Final Edited 4t3c3y

1

CHAPTER I THE PROBLEM AND SCOPE Introduction With the uprising population and commodities in the community, morbidity and mortality: it is hard to avoid since the people in the society are also experiencing phenomenal calamities like flood. Floods may carry microorganisms which can contribute to poor wound healing. The researchers conducted this study since the Carabao Grass (Paspalum conjugatum) is known to have an antimicrobial property and is widely spread in local areas and in most communities in Philippines. In addition, the researchers want to help minimize the expenses in health services most especially to the medications being bought. There are number of people living in far flung areas that are unfamiliar to this kind of grass which can be a possible alternative antimicrobial regimen. This research aims to prove that Carabao Grass (P.conjugatum) has an antimicrobial agent. Through this, the researchers will be of help in disseminating information to the people not only in remote areas but also to those people living in the community. The researchers aim to know more about the phytochemical analysis that are found in Carabao Grass (P.conjugatum) and its potential action as an antimicrobial agent. People are not aware about the idea of Carabao Grass as an antimicrobial agent (Ebadi, 2007) and through this research; people in the community are beneficial with regard to the availability of resources.

2

Theoretical Framework The study consists of antimicrobial assay, which then would determine the attribute of P. conjugatum. The researcher must endeavor and analyze things to equipped themselves and be able to discover the significance of the study. The Kirby-Bauer agar diffusion method of antimicrobial assay is a convenient and efficient procedure to conduct the study. It is placing of filter paper disk with a concentration of treatment and control extract. The diffusion will depend on the agar properties and the molecular weight of the filter paper disk. Antimicrobial activity may be observed based on the presence of the zones of inhibition within the span of 24 hours. The slower the diffusion, the larger the molecular weight present in the filter disk. The size of the inhibition zone will depend on the depth of the agar. There are some theories that can the study of the researchers. Theories like the ‘Environmental Model’. It focuses on the manipulation of the availability of resources in the environment. It is in this theory that focusing on changing and manipulating the environment in order to put the patient in best possible conditions for nature to act. The said study has relation to this theory for it benefits the community to lessen their expenses in health services and can be an alternative antimicrobial regimen for people living far away from health centers and pharmacies.

3

Objectives of the Study This study aims to pursue the following objectives: 1. To evaluate the antimicrobial activity of Paspalum conjugatum based on the zones of inhibition against the test organism such as Staphylococcus aureus (Grampositive), Pseudomonas aeruginosa and Escherichia coli (Gram-negative). 2. To determine the significant relationship of antimicrobial activity of Carabao

Grass Decoction against the Chloramphenicol as positive control.

Significance of the Study Over the years, plants have been primary source for development of drugs and some are beneficial in an extensive form to gauge in clinical use (Kala, 2011). This study will provide information about antimicrobial activity of the Carabao Grass (P. conjugatum) through Kirby-Bauer disc diffusion test against the bacteria namely, Staphylococcus aureus, Pseudomonas auruginosa and Escherichia coli. The gathered data for antimicrobial activity of P. conjugatum will be useful to gain alternatives in combating microbial infection. This study would be beneficial in the following groups and institutions: Community: The study is primarily for the beneficial of the community that would be able to have easily access of antimicrobial agent and that is easily prepared and will much cost lesser than other antimicrobial drugs. The outcome of the study would be greatly

4

helpful as it is will be payless to access too. Complication of microbial agent would be easily tended. Pharmaceutical Institutions: Delivering result for antimicrobial agent of P. conjugatum will pull the curiosity of institutions and that would lead to advance study and development of the plant and pursue an affordable antimicrobial drug. Government and non-government Health Services Institutions: Of the new discovery of the antimicrobial agent in P. conjugatum institution like DOH and NGO organization would be able to enhance and developed the ability of P. conjugatum as antimicrobial and informed the public about the effectiveness of the drug which then useful in health services. Future Researchers: researchers may improve, innovate or enhance a more comprehensive study. They would be able to create a more potent antimicrobial agent with the aid of this study. This study would be a tool and be useful as reference in the study of P. conjugatum other medicinal uses.

