the antimicrobial properties of plants
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UNIVERSITI TEKNOLOGI MARA
INTERNATIONAL EDUCATION CENTRE
(INTEC)
BIOLOGY LABORATORY REPORT
A LEVEL PROGRAMME
2014
17 MARCH 2014
NAME: MAISARAH BINTI ZAINUDDIN
IC NUMBER: 950201-14-5724
GROUP: 11SC6
SID NUMBER: 1311171707
TITLE: THE ANTIMICROBIAL PROPERTIES OF PLANTS
1.0 TITLE
The Antimicrobial Properties of Plants
2.0 OBJECTIVES
To identify the plant that has the greatest antimicrobial properties
To study the effects of microbial properties in plants
3.0 INTRODUCTION
Escherichia coli
Escherichia coli which is commonly abbreviated as E. coli is a Gram-negative,
facultative anaerobic and is a rod-shaped bacterium that is commonly found in the lower
intestine of warm-blooded organisms. There are many different types of E. coli where
some E. coli strains live in the intestine quite harmlessly while some serotypes can cause
serious food poisoning in their hosts, and are occasionally responsible for product recalls
due to food contamination. The harmless strains are part of the normal flora of the gut,
and can benefit their hosts by producing vitamin K2 and preventing colonization of the
intestine with pathogenic bacteria.i
Image 1: Scanning electron micrograph of Escherichia coli, grown in culture and adhered to a
cover slip.
The commonest infection caused by E. coli is infection of the urinary tract, the
organism normally spreading from the gut to the urinary tract. E. coli is also the
commonest cause of cystitis (infection of the bladder), and in a minority of patients the
infection may spread up the urinary tract to the kidneys, causing pyelonephritis.
Otherwise healthy patients in the community may develop cystitis, and patients in
hospital who have catheters, or tubes, placed in the urethra and bladder are also at risk. E.
coli is also present in the bacteria that cause intra-abdominal infections following leakage
from the gut into the abdomen, as for example with a ruptured appendix or following
traumatic injury to the abdomen.ii
Image 2: Disease caused by E. coli
E. coli bacteria may also cause infections in the intestine. Diarrhoeal infections
(intestinal) are caused by a group of E. coli known as 'enter virulent' (harmful to the
intestines). Overspill from the primary infection sites to the bloodstream may cause blood
poisoning (E. coli bacteraemia). In rare instances, E. coli may cause meningitis in very
young children.
Turmeric
Turmeric (Curcuma longa) is a rhizomatous herbaceous perennial plant of the ginger
family, Zingiberaceae. India is a significant producer of turmeric which has regional
names based on language and country. It is also native to tropical Tamil nadu, in
southeast India, and needs temperatures between 20 °C and 30 °C (68 °F and 86 °F) and a
considerable amount of annual rainfall to thrive. Plants are gathered annually for their
rhizomes, and propagated from some of those rhizomes in the following season.
Turmeric has a peppery, warm and bitter flavour and a mild fragrance slightly
reminiscent of orange and ginger, and while it is best known as one of the ingredients
used to make curry, it also gives ballpark mustard its bright yellow colour. It also has a
tough brown skin and a deep orange flesh.
Image 3: Turmeric
Curcumin is also a powerful antioxidant. Antioxidants scavenge molecules in the
body known as free radicals, which damage cell membranes, tamper with DNA, and even
cause cell death. Antioxidants can fight free radicals and may reduce or even help prevent
some of the damage they cause. In addition, curcumin lowers the levels of two enzymes
in the body that cause inflammation. It also stops platelets from clumping together to
form blood clots.iii Its anti-inflammatory also makes it able to cure many other diseases
such as flatulence, jaundice, menstrual difficulties, bloody urine, haemorrhage, toothache,
bruises, chest pain, and colic.iv
Fennel
Image 4: Fennel
Fennel (Foeniculum vulgare) is a plant species in the genus Foeniculum (treated as
the sole species in the genus by most botanists). It is a member of the family Apiaceae
(formerly the Umbelliferae). It is a hardy, perennial, umbelliferous herb, with yellow
flowers and feathery leaves. It is indigenous to the shores of the Mediterranean but has
become widely naturalized in many parts of the world, especially on dry soils near the
sea-coast and on riverbanks. It is a highly aromatic and flavorful herb with culinary and
medicinal uses and, along with the similar-tasting anise, is one of the primary ingredients
of absinthe.
