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Frequently Asked Questions

Frequently Asked Questions

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 contact us if you desire a more in-depth explanation of any of the topics 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 presence of cells that do not grow on the selected plates such as stressed or dormant cells, non-culturable cells or cells that prefer other growth conditions – these are counted by BactoBox and not by CFU
  • The cells’ tendency to cluster – our data indicates that cell aggregates are separated by fluidic shear forces in our flow cell, which has to be specifically addressed in sample preparation when measuring CFU

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:

  • Bacteria may be viable and still not grow on the plates. This can be caused by bacteria being in a stressed or dormant state. Or the bacteria in the sample can be very selective about when they grow, e.g. by requiring an environment without oxygen, only growing at specific temperatures, requiring specific nutrient compositions or requiring a long time to multiply enough to be visible on plates.
  • One colony is not necessarily formed by one bacterium but can be started by multiple bacteria that are clustered together.

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 measures intact cells pr. mL (intact cells/mL) in a sample. Our electrical detection principle measures each individual particle that passes through our detection sites for an intact bacterium cell membrane structure. A bacterium with an intact cell membrane has unique electrical properties, and BactoBox can detect and count such cells very fast.
For most bacteria that can be cultivated in the laboratory, the intact cell concentration is a good approximation for colony forming units, CFUs. On the other hand, something like a water or soil sample would have much higher intact cell concentrations than CFU/mL, because only a fraction of the bacteria in a water or soil sample are culturable on standard media.

What are the detection limits?
In practice, the detection range for BactoBox is typically 10,000 to 5,000,000 intact cells/mL for actively growing cultures, where the number of dead cells is negligible. BactoBox's upper limit of detection is dependent on the number of total particles/mL, i.e. the number of intact cells/mL plus the number of other particles/mL. The maximum is 5,000,000 total particles/mL.
BactoBox will show < 5,000 intact cells/mL for samples that have few or no intact cells. It will show 5,000 - 10,000 intact cells/mL for samples within this range, but not report an actual number for the concentration as the measurement variance is too high in this range. For samples in the measurement range above 10,000 intact/mL, the actual intact cell concentration will be reported and likewise, the total particle concentration.

How much sample do I need?
The typical sample consumption is ~100µL. In most cases, at least a 1:100 dilution is prepared and the 100 µL primary sample is transferred to 9,900 µL BactoBox diluent. The measured sample volume is therefore typically 10 mL. Minimum volume for a measurement is 4 mL, but we generally recommend to use 10 mL final sample volume.

What are the prerequisites for BactoBox measurement?
 ·         Conductivity: BactoBox measures the electrical signatures of bacteria directly without any need for the addition of labels or tags like fluorophores. But in order to discriminate intact cells from other types of particles (e.g. dead cells), it is very important that the conductivity is between 1,700-2,100 µS/cm in the final sample. This is easily achieved by diluting the primary sample in BactoBox diluent, a diluted form of phosphate buffered saline, PBS.
 ·         Single-cell suspension: BactoBox has a measurement channel that only permits particles with a diameter <5 µm to enter. Bacteria have a tendency to auto-aggregate and form clumps. These must be dispersed for a valid BactoBox measurement (or plate count). Along with BactoBox, we supply fast sample-workup protocols that are effective at disaggregating without rupturing bacterial membranes. 
 ·         Concentration: The total particle concentration must be below 5,000,000 total particles/mL and the intact cell concentration must be between 10,000-5,000,000. If the concentration is above this range, simply dilute the sample. If it is below, move to a more concentrated sample in the dilution series or concentrate the bacteria e.g. by centrifugation or filtration.

What are the dilution requirements for BactoBox?
We recommend diluting the sample below 5,000,000 total particles/mL. For a typical overnight culture of ~109 CFU/mL, this is typically achieved by diluting the samples 1:10,000 i.e. two sequential 1:100 dilutions.

Which sample matrices are compatible with BactoBox?
BactoBox has successfully been demonstrated for samples as diverse as fermentation broths, glycerol freeze-stocks, overnight cultures, agar plate harvests, stool/intestinal samples, saliva, sourdough, water/waste-water/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 biotherapeutics products (BPLs), 
Our device has very broad applicability as long as a “close-to” single-cell suspension is created. Single-cells suspensions are usually created using aggressive vortexing or bead-beating. It may also be necessary to remove clogging substances from dirty samples, and these are typically removed using syringe/cell-strainer filtration through 5 or 10 µm filters.
The easiest workflow is currently with samples that have a relatively high concentration of bacteria, typically >100,000 CFU/mL, but there are excellent sample workup workarounds if the concentration is lower than this.

Does BactoBox work for strictly anaerobic bacteria? 
Yes! Because of the compact size, BactoBox and the diluents can easily be placed inside an anaerobic chamber. Several users are currently using this approach to quantify strictly anaerobic microorganisms.

