Alternative Gram Staining Technique in Identification of Gram-Negative

Microorganism Using Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals

Senior High Thesis Proposal Presented to the

Faculty of Senior High School Department
In DMMC Institute of Health Science
Tanauan City, Batangas

In Partial Fulfillment of this Requirements

For Senior High School

11 - Newton

Shaina Chavez

Felicity Opulencia
Rencelle Arnego
Alieson Ferrer
Joana Dimayuga

2019
 ACKNOWLEDGEMENT

The researchers greatly acknowledge their gratitude and appreciation to the

following people who made the completion and accomplishment of this thesis possible:

To Sir Jon Paul Reyes, the researcher’s thesis adviser, for assisting the researchers

throughout the conduct of the study, bestowing strong support in the experiment, sharing

his knowledge, and imparting inspiration that continually complete this study;

To Sir Menardo Bautista, the researcher’s Practical Research 2 Teacher, for the

encouragement to pursue this study from the start;

To Ms. Michelle Opulencia, the researcher’s Capstone Teacher, for the incessant

support and persistent guidance;

To Ms. Wennie Rose Matira, the researcher’s statistician, for lending her busy

hours and lending an effort to give her guidance in interpreting the outcome of the

experiment;

To the beloved parents, for giving emotional, technical, and financial support that

greatly encouraged the researchers to push through even with difficulties encountered;

And most importantly, to the almighty God, for the blessings, knowledge and

inner strength the researchers highly needed to accomplish this research study.
 DEDICATION

With our deepest love and gratitude, we humbly dedicate this study to

The researchers’ beloved family

The researchers’ thesis adviser Jon Paul Reyes

The researcher’s Research 2 teacher Menardo Bautista

The researchers’ Capstone teacher Michelle Opulencia

The researchers’ statistician and class adviser Wennie Rose Matira

The researchers’ friends Nicole Valencia, Kirstin Jan Manimtim, Edward Molinyawe,

Jhammy Pearl Luna, Jhanna Mae Manalo and Jill Diamaro

The DMMC Institute of Health Sciences

The future researchers

And lastly, to Almighty God

 4

Table of Contents
Acknowledgement 2
Dedication 3
Table of Contents 4
Chapter I: The Problem and Its Background 6

Introduction 6
Statement of the Problem 9

Null Hypothesis 10

Review of Related Literature 11

Significance of the Study 24

Scope and Limitations of the Study 25

Definition of Terms 25

Theoretical Framework 26

Operational/Conceptual Framework 28

Chapter II: Methodology 29

Research Design 29
Sources of Data 30
Research Locale 30
Research Workflow 31
Preparations of Material Needed in Gram Staining 32
Specimen Collection and Plant Extraction 32
Positive Control 33
Testing Process 34
Proper Disposal and Handling of Specimen 34
Statistical Tool 34
Interpretation of Results 35
Chapter III: Presentation, Analysis and Interpretation of Data 36
 5

Discussion 41
Chapter IV: Summary of Findings, Conclusions and Recommendations 43
Summary of Findings 44
Conclusions 46
Recommendations 47
Appendices 49
References 56
 Chapter I

THE PROBLEM AND ITS BACKGROUND

Introduction

This study is all about an alternative gram staining technique in identification of

gram-negative microorganism using Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals. Each flower has

capability to use in gram staining technique in identification of gram-negative

microorganisms.

The rose petals contain well below one percent of ethereal oil. On account of its

volatility, the rose oil content reduces steadily in the course of the flowering season; rose

flowers for distillation must therefore be gathered by hand every day, and only from the

first crack of dawn until sunrise at the latest. The components of rose oil that determine

its character are the acyclical monoterpene alcohols geraniol (up to 75%), citronellol

(20%) and nerol (20%); in addition to these, long-chain hydrocarbons like nonadecane

and heneicosane have been found. (Büchner, 2020)

Gumamela (Hibiscus) flower varies in different color: red, yellow, orange, white,

purple, pink, and other color combination. But the researchers are just focusing in the

Gumamela red flower, known as the Hibiscus rosa- sinensis, the flower is used as the

medicinal plant in the Philippines. According to Gilmann(1999), “It is said to be one of

the sweetest - smelling flowers in the country. It is also considered as medicinal plant that

represents a rich source of antimicrobial agent. Hibiscus rosa-sinensis common name is

Chinese hibiscus from the family of Malvaceae. It has broadleaf evergreen, flowering pot
 7

plant and indoor foliage plant. This popular landscape shrub creates a bold effect with its

medium-textured, glossy dark green leaves and vibrantly colored, four to eight-inch-

wide, showy flowers, produced throughout the year.” The Hibiscus rosa-sinensis known

to treat many illnesses like expectorant, diuretic, anti-infectious, anti-inflammatory,

antipyretic anodyne, and refrigerant. It also used as treatment of Bronchitis- as an

expectorant, coughs, sore throat, fever- as refrigerant drink, treats dysentery, urinary tract

Infection, bladder Infections, High Blood Pressure, prevention of constipation,

headaches, boils, swelling, abscesses and mumps, in Venezuela, used to treat tumors, this

is according to Tiffany Annetan (2015).

Mirabilis jalapa Linn. (Nyctaginaceae) or four o’clock flower is a popular

ornamental plant grown worldwide for the beauty of its flowers, sweet fragrance and

folklore remedies around the world for treating a variety of conditions. It is commonly

called as four o’clock. It has been well characterized with respect to its chemical

components. This plant contains several compounds and some are having been isolated

from its parts, such as contains alkaloids, glycosides, carbohydrates, flavonoids,

phytosterols (beta-sitosterol and stigmasterol), ursolic acid, oleanolic acid, brassicasterol,

trigonelline and others. Regarding its biological activity, this plant expored for its

cytotoxic, hypoglycaemic, anti-hyperlipodemia, anti-nociceptive, anti-inflammatory, anti-

histamine, anti-oxidant, antimicrobial (antiviral, antibacterial and antifungal), and anti-

spasmodic activities, and also used as a reductant (reducing agent) for the production of

gold nanoparticles. (Nidavani, Ramesh & AM, Mahalakshmi, 2014)

 8

Gram staining is a common technique used to differentiate two large groups of

bacteria based on their different cell wall constituents. The Gram stain procedure

distinguishes between Gram positive and Gram-negative groups by coloring these cells

red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of

peptidoglycan in their cell walls, which retains the crystal violet these cells are stained

with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner

peptidoglycan wall, which does not retain the crystal violet during the decoloring process

(Bruckner, 2016).

