Ramon M. Durano Sr.

Foundation Science and Technology Education Center

Banaba, Guinsay, Danao City

The Efficacy of Glycerol (Propane-1,2,3-triol) as a Detection

Substance for Gram-negative Bacteria

(Vibrio Cholerae)

Researchers:

Tancinco, Shantal Shean D.

Lugas, Janah Mae H.

Balais, Kristel Ane H.

Batalon, Jenny Rose L.

Canson, Lynelle Valere M.

Montecillo, Sheriel Christes V.

Research Adviser

Mrs. Matiga, Lilet T.

 Outline of Rationale of the Study

Water has been naturally essential not only in surviving but also in daily

households like washing and cleaning. One of the facts that the professional had

spoken legitimately from the start was that the world is made from 71% of water and

known as the universal solvent, these statements prove that water is crucial, but the real

problem is, it is not free nor easy to find mainly the safe ones especially in those places

which are known as rural regions where there they don't have enough source of safe

and clean water. One of the prominent and deadly bacteria is the Vibrio Cholerae, a

member of the Gram-negative Bacteria who is responsible for the death of over 143,000

worldwide with 4.0 million cases each year. This problem can be avertible right from the

start if people had the knowledge, information, and tools to detect these bacteria right

from the beginning. Researchers came-up with this study which is the main objective is

to detect the presence of Vibro Cholerea in water substances. Water, Glycerol Solution,

and Detergent are the main components behind this study which aim to help and

prevent the Cholerea outbreak. Therefore, this would help the community in living a safe

and stable environment.

Concept Description
• Glycerol (Propane-1,2,3-triol) Is a colorless, odorless, sweet-tasting,

and non-toxic viscous liquid polyol

compound. The alcohol can be found in

the triglyceride structure of oils/fats, and

 the content ranges from approximately 9

to 13.5% and is commonly used for

constipation, improving hydration, certain

skin conditions and more.

•Antimicrobial and Antiviral Glycerol contains antimicrobial and

antiviral properties and is approved for

wound and burn treatments (FDA,1959).

•Detection Due to its properties, the detection

among bacteria is possible specially

among Gram-negative Bacteria which is

well known to be susceptible in

antimicrobial compounds.
•Health Problems and Sanitations Gram-negative Bacteria can cause many

serious infections if left inside the body

one example is Cholera disease that is

caused by Vibrio Cholerae. This

bacterium is commonly found in water

and food with less sanitations applied;

effects can be fatal if left untreated.

• Epidemiology, Genetics, and Ecology of Cholera toxin, comma-shaped bacterium.

Toxigenic Vibrio Cholerae The bacterium's natural habitat is

brackish or saltwater where they attach

themselves easily to the chitin-containing

shells of crabs, other shellfish. which is

responsible for the profuse diarrhea, is

 encoded by a lysogenic bacteriophage

designated. Cholera patients should be

evaluated and treated quickly. With

proper treatment, even severely ill

patients can be saved.

(Mekalanos,1998).
• Epidemiology, Genetics, and Ecology of Cholera toxin, comma-shaped bacterium.

Toxigenic Vibrio Cholerae The bacterium's natural habitat is

brackish or saltwater where they attach

themselves easily to the chitin-containing

shells of crabs, other shellfish. which is

responsible for the profuse diarrhea, is

encoded by a lysogenic bacteriophage

designated. Cholera patients should be

evaluated and treated quickly. With

proper treatment, even severely ill

patients can be saved.

(Mekalanos,1998).
• Global Climate and Infectious Disease: On a global scale, cholera epidemics can

The Cholera Paradigm now be related to climate and climatic

events, such as El Niño, as well as the

global distribution of the plankton host.

Remote sensing, with the use of satellite

imagery, offers the potential for predicting

 conditions conducive to cholera

outbreaks or epidemics.

Outline of Significance of the Study

Identified Benefits Description

• Poverty This study will help people living in

poverty, having this will serve as a guide

of what water to drink.

• Future Researchers and Laboratory This study can improve services and

Staffs treatments that are not yet readily

available to the public.

• Health Organizations in a greater scale the health organizations

can be able to refine and innovate more

for their future studies in maintaining

healthy community.
•Travelers This study will help travelers to identify

the proper sources of water in their

escapade.

Statement of the Problem

Main Problem:
How effective is the Glycerol (Propane-1,2,3-triol) in detecting Gram-negative bacteria

(Vibrio Cholerae) in water?

Sub-Problems:

1.What is the specific amount of glycerol (Propane-1,2,3-triol) to be applied?