Does BactoBox differentiate between viable & non-viable bacteria?
Bacteria with an intact cell membrane have an electrical signature that differs substantially from other particles or objects because i) the phospholipid membrane acts as an electrical insulator and ii) the cytoplasm has a conductivity interior, i.e. salty-water, that differs from the external diluent. If a bacterium has a perforated/ruptured cell membrane, it will not be counted as an intact cell by BactoBox. 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 died. Inactivation by UVC light is an extreme example of a disinfection method where BactoBox can not “see” a difference right after treatment, while on the other hand, the plate count method will show a loss in the number of colony-forming units, CFU’s. This is because UVC germicidal irradiation mainly works by creating pyrimidine dimers in DNA so the cells can no longer replicate and form “offspring”. Of note, if the same UVC-exposed sample is measured on BactoBox 24-48 hours after the initial exposure a clear drop in the number of intact cells will be observed because the cells lose the ability to repair themselves and the membranes disintegrate.   

Can BactoBox provide live/dead information during a growth curve? 

Yes, partially. BactoBox reports the intact cells concentration (viable) as a subset of the total particles detected. When the intact cells are subtracted from the total particles this subset will include all ~0.5-5 µm-sized particles detected by BactoBox, e.g. dead cells, insoluble substances, salt crystals, microplasts, etc. During a growth curve, the non-bacterial background particles will be relatively constant and therefore the ratio (or percentage) between the intact cells and total particles will provide actionable information on the “well-being” and status of the culture. For an E. coli growth curve this would typically manifest as exemplified below:
·         Lag phase: Intact cell concentration will be relatively low and the contribution from other particles will be pronounced;
·         Exponential phase: Intact cell concentration will increase rapidly and will virtually constitute all the detected particles, i.e. intact cell concentration ~ total particles;
·         Stationary phase: Intact cell concentration will be very high, but total particles will gradually become considerably higher than intact cell concentration;
·         Death phase: The intact cell concentration is gradually dropping and converting to “non-intact” total particles. 

How does BactoBox correlate with FACS measurements? 

Our customers have performed numerous comparisons between BactoBox and FACS instruments and found excellent correlations between the two methods. Especially for stool samples, the researchers have found that the sample-workup is much faster for BactoBox (below 30 min) compared to the lengthy protocols needed for sample-workup for FACS analyses (4 hours sample prep. time).
The measurement principle of BactoBox is in many ways analogous to fluorescence-activated cell sorting (FACS), but because the read-outs are based on impedance measurements (electricity), BactoBox is much more portable, robust and inexpensive compared to the pristine lasers in the FACS that result in delicate, expensive and bulky instruments. Also, BactoBox measurements are very easy to perform even for an inexperienced user, while the FACS typically requires a long learning curve to master the technology. 
NB! It must be mentioned, that BactoBox can’t be used for cell sorting. An additional difference is that most FACS instruments have walk-away instrumentation with autosamplers for the injection of samples which can be convenient in the case of high-throughput analyses. BactoBox does presently not have an autosampler, but typically this is not a problem because the time-to-result is so fast (often below 1 min) that it just leaves enough time to do the sample workup for the subsequent sample.  

Can BactoBox provide 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 concentration of bacteria in a sample.

Does BactoBox also measure yeast, molds, and human cells? 

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 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 are the main parameter of interest and if bacteria cannot be used as a proxy.
As in the case with yeast and molds, human cells are too big to enter the measurement channel and are therefore not counted by BactoBox.

Does BactoBox measure bacterial spores? 

Yes. BactoBox has successfully been tested for Actinomyces and Bacillus endospores. 

Can BactoBox provide abundance in metagenomics studies? 

Yes. BactoBox is currently being used by several end-users to provide information on absolute bacterial concentrations. While techniques like NGS and qPCR methods give an excellent description of what bacteria are present in the sample they are often hampered by the lack of how many, i.e. the total abundance of bacteria in the system. As in the case of e.g. Campylobacter and human disease, quantity matters, and therefore the intact cell concentration provided by BactoBox can be crucial for the right conclusions to be drawn.

How does BactoBox correlate with CFU? 

When bacteria are grown under controlled conditions, the intact cell concentration provided by BactoBox correlates very closely with CFUs. This is the case for fermentation and biotech applications.
For other sample types - e.g. soil, water, and clinical samples – the correlation with CFUs is not as simple because the chosen cultivation conditions are only suitable for some of the bacteria and because viable but non-culturable cells (VBNC) may exist in these matrices. It is therefore well-known that CFUs are not always a meaningful readout for these sample types and that cultivation-independent methods should be used to characterize these samples.
It must be mentioned that effective homogenization is crucial for reliable BactoBox (and CFU) determinations. If cells are present as clumps there is a risk that they are either too big to enter the measurement channel or that several bacterial cells are counted as one. For the CFU determinations, a clump consisting of several bacteria are likewise counted as a single colony. Fortunately, in most cases, it is relatively fast and straight-forward to disperse bacterial clumps, e.g. by bead-beating or blending.

What are the consumables for the system? 

The flow cell in BactoBox is a consumable. It lasts for 250 measurements after which no more measurements can be performed. Apart from the flow cell, the consumables include standard lab items such as 15 mL centrifuge tubes, PBS buffer, and high-quality, low-conductivity water (e.g. MilliQ, WFI, or similar). We also provide a range of other consumables like syringe filters that can ease your workflow, but these are all optional.

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SBT Instruments A/S
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2730 Herlev
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SBT Instruments
Symfonivej 37
2730 Herlev
Denmark