BactoBox® Application Note

Why You Should Use BactoBox® to Monitor Bacterial Productions

If you’re a microbiologist, you’re probably using plate counts frequently when enumerating bacterial cells. The plate count method is well established and often a customer or regulatory requirement. But it doesn’t give fast, actionable data, and therefore prohibits fast learning loops.

BactoBox is a small benchtop device that is used to perform accurate enumeration of the intact cell concentration of your samples in minutes. The ease-of-use and short time-to-result makes it highly relevant for optimizing, validating and monitoring every critical step of your processes.

While BactoBox does not necessarily replace plate counts, this article shows how well BactoBox correlates to plate counts and justifies its use as in-process monitoring tool that can help you achieve the most efficient process possible without compromising quality.

Keywords: Total viable count (TVC), cell counter, cell counts, aerobic colony count, aerobic plate count, standard methods agar (SMA), plate count agar (PCA), Drigalsky, microorganism, rapid microbiology methods, serial dilution, agar plate, aerobic plate count (APC), good manufacturing processes (GMPs), growth, bacterial concentration, in-process samples, pour plate, spread plate, spiral plating, colony counter rapid microbiology methods, microbiome research, bacteria detection device.

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?

Plate counting is the most frequently used method for microbial enumeration

Plate counts or colony forming units, CFUs, are considered the golden standard for enumeration of microorganisms [1]. The method has a very low detection limit and it is inexpensive to establish because it only needs modest equipment and reagents. An additional benefit is that selective and/or chromogenic media can be exploited to quantify a specific microorganism.

Drawbacks with the plate count method

Even though millions of plate spreads are done every day, the plate count method has obvious shortcomings which makes it difficult to use in-process monitoring:

Time-to-results is days or even weeks: For anaerobic bacteria it can take a week for colonies to appear and in extreme cases like the 10-16 hour generation times of Mycobacterium tuberculosis, time-to-results can be more than 2 weeks [2].
Plate count results are influenced by the analyst: It takes a great deal of practice and skill to get consistent plate spreads. Contamination can be a serious issue.
One-size-fits-all growth conditions do not exist: Bacteria are extremely diverse. Some require highly specific growth factors such as certain combinations of acid/base, temperature, and anaerobic/aerobic conditions. Another challenge is that some bacteria grow fast on standard media and can completely overshadow slow-growing bacteria. These two general problems explains the often extreme underestimation of bacteria also referred to as “the great plate count anomaly” [3].
Bacteria can be dead and also “viable but non-cultureable, VBNC”: Plate counts does not enumeration dead bacteria in the sample. Also, bacteria can enter a VBNC state e.g. as a long-term survival mechanism to environmental stressors like temperature and chemicals [4], [5]. VBNC bacteria do not form colonies on plates and therefore the plate count may underestimate the true cell count.
Plate counts are associated with analytical imprecision: There is high technical variance associated with the plate count method. For concentrated bacterial suspensions, it is normal to have coefficient of analytical variation, CV, of 15-35% [6].
Plate counts are expensive and laborious: Even though sophisticated equipment and reagents are not required, it takes a long time to prepare reagents, dilute samples and perform plating. When adding up reagents (excluding working hours), the price is often around 15 EUR per sample. In addition, plate counts are low throughput, so it typically requires the analyst an entire day to prepare, dilute and plate e.g. 100 samples. Colony counters can speed up the counting process, but manual handling is still required.

Advantages of BactoBox measurements

BactoBox is a fast cell counter for bacteria and was developed to address the well-known issues associated with the more than 100-year-old plate count method. BactoBox gives the total bacterial concentration within 2 minutes, so that you get actionable answers for your process and accelerated learning loops. Because BactoBox is a cultivation-independent method based on impedance flow cytometry, it is not biased by specific growth condition requirements. VBNC bacteria are detected as a subset of the intact cells. Dead bacteria are also detected but as other particles than intact cells. BactoBox has minimal dependency of the analyst because the measurement is operated by the push of a button and the various built-in flow-, conductivity- and current sensors will give alerts and provide guidance if anything is wrong with the sample. Finally, contamination during sample handling is negligible because the bacteria have no time to grow during the fast measurements.