The gram staining procedure begins with the application of a basic dye, crystal

violet. A solution of iodine is then applied; all bacteria will be stained blue at this point in

the procedure. The cells are then treated with alcohol. Gram positive cells retain the

crystal violet-iodine complex, remaining blue while gram negative cells are completely

decolorized by alcohol. As a last step, a counterstain (such as the red safranin) is applied

so that decolorized gram-negative cells will take on a contrasting color and the gram-

negative cells now appear purple. (Bautista, 2006)

The ingredients or materials that should be use in identification are hard to find

and extravagant. So that, the researchers seek for inexpensive Safranin O (positive

control) which is alternative natural resources such as Rosa Gallica (Rose), Hibiscus

rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals as

alternative way for identification of gram-negative microorganisms. These flowers are

easy to find somewhere and more convenient to use than the positive control (safranin

O).
 9

The purpose of this study is to use an alternative way for the last step in gram

staining which is counterstain (such as the red safranin) that decolorize gram-negative

cells will take on a contrasting color and the gram negative cells will appear purple color

using Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn. (Gumamela), and Mirabilis

jalapa (Four O'clock Flower) petals.

The significance with the researchers of this study is they will be able to use this

in the near future because some of the researchers are going to take medical or allied

health field course, specifically the Medical Technology course. Thus, if ever the future

Medical Technologists is in needed to use to identify the microorganisms whether it is

gram-negative or gram-positive, they can use this study as an alternative way if they can’t

find the Safranin O (positive control) or to lessen their expenses. Furthermore, the

researchers enabled to explore and have knowledge about this study.

Statement of the Problem

This study was conducted to determine the effectiveness of Rosa Gallica (Rose),

Hibiscus rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower)

Petals as an alternative gram staining technique in identification of gram-negative

microorganism:

The following questions need to be answered:

1. What is the level of effectiveness of Rosa Gallica (Rose), Hibiscus rosa-

sinensis  Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals as

an alternative gram staining technique in identification of gram-negative

microorganism using the following concentrations?

 10

a. 100%

b. 75%

c. 50%

d. 25%

2. Is there any significant difference on the level of effectiveness of Rosa Gallica

(Rose), Hibiscus rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four

O'clock Flower) Petals as an alternative gram staining technique in identification

of gram-negative microorganism?

3. How may the findings of the study become helpful on the identification of

microorganism in Microbiology?

Null Hypothesis

The null hypothesis was conducted to know the significant difference from the

positive control. H0 is the commonly accepted fact so, the researchers work to reject,

nullify or disprove the null hypothesis.

Ho: There is no significant difference on the effectiveness of Rosa Gallica (Rose),

Hibiscus rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock

Flower) Petals as an alternative gram staining technique compared to red safranin

which applied for counterstain (positive control) in identification of gram-negative

microorganism.
 11

Review of Related Literature

The literature review discusses the relevant study that is useful to the objectives of

this research project. Several research studies investigating the topic under review are

found recently.

Components of Rose (Rosa Gallica), Gumamela (Hibiscus rosa-sinensis Linn.), and

Four o’clock (Mirabilis jalapa) Flowers

The rose petals contain well below one per cent of ethereal oil. On account of its

volatility, the rose oil content reduces steadily in the course of the flowering season; rose

flowers for distillation must therefore be gathered by hand every day, and only from the

first crack of dawn until sunrise at the latest. The components of rose oil that determine

its character are the acyclical monoterpene alcohols geraniol (up to 75%), citronellol

(20%) and nerol (20%); in addition to these, long-chain hydrocarbons like nonadecane

and heneicosane have been found. (Büchner, 2020)

The Hibiscus rosa-sinensis or gumamela known to treat many illnesses like

expectorant, diuretic, anti-infectious, anti-inflammatory, antipyretic anodyne, and

refrigerant. It also used as treatment of Bronchitis- as an expectorant, coughs, sore throat,

fever- as refrigerant drink, treats dysentery, urinary tract Infection, bladder Infections,

High Blood Pressure, prevention of constipation, headaches, boils, swelling, abscesses

and mumps, in Venezuela, used to treat tumors, this is according to Tiffany Annetan

(2015).
 12

Mirabilis jalapa Linn. (Nyctaginaceae) or four o’clock flower is a popular

ornamental plant grown worldwide for the beauty of its flowers, sweet fragrance and

folklore remedies around the world for treating a variety of conditions. It is commonly

called as four o’clock. It has been well characterized with respect to its chemical

components. This plant contains several compounds and some are have been isolated

from its parts, such as contains alkaloids, glycosides, carbohydrates, flavonoids,

phytosterols (beta-sitosterol and stigmasterol), ursolic acid, oleanolic acid, brassicasterol,

trigonelline and others. Regarding its biological activity, this plant expored for its

cytotoxic, hypoglycaemic, anti-hyperlipodemia, anti-nociceptive, anti-inflammatory, anti-

histamine, anti-oxidant, antimicrobial (antiviral, antibacterial and antifungal), and anti-

spasmodic activities, and also used as a reductant (reducing agent) for the production of

gold nanoparticles. (Nidavani, Ramesh & AM, Mahalakshmi, 2014)

An optimized staining technique for the detection of Gram positive and Gram-

negative bacteria within tissue.

According to Becerra SC (2016), gram stain was developed and tested as an

alternative to Gram stain that improves the contrast between Gram positive bacteria,

Gram negative bacteria and host tissue. Initially, clinically relevant strains of

Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in

biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry

techniques involving bacterial strain-specific fluorescent antibodies as validation tools.

 13

H&E and Gram stain of serial biopsy sections were then compared to a modification of

the Gram stain incorporating a counterstain that highlights collagen found in tissue.

Alternative fluorescent labeling strategies for characterizing gram-positive

pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome

analysis of Staphylococcus aureus retrieved from infected host cells.

According to Hildebrandt (2016), staphylococcus aureus is a Gram-positive

opportunistic pathogen that is able to cause a broad range of infectious diseases in

humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host

cell which serve as a niche for the pathogen to hide from the immune system and

antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate

host-pathogen interactions upon internalization.

The Use of Plant Dyes for Microbial Staining and Identification: An Eco-friendly

and Non-Toxic Alternative Method

According to Adeyemo, Stella & Akinloye, Akinwumi & Adekanmi, G. (2018),

stated that the dye from the four plants extricate when oxidized, may well be utilized as

an appropriate substitute for the regular stains utilize in Gram recoloring method in

Nigeria and other nations where these plants developed. The objective of this research is

to investigate the effectiveness of common colors from four diverse plants which can be

utilized to recolor bacterial cells. Through this method, plant extricates which has been

handled into colors to be specific Enantia chlorantha, Harungana madagascariensis,

 14

Sphenocentrum jollyanum and Sarcocephalus latifolius were gotten from Botany Office,

Obafemi Awolowo College, Nigeria.

The significance of bacterial engulfment in Gram-stained sputum in patients with

respiratory infections.

As stated by Shimoda (2018), physicians believe that the presence of bacterial

engulfment in white blood cells (WBCs) on Gram-stained sputum is a hallmark of lower

respiratory infection. However, no studies have described the significance or diagnostic

accuracy of engulfment in lower respiratory tract infections. We prospectively studied

sputum samples by Gram staining (Favor method) for their quality and engulfment score

in WBCs obtained from patients with respiratory symptoms at inpatient and outpatient

settings at Kyorin University Hospital between December 2012 and April 2015.

Usefulness of sputum gram stain for etiologic diagnosis in community-acquired

pneumonia: a systematic review and meta-analysis.

