I have three isolates (representing possibly two strains) of a novel species of bacteria that thus far only appears to grow attached to the culture vessel, not growing in the broth itself. It grows on plates easily, albeit rather slowly. Typically, characterisation for the purpose of describing a new species involves liquid cultivation for determination of growth rates, getting biomass to perform various analyses etc. As my strains cannot (at least at present) be cultivated very successfully in liquid, I'd like to hear thoughts from fellow microbiologists on how they would proceed, with a view to eventual publication. Thanks in advance
Successive rounds of sub-culturing in vitro on solid media can result in "transformation" into a non-adherent type that exhibits planktonic growth in broth . . .
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This scheme worked for Aggregatibacter actinomycetemcomitans (Klinger 1912) comb. nov.
Nørskov-Lauritsen and Kilian (2006) Int J Syst Evol Microbiol 56: 2135-46.
https://www.ncbi.nlm.nih.gov/pubmed/?term=16957111
Hi Laura ^^
Well I would say that you can do the required growth tests (pH/Temperature/NaCl) on agar plates. That would give you at least the range (a lot of description's articles are doing it like this). To get the optimum, I think I would try to just count the colonies and run statistical analyses on the results... After for the chemotaxonomical tests (quinones, polar lipids and so on) I think it should be fine to harvest the biomass on agar plates but I can have a look if you like.
Cheers
Cendrella
As I can understand you are facing problems with monitoring the growth rate of your strains in liquid medium, I think you need not to do that and as suggested by Cendrella you can observe the growth on agar medium, if you want to have a quantitative value you can measure the size of colonies.
If you want to harvest biomass for chemo-taxonomy you can grew your strain in broth and scrap-out the biomass form the surface by using a sterilized knife or glass rod,
Best wishes,
I think you will be fine. There are lots of species that can't be cultured easily.
If you are trying to do growth experiments to obtain growth curves under various conditions, perhaps consider a less conventional means to plot growth: Maybe grow your bacteria in liquid culture in plastic 96-well plates and crystal violet stain the cultures while they are attached to the plate? (Your bacteria sound like they are growing in a biofilm in your culture vessel, and biofilms can be readily quantified by crystal violet staining) Although this will mean you will have to set up lots of cultures in the 96 well plate, since at each time point you will basically "lose" the culture when you quantify it. I hope that makes sense.
Crystal violet assay is a standard assay for a quantification of the biofilm growth. This protocol was streamlined for a 96 well plate, but in principle can be applied to other surfaces. You would need to do a time course experiment to determine a biomass increase rate.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/
You could monitor growth by decanting the media and weighing the vessel. Alternatively, does your media contain detergent? If the bacteria are not sensitive to detergent you could add some to the media to encourage planktonic growth. For growth of mycobacteria, we routinely use 0.05% tween 80 or tyloxapol to prevent clumping.
You can increase the surface area vastly by using microcarrier beads. Try growing the novel species on agarose beads suspended in their usual medium. It might be a good idea to preincubate the beads in the medium broth to infuse them with nutrients before inoculation with bacteria. In any case this should increase the biomass compared to agar plate colonies.
There are many species that appear to be obligate biofilm species but often under certain conditions they become "swimmers" and can be grown in suspension. Sometimes this can happen in nutrient-depleted environments, sometimes in nutrient-rich ones. You'll have to carry out a systematic study for that.
Exciting times! Look forward to seeing the publications resulting from this. Good luck!
Isothermal microcalorimetry (IMC) is a proven method for measuring bacterial growth rates in vitro, regardless of whether the bacterial are in suspension, on surfaces, in scaffolds, etc. It works by dynamically quantifying growth-related metabolic heat continuously in real time. See; https://en.wikipedia.org/wiki/Isothermal_microcalorimetry#Microbiology
and for a general introduction to the method, see the entire article (with 60+ references) at
https://en.wikipedia.org/wiki/Isothermal_microcalorimetry
A person with extensive expertise and experience in using iMC to measure growth rates of bacteria (including those on surfaces) is Olivier Braissant PhD at University of Basel.
Thank you all for your helpful suggestions thus far, I shall be looking into them all. Cendrella, I've been doing or at least trying to do these tests on agar thus far. The problem with counting colonies is that individual colonies aren't visible as such. Colonies are tiny, flat, transparent and tend to grow into each other, making quantification extremely difficult. Plus biomass could be an issue based on what I've been told the requirements are for quinones etc. One idea I've had of late is to grow on microscope slides, thus making the retrieval of biomass easier!
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