What is it?

Method suitability testing, simply put, is a process by which your microbiology testing lab determines the most suitable way to test your unique formulation to ensure accurate results. There are standardized methodologies available for routine microbiological testing, such as USP <61> and <62>. These standards are intentionally vague in some areas to allow flexibility to suit unique product formulations that may require unique testing protocols. These standardized testing protocols are a great starting point, but they may not always capture the full microbiological picture. That is where method suitability testing comes in.

Who needs it?

Regulatory bodies like the FDA require method suitability testing as a precursor to antimicrobial effective testing (USP <51>) or as a precursor to sterility testing (USP <71>) in aqueous pharmaceutical products. BUT (and this is a big but) these are not the only products that can benefit from this type of testing. Method suitability can be used to validate microbiological testing on any product from cosmetic mineral oil to turmeric capsules to birthday cake protein powder, and is called “preparatory testing” when it is performed for dietary supplement products. 

Perhaps you are asking yourself why you would perform (and pay for) testing that is not required regulatorily. Let’s take the cosmetic mineral oil as an example:

• Mineral oil does not dissolve in watery solutions (we’ll spare you from a deep-dive into polar vs. nonpolar solutions) and instead floats on the surface, just like you see with oil and vinegar. Let’s say that mineral oil, unbeknownst to you, is chalk full of E. coli. Daane Labs received a sample of mineral oil with no prior method suitability in place. The mineral oil is diluted in a buffer or liquid culture media, plated, and incubated. If oil and water don’t mix, neither will mineral oil and culture media. And if the mineral oil isn’t mingling with the culture media, neither is the E. coli. This could result in a false negative, or bias-low quantitative values.
• Alternatively, Daane Labs receives a sample of mineral oil that has had method suitability/ preparatory testing performed. In addition to the culture media, we know to add Tween80 to the culture media to get the mineral oil to go into solution. The E. coli in the sample mingles readily with the culture media and we report that your sample is positive for E. coli.

This type of interaction, where the nature of the product interferes with the ability to test it, is called matrix interference and it can create downstream challenges if standard test methods are ineffective in testing a product. We advocate for performing method suitability on any new formulations in a product due to the possibility of unexpected matrix interference. 

How does it work?

Method suitability testing begins with diluting your product to different strengths with a neutralizing buffer. The idea is that at least one of these dilutions will hit the sweet spot where antimicrobial compounds or properties have been neutralized. Next, a known quantity of several microorganisms are added to the diluted product. USP-compliant method suitability testing requires the use of the organisms in the table below. 

Organism Name	Organism Type	Gram Stain
Staphylococcus aureus (ATCC 6538)	Bacteria	G+ Cocci
Escherichia coli (ATCC 8739)	Bacteria	G- Rod
Pseudomonas aeruginosa (ATCC 9027)	Bacteria	G- Rod
Aspergillus brasiliensis (ATCC 16404)	Fungus (mold)	N/A
Candida albicans (ATCC 10231)	Fungus (yeast)	N/A

After the diluted product has been inoculated with the organisms indicated above, we go about testing the product the same way we would during routine testing. We want to mimic the “real world” as much as possible to ensure the method suitability testing translates seamlessly into routine testing down the road. The sample is plated on selective and non-selective culture media and incubated at appropriate conditions for microbial recovery (30-35C for bacterial cultures, 20-25C for fungal cultures). After the incubation period is complete, the apparent growth in the samples is compared to saline control cultures. According to the USP, “a suitable recovery scheme is one that provides at least 50% of this saline control count”.

Once the laboratory has developed a testing scheme which delivers at least 50% recovery of the control, the method suitability is done and you can begin moving to routine testing. Daane Labs staff have been performing method suitability testing and preparatory testing for over a decade and can confidently say that no matter what type of product you are manufacturing, we can find a suitable method for microbial testing. As always, reach out to Daane Labs for inquiries on method suitability and routine microbial testing. We are here for you.

