Dear Shakeel,

Please read the following text taken from a publication entitled " Antifungal susceptibility and growth inhibitory response of oral Candida species to Brucea javanica Linn. extract" published in BMC Complement Altern Med. 2013; 13: 342. The experimental part should be similar to that you are planning to conduct. However, please note that the concentrations of the extracts is in the range of mg/mL (you indicated in your question a range of mg/ul).

Antifungal susceptibility

The antifungal activity of the extract was carried out based on the disc diffusion concept of the Kirby-Bauer sensitivity test [15]. Sterile blank discs of 6 mm diameter were impregnated with a concentration of 100 mg/ml. The discs were air-dried prior to firm placement on the agar surface which had earlier been seeded with the respective candidal. Throughout this experiment, a blank disc impregnated with sterile distilled water represented as negative control while a disc impregnated with a mouth rinse containing 0.12% w/v chlorhexidine digluconate (CHX) represented as the positive control. The volume of the test extracts, positive and negative controls impregnated onto the discs were standardized at 100 μl. All plates were incubated overnight at 37°C (except for C. parapsilosis which required incubation temperature of 35°C). The susceptibility of candidal species was determined by the diameter of the growth inhibited zone surrounding the discs. The experiment was carried out three times in triplicate to ensure reproducibility of observations.

Determination of minimum inhibitory concentration (MIC)

Two-fold microdilution broth method was used to determine the MIC value [16]. The MIC is the lowest concentration of the samples that visually shows absence of growth. 100 μl of YPD broth was dispensed into wells marked as Well 1 (W1) to Well 7 (W7). Following this, 100 μl of stock solution (200 mg/ml) was added into W1 and two-fold serial dilution was repeated for W2 through W5. Hence, the final concentrations of B. javanica extract in W1, W2, W3, W4 and W5 were 100, 50, 25, 12.5, 6.25 and 3.13 mg/ml, respectively. CHX was used in place of the plant extract as positive control in W6, while W7 which only contain the mixture of YPD broth and the extract represented the negative control. 20 μl of candidal suspension (106 CFU/ml) was added to W1 through W6, except for W7. Triplicate samples were performed for each test concentration. The microdilution plates were incubated overnight at 37°C (except C. parapsilosis, 35°C). Following this, the growth inhibition of the candidal cells in microdilution wells was observed.

Determination of minimum fungicidal concentration (MFC)

A standard procedure described by Espinel-Ingroff et al.[17] was applied to determine the MFC. The MFC criteria value considered in this work was the concentration where no growth or fewer than three colonies were obtained to give an approximately 99 to 99.5% killing activity. Briefly, 50 μl was taken from the wells of the MIC assay in which no indication of growth was observed for all respective Candida species, was sub-cultured onto fresh YPD agar plates. The plates were incubated at 37°C (C. parapsilosis at 35°C) for 24 to 48 h following which any visible sign of growth.

Determination of the percentage inhibition of diameter growth (PIDG)

PIDG provides an indication with regards to the strength of antifungal activity of the extract in comparison to the positive control (0.12% w/v CHX). The percentage inhibition of diameter growth (PIDG) values was estimated according to the equation as below [14]:

PIDG(%) = Diameterofsample‒DiameterofpositivecontrolDiameterofpositivecontrol×100

Growth profiles of Candida species

Five millilitre of candidal suspension (106 cells/ml) was dispensed into three sterile conical flasks, each containing 40 ml of YPD broth. 5 ml of sterile distilled water was added to give a total volume of 50 ml in each flask. The flasks were incubated at 37°C (C. parapsilosis at 35°C) for 18 h in a shaking water bath to continuously agitate the suspension. The growth of each species was elucidated by viable cell counts (CFU enumeration) which were estimated at 2, 6, 10, 14 and 16 h interval. The cell suspension was first diluted by serial dilution in a nontoxic diluent (e.g. phosphate-buffered saline, pH 7.2-7.4) before plating. Spectrophotometric assay [18] which was based on continuous monitoring of changes in the optical density of cell growth was employed. Cell growth was measured periodically at every one hour interval over a period of 18 h at an on optical absorbance of 550 nm. The growth of different candidal species can be distinguished by measuring the changes of specific-growth rate (μ) and doubling time (g) following equations previously described [19,20]:where, Nt represented the number of cells at log phase, No represented the number of cells at zero time, t2 was the time taken to reach plateau, and t1 zero time when the cells enter the log phase. Throughout of the study, CHX was used in place of the extract as a positive control.

(i) Specific-growth rate: μ=In(Nt/No)t2−t1

(ii) Doubling time: g = log10(Nt/No)/log102

Growth inhibitory activity of Brucea javanica extract

Brucea javanica extract was prepared into stocks of 10, 30 and 60 mg/ml. Five mililiter of each stock concentration was dispensed into sterile conical flasks containing 40 ml of YPD broth, followed by 5 ml of the respective candidal suspension (106 cells/ml) to give a final concentration of 1, 3 and 6 mg/ml of the extract. In a similar manner, the culture flasks were placed in a shaking water bath at 37°C (C. parapsilosis at 35°C) and the growth of cells in presence of the extract was measured periodically at every one hour interval over a period of 18 h. Changes in specific-growth rate (μ) and doubling time (g) were calculated and the findings were compared with that of the standard. The inhibitory effect of the extract was also determined based on viable cell counts.

Statistical analysis

All results were computed and expressed as mean ± standard deviation (SD) from three determinations performed in triplicate (n = 9). Statistical analysis was performed using SPSS software (version 18.0) with analysis of variance (One-Way ANOVA) and post-hoc test Dunnett’s T3 were used to compare the significant difference between the groups. A ρ-value of