According to Triana (2019), implementation of sputum Gram stain in the initial

assessment of community-acquired pneumonia (CAP) patients is still controversial. We

performed a systematic review and meta-analysis to investigate the usefulness of sputum

Gram stain for defining the etiologic diagnosis of CAP in adult patients.

Diagnosing periprosthetic infection: false-positive intraoperative Gram stains.

Oethinger (2011) stated that intraoperative Gram stains submitted from revision

arthroplasty cases during a 3-month interval using microbiologic cultures of the same
 15

samples as the gold standard. Methods of specimen harvesting, handling, transport,

distribution, specimen processing including tissue grinding/macerating, Gram staining,

and interpretation were studied.

Is Gram staining still useful in prosthetic joint infections?

According to Wouthuyzen-Bakker (2011), Staphylococcus aureus is an

independent risk factor for DAIR failure in patients with a late acute prosthetic joint

infection (PJI). Therefore, identifying the causative microorganism in an acute setting

may help to decide if revision surgery should be chosen as a first surgical approach in

patients with additional risk factors for DAIR failure.

Direct Gram staining and its various benefits in the diagnosis of bacterial infections

As stated by Boyanova (2018), in the era of rapid development of molecular and

other diagnostic methods, direct Gram staining (DGS) tends to remain in the background,

although it can provide both microbiologists and clinicians numerous benefits. The DGS

can provide early information for a timely diagnosis of infections, can reveal the

causative agents of the infections even under suboptimal conditions of specimen

collection, transport or identification methods, can detect the presence of rare/unusual

pathogens, moreover, the method shows the specimen quality, by distinguishing between

contamination and true infection, it can direct or change initial antibiotic treatment before

the availability of culture results, can indicate the need of other methods for pathogen

identification and, in some cases, can show the need for emergency attention such as

urgent antibiotic therapy and surgical measures.

 16

Improving Gram stain proficiency in hospital and satellite laboratories that do not

have microbiology.

According to Guarner (2017), consolidation of laboratories has left many

hospitals and satellite laboratories with minimal microbiologic testing. In many hospitals

and satellite laboratories, Gram stains on primary specimens are still performed despite

difficultly in maintaining proficiency.

Usefulness and limit of Gram staining smear examination

As stated by Nagata (2010), gram staining is one of the simplest and inexpensive

methods for the rapid diagnosis of bacterial and fungal infections. The Gram staining

conditions, and morphology of bacteria sometimes change due to antimicrobial therapy.

Species of Gram-negative rods sometimes become filamentous and pleomorphic. Gram-

positive bacteria may become gram variable (change in staining condition) after

antimicrobial therapy. Enterococcus faecalis is a Gram-positive diplococcus, forming

Gram-positive clustered cocci in specimens from blood culture bottles, resembling

Streptococcus pneumoniae.

Clinical utility of an automated instrument for gram staining single slides.

Baron (2010) stated that gram stains from either heat- or methanol-fixed slides

stained with the new instrument were easy to interpret, and results were essentially the

same as those from the methanol-fixed slides prepared as a part of the routine workflow.

This instrument is well suited to a rapid-response laboratory where Gram stain requests

are commonly received on a stat basis.

 17

Multicenter Assessment of Gram Stain Error Rates

Gram stains remain the cornerstone of diagnostic testing in the microbiology

laboratory for the guidance of empirical treatment prior to availability of culture results.

Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are

no comprehensive studies that have evaluated the reliability of the technique and there

are no established standards for performance. In this study, clinical microbiology

laboratories at four major tertiary medical care centers evaluated Gram stain error rates

across all nonblood specimen types by using standardized criteria. (Samuel, 2016)

An optimized staining technique for the detection of Gram positive and Gram-

negative bacteria within tissue

Sanchez (2016) stated that bacterial infections are a common clinical problem in

both acute and chronic wounds. While newer costly bacterial identification methods are

being explored, a simple and inexpensive diagnostic tool would aid in immediate and

accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E)

and Gram stains have been employed but are far from optimal when analyzing tissue

samples due to non-specific staining. The goal of the current study was to develop a

modification of the Gram stain that enhances the contrast between bacteria and host

tissue.
 18

Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of

Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram-Negative

Bacteria with Transferable Multiple Drug Resistance

According to Valle DL, Jr., Cabrera EC, Puzon JJM, Rivera WL (2016), Piper

betle L. has traditionally been used in alternative medicine in different countries for

various therapeutic purposes, including as an anti-infective agent. This study determined

the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on

clinical isolates of multiple drug resistant bacteria which have been identified by the

Infectious Disease Society of America as among the currently more challenging strains in

clinical management. The extracts proved to be more potent against the Gram-positive

MRSA and VRE than for the Gram negative test bacteria.

Assessment of airborne bacteria in selected occupational environments in Quezon

City, Philippines

Rendon, Garcia & Vital (2017) stated that exposure to bioaerosols has been

associated with health deterioration among workers in several occupational

environments. This highlights the need to study the microbiological quality of air of

workplaces as no such study has been conducted yet in the Philippines. To detect and

characterize the culturable mesophilic airborne bacteria in selected occupational

environments we used passive sedimentation technique. It was observed that the number

of colony-forming units was highest in junk shop, followed by the light railway transit
 19

station and last the office. These findings suggest that the presence of airborne bacteria

may be a potential health hazard in urban occupational environments in the Philippines.

Antibacterial activities of ethanol extracts of Philippine medicinal plants against

multidrug-resistant bacteria

According to Valle, Andrade, Puzon, Cabrera, & Rivera (2015), the leaf extracts

of Psidium guajava, Phyllanthus niruri, Ehretia microphylla and Piper betle (P. betle)

showed antibacterial activity against the Gram-positive methicillin-resistant

Staphylococcus aureus and vancomycin-resistant Enterococcus. P. betle showed the

highest antibacterial activity for these bacteria in the disk diffusion (16–33 mm inhibition

diameter), minimum inhibitory concentration (19–156 μg/mL) and minimum bactericidal

concentration (312 μg/mL) assays.

Proficiency Testing of Clinical Laboratories for Bacteriology in the Philippines,

2009–2015

According to Mondoy, M., Rapanut, J. M., Bugayong, M., Aguinaldo, R.,

Navarro, R., Yap, K.J., Kapawan, M.T., Almonia, D.J., Esparar, G., Sadiasa, A.,

Macalalad, N., Sombrero, L., Capeding, M.R., Lupisan, S. (2017), the National External

Quality Assessment Scheme (NEQAS) has been established by the Department of

Health–Philippines (DOH) to provide DOH-approved external quality assessment

programs, including the Proficiency Test (PT) for Bacteriology to clinical laboratories.

The PT for Bacteriology aims to monitor and evaluate laboratory capabilities in the

identification of clinically important pathogens through proficiency testing. Since then,

 20

participation in the NEQAS has been a requirement for clinical laboratories to obtain a

license to operate from the DOH–Health Facilities and Services Regulatory Bureau

(HFSRB).

