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Because spores are plated directly on the selective medium, each colony that survives the selection represents a unique meiotic product that can be individually genotyped for QTL mapping and molecular karyotyping.
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Care was taken to maintain similar-sized patches of brood (roughly one side of one medium frame) and stored honey (1 medium frame) in each colony to control for foraging motivation via food storage quantity and brood nutritional needs.
A loopful of cells from each colony growing on solid medium was placed in 100 μl of sterile water.
Portions of individual colonies from the stock plate were tested for the presence or absence of plasmid: Cells from each colony were spotted onto plates containing non-selective medium or selective medium, and a colony whose cells could not grow on selective medium was chosen.
This cell suspension was plated on 50 large, square dish plates (245 mm × 245 mm × 25 mm; ThermoFisher Scientific, Kanagawa, Japan) containing 2xYT agar medium, and colony formation was monitored for 21 days.
Microscope observation of 10 isolates representative of each colony morphology revealed the presence of long, filamentous, nonmotile rods (large and smooth colonies), short nonmotile rods (large and rough colonies), and ovoid forming cocci (medium colonies) (data not shown).
After PDT treatment, cells were trypsinised and plated in growth medium for colony formation.
After treatment, cells were washed 3 times with ice-cold PBS and restored in fresh growth medium for colony formation.
Each run of preculture/culture was separated by a plating on YMD medium, and a colony of this plating was used to inoculate the next run of preculture/culture in increasing IL%.
The cultured medium of 96 colonies each was collected by means of a BIOMEK pipetting robot (Beckman) and pooled in one well of a 96 deep well plate.
Cell lines derived in LBX medium had higher colony formation ability than that derived in KOSR medium.
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