Bacterial Cultures for Phage Work

From:
BACTERIOPHAGES
Biology and Applications

EDITED BY
Elizabeth Kutter
Alexander Sulakvelidze

Written by Karin Carlson
Department of Cell and Molecular Biology,
University of Uppsala, Uppsala, Sweden


Bacterial Stocks


For most bacteria, stocks are most conveniently maintained at –70°C.

To prepare bacterial stocks, a cryoprotectant such as glycerol (10%–30%, v/v) or dimethyl sulfoxide (DMSO, 5%–7%, v/v) is added to an exponential culture, and the samples then flash-frozen in ethanol-dry ice or liquid nitrogen for storage at -70°C.

Alternatively, bacterial strains may be lyophilized (after suspension in an excipient such as 20% skim milk or 12% sucrose).

Lyophilized samples can be stored (either refrigerated, frozen, or at room temperature) for several years without a noticeable decrease in titers.


Preparing Bacterial Cultures for Phage Infection


For phage work, the growth medium is a bacterial culture, generally in mid-exponential phase.

A bacterial culture intended for infection should be monitored for a couple of generations before it is infected to ensure that it is growing exponentially, using a spectrophotomer.

Alternatively, cells can be counted in special microscope counting chamber

Starter cultures may be initiated with a single colony and grown overnight or longer under whatever conditions the bacteria favor.

Such cultures normally enter stationary phase, and after dilution they will lag for an irreproducible period of time before resuming growth. Therefore, they need to be diluted at least 100-fold and go through several divisions to ensure that all cells are in the same growth phase at the time of infection.

An alternative approach works well with bacteria that do not lose viability upon rapid chilling: a starter culture grown to mid-logphase is immediately chilled in an ice bath and kept cold.
Such cultures need only a 20-fold dilution for regrowth the next day, and the cells usually resume growth in a more reproducible manner than when stationary starter cultures are used.

Determination of Bacterial Titers

to avoid unpleasant surprises, it is prudent to use microscopy to determine the total number of cells before infection whenever possible. This also provides potentially relevant information about the state of the bacteria; the morphology and degree of aggregation of cells may vary significantly with growth conditions or other stresses.

Infection with Phage

When a liquid bacterial culture intended for phage studies reaches the required cell density, it should either be chilled rapidly (to be used as a plating indicator or starter culture) or, preferably, infected immediately.

Both phage and bacterial titers must be known reasonably well at the time of infection to achieve the desired ratio between infecting phage and infected bacteria (defined as the multiplicity of infection or MOI).