Page 212 - 80_01

Krishna
Naik&
col.

2.2.
Antimicrobial
activity

a.
Antibacterial
activity

The antibacterial activity of synthesized compounds (250 µg/mL in DMSO) was
preliminarily studied by disc diffusion method. The procedure followed for the disc
diffusion method is given below.

A suspension of Staphylococcus aureus was added to sterile nutrient agar at
45oC. The mixture was transferred into sterile petri-dishes to a depth of 3 mm and
allowed to solidify. Sterile discs of 5 mm in diameter (made of Whatmann Filter
paper)were immersed in solutions of synthesized compounds. Sterile discs immersed in
DMSO were used as control. Both chemical-treated and DMSO-treated discs were laid
down onto bacteria mixed agar plates. The plates were allowed to stand for 1 hour at
room temperature followed by incubation at 37oC for 24 hours and observed for
antibacterial activity. The diameter of the zone of inhibition was measured in each plate.
The average zone of inhibition was calculated. A similar procedure was adopted for the
antibacterial activity studies against other organisms.

b.
Antifungal
activity

The
procedure
described
above
was
followed
for
antifungal
activity
against

Aspergillus
niger
NCCS
1196
and
Candida
albicans.
Compounds
were
treated
at

several
different
concentrations
using
DMSO
as
a
solvent.

c.
Determination
of
Minimum
Inhibitor
Concentration

The
procedure
followed
to
find
out
MIC
by
Broth
Dilution
Method
is
given

below.

Standardized
inoculum
(matched
to
McFarland
BaSO4
standard)
of

suspension
of
organisms
was
prepared.

A
series
of
glass
tubes
containing
different

concentrations
of
test
compounds
dissolved
in
DMSO
and
spiller
in
nutrient
broth

were
incubated
with
one
drop
of
inoculum
and
shaken
gently
to
mix
the
contents.

Two
growth
control
tubes
were
also
prepared
by
mixing
0.1
mL
of
control
and
0.9

mL
of
sterile
saline
and
its
optical
density
was
determined.

The
control
contained

1
×
10-­-5
colony
forming
units
/mL
which
is
equivalent
to
20
colonies.

Tubes
were
incubated
for
24
hours
at
37oC
in
air.

The
turbidity
developed

in
each
tube
was
recorded
by
UV-­-Visible
spectrophotometer.

The
turbidity

produced
by
the
broth
(without
inoculum)
was
considered
as
100
%
transparency.

The
minimum
inhibitory
concentration
(MIC)
was
noted
as
the
concentration
of

the
test
sample
which
completely
inhibits
the
growth
of
the
microorganism
i.e.

100
%
transparency.

2
06