The Strengths and Weaknesses of Sterility Testing Methods
A look at some of the common methods for microbial QC testing. Which works best and why.
March 17, 2022
In the first two blogs in this series, we reviewed the Membrane Filtration Method for microbial QC testing and looked at why it is often the method of choice, particularly in sensitive environments such as pharmaceuticals sterility only really used in pharma where sterility is critical and the risk of a false negative needs to be as close to zero as possible. In this third and final part of the series, we discuss in greater depth the three main alternative testing methods and highlight the limitations of each. You can find links to the first two blogs in this series at the end of this article.
Microbial QC and sterility testing.
As a brief recap, the reason that the membrane filtration method (MFM) is so frequently used for sterility testing is a combination of ease and speed of use, as well as accuracy and sensitivity. Membrane filtration uses the entire sample, rather than just a small portion of it and the protocol is quick and simple. Full details on MFM can be found in blog 1 in this series.
In contrast to MFM, Pour Plate requires the serial dilution of a sample in a series of sterile water blanks of known volume. This creates a series of samples, each at a known lower concentration than the last. These diluted samples are then poured into a petri dish and molten growth media is added. This method is useful in the case of highly concentrated samples where a high number of colonies are expected. The serial dilutions ensure that at least one of the plates contains a sample of sufficient dilution that individual colonies can be counted to determine the concentration in the original sample. However, the method is labor-intensive (making up serial dilutions) and is not as well suited to the testing of samples where a low concentration of organisms is expected, such as drinking water, cosmetics, pharmaceuticals, etc. Pour plate is also susceptible to underestimating the true level of microorganisms present. Firstly, the use of hot agar risks killing some of the organisms present and the fact that the sample is mixed into the agar (not spread on top) means that small colonies can be obscured and not counted. Secondly, any obligate aerobe species buried in the agar may not grow due to the lack of oxygen, both factors which serve to reduce the total number of colonies counted, and underestimate the true contamination of the sample. Discreet extraction of individual colonies from within the agar is also challenging, making it difficult to pick a single species for further study.
Spread Plate has the advantage over Pour Plate in that the colonies are spread onto the surface of the plate, making counting and retrieval of individual colonies easier. Spread Plates made up using serial dilutions are a useful tool for determining microorganism counts in relatively concentrated samples. The disadvantage of Spread Plate is that it can only tolerate the smallest volume of any of the available techniques, limited to a maximum of around 0.51 mL per plate, as the liquid sample cannot be allowed to pool or run on the surface of the agar. Due to this low volume requirement, the microorganisms present must be at a sufficiently high concentration to be accurately detected, and there is always a risk of sampling bias when removing only 0.1 mL from a tube that may contain 10 mL in the case of serial dilutions.
Both Pour Plate and Spread Plate suffer by the fact that only very small volumes can be applied to the plate, and consequently, only a fraction of the total collected sample can be tested. Membrane Filtration by contrast can process the entire sample, up to 100 mL or more in the case of drinking water testing.
Most Probable Number (MPN)
MPN is a lengthy but exhaustive method that integrates three separate experiments on the same sample to determine the presence or absence of a microorganism: the Presumptive Test, the Confirmed Test, and the Completed Test. The protocol itself is too lengthy to go into here, but involves the inoculation of a series of test tubes and media plates. The tests look for a variety of indicators of life including the production of gas, changes in turbidity of the liquid growth media, and the presence of colonies on the media plates. By varying the growth media to favor or inhibit certain organisms the test can make assumptions about the nature of the organism present.
However, MPN is only able to indicate the presence or absence of an organism and allows an approximation of the number of organisms present. It also suffers from low accuracy and precision in comparison to all of the other techniques discussed and is the most labor and time-intensive to perform. MPN can provide an alternative to membrane filtration for specific sample types such as mud and sediments that can be challenging for membranes, although there are an increasing number of specialized membrane filtration protocols available that allow the use of the membrane filtration technique with these challenging samples, as we discussed in blog 2 in this series.
While there is undoubtedly a place for these alternative testing methods in the microbiologist’s toolbox, the best choice for front-line testing in a microbial QC environment is clearly membrane filtration. You can read more about MFM and how advances in membrane technology and testing protocols have led to improvements and extended the use of MFM to previously challenging samples in the previous two blogs in this series. You can also learn more about the available MFM specific membranes on the Pall Website.
Blog 1. Is Your Microbial QC Testing Stuck in the Past? A guide to Membrane Filtration Method
Blog 2. Sample Dilution in Microbial QC. How, Why, and When? An example of how modern membrane filtration can be used with challenging sample types.