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Antibacterial Activity of Agavaceae plant extracts

against Pseudomonas Aeruginosa

SCIENCE INVESTIGATORY PROJECT

Submitted by:
Kate Pamela T. Bialen
Researcher
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Antibacterial Activity of Agavaceae plant exracts against

Pseudomas Aeruginosa

Researcher:
Kate Pamela T. Bialen

ABSTRACT

This research aimed to determine the antibacterial activity

of the agavaceae species, specifically, buntot-tigre (Cordyline
roxburghiana (Schultes) Merr.) and tigre (Sansevieria
trifasciata Prain) against Pseudomonas aeruginosa. The
investigation stresses the possibility of extracting a new,
cheaper and readily available herbal medicines form the
Agavaceae family which is not ever known for its any
pharmacological importance in the country, therefore provide
great savings on the part of Filipinos mostly in poverty line.
This study has four treatments. 100% combined agavaceae plant
extracts; 75% combined agavaceae plant extracts and 25%
distilled water; 50% combined agavaceae plant extracts and 50%
distilled water; and the positive control which is the
chloramphenicol.

This study also aimed to determine if there are significant

differences in the zones of inhibition with the different
concentrations of the combined agavaceae plant extracts.

At the end of the study, the researcher concludes that the

agavaceae leaves extracts, specifically, buntot-tigre (Cordyline
roxburghiana (Schultes) Merr.) and tigre (Sansevieria
trifasciata Prain) are effective antibacterial against
Pseudomonas aeruginosa.
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RESEARCH PLAN

A. Materials and Tools

Materials:
Agavaceae plants (buntot-tigre and tigre), pure
cultured Pseudomonas Aeruginosa, positive control
(chloramphenicol), swab/cotton, marking pen, laboratory
gown, disposable gloves and masks.

Tools:
Incubator, mortar and pestle, alcohol lamp,
refrigerator, beakers, test tubes, cheese cloth, and amber
bottles.
B. Treatments / General Procedure
Extraction Procedure
1. Pound mg of Agavaceae plants separately using
mortar and pestle.
2. Squeeze, strain and filter the pounded leaves of
Buntot-tigre into a beaker with the use of the
cheese cloth. The same procedure implies with the
pounded leaves of Tigre. This procedure is repeated
until the desired amount of extract is acquired.

Treatments:
Treatment A= 100% combined Agavaceae plant extracts
Treatment B= 75% combined Agavaceae plat extracts and
25% distilled water
Treatment C= 50% combined Agavaceae plant extracts and
50% distilled water
Treatment D= Positive control (chloramphenicol)
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Procedure:

1. Preparation of Mueller- Hinton Agar Plates

38 grams of dehydrated Mueller-hinton agar was placed
on a weighing scale with aluminum foil and transferred
in an Erlenmeyer flask. It was then rehydrated with
1000 ml distilled water and heated in a boiler for it
to melt. The medium was sterilized in the autoclave at
121 c for 15 minutes at 15 psi. the sterile medium is
cooled at at the temperature between 40-45 C and
transferred into sterile petri dishes at 20 ml each.
The plates were allowed to cool and harden and were
set aside until ready for use.

2. Bioassay Proper
Mueller-Hinton agar plates and test plates in 4
replicates were divided in to four equal parts each by
means of using a marking pen and were respectively
labeled.
All plates were heavily streaked with the bacterial
suspension of pseudomonas aeruginosa. The four
replicates of the four replicates of the streaked
Mueller-Hinton agar plates. The four parts were then
applied with the corresponding treatments with the use
of a dropper. The same procedure was used with the 3
other replicates. These plates were allowed to stay at
room temperature for a few minutes, then these plates
were incubated at 37 C for 24 hours.
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After an overnight incubation, the plates were

examined for formation of the zones of inhibition
around the area where the extracts have been applied.
The diameters were then measured by means of a ruler
and average of the data was taken from the data in the
y and x axis of the zone.
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C. Procedural Design

Gathering of Agavaceae plants

Washing and air-drying

Squeezing, straining, and filtering of pounded

agavaceae plants

Preparation of

Treatments Cultured Pseudomonas aeruginosa

Treatment A= Treatment B= Treatment C= 50% Treatment D=

100% combined 75% combined combined Positive control
agavaceae plant agavaceae agavaceae plant (chloramphenic
plant extracts extracts and 50%
extracts ol)
and 25% distilled water
distilled water

Labeling of Mueller-Hinton agar plates with the

corresponding treatment and dividing them into 4 equal
parts.

