Nicole Cotta (00:04):

Hello, and thank you very much for joining us today for the first in our series of webinars focused on some of the analyses for DMR-QA. Today we will talk about wastewater microbiology and some tips to improve your analysis. I'm your moderator, Nicole Cotta. Your speaker today will be Dr. Christy Abbas. Before we get started today, I just want to take a couple of minutes and highlight some of the features on the console page for the webinar. In the middle, you will have the slides that Christy will speak through. In the top left, you have the question and answer box where you can submit your questions that we will answer after the presentation. The bottom left, we have a list of resource links related to this topic and some other related topics. In the top right, we have Christy's bio if you would like to learn more about Christy. Then in the bottom right we have a survey, and we would love to get your feedback about the webinar, about future topics and other areas of interest.

 

(01:31):

Now, our focus for today's webinar will be to discuss some overall tips to improve your microbiology analysis. Some key topics we'll focus on will be the evaluation criteria for wastewater microbiology. We'll talk about proper microanalysis techniques and some best practices and recommendations for accurate results. Our speaker today is Dr. Christy Abbas. She holds a PhD in applied chemistry from the Colorado School of Mines. She's been with Waters ERA for almost five years. Previously, she worked for an analytical instrumentation manufacturer, and she's held positions in the nuclear, pharmaceutical and chemical industries. In her role with the Waters ERA, her job is to expand our product line to better serve the needs of our customers. With that, I will turn it over to Christy.

 

Christy Abbas (02:35):

Thank you, Nicole. Hello everyone and welcome to ERA's webinar on getting better PT results for wastewater micro samples. Today on the agenda we're going to cover four main areas. We'll go over the NELAC criteria for wastewater micro samples. We'll go over calculating PT limits using the NELAC criteria, and we'll cover ERA sample design and our intended use. These three areas are really just to give you a general understanding of why we make our products the way that we do, how are they evaluated, and what is ERA's product. This is simply to give you a good foundation and a basic understanding so that when we do move on to our main focus, which is keys to improved results, everyone will have a similar background. Hopefully the presentation is going to cover most of the issues. But at the end, we'll have a Q&A session where Matt Graves will join us. Matt is our organic technical lead at ERA.

 

(03:37):

But before we get into the meat of the webinar, I would like to introduce you to ERA. ERA was established in 1977. We're headquartered in Golden, Colorado just 10 miles west of Denver. Our facility here in Golden includes more than 25,000 square feet of dedicated laboratory space with areas dedicated to inorganics, organics and customs. ERA is a provider of proficiency testing, products and certified reference materials to more than 9,000 environmental laboratories in more than 80 countries. Our products are available in a variety of matrices, including wastewater, drinking water, air and emissions, soils, and more. ERA is a TNI and ISO 17043 accredited PT provider. We are also a certified reference material provider and a chemical testing laboratory by A2LA accreditation. We are registered to ISO 9001 by the National Quality Assurance Organization. In addition to serving environmental laboratories, we also manufacture TOC and conductivity reference materials and calibration standards for pharmaceutical medical devices and biotech manufacturers.

 

(04:57):

Let's start with the NELAC criteria. The National Environmental Laboratory Accreditation Conference, sometimes referred to as the NELAC Institute, is the body that defines the required target analytes, manufacturing ranges, acceptance criteria and proficiency testing reporting limits that are used by all approved PT providers to design their samples and evaluate the results. These are all communicated to the PT providers via what we refer to as fields of proficiency testing tables or FOPT tables. The one shown here is for the non-potable water table, which covers primarily wastewater. These are posted on TNI's website and on ERA's website. These tables are put together by communities that were formed by TNI. They are groups of experts representing the EPA state agencies, the PT providers and the laboratory community. They get together and actually define these parameters, not just for microbiology but for chemistry too. This is really the skeleton of the criteria that drive the proficiency testing samples.

