Plate smarter

MPD-8: Use BactoBox® to find the right dilution for plate counts

Plate counts are often done "blind" because the cell concentration is unknown. This leads to excessive and tedious workload. With BactoBox®, you eliminate the guesswork. By knowing the cell concentration, you can target the optimal dilution for plate counts.

Hit the right dilution for plate counts

The illustration below is a typical dilution series for plating.

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 lower numbers shows the cells/mL for the individual vials. Eight 1:10 dilutions are needed to get 100 cells per mL. We recommend Bracketing dilutions. Use the cyan bracket when plating 1 mL. Use the magenta bracket when plating 0.1 mL.

Petrifilm, compact dry or pour plate method: Plate 1 mL

With a culture that has a concentration of 1×10¹⁰ cells/mL you should do eight 1:10 dilutions to get the concentration down to 100 cells/mL. This dilution works for petrifilm, compact dry, and pour plates where 1 mL is added.

Rule of thumb for dilutions before petrifilm, compact dry, or pour plate method

Use the cells/mL× dil from the BactoBox® measurements to determine the required sample dilution.

Write the number with thousand separators and determine how many zeroes to remove to get 25–250 cells per mL. In the example above 8 zeros are removed from 10 000 000 000 to get to 100. That is use 8 dilutions (10^-8) prior to plating

Spread plate method: Plate 0.1 mL

The spread plate method typically only uses 0.1 mL. If you're using this method do one dilution less, i.e. use the seventh dilution instead of eight dilutions.

Rule of thumb for dilutions before spread plate method

Use the cells/mL× dil from the BactoBox® measurements to determine the required sample dilution.

Write the number with thousand separators and determine how many zeros to remove to get 25–250 cells per mL. In the example above 8 zeros are removed from 10 000 000 000 to get to 100.

Finally, reduce the number of dilutions by one because only 0.1 mL is plated. That is, use 7 dilutions (10^-7) prior to plating.

Bracketing dilutions

With the above dilution series for plating we assume that the Bactobox® cells/mL is similar to the CFU/mL. As discussed in Correlation with plate counts this is usually a valid assumption. But there are exceptions where BactoBox® cells/mL is either higher or lower than the CFU. Therefore it is often a good idea to "bracket" the dilution series. That is, also do plate counts on the vials next to the assumed optimal dilution. This assumes that the concentrations are within the same log

This is exemplified in the above illustration.

• When plating 1 mL use the 3 vials in the cyan bracket.

• When plating 0.1 mL use the 3 vials in the magenta bracket.