We frequently receive insightful inquiries from our users, and this list aims to address some of the most common questions. While we have provided concise answers to these questions, we understand that each topic may have multiple layers of complexity. Please do not hesitate to contact us if you require a more detailed explanation of any subjects discussed below.
It is always a pleasure to engage with customers and satisfy your curiosity. We are often asked very interesting questions, and this list is an attempt to answer some of the most common ones. However, there are always multiple layers to a certain topic and question, so do not hesitate to follow up if you desire a more in-depth explanation to any of the topics below!
Is the measurement real-time or how fast is it?
When a bacterium passes over the electrodes in our flow cell it will be detected. This detection happens in real-time. However, we only measure a subset of the sample inserted in BactoBox. We need to measure for a certain time period in order to build sufficient statistical evidence to provide an accurate concentration measurement. This typically takes a few minutes depending on the application.
Does BactoBox differentiate between viable & non-viable bacteria?
Differentiating viable and non-viable bacteria is a technical challenge for any fast bacteria measurement. With traditional plate counts, the bacteria are given time to grow, which helps determine which are viable and culturable, at least at the given conditions. However, due to the slow growth process this is not an option for a fast measurement. Instead, BactoBox uses membrane intactness to assess viability by probing for intact cell structures electrically and omitting non-intact cells from the count. Thus, the unit of BactoBox is “intact cells/ml”. So, is membrane intactness a good estimation of viability? Yes – in the vast majority of cases. Viable cells have intact cell membranes, and most non-viable cells do not have an intact cell membrane – although this can vary depending on how the bacteria have been killed.
Can BactoBox do species differentiation?
No. At this stage in our technology maturation we are not able to differentiate between different species of bacteria. BactoBox measures the total number of bacteria in a sample.
Does BactoBox also measure yeast and molds?
BactoBox measures intact cells/ml. However, BactoBox only measures intact cells in the size range 0.5 µm to 5 µm due to the design of the microfluidic flow cell. This size range covers by far most bacteria and excludes by far most yeast and mold cells. However, in microbiology there will always be edge cases. If a yeast or mold cell is smaller than 5 µm there is a chance it will be counted as an intact cell. We do not recommend using BactoBox to measure yeast and mold cells if yeast and mold counts is the main parameter of interest and if bacteria cannot be used as a proxy. We have vague plans about developing a yeast and mold version of BactoBox in the future and we are still very interested to get input from potential users on where they would want to use such a device.
Is BactoBox an online system or can you make it online?
BactoBox is not an online sensor in the sense it cannot do automatic. BactoBox is a grab sample system that you can use to measure samples from many different sampling points. Regular cleaning of BactoBox is necessary and this feature has not been automated and we are not planning to make a fully online sensor anytime soon. There are many reasons for this, but the primary being that making a fully functional online sensor is much more difficult than what you would think. And believe us, we did try in 2014-2016. However, never say never. The technology is very suitable for being online. It is just not on the roadmap yet.
Is there a consumable?
Yes. The flow cell in BactoBox is a consumable. It lasts for several hundred measurements, but it has to be exchanged eventually. We also provide a range of other consumables that can ease your workflow, but these are all optional.
How does BactoBox correlate with (non-specific) CFU such as standard plate count/aerobic plate count/total plate count?
The answer to this question is unfortunately much more complicated than what we would have preferred it to be. We will start by giving the short explanation for experts in the field, and then we also have a more elaborate explanation for the rest of us.
The short version: BactoBox’ measures intact cells which correlates with CFU, but can be skewed by:
The slightly longer version: First, it is important to align on what a colony forming unit (CFU) is. A CFU is a unit used in microbiology to estimate the number of viable bacteria in a sample. You only count the bacteria that decide to grow and multiply when you count CFU. This means that CFU does not always correlate with the true number of viable bacteria in a sample. Two effects contribute to the true number of viable cells being different from the CFU count:
In contrast to CFU, the technology in BactoBox counts all bacteria in the sample as long as the bacteria have intact cell membranes. Like any other fast method, BactoBox is not dependent on growth and multiplication of bacteria. This means BactoBox also counts stressed or dormant bacteria cells, or bacteria cells which require exotic growth conditions.
