( XL1-Blue bacteria containing thepB...)High-Throughput Cultivation of Bacterial Clones

XL1-Blue bacteria containing the pBluescript plasmid. One ml of LB medium, containing 100 mg/ml ampicillin, was added to each 2-ml well of the 96-well plate. Bacteria were inoculated into alternate wells of the plate at a density of 2 10⁵ cells/ml. The plate was sealed with its flexible plastic lid, which is designed to seal each well to prevent cross contamination. Perforations were made in the lid over the first 48 wells to allow an exchange of gases between the air space above the culture medium and the atmosphere. The 96-deep-well plate was then incubated at 37C in the thermoshaker (600 rpm).

Bacteria at the same cell density were inoculated into a 2-liter flask, which held 1 liter of LB medium containing 100 g/ml ampicillin. The flask was covered with a piece of sterile aluminum foil and incubated in a New Brunswick Scientific Model G25 Series floor incubator-shaker at 300 rpm.

Bacterial growth was monitored at various time points by removing samples from the aerated and nonaerated wells of both the plate and flask, then measuring absorption at 550 nm.¹ Bacteria cultured in nonaerated wells of the plate were removed by perforating the plate lid with a hot needle just prior to sampling. This method prevented the need to remove the lid from the plate and introduce air into the nonaerated wells of the plate.

Bacterial Growth Rates

Figure 2 demonstrates equivalent growth rates of bacteria in early- and mid-log phases in aerated and nonaerated wells of the plate. Late-log phase growth rates of bacteria cultured in nonaerated wells were only slightly slower than in aerated wells. Cell densities at the end of the incubation period in aerated and nonaerated wells were identical. Flask-cultured bacteria demonstrated a slower initial rate of growth compared to plate-cultu
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