BactoBox® Application Note

BactoBox® for Freeze-dried Bacterial Products

Freeze-dried products are significantly easier to stabilize than their liquid counterparts. Nonetheless, freeze-drying can induce sublethal damage to bacteria and it can be difficult to revive all the viable bacteria in a sample with the standard plate count method. Here we give a simple example with Lactobacillus rhamnosus PB01 (DSM 18470) reconstituted in either peptone salt diluent or MRS medium to show how BactoBox can be used to screen for the proper reconstitution medium. The choice of proper reconstitution medium will aid greatly in reducing the variability associated with subsequent plate counts. Because BactoBox has high precision (low variation) and is virtually operator-independent, fewer replicates are needed to achieve statistical significance. In addition, the learning loops are accelerated because results are available immediately instead of the usual days of waiting for colonies to appear.

Keywords: Freeze-dried powder, lyophilized, bacteria, Lactic acid bacteria, viability, rehydration, resuscitation, impedance spectroscopy, flow cytometry, lyoprotectant, anaerobic microorganisms, live biotherapeutic products, probiotics, postbiotics.

Resuscitation of bacteria from freeze-dried powders can be difficult

Freeze-drying (lyophilization) is beyond doubt the most frequent preservation method for maintaining the viability of bacteria during storage. But even though freeze-drying is one of the gentler drying methods, it is still tough for microbes to be deprived of water and it can be difficult to resuscitate (revive) bacteria after freeze-drying. In the case of a newly isolated strain, it can be difficult to predict the ideal reconstitution parameters and therefore investigations require lengthy trial and error approaches or rigorous statistical testing like design of experiments.

Often it is necessary to screen several parameters to ensure efficient resuscitation. These parameters include 1) pH, 2) availability of a metabolizable sugar, 3) reconstitution duration, 4) powder matrix and 5) ratio of powder to reconstitution solution¹. The workload for doing these studies is immense. The effort required is further aggrevated by the fact that plate counts are associated with very high analytical imprecision (noise) and therefore several replicates are needed to gain statistical confidence in the results.

But the frustrations associated with plate counts are not just a matter of workload. The time to answer for e.g. anaerobic bacteria is usually several days and even up to a week. This means that the learning loops are slow and frustrating.

In addition, it is known that physical conditions like starvation and low temperature can trigger bacteria to enter a “viable but non-cultureable” (VBNC) stage or at least a “difficult to resuscitate” stage. Some species can be salvaged on solid medium by the proper choice of parameters, but then again other sub-lethally injured bacteria may require liquid media for resuscitation^2–4. These findings mean that even though plate counts are often observed as the golden standard for a viability, it is not always a valid representation of the viability status for the bacteria in a sample.

Within spectrophotometry, OD600 is often used as a time-saving proxy measurement for biomass concentration2. The measurement principle is based on light-scattering as shown in the illustration below: Light is passed through the liquid medium and collides with individual bacteria and other particles in the suspension. Higher bacterial concentrations will result in less light reaching the detector.
The wavelength 600 nm is typically chosen because it offers an acceptable tradeoff between signal strength and specificity where most of the light “loss” is caused by light scattering and not by pigment absorption. While OD600 is possible to multiplex in 96-well plates, extremely cheap, simple, and can be performed on-line, there are obvious pitfalls and drawbacks for quantification of microorganisms:

OD600 provides a number with an arbitrary unit. It does not provide a bacterial concentration unless laborious OD600/CFU calibration curves are carried out.
Spectrophotometer configurations vary and therefore the OD600 measurements from one device can’t be compared directly with another device unless a calibration with measurement standards is performed3.
OD600 does not differentiate between bacteria and other particles. If the medium contains a background of non-bacterial particles or a high background of dead cells, this could result in misleading results.
Multiple scattering (Figure 1A, red arrow) can lead to non-linear effects where the incident light reaches the detector because it “bounces off” several particles in solution.
Many bacteria produce pigments that absorb in the OD600 range. The pigment production can vary over time and therefore the OD600/CFU ratio will be inconsistent
Bacterial cell size typically vary during a growth curve and because larger cells spread light more than smaller bacteria, this will also give a variable OD600/CFU ratio during the growth curve.
Some bacteria – especially photosynthetic – transmit light very effectively through the cells and are therefore sometimes referred to as “OD-transparent” because even very high concentration of cells (>109 CFU/mL) result in very little light scattering2. This means that they are difficult to detect by OD600 measurements.

