The Importance of Suitability Testing

Posted by Caron Ockerman

Many pharmaceutical, cosmetic, and toiletry products contain preservatives that help extend the product’s shelf life, keeping spoilage at bay for a certain period of time, and also act with antimicrobial properties if microbes are introduced.  A consumer dipping a finger into a jar of facial cream has introduced microbes into the cream, and the preservative should effectively kill or reduce the number of microorganisms (for more information on preservative effectiveness, please read the blog “Preservative Efficacy Testing: Guidelines to an Automated Simplified Testing System”).

Why we need suitability testing? The measurement of preservative test requires the ability to count the number of surviving microorganisms after exposure to the antimicrobial agent in the product.  Bioburden (also called microbial content) testing has the same requirement because the assay depends on the ability to recover viable microorganisms in the presence of potentially antimicrobial in the product or raw materials. Carryover of residual inhibitors from the product could inhibit growth in the recovery medium, leading to poor microbial recovery. This potential residual activity must be neutralized and it is necessary to demonstrate the adequacy of neutralization for the test. This demonstration of neutralization in compendial microbiological tests is known as demonstration of method suitability.

Therefore, before conducting any microbiological test, it is important to find the most effective neutralizing agent or dilution that will create a suitable environment for growth, allowing for accurate results during microbial content and preservative efficacy testing.

How to test for Suitability- Suitability in the presence of product is the ability of microorganisms to grow when introduced in low numbers.  Following the plate count method for suitability in USP Chapter <61> MICROBIOLOGICAL EXAMINATION OF NONSTERILE PRODUCTS: MICROBIAL ENUMERATION TESTS, the appropriate growth media alone (control) and the product (test) are inoculated with 10-100 cfu.  The inoculated product is then diluted 1 to 10.   If growth occurs on control plates, but does not grow on test plates, or counts are half that of the control plates, neutralization or further dilution may be necessary to show suitability of product.

How to Neutralize Inhibitors- Depending upon the type of product and the inhibitory system being tested, different diluents should be used as some products may require neutralization of a strong preservative that would suppress growth of naturally occurring microbes.  Table 1 below summarizes some neutralizing agents used in common neutralizing broths such as Modified Letheen Tween (MLT) broth, D/E Neutralizer, and Tryptone Azolectin Tween (TAT) broth among others.

Sometimes, even the use of a neutralizing agent may not be enough, and dilution further than 1 to 10 may be necessary.  It is important to keep in mind that five different organisms are being tested in one product, and what allows one organism to grow might not work as well for another.  Essentially, it is important to find any and all different parameters that make a product suitable for inoculation.

Once a proper neutralizer or dilution is found for each product and organism, microbial content testing and PET testing can proceed.  Without performing suitability testing first, microbial content testing and PET may yield false negative results; bacteria, yeast or mold may be present, but simply remain dormant until the optimal growth conditions are introduced.

The suitability test can be performed very simply and quickly with the BioLumix system.

Use of BioLumix System for suitability testing- The BioLumix system is capable of determining whether a product meets suitability standards.  Products and controls are inoculated, then 1.0 mL is added to the BioLumix vials, entered into the instruments, and initial results may be observed within 18-24 hours.  The figure shows examples of curves obtained from the BioLumix system using Staphylococcus aureus with various preservative systems.  The top picture shows a product that is properly neutralized. While as the bottom picture demonstrates that without proper neutralization, product can hinder the growth of organisms.

In the BioLumix instrument, if the detection time (DT) of the media without product is within a couple of hours of the product containing media it means that enough neutralization is achieved. If there is a DT without the product in the positive control; but the sample containing vial does not have a DT it means that the product prevents the growth of bacteria and a different neutralization scheme needs to be employed.

What are the advantages of the BioLumix system?
Time Saving: The results are available much faster, for example, the results of the Yeast and Mold vials occurred in less than 48 hours, instead of five days for countable colonies. All products tested with bacteria using the automated BioLumix assay yielded results typically within 10-18 hours, instead of 48 hours for the plate count methodology. The advantage using the BioLumix system is that you can see results an entire day earlier if the product is exhibiting any sort of inhibition.

Labor Saving: The setup of the assay can be done much faster using the BioLumix system as opposed to traditional plating methods, saving significant hands-on labor due to its automation and simplicity of use.

High Correlation with USP: The BioLumix System showed a high correlation between the instrument results and the USP methodology.