References

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

So, you’re looking at your fungal certificate of analysis and you’ve hit the portion of the report that lists Insects Fragments, Pollen, and Hyphal Fragments. We know you don’t need a lot of help figuring out those first two, but Hyphal Fragments? That’s probably a new one for you and that’s exactly what this blogpost is for.

First, let’s get some vocabulary out of the way:

• “Hyphae” (pronounced HI-fee) make up the fungal structures that produce spores. 
• “Hyphal Fragments” (pronounced HI-full) are what we call broken up Hyphae. 

We like to explain that Hyphae and Hyphal Fragments are the “smoking gun” for the presence of whatever spores may be in your home. (Fun fact – Penicillium is named for the Latin word for “paint brush” because of its paintbrush-like appearance under a microscope.) So, picture a paint brush with us and imagine spores are produced then released from the tip of every bristle. That entire paintbrush you’re picturing would be considered Hyphae. Smash the paintbrush and you’ve got Hyphal Fragments. 

Most molds, including that paintbrush-like Penicillium, go through a growth phase where they aren’t making any spores at all. It’s all Hyphae. It’s totally possible… even probable, then… that air and surface samples of a moldy environment will show the presence of Hyphal Fragments. And that may occur with or without spores. Microbiologists typically need to see some spores to be able to identify which mold is growing, but the presence of Hyphal Fragments can be enough to say that mold is growing (but check with your home inspector, assessor, or environmental consultant for a full interpretation of your reported results).

We get a lot of questions asking why our report indicates “Growth Likely” when there are only a handful of spores and significant numbers of Hyphal Fragments present. We hope this analogy- and imagery-packed blog post clarifies some of that for you. If not, Daane Labs microbiologists are just one email or phone call away.

Burkholderia cepacia

An Emerging Challenge for Liquids Manufacturers

Burkholderia cepacia (pronounced burr-kuld-AIR-eeyah see-PAY-sheeyah) is not like your E. colis and Salmonellas of the world. Few people outside of healthcare settings have ever heard of this bacterium, and that’s why we’re here. We want to fill in some gaps for this emerging pathogen that is often overlooked when designing product testing protocols and specifications.

We’ll start with some recent history. There were two major multi-state outbreaks of Burkholderia cepacia complex in 2016 and 2017 that were traced back to liquid docusate, a medication used both in healthcare settings and home environments. The FDA issued product recalls following these outbreaks and this put drug manufacturers on notice: comprehensive microbiological testing is non-negotiable. While Daane Labs is not yet in the drug-testing game, we have seen increasing interest in Burkholderia cepacia testing among medical device manufacturers, personal care product manufacturers, and even some bottled water companies. Since no standard laboratory protocol existed for detecting this organism in non-drug substances, Daane Labs actually developed and validated our own, proprietary method to detect the presence of B. cepacia in customer samples. It’s something we are very proud of.

Alright, enough tooting our own horn and moving on… We can’t let a whole blogpost about Burkholderia cepacia slip by without diving into the actual bacterium itself. Science nerds, this is for you! Burkholderia cepacia is a non-fermentative Gram-negative bacilli that behaves as an opportunistic pathogen. It is multi-drug resistant, gamma-hemolytic, and mesophilic with an incubation time of 2-3 days. B. cepacia has extraordinary metabolic capabilities – it can reproduce in distilled water, for example. This bacterium was grouped with Pseudomonas species as Pseudomonas cepacia until 1981 because of their many shared morphological and biochemical characteristics. Once genomic sequencing became more widely-used, many Pseudomonas species were transferred to the novel Burkholderia genus. 

We’ll preface this conclusion by saying that Daane Labs isn’t in the business of telling you how you should test your product. You know your product and manufacturing process better than we do, so you really are the best person for that job. What we want to stress here is that microbial testing often goes beyond a simple Yeast & Mold count or a E. coli screen. Sometimes your product or process is unique enough that you might have to think outside the box and test beyond the typical indicator organisms. Maybe that means you look into testing your products for B. cepacia, or maybe it means you give consideration to some other facet of your operation. 

