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December 3, 2013

Traditional Culture Methods or PCR: Which is Right for You?

  • Food Safety

By InstantLabs

By Lauren Bambusch

Today’s food safety microbiologists and quality control managers have their pick of technologies to perform bacterial testing. From culture plates to immunoassays to PCR and Real-Time PCR, there is a wide range of test types to choose from. This post will compare the two most accurate methods: traditional culture methods and Real-Time PCR. Which one is right for you?

Traditional Culture Methods

Traditional culture methods (TCM) are how most people picture microbiology; Petri dishes spread around and red, blue and yellow bottles of broth on the shelves, maybe even a microscope in the corner. Technicians plate the bacteria on the culture plates, place it in an incubator, and wait to see what grows up. This is the process used today when looking for foodborne pathogens on culture plates. TCM is all about growing what’s there, slowly but surely. It often involves a process of trial and error, ruling out species after species until you have only one option left using observable characteristics. For instance, both E. coli and Pseudomonas aeruginosa have gram negative, rod-shaped cells and look similar under the microscope (click here for more information on Gram straining), but only E. coli can metabolize lactose. Simply growing the culture in a lactose medium can tell you which organism is which. While growing plates does not require great skill, reading and interpreting the results can require deeper knowledge when presented with non-textbook results.

Real-Time PCR

In an earlier post (What is PCR?), I talked briefly about the history and mechanics of Real-Time PCR. The major advantage of Real-Time PCR over TCM is the ability to obtain results in a drastically shorter time frame without sacrificing accuracy. This can be especially important if you are testing foods that are perishable and have a short shelf life.

The Process

When hunting for foodborne pathogens, TCM and Real-Time PCR start the same way — enrichment of the sample to make sure there’s enough detectable bacteria. Enrichment times vary based on the target organism, method used, and specific media, but the process is the same: mix the food or environmental sample with specially-formulated growth media containing sugars, proteins, and other building blocks, and incubate for hours or days. This is called a primary enrichment. Some organisms grow fast — E. coli can double its population in just 20 minutes — but some take much longer to grow, like Listeria. This is why the enrichment times for Listeria are typically much longer.

TCM normally then requires an additional enrichment with a second growth media that contains growth pressures that will restrict or inhibit the growth of non-target organisms. This secondary enrichment is needed so enough of the target can be seen with your eyes without it being crowded out by other microbes. After the secondary enrichment has incubated for a day or two, the primary and secondary enrichments are spread onto Petri dishes containing yet another growth medium or two, and put back into the incubator to allow colonies to form on the plates (typically this requires an additional 1 – 2 days). Depending on what organism you’re looking for, this may be enough, but if it’s a more specific organism like E. coli O157:H7 or Listeria monocytogenes, more steps may be required to completely identify those targets.

On the other hand, using InstantLabs Hunter® Real-Time PCR system, the sample goes directly from the primary enrichment into the Real-Time PCR process, shortening the time to result to hours instead of days. After the primary enrichment, the next step with Real-Time PCR is sample prep. The bacterial cell wall is opened using a method such as heat, chemical, or mechanical force and the genomic DNA is extracted. It may sound complicated, but it is pretty easy – all you need is a heat block and maybe a fast whirl in a small centrifuge. Once the sample is prepared, it is mixed with the Real-Time PCR reagents and the thermocycling is started. On the Hunter® system, sample prep to results takes less than 2 hours to complete.

Which is Right for Me?

When considering which method to use, there are a few things that you’ll want to consider.

