As a food, dietary supplement, or personal care product manufacturer, your products are expected by the public and by regulatory bodies to be safe for use and consumption. But what is safe, exactly? Product safety can involve everything from how the product is labeled (if it contains gluten, does your label state that?) to whether pathogenic microorganisms are present in your product. There are entire publications dedicated to ensuring a high-quality finished product, so we are going to focus here on how to determine whether your product meets microbiological guidelines for human use and consumption.

This is one area where we at Daane Labs field a ton of questions because the guidance available from regulatory bodies like the FDA is pretty vague. Our clients want a list of organisms to test for and the acceptable levels. We hate to be the bearer of bad news, but no such lists exist. It’s up to you to develop that list and those levels, called product specifications, for your product yourself. But don’t worry! This blog post isn’t over yet and we’re here to help you develop those specifications for your products.

Big picture: your individual product specifications should be a reflection of the nature of your product. This includes everything from raw ingredient quality, manufacturing process, packaging, storage conditions, intended use, etc. You should have a solid understanding of what the lifecycle of your product is to be able to make the most comprehensive, competent decisions about what to test for and how. Let’s start with a a couple examples of what product specifications may look like and we’ll work our way backwards on how we got here:

Example 1 – Turmeric Capsules
Organism	Specification
Total Aerobic Count	<10,000 CFU/ gram
Total Yeast & Mold	<1,000 CFU/ gram
E. coli	Negative in 10 grams
Salmonella spp.	Negative in 10 grams

In the above Example 1, this product would be “allowed” to have up to 10,000 CFU/g total aerobic organisms, up to 1,000 CFU/mL yeast and mold, and no E. coli or Salmonella spp. at all. 

Example 2 – Eye Drops
Organism	Specification
Total Aerobic Count	<10 CFU/ mL
Total Yeast & Mold	<10 CFU/ mL
Total Coliform	<10 CFU/ mL
E. coli	Negative in 10 mL
Salmonella spp.	Negative in 10 mL
Pseudomonas aeruginosa	Negative in 10 mL
Burkholderia cepacia	Negative in 10 mL

In Example 2, the levels are much more stringent for the quantifiable organisms and there are additional pathogens, Pseudomonas aeruginosa and Burkholderia cepacia, that must be confirmed absent in the product.

So — why the difference? You would think that if Pseudomonas aeruginosa is a no-go in one product it should be a no-go in all products, right? You’d be right, but with a caveat. The nature of your product will determine the likelihood certain organisms will be found there, thereby often rendering testing for certain organisms wasteful. For example, Pseudomonas aeruginosa and Burkholderia cepacia are both Gram-negative rods heavily associated with industrial water systems. If your entire manufacturing process is void of such a system, such as dry-blending and filling turmeric capsules, you would waste a lot of money testing for waterborne organisms. Manufacturing eye drops, on the other hand, would require industrial deionized water systems and would therefore risk introducing waterborne organisms into the product.

Now that we’ve covered how to determine which organisms to test for, we’ll get into how to set those levels for things like Total Plate Count. This will ultimately be decided, again, by the nature of your product. Herbal dietary supplements are an excellent example of this principle. The American Herbal Products Association (AHPA) has specification recommendations for “powdered botanic extracts” as high as 10,000,000 CFU/ gram for total aerobic plate count. Such a high level of microbial activity is considered acceptable because the product contains “dirty” raw plant material. Setting the quantitative specifications depends on the microbiological activity of the ingredients going into the product, whether the product undergoes any sort of pasteurization or sterilization, at what temperature the product is stored and used, etc. You may even find it useful to run several repeat tests on your ingredients and finished product to get baseline values for microbes in the product, and set specifications accordingly. 

Whether the presence of bacteria or fungus is an inherent property of the product, such as turmeric capsules, or an acceptable risk that can be mitigated, it is important to understand all of the challenges your product faces. There are a variety of quantitative and qualitative methods that have been published by the USP, FDA/ BAM, AOAC, and more, and we perform many of these methods here at Daane Labs. If you are struggling with method selection, the team at Daane Labs are experts in current methodologies and we are always happy to help our readers make the best scientific decisions you can for your business.

Since Daane Labs is a testing laboratory rather than a testing consultant, we are unable to direct you on what your product specifications should be. What we really want you to know is that as long as your specifications are guided by a comprehensive understanding of your product and you have documented reasons for testing plan, you’re on the right track. Regulatory bodies want to see that you know the risks to your product and that you are doing your due diligence to prevent those risks from reaching the public. 

Disclaimer: Daane Labs does not set product specifications and the content of this blog post is not intended to set specifications for any existing or future products.

