Proposal of a low-cost protocol for colorimetric semi-quantification of secretory phospholipase by Candida albicans grown in planktonic and biofilm phases Lisa Taniguchi a , Berenice de Fátima Faria a,b , Rosimeire Takaki Rosa a , Alessandra de Paula e Carvalho a , Lauren Christine Gursky a,c , Selene Lobo Elifio-Esposito d , Nipuna Parahitiyawa e,f , Lakshman Perera Samaranayake f , Edvaldo Antonio Ribeiro Rosa a, ⁎ a Laboratory of Stomatology, Faculty of Dentistry, The Pontifical Catholic University of Paraná, Rua Imaculada Conceição 1155, Curitiba, Brazil b Formédica Pharmacy Co. Rua Coronel Dulcídio 436, Curitiba, Brazil c Department of Periodontology, The Forsyth Institute, 140 The Fenway, Boston, USA d Laboratory of Physiology and Biophysics, Faculty of Biological Sciences, The Pontifical Catholic University of Paraná, Rua Imaculada Conceição 1155, Curitiba, PR 80215-901, Brazil e Division of Microbiology, Faculty of Dental Sciences, University of Peradeniya, Peradeniya 20400, Sri Lanka f Oral Biosciences Unity, Prince Philip Dental Hospital, University of Hong Kong, 5F/34 Hospital Road, Sai Ying Pun, Hong Kong abstract article info Article history: Received 30 January 2009 Received in revised form 14 May 2009 Accepted 15 May 2009 Available online 20 May 2009 Keywords: Biofilm Candida albicans Colorimetry Phospholipase Planktonic Biofilms are aggregates of microorganisms living in multilayered structures inside polymeric matrices onto surfaces. These biofilms may subvert the physiological properties of adjacent tissues causing morphofunc- tional failure. Many studies have shown that the expression of virulence attributes is maximized when microbes form such communities. This study evaluated the differential phospholipasic activity of Candida albicans SC5314 grown in planktonic phase and in biofilm. We propose two distinct protocols for the colorimetric evaluation of phosphatidylcholine hydrolysis in neutral and acidic conditions. The results showed that both protocols are suitable for the proposed intention and that 72 h-old planktonic cultures of C. albicans SC5314 secrete higher quantities of neutral (6.42-fold) and acidic (3.85-fold) phospholipases than biofilms. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Among the virulence factors that confer pathogenic behavior to Candida albicans, the phospholipases deserve special attention. Phospholipases form a heterogeneous group of enzymes that share the ability to hydrolyze one or more ester linkages (Ghannoum, 2000). Such enzymes facilitate the penetration of true hyphae into the cells and tissues (Ibrahim et al., 1995). Different strains produce variable amounts of enzymes depending on their genetic patrimony, environmental stimuli (Mukherjee et al., 2003; Samaranayake et al., 2005), and other conditions (Samaranayake et al., 2006). The predominant extracellular phospholipases in C. albicans are phospholipase B (PLB) and phospholipase D (PLD). Noteworthy, PLB is an enzyme that can remove both sn-1 (PLB1) and sn-2 (PLB2) fatty acids from phospholipid backbones. During the depolymerizing events the fatty acid release promotes a drop in surrounding pH that may be detected by color-based pH indicators. The color shifts may be useful for relative measurement of enzymatic activity in buffered systems by colorimetry (Price, 2007). The mostly used diagnostic method for phospholipase determina- tion is based on the growth onto egg yolk agar plates (Samaranayake et al., 1984). However, this method is not suitable for investigations involving biofilms. Albeit more accurate and proper for the quantifica- tion of soluble phospholipases (Ghannoum, 2000), substrates for colorimetric assays are quite expensive (Price, 2007) and new strategies must be designed. In this study, we compared the phospholipasic activity of biofilm and planktonic cells of C. albicans SC5314, an oral isolate widely employed for biochemical, genetic, and molecular purposes. For this, we developed two protocols based on the enzymatic degradation of pharmaceutical grade phosphatidylcholine and subsequent semi-quantification by pH indicator color shifts under neutral and acidic conditions. 2. Materials and methods 2.1. Chemicals Excepting those whose origin is indicated, all chemicals used in this study were purchased from Merck KGaA (Darmstadt, Germany) Journal of Microbiological Methods 78 (2009) 171–174 ⁎ Corresponding author. Pontifícia Universidade Católica do Paraná, Centro de Ciências Biológicas e da Saúde, Laboratório de Estomatologia. Rua Imaculada Conceição 1155, Curitiba, 80215-900, Brazil. Tel.: +55 41 32711497; fax: +55 41 32711405. E-mail address: edvaldo.rosa@pucpr.br (E.A.R. Rosa). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.05.012 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth