C CFU assay

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1 Add 0.1 ml of Candida suspension (containing 1 x 10" yeasts) to wells containing washed monocytes/macrophages (effector-to-target ratio 10:l)a and to six wells not containing macrophages to be used as control cultures.

2 Add 0.1 ml of complete medium to each well to yield a final volume of 0.2 ml/well.

3 Incubate the plates containing macrophages and Candida for 3 h at 37°C.

4 Remove the plates from the incubator and shake vigorously on a shaking platform.

5 Stop the intracellular Candida killing by lysing the phagocytic cells with 20 |xl/well of a solution of 10% Triton X-100 in water.

6 Carefully mix each well by pipetting and make serial dilutions in distilled water (from 1:20 to 1:100 final dilution).

7 Plate 0.1 ml of serial dilutions on Sabouraud dextrose agar (triplicate samples).

8 Count the surviving colony-forming units (CFU) after 24 h of incubation at 37°Cand compare them to control cultures consisting of C. albicans incubated without effector cells.

9 Express the results as the percentage of anti-Candida activity according to the formula:

{1 - (CFU of experimental group/CFU of control culture)} x 100.

3 Different effector-to-target ratios should be tested (e.g. from 20:1 to 2.5:1). Usually, the number of target cells is kept constant and an effector-to-target ratio titration Is achieved by varying the number of effector cells.

To optimize this test, it is important to use an agerminative strain of C. albicans that grows as a pure yeast form in vitro at 28°C or 37°C in conventional media. This will avoid bias due to hyphae formation during the incubation (49).

4.2.3 Extracellular killing of C. Albicans

Protocol 9 describes a method for assessment of extracellular killing of Candida (hyphal form) using the (3-(4,5-dimethy 1thiazol-2-yl)-2,5-di-phenyIterrazolium) MTT colorimetric assay.

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