Comparison of methods to detect biosurfactant production by diverse microorganisms Noha H. Youssef a , Kathleen E. Duncan a , David P. Nagle a , Kristen N. Savage a , Roy M. Knapp b , Michael J. McInerney a, * a Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval Norman, OK 73019, USA b Department of Petroleum Engineering, University of Oklahoma, Norman, OK, USA Received 22 September 2003; received in revised form 3 November 2003; accepted 3 November 2003 Abstract Three methods to detect biosurfactant production, drop collapse, oil spreading, and blood agar lysis, were compared for their ease of use and reliability in relation to the ability of the cultures to reduce surface tension. The three methods were used to test for biosurfactant production in 205 environmental strains with different phylogenetic affiliations. Surface tension of select strains that gave conflicting results with the above three methods was also measured. Sixteen percent of the strains that lysed blood agar tested negative for biosurfactant production with the other two methods and had little reduction in surface tension (values above 60 mN/m). Thirty eight percent of the strains that did not lyse blood agar tested positive for biosurfactant production with the other two methods and had surface tension values as low as 35 mN/m. There was a very strong, negative, linear correlation between the diameter of clear zone obtained with the oil spreading technique and surface tension (r s = À 0.959) and a weaker negative correlation between drop collapse method and surface tension (r s = À 0.82), suggesting that the oil spreading technique better predicted biosurfactant production than the drop collapse method. The use of the drop collapse method as a primary method to detect biosurfactant producers, followed by the determination of the biosurfactant concentration using the oil spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant production. The large number of false negatives and positives obtained with the blood agar lysis method and its poor correlation to surface tension (r s = À 0.15) demonstrated that it is not a reliable method to detect biosurfactant production. D 2003 Elsevier B.V. All rights reserved. Keywords: Biosurfactant production; Screening; Oil spreading; Drop collapse; Blood agar lysis 1. Introduction Biosurfactants are a diverse group of surface-active chemical compounds that are produced by a wide variety of microorganisms (Banat, 1995). The types of biosurfactants include lipopeptides synthesized by many bacilli and other species, glycolipids synthe- sized by Pseudomonas species and Candida species, phospholipids synthesized by Thiobacillus thiooxi- dans, polysaccharide – lipid complexes synthesized by Acinetobacter species, or even the microbial cell surface itself (Van Dyke et al., 1991; Bodour and Miller-Maier, 2002). Having both polar and nonpolar domains, biosurfactants are able to partition at water – 0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2003.11.001 * Corresponding author. Tel.: +1-405-325-6050; fax: +1-405- 325-7619. E-mail address: mcinerney@ou.edu (M.J. McInerney). www.elsevier.com/locate/jmicmeth Journal of Microbiological Methods 56 (2004) 339 – 347