Tlc immersion device

The chromatogram was immersed (immersion speed 3.5 cm/s and immersion time 2 s, TLC Immersion Device, CAMAG) in an Aliivibrio fischeri culture. The readiness of the bacterial growth prepared according to [43 ,44 (link)] was visually controlled for an emitted brilliant green-blue light by shaking the culture flask in a dark room. The bioluminescence was recorded with an exposure time of 50 s (time interval 3 min, BioLuminizer, CAMAG). Dark zones indicated antibacterial compounds.

The developed TLC plate was immersed for 8 s in the bacterial suspension (8.0 × 10⁷ CFU/mL) using the TLC Immersion Device (CAMAG, Muttenz, Switzerland). Next, the plate was placed in a moistened plastic box and incubated at T = 37 °C for 17 h. Then, for visualization, the bioautogram was sprayed with a 0.2% MTT aqueous solution. To improve the intensity of the color, a drop of Triton X-100 was added per 10 mL of aqueous MTT solution. After reincubation at 37 °C for 0.5 h, white zones of bacterial growth inhibition were visible against a purple background. The bioautogram was digitized by the Visualizer [40 (link)].

Lyophilized plant powder samples (100 mg each) were weighed accurately, and each was dissolved in 2 mL ethyl acetate–ethanol–water 1:1:1, V/V/V, ultrasonicated for 30 min (Sonorex Digiplus, Bandelin, Berlin, Germany), and centrifuged at 3000× g for 15 min (Labofuge 400, Heraeus, Hanau, Germany). Each supernatant (50 mg/mL) was transferred to an autosampler vial. The plant extracts (5 µL/band, if not stated otherwise) were applied (ATS 4, CAMAG) on HPTLC plates silica gel 60 F₂₅₄ (without F₂₅₄ indicator for SOS-Umu-C bioassay), separated with ethyl acetate–toluene–methanol–water 4:1:1:0.4, V/V/V/V, or toluene–ethyl acetate 7:3, V/V, up to a migration distance of 70 mm (about 20 min, Twin Trough Chamber, CAMAG) and detected under white light illumination (reflection and transmission mode), UV 254 nm, and FLD 366 nm (TLC Visualizer, CAMAG). Immersion (3 cm/s, 2 s, TLC Immersion Device, CAMAG) or piezoelectric spraying (blue nozzle, level 3, Derivatizer, CAMAG) was used for the effect-directed assays as follows.