 

References

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192668/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC88367/

https://www.fda.gov/drugs/drug-safety-and-availability/fda-advises-drug-manufacturers-burkholderia-cepacia-complex-poses-contamination-risk-non-sterile

https://www.frontiersin.org/articles/10.3389/fbioe.2020.00630/full

http://medcraveonline.com/IJVV/IJVV-03-00064.pdf

https://bacdive.dsmz.de/index.php?search=1906&submit=Search

Whether you are a Quality Manager reading a Microbial COA or a homeowner reading the results of your in-home mold test, you may find yourself interpreting quantitative data, qualitative data, or perhaps both. You may not realize you’re even looking at these kinds of data, but that’s why we’re here. Understanding the differences between qualitative and quantitative data can provide the end-user of that data (you!) with context and guidance on how the data should be interpreted. 

Microbiologists utilize quantitative and qualitative tests for different organisms for different reasons. This can be tedious to explain without some foundation, so for now let’s use pregnancy tests as an example to compare and contrast qualitative and quantitative testing:

Typically, the first pregnancy test a woman would take is an at-home urine test. The result is either Positive or Negative. Pregnant or Not Pregnant. This is a qualitative test. It describes a quality of the woman via urinalysis. It is an excellent screening tool that provides a very basic amount of information. Next, the woman may visit a healthcare practitioner and have her hCG (pregnancy hormone) levels checked to confirm pregnancy and get an exact measurement of  her hCG. This is a quantitative test because her hCG level is quantified.

In summary:

Test:	At-Home Urinalysis	Blood Draw for hCG
Hypothetical Result:	Positive	425 mIU/ mL
Test/ Result Type:	Qualitative	Quantitative

We would expect this hypothetical pregnant woman to use these pieces of data differently, right? The Positive urinalysis might mean she stops drinking alcohol, begins taking prenatal vitamins, and exchanges salads for fast food. And since hCG levels increase at a known rate during the early stages of pregnancy, her hCG levels may be interpreted to help provide a due date. She didn’t need to know her hCG levels to decide to stop drinking alcohol. Vice versa, a Positive pregnancy test does not provide a due date. It was all important information for her to make time-sensitive, but different decisions.

Ya still with us? I hope so because we’re just about to get into the microbiology side of things.

Microbiologists typically use qualitative test methods when testing for microorganisms that cause disease, referred to as pathogens. This is common practice because the goal is always to have zero pathogens. One is too many. And why bother counting if one is too many? That is an oversimplification, but it fits. A peanut butter manufacturer never, ever, ever wants E. coli O157 in their product. Not one cell, not one million cells. It is a nasty bug that will make lots of people very sick, and so they would likely choose a qualitative testing approach. If even one cell is present, the test will be Positive for E. coli O157 and the manufacturer would have to destroy/ dispose of the tainted product. There is a lot of tedium in exactly how pathogens are tested for qualitatively and why such methods are chosen, but we’ll save that for another blog post.

Quantitative tests, on the other hand, are typically used to test for organisms which do not often cause disease. While it is industry dependent, many consumer products have an expected and accepted level of microbiological activity. Unless a product or area is sterile, there are bound to be microorganisms present. Quantifying what is present provides the data end-user with more precise information that can be compared to expected or previous results. Consider the following examples.

It would not be very helpful if your in-home mold test result simply said “Living Room – Positive” and “Outside – Positive”. Mold grows outside, you open a door or window, and mold comes inside. It is expected to be found inside and outside, so simply knowing that the mold is there doesn’t do much for you. What is important to know is how much mold is in your Living Room compared to how much mold is Outside. Quantitative testing is critical for this reason. Similarly, a certain level of microbiological activity would be expected in a cold-pressed raw juice product.  Knowing whether there are 100 CFU/mL or 100,000,000 CFU/mL is a more useful set of data than simply knowing there are microorganisms present. Simply stated, quantification allows for a baseline against which data can be compared. This may be in the form of product specifications, the Healthy Home Standard, or other pre-set guidelines on acceptable microbiological activity in a product or environment.