 

(06:10):

The table that you're looking at right now currently defines four analytes. You'll see total coliform, fecal coliform, E. coli, and enterococci. You'll see that they're listed by two separate technologies. MF is membrane filtration, and MPN is most probable number. This gives us eight separate analytes that the PT providers must treat individually. When I say that, what I mean is that we must collect the data separately and evaluate them independently from each other. The primary reason that NELAC split these analytes apart was because typically different methods produce different results with different standard deviations. In order to treat everyone fairly, they're split apart. Everyone isn't lumped together. To add a little bit more clarification to that, if you were running method 9222B for total coliforms by membrane filtration, you'd be reporting the result as total coliform MF. If you were doing 9222D, fecal coliform by membrane filtration, it would be reported as fecal coliform, MF.

 

(07:23):

Now also on this table, you have a design range that's given for each of the analytes. This is a range that the PT providers will target for each of the lots that are manufactured. It's required for us to vary it from study to study over the studies that we produce in a given year. You can see that the range is 20 to 2400. In any given one study, we may be designing the PT sample to fall towards the low end of that range, the mid or the high. It's a requirement that our accreditors will look at to make sure that in fact we're doing that. We can't always make it at the same level. Just depending on what study you're enrolled in, you may find something that's lower or higher. It's something that's varied. Also on its design range, it's important to keep in mind that it is in fact just a range that we're designing our product to. It's not a range of acceptable results.

 

(08:24):

A study designed at the lower end of the range may have a range of acceptable results which actually go outside of that design range. If we make something at 20, it's very possible that the range of acceptable values will be below that. Or conversely, if we make it on the very high end, it's possible that the range of acceptable results would go above the high end of the range. The other thing that you see presented on this table is the PTRL. It's presented as the number two CFU or MPN per 100 milliliters. This is given to us by NELAC basically as a guide for the laboratory for PT samples. It gives you a lower level. Well, I should say it's the level at the lowest acceptable result could be obtained from the lowest spike level. If we were to manufacture a given lot at the very low end of the range, you should not expect to see an acceptance range that would go below two.

 

(09:31):

It's never to go below... Excuse me. It's never going to go down to zero or one. The other area that's covered on that table is the acceptance criteria. It's listed as a log transformed plus or minus three standard deviations. That's the acceptance criteria that all the PT providers have to follow when it comes to non-potable micro samples for all of these eight analytes. Let me try to explain that. Essentially what that means is that all the data reported by the participants needs to first be log transformed. Once you've log transformed the data, you'll calculate the mean, the standard deviation, and then you'll calculate an acceptance interval plus or minus three standard deviations. Then you'll evaluate all of those results, and then they all get converted back to the whole numbers before the reports are sent out to customers.

 

(10:36):

Now, you may be asking yourself, "Why would they do that?" I've included a graph on this slide to try to illustrate why you want to log transform microdata. Essentially, this graph is a frequency plot. You can see two lines on it, one is labeled as non-log data, and the other is labeled as log transform data. Generally when you deal with chemical analysis, the distribution of analytical results that you get follow a Gaussian curve or what people refer to as a symmetrical distribution of values about the mean. Kind of the bell curve as you would. The non-log transform data you can see is not bell-shaped. Generally, microdata is not symmetrical. It's very rarely that you do get a symmetrical data set. The bacterial counts often are characterized as having what they call the skewed distribution. You get a large number of low values and a few high ones, and really this is the nature of the test methods when you're sampling a biological population.

 

(11:37):

It has a lot to do with the selectivity and differential nature of the test themselves. What you get is a data set like this where the mean is considerably larger than the median. If you're going to evaluate this data set, in order to treat everyone fairly you need to somewhat normalize the data set before you apply the plus or minus three standard deviation limit. The non-log transform data you can see starts to take a much more bell-shaped curve. Again, all data is treated exactly the same way, but you get to treat both the high and the low data points that are resulted more fairly. It is critical that you do this. If you don't do that, you're really going to discriminate against certain data points. It's important to keep in mind that all the PT providers are evaluating the data by the NELAC criteria that we're given. This is specifically what needs to be done with the data.

