Inhibiting characteristics of Ipil-Ipil Seed Extract to Trichophyton Mentagrophyte, Enterococcus faecalis, Proteus vulgaris, Shigella and Clostridium perfringens
A Science Investigatory Project Plan
Submitted as Partial Fulfillment
Of the Requirements in Research IIA
ARIAS, Nicole R.

FERNANDEZ, Nicole Ann P.

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PUA, Danielle Marie M.

Quezon City Science High School
July 23, 2018
RATIONALE:
Leucaena leucocephala or Ipil-Ipil is a small tree that can grow up to 8 meters high. It is from kingdom plantae under the classification of spermatophyta. It is from class dicotyledonae and order fabales and came from a family fabaceae.

Ipil-Ipil tree is originally from Southern Mexico and Northern Central America. It is known to have the ability to spread easily across many tropical locations. Although Ipil-Ipil is not from the Philippines, it has been spreading across the country. It has adapted to the Philippines very well and can be found easily anywhere the country because it is a type of a tropical rainforest plant.

In some provinces of the Philippines, the Ipil-Ipil seeds are used as a coffee substitute. Leaves and seeds are also used as food in Central America and Thailand, and eaten processed or unprocessed in Java, Indonesia. It is also used for agriculture and animal feed.

Nowadays, Ipil-Ipil seeds are not fully utilized for medicinal purposes.
Studies were carried out to characterize the oil and seed meal of Ipil-Ipil (Leucaena leucocephala) seed. The GLC analysis revealed that the oil is composed of palmitic acid (15.7%), palmitoleic acid (0.2%), stearic acid (3.6%), oleic acid (15.5%), linoleic acid (63.2%) linolenic acid (0.4%), arachidic acid (0.5%), and lignoceric acid (0.9%). Ipil-Ipil seed is known to have oleic acid. Oleic acid is a fatty acid which is known to be antifungal.
Based from the phytochemical analysis of the Ipil-Ipil seeds oleic acid is to be found and known to be antifungal.

Enterococcus faecalis is a lactic corrosive microorganisms. Enterococci is Gram-positive cocci. Enterococcus species are facultative anaerobic living beings. E. faecalis causes around 80% of human diseases. It may cause bacteremia, abdominal and pelvic infections, urinary tract infections, oral infections and septicemia.
Proteus vulgaris has been accounted for to cause urinary tract infections, circulatory system diseases, respiratory tract infections as well as wound infections. Proteus vulgaris is known to be an aerobic, rod-shaped, Gram-negative bacterium.

Clostridium perfringens is a gram-positive bacillus that is known produce extracellular toxins C. perfringens type A produces an alpha toxin that can cause acute foodborne illness in human that can result to diarrhea and abdominal discomfort. C. perfringens type A is the cause of necrotizing enteritis in chickens. C. perfringens can be found on raw meat and poultry. It also prefers to grow in conditions with very little or no oxygen, and under ideal conditions can multiply very rapidly
Shigella is gram-negative, facultative intracellular pathogens. Shigella infection (shigellosis) is an intestinal disease caused by a family of bacteria known as Shigella. The main sign of Shigella infection is diarrhea, which often is bloody.

The findings of this study will benefit the society considering that microbiology plays an important aspect in the field of medicine. With the greater demand for effective medicines and treatments having this study justifies the need for more substitutes discovering possible ingredient for medicine.

RESEARCH QUESTION:
Which of the following microorganisms can be inhibited by the Ipil-Ipil seed extract?
HYPOTHESIS:
Alternative: The Ipil-Ipil seed extract will has a significant effect against the different microorganisms.

Null: The Ipil-Ipil seed extract will has no significant effect against the different
microorganisms.

EXPECTED OUTCOME
In this study, the Ipil-Ipil seed extract is expected to inhibit the various microorganisms namely Trichophyton mentagrophyte, Enterococcus faecalis, Clostridium perfringens, Proteus vulgaris and Shigella. The extract has an antimicrobial or antifungal property that is capable of inhibiting the different microorganisms.

RISK AND SAFETY
Biological hazards can be a threat to the health of living organisms, primarily that of humans. This can include samples of a microorganism like virus or toxin. Doing anything related to microbiology poses a safety hazard. Some examples are skin infection and inhalation of airborne chemicals. In this research project, glassware, bacterium, and flammable materials will be utilized. Biosafety level two was considered in dealing with the microorganisms. Since fungus and bacteria are going to be used, safety precautions were observed when handling it. All the equipments used are going to be sterilized after work.