Application of the different treatments

on the pure cultured Pseudomonas
Aeruginosa.

Incubation of the agar plates.

Measuring of the zones of inhibition, recording and analysis of data acquired.

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I. Title Page 1

II. Abstract 2

Research Plan 3

Pr o c e d u r a l d e s i g n 4

I I I . In t r o d u c t i o n 8

Ba c k g r o u nd o f t h e Study 8

St a t e m e n t o f t h e problem 8

Statement of the Hypothesis 9

Objectives 9

Significance of the Study 9

Scopes and Limitations 10

Definition of Terms 10

IV. Review of Related Literature 12

V. Presentation, Analysis and, Interpretation 18

VI. Conclusions, and Recommendations 20

VII. Plates 22

VIII. Reference/Bibliography 27

IX. Appendices 28

X. Acknowledgements 32
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INTRODUCTION
A. Background of the Study

These days, many studies were proposed and done regarding

the utility of plant extracts as an antibacterial. Many plants
have been used and tested. But people still find more treatments
that are effective and more unique.
The Agavaceae family is abundant in our country and these
are typical houseplants. Better known as snake plants. Several
species are used as a source of fiber and as medicinal plants.
(Content copyright © 2013 by Connie Krochmal)
These plants are known by various other common names. These
include crocodile’s tongue, mother-in-law’s tongue, spear plant,
and zebra lily. Another of their common names is bow-string
hemp. Despite the name, this shouldn’t be confused with true
hemp fiber.
Ceylon bow-string hemp (Sansevieria zeylanica) is native to
Sri Lanka. In the Philippines, this is called buntot-tigre. This
is a source of fiber. It is used for making sails and for a type
of paper. The sturdy, white fiber is a favorite for twine, and
cloth as well. The fiber is extracted from the leaves.
The Abyssinian bow-string hemp (Sansevieria abyssinica) is
native to east Africa. The leaves are also the source of a
fiber. This is often used in clothing.

B. STATEMENT OF THE PROBLEM

 Are the species of the agavaceae species effective as an
antibacterial against Pseudomas Aeruginosa?
 Is there a significance difference in the mean zones of
inhibition on Pseudomas Aeruginosa using the different
concentrations of the combined extracts of the Agavaceae
species?
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C. STATEMENT OF THE HYPOTHESIS

 The species of the agavaceae species are not effective
as an antibacterial against Pseudomas Aeruginosa.
 There are no significant differences in the mean zones
of inhibition on Pseudomas Aeruginosa using the
different concentrations of the combined extracts of
the agavaceae species.

D. OBJECTIVES
 To determine the antibacteriall activity of the
Agavaceae family.
 To determine which specie of Agavaceae is most
effective as an antibacterial.
E. SIGNIFICANCE OF THE STUDY

Plants are important to lives of people throughout the

world. We depend upon plants to satisfy some of our basic human
needs as food, clothing, shelter, and health care. Increasing
attention has also been given to the role of plants in disease
management and alternative to synthetic medicines.
Different bacteria are now immune to a wide range of
synthetic medicines, thus alternative medicines from plants are
in the greatest concern to replace it. The investigation
stresses the possibility of extracting a new, cheaper and
readily available herbal medicines form the Agavaceae family
which is not ever known for its any pharmacological importance
in the country, therefore provide great savings on the part of
Filipinos mostly in poverty line. Similarly, it could provide a
potential source of income when large scale production is
feasible.
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F. SCOPE AND LIMITATION

This study is focused on the utilization of the combined

extracts from two different Agaveae species against Pseudomas
Aeruginosa and comparing the zones of inhibition.