 

(12:34):

I wanted to talk a little bit about the sample design and the attended use. ERAs, QCs and PTs are delivered to our customers as lyophilized pellets in glass vials. They're accompanied with a phosphate rehydration fluid. ERA has been supplying product in this particular format for many years. For the non-potable water, ERA has the coliform sample, which is designed in the 20 to 2400 CFU or MPN per 100 milliliter range. Remember that these are the design ranges. They're not the range of acceptable results, and the product numbers are shown here on the slide. I also wanted to mention that the organisms that are used in ERA's wastewater coliform product, just so that everyone was aware, for total fecal and E. coli analytes are accomplished using E. coli as our target organism in these samples. The specific culture collection that we use for that sample design is ATCC number 51813.

 

(13:44):

All of that information comes on the certificates that we supply with the product. It also is disclosed on any proficiency testing report that you get. We also have an enterococci sample for non-potable water that is designed within the 20 to 1000 CFU or MPN per 100 milliliter range, which can be analyzed for enterococci and or fecal streptococci, MF or MPN. ERA's quantitative products have been validated to be unaffected by ambient temperature shipping conditions up to seven consecutive days. ERA's normal shipping service is the second day service, but the samples are stable well beyond that normal shipping service. We routinely ship things ground. We ship these all over the world these days. Having a sample that is designed in a way that allows it to be very stable at ambient temperatures is really important in order to supply all the participants an equivalent challenge. Now, once you receive the sample, the pellet does need long-term storage at four degrees C. That's just to ensure the integrity of the sample until you get ready to analyze it.

 

(15:03):

As a PT, that may be analyzing it on the 44th day of a 45-day-long study. Or if it's a QC, it might be 6, 8, 9 months after you receive it. You are putting it in the fridge to give it a long-term stability. The phosphate buffer that comes along with all the samples is your typical phosphate buffer with mag chloride. It doesn't require any refrigeration. You can just store it at room temperature. That's how the instructions read. People have put it in the fridge. It's not going to cause any problem, but really there's no reason to have it there. When you get ready to use it, you have to warm it up to room temperature anyway, so please just leave it out at room temperature. Now, the expiration date on ERA's products is three years from the actual manufacturing date of the pellet. Obviously, that indicates that it's a very stable, very robust product.

 

(15:59):

The reason we lyophilize our samples is because it is one of the truly recognized techniques that ensures the integrity of the organism over long periods of time. Doing it in any kind of modified process, whether that's a controlled dry process like a vitro or a lenticule or it's a whole volume, all of those do involve some preservation, but they're less recognized as really ensuring the long-term integrity of not just the viability of the organism but the integrity. Anything shy of true lyophilization opens you up to the possibility of the organisms actually going through some changes that you want to avoid. Definitely for a QC and a PT sample. The product that we're providing is intended to be used as a proficiency testing sample or a quality control check of the entire analytical process. It's not necessarily intended to be used to start stock cultures to rehydrate directly onto media.

 

(17:07):

It's really designed to create a sample and challenge your overall method. That's how we intend it to be used. I also wanted to give you a general overview of the instructions on how we ask you to use our product. This is an example of an instruction sheet. The product is quite simple to use. Basically all we ask you to do is remove the pellet from the fridge that you've been storing at four degrees, warm it to room temperature. It generally will take about 30 minutes to do that, assuming that you have a fairly comfortable laboratory. Once you warm that to room temperature, you should already have your buffer solution at room temperature. All you need to do is aseptically transfer the pellet from its glass vial over to the buffer. You can accomplish this in a variety of ways. You can just pour it and that is quite simple to do, or you could use a pair of sterile tweezers. I've heard of a variety of techniques over the years, but you basically want to make sure that you don't contaminate the sample when you transfer it.

