Hi all,
I am trying to locate a standard method for culturing bacteria (Escherichia coli and Staphylococcus epidermidis probably) in a liquid suspension, rather than on agar plates.
We want to incubate them with something (most likely for 24 hrs) and then do a viable count.
We want to use optical density (spectrophotometry) as a way of measuring the viable count also.
Has anyone had any success or has any ideas?
I would suggest you to use LB broth for such incubation study. It can be easily done. 1st you have to standardize the growth curve of the bacteria. You can inoculate the bacteria in LB broth and incubate. Every 3-6 hours you should note the count by serial dilution spread plating in agar plate and note the OD values each case. Thus you can get the standard curve for estimating viable count corresponding to different OD value. Use sterile broth for reference OD.
Good luck.
Culturing E. coli and S. epidermidis will be trivial, but correlating OD after 24h to the original CFU was not trivial. if you look at classical growth curve you will notice that you can reach the same OD starting from different initial ODs, simply because you reach the stationary stage.
What you can do, and indeed is often done for water microbiology, is use the Most Probable Number approach (MPN). Basically you serially dilute your original sample into a selective media, incubate, and test for growth/no growth (or in some cases growth and some specific activity). Once you see in which dilution there is no growth there are tables that allow you to estimate the original number of bacteria in your sample.
Yoram
do not waste your time with growth dependent counting. Use a flow cytometer and count all bacteria and assess their functional status. If the chaps in your department are afraid of having live bugs in their instrument do a live / dead staining such as CFDA-SE and PI before killing them if you are interested in intact and permeabilised cells, but live / dead is more complex than that.
Article Functional single-cell analyses: Flow cytometry and cell sor...
https://www.aphis.usda.gov/animal_health/vet_biologics/publications/BBSOP0019.pdf
Gerhard, what are you trying to do this for. This will determine the media. As stated above, I would use an MPN like we did in Colworth. 96-well plate per dilution would mean that you could detect a difference of 8% or more. I'd suggest using Bland Altman statistic and Demming regression or Passing Bablok. I work in Birmingham Mon to Thurs so we could catch up and discuss this in person
96-well Plate. Eight dilutions 12-well per dilution. Use Tryptic soya broth. It is buffered and has a little glucose in it too. This is a better all round medium for both organisms and is used in some clinical labs in blood cultures. LB broth is designed for E. coli strains used in molecular biology. It is not used in microbiology labs outside universities and its use would mean that your findings would have limited relevance outside of your own work
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