G. Definition of terms
 Chloramphenicol- Chloramphenicol is a man-made
antibiotic. It slows growth of bacteria by preventing
them from producing important proteins that they need
to survive.
- it refers to the positive control usesd in
the study
 Mueller- Hinton Agar-is a medium very rich in
nutrients that was originally recommended for the
isolation and development of gonococci and
meningococci. It is used primarily for sensitivity
testing of microorganisms.
 Agavaceae- the flowering plant order Asparagales,
consisting of 23 genera and 637 species of short-
stemmed, often woody plants distributed throughout
tropical, subtropical, and temperate areas of the
world. Members of the family have narrow, lance-
shaped, sometimes fleshy or toothed leaves that are
clustered at the base of each plant. Most species have
large flower clusters containing many flowers. The
fruit is a capsule or berry.
- In this study, buntot-tigre (Cordyline
roxburghiana (Schultes) Merr.) and Tigre
(Sansevieria trifasciata Prain) was used in
the study.
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 Pseudomonas Aeruginosa- Pseudomonas aeruginosa is member of

the Gamma Proteobacteria class of Bacteria. It is a Gram-
negative, aerobic rod belonging to the bacterial family
Pseudomonadaceae. Since the revisionist taxonomy based on
conserved macromolecules (e.g. 16S ribosomal RNA) the
family includes only members of the genus Pseudomonas which
are cleaved into eight groups. Pseudomonas aeruginosa is
the type species of its group. which contains 12 other
members.
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REVIEW OF RELATED LITERATURE

Man in solving its numerous medical challenges for ages

depends on his immediate environment taking advantages of
nature’s provisions of its beauty for life and survival. They
learned to depend on plants and in some cases animals in
providing solutions to the myriad of their health problems.
Aside from these, there has been an increasing incidence of
multiple resistances in human pathogenic microorganisms in
recent years, largely to indiscriminate use of commercial
antimicrobial drugs commonly employed in the treatment of
infectious diseases. This has forced scientist to search for new
antimicrobial substances from various sourceslike medicinal
plants. (Antessa 2008).

Pseudomonas Aeruginosa

Pseudomonas aeruginosa is member of the Gamma

Proteobacteria class of Bacteria. It is a Gram-negative, aerobic
rod belonging to the bacterial family Pseudomonadaceae. Since
the revisionist taxonomy based on conserved macromolecules (e.g.
16S ribosomal RNA) the family includes only members of the genus
Pseudomonas which are cleaved into eight groups. Pseudomonas
aeruginosa is the type species of its group. hich contains 12
others.

Like other members of the genus, Pseudomonas aeruginosa is a

free-living bacterium, commonly found in soil and water.
However, it occurs regularly on the surfaces of plants and
occasionally on the surfaces of animals. Members of the genus
are well known to plant microbiologists because they are one of
 P a g e | 13

the few groups of bacteria that are true pathogens of plants. In

fact, Pseudomonas aeruginosa is occasionally a pathogen of
plants. However, Pseudomonas aeruginosa has become increasingly
recognized as an emerging opportunistic pathogen of clinical
relevance. Several different epidemiological studies track its
occurrence as a nosocomial pathogen and indicate that antibiotic
resistance is increasing in clinical isolates.

Pseudomonas aeruginosa is an opportunistic pathogen, meaning

that it exploits some break in the host defenses to initiate an
infection. In fact, Pseudomonas aeruginosa is the epitome of an
opportunistic pathogen of humans. The bacterium almost never
infects uncompromised tissues, yet there is hardly any tissue
that it cannot infect if the tissue defenses are compromised in
some manner. It causes urinary tract infections, respiratory
system infections, dermatitis, soft tissue infections,
bacteremia, bone and joint infections, gastrointestinal
infections and a variety of systemic infections, particularly in
patients with severe burns and in cancer and AIDS patients who
are immunosuppressed. Pseudomonas aeruginosa infection is a
serious problem in patients hospitalized with cancer, cystic
fibrosis, and burns. The case fatality rate in these patients is
near 50 percent.

Pseudomonas aeruginosa is primarily a nosocomial pathogen.