 

(18:15):

At that point, the pellet will almost dissolve upon contact with the surface of the buffer. Close the top, shake for a few minutes. Generally 10 to 20 seconds is more than sufficient and it's ready to perform your analysis. Now we want to get into the meat of the webinar, and that's tips to improve analysis. Hopefully, those explanations of our products and the evaluation criteria have given you just a basic background of how our products are used and intended to be used. I want to get into some of the areas that we've seen over the years that if people don't have in place, they can have trouble. The first thing I wanted to start with is just a general disclaimer. I want everyone to be aware that the issues that I'm going to cover are intended to get you to think about what's important in microanalysis. I'm not bringing them up nor do I intend them to supersede any of your current procedures that you have in place.

 

(19:19):

My real intent is to make sure that you're thinking about some of the issues that we've seen come up with people that are getting poor results and give you tips to improve analysis. Let's start out first with just some general quality control criteria. Laboratories should perform sterility checks for the procedures that they have in use. It goes without saying but I need to say it, for membrane filtration procedures, you should check the sterility of your media, you should check the membrane filters, you should check any buffer dilution water that you're using. If you're using pipettes, flasks, Petri dishes, basically all the equipment involved in your process, you should be checking. Generally, it would be good practice when you're doing the analytical procedure. Depending on which procedure you're doing, you may want to check for sterility at both the beginning and the end of the series. You could do this with using buffer dilution water as the sample. An example would be if you were doing a membrane filtration and you only had one or two filter flasks and you were going to use those filter flasks throughout the process with just rinsing in between.

 

(20:29):

You want to make sure that that's an effective technique, so you incorporate some sterility checks to make sure that you're not getting any carryover from one sample to the next. For multiple tube procedures, again, you want to check sterility of media, buffer dilution, your glassware. Each new batch of any of these materials, when they show up, you should check before you put them into use. The next area would be positive controls. For each new batch of media and method of apparatus, you should check the analytical procedure by testing a positive control to demonstrate that the media produces the expected reaction to the organisms under test. You really want to ensure identity traceability reference culture that you're using. You need to make sure that you're coming or that they're coming from some recognized traceable source. There are folks that will use in-house stock cultures that they are maintaining. There's limits to that.

 

(21:35):

You want to make sure that you're not passing that too many times, you can have issues that will come up. If you can't start with something that is ideally fresh each and every time and perfectly identifiable traceable reference culture. You just want to remember that this could be an issue. You should always be challenging media and new equipment to make sure that you don't have any issue. The cultures could be like a single use preparation organ cultures that you maintain. You just want to make sure that you have good purity and good viability of the organism. Many companies sell QC samples that are intended for that purpose. The important thing for you to remember is you need to be checking all of these things before you move forward. If you're assuming that your media is just as good as what it was the last time relying solely on the manufacturer, you may be making a mistake. A little bit more about positive controls. Standard Methods has a variety of cultures that they recommend. You may want to look at those.

 

(22:43):

I've mentioned to you the strain that ERA uses for RPTs and QCs. That may be what you want to use. Your state agency may tell you what you should use. You may be using some of the commercially available methods out there. If you are, you may want to follow that manufacturer's recommendations. On negative controls, for each new batch of media and the method apparatus you need to check the analytical procedure by testing a negative control to demonstrate that the media does not demonstrate the typical positive reaction for that target organism. If you're expecting it to produce an atypical colony, it should produce an atypical colony. Your negative control should be an organism. It should be something that either recommended by Standard Methods or recommended by a standard manufacturer. But generally it would be something that you would get some kind of reaction with your media, either turbidity with no gas production, an atypical colony, something along that line.

