham et.al., 2012, uses a
legume seeds that have found to have a good protein source for nutritional
purposes. Different protein source is also use in the study for growing a
bacteria such as E.Coli, Bacillus sp., Klebsiella sp., Staphylococcus sp., and
Pseudomonas sp.
13
Independent Variable Dependent Variable
Control
Positive:
Mannitol Salt Agar
Negative:
Staphylococcus Growth of bacteria
epidermidis
Experimental group
Cauliflower Agar
Effectiveness of
Cauliflower as substitute
to Mannitol MSA
Figure 1. Conceptual Framework

The figure above describes the framework of the study. It shows two
groups: the control group which is the MSA and Staphylococcus aureus
(Positive control) and Staphylococcus epidermidis (Negative control),
and the experimental group, the Cauliflower Agar. From these groups,
it is expected to identify if Cauliflower Agar will be able to differentiate
the Staphylococcus aureus in the agar plates. Results would reveal
whether or not Cauliflower may be used as an alternative to Mannitol
Salt Agar.
14
Chapter 2
METHODS AND PROCEDURES
This section deals with the procedures and methods of research
applied in this study. The manner how the study was designed and
executed were also presented.
RESEARCH DESIGN
The design used in this study is Quasi-experimental method, a non-
randomized study. In the experiment only the post test is measured.
 CONTROL GROUP: Mannitol Salt Agar (Y)
 EXPERIMENTAL GROUP: Cauliflower Agar (x)
GROUP A: Staphylococcus aureus (no pretest) > X > O
GROUP B: Staphylococcus aureus (no pretest) > Y> O
GROUP C: Staphylococcus epidermidis (no pretest) > X > O
GROUP D: Staphylococcus epidermidis (no pretest) >Y > O
Figure 2. Control and Experimental Group Relationship
There is only one experimental group used in the experiment, the
plate with the cauliflower powder substitute for mannitol where the
Staphylococcus aureus and Staphylococcus epidermidis was cultured and

15
observed. The Mannitol Salt Agar plate serve as the control group assessed
during the experimentation to obtain desired result.
RESEARCH SETTING:
The dried Cauliflower was pulverized and was collected from
Cabanatuan City Public Market. It was bought only in one stall at the same
day. The culture of organism to be tested was ordered from University of
Santo Tomas, España, Manila and also the pulverization process was held
at the Medical Technology Laboratory in Wesleyan University-Philippines.
The streaking of Staphylococcus aureus and Staphylococcus epidermidis
was done at Wesleyan University- Philippines Laboratory and there it was
observed and results were analyzed.
Subject, Materials and Equipment
Figure 3. Materials and Equipment; Left: Ingredients, Right: Oven

16
Figure 3. Materials and Equipment; Left: Autoclave, Right: Hotplate
Collection of the sample
10kg of Whole Cauliflower were purchased from the Cabanatuan
City Public Market and was cut into small pieces before it was used in the
experiment.
Figure 4. Fresh Whole Cauliflower; Bureau of Plant Industry
The researcher brought the plant sample at the Bureau of Plant
Industry, Manila to ensure the authentication of the plant. (Figure 4)

17
Source of Pure Bacterial Culture
The pure culture of Staphylococcus aureus and Staphylococcus
epidermidis was obtained at the University of Santo Tomas, Manila. (Figure
5)
Figure 5. University of Santo Tomas, Manila.
Culture media
Mannitol Salt Agar serve as the control medium which contains
7.5% NaCl, 1% Mannitol 1.5% Agar, and Phenol Red, the pH is adjusted
to 7.4 at 25°C. (Figure 6)
Figure 6. Mannitol Salt Agar

18
Sampling Procedure:
The cauliflower used in the experiment was bought on one stall of
Cabanatuan City Public Market within one day (morning). The samples
were cut into very thin slices, air dry and put in the blender until finely
powdered.
General Procedure:
PREPARATION OF CAULIFLOWER POWDER
The researchers used a 10kg of Whole Cauliflower, it was washed and
weighed using a triple beam balance. It was cut into a smaller pieces and
air dried. A grinder to turn it into a powdered form and was kept in an
airtight container for the next procedure. (Figure 7, Figure 8)
Figure 7. Air drying of cauliflower

19
Figure 8. Pulverize Cauliflower
PREPARATION OF THE CULTURE MEDIA
The researchers used sterilized laboratory glasswares by
autoclaving at 121 Celsius for 15 minutes at 15 psi. The 100 grams
powdered Cauliflower was mixed with 15 grams of Agar, .25 grams of
Phenol Red 75 grams of NaCl and 1000ml distilled water. The prepared
agar mix was gently heated until it dissolved and then autoclaved. (Figure
9)
Figure 9. Heating of Cauliflower agar mixture

