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Frequently Asked Questions

Frequently Asked Questions

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?

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?

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SBT Instruments
Symfonivej 37
2730 Herlev
Denmark

SBT Instruments
Symfonivej 37
2730 Herlev
Denmark

SBT Instruments © 2020

SBT Instruments © 2020