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Anaerobic and aerobic growth was evaluated by aseptically dispensing 3 mL of Luria-Bertani (LB), tryptic soy broth (TSB) supplemented with 0.1% cysteine, or Chamberlain's media into 13×100 mm glass culture tubes then inoculating using a sterile loop.
For each isolate, a single colony grown on 5% sheep blood agar (Biotechnology Appliquée, Dinan, France) was taken using a sterile loop, mixed with freezing beads for storage at −20°C prior to inactivation as previously described [32] and DNA extraction using Qiagen kit (Qiagen, Courtaboeuf, France).
Fresh TOP10 cells containing the appropriate expression plasmid were collected from a LB/Ap plate using a sterile loop and used to inoculate LB/Ap medium (15 mL).
Biofilm cells were removed from the tubing using a sterile loop to detach the adherent cells into 20 ml of cold (4°C) phenol ethanol solution.
2.5 ul of this broth was then streaked onto plates of (a) MacConkey #3 agar and (b) Bile Aesculin agar using a sterile loop.
Approximately 0.1 mL of each of the other samples was placed directly onto a 7H10 Middlebrook medium plate and spread over the plate by using a sterile loop.
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Overnight grown individual colonies were removed using a sterile, plastic loop.
The spores were dislodged using a sterile inoculation loop under strict aseptic conditions, and the number of spores in the suspension was counted using a Neubauer chamber.
Conidial suspensions were prepared by adding 10 ml of 0.1% Tween 80 (AppliChem) into the 4-week-old Petri dishes, and the conidia were taken from the agar surface, using a sterile inoculation loop to dislodge.
The bacteria were harvested and resuspended in phosphate-buffered saline (pH 7.4) using a sterile inoculating loop.
A sample of culture was selected using a sterile plastic loop and re-suspended in 200 μl of 5 % Chelex 100 (BioRad) in a microcentifuge tube.
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