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.

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.