RESEARCH PLAN

RATIONALE

Over the past few decades, noble metals such as silver exhibited significantly

distinct physical, chemical, and biological properties from their bulk counterparts

(Khouloud et al., 2010). These significant and useful inorganic compounds are currently

attracting attentions because of its wide range of application in the field of medicine and

pharmaceuticals. Discoveries in past decades also demonstrated how these compounds

are in relationship of antibacterial properties, and the use of silver nitrate (AgNO3) as

antibacterial agents is new (Guzman, Dille, & Godet, 2009). With the increasing and

wide-range application of this compound, it is becoming necessary and required for a

broad and deeper range of understanding on the mechanisms of biological interaction (Ge

et al., 2014).

Silver nitrate is an inorganic compound with a chemical formula of AgNO3. This

compound is a versatile precursor to many other silver compounds. Silver nitrate is

considered to have many medical effects on the human body, and silver salts have also

been recorded to have anti-septic and anti-bacterial properties.

Some good examples of common but dreadful bacteria are the bacteria

Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. These bacteria

are all human pathogens, meaning they are found in the human body. They are also found
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in soil, water, skin flora, and most man-made environments throughout the world. They

use a wide range of organic material for food in animals; its versatility enables the

organism to infect damaged tissues or those with reduced immunity. These bacteria are

also held responsible for many infections. Due to upheaval in the resistance of bacteria to

the existing antimicrobial agents, plants are being used as alternative sources for the

development of safe, effective, and inexpensive new agents to treat and prevent bacterial

infections (Pumirat, & Luplertlop, 2013). Recent studies have shown that rice (Oryza

sativa L.), an important source of nutrients and a very great agent in suppressing bacteria,

are very abundant and are greatly consumed by Filipino families, consuming at least 8.9

kg of rice a week, making it 463 kg of rice a year in 2016 (Philippine Statistics Authority,

2010).

Some conventional methods for synthesizing and creating a solution such as

evaporation-condensation and laser ablation are the most important physical approach,

meanwhile chemical reduction is the most commonly used technique, which organic and

inorganic agents such as extracts and solutions were gathered then mixed with the

inorganic compound (Iravani et al., 2014). These methods are very effective, however,

they can be very expensive for the sake of some rural areas, and some developing

communities as well.

The need for a lower cost, sustainable, efficient, yet very effective alternatives

have fostered research on creating a solution that can kill bacteria. These natural

materials and inorganic compounds, specifically asian rice (Oryza sativa L.), and silver
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nitrate, when mixed and experimented correctly, can be the answer to the growing

infestation of the bacteria Pseudomonas aeruginosa, Staphylococcus aureus, and

Escherichia coli.

The study will be beneficial to many sectors of the community, specifically

pharmaceutical scientists, pharmaceutical companies, to the urban community, and for

future researchers.

Firstly, pharmaceutical scientists will be able to create a solution that can kill

Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli and cure the

diseases that they cause. They can also enhance the effectiveness of the antibacterial

properties of these created solutions. Secondly, pharmaceutical companies will also be

greatly benefitted since they will be able to finance and invest to it once it was proven

that these created solutions can be actually used as an anti-bacterial agent. Thirdly, the

urban community will be free from the destructive effect of these harmful bacteria.

Useless and excess silver can now be also used as an anti-bacterial agent. Lastly, future

researchers can use the research study as a basis and reference for their future

investigative projects.

RESEARCH QUESTIONS AND HYPOTHESES

The research study aims to determine the antimicrobial activity of the created

silver nitrate (AgNO3)-induced asian rice (Oryza sativa L. cv. NFA Rice) stock/wash
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solution against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.

Specifically, it seeks to find answer to the following questions:

1. What is the effect of AgNO3-induced Asian rice wash solution to the growth of

Pseudomonas aeruginosa?

2. What is the effect of AgNO3-induced Asian rice wash solution to the growth of

Staphylococcus aureus?

3. What is the effect of AgNO3-induced Asian rice wash solution to the growth of

Escherichia coli?

Following are the tentative answers to the research questions:

1. AgNO3-induced Asian rice wash solution will inhibit the growth of Pseudomonas

aeruginosa.

2. AgNO3-induced Asian rice wash solution will inhibit the growth of

Staphylococcus aureus.

3. AgNO3-induced Asian rice wash solution will inhibit the growth of Escherichia

coli.

PROCEDURES

Preparation of Materials. The primary materials that will be used in this study

are the rice stock/wash solutions and the 0.01M silver nitrate (AgNO3). In preparing the
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0.01M silver nitrate (AgNO3), one gram of silver nitrate (AgNO3) salt were mixed in 100

mL water. After mixing, 50 mL of the solution were measured and poured into the two 50

mL beakers, having two beakers with 50 mL of 0.01M silver nitrate (AgNO3) each. In

preparing the rice stock/wash solution, Asian Rice (Oryza sativa L.) cultivars- NFA Rice

will be washed in tap water. The 50 mL of sample of rice stock/wash from each were

then poured on two 50 mL beakers respectively.

