First, the indicator organisms are supposedly representative of the types of organisms that will be encountered during the
testing, but this is not necessarily true. The indicator organisms are a subset of organisms that are known to grow on properly
prepared media, but the organisms contaminating a manufacturing process may not belong to that subset. As a result the quality
control laboratory may repeatedly face what appears to be a microbial contamination event despite monitoring cultures that
show no growth. It is very important to know what organisms are normally present in the working environment and to include
these environmental isolates in a validation program. There is little value in proving that a medium will support the growth
of indicator organisms if the environment is full of organisms with very different cultivation requirements.
The second issue involves media handling. The qualification or validation study may require autoclaving the medium and then
pouring culture plates as the autoclaved material cools. In laboratories with a low testing load, the excess material is often
poured into large tubes or culture flasks to cool and solidify and then stored for future use, usually in a refrigerator.
However, when future testing is done, the second heating of the medium may not be captured in the qualification or validation
check and may not even be mentioned in the test procedure. If the agar is melted under gentle conditions and quickly poured,
there may be no problem, but in some cases, technicians have placed the flasks in microwave ovens to heat the medium while
taking a short break. With a powerful microwave oven it is easy to boil the medium for an unknown period of time. This can
destroy nutrients or produce toxic or inhibitory substances. Consequently, in laboratories where this second heating is a
common practice, this procedure must be captured in the validation and described exactly in the test procedures.
When preparing the validation protocol, the analyst should specify the recovery level expected for each of the indicator organisms.
Generally, recovery of at least 80% of the inoculum or control is desirable. Recovery of less than 50% is usually unacceptable
and should raise questions about the presence of inhibitory substances, especially when the testing is taking place in the
presence of a raw material or product intermediate. It may be necessary to introduce — and validate the performance of — an
agent that inactivates the inhibitor. It is important to set the specifications before the study is conducted and to hold
to these specifications. If specifications are not pre-set and the test system cannot meet general acceptance specifications,
it is very easy to set "acceptable" specifications that would otherwise have been unacceptable. The other problem is the "specification
creep" that occurs when a recovery of 78% is found and the specification is 80%. A quality assurance or quality control worker
who allows the 78% to pass will soon face the expectation that 75% should pass because it is "only slightly different from
the other one." Over the course of a few years, an 80% specification can gradually turn into a 70%, then 65%, specification.
The incubation temperature can have a major effect on the ability of an organism to grow in a given medium. It is well known
that yeasts and molds require a different incubation temperature than bacteria in a sterility test. Similarly, cells in tissue
culture are often extremely sensitive to small changes in temperature, not only for their growth but also in their susceptibility
to being infected or lysed by viruses. The analyst may need to develop temperature curves to justify the incubation temperatures
used for the test. It is also important to verify the incubator's ability to maintain the set temperature within the specified
range. If a four-degree temperature variation can cause a significant change in the test results, the incubator's ability
to hold a ±1° C range at all internal locations is critical. This may not be covered in a validation study, but it should
be included in the incubator's qualification studies.
In addition to the usual range from 20 – 40° C, it may be necessary to demonstrate the ability to grow organisms at extreme
temperatures. If it is necessary to monitor the presence of microbes in a hot or cold room, it will be necessary to demonstrate
an ability to cultivate thermophiles or psychrophiles in addition to organisms that grow under more normal conditions. While
the significance of these extremophiles may be open to question, their presence and the possibility that they may leave residues
such as endotoxins must be considered.