Serial dilution is a foundational laboratory technique used to systematically reduce the concentration of a substance in a solution. This sequential process involves mixing a small volume of a concentrated sample with a larger volume of a solvent, then repeating this action using the newly diluted solution. By consistently applying the same reduction ratio at each stage, scientists create a series of solutions with concentrations that decrease in a predictable, geometric progression. This controlled reduction allows for precise measurements that would be impossible to obtain from the highly concentrated original sample.
Defining the Purpose and Key Terminology
The primary goal of performing a serial dilution is to bring a sample’s concentration into a range measurable by laboratory instruments or techniques. For instance, a microbial culture may contain billions of cells per milliliter, which is too dense to count directly. Diluting the sample ensures the final result will be statistically manageable, such as producing a number of bacterial colonies that can be counted accurately.
The initial, highly concentrated liquid is the Stock Solution. The liquid used to dilute the stock is the Diluent, often a sterile buffer or water. A small, measured volume removed from one solution and transferred to the next tube is called the Aliquot.
The Dilution Factor (DF) represents the ratio of the initial volume of the sample to the final volume of the diluted solution. For example, mixing one part of the stock solution with nine parts of diluent results in a 1-in-10 dilution, or a dilution factor of 10.
Step-by-Step Guide to Performing a Standard Serial Dilution
Before beginning the process, necessary labware, such as test tubes, must be prepared and clearly labeled with the intended final dilution factor. For a standard ten-fold dilution series, each tube is filled with 9 milliliters (mL) of the sterile diluent. The initial Stock Solution is set aside as the starting material for the first transfer.
The first step involves transferring 1 mL of the Stock Solution into the first tube containing the 9 mL of diluent. This aliquot transfer is typically performed using a calibrated instrument like a micropipette to ensure volume accuracy. Once transferred, the solution is thoroughly mixed, either by gently vortexing or by repeated aspiration with the pipette, to ensure the substance is evenly distributed. This mixed solution now represents a 1:10 dilution of the original stock.
To create the next step, a new, clean pipette tip is attached to avoid carrying over residual, higher-concentration material. A 1 mL aliquot is then taken from this first, 1:10 dilution and transferred into the second tube, which also contains 9 mL of diluent. This second tube is mixed completely, resulting in a 1:100 dilution of the original stock.
The sequential process is repeated for as many steps as required to reach the desired final concentration, always using the solution from the previous tube as the source for the next aliquot. The consistency of the transfer volume (1 mL) and the diluent volume (9 mL) at each stage ensures that every step reduces the concentration by the same factor of ten.
Calculating Concentration and Dilution Factors
The Dilution Factor for a single step is mathematically determined by dividing the final total volume by the volume of the sample added. In the common example of adding 1 mL of sample to 9 mL of diluent, the total volume is 10 mL, making the single-step dilution factor $10\text{ mL} / 1\text{ mL} = 10$. This factor is often expressed as a ratio, such as 1:10, or as an exponent, $10^{-1}$.
To find the cumulative dilution for any tube in the series, the dilution factors of all preceding steps are multiplied together. For instance, a third tube in a ten-fold series has a cumulative dilution factor found by multiplying $10 \times 10 \times 10$, which equals 1,000. This means the solution in the third tube is one-thousandth the concentration of the original stock.
Calculating the final concentration of a specific tube requires multiplying the initial concentration of the Stock Solution by the inverse of the cumulative dilution factor. If the stock concentration was 100 milligrams per milliliter (mg/mL), and the cumulative dilution factor is 1,000, the final concentration is $100\text{ mg/mL} \times (1/1000)$, resulting in $0.1\text{ mg/mL}$.
Common Applications in Science
Serial dilution is used in microbiology to estimate the concentration of microorganisms in a sample, a process often called microbial enumeration. By plating a small volume of several dilutions onto agar and incubating them, a plate can be obtained where the resulting colonies are numerous but not too crowded. Counting the Colony Forming Units (CFU) on this plate and multiplying by the corresponding cumulative dilution factor allows for the calculation of the original sample’s total bacterial count.
In biochemistry and analytical chemistry, serial dilutions are used for creating Standard Curves. A standard curve is a graph generated by measuring the response of an analytical instrument, like a spectrophotometer, to a series of solutions with precisely known concentrations. Using solutions created through serial dilution ensures a wide range of accurately known concentrations to calibrate the