Colonies were counted after 24-h incubation at 37 °C and the number
of CFU was calculated. A suspension of C. albicans ATCC 14053 containing 1 × 106 cells mL−1 in RPMI 1640 was mixed with different dilutions of allicin and fluconazole (1/2 × MIC, 1 × MIC and 10 × MIC) and incubated at 35 °C for 24 h. Cells were fixed in 2% v/v glutaraldehyde in phosphate-buffered saline (pH 7.2) and washed with sodium cacodylate buffer. For postfixation, samples were rinsed in 1% osmium tetroxide for 2 h at 4 °C, washed again with sodium cacodylate buffer and then dehydrated with ascending ethanol series. After that, samples GDC-0068 order on coverslips were put into a critical point dryer and then stuck onto the stub. The specimens were coated with gold and observed through a JEOL JSM 6400 scanning electron microscope. For the experiments on the animal model of systemic candidiasis, 4–6-week-old female BALB/c mice were infected intravenously through the tail vein with 200 μL per mouse of C. albicans ATCC 14053 (5 × 106 yeast cells mL−1). The mice were divided into five experimental groups of 12 mice each. In the first two groups of mice, allicin (200 μL per mouse) was administered intravenously once daily for 5 days beginning 1 h after Candida injection (postinfection) at 1 and 5 mg kg−1 day−1, respectively (Shadkchan et al., 2004). For the third and fourth
groups of mice, fluconazole (200 μL per mouse) was administered via the intraperitoneal route once daily for 5 days starting 1-h postinfection at 1 and 5 mg kg−1 day−1, respectively (Rex et al., 1998). For the untreated control group, 200 μL of normal saline was injected into each mouse at 1-h postinfection. buy AZD2281 The infection was followed up for 28 days and evaluated in terms of mortality and morbidity. For studies of tissue burden, two randomly chosen mice were sacrificed from each experimental group on days 2, 4, 7, 10, 14 and 28 after infection (Shadkchan et al., 2004). Kidneys, liver and spleen from each mouse were aseptically removed and homogenized in 1 mL of sterile normal
saline and cultured on Sabouraud dextrose agar Adenosine plates as explained in the time–kill study, and assessed by determination of fungal colonization of viscera. Histopathologic analyses were performed for a qualitative confirmation of the result. Tissues were fixed in 10% formalin, then blocked by paraffin wax and cut with a microtome (Leica, model RM2025) in 4-μm thickness. Hematoxylin and eosin and periodic acid-Schiff staining were used to observe the tissue and presence of fungal elements. All animal care procedures were supervised and approved by the University of Putra Malaysia Animal Ethics Committee (ACUC NO.: UPM/FPSK/PADS/BRUUH/00278). For quantitative statistical analysis of inhibitory effects of drugs in vitro and also reduction of fungal load in tissues of mice, data were analyzed in terms of normality and one-way anova was carried out.