References

http://www.ahpa.org/Portals/0/PDFs/Policies/14_0206_AHPA_micro_limits_comparisons.pdf

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

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:

Category	Product Description
1	Injections; other parenterals including emulsions, otic products, sterile nasal products, and ophthalmologic products made with aqueous bases or vehicles
2	Topically used products made with aqueous base or vehicles; nonsterile nasal products and emulsions, including those applied to mucous membranes
3	Oral products other than antacids, made with aqueous bases or vehicles
4	Antacids made with an aqueous base

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:

Category 1
Bacteria	No less than (NLT) 1.0 log reduction from the initial calculated count at 7 days, NLT 3.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count to 28 days
Yeast and Mold	No increase from the initial calculated count at 7, 14, and 28 days
Category 2
Bacteria	NLT 2.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days
Yeast and Mold	No increase from the initial calculated count at 14 and 28 days
Category 3
Bacteria	NLT 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days
Yeast and Mold	No increase from the initial calculated count at 14 and 28 days
Category 4
Bacteria, Yeast, and Mold	No increase from the initial calculated count at 14 and 28 days

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)

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.

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.

Air sampling for fungal spores has been in the limelight for a long time. The yin to air sampling’s yang is surface sampling and we think it deserves some attention as well. Something that often gets overlooked with air sampling is that the mold isn’t actually growing in the air – that’s just where evidence of fungal growth is most easily found. Fungal growth occurs on surfaces and releases spores into the air, and then those spores land in the air sample and “Ah ha!”, mold has been found. But what about that surface? Where exactly is that mold growing? That is where surface sampling comes in. Surface sampling may not be required for every project, but it can add value to any project when done correctly and purposefully. Here, we explore some of the most common techniques and materials used by industry professionals for surface sampling for mold.

 

Bio-Tape™  Slides

Bio-Tape™  slides are sterile, flexible, plastic microscope slides that consist of an adhesive sampling area and label, and are the ideal tool for sampling flat surfaces such as walls, tables, joists, etc. The adhesive area should be lightly and evenly pressed to the surface in question to collect surface particulate. These are some of Daane Labs’ favorite surface sampling products because we can stick them straight under a microscope for analysis with very minimal prep time (read: faster analysis… read: faster results). Oh, and what’s that? Daane Labs provides these for free? Yes, yes we do. 

 

Copan Swabs 

Copan swabs are another product that Daane Labs provides to clients free of charge. These swabs are sterile (like the Bio-Tape™ slides) and are best used for sampling surfaces you can’t quite get to with a Bio-Tape™ slide. These could be areas like the corner of a windowsill, inside the slats of an A/C vent, between bathroom tiles, etc. Roll the swab evenly across the surface you are sampling, ensuring all sides of the swab come in contact with the surface. 

 

Office Tapes

While we recommend using a product specifically designed for surface sampling, we understand that won’t always be the best option for our clients. Maybe you ran out of supplies (Let us know and we’ll get them to you for free!) or maybe you have a personal preference after years in the field. Regardless of which type of office tape is used, the sampling technique should be about the same. We recommend holding the tape with your thumb and pointer finger, sticky side out. Lightly touch the tape to the surface you are sampling, then stick the tape to the inside of a plastic sandwich bag or directly to a microscope slide. Here are the types of office tape we’ve seen used over the years and what we have to say about them:

 

• Transparent Scotch® Tape
  • This type of tape is a good replacement option for Bio-Tape™ slides because it is transparent, narrow (fits on a microscope slide), and affordable. 

• Frosted Scotch® Tape
  • Frosted tape appears frosted to the naked eye because of microscopic bubbles and imperfections in the tape. These bubbles and imperfections make it very difficult to get a clear picture of what is actually on the sample, and is not an alternative we would suggest using. We can and have and will analyze frosted tape samples, but we still think it is important for you to understand its shortcomings as a surface sampling product.

• Packing Tape
  • Packing tape has similar benefits to Transparent Scotch® Tape, except that it is much wider and therefore requires additional processing by the lab. We are unable to analyze an entire piece of packing tape and must either cut or sub-sample the tape to get it under the microscope. 

 

Q-Tips

Q-tips are non-sterile cotton swabs that can be used in a pinch to collect a surface sample, but it is not something Daane Labs recommends doing regularly. Any non-sterile product used for sampling risks having something already on it, so there can be no telling if what is on the swab was there before or after sampling. Additionally, Q-tips tend to be softer and more delicate than Copan swabs and can deteriorate during processing (i.e., cotton tip can somewhat come apart when exposed to lactophenol cotton blue staining agent).

 

Bulk Samples

A bulk sample is essentially when the material suspected of growing mold is itself submitted rather than taking a surface sample. Examples of bulk samples we have received are cutouts of drywall, HVAC insulation, wallpaper, roof shingles, baseboards, etc. It is difficult to provide a lot of guidance on this sample type simply due to the fact that these samples can vary so widely. Ideally, bulk samples should be small and representative of the area of concern. 

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.

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.