ALUGBATI(Basella alba) BERRIES AND GUMAMELA (Hibiscus rosa sinensis) FLOWER EXTRACTS AS ALTERNATIVE DYES FOR GRAM STAINING research paper

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CHAPTER I

INTRODUCTION

Gram staining has been widely used in medicine to diagnose certain diseases by

identifying their causative agents. Crystal violet and Safranin, both dyes used in the Gram

staining method, are synthetic dyes. They give purple and red color, dividing bacteria to Gram

positive and Gram negative making identification easy. Through this, doctors can diagnose

correctly and can provide an efficient treatment. However, both of these synthetic dyes have

toxic effects to people who are constantly in contact with them. Most of these people at risk are

medical technologists.

According to Brit (2008), synthetic dyes contain the chemical Aniline which is the basis

for Azo dyes and is considered as a deadly poison, giving off carcinogenic amines. Crystal violet

and Safranin are both classified as IARC Group 1: carcinogenic to humans ( International

Agency for Research on Cancer of the World Health Organization, 2016). Crystal violet is

considered a hazardous substance in the statement of hazardous nature of the Occupational

Safety and Health Administration 29 Code of Federal Regulation 1910.1200 (Chemwatch, 2010).

Safranin red has also been found to be carcinogenic and can cause malformations in embryo or

fetuses of pregnant women (Summers, Texley, & Kwan, 2006).

Because of this, a lot of people are now leaning towards using natural dyes as they are

less toxic, less health hazardous, non-carcinogenic, and non-poisonous (Roberts, 2015). Plants

have anthocyanins that are responsible for the bright attractive red, orange, purple and blue

colors of most fruits and vegetables (Giusti, 2008). In the Philippines, plants like Alugbati

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(Basella rubra) and Gumamela (Hibiscus rosa sinensis) have been found out to be potential dyes

for biological staining as they both contain anthocyanins.

Deshmukh and Gaikwad (2013) used Alugbati berries as a biological stain for plant

nuclei and organelles. Alugbati berries extract turned out to be a favorable substitute for crystal

violet (Enerva, Utilization of an Indigenous dyestuff from Basella rubra (alugbati) as

microbiological stain, 2006; Stuart, 2015). On the other hand, Gumamela flower extract has been

found out to be an alternative dye for eosin (Ampana, et al., 2013). Another similar study by

Castor, Fabile, Jimenez, San Jose, Sumarago et al (2014) used Alugbati extract as primary stain,

kalamansi extract as mordant and Gumamela flower extracts as counter stain as alternative for

Gram staining and established a conclusion that these were comparable to Gram stain.

With these new developments, the proponents of this study aim to find safer, alternative

stains for the Gram staining using Alugbati berries and Gumamela flowers as primary and

secondary stains, respectively and to find locally-made dyes from natural and organic materials.

The researchers will perform this experiment with the recommendation of increasing

concentrations of both extracts to improve staining and color.

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Review of Related Literature

Gram Stain

Nowadays, Gram staining is the principal stain used for the microscopic examination of

clinically important bacteria. This was first devised by Hans Christian Gram in the late

nineteenth century, and has been used until now to divide most bacterial species into two large

groups: gram-positive, – those that take up the basic dye, crystal violet and gram-negative, –

those that allow the crystal violet dye to be washed out easily with the decolorizer alcohol such

as acetone and take up the red dye, safranin red. Its principle lies on the thickness and

composition of the bacterial cell walls. Gram-positive cell walls contain thick peptidoglycan with

numerous teichoic acid cross-linkages, while gram-negative cell walls contain a thinner layer of

peptidoglycan. The teichoic acid cross-links contribute to the ability of gram-positive organisms

to resist alcohol decolorization. Gram reaction coupled with the determination of cell size,

morphology, and arrangement are key aspects in accurately identifying bacteria (Brooks, 2013;

Forbes, Sahm, & Weissfeld, 2007).

Stains used in grams staining are both basic dyes, therefore carrying positive ions. The

positive ion in a basic dye is attracted to the slightly negative charged bacterial cell (Tortora,

Funke, & Case, 2002).

Crystal Violet as Primary Stain

Crystal Violet is a triarylmethane dye which can be used as a primary dye in Gram

staining to facilitate classification of bacteria or for simple histological staining procedure.

Furthermore, Crystal Violet also has antibacterial, antifungal and anti-helminthic properties and

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was used in the past as a topical antiseptic. The medical use of the dye has been superseded by

more modern drugs, although it is still listed by the World Health Organization (Crystal Violet,

2013).

Consequently, various studies have reinforced that Crystal Violet has a hazardous effect

to humans and the environment. It has been reported as a recalcitrant dye molecule that persists

in environment for a long period of time and can pose toxic effects. It acts as mitotic poison,

potent carcinogen and a potent clastogene promoting tumor growth in some species of fish.

Crystal Violet is thus regarded as a biohazard substance (Mani & Bharagava, 2016). It is also

considered a hazardous substance in the statement of hazardous nature of the Occupational

Safety and Health Administration 29 Code of Federal Regulation 1910.1200 (Chemwatch, 2010).

In one study, it demonstrated dose-related carcinogenic potential at several different organ sites

in mice (The Carcinogenic Potency Project, 2007; Littlefield, Blackwell, Hewitt, & Gaylor,

1985).

Safranin as Secondary Stain

Safranin (also Safranin O or basic red 2) is a biological stain employed in microbiology,

histology and cytology. It is used as a counter stain in some staining protocols, imparting color to

all cell nuclei red. This is the classic counter stain in both Gram stains, and endospore stains. It

can also be used for the detection of cartilage, mucin and mast cell granules. Safranin typically

has the chemical structure sometimes described as Dimethyl safranin. There is also

Trimethylsafranin, which has an added methyl group in the ortho- position of the lower ring.

Both compounds behave essentially similar in biological staining applications, and most

manufacturers of safranin do not distinguish between the two. Commercial safranin preparations

often contain a blend of both types. Safranin is also used as redox indicator in analytical

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chemistry (Rosenberg, 1971).

Safranin is also known by the names of safranin O, safranin Y, safranin T, safranin A,

basic red 2, gossypimine and cotton red. It consists of dark red crystals available as a dimethyl or

trimethyl derivatives; both show the same staining properties. Commercial safranin stains may

contain either of these two or a combination of both derivatives. Futhermore, it also used in

histological and cytological staining procedures to stain cellular nuclei red and used as a

component in the Flemming triple stain to stain chromosomes. Safranin has also been used in the

staining of cartilage, where the nuclei appear red against a pink background (Stewart, 2006).

The stain can cause both skin and eye irritation, and may be harmful if swallowed,

absorbed through the skin, or inhaled. Safranin O is also considered an irritant of mucous

membranes and the upper respiratory tract, but is not flammable or explosive under typical

laboratory conditions (Davidson, 2015). It is also found to be carcinogenic and causing

malformations in embryo or fetuses of pregnant women (Summers, Texley, & Kwan, 2006). In

addition, exposure to these chemicals can be irritating to the skin, harmful to the respiratory

system when inhaled, and to the digestive tract when ingested (Mohammed, Ibrahim, & Shitu,

2014).

