Antimicrobial effect of different aromatic material extracts of Pandan leaves (Pandus tectorium), Basil leaves (Ocimum basilicum) on selected

pathogenic gram-positive Bacillus subtilis and gram-negative E-coli microorganisms

Presented by: Bigot, Florence Mae Juezan, Ervin Mariano, Nicole Quitalig, Roland

Presented to: Mr. Ranino Rivera

Table of Contents

I.

ACKNOWLEDGMENT----------------------------------------------------------

II.

INTRODUCTION------------------------------------------------------------------

III.

PROCEDURES---------------------------------------------------------------------

IV.

FLOWCHART----------------------------------------------------------------------

V.

ILLUSTRATION-------------------------------------------------------------------

VI.

TABLE(RESULTS-----------------------------------------------------------------

VII.

RESULTS AND DISCUSSION-------------------------------------------------

VIII. PICTORIALS----------------------------------------------------------------------

IX.

INSIGHTS--------------------------------------------------------------------------

Acknowledgement
No one walks alone on the journey of life. Just where you start to thank those that joined you, walked beside you, and helped you along the way. To God the father of all, we are thankful for the strength that keeps us standing and for the hope that keeps us believing that this affiliation would be possible and more interesting. We, the researchers are deeply indebted to our principal, Sr.Mailyn Bolivar O.P, who modeled us both technically and morally for achieving greater success in life. We would like also to express our deepest gratitude to Mr. Ranino Rivera for giving us knowledge and invaluable guidance that made this research successful. Without him, we wouldnt be able to achieve our goals in making this experiment successful and worth remembering. He inspired us greatly to work in this project. His willingness to motivate us contributed tremendously to our project. We also would like to thank him for showing us some example that related to the topic of our project We also express our sincere thanks to Mrs. Sophia Valencia for providing us with an environment and allowing us to use different lab apparatuses. Besides, we would like to thank the authority of Notre Dame-Siena School of Marbel for providing us with a good environment and facilities to complete this project. To the authority of Notre Dame Marbel University that provided us the bacterias that were needed for the experiment, We thank you. Finally, We want to express our gratitude to all the people who have given their heart whelming full support in making this compilation a magnificent experience. We also wanted to thank our families who inspired, encouraged and fully supported us in every trial that came our way. Also, we thank them for giving us not just financial, but moral and spiritual support. To our group mates who willingly helped us gather the necessary data and information needed for this compilation, we thank you.

Introduction
Aromatic materials are very important in our lives. They used as a fragrant and some used as medication, characterized by a fragrant smell, and usually by a warm, pungent taste, as ginger, cinnamon, spices. Various kinds of aromatic extracts from plants have been utilised for their health giving properties long before humans were around. Animals and insects can be powerfully affected by the smells from plants; just think of what catmint does to cats. For many insects and animals life revolves around smell.

Fragrance is a key part of everyone's lives, whether that smell is good or bad, and even if we are not conscious of the smell. For example, a lot of research has been undertaken on pheromones (animal/human natural fragrances). These odiferous molecules seem to play a key role in our own biological functions - particularly where fertility and reproduction are concerned. Perhaps it is not unexpected that many of the fragrance molecules found in plants also occur in insect and human pheromones.

Pandan leaves are used widely in Southeast Asian cooking as a flavoring. The plant is rare in the wild, but is widely cultivated. It is an upright, green plant with fan-shaped sprays of long, narrow, bladelike leaves and woody aerial roots. The plant is sterile, flowers only very rarely, and is propagated by cuttings.

The pandan leaf is found on the screwpine, a tropical plant that grows in certain European and Asian regions and is particularly popular in Southeast Asia. The leaves are approximately 4 inches (10 cm) long and are green, slender, shiny, and pleated. Commonly used to wrap foods like fish or shrimp, pandan leaf paste can imbue a dessert with sweetness and bright green coloring. Pandan leaves have been used to make thatched roofs, baskets and grass skirts. Additionally, this plant is believed to have medicinal properties and is an effective and natural cockroach repellent.

