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 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.