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Forgive my naivety, I'm an undergraduate Chemical Engineering student doing research this summer into growth rates of various combinations of bacterial isolates.

I have about 9 isolates from a toxic leachate, and I am looking into finding combinations of these 9 isolates which degrade the leachate the fastest. I have also have the general consortium of what was growing in the leachate.

I am trying to compare the growth rates of my isolates (and combinations) to this growth rate in order to demonstrate some increase in treatment efficiency. (The idea is that a faster growth rate in the leachate demonstrates a faster breakdown of the leachate)

My current way of measuring growth is to fill a 96-well plate with some known volume of leachate and inoculate the appropriate isolates into the appropriate wells. This is my community matrix. Then I run my plate reader (TECAN) for 72-hours in order to measure the turbidity change over time, as an indirect measure of leachate breakdown.

The problem is that many of my isolates and combinations of isolates, including the consortium clump when they grow, meaning that the plate reader I am using does not measure the growth the way I'd like it to.

Here is an example of a good growth curve of Brevundimonas diminuta growing in my leachate, which I consider good: enter image description here

Here is my "growth curve" of my consortium, the bacteria have a tendency to clump here: enter image description here

Essentially, I am asking if there is a way for "force" my bacteria to grow in a planktonic fashion?

More information: These are my isolates:

  • Brevundimonas diminuta
  • Streptomyces sp.
  • Lysinibacillus sp
  • Alcaligenes sp.
  • Pseudomonas sp.
  • Pseudomonas fluorescens
  • Paenibacillus sp
  • Pseudomonas fluorescens
  • Stenotrophomonas chelatiphaga

These and the consortium grow the way I want them to in M9 salts supplemented with acetate, all planktonic, and measuring their growth in that is very easy, but I want to demonstrate a growth curve in my leachate. I don't know a way to do this on a scale which the 96 well plate reader allows me to do. I can get 32 different combinations out in a single plate if I grow them in triplicates.

A post-doc has been trying to measure a decrease in ammonia flourescence in the plate reader, but has had less luck than me.

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  • $\begingroup$ Have you tried to see if light vortexing of the plate will break up the clumps? Pipetting up and down often will provide enough shear stress as well. Lastly, you could sacrifice the experiment at different time points, and do a high speed centrifugation to pellet the bacteria, and remove the liquid to another 96 well plate to read on your plate reader. $\endgroup$ – akaDrHouse Aug 4 '16 at 14:55
  • $\begingroup$ @akaDrHouse Yes. The plate reader shakes the plate over the 72 hour reading period, but clumps still form over that time. I pipette up and down when inoculating in order to break up whatever clumps there may be too. $\endgroup$ – Georgeos Hardo Aug 4 '16 at 14:56
  • $\begingroup$ Good question. Some bacteria would grow that way in certain media. By preventing that you would essentially be disrupting their growth. You can possibly adopt a different method of growth estimation. Perhaps you can measure glucose consumption but even that would not be very accurate. Growth dynamics is not always straightforward. $\endgroup$ – WYSIWYG Aug 4 '16 at 19:02
  • $\begingroup$ @WYSIWYG Thank you for the suggestion, the problem is that glucose is not the carbon source in my leachate. It's likely a bunch of aromatics. $\endgroup$ – Georgeos Hardo Aug 4 '16 at 23:00
  • $\begingroup$ Well, if the idea is that a faster growth rate in the leachate demonstrates a faster breakdown of the leachate, then why not just measure the components of the leachate? If the composition is not fixed or if there are many components then the task would be difficult. Alternatively, you can image the plates at different time points and analyse the images. You may have to develop an algorithm to differentiate between the bacteria as each would behave differently. For example, cell-1's growth may be measured using turbiditty whereas for cell-2 the size of the clump should be measured. $\endgroup$ – WYSIWYG Aug 5 '16 at 4:42

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