20
PREPARATION OF MANNITOL SALT AGAR
111 grams of Mannitol Salt Agar was dissolved in 1000 mL distilled
water. Gently heated to completely dissolve the medium for 5 minutes.
Then, sterilized by autoclaving at 121 Celsius for 15 minutes at 15 psi.
PREPARATION OF AGAR PLATES
The prepared culture medium were extracted on the disposable petri
dish and allowed to stand for a minute, then it was refrigerated at 4°C.
Researchers did a triplicate sample of each Agar Plate. (Figure 10, Figure
11)
Figure 10. Extraction of Cauliflower Figure 11. Cauliflower Agar
STREAKING OF BACTERIA IN THE AGAR PLATES
The pure culture of Staphylococcus aureus and Staphylococcus
epidermidis were inoculated from the Nutrient Agar. Using the overlapping
streak plate method, the bacteria from the pure culture was streaked to
21
the prepared Cauliflower agar and Mannitol Salt Agar using a disposable
inoculating loop. (Figure 12)
Figure 12. Streaking of Bacteria
INCUBATION
After streaking of the bacteria into the prepared agar plates, it was
placed inside the incubator with an optimum temperature of 37°C. Direct
observation and monitoring of the agar plates were done in 12th and
24thhours of incubation, which follows the methods of the past studies.
(Figure 13)
Figure 13. Incubator

22
BIOCHEMICAL TESTING
The incubated agar plates undergo biochemical testing to identify
the species that grow on the agar plates. Researchers first describe the
morphology on the agar plate then perform the Gram Staining to identify
if it is a Gram Positive Bacteria and BBL Crystal Identification, for
identification of the Staphylococcus aureus and Staphylococcus
epidermidis. (Figure 14, 15)
The principle of BBL Crystal ID System is that it serve as a
miniaturized identification method employing modified conventional
fluorogenic and chromogenic substrates. It is intended for identification of
frequently isolated aerobic gram-positive bacteria.
Figure 14. Gram Stain
Figure 15. BBL Kit, Inoculum Fluid, Incubation, Readings

23
DATA GATHERING TOOLS
These were the ingredients, instrument and apparatuses used
during the experimentation phase: Analytical Balance was used to
measure /weigh the desired amount of agar. Beaker was used as a
container for the agar while heating. Biosafety Cabinet was used as the
workplace for streaking of the bacteria, Erlenmeyer flask it is the container
suitable for heating liquids. It was also used for the preparation of
microbial cultures, Graduated cylinder was used to measure the volume
of distilled water, Hot plate was used to heat and dissolve the agar,
Incubator was the device used to grow and maintain the bacterial culture
and it maintains the optimal temperature, humidity, and other conditions
for the bacteria to grow, Inoculating loop was used to transfer and streak
an inoculum from the cultured bacteria to another agar plate, Petri dish A
flat dish with a lid which held the prepared solid agar, It was used for
culturing the bacteria Staphylococcus aureus and Staphylococcus
epidermidis, Refrigerator was used for cooling the agar, Staphylococcus
aureus This bacteria was used to grow in Cauliflower Agar for Positive
Control, Staphylococcus epidermidis was the bacteria used to grow in
Cauliflower Agar for Negative Control, Sterile container was used to
contained the powdered cauliflower, Stirring rod was used to stir the agar
while being heated. BBL™ CRYSTAL™ ID KIT was used to determine if the
colony that grew on the agar were S. aureus and S. epidermidis.

24
DATA MANAGEMENT AND ANALYSIS
Upon finishing and gathering the data, the researchers used
descriptive statistic. According to Jackson, (2009), descriptive statistics are
pretty much as they sound — they describe situations. There are three
main types of descriptive statistics but the observational methods were
use under the laboratory observation.
ETHICAL CONSIDERATIONS
Researchers ensure to maintain the ethics during the
experimentation period. Ethics Committee and Adviser’s approval and
guidance are asked prior in conducting this study. Permission to use
laboratory tools and equipment were also done. Data and document report
were all in accordance to what have been observed during the period. There
is no fabrication, falsification, nor plagiarism in performing this study.
Moreover, there is no human – physically, emotionally or psychologically
harmed during the conduct of this study. The internal validity is also
ensured by maintaining the optimum temperature, sterility of the
environment and proper instrumentation.