Fennel is used for various digestive problems including heartburn, intestinal gas,
bloating, loss of appetite, and colic in infants. It is also used for upper respiratory tract
infections, coughs, bronchitis, cholera, backache, bedwetting, and visual problems. Some
women use fennel for increasing the flow of breast milk, promoting menstruation, easing
the birthing process, and increasing sex drive. Fennel powder is used as a poultice for
snakebites. In foods and beverages, fennel oil is used as a flavouring agent. In other
manufacturing processes, fennel oil is used as a flavouring agent in certain laxatives, and
as a fragrance component in soaps and cosmetics.
Cinnamon
Cinnamon is a spice obtained from the inner bark of several trees from the genus
Cinnamomum that is used in both sweet and savoury foods. While Cinnamomum verum is
sometimes considered to be "true cinnamon", most cinnamon in international commerce is
derived from related species, which are also referred to as "cassia" to distinguish them from
"true cinnamon".
Image 5: Cinnamon
Numerous studies show that cinnamon regulates blood sugar, making it a great choice
for diabetics and hypoglycaemic alike. It also reduces LDL cholesterol levels. LDL is also
known as the harmful cholesterol. Reducing it may help reduce the risk of cardiovascular
disease. Besides, it has natural anti-infectious compounds. In studies, cinnamon has been
effective against ulcer-causing H. pylori bacteria and other pathogens.v
Mint
Mint, the well known mouth and breath freshener that is scientifically known as
Mentha, has more than two dozen species and hundreds of varieties. It is an herb that has
been used for hundreds of years for its remarkable medicinal properties. The market is full of
products like tooth paste, chewing gum, breath fresheners, candy and inhalers which have
mint as their base element. Most of us are familiar with the refreshing application of mint, but
it has far more to offer than that.
Image 6: Mint
Mint is a great appetizer or palate cleanser, and it promotes digestion. It also soothes
stomachs in cases of indigestion or inflammation. When you feel sick to your stomach,
drinking a cup of mint tea can give you relief. Also, if you are someone who travels long
distances via plane or boat, the menthol oil derived from mint can be very soothing for
nausea and related motion sickness. viSome more, the strong aroma of mint is very effective
in clearing up congestion of the nose, throat, bronchi and lungs, which gives relief for
respiratory disorders that often result from asthma and the common cold. As mint cools and
soothes the throat, nose and other respiratory channels, it relieves the irritation which causes
chronic coughing. This is the main reason why so many balms are based on mint.vii
Ginger
Ginger or ginger root is the rhizome of the plant Zingiber officinale, consumed as a
delicacy, medicine, or spice. It lends its name to its genus and family (Zingiberaceae). Other
notable members of this plant family are turmeric, cardamom, and galangal. The distantly
related dicots in the Asarum genus have the common name wild ginger because of their
similar taste. Ginger cultivation began in South Asia and has since spread to East Africa and
the Caribbean.
Image 7: Ginger
According to the National Library of Medicine, part of the NIH (National Institutes of
Health), ginger is widely used throughout the world for treating loss of appetite, nausea and
vomiting after surgery, nausea resulting from cancer treatment, flatulence, stomach upset,
colic, morning sickness and motion sickness. Some people find ginger helps them with the
symptoms of upper respiratory tract infection, bronchitis, cough, menstrual cramps, and
arthritis and muscle pain. In some parts of the world, ginger juice is applied to the skin to
treat burns. Ginger is also used as flavouring by the food and drinks industry, as a spice and
flavouring in cooking, and for fragrance in soaps and cosmetics.viii
Garlic
Allium sativum, commonly known as garlic, is a species in the onion genus, Allium.
Its close relatives include the onion, shallot, leek, chive and rakkyo. With a history of human
use of over 7,000 years, garlic is native to central Asia and has long been a staple in the
Mediterranean region, as well as a frequent seasoning in Asia, Africa, and Europe. It was
known to Ancient Egyptians, and has been used for both culinary and medicinal purposes.