Natural Dyes

Plants containing anthocyanins are responsible for the bright attractive red, orange,

purple and blue colors of most fruits and vegetables (Giusti, 2008). Anthocyanins are versatile

flavonoid pigments found in red/purplish fruits and vegetables, hence, have a potential of

becoming a natural dye for staining. This pigment occurs naturally in plants in the form of

glycosides, in which an anthocyanidin molecule is paired with a sugar. The part of the pigment

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that exists free of sugar (generically known as aglycone) is called an anthocyanidin (Webb,

2014). The most common sugars present in these natural pigments are glucose, fructose,

galactose, xylose, arabinose and rhamnose (Tazzini, 2014).

Anthocyanin changes color, ranging from red, under very acidic conditions to purple-

blue, in intermediate pH conditions until yellow-green, in alkaline conditions. In addition to the

pH, the color of these flavonoids can be affected by the degree of hydroxylation or methylation

pattern of the A and B rings, and by glycosylation pattern (Tazzini, 2014). At pH 8, anthocyanin

remaind purple color and then changed to violet, blue respectively when pH rose from 9-11 due

to the presence of quinoidal ion (Nghia, 2014). To obtain the best yield of anthocyanin

extraction, weak organic acids such as acetic acid and citric acid and low concentration of strong

acids such as hydrochloric acids at less than <1% (Dai & Mumper, 2010), are used.

Anthocyanin is rapidly degraded in the presence of light and high temperature

(Bakowska-Barczak, 2005). Therefore, crude extracts which will be obtained in this study will be

stored in amber bottles to prevent interference of light, and refrigerated at 4˚C so as to prevent

anthocyanin degradation (Zozio, Pallet, & Dornier, 2011).

Alugbati (Basella alba) berries

Figure 1. Basella alba (Alugbati) BerriesRetrieved: January 26, 2016 from https://en.wikipedia.org/wiki/Basella_alba

https://en.wikipedia.org/wiki/Basella_alba

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Local Name: Alugbati

Kingdom- Plantae

Phylum- Magnoliophyta

Class- Magnoliopsida

Order- Caryophyllales

Family- Basellaceae

Genus- Basella

Species- alba

(Adhikari, Kumar, & Shruthi, 2012)

Alugbati is a succulent, branched, smooth, twining herbaceous vine, several meters in

length. The fruit is fleshy, stalkless, ovoid or nearly spherical, 5 to 6 millimeters long, and purple

when mature. Phytochemical screening of various extracts yielded chemicals, specifically

anthocyanin. This chemical is a water-soluble vacuolar pigment that may appear red, purple, or

blue depending on the pH (Stuart, 2015).

There are four varieties of alugbati. One, Basella rubra has purple pigment on the stem,

leaves and as well as the petioles. The leaf is ovate with a cordate base. Two, Basella alba has

green stems, leaves and petioles. The leaf is ovate and cordate at the base. Three, Basella

cordifolia has a green stem, leaves and petiole with elongated heart shaped or cordate leaves.

This type has pigmentation on the node as well as the base of the petioles and the first two

internodes above the soil level. Four, Basella alba variety has green stem, leaves and petioles

with oval to almost round leaves (T.A. & Mabel, 2015).

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It is considered an important green leafy vegetable found commonly in the tropical

regions of the world and also has great ethno-medicinal importance. The plant is rich in Vitamin

A and Vitamin C along with flavonoids, saponins, carotenoids, many amino acids and organic

acids. It is known to be androgenic, anti-diabetic, anti-inflammatory, antimicrobial, antioxidant,

antiulcer, antiviral, central nervous system depressant, hepatoprotective and can heal wounds.

Also, the plant possesses a valuable ethno-medicinal importance and is used to cure

digestivedisorders, skin diseases, bleeding piles, diarrhea and many more (Deshmukh &

Gaikwad, 2013).

Gumamela (Hibiscus rosa sinensis) flower

Figure 2. Hibiscus rosa sinensis (Gumamela) flowerRetrieved: March 15, 2016 from http://goo.gl/UZnnJE

Scientific Name: Hibiscus rosa-sinensis

Local Name: Gumamela

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Malvales

Family: Malvaceae

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Genus: Hibiscus

Species: rosa-sinensis

(Chua, 2010)Gumamela is a shrub that grows from 1 meter up to 4 meters high. It is also known as

Hibiscus, China Rose, and Shoeflower. In the Philippines, it is cultivated as an ornamental plant

and it comes in many colors: red, yellow, orange, white, purple, pink and other color

combinations (Philippine Herbal Medicine, 2). Its flowers are solitary, axillary, very large about

10 cm long and 12 cm in diameter. Petals are obovate, entire, rounded tip and imbricate. Flowers

are best collected from May to August (Stuart, 2015).

Gumamela contains taraxeryl acetate, beta-sitosterol, campesterol, stigmasterol,

ergosterol, lipids. Arachidic acid, behenic acid, oxalic acid, palmitic acid, octanoic acid, stearic

acid, sterculic acid, tricosanoic acid, tridecanoic acid, undercanoic acid, citric acid, tartaric,

fructose, glucose, sucrose, flavonoids and flavonoid glycosides, hibiscetin, alkanes,

hentriacontanecyanidin, cyanidin chloride and cyaninglucosides (Lim, 2014).

Test Organisms

Escherichia coli (E. coli)

Figure 3. Escherichia coli (E. coli)Retrieved: July 8, 2016 from Jawetz, Melnick & Adelberg's Medical Microbiology 25th Edition

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Escherichia coli is a Gram-negative, facultative anaerobe bacilli or rod-shaped, normally

found in the intestines (Hale, 2013). E. coli will take the color red after the addition of safranin

since the crystal violet is decolorized by the acetone alcohol (Brooks, 2013). E. coli bacteria

were discovered in the human colon in 1885 by German bacteriologist Theodor Escherich. He

showed that certain strains of the bacterium were responsible for infant diarrhea and

gastroenteritis (Marler & Clark, 2016).

Staphylococcus aureus (S. aureus)

Figure 4. Staphylococcus aureus (S. aureus) Retrieved: July 8, 2016 from Jawetz, Melnick & Adelberg's Medical Microbiology 25th Edition

Staphylococcus aureus is a gram positive, non-moving small round shaped or non-motile

cocci, usually found in grape-like (staphylo-) clusters. S. aureus retain the crystal violet-iodine

complex thus remaining blue then becoming purple after addition of safranin (Brooks, 2013).

S. aureus was discovered in Aberdeen, Scotland in 1880 by the surgeon Sir Alexander

Ogston in pus from surgical abscesses. It belongs to family Staphylococcaceae. Staphylococcus

is one of the five most common causes of infections after injury or surgery. It affects around

500,000 patients in American hospitals annually. It affects all known mammalian species,

including humans. Furthermore, due to its ability to affect a wide range of species, S. aureus can

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be readily transmitted from one species to another. This includes transmission between humans

and animals (Mandal, 2012).