Clustering of bacteria is a well-known problem when measuring CFU as it will typically be several clustered bacteria that result in one colony. Our data indicates that BactoBox break up clusters of bacteria due to the fluidic shear forces in the flow cell. As a result, four bacteria might result in one CFU, but in four intact cells when measured with BactoBox.
Therefore, the correlation between BactoBox and CFU will be linear for samples that:
(1) only contain bacteria in a culturable state, i.e. ready to grow on plates – typically single species samples
(2) cluster minimally
This will often be the case in fermentations and biotech applications where bacteria are grown under controlled conditions.
It is a different story when measuring samples with complex mixes of bacteria in different states and with different preferred growth conditions. These cases include environmental, product or water samples. BactoBox measures the actual cell count in the sample regardless of the mix of bacteria. In contrast, the CFU result strongly depends on the fraction of bacteria which prefer the growth conditions provided to them. To illustrate this point, if you take a sample, split it into two and incubate it on two growth media containing different nutrients, then you will get two different CFU results. Therefore, when measuring on complex bacteria mixes, CFU will usually measure much fewer bacteria than BactoBox – in some cases up to 200x fewer. This makes the correlation between the two methods less apparent, and instead you must consider if measuring the true cell count provides value to your process. However, why not measure everything if you are trying to assess the total bacteria count?
What is the measurement output of BactoBox?
BactoBox provides the concentration of intact cells per milliliter (intact cells/mL) and a separate result for the total particles per milliliter (total particles/mL) in a given sample. The technology within BactoBox is designed to detect each individual particle as it passes through the detection unit. Subsequently, each particle is identified as either an intact bacterial cell or a different type of particle. This distinction is made possible due to the unique electrical properties of bacteria with intact cell membranes.
What are the detection limits?
The BactoBox is capable of detecting a range of 5,000 to 5,000,000 intact cells/mL in actively growing cultures, assuming a negligible presence of dead cells. The upper detection limit of the BactoBox depends on the total number of particles/mL, which includes both intact cells/mL and other particles/mL, with a maximum of 5,000,000 total particles/mL.
For samples with few or no intact cells, BactoBox will indicate a value of less than 5,000 intact cells/mL. For samples containing 5,000 to 10,000 intact cells/mL, BactoBox will display a value within this range, but it will not provide an exact concentration due to the high measurement variance in this range. However, for samples with more than 10,000 intact cells/mL, BactoBox will accurately report the concentration of intact cells and the total particle concentration.
How much sample do I need?
The standard sample volume used for BactoBox is approximately 100µL. In most instances, a 1:100 dilution is prepared by transferring the 100µL primary sample to 9.9mL of BactoBox diluent. Consequently, the typical measured sample volume is around 10mL. Although the minimum volume required for a measurement is 4mL, it is generally recommended to use a final sample volume of 10mL for optimal results.
What are the prerequisites for a successful BactoBox measurement?
- Conductivity: BactoBox detects the electrical signatures of bacteria without requiring labels or tags, such as fluorophores. However, to differentiate intact cells from other particle types (e.g., dead cells), it is crucial to maintain a conductivity between 1,600-2,100 µS/cm in the final sample. This can be easily achieved by diluting the primary sample in BactoBox diluent, which is a diluted form of phosphate-buffered saline (PBS).
- Single-cell suspension: BactoBox features a measurement channel that allows only particles with a diameter smaller than 5 µm to enter. Bacteria often auto-aggregate, forming clumps that must be dispersed for accurate BactoBox measurements (or plate counts). To assist with this process, we provide efficient sample workup protocols that effectively disaggregate clumps without rupturing bacterial membranes.
- Concentration: The total particle concentration must not exceed 5,000,000 total particles/mL, and the intact cell concentration should range from 10,000 to 5,000,000 intact cells/mL. If the concentration is higher than the recommended range, simply dilute the sample. If the concentration is lower, consider using a more concentrated sample in the dilution series or concentrating the bacteria through methods such as centrifugation or filtration.
What are the dilution requirements for BactoBox?