2. What is the efficacy of the glycerol in detecting when exposed to other temperature

rather than a room temperature?

3. What is the significant relationship between the amount of glycerol (Propane-1,2,3-

triol) with the substance of the Gram-negative Bacteria (Vibro Cholerae)?

Review of the Related Literature

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, has ravaged

humanity from time immemorial. Although the disease can be treated using antibiotics

along with administration of oral rehydration salts and controlled by good sanitation,

cholera is known to have produced mayhems in ancient times when little was known

about the pathogen. By the 21st century, ample information about the pathogen, its

epidemiology, genetics, treatment, and control strategies was revealed. However, there
is still fear of cholera outbreaks in developing countries, especially in the wake of

natural calamities. Studies have proved that the bacterium is mutating and evolving,

outcompeting all our efforts to treat the disease with previously used antibiotics and

control with existing vaccines. In this review, the major scientific insights of cholera

research are discussed. Considering the important role of biofilm formation in the V.

cholerae life cycle, the vast availability of next-generation sequencing data of the

pathogen and multi-omics approach, the review thrusts on the identification of suitable

biofilm-inhibiting targets and the discovery of anti-biofilm drugs from nature to control

the disease. (Lekshmi, N., Joseph, I., Ramamurthy, T., & Thomas, S. 2018).

Cholera (meaning being ‘a gutter’) is the right name for the disease, caused by

the ingestion of contaminated water. The causative organism is a comma-shaped,

flagellated bacterium, Vibrio cholerae. From 1817, there have been seven pandemics of

cholera till date. High mortality rate and its spread in all major continents such as Asia,

America, Europe and Africa make it important for research (Lekshmi. N. et al 2018). The

Indian subcontinent is the homeland of cholera, and from here, it had spread rapidly to

other countries of the world. Symptoms that distinguish cholera from other diarrheal

illnesses are the typical rice watery stool and severe vomiting that can lead to

dehydration and death within 48 h if left untreated. According to the World Health

Organization (WHO) report, 3.5 million people get infected and 100,000-120,000 people

die due to cholera each year in the developing countries1. In 2014, the case fatality due

to cholera was 1.17 per cent in 42 countries, which represented a 47 per cent increase

as compared to 20131. Cholera pandemics are ranked sixth amongst the world's worst
ten pandemics. The huge death rate is particularly reported by countries that suffer from

poor sanitation facilities and lack of proper water distribution systems. Cross-border

cases of cholera have been a serious issue in the Sub-Saharan African countries since

the past many years raising the case-fatality rate to 5 per cent2. School-going children

and children below five years are most affected in cholera-endemic countries. Cholera

cases and deaths in African and South Asian countries have accounted for 99 per cent

of the total cholera cases worldwide.

(Brad Lobitz, Louisa Beck, Anwar Huq, Byron Wood, George Fuchs, A. S. G.

Faruque, and Rita Colwell, 2000) stated that it has long been known that cholera

outbreaks can be initiated when Vibrio cholerae, the bacterium that causes cholera, is

present in drinking water in sufficient numbers to constitute an infective dose, if ingested

by humans. Outbreaks associated with drinking or bathing in unpurified river or brackish

water may directly or indirectly depend on such conditions as water temperature,

nutrient concentration, and plankton production that may be favorable for growth and

reproduction of the bacterium. Although these environmental parameters have routinely

been measured by using water samples collected aboard research ships, the available

data sets are sparse and infrequent. Furthermore, shipboard data acquisition is both

expensive and time-consuming. Interpolation to regional scales can also be

problematic. Although the bacterium, V. cholerae, cannot be sensed directly, remotely

sensed data can be used to infer its presence. In the study reported here, satellite data

were used to monitor the timing and spread of cholera. Public domain remote sensing

data for the Bay of Bengal were compared directly with cholera case data collected in
Bangladesh from 1992–1995. The remote sensing data included sea surface

temperature and sea surface height. It was discovered that sea surface temperature

shows an annual cycle like the cholera case data. Sea surface height may be an

indicator of incursion of plankton-laden water inland, e.g., tidal rivers, because it was

also found to be correlated with cholera outbreaks. The extensive studies accomplished

during the past 25 years, confirming the hypothesis that V. cholerae is autochthonous to

the aquatic environment and is a commensal of zooplankton, i.e., copepods, when

combined with the findings of the satellite data analyses, provide strong evidence that

cholera epidemics are climate-linked.

