Tissue culture plate

Biofilm formation assay was performed according to the method described previously (Stepanović et al., 2007 (link)), with modifications. Wells of 96-well microtiter plates (Tissue Culture Plate, Sarstedt, Germany) were filled with 180 μl Luria-Bertani broth medium and then 20 μl aliquotes of overnight cultures of Achromobacter spp. strains (adjusted to the 0.5 McFarland standard) were added. All strains were tested in triplicate. Sterile medium tested in triplicate was used as negative control. Microtiter plates were incubated aerobically for 48 h at 37°C. After incubation and washing (three times with phosphate-buffered saline, PBS; pH 7.2) remaining bacteria were fixed by drying at 65°C for 30 min. For staining and visualization of biofilm, 0.1% crystal violet (HiMedia Labs Pvt. Ltd., India) was used (30 min at room temperature). The stain was rinsed by washing three times with 1X PBS and then resolubilised with 96% ethanol and acetone (4:1). Quantification of biofilm formation was done by measuring absorbance at 595 nm using Plate Reader Infinite 200 pro (MTX Lab Systems, Austria).
On the basis of biofilm formation, Achromobacter spp. isolates were divided into four classes: no biofilm producer (N), weak (W), moderate (M), and strong (S) biofilm producer (Stepanović et al., 2007 (link)).

The ability of phages SJM3 and LASTA to lyse host cells in preformed biofilm on porous glass beads was investigated as previously described [22 (link),38 (link)] with minor modifications. Glass beads (diameter 4 mm, pore size 60 μm, and surface area of ~60 cm²; VitraPor; ROBU, Hattert, Germany) were statically incubated with 100-times-diluted overnight culture of Ni9 for 24 h at 37 °C. Further on, beads were washed three times with saline solution and separated in single wells of a 24-well plate (Tissue Culture Plate; Sarstedt). Each was treated with 10⁹ PFU/mL phage suspension (LASTA, SJM3 or cocktail) at predicted MOI 100 in LB medium for 24 h. After 24 h, treated beads and beads from the untreated control were washed with saline, vortexed in microtubes and, subsequently, sonicated in an ultrasound water bath and vortexed once again in order to detach biofilm-embedded bacteria [39 (link)]. CFUs of biofilm before treatment, biofilm after phage treatments and biofilm with fresh medium were counted on LA plates.

Granulosa cells from immature mice were collected, as described above, at 40h after eCG administration under aseptic conditions. Granulosa cell suspension was centrifuged at 1000g for 5 min at 37°C and pellet was re-suspended in electroporation medium (DMEM/F12 glutamax, Gibco 10565–018). Following cell counting using a hemocytometer, granulosa cells were diluted to a final concentration of 0.2 X10⁶ cells in 10μl of DMEM/F12 media. Homogenous granulosa cell suspension was split into three parts. Each part was then mixed with either medium or control siRNA (at 20nM conc, Dharmacon, D-001210–05–05) or Egr1 siRNA smart pool (20nM, Dharmacon, M-040286–01). Electroporation was done using the Neon Transfection System (MPK 1096–772) and MBI microporator (Digital Bio) with the settings of 1000 volts, 30 millisecond and 3 pulses. Electroporated granulosa cells were plated in 24-well tissue culture plate (Sarstedt; 0.2 X10⁶ cell/well) containing pre-warmed medium and incubated for 6 h at 37°C and 5% CO₂. At the end of 6h incubation period, granulosa cells were treated with either forskolin (Fo; 10μM, Sigma, P3917) and phorbol-12-myristate (PMA; 20μM, Sigma, P1585) or medium for 4 h. Combined treatment with Fo and PMA (Fo+PMA) was used to mimic LH or hCG treatment in vivo. Following this treatment period, granulosa cells were harvested for either transcript or protein analysis.

Characterization of phage-planktonic bacteria kinetics was performed as follows: overnight culture of bacterial strain A. baumannii 6077/12, grown in MH, was adjusted to density of 0.5 McFarland scale and further on diluted 100 times into 200 μL MH broth without (growth control) or with ISTD phage suspension in a 96-well-microtiter plate (Tissue Culture Plate; Sarstedt). Phages were applied at MOIs 0.01, 0.1, 1, and 10. At 3-, 6-, 9-, and 24-h time points after treatment, samples were taken, serially diluted in saline, and plated on MH agar. Viable cell counts were calculated from triplicate assays.

Interaction dynamics of phage and planktonic bacteria was tested in a 96-well microtiter plate (Tissue Culture Plate; Sarstedt) in triplicate, according to the protocol described by Vukotic et al. [22 (link)]. The K. pneumoniae Ni9 overnight culture was adjusted to 0.5 McFarland units and further diluted 100 times in 200 μL LB distributed in wells. Phages SJM3 and LASTA were applied separately, as well as in a cocktail suspension (mixture of 50:50), at final MOI 10 (growth control excluded from treatment with phage). At 3, 6, 9 and 24 h after the onset of the experiment, viable cell count was enumerated by spotting dilutions on LA plates [37 (link)]. A total of 80 colonies (ten from each time point for each phage) were collected, propagated and analyzed for sensitivity to both phages using spot testing [23 ]. Among colonies that appeared resistant, eleven were selected for further investigation of phage-resensitization phenomena and designated Ni92–Ni912. They were chosen from each time point and each infection setting.

Arabidopsis seedlings (5-day old) were transferred using sterile forceps into individual wells of a sterile 24-well plate (Sarstedt^® Tissue Culture Plate) containing 1 ml SDW. Seedlings were handled with care during the transfer process to avoid mechanical damage to root hairs and elevated background death levels. 24-well plates were sealed using autoclave tape, placed in a Grant SUB Aqua Pro 26 water bath already stabilized at the desired heat stress temperature (25, 35, 45, 50, 55, 65, 75, or 85°C), and heat stressed for 10 min. Seedlings were returned to the 21°C growth chamber and scored 14–16 h after stress application to allow PCD morphology to fully develop. The RHA was used to quantify the stress response in terms of viability, PCD and necrosis as described by Hogg et al. (2011) (link).