Antibacterial activity of extracts of twelve common medicinal plants from the

Philippines

Penecilla, G. & Magno, C. (2011) stated that the antibacterial activity of the n-

hexane, acetone/dichloromethane, ethanol and aqueous extracts of twelve common

medicinal plants from the Philippines obtained through pounding and solvent extraction

was evaluated using disc Agar diffusion. The common medicinal plants which showed

highly positive activity were Psidium guajava (guava), Eucalyptus globulus, Mangifera

indica (Indian mango), Nasturtium officinale (Watercress), Pterygospermum oleiferum

(Moringa), Carmona retusa (Wild tea), Citrus aurantifolia (Lemon), Citrus sinensis

(Orange), Allium sativum (garlic), and Allium cepa (onion). The results suggest that the

different plant extracts contain bioactive constituent(s) particularly tannins, flavonoids,

terpenoids and other glycosides with very strong antibacterial activity and validates the

ethno-medical use in the treatment of bacterial skin diseases and other forms of bacterial

infections.

Antibacterial potential of some medicinal plants of the Cordillera Region,

Philippines

Eleven medicinal plants of the Cordillera Region, in the Philippines were

evaluated for their antimicrobial properties by the standard disc diffusion assay method
 21

using test bacterial organisms: Escherichia coli, Bacillus subtilis, Staphyloccocus aureus

and Proteus vulgaris, while test fungal organisms used were Candida albicans and

Aspergillus flavus. Only four plants namely, Agathis dammara, Eupatorium triplenerve,

Citrus aurantifolia and Tithonia divserifolia were found to have antibacterial properties.

Results of MIC determination revealed that the gram-positive organisms, Bacillus subtilis

and Staphylococcus aureus, were more sensitive to the four plant extracts, since they

scored the lowest value of MIC which ranged from 62.5 to 125 μg/ml. The gram-negative

organisms Escherichia coli and Proteus vulgaris scored higher MIC values ranging from

62.5 - 250 μg/ml. Phytochemical screening of the four plants with antibacterial activities

were also undertaken. (Gutierrez, Rosemary & Baculi, Ronan & Pastor, Nardo & Puma-

at, Timothy & Balangcod, Teodora, 2013)

Antibacterial Activity of Ethanolic Extract of Hibiscus Rosa-Sinensis Flower against

Staphylococcus Epidermis and Staphylococcus Saprophyticus

Bacteria are one of the major causes of urinary tract infection. To ease and

prevent the wide spread of it, the researchers test a flowering plant, Hibiscus rosa-

sinensis (Gumamela red flower) to fight against Staphylococcus epidermidis and

Staphylococcus saprophyticus. The researchers used in vitro testing of gumamela flower

ethanolic extract to know its effectiveness against Staphylococcus epdermidis and

Staphylococcus saprophyticus. It was tested at different concentrations to identify its

capacity to fight against the said causative agent of urinary tract infection Concentration

used was 80% and 40% ethanolic extracts. Each ethanolic extract was diluted into 1:1,
 22

1:10, 1:100 and 1:1000 using serial dilution to perform the MIC. Results revealed that

gumamela extract is not potent as treatment for the two bacteria. There is no visible zone

of inhibition in different concentrations. (Barraquia, A. R., Cabusao, A. P., Narvacan, C.,

2017)

Isolation and identification of Hibiscus Chlorotic Ringspot Virus (HCRSV) infecting

Gumamela (Hibiscus rosasinensis) in the Philippines

According to Gonzales, M., Retuta, Y., Magdalita, P. (2016), gumamela plants

(IPB Accessions No. 95 and 97 and the variety “Superstar”) showing chlorotic ringspots

and the symptomatic indicator plants tested positive for HCRSV by Enzyme-linked

immunosorbent assay (ELISA). The Reverse Transcriptase Polymerase Chain Reaction

(RT-PCR ) using primers that amplify a conserved region in the coat protein (CP) gene of

HCRSV giving an amplification with a size of 557bp further confirmed the results.

Nucleotide sequence analysis of the CP gene of the HCRSV Philippines showed 97.5 to

97.9% similarity to the HCRSV isolates of Iran, New Zealand and Singapore. It is most

related to the Israel isolate with 98.1% identity and less similar with HCRSV-Taiwan

with only 93% sequence identity. To our knowledge, this is the first report of HCRSV in

gumamela in the Philippines.

A Study on the Mucolytic Activity of the Extract of Gumamela Flower [Hibiscus

rosa-sinensis (Linn.)].

 Lewis, J (2011) stated that the effect of gumamela extract on the relative

viscosity change of mucin was determined from the time of flow of mucin solution after
 23

treatment with water, with carbocysteine as positive control and with gumamela extract.

There is no significant effect on the viscosity of purified mucin after treatment with

gumamela extract since the result parallel with that after water treatment. However, the

initial decrease in the time of flow one hour after treatment with gumamela may possibly

be due to a limited effect of the extract.

Antibacterial Activity of Premna odorata Blanco (ALAGAW) Leaf Extract on

Bacillus subtilis, Escherichia coli, AND Staphylococcus aureus

According to Pangilinan (2013), alagaw (Premna odorataBlanco) is an important

medicinal plant that possesses cardio tonic, antibiotic, anti-coagulant, carminative,

hepatoprotective, and anti-tumor properties. It is proven to loosen phlegm and coughs.

Literature showed that the genus Premna contains antimicrobial and antibacterial

properties. The present study aims to evaluate the antibacterial activity of the leaves

extract of P. odorata against the selected human pathogens (Bacillus subtilis, Escherichia

coli, and Staphylococcus aureus). Based on the results, it can be concluded that P. odorata

Blanco has antibacterial property.

Characterization and Identification of Congo Red Decolorizing Bacteria from

Monocultures and Consortia

Congo red is a carcinogenic direct diazo dye used for the coloration of paper

products. It is recalcitrant and is thus found in effluents of paper factories. Bacteria in

consortia capable of decolorizing Congo red were isolated from polluted and non-

polluted sites. The toxicity of decolorized medium and undegraded Congo red was tested
 24

on bacteria, yeast, rice, and mungbean plants. The monocultures were characterized and

identified. The decolorizing activities were 97% for SB13B, 96% for SB12D, 92% for

IRRI-1C and 96% for S22B. S22B and SB13B were able to degrade Congo red while

reducing nitrate to nitrogen gas and nitrite. SB13B was identified as Escherichia coli by

API 20 E, fatty acid analysis and 16S rRNA sequencing. [ CITATION Ail10 \l 1124 ]

Significance of the Study

The researchers believe that the study will be beneficial to the following:

Researchers. They will be able to explore and know about gram stain from gram-

positive to gram-negative. Also, this research may serve as their reference for the next

researchers.

Future Researchers. The study will give them the correct information to make

their research in the future.

Medical Students. This study will be able to help them utilize this research in

their study and to know that there are some alternative ways for identification of gram-

negative microorganisms.

College Students of Allied Health Medical Technology. This study will be

beneficial to them to help them utilize this research that there are alternative ways for

identifying gram-negative microorganisms.