25
CHAPTER 3
PREPARATION, ANALYSIS and INTERPRETATION
This chapter presents and discusses the data collected based from
the experiment conducted. Within 24 hours, the Staphylococcus aureus
and Staphylococcus epidermidis plated on the Cauliflower agar and
Mannitol Salt Agar and incubated at 37°C were observed.
CONTROL GROUP AND EXPERIMENTAL GROUPS
I. CONTROL GROUPS
AGAR PLATE Staphylococcus aureus growth in every 12 COLOR
hours interval CHANGE
Mannitol Salt 12th hour 24th hour
Agar 2 3 A
Table 1.1 GROWTH OF POSITIVE CONTROL GROUP
1- Without Growth
2- Few Growth
3- Moderate Growth A- Yes
4- Heavy Growth B- No
Table 1.1 shows the result of trial under the controlled conditions
after the 12th and 24thhours of observation of Staphylococcus aureus in
Mannitol Salt Agar. The Staphylococcus aureus shows an increase in
growth through time in the Mannitol Salt Agar and a change in color from
red to yellow.
26
AGAR PLATE Staphylococcus epidermidis growth in Color
every 12 hours interval Change
Mannitol Salt 12th hour 24th hour
Agar 2 3 B
Table 1.2 NEGATIVE CONTROL GROUP

1- Without Growth
2- Few Growth
3- Moderate Growth A- Yes
4- Heavy Growth B- No
Table 1.2 shows the result of the trial under the controlled conditions
after the 12th and 24thhours of observation off Staphylococcus epidermidis
in the Mannitol Salt Agar. This shows that Staphylococcus epidermidis
shows an increase in growth through time but no change in color at all in
Mannitol Salt Agar.
II. EXPERIMENTAL GROUPS

AGAR PLATE Staphylococcus aureus growth in COLOR
every 12 hours interval CHANGE
Cauliflower 12 hour
th 24th hour
Agar 2 2 B
Table 2.1 EXPERIMENTAL GROUP (A)
1- Without Growth
2- Few Growth
A- Yes
3- Moderate Growth
B- No
4- Heavy Growth
27
Table 2.1 shows the result of the trial under the controlled
conditions after the 12th and 24th of observation of the Staphylococcus
aureus onto the Cauliflower Agar It shows that Staphylococcus aureus has
the same growth through time and it does not show any color changes at
12th and 24th hours.
AGAR PLATE Staphylococcus epidermidis growth in COLOR
every 12 hours interval CHANGE
Cauliflower 12th hour 24th hour
Agar 2 2 B
Table 2.2 EXPERIMENTAL GROUP (B)
1- Without Growth
2- Few Growth A- Yes
3- Moderate Growth B- No
4- Heavy Growth
This Table 2.2 shows the result of the trial under the controlled
conditions after the 12th and 24thhours of observation of
Staphylococcus epidermidis on the Cauliflower Agar. It shows that
Staphylococcus epidermidis has the same growth through time but
does not show any changes in its color at 12th and 24th hours of
observation.
28
According to a study by Wolff in 2016, Staphylococcus sometimes
grow a little slower and 48 hours might be the appropriate time to check
for growth.
III. IDENTIFICATION OF BACTERIA
I. Morphology
CAULIFLOWER Staphylococcus Staphylococcus
AGAR aureus (A) epidermidis (B)
R1 1.0-1.5 mm 0.8-1.2 mm
diameter, diameter
Round and Slightly Round, White
Yellow Colonies Colonies
R2 1.0-1.5 mm 0.8-1.2 mm
Round and Slightly diameter
Yellow Colonies Round, White
Colonies
R3 1.0-1.5 mm 0.8-1.2 mm
Round and Slightly diameter
Yellow Colonies Round, White
Colonies
Table 3.1 Morphology of the Bacteria cultured on the
Cauliflower Agar
29
Table 3.1 shows the Morphology observed at 24th hour incubation of
Cauliflower agar for the identification of the colonies. As the table shows,
the colonies that grow on the agar are Staphylococcus aureus and
Staphylococcus epidermidis based on observed morphology.
Staphylococcus aureus colony has a circular or round shape has a
pigment of yellow because of staphyloxanthin and has a 1-4 mm in
diameter. Staphylococcus epidermidis is describe as white, round colonies
with a diameter of 1-2mm after overnight incubation (Newman,2011).
II. Gram Stain
CAULIFLOWER Staphylococcus Staphylococcus
AGAR aureus (A) epidermidis (B)
R1 Gram Positive Gram Positive
Table 3.2 Gram Stained Bacteria cultured on the Cauliflower

Agar
Table 3.2 shows the result of the Gram Stain bacteria from the
Cauliflower Agar Plate. This shows that the organism on the plate are a
Gram positive Bacteria.
Gram Positive bacteria have a very thick cell wall made of protein
called peptidoglycan. These bacteria retain the crystal violet dye (chemical
use in for gram staining) (Wells,2014).

