Pseudomonas isolation agar

Escherichia coli BCRC 11,634 and Staphylococcus aureus BCRC 10,451 were purchased from the Biosources Collection and Research Center (Hsinchu, Taiwan). EDTA, sodium bicarbonate (NaHCO₃), and nisin (10⁶ IU) were obtained from Sigma Chemical Co. (Gillingham, UK). Chitosan powder was obtained from Applied Chemical Co., Ltd. (Kaohsiung, Taiwan). Bacto agar, nutrient broth (NB), nutrient agar, plate count agar (PCA), Pseudomonas isolation agar, starch ampicillin agar (SAI), thiosulphate-citrate-bile salts-sucrose, tryptic soy broth, and violet red bile agar were supplied by Becton Dickinson (Sparks, MD, USA). Polylactic acid (PLA, manufactured by Natureworks@ (4032D), Minnetonka, MA, USA) had a weight-average molecular weight (Mw) of 1.96 × 10⁴ Da, as determined by gel permeation chromatography.

P. aeruginosa strain DWW2 with a mucoid phenotype and from CF sputum (29) was routinely grown on LB agar (Invitrogen, United Kingdom) at 37°C. AGS (Streptococcus anginosus) strain 3a from CF sputum during exacerbation (33) was maintained at 37°C on blood agar (Blood Agar Base no.2, Oxoid, United Kingdom) containing 6% (vol/vol) defibrinated horse blood in an anoxic atmosphere (80% nitrogen, 10% hydrogen and 10% carbon dioxide). Liquid cultures for inoculating filters for biofilm growth were in Todd Hewitt Broth (Oxoid, United Kingdom) supplemented with 0.5% Yeast Extract (Becton Dickinson & Co., USA) (THY) incubated under normoxic conditions (in air) at 37°C with agitation (strain DWW2) or incubated under anoxic conditions (strain 3a).
For selection of DWW2 from co-cultures bacterial mixtures were plated onto Pseudomonas Isolation Agar (PIA) (Difco Laboratories, Becton Dickinson and Co., USA) and incubated under normoxic conditions at 37°C. For selection of 3a from co-cultures bacterial mixtures were plated onto a semi-selective agar (NAS) containing 1.1 gm per litre sulfamethazine and 37.5 mg / L nalidixic acid [33 (link)].

Strains and plasmids used for this study are listed in Tables S1 and S3, respectively. Strains were grown at 37 °C in LB medium or in M63 medium supplemented with 1 mM MgCl₂, 0.5% casa-aminoacids, 0.2% glucose. Recombinant plasmids were introduced into P. aeruginosa genome through conjugative transfer using pRK2013. Transconjugants were selected on Pseudomonas isolation agar (PIA;Difco Laboratories) supplemented with appropriate antibiotics. Kanamycin (Km) at 50 µg/ml and tetracycline (Tc) at 150 µg/ml supplemented with streptomycin^{47 (link)} at 2 mg/ml were used for E. coli and P. aeruginosa strains, respectively.

Microbes from water samples were isolated according to the dilution plate method (Warcup, 1960 ), using: Potato Dextrose Agar (PDA; Fluka Analytical, Sigma-Aldrich, St. Louis, MO, United States) and Corn Meal Agar (CMA; Fluka Analytical, Sigma-Aldrich, St. Louis, MO, United States) for fungi, and Pseudomonas Isolation Agar (PIA; Difco Laboratories, Sparks, MD, United States) for bacteria, following the manufacturer’s instructions. We chose these media based on literature reports on the transient aquatic fungal diversity (Velez et al., 2016 (link)), and the cultivable prokaryotic community(Ponce-Soto et al., 2015 (link)).
Plates were prepared using 100 μl of each water sample at 10^-1–10^-6 dilutions in test tubes with sterilized distilled water. Three replicates per dilution were plated, and incubated for 2 (bacteria) and 7 (fungi) days at 25°C with a 12 h photoperiod in case of fungi. The plates were examined daily, and each colony that developed was subsequently transferred to PDA for fungi and Luria Bertani agar (LB; Lennox L Agar, Invitrogen, Carlsbad, CA, United States) for bacteria.

Cells were seeded on a six-well plate and then infected with PA. After 1 h infection, cells in one well were treated with gentamicin (at the concentration of 300 µg/ml for 30 min, Sigma) to erase the extracellular bacteria, washed with ice-old PBS, and then lysed with 0.1% Triton-X. Cells in the other duplicate well were incubated for another 1 h and then lysed according to the same procedure. A series of 10-fold dilutions were plated on Pseudomonas isolation agar (BD Difco Laboratories) in triplicate. Intracellular bacterial load was reported as CFU per 10⁶ cells ± SEM.

Corneas from siSTING versus siNC-treated BALB/c mice (at 5 days p.i.) or from cGAMP versus control-treated C57BL/6 mice (at 5 days p.i.) were pooled (n = 5/group/time). The number of viable bacteria was calculated as described before (30 (link)). Briefly, individual corneas were homogenized, diluted in a series, and seeded on Pseudomonas isolation agar (BD Difco Laboratories) in triplicate. Results are reported as log10 CFU per cornea ± SEM.

Samples were homogenized in PBS (agitation speed 5500 min^-1, 3x30 s) using the Precellys Lysing Kit (Bertin Industries, Île-de-France, France). The resulting solution was diluted 1:5 and 1:100 with PBS. 50 µl of each sample were transferred on columbia agar + 5% sheep blood (COS), columbia agar CNA + 5% sheep blood agar plates (Biomerieux, Marcy l’Etoile, France) and pseudomonas isolation agar (BD, Heidelberg, Germany). After 48 hours of incubation at 37°C, the number of colony forming units (CFU) was determined and multiplied by the respective dilution factors. Species were identified using MALDY-TOF-mass spectrometer (Bruker Daltonik MALDI Biotyper, Bruker, Billerica, MA, USA).