Related Studies

Lin et al., (2010) studied structural identification and bioactivities of red violet pigments

present in Basella alba fruits and clarified that the fruit possessed gomphrenin-I as major red

pigment along with betanidin dihexose, betalins and isobetanidin dihexose. The study also

reported antioxidant properties and anti-inflammatory function of gomphrenin I and mentioned

the potential use of fruit in the development of food colorants and nutraceuticals.

Amon & Pladio (2012) studied Basella rubra fruit extracts for their potentiality as a food

colorant and that the extract showed the presence of anthocyanin and also exhibited DPPH

radical scavenging activity. The extract was proved to be non toxic and can also be implied in

food coloring industries to impart reddish colour.

In a study by Obi and Uneh (2003) and Obi, Osenu and Osayande (1998), gumamela

petals were treated with formic acid, ethanol and water as extracting agents for anthocyanin.

Futhermore, in the study conducted by De Leon, Latoza, Nues, Pilac, Soliman, Sistoza (2016),

alugbati was extracted by adding 5ml of methanol to 5grams of berries, macerated and then

centrifuged at 3000 rpm for 3-5 minutes. Another study used 80% ethanol to extract air-dried

Rhus coriaria L. berries (Abu-Shanab, Adwan, Abu-Safiya, Adwan, & Abu-Shanab, 2005) and

air-dried Moringa pterygosperma Gaertn flowers (Bargah, 2015) using 95% ethanol

(Maisuthisakul, Pasuk, & Ritthiruangdej, 2008).

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Alugbati extract turned out to be comparable with synthetic stains in a study conducted

by Enerva (2006) where alugbati berries were macerated in a blender and extracted with 1% HCl

in 95% methanol and then filtered, the crude extract was used as to stain for S. aureus and E.coli.

Ampaña et al (2013) found out that Gumamela could be an alternative dye for eosin. Castor et al

(2014) used Alugbati extract as primary stain, kalamansi extract as mordant and Gumamela

flower extracts as counter stain as alternative for Gram staining using 100 grams of berries and

gumamela petals and extracted it using 50ml of 95% methanol and 50ml of 1% Hydrochloric

Acid. Results of their study were comparable to Gram stain but increasing concentration was

recommended to prolong the stain as that of the gram stain.

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Conceptual Framework

Independent Variable Dependent Variable

Staining ability of alugbati + gumamela

1. Retention of staina. 1 day after

stainingb. 2 days after

stainingc. 4 days after

staining

2. Morphologic characteristic of the stained organisms in terms of:a. colorb. shape

Experimental GroupDyes & Concentration

A. Alugbati berries extract (primary dye) and Gumamela flower extract (secondary dye)

a. 50% concentrationb. 60%concentrationc. 75%concentration

B. Time Exposure

Primary Dye a. 1 minuteb. 3 minutesc. 5 minutes

Secondary Dye a. 30 secondsb. 1 minutec. 2 minutes

Test OrganismsA. Staphylococcus aureusB. Escherichia coli

Positive ControlOriginal Gram Stain

Negative controlA. No primary and / or secondary

stain

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Figure 5. Relationship of Variables

Figure 3 shows the relationship of the variables that will be used in the study. Alugbati

berries and Gumamela flowers crude extracts as the independent variable while the standard

Gram stain will serve as the positive control and no application of primary and secondary dye

will serve as the negative control. The dependent variable for this study is the staining ability of

alugbati and gumamela extracts in terms of stain retention and morphological characteristics of

the test organisms.

Statement of the Problem

1. What is the retention capacity of Alugbati berries crude extract and Gumamela flower

crude extract as compared to the positive control after:

a. 1 day

b. 2 days

c. 4 days?

2. What is the resulting color and shape of Escherichia coli and Staphylococcus aureus

when exposed to the different concentrations of Alugbati berries crude extract at different

time exposure of

a. 1 minute

b. 3 minutes

c. 5 minutes?

3. What is the resulting color and shape of Escherichia coli and Staphylococcus aureus

when exposed to the different concentrations of Gumamela flowers crude extract at

different time exposure of

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a. 30 seconds

b. 1 minute

c.[b.] 2 minutes?

4.[3.] Is there a significant difference in the retention of Alugbati berries extract and

Gumamela flower crude extract compared to the positive control?

5.[4.] Is there a significant difference in the resulting color and shape of Escherichia coli and

Staphylococcus aureus at different concentration and time exposure to experimental dyes

with that of the positive and negative control?

[5.]

Hypothesis

HA1 The retention of Alugbati berries extract and Gumamela flower crude extracts on the test

organisms are comparable to that of the positive control.

HA2 The color and the shape of Escherichia coli and Staphylococcus aureus are comparable to

that of the positive control.

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CHAPTER II

METHODS

Presented in this chapter are the research design, locale, subjects, measures and

procedures that will be used in the study.

Research Design

This study utilizes an experimental design to determine the efficiency of Alugbati

(Basella alba) berries and Gumamela (Hibiscus rosa sinensis) flower crude extracts dyes as

alternative primary and secondary stain, respectively, for Gram staining in the context of its

characteristic morphology in terms of color and shape. A positive and negative control will be

used and outside variables like temperature will be controlled as not to affect the result of this

study. Making sure that exposure of the experimental treatments, positive and negative control

will be the same throughout the duration of the experiment.

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Setting

The preparation and extraction of plant will be executed in a Dominican Learning

Community founded by Dominican Sisters at Clovis Thibault building, room 306 of San Pedro

College located at C. Guzman Street, Davao City.

Test Organisms

The test organisms that will be used in the study are Staphylococcus aureus as the gram

positive bacteria and Escherichia coli as the gram negative bacteria. The organisms will be

purchased from the Department of Science and Technology, Davao City.

Plant Subjects

Alugbati berries should be ripe and purple. Gumamela flowers should be in bloom and

petals are red. Plants will be purchased in an Alugbati garden in Barangay Mua-an, Kidapawan

City and a Gumamela Farm in Tugbok, Davao City. The requirements needed for Alugbati

cultivation are the following: adequate water supply; soil rich in organic matter; and hot, humid

climate (Holmer, et al., 2008). Barangay Mua-an, Kidapawan City is a tropical environment with

mild climate change and the garden soil is filled with organic matter from decaying sawdust and

is near from a water source which is suitable for the growth and development of the Alugbati

plant (Patricio Jr., 2013). Meanwhile, the requirements needed for cultivation of Gumamela

flowers are the following: full sun, loamy soil, and adequate water supply and water should not

be in excess to prevent rotting of roots (Forsling, 2016). Tugbok, Davao City has a humid

atmosphere and provides an adequate water supply. The area has an open field that ensures the

plant’s sustenance of light which is highly recommended for the plant growth and development

(Diansay, 2003).

