For example: if one strain has starting inoculum (CCU/ml) of log10 3.5, second with 4 and third with 3, is it still possible to measure and quantitate the difference in their growth rate? If yes then how? One way is to measure doubling time for all strains at some specific time interval. But main question is whether slight variation, as mentioned above, has any importance on the measured doubling time? Sometimes it is very difficult to standardize all cultures at exactly the same concentration especially mycoplasma.
I think the DO is what matters, all the strains at the same DO, then you quantify the CCU/ml at various
Dear Artur, thanks for your reply. Can you please suggest how to decide the best time point for calculating the doubling-time during log phase? I mean, say there are 4 time points in log phase for these strains, should I calculate doubling time for each of these intervals or choose some optimum time interval? And what should be the criteria for that "optimum" time interval?
Thanks in advance.
After plating at different time points since inoculation and counting the number of colonies, you get the doubling time from the slope of the curve since this is linear during log phase; the more points there are, the more reliable the interpolation. The size of initial inoculum is irrelevant, as pointed out.
After inoculation of bacteria into liquid medium they go into lag phase then log phase then stationary phase. To know doubling time of any bacteria in certain liquid medium in certain conditions (volume of flask, volume of medium, rpm of your shaker, temperature) you need to keep your strains in log phase as long as needed to find the difference in the growth rate. Normally, if the doubling time of your bacteria is within 30-45 min, you have to monitor your cultures for 5-6 hour constantly keeping them in log phase. For example, you know that your type of cultures start transitioning to stationary phase at OD=0.7. Therefore, when your culture gets to OD=0.4 or so, you take e.g. 1 ml of this culture and inoculate it in 99 ml of preheated and preshaken flask. You will need 3-5 flasks per culture. On a graph, you build OD of the culture over time taking OD dilutions into consideration. The ODs will be artificial. You have no need to do any plating.
Don't know if this is much help, but if doing a number of strains, you can do growth curves in microtitre plates and measure OD 600 on a plate reader. This enables the rapid accumulation of a number of data points over a period of time. I used it for looking at the effect of different drug concentrations on different bacterial strains over a period of 6-8h at 30 min intervals and got reasonable growth curves. I accept that the growth conditions are not ideal, in that you are not getting maximal aeration and that growth rates may be slower, but with gentle agitation on a shaker it seems to work. At the time, the prospect of growing 96 flasks on shakers and manually measuring ODs did not appeal. The doubling times can be calculated as described above from an excel spread sheet.
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