Scope and Limitation of the study This research was conducted to determine if

P. conjugatum as a promising

antimicrobial agent. It is an in vitro study in the settings of College of Nursing laboratory in Mindanao State University-Iligan Institute of Technology (MSU-IIT), Iligan City. The Kirby-Bauer antimicrobial diffusion method of antimicrobial assay was conducted in demonstrating the presence of any possible antimicrobial property. The test microorganisms were obtained in the microbiology laboratory of biology department. Gathering of Paspalum conjugatum took place in Iligan City. Then, a Professor of Botany of the Department of Biological Sciences of Mindanao State University–Iligan

5

Institute of Technology (MSU-IIT) checked it. The preparation of the extract was in the College of Nursing Microbiology Laboratory in Mindanao State University-Iligan Institute of Technology (MSU-IIT), Iligan City. The stem leaves and roots were extracted prior to assay.

Definition of Antimicrobial – is an agent that kills microorganism or inhibits their growth. Antimicrobial Assay- a method for quantitatively determining the concentration of an antibiotic by its effect in inhibiting the growth of a susceptible microorganism. Bacteria –are microscopic living organism, usually one-celled, that can be found everywhere. Kirby-Bauer agar diffusion method -is a test which uses antibiotic-impregnated wafers to test whether bacteria are affected by antibiotics Minimum inhibition concentration – is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Mueller Hinton Agar – is a microbial growth medium that is commonly used for antibiotic susceptibility testing. Zone of inhibition – is an area of growth inhibition around a point source, within a cultured organism on a solid medium, due to the action of growth-inhibitory substance such as antimicrobial present at the source.

6

7

CHAPTER II REVIEW OF RELATED LITERATURE For over a thousand years, human beings’ primary sources for medicine are the plants, until now people are developing newer medicines that are basically derived from plants. Then and now has no apparently significant changes that are worthy to say that people no longer use plants as a one of sources for medicines (Kala, 2011). On the 20 th century plant become the emerging medicinal products that have been discovered using extract and some modern medicines comes from pure compounds isolated from plants. Ethno medicine helps researcher to conduct new study with basis of this traditional plant (Bernhoft, 2008). The medicinal effect of the plants that is used as ethno medicines or for the development of new medicine is the reason that plant produces secondary metabolites that are no longer needed but these metabolites are the ones that give effect for medicinal use (Kala, 2000& Parr, et al, 2000). Developed countries are using this secondary by isolating the compound to create a more potent and effective medicine however, developing countries like our country opted for herbal medicines, due to the lack of facilities and fund to able isolate pure compounds (Hudzicki, 2009). The medicinal benefits of this herb are dated in all the early records of the human existence. It started during the early Chinese era, which is dated more than 4000 years ago up to our generation today, the extracts of these herbs and plants are still being used to treat diseases today. Medicinal herbs are being used extensively in our generation and are being expanded into much noncomplex forms. This study is then focused on the

8

application of Paspalum conjugatum extracts to determine its biological activity through putting it to test for its antimicrobial properties.

Developing herbal medicine rather than pure isolation of pure compound is sought out to this study, a grass known as Paspalum conjugatum.Itis a type of grass that grows about 20-40 cm and some might reach 60 cm that grows in an open area and spread quickly, due to its feature as a creeping grass (Rao, 2000). The grass is known as ‘buffalo grass’;’ sour grasses in Jamaica, ‘Hilo’ in Hawaii and locally known as ‘pad pad’ (Burkill, 1985). Traditionally in some countries, they use this type of grass as a means of treatment it is known as Paspalum conjugatum. It is used to help these indigenous people from Amazon area to relieve their headache. These people use the grass by infusion (Russo, 1990). Also, in other country like Cuba, they apply this by mixing the grass in their bath to relieve malaria; others create a medicine by grinding the grass to formulate an alternative medicine for pleurisy, pneumonia, flu, fever, and fatigue. The Bahamian people prepared it with prickly pear (Opuntia) and wood ashes as a medicine for tuberculosis (Austin, 2004). In the local settings, indigenous people use the grass in relieving diarrhea problems, fever and wound, the fact that this kind of grass can be found elsewhere in the local areas (Carabao Grass for Kidney Disease, 2014). Aside from the phytochemicals that was found on the grass, the plant has been traditionally used as a medicine for variety of ethnicity all over the globe (Cambie, et al. 1994).Paspalum conjugatum is one of ethno medicine that is used by variety of people all over the globe. In Trinidad, people use it as a cure for pleurisy, pneumonia, fever, flu and fatigue (Austin, 2004). Furthermore, flavonoids have compounds that are known to

