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The headspace of the sample flasks was flushed with nitrogen prior incubation.
Currently, to monitor the environmental variables, several sample flasks are prepared and cultured simultaneously, and a single flask is retrieved from the shaking table platform periodically and sampled aseptically to measure the time course of the shake-flask culture of microbes.
At 0, 1, 2, and 3 h, 1 mL aliquots were taken from control and sample flasks and spun in a microcentrifuge at 1400 rpm for 3 min, resulting in a pellet.
The water in the round-bottomed flask was then dried in the oven at 105°C for 24 h to determine the weight of extractives present in the sample flasks.
Human melanoma cell line and C8161 cells (which have a doubling time of 24 and 20 h, respectively) were cultured in two 25 cm sample flasks until 90% confluent, and incubated with TNF- α or α-MSH (alone or in combination) for 4 32 h.
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The oil which was remained in the sample flask was weighed after the process was completed.
These tests identified the ideal position of the sample flask and indicated that the maximum time that the sample could be analyzed without arsenic lost due to increase of the temperature was 180 minutes.
Optimal conditions for ultrasound assisted extraction were obtained by evaluation of parameters such as the position of the sample flask inside the ultrasound bath and temperature as a function of the immersion time.
After drying of the samples, a few boiling stones (see section on preparation of STRIP reagent) are added to the sample flask and the distillation apparatus is assembled, cooling water and N2 gas flow started.
For MPTMS-RSNO coatings, thermal decomposition of the S-nitrosothiol moieties was achieved using a light-shielded sample flask and the addition of DTPA to chelate trace copper.
For films containing S-nitrosothiol NO donors (i.e., MPTMS particles), the sample flask was shielded from light, and 500 μM DTPA was added to the PBS buffer to chelate trace copper.
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