Image 8: Garlic
Garlic is used for many conditions related to the heart and blood system. These
conditions include high blood pressure, high cholesterol, coronary heart disease, heart attack,
and atherosclerosis. Garlic actually may be effective in slowing the development of
atherosclerosis as well and seems to be able to modestly reduce blood pressure. Some people
use garlic to prevent colon cancer, rectal cancer, stomach cancer, breast cancer, prostate
cancer, lung cancer and might as well cure diabetes, osteoarthritis, hay fever, high blood
pressure late in pregnancy (pre-eclampsia), cold and flu. It is also used for building the
immune system, preventing tick bites, and preventing and treating bacterial and fungal
infections. Other uses include treatment of fever, coughs, headache, stomach ache, sinus
congestion, gout, rheumatism, haemorrhoids, asthma and bronchitis, shortness of breath, low
blood pressure, low blood sugar, high blood sugar, and snakebites. It is also used for fighting
stress and fatigue, and maintaining healthy liver function.ix
4.0 PROBLEM STATEMENT
Different plant extracts have different antimicrobial properties. This experiment is
conducted to determine which ingredient disc that has the biggest clear zone around
the disc. This is because; having big clear zone indicates that the plant extract has
better ability to inhibit the bacterial growth around it thus, proving that the plant has
greater antimicrobial properties.
5.0 HYPOTHESIS
Garlic has the greatest antimicrobial properties compared to other plant extracts which
is turmeric, fennel, cinnamon, mint and ginger.
6.0 VARIABLES
Manipulated Variable:
Type of plant extract used
Use different plant extracts namely turmeric, fennel,
cinnamon, mint, ginger and garlic.
Responding Variable:
Diameter of clear zone
Measure and record the diameter of the clear zone after
2 days.
Constant
Variable:
Concentration of
plant extract
For every plant extract, 30% solution concentration was
prepared y diluting 3g of plant extract in 10cm3 of
distilled water.
Temperature at
which the petri
dish was kept
Keep the petri dish in the incubator with temperature
60°C.
Volume and type
of bacterial
culture being
used
For every petri dish, 200µl of E. coli was poured into
them.
7.0 APPARATUS
Sterile petri dish, micropipette with sterile tips, test tubes, mortar and pestle, sterile
forceps, cellophane tape, marker pen, Bunsen burner, laminar flow chamber,
weighing machine, incubator, small measuring cylinders, metre rule and small
beakers.
8.0 MATERIALS
Agar warmed at 60°C, ethanol, distilled water, plant extracts (turmeric, fennel,
cinnamon, mint, ginger and garlic) and E. coli kept in sterile bottles.
9.0 TECHNIQUES
Measure and record the diameter of the clear zone by using metre rule after the
ingredient disc was placed into agar with E. coli mix with it after 2 days.
10.0 PROCEDURES
Preparation of the plant extract solution
1. Six types of plant extracts were chosen namely turmeric, fennel, cinnamon, mint,
ginger and garlic.
2. Garlic, fennel, mint, ginger and garlic were pounded by using mortar and pestle
while the cinnamon powder was readily prepared in powder form.
3. The plant extract had to be prepared in a 30% solution. This was done by
dissolving 3g of each extract in 10ml of distilled water.
4. 3g of each plant extract was weighed by using weighing balance while 10ml of
distilled water was measured by using small size of measuring cylinder.
5. Both substances (3g of plant extract and 10ml of distilled water) were mixed
thoroughly in a small beaker respectively.
Image 9: Plant Extracts
Preparation of the agar plates
1. Hand must first be washed wished ethanol before preparing the agar plates.
2. Two sterile petri dishes were labelled with 1, 2,3,4,5,6,7,8 and 9. Each of the
number represented different type of plant extracts as shown below:
1 Turmeric
2 Fennel
3 Cinnamon
4 Distilled Water
5 Mint
6 Ginger
7 Garlic
8 Distilled Water
Table 1: Plant Extracts and Its Respective Quadrant
Image 10: Quadrants of the Petri Dishes
3. Agar solution which had been kept in incubator of 60°C earlier was poured into
both of the sterile petri dishes until half-filled (at least covered the whole surface
of the petri dishes).
4. Starting from this procedure, everything must be conducted in the laminar flow
chamber to ensure that the surrounding air was also well-sterilised.
5. Using aseptic technique, 200μl of the E. coli broth was transferred in sterile
condition inside a laminar flow chamber.
