[Microbiology] Re: Measuring Rhizobium growth

Lesley Robertson l.a.robertson at tnw.tudelft.nl
Mon Jun 26 03:34:12 EST 2006


<dkafkewitz at aol.com> wrote in message 
news:1151011212.675029.207030 at g10g2000cwb.googlegroups.com...
> I've never worked with Rhizobia, but in the past when faced with this
> sort of problem we did the following:
>
> Put an aliquot of the liquid culture  in a 13 X100 mm tube. Spin down
> the cells. Add about 5 ml 1.0N NaOH, put in boiling water bath for
> about 30 minutes, cool, add Biuret reagent. Wait however many of
> minutes the Biuret reaction takes ( I don't remember right now) and
> then read in a spectrophotometer. Standardized against a known protein
> like bovine serum albumin of lysozyme, this gives you total protein in
> your culture aliquot.
>
> That is not cell number but all things considered a reasonable
> surrogate.  Protein content varies by about a factor of two, and cells
> usually don't accumulate extracellular proteins, so It is a
> reasonable and quick (and cheap approach).   Whether it is applicable
> to Rhizobia, I do not know.


This is pretty much what we did - it has one advantage over dry weight in 
that you know what it's measuring. Some of out bugs can produce as much as 
50% of their cell weight as PHB, etc, which made dry weight as a measure of 
growth of little use. Extracellular polysaccharides might also be a factor 
here - something must be sticking the cells together.
One thing extra that we did was to have a wash step before adding the Biuret 
(or in our case micro-Biuret) reagent as there was so much ammonium in our 
medium that it would interfere.
We did some studies on our bugs (Paracoccus/Thiobacillus/Pseudomonas 
species) and found that in log phase (or rate-controlled growth in 
chemostats) they were around 50% protein. By the end of stationary phase, 
they were up to 75%.
Lesley Robertson




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