Basil, also known as St. Josephs Wort and sweet basil, is one of the most popular herbs used in cooking. It is a low growing herb with tender, light green large leaves. The leaves grow with a silky look to them, while the flowers on the basil leaves grow to be very large and with a wonderful pure white color to them. Basil, which is native to Asia, and more specifically India, has more history then you could imagine. It has been around for thousands of years and over the years the plant has been linked with closely with folklore and mythology. It has been linked in many ways, but not more so than the link between household and love.

Basil is commonly used fresh in cooked recipes. In general, it is added at the last moment, as cooking quickly destroys the flavor. The fresh herb can be kept for a short time in plastic bags in the refrigerator, or for a longer period in the freezer, after being blanched quickly in boiling water. The dried herb also loses most of its flavor, and what little flavor remains tastes very different, with a weak coumarin flavor, like hay. Basil is one of the main ingredients in pestoa green Italian oil-and-herb sauce. Its other main ingredients are olive oil, garlic, and pine nuts.

The leaves of basil are specific for many fevers. During the rainy season, when malaria and dengue fever are widely prevalent, tender leaves, boiled with tea, act as preventive against theses diseases. In case of acute fevers, a decoction of the leaves boiled with powdered cardamom in half a liter of water and mixed with sugar and milk brings down the temperature. The juice of tulsi leaves can be used to bring down fever. Extract of tulsi leaves in fresh water should be given every 2 to 3 hours. In between one can keep giving sips of cold water. In children, it is every effective in bringing down the temperature. Water boiled with basil leaves can be taken as drink in case of sore throat. This water can also be used as a gargle. Basil has strengthening effect on the kidney. In case of renal stone the juice of basil leaves and honey, if taken regularly for 6 months it will expel them via the urinary tract. Basil has a beneficial effect in cardiac disease and the weakness resulting from them. It reduces the level of blood cholesterol.

The purpose of this study is to know the antimicrobial efficacy of The Pandan and Basil leaves extracts on the selected Pathogenic Gram Positive Basillus subtilis and Gram Negative Escherichia coli Microorganisms. Although Pandan and Basil leaves are known for their aroma and as fragrant, we want to open our true intention in the study. We want to know with which bacterias(E.coli and B.subtilis) is more effective in determining which of the two aromas are more effective in killing bacterias.Like alchohol, soaps, and many other products have a labeled seal at the back which we can see in different commercials on television. Some alcohols have 99% in killing microorganisms. In our study we want to try different aromas in knowing how many percentages of aroma products can kill microorganisms.

PROCEDURES

1.) Preparation of chemicals and lab equipments.

lab equipments

Chemicals

12 petridishes 2 test tubes 6 small beakers Inoculating loop Erlenmeyer flask (1000 ml) Bunsen burner/alcohol lamp Water bath Vernier caliper Incubator Weighing scale Filter paper Stirring rod Mortar and pestle Funnel

15 grams of agar 5 grams of peptone 3 grams of beef extract 1000 ml distilled water 70 grams of Pandan leaves 70 grams of Basil leaves culturing microorganisms(Bacillus subtilis and Escherichia coli) Penicillin(Penicillium chrysogenum)

We also have to prepare our personal laboratory needs and wears like lab coats, surgical gloves, disinfectant, tape, cotton, pentel pen, rag, puncher, filter paper, newspapers and camera. In preparing materials, we should always remember one thing:BE ORGANIZED.

2.) Disinfection of Area Use disinfectant (Lysol) in cleaning. Once cleaned, you may start sterilizing the materials to free from microorganisms. TIP: (CLAYEX) CLEAN AS YOU EXPERIMENT.

3.)Sterilization of equipments/apparatuses Wrap on a newspaper each of the equipments or glasswares, the 12 petridishes, 2 inoculating loop, 2 test tubes, 6 small beakers, Erlenmeyer flask(500 ml), filter papers, cheese cloth and sterilize on an autoclave for about 121oC for 15 psi minutes.