6. Using last finger, the bottle containing E. coli broth was opened. The mouth of
bijou bottle was then flamed with Bunsen burner to kill any pathogens that may
present.
7. The E. coli broth was then pipette using micropipette that has been calibrated to
200μl. The knob of the micropipette was gently pressed until the first pressure
exerted felt and released inside the bijou bottle (in the middle of the bacterial
broth solution) so that no air bubble will be formed.
8. The mouth of bottle flamed again before closed to avoid contamination.
9. The E. coli broth was dispensed into the Petri dish (lid of Petri dish only lifted
enough to allow entry of the pipette tip) and closed quickly by putting the lid onto
the Petri dish to avoid contact with air too long.
10. Step 5-8 were repeated on another petri dish.
11. Both of the petri dishes were moved sideways slowly to allow the E. coli broth to
be scattered in the agar solution evenly.
12. The petri dishes then were allowed to solidify within 10 minutes.
13. Once it had been solidified, placed one paper disc into each beaker containing the
plant extracts earlier by using a pair of sterilised forceps.
14. All of the paper discs that have been immersed in the respective plant extracts
were then placed onto the solidified agar at their respective quadrant in the petri
dish.
15. The Petri dishes were closed and cellophane tape was used to tape it sideways to
prevent growth of anaerobic bacteria and enable entering of oxygen for the
bacteria to respire.
16. The Petri dish containing agar and paper discs with plant extracts were then
inverted and placed into incubator and left for two days to enable bacterial growth.
17. Observations were done on the next day and the area of clear zone (area of
inhibition of bacterial growth) was calculated.
11.0 RESULTS
Petri Dish QuarterType of Plant
Extract
Diameter of Bacterial Growth Inhibition
Zone (cm)
1 2 Average
A
1 Turmeric 0.8 0.8 0.80
2 Fennel 1.0 1.3 1.15
3 Cinnamon 0.7 0.8 0.75
4 Distilled Water 0.6 0.6 0.60
B
5 Mint 1.0 1.4 1.20
6 Ginger 1.1 1.1 1.10
7 Garlic 2.0 2.4 2.2
8 Distilled Water 0.6 0.6 0.60
Table 2: Diameter of Bacterial Growth Inhibition for every Plant Extracts
Turmeric Fennel Cinnamon Mint Ginger Garlic Control0
0.5
1
1.5
2
2.5
0.8
1.15
0.750000000000001
1.2
1.1
2.2
0.600000000000001
Graph of Diameter of Bacterial Growth Inhibition Zone against Types of Plant
Extract
Type of Plant Extract
Diam
eter
of B
acte
rial G
row
th In
hibi
tion
Zone
(cm
)
Graph 1: Graph of Diameter of Bacterial Growth Inhibition Zone against Different Types of Plant
Extracts
12.0 DISCUSSION
Table 2 above shows the area of clear zones (inhibition zone) surrounding the paper
discs that were soaked into respective plant extracts. This data will be analysed by measuring
the diameter of the clear zone instead of the cloudy zone after at least 24 hours being kept in
incubator of 25°C. Image below shows the difference between the clear zone and the cloudy
zone:
Image 11: Inhibition Zone
The clear region is an indication of the effective inhibition of microbial growth by the
antimicrobial agent. This method is preferable since it is quite fast and inexpensive relative to
other laboratory tests for antimicrobial activity.
Based on this table, every plant extracts show different diameter of the clear zone thus
proving that different type of plants has different antimicrobial properties. At the end of this
experiment, garlic has the longest diameter followed by mint, fennel, ginger, turmeric and
cinnamon. On the other hands, paper discs in Quadrant 4 and 8 have been immersed in
distilled water to act as a point of comparison (controlled variable) to the others that have
been immersed in plant extracts.
Theorically, ginger should have longer diameter compared to mint and fennel
however, it does not turn out that way when at the end, mint and fennel are having larger
diameter than ginger. This is due to a few limitations. During the preparation of mixing the
ginger powder into distilled water, it does not seem to mix well with the denatured alcohol
and formation of clump can be observed. This might overall affect the concentration of the
plant extracts in the distilled water thus causing errors in diameter of the clear zone.
