Cfu and serial dilutions - (Dec/10/2005 )
Hi! I need to grow Vibrio anguillarum wild type over night and then make serial dilutions so taht after I plate the calculated amount, I get 100-200 cells. How can I perform that? How do one make serial dilutions?
It is hard to know the amount of viable cells in a culture unless you have good standard for a certain turbidity (OD) value. For example, for my organism I know at an OD(600nm) of 0.500, I have around 500 000 000 viable organism per mL. So if I want 100-200 cells per plate I need to dilute my culture to 1000 cells per mL and plate 100 uL. So basicly I would centrifuge my cells and resuspend my cell such that OD(600nm) equal 0.1 which will equal around 100 000 000 cells per mL. The following step is to serial dilution untill I reach a dilution factor of 1/100 000.
For serial dilution, this image in the link below does explain the basic.
http://biology.kenyon.edu/courses/biol09/t...%20dilution.jpg
For an overnight value, it is likely that your culture will reach 1 000 000 000 organism per mL.
Thanks, it tottaly gives me an idea now:) What do you dilute them with though, the broth or water? And I think my Vibrio's O/N yields 1 000 000 000 cells, but is that OD 0.5 or OD 1 or what do you think? I need to do this tomorrow, any help is welcome!
Do not dilute you bacteria with water, you will kill your bacteria. You need to keep osmotics pressure the same so you use sterile saline (145 mM NaCl) solution, media or any solution that you use to wash or resuspend bacteria. Media is a bit expensive so I personally use a saline solution.
For an overnight, the OD(600 nm) is likely to be above 1.0. It is a good thing to measure your OD before start your dilution.
For an overnight, the OD(600 nm) is likely to be above 1.0. It is a good thing to measure your OD before start your dilution.
Ok, so what when you know that at OD600 you have 10 000 000 000 cells and you have measured OD600=1.4? What does that mean and how do you proceed?
Do not dilute you bacteria with water, you will kill your bacteria. You need to keep osmotics pressure the same so you use sterile saline (145 mM NaCl) solution, media or any solution that you use to wash or resuspend bacteria. Media is a bit expensive so I personally use a saline solution.
For an overnight, the OD(600 nm) is likely to be above 1.0. It is a good thing to measure your OD before start your dilution.
Ok, so what when you know that at OD600 you have 10 000 000 000 cells and you have measured OD600=1.4? What does that mean and how do you proceed?
Forget about that, I just found the answer:)
It means that an OD600=1.4 there is 10 000 000 000 viable cells per mL in your culture. Base on your requirement (100 to 200 cells per plate), you just need to dilute your cells to 1000-2000 cells per mL and plate 0.1 mL. The dilution with 1000-2000 cells per mL should be 1 in 10 000 000 but to be on the safe side you dilute to 1 in 100 000 000 and maybe to 1 in 1 000 000 000. The second t last dilution should have 1 or 2 colony per plate and the last should not have any colonies.
The easiest way to get a 100-200 CFU count is by starting with an overnight TSA plate that you have harvested into sterile saline from this remove 1ml and place into 9ml of sterile saline to give a 1 in 10 dilution of 10-1 continue with this 1 fold dilution down to about 10-9 depending on how concentrated the starting culture was. From each dilution plate out 2 plates of TSA with 0.1ml of the suspension and count the resulting colonies after 24 hours.