According to the CDC, the overall incidence of P.
aeruginosa infections in U.S. hospitals averages about 0.4
percent (4 per 1000 discharges), and the bacterium is the fourth
most commonly-isolated nosocomial pathogen accounting for 10.1
percent of all hospital-acquired infections.
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Characteristics

Pseudomonas aeruginosa is a Gram-negative rod measuring 0.5 to

0.8 µm by 1.5 to 3.0 µm. Almost all strains are motile by means
of a single polar flagellum.

The bacterium is ubiquitous in soil and water, and on surfaces

in contact with soil or water. Its metabolism is respiratory and
never fermentative, but it will grow in the absence of O2 if
NO3 is available as a respiratory electron acceptor.

The typical Pseudomonas bacterium in nature might be found in a

biofilm, attached to some surface or substrate, or in a
planktonic form, as a unicellular organism, actively swimming by
means of its flagellum. Pseudomonas is one of the most vigorous,
fast-swimming bacteria seen in hay infusions and pond water
samples.

In its natural habitat Pseudomonas aeruginosa is not

particularly distinctive as a pseudomonad, but it does have a
combination of physiological traits that are noteworthy and may
relate to its pathogenesis.

• Pseudomonas aeruginosa has very simple nutritional

requirements. It is often observed "growing in distilled water",
which is evidence of its minimal nutritional needs. In the
laboratory, the simplest medium for growth of Pseudomonas
aeruginosa consists of acetate as a source of carbon and
ammonium sulfate as a source of nitrogen.

• P. aeruginosa possesses the metabolic versatility for which

 P a g e | 15

pseudomonads are so renowned. Organic growth factors are not

required, and it can use more than seventy-five organic
compounds for growth.

• Its optimum temperature for growth is 37 degrees, and it is

able to grow at temperatures as high as 42 degrees.

• It is tolerant to a wide variety of physical conditions,

including temperature. It is resistant to high concentrations of
salts and dyes, weak antiseptics, and many commonly used
antibiotics.

• Pseudomonas aeruginosa has a predilection for growth in moist

environments, which is probably a reflection of its natural
existence in soil and water.

These natural properties of the bacterium undoubtedly contribute

to its ecological success as an opportunistic pathogen. They
also help explain the ubiquitous nature of the organism and its
prominence as a nosocomial pathogen.

P. aeruginosa isolates may produce three colony types. Natural

isolates from soil or water typically produce a small, rough
colony. Clinical samples, in general, yield one or another of
two smooth colony types. One type has a fried-egg appearance
which is large, smooth, with flat edges and an elevated
appearance. Another type, frequently obtained from respiratory
and urinary tract secretions, has a mucoid appearance, which is
attributed to the production of alginate slime. The smooth and
mucoid colonies are presumed to play a role in colonization and
virulence. (Cowan and Talaro, 2006)
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Buntot-tigre

Buntot-tigre is a herbaceous perennial with short stem and

a rootstock that is very stout, branching and stoliniferous.
Leaves are erect, fleshy, fibrous and flat ( in other varieties
cylindrical or concave above, rounded dorsally), sub-erect,
dagger-shaped, rigid, pale-green with transverse bands of dark
green, or dark-green with gray mottles, 0.4 to 1.5 meters long,
4 to 7 centimeters wide. Scape is erect, 30 to 80 centimeters
long. Flowers are pale-colored, numerous, in fascicles of 3 to
6, sweet-scented, 2.5 to 3 centimeters long, with the perianth
segments nearly twice as long as the tube. Fruit is sparingly
produced, globose, about 8 millimeters in diameter. Seeds are
broadly ovoid and white, with horny albumen.

Buntot-tigre (Sansevieria roxburghiana) is often confused with

Tigre (Sanseviera trifasciata). Some compilations list them as
synonyms. (http://www.stuartxchange.com/Buntot-tigre.html)

The plant contains, besides other components, an active

constituent: an alkaloid, sansevierine.

