Pre - Report

Investigating Antimicrobial Activity of Plant Extracts

2016 / 04 / 04
Section 1 / 2015101109 / Rahman Tony Nur

1. Title
Investigating Antimicrobial Activity of Plant Extracts.

2. Purpose
Investigation of antimicrobial activity of plant extracts on the Escherichia coli.

3. Theory
a. Culture method: Spreading
One way to isolate pure colonies is the spread
plate technique. Starting from a liquid culture of
bacteria, a series of tenfold dilutions is made, and
a small amount of each dilution is placed directly
on the surface of separate agar plates. The
sample is spread over the surface of the plate with
a heat-sterilized, bent glass rod. The early
dilutions, those containing the most bacteria, will
produce confluent growth that covers the entire
agar surface. Later dilutions, containing fewer
and fewer organisms, yield individual colonies.
As we shall see later, spread plates not only enable us to isolate pure cultures but also can be
used to enumerate the number of viable bacteria in the original growth tube. A viable organism
is one that successfully replicates to form a colony. Thus, each colony on agar plate represents
one viable organism present in the original liquid culture.

b. Strain: E. coli (BL21)

Protease Deficient ([lon] ompT): E. coli B strains are "naturally" deficient in the lon protease
which in K-12 strains serves to degrade misfolded proteins and to prevent some cell cyclespecific proteins from accumulating. The OmpT protease resides at the surface of wild type E.
coli in both K-12 and B strains, presumably helping the cells to derive amino acids from their
external environment. Cells deficient in both these proteases are much more amenable to the
production of proteins from cloned genes.
T1 Phage Resistant (fhuA2): T1, an extremely virulent phage requires the E. coli ferric
hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this
type of phage, but does not significantly affect the transformation or growth characteristics of
the cell.

c. Clear zone (Zone of Inhibition)

On this agar plate, a bacterial isolate is tested for

resistance to each of twelve different antibiotics. The
clear zones around each disc are the zones of inhibition
that indicate the extent of the test organisms inability to
survive in the presence of the test antibiotic. (A)The disk
shows a large zone of inhibition; whereas (B) shows no
zone of inhibition, indicating resistance of the isolate to
the test antibiotic. Presence of zone of inhibition is not
automatically interpreted as susceptibility to the
antibiotic; the zone width has to be measured and compared against a reference standard which
contains measurement ranges and their equivalent qualitative categories of susceptible,
intermediately susceptible or resistant.
For example, this E.coli isolate on the right has a zone of
inhibition of 10.1mm around ampicillin (AM); since the
zone diameter interpretation chart is as follows:
Resistant: 13mm or less
Intermediate: 14-16 mm
Susceptible: 17 mm or more
This particular E.coli isolate is read as resistant to ampicillin.

d. Characteristics and mechanism of the anti-bacterial activity of S. baicalensis and

C. chinensis.
Scutellaria baicalensis: 1.82g dissolved in 4ml
DW () - Coptis chinensis: 1.56g dissolved
in 4ml DW ().
The Chinese name for Coptis sp. Franch, from the
Ranunculeae family, is Huang lian. There are 16
species worldwide, 21 six of these grow in mainland
China, and only Coptis quinquefolia in Taiwan. Of
these, C. chinensis, C. deltoidea and C. tetoides are
typically found in herbal markets in China and
Taiwan. The rhizome has been found to be a rich source of the alkaloid berberine. In reference
books, Huang Lian is specified most often as C. chinensis. It is mainly used traditionally as a
heat purgative, anti-inflammatory, de-toxicant and to clear damp. The Chinese name for S.
baicalensis Georgi, from the Lamiaceae family is Huang Qin and has been found to contain
high levels of the flavones wogonoside and baicalin. Both these herbs are traditionally used to
treat conditions brought about from excess heat and damp in the body, including bacterial
gastroenteritis.

e. Method of extracting of the antimicrobials in plants.

i.

Solvent extraction
The plant parts are dried immediately either in an artificial environment at low
temperature (50-60C) or dried preferably in shade so as to bring down the initial large
moisture content to enable its prolonged storage life and . The dried berries are pulverised
by mechanical grinders and the oil is removed by solvent extraction. The defatted material
is then extracted in a soxhlet apparatus or by soaking in water or alcohol (95% v/v). The

resulting alcoholic extract is filtered, concentrated in vacuo or by evaporation, treated with

HCl (12N) and refluxed for at least six hours. This can then be concentrated and used to
determine the presence of phytoconstituents. Generally, the saponins do have high
molecular weight and hence their isolation in the purest form poses some practical
difficulties. The plant parts (tubers, roots, stems, leave etc) are washed sliced and extracted
with hot water or ethanol (95% v/v) for several hours. The resulting extract is filtered,
concentrated in vacuo and the desired constituent is precipitated with ether.

ii.

Supercritical fluid extraction (SFE)

Super Critical Fluid Extraction (SFE) involves use of gases, usually CO2, and
compressing them into a dense liquid. This liquid is then pumped through a cylinder
containing the material to be extracted. From there, the extract-laden liquid is pumped into
a separation chamber where the extract is separated from the gas and the gas is recovered
for re-use. Solvent properties of CO2 can be manipulated and adjusted by varying the
pressure and temperature that one works at. The advantages of SFE are, the versatility it
offers in pinpointing the constituents you want to extract from a given material and the fact
that your end product has virtually no solvent residues left in it (CO2 evaporates
completely).

iii.

Microwave-Assisted extraction
MAP applications include the extraction of high-value compounds from natural sources
including phytonutrients, nutraceutical and functional food ingredients and pharmaceutical
actives from biomass. MAP technology offers some combination of the following
advantages: 1. Improved products, increased purity of crude extracts, improved stability of
marker compounds, possibility to use less toxic solvents; 2. Reduced processing costs,
increased recovery and purity of marker compounds, very fast extraction rates, reduced
energy and solvent usage. Examples include antioxidants from dried herbs, carotenoids
from single cells and plant sources, taxanes from taxus biomass, phytosterols from
medicinal plants, polyphenols from green tea, and many more.

4. Experimental Method (Reagent & Apparatus)

a. Reagent & Apparatus
Alcohol lamp, plate, loop, ethanol, spreader, micropipette, 5mm paper discs, tweezer,
deionized water, agar plate, pre-cultured E. coli broth, plant extract solutions (S, C), diluted
extract solutions (1/10 S, 1/10 C), incubator.

b. Experimental Methods
1. Label at the bottom of the plate (1/10 S, 1/10 C, S, C, DDW).
2. Pour 95% ethanol to a beaker and immerse the spreader. Sterilize the spreader using flame
and chill it to ambient temperature.
You should handle the spreader carefully when using ethanol with flame.
Sterilize the spreader and cool it down in the air for 15 seconds.
3. Drop 200 of the pre-cultured E. coli broth on agar plate using micropipette and
micropipette tips.
4. Spread the broth on agar plate.
5. Put 5 paper discs (5 mm) on the agar plate using flame-sterilized tweezer.
6. Drop 10 of deionized water (control), plant extract solutions (S, C), diluted extract
solutions (1/10 S, 1/10 C) on each paper disc.
Drop the solution slowly.
7. Incubate the agar plate for overnight at 37 and observe the formation of the clear zone.
(measure the size)

 Do not let the plate be upside-down

5. Reference
1. Joan L. Slonczewski, John W. Foster; Microbiology: An Evolving Science 2nd ed. ; W. W.
Norton & Company; 2011; p126-128
2. Francesca S. Leach; Anti-microbial properties of Scutellaria baicalensis and Coptis chinensis,
two traditional Chinese medicines; http://biohorizons.oxfordjournals.org/content/4/2/119
3. http://amrls.cvm.msu.edu/microbiology/detecting-antimicrobial-resistance/testmethods/examples-of-antibiotic-sensitivity-tesing-methods
4. https://www.neb.com/products/c2530-bl21-competent-e-coli