Bacterial colonies were isolated on Difco Pseudomonas Isolation Agar (BD, Sparks, MD). Genomic DNA was extracted from overnight cultures using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). Genomic DNA (500 ng) was mechanically fragmented for 40 s using a Covaris M220 (Covaris, Woburn, MA) with default settings. Fragmented DNA was transferred to a polymerase chain reaction tube and library synthesis was performed with the Kapa Hyperprep kit (Kapa Biosystems, Wilmington, MA) according to the manufacturer’s instructions. TruSeq HT adapters (Illumina, SanDiego, CA) were used to barcode the libraries, which were each sequenced in 1/48 of an Illumina MiSeq 300-bp paired-end run at the Plateforme d’Analyses Génomiques of the Institut de Biologie Intégrative et des Systèmes (Université Laval, Québec, Canada). Each data set was assembled de novo with the A5 pipeline version A5-miseq 20140521. Raw reads, assembly data and metadata were uploaded on IPCD (https://ipcd.ibis.ulaval.ca ).
Difco pseudomonas isolation agar
Manufactured by BD
Sourced in United States
Difco Pseudomonas Isolation Agar is a culture medium used for the isolation and identification of Pseudomonas species from a variety of samples. It contains selective agents that inhibit the growth of non-Pseudomonas bacteria, allowing for the isolation of Pseudomonas colonies.
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Lab products found in correlation
4 protocols using difco pseudomonas isolation agar
1
Bacterial Genome Sequencing and Assembly
2
Quantifying Bacterial Burden in Cornea Infection
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Viable bacteria were quantitated in the cornea of KEI 1025- and MDR9-infected mice at 3 and 5 days p.i. (n = 5/group/time/experiment). Each cornea was homogenized in 1 ml of sterile saline (0.85% NaCl, pH 7.4) containing 0.25% BSA. A 100-μl aliquot of the corneal homogenate was serially diluted (1:10) in sterile saline containing 0.25% BSA, and selected dilutions were plated in triplicate on selective culture medium (Difco Pseudomonas Isolation Agar, BD Biosciences, Inc., Franklin Lakes, NJ, USA). Plates were incubated overnight at 37°C and viable bacteria manually counted. Results are reported as log10 CFU/cornea ± SEM (Hobden et al., 1997 (link); Ekanayaka et al., 2016 (link)).
3
Murine Model of Pseudomonas Infection
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Whole corneas from the murine model of P. aeruginosa PAO1 infection were placed in 1 ml of sterile saline (0.85% NaCl, pH 7.4) containing 0.25% bovine serum albumin (BSA) and homogenized. Serial 10‐fold dilutions were prepared and plated in triplicate on a selective culture medium (Difco Pseudomonas Isolation Agar; BD Biosciences, Inc.). The plates were then incubated at 37°C for 18–24 h, and the CFU number was determined by direct counting.
4
Quantifying Bacterial Load in Corneas
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Mice were sacrificed at 3 and 5 days PI and KEI 1025 or ATCC 19660–infected corneas from GLY-, CBX- or PBS-treated (or GLY+/– rHMGB1) B6 mice were harvested (n = 5/group/time). Each cornea was homogenized in 1 mL of sterile saline (0.85% NaCl, pH 7.4) containing 0.25% BSA. A 100 μL of the corneal homogenate was serially diluted (1:10) in sterile saline containing 0.25% BSA. Selected dilutions were plated in triplicate on selective culture medium (Difco Pseudomonas Isolation Agar, BD Biosciences, Inc., Franklin Lakes, NJ, USA). Plates were incubated overnight at 37°C and viable bacteria manually counted. Results are reported as log10 CFU/cornea ± SEM.
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