Multiskan spectrum microplate spectrophotometer

Minimum inhibitory concentrations (MICs) of AHL-degrading bacteria were determined using microtiter plate dilution assays in R2A broth with about 1 × 10⁵ cells/well, as previously described (Yajko et al., 1987 (link); Riesenfeld et al., 2004 (link)). MICs were determined after 1, 2, and 3 days of incubation at 30°C in the dark. MICs were read using a Multiskan^® Spectrum microplate spectrophotometer (Thermo Labsystems, Vantaa, Finland) and was defined as the lowest concentration of an antimicrobial agent at which the organism showed no visible growth (Yajko et al., 1987 (link)). E. coli strain EPI300^TM (Epicentre, Madison, WI, United States) was used as a negative control. The criteria used for the interpretation of antimicrobial susceptibility were based upon the achievable levels of antimicrobial agents. The tested antibiotics were PENG, AMP, AMO, carbenicillin (CAR), piperacillin (PIP), CEFL, and CEFD. All these antibiotics are in common use, and the environmental samples used in the present study were polluted with wastewater from PENG, AMP, AMO, CEFL, and CEFD production. The antibiotics were tested at concentrations of 8, 16, 32, 64, 125, 250, and 500 μg/mL.

Forty-four HLCR K. pneumoniae mutants with different mutations, including equal numbers of mcr-1-negative and mcr-1-positive derivatives, were selected incubated in fresh MH broth under shaking (180 rpm) conditions at 37°C. The overnight cultures were inoculated in fresh MH broth, and OD₆₀₀ was measured using the Multiskan Spectrum microplate spectrophotometer (Thermo Labsystems, Franklin, MA, United States). The growth rates were determined by plotting the logarithm of OD₆₀₀ versus time. Data analysis was performed using a non-parametric Mann–Whitney U-test, followed by Dunn’s multiple comparison test (Zwietering et al., 1990 (link)).

The cytotoxic effect of Vin on BMM cell viability was examined using the CCK-8 Assay Kit. Briefly, BMMs seeded in 96-well plates in triplicates were treated without (mock control) or with different concentrations of Vin (1.25, 2.5, 5, 10, 20, or 40 μM) for 48 h. The cells were then incubated with CCK-8 reagent for 2 h, and the absorbance at the wavelength of 450 nm was measured using a Multiskan Spectrum microplate spectrophotometer (Thermo Fisher Scientific).

For the comparative quantification of endogenous hormones such as IAA, GA, CYT and ETH, samples were collected at the tillering, grand growth and maturing phases respectively. Samples of top visible dewlap (TVD) leaf (leaf +1) were taken in triplicates for endogenous hormones quantification. The leaf samples were immediately shifted to liquid nitrogen and stored at -80°C for further analysis. For extraction of hormones, two grams of sugarcane leaf samples were weighed, and ground under liquid nitrogen using a pestle and mortar. The powdered samples were shifted to centrifuge tube, and 700 μL PBS buffer was added. The mixture was centrifuged at 12,000 rpm for 12 min at 4°C, and the supernatant was collected in Eppendorf tube. Measurements of entire hormones concentrations were carried out by using plant enzyme-linked immunosorbent assay (ELISA) kits (Wuhan Genemei Biotechnology Co., Ltd., China) according to the guidance of manufacturer and OD absorbance at 450 nm was measured with Multiskan Spectrum Microplate Spectrophotometer (Thermo Scientific, USA).