 25

Scope and Limitation of the study

This study focuses on making Alternative Gram Staining Technique in

Identification of Gram-Negative Microorganism using Rosa Gallica (Rose), Hibiscus

rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals. This

study can help us in terms of detecting the microorganism in an inexpensive way that

may not use red safranin to decolorize the gram-negative.

This research limits itself on using other flower petals to prove that Rosa Gallica

(Rose), Hibiscus rosa-sinensis  Linn. (Gumamela), and Mirabilis jalapa (Four O'clock

Flower) petals are effective as an alternative way for identification of gram-negative

microorganism.

Definition of Terms

The following terms are conceptually and operationally defined for better

understanding of the readers.

Staining is a technique in which cells or thin sections of biological tissue that are

normally transparent are immersed in one or more colored dyes (stains) to make them

more clearly visible through a microscope. (ELITE CAFEMEDIA, 2019)

Counterstain is a stain of a contrasting color used to color the components in a

microscopic specimen that are not made visible by the principal stain. (Houghton Mifflin

Harcourt Publishing Company, 2018)

Crystal Violet a triphenylmethane dye found in gentian violet. (STANDS4 LLC, 2019)
 26

Decolorizer is an agent that removes color or bleaches. (Farlex, Inc, 2020)

Gram-negative bacteria lose the crystal violet stain (and take the color of the red

counterstain) in Gram's method of staining. (Shiel, 2020)

Gram-positive bacteria have a very thick cell wall made of a protein called

peptidoglycan. (Byers, 2016)

Gram stain test is the most extensively performed procedure in diagnostic

microbiology. (Harr, 2019)

Iodine (I− or I3−) acts as a mordant and as a trapping agent. (Thairu, Nasir, Usman,

2014)

Safranin O is another commonly used histological stain capable of staining nuclei black,

cytoplasm a grayish green, collagen green and cartilage, mucin and mast cell granules a

reddish orange color (Lillie and Fullmer, 1976; Lillie et al., 1977).

Theoretical Framework

Biological Staining Theory

According to Horobin (2002), new staining techniques continue to be introduced,

and older ones continue to be used and improved. Several factors control specificity,

selectivity and visibility of the end product in any procedure using dyes, fluorochromes,

inorganic reagents or histochemical reactions applied to sections or similar preparations.

Local concentration of the tissue target often determines the intensity of the observed
 27

color, as does the fine structure within the object being stained, which may facilitate or

impede diffusion of dyes and other reagents. Several contributions to affinity control the

specificity of staining. Nonionic forces can also increase visibility of stained sites by

causing aggregation of dye molecules. Covalent bonds between dye and tissue result in

the strongest binding, such as in methods using Schiff's reagent and possibly also some

mordant dyes. Selective staining of different organelles within living cells is

accomplished mainly with fluorochromes and is controlled by mechanisms different from

those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such

reagents can be derived from such molecular properties as hydrophilic-hydrophobic

balance, extent of conjugated bond systems, acid-base properties and ionic charge. The

QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria,

DNA, or the plasma membranes of living cells.

 28

Operational/Conceptual Framework

This model shows the summary of this study. It can be expressed through
the following framework format:

Independent Variable Dependent Variable

The amount of Rosa Result of detected

Gallica (Rose), Gram-Negative
Hibiscus rosa- Microorganisms
sinensis Linn. using Rosa Gallica
(Gumamela), and (Rose), Hibiscus
Mirabilis jalapa (Four rosa-sinensis Linn.
O'clock Flower) (Gumamela), and
Petals as an Mirabilis jalapa
alternative gram (Four O'clock
staining technique in Flower) Petals in
identification of gram- Gram Staining
negative Technique.
microorganism.

Figure 1. The Conceptual Framework of the Study

Figure 1 shows the conceptual framework of the study. Independent variable is

the variable that is changed or controlled in a scientific experiment to test the effect on

the dependent variable. Dependent variable is the variable being tested and measured in a

scientific experiment. We manipulate the Rosa Gallica (Rose), Hibiscus rosa-

sinensis  Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals to know

the exact concentration of Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals to use as an alternative

gram staining technique in identification of gram-negative microorganism.

 Chapter II

RESEARCH METHODOLOGY

This chapter shows the method of the effectiveness of Rosa Gallica (Rose),

Hibiscus rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower)

Petals as an alternative gram staining technique in identification of gram-negative

microorganism and procedure on performing culture and gram staining. Proper waste

management and precautions were also included.

Research Design

A trial and error method experimental design was used in the study. Trial and

error is a problem solving method in which multiple attempts are made to reach a

solution. It is a basic method of learning that essentially all organisms use to learn new

behaviors. Trial and error is trying a method, observing if it works, and if it doesn't, try a

new method. This process is repeated until success or a solution is reached (AlleyDog,

2020). The experimental method of research will be utilizing in the study for it is the best

method of research to establish the cause and effect relationship within the given

hypothesis. The study thrives to assess and observe the effectiveness of Rosa Gallica

(Rose), Hibiscus rosa-sinensis  Linn. (Gumamela), and Mirabilis jalapa (Four O'clock

Flower) Petals as an alternative gram staining technique in identification of gram-

negative microorganism.
 30

Sources of Data

The primary source of data was used in the study including books, related studies,

and published journals. The Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals were used in the study.

This includes to be extract through ethanol. Plant were sent at DMMC Institute of Health

Sciences Medical Technology Laboratory for testing.

Research Locale

This is about identifying the gram negative using Rosa Gallica (Rose), Hibiscus

rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals for

alternative gram staining to know which organism is gram negative. We chose these

flowers because if when you rub, it will stain to your hand which is applicable for gram

staining to know whether it is gram negative or gram positive.

 31

Research Workflow

Here, the researchers will follow the step by step procedure which indicates the

workflow of the study.

Collection of Flower Petals

Preparation of Materials

Extraction

Result

This research workflow shows the step by step procedure of the study. The

researchers collected first the flower petals such as Rosa Gallica (Rose), Hibiscus rosa-

sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) at Payapang

Burol, Malvar, Batangas. Next is the preparation of materials. The following materials

that were used in the study are clean glass slides, cotton swab, inoculating loop, Bunsen

burner, Bibulous paper, microscope, lens paper, lens cleaner, immersion oil, and distilled

water. The third step is extraction. With the used of ethanolic extract, it should evaporate

then the extract would flow. The extract diluted using distilled water into 100%, 75%,

50%, and 25% to determine which concentration was effective. Lastly, the result

determines if which petals is effective for identifying gram-negative microorganisms.

 32

Preparation of Materials Needed in Gram Staining

The following materials that were used in the study are clean glass slides, cotton

swab, inoculating loop, Bunsen burner, Bibulous paper, microscope, lens paper, lens

cleaner, immersion oil, and distilled water. They have different uses and/or functions in

identifying gram staining if it is gram-negative or gram-positive. Clean glass slides use a

lint-free wipe and cotton swab to gently rub the surface clean of dirt and residues.