For BactoBox, it is required to dilute the sample to a concentration below 5,000,000 total particles/mL. In the case of a typical overnight culture with approximately 10⁹ CFU/mL, this can be achieved by performing a 1:10,000 dilution, which involves two sequential 1:100 dilutions.
Which sample matrices are compatible with BactoBox?
BactoBox is compatible with a wide variety of sample matrices, including fermentation broths, glycerol freeze-stocks, overnight cultures, agar plate harvests, stool/intestinal samples, saliva, sourdough, water/wastewater/sludge samples, Actinomycetes/Bacillus spore suspensions, freeze-dried/lyophilized probiotic cultures, skin swabs, swab samples for CIP-validation, contaminated enzyme products, contaminated eukaryotic cell cultures, and live biotherapeutic products (LBPs).
The device exhibits broad applicability as long as a near-single-cell suspension is prepared. Single-cell suspensions can typically be created using vigorous vortexing or bead-beating. In some cases, it may be necessary to remove substances that could cause clogging in dirty samples, which can be achieved using syringe/cell-strainer filtration through a 10 µm filter.
BactoBox is most efficient with samples containing a relatively high concentration of bacteria, typically >100,000 CFU/mL. However, effective sample preparation workarounds are available if the concentration is lower than this.
Does BactoBox work for strictly anaerobic bacteria?
BactoBox is suitable for use with anaerobic microorganisms due to its compact size. Both the BactoBox device and diluents can be conveniently placed inside an anaerobic chamber. This approach is currently employed by several users to accurately quantify strictly anaerobic microorganisms.
Does BactoBox differentiate between viable & non-viable bacteria?
BactoBox can differentiate between viable and non-viable bacteria based on the integrity of the cell membrane. Bacteria with intact cell membranes exhibit a unique electrical signature due to the insulating properties of the phospholipid membrane and the conductivity difference between the cytoplasm and external diluent. BactoBox will not count bacteria with perforated or ruptured cell membranes as intact cells.
Viable cells possess intact cell membranes, while most non-viable cells have compromised membranes, although this can vary depending on the cause of cell death. For example, BactoBox cannot immediately distinguish between viable and non-viable cells following UVC light treatment, as UVC germicidal irradiation primarily inactivates cells by creating pyrimidine dimers in DNA, preventing replication and subsequent colony formation. However, when measuring the same UVC-exposed sample 24-48 hours after the initial exposure, BactoBox will detect a significant drop in the number of intact cells, as the cells lose their ability to repair themselves and their membranes disintegrate.
Can BactoBox provide live/dead information during a growth curve?
BactoBox can partially provide live/dead information during a growth curve. It reports the concentration of intact cells (viable) as a subset of the total particles detected. By subtracting the intact cells from the total particles, the remaining subset will include all ~0.5-5 µm-sized particles detected by BactoBox, such as dead cells, insoluble substances, salt crystals, microplastics, etc. During a growth curve, the non-bacterial background particles generally remain constant, so the ratio (or percentage) between intact cells and total particles can offer valuable insights into the culture's health and status. For an E. coli growth curve, this would typically manifest as follows:
- Lag phase: The intact cell concentration will be relatively low, and the contribution from other particles will be more pronounced.
- Exponential phase: The intact cell concentration will increase rapidly and will constitute nearly all detected particles, i.e., intact cell concentration ~ total particles.
- Stationary phase: The intact cell concentration will be very high, but total particles will gradually become significantly higher than the intact cell concentration.
- Death phase: The intact cell concentration will gradually decrease and convert to "non-intact" total particles.
How does BactoBox correlate with fluorescence flow cytometry?
Customers who have compared BactoBox with fluorescence flow cytometry instruments have found strong correlations between the two methods. Notably, for stool samples, researchers have discovered that BactoBox's sample preparation is much faster (less than 30 minutes) compared to the extensive protocols required for fluorescence flow cytometry analysis (4 hours of sample preparation).
BactoBox's measurement principle is somewhat analogous to fluorescence flow cytometry; however, since BactoBox relies on impedance measurements (electricity), it is more portable, robust, and cost-effective than fluorescence flow cytometry, which uses delicate lasers, resulting in expensive and bulky instruments. Additionally, BactoBox measurements are easy to perform, even for inexperienced users, while fluorescence flow cytometry typically requires a longer learning curve to master the technology.