 

(23:49):

Some of the negative controls or negative cultures that ERA uses in some of their products are pseudomonas species. Generally fairly good for most of the various wastewater methods that are out there. But again, you want to use something that's appropriate for the method that you are using. Again, it could be a single use preparation or it could be something that you maintain a stock culture, but the key is that you need to maintain the purity and viability of it. You don't want that organism changing over time and giving you a false reading of some sort. Let's continue with tips for general quality control and start with method evaluation or validation. Does the procedure produce the expected quantitative result? All of the challenges that you're getting in non-potable water are quantitative challenges. When you do evaluations, when you first put a method into place, you should be challenging it quantitatively.

 

(24:48):

You want to make sure that it's giving you a result that would pass if it were a proficiency testing sample. You can do that by either buying a quality control sample or if you're using a stock culture, you could come up with a dilution scheme that would ultimately dilute down into a water sample that would challenge it at an appropriate level. An appropriate level for wastewater type testing, at least when it comes to the PTs is 20 to 2400. If you are challenging your method with basically a sample that is in the 10 to seventh range, you're not really evaluating it at the level that would be appropriate for passing a PT. You may have issues with your method or your media that you would see a too numerous to count plate. But if you were to dilute that down into say 100 counts, you may see that the media may not be up to snuff.

 

(25:42):

Method evaluation is really important, and you want to challenge it appropriately. Next, hydration of pellet. I wanted to touch on this to make sure that everyone understands that you should follow the instructions that you're provided with for PTs and QC samples. The reason you don't want to deviate from that is because, as we've discussed with the evaluation criteria, when it comes to a PT you're evaluated based on the mean of the study data plus or minus three standard deviations. The assumption is that everyone is following the instructions in the same fashion. If you decide to do it in a way that is completely different from your peers and you're a participant in that study, it's possible what you do could produce a result that is different just because you were off on your own doing something different. It's really important to follow the instructions that you're given. Our sample comes to you, as I've discussed, lyophilized with a phosphate buffer. Once you rehydrate it, you've got about 30 minutes to perform your analysis. The reason I say that is because the pellet does have certain carbon sources in it.

 

(26:57):

When you've dissolved that in the phosphate buffer, there is the possibility that the organisms once they are somewhat resuscitated could use the source of carbon in the pellet as something that will cause them to grow. You definitely have to be somewhere beyond 30 minutes before that becomes a concern, but it is potentially a concern. Don't rehydrate your sample and then go out to lunch and then come back and cross your fingers. It's an area that you'd need to follow the instructions. Next, culture media. The quality of the media when it comes to getting the right answer on the sample is probably one of the most critical aspects of getting the PT or QC to work out. You should never prepare your media from raw ingredients if the source of dehydrated media is available. If you're preparing it from dehydrated media, follow the instructions closely. Critical aspect, if you're told to check the pH, you really need to check the pH and make any adjustments.

 

(28:02):

Organisms are very finicky when it comes to pH, you want to make sure that you're checking it. You always want to evaluate the media before you put it into first use. You never want to use the media outside of its expiration date. You never want to use media that hasn't been stored according to the manufacturer's specifications. If they store it out of the light at ambient temperature, you need to do that. If they say store it in the fridge, you need to do that. These are the source of carbon that you're going to grow your organisms from. If you do anything that starts to break down this media, you're going to have trouble. Make sure that you follow the instructions from the manufacturer both when it comes to manufacturing it and storing it. Next, proportions. Make absolutely sure that if you're supposed to weigh out a certain number of grams to a certain volume, follow it very, very closely. If you start deviating from it, you're basically creating conditions that are going to be different from you than they are from other participants in the study or the folks that analyze the QC samples.

 

(29:19):

Next, let's talk about dilutions and rinse water. As I mentioned on the prior slide pH conditions are critical. You only want to rehydrate the pellet that we provide you with the buffer that we provide you. All of our quality control, all the other participants, everyone is using that same buffer. You want to make sure that you use it as well. If you're doing any kind of dilutions or rinsing of your apparatus, you don't want to use deionized water or distilled water. ERA Standard Methods, general good micro practice is going to recommend that you're using phosphate buffer or peptone water. The reason is it's not uncommon for the pH of deionized or distilled water to be, well, something other than what would be ideal. That can cause inhibitory effects on the organisms. Take a look at Standard Methods, section 9050C. It'll give you the instructions on how to make buffered water or peptone water. The other general quality control criteria that I wanted to mention was thermometers.

