Schilke Lab

Ingredient List (see Recipes for additional instructions)

• MRS Broth
• 10-mM potassium phosphate buffered saline (PBS)
• Pediococcus pentosaceous culture (frozen)
• Disposable loops
• 10 – 100 microliter pipette and tips
• 500 – 5000 microliter pipette and tips

Technique

The serial dilution technique can greatly amplify small errors in transfer of fluids. Use good technique and “reverse pipetting” when possible — see the following articles for some excellent suggestions on high-accuracy pipetting:

• 17 Ways to Stop Pipetting Errors from Ruining Your Experiments
• Thermo Scientific’s Pipetting Guide

You’ll be glad you did.

Preparation

1. Decide on the solutions to be tested and the series of dilutions you will be using (starting with 10x, 100x, et cetera).
2. Make the MRS broth you will be using. The amount needed is dependent on the number of dilutions to be made and the number of sample tubes. You will need 10 mL of MRS broth per sample tube (a sample tube is a large Falcon tube used to grow P. pentosaceous, and there should be one for each different solution you are testing along with one or two spare), and additional broth to dilute tubes to a specific absorbency. 100 mL of broth is usually sufficient, unless you are making a large number of samples.
3. Make and filter 7.4 pH PBS buffer. You will need approximately 5 mL for each dilution tube, and a small additional amount for control.
4. Pour 10 mL of MRS broth into each sample tube. Label these tubes appropriately.
5. Acquire frozen P. pentosaceous culture from the -80 degree freezer. Using a new loop each time, take samples of the culture and mix into the MRS broth in each tube. Return the culture to the freezer as quickly as possible, as many bacteria will die if thawed and refrozen.
6. Incubate the sample tubes at 37 degrees Celsius for approximately 16 hours. During this time, label your MRS broth and PBS buffer and store them in the cold room.
7. Once incubation is complete, remove the sample tubes and test the absorbency of each. Choose which sample tubes you will be using and dilute these to the same absorbency using blank MRS broth. This absorbency should be between 0 and 1 A — 0.7 A is commonly chosen.
8. Set up and label racks of small Falcon tubes for dilution, as separate series in order of dilution.

Dilution Procedure

1. Mix an appropriate amount of incubated broth from the sample tubes with the solution to be tested in an empty tube. The final volume should equal 5 mL. Repeat for each solution to be tested (using a different sample tube each time); these are the ‘master’ tubes. *Note: Make VERY sure that the master tubes are labeled correctly!
2. If necessary (as in the case of nisin or TFA), incubate the master tubes. Once incubation is complete, remove the tubes.
3. Combine appropriate amounts equaling 5 mL in total of broth from a master tube and PBS buffer in the first dilution tube in a series and mix by swirling. The final mixture should be appropriately dilute. Repeat for each series. For example: If the first dilution is 10x, add 4500 microliters of PBS and 500 microliters of master solution. If the first dilution is 100x, add 4950 microliters of PBS and 50 microliters of master solution.
4. Add 4500 microliters of PBS to all other dilution tubes.
5. Transfer 500 microliters from the first dilution tube in a series to the second. Mix, then transfer 500 microliters from the second tube to the third. Continue until the end of the series.
6. Repeat step 5 for all series.
7. Label the racks of dilution tubes, then store carefully until ready for plating?.