Advantages with BactoBox when studying freeze-dried bacterial products

Advantages with the BactoBox

BactoBox is cultivation-independent and label-free. This means that the sample preparation is fast and the whole process from sample preparation to the final measurement is usually within 5 minutes. With plate counts, you would have to wait for days and even with traditional flow cytometry, it is necessary to label the cells with fluorescent dyes, load sample and perform data-analysis; typically, this takes roughly 30 minutes.

Dissolution of bacteria from freeze-dried powders is a complicated process, and the benefit of having immediate results is that you can monitor this process as it is progressing. Initially, the exterior part of the individual powder particles dissolves, while the inner bulk can still be in particulate form. If the rehydrated powder is plated on solid medium too early the colony forming units (CFUs) may be underestimated because some a proportion of the particulates still contain several CFUs that just haven’t been dispersed yet.

The total particle concentration reported by BactoBox gives insight into the necessary rehydration time for maximal dissolution. BactoBox also reports the intact cell concentration, which indicates if the rehydration medium is sufficient for resuscitation or if membrane integrity is lost as a function of rehydration time.

A simple case study using BactoBox to study resuscitation of bacteria

As an analyst you aim to get a reliable representation of the viable bacteria in the sample. With a newly isolated strain it can be difficult to predict the best resuscitation buffer/medium and because plate counts for anaerobic bacteria take several days, each learning loop will typically require at last a week of planning, experimenting, waiting, counting, and performing data analysis. With BactoBox you get immediate results and because the results have higher precision than plate counts, you can also skip replicates and focus on frequent sampling.

Here, we performed a simple experiment to illustrate the benefits of using BactoBox instead of plate counts for assessing resuscitation from freeze-dried powder. As shown in the workflow illustration, freeze dried powder was added to MRS medium and peptone salt diluent (PSD). Subsequently, 3mm glass beads were added and aggressive bead beating (speed 4800) was performed for 2 minutes to aid in the rehydration procedure. Two consecutive 1:100 dilutions were performed in BactoBox diluent for a final dilution factor of 1:10,000 (10^-4) and the final tube was analyzed by BactoBox. In the meantime, the rehydration tube was placed back on the bead beater for gentle agitation (speed 500) between sampling points.

Illustration of sample workup and for the rehydration of Lactobacillus rhamnosus freeze dried powder and subsequent BactoBox measurements.

BactoBox gives a here-and-now representation of the rehydration process

The charts below show the data from the rehydration test in PSD and MRS. The left chart shows the intact cell concentration measured by BactoBox for the first ~2 hours. With the peptone (black curve) the intact cell concentration drops over time showing that the bacterial membranes are not stabilized properly in PSD. On the other hand, with MRS (violet curve), the intact cell concentration increases within the first ~30 minutes of rehydration. The total particle concentration is relatively constant in this time frame (results not shown). When the impedance data are inspected in detail, the bacterial populations shift slightly, but consistently towards more conductive properties, i.e. into the intact cell range. The size of the object is also increasing (results not shown). We interpret this as an initial swelling (wake-up) of metabolically dormant freeze-dried bacterial within the first 30 min. To get reliable results it is important to wait ~30 minutes before BactoBox analysis otherwise the bacterial concentration would be underestimated. For example, 5 minutes after the aggressive bead beating, the bacterial concentration would still only be 40% of the correct value.

