h

p p ? F

Burst Size Determination:

A One-Step Growth Curve

exerclse
The average number of mature phage particles (virions) released by the lysis of a single cell is called the burst size. This number can vary between 20 and 200. The burst size can be determined by adding a small amount of phage to a known quantity of bacterial host cells and lysing the cells at 5-minute intervals by adding chloroform to the culture. The lysed cell material, containing virions, is then mixed with new bacterial host cells, plated out and incubated. By counting the number of plaques in the various time samples, it is possible to determine the burst size of coliphage Ta when it infects E. coli strain B. this period, which lasts approximately 12 minutes for Ta, the entire physiology of the host cell is reoriented toward the production of phage components: capsids, tail fibers, base plates sheaths, and viral nucleic acid. During this period only phage components are present with no mature virions in the lysate. If the lysate were assayed for phage, no plaques would be formed.

Late in the eclipse period, the phage components come together by self-assembly to form mature infective virions. The phage now enters the maturation stage of replication. The lysis of cultures with chloroform beyond 12 minutes after time zero will reveal the presence of mature virions that can produce plaques
on assay plates. Lysis by the phage of the population of infected cells does not occur all at once, but rather the number ofphage particles produced is characterizedby a normal distribution curve with a rise period. The rise period lasts for several minutes and represents the increase in the numbers of mature phage produced. The peak of the curve is the burst size for the phage

Figure 26.1 illustrates the procedure of this experiment. The first step is to add the phage to the susceptible bacteria. As soon as the two are mixed, adsorption begins. The phages collide in random fashion with the bacterial cells and attach their tails to specific receptor sites on the surfaces of host cells. The adsorption process can be stopped at any time by dilution. Time zero of adsorption is the time of mixture of phage and
bacteria.

Note in figure 26.I that 0.1 ml of coliphage To(2 x 10Vml) and2 ml of E. coli (5 x 108/ml) are mixed in the first tube, which is labeled ADS. The ratio of phage to bacteria in this case is 0.02, which
calculates out in this manner:

infection. In this exercise, you burst size for To.

will

determine the

0.1x2x108

2x5x108
or m.o.i.

0.2x108

10xI08

= 0.42,

or 1/50

This ratio is called the multiplicity of infection,

By referring back to figure VI.1 on page 000, we can see what is occurring in this experiment. Note that during the adsorption stage, DNA in the capsid passes down through the tail into the host through a hole produced in the cell wall by enzymatic action at the tip of the phage tail.

To accommodate the availability of time and materials, there are two options for performing this experiment. The first option is for students to work in pairs to perform the entire experiment. Figure 26.1 illustrates the procedure for this method. The other option, which requires much less media and time, utilizes a team approach in which students, working in pairs, do just a portion of the experiment; in this case, data arc pooled to complete the experiment. Figure 26.3 illustrates the procedure for this method. Your instructor will indicate which method will be used.

IIIr;EIII$I:IIL!
As soon as the phage DNA is injected into the host cell, the phage enters the eclipse period. During
To perform the experiment in its entirety, follow the procedures that are shown in figure 26.1.

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