I have some isolates which I am trying to grow in some toxic leachate in order to measure their growth rate I also want to measure the growth rates of certain combinations of these bacteria.

I've tried a few ways to measure their growth rate in leachate (mostly turbidity), all of which have been unsuccessful. My supervisor has therefore recommended I try wet weight measurements.

I am a chemical engineering undergraduate, and I have never done wet weight measurements before, so please forgive my naivety!

My set up is as follows:

  1. I have overnight cultures of the isolates which are being grown up in leachate (themselves inoculated from agar plates) in falcon tubes as an inoculum for my wet weight cultures.
  2. I plan on having several (~20) 250ml conical flasks with leachate in them. I will innoculate these from my overnight cultures, so that different flasks contain different isolates
  3. I want to take daily kinetic measurements in order to get the wet weight of the bacteria growing in leachate. This will be done over a period of 5 days.
  4. I will take an aliquot each day for 5 days from the conical flasks, centrifuge in order to pellet the cells, then remove the supernatant and weigh the biomass.

So what I don't know are the specifics of how I might set this up.

I know that my isolates grow slowly in leachate, and therefore I will need a relatively large volume of liquid (around 5-10ml) in order to get a meaningful change in biomass.

So my queries are;

  1. How many replicates is sensible for this sort of wet weight measurement? I know that they can be quite varied from replicate to replicate, but I have limited room in my incubator. I think 4 replicates might be too many, but I don't know the extent of the variability when it comes to wet weight measurements.
  2. In 250ml conical flasks, what is the maximum volume of liquid I can put in them before there is not enough head-space remaining?
  3. What volume of an inoculum would be sensible for this experiment? And if I am wanting to get a growth rate, then does it even matter? As I am only concerned with the rate of change of biomass over time and not the final amount.
  4. I want to take large enough aliquots to demonstrate an increase in biomass, but obviously not too much that it begins to affect the culture growth. As this is destructive sampling, what volume of my culture can I afford to lose? I have demonstrated that in a volume of 5ml of leachate, and given 72 hours, my isolates' biomass increases by approximately 8,000 μg - 10,000 μg. So by this logic, is it right to say for the daily measurements, aliquots might should be 15ml so that I can get meaninful readings?

I already know it is not feasible for me to do dry-weight measurements simply because of time and the system I'm working with.


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