4.) Getting of extracts (Pandan leaves and Basil leaves)

Weigh separately 70 grams of pandan leaves (Pandus tectorium), 70 grams of Basil leaves (Ocimum basilicum). Grind separately the materials with the use of mortar and pestle. Squiz the extracts with the use of cheese cloth. Put the extracts into 6 small beakers, 3 for pandan leaves and 3 for basil leaves. Divide each extracts into 100%, 75%, and 50%. For 100%= 1 ml extract, 75%= 0.75 extract 0.25 distilled water, 50%= 0.5 extracT and 0.5 distilled water. For our control, grind the penicillin and dissolve in 0.9 distilled water.

5.) Preparation of the media (Nutrient Agar) Weigh separately 5 grams of peptone, 3 grams of beef extract and 15 grams of agar. Dissolve in a 1000 ml of distilled water. Mix with the use of a stirring rod and boil in a water bath for 20 minutes.

6.) Cultivation of microorganisms. Prepare the 2 sterilized test tubes. Pour the melted agar media (nutrient agar) in the test tubes. Sterilize the inoculating loop and get the microorganism (gram-negative E.coli) in the first test tube containing the nutrient agar. Sterilize the neck of the tube before closing, then shake. Repeat the same process with the other microorganism( gram-positive Bacillus subtilis). Get the 12 petridishes. Pour the first test tube containing agar with gram-negative E.coli microorganism on 6 petridishes and the other test tube containing agar with gram-positive Bacillus subtilis on the other 6 petridishes.

7.) labelling Punch 48 circular pieces of filter papers . Dip 3 filter papers in each petridishes with 3 different replicas of different extracts(pandan leaves and basil leaves) and microorganisms. For the positive control(Bacillus subtilis), dip filter papers on dissolved penicillin (Penicillium chrysogenum)and put on the center on the 6 petridishes. For the negative control(Escherichia coli), dip filter paper on distilled water and put on the center of the other 6 petridishes.

8.) Incubation After doing the above procedures, incubate the petridishes in a 37oC temperature for 2 days.

9.) Measuring the zone of inhibition Measure the zone of inhibition with the use of vernier caliper. In this, you can determine which of the selective pathogenic gram-positive B. Subtilis and gram-negative E.coli has antimicrobial effect on 2 different aromatic materials.

10) Gathering of results.

FLOWCHART

Preparation of chemicals and lab equipments/ apparatuses

Disinfection of Area

Sterilization of equipments/apparatuses

Getting of extracts

Preparation of the media( Nutrient Ager

Cultivation of Microorganisms

labelling

Incubation

Measuring(Zone of Inhibition)

Gathering of results

ILLUSTRATION
Pandan leaves extract 100% 75% 50%

Bacillus Subtilis (+ control) Penicillin(P)

R2

R1 P R3 R2

R1 P R3 R2

R1 P R3

Escherichia coli(- control) distilled water (D)

R2

R1 D

R3

R1 D

R1 D R3 R2 R3

R2

R3

R2

Basil leaves extract

R2 R3 R1 P R3 R2 R3 R2 R2 R1 P R3 R3

Bacillus subtilis (+ control)

R2

R1 P

Escherichia coli

R2

R1 D

R3

R2

R1 D

R3

R2

R1 D

R3

(- control)

R2

R3

R2

R3

R2

R3

R2

R3

R2

R3

R2

R3

TABLE(THE RESULTS)

Basil Leaves Penicillin R1 R2 R3

Basillus subtilis (+) 100% 75% 50% 18.30mm 21.75mm 22.35mm 9.30mm 13.30mm 12mm 12.50mm 12.15mm 11mm 22mm 8.50mm Escherichia coli (-) 75% 7.10mm 10.10mm 13.50mm 10.25mm 9.05mm

Basil Leaves Distilled Water R1 R2 R3 100% 7.30mm 38.55mm 25.25mm 32.60mm

50% 7.25mm 10.25mm 16mm 8.05mm

Pandan Leaves Penicillin R1 R2 R3

Basillus subtilis (+) 100% 75% 50% 17.05mm 25.30mm 20.75mm 13.55mm 11.20mm 14.40mm 9.25mm 9.10mm 10.75mm 12.20mm 9.20mm 8.40mm Escherichia coli (-) 75% 9.10mm 7.60mm 7.20mm 7.25mm

