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All the microbial cultures were adjusted to 0.5 McFarland standard, which is visually comparable to a microbial suspension of approximately 1.5 × 108 cfu/ml.
0.5 McFarland microbial suspension of C. albicans colonies was formed in Brain Heart Infusion Broth.
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Microbial suspensions of 1.5 × 108 CFU/ml corresponding to 0.5 McFarland density obtained from an overnight culture of bacteria developed on solid media were used.
Microbial suspensions of 1.5 × 108 CFU mL−1 (0.5 McFarland density) obtained from 15 to 18 h of bacterial cultures developed on solid media were used.
Microbial suspensions of 1.5 × 108 colony-forming unit (CFU /ml corresponding to 0.5 McFarland density obtained from 15- to 18-h bacterial cultures developed on solid media were used in our experiments.
Microbial suspensions of 1.5 × 10 CFU/mL corresponding to a 0.5 McFarland density obtained from 15 18 h bacterial cultures developed on solid media were used in our experiments.
Cyclic operation is imposed by the periodic harvesting of a fraction of the microbial suspension and replenishment of the harvested volume with an equal volume of fresh medium.
The microdilution method was used to determine the Minimum Inhibitory Concentrations (MICs) of A. salicina extracts; 100 μL of microbial suspension containing, approximately 105 cells/mL, was added to 100 μl of the extract dilution (concentrations ranging from 62.5 μg/mL to 10 mg/ml in water).
50 µl of microbial suspension was added to each corresponding well of a 96-well microtiter plate (polypropylene) together with 50 µl of peptide solution.
Briefly, 75 µL of an agar/mucin solution [5% porcine mucin type III (Sigma-aldrich, Diegem, Belgium), 0.8% agar (BD, Erembodegem, Belgium)] was brought on the porous membrane (0.4 µm) of a 24-well plate Transwell® system (Corning Inc., NY, USA) and allowed to solidify for at least 30 min after which 20 µL of a microbial suspension was spotted on top of this agar/mucin layer (apical compartment).
Next, 500 μl of hydrocarbon was added to 5 ml of microbial suspension and vortexed for 2 min.
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