According to Guerrero (Guerrero, 1921) the leaves when roasted,

are used as emollient. Kirtikar and Basu (Kirtikar and Basu,
1918) declares that the rootstocks are prescribed as a cough
medicine in India. The juice of the tender shoots is
administered to children to clear their throats of viscid
phlegm. (Quisumbing, 1978)
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Tigre

Sanseviera trifasciata is an herbaceous, succulent,

perennial plant, growing to a height of 90 centimeters. Leaves
form a basal rosette, are flat, thick, leathery, sword-shaped,
and variegated with grayish white transverse markings Flowers
are whitish green, up to 5 centimeters long.
(http://www.stuartxchange.com/Buntot-tigre.html)

Chopra reports that it is used as a purgative, a tonic, and

a febrihuge in India. (Chopra, 1933)
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Presentation, Analysis and, Interpretation

Inhibitory effect of the different concentrations of Agavaceae
plants against Pseudomonas Aeruginosa.

Treatment Zones of Inhibition in millimeters (mm)

R1 R2 R3 R4 Mean

A 30 20 30 35 28.75

B 25 25 25 28 25.75

C 15 18 13 18 16

Positive
15 18 14 20 16.75
Control

Results show that the Treatment A got the highest mean among the
4 treatments with 28.75 followed by Treatment B with the mean of
25.75, Treatment D or the Positive control with 16.75 and the
treatment C with the mean of 16.
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ANOVA
Inhibition
Sum of Df Mean F Sig.
Squares Square
Between
427.500 3 142.500 13.959 .000
Groups
Within
122.500 12 10.208
Groups

Total 550.000 15

ANOVA shows the summary of the different zones of inhibition of

Agavaceae plants using different concetrations on Pseudomonas
Aeruginosa. The null hypothesis is rejected.

Results showed that the Agavaceae plants contain antibacterial

properties against the growth of Pseudomonas Aeruginosa.
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CONCLUSION AND RECOMMENDATIONS

CONCLUSION
Based on the executed experimentation, the researcher
therefore concludes that:
 The constituents of the agavaceae species are
effective as an antibacterial against Pseudomas
Aeruginosa.
 There are significant differences in the mean zones of
inhibition on Pseudomas Aeruginosa using the different
concentrations of the combined extracts of the
agavaceae species.
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RECOMMENDATIONS
Based on the conclusion and results, the following
proposals are given:
1. Supplementary study about the utilization of the other
parts of the plants used in this study.
2. Supplementary studies are recommended regarding the use of
the similar plants.
3. Additional studies should be conducted if the agavaceae
species have an antibacterial effect on other bacteria.
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PLATES

Gathered materials and tools

Pounding of leaves with mortar and pestle

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Mueller-Hinton agar plates

Divided and labeled agar plates

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Cultured Pseudomonas Aeruginosa

Application of Pseudomonas Aeruginosa on the agar plate by beans

of a cotton swab
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Putting of holes unto the agar plate for the extract to fully
penetrate.

Application of treatments.
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Incubation
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REFERENCE/ BIBLIOGRAPHY
A. Website
http://www.stuartxchange.com/Tigre.html
http://www.stuartxchange.com/Buntot-tigre.html

B. Books

Dr. Quisumbing, Eduardo (1978). Medicinal Plants of

the Philippines. Quezon City: Katha Publishing Co.,
INC.
Chopra, R. N. (1933). Indigenous drugs of India.

Guerrero, Leon Ma. (1921). Medicinal uses of

Philippine plants.
Kitikar, K. R. and Basu, B. D. (1918). Indian
Medicinal plants.

Cowan, Marjorie Kelly and Talaro, Kathleen (2006).

Microbiology, A systems approach.
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APPENDICES
A. Statistical Tool
ANOVA

Inhibition
Sum of df Mean F Sig.
Squares Square
Between
427.500 3 142.500 13.959 .000
Groups
Within Groups 122.500 12 10.208
Total 550.000 15

