A selective medium for recovery and enumeration of endolithic bacteria Kaushik Bhattacharjee, S.R. Joshi ⁎ Microbiology Laboratory, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong 793022, India abstract article info Article history: Received 9 May 2016 Received in revised form 29 July 2016 Accepted 29 July 2016 Available online 30 July 2016 The study of lithic microbial communities, inhabiting rock substrates has been gathering momentum due to a growing attention of their wide importance as model systems in ecological studies and for their community structure. It is generally accepted that the success of cultivation-based technique is primarily based on suitable culture medium for isolation. The media available for enumeration and recovery of endolithic bacteria are mainly specific to particular type of rock which may not be suitable to isolate endolithic bacterial community from di- verse lithobiontic niches. In this study, a new unoptimized medium was formulated, designated LM10 (unoptimized) for enumeration and recovery of endolithic bacteria by addition and/or omission of media com- ponents to the basal medium R2G, which was selected after experimental evaluation of five different existing media. The endolithic bacterial count in LM10 medium (unoptimized) was significantly higher than the R2G me- dium (t = -12.57, p b 0.0001). The culture and nutritional parameters associated with unoptimized LM10 me- dium were optimized using statistical approach to maximize the recovery and enumeration of endolithic bacteria. The first phase of the study comprised of a Plackett-Burman (PB) design experiment conducted to screen thirteen medium components and two culture parameters as variables with effect on bacterial enumera- tion and recovery. Out of these, Yeast extract, Casein hydrolysate, Glucose, Starch and Sodium thiosulphate were found to be significantly affecting the bacterial count (p b 0.05) based on PB design. On keeping rest of the media components and culture conditions at fixed value as per the PB design analyses (p N 0.05 and coefficients), further optimization was carried out for significant factors using Box-Behnken design (BBD) of response surface meth- odology (RSM). Optimized media components obtained by BBD were Yeast extract, Casein hydrolysate, Glucose and Starch in 0.05 g/l each and Sodium thiosulphate in 0.047 g/l concentrations. The composition of optimized LM10 medium formulated (per litre) is 0.05 g Yeast extract, 0.05 g Casein hydrolysate, 0.05 g Glucose, 0.05 g Starch, 0.01 g K 2 HPO 4 , 0.02 g Sodium pyruvate, 0.2 g MgSO 4 , 0.001 g FeSO 4 ·7H 2 O, 0.285 g NH 4 Cl, 0.039 g CaCl 2 ·2H 2 O, 0.047 g Na 2 S 2 O 3 ·5H 2 O, 0.002 g NaHCO 3 and 11 g Gellan gum (pH = 7.4). Validation of optimized LM10 medium using nine different rock samples from Meghalaya clearly indicated that optimized LM10 medium was better suited for higher recovery and enumeration of endolithic bacteria under both aerobic and anaerobic conditions. © 2016 Elsevier B.V. All rights reserved. Keywords: Endolithic bacteria Colony forming unit Plackett-Burman Box-Behnken 1. Introduction The study of lithic microbial communities, inhabiting rock substrates has been gathering momentum due to a growing attention of their wide importance as model systems in ecological studies and for their commu- nity structure (Warren-Rhodes et al., 2007). They are also relevant to as- trobiologists due to their potential as analogs for possible life on Mars (da la Torre et al., 2003). Lithic microbial communities also play a vital role in microbe-mineral interactions (Blackhurst et al., 2004). The rock inhabiting microorganisms were first observed and described about 100 years ago (Horath and Bachofen, 2009). Subsequently several stud- ies were reported of the presence of lithic microorganisms in sandstone (Bell, 1993), limestone (Gerrath et al., 2000), gypsum (Hughes and Lawley, 2003; Stivaletta et al., 2010; Rhind et al., 2014), dolomite (Sigler et al., 2003; Horath and Bachofen, 2009), carbonates (Hoppert et al., 2004), ignimbrite (Wierzchosa et al., 2013) and granite (Li et al., 2013). Among the different lithobiontic habitats, the microorganisms which are present in interior of rock are termed as endoliths (Olsson- Francis et al., 2010). They are usually the dominant form of life in ex- treme environment and bacteria represent the main component of that niche (McNamara et al., 2006; Olsson-Francis et al., 2010). It is generally accepted that the success of cultivation-based tech- nique is primarily based on suitable culture medium to isolate them. It is an axiom of microbiology that most culture media are selective for subsets of the total bacterial community; therefore, advancement of a variety of culture media is required to maximize the recovery of diverse microorganisms from different niches (Stevens, 1995). To isolate the endolithic bacteria, researchers have experimented various media Journal of Microbiological Methods 129 (2016) 44–54 ⁎ Corresponding author at: Department of Biotechnology & Bioinformatics, North- Eastern Hill University, Mawlai, Shillong 793022, Meghalaya, India. E-mail address: srjoshi2006@yahoo.co.in (S.R. Joshi). http://dx.doi.org/10.1016/j.mimet.2016.07.026 0167-7012/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth