A simple medium modification for isolation, growth and enumeration of Acidithiobacillus thiooxidans (syn. Thiobacillus thiooxidans) from water samples Janeta Starosvetsky a, ⁎, Udy Zukerman b , Robert H. Armon a a Faculty of Civil & Environmental Engineering, Department of Environmental, Water and Agricultural Engineering, Technion, 32000, Israel b Mekorot Water Company Ltd., Israel abstract article info Article history: Received 14 November 2012 Accepted 14 November 2012 Available online 23 November 2012 Keywords: Acidithiobacillus thiooxidans Thiobacillus agar Bromocresol green Phenol red Spread Plate method MPN technique High concentrations of H 2 S in groundwater are commonly removed using Biological Trickling Filter (BTF) that contains high numbers of biofilm immobilized sulfur oxidizing bacteria (mainly Thiobacillus thiooxidans). BTF performance requires continuous monitoring of these bacteria at several sampling points. The Most Prob- able Number (MPN) technique is at the moment the method of choice to enumerate viable T. thiooxidan cells under the above conditions. However, this method is extremely time-consuming (7–10 days) and not always suitable for environmental monitoring. In the present study, Thiobacillus agar recommended for isolation and cultivation of Thiobacillus species by Spread plate method was modified by addition of bromocresol green (BCG) in order obtain a clear-cut resolution of the growing colonies resulting in similar or higher numbers compared to other methods. Visual emergence of bacterial colonies on the 3rd and 4th days, from the initial plating, was associated with sulfuric acid production, resulting in an unambiguous color change from blue to yellow, around each colony. This study revealed that BCG modified Thiobacillus agar is substantially time saving and much easier to infer compared to MPN technique. Published by Elsevier B.V. 1. Introduction As a result of potable water scarcity in southern part of Israel, groundwater containing high concentrations of H 2 S (~20 ppm) is used as a water supply source for drinking water. H 2 S removal pro- cess combines an air degasifying tower releasing around 600 ppm H 2 S/m 2 conveyed to a cooling tower. The processed air is conveyed to a BTF through several polyurethan foam layers as media for sulfate oxidizing bacteria (SOB) (mainly Acidithiobacillus thiooxidans syn. Thiobacillus thiooxidans). H 2 S emissions are controlled and regulated by the ministry of environmental protection and according to current regulation for ambient air a daily average of b 5 ppm/m 2 is demanded. As much as 98% of the produced H 2 S is removed from the air stream by this method. Optimization of the BTF performance involves monitoring of SOB at several sampling points. The main goal of the present study was to develop the most appropriate growth medium to enumerate viable T. thiooxidan cells, in order to support rapid and effective operational data of the BTF. Thiobacilliaceae, effectively oxidize reduced forms of sulfur (H 2 S, metal sulfides, thiosulates and elemental sulfur — S 0 ) in order to obtain energy and fix atmospheric CO 2 for growth requirements (Ehrlich, 1996). Typical members of this group are T. thiooxidans, Thiobacillus albertis, Thiobacillus ferrooxidans (all strict autotrophs) that oxidize reduced forms of sulfur to sulfuric acid (termi- nologically acidophilic) to drop pH close to 0 value. In order to estimate Thiobacillus species, the MPN technique with liquid media is commonly recommended and widely applied (Andrew et al., 2005; Southam and Beveridge, 1992; Shinabe et al., 1995, 2000; Knickerbocker et al., 2000). It is still the most precise method allowing detection of viable cells with the accuracy of 1 colony forming units (CFU)/100 ml is MPN. Regrettably, this technique is time consuming both for medium preparation (sterilization by intermittent steam for each of the three consecutive days) and growth detection (measurement of pH or optical density of each tube) therefore not suitable for extensive monitoring use. Occasionally monitoring of Thiobacillus cells is performed by mea- suring optical density of growth culture or bacterial suspension that allows easy and adequately fast tracking of bacterial growth dynamic or biofilm development (Crescenzi et al., 2006; Kurosawa et al., 1991; Lee et al., 2005, 2006). However the precision of the method does not extend over one to two orders of magnitude. Occasionally, hemocy- tometer — a device originally designed for blood cell count that is used for quantification of T. thiooxidan cells (Chen et al., 2002). Nevertheless this method has two shortcomings: 1) it is impossible to differentiate between viable and dead cells and 2) at very low initial concentration the count is highly inaccurate. The use of Spread Plate method should be the most appropriate technique for CFU detection, but it requires a solid nutrient medium (with agar). Culture of acidophilic microorgan- isms was extensively performed in liquid media (Johnson, 1995), but Journal of Microbiological Methods 92 (2013) 178–182 ⁎ Corresponding author. E-mail address: starjean@tx.technion.ac.il (J. Starosvetsky). 0167-7012/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.mimet.2012.11.009 Contents lists available at SciVerse ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth