The Meaning of Bacterial Death
For a microbial cell, death means the irreversible loss of the ability to reproduce (grow and divide). Noting the exception of VBMC organisms described previously, the empirical test of death is culture of cells on solid media: A cell is considered dead if it fails to give rise to a colony on appropriate medium. Obviously, then, the reliability of the test depends on the choice of medium and conditions: For example, a culture in which 99% of the cells appear “dead” in terms of the ability to form colonies on one medium may prove to be 100% viable if tested on another medium. Furthermore, the detection of a few viable cells in a large clinical specimen may not be possible by directly plating a sample because the sample fluid itself may be inhibitory to microbial growth. In such cases, the sample may have to be diluted first into liquid medium, permitting the outgrowth of viable cells before plating.
The conditions of incubation in the first hour after treatment are also critical in the determination of “killing.” For example, if bacterial cells are irradiated with ultraviolet light and plated immediately on any medium, it may appear that 99.99% of the cells have been killed. If such irradiated cells are first incubated in a suitable medium for 20 minutes, plating may indicate only 10% killing. In other words, irradiation determines that a cell will “die” if plated immediately but will live if allowed to repair radiation damage before plating. A microbial cell that is not physically disrupted is thus “dead” only in terms of the conditions used to test viability.
The Measurement of Bacterial Death
When dealing with microorganisms, one does not customarily measure the death of an individual cell but the death of a population. This is a statistical problem: Under any condition that may lead to cell death, the probability of a given cell’s dying is constant per unit time. For example, if a condition is used that causes 90% of the cells to die in the first 10 minutes, the probability of any one cell dying in a 10-minute interval is 0.9. Thus, it may be expected that 90% of the surviving cells will die in each succeeding 10-minute interval, and a death curve can be generated. The number of cells dying in each time interval is thus a function of the number of survivors present, so that death of a population proceeds as an exponential process according to the general formula:
where S0 is the number of survivors at time zero and S is the number of survivors at any later time t. As in the case of exponential growth, −k represents the rate of exponential death when the fraction ln (S/S0 ) is plotted against time.
The kinetics of bacterial cell killing is also a function of the number of targets required to be hit by a particular agent to kill a specific planktonic microbe. For example, a single “hit” could target the haploid chromosome of a bacterium or target its cell membrane. By contrast, a cell that contains several copies of the target to be inactivated exhibits a multihit curve. This analysis is graphically shown in Figure1.
Fig2. Death curve of a suspension of 106 viable microorganisms per milliliter. A: Single-hit curve. The one-hit curve is typical of the kinetics of inactivation observed with many antimicrobial agents. The fact that it is a straight line from time zero (dose zero) as opposed to exhibiting an initial shoulder, means that a single “hit” by the inactivating agent is sufficient to kill the cell (ie, only a single target must be damaged for the entire cell to be inactivated). B: Multihit curve. A cell that contains several copies of the target to be inactivated. The straight line portion extrapolates to 6.5, corresponding to 4 × 106 cells. The number of targets is thus 4 × 106, or four per cell.