13.0 FURTHER STUDIESAnother experiment can be carried out using different type of bacteria which can be
staphylococcus aureus. Different bacteria may have different effect towards the antimicrobial
actions. All of this depends on its resistance towards the chemical it encounter.
14.0 SAFETY PRECAUTIONIn order to avoid and prevent any kind of accidents or injuries from happening while
conducting this experiment, there are a few common safety precautions that everyone must
follow. First and foremost, wearing a lab coat and a pair of suitable and closed shoes are the
very first things everyone must do once entering the laboratory. This is to protect our clothes
and exposed skin from spillage of any chemical substances or bacteria culture. Wearing hand
gloves is also very crucial since we were handling bacteria throughout this experiment.
However, wearing hand gloves alone does not protective enough so we still have to rinse and
wash our hands with clean water before and after conducting the experiment. These are all
very important to protect ourselves from getting any kind of infections due to the presence of
microorganisms and bacteria. Besides, all of the glass wares that being used in this
experiment must be handled with full care since they are very fragile. All of the samples and
apparatus must also be placed back to their original position.
Since we are dealing with bacteria throughout this experiment, all of the apparatus
used must be sterilised. The sterilising process can be done by heating the apparatus for a
while at the flame of the Bunsen burner such as the forceps and the mouth of the bottle that
stored E. coli broth. The tip of the micropipette also must be sterilised by using a steriliser
machine (since it cannot be heated or else the tip will melt) and must be changed when we are
using other solutions. Almost all of the procedures must be conducted in the laminar flow
chamber because in this chamber, the surrounding air is well-sterilised so contaminations of
the agar solution or the plant extracts are unlikely to occur. If there is no laminar flow
chamber, we can use flame to sterilise the air by lighting up Bunsen burner and allow it to be
lighted up a few minutes before conducting the experiment.
For the agar solution, since it can easily solidified once it does not exposed to heat, it
is advisable to use it immediately after it has been taken out from the vortex machine. This is
to avoid the agar from being solidified too fast even before the bacteria mere mixed and also
to prevent wastage of the agar solution. Once the E. coli broth had been successfully placed
into the agar solution in the petri dish, the bacteria and the agar solution must be mixed well
as this will affect bacterial growth across the agar.
When the paper discs were placed onto the agar, the lid of Petri dish was only lift up
enough to let the forceps to place the discs. This is to prevent contamination of other bacteria.
The Petri dish may open when it is inverted before placed into incubator. This was prevented
by taping the sides of Petri dish. The Petri dishes were not taped all round as it will encourage
growth of anaerobic bacteria, some which may be harmful and also exclude oxygen needed
for growth of bacteria in the agar.
After done all of the procedures, both of the petri dish must be placed in incubator for
at least 24 hours. The temperature of the incubator must be kept constant which is at 25°C
because at this point only E. coli that able to grow. However, both of the petri dish cannot be
kept at 37°C because at this temperature, other harmful bacteria are also able to grow as well
as E. coli. By keeping the temperature at 25°c, it is not only protecting us from any other
harmful bacteria but also important in accuracy of the results.
15.0 CONCLUSIONTheoretically, each and every plant has its own strength of antimicrobial property in
order to fight against bacterial infection. From this experiment, it can be concluded that garlic
has the highest antimicrobial property toward E. coli followed by cinnamon powder, ginger
and mint. Thus, the hypothesis is accepted.
16.0 REFERENCES
i http://www.mayoclinic.org/diseases-conditions/e-coli/basics/definition/con-20032105ii Public Health England at
http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/EscherichiaColi/GeneralInformation/iii Turmeric | University of Maryland Medical Center
http://umm.edu/health/medical/altmed/herb/turmeric#ixzz2vwZhrO3Yiv http://www.whfoods.com/genpage.php?tname=foodspice&dbid=78v http://www.care2.com/greenliving/10-surprising-health-benefits-of-cinnamon.htmlvi http://www.organicfacts.net/health-benefits/herbs-and-spices/health-benefits-of-mint.htmlvii http://timesofindia.indiatimes.com/life-style/health-fitness/diet/Health-benefits-of-mint-leaves/
articleshow/10452719.cmsviii http://www.nlm.nih.gov/medlineplus/druginfo/natural/961.htmlix http://www.nlm.nih.gov/medlineplus/druginfo/natural/300.html
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