Does someone know a methodology which does not require overnight to standardize the bacterial inoculum but wait 6 hours or so to complete the absorbance reading?
Hi Marcio! That would depend on the species you are testing, you should make a growth curve for each one using your medium to know that. Imagine you are using an E. coli culture, that you have in a solid medium. In the morning (like 8:00 AM) inoculate an 250 mL Erlenmeyer flask containg 75 mL with the bacteria, scrapping your loop in the colony (inoculate with the entire loop filled with bacteria). After, lets say 3 hours, take samples every each hour and measure your OD and make serial dilutions CFUs.
That's the only way you will know for sure.
For instance, I work with Pseudomonas fluorescens and Bacillus cereus. With P. fluorescens, after 5-6 hours I have OD(610nm)=0.80+- and B. cereus is a little slower.
is exactly what I mean. I will not wait all night. My experiment is on the evaluation of plant extracts on bacterial nosocomial bacteria. and am using a standardized inoculum 0.08-0.13 x 10e6 cfu / ml to an absorbance at 630 nm. but it is possible to standardize the bacterial inoculum in 6 hours?
Hi Marcio! That would depend on the species you are testing, you should make a growth curve for each one using your medium to know that. Imagine you are using an E. coli culture, that you have in a solid medium. In the morning (like 8:00 AM) inoculate an 250 mL Erlenmeyer flask containg 75 mL with the bacteria, scrapping your loop in the colony (inoculate with the entire loop filled with bacteria). After, lets say 3 hours, take samples every each hour and measure your OD and make serial dilutions CFUs.
That's the only way you will know for sure.
For instance, I work with Pseudomonas fluorescens and Bacillus cereus. With P. fluorescens, after 5-6 hours I have OD(610nm)=0.80+- and B. cereus is a little slower.
There are quite a few mothods to change the growth rates of bacteria during culturing. I agree that a more densely inoculated culture will provide a larger number of organisms in a shorter period of time, However, this comes at a cost of higher diversity of generation 0 bacteria with fewer generations. For DNA related studies, this is not optimal.
Additionally, for a short inoculation time, you run the risk of having cryopreservatives still be a part of your culture solution (unless you have prior passages of bacteria that are not explained above). If there is a prior passage of bacteria from the frozen stock, then you can place exponential phase bacteria as your starting bacteria, thus eliminating the lag time prior to your 6 hour time point.
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