Lab lemco powder

F. johnsoniae (DSM 2064^T) and P. saltans (DSM 12145^T) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig, Germany. F. johnsoniae was grown in liquid CYE medium (g per liter of distilled water): Casitone, 10.0; yeast extract, 5.0; MgSO₄, 0.1; Tris buffer, 1.2; pH 7.2; 25°C. P. saltans was grown in DSMZ Medium 948 (g per liter of distilled water): Lab-Lemco powder (Oxoid), 1.0; yeast extract, 2.0; peptone, 5.0; NaCl, 5.0; pH 7.2; 25°C. Additional P. saltans cultures were grown in triplicate at 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9 in temperature controlled rooms to determine changes in IPL fractional abundance due to different environmental conditions. The temperatures and pH levels were chosen based on temperature growth range reported by Liolios et al. (2011 (link)) and initial growth experiments, in which P. saltans cultures did not readily grow at temperatures below 15°C or below pH 7. The pH was adjusted by addition of HCl or NaOH solutions to achieve the desired values. Biomass from 250 ml cultures was collected by centrifugation at the stationary growth phase and freeze dried for lipid extraction and further analysis.

The following wound dressings were selected for this study: alginate (Suprasorb^® A, Lohmann & Rauscher GmbH & Co. KG, Germany) and alginate + ionic-Ag (Suprasorb^® A + Ag, Lohmann & Rauscher GmbH & Co. KG, Germany), alginate + nano-Ag (Acticoat^◊ Absorbent, Smith & Nephew GmbH, Germany), sodium carboxymethylcellulose (CMC) (AQUACEL^®, ConvaTec GmbH, Germany) CMC with Ag⁺ (AQUACEL^® Ag, ConvaTec GmbH, Germany) as well as polyurethane (PU)-foam with TLC (UrgoCell^®, URGO GmbH, Germany) and PU-foam with TLC/Ag⁺ (UrgoCell^® silver, URGO GmbH, Germany). Active ingredients and wound dressing basic materials are specified in Table 2 according to the manufacturers’ descriptions.
Staphylococcus aureus ATCC 6538 and P. aeruginosa ATCC 27853 were obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany). For cultivation of bacteria, special peptone and “lab-lemco” powder for preparation of caso-bouillon and bacteriological agar were purchased from Oxoid (UK). Columbia agar plates with 5 % sheep blood and MH2 agar plates were acquired from Biomeriéux (France).
0.9 % NaCl solution was purchased from Fresenius Kabi Deutschland GmbH (Germany). 1 N HCl, 1 N NaOH, and Tween 20 were obtained from Carl Roth GmbH (Germany).

To determine the biological activity of substances 1–5 possessing anti-tuberculosis properties in the M. smegmatis mc²155 test system, the paper disk method was used. The technique involved determining the size of the zone of inhibition of the growth of the strain seeded as a lawn on an agar medium, around paper disks containing the compound in various concentrations. The bacteria washed off Petri dishes with tryptone soya agar M-290 medium (Himedia) were grown overnight in Lemco-TW liquid medium (Lab Lemco' Powder 5 g L⁻¹ (Oxoid), peptone special 5 g L⁻¹ (Oxoid), NaCl 5 g L⁻¹, Tween-80) at +37 °C until the average logarithmic growth phase at optical density OD600 = 1.5, then mixed with molten agar medium M-290 in a ratio of 1 : 9 : 10 (culture : Lemco-TW : M-290) and the resulting mixture was poured as a top layer onto Petri dishes, 5 ml per dish, with 20 ml already solidified M-290 agar medium. After the agar in the top layer solidified, paper disks soaked with a solution of the test substance were placed on the plate surface. The culture was incubated for 24 hours at +37 °C. The diameter of the zone of inhibition of M. smegmatis mc²155 growth around the paper disk impregnated with the compound was determined. The MIC (minimum inhibiting concentration) was taken as the concentration of the compound where the zone of growth inhibition was the smallest.

Four different growth media were tested. The first was standard “MRS media” which was used as a positive control. This contained 10 g/L proteose peptone no. 3 (Oxoid, Scoresby, Victoria, Australia), 10 g/L lab-lemco powder (Oxoid), 5 g/L yeast extract (Sigma Aldrich), 20 g/L glucose (Merck, Bayswater, Victoria, Australia), 1 mL/L Tween 80 (The Melbourne Food Depot, Brunswick, Victoria, Australia), 2 g/L ammonium citrate (Sigma Aldrich), 5 g/L sodium acetate (Merck, Bayswater, Victoria, Australia), 0.1 g/L magnesium sulfate (Merck), 0.05 g/L manganese sulfate (Sigma Aldrich), and 2 g/L dipotassium phosphate (Merck). The second medium was termed “Tween 80 stress media” and was formulated as per the standard MRS described above but without Tween 80. The third medium was named “glucose stress media”; this was standard MRS medium without glucose. The fourth medium, “double stress media,” was formulated as standard MRS but this time without glucose and Tween 80. glucose and Tween 80 were chosen as the main two stressors investigated in this study as it is known that both compounds are needed for high growth rates in Lactobacilli (De Man et al., 1960 (link); Terraf et al., 2012 (link)).

E. coli strain 506 (O78, K80), originally isolated from a commercial broiler with colibacillosis46 (link) was prepared by plating one frozen bead of E. coli 506 stock on sheep blood agar. The next day a single colony was transferred to 0.1% glucose broth (0.5% Lab Lemco powder (Oxoid, Basingstoke, UK), 1% bacteriological peptone (Oxoid), 0.5% NaCl) Overnight culture was diluted to a concentration of 10⁷ colony forming units (CFU)/ml with phosphate buffered saline (PBS) and kept on ice until inoculation. Bacterial concentration was verified by plating the diluted culture on sheep blood agar plates and performing a colony count.