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Measures

The test would be measured through grading the degree of retention of the stain and

similarity on the cell morphology, in terms of shape and cell color which is microscopically

determined with the positive control. Tool or criteria in grading seen in Appendix D.

Procedure

Plant collection (Alugbati berries and Gumamela flowers)

Purchase and verification of test organisms

(Staphylococcus aureus and Escherichia coli)

Plant extraction

Plant authentication

Submission of transmittal letters

Experimentation Proper

SmeSmearingSubculture Staining

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Figure 6. Schematic Diagram showing the protocol of the study

Pertinent letters will be submitted to the laboratory stockroom head Helen J. Ancla, and

to the BMLS Department Dean, Dr. Josephine Bandalan, by the researches to ask permission for

the conduct of the study in this institution. A letter of request will also be given to the

Department of Science and Technology for the purchase of the test organisms and to the farms

where the plants will be purchased (See Appendix A).

Plant Material Collection

The berries of the Alugbati and the flowers of Gumamela plant are the particular parts

needed for this study. Plant parts will be picked by hand and placed in a black plastic bag

container to prevent exposure to direct sunlight and cooled so as not to accelerate degradation of

anthocyanin (Bakowska-Barczak, 2005). The black plastic bag containing the berries should be

put in a plastic container and transported in a Styrofoam box with an ample amount of ice to

prevent chilling or freezing injury. (Vigneault, Thompson, Wu, Hui, & LeBlanc, 2009). Black

plastic bags containing gumamela flowers should be placed in a plastic container, properly

arranged to avoid damage of the petals, and transported in a cooler. Low temperature and high

humidity is important to reduce water loss and maximize shelf life (Nell, 2012). To maintain

Analysis of Data Gathered

Gathering and Evaluation of Data

Waste Disposal and Management

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humidity, flowers shall be sprayed constantly with a fine spray of water since the effect is

temporary (Carroll, 2015).

Plant authentication

Plants will be authenticated at Ateneo de Davao University Biology Department, Davao

City. (See Appendix B)

Plant extraction

The researchers will prepare the Alugbati berries and Gumamela flower petals for plant

extraction. Both plant parts will be air dried and pulverized using a mechanical grinder.

The powdered Alugbati berries will be macerated for 24 hours with 80% ethanol and

then filtered. Filtrate will undergo rotary evaporation to remove ethanol. The extract obtained

will be dried and stored in amber bottles (Abu-Shanab, Adwan, Abu-Safiya, Adwan, & Abu-

Shanab, 2005) at 4˚C (Zozio, Pallet, & Dornier, 2011). The amber bottles will protect the crude

extracts from possible chemical alterations by light and the low temperature will prevent

degradation of anthocyanin (Bakowska-Barczak, 2005).

Powdered gumamela flowers will be macerated with 95% ethanol (Maisuthisakul,

Pasuk, & Ritthiruangdej, 2008) and properly sealed with aluminium foil to prevent

contamination and left for 72 hours. The solution will then be filtered and the extracts obtained

will be concentrated using rotary evaporator. The extracts will be stored in amber bottles and

refrigerated (Bargah, 2015) at 4˚C (Zozio, Pallet, & Dornier, 2011). (See Appendix C)

Perform Iodoform Test to confirm the removal of ethanol to both extracts (See Appendix

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C). Dilute the extracts with distilled water in different concentrations (50%, 60%, 75%) when

needed.

Qualitative Phytochemical Screening for Anthocyanin

Presence of anthocyanins can be demonstrated by adding 2mL of 2 N HCl to 2mL

aqueous plant extract. The appearance of pink-red color that turns purplish blue after adding

ammonia indicates presence of anthocyanin (Obouayeba, Diarrassouba, Soumahin, & Kouakou,

2015; Harsha, Sridevi, Chandana Lakshmi, Rani, & Divya Satya Vani, 2013).

Purchase and Verification of Test organisms

The organisms will be purchased from the Depatment of Science and Technology, Davao

City.

Experimentation Proper

Subculture

The Blood Agar and MacConkey Agar plates will be used to culture Staphylococcus

aureus and Escherichia coli species respectively. The Agar plates will be prepared by following

the manufacturer’s protocol. Bacteria from the broth culture will be inoculated to the Agar plates

using the Quadrant Streak Technique. The prepared subcultures will be incubated for 24 hours at

37̊ C (See Appendix C).

Smear preparation

Standard smearing technique from broth culture will be followed (See Appendix C).

Staining proper

For the preparation of the positive control, standard procedures for Gram staining will be

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followed. (See Appendix C).

For the alternative staining procedure, the Gram stain protocol will still be adapted but

the standard dyes, Crystal violet and Safranin, will be replaced by Alugbati crude extracts and

Gumamela extracts respectively. Gram’s Iodine and Acetone Alcohol will still be used. (see

Appendix C).

Evaluation of stained smears

Stained smears will be evaluated by registered medical technologists or bacteriologists. They

will be given a check list on how to evaluate and compare the morphology and staining reaction

of the test organisms after exposure to experimental stains against the positive and the negative

controls. (See Appendix D)

Ethical Considerations

The proponents of this study shall ensure the safety of every individual by following the

school’s laboratory protocol. The researchers shall wear personal protective equipments such as

gloves, masks, head caps, goggles, laboratory gowns and shoes. Test organisms will be placed in

leak-proof containers and properly labelled during transport and storage. Stained slides must not

be drained in the sink, a separate waste bottle for the residue shall be provided. Smearing shall be

done inside the biosafety cabinet. Alcohol lamps will not be put inside the biosafety cabinet.

Heat fixing of the smear will be done outside of the biosafety cabinet. In case of accidents and

other unexpected outcomes such as catching fire of laboratory gowns, spilling of hazardous

reagents to skin or other body parts, the researchers shall immediately notify the mentor or any

laboratory stockroom personnel present to address the problem. Working tables and equipments

should be disinfected using Lysol or bleach and then alcohol. Inoculating loops should be heated

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before and after using as a form of sterilization process. Agar plates and test tubes must be sealed

and will be given to the laboratory stockroom for autoclaving and disposal as stated in the San

Pedro College Laboratory Stockroom Protocol (See Appendix E).

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Amon, M. F., & Pladio, L. P. (2012). Potential Food Colorant from the Extracts of Alugbati (Basella rubra L.). Retrieved February 4, 2016, from http://goo.gl/KjsjD2

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Bakowska-Barczak, A. (2005). Acylated Anthocyanins as Stable, Natural Food Colorants-Review. Polish Journal of Food and Nutrition Sciences, 14/55, 107-116. Retrieved July 8, 2016

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32

APPENDIX ASan Pedro College

Davao City

July 11, 2016

DR. JOSEPHINE BANDALANDean of the Department of Medical Laboratory Science

Dear Dr. Bandalan:

The following undersigned are 4th year students of San Pedro College, Guzman St., Obrero, Davao City as course requirements for Bachelor in Medical Laboratory Science and presently conducting a research entitled: “Basella alba (Alugbati) Berries and Hibiscus rosa sinensis (Gumamela) Flower Crude Extracts as Alternative Dyes for Gram Staining”. The objectives of the study are:

a) To determine the efficiency of Basella alba (Alugbati) Berries and Hibiscus rosa sinensis (Gumamela) flower crude extracts as alternative primary and secondary stain, respectively, for Gram Staining in the context of its characteristic morphology in terms of color and shape.

b) To find locally-made dyes from natural and organic materials that is not carcinogenic.

