KrishnaNaik&col.
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2.2. Antimicrobial activity
a. Antibacterial activity
The antibacterial activity of synthesized compounds (250 µg/mL inDMSO) was
preliminarily studied by disc diffusion method. The procedure followed for the disc
diffusionmethod is given below.
A suspension of
Staphylococcus aureus
was added to sterile nutrient agar at
45
o
C. 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
DMSOwere 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 37
o
C for 24 hours and observed for
antibacterial activity. The diameter of the zone of inhibitionwasmeasured in each plate.
The average zone of inhibitionwas calculated. A similar procedurewas adopted for the
antibacterial activity studies against other organisms.
b. Antifungal activity
The procedure described abovewas followed for antifungal activity against
Aspergillus niger
NCCS 1196 and
Candida albicans
. Compounds were treated at
several different concentrationsusingDMSOasasolvent.
c.DeterminationofMinimumInhibitorConcentration
The procedure followed to find out MIC by Broth DilutionMethod is given
below. Standardized inoculum (matched to McFarland BaSO
4
standard) of
suspensionof organismswasprepared. Aseriesof glass tubes containingdifferent
concentrations of test compounds dissolved inDMSOand spiller innutrient broth
were incubatedwith one drop of inoculumand shaken gently tomix the contents.
Two growth control tubeswere alsopreparedbymixing0.1mLof control and0.9
mLof sterile saline and its optical densitywas determined. The control contained
1×10
-‐5
colony formingunits/mLwhich isequivalent to20colonies.
Tubeswere incubated for 24 hours at 37
o
C in air. The turbidity developed
in each tube was recorded by UV-‐Visible spectrophotometer.
The turbidity
producedby thebroth (without inoculum)was consideredas100%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.