30
III. BBL Crystal Identification
CAULIFLOWER Identified species on agar plate
AGAR
R1A Staphylococcus aureus
R1B Staphylococcus epidermidis
Table 3.3 BBL Crystal Identification of the Bacteria cultured on
the Cauliflower Agar
Table 3.3 shows the species of bacteria from the Cauliflower agar.
Using the BBL Crystal Identification it is confirmed that the cultured
bacteria from the Cauliflower agar are Staphylococcus aureus and
Staphylococcus epidermidis.
31
FIGURE 16. 12th hours A) (Left) Mannitol Salt Agar streak with
Stapylococcus aureus B) (Right) Cauliflower Agar streak with
32
FIGURE 17. 12th hours C) (Left) Mannitol Salt Agar streak with
Stapylococcus epidermidis D) (Right) Cauliflower Agar streak with
33
FIGURE 18. 24th hours A) (Left) Mannitol Salt Agar streak with
Stapylococcus aureus B) (Right) Cauliflower Agar streak with
Staphylococcus aureus.
34
FIGURE 19. 24th hours C) (Left) Mannitol Salt Agar streak with
Stapylococcus epidermidis D) (Right) Cauliflower Agar streak with
35
Figure 20. Staphylococcus aureus (Left) Staphylococcus epidermidis
(Right)
Figure21. BBL Crystal ID Encoding

36
Chapter 4
SUMMARY, CONCLUSION AND RECOMMENDATIONS
This chapter presents the three important section. The summary
presents the key points of this study. The conclusion answers the
statement of the problem. Lastly, the recommendation provides some
reasonable suggestions to improve this study or the discipline in general.
SUMMARY
This study aimed to prove the effectiveness of Cauliflower as a
differential media for the Staphylococcus species in an agar plate. The
Quasi-Experimental research design was used which comprised of
Cauliflower agar as the experimental group; Mannitol Salt Agar as the
control group. Staphylococcus aureus as the positive control and
Staphylococcus epidermidis as the negative control which was ordered
from the University of Santo Tomas, Thomas Aquinas Research Center
(TARC). Data collection and experimentation was done last February to
April 2018 at Wesleyan University- Philippines, Medical Technology
Laboratory. Ethics in conducting the experimental research and the
internal validity were ensured.
Observation analysis revealed that cauliflower leads to bacterial
growth, however, due to its inability to differentiate the type of bacterial
growth, it is not a suggested alternative to Mannitol salt agar.

37
CONCLUSION
Based on the actual experiment on Cauliflower agar, the researchers
therefore concluded that:
1. Staphylococcus aureus grows in the Cauliflower Agar which means
it is able to provide enough nutrient for the both bacteria for it to
grow.
2. The Cauliflower agar is not an effective alternative to Mannitol in
Mannitol Salt agar because it could not differentiate Staphylococcus
aureus from Staphylococcus epidermidis.
RECOMMENDATIONS
The following statement are some suggestions that could be applied
for further research of this study for improvement:
1. Future researchers may use the cauliflower agar on culturing other
organism like gram negative bacteria.
2. The future researchers may use a particular inhibitors to lessen or
inhibit undesirable growth of possible contaminant like fungi in
their media.
3. Using other improved methods that could collect a pure mannitol in
from the Cauliflower or in different fruits and vegetables.

38
4. Finding out the components that could promote a growth and ability
to differentiate Staphylococcus aureus and Staphylococcus
epidermidis or other certain bacteria.
5. The researchers can use the cauliflower as a Broth instead of an
agar.
6. The future researcher may try to use an enhancer to improve the
cauliflower or another fruit vegetable’s differential component.

39
APPENDICES
I. University of Santo Tomas

April 6, 2018
Prof. Gina R. Dedeles, PhD

Curator
Greetings!
We are students from Wesleyan University-Philippines, and currently conducting our
undergraduate research study entitled “Cauliflower as a substitute for Mannitol Salt
Agar, a differential media for isolation of Staphylococcus aureus”. As part of our
research, we need a bacterial strain of Staphylococcus aureus and Staphylococcus
epidermidis that serves as ourr positive and negative control.
We are hoping for your response regarding this matter. Thank you and God bless!
Respectfully yours,
Andres, Kristel Joy

Dela Paz, Crisha Darryale
Dela Paz, Cristian Deo
Miralles, Marjorie Noted by:
Sigue, Rachelle Sharey R. Capati, RMT, MLS(ASCPi)CM
Teano, Cristiana (Research Adviser)
(Researchers)
40
II. Bureau of Plant Industry

