Tese_Tânia Vieira.pdf - Ubi Thesis

Chapter II – Materials and Methods
2.2.6. Nanoparticles Fourier Transform Infrared Spectroscopy Analysis
The different nanoparticles were also analyzed by FT-IR. This spectroscopic tool gives
information about the molecular structure of chemical compounds, and it is useful for the
characterization of biopolymers (Lawrie et al. 2007). The spectra was acquired in a Fourier
transform infrared spectrophotometer Nicoletis 20 (64 scans, at a range of 4000 to 400 cm -1 )
from Thermo Scientific (Waltham, MA, USA) equipped with a Smart iTR auxiliary module.
2.2.7. Determination of antibacterial activity
2.2.7.1. Minimum Inhibitory Concentration and Minimal
Bactericidal Concentration
The antimicrobial activity of the synthesized AgNPs was assessed through a standard
microdilution method, in order to determine the Minimum Inhibitory Concentration (MIC), which
is defined as the lowest concentration of material that inhibits the growth of an organism. This
assay was performed in agreement with the standards recommendations from the National
Committee for Clinical Laboratory Standards (NCCLS). The antimicrobial activity of AgNPs was
scrutinized using E. coli as a model of Gram-negative bacteria, in a 96 wells plate. The AgNPs
were diluted several times with 100 µL of culture medium (LB Broth). After that, 100 µL of
culture medium inoculated with the tested bacteria at a concentration of 2.5×10 5 colony-forming
unit (CFU)/mL was added to the plate. A negative control was prepared with 200 µL culture
medium and a positive control was set by using 100 µL of culture medium inoculated with 100 µL
of the bacteria at a concentration of 2.5×10 5 CFU/mL. The plate was incubated 24 h at 37 °C and
then the MIC of the tested substance was determined (Panác ek et al. 2006). This test was done
in triplicate.
The Minimal Bactericidal Concentration (MBC) may be characterized as the minimum
concentration of the sample required to achieve bacterial dead after 24 h of incubation. The
MBC was examined by a modified imprinted method. 5 µL of the tested samples with defined
concentrations were transferred from the plate wells and imprinted on the surface of a plate of
LB agar without antimicrobial agents. Then the petri plate was incubated for 24 h at 37 °C and
the MBC was determined as the lowest concentration that inhibited the bacterium visible growth
(Panác ek et al. 2006). The nanoparticle concentration causing bactericidal effect was
determined based on the absence of colonies on the agar plate (Ruparelia et al. 2008).
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