Luria bertani medium lb

An E. coli strain was isolated from blood of wild type C57Bl76 mice after 24 h of sepsis induced by cecal ligation puncture (CLP) procedure. This strain was the most frequent Gram-negative bacteria isolated from blood of this septic mouse. The E. coli strain was stored at -80 °C in Luria-Bertani medium (LB; Sigma) containing 10% glycerol. To prepare the inoculum for sepsis induction, 10 µl of E. coli stock was cultured in LB medium at 37 °C to exponential growth phase and washed twice with cold phosphate-buffered saline (PBS). The absorbance at 600 nm was measured in a spectrophotometer to estimate the number of bacteria in the culture. The bacterial density was adjusted to 1x10⁹ bacteria/mL. Sepsis was induced in WT, GzmA^-/- and GzmK^-/- mice by intraperitoneal injection of 2×10⁸ bacteria in 200 µl of PBS.

The most frequent Gram-negative bacteria, an E. coli strain, was isolated from blood of WT mice after 24 h of sepsis induced by CLP. Bacteria was stored at -80 °C in Luria-Bertani medium (LB; sigma) with 10% glycerol. The inoculum for sepsis induction was prepared from 10 μL of E. coli cultured in LB medium at 37 °C to exponential growth phase and washed twice with phosphate-buffered saline (PBS). Absorbance at 600 nm was measured in a spectrophotometer to estimate the number of bacteria in the culture. The density of bacteria was adjusted to 1x10⁹ bacteria/mL. Sepsis was induced in WT, GZMA^-/-, and GZMA (^+/+, ^-/-) littermate mice by i.p. of 2x10⁸ bacteria in 200 μL of PBS.
Mouse survival was monitored for 5 days. Mice were observed three times a day and if there were signs of respiratory distress, pain when touching, vocalisations, hunched posture or inability of a supine animal to stand, the human endpoint was applied.

Commercially available strains Staphylococcus aureus (S. aureus, pathogen, ATCC 43300), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans, pathogen, ATCC 33384) and Escherichia coli (E. coli, non-pathogen, ATCC BAA-1427) were purchased from the American Type Culture Collection (ATCC, MA, USA) and cultivated following the manufacturer’s instructions. A clinical isolate of Staphylococcus epidermidis (S. epidermidis, pathogen) was collected at the Clinical Microbiology Unit at the Novara Maggiore Hospital (Novara, Italy). The clinical isolate was obtained after patient’s informed consent in full accordance with the Declaration of Helsinki. Clinical strain was cultivated in Luria-Bertani medium (LB, Sigma-Aldrich, Milan, Italy) at 37 °C. For experiments, a single colony from each strain was collected and inoculated in 9 mL of LB broth at 37 °C overnight (18 h). After incubation, a new fresh LB tube diluted 1:10 was prepared and incubated at 37 °C for 3 h to achieve the logarithmic growth phase. Finally, broth cultures were diluted in LB broth until the optical density was 0.001 at 600 nm, corresponding to a final concentration of 1 × 10⁵ cells/mL.