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Vesicular liquid swabs were added to 200 µL of PBS and centrifuged at 2000×g for 3 min. Viral isolation was performed by inoculation of 200 µL of processed samples onto CEF monolayers, followed by 72 hours of incubation.
Deep-throat swabs were added to lysis buffer from the AllPrep DNA/RNA extraction kit (Qiagen, CA) in a lysing matrix B tube (Qbiogene, CA) and lysed by bead-beating using a FastPrep system (Qbiogene, CA) for 30 seconds at 5.5 m sec−1.
Then the swabs were added to brain heart infusion broth medium for 48 h at 37°C.
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The remainder of the fluid and the swab were added to 8 mL of mannitol salt broth containing 4 mg/L of cefoxitin and incubated for 48 hours at 37°C in air.
One 200-µL aliquot of suspended nasal swab was added to a 2 mL tube containing 200 µl of AVL buffer and 10 µl of MS2 phage internal control and incubated for 10 minutes at room temperature for inactivation.
Swab samples were added to MDCK or swine testicle (ST) cells (American Type Culture Collection) as described (27, 28 ).
Rectal swab specimens were added to Campylobacter blood agar plates containing 10 μg/mL vancomycin (Becton Dickinson Diagnostic Systems, Sparks, MD, USA).
Because clinical samples from the 2 patients were negative for all pathogens tested, urine and throat swab specimens were added to epithelial cells, and virus isolates detected were characterized by molecular analysis and electron microscopy.
Briefly, the cotton swab and ST solution were added to 500 μL of extraction buffer (200 mM NaCl, 200 mM Tris, and 20 mM EDTA; pH 8), 500 μL of phenol chloroform:isoamyl-alcohol (25:24:1; pH 7.9) (Sigma, Steinheim, Germany), 210 μL of 20 % SDS, and 500 μL of zirconia-silica beads (0.1 mm in diameter; Biospec Products Inc., Bartlesville, OK).
Urine and throat swab specimens from each patient were added to MRC5, MDCK, and Vero cells.
After thawing the swabs at room temperature for 30 minutes, 1250 µl diluted PBS [pH 7.4] were added to the swab and gently vortexed for at least 15 seconds.
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