Polystyrene tubes (12 mm × 75 mm) (Sarstedt) were filled with 3 mL of 0.45% sterile saline (CareFusion). Homogenous suspensions of bacteria were prepared in the 3 mL 0.45% sterile saline. Suspensions were examined using the DensiCheck Plus (BioMérieux) device to ensure that the suspensions were within the turbidity range of 0.5–0.63 (McFarland standard) for both Gram‐positive and Gram‐negative bacteria. The appropriate Vitek cards (Vitek GP and Vitek GN) (BioMérieux), which contained 64 wells with miniaturized selective media and biochemical reagents, were used for bacterial identification. The Vitek system identified the bacteria by filling the Vitek wells with the standard turbidity bacteria suspension in 3 mL of 0.45% sterile saline. The colour codes from the miniaturized selective media as well as the results of the miniaturized biochemical tests were analysed by the Vitek 2 Compact system software (Version 08.01, BioMérieux) to identify the bacteria species.
Densicheck plus
Manufactured by bioMérieux
Sourced in France, United States, United Kingdom
The DensiCheck Plus is a compact and versatile instrument designed for optical density measurements. It provides accurate and reproducible results for a wide range of applications, offering a reliable solution for various laboratory settings.
Automatically generated - may contain errors
5 protocols using densicheck plus
1
Bacterial Identification Using Vitek System
2
Automated MIC Determination for Antibiotic Susceptibility
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Susceptibility to antibiotics based on an automated minimal inhibitory concentration (MIC) was determined on a VITEK® 2 Compact System instrument (bioMérieux, Marcy-l’Étoile, France) using VITEK® AST N330 susceptibility card for Gram negative organisms, according to the manufacturer’s instructions. Briefly, bacterial inoculum was prepared from pure overnight culture grown on Trypticase Soy Agar (Merck, Darmstadt, Germany) by suspending single colonies in sterile saline solution to an optical density of 0.5–0.63 McFarland units as measured by DensiCheck Plus (bioMérieux, Marcy-l’Étoile, France). Tube with suspension was then placed in the appropriate slot of the VITEK 2 Compact system and analysis was performed by the instrument. The card contained 17 antibiotics: Amikacin (AK), Cefotaxime (CTX), Cefoxitin (FOX), Cefalexin (CN), Meropenem (MEM), Cefuroxime (CXM), Gentamicin (GEN), Ciprofloxacin (CIP), Norfloxacin (NOR), Ceftazidime (CAZ), Cefepime (FEP), Ertapenem (ETP), Nitrofurantoin (FT), Piperacillin/Tazobactam (TZP) and Trimpethoprim/sulphametaxazole (SXT). Interpretation of MIC values obtained on VITEK 2 was made according to the CLSI M100 guidelines [21 ]. Quality control was performed according to the manufacturer’s instruction for VITEK testing with reference strain Escherichia coli ATCC 25922.
3
Microdilution Assay for Antimicrobial Potency
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The MIC was measured
using a broth microdilution method following the Clinical and Laboratory
Standards Institute (CLSI) guidelines, as previously described.16 (link) Briefly, bacteria were cultured in 3 mL of Muller
Hinton (MH) II broth in 15 mL round-bottom tubes with shaking at 180
rpm at 37 °C overnight. The peptides and drugs were prepared
by serial 2-fold dilution. Bacterial cells were adjusted to 0.5 McFarland
standard using a densitometer (Densicheck plus, Biomerieux) and 10-fold
diluted in MH II to make a bacterial suspension. 10 μL of the
bacterial suspension was added to each well in the 96-well plate.
