1162 A. Brand, K. Lee, V. Veses and N. A. R. Gow Table 1. C. albicans strains used in this study. Strain Genotype Description NGY152 CAI4/CIp10-URA3 Control strain Brand et al. (2004) 3153A Wild type Clinical isolate Mackenzie and Odds (1991) NGY166 cch1D Mutant lacking a plasma-membrane, voltage-gated calcium channel Brand et al. (2007) NGY167 mid1D Mutant lacking a plasma-membrane, stretch-activated calcium channel Brand et al. (2007) NGY368 cch1D/mid1D Mutant lacking the Cch1-Mid1 plasma-membrane calcium channel complex Brand et al. (2007) NGY468 mid1D+MID1 mid1D mutant with 1 copy of MID1 re-integrated at the RPS1 locus Brand et al. (2007) NGY355 pmr1D Golgi-ATPase mutant, partially deﬁcient in O- and N-glycosylation Bates et al. (2005) NGY356 pmr1D+PMR1 pmr1D mutant with 1 copy of PMR1 re-integrated at the RPS1 locus Bates et al. (2005) MKY380 crz1D Mutant lacking the calcium-dependent, calcineurin-activated transcription factor Karababa et al. (2006) MKY381 crz1D+CRZ1 crz1D mutant with 1 copy of CRZ1 re-integrated at the RPS1 locus Karababa et al. (2006) CACB8B-5 chs3D Mutant lacking the chitin synthase responsible for the majority of chitin in the Bulawa et al. (1995) lateral cell wall NR3 fks1D Golgi ATPase mutant, partially deﬁcient in O- and N-glycosylation Douglas et al. (1997) HMY5 mns1D Mutant lacking outer-chain N-glycans and phosphomannan Mora-Montes et al. (2007) HMY6 mns1D+MNS1 mns1D with 1 copy of MNS1 re-integrated at the RPS1 locus Mora-Montes et al. (2007) NGY337 mnt1/2D O-glycosylation double mutant lacking terminal mannan residues Munro et al. (2005) NGY335 mnt1/2D+MNT1 mnt1/2D double mutant with 1 copy of MNT1 re-integrated at the RPS1 locus Munro et al. (2005) Reference Experimental procedures Strains, media and growth conditions The C. albicans strains used in this study are listed in Table 1. Strains were maintained and grown overnight at 30°C in YPD [1% w/v yeast extract (Oxoid, Unipath, Basingstoke, UK), 2% w/v mycological peptone (Oxoid), 2% w/v glucose (Sigma, Poole, UK)], solidiﬁed as appropriate with 2% agar (Oxoid). Hyphae were grown on solid medium containing 20% (v/v) FBS (Biosera, Ringmer, Sussex, UK) in ddH2O, solidiﬁed using 1%, 2%, 4% or 6% (w/v) puriﬁed agar (Oxoid) (see Results). Solid medium was supplemented with 2% (w/v) glucose, maltose or sorbitol (Fisher Scientiﬁc, Loughborough, UK), 1.5 mM BAPTA (a Ca2+ chelator) tetrapotassium salt (Sigma) or 1.5 mM CaCl2 (Sigma), as required. Alternatively, the medium was supplemented with 50 mg ml-1 Calcoﬂuor White, 200 mg ml-1 Congo Red, 500 mM gadolinium or 250 mM verapamil (Sigma). The Ca2+ concentration of 20% fetal calf serum (FCS) was determined as 0.7 mM using a QuantiChrom Calcium Assay Kit (DICA-500) (BioAssays, Hayward, USA) according to the manufacturer’s instructions. try, UK). More than 100 hyphae were observed per strain per experiment. Each experiment was performed on three or more independent occasions. Statistical analysis was per- formed using the Dunnett’s t-test as part of SPSS statistical software (Woking, UK). Fluorescence microscopy Yeast cells were adhered to poly-L-lysine slides in sterile water for 20 min and non-adhered cells washed off. Hyphae were induced by incubation in 1% (v/v) serum at 37°C for 5 h. The supernatant was removed and hyphae pulsed with 1 ml of FM4-64 (from 16 mM in DMSO stock) in 1 ml of the retained supernatant by incubation at 37°C for 45 min in the dark. Slides were rinsed with water and incubated for a further 2 h in the dark at 37°C in fresh medium. After further rinsing, 10 ml Calcoﬂuor White (from 10 mg ml-1 stock) in 990 ml ddH2O was added and the slide viewed under an Axioplan 2 micro- scope (Carl Zeiss Ltd, UK) ﬁtted with an ORCA-ER camera (Hamamatsu Photonics, Hamamatsu, Japan). Images were captured using Openlab 5.0 software (Improvision, Coventry, UK). Characterization of sinusoidal growth in hyphae Confocal microscopy Overnight cultures of yeast cells were harvested by centrifu- gation and washed twice in ddH2O. Cells were diluted 1/2500 and 200 ml was spread onto the surface of 20% (v/v) FCS solid medium and incubated at 37°C for 10 h or until the mean length of hyphae was > 80 mm. Coverslips were placed on the surface of the medium and hyphae were viewed by light microscopy using an Olympus BX50F4 microscope ﬁtted with an Olympus DP11-P camera. The number of primary hyphae over > 80 mm in length that formed two or more consecutive sinusoidal waves was expressed as a percent- age of all primary hyphae formed. The morphology of sinu- soidal hyphae was characterized by analysing the wavelength (l) (distance between helical apices), amplitude (A) and cell length (length of cell compartment bounded by two septa) using Openlab 5.0 software (Improvision, Coven- Cells were grown on solid medium (as above) in 60 mm Petri dishes. Hyphae were stained by the addition of the amphiphilic styryl dye FM4-64 (16 mM in 3 ml dH2O from 16 mM stock in DMSO) (Molecular Probes Europe BV) and incubation in the dark for 30 min. Dishes were positioned on the stage of a Zeiss LSM510 META confocal microscope and 3 ml dH2O added to the staining solution above the agar. Hyphae were viewed with a ¥63 (NA 0.9) water immersion objective using an Argon 488 laser. Images were captured and manipulated using Zeiss LSM510 software (Ver 3.2). Acknowledgements We gratefully acknowledge the imaging assistance of Mr Kevin Mackenzie. We acknowledge ﬁnancial support from the © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1155–1164