Biolog phenotype MicroArrays

Stress responses of E. coli FAM21805 wild-type and its LHR mutants, as well as E. coli K-12 transconjugants, were assessed using the Phenotype MicroArrays (PM) 9 and 10 of the Biolog system screening for growth depending on osmolytes and pH (BIOLOG, Inc., Hayward, CA, USA). Strains were prepared and plates inoculated as follows: Strains from glycerol stocks were re-activated by two overnight incubations at 37°C on blood agar plates (Columbia agar + 5% sheep blood, bioMérieux, Geneva, Switzerland). The second plate was less than 24 h old when used for inoculation of the PM plates. Inoculating fluids (IF) were prepared by addition of 25 ml sterile water to 125 ml IF-0a and addition of 1.5 ml dye mix A and 23.5 ml water to 125 ml IF-10b. Cells were inoculated into IF-0 to a transmittance of 42 ± 2% using a sterile cotton swab and diluted (1:5) into fresh IF-0 resulting in a final transmittance of 85 ± 2%. This suspension was diluted 1:200 into IF-10b + dye mix A and used to inoculate PM plates (100 μl per well). Metabolic activity was assessed at 37°C for 72 h under standard atmosphere. PM plates, IF-0 and IF-10b were obtained from Endotell AG, Allschwil, Switzerland.