Real-time monitoring of biofilm metabolism using microcalorimetry
Direct measurements of metabolism can add a new dimension to research into biofilms. While there are different methods, microcalorimetry has a number of advantages that allow it to fit into various biofilm workflows. It is non-destructive, label-free and performs measurements in real-time which can produce a very dynamic picture of how biofilm samples are really behaving. Once the microcalorimetry has been done, the biofilm sample can then be used for other assays such as gene expression or microscopy which is a powerful approach to correlate results using different methods.
Biofilms are dynamic
The more research is published, the more we see that biofilms are incredibly dynamic structures. Gene expression, physiology, and biofilm morphology are changing all the time. Standard density measurements for growth fail to capture this and more complex methods often require end-point assays or the use of selective dyes which limit the information that can be gathered. Microcalorimetry offers a way to see the dynamic behaviour of a biofilm in a very simple way.
Microcalorimetry captures dynamic metabolic information in a simple way
While DIY microcalorimetry is not simple, commercial instruments usually are. They are typically benchtop machines that read microcalorimetry plates consisting of a number of small wells. In other words, if you have an experimental workflow that uses 96-well plates, then you will be able to use microcalorimetry. Most of the people who demo our microcalorimetry instrument, the calScreener are surprised how easy it is to set up an experiment. They are surprised because the setup doesn’t look quite like a 96-well plate. The main difference is that each well has to be sealed with a cap with a very specific tension. This looks tricky at first but it’s easy with our torque wrench.
The fact that all the wells are sealed gives another significant advantage to microcalorimetry, it can easily measure biofilms in different hypoxia conditions.
Measure biofilms anaerobically
Adding anaerobic conditions to an experimental setup can impose a lot of restrictions. Special chambers and seals have to be made to maintain an anaerobic atmosphere throughout the course of the experiment. Due to the tight seal of the wells in our calScreener plates, if the wells are prepared in an anaerobic chamber, they will remain anaerobic even when the plate is removed. This means that you can prepare any atmosphere in your anaerobic cabinet and this will be maintained automatically throughout the course of your metabolism experiment.
Measure with real samples
Many reviewers complain that experiments presented in manuscripts do not represent the “real” biofilm environment. As a method, microcalorimetry can work on any sample. Research groups have run it with agar plugs at the bottom of the well, with pellicle biofilms or with beads floating in suspension. The microcalorimetry technique is extremely flexible when it comes to the matrix where biofilms are formed. As an example, if you’re studying clinical biofilms, microcalorimetry could even be done directly with patient samples on anything from bone fragments to colonised catheters. The real-time microcalorimetry traces act like a reproducible “fingerprint” for an individual microorganism, this means it can also be used for diagnostics.
Diagnostics and point-of-care
Microcalorimetry is an interesting diagnostic technique as almost any patient sample can be used. Given that the technique can be done in real-time and that microorganisms produce a unique fingerprint, it makes sense that clinical microbiology groups are using microcalorimetry to test both purified patient isolates as well directly on patient samples. A strength of microcalorimetry is that it measures metabolism and not growth. Many diagnostic assays used to measure if bacterial cells are dividing, however cells in a biofilm may be alive and metabolically active without necessarily growing.
Metabolism does not equal growth
The simplest application of microcalorimetry is to determine if a biofilm is alive or dead after a specific experimental treatment. We know that explicitly monitoring growth is a poor proxy for whether an organism is viable or not, and this is especially true in biofilms. By monitoring the bulk metabolic activity, it is possible to detect as soon as a microbial community starts to exhibit signs of metabolic activity, even if they are not growing.
However, the data from microcalorimetry is so rich that it is possible to see much more than if an organism is alive or dead. As the measurements are done in real-time, it is possible to see precisely when metabolism shifts and by how much which has implications for microbial physiology and gene expression.
Unlike other methods, microcalorimetry provides a direct measurement of the total metabolic output of a sample by detecting small changes in heat production. It does not rely on proxies such as respiration and it is not limited by any specific media or set up conditions. In any experimental setup, microcalorimetry will deliver a reproducible picture of the total metabolism of the biofilm over time.
To see how researchers are using microcalorimetry to study biofilms, watch the webinar we recorded earlier this year with Thomas Bjarnsholt and Mads Lichtenberg.
Which microorganisms can be tested using microcalorimetry?
The flexibility of microcalorimetry means that the only limit of what can be measured is if it is cultivable or not. It’s certainly possible to assay both Gram-positive and Gram-negative organisms. We’ve tried a number of different organisms, here is an example list which is by no means exhaustive:
○ Escherichia coli
○ Pseudomonas aeruginosa
○ Klebsiella pneumoniae
○ Acinetobacter baumannii
○ Salmonella spp
○ Proteus mirabilis
○ Staphylococcus spp
○ Strepctococcus pneumoniae
○ Bacillus subtilis
○ Mycobacterium marinum
○ Candida albicans
○ Saccharomyces cerevisiae
It has also been possible to monitor phage infections using the calScreener which produced a unique fingerprint for the lifecycle of an infecting phage as well as multispecies biofilms.
We have some example data but most of it is being prepared for publication. If you’d like to discuss specific examples related to your organism, please contact one of our application scientists.
Microcalorimetry for biofilms
Microcalorimetry is a simple way to add an extra dimension to biofilm research. Metabolism is an important feature of microbial communities which is often measured using proxies that give an incomplete picture of the real situation. The technique measures the production of heat via metabolism in real-time, meaning that it is completely label-free and easy to apply to any organism or experimental setup.
If you’re interested in the technique but are unsure of how it would work with your setup, we are currently taking applications for demos so contact us if you are interested.
For more information on how other researchers are using microcalorimetry in biofilm research, watch our recent webinar with Thomas Bjarnsholt and Mads Lichtenberg.