Is Your Microbial QC Testing Stuck in the Past?
Pour Plate and Most Probable Number tests have changed little in 50 years, while new technology has advanced Membrane Filtration Tests for microbial QC
February 4, 2022
The Membrane Filtration Method has come a long way in recent years, with improvements in membrane technology and new protocols to handle challenging samples the Membrane Filtration Method is rapidly becoming the simplest and most comprehensive test for contamination in everything from drinking water to pharmaceuticals, and from food to environmental samples.
Alternatives to Membrane Filtration have changed little since their original inception
Testing methods such as Most Probable Number, Pour Plate, and Spread Plate techniques are all used for determining the presence of microbes in liquid samples, however while membrane technology has moved on, expanding the scope and utility of the Membrane Filtration Method, these other techniques are essentially the same now as they were 50 or more years ago. Many labs continue to use these methods simply because it’s what they’ve always done. In this article, we look at some of the critical performance advantages of modern Membrane Filtration Method tests.
The Membrane Filtration Method tests the whole sample, not just part of it
Perhaps the most important advantage of membrane filtration is its accuracy and sensitivity, driven by the fact that it uses filtration as the first step in the protocol and therefore tests the entire sample. This is critically important for testing samples such as pharmaceuticals, food, or drinking water where even low levels of contamination could be a danger to human health. It is also important where large volumes are required to be tested, such as for Environmental Protection Agency (EPA) water purity tests in the US where a volume of at least 100 mL is required, way beyond the maximum volume that can be processed by Spread Plate (0.5 mL) or Pour Plate (2 mL) techniques. Even the much more elaborate and time-consuming Most Probable Number protocol will only test a fraction of such a large sample. This bias leaves the other test methods vulnerable to type 2 errors, in cases of low-level contamination there is an increased chance of missing the contamination and returning a false negative result.
The Membrane Filtration Method is simple and rapid
Using Membrane Filtration, the full volume of a sample is passed through the filter. Any organisms present in the sample will be captured and concentrated on the surface of the filter membrane, which is then placed directly onto growth media in a petri dish. Nutrients and water in the growth media will pass easily back up through the filter to support the growth of any organisms present on the surface of the membrane. By contrast both Pour and Spread Plates are dependent on operator skill to accurately disperse the sample onto or into the growth media, and Most Probable Number requires a series of three separate tests with multiple outcomes to be measured in parallel.
Membrane Filtration allows accurate identification of any microbes detected
If any colonies do grow on the filter, these can be sampled and grown-up individually on new media to allow the identification and characterization of each discrete colony. Material from these isolated colonies is also amenable to examination with the tools of modern molecular biology, including whole-genome sequencing. A clear picture of not just the total concentration of microorganisms in the original sample, but exactly which species and the relevant abundance of each, is then possible. While the Spread Plate technique will also provide colonies that can be picked and analyzed in the same manner, it must be remembered that Spread Plate is limited to a maximum of 0.5 mL of sample per plate and cannot possibly assay the entire sample. Neither Pour Plate nor Most Probable Number techniques provide colonies amenable to picking and isolation in the same manner.
Advances in membrane technology make Membrane Filtration more broadly applicable
Membrane selection is a critical part of protocol design for the Membrane Filtration Method and membranes can be selected to suit a particular application where the size of the target organism is known. 0.45 mm GN6 Metricel® membrane or 0.4 mm polycarbonate are common choices for general applications to collect microbes for growth on media and further analysis. Alternatively, pore sizes as small as 0.2 mm can be used to catch any contamination present for critical applications such as pharmaceutical testing. In the past, such extremely small pore sizing could create problems of backpressure and reduced flow rates. Advances in membrane technology, such as the 0.2 mm Supor® membrane disc filters, enable these membranes to maintain a flow rate closer to a traditional 0.45 mm filter, making 0.2 mm filtration a possibility and Membrane Filtration a practical solution in applications that would previously require an alternative technique. Some membranes are also available in black to make light-colored colonies easier to see.
Membrane Filtration addresses difficult sample types
There are some sample types that can be difficult to assay. Both Pour and Spread Plate techniques struggle with viscous samples which are hard to distribute evenly across or through the growth media. While such viscous samples can be diluted, this only serves to compound the problem of false negatives when not testing the whole sample. Only Membrane Filtration allows for dilution of the sample to reduce its viscosity, while still enabling filtration of the whole sample and identification of any contamination present.
The simplicity and sensitivity of the Membrane Filtration Method, together with its ability to deal with large sample volumes and difficult sample types, make it an increasingly popular choice for Quality Control and other applications where the identification and characterization of microbes in liquid samples is critical. New and specialized membranes are making Membrane Filtration practical for a broader range of applications, for which the older more outdated techniques such as Pour Plate, Spread Plate, and Most Probable Number techniques can be replaced with simple, efficient and accurate filtration.
Learn more about Membrane Filtration Method specific membranes and applications.