The samples were incubated at 37 °C for 18 h. The UV absorbance
at 600 nm (OD600) was measured using an EPOCH2 microplate
reader (BioTek, Winooski). The growth of the bacterial cells was normalized
with 200 μL of 2.5% DMSO MH II broth with bacterial cells (100%
growth) or 200 μL of 2.5% DMSO MH II broth without bacterial
cells (0% growth). The MIC value was defined as bacterial growth inhibition
<10%. All experiments were done in triplicate independently.
using a broth microdilution method following the Clinical and Laboratory
Standards Institute (CLSI) guidelines, as previously described.16 (link) Briefly, bacteria were cultured in 3 mL of Muller
Hinton (MH) II broth in 15 mL round-bottom tubes with shaking at 180
rpm at 37 °C overnight. The peptides and drugs were prepared
by serial 2-fold dilution. Bacterial cells were adjusted to 0.5 McFarland
standard using a densitometer (Densicheck plus, Biomerieux) and 10-fold
diluted in MH II to make a bacterial suspension. 10 μL of the
bacterial suspension was added to each well in the 96-well plate.
The samples were incubated at 37 °C for 18 h. The UV absorbance
at 600 nm (OD600) was measured using an EPOCH2 microplate
reader (BioTek, Winooski). The growth of the bacterial cells was normalized
with 200 μL of 2.5% DMSO MH II broth with bacterial cells (100%
growth) or 200 μL of 2.5% DMSO MH II broth without bacterial
cells (0% growth). The MIC value was defined as bacterial growth inhibition
<10%. All experiments were done in triplicate independently.
4
Antagonistic Screening of Endophytic Bacteria
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A modification of the agar disk diffusion method for detecting antagonism was used against the four common bacteria listed in Table 1 (Nie et al., 2012 (link)). The common bacteria and endophytes were each pre-cultured overnight, and 5 mL-1 of each culture was centrifuged at 604 × g for 5 min. The pellets were resuspended in sterile phosphate buffered saline (PBS) in a laminar air flow cabinet and density adjusted to 108 colony forming units (CFU) mL-1 by using Densicheck plus (BioMérieux, United States). A total of 200 μL of the common bacteria cell concentrate was inoculated and evenly spread by sterile cotton swaps onto the surface of the medium, and then four 5-mm-diameter pieces of sterile filter paper were placed on each corner of the petri dish. A total of 10 μL of each endophyte strain was then added dropwise to the filter paper. All plates were wrapped with parafilm, incubated at 37 ± 2°C for 24 h and observed for the inhibition of the common bacteria (Cho et al., 2007 (link)). Antibacterial activity was assessed by measuring the diameter of the clear zone of growth inhibition. An equivalent volume of sterile phosphate buffered saline (PBS) instead of the endophytic bacteria was used as a negative control.
5
Optimized Microbial Susceptibility Assay
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MICs had previously been determined for each isolate using a 2-fold dilution series according to the broth microdilution method described in the European Committee for Antimicrobial Testing (EUCAST) guidelines and in accordance with ISO 20776 (38 , 39 ). This method was adapted to 5-overlapping 2-fold dilution series to increase accuracy to within 20% of the dilution (compared to standard 2-fold dilution series) as previously described (40 (link)– (link)42 (link)).
Bacterial suspensions were prepared from individual colonies suspended in phosphate-buffered saline (PBS) with comparison to 3 McFarland standards using DensiCheck Plus (bioMérieux, Hampshire, UK). Suspensions were diluted in CAMHB to achieve a final in-plate inoculum of 5 × 105 CFU/mL. The MIC was recorded following overnight static incubation at 37°C. Two control isolates (mcr-1 negative [NCTC 12241] with an expected MIC of 0.5 or 1 mg/L and mcr-1 positive [NCTC 13846] with an expected MIC of 4 mg/L) were included in each plate, with MICs accepted within one dilution of the expected range.
Bacterial suspensions were prepared from individual colonies suspended in phosphate-buffered saline (PBS) with comparison to 3 McFarland standards using DensiCheck Plus (bioMérieux, Hampshire, UK). Suspensions were diluted in CAMHB to achieve a final in-plate inoculum of 5 × 105 CFU/mL. The MIC was recorded following overnight static incubation at 37°C. Two control isolates (mcr-1 negative [NCTC 12241] with an expected MIC of 0.5 or 1 mg/L and mcr-1 positive [NCTC 13846] with an expected MIC of 4 mg/L) were included in each plate, with MICs accepted within one dilution of the expected range.
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