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 intact cells/mL is a subset of the total particles/mL. 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-compatible 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-compatible diluent, which is a diluted 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 range of sample matrices, including: fermentation broths, glycerol freeze-stocks, overnight cultures, agar plate harvests, stool/intestinal samples, saliva, sourdough, wastewater and sludge samples, Actinomycetes/Bacillus spore suspensions, freeze-dried/lyophilized probiotic cultures, contaminated enzyme products, contaminated eukaryotic cell cultures, and live biotherapeutic products (LBPs).
The device exhibits broad applicability, provided a near-single-cell suspension is prepared. Such suspensions can typically be achieved using vigorous vortexing or bead-beating. In some cases, especially with dirty samples, it may be necessary to remove substances that could cause clogging. This can be achieved using syringe/cell-strainer filtration through a 10 µm filter.
BactoBox is most efficient with samples containing a high concentration of bacteria, typically more than 100,000 CFU/mL (Colony-Forming Units per milliliter). However, if the concentration is lower than this, effective sample preparation workarounds, such as concentration steps or enrichment cultures, are available.
Does BactoBox work for strictly anaerobic bacteria?
Yes, BactoBox is well-suited for use with strictly anaerobic bacteria. BactoBox exhibits consistent efficiency in enumerating both anaerobic and aerobic bacteria, reflecting its adaptability across a diverse range of microbial strains. Its design features, such as compact size and ease of handling, make it an ideal choice for working in anaerobic conditions. The BactoBox device, along with the necessary diluents, can be conveniently placed inside an anaerobic chamber. This capability is crucial, as it ensures that the anaerobic environment is maintained throughout the sample preparation and measurement process.
Does BactoBox differentiate between viable & non-viable bacteria?
Yes, 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 is capable of providing partial live/dead cell differentiation during a growth curve. It does this by reporting the concentration of intact cells, which are typically viable, as a subset of the total particles detected. To discern the proportion of live and dead cells, one can subtract the count of intact cells from the total particle count. The remaining subset includes all particles within the ~0.5-5 µm size range detected by BactoBox, such as dead cells, insoluble substances, salt crystals, microplastics, etc. Throughout a growth curve, non-bacterial background particles generally stay constant. Therefore, the ratio or percentage of intact cells to total particles can provide valuable insights into the culture's health and viability.
Consider an E. coli growth curve as an example:
- 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 equals total particles.
- Stationary phase: The intact cell concentration will be 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?
Comparative studies, including a peer-reviewed ring test, have shown a high level of agreement between BactoBox's impedance flow cytometry and traditional fluorescence flow cytometry. While fluorescence flow cytometry typically utilizes fluorescent dyes to differentiate live and dead cells based on membrane integrity, BactoBox focuses on enumerating membrane-intact bacterial cells through their electrical properties. Despite these differences in approach, the outcomes in terms of bacterial concentration and live/dead ratio assessments are consistent between the two methods.
Both BactoBox and traditional fluorescence flow cytometry are effective for enumerating bacteria. BactoBox stands out with its ease of use, rapid processing time, and label-free technique, enhancing its simplicity, ease of standardization, and cost-effectiveness. Conversely, fluorescence flow cytometry typically requires more complex setups, including lasers and intricate sample preparation, and demands greater expertise for operation. While BactoBox does not offer cell sorting capabilities like some fluorescence-activated cell sorting (FACS) instruments, its quick and straightforward operation makes it a valuable tool in various applications, including probiotics, postbiotics, crop sciences, and live biotherapeutics.
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 CFUs?
The correlation between BactoBox's intact cell concentration readings and Colony-Forming Units (CFUs) varies depending on the sample type. In scenarios where the sample predominantly contains culturable bacteria, such as in many fermentation and biotech applications, BactoBox's intact cell count tends to closely align with CFU measurements. However, this correlation can be less direct in other types of samples, like soil, water, and clinical specimens. This discrepancy arises because cultivation conditions may favor certain bacteria, and some viable but non-culturable cells (VBNC) might be present. In these cases, CFUs might not provide a complete representation, making cultivation-independent methods a more accurate choice for characterizing these samples.
Effective homogenization is a critical factor for both reliable BactoBox readings and CFU determinations. Clumped cells can lead to inaccuracies; they might be too large for the measurement channel of BactoBox or get counted as a single entity, although comprising multiple bacterial cells. This issue also affects CFU counts, where a clump of bacteria might be mistaken for a single colony. Most often, bacterial clumps can be efficiently dispersed using techniques like bead-beating or blending, facilitating more accurate measurements.
What consumables are required for operating BactoBox?
For operating BactoBox, the primary consumable is the flow cell, which has a lifespan of approximately 250 measurements. Once this limit is reached, the flow cell needs to be replaced to ensure continued, accurate operation of the device. Besides the flow cell, BactoBox operation also requires standard laboratory consumables. These include 15 mL centrifuge tubes, phosphate-buffered saline (PBS), and high-quality, low-conductivity water, such as Milli-Q, Water for Injection (WFI), or similar types.
Additionally, while we offer various optional consumables like syringe filters to enhance and streamline the user experience, they are not essential for the basic functioning of BactoBox. These optional items can provide convenience and efficiency but are not a necessity for the device’s operation.
Interested in experiencing BactoBox® firsthand or have questions regarding SBT Instruments' innovative technology? We invite you to connect with us through our convenient contact form below. A specialist from our team will contact you within one business day.