 

(30:33):

Temperature is critical. You want to make sure that you're using thermometers that are calibrated and are incremented that are appropriate for whatever method you happen to be using. You want to check the accuracy semi-annually against a NIST thermometer. You want to use any correction factors if there are any related to the thermometer you're checking it against. Water bath incubators. You want to verify that the water bath is at the desired test temperature. If it says it's supposed to be within plus or minus two degrees C, it absolutely needs to be within that tolerance. You probably want to record it. Monitor it at least twice daily, generally four hours apart. You want to make sure that it's stable and uniform. Water baths, you want to use ones that have a gable cover, you want to use circulators. You want to place the incubator in an area of the room where the temperature is relatively stable. You don't want to put it next to the air conditioning vent or you don't want to put it on a window.

 

(31:42):

Those are all things that are going to cause you havoc when you're trying to maintain such a tight temperature tolerance. The air incubators, again, you want to make sure that you're staying within the tolerance. You want to make sure that cold samples are incubated at the test temperature for the required time. You want to record temperature on the various shelves if it's a fairly large incubator. It's very possible that the temperature at the top is not the same as the temperature at the bottom. You want to make sure that you know that it's at the right temperature for where you're incubating the samples. Opening and closing the doors of incubators. Incubators, depending on what size they are, are going to have a refresh time. When you open the door, the temperature's going to drop, and it's going to take certain amount of time before it ever gets back to the incubation temperature.

 

(32:44):

You want to have an idea of what that time is so that you can compensate your incubation time based on the period of time where it's not actually at the incubation temperature. Get an idea, avoid opening up the doors as much as possible. Now, let's get into some method-specific issues starting with membrane filtration methods. One of the most important things you need to know about membrane filtration PT results is PT results that are reported as too numerous to count will be evaluated as no evaluation. It's therefore critical that you run the appropriate dilutions. We'd also recommend if you're running QC that you routinely run them at various dilutions to simulate the PT scenario. Proper dilution scheme. When you're performing membrane filtration, basically the sample size will be governed by the expected bacterial density. The assigned value for all ERA QC and PT lots will fall within the range of 20 to 2400 CFU or MPN in per 100 milliliters. Given that range, you want to select sample portions that will most likely yield a membrane filter count in the ideal range.

 

(34:18):

Depending whether it's a total method or a fecal method, that may be 20 to 80 or 20 to 60. What we would recommend is that you run the entire volume of the sample in portions to cover that range, and of course the sum volume of all of those should be equal to the total volume of the sample that's provided. That would generally give you a result on one of those plates that falls within your ideal count range. Then you just need to make the adjustment based on which plate you selected. If in the event it's a lot that's manufactured in the lower portion of the range, it may be possible that none of the plates fall within the ideal counting range. If you look at Standard Methods, you'll see a section that will actually guide you through what do you do in that instance. Basically it just involves looking at all of the plates that you ran to come up with what the number is that you're going to report.

 

(35:24):

A general example that we give folks when they're running this sample, you could run a 50 milliliter, a 30 milliliter, a 12 milliliter, a 5 milliliter, a 2 milliliter, and a 1 milliliter. If you're going to dilute those volumes with any additional volume of water, you want to make sure that it's also buffered water. Just if you're doing a filtration test, you don't want to pull too low of a volume through your filters. Most of your procedures probably will give you guidance as to what that volume can go down to before you need to add additional diluent. Now more on membrane filtration methods. First, filter paper. Generally folks are using 47 millimeter diameter filter paper or 0.45 micron pore size. Filter paper can be a real issue for poor recoveries. Generally, when you've got an issue with your filter paper, you're going to see the colony development be out of the ordinary. You may have a colony that'll go to a grid line. If it's a grid line paper and it changes direction, that's a good indication that there's something wrong with your filter paper.