9

synthesize as an antioxidant, antimicrobial, and many more. Other studies found out those flavonoids are just not antioxidants but also exhibits antiallergenic, antiviral, antiinflammatory and vasodilation actions (Pietta, 2000). However, to test the antimicrobial activity Paspalum conjugatum and is to conduct an in vitro study by which testing it through susceptibility of common bacteria that are commonly the causes of infections such as Staphylococcus aureus (Uwaezuoke, & Aririatu, 2005). Through Kirby-Bauer agar diffusion method,the in vitro study is used to test the susceptibility of bacteria against the extracts of PaspalumConjugatum, which is efficient and reliable in screening compound of natural extract of the plant (Sarker, et al, 2007). It has been alarming that more and more of bacteria are resistant to drugs including Staphylococcus aureus (Locksley, et al, 1982). As antibacterial becomes to falter from its efficacy, it also has never ending complaints of the side effects that brought together upon taken (Uwaezuoke, &Aririatu, 2005). It is also the same in the locality but rather the inevitable growing population and poverty, that drags health services to implacably unavailable to those who needed it most (Chan, 2010 & WHO, 2011). With the majority found on common damp environments, Pseudomonas aeruginosa is a rod-shaped, strict aerobe. Furthermore, it is regularly found in soilbut more in waterborne creatures. However, colonization has been found in numerous different plants and organisms, thus it appeared to bring a colony of more than fifty percent of the people. Pseudomonas aeruginosa will be an entrepreneurial pathogen that camwood actuate infections that regularly bring about hospitalization and are frequently lives

10

undermining. This organism exhibits intrinsic imperviousness to large portions of antibiotics and could adjust when laid open on antimicrobial operators to create new safety measures. This permits Pseudomonas aeruginosa to flourish and further develop. With such survivability, these microscopic organisms may not be best deliberately monitored alongside hospitals. Its genome will be continually updated under a database, and it will be comprehensively monitored because of its possibility similarly to as antibiotic weapon (Joey Silhavy, 2009). Bacillus subtilis may be a Gram positive, Pole molded bacteria and it is ordinarily found in soil. It might have been initially named “Vibrio subtilis” asChristian Gottfried Ehrenberg might have found it previously during the year 1835. It might have been renamed “Bacillus subtilis” clinched alongside 1872 by Ferdinand Cohn. This bacterium will be also known toward the names feed bacillus, grass bacillus, or Bacillus globigii.Bacillussubtilis is a endospore framing bacteria, and the endospore types. Itpermits to withstand an amazing temperature and additionally dry situations. Bacillussubtilis may be acknowledged and commit aerobicity. The point when over the vicinity for nitrates or glucoseBacillussubtilis is not recognized pathogenic or harmful. Furthermore,it will not be an agonizing sickness. Bacillussubtilis needs aflagellum, which makes motility quicker (Ellen Kirk, 2009). Escherichia coli (E. coli) microbes ordinarily live in the digestion tracts of individuals and creatures. Generally, E. coliis innocuous and really is a critical part of the human intestinal tract. Some E. coli are pathogenic, which means they can bring sickness, either loose bowels or disease outside of the intestinal tract. The sorts of E. coli that can bring about loose bowels can be transmitted through tainted water or sustenance, or

11

through with creatures or persons (Center for Disease Control and Prevention, 2014). E. coli comprises of a various gathering of microbes. Pathogenic E. coli strains are sorted into pathotypes. Six pathotypes are connected with looseness of the bowels and all things considered are alluded to as diarrhea genicE. coli.

Justification of the Study The selections of Paspalum conjugatum are as follows. (1) It is commonly used in other ethnicity and areas; (2) it has been used as a medicinal plant. (3) It is widespread in the local settings especially in rural areas (4) the extracts of the plant were traditionally used to treat skin conditions such as wounds and in addition,(5) it can be easily accessible aside from itswidespread distribution in the country, (6) it can be easily prepared. There are no journals about the study of Paspalum conjugatum for its antimicrobial activity. This is the first study to extract the compounds of Paspalum conjugatum, which will then be used as an effective anti-microbial alternative. With this, researchers are determined to conduct a novel study regarding its anti-microbial activity.

Synthesis Treatment of infectious diseases in this millennium shows significant side effects. Too much intake of this medicine might cause the bacteria to produce resistance to this drug, thusthis dilemma is inescapable. To help and minimize this problem, an in vitro study was conducted that would determine the susceptibility of bacteria against the extract of Paspalum conjugatum.

