Enumeration of viable Candida albicans blastospores using tetrabromofluorescein (Eosin Y) and flow cytometry

Abstract

Key terms: Colony forming units, viability testing, systemic candi-is, cell respirationInvestigation of the immune responses in a murine model of systemic candidiasis is currently being performed in our laboratory. AKR, BALB/c, CBNH, C57Bl/6J, and DBA/2J mice vary in their ability to resolve a systemic infection with Candida albicans (1,5,7). The mice are routinely inoculated intravenously (iv) with a sublethal dose of C albicans blastospores. Following inoculation the number of viable cells in the inoculum is usually estimated by determining the number of colony forming units (cfu) per milliliter. To ensure that the mice receive uniform numbers of viable C. albicans blastospores, the viability of C albicans in overnight cultures needed to be determined accurately prior to iv inoculation.Enumeration of cfu on solid phase culture medium is a common method of ascertaining the viability of C albicans in suspensions (2,4,13,15,17). Optical density ( 15), fluorescence microscopy ( 14,16), and/or a hemocytometer have been used to determine the viability of C albicans blastospores. More efficient methods of estimating C albicans viability have been developed by using fluorescent dyes and flow cytometry (3,6, Tetrabromofluorescein (eosin Y) is a vital stain which fluoresces green with an absorption maximum between 5 15 and 5 18 nm (9). The present study was performed to assess whether eosin Y, a cheap and readily available dye, could be used in conjunction with flow cytometry to assess the viability of C. albicans. Eosin Y and flow cytometry were utilized in combination to develop a rapid and reproducible method of accurately evaluating the viability of C. afbicans blastospores. 10,12). MATERIALS AND METHODS Culture ConditionsA single clinical isolate of C. albicans (KEMH 5) was obtained from the Department of Pathology, King Edward Memorial Hospital, Subiaco, Perth, Western Australia. The organism was cultured in yeast extract peptone dextrose (YEPD) broth (1% yeast extract, 2% peptone, 2% glucose) overnight at 30°C in a shaking water bath. A 1 ml aliquot of the overnight culture was pelleted by centrifugation at 2,OOOg for 5 min and washed twice with 0.15 M sodium chloride (saline). The cell pellet was resuspended in 10 ml of saline. To ensure that the viable control suspensions contained 100% viable organisms, cultures in mid to late log phase were used. Staining ConditionsWashed C albicans blastospores were resuspended in 0.01% tetrabromofluorescein in 0.1 5 M sodium chloride (eosin Y solution) and incubated at 25°C for 10 min. VIABILITY TESTING O F C. ALBICANS USING EOSIN Y 3714°C. Fluorescence activity and cell staining characteristics of the stock eosin Y solution were found to decrease with time (unpublished observations). Flow CytometryPrior to analysis of C. albicans suspensions, the flow cytometer was standardized using Immunocheck beads (Cbulter Cytometry, Hialeah, FL). Stained suspensions of C. albicans were measured for fluorescence on a Coulter EPICS-PROFILE I1 using an air-cooled argon ion laser operati...

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice

“…In the present study, we performed rapid in vitro determination of lasioglossin anti‐ Candida activity by eosin Y staining, as described in detail by Constantino et al . . We detected significant inhibitory effects for both 11.5 µM and 21 µM of LLIII or LLIII‐D in vitro , which is comparable to the results of Slaninova et al .…”

“…These methods use acridine orange, ethidium bromide, fluorescein diacetate or tetrabromofluorescein (eosin Y) to non-specifically stain non-viable or viable blastoconidia (23,24). In the present study, we performed rapid in vitro determination of lasioglossin anti-Candida activity by eosin Y staining, as described in detail by Constantino et al (24). We detected significant inhibitory effects for both 11.5 mM and 21 mM of LLIII or LLIII-D in vitro, which is comparable to the results of Slaninova et al (3).…”

“…Rapid methods for susceptibility testing of C. albicans by flow cytometry have been developed. These methods use acridine orange, ethidium bromide, fluorescein diacetate or tetrabromofluorescein (eosin Y) to non‐specifically stain non‐viable or viable blastoconidia . In the present study, we performed rapid in vitro determination of lasioglossin anti‐ Candida activity by eosin Y staining, as described in detail by Constantino et al .…”

“…10 4 Candida cells resuspended in buffered RPMI were mixed with 1/10 vol. of LLIII or LLIII-D peptides in PBS to reach final concentrations of 11.5 mM (20 mg/L) or 21 mM (37 mg/L), respectively, transferred to the wells of 96-well plates and incubated at 35°C (to prevent hyphae formation) for 24,48 or 72 hr to test the peptides' antifungal activity. The same conditions except for a temperature of 37°C were used for testing the LLIII and LLIII-D peptides potency in inhibiting morphological transition of Candida toward pseudohyphal or hyphal forms.…”

Microtubule Motor Protein Kar3 Is Required for Normal Mitotic Division and Morphogenesis in Candida albicans

“…As loss of Kar3p causes a significant proportion of ⌬kar3/⌬kar3 cells to be nonviable (Fig. 4C), live cells from log-phase cultures were selected with the dye eosin Y. Eosin Y stains dead C. albicans cells (15) and allowed the sorting of live (unstained) cells, which were then subsequently fixed and stained with the DNA stain SYTOX Green. Cell cycle stages were followed by using flow cytometry (as described in Materials and Methods).…”