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11 days post-inoculation (dpi), infected tissue was collected, powdered in liquid nitrogen and inoculation buffer was added (0.71 g of infected tissue per mL of inoculation buffer).
Local lesions typical of PMMoV infection were excised and crushed into 0.6 ml of inoculation buffer, and 350 µl of this solution were tested for the presence of PMMoV by RT-PCR using primers published by Hamada et al. [10], by electron microscopy, and by inoculation to one plant of Capsicum annuum var Yolo Wonder or Nicotiana benthamiana, two susceptible hosts for PMMoV.
In both cases, the inoculated leaf was removed and a sap was prepared with 150 mg of infected tissue in 1 mL of inoculation buffer.
Two mL of this mixture was placed in 10 mL of inoculation fluid (IF) containing 24 μL of tetrazolium-based dye A (Biolog inc)., resulting in a final OD600 of ~0.07.
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Therefore, a stock solution of 450 g/L glucose was prepared (23 mL) for inoculation after complete glucose consumption.
The cells were centrifuged and washed with 5 ml of water before inoculation.
The starter culture was used to inoculate 50 mL of LB (2 % inoculation) followed by incubation at 37 °C with shaking for 2 h.
Collected aliquots were diluted in LB medium to a final volume of 100 mL before inoculation in 900 mL of SD medium for bioreactor cultivation.
The media were inoculated with 0.4 mL of a standardized bacterial inoculation.
The media were dispensed in flasks of 250 mL capacity each containing 50 mL and then autoclaved at 121 °C for 15 min, then inoculated with 0.4 mL of a standardized bacterial inoculation.
For the collection of zoospores for the experimental inoculation, one ml of a 5 day old broth culture was inoculated on TGhL agar (16 g tryptone + 2 g hydrolysed gelatin + 4 g lactose + 10 g agar in 1 l distilled water) and incubated for 5 to 7 days at 20°C.
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