12

CHAPTER 3 METHODOLOGY

Research Design The study was carried out through in vitro study where a strict and proper observation is required in conducting the antimicrobial assay and keeping it from contamination wherein samples are cultured bacteria and isolated in a prepared agar, it included two types of bacteria. Two from Gram-positive bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) and one from Gram negative bacteria (Escherichia coli). The bacteria were cultured and obtained in the microbiology laboratory of the Department of Biological Sciences. Kirby-Bauer zone of inhibition test was used in determining antimicrobial activity of the decoction extract. In the Kirby-Bauer antimicrobial test, microbial suspension containing bacteria were spread evenly onto the surface of the solidified medium. The plates were allowed to

13

solidify for 15 minutes before the test discs were placed at equidistance from each other. The top cover of plates was marked as each filter disc paper was placed to create zones of inhibition. The petri dish of culture group bacteria consists of four filter paper, two (2) for the plant extract and two (2) for standard group. The assay was left for 18 to 24 hours. For the zones of inhibition, Vernier caliper was used in measuring.

Local of the Study

Image source: Google Maps

14

Fresh plant parts were collected near the Andrada heights subdivision, Iligan City.Fresh plant materials were washed and cleaned thoroughly under running tap water, and then brought to the College of Nursing (CON) of Mindanao State University – Iligan Institute of Technology (MSU-IIT)afterdecoction.

Extraction and Preparation of Crude Extracts Decoction Prepared the sample of Carabao grass and preheated the pot to 50-60 degree Celsius. Placed the Carabao grass into the preheated pot for 15 minutes and regulated the temperature to 50-60 degrees.

Screening of Antimicrobial Activities The antimicrobial activities of the extracts were carried out in a Kirby-Bauer agar diffusion method. Sterilized molten agar (20 ml) were dispensed to each sterile disposable Petri dish (diameter 9 cm) and allowed it to solidify. Nutrient broth with agar was used in bacterial culture. During the transfer of bacteria, medicine dropper was used to drop alcohol and inoculation of the string to obtain sample of bacteria in the culture after which, the string with bacteria were evenly distributed on each corner of the petri dish. Microbial suspension containing bacteria were spread evenly onto the surface of the solidified medium. The plates were allowed to dry for 15 minutes before the filter paper discs was placed at equidistance from each other. Each plate consisted of standard group,

15

the negative and positive group and two other filter paper discs impregnated with various extracts. After the assay procedure is completed, the Petri dishes were incubated in an inverted position at 37°C for 18 to 24 hours for bacteria to allow growing. The following day, observation was made.

Zones of Inhibition The diameters of the zone of inhibition (ZOI), defined by the clear area devoid of growth, were measured twice. The anti-microbial activities were determined by the ratio of the ZOI diameters of the extracts to that standard antibiotic is in the same petri dish. This was measured through Vernier caliper of the distance on the inhibition from filter paper disc.

16

CHAPTER 4 RESULTS AND DISCUSSION

Table 1 displays the results of the antimicrobial assay done with the Carabao Grass decoction on the four types of organisms namely: Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosaand Staphylococcus aureus. The samples given were tested against a positive control, which is chloramphenicol, a broad-spectrum antibiotic drug, and a negative control, which is sterile water. The results showed that Carabao Grass decoction has no antimicrobial activity on all of the test organisms employed in the study. The samples given did not yield Zones of Inhibition (ZOI). Table 1 Average Zones of Inhibition (ZOI) in millimeters (mm)

R1

R2

R3

Negative control (Distilled Water)

Bacillus subtilis

0

0

0

0

22.24

Staphylococcus aureus

0

0

0

0

17.08

Escherichia coli

0

0

0

0

12.00

Pseudomonas aeruginosa

0

0

0

0

4.25

Decoction Extract Test Organism

Positive control (Chloramphenic ol)

*Unit of measurement is in millimeter.

Based on the result, the table showed no antimicrobial activity of the Carabao decoction extract. It yielded no zones of inhibition (ZOI) from replicates one, two, and three.

