In the previous step, you calculated which dilutions to use for plating. Now you will prepare the dilution series and do the plating.
We assume that you will use pour plates, Compact Dry, or Petrifilm®. These techniques use 1 mL for plating. Please refer to Plate smarter if you are using only 0.1 mL for the Drigalsky or similar technique.
Dilution series for plate counts. The 15 mL centrifuge vial has a concentration of 1×1010 cells/mL. A 1:10 dilution is prepared by transferring 1 mL sample to 9 mL buffered peptone water (or similar). The lowest numbers shows the cells/mL for the individual vials. Eight 1:10 dilutions are needed to dilute to 100 cells/mL. We recommend Bracketing dilutions. Use the cyan bracket when plating 1 mL. Use the magenta bracket when plating 0.1 mL.
Always make a new dilution series for each plate count repeat
It is tempting to use the same dilution series for the replicates, but always make a new dilution series as this provides a more reliable average and standard deviation of the method.
Step by step
1
Rack and label tubes
Place sufficient number of 9 mL buffered peptone vials in a rack.
In the above illustrated example you would need 9 vials for each plate count repeat.
Label the vials. Since the culture vial is 100, the subsequent vial will be 10-1. Leave out the base number (10) to save time.
2
Vortex briefly
Vortex the sampling vial 5 seconds at max speed to ensure even dispersion of cells. This assumes that the sample was already aggressively disaggregated in the step Retrieve culture sample.
3
Dilute 1:10
Transfer 1 mL sample to 9 mL buffered peptone water or equivalent suitable sterile dilution medium for your culture. Vortex 10 seconds at max speed.
4
Continue dilutions
Repeat step 3 until the cell concentration in the diluted vial is within ~2 - 25 cells/mL.
In the above illustrated example, this would be a total of 9 dilutions.
5
Select the vials for plating
Pick the vials bracket that covers the expected optimal dilution and the two adjacent vials.
In the above illustrated example, you should remove vials -1 to -6 so only vial -7, -8, and -9 is left in the rack. This represents the cyan bracket.
6
Label agar plate / Compact Dry / Petrifilm®
Label each plate with the corresponding dilution and repeat.
In the above illustrated example, we would write -7#1, -8#1, and -9#1.
7
Plate the dilutions
Plate 1 mL of each vial to the corresponding agar plate.
8
Do two more dilution and plating repeats
Repeat step 1-7 two times for two additional plate count repeats. Once you are done, you should have three plates of each dilution bracket.
9
Incubate
Incubate at preferred growth conditions. For E. coli this would be 37 °C.
Summary
You have now finalized the plating. Wait for the plates to incubate and proceed to Count colonies.
