By Contributor
Updated Aug 30, 2022
Mastering the art of dilution is essential for anyone working in chemistry or microbiology. Accurate dilutions improve laboratory precision, reduce variability, and ultimately lead to more reliable data. The following step‑by‑step guide is grounded in laboratory best practices and safety standards, ensuring you perform each dilution with confidence and accuracy.
For chemistry or analytical dilutions, use volumetric glassware such as a volumetric pipette and a volumetric flask. In microbiology, serological pipettes and graduated cylinders are appropriate for transferring small volumes while keeping the solution countable.
Begin with a liquid stock solution—either a pure liquid sample or a solution prepared from a powder or liquid that has been diluted to a known concentration.
Measure the exact volume of stock using a volumetric pipette and transfer it into a volumetric flask that holds the desired final volume. For a 1:100 dilution, pipette 1.0 mL of stock into a 100 mL flask and add 99 mL of diluent to reach a total of 100 mL.
Transfer the stock solution with a serological pipette into a beaker, then add the diluent using a graduated cylinder. For a 1:100 dilution, add 1 mL of stock to 100 mL of diluent, yielding a final volume of 101 mL.
Use the diluent specified for your method. In microbiology, common choices include media, buffers, and sterile water. In chemistry, diluents may be solvents, acids, bases, or deionized water, depending on the analyte.
Swirl the flask halfway through the addition of diluent to ensure homogeneity, then continue adding until the final volume is reached.
For the last few milliliters, use a dropper to add diluent in single drops, ensuring the final volume is measured accurately.
Hold the flask or beaker at eye level and read the lower edge of the meniscus— the concave curve formed by the liquid surface. Align this point with the scale on the container for precise measurement.
Insert a magnetic stir bar into the dilution and place the container on a stir plate, or, if a stir plate is unavailable, stop the flask, swirl, and flip it upside down repeatedly to mix.
When a large final volume is required, perform serial dilutions. For a 1:10,000 dilution, first make a 1:100 dilution (1 mL stock into 100 mL diluent). Then transfer 1 mL of this intermediate solution into another 100 mL of diluent. The cumulative effect yields a 1:10,000 dilution.
Always add acid to water, not the reverse, to avoid exothermic reactions. Begin by adding a small volume of water to the flask before introducing the acid, then dilute to the required volume.
Label dilutions clearly, avoid replacing liquid lost during mixing, and always add acid to water to ensure safety and accuracy.
Adding water to acid can produce violent reactions and serious injury. Use a pipette bulb, never mouth pipet, and always add acid to water.