Inoculating loop is a simple tool used mainly by microbiologist to pick up and transfer a

small sample (inoculum) from a culture of microorganisms. Bunsen burner a small

adjustable gas burner used in laboratories. Bibulous paper is used to absorb an excess of

liquid substances (such as ink or oil) from the surface of writing paper or objects.

Microscope is an instrument used to see objects that are too small to be seen by the naked

eye. Lens paper used for cleaning microscope slides while Lens Cleaner Clean smudged

and dirty lenses at your convenience with these handy individual packets. Immersion oil a

technique used to increase the resolving power of a microscope. Distilled water a

technique used to increase the resolving power of a microscope, 18 to 24 hour cultures of

organisms.

Specimen Collection and Plant Extraction

Fresh Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn. (Gumamela), and

Mirabilis jalapa (Four O'clock Flower) petals were collected in Payapang Burol, Malvar

Batangas. The plants were sent at DMMC Institute of Health Sciences Medical

Technology Laboratory for testing.

 33

The method was adapted to Olowa and Nuñeza (2013). The fresh petals were air

dry for about one week and ground into fine powder using a mechanical grinder. Twenty

grams of the fine powder of each petals samples will weigh and add into an Erlenmeyer

flask containing 250 mL of 95% ethanol. The solution covered and shake every 30 min

for about six (6) hours and allow to stand for about 48 hours in room temperature. Then,

it was shake and filtered with the used of Whatman filter paper (No.1). After filtration,

the solvent removed by evaporation using a rotary evaporator under reduce pressure at

temperature below 55°C. The extracts were reconstituting by dissolving in normal saline

solution to the required concentrations (100%,75%,50% and 25%). The reconstituting

extracts were maintaining at 2–8 ºC. The procedure was sent to Medical Technology

Laboratory at DMMC Institute of Health Sciences.

Positive Control

The gram stain procedure has been performed properly and the reagents are fresh.

The researchers would use an alternative way such as Rosa Gallica (Rose), Hibiscus

rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals in

safranin for dyeing as a biological stain, in the positive control (gram-positive). It should

appear as a purple to lavender and the negative control (gram-negative) should appear as

a pink to red.

Testing Process

The organism isolated from the experiment was transferred into slide using

inoculating loop. It was flooded using crystal violet staining reagent for about 1 minute.
 34

The slide was wash into gentle by tap water for about 2 seconds and stained with a

mordant (gram’s iodine). The slide was wash again by tap water and decolorized for 15

seconds using methanol. The slide would be flooded with the alternative gram staining

reagent which is the Safranin O as the counterstain for 30 seconds to 1 minute.

Microorganisms were visualized under the microscope and would be compared to the

positive control.

Proper Disposal and Handling of Specimen

Original cultures and sub-cultures and contaminated material would autoclave at

121 º Celsius for 15-30 minutes before disposal as regular waste material. Bacteria that

were used in the study were treated as if they are all pathogenic. Proper handling of

specimen was observed by using safety equipment such as PPEs (Personal Protective

Equipment). Cultures are manipulating carefully to avoid uncontrolled release of aerosols

or the generation of large droplets or spills. Loading, removing, opening tubes and plates,

streaking, sub-culturing was done within a biological safety cabinet level 2.

Statistical Tool

For S.O.P 1 a weighted mean and a frequency distribution table was used for the

level of effectiveness as Rosa Gallica (Rose), Hibiscus rosa-sinensis  Linn. (Gumamela),

and Mirabilis jalapa (Four O'clock Flower) petals as an alternative gram staining

technique in identification of gram-negative microorganism. The weighted mean is a type

of mean that is calculated by multiplying the weight (or probability) associated with a

particular event or outcome with its associated quantitative outcome and then summing

all the products together. A frequency distribution is a list, table or graph that displays the
 35

frequency of various outcomes in a sample it is used to organize and summarize the data.

Specifically, it is a list of either qualitative or quantitative values that a variable takes in a

data set and the associated number of times each value.

For S.O.P 2 T-test was used to determine if there any significant difference on the

level of effectiveness of as Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals as an alternative gram

staining technique in identification of gram-negative microorganism. A t-test is a type of

inferential statistic used to determine if there is a significant difference between the

means of two groups, which may had related in certain features.

Interpretation of Results

Results were interpreted by counting the total number of gram positive/negative

cocci using the alternative stain on at least 20 different fields on different trials and would

compared to the existing gram stain reagents. Statistical tools were used for the

determination of the level of significance.

 Chapter III

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter deals with the gathered data which were analyzed and interpreted for

the better understanding of the study. The framework of the analysis and interpretation is

guided by the problem stated in Chapter 1.

100% 75% 50% 25%

(Code) Frequency Percent Frequency Percent Frequency Percent Frequency Percent
Rose 1 33.3 2 66.7 1 33.3 1 33.3
Rose 2 66.7 1 33.3 1 33.3 1 33.3
Rose 1 33.3 1 33.3
Total 3 100% 3 100% 3 100% 3 100%
1. What is the level of effectiveness of Rosa Gallica (Rose), Hibiscus rosa-

sinensis  Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals as

an alternative gram staining technique in identification of gram-negative

microorganism using the following concentrations?

a. 100%

b. 75%

c. 50%

d. 25%

Table 1

Level of Effectiveness

In Rosa Gallica (rose) extract shows that every concentration there are differences

in effectiveness of rose petal as an alternative gram staining technique in identification of

gram-negative microorganism using the following concentrations. In using 100%

 37

concentration of rose, there is slight gram-negative bacilli seen. Then using 75%

concentration of rose, there is moderate gram negative seen. By using 50% concentration

of rose, there is slight to heavy gram-negative bacilli seen. Lastly, the 25% concentration

of rose is there still slight to heavy gram-negative bacilli seen.

100% 75% 50% 25%

(Code) Frequen Perce Frequen Perce Frequen Perce Frequen Perce
cy nt cy nt cy nt cy nt
Gumame 1 33.3 1 33.3 1 33.3 1 33.3
la
Gumame 1 33.3 2 66.7 1 33.3 1 33.3
la
Gumame 1 33.3 1 33.3 1 33.3
la
Total 3 100% 3 100% 3 100% 3 100%

In Hibiscus rosa-Sinensis Linn. (gumamela) extract shows that every

concentration there are differences in effectiveness of gumamela petal as an alternative

gram staining technique in identification of gram-negative microorganism using the

following concentration. The 100% concentration of gumamela, there is slight to

moderate gram-negative bacilli seen. By using 75% concentration of gumamela, it shows

there is slight gram-negative bacilli. Next, using 50% concentration of gumamela, there is

slight to heavy gram-negative seen. And in 25 % concentration of gumamela, it shows

there is likely slight to heavy gram-negative.

