Sample Dilution in Microbial QC. How, Why, and When

Some microbiology QC samples are more easily handled when diluted. Find out how to dilute challenging samples and still meet the regulatory requirements.

February 15, 2022

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As we discussed in our last blog on this topic, the Membrane Filtration Method (MFM) is a simple and accurate method for sterility testing and quality control that has a number of advantages over alternative methods such as Most Probable Number, Pour Plate, and Spread Plate techniques in terms of accuracy, sensitivity and reproducibility as well speed and simplicity. Improvements in materials (particularly new purpose-designed filter membranes) and testing protocols are making MFM a viable option for an ever-wider array of sample types and enabling labs to move away from outdated testing methods in favor of MFM. In the second blog in this series, we focus on one such challenging sample type and discuss the handling of viscous samples via MFM.

 

Viscous samples. Traditionally challenging for Microbial QC

 

Many of the traditional techniques of microbial QC struggle with viscous samples. Spread Plates and Pour Plates both suffer from problems of adequately distributing the sample respectively over or through the growth media. While it is possible to dilute the sample with a suitable solvent prior to testing this also has the effect of diluting any microbial contamination and reducing the chances of accurately detecting a contaminated sample. Alone amongst the competing methods of Microbial QC, MFM is able to utilize dilution as a strategy to overcome the challenge of sample viscosity, while still testing the entire sample for contamination. This makes MFM the only truly accurate and reliable testing method for handling these challenging samples while ensuring an accurate result.

 

The problem of viscosity is further compounded with products such as oils, creams, and ointments, all of which are common formulations for both pharmaceuticals and cosmetics, as many of these oil-based formulations are not readily solvated in water and require a suitable organic solvent for dilution prior to testing. The solution to this problem as well as the detailed guidelines laboratories are required to follow for sterility testing of critical products such as pharmaceuticals and cosmetics are covered in the United States Pharmacopeia (USP)[1] chapter 71, (typically denoted USP <71>) which provides a standardized framework for sterility testing that is acceptable to many regulatory agencies across the globe.[2]

 

Diluting viscous samples within the framework of USP <71>

 

In a recent study, Pall scientists tested a protocol for the dilution of high viscosity oil-based products following the requirements of USP <71> for sample preparation and testing. USP <71> allows for the dilution of viscous oil-based products with a ‘suitable sterile diluent’ prior to testing. The choice of diluent however must take into account any anti-microbial action of the diluent which could influence the results of the testing. The scientists evaluated the performance of sterile isopropyl myristate (IPM), a diluent that has been shown not to have antimicrobial activity under the conditions of the test. The study provides a useful evaluation of the effectiveness of IPM as a diluent, as well as demonstrating the utility of the membrane filtration method by comparing the filtration times of diluted and undiluted oils.

 

Testing samples with the Membrane Filtration Method

 

The researchers selected three different oils for testing, evaluating the filtration times for both undiluted and IPM diluted oil samples to establish the efficacy of IPM as a tool to enable the effective sterility testing via MFM of high viscosity oil-based products. In each case, the researchers were able to test a relatively large sample size (50 ml of oil sample) which was either filtered without dilution or diluted 1:1 with sterile IPM. The results are shown in Table 1, below.



Table 1. Mean filtration times for diluted and undiluted oil samples

 

 

Filtration time (min:sec)

Sample

Diluted

Undiluted

Oil 1 (Canola)

1:40

10:08

Oil 2 (Sesame)

2:54

Unfinished at 2 hours

Oil 3 (Olive)

1:39

18:49

 

Conclusion and benefits for Membrane Filtration

 

The results of the comparative testing show the utility of the membrane filtration method when used with a suitable USP <71> approved dilution protocol to handle even the most challenging samples. In all three cases, the diluted samples were filtered in a short time easily managed within any laboratory workflow. Conversely, the undiluted samples were highly variable taking over 10 minutes, and over 18 minutes respectively for oil samples 1 and 3, and in the case of Oil 2 remaining incomplete after several hours.

 

You can find the full application note on which this blog is based on the Pall website, including materials and methods and full results data as a useful tool to understanding the process and benefits of dilution for viscous materials in microbial QC.   You can read the first blog in this series on Microbial QC testing and membrane filtration on the Pall website.

 

 

References

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