Biofilm Disinfectant Testing

Bacteria that are on a hard surface commonly exist together in a biofilm.

Bacteria that exist in a biofilm are particularly challenging to disinfect against due to the protection that these biofilms provide the bacteria that reside in them. Biofilms regularly exist in a matrix made up of highly sticky, glue-like substrates that help facilitate their formation by helping bacteria adhere to a surface, while also providing a protection barrier to the bacteria residing in them. As a result, being able to disinfect surfaces with biofilms is a particularly challenging, but lucrative, endeavor for products to be able to claim.

Two testing methodologies have been developed by ASTM for the culturing of and assessment of biofilms, respectively: ASTM E3161 and ASTM E2871. ASTM E3161 describes on how to culture biofilms using a CDC bioreactor onto borosilicate carriers, and ASTM E2871 describes the conduct of the efficacy assessment. Efficacy is determined by assessing whether the test substance can attain at least a 6 log₁₀ reduction when compared back to a parallel positive control.

Summary of both the ASTM E3161 and ASTM E2871 Test Methods:

• The products tested must already demonstrate disinfection efficacy against both S. aureus ATCC 6538 and P. aeruginosa ATCC 15442. The method-specified organisms for substantiating a biofilm disinfection claim are S. aureus ATCC 6538 and P. aeruginosa ATCC 15442.
  • EPA allows for claims against liquid formulations; spray formulations are evaluated as a liquid by removing an appropriate amount of liquid from the spray product. For towelette products, the EPA should be consulted in advance.
• An 18-24 hour culture is used to inoculate a fully assembled bio-reactor that contains an appropriate amount of growth medium and test carriers. The reactor is set up such that the incubation conditions are maintained for the specific test microorganism.
• A baffled stir bar inside of the reactor is turned on to initiate the first phase of biofilm growth (Batch phase) for 24 hours.
• Following the conclusion of the batch phase, the reactor is set up to allow for a continuous flow of fresh growth media into the reactor, while allowing old media to drain out. This phase of growth (CSTR phase) is allowed to run for an additional 24 hours.
• Following the conclusion of the CSTR phase, the carriers are harvested into sterile conicals and tested by applying a volume of the product (test substance) to the carrier for the sponsor-requested contact time. The carriers are then neutralized at the conclusion of this contact time.
  • Control carriers are treated and neutralized in the same manner as in the test with an inert substance.
• The neutralized carriers are then enumerated and a reduction is calculated from the results of the assay.

To obtain a biofilm disinfection claim, three batches of the product at or below the lower certified limit (LCL) must demonstrate at least a 6 log₁₀ reduction in ≤10 minutes when the test substance treated carriers are compared to the control treated carriers.

Strengths of the ASTM E2871 Test Method

• The method is quantitative, allowing for clear interpretation of results.
• The method allows for the substantiation of a highly marketable biofilm disinfection claim.

Weaknesses of the ASTM E2871 Test Method

• Culturing of biofilms, especially with organisms that have very particular growth requirements for biofilm formation, can be especially challenging to elicit healthy biofilm growth.
• Due to the nature of how the biofilm is cultured, variability between carrier replicates is sometimes observed.
• The limit of detection can be somewhat high for potent products due to the volume of neutralization agent that may be required to inactivate the product.
• The cultivation of a biofilm requires considerable technical expertise in the successful operation of equipment and generation of viable biofilm for testing, decreasing the number of laboratories that may be able to perform the test and increasing the likelihood of lab-to-lab variability.

Microchem Laboratory is one of a handful of labs in the world with skill and expertise in this particular method. If you are interested in setting up a study with the lab, please contact the laboratory for a quote.