## Sunday, August 10, 2014

### Serial Dilution of Bacterial culture / Stock solution - Method and Calculation

Serial Dilution is a series of sequential dilution of a substance in solution.

How to do serial Dilution?

To dilute a stock solution, Mix the stock solution with diluent. The ratio of the Final volume to the aliqout volume of the stock is known as Dilution Factor (DF). Dilution is the inverse of Dilution Factor. 10 fold dilution is actually 10^-1 dilution (Inverse of Dilution Factor: 1/10).

Example:

Diluting a 1 M (Molar) Stock solution 1000 fold by serial Dilution.

1000 fold serial dilution can be done by three 10 fold dilutions. (10*10*10 = 1000 fold).

Lets see how to serially dilute 1 M stock solution to a final volume of 100 mL.

Dilution Factor = Final Volume / Volume of Stock.

Since we need to do 10 fold dilution, dilution factor is 10.

Final Volume - 100 mL
X - Stock Solution required.

so; 10 = 100 mL /X mL;

10X = 100; X= 100/10 = 10 mL.

Diluent Volume required = [Final Volume - Stock Volume] = 100mL - 10mL = 90mL.

To do a 10 fold dilution of 1M Stock solution to 100mL,

Mix 10mL of 1M stock with 90mL of Diluent. Label the tube as Dilution 1.

From Dilution 1 tube take 10mL and mix 90mL of diluent and Label it as Dilution 2.

From From Dilution 2 tube take 10mL and mix 90mL of diluent and Label it as Dilution 3.

As we have done 10 fold dilutions, the tube 3 (Dilution 3) will be 1000 fold diluted than the stock.

Stock solution is 1 M, after 1000 fold dilution it becomes - 1/1000 = 0.001 M.

Dilution 1 - 0.1 M
Dilution 2 - 0.01 M
Dilution 3 - 0.001 M

How to do Serial Dilution of Bacterial Culture?

Dilution of bacterial culture can be performed the same way as explained above. Serially dilute the bacterial culture, Bacterial culture is generally serially diluted for plating (for bacterial isolation), for re-inoculation or for other applications like checking optical density, etc.

Below is the Overnight incubated plate, plated by serial dilution of the sample. Note the plate with lesser number bacterial colonies that is the one which is plated from a more diluted solution.

Bacterial Culture Dilution & Calculation

Example:
You have a bacterial culture, which has 10^6 bacterial cells  and you want 100 cells/mL how to dilute the culture to get 100cells/mL

Solution:
Inital Concetration - 10^6 cells
Final Concentration Required - 100 cells/mL or 10^2 cells/mL

You need to do Four 10 fold dilution [10*10*10*10 = 10000 fold dilution] to a volume of 1mL to get  10^2 cells/mL final concentration.

here is how you can do it,

Stock of Bacterial Culture - 10^6 Bacterial Cells

Tube 1 (Dilution 1) - Take 900uL of sterial water in tube 1 and add 100uL of bacterial stock - so tube 1 will have10^5 cells/mL.

Tube 2 (Dilution 2) - Take 900uL of sterial water in tube 2 and add 100uL from Tube 1 - so tube 2 will have10^4 cells/mL.

Tube 3 (Dilution 3) - Take 900uL of sterial water in tube 3 and add 100uL from Tube 2 - so tube 3 will have10^3 cells/mL.

Tube 4 (Dilution 4) - Take 900uL of sterial water in  tube 4 and add 100uL from Tube 3 - so tube 4 will have10^2 cells/mL or 100 cells/mL.

Serial Dilution Calculation: Problems and Solutions

You do a dilution by combining 100 ml volume of NaCl plus 700 ml unit volumes of Distilled water.  What is the dilution factor, i.e, how many more times dilute is it than the original concentration?