The proponents of this research study are hereby seeking your consent to allow us to conduct our research in this institution. With your expertise, we are humbly asking your permission to validate the following attachments to this letter to assist you in reaching a decision.

(a) A copy of an ethical clearance certificate issued by the Institution(b) A copy of the research instruments which I intend using in my research

Should you require any further information, please do not hesitate to contact us. Our contact details are as follows:

E-mail: [email protected]

33

Mobile number: 09329740053

Upon completion of the study, the proponents of this research study shall undertake to provide you with a bound copy of the dissertation.

Your permission to conduct this study will be greatly appreciated. Thank you and God Bless!

Respectfully yours,

Aballe, Loreyleine M.Ara, Janessa M.Boctoto, Rejoice D.Dimacuta, Shaquillah S.Estrada, Ian Mark R.Galido, Marie Remcy Kyle B.Millan, Maria Monica C.Mokamad, Aira Ann A.Pacete, Shaira Leigh V.Questo, Irel Anne R.Villegas, Hazel Mae P.BMLS – 4D Research students

Conforme:

Aileen Grace L. Ang, RMT, MAST-Bio, MSMTResearch Adviser/Mentor

34

San Pedro CollegeDavao City

July 11, 2016

MS. HELEN J. ANCLALaboratory Stockroom Head

Dear Ma’am Gustillo:

The following undersigned are 4th year students of San Pedro College, Guzman St., Obrero, Davao City as course requirements for Bachelor in Medical Laboratory Science and presently conducting a research entitled: “Basella alba (Alugbati) Berries and Hibiscus rosa sinensis (Gumamela) Flower Crude Extracts as Alternative Dyes for Gram Staining”.

In connection with this, we would like to ask your help to provide the necessary list of things needed for conducting our experiment which is attached to this letter. We would like to appreciate your assistance and support in this particular research endeavor.

We are hoping for your positive response. Thank you and God bless!

Respectfully yours,

Aballe, Loreyleine M.Ara, Janessa M.Boctoto, Rejoice D.Dimacuta, Shaquillah S.Estrada, Ian Mark R.Galido, Marie Remcy Kyle B.Millan, Maria Monica C.Mokamad, Aira Ann A.Pacete, Shaira Leigh V.Questo, Irel Anne R.

35

Villegas, Hazel Mae P.BMLS – 4D Research students

Conforme:



July 11, 2016

DR. ANTHONY C. SALES, CESO IIIRegional DirectorDepartment of Science and Technology

Dr. Sales:

Greetings!

We, the 4th year Medical Laboratory Science students of San Pedro College are currently embarking a study on the entitled “Basella alba (Alugbati) Berries and Hibiscus rosa sinensis (Gumamela) Flower Crude Extracts as Alternative Dyes for Gram Staining”. This is in partial fulfilment of the requirement of the abovementioned course

In this regard, the group would like to seek your permission to inquire and purchase for the availability of the following microorganisms to be used in the study.

Pure culture of Staphylococcus aureus Pure culture of Escherichia coli

We assure you to perform the proper protocol in handling, processing and disposal of such infectious organisms.

We are hoping for your positive response. Thank you and God Bless!

Respectfully yours,

Loreyleine AballeBMLS 4D Group Representative

Noted by:

36


Approved by:

Dr. Anthony C. Sales, CESO IIIRegional Director-Department of Science and Technology


July 12, 2016

DR. OSCAR P. GRAGEDA, MD, PSP, PSMIDChief Pathologist, San Pedro Hospital

Dr. Grageda:

Greetings!

We, the 4th year Medical Laboratory Science students of San Pedro College are currently embarking a study on the entitled “Basella alba (Alugbati) Berries and Hibiscus rosa sinensis (Gumamela) Flower Crude Extracts as Alternative Dyes for Gram Staining”. This is in partial fulfilment of the requirement of the abovementioned course


Culture of Staphylococcus aureus Culture of Escherichia coli

We assure you to perform the proper protocol in handling, processing and disposal of such infectious organisms.


Respectfully yours,

Loreyleine AballeBMLS 4D Group Representative

Noted by:

Aileen Grace L. Ang, RMT, MAST-Bio, MSMT

37

Research Adviser/Mentor

Approved by:

Dr. Anthony C. Sales, CESO IIIRegional Director-Department of Science and Technology


July 11, 2016

To whom it may concern:

Greetings!

We, the 4th year Medical Laboratory Science students of San Pedro College are currently embarking a study on the entitled “Basella alba (Alugbati) Berries and Hibiscus rosa sinensis (Gumamela) Flower Crude Extracts as Alternative Dyes for Gram Staining”. This is in partial fulfilment of the requirement of the above mentioned course.


Alugbati Berries Gumamela flowers

We assure you to perform the proper protocol in handling, transport, processing and disposal of such plants.


Respectfully yours,

Loreyleine AballeGroup Representative

Noted by:


38

APPENDIX B

39

APPENDIX C

Preparation of Blood Agar Plate

1. Suspend 40.0 grams in 1000 mL of distilled water.

2. Heat to boiling to dissolve the medium completely.

3. Sterilize by autoclaving at 15 lbs pressure (121C) for 15 minutes.

4. Cool to 50C and aseptically add 5% v/v sterile defibrinated blood.

5. Mix well and pour into sterile Petri plates. Allow to cool and solidify and invert the dish

which is now available for inoculation (HiMedia Laboratories, 2012)

Preparation of MacConkey Agar Plate

1. Suspend 50 grams in 1 litre of demineralized water by heating in a water bath or in a

current of stream.

2. Autoclaving at 15 lbs pressure (121C) for 15 minutes.

3. ssCool to 50C and aseptically add 5% v/v sterile defibrinated blood.

4. Mix well and pour into sterile Petri plates. Allow to cool and solididy and invert the dish

which is now available for inoculation (Merck, 2016)

40

Subculture

1. Select an agar (Blood Agar Plate for Staphylococcus aureus and MacConkey Agar Plate

for Escherichia coli). Label the plate with the group number and section, test organism

and date.

2. Sterilize the loop by heating it to redness.

3. Obtain a loopful of Staphylococcus aureus and Escherichia coli colony from their

respective broth cultures and perform Quadrant Streaking. Avoid air contamination by

lifting the lid just enough to permit entry of the loop.

4. Replace the lid then sterilize the loop to destroy remaining bacteria.

5. Pick another sterilized loop. Pass the loop one time across the previous streak to pick up

some bacteria and continue streaking into the second quadrant. Distance of the streak

from the previous should be a bit farther. Replace the lid and sterilize the loop.