FLOWCHART:
center7620Collection and preparation of Ipil-Ipil seeds
00Collection and preparation of Ipil-Ipil seeds

center25971500
center454025Culturing of Bacteria
00Culturing of Bacteria

center25527000
center447040Antimicrobial testing
00Antimicrobial testing

center24130000
center433705Gathering of Data
00Gathering of Data

center23241000
center440055Data Analysis
00Data Analysis

Collection of Materials
A kilogram of Leucaena leucocephala seeds were to be handpicked in San Jose Del Monte, Bulacan and will be brought to UP Institute of Biology for proper authentication. Trichophyton Mentagrophyte will be bought and used for experimentation at the Natural Sciences Research Institute of UP Diliman.

Preparation of Materials
Leucaena leucocephala seeds will be separated from its seeds and soaked for 3 days in 95% ethyl alcohol. After 3 days, the seeds will be filtered out using a cheese cloth to collect the liquid residue and be sent to the UP Institute of Chemistry to undergo the process of rotary evaporation. The microorganisms Trichophyton Mentagrophyte, Enterococcus faecalis, Clostridium perfringens, Proteus vulgaris and Shigella will be kept and supervised in the Natural Sciences Research Institute and BGR hydrolab until experimentation begins.

Experimentation
For Trichophyton Mentagrophyte microbial suspension was prepared from 7-day old culture of the fungus. The suspending medium used was 0.1% peptone water.

Pre-poured Potato Dextrose Agar (PDA) plates were going to be inoculated with the microbial suspension by swabbing the agar surface. The cotton swab on an applicator stick is supposed to be dipped into the microbial suspension, rotated several times and pressed firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab. The swab will be streaked over the entire agar surface. This procedure is going to be repeated two more times, rotating the plate 60° each time to ensure even distribution of the inoculums. Three (3) equidistant wells will be made on the agar plate using a cork borer (10 mm diameter). Two hundred (200) ?l of the sample will be placed in each well.

For the different kinds of bacteria. The cultured bacteria will be placed in a petri dish using multiple swabbing. The disc will be placed in the center of the petri dish then a drop of the Ipil-Ipil seed extract for positive control and distilled water to serve as the negative control was placed on the disc. The petri dish is going to be incubated for 24 hrs.
The PDA plates is going to be incubated at room temperature for 3 days. The clearing zone is going to be measured in millimeters and the average diameter of the clearing zones will be using this formula:
AI = Diameter of clearing zone-Diameter of wellDiameter of wellThis study is to determine the effectiveness of the Ipil-Ipil extract against the stated microorganisms in the BGA hydrolab. The measurement of inhibition zones and antimicrobial indexes of each sample will be recorded within a week. The usage of bacteria was considered due to insufficient time. 100% concentration of the extract was used for the same reason.

Gathering Data
The data will be collected by observing the physical change of the microorganisms. The data that will be included in the experiment are the reactions, presence, changes in physical appearance, texture, and smell of the microorganisms to the extract.

Data Analysis
This research will focus on the effectiveness of the Ipil-Ipil seed extract on each microorganism. The type of statistical test this research will be using is Independent T-test. The inhibition zones of the microorganisms will be treated as the dependent variable and the extract from Ipil-Ipil seeds will be treated as the independent variable

BIBLIOGRAPHY
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Bottone, Edward J. 2010. Bacillus cereus, a Volatile Human Pathogen. April 23. Accessed September 21, 2018.

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n.d. Health line. Accessed September 21, 2018.

Hossain, M.A. 1998. Studies on the composition of Ipil-Ipil. Accessed July 21, 2018.

2004-2015. Ipil-Ipil scientific name: Leucaena Leucocephala. Accessed January 18, 2018.

Jaya Sureshbabu, Russell W Steele. n.d. Medscape. Accessed September 25, 2018.

Peter M. Rabinowitz, Lisa A. Conti. 2010. Clostridium perfringens. Accessed September 21, 2018.

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n.d. Environmental Challenges of the Philippines. Accessed September 25, 2018.

BIBLIOGRAPHY m sci14 l 1033 2014. sciencedirect. August. Accessed September 14, 2018.