We are often asked how BactoBox results compare to the plate count method. To investigate this in detail, we performed a head-to-head comparison of five different bacterial species. These were selected to represent different combinations of Gram+ and Gram- envelopes as well as different shapes in the form of coccoid and rods. All measurements were performed in technical triplicates with repeated dilution series from the homogenized primary sample.

BactoBox follows CFU closely for actively growing cultures

The figure below shows the colony forming units (CFUs, dashed lines) as well as the simultaneous BactoBox intact cell concentrations for the five bacterial species (ICC, normal lines). All species were grown in Erlenmeyer shake flasks. Samples were homogenized to single cell suspensions and diluted to bring them into the measurement range for the BactoBox method (10,000 – 5,000,000 total particles/mL). For the spread plate method, the ICC concentration was used to determine the necessary dilution factor needed to get between 30 to 300 colonies per plate.

Generally, BactoBox results track the results of the plate count method even though the studied bacteria have very different morphologies and cell wall compositions. These results show that it is indeed possible to have fast, CFU-like measurements with very little sample handling. For the plate count results, most colonies could be counted within 1-2 days, but there were also several cases where additional colonies had appeared after 3-4 days.

BactoBox intact cell concentrations follow CFU for actively growing cultures. Growth curves for five bacterial species Plate counts (CFU/mL) are depicted with dashed lines while BactoBox measurements while intact cells/mL (ICC) are depicted with normal lines.

BactoBox has great precision for high bacterial concentrations

Several 1:10 dilutions are needed to bring the bacterial concentration into the 30-300 measurement range of the plate count method. Every pipetting and vortexing step will inevitably introduce variation which will result in high variance when multiplying with the dilution factor. The tables below clearly show this trend, especially for the later stages of growth. One example is the E. coli growth curve at the 6.1-hour sampling point. In practice the number of colonies per plate were relatively similar for the triplicate dilution series (105, 81 and 136), but because 8 serial dilutions were performed, this plate count is multiplied by a factor 10⁸ and the CV is 26%. BactoBox, on the other hand, likes to work with relatively high bacterial concentration and for the same time point (7.3 hours), a 2-step 1:100 dilution procedure was performed for a final 1:10,000 dilution of the primary sample. This resulted in a CV of 6%.

One interesting case where both the CFU method and BactoBox have relatively high variation is A. baumannii. This species is known to have “sticky fingers” and prone to adhere strongly to polymer surfaces and form biofilms [7]. It is therefore likely that the high variation is caused by adhesion to pipette tips and vials during the preparation of the dilution series.

The green numbers in the table indicate the measurements, where the CV was below 10%. For the plate count method, 36% of the sampling points fulfill this criterium, while the same percentage for the BactoBox is 69%. Low variation is key to make right decisions and with lower “noise” in the measurements, it is more likely to see if a culture is increasing or decreasing in bacterial concentrations and make reliable decisions. Also, fewer replicates are needed to achieve statistically significant results when investigating experimental parameters. Summing up, BactoBox provides results here-and-now, less tedious work and more precise data.

BactoBox has higher precision than the plate count method. Summary of coefficients of variation of the mean (CV%) for both BactoBox and CFUs. CVs below 10% are green. 36% of plate counts are green while the same number for BactoBox is 69%.

Use BactoBox to avoid extensive plating

BactoBox can be used as a novel method to monitor your processes, and for the final QC analysis. Any analytical method has its strengths and weaknesses. In the present context, the strengths of BactoBox are speed, ease-of-use, and precision. Nonetheless, in many regulatory aspects plate counts is a legislative norm that are difficult to substitute by an alternative method without validation efforts. Admittedly, plate counts are indispensable in some cases, but one major issue is that plate counts are often performed “in the dark”, because the exact concentration of the primary bacterial sample is unknown. The colonies pr plate should typically be 30-300 and if the proper dilution is missing, the plates could be useless either because they’re i) completely overgrown and impossible to count or ii) devoid of colonies.

