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%).