IGEM:IMPERIAL/2009/M3/Assays/ColonyFormingUnits

Aims
By performing this colony forming units assay, we aim to determine the % efficiency of our killing strategy by counting the ratio of colonies counted on plates containing the inducer for killing, and plates without the inducer.

Using this induction method, we aim to kill the maximum number of bacteria, which will mark the success of M3.

Colony Forming Units Assay
We will be using serial dilutions, plating and counting of live bacteria to determine the number of bacteria in a given population. To this end we will make serial dilutions of a solution containing an unknown number of bacteria, plate these bacteria and determine the total number of bacteria in the original solution by counting the number of colony forming units and comparing them to the dilution factor. Each colony forming unit represents a bacterium that was present in the diluted sample. The numbers of colony forming units (CFU’s) are divided by the product of the dilution factor and the volume of the plated diluted suspension to determine the number of bacteria per mL that were present in the original solution.

Rationale for Modification
In order to prevent cells dividing during the time lag between inducing killing and plating the cells, we have decided to induce killing by plating the cells on a plate containing the arabinose inducer (and no other carbon source present). We have a single cell suspension from which we perform serial dilutions as seen below.

When plating, two separate plate types will be used, one with just the arabinose inducer and one without the inducer (only agar as the control). This way newly divided cells are separate from killed ones. It is expected that the cells on the control will grow and form visible colonies, which can then be counted using the method below. Cells plated onto the inducer are not expected to form colonies as the agar does not contain glucose and will be forced to take up the inducer. If any colonies do appear, this would clearly indicate that the cell was not killed off. However, we believe that the only thing to survive will be escape mutants and they will be rare. By comparing the CFUs on both plates we can determine a % efficency of our killing strategy. (See below and output data).



Equipment
1) 20 sterile glass test tubes / 5mL falcon tubes

2) Test tube rack

3) Pipette

4) 20 sterile TSA plates

5) Beaker with "hockey stick"

6) Bunsen burner

Reagents
1) 70% ethanol

2) M9 salts. If unavailable, then obtain


 * 6.4 g Na2HPO4.7H2O
 * 1.5 g KH2PO4
 * 0.25 g NaCl
 * 0.5 g NH4Cl
 * 500mL DI water

3) Agar with arabinose inducer only

4) Agar with glucose only

5) Bacterial solution

6) 500mL glass bottle

7) Magnetic stirrer

Colony Forming Units Protocol

 * Preparing 1X M9 salts

1. Dissolve:


 * 6.4 g Na2HPO4.7H2O
 * 1.5 g KH2PO4
 * 0.25 g NaCl
 * 0.5 g NH4Cl

in 500mL DI water, to make 500mL of 1X M9 salt solution.

2. Stir well with a magnetic stirrer.


 * Serial dilutions

1. Label 10 small, sterile test tubes 1 through to 10 and add 4.5 mL of M9 salts to each test tube. (Bacteria will not grow in this media but will remain in a state of stasis until the diluted cells are plated on media containing a carbon source).

2. Pipette 0.5 mL of the original bacterial solution into test tube 1. Mix this bacterial suspension thoroughly by votexing.

3. Using a clean pipette withdraw 0.5 mL of the diluted bacterial suspension from the first test tube and pipette into the second test tube. Continue in this fashion until you have serially diluted the original bacterial suspension into test tube 10.

(So tube 1 contains 4.5 mL of sterile media; you will add 0.5 mL of the undiluted bacterial suspension to yield a total volume of 5.0 mL. Tube 2 contains 4.5 mL of sterile media; you will add 0.5 mL of the 1:10 diluted bacterial suspension to yield a total volume of 5.0 mL).


 * Plating the serially diluted cells:

Note: This will be done twice, once for the 10 control plates, and once for the plates containing the inducer.

1. Label 10 TSA plates. (In this case we will plate the following dilutions: 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, 1 x 10-6, 1 x 10-7, 1 x 10-8, 1 x 10-9 and 1 x 10-10).

2. Obtain a beaker containing a “hockey stick”. This is to spread the diluted bacterial suspension evenly over the surface of the plate. Spray a small volume of 70% ethanol into the beaker for sterlisation.

3. For the cell suspension that will be plated onto the TSA plate labeled 1 x 10-1, pipette 0.5 mL of the diluted suspension from the appropriately diluted test tube onto the surface of the plate.

4. Dip the hockey stick into the 70% ethanol and flame the stick until the ethanol has burned off. Do not heat the stick to long; you only need to flame the loop to burn off the ethanol—that will be sufficient for sterilizing the hockey stick.

5. After sterilizing the stick, use the hockey stick to spread the bacterial suspension evenly over the entire surface of the plate.

6. Allow the plate to dry. Repeat steps 1 - 5 for the remainder of the bacterial dilutions.

7. Tape all the plates together and incubate upside down, at 37°C for 24 hours.

Note:Repeat steps 1 - 7 for the control (plate without inducer. You will now have 20 plates in total.

Results Processing

 * Counting colony forming units and calculating the amount of bacteria in the original solution.

1. For each dilution, count the number of colony forming units on your plates. (For the control plates, 30 and 800 are considered statistically accurate. For plates containing the inducer it is expected that fewer or no colonies will be visable).

2. If the number of CFUs on your plate are greater than 1000, you may record in your table TNTC (too numerous to count). Alternatively, if your numbers are greater than 1000 AND you have evenly distributed the diluted bacterial suspension on the surface of the plate AND you can discern individual colonies; divide the plate into 4 sectors, count the number of bacteria in one sector and multiply by four. If the number of CFUs on your plate is below 10, record the number of CFUs, but do not use this in the calculations.

3. Enter results in the following table.




 * Calculating the number of bacteria per mL of serially diluted bacteria:

1. To calculate the number of bacteria per mL of diluted sample one should use the following equation:



(For example, if for the 1x 10-8 dilution plate you plated 0.1 mL of the diluted cell suspension and counted 200 bacteria, then the calculation would be: 200/0.1 mL x 10-8   or 200/10-9 or 2.0 x 10^11 bacteria per mL.)

2. We can then calculate % efficiency of our killing strategy. We use the calculation below for each dilution plate.