Fear of missing out of the right dilution leads to extensive dilution series and an unnecessarily high number of plated samples, i.e. more workload and higher costs on reagent and personnel hours. Multiplying with the the replicates needed to get a precise average, this results in excessive workload. A solution is to use BactoBox to determine the concentration in the culture prior to plating. As shown in the illustration below, by knowing the bacterial concentration, it is easy to determine the dilution factor necessary to get 30-300 colonies per plate. With this BactoBox-guided plating workflow, it will often be sufficient to plate a single dilution series instead of the typical plating of 2-9 dilutions and plate spreads.

In short – when plate counts are indispensable – you can use BactoBox to avoid plating in the dark. The BactoBox-guided dilution workflow will cut costs, save time, and make the plate counting method much less frustrating.

BactoBox-guided plating workflow saves time, costs, and frustrations. Initially a dilution series is performed in BactoBox diluent and 1 mL of the final dilution is used to extend the dilution series to the plate count concentration range. Knowing the concentration of bacteria means that unnecessary dilution and plating is avoided.

BactoBox saves time, money, and frustrations

As the data for the five growth curves implies, BactoBox correlates closely with plate counts for the lag-phase, exponential phase, and stationary phase. The main advantage of BactoBox is that you get actionable, here-and-now measurements to make preventative measures if bacteria are behaving as they should. The benefits of BactoBox are summarized below.

BactoBox provides here-and-now, actionable results: BactoBox measurements are completed in rougly 2 minutes. Including the sample workup, results are typically available within 5 minutes.
BactoBox has high precision: While the plate count method typically has CVs of 15-35% for high bacterial concentrations, BactoBox works best with concentrated samples and typically has CVs below 10%. In addition, the BactoBox results are not influenced by the hands-on work of the analyst because the measurement happens automatically by the push of a button.
BactoBox is free of cultivation-biases: BactoBox counts bacteria in the sample with no need for pre-cultivation. This eliminates the well-known issues with missing colony formation due to specific requirements like medium composition, growth conditions and domination by fast-growing microorganisms. Also, BactoBox will also detect VBNC bacteria even though they wouldn’t easily form colonies on a plate.
Cut costs and save manual work: As an operator, you want to apply corrective actions for your process as soon as possible to save resources and potentially prevent further processing of a faulty batch. BactoBox can save you production capacity, resources, and time by enabling fast and accurate bacterial cell counts throughout your processing.
Makes your plate count easier: With plate counts there is a risk that plates are impossible to count either because they are overgrown with a lawn of bacteria or have too few colonies for statistically valid results. By immediately revealing the concentration of bacteria in the sample, BactoBox-guided dilution workflow complements the plate count method to avoid excessive plating.

Or to cut this short and simply quote one of our favorite customer statements on LinkedIn: “After trying the BactoBox, plating seems like a waste of time”.

References

H. M. Davey, “Life, Death, and In-Between: Meanings and Methods in Microbiology,” Appl. Environ. Microbiol., vol. 77, no. 16, p. 5571, Aug. 2011, doi: 10.1128/AEM.00744-11.
R. Ghodbane, D. Raoult, and M. Drancourt, “Dramatic reduction of culture time of Mycobacterium tuberculosis,” Sci. Reports 2014 41, vol. 4, no. 1, pp. 1–4, Feb. 2014, doi: 10.1038/srep04236.
J. T. Staley and A. Konopka, “Measurement of in situ activities of non-photosynthetic microorganisms in aquatic and terrestrial habitats.,” Annu. Rev., vol. 39, pp. 321–346, Nov. 2003, doi: 10.1146/annurev.mi.39.100185.001541.
J. R. Stokell and T. R. Steck, “Viable but Nonculturable Bacteria,” eLS, Oct. 2012, doi: 10.1002/9780470015902.A0000407.PUB2.
J. D. Oliver, “Recent findings on the viable but nonculturable state in pathogenic bacteria,” doi: 10.1111/j.1574-6976.2009.00200.x.
USP, “<1223>: Validation of Alternative Microbiological Methods,” United States Pharmacop., vol. 04-Oct-202, pp. 4–6, 2021.
N. Pakharukova et al., “Structural basis for Acinetobacter baumannii biofilm formation,” Proc. Natl. Acad. Sci. U. S. A., vol. 115, no. 21, pp. 5558–5563, May 2018, doi: 10.1073/PNAS.1800961115.

Optimise Your Bacteria Enumeration with BactoBox®

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