Creation of the AgNO3-induced Asian rice wash solution. The first 50 mL

0.01M silver nitrate (AgNO3) will be mixed together with the 50 mL NFA Rice

stock/wash solution on one of the 100 mL graduated cylinders. The prepared solution

will be left to settle for eight hours.

Evaluation of the Antibacterial Activity of the AgNO3-induced Asian rice

wash solution. The evaluation of the antibacterial activity of the AgNO3-induced Asian

rice wash solution solution against Escherichia coli, Staphylococcus aureus, and

Pseudomonas aeruginosa will be conducted and done at the Department of Science and

Technology–Industrial Technology Development Institute: Standards and Testing

Division Laboratory. The zone of inhibition in millimeter (mm) will be measured and its

reactivity will be determined using the disk diffusion method.

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RISK AND SAFETY

According to The National Institute for Occupational Safety and Health [NIOSH]

(2015), silver nitrate poses chemical danger when exposed to high temperature for it

forms toxic fumes including nitrous oxide. The substance is toxic to human when the

aerosol will be inhaled or the physical substance will be ingested. To ensure the safety

during the preparation of the rice wash solution, the procedure will be done in a location

far from combustible materials. Also, protective body covering will be worn such as

gloves and face mask.

There are numerous potential hazards involved in culturing bacteria like

Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa for these are

known to be pathogenic. The evaluation of the antibacterial activity of the prepared rice

wash solution will be done by a qualified scientist from the Department of Science and

Technology’s Industrial Technology Development Institute: Standards and Testing

Division Laboratory following the standard safety precautions by the service laboratory.

DATA ANALYSIS

For the quantitative data to be collected, zone of inhibition (mm), mean will be

obtained. For the qualitative data, reactivity and activity of inhibition, verbal descriptions

will be used. Tables 1 and 2 contain the bases to be used in evaluating reactivity and

activity of inhibition.
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Table 1. The basis for the reactivity rating of the created solutions against the bacteria
Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.
Reactivity Rating Description
Severe 4 Zone extends more than 10 mm beyond the specimen
Moderate 3 Zone extends 5 to 10 mm beyond the specimen
Mild 2 Zone limited under the specimen
Slight 1 Some malformed or degenerated cells under the specimen
None 0 No detectable zone under the specimen

Table 2. The basis for the activity of inhibition of the bacteria Escherichia coli,
Staphylococcus aureus, and Pseudomonas aeruginosa.
Activity Rating Description
Complete +++ Absence of microbial growth within zone of inhibition
Partial ++ Few microbial growth within zone of inhibition
Slight + Moderate microbial growth within zone of inhibition
Negative - Severe microbial growth / No zone of inhibition

BIBLIOGRAPHY

Ge, L., Li, Q., Wang, M., Ouyang, J. (2014). Nanosilver particles in medical applications:

synthesis, performance, and toxicity. Retrieved July 20, 2016 from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037247/

Guzman, M. G., Dille, J., & Godet, S. (2009). Synthesis of silver nanoparticles

bychemical reduction method and their antibacterial activity. International Journal

of Chemical and Biomolecular Engineering 2(3). Retrieved July 20, 2016 from

http://www.omnis-mg.hr/radovi/4bak-4siz-peru.pdf
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Iravani, S., Korbekandi H., Mirmohammadi, S. V., & Zolfaghari, B. (2014). Research in

Pharmaceutical Sciences. Retrieved March 8, 2016 from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326978/

Khouloud, M. M., El-Nour, A., Eftaiha, A., Al-Warthan, A. (2010). Synthesis and

Applications of Silver Nanoparticles. Arabian Journal of Chemistry 3(3).

Retrieved March 8, 2016.

http://www.sciencedirect.com/science/article/pii/S1878535210000377

Philippine Statistics Authority. (2010). A Filipino family consumed 8.9 kg of ordinary

rice a week in 2006 (Results from the 2006 Family Income and Expenditure

Survey (FIES). Retrieved July 20, 2016 from https://psa.gov.ph/content/filipino-

family-consumed-89-kg-ordinary-rice-week 2006-results -2006-family-income-

and

Pumirat, P., & Luplertlop, N. (2013). The In-vitro Antibacterial Effect of Colored Rice

Crude Extracts against Staphylococcus aureus Associated with Skin and Soft

Tissue Infection. Journal of Agricultural Science 5(11). Retrieved July 20, 2016

from http://www.ccsenet.org/journal/index.php/jas/article/viewFile/29040/18297
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The National Institute for Occupational Safety and Health [NIOSH]. (2015). Silver

Nitrate. Retrieved July 20, 2016 from

https://www.cdc.gov/niosh/ipcsneng/neng1116.html