6. Pick another sterilized loop. Pass the loop one time across the previous streak and streak

to the next area. Streaks should be far now to obtain isolated and separated colonies.

Replace the lid and sterilize the loop.

7. Invert the plate and incubate for 24 hours at 37°C.

41

Quadrant streak

1. Loosen the cap of the bottle containing inoculums.

2. Hold an inoculation loop in your right hand.

3. Flame the loop and allow it to cool.

4. Lift the test tube containing inoculums with your left hand.

5. Remove the cap/ cotton wool plug of the test tube with the little finger of your right

hand.

6. Flame the neck of the test tube.

7. Insert the loop into the culture broth and withdraw. At all times hold the loop as still

as possible.

8. Flame the neck of the test tube again.

9. Replace the cap/cotton wool plug of the test tube using little finger of your right

hand. Place the test tube in a rack.

10. Partially lift the lid of the perti dish containing solid medium. Touch an unused part

of the agar surface close to the periphery of the plate and then drag it several times

across the first quadrant.

11. Close the petridish and reflame and cool the loop.

12. Turn the dish 90 degrees clockwise then the touch the loop on a corner of the culture

in quadrant 1 and drag it several times across the agar in quadrant 2, hitting the

42

original streak a few times.




original streak a few times.




original streak three times.


18. Incubate the plate in inverted position in an incubator for 24-48 hours (Ministry of

Human Resource Department, 2011).

43

Plant Extraction

Alugbati berries extraction

1. Prepare 1 kilogram of the Alugbati berries. Wash thoroughly to remove debris. Air dry the

berries under a shade.

2. Pulverize the dried berries using a mechanical grinder. Deposit the pulverized berries in a

large 4-liter capacity liquid container. Note: Transparent container should be wrapped in

black cellophane to prevent possible degradation of the extract by light.

3. Macerate powdered berries in 4 liters of Ethanol. This acts as the extracting agent. Store

mixture away from light at 4̊ C for 24 hours.

4. Carefully filter the contents through a Whatman No. 2 filter paper under suction using a

Buchner funnel.

5. Perform Rotary Evaporation to the filtrate.

6. Measure the resulting extract t in a weighing scale.

7. Store in Amber-colored bottles at 4̊ C away from light. (Abu-Shanab, Adwan, Abu-Safiya,

Adwan, & Abu-Shanab, 2005)

Gumamela flowers extraction

1. Gumamela flowers will be air dried at room temperature followed by pulverization to

powder using a mechanical grinder.

44

2. Weigh 500 grams of the powdered gumamela flowers and mix with 1500 mL of ethanol

then filter.

3. Cover the container with aluminium foil to prevent contamination and macerate for 72

hours at 4˚C.

4. The solution was then filtered using funnel and filter paper.

5. The extracts obtained will be concentrated using rotary evaporator.

6. Extracts will be stored in amber bottles and stored at 4˚C (Bargah, 2015).

45

Rotary Evaporator Procedure

1. Remove the round bottom flask from the base of the condenser, directly above the hot

bath. Inspect the flask to ensure it is clean.

2. Load your sample into the cleaned round bottom flask.

3. Attach the round bottom flask to the condenser. If need be, use vacuum grease located

next to the rotary evaporator to create a vacuum seal between t he condenser and the

round bottom flask. Be sure to use a Keck clamp to secure the round bottom flask to the

condenser.

4. Inspect the collection flask located to the left of the hot bath, and below the condenser.

Make sure the flask is clean before use.

5. Open the cabinet doors directly below the rotary evaporator. Since the chiller is turned

sideways, the back of the chiller is on the right side of the cabinet and the front of the

chiller is located on the left side of the cabinet.

6. Make sure the tubing is securely connected to the back of the chiller and the condenser.

7. Turn on the chiller using the power switch located in the back of the chiller. The

condenser should fill with water, if not already full.

8. The chiller is normally set to 20 °C. If you need to change this temperature for some

reason, do not set it below 15 °C. To change the temperature, rotate the knob located on

the front of the chiller.

46

9. Make sure the vacuum pump located next to the rotary evaporator is connected to the

condenser. Make sure the release valve at the top of the condenser is turned to the closed

position.

10. Turn on the vacuum. The gauge on the vacuum pump should read 27 in Hg if the system

is not leaking.

11. Fill the hot bath with enough water for the round bottom flask to sit it.

12. Lower the round bottom flask into the hot bath by either turning the condenser column, or

lowering the entire setup.

a. To turn the condenser, locate the dark grey knob on near the top of the support. Turn

the knob counter clockwise until the knob pops out about an inch. Gently turn the

condenser with your hands to the satisfactory position, then push the grey knob back

in, and turn clockwise to lock it back into place.

b. To lower the entire setup, press the power button on the rotary evaporator control

panel. Use the Up and Down keys to raise or lower the setup.

13. Turn on the hot bath using the switch located on the right side of the bath towards the

back. Use the dial on the hot bath control panel to set the temperature of the hot bath.

Since water is typically present in the hot bath, the heating system will not heat above

100 degrees.

14. If you have not already turned on the rotary evaporator, press the power button on the

rotary evaporator control panel.

15. Use the dial on the rotary evaporator control panel to set the desired spin speed of the

condenser.

16. If the desired evaporation should be timed, press the timer button on the rotary evaporator

47

control panel and use the dial to set a time in minutes, or press the into button and use the

dial to set a time in seconds.

17. When all the specifications have been made, press the dial on the rotary evaporator

control panel in. The rotary evaporator will begin to rotate.

18. To stop the rotary evaporator, push the dial on the rotary evaporator control panel in

again, or wait until the specified length of time passes. The rotary evaporator will stop

spinning then.

19. If the separation was successful, the higher boiling point substance will be left in the

original round bottom flask, and the lower boiling point substance will be collected in the

collection flask.

20. Press the power button to turn the rotary evaporator off. If the entire system was lowered

in step 12b, the system will raise to its original height.

21. Turn off the hot bath using the switch on the right hand side, near the back. Make sure the

temperature dial is set to zero.

22. Turn off the chiller using the power switch located in the back of the chiller.

23. Turn off the vacuum pump.

24. Slowly vent the system by turning the release valve at the top of the condenser to the

open position. Make sure the round bottom flask is cool before removing it from the

condenser.

25. Remove your product from the round bottom flask and clean the flask.

26. Remove the collection flask from the condenser setup. Remove your product from the

collection flask and clean the flask.

27. Replace the collection flask and the round bottom flask for the next user.

48

28. If the area surrounding the chiller is cool enough, close the wooden cabinet doors.

29. If the area surrounding the chiller is not cool enough, leave the doors open, and someone

will close them (Sepos, 2012).

