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Using this CryoSEM technique, the inoculation surface of the frozen leaf segments was viewed directly while being maintained at – 135°C.
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For the direct seed inoculation surface-sterilized seeds were placed on wet filter paper for 3-5 days in an incubator at 25°C with 16 h photoperiod (white fluorescent bulbs at 67 μmol m-2 s-1) followed by soaking in PsJN solution (0.5 of OD600) for 1 min.
P. viticola was collected from sporulated field leaves and used for the artificial inoculations of surface-sterilized leaves.
P. viticola inoculum was collected from sporulated field leaves and used for the artificial inoculations of surface-sterilized leaves.
Mycelia from sawdust cultures were harvested 2 weeks after inoculation from the surface of the filter membrane.
For leaf spray inoculation, the soil surface was covered with aluminum foil in order to avoid that the inoculant reached the soil.
A range of surface inoculation levels were evaluated, but higher inoculum (e.g. ≥ 103 cfu/site) of C. difficile spores produced growth that was too numerous to count when sampled using Rodac plates.
As a demonstration that this reversion was a result of the presence of an extracellular protein, the co-incubations C1 and C2 were treated with proteinase K (PK) before the inoculation of leaf surfaces.
Before inoculation leaves were surface-sterilized with bleach, followed by three washes in sterile water.
Rice roots were collected in two different experiments at various times upon inoculation and were surface-sterilized prior to processing.
For the final volume of liquid for both leaf and soil surface spray inoculation was 1 mL (water + inoculant) per pot containing two plants, and inoculants were diluted with sterile distilled water at 1 1000 (v:v) and 1 7500 (v:v) for spraying maize and wheat, respectively.
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