Kill the Bioburden, Not the Biological Indicator

Understanding the purpose of the biological indicator can guide the development of an
effective sterilization process.
Apr 01, 2017 By James P. Agalloco BioPharm International Volume 30, Issue 4,
pg 50–52

Satirus/Shutterstock.comSterilization processes are used to

ensure the safety of patients treated with products and
materials expected to be sterile at time of use. The objective
is to eliminate microorganisms in and on products that are
introduced into the body in a manner that defeats the
ordinary protections of skin, intestines, and other safeguards
present. In considering patient safety with respect to sterility,
a minimum requirement of one contaminated unit in a million units is considered acceptable
for sterilized materials (1). The original term for this value, sterility assurance level (SAL), is non-
intuitive and defining it usually entails the use of the word ‘probability’. Increasingly, this value
is being called the probability of a non-sterile unit (PNSU). In routine practice, additional
precautions are taken so that this minimum expectation is substantially exceeded.
The calculation of PNSU uses Equation 1, in which the lethality delivered, D-value, and initial
population of the microorganism are inserted.
The equation is simple enough; however,
there is a common misconception in its use.
The problem lies in the incorrect use of
values for population and resistance from
the biological indicator rather than for the
bioburden. The safety expectation relates
to the routine use of a sterilizer where the bioburden is present, rather than the initial or
periodic validation of the sterilization process when a biological indicator is employed. In the
majority of instances, materials sterilized in conjunction with the validation exercise are not
intended for patient use. The minimum PNSU as derived from the bioburden present is the
critical concern.
Equation 2 estimates the PNSU for a 3-
minute process at 100 °C with a starting
population of 100 CFU/unit and an
estimated D100 of 0.0003 minutes (2).
It should be immediately evident that this
extremely short and low-temperature sterilization process provides an overwhelming margin
of safety that is nearly 10,000 times greater than the minimum expectation. The moist heat
resistance of the bioburden is so minimal at these conditions that there is essentially no
chance for its survival (3). This is true even though the process is 3 minutes at 100 °C, not the
more commonly (and wrongly expected) process performed in excess of 121 °C. The lethality
of this low temperature process cannot be established with the conventional biological
indicator of Geobacillus stearothermophilus, whose resistance is such that the assumed
process would have no meaningful impact on its population.
Requiring destruction of a 106 population
of G. stearothermophilus to the minimum
PNSU expectation of 6 would require a
process at 121 °C and an F0>10 minutes.
Such a process offers no benefit to the
patient because the bioburden will already have been killed well beyond minimum
expectations at the lesser condition. If a 121 °C process delivering an F0=10 minutes were
utilized instead, the PNSU would be as shown in Equation 3.
The estimated PNSU in this example would be extreme: not more than one positive in more
than three million times the minimum requirement. The only justification for using such a cycle
is to destroy a bioindicator that has no resemblance to the native bioburden, is present at a
concentration that exceeds any reasonable real-world situation, and has extreme moist heat
resistance. Killing the bioindicator is certainly safe, but this approach arbitrarily increases the
adverse process impact on the product. The real target in sterilization is always the
bioburden, which is generally far easier to kill. Therefore, the sterilization process should be
developed with that as the objective.

The purpose of the biological indicator in sterilization is not to define the process, but rather
to measure it. The steps involved in sterilization process development are outlined in Figure 1.

Figure 1. Establishing a
bioburden-based sterilization process. Figure is courtesy of the author.