Another interesting observation with MRS is that the concentration is relatively stable from 30-120 minutes. This shows that it is not that critical if the sample is measured immediately after 30 minutes or if delays occur. With CFU-determinations there would likely have been large error bars (typical deviations of up to 35% with respect to the average⁵ and therefore it would be difficult to see this highly stable trend. The measurements are much more reproducible with BactoBox and therefore the stable trend is significantly more pronounced.

BactoBox gives fast and reliable insights into the dissolution of the freeze-dried bacterial powders and shows that MRS medium protects lactic acid bacteria much better than peptone/salt diluent (PSD). Left chart represent the intact cell concentrations (ICC) as a function of rehydration time, while the chart on the right represents the intact cell concentration divided by the measured concentration of total particles. Time zero indicates the time where aggressive bead beating was initiated.

The chart on the right shows a wider timespan for the rehydration process and the y-axis depicts the ratio of the intact cell concentration (ICC) and total particle concentrations detected by BactoBox. In this experiment, this can be used as a magnifying glass into the live/dead status of the bacteria in the sample. Looking at the black curve (PSD) the drop in intact membranes over the first 60 minutes becomes even more apparent and it is also interesting to see that the ratio increases again after 120 minutes. L. rhamnosus is a slow-grower and also typically needs anaerobic conditions for substantial growth and it is therefore unlikely that the increase in intact and total cells is due to this bacterium. Instead, the increase can likely be explained by a contaminating bacterial species that thrives in the PSD medium.

With the MRS rehydration (violet curve), no increase in ICC/Total ratio occurs. These data give the analyst the confidence that the concentration in the MRS medium is relatively stable and therefore the analyst is free from worrying about falsely high bacterial CFU concentrations due to growth in the rehydration step. A final observation is that with the wide timespan, bacteria in MRS also lose some membrane intactness after the 120 minutes of rehydration given the fact that the total particle concentration is constant (data not shown).

BactoBox as an indispensible tool for screening resuscitation parameters

As shown in the simple example above, BactoBox provides fast insight into the rehydration/resuscitation parameters for bacteria in freeze-dried powders. Some of the key take-aways are summarized below:

BactoBox aids in finding the proper resuscitation medium. In the MRS/PSD example above, BactoBox measurements clearly show that MRS is the wiser choice. Firstly, MRS protects the bacterial from membrane lysis. Secondly, MRS reduced the risk of false-positive growth by environmental contamination.
BactoBox gives easy-to-interpret live/dead insights. The label-free impedance principle underlying the BactoBox technology can distinguish intact bacterial membranes from other “stuff”. Therefore, the percentage intact (relative to total particles detected) gives critical insight into the percentage of bacteria “salvaged” or resuscitated from the freeze-dried powder.
BactoBox has high precision. The effect of the low relative standard deviation given by BactoBox means that the trends are significantly easier to interpret than if you were using plate counts. With plate counts, you would typically need several technical replicates for the same level of consistency, which means more expenses and (tedious) time spent.
BactoBox gives results immediately. This means that experiments can be terminated when change is absent, and it is no longer meaningful to extend the experiments. Also, learning loops are accelerated because you don’t have to wait for days or weeks for colonies to appear on a plate.

References

Muller, J. A., Stanton, C., Sybesma, W., Fitzgerald, G. F. & Ross, R. P. Reconstitution conditions for dried probiotic powders represent a critical step in determining cell viability. J. Appl. Microbiol. 108, 1369–1379 (2010).
Wai, S. N., Mizunoe, Y., Takade, A. & Yoshida, S. I. A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila. Arch. Microbiol. 173, 307–310 (2000).
Parthuisot, N., Catala, P., Lebaron, P., Clermont, D. & Bizet, C. A sensitive and rapid method to determine the viability of freeze-dried bacterial cells.
Zeng, B. et al. Formation and resuscitation of viable but nonculturable Salmonella typhi. Biomed Res. Int. 2013, (2013).
USP. <1223>: Validation of Alternative Microbiological Methods. United States Pharmacop. 04-Oct-2020, 4–6 (2021).

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