Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development

Abstract

As there are sparse data on the impact of growth media on the phenomenon of biofilm development for Candida we evaluated the efficacy of three culture media on growth, adhesion and biofilm formation of two pathogenic yeasts, Candida albicans and Candida tropicalis. The planktonic phase yeast growth, either as monocultures or mixed cultures, in sabouraud dextrose broth (SDB), yeast nitrogen base (YNB), and RPMI 1640 was compared, and adhesion as well as biofilm formation were monitored using MTT and crystal violet (CV) assays and scanning electron microscopy. Planktonic cells of C. albicans, C. tropicalis and their 1:1 co-culture showed maximal growth in SDB. C. albicans/C. tropicalis adhesion was significantly facilitated in RPMI 1640 although the YNB elicited the maximum growth for C. tropicalis. Similarly, the biofilm growth was uniformly higher for both species in RPMI 1640, and C. tropicalis was the slower biofilm former in all three media. Scanning electron microscopy images tended to confirm the results of MTT and CV assay. Taken together, our data indicate that researchers should pay heed to the choice of laboratory culture media when comparing relative planktonic/biofilm growth of Candida. There is also a need for standardisation of biofilm development media so as to facilitate cross comparisons between laboratories.

Fig. 1. : growth curves of planktonic Candida in three different culture media: yeast nitrogen base (YNB), sabouraud dextrose broth (SDB) and RPMI 1640. C. albicans (A), C. tropicalis (B) and 1:1 mixed species (C) growth in YNB, SDB and RPMI. Data are presented as mean (± standard deviation) of three independent experiments performed in triplicates.

Fig. 2. : adhesion of Candida albicans, C. tropicalis and 1:1 mixed species in yeast nitrogen base (YNB), sabouraud dextrose broth (SDB) and RPMI 1640 media. (A) MTT assay (B) crystal violet (CV) assay. Adhesion in phosphate buffered saline (PBS) served as the control. All error bars represent the standard deviations (SD). For comparison of relative adhesion of Candida species in different culture media, a two-way ANOVA was performed followed by a Bonferroni post hoc test. *: indicates a significant difference in adhesion between three culture media for given organism, with a p < 0.05 using ANOVA.

Fig. 3. : relative biofilm formation of Candida species in three different culture media over a 96 h period. (A) MTT assay (B) crystal violet (CV) assay. Data are mean ± standard deviation of three independent experiments performed in triplicates.

Fig. 4. : (A) scanning electron micrograph of a 48 h old, Candida albicans biofilm in RPMI 1640 medium. Note the architecture of 72 h old mature biofilm with profuse extracellular matrix (M), hyphal elements (white solid arrow) blastopores (★), some bearing bud-scars (+) (Scale indicates 50.0 µM); (B) scanning electron micrograph of a 48 h old, C. tropicalis biofilms in RPMI 1640 medium. Note the architecture of 72 h old mature biofilm devoid of extracellular matrix and relatively sparse growth compared to Fig. 4A above (Scale indicates 50.0 µM); (C) scanning electron micrograph of a 48 h old, 1:1 mixed species biofilm of C. albicans and C. tropicalis in RPMI 1640 medium. Note the architecture of 72 h old mature of 1:1 mixed species biofilm in RPMI 1640 medium devoid of extracellular matrix but clearly showing hyphal elements of C. albicans intermixed with C. tropicalis blastospores devoid of hyphae (Scale indicates 50.0 µM).