Iodoform test

1. Put 4 drops of unknown into the test tube

2. Add to this 0.5ml distilled water

3. Add 0.25ml 6M NaOH and 0.25ml of water

4. Add 5 drops of KI3 solution

5. Note observations at beginning and after 10 minutes

6. Do not collect precipitate

Result:

Postive Test= Yellow cloudy bottom layer

Negative Test= white cloudy, clear (Ladziata, 2007)

Qualitative Phytochemical Screening for Anthocyanin

1. Prepare 2 mL of the aqueous plant extract.

2. Add same amount of 2N HCl. An appearance of pink-red color should be visible.

3. Add ammonia by drops to the solution. A change of color to purplish blue indicates

presence of anthocyanin (Obouayeba et al, 2015; Harsha et al, 2013).

49

Smear preparation

a. Clean the glass slide using lens paper to remove dirt, dust or other contaminants. And

label each slide with the test organism.

b. Sterilize the inoculating loop by heating to redness the entire wire portion over the

flame. Allow to cool before use.

c. Place a drop of sterile normal saline solution on a clean glass slide by means of the loop.

d. Sterilize the loop and pick-up a small amount of bacterial growth by touching lightly

upon the surface colony and transfer to the slide.

e. Emulsify and spread to about 1 square centimetre area. Re-sterilize the inoculating loop.

f. Allow the smear to dry.

g. Fix the dried smear by passing the slide rapidly just over the flame, film side up, 2 or 3

times. This prevents the film from being washed away and also kills the bacteria. Avoid

excessive heating since it will alter the composition of the organisms.

50

Staning Proper

A. Gram’s Stain

1. Prepare and fix the specimen to the microscope slide before staining.

2. Cover the smear with crystal violet, the primary stain, for 1 minute

3. Gently rinse off the stain with water.

4. Cover the smear with Gram’s iodine, the mordant, for 1 minute.

5. Pour off the excess Gram’s iodine.

6. Run the acid-alcohol decolorizer over the smear until the solution appears clear.

7. Gently rinse with water.

8. Cover the smear with safranin, the secondary or counter stain, for 20 seconds.

9. Gently rinse the stain with water.

10. Blot dry with bibulous paper. (Brooks, 2013)

Expected Results:

Gram-positive organisms will appear dark purple to a deep blue.

Gram-negative organisms will appear pink to a deep magenta. (Tille, 2014)

B. Alternative Stains

a) Stain the heat-fixed smear with the Alugbati berries dye for one minute and wash gently

with running water for not more than 5 seconds.

51

b) Cover the stain with Gram’s iodine for one minute and wash again with running water

for not more than 5 seconds to form a bridge between the cell and initial stain so the cell

will retain the stain.

c) Wash the smear with acetone alcohol for 30 seconds and rinse with running water for

not more than 5 seconds to remove the initial stain.

d) Stain with Gumamela flowers dye for 10-30 seconds and wash gently with running

water for 5 not more than 5 seconds to stain the decolorized cells. Then blot or air dry.

52

APPENDIX DMicroscopic Observations of Gram Stained Bacteria

Name of Evaluator: ________________________________Date of Evaluation: ________________________________CHECKLIST:

A. Organism: Staphylococcus aureus

Alugbati berries extract (primary dye)

andGumamela flower extract

(secondary dye)

Concentration Time exposure to experimental dye

50% 60% 75% Primary Dye Secondary Dye1 minute 3 minutes 5 minutes 30

seconds1 minute 2 minutes

Results: 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0Cell morphology

ShapeCoccus:Bacillus:

Cell color:Red:Purple:

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B. Organism: Escherichia coli

Alugbati berries extract (primary dye)

andGumamela flower extract

(secondary dye)

Concentration Time exposure to experimental dye

50% 60% 75% Primary Dye Secondary Dye1 minute 3 minutes 5 minutes 30

seconds1 minute 2 minutes

Results: 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0 5 3 0Cell morphology

ShapeCoccus:Bacillus:

Cell color:Red:Purple:

Criteria:5 – Strongly comparable3 – Moderately comparable0 – Not comparable

Legend:

Shape E.coli S.aureus5 Rod shape Oval shape3 Irregular rod shape Irregular oval shape0 Unidentifiable Unidentifiable

Color E.coli S.aureus5 Red Purple3 Light pink Light blue0 No color No color

54

Retention of StainA. Organism: Staphylococcus aureus

1 day after staining 2 days after staining 4 days after stainingRetention of stain 5 3 0 5 3 0 5 3 0

a. Clarity/ visibility of stained organism

b. Intensity of color

c. Quality of shape

1 day after staining 2 days after staining 4 days after stainingRetention of stain 5 3 0 5 3 0 5 3 0

d. Clarity/ visibility of stained organism

e. Intensity of color

f. Quality of shape

B. Organism: Escherichia coli

55

Criteria:5 – Strongly comparable3 – Moderately comparable0 – Not comparable

Legend:

Clarity/ visibility of stained organism

E.coli S.aureus

5 Very visible Very visible3 Slightly visible Slightly visible0 Not visible Not visible

Intensity of color E.coli S.aureus5 Complete retention of color as

comparable to the gram stained specimen

Complete retention of color as comparable to the gram stained specimen

3 Partial retention of color as comparable to the gram stained

Partial retention of color as comparable to the gram

56

specimen stained specimen0 No retention of color No retention of color

Quality of shape E.coli S.aureus5 Complete retention of shape as

comparable to the gram stained specimen

Complete retention of shape as comparable to the gram stained specimen

3 Partial retention of shape as comparable to the gram stained specimen

Partial retention of shape as comparable to the gram stained specimen

0 No retention of shape No retention of shape

57

APPENDIX E

Ethical considerations

a. Wearing personal protective equipments such as gloves, masks, head caps, goggles,

laboratory gowns and shoes.

b. Placing test organisms in leak-proof containers and properly labelled during transport

and storage.

c. Rinsing stained slides must not be drained in the sink. Provide a separate waste bottle

for the residue.

d. Smearing inside the biosafety cabinet. Alcohol lamps will not be put inside the biosafety

cabinet. Heat fixing of the smear will be done outside of the biosafety cabinet.

e. Disinfecting working tables and equipments using Lysol or bleach and then alcohol.

Inoculating loops should be heated before and after using as a form of sterilization

process.

f. Sealing and autoclaving at 121°C for 30 minutes agar plates and test tubes containing

bacteria before disposal and will be given to the laboratory stockroom for autoclaving

and disposal.

58

Laboratory stockroom protocol in disposal

a. Place items to be discarded such as culture tubes, culture plates, swabs, toothpicks, wipes,

disposable transfer needles, and gloves, in a biohazard autoclave bag.

b. Submit contaminated agars to the stockroom.

c. Autoclave 30 to 40 minutes at 121° C at 20 pounds of pressure (James, 2008).

Wrap autoclaved agars in a yellow bag.

d. Dispose into a black trash can.

e. CENRO personnel collect waste for disposal.

