Hamilton microsyringe

The rats were treated icv. in a volume of 2 µL over 30 s using a Hamilton microsyringe (Merck KGaA, Darmstadt, Germany). The doses applied were 0.1 or 1 µg of Kp-8 dissolved in 0.9% saline. Control animals were injected with 2 µL of 0.9% saline alone. The animals were treated 30 min prior to the behavioral tests. Collection of trunk blood for LH ELISA, corticosterone ELISA and serum corticosterone measurement were carried out 15 min and 30 min after icv. treatment, respectively.

Strains were inoculated from GYM plates to GYM liquid broth and grown on a rotary shaker for 24-48 h at 28°C, 250 rpm. Fresh GYM broth was inoculated with 5% of these cultures and cultivation continued for next 10 days at 28°C, 250 rpm. Cells were separated by centrifugation for 10 min at 10 000 × g, 4°C, and cultivation broth was used for TLC analyses. Thirty μl of cultivation broth were applied on silica plates (Merck, Germany) by Hamilton micro syringe and the plates were placed in a chromatographic chamber pre-saturated for minimum 1 h with a mobile phase consisting of chloroform : methanol : ammonia (28%), 12:3:0.1. Detection of low-molecular weight substances was performed using a UV lamp at 254 and 366 nm, and by staining with coloring reagent. For this purpose developed plates were dried for 30 min at room temperature prior to spraying by orcinol reagent (orcinol 250 mg, ethanol 44 ml and sulphuric acid 6 ml). The plates were subsequently heated at 110°C for 2 min and spot appearance, position, and color were determined. Retention factor (R_f) values were determined by dividing distance moved by compound by distance moved by solvent.

A modified version of the assay described by Ramarao et al. (2012) (link) was performed. Larvae of G. mellonella were incubated for 30 min at room temperature before injection. Overnight cultures of each microorganism were centrifuged (3220 × g, 15 min) and suspended in PBS. This was repeated twice. Cultures were adjusted to an OD_{600 nm} of 0.1. For intrahemocoelic injection, bacterial suspensions were prepared with final concentrations in the range of 10⁴ CFU/ml to 10⁸ CFU/ml. Volumes of 5 μl of each strain, cell-free extract or lysate were delivered directly to the hemocoel through an injection in the rear left pro-leg using a 26-gauge needle Hamilton microsyringe (Sigma, United Kingdom). Sterile PBS (5 μl) was injected into the “trauma” control group and additionally, a “no treatment” control group was added. The right pro-leg was used as the injection site. Different sites were used for pathogenic and probiotic strains to reduce the risk of injection site infection. Infected larvae were incubated in a petri dish in groups of 10 at 37°C in the dark for the duration of the experiment (5–7 days).