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. 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. Most samples analyzed on BactoBox are diluted prior to being measured.

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.

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.

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.

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.

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.

Does BactoBox also measure yeast, molds, and mammalian 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 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.

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 measure bacterial spores? 
BactoBox can measure bacterial spores, but only in pure cultures and it requires the user to modify the default gating settings used for detecting intact bacterial cells. This can be accomplished with help from SBT.

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. By delivering precise bacterial abundance data, BactoBox complements other analytical methods, ensuring a more comprehensive understanding of the bacterial landscape in a given sample.

What consumables are required for operating BactoBox? 
For operating BactoBox, the primary consumable is the flow cell, which has a lifespan of 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.