Glycerol is a fatty acid monoester that is recognized as a safe, natural compound

by the FDA and is commonly used as an emulsifier and preservative in the cosmetic

and food industries. It has also been recognized as a broad-spectrum antibacterial

agent, particularly against gram-positive pathogens, including gram-positive cocci,

Bacillus anthracis, and clostridia by interfering with surface signal transduction systems

[12, 13]. However, the compound likely has multiple mechanisms of action. GML inhibits

gram-positive exotoxin production and removes pre-formed S. aureus biofilms at sub-

growth-inhibitory concentrations [12–14]. Additionally, it has been documented to have

anti-inflammatory activity in tissue culture and in vivo at host mucosal surfaces, which

reduces infection establishment by gram-positive bacteria and enveloped viruses,

including simian immunodeficiency virus (SIV), without altering the normal microflora

[15–18]. Combined, these properties make GML a potential candidate as a safe tropical

microbicide. (Mueller E.A., Schlievert P.M. 2015)

 (Jubala, Aneta. May 2006). Cholera is an important enteric disease, which is

endemic to different regions of the world and has historically been the cause of severe

pandemics. Vibrio cholerae is a natural inhabitant of the aquatic environment and the

toxigenic strains are causative agents of potentially life-threatening diarrhea. A

multiplex, real-time detection assay was developed targeting four genes characteristic

of potentially toxigenic strains of V. cholerae, encoding: repeat in toxin (rtxA),

extracellular secretory protein (epsM), mannose-sensitive pili (mshA) and the toxin

coregulated pilus (tcpA). The assay was developed on the Cepheid Smart Cycler using

SYBR Green I for detection and the products were differentiated based on melting

temperature (Tm) analysis. Validation of the assay was achieved by testing against a

range of Vibrio and non-Vibrio species. The detection limit of the assay was determined

to be 103 CFU using cells from pure culture. This assay was also successful at

detecting V. cholerae directly from spiked environmental water samples in the order of

104 CFU, except from sea water which inhibited the assay. The incorporation of a

simple DNA purification step prior to the addition to the PCR increased the sensitivity

10fold to 103 CFU. This multiplex real-time PCR assay allows for a more reliable, rapid

detection and identification of V. cholerae which is considerably faster than current

conventional detection assays.

(J A Hasan, A Huq, M L Tamplin, R J Siebeling, R R Colwell, 2000) contributes a

report on the development and testing of a novel, rapid, colorimetric immunodiagnostic

kit, Cholera SMART, for direct detection of the presence of Vibrio cholerae O1 in clinical
specimens. Unlike conventional culture methods requiring several days to complete, the

Cholera SMART kit can be used directly in the field by untrained or minimally skilled

personnel to detect V. cholerae O1 in less than 15 min, without cumbersome laboratory

equipment. A total of 120 clinical and environmental bacterial strains, including both O1

and non-O1 serotypes of V. cholerae isolated from samples collected from a variety of

geographical regions, were tested, and positive reactions were observed only with V.

cholerae O1. Also, results of a field trial in Bangladesh, employing Cholera SMART,

showed 100% specificity and 96% sensitivity compared with conventional culture

methods. Another field trial, in Mexico, showed that Cholera SMART was 100% in

agreement with a recently described conglutination test when 108 stool specimens were

tested.

(Else M. Fykse, Gunnar Skogan, William Davies, Jaran Strand Olsen, Janet M.

Blatny 2006). A multitarget molecular beacon-based real-time nucleic acid sequence-

based amplification (NASBA) assay for the specific detection of Vibrio cholerae has

been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated

pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and

the 60-kDa chaperonin product (groEL) were selected as target sequences for

detection. The beacons for the five different genetic targets were evaluated by serial

dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated

all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA

was amplified. The specificity of the assay was investigated by testing several isolates

of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and
Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae

isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates.

The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker

groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell

viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA

isolated from different environmental water samples spiked with V. cholerae was

specifically detected by NASBA. These results indicate that NASBA can be used in the

rapid detection of V. cholerae from various environmental water samples. This method

has a strong potential for detecting toxigenic strains by using the tcpA and ctxA

markers. The entire assay including RNA extraction and NASBA was completed within

3 h.

