# Characterization and Selection of _Lactobacillus_ Strains with Potential Robotic Applications
## 2 Materials and Methods
Test microorganisms with IZ \(\geq\) 18 mm were considered sensitive (high antimicrobial activity); moderately sensitive were those with IZ between 12 and 18 mm (moderate antimicrobial activity); and resistant were those with IZ \(<\) 12 mm (low antimicrobial activity) or completely missing. **Antibiotic susceptibility test**  
Antibiotic susceptibility test was performed by the disk diffusion method [18] with impregnated paper disks of 23 antibiotics (Bul Bio--NCIPD Ltd., Sofia, Bulgaria). The strain suspensions (0.1 mL) were spread plated onto MRS agar medium, and then four disks of different antibiotics per Petri dish (d = 90 mm, Isolab GmbH, Germany) were put on the agar medium surface. The dishes were incubated at 37 \({}^{\circ}\)C for 48 h. Zones of inhibition were measured and recorded at 24 th and 48 th hour of incubation. Strains with no inhibition zones were considered resistant (R), strains with inhibition zones from 7 to 16 mm were considered intermediate sensitive (SR), and strains with zones \(>\) 16 mm were considered sensitive (S). **Adhesion ability to intestinal cells**  
Adhesion test was performed as previously described [19] with minor modifications. _Cell line_  
To determine the adhesion ability of the _Lactobacillus_ strains to intestinal cells, 24 h monolayers of IPEC-J2 cell line (intestinal porcine enterocytes isolated from the jejunum of a neonatal unsuckled piglet) grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) and antibiotics (Penicillin, Streptomycin) at 37 \({}^{\circ}\)C under 5% CO\({}_{2}\) atmosphere were used. _Preparation of the Lactobacillus strains_  
A quantity of 1 mL from each _Lactobacillus_ culture in MRS broth was centrifuged at 2500 rpm for 5 min. The supernatant was discarded, and the pellet was suspended in 1 mL of sterile PBS. The optical density (OD) was determined at 600 nm, and each sample was adjusted to 2 \(\times\) 10\({}^{8}\) CFU/mL (by the drop plate method) in the infection media. _Preparation of cell-Lactobacillus culture_  
The monolayer cell culture in a 24-well plate with a lid was washed with 0.5 mL of sterile PBS per well. DMEM (without FBS and antibiotics) and MRS, each 200 \(\upmu\)L, were added to the wells. Then, 100 \(\upmu\)L of bacterial suspensions (2 \(\times\) 10\({}^{8}\) CFU/mL) was added to the appropriate wells, and the plate was incubated at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\) for 2 h. After the incubation, the medium was aspirated from the infected wells, and then the monolayers were washed gently three times with PBS (0.5 mL/well). A 1% Triton X-100 in PBS solution, 200 \(\upmu\)L per well, was added, and the plates were incubated on a rocker at 37 \({}^{\circ}\)C for 10 min. Next, 0.8 mL/well of PBS was added and pipetted up and down 10-15 times to remove all attached cells. The samples were serially diluted using a 96-well microplate (180 \(\upmu\)L of PBS per well was added and then mixed with 20 \(\upmu\)L from each repetition to a well in the first row of the plate). A quantity of 5 \(\upmu\)L of each dilution was dropped on pre-prepared plates containing MRS agar, incubated at 37 \({}^{\circ}\)C for 48, and then counted for CFU calculation. **Statistical analysis**  
Data from triplicate experiments were processed with MS Office Excel 2010 software using statistical functions to determine the standard deviation (\(\pm\)SD) and maximum estimation error at significance levels \(p<\) 0.05. A multiple comparison test was performed by ANOVA using the LSD (Least Significant Difference) method.