 

(36:45):

You may get abnormal spreading of the colonies. You may get poor or absent colony sheen. Keep in mind that I've told you the organism that we use every time for the wastewater sample. You should never expect to see any kind of atypical results. These samples are not tricky in that way. You're always getting the same organism every time. If you can't get good sheen, sometimes it can have to do with the filter paper itself. You may see brittleness in the paper. When you do your initial evaluation, you may see decreased recoveries in what you normally see. For that quantitative challenge that I talked about earlier, you may get wrinkling on the paper, you may get areas of the paper where you get absolutely no absorption of your media. If you don't get any absorption of the media, that means that an organism that's seated on that filter in that area does not have any food to grow. That obviously will affect your results. It's not going to develop a colony that's going to be countable. If you get a tie-dyed look to your filter paper, it's very possible that you got a bad batch.

 

(38:03):

Another little trick that's in Standard Methods, they talk about taking a filter and floating it on reagent water. You can take a Petri dish, put in a small amount of DI water, take a pair of tweezers, float the filter on top of the water, and you should see it uniformly diffuse in about 15 seconds. If your filter floats like a little boat there for a minute, you've got something wrong with your filter paper. It won't absorb the nutrients in a way that are going to get the organisms to grow. Now let's talk about media broth for broth and pad. Pads must retain between 1.8 and 2.2 milliliters of broth, that's defined in Standard Methods. You need to confirm that if you are getting media that's premixed in vials and you're cracking the vials and dumping them out, make sure that when you're delivering to the pad that it falls within that tolerance.

 

(39:04):

You can do that by just measuring how much are in the ampules to begin with. Pour it onto the filter. If you have broth that doesn't get absorbed in the filter, measure how much of that. If it puts you outside of that tolerance, it's possible that those pads have become compressed over time and have absorbed moisture. If that happens, it's going to make it difficult for you to get good recovery because you're not delivering the amount of nutrients to the organism that really is going to be ideal for growth. Next, application of the filter. It's really important that you get good contact between the filter where your organisms are seated and the pad that has the absorbed nutrients. If you don't make a good contact between these two, there's no nutrients for your organisms. You need to put it on very carefully. Generally, what's recommended is to get maximum contact as you roll it from one edge to the other. You take one edge, you put it on the pad after you've put your media on. You slowly roll it onto the pad, making sure that you don't trap any air underneath it.

 

(40:17):

Once you've completed rolling it on, a good practice is to tap the Petri dish on the lab bench. That'll add an additional contact between the two. If you don't have a good contact between your filter and the pad, your organisms will not grow. They don't have the nutrients. They're sitting up there with no lunch so to speak. Now let's talk about keys to improve analysis related to multi-tube methods. I wanted to get into a few issues here. Again, I also wanted to mention if you're doing a tube method that involves a pre-enrichment with some type of membrane filtration technique, refer to what I was talking about with the membrane filtration techniques related to that portion of it. This is primarily focused on doing tubes. One of the most important things that people need to know when it comes to multi-tube fermentation is a greater than or equal to value will be scored as that value.

 

(41:24):

If you're doing a method with say, 15 tubes where your maximum value if you get five positives in each of your three series of tubes is 1600. If you report greater than or equal to 1600 it'll be scored as 1600. If you recall, the manufacturing range that PT providers are required to use is 20 to 2400. You're basically using a method that doesn't cover the entire manufacturing range. If you're analyzing a PT that happens to be in the higher portion of the range, you may have cut yourself short. You basically aren't covering the entire range. Now dilution scheme. If you're performing multiple tube fermentation, you want to run a series of results that's consistent with that range. As I mentioned earlier with the 15 tube Standard Methods, you are allowed to run an additional series of tubes to increase the range that you can cover. You may want to consider that given how the PT samples are designed. You can basically, instead of just running 10, 1, 0.1, you can do an additional 0.01, and Standard Methods will give you guidance on how you shift the interpretation of your positive tubes.