Another distinction is that certain fluorescence flow cytometers provide walk-away functionality with autosamplers for sample injection, which is advantageous for high-throughput analyses. Although BactoBox currently lacks an autosampler, this is generally not an issue, as the rapid time-to-result (often less than 1 minute) allows sufficient time for preparing the next sample. It is important to note that BactoBox cannot be utilized for cell sorting, unlike fluorescence-activated cell sorting (FACS) instruments.
Can BactoBox provide species differentiation?
At this stage in our technology maturation, we are not able to differentiate between different species of bacteria. BactoBox measures the total concentration of bacteria in a sample.
Does BactoBox also measure yeast, molds, and human cells?
BactoBox measures intact cells/ml, but it is specifically designed to measure intact cells within the size range of 0.5 µm to 5 µm due to the microfluidic flow cell configuration. This size range primarily encompasses bacteria and excludes the majority of yeast and mold cells. However, there may be exceptions in microbiology. If a yeast or mold cell is smaller than 5 µm, it could potentially be counted as an intact cell. We do not recommend using BactoBox to measure yeast and mold cells if their counts are the primary parameter of interest and bacteria cannot serve as a proxy.
Larger cells, such as human cells, can be present in the sample, but they simply won't be counted by BactoBox, as they are too large to enter the measurement channel.
Does BactoBox measure bacterial spores?
BactoBox can measure bacterial spores, but it requires the user to modify the default gating settings used for detecting intact bacterial cells. This can be accomplished using the free software provided by SBT. By adjusting the gating parameters, users can adapt BactoBox to accurately detect and quantify bacterial spores in their samples. Once the new gating settings have been determined, they can be uploaded to BactoBox as a permanent setting, allowing for consistent and accurate measurement of bacterial spores in future experiments.
Can BactoBox provide abundance in metagenomics studies?
BactoBox is currently utilized by numerous end-users to obtain information on absolute bacterial concentrations. While techniques like NGS and qPCR methods offer exceptional insight into the types of bacteria present in a sample, they often fall short in providing data on the total abundance of bacteria in the system. In cases like Campylobacter and human disease, the quantity of bacteria plays a critical role, making the intact cell concentration provided by BactoBox essential for drawing accurate conclusions. By delivering precise bacterial abundance data, BactoBox complements other analytical methods, ensuring a more comprehensive understanding of the bacterial landscape in a given sample.
How does BactoBox correlate with CFU?
When the sample only contains culturable bacteria, such as in most fermentation and biotech applications, the intact cell concentration provided by BactoBox correlates closely with CFUs. However, in other sample types, like soil, water, and clinical samples, the correlation with CFUs may not be as straightforward. This is because the chosen cultivation conditions may only be suitable for certain bacteria, and viable but non-culturable cells (VBNC) can exist in these matrices. As a result, CFUs are not always a meaningful indicator for these sample types, and cultivation-independent methods should be employed to characterize them more accurately.
It is essential to note that effective homogenization is crucial for reliable BactoBox (and CFU) determinations. If cells are present as clumps, they might either be too large to enter the measurement channel or counted as a single entity despite being composed of several bacterial cells. Similarly, for CFU determinations, a clump consisting of multiple bacteria is often counted as a single colony. Fortunately, in most cases, it is relatively quick and straightforward to disperse bacterial clumps using methods like bead-beating or blending.
What are the consumables for the system?
The flow cell in BactoBox is a consumable item with a lifespan of 250 measurements. After reaching its limit, it must be replaced for continued use. In addition to the flow cell, other consumables required for BactoBox operation include standard laboratory items such as 15 mL centrifuge tubes, phosphate-buffered saline (PBS), and high-quality, low-conductivity water (e.g., Milli-Q, WFI, or similar). While we offer a variety of optional consumables, such as syringe filters, to streamline your workflow, these items are not mandatory for the BactoBox to function properly.
Would you like to request a demo, on-site trial, or do you just have a general question about BactoBox® and SBT Instruments' technology? Please get in touch via our quick contact form below, or, reach out directly to our CEO, Gustav.
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