59

APPENDIX F

Time Table

Month ActivityJuly (week)

1-2

3-4

July 12, Tuesday; Submission of proposal to the ethics committee

Locate possible sources of Alugbati berries and Gumamela flowers in the vicinity

August (week)1-2

3-4

Retrieval of results from the Ethics Committee. Later revisioning.

Plant preparation and extraction (Rotavap) and purchase of organisms, Subculture, Experimentation proper

September (week)1-3

4

Experimentaation proper (continuation)

Gathering and Evaluation of data, Analysis of dataOctober (week)

1-2

3

4

Analysis of data (continuation)

Preparation for defense

Final DefenseNovember

Submission of hardbound copy of Thesis

60

Curriculum Vitae

Personal Information

Name: Loreyleine Moncal Aballe

Age: 19

Gender: Female

Date of Birth: December 22, 1996

Citizenship: Filipino

Marital Status: Single

Father’s Name: Reynaldo L. Aballe

Mother’s Name: Lorellie M. Aballe

Contact Information

Address: St. Michael Village Daliao Toril, Davao City

Telephone: N/A

Mobile: 09329740053


Education

Pre-School: Little Sunbeam Pre-School of Toril

Primary: Don Juan dela Cruz Central Elementary School (2009)

Secondary: Davao Central College (2013)

Tertiary: San Pedro College

Awards and Honors: 1st Honorable Mention (2009 & 2013)

61

Personal Information:

Name: Irel Anne Rebuta Questo

Age: 19

Gender: Female

Date of Birth: September 11, 1996



Father’s Name: Irwin R. Questo

Mother’s Name: Eloisa R. Questo

Contact Information:

Address: B11 L11 Mt. Makiling Street, Dinaville Subdivision, Ma-a Davao City

Telephone: N/A

Mobile: 09997244503


Education:

Pre-School: Rotary Anns Pre-School of Davao

Primary: Kapitan Tomas Monteverde Senior Central Elementary School (2009)

Secondary: Davao City National High School (2013)


62


Name: Shaquillah Salic Dimacuta

Age: 19

Gender: Female

Date of Birth: August 23, 1996



Father’s Name: Tagoranao L. Dimacuta

Mother’s Name: Sittie S. Dimacuta


Address: Sto. Domingo Village 2, Davao City

Telephone: N/A

Mobile: 09233057284


Education:

Pre-School: Project Hope Learning School

Primary: Doña Asuncion Elementary School (2009)

Secondary: Philippine Nikkei Jin Kai International School (2013)


63


Name: Rejoice Dela Cruz Boctoto

Age: 19

Gender: Female

Date of Birth: June 5, 1996



Father’s Name: Rolando P. Boctoto

Mother’s Name: Grace D. Boctoto


Address: Blk 17 Lot 16, Pearl St., Country Homes, Cabantian, Davao City

Telephone: N/A

Mobile: 09053040016


Education:

Pre-School: Project Hope- 2003

Primary: Buhangin Central Elementary School- 2009

Secondary: Cabantian National High School- 2013


64


Name: Ian Mark Ricamara Estrada

Age: 19

Gender: Male

Date of Birth: September 25, 1996



Father’s Name: Winebaldo O. Estrada

Mother’s Name: Maria Dolores R. Estrada


Address: Km. 18, Tibungco, Davao City

Telephone: N/A

Mobile: 09223935458


Education:

Pre-School: Immanuel School of Davao Inc.

Primary: Immanuel School of Davao Inc. (2009)

Secondary: Immanuel School of Davao Inc. (2013)


Awards and Honors: Drummer of the Year Award (2009); Loyalty Award (2013)

65


Name: Janessa Mala Ara

Age: 20

Gender: Female

Date of Birth: August 26, 1995



Father’s Name: Abdulnasser Alongan Ara

Mother’s Name: Noria Mala Ara


Address: Blk 12 Lot 12 Don Isidro Village, Madapo Hills, Davao City

Telephone: 082-222-9030

Mobile: +639162993567


Education:

Pre-School: Lugay-lugay, Cotabato City

Primary: Cotabato City Central Pilot School (2009)

Secondary: Notre Dame-RVM College of Cotabato (2013)


66


Name: Aira Ann Alan Mokamad

Age: 19 years old

Gender: Female

Date of Birth: May 30, 1996



Father’s Name: Abdulrakman A. Mokamad

Mother’s Name: Melinda A. Mokamad


Address: D11 Lacson St. Extension, Bo. Obrero, Davao City

Telephone: (082) 327 2697

Mobile: 09065208798


Education:

Pre-School: Notre Dame-RVM College of Cotabato

Primary: Notre Dame-RVM College of Cotabato (2009)

Secondary: Notre Dame-RVM College of Cotabato (2013)


67


Name: Maria Monica Castillon Millan

Age: 19

Gender: Female

Date of Birth: January 7, 1997



Father’s Name: Rafael Mejos Millan

Mother’s Name: Josephine Castillon Millan


Address: #47 Mercedez Street, Sarphil Village, Davao City; Aguinaldo Street, Poblacion,

Lupon, Davao Oriental

Telephone: N/A

Mobile: 09998031721


Education:

Pre-school: Lupon Christian Learning Center (2003)

Primary: Maryknoll School of Lupon (2009)

Secondary: Maryknoll School of Lupon (2013)


68


Name: Hazel Mae Palmes Villegas

Age: 19

Gender: Female

Date of Birth: October 25, 1996



Father’s Name: Herminio Villegas

Mother’s Name: Ma. Victoria Villegas


Address: 980 Purok 28 Ma-a Davao City

Telephone: 082-244-1752

Mobile: +639996795452


Education:

Pre-School: San Isidro (Batangas City) Elementary School

Primary: Maa Central Elementary School (2009)

Secondary: Davao City National High School (2013)


69


Name: Shaira Leigh Vallejos Pacete

Age: 19

Gender: Female

Date of Birth: November 20, 1996



Father’s Name: Enrico C. Pacete

Mother’s Name: Estrella V. Pacete

Contact Information

Address: 12 D. Guzman Street, Davao City

Telephone: N/A

Mobile: 09101843842


Education

Pre-School: St. Mary’s Academy of Kidapawan

Primary: Kidapawan City Pilot Elementary School (2009)

Secondary: Kidapawan City National High School (2013)


Awards and Honors: First Honor (Pre School); Top 10 (2009); COMELEC President (2013)

70


Name: Marue Remcy Kyle Buenaflor Galido

Age: 19

Gender: Female

Date of Birth: November 20, 1996



Father’s Name: Ricky C. Galido

Mother’s Name: Dinah Marie B. Galido

Contact Information

Address: NHA Bangkal, Phase 1, Block 1, Lot 18

Telephone: N/A

Mobile: 09126865660


Education

Pre-School: Patrick Ryan School

Primary: Salaman Central Elementary School

Secondary: Notre Dame of Salaman College


71

Health & Medicine

ALUGBATI(Basella alba) BERRIES AND GUMAMELA (Hibiscus rosa sinensis) FLOWER EXTRACTS AS ALTERNATIVE DYES FOR GRAM STAINING research paper