# Help with calculating CFU prior to infecting mice - (May/11/2018 )

Hi,

My bacteria stock is at a concentration of 1x10^{8 }CFU/ml (it's in 1ml total volume). I would like to infect mice with different CFU concentrations: 5x10^{7} 5x10^{6 }5x10^{5 }5x10^{4 }each in a 40ul volume (in PBS). Could you please help me with the calculations?

1. Is this correct?

1x10^{8 } to 5x10^{7 }-> dilution factor is 2, 40ul/2= 20ul, so if I take 20ul from the 1x10^{8 }CFU/ml stock into 20ul of PBS I will get 5x10^{7 }

2. When I take volume from my original stock = 1x10^{8 }CFU/ml, in order to get the new concentrations, am I changing the original 1x10^{8 }CFU/ml concentration?

Thanks!

1) If you want those as a concentration (e.g 5 x10^{7} CFU/ml), as you say in your post then use C1V1 = C2V2

C1 = 1x10^{8}, V1=?, C2=5x10^{7}, V2= 0.04 ml

V1= (5x10^{7 }x 0.04) / 1x10^{8}

^{V1=...}

If you want the amount (i.e you want to take 5x10^{7} PFU total) to take from your stock (which is what your calculation suggests) then you use V=N/C

V= 5x10^{7 }/ 1x10^{8}

V=... Note the answer is in ml.

Note that if you want these as amounts in 40 ul, you can not do it - the concentration of your stock is too low. You can only do this if you want your working solutions to be concentrations.

2) The concentration of your stock doesn't change, just the amount of PFU left. For example, if you took out 500 ul from your ml, you will have taken out 5x10^{7} PFU in total. However the remaining stock is still at 10^{8} PFU/ml, so you could take out. This is how concentrations work - they stay the same. This is why you can make a 1 M tris stock in 100 ml using 12.14 g of tris or use 121.14 g of tris for a 1 L stock - but the concentration of the final solutions is the same.

Make sure you do not confuse concentration and amount!

Thank you very much for your detailed explanation!

I have more than 1 tube of the 1x10^{8 }CFU/ml (1 mL per tube) and I am intrested in the second option that you mentioned (the amount = CFU).

So if I understand correctly, for the 5x10^{7} CFU I will take (5x10^{7}/1x10^{8}=0.5=) 500ul from the 1x10^{8 }tube. I can then centrifuge this and reconstitute it in 40ul PBS and I will be able to infect the mice with this 40ul volume?

Thank you!

Possibly, you will lose some titre with the pelleting and resuspension - I would also expect that amount of CFU to take up a significant proportion (i not all) of the 40 ul

Thanks!! I will test it out.