    • Skill Level Required: Knowing what to look for can be tough when it comes to TCM. While some tests are cut and dry — the organism either grew up or it didn’t — others require a bit of interpretation in order to be correctly read. Bacterial identification is an art form that can take years to perfect.
      Until recently, Real-Time PCR has been much the same, requiring a seasoned eye with the technical skills to interpret results. However, as science melds with technology, new instruments such as the Hunter® greatly simplify the process and can consistently and automatically interpret the results.
    • Time to Result: Bacteria grow on their own schedule; they don’t care about your company’s bottom line. More time spent waiting for results means more time a product spends in a holding pattern – this can be a significant cost and logistical issue for many products.TCM can take anywhere from 3 to 7 days for the most common food contaminants, while Real-Time PCR—including primary enrichment—can often be run in 10-24 hours.
    • Accuracy: There’s always one oddball out in a crowd, and bacteria are no different. There’s always one cell that just won’t conform to the standardized norm for a species—an E. coli that doesn’t metabolize lactose, or a Citrobacter freundii that no longer produces hydrogen sulfide. Because many of the traits that TCM look for are considered non-essential traits by the organism in question, there’s always the possibility that a pathogenic organism could be missed even by the most seasoned technician.
      Real-Time PCR, on the other hand, usually hunts for highly conserved genes or essential genes that the cell would likely die without were they damaged or mutated. This allows Real-Time PCR to detect even those non-conforming organisms.
      And because of the technology behind Real-Time PCR, many of the new instruments feature automatic electronic report-generating. Simply print it out and store for your records. No more worrying about incomplete record-keeping or trying to decipher Ted’s chicken-scratch.
    • Waste Generated: At first glance, the amount of waste each test generates per sample doesn’t seem like an important concern, but it is. Consider this: each pound of waste generated has to be disposed of properly. This could require treating with household bleach or autoclaving in some cases. And because TCM often require both the primary and secondary enrichment to be plated in replicates of 5, all of those plates add up to quite the pile. For example, to test one slice of deli turkey for Listeria monocytogenes, two different broth media and 24 plates (of 3 different types) are required. And that doesn’t even count the biochemical test plate! Imagine running even just 20 samples a month.
      Because Real-Time PCR can hunt for the correct DNA segment without all the trial and error, there is substantially less waste to be considered; typically only a few small tubes beyond the primary enrichment.
    • Cost: It’s not just the cost of reagents that needs to be considered. We’ve already touched on the fact that TCM require a more advanced skillset, and that can translate into higher wages and more cost per sample. Also, we’ve talked about how much time is taken by each sample, which pulls your staff from doing other important tasks, and the differences in the amount of reagents needed. Sure, a single plate may cost only $0.25, but when you’re going through 24 at a time, that can add up quickly. It is estimated that, even with the increased cost of the technology, PCR is 38% less expensive to run than TCM. See Real-Time PCR for the Detection of Salmonella.
      Perhaps most important to consider is the cost of delaying the clearance of food products before they can be shipped to stores and consumers. Waiting for a negative result from TCM can mean reduced remaining shelf-life for the product and increased warehousing costs. It costs a lot to keep huge amounts of food refrigerated, or especially frozen! Real-Time PCR means faster release of product, which decreases your bottom line.
    • Diversity of Available Tests: This is where TCM beats out Real-Time PCR. Virtually all of the common foodborne pathogens including Salmonella, E. coli O157, Listeria monocytogenes, Vibrio, and Campylobacter can now be tested rapidly and accurately using Real-Time PCR. However, there are still some very rare and emerging pathogens where Real-Time PCR tests don’t yet exist – at least not commercially available and approved versions. For these very rare pathogens, TCM is still the method of choice. Luckily, it is highly unlikely that you will need to look for these rare bugs unless you are in a specific part of the world and are producing a very specific product.

Because of the number of advantages inherent with real-time PCR, especially over an older technology like TCM, much of the industry is moving toward implementing real-time PCR machines in their own facilities. What about you? What tests are you using, and why? We’d love to hear from you—leave us a comment on what you use and why!

Lauren Bambusch is a microbiologist by trade as well as a writer and baker by hobby. She lives in Baltimore with her husband, two cats, a super-sized mutt, and a school of fish, all of whom root for her Alma mater, Michigan State. Go Green!