 

(42:56):

Then you go to the MPN table and you just adjust that value accordingly. That may be something that people doing the old 9221 method may want to consider when they're running unknowns, PTs. More on multi-tube fermentation methods. Another issue that comes up periodically is degassing of broth. To avoid false positive results due to dissolved gas in the media, you want to make sure that you're at a minimum pre-warming your tubes up to room temperature. Then if you do get any outgassing of the media, you want to get that out of your Durham tube before you actually do your inoculation or out of your fermentation tubes. You don't want to inoculate the sample. Put it in say a water bath. Have it warm up the media. Have oxygen come out of the media. You end up with this little bubble in your tube, and you interpret that as a positive result.

 

(44:03):

You want to make sure that you're eliminating that possibility. Therefore, degas. The other thing you may want to consider is before you inoculate samples, you get the tubes to your incubation temperature. That will avoid that significantly. The other issue I wanted to mention has to do with broth concentration. If you're doing an MPN method, you want to make sure that you're doing broth concentration. You want to make sure that you're making it at the appropriate strength for the method that you're using. If it's double strength that's required, you want to make sure that you're doing it. If you're not doing it at the right strength, it's very possible that the aliquot of sample that's being used you can end up with a negative result just because essentially you're not following the method.

 

(45:00):

Finally, let's discuss keys to improved results specifically related to enzyme substrate methods. A lot of you are probably familiar with Quanti-Tray. This is an area that I want to try to address some of the issues. The first area when you're dealing with some enzyme substrate methods is that there are a ton of them out there. There are a variety. They have interpretation time windows when the results are considered definitive. An example, Colilert 18 uses an 18 to 22 hour window. Colilert uses a 24 to 28 hour window. Basically, what that is saying to you is that Colilert 18 is not really an 18-hour test. At the end of 18 hours if the result has changed, it's yellow or it fluoresces. That's a definitive result. If it is not, then it means that the result is still or still could potentially be definitive out through whatever that read time is.

 

(46:13):

Even though you get these products and your standard practice may be always read them at 18, you may be cutting yourself short because technically the manufacturer still considers the result to be a positive result if it changes within a certain time window. It's not until you get outside of that window that a negative result is considered definitively negative. Just make sure that you're aware of that. Another issue when you're dealing with Quanti-Tray, there's a couple of different formats out there. One is the 51 well, the other is a 97 well. You may want to make sure that the range that's covered by the number of wells for the format that you're using covers the manufacturing range. Now, if you use a 51 well, you're not actually covering the entire manufacturing range. It has an upper capacity. The 97 well actually has a capacity that'll cover the entire range.

 

(47:18):

It doesn't necessarily prevent you from using the different formats, you just need to be aware that you're not completely covering the range if you run the sample at a 100%. Comparators. Ideally, every manufacturer of an enzyme substrate method would be required to have a comparator. Unfortunately, they're not. Not all of them do. If there is a comparator, you need to use it. As most of you know, a comparator really distinguishes the threshold of what they consider to be a positive result from a negative result. You should use them if they're available. If you don't have them available, what we recommend is that you use a quality control sample to run along with it. You use that as a point of reference as to what constitutes a chromogenic or fluorogenic change.

 

(48:24):

Well, that brings us to the end of today's webinar. In conclusion, evaluation criteria is set by NELAC and all PT providers follow that standard. I hope I have taken some of the mystery out of calculating limits by going over the log transform process. Just remember, it is used to reduce skewness of a measurement variable. Culturing of microbes requires adherence to specific and defined criteria to ensure accurate analytical results, and there are many variables to consider. I hope this webinar has provided you with a deeper understanding of the requirements for microanalysis and has provided helpful information to improve your results. With that, I will turn it back over to Nicole.