Spectrofluorimetric method for the estimation of total lipids in Eremothecium ashbyii fungal filaments using Nile blue and avoiding interference of autofluorescent riboflavin S. Vijayalakshmi, T.N. Karthika, A.K. Mishra, T.S. Chandra * Department of Chemistry, Indian Institute of Technology, Madras, Chennai 600036, India Received 3 July 2002; received in revised form 30 January 2003; accepted 1 April 2003 Abstract A rapid, simple and sensitive spectrofluorimetric technique was developed for monitoring total lipids in hyphae of the riboflavin-overproducing fungus Eremothecium ashbyii using the fluorescent probe Nile blue in an aqueous system, avoiding the interference due to autofluorescent riboflavin. The existing methodologies for lipid estimation are tedious, requiring large biomass, solvent extraction and gravimetry. E. ashbyii is a hemiascomycete fungus which accumulates lipids in its mycelia prior to flavinogenesis. This study defines the conditions (wavelength selection and sensitivity) for the spectrofluorimetric quan- tification of lipids in situ in the macerated mycelia of this fungus in the presence of intracellular autofluorescent riboflavin without the need to extract the lipids from the mycelia. The fluorescent intensity was linear with the lipid concentration of the mycelia (by gravimetry) under three different growth conditions using glucose, olive oil and sunflower oil as carbon sources. This spectrofluorimetic method of lipid estimation can be applied to other fungi and microorganisms. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Nile blue; Phenoxazine; In situ lipid estimation; Fluorescent probe; Spectrofluorimetry; Eremothecium ashbyii; Mycelia; Riboflavin; Gravimetry 1. Introduction Eremothecium ashbyii is a filamentous hemiasco- mycete fungus overproducing riboflavin (vitamin B 2 ) and is similar to the flavinogenic Ashbya gossypii. Riboflavin has various applications in pharmaceutical, animal feed and food industry (Stahmann et al., 2000). Both E. ashbyii and A. gossypii accumulate lipids prior to riboflavin overproduction which were detected in the filaments by Nile blue and Nile red staining and fluorescent microscopy (Stahmann et al., 1994; Vijayalakshmi and Chandra, 1999, 2000). The lipids decrease when riboflavin production increases. In order to understand the biochemical relationship between lipid metabolism and riboflavin overproduc- tion in these fungi, it is necessary to follow the time course of intracellular total lipid accumulation in their filaments under different growth conditions using glucose, olive oil and sunflower oil as carbon sources. Therefore, a very rapid and sensitive method is needed for estimation of total lipid in such filamentous fungi. The conventional method of lipid estimation by gravimetry is tedious and involves solvent extraction 0167-7012/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(03)00137-4 * Corresponding author. Tel.: +91-44-22578245; fax: +91-44- 22578242. E-mail addresses: chandra@iitm.ac.in, tschandra@lycos.com (T.S. Chandra). www.elsevier.com/locate/jmicmeth Journal of Microbiological Methods 55 (2003) 99 – 103