Page 609 - e-Book

Inoculation of bacteria

5 ml of nutrient broth was added into each test tube. Loopful of bacteria was taken from pure
culture of clinical isolates and was inoculated into it. The inoculated test tubes were

incubated for 24hrs.
Preparation of plant extracts

Absolute ethanol was used to extract the phytochemicals from green tea and black tea using
Soxhlet apparatus. 3 grams of green tea and black tea leaves were weighed and its

phytochemicals were Soxhlet extracted using 160ml of 100% ethanol. After the

extraction, the ethanol containing phytochemicals was transferred to rotary evaporator to
evaporate the ethanol solvent. The tea extract which is obtained after evaporation was made

into 4 different concentrations like 0.2%, 0.3%, 0.4%, 0.5% using the same solvent (A. Tahir

et al., 2011).
Agar well diffusion assay

Antibacterial activity was done by agar-well diffusion method. Nutrient agar was poured onto
the Petri plate and made it to solidify. Wells were made using 8mm cork borer. On the

surface of media, bacteria were inoculated by smearing through cotton swab. 10µl of leaf
extract was put into the well using a micro pipette. These inoculated plates were incubated at

37°C and checked for zone of clearance after 24hrs and diameter was measured. Only ethanol

was taken as negative control and antibiotic streptomycin was taken as positive control (A.
Farjana et al., 2014).

Qualitative estimation of phytochemicals

Test for Tannins
1ml of the tea extract was added to 2ml of 5% ferric chloride. Dark blue or greenish

black formation indicates the presence of tannins.

Test for Saponins
2ml of tea extract and 2 ml of distilled water were mixed and shaken. Formation of 1cm

layer of foam indicates saponins.
Test for Alkaloids

To 2ml of tea extract, 2ml of concentrated hydrochloric acid and Mayer’s reagent were
added. Green color or white precipitate indicates the presence of alkaloids.

Test for Flavonoids

To 2ml of the tea extract, 1ml of 2N sodium hydroxide was added. Formation of yellow
color indicates the presence of flavonoids.

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