(A Huq, B Xu, M A Chowdhury, M S Islam, R Montilla, R R Colwell, Applied and

Environmental Microbiology Jul 1996), Plankton to which cells of Vibrio cholerae O1

and/or O139 were attached was introduced into 0.5% Instant Ocean microcosms

maintained at 25 degrees C. The bulk of the plankton and associated particulates was

removed with a filter constructed from either nylon net and one of several different types

of sari material, the latter being very inexpensive and readily available in villages in

Bangladesh, where V. cholerae is endemic. V. cholerae was enumerated before and

after filtration to evaluate the efficiency of the filtration procedure. The results obtained

indicate that 99% of V. cholerae, i.e., those cells attached to plankton, were removed

from the water samples. Epidemic strains of V. cholerae O1 and O139 from various

geographical sources, including Bangladesh, Brazil, India, and Mexico, were included in
the experiments. Removal of vibrio from water by this simple filtration method was found

to yield consistent results with all strains examined in this study. Thus, it is concluded

that a simple filtration procedure involving the use of domestic sari material can reduce

the number of V. cholerae attached to plankton in raw water from ponds and rivers

commonly used for drinking. Since untreated water from such sources serves as

drinking water for millions of people living in developing countries (e.g., Bangladesh),

filtration should prove effective at reducing the incidence and severity of outbreaks,

especially in places that lack fuel wood for boiling water and/or municipal water

treatment plants. The results of this study provide the basis for determining such

reductions, which are to be carried out in the near future.

References:

•A Huq, B Xu, M A Chowdhury, M S Islam, R Montilla, R R Colwell Applied and

Environmental Microbiology Jul 1996, 62 (7) 2508-2512; DOI:

•Aneta J. Gubala “Multiplex real-time PCR detection of Vibrio cholerae” Journal of

Microbiological Methods Volume 65, Issue 2, May 2006

•Brad Lobitz, Louisa Beck, Anwar Huq, Byron Wood, George Fuchs, A.S.G. Faruque,

Rita Colwell Proceedings of the National Academy of Sciences Feb 2000, 97 (4)

14381443; DOI: 10.1073/pnas.97.4.1438

•Else M. Fykse, Gunnar Skogan, William Davies, Jaran Strand Olsen, Janet M. Blatny

Applied and Environmental Microbiology Feb 2007, 73 (5) 1457-1466; DOI:

10.1128/AEM.01635-06

•J A Hasan, A Huq, M L Tamplin, R J Siebeling, R R Colwell Journal of Clinical

Microbiology Jan 1994, 32 (1) 249-252; DOI:

•Lekshmi, N et al. “Changing facades of Vibrio cholerae: An enigma in the epidemiology

of cholera.” The Indian journal of medical research vol. 147,2 (2018): 133-141.

doi:10.4103/ijmr.IJMR_280_17

•Masaki H, Asoh N, Tao M, et al. [Detection of gram-negative bacteria in patients and

hospital environment at a room in geriatric wards under the infection control against

MRSA]. Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious

Diseases. 2001 Feb;75(2):144-150. DOI:

10.11150/kansenshogakuzasshi1970.75.1444.

•Mueller E.A., Schlievert P.M. ‘‘Non-aqueous glycerol monolaurate gel exhibits

antibacterial and anti-biofilm activity against gram-positive and gram-negative

pathogens” (2015) PLoS ONE, 10 (3) , art. no. e0120280

RESEARCH METHODOLOGY

Research Design
This study can be used particularly in water bodies and on other substances like food in

the future. A quantitative case study is good for gaining in-depth understanding and

information of this chosen study since this product contain experiments and

generalization across groups of people. This study is considered as Physical Science

due to its chemical compositions used. Researchers have planned on getting the kind of

data that deals on the cleanliness or the level of exposure of Gram-negative bacteria on

water sources that can be seen naturally like tap waters, rivers, wells and more. During

the experiment 2 Setups are used, Setup A with the presence of the V. cholerae and

Setup B for the absence of V. cholerae, with the Glycerol (Propane-1,2,3-triol) being the

major material for the study. This can be collected through visiting the said places for

getting the water samples for testing and in local drug or synthetic stores.

Research Strategy

To successfully conduct this research numerous plans has been formulated this

includes collecting, executing, and monitoring the said experiment with the main goal to

produce a safe, nontoxic, less expensive, and less time-consuming detection sheets for

the Gram-negative Bacteria, Vibrio Cholerae that has infested millions of health

worldwide. In a single dip, consumers will be able to determine if their water source is

safe. The proposed study took the form of a new research but inspired on an existing

study about detecting bacteria.

Research Environment
These days, pollution has been one of the major problems. Due to these pollutions,

harmful bacteria may grow uncontrollably in many forms of water with less sanitation

which may result to more complicated things like diarrhea, nausea, cholera and even

worse, death. Researchers will test this byproduct to areas that are known to have poor

drinking water source or in the areas that can easily be infected with the diseases via

contaminated water, communities like in Sitio Bulahan and Sitio Amoy places that are

within the Danao City Cebu or in the researcher’s residents water source.

