Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 138

3.3.1.3 Determination of Inhibitory Zone Diameters [128]

A total of 150 μL of 0.5 McFarland standard bacterial suspensions was spread onto the agar
plate. Sterilized stainless steel tubes of 8.0 × 1.0 × 10 mm (inner diameter 6 mm) were
added to the surfaces of the media and filled with 100 μL of sterilized antimicrobial solu-
tion. The blood plates were incubated in an anaerobic chamber under an atmosphere of
80% N 2 , 10% H 2 , and 10% CO 2 . The inhibitory zone was considered to be the shortest dis-
tance (mm) between the outer margin of the cylinder and the initial point of microbial
growth. Six replicates were made for each microorganism. Each assessment was performed
three times to ensure the reproducibility of the results.

3.3.2 Antimicrobial Chitosan Derivatives

3.3.2.1 Preparation of N-[1-hydroxy-3-(trimethylammonium)propyl] Chitosan Chloride

N -[1-hydroxy-3-(trimethylammonium)propyl] chitosan chloride (HTCC) was synthesized
by reacting chitosan with glycidyltrimethylammonium chloride (GTMAC) [129]. Briefly,
6 g of chitosan was mixed and dispersed in 225 mL of 2-PrOH. The reaction was carried
out with stirring at 80-90°C for 1 h. GTMAC was dissolved in deionized water (30% w/v)
to form a solution. The GTMAC solution was added to the chitosan suspension slowly
under continuous stirring. The molar ratio of GTMAC to the amino groups of chitosan was
4:1. After 4 h of reaction at 80°C, any precipitate that formed was filtered. The product was
then poured into EtOH and washed three times. Quaternized CS was obtained by drying
at 80°C for 48 h. The degree of quaternization (DQ) was determined by titrating the amount
of Cl ions on HTCC with 0.1 M AgNO 3 [130].

3.3.2.2 Antibacterial Activities of HTCC

P. gingivalis, P. intermedia , and A. actinomycetemcomitans are Gram-negative strains, and
S. mutans is a Gram-positive strain. MIC was quantified for all the standard bacterial strains
selected, and the results are shown in Table 3.3 and Figure 3.12. MIC values for each bacte-
ria strain ranged from 0.25 to 2.5 mg/mL. The lactic acid (LA) solution of HTCC exerted
higher antibacterial activities against P. intermedia , A. actinomycetemcomitans , and S. mutans
(MICs were 1, 1 and 0.5 mg/mL, respectively) than the LA solution of chitosan (MIC
2.5 mg/mL). However, the LA solution of chitosan and HTCC had the same MIC value
against P. gingivalis (MIC 0.5 mg/mL). The aqueous solution of HTCC exhibited relatively
lower antibacterial activity against P. gingivalis and S. mutans than the LA solution of
HTCC. In addition, Gram-negative and Gram-positive strains were all susceptible to chito-
san and HTCC, and there was no significant difference between them.

TAble 3.3
MIC Values of CS and HTCC for Four Oral Bacterial Strains (mg/mL)

Samples P. gingivalis P. intermedia A. actinomycetemcomitans S. mutans

HTCC(LA) 0.5 1 1 0.5

HTCC(H 2 O) 1 1 1 1

CS(LA) 0.5 2.5 2.5 2.5

CS + HTCC 0.25 0.25 0.25 0.25

Note: (LA): lactic acid solution, (H 2 O): aqueous solution.