Pandan Leaves Distilled Water R1 R2 R3

100% 7.20mm 7.90mm 7.70mm 11.10mm

50% 7.30mm 8.55mm 7.90mm 7.20mm

RESULTS AND DISCUSSION

We, The researchers have discussed the final results. And from the table of results, you can see that in the Treatment 1: Basil leaves with Penicillin as the controller and Bacillus subtilis as the microorganism, The biggest zone of inhibition can be found in Replication 2 with the filter disk soaked in 100% Basil leaves extract. In 100% extract, Replication 1 has 9.30mm as the zone of inhibition, In Replication 2, we have 12.50mm and while in Replication 3, we have exactly 22mm. For 75% extract and 25% distilled water, Replication 1 has 13.30mm, Replication 2 has 12.15mm and in replication 3, we have 8.50mm as their zone of inhibition. Finally for 50% extract and 50% distilled water, In replication 1, we have exactly 12mm, Replication 2, we have 11mm and in replication 3, we have 9.05mm. Also in this treatment, we also measured the Zone of inhibition of the Penicillin. In the 100% extract, the zone of inhibition is 18.30mm while in 75% extract and 25% distilled water, The zone of inhibition is 21.75mm and for the 50% extract and 50% distilled water, we have 22.35mm as the zone of inhibition. For the other extract in Treatment 1 which is Panadan leaves and Penicillin as also the controller and Basillus subtilis as the microorganism. The biggest zone of inhibition can be found in Replication 1 with the filter disk soaked in 50% extract and 50% distilled water. In 100% extract, Replication 1 has 913.55mm as the zone of inhibition, In Replication 2, we have 9.25mm and while in Replication 3, we have 12.20mm. For 75% extract and 25% distilled water, Replication 1 has 11.20mm, Replication 2 has 9.10mm and in replication 3, we have 9.20mm as their zone of inhibition. In this treatment, we measured the zone of inhibition of the Penicillin. In the 100% extract, the zone of inhibition is 17.05mm while in 75% extract and 25% distilled water, The zone of inhibition is 23.30mm and for the 50% extract and 50% distilled water, we have 20.75mm as the zone of inhibition.

For Treatment 2: Basil leaves with distilled water as the controller and Escherichia coli as the microorganism. The biggest zone of inhibition can be found on Replication 1 with the filter disk soaked in 100% Basil leaves extracts. In 100% extract, Replication 1 has 38.55mm as the zone of inhibition, In Replication 2, we have 9.25mm and while in Replication 3, we have 32.60mm. For 75% extract and 25% distilled water, Replication 1 has 10.10mm, Replication 2 has 13.50mm and in replication 3, we have 10.25mm as their zone of inhibition. Finally for 50% extract and 50% distilled water, In replication 1, we have 10.25mm, Replication 2, we have 16mm and in

replication 3, we have 8.05mm. In this treatment, we measured the zone of inhibition of the water. In the 100% extract, the zone of inhibition is 7.30mm while in 75% extract and 25% distilled water, The zone of inhibition is 7.10mm and for the 50% extract and 50% distilled water, we have 7.25mm as the zone of inhibition. For the other extract in Treatment 1 which is Panadan leaves and Distilled water as also the controller and Escherichia coli as the microorganism. The biggest zone of inhibition can be found in Replication 3 with the filter disk soaked in 50% extract and 50% distilled water. In 100% extract, Replication 1 has 7.90mm as the zone of inhibition, In Replication 2, we have 7.70mm and while in Replication 3, we have 11.10mm. For 75% extract and 25% distilled water, Replication 1 has 7.60mm, Replication 2 has 7.20mm and in replication 3, we have 7.25mm as their zone of inhibition. In this treatment, we measured the zone of inhibition of the distilled water. In the 100% extract, the zone of inhibition is 7.20mm while in 75% extract and 25% distilled water, The zone of inhibition is 9.10mm and for the 50% extract and 50% distilled water, we have 7.30mm as the zone of inhibition.

PICTORIALS

INSIGHTS