Challenges of Microbiological Testing: Part 1- Controls and Validation

In the evaluation of bioburden, microbial limits testing, or any microbial examination of non-sterile products (the theory also applies to sterility testing and other microbiological assays), the objective is generally to enumerate or otherwise understand the quantity of microorganisms present in a product. This may be evaluated generally, as in a Total Aerobic Plate Count (TAMC), Heterotrophic Plate Count (HPC), or more specifically as in a Total Yeast and Mold Count (TYMC), Total Coliforms, Pseudomonas Count, Burkholderia Cepacia count, or Quantitative Bile-Tolerant Gram Negative Test. It can also be applied where the objective is to determine absence or presence of a specific organism in product, in other words, enumerating <1 or >1 Colony Forming Unit, as in the enrichment tests of USP<62> Tests for Specified Microorganisms.

In order to achieve this objective, some of the basics of scientific experimentation must be applied. This includes positive and negative controls. Most pharmaceutical compendia, e.g. pharmacopoeia, provide some guidance regarding negative controls, as well as a good set of parameters for positive controls.

Negative controls are employed to demonstrate any recovered microorganisms in the test solutions were not a result of contamination from a source other than the sample. This can include environmental controls, such as settling plates, contact plates, active air samples, and/or particulate counts. The media utilized (if applicable) in these controls should be the same as those of the actual test, but may be slightly different either in format or content. For instance, a USP<61> test would use Tryptic Soy Agar, a.k.a. Soybean-Casein Digest Agar, for TAMC. If one were to employ a contact plate as a control, it may contain Lecithin and/or Polysorbate, and would be in a contact plate format.

Another negative control commonly employed is a media negative control. While microbiological media has its own set of quality control tests, it is a good practice to include diluent and agar controls. The diluents used may be plated in agar in the same quantity as the sample solution, while the agar controls are generally just the agar plated with no other components of testing (aside from the petri plate). Diluent controls can also be employed as enrichments, where an entire bottle, or an aliquot of the same volume used in sample solution, may be incubated and evaluated for growth either by turbidity or subsequent streaking to a subculture medium.

Negative controls can go further, to encompass materials used in testing, such as pipettes, gloves, petri plates, weighing containers, weighing implements, tape and other secondary, tertiary, or quaternary packaging components for any testing supplies or media, disinfectants or sanitizers used, and expand to just about anything in or around the test and storage areas. However, outside of sterility failures and other high-risk investigations, these are rare. I only mention them to highlight the necessity for good controls and aseptic technique, as microbiological investigations rarely succeed in determining a definitive root cause, because the number of variables, and the resources required to investigate them fully, are prohibitively extensive.

Positive controls are a bit easier and well-defined in most procedures. As mentioned earlier, media QC is an integral part of testing. Positive controls are employed to demonstrate that media is suitable for use. This is known as growth promotion testing. In growth promotion testing, the test media is inoculated with a low level of microorganisms of interest. For instance, the general growth media employed in USP<61>, TSA, is inoculated with low levels of Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Aspergillus brasiliensis. The recovery is then compared to either a lot that is already qualified, to a known culture concentration value, or a suitable alternative. The growth promotion test is also employed for selective and general growth media, such as Sabouraud Dextrose Agar, Sabouraud Dextrose Broth, Soybean Casein Digest Broth a.k.a. Tryptic Soy Broth (TSB), Fluid Thioglycollate Medium, Rappaport Vasiliadis Broth, MacConkey Broth, MacConkey Agar, Mannitol Salt Agar, Cetrimide Agar, and Reinforced Medium for Clostridia.

Positive controls can also be employed in the test solution, either during routine testing, or more commonly in advance, to prove no enhancement or interference to recovery of microorganisms of interest. This is referred to as suitability testing, and will be covered in Part II of this series. Successful repetition of this test (and its results) on 3 representative lots of a product is often considered the industry standard for validation.