Considerations in Formulating Freeze Dried (Lyophilized) Quality Control Cultures

Operating a contract freeze drying laboratory allows our scientists to work with many different microbial strains. Unique or specific requests for freeze dried cultures, including vials of mixed cultures and/or vials with quantifiable viable strains, has given us valuable knowledge of the process. Here are some helpful insights, when setting up your own lyophilized quality control (QC) cultures.

The preparation of freeze-dried microbial cultures as quality control standards and for proficiency testing can be economical and useful. Depending on their use, freeze drying bacteria and fungi, either as pure or mixed cultures, can yield a ready supply of tailored positive controls or test samples at a reasonable cost. However, the feasibility of producing such lyophilized cultures is dependent upon variables that may be inherent to the microorganisms.

When producing freeze dried quality control cultures, there are several factors that should be considered:

  • Application - Are the cultures being used for assays that require growth or for nucleic acid detection,
  • Pure or Mixed - Does the culture have one or more microbes in it,
  • Distribution - Will the cultures be distributed or used in-house
  • Quantitation - Will the cultures be used for a qualitative test (e.g., gram stain) or quantitative test (e.g., bacterial counts)

Before exploring these factors in greater detail, some background on freeze drying microbes is useful. Foremost, not all prokaryotes and fungi survive the freeze-drying process; this can be strain dependent. Secondly, storage conditions after lyophilization can dramatically impact viability. Stability studies have shown that some bacterial strains can rapidly die off when stored at temperatures above 4°C. Another factor about stability is that some cultures are not viable for prolonged periods. Finally, some groups of fastidious microbes can be very difficult to lyophilize because of nutritional and culturing parameters. In short, if you have a choice, pick an easy to grow, easy to freeze dry, long-term stable, microbe.

Application of Freeze Dried QC Cultures

Quality control cultures can be used to verify a test process or validate a lab. In many cases, such as with membrane filtration protocols, the microorganisms must be viable and grow to visible colonies once rehydrated and tested. In other circumstances, the freeze-dried microbes can be used as positive controls for nucleic acid based assays. For DNA based assays, the cells do not have to be viable, while for RNA assays, intact transcripts may be needed, and therefore viability is necessary

For applications requiring viable cells, shelf-life stability studies are needed for strains. As noted, most strains survive very well when freeze dried, but it is not uncommon to find cells that die off. The most accurate method of assessing stability is to hold the cultures at realistic temperatures and humidity; in real-time. Plotting out the number of viable cells over a month should provide some insight into the longer-term survivability of the cultures. Testing viability can be made simple using a dilution to extinction protocol.

Pure or Mixed Lyophilized Controls

Off-the-shelf cultures are usually pure and used mostly for positive controls or identification testing. However, when designing cultures, it is possible to mix organisms for differential testing. The preparation of pure cultures is straight forward and does not pose all the same challenges as mixed cultures, however there are some parameters which need to be optimized. Mixed cultures can be relatively easy to produce, such as by growing up microorganisms independently and then mixing the cultures prior to freeze drying. However, depending upon how the mixed cultures are used, incompatibility between strains needs to be considered. For instance, if the mixed culture is to be spread plated on an agar that differentiates, all species need a common media, temperature, pH, and atmosphere. Furthermore, if any of the strains secrete inhibitory substances, that may also be an issue if they are cultivated together. Finally, there is a very real possibility that different strains may have different die off rates, which needs to be considered if mixed cultures are used for quantitative testing.

Distribution of Freeze Dried Cultures

Quality control cultures that are shipped between laboratories risk losing viability unless the cultures are shipped on ice, as shipping conditions can vary drastically. Though lyophilized microorganisms are normally very stable, there can be significant loss of viability if cultures are not maintained at 4°C. Consequently, if large number of cultures require distribution, shipping costs for overnight service or ice charges may be significant. With cultures used in-house, the additional costs are not an issue.

Quantitation

Lyophilized QC microbes are useful for testing proficiency of laboratories and personnel, and for controls for quantitative testing, such as membrane filtration. There are two main issues with preparing and using lyophilized QC cultures, with the first being the preparation and the second being a drop in viability over time. During preparation of cultures, the issue of die off has already been discussed, especially if mixed cultures are used. If a population of preserved microbes has a predictable death rate, then anticipated counts can be calculated.

The preparation of cultures for freeze drying so that each vial has a countable number of cells is very laborious. For instance, standard freeze drying can include the simple technique of flooding a petri dish with Microbial Freeze-Drying Buffer ), suspending the cells, and transferring the suspension to glass vials. Using liquid cultures isn't much more difficult in that a culture is centrifuged, and the pelleted cells are resuspended in freeze drying buffer. However, preparing cultures of a known number of cells in each vial requires a significant amount of basic microbiology. Growth curves need to be established for each organism so optical densities can be used to estimate diluting cultures to desirable concentrations. Unlike making dilutions for plate counts, it is impractical to bracket the desired concentration of cells as it limits the number of vials that can be processed.

One strategy for reaching the desired concentration of cells is to freeze dry at a concentration above target and then dilute the cells when rehydrating. With this approach, vials containing known cell concentrations can be used for longer periods of time as die off becomes less of a factor. As cells become non-viable, the dilution level decreases.

Conclusion

Freeze drying microbial cultures can provide a readily available and economical source of quality control samples. Though available commercially, producing freeze dried cultures in-house or by contract labs can lower costs and allow for customization of the product. By controlling the design of the cultures, i.e., type of bacteria or fungi, and concentration, customized freeze-dried cultures can be used for quantitative quality control applications, including proficiency testing. With an understanding of a microorganism's stability (i.e., shelf-life), bacteria and fungi can be mixed and lyophilized for more dynamic controls. For laboratories that use a large number of lyophilized microorganisms for quality control, customization is a good option to control cost and availability.

For more information on customized quality control cultures, please contact us at (908) 253-3444 or by email.

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