Serum resistance was analyzed following the procedure described by (Podschun et al., 2016 (link)). Briefly, 25 µL of inoculum of 2.5 x 106 colony forming units (CFU)/mL of a mid-log phase culture were mixed with 75 µL of normal human serum obtained from healthy volunteers in 96-well polystyrene, round-bottomed microtiter plates (Greiner bio-one). The mixture was incubated at 37°C for 1, 2, and 3 h. After incubation, the cell count was determined using 10-fold serial dilutions and conventional plating in LB agar (Miller’s LB AGAR, Condalab). The test was graded as shown in Table 2
Miller s lb agar
Manufactured by Condalab
Sourced in Spain
Miller's LB AGAR is a culture medium used for the growth and maintenance of bacterial cultures. It provides the necessary nutrients and solidifying agent for the cultivation of a wide range of bacterial species. The medium is formulated to support the general growth requirements of microorganisms.
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Lab products found in correlation
3 protocols using miller s lb agar
1
Serum Resistance Assay Protocol
2
Preparing Electrocompetent E. coli Cells
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The E. coli ATCC 25922 reference strain, used as the wt strain in this study, was grown for 24 h at 37 °C in Luria Bertani (LB) agar (Miller’s LB AGAR, Condalab, Madrid, Spain). Electrocompetent cells were made after the overnight culture of a single colony in LB broth (Miller’s LB Broth, Condalab) at 37 °C with shaking at 180 rpm. One millilitre of the overnight culture was used to inoculate 100 mL prewarmed (37 °C) LB broth and incubated in aerobic conditions at 37 °C under shaking at 180 rpm. When bacteria reached the mid-log phase (optical density (OD) at 600 nm = 0.6 ± 0.2), the cells were incubated on ice for 20 min. Bacterial cells were pelleted at 7000× g for 10 min, and washed three times: first with 50 mL of ice-cold water, second with 25 mL of ice-cold water, and lastly with 2.5 mL of ice-cold 10% glycerol. Electrocompetent cells were aliquoted and stored at −80 °C.
3
Enumerating Bacterial Biofilm Cells
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The number of cultivable cells from disrupted biofilms was obtained by colony counting. In brief, after the aspiration of supernatants, the wells were rinsed once with 1x PBS to remove non-attached cells. The plates were then sonicated at 40 kHz for 1 min, following the protocol described by Iñiguez-Moreno et al. (Iñiguez-Moreno et al. 2017) . Later, the biofilms were scraped with a cell scraper (VWR international) and serially diluted for colony counting. In monomicrobial biofilms, K. pneumoniae and E. faecalis were plated on Luria Bertani agar (Miller's LB AGAR, Condalab) and BD Columbia agar with 5 % Sheep Blood (Becton Dickinson), respectively. In polymicrobial biofilms, aliquots were plated both on selective media MacConkey II agar (Becton Dickinson) for selection of K. pneumoniae cells and on Enterococcosel agar (Becton Dickinson) for E. faecalis. Agar plates were incubated at 37 °C for 18 -24 h. Assays were performed in triplicate.
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