Many microbiology processes involve:
1. Growing a starter culture to high density
2. Back diluting into fresh media by some dilution factor, eg, 1:100
3. Growing to OD X (eg, OD=0.5)
4. Harvesting for some assay, extraction, or process
My question is: How can you predict/understand the dilution factor necessary?
In this case, I'm asking about RNA extraction for qPCR, but I'm interested in the question more broadly.
https://www.phe-culturecollections.org.uk/technical/cell-culture-protocols/cellcounting.aspx .
Dear Gen,
During the growth of a bacterium, the concentration of cells increases by time in a batch medium. This increase in cell concentration is linked with a change in the growth phase i.e. lag, logarithmic, stationary and death. The maximum cell concentration is achieved at the stationary phase. So, the logarithmic phase is indicated between the end of the lag phase and the beginning of the stationary phase.
On the other hand, the cell concentration could be followed by different methods such as the bacteria cell counts, the absorbance (Optical Density, OD), and protein concentration. So, for determining the log phase of a bacterium, we can measure the OD of the growth medium. This log phase is located for many bacterial strains when the OD value of growth medium ranges between about 0.1 and 1.0.
So, if the OD is indicated by a value of 0.5, this means that the bacteria is in the mid range of the logarithmic phase.
Best wishes
Duried
Hi Gen,
Druid is right. I did want to add a few points that might be important to pay attention to if you are trying to get a standard procedure to use. For practical reasons you may really want to get the cells to reach 0.5 within so many hours so that you can start the regrowth, monitor density, harvest and process in a single day.
When you grow starter culture to high density is that overnight from the day before, or are the high density cultures grown and kept in a refrigerator.
This can impact the level of still viable bacteria.Usually the more important impact is on the recovery (lag) time required for the cells to start dividing when you dilute them into fresh media.
If your worried that metabolites from the high density growth might inhibit growth you can spin down the cells aspirate of the media and resuspend in fresh media before dilution for regrowth.
For most routine work I have found that 1:100 is a good dilution, but it is not universal.
Good Luck,
Frank
https://www.phe-culturecollections.org.uk/technical/cell-culture-protocols/cellcounting.aspx .
yes,it is very important and it effect to cut off from result
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