Strains were routinely
grown in Luria Bertani (LB) broth under shaking conditions at 37°C. To analyze the development of biofilm-like structures, bacterial strains were grown in the previously described ASM+ . To attain consistency from batch to batch of medium ASM+ was made in a quantity sufficient for each planned set of experiments, a stringent method of preparing the medium was used. The components were added into sterile water in exact selleckchem order with vigorous vortexing for 10–30 seconds after each addition: mucin (Sigma-Aldrich, St. Louis, MO), 0.5% (w/v); unsheared salmon sperm DNA (Sigma-Aldrich), 0.4% (w/v); NaCl, 0.5% (w/v); KCl, 0.2% (w/v); casamino acids (Amresco, Solon, OH), 0.5% (w/v); egg yolk emulsion (source of lecithin; sterile; Remel, Lenexa, KS), 0.25% (v/v); diethylene triamine pentaacetic acid (1 mg/ml stock in 1 M NaOH; sterile; Sigma), 0.0005% (w/v). Finally, the pH was adjusted to 6.8. Antibiotics were then added to maintain sterility and for maintenance of plasmids: 300 μg carbenicillin/ml AZD5363 cell line and/or 50 μg tetracycline/ml for P. aeruginosa; 10 μg erythromycin/ml for S. aureus. The completed medium was then vortexed
for 2 minutes and again prior to pipetting. To induce biofilm formation on the substrate surface, we used trypticase soy broth dialysate (TSBDC) to which glycerol (1% v/v) and monosodium glutamate (0.5 M) were added . Table 6 Strains and plasmids used in this study Strain Description Source
Plasmids pCM11 Plasmid stable in S. aureus that constitutively expresses green fluorescent protein (GFP); Emr Alexander Horswill, personal communication pMRP9-1 pUCP-18 cloning vector carrying a GFP cassette; Cbr  pMP7605 pBBR1MCS-5 cloning vector carrying the mCherry gene under the control Histamine H2 receptor of the tac promoter; Cbr  Pseudomonas aeruginosa PA103 Human isolate  PAK Prototroph; human isolate  PAO1 Prototroph; human isolate  PAO-R1 ΔlasR derivative of PAO1; Tcr  CH5424802 PAO-JP1 ΔlasI derivative of PAO1; Tcr  PDO111 rhlR::Tn501 derivative of PAO1; Hgr  PDO100 ΔrhlI::Tn501 derivative of PAO1; Hgr  PW2798::pqsA-lacZ pqsA-H05::ISlacZ/hah derivative of PAO1; Tcr ; University of Washington Genome Center CI-4 Human isolate from chronic lower respiratory infection; ΔlasR, ΔrhlR  Staphylococcus aureus AH133 RN4220 carrying pCM11; Emr ; Alexander Horswill, personal communication Em, erythromycin; r, resistant; Cb, carbenicillin; Hg, mercury; Tc, tetracycline. To allow visualization of the bacteria, all P. aeruginosa strains were transformed by electroporation  with pMRP9-1 from which the gene for green fluorescent protein (GFP) is constitutively expressed . To visualize PAO1 grown together with AH133, PAO1 was transformed with pMP7605 in which the mCherry gene that codes for red fluorescent protein (RFP) is expressed from the tac promoter . The S.