The evolution experiment
Thirty low-temperature (10°C) microcosms were set up as edge-habitat populations, six replicates for each of the following treatments: bacterial populations (B), bacterial populations with immigration (B+IB), bacteria/phage populations (BP), bacteria/phage populations with immigration of phages (BP+IP) and bacteria/phage with immigration of bacteria and phages (BP+IBP). Immigrants to B+IB were from six source bacterial microcosms grown at 28°C (SB); and those to BP+IP and BP+IBP were from six source bacteria/phage microcosms grown at 28°C (SBP). Therefore, we set up a total of 42 microcosms.
Each microcosm was initially inoculated with 60 µL of bacterial culture that had been acclimated at an appropriate temperature, and about 103 phage particles for the microcosm designed to include phages. The bacterial acclimation procedure involved transferring 60 µL of cultures reconditioned overnight at 28°C to fresh medium and grown at relevant temperatures (10 or 28°C) for 48 h. Cultures were then propagated for 20 serial transfers (one transfer every 48 h). At each transfer, 60 µL of culture from each microcosm was transferred to fresh media. Microcosms with immigration received a further 3 µL of proper cultures from the source microcosms. To obtain phage populations from the source bacteria/phage microcosms, samples of the cultures were mixed with chloroform (10:1 volume ratio), vortexed and centrifuged at 13000 g, with phages remaining in the supernatant (Buckling & Rainey 2002). Optical density at 600 nm wavelength (mOD600) of each culture (200 µL of sample) was measured using a plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) before each transfer, as a proxy for the density of P. fluorescens . Samples of cultures at transfer 8 and 20 were frozen at -80°C with glycerol (final glycerol concentration 25%).