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Quantitative Methods: Part 2. Diluting Solutions

Author(s): David R. Caprette, PhD

Serial Dilutions

It often is convenient to make dilutions of a stock solution. For example, to conduct an assay on a substance when nothing is known about the starting concentration, one can make several dilutions and determine which dilution gives a concentration that falls within the sensitive range of the assay. Serial dilutions, in which one systematically prepares dilutions sequentially by the same factor, accomplish this purpose very well. Other uses for serial dilutions include determining the effective concentration of an antibody solution, the best dilution for conducting bacterial counts, or the best dilution for an enzyme assay.

Start your serial dilutions by deciding how much to dilute each successive sample. It is common to make ten-fold dilutions. Then, decide on a container. Larger volumes tend to reduce error introduced by pipetting inaccuracies.

Place a predetermined volume of solvent in each container. To make 5 serial dilutions of a bacterial sample, you might start with five 125 ml Erlenmeyer flasks and aseptically put 90 ml of culture medium into each flask. Next, transfer 10 ml of liquid bacterial culture to the first flask and mix completely to make a 10 fold dilution. Transfer 10 ml from that flask to the next one and mix again, repeating for the remaining five flasks. The fifth flask should contain a 105 dilution of the starting material.

The pipette used for transfer must be rinsed each time, or even replaced, since any remaining liquid is ten times concentrated, compared to the solution you will transfer next. Errors will be compounded, so it is crucial to use an accurate transfer technique.