Synthesis, characterization and biological activity of Schiff bases based on chitosan and arylpyrazole moiety

3.6. Minimum inhibitory concentration (MIC) measurements 
The MIC was determined by the broth micro dilution method using 96-well micro-plates [44, 45]. 
The inoculate of the microbial strains was prepared from 24 h broth cultures and suspensions were 
adjusted to 0.5 McFarland standard turbidity. Each sample (1.0 mg) was suspended in DMSO (1 
mL
) to obtain 1000 
µg mL
-1
stock solution. A number of wells were reserved in each plate for 
positive and negative controls. Sterile broth (100 µL) was added to the well from row B to H. The 
stock solutions of samples (100 µL) were added to the wells in rows A and B. Then, the mixture of 
samples and sterile broth (100 µL) in row B was transferred to each well in order to obtain a 
twofold serial dilution of the stock samples (concentration of 500, 250, 125, 62.5, 31.3, 15.6 and
7.81, 3.9, 1.95, 0.98 and 0.49 
µg mL
-1
). The inoculums (100 µL) were added to each well and a 
final volume, 200 µL, was obtained in each well. Plates were incubated at 37°C for 24 h in case of 

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antibacterial activity and 48 h at 25°C for antifungal activity. Microbial growth was indicated by the 
presence of turbidity and a pellet at the bottom of the well. 
4. Results and discussion 
4.1. Characterization of the prepared chitosan Schiff bases 
4.1.1. FT-IR Spectra Studies 
 
Fig.1.
FT-IR spectra of pure chitosan (Ch) and that of the Schiff bases (ChBs) are illustrated in Fig. 1. The 
spectra of pure chitosan showed the typical absorption bands of chitosan situated at 1660 and 1597 
cm
-1
, corresponding to the amide I & II, respectively, and a broad band appearing 
around 3444 
cm
−1
due to the stretching vibration of O–H and N–H [46]
as well as inter
−
and intra
−
molecular 
hydrogen bonding of chitosan molecules. The peaks at 2948 and 2866 cm
−1
are attributed to C-H 
stretching vibration. The absorption bands around 1154 (asymmetric stretching of the COC bridge), 
∼1088 and ∼1022 cm
−1
(skeletal vibrations involving C―O stretching) are characteristic of 
chitosan’s saccharide structure. For FT-IR spectra of ChBs, new absorption peaks appeared at 
1639cm
−1
corresponding to the C=N characteristic vibration of imines [47]. 
The bands appear at 
1540, 1500, 1446, and 1400 cm
-1
corresponding to the phenyl groups and the peaks appear at 1348, 
732 cm
-1
are due C―NO
2
and C―Cl stretching vibration, respectively. Moreover, 
the broad peak at 
around 3444 cm
-1
corresponds to the stretching vibration of N–H and O–H bands shifted to higher 
frequency. In addition, the characteristic absorption peak at 1597 cm
-1
almost disappears, 
representing a decrease in –NH
2
group content, and there is no evidence of the characteristic band 
related to free aromatic aldehyde near to 1680 cm
−
1
, indicating that the amino groups on chitosan 
reacted with the aldehyde to form a Schiff base under these experimental conditions. The results of 
the elemental analyses of ChBs, are shown in Table 1, are quite different from those expected for a 
100% degree of substitution (DS), suggesting that the substitution is not complete. As shown in 
Table 1, the DS are found to be 0.71, 0.67, 0.63, 0.62 and 0.52 for Ch-H, Ch-CH
3
, Ch-OCH
3
, Ch-Cl 
and Ch-NO
2
, respectively.