Research Instrument

In conducting the qualitative research, the instrument to be used will be a researcher –

based structured observation. Since this research is based on experiments, it will be a

perfect example of a based structured information. To ensure the effectiveness and

reliability of the research outcome. Numerous pre-examinations and trials will be

conducted, to gain an in dept understanding of the said research.

Research Procedures

While choosing the medium of detection researchers have consider the factors like

convenience. Test kits and detection sheet are the best contenders, but detection

sheets have more rate of convenience while still maintaining the effective results like the

Test Kits. There are 2 procedures on creating the product:

A. How to create the Glycerol Solution (Propane-1,2,3-triol)

B. How to fuse sheets to the solutions.

A. Procedures on creating the Glycerol Solution (Propane-1,2,3-triol)

1. Prepare a solution of 30% glycerol (v/v) by mixing 30 ml of glycerol with 70 ml

of water.

2. Transfer the solution to a screw cap glass bottle and sterilize by autoclaving at

121°C for 15 min, pressure cookers can be used as a substitute for the

autoclaving process.

3. Loosen the cap during autoclaving.

B. Procedure on fusing the sheets to the solutions.

1. Prepare all the materials needed.

2. Mix 10ml of distilled water with 10ml detergent and 50% glycerol solution

Propane-1,2,3-triol as alternative for Sodium Azide for the Glycerol Solution in

a container.

3. Put the solution in the container and rest it for 10 minutes in a room

temperature.

4. Prepare the 1% tetramethyl-p-phenylenediamine dihydrochloride (7ml) in a

container which can be bought in the local chemical store.

5. Prepare the blotting paper to be soaked in the Glycerol Solution and in the

tetramethyl-p-phenylenediamine dihydrochloride.
6. Soak the Sheet first in the Glycerol Solution and leave it for 10 minutes for

total absorption. Remember to always wear PPE in executing this part,

irritations and skin allergies may occur.

7. After 10 minutes let the sheets dry up for the next stage of soaking.

8. If dried already soak the sheets in the 1% tetramethyl-p-phenylenediamine

dihydrochloride in the container this is for the Violet/Blue indication.

Remember to always wear PPE in executing this part, irritations and skin

allergies may occur.

9. Let the sheets dry under a room temperature for total absorption.

10. While waiting prepare the container for the byproduct.

11. Spray the insides of the container with glycerol solution for longevity.

12. If the products are dry enough soak the upper part of the sheets in a distilled

water again for 10 minutes this will be the touchable side, it is done to remove

impurities and solution that may cause allergic reaction to users due to the

substance used, then dry again.

13. After the process dyeing is now is possible, use yellow natural dye as an

indication for the touchable side.

14. After 10 minutes, contain the sheets in the container made specially for it.

15. Label the back of the container for the instruction of the users.

16. Usage is done by pulling the visible yellow sheets outside the container and

then dipping the brown like part of the sheet to a water solution with unknown

properties while holding the yellow part. Results can be seen on 5-10 minutes
 of waiting. No changes in color of the sheets may determine the absence of

the bacteria while Violet/Blue may indicate the presence of Vibrio Cholerae.

The container of the byproduct Is in the form of a gum-like container exposing the

yellow part for easy use.

F1: Materials Used for The Detection Paper for Vibrio cholerae

Materials Uses
10 ml Distilled water A product that does not contain any

impurities which is ideal for bacterial

detection. Product used for glycerol

solution.
50% Glycerol solution Contains the same level of Sodium

Azide but less toxic. Acts as the

detector for Vibrio cholerae.

10 ml Pure glycerin Product used for glycerol solution.

Ideal for detection due for chemically

pure structures.
Commercially prepared paper disk or Absorbs the solution. Contains 2 parts,

Blotting papers the white for detection and the yellow

part for the touchable side.

Containers Contains the sheets with glycerol
 solution.
Detergent Contains anti-microbial properties

needed in the production of Glycerol

Solution.
Labels For the instructions for the users.
Moist filter paper with substrate (1% Is an oxidable compound that serves

tetramethyl-p-phenylenediamine as a reducing co-substrate for heme

dihydrochloride) peroxidases. Acts as an artificial

electron acceptor for the enzyme

oxidase and is oxidized to form the

color compound Wurster’s blue (is the

one that brings out the color violet).

Pressure Cooker Substitute for steam sterilizers.
Stirring Rods Tool used for mixing ingredients.
Yellow Natural Dye Indication for the Touchable side.