17

The Carabao grass contains chemical compounds such as the flavonoids that are capable of killing microorganisms. However, the data is in contrary to the study of Antimicrobial Activity of Betula pendula ,in which the decoction of buds and flowers of the plant yielded positive inhibition in the Zone of Inhibition with the same bacteria sample.(Duric et.al., 2013) in which it has antimicrobial effects. The distilled water on the other hand as negative control yielded an expected zeros result because it does not contain any antimicrobial properties. The positive control, which is the Chloramphenicol, yielded antimicrobial activity against the four test organisms such as Bacillus subtilis (22.24), Staphylococcus aureus (17.08), Escherichia coli (12.00), and Pseudomonas aeruginosa (4.25 mm).The positive control is a broad-spectrum antibiotic that is commonly used in the hospital due to its known killing effects on microbes, availability in the pharmacy, and affordability. In comparison, the decoction extract is no match to the positive control.

18

CHAPTER 5 SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS Summary The study of the Antimicrobial Activity of Carabao Grass (Paspalumconjugatum) is a quantitative experimental research. It was done to gather data and facts in the management of various infections and at the same time a cost-saving intervention to people in the community that are not financially stable. The antimicrobial potency of Carabao grass decoction extract was assessed through the Kirby-Bauer Disc Diffusion Test. The study was conducted within the period of January to May 2016 in Iligan City, specifically at MSU- IIT College of Nursing and the Department of Biological Sciences (DBS) in the College of Science and Mathematics (CSM). As to sample, plants were gathered in the local area of Iligan City, cleansed from roots to leaves before subject to decoction. Upon obtaining the sample, extract of Carabao Grass were then forwarded to laboratory for antimicrobial assay. The samples given were tested against a chloramphenicol, a broad-spectrum antibiotic as a positive control and distilled water as negative control. Results of the antimicrobial assay were collected after 24 hours of determining Zones of Inhibition (ZOI).

19

Conclusion The results showed that the Carabao grass (Paspalum conjugatum) had no antimicrobial activity in all three replicates of the four test microorganisms such as the Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The decoction extract was of no match against the broad-spectrum antibiotic, which is the Chloramphenicol that yielded antimicrobial activity. In this finding, it could be the said herbal plant usually used and practiced in the community were not that efficient in deterring the microbes as used for wound healing. Despite the findings, it was not conclusive to say that all Carabao grass decoction extract has no effect on killing microorganisms. Further investigation should be done before we can conclude that the said sample is not potent for antimicrobial effect.

Recommendation The following recommendations are drawn on this study based on the results: 1. To conduct a more extensive research and experimentation for Carabao Grass (Paspalum conjugatum) not only for antimicrobial testing but also for the antifungal testing and phytochemical analysis. 2. To obtain bigger sample of the Carabo grassfor a better antimicrobial activity . We also recommend the future researchers to reconduct the antimicrobial testing and if the result will still be zero they should inform and conduct an awareness program to the community that this herb or grass is not a potent herb to be used as an alternative antimicrobial agent to treat their diseases.

20

BIBLIOGRAPHY BOOKS Austin, D. F., FL. (2004).Florida Ethnobotany, pp. 482. Suite 300 Boca Raton, CRC press. 6000 Broken Sound Parkway, Norway. Cambie, R.C., Ash, J., (1994). Fijian Medicinal Plants. pp. xvi. CSIRO. Australia. KALA, (2006). Medicinal Plants and Sustainable Development. pp. 43-56. Nova Science Publishers Inc. New York. ARTICLE Perry, L.M., (1980) Medicinal plants of East and South East Asia. 620 pp. MIT Press. Cambridge. Massachusetts.

JOURNAL

21

Hollman PCH (2004): Absorption, Bioavailability, and Metabolism of Flavonoids. Pharmaceutical Biology. 42:74–83. Retrieved May 20, 2015 from http://www.tandfonline.com/doi/pdf/10.3109/13880200490893492 Bernhoft, A. (2013, August). Highlights through the History of Plant Medicine: Journal of Food Engineering.The Norwegian Academy of Science and Letters, Oslo,Norway Locksley, R M, Cohen, M I, Quinn, T C, Tompkins, L S, Coyle, M B, Kirihara, J M,Counts.(1982).

