Hi everyone

I work at a project contain detecting AHLs from an activated sludge process (lab-scale) but despite all efforts I could not see any color on the TLC plates. My protocol is including:

1- Extract AHLs by ethylacetat. centrifuge the sample (10 ml), remove the supernatant, add 5 ml ethylacetat, vortex for 2 hours, centrifuge and take supernatant as AHLs solution, and then dried at 60 centigrade.

2- Add 100 microliters methanol and spot 3-5 microliters on TLC plate (Silica gel RP-18 Merck) and then develop in a glass tank with methanol/water (65:35), and then dried at room temperature.

3- The dried plates were overlaid with a culture of the indicator bacterium prepared as follows. For 20 × 20 cm plates, a 5-ml overnight culture of NT1(pDCI41E33) was used to inoculate 50 ml of LB and the new culture was grown 3-4 hours. The entire 55 ml of culture was added to 100 ml of the same medium containing 1 g of melted agar and 60 μg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal) maintained. The culture was mixed thoroughly and immediately spread over the surface of the developed plate held in a Plexiglas jig designed to produce a uniform layer of agar about 3 mm thick. After the agar solidified, the coated plates were incubated at 28°C for 24 hr in a closed plastic container.

These were the method for detecting quorum sensing signals in detail in our laboratory.

I don't know what is the problem!!