Morphological and growth altering effects of Cisplatin in C. albicans using fluorescence microscopy
© Kesavan et al; licensee BioMed Central Ltd. 2005
Received: 24 February 2005
Accepted: 25 April 2005
Published: 25 April 2005
Changes in morphology and growth curve of Candida albicans in response to treatment by Cisplatin has been studied using fluorescence staining with ethidium bromide. Treatment with Cisplatin was found to markedly inhibit hyphae and ovoid growth as revealed by ethidium bromide staining of drug treated cells. These changes were concomitant with inhibitory effects on the growth curve with respect to untreated cells
Presence of Cisplatin not only caused suppression in the limiting values in the growth curve, but also caused a slight left shift in the EC50 values. Some of the ovoid cells undergoing poisoning with cisplatin were found to be unusually enlarged before undergoing their natural fate thus suggesting formation of similar cytotoxic end products with DNA.
Candida albicans is a yeast pathogen that causes mild, chronic, superficial systemic infections in immunocompromised and cancer patients [1–3]. Research, worldwide is broadly focused on 1) search of new targets in C. alibcans, 2) Screening inhibitory activities with therapeutic agents, and 3) designing or modifying novel agents, to overcome treatment resistance . C. alibcans has been shown to harbor a self splicing group-I intron in the nuclear 25s rRNA genes . Presence of this gene in C. alibcans, but not in the human genome, suggests that splicing inhibitors could potentially act as alternate targets in controlling the proliferation of fungal pathogens. Recent screening studies using disk diffusion have shown that cis-diamminedichloro platinum (Cisplatin), a well-established antitumor agent, inhibits the growth of C. alibcans at lower concentrations . Despite its prevalent use in cancer treatment, very little work has been done [7–10] to document the effects of Cisplatin and related metal complexes in controlling the proliferation behavior of C. alibcans. With the molecular mechanisms remaining largely unknown, this provided us a basis to study the effects of Cisplatin treatment on the morphology and growth curve of C. alibcans. Morphological changes in Candida can be followed by direct fluorescence staining with suitable dyes to selectively visualize different parts/aspects of cellular activities [11–13]. Though the use of Ethidium Bromide for fluorescence staining is well known, there is no report to show the use of this technique in studying the morphology of Candida, especially in response to treatment with anticancer agents such as Cisplatin. Herein, we report the morphological changes of C. alibcans in response to cisplatin using fluorescence microscopy with ethidium bromide staining.
Materials and methods
Media and Inoculums
Yeast isolates (clinical strains) were obtained from patients, Royapettah Hospital, Chennai. Sub-cultured isolates were maintained in Sabouraud dextrose agar (B.B.L, Cockeysville, MD) at 1–5 × 106 CFU/10 ml media. 30 μl of each sample (log phase) was used for slide preparation to study the morphological changes.
A 2 mg/ml stock of Cisplatin (TNDPL, Chennai) was prepared in sterile Millipore water. Stock was diluted to 60 μg/10 ml culture for treatment purposes.
Ethidium bromide staining was performed using a freshly prepared stock of ethidium bromide (1 mg/ml water) from which 50 μl/100 ml concentration was used for staining the slides. Gram staining was performed from commercially available kits as per instructions.
Morphological studies using Fluorescence Microscopy
30 μl of cultured C. alibcans (Drug treated/non-treated cells) was taken and spread over the clean glass slide and dried at 37°C for 5 minutes. Slides were dipped into the EtBr staining solution for 30–40 seconds followed by immersion in water for 15–20 seconds to remove excess stain. Gram staining was performed in parallel on a similar set of slides to validate the results. Both the set of slides were viewed using NFM under the 100× magnification.
Results and Discussion
K.C thanks the Lady Tata Memorial Trust for the fellowship in pursuing this research work.
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