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