Multiple Comparisons
Dependent Variable: Inhibition
Tukey HSD

(I) (J) Mean Std. Sig. 99% Confidence Interval

Treatment Treatment Difference Error Lower Upper
(I-J) Bound Bound
2 1.75000 2.25924 .864 -7.0390 10.5390
1 3 11.50000* 2.25924 .001 2.7110 20.2890
4 10.75000* 2.25924 .002 1.9610 19.5390
1 -1.75000 2.25924 .864 -10.5390 7.0390
2 3 9.75000* 2.25924 .005 .9610 18.5390
4 9.00000* 2.25924 .008 .2110 17.7890
1 -11.50000* 2.25924 .001 -20.2890 -2.7110
3 2 -9.75000* 2.25924 .005 -18.5390 -.9610
4 -.75000 2.25924 .987 -9.5390 8.0390
1 -10.75000* 2.25924 .002 -19.5390 -1.9610
4 2 -9.00000* 2.25924 .008 -17.7890 -.2110
3 .75000 2.25924 .987 -8.0390 9.5390
*. The mean difference is significant at the 0.01 level.
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B. Time table
DA T E TASK REMARKS

May 26, 2014 S tudy proposal The task was don e

June 17, 2014 F i nalization of The task was don e
the title.
June 30, 2014 C o nstruction of The background of
t h e background of the study was
t he study and evaluated and the
r e v iew of related researcher made
literature. some corrections.
Ju l y 2 , 2 0 1 4 R e v ision of the The task was don e
b a c kground of the
study and rev iew
of related
li t erature.
July 8, 2014 A p p roval of the The task was don e
r e v ised
b a c kground of the
study and rev iew
of related
l i t erature.
Ju l y 1 6 , 2 0 1 4 C o nstruction of The significance
t h e significance of the stu dy,
o f the study, statement of th e
s t atement of the problem, the
problem, the hypothesis,
hypothesis objectives and
objectives, the scope and
scopes and limitations of
limitations the study w ere
 P a g e | 30

evaluated and th e
researcher mad e
some corrections
Ju l y 2 1 , 2 0 1 4 C o r rection of the The task was don e
s i gnificance of
the study,
s t atement of the
problem, the
hypothesis
objectives,
scopes and
limitations
Ju l y 2 8 , 2 0 1 4 A p proval of the The task was done
s i gnificance of
the study,
s t atement of the
problem, the
hypothesis
objectives,
scopes and
limitations
Au g u s t 1 5, 2 0 1 4 P r eparation and The task was done
g a thering of the
tools and
m a terials needed
in the
e x perimentation
Au g u s t 1 6 , 2 0 1 4 A pplication of The task was done
extracts.
Au g u s t 1 7 , 2 0 1 4 The task was done
M e asuring of the
zones of
 P a g e | 31

inhibition
Au g u s t 2 5, 2 0 1 4 P hytochemical The task was done
a n alysis of the
p lant samples.
Se p t e m b e r 1, 2 0 1 4 Analysis and The task was done
c o nstruction of
the acquired
data.
Se p t e m b e r 1 0 , F i nalization of The document wa s
20 1 4 t he write ups. submitted to be
corrected.
Se p t e m b e r 1 3 , R e vision of the The task was
20 1 4 write ups. done.
Se p t e m b e r 1 6 , A p proval of the The task was
20 1 4 write ups. done.
 P a g e | 32

ACKNOWLEDGEMENT

The researcher would like to extend h er genu ine

g r a t i t u d e a n d c a n d i d g ratitude to the following:

To our science research adviser and at the s ame

time our mother, Mrs. Elena F. Frio for her limitless
encouragement and aid for the accomplishment of this
study.

To Ms. April Anisco , our M athematics adviser for

sharing her skills and knowledge regarding the
s t a t i s t i c a l t o o l i n t h is study.

Mr. Normandy Balasa for sh aring his pre cious time

i n c o r r e c t i n g t h e w r i t e ups.

To the researcher’s dearest friends especially

T r a s h b i n s a n d the Angelics - IV for the non - stop backing
a n d f o r t h e s u p p o r t t h ey gave.

To the researcher’s parents, Mrs. Dianesa Bialen

a n d Mr. Ronie Bialen for the ir time, consi deration, and
financial support.

La s t l y , t o G o d . F or never le aving the rese archer’s

s ide , f o r g u i d a n c e , a nd strength to work on this study.

THANK YOU FOR EVERYTHING.