Inhibiting characteristics of Ipil-Ipil Seed Extract to Trichophyton Mentagrophyte, Enterococcus faecalis, Proteus vulgaris 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
October 9, 2018
Leucaena leucocephala, or Ipil-ipil, is a small tree that can grow up to 8 meters high.Leucaena leucocephala is from kingdom plantae and the phylum classification is Tracheophyta. It from class leguminosae andorder fabales and came from a family fabaceae..

The 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 were used as a coffee substitute. Leaves and seeds are also used as food in Central America, Indonesia and Thailand, and eaten in processed or unprocessed forms in Java, Indonesia. Ipil-Ipil seeds are also used for agriculture and animal feed.
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 (the presence of bacteria in blood), abdominal and pelvic infections, urinary tract infections, oral infections (particularly with root canals), and septicemia (blood poisoning).
Proteus vulgaris, previously considered biogroup 2, has been accounted for to cause UTIs, wound contaminations, circulatory system diseases, and respiratory tract contaminations. 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.

Will the Ipil-ipil seed extract be able to inhibit Trichophyton Mentagrophyte, Enterococcus faecalis, Proteus vulgaris and Clostridium perfringens?
Alternative: The Ipil-ipil seed extract will have a significant effect against the different microorganisms.

Null: The Ipil-ipil seed extract will not have a significant effect against the different
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 as antimicrobial property that is capable of inhibiting the different microorganisms.

Biological hazards can be a threat to the health of living organisms, primarily that of humans. This can include samples of a microorganism, virus or toxin (from a biological source) that can affect human health. 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 were utilized. Biosafety level two was considered in dealing with the microorganisms.

center7620Collection of Materials
00Collection of Materials

center454025Preparation of the Materials
00Preparation of the Materials


center433705Gathering of Data
00Gathering of Data

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 authenticated at the Natural Sciences Research Institute of UP Diliman.

Preparation of Materials
Leucaena leucocephala seeds will be separated from its pods and soaked for 3 days in 100% 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 until experimentation begins.

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.

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 is going to be calculated. The antimicrobial index (AI) is going to be computed using the following formula:
AI = Diameter of clearing zone-Diameter of wellDiameter of wellFor 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.
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 n.d. Bode science center. Accessed October 25, 2018.

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

n.d. Center for disease control and Prevention. Accessed October 25, 2018.

n.d. Enterococcus Faecalis. Accessed October 21, 2018.

n.d. Health line. Accessed October 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 October 25, 2018.

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

Nader Shetta, Mohamed Zaky Zayed, Sobhy Sallam. n.d. ResearchgAte. Accessed October 25, 2018.

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


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