( Cultur...)High-Throughput Cultivation of Bacterial Clones

Culture 192 clones simultaneously with the tempest thermoshaker

Jeff Braman
Stratagene

Figure 1: The tempest Thermoshaker

The tempest thermoshaker and a 96-deep-well round-bottom plate were used to cultivate bacterial clones in a high-throughput format. Early- and mid-log phase rates of bacterial growth were equivalent in aerated and nonaerated wells of the plate. Late-log phase growth rates of bacteria cultured in nonaerated wells were only slightly slower than growth rates measured in aerated wells. Final cell densities in aerated and nonaerated wells were identical. Flask-cultured bacteria demonstrated a slower initial rate of growth compared to plate-cultured bacteria, however, mid-log phase growth and final cell density were equivalent to plate-cultured bacteria. Well-to-well plate contamination was undetectable. Equal quantities of plasmid DNA were recovered from equal volumes of plate- and flask-cultured bacteria sampled at the end of the incubation period.

The Tempest thermoshaker is a compact benchtop incubator and shaker that is used for regulated temperature incubation (0 to 40C) and rotary mixing (200 to 1500 rpm) of multiple samples. The unit is supplied with a holder to accommodate fifty six 1.5-ml tubes. With Stratagenes minitube adapters, the thermoshaker can also hold 0.5- or 0.6-ml tubes. A microplate platform, also available, holds four, 96-well microtiter plates or two, 96-deep-well plates. The latter plates are particularly useful for cultivating bacteria in a high-throughput.

96-Well Plate vs. Flask Cultivation of Bacteria

Growth rates and final cell densities were measured for plate- and flask-cultured Epicurian Coli 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-cultured bacteria; however, mid-log phase growth and final cell density were equivalent to plate-cultured bacteria. No 550-nm absorption above background was observed in noninoculated wells of the plate.

A sample from every well of the plate was streaked onto an LB-ampicillin agar plate with a sterile toothpick and the plate was incubated at 37C for 24 hours. Bacterial growth was observed only from wells initially inoculated with bacteria. Bacterial growth was not observed in samples retrieved from noninoculated wells (data not shown). Therefore, cross contamination to alternate noninoculated wells did not occur in the aerated and nonaerated wells of the plate.

Plasmid Yield from Plate- and Flask-Cultured Bacteria

Plasmid DNA was isolated from 1 ml of plate- (aerated and nonaerated wells) and flask-cultured bacteria collected at the end of the incubation time period using the StrataPrep plasmid miniprep kit. Plasmid DNA yields were equivalent in plate- and flask-cultured bacteria, as judged by gel electrophoresis (Figure 3).

Fig.3

Conclusions

The Tempest thermoshaker allows 192 bacterial clones to be cultivated simultaneously in two, 96-deep-well plates. A special lid seals each well and prevents cross contamination. Bacterial growth rate, final cell density, and plasmid yield are virtually identical in aerated and nonaerated plate- and flask-cultured bacteria. The Tempest thermoshaker is a convenient and valuable tool when high-throughput capability is required.

REFERENCES

1. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. pp. 32-33.