Multiple

antibiotic

resistant

Staphylococcus

aureus:Introduction,transmission and evolution of nosocomial infection. Annals of Internal Medicine, 9:317 – 324. Department of Medicine, RM-16, University of Washington; Seattle, WA 98195. Duric, K., Kovac-Besovic, E., Niksic, H., Sofic, E., (2013): Jounal of Plant Studies Vol.2: Antimicrobial Activity of Methanolic Extracts, Decoction and Isolated Triterpene Products From Different Part of Birch, Bertula pendula, Roth. Page 69. Candian Center Science and Education Finchurst Plaza, 2235 Sheppard Ave E, Toronto, ON M2J 5B5, Canada Maeda, H. and Akaike, T.(1998). Biochemistry (Moscow): Nitric oxide and oxygen radicals in infection, inflammation and cancer. Vol. 63, no. 7 pp. 854–865.). Department of Microbiology, Kumamoto University School of Medicine, 2-21 Honjo, Kumamoto 860-0811, Japan Parr AJ, Bolwell GP (2000). J SciFood Agric: Phenols in the plant and in man.The potential for possiblenutritional enhancement of the diet by modifying the

22

phenols content or profile. 80: 985–1012. Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Pietta PG: (2000). Journal of Natural Products: Flavonoids as antioxidants. 1035–1042. American Chemical Society. 1155 Sixteenth Street, NW, Washington, DC 20036, USA Ramawat, K.G., Dass, S., & Mathur, (2009). Herbal Drugs: Ethnomedicine to Modern Medicine., p. 402. Springer. New York City Rao, V.S. NH, USA. (2000), Principle of Weed Science: Second edition., pp. 437. Science Publisher, Inc. New York, New York Sarker SD, Nahar L, Kumarasamy Y (2007): Methods: Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitroantibacterial screening of phytochemicals. 42:321–324. Uwaezuoke JC, Aririatu LE (2004): Journal of Applied Sciences and Environmental Management Vol. 8: A Survey of Antibiotic Resistant Staphylococcus Aureus Strains from Clinical Sources in Owerri. P. 67 – 69.

WEB SOURCE Pseudomonas

aeruginosa.

Retrieved

from

http://web.mst.edu/~microbio/BIO221_2010 /P_aeruginosa.html May 25, 2016 Bacillus

subtilis.

Retrieved

http://web.mst.edu/~microbio/BIO221_2009/B_subtilis.html

from May

25,

23

2016Escherichia

coli

retrieved

from

http://www.cdc.gov/ecoli/general/index.html May 25, 2016 Chan, M. (2010, November 18). Health promises and perils in an Independent World: Break the cycle of poverty, misery, and illness. Retrieved from http://www.who.int/dg/speeches/2010/tokyo_20101118/en/ Hudzicki, J: Kirby (2009). Bauer Disk Diffusion Susceptibility Test Protocol. Retrieved from

http://www.microbelibrary.org/component/resource/laboratory-

test/3189-kirbybauer- disk-diffusion-susceptibility-test-protocol Carabao Grass for Kidney Disease. (2014, December 20). Retrieved from http://www.pkdclinic.org/chinese-herbs/2140.html Burkill, H.M. (1985). The useful plants of west tropical Africa, Vol 2. Retrieved from http://plants.jstor.org/stable/10.5555/al.ap.upwta.2_659 Russo, E. B., Headache Treatments by Native People of the Ecuadorian Amazon: A Preliminary Cross-Disciplinary Assessment. Retrieved from http://manu.montana.com/paper.html

24

25

APPENDIX A

26

Mindanao State University Iligan Institute of Technology College of Nursing

November 23, 2015 Prof. Muhmin Michael E. Manting Faculty Department of Biological Sciences

Dear Sir, Good Day! The students listed below from MSU-IIT College of Nursing will be conducting an experimental research in partial fulfilment of the requirements for the degree of Bachelor of Science in Nursing. In line with this, we would like to request from your department to authenticate the provided herbal plant to be used of the said experiment. This will help us fulfil our Nursing Research and hopefully present a research paper on the same school year. Hoping for your kind consideration and approval. More power and God Bless! Truly yours,

Laeh Mae A. Abugan Azim M. Baruang Dieza May O. Teves Noted By:

27

Prof. Clowe D. Jondonero, MAN, MSciEd Bernie A. Casera, RN MSU-IIT, College of Nursing, Dean Adviser

APPENDIX B

28

Fig. 1. Determine the location of the inhabited grass

29

Fig. 2. Picking of grass including the roots

Fig. 3. Decoction of the carabao grass

30

Fig. 4. Carabao Grass Escherichia coli 100%

Fig 5. Carabao Grass Bacillus subtilis 100%

31

Fig. 6. Carabao Grass Paseudomonas aeruginosa 100%

Fig. 7. Carabao Grass Staphylococcus aureus 100%