We hope this blog post was informative and helpful for our readers, as we hope for all of our blog posts so far. We welcome any feedback or topics you would like covered. After all, this is a blog for you. Don’t forget to check back next month for another blog post!

If you are a quality control professional testing your products for pathogens, a Positive test result is probably the last thing you want. A Positive test result can mean that your entire production batch must be disposed of or destroyed, costing you production time, money, and personnel. But, what if we told you that not all Positive test results were created equally? Let’s dive into some of the lingo you may hear around a Positive test result, what it means, and what your next steps could be.

Potential Positive
While it is unlikely you will ever find “Potential Positive” or “Potentially Positive” as a reported result on a Certificate of Analysis, you may receive a phone call from your lab letting you know that you have a test result that is potentially positive (That’s what we do over here at Daane Labs, anyway) What this means is that the first screening that was used to test for that specific bug, say E. coli, was Positive. The AOAC, USP, and FDA methods for pathogens like E. coli are agar-based methods and can yield some false positives. This is because those agar-based methods aren’t so selective that they are only testing for E. coli.

MacConkey Agar, for example, is the principal media in USP methods for E. coli. This agar selectively grows Gram-negative, fermentative rods. E. coli is a Gram-negative, fermentative rod… but so is Klebsiella pneumoniae, Enterobacter aerogenes, and many many more. These organisms can provide a false positive in your E. coli test because they are very similar bacteria with lots of characteristics in common. This is why you may hear the term “Potential Positive”. The test appears positive, but more testing is needed.

Once you have received word that you have a Potential Positive result on your hands, the next step should be confirming these results. This is where some decision making will have to happen on your end, and why it’s beneficial to receive a call from your lab when you find yourself in this position. There are essentially two routes to take from here: (1) secondary growth promotion testing and (2) genetic or biochemical confirmation. The first option is less expensive and faster, but not as selective as option two. The second option is to have the organism identified using PCR (genetic testing) or biochemical testing. We’ll get into these two options next.

Presumptive Positive
A Presumptive Positive result is one which has been confirmed using a secondary growth promotion. What does that mean? We’re basically talking about re-plating and re-incubating the suspect organism on different media. Let’s keep using E. coli for our example and say you had a Potential Positive result on MacConkey agar. The next step is typically to re-plate what grew on MacConkey onto a different agar, maybe an AOAC-approved agar like REC2 (Rapid E. coli 2). The idea here is to use a second selective media that can lend some credence to the original result. If it is Positive on MacConkey and Positive on REC2, then you’ve got a Presumptive Positive.

What now? Well, that will be up to you. You can accept this result as-is and make decisions using this information alone. That isn’t something we have seen many people do because the cost of getting a Confirmed Positive compared to the cost of destroying a batch of product is very low. If you choose to destroy your product over a Presumptive Positive, you are committing to throwing away tens-of-thousands of dollars over a “Probably”. Might as well go for the Confirmed Positive, right?

Confirmed Positive
A Confirmed Positive is simply one which is backed up with genetic or specific biochemical tests. This is what you want. You want certainty if you are going to destroy production batches. We have seen a sample test Positive multiple times for Salmonella. No matter what test we threw at it, it was Salmonella every time. The client asked us to move forward with confirmatory genetic testing, and surprise! It was something called Morganella. While super, duper similar to Salmonella, it was something else. Luckily she had a report that ultimately said “Salmonella – Negative” and not “Salmonella – Presumptive Positive”.

Obviously no one wants to be faced with an unexpected Positive result when testing for pathogens, but we’re here to help keep things on the rails when a Positive does pop up. Daane Labs has walked through these steps thousands of times: test, confirm, test, confirm, test, confirm. We know the drill. We know how others in different industries have handled these scenarios and are happy to help guide you during your decision making.