Bactiter glotm

Adenosine-5’-triphosphate (ATP) levels of the samples were improved by a luciferase-luciferin enzymatic assay kit BacTiter-Glo^TM from Promega (France). The BacTiter-Glo^TM reagent is directly added to microalgae cells in medium and triggers cell lysis. The luminescence can be measured without washing cells or removing medium. ATP concentration was performed using a standard curve. All experiments were done in triplicate.

To measure the antimicrobial susceptibility of biofilms, the MBEC Assay (Innovotech) was used as described [30 (link)]. Briefly, an overnight culture was diluted and seeded into a MBEC plate in 10% MHB v/v diluted in DPBS and incubated for 24-hrs at 35°C with agitation at 150 RPM. The MBEC lid was then washed to remove non-adherent cells, transferred to a 96-well treatment plate, and incubated for the indicated time at 35°C without agitation. Following treatment, the MBEC lid was washed and transferred to a black 96-well ViewPlate (PerkinElmer) filled with 40% (v/v) BacTiter-Glo^TM (Promega) diluted in DPBS to enumerate cell viability using luminescence by an EnVison Multilabel Plate Reader (PerkenElmer, Waltham, MA). The BacTiter-Glo^TM Microbial Cell Viability Assay is a luminescent assay that determines the number of viable cells based on quantification of ATP concentration. A calibration curve was previously performed and it was found using a linear regression the coefficient of determination was r² = 0.9884 for luminescence versus CFUs/mL [30 (link)].

Adenosine-5′-triphosphate (ATP) levels were analyzed using BacTiter-Glo^TM (Promega, Madison, WI, USA). All the steps were performed in accordance with the protocol provided by the manufacturer. Shortly after contact with materials, 100 µL of bacteria cell suspension were mixed with the same volume of a reagent. The samples were then incubated on an orbital shaker for 5 min. The luminescence of the samples was measured using Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA). The results are presented as percent values using control as 100%.

Exponentially growing E. coli cells were resuspended in Seine river fresh water and incubated at 37°C in a shaking bath (122 rpm) for 3 h with 0, 50 or 100 ppm of P25 nano-TiO₂ under room light. ATP release in the samples was determined by a luciferase-luciferin enzymatic assay kit BacTiter-Glo^TM from Promega in a microplate reader (Infinite 2000, Tecan). All experiments were done in triplicates in 96 well-microplates for chemiluminescence (Eppendorf). The BacTiter-Glo^TM reagent is directly added to bacterial cells in medium and triggers cell lysis and ATP recovery at the same time. The luminescence can be measured without washing cells or removing the medium. A series of blank as well as control are realized on Seine river fresh water and bacterial cells without nano-TiO₂, respectively. Determination of ATP concentration in the medium was performed using a standard curve established on serial dilutions of ATP as recommended by Promega.

2× Mueller-Hinton broth was inoculated with 2 × 10⁶ CFU mL^–1 MRSA or K. pneumoniae and challenged with equal volume of the nanocapsule dispersions (diluted in PBS to yield eugenol or clove oil concentrations of 0.625, 0.25, 0.1875, 0.125, and 0.025 mg mL^–1) for 16 h allowing growth curves and IC₅₀ values to be determined (ESI,† Section 10). Bacterial viability in nanocapsule-challenged media was compared with PBS (negative control) and hydrogen peroxide (8% w/w) (positive control) using the BacTiter-Glo^TM (Promega) microbial viability assay (ESI,† Section 11).

The viability of biofilms remaining post-treatment was evaluated using the BacTiter-Glo^TM (Promega, Madison WI) assay, which measures the production of adenosine triphosphate (ATP) by metabolically active bacterial cells using a luminescent reporter. As ATP production is relatively constant across many different growth conditions (Schneider and Gourse, 2004 (link)), ATP-based assays are used routinely to monitor the presence of active bacteria and have been applied to biofilms (Takahashi et al., 1988 (link); Monzón et al., 2001 (link); Romanova et al., 2007 (link); Sule et al., 2009 (link); Lu et al., 2014 (link)).
After honey and antibiotic treatment of established biofilms as outlined above, BacTiter-Glo reagent (20 μl) was added into each well followed by 100 μl of TSB. The bioluminescence reaction was started by the addition of the BacTiter-Glo reagent and incubation in the dark for 10 min at 37°C. Bioluminescence was determined in a Tecan Infinite 200 PRO series microplate reader (Tecan Group, Switzerland). To allow for consistency in incubation times, a single 96-well microtiter-plate was processed at a time. Statistical analysis to determine significant differences in viability following treatments was performed using one-way ANOVA with the Tukey test in GraphPad Prism (version 6; CA, USA) with statistical significance set at p <0.05.

ATP content of enrichment cultures was determined via luminescence using BacTiter-Glo^TM (Promega, Madison, WI, United States) as previously described (Napieralski et al., 2019 (link)).