100% 75% 50% 25%

(Code) Frequenc Perce Frequenc Perce Frequenc Perce Frequenc Perce
y nt y nt y nt y nt
4 1 33.3 2 66.7 1 33.3 1 33.3
O’cloc
 38

k
4 2 66.7 1 33.3 1 33.3 1 33.3
O’cloc
k
4 1 33.3 1 33.3
O’cloc
k
Total 3 100% 3 100% 3 100% 3 100%

In Mirabilis jalapa (Four O'clock Flower) extract shows that every concentration there

are differences in effectiveness of Four O'clock Flower petal as an alternative gram

staining technique in identification of gram-negative microorganism using the following

concentrations. In using 100% concentration of Four O'clock Flower, there is slight

gram-negative bacilli that be seen. Then in 75 % concentration of Four O'clock Flower,

there is moderate gram-negative bacilli that be seen. Using 50 % concentration of Four

O'clock Flower shows that there is slight to heavy gram-negative bacilli that be seen.

Lastly, in 25% concentration of Four O'clock Flower, there is slight to heavy gram-

negative bacilli that be seen.

2. Is there any significant difference on the level of effectiveness of Rosa Gallica

(Rose), Hibiscus rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four

O'clock Flower) Petals as an alternative gram staining technique in identification

of gram-negative microorganism?

Table 2

Significance difference using T-test

t-value p-value Interpretation Decision

Rose Control (100%) .000 1.000 Not Significant Reject H0
 39

Rose Control (75%) 8.000 .015 Significant Failed to Reject H0

Rose Control (50%) 7.000 .020 Significant Failed to Reject H0
Rose Control (25%) 7.000 .020 Significant Failed to Reject H0

Based on the result of using rose petals in 100% concentration of rose, the

researchers conclude that the most effective as an alternative gram staining technique in

identification of gram-negative microorganisms is 100% concentration. However, 75%,

50%, and 25% concentration of rose are not effective as an alternative gram staining

technique.

t-value p-value Interpretation Decision

Gumamela Control .000 1.000 Not Significant Reject H0

(100%)
Gumamela Control -.500 .667 Not Significant Reject H0

(75%)
Gumamela Control 3.464 .074 Not Significant Reject H0

(50%)
Gumamela Control 3.464 .074 Not Significant Reject H0

(25%)
 40

By using 100%, 75%, 50%, 25% concentration of gumamela they are all

effective. So that the gumamela is able to use as alternative gram staining to identify

gram-negative bacilli.

t-value p-value Interpretation Decision

4 O’clock Control .000 1.000 Not Significant Reject H0

(100%)
4 O’clock Control 8.000 .015 Significant Failed to Reject H0

(75%)
4 O’clock Control 7.000 .020 Significant Failed to Reject H0

(50%)
4 O’clock Control (25%) 7.000 .020 Significant Failed to Reject H0

Based on the result, 100% concentration using Four O'clock Flower petal the most

effective. However, 75%, 50%, and 25% of concentration are not effective.

DISCUSSION

This research entitled “Alternative Gram Staining Technique in Identification of

Gram-Negative Microorganism Using Rosa Gallica (Rose), Hibiscus rosa-Sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals” was chosen by the

researchers to determine if the petals (rose, gumamela & 4 O’clock) could be used as an

alternative gram staining technique in identifying gram-negative microorganisms and to

test its potential that can help to lessen expenses in using safranin. By using different

concentrations such as 100%, 75%, 50%, and 25%, the researchers had proven its

effectiveness. Based on the results, for rose in using 100%, there is slight gram negative

bacilli seen, in 75% there is moderate gram-negative bacilli seen, using 50% and 25%
 41

there is slight to heavy gram-negative bacilli seen. In using 100% of Gumamela, some

area of the slide doesn't show any gram-negative bacilli. However, there is some area in

the slide that have slight to moderate gram-negative bacilli. In 75% there is slight gram-

negative bacilli; for using 50% and 25%, it shows that there is slight to heavy gram-

negative bacilli. And in using 100% of 4 O’clock, there is slight gram-negative bacilli

seen; in 75%, there is moderate gram-negative bacilli seen. In 50% and 25%, there is

slight to heavy gram-negative seen.

Furthermore, the microorganism that had been used was Escherichia Coli, it is gram-

negative bacilli. The study will be helpful to the Microbiologist, as the concentrations of

Gumamela in 100%, 75%, 50%, and 25% have the ability to use as an alternative gram

staining technique in identifying of gram-negative microorganism. As well as the

concentrations of 4 O’clock and rose in 100% to lessen expenses instead of using the

positive control (safranin). The study could be beneficial to the future researchers to

continue the research and provide other solutions in doing the experiment.
 Chapter IV

SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS

This chapter includes the summary of findings, conclusions, and recommendation

made by the researchers. This study was based on the results of the data gathered through

experiment. An analysis of the content was done with the use of the statistical tool as

frequency table and SPSS Annotated Output T-Test.

This study aims to determine the effectiveness of Rosa Gallica (Rose), Hibiscus

rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals as an

alternative gram staining technique to identify gram-negative microorganism. This study

is utilized the pretest-posttest research design because it is the most effective from the

other experimental research design which measures the degree of change that serves as

the result of the study.

This study aimed to determine the effectiveness of Rosa Gallica (Rose), Hibiscus

rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals as an

alternative gram staining technique in identification of gram-negative microorganism:

Specifically, this sought to answers to the following sub-problems:

1. What is the level of effectiveness of Rosa Gallica (Rose), Hibiscus rosa-

sinensis  Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals as

an alternative gram staining technique in identification of gram-negative

microorganism using the following concentrations?

a. 100%
 43

b. 75%

c. 50%

d. 25%

2. Is there any significant difference on the level of effectiveness of Rosa Gallica

(Rose), Hibiscus rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four

O'clock Flower) Petals as an alternative gram staining technique in identification

of gram-negative microorganism?

3. How may the findings of the study become helpful on the identification of

microorganism in Microbiology?

SUMMARY OF FINDINGS

The researchers conduct a study about “Alternative Gram Staining Technique in

Identification of Gram-Negative Microorganism Using Rosa Gallica (Rose), Hibiscus

rosa-sinensis Linn. (Gumamela), and Mirabilis jalapa (Four O'clock Flower) Petals”.

The said flowers have the ability to use for alternative gram staining in identifying gram-

negative microorganisms because of its components to stain. As the purpose of their

study, the researchers used the following petals as an alternative gram staining technique

in identifying gram-negative microorganisms that can help to lessen expenses instead of

using safranin (positive control).

In the process of their experiment, the plants were submitted to DMMCIHS

Medical Technology Laboratory to test the effectiveness of rose, gumamela, and four

o’clock flower petals with different concentrations such as 100%, 75%, 50%, 25%. The

microorganism that had been used was Escherichia Coli, it is gram-negative bacilli.
 44

The following are the summary findings of the researchers:

1. The level of effectiveness of Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals as an

alternative gram staining.

Based on the results, for rose in using 100%, there is slight gram-negative bacilli

seen, in 75% there is moderate gram-negative bacilli seen, using 50% and 25% there

is slight to heavy gram-negative bacilli seen. In using 100% of Gumamela, some area

of the slide doesn't show any gram-negative bacilli. However, there is some area in

the slide that have slight to heavy gram-negative bacilli. In 75% there is slight gram-

negative bacilli; for using 50% and 25%, it shows that there is slight to heavy gram-

negative bacilli. And in using 100% of 4 O’clock, there is slight gram-negative bacilli

seen; in 75%, there is moderate gram-negative bacilli seen. In 50% and 25%, there is

slight to heavy gram-negative seen.

