Germination and growth considerations
Out growth of a nonsterile BI is a function of:
- Initiation of spore germination
- Conversion to the cell form
- Cellular metabolism
- Cell division and multiplication.
Cell division and multiplication are required for identification of a nonsterile BI whether the determination of nonsterility
is based upon the visual detection of turbidity and/or a metabolic reaction that may result in, for example, a change in pH.
Visual detection of turbidity requires cellular metabolism and abundant growth of the microorganisms in the medium. For this
growth to occur, initiation of germination, conversion to the cell form, cellular metabolism, and cell division must first
occur. As illustrated in Figure 1, the time required to first discern that a BI is nonsterile, by turbidity or pH change,
is a function of both the time it takes for a spore to germinate and become a cell form and the cell division or generation
time. As can be seen (left panel), if the time for a spore to germinate and turn into a cell form is delayed by 1 hour but
the generation time is unaffected, the time delay for detection of nonsterility would also be delayed by 1 hour. If the generation
time of the cell form is affected (right panel), the delay in the time to detect nonsterility is related to the magnitude
of the increase in the generation time.
Figure 1: Effects of delayed germination and increased generation time on the detection of a positive test. (ALL FIGURES ARE
COURTESY OF THE AUTORS)
Visual detection of turbidity due to microbial growth requires on average a cell density greater than 106 cells/mL with smaller cells requiring a somewhat higher number. Table III gives the number of generations required to attain
greater than 106 cells/mL for starting numbers of cells ranging from 1 to 105; bacterial growth is by binary fission, therefore, each generation is a doubling of the population. Approximately 20 generations
(doublings) are required for one cell to provide detectable turbidity; i.e., greater than 106 cells/mL. The time for one cell to produce detectable turbidity would, therefore, be 20 times the generation time. BIs that
have been exposed to an ineffective sterilization process, with most or all BIs having a large number of surviving spores,
will yield visible turbidity in a sterility test sooner than those exposed to a process that yields a low percentage of nonsterile
BIs with most having 0 or one surviving spore.
Table III: Generations required to achieve greater than 1 million cells with starting cell numbers 1 to 100,000.
Considerable research has been conducted with different sterilization methods that demonstrate that the processes can adversely
impact spore germination (21). One could predict that for stressed populations of spores, delayed outgrowth would generally
be the result of longer germination times rather than longer generation times. To result in the latter, a process must inflict
an inherited mutation producing a rate-limiting effect on reproduction without it being a lethal mutation. However, many adverse
effects on the spore coat, spore cortex, or ionic constitution, for example, have been shown to delay germination without
resulting in longer generation times of the offspring (22). Therefore, merely on the basis of probability, one may conclude
that most outgrowth delays result from delayed germination time.
Targeting an average number of one surviving spore per BI appears to be a crucial aspect of the RIT protocol; the 30 to 80
nonsterile BI "window" provides a MPN average of 0.357 to 1.609 spores per BI. Therefore, the protocol is suited to accomplish
its intended purpose. When exposure to a sterilization process yields a BI with a single viable but possibly damaged spore,
variations in grow-out time can be ascribed to the metabolic/genetic constitution of that spore. However, if multiple spores
are present on an exposed BI, the resulting nonsterile outcome will be the result of the most rapid out-grower in the group.
This is true whether the spore driving the nonsterile outcome does so by faster germination and conversion to a cell form,
shorter generation time, or a combination of both. The fact that the outgrowth characteristics of individual spores can be
masked when multiple spores survive on a given BI is important for the subsequent discussion here; BIs that exhibit prolonged
grow-out times likely have one surviving but damaged spore.