Antimicrobial effectiveness testing is a protocol which tests the effectiveness of the antimicrobial compounds in non-sterile, aqueous pharmaceutical products. This type of testing is done to ensure that the antimicrobial compounds present in the product are capable of inhibiting the growth of microorganisms that may be introduced during or after the manufacturing process. Microorganisms may be introduced to these types of products in any number of ways – one of the most common is reusing a jar of lotion which requires the consumer to dip their fingers into the jar. Even just-washed hands aren’t sterile, so it is inevitable that microbes will be introduced. What the testing here aims to do is provide evidence that the antimicrobial compounds can inhibit those microbes over a period of time.

So, where do we start? If you haven’t read our blog post about Method Suitability Testing, head there first to understand the true beginning of the process. If you’re already versed in method suitability, keep reading…

Once Daane Labs has performed method suitability and understands the strength of your preservative system, we are ready to begin challenging that system by introducing known levels of bacteria and fungus. The challenge organisms used in USP 51 Antimicrobial Effectiveness Testing are the same as those used for method suitability testing: Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Aspergillus brasiliensis (ATCC 16404), and Candida albicans (ATCC 10231). 

Ideally this testing is performed in five original containers (i.e., the final manufactured package) to mimic real-world conditions as closely as possible. The challenge organisms are inoculated directly into the five containers and we perform a “Day 0” plate count which gives us our baseline. The inoculated containers are incubated at 20-25C for up to 28 days and are plated periodically during that time. 

USP has categorized non-sterile aqueous products into four categories which are tested at different intervals and have to meet slightly different criteria for antimicrobial effectiveness testing. The four categories are summarized in the table below:

What we expect to see with antimicrobial effectiveness testing is, at minimum, no increase in microbial growth and, at best, a microbial population decline over time. The four categories indicated above have unique requirements for the antimicrobials to be considered effective. The table below describes the criteria:

As you can see, there is a lot that goes into antimicrobial effectiveness testing from method suitability to final calculations. Luckily, we’ve been doing this for a long time and we are experts at compendial USP methods, USP 51 included. If this is a type of testing you need, or think you need, reach out! Daane Labs staff are always happy to help new and existing clients with antimicrobial effectiveness testing, method suitability, and routine testing.

References

https://latam-edu.usp.org/wp-content/uploads/2021/01/USP-NF-51.pdf (USP 51)

Daane Labs designed our mold reports to be understandable by everyone from highly-specialized industrial hygienists who take mold samples to homeowners with allergies and questions. Our hope here is that regardless of where in that spectrum you fall, this blog will help you understand that report a little bit better. If you’re looking at a Daane Labs mold report and have some questions about the Background, this one is for you. But before we get into that, you’ll need to know a bit more about how exactly your mold samples are collected and analyzed.

Samples are collected by using a pump to pass a known amount of air over an adhesive surface inside a disposable device called a “cassette”. As the air is pumped through the cassette and over the adhesive surface, the particulates in the air get stuck to the adhesive. These samples are given to the lab for processing, where we open the cassette and use a microscope to look at that adhesive surface where all of the particulates have gotten stuck. We count and identify the mold spores on the adhesive, but there’s usually a lot more than just mold in each sample.

All that other “stuff” in the sample is better known as the Background, and we assign a value to give you an idea of how much “stuff” is in the sample. Short of some extraordinary event that severely impacts outdoor air quality, such a wildfire or nearby building demolition, the Background of outdoor samples is typically pretty low and does little to impact the analysis. The Background of indoor samples, however, can vary greatly from room to room within the same house and frankly, it’s what we get the most questions about. So let’s get into it.

The Background of indoor samples is usually composed of the typical components of house dust: human skin, pet dander, insect fragments, fibers from clothing, etc. The value of the Background directly correlates to the amount of “stuff” in the sample: 1 indicates there are almost no particulates in the sample and 5 indicates that the sample has essentially been overrun by particulates. So what does that mean? We’ll use an analogy we’ve leaned on for years to explain why the Background matters and how it impacts your test results.

Imagine you are looking at an old oak tree, full of large branches and lush leaves. You are asked to count the number of birds in the tree. You could probably count the birds on the lower branches, maybe some of the birds sitting right on the outside of the tree. But you could assume there are birds in the tree that are hidden by the branches and leaves that you are unable to count. So even if there are 100 birds in that tree, maybe you can only see 8 or 12 of them. Compare that to counting the birds in a dead tree with no leaves and only a handful of branches. You could probably see just about every bird in that tree and you would be more confident in that bird count, right?

Similarly, we can see just about every single spore in a sample with a low Background and we can make the assumption that we cannot see every single spore in a sample with a high Background. This is called a negative bias and it increases as the Background value increases. This doesn’t mean that a report with a high Background is wrong or that the lab report is any less useful… not at all. The Background simply aids the investigator in determining how to interpret the report and next steps. The investigator may have the expertise and project-specific knowledge to apply the data to that project, or they may decide to collect additional samples. While Daane Labs staff is always more than happy to provide insight into the data within the report, interpreting how a high Background impacts next steps is left to the investigator who collected the samples, such as a building inspector or mold assessor.

Stachybotrys (pronounced stacky-BOT-riss) is known colloquially as the “Black Mold”. This particular mold rose to fame, or infamy rather, following reports in the mid-1990s of acute pulmonary illness in children living in a home where this mold was found. Stachybotrys gained further notoriety in 2004 when a multimillion dollar lawsuit was awarded in favor of homeowners whose Stachybotrys remediation insurance claim was mishandled.

The presence of Stachybotrys inside of a structure is typically indicative of long-term water damage. Stachybotrys is known as a tertiary colonizer, which means it is a mold that is likely to colonize a surface after multiple organisms (primary and secondary colonizers) have already done so. These primary and secondary colonizers have lower moisture requirements than tertiary colonizers. This means that the longer something is water damaged and the more water intrusion that is allowed to occur, the more likely you will see Stachybotrys colonize the area.

Stachybotrys appears black both macroscopically (to the naked eye) and microscopically. Stachybotrys spores are pill-shaped, rough-textured, and black when they are fully mature. Oftentimes, Stachybotrys appears as a slick, black biofilm when growing on cellulose materials such as gypsum, wallpaper, books, or baseboards.

Interestingly, because Stachybotrys has such a high affinity for water, the spores tend to remain on the surface where the mold is growing. Most indoor mold types readily release spores into the air, making these molds easy to detect with air sampling. Stachybotrys, on the other hand, could be flourishing on indoor surfaces and remain undetectable in the air. This is why it is important to take surface samples in addition to air samples. Surface sampling, such as tape lifts, swab samples, and bulk samples can provide a definitive answer on what is growing where. Airborne spores can come from anywhere, and surface sampling data paints a more complete picture of the health of a building.