March 16, 2018
George Y. Culaste
OIC- Director
Bureau of Plant Industry
692 San Andres St., Malate, Manila
466
525-79-09
Sir:
Greetings!
We are students from Wesleyan University-Philippines, and we are currently conducting our
undergraduate research study entitled “Cauliflower as a substitute for Mannitol Salt Agar, a
differential media for isolation of Staphylococcus aureus”. In the line with this, we are tasked to
certify our plant samples under the National Museum Botany Department. This is to guarantee the
authenticity of the sample we are about to use in our experimentation.
An attachment to this letter includes images of our sample and the sample to be submitted to the
National Museum Botany Department.
We are hoping for your response regarding this matter. Thank you and God bless!
Respectfully yours,
Andres, Kristel Joy
Dela Paz, Crisha Darryale
Dela Paz, Cristian Deo
Miralles, Marjorie Noted by:
Sigue, Rachelle Sharey R. Capati, RMT
Teano, Cristiana (Research Adviser)
(Researchers)
41
41

The Problem and Its Setting: Wesleyan University-Philippines

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

The Problem and Its Setting: Wesleyan University-Philippines

Încărcat de

Drepturi de autor:

Formate disponibile

1

THE PROBLEM AND ITS SETTING

Culture media contains nutrients and physical growth parameters

necessary for microbial growth. It could be a solid or liquid preparation for

the growth, transport and storage of microorganism. It is important for

most microbiological tests to obtain pure cultures, to grow and count

microbial cells, and to cultivate and select microorganisms. Differential

culture medium contain compounds that allow groups of microorganisms

to be visually distinguished by the appearance of the colony or the

surrounding media. Selective media contain ingredients that inhibit the

growth of some organisms but allow others to grow (Acharya, 2010).

Mannitol Salt Agar (MSA) as a selective, differential and

indicator medium used to isolate and identify Staphylococcus aureus from

carbohydrate mannitol as a carbon source. This is a medium that is

composed of Peptone, as the source of Nitrogen, Vitamin and Carbon,

Sodium Chloride which is 7.5% concentration can inhibit the other

Staphylococcus aureus is a gram-positive bacterium that is found on

the skin and in the nasal passages of about a quarter of humans. It is a

facultative anaerobe, meaning it can produce energy either in the presence

a Laboratory Testing, this bacteria is isolated in the mannitol salt agar,

with a presence of Growth and Yellow Medium that indicates S. aureus a

Mannitol Fermenter (Al-Araji, 2015).

Brassica oleracea var. botrytis also known as Cauliflower is a flower

contain a variety of components, such as polysaccharides, proteins, some

lectins and polyphenols like mannitol (Scott, 2016).

This research study aims to produce a differential culture medium from

STATEMENT OF THE PROBLEM

This study generally aimed to test or determine the effectiveness of

Cauliflower as substitute ingredient for Mannitol in Mannitol Salt Agar to

differentiate Staphylococcus aureus to other Gram Positive bacteria.

The specific problems were:

1. Does Staphylococcus aureus grows in the Cauliflower agar?

2. Does the use of Cauliflower as Mannitol in MSA provide a more

effective differential culture medium for growing Staphylococcus

aureus than Mannitol Salt Agar?

The following hypotheses were stated in null form

1. The Staphylococcus aureus did not grow on the Cauliflower agar.

2. The use of Cauliflower as a substitute mannitol in Mannitol Salt agar

does not provide a more effective differential culture medium for

growing Staphylococcus aureus than in Mannitol Salt Agar.

SIGNIFICANCE OF THE STUDY

In this study, the researcher will be able to produce a substitute

Mannitol for differentiating Staphylococcus aureus from other Gram

The result will be focused on the following benefits:

The Researchers. They will be able to enhance their observing skills

in terms of experimental research design and in discovering new culture

medium that can provide nutrient and differentiate Staphylococcus aureus

from other Gram positive bacteria.

The Future Researchers. They can use this study as a guide in

pursuing a deeper study.

The Medical Technology Students. They will be knowledgeable

about the capability of fruits and vegetables to provide nutrient and

Registered Medical Technologist. They will be able to increase

their knowledge in making a substitute culture medium that are used to

differentiate different types of microbes.

SCOPE AND LIMITATION OF THE STUDY

This study is focused in identifying the effectiveness of Cauliflower

as a substitute for Mannitol in Mannitol Salt Agar on isolating

Staphylococcus aureus only. This study was conducted at Wesleyan

University-Philippines Medical Technology Laboratories on month of

March and April 2018.

The researcher limited the study on observation and documentation

to obtain the desired result. Determination of the specific component