2. The significant difference of Rosa Gallica (Rose), Hibiscus rosa-sinensis Linn.

(Gumamela), and Mirabilis jalapa (Four O'clock Flower) petals to the

standard control.

Using the table of paired sample test having Sig. (two-tailed) for rose of 1, 0.015,

0.020, and 0.020, then for gumamela of 1, 0.667, 0.074, and 0.074; and for four

o’clock flower of 1, 0.015, 0.020, and 0.020, it presented the significance difference

among the concentration. As shown in the data, for gumamela, by using the

concentrations 100%, 75%, 50%, and 25% have no significant difference to the standard
 45

control. Then, 4 O'clock and rose has the same result in 100% concentration wherein

there is no significant difference to the standard control. But, 75%, 50%, and 25% has a

significance difference because it is less than 0.05 level of significance.

3. The contribution of the study in Microbiology.

The findings of the study showed that the concentrations of gumamela in 100%,

75%, 50%, and 25% have the ability to use as an alternative gram staining technique

in identifying of gram-negative microorganism. As well as the concentrations of 4

O’clock and rose in 100% to lessen expenses instead of using the positive control

(safranin).

CONCLUSION

From the summarized findings, the following conclusions were drawn.

1. The use of 100%,75%, 50%, and 25% concentrations of gumamela extract, they

are no significant difference to the positive control which only means it can be

used as an alternative gram staining technique to identify gram-negative

microorganisms.

2. The use of 100% concentrations of 4 O’clock and rose extract showed no

significant difference to a positive control, making the usage of this concentration

of 4 O’clock and rose as an alternative gram-staining techniques in identifying

gram-negative microorganisms comparable to existing positive control (safranin)

in the market.
 46

3. The use of 75%, 50%, and 25% of 4 O’clock and rose extract to identify the

gram-negative bacilli proved that have significance difference from the positive

control (safranin).

4. As the use of concentrations of gumamela in 100%, 75%, 50%, and 25% proved

to be effective as well as the concentration of 4 O’clock and rose in 100%

concentration. The findings of the study would be very effective as an alternative

gram staining technique in identifying of gram-negative microorganism to lessen

expenses instead of using the positive control (safranin).

RECOMMENDATIONS

From the conclusion drawn, the following recommendation are hereby endorsed:

1. The reagents are expensive, so that the concentrations of gumamela in 100%,

75%, 50%, and 25% as well as the concentration of 4 O’clock and rose in 100%

concentration recommended as an alternative gram staining technique in

identifying of gram-negative microorganism to lessen expenses instead of using

the positive control (safranin).

2. Microbiologists can use the concentrations of gumamela in 100%, 75%, 50%, and

25% as well as the concentration of 4 O’clock and rose in 100% concentration as

an alternative gram staining technique in identifying of gram-negative

microorganism to lessen expenses instead of using the positive control (safranin).

3. Medical Students can use the concentrations of gumamela in 100%, 75%, 50%,

and 25% as well as the concentration of 4 O’clock and rose in 100%

 47

concentration as an alternative gram staining technique in identifying of gram-

negative microorganism to lessen expenses instead of using the positive control

(safranin).

4. Future researchers are recommended to conduct the study with different gram-

negative microorganisms to have more accurate results.

APPENDICES
 49

Letter of Approval to Conduct the Study to Medical Technology Laboratory

 50

CHAVEZ, SHAINA P.
STEM 12

Bagumbayan, Tanauan City, Batangas

0965-155-9721

shaina.chavez123@gmail.com

PERSONAL INFORMATION

Age: 18
Date of Birth: December 16, 2002
Place of Birth: Tanauan City Batangas
Gender: Female
Nationality: Filipino
Religion: Roman Catholic
Civil Status: Single

EDUCATIONAL BACKGROUND

Secondary Education:
Senior High School
DMMC Institute of Health Science
S.Y. 2019- Up to Present

Junior High School

Tanauan Institute Inc.
S.Y. 2015- 2019

Primary Education:
Tanauan North Central
 51

OPULENCIA, FELICITY A.
STEM 12

Bagumbayan, Tanauan City, Batangas

0915-699-4075
felicityopulencia10@gmail.com

PERSONAL INFORMATION

Age: 18
Date of Birth: April 10, 2002
Place of Birth: Tanauan City Batangas
Gender: Female
Nationality: Filipino
Religion: Roman Catholic
Civil Status: Single

EDUCATIONAL BACKGROUND

Secondary Education:
Senior High School
DMMC Institute of Health Science
S.Y. 2019- Up to Present

Junior High School

Tanauan City Integrated Highschool
S.Y. 2015- 2019

Primary Education:
Bagumbayan Elementary School
S.Y. 2009-2015
 52

ARNEGO, RECELLE H.

STEM 12

San Vicente, Sto. Tomas, Batangas

0948-851-5603

Renz.celle@gmail.com

PERSONAL INFORMATION

Age: 18
Date of Birth: October 21, 2002
Place of Birth: Mangagoy Sug-ubon Tabon, Bislig City
Gender: Female
Nationality: Filipino
Religion: Jehova’s Witnesses
Civil Status: Single

EDUCATIONAL BACKGROUND

Secondary Education:
Senior High School
DMMC Institute of Health Science
S.Y. 2019- Up to Present

Junior High School

San Pedro National Highshool
S.Y. 2015- 2019

Primary Education:
San Pedro South Central Elementary School
S.Y. 2009-2015
 53

FERRER, ALIESON R.

STEM 12

Janopol Oriental, Tanauan City, Batangas

0945-493-8657
aliesonroxasferrer@gmail.com

PERSONAL INFORMATION

Age: 18
Date of Birth: July 20, 2002
Place of Birth: Santor, Tanauan City
Gender: Male
Nationality: Filipino
Religion: Jehova’s Witnesses
Civil Status: Single

EDUCATIONAL BACKGROUND

Secondary Education:
Senior High School
DMMC Institute of Health Science
S.Y. 2019- Up to Present

Junior High School

Janopol Oriental National Highshool
S.Y. 2015- 2019

Primary Education:
Janopol Oriental Elementary School
S.Y. 2009-2015
 54

DIMAYUGA, JOANA PAULA G.

STEM 12

Santiago, Malvar, Batangas

0915-699-4075
Joanapaula030@gmail.com

PERSONAL INFORMATION

Age: 18
Date of Birth: September 30, 2002
Place of Birth: Malvar, Batangas
Gender: Female
Nationality: Filipino
Religion: Roman Catholic
Civil Status: Single

EDUCATIONAL BACKGROUND

Secondary Education:
Senior High School
DMMC Institute of Health Science
S.Y. 2019- Up to Present

Junior High School

Santiago National Highschool
S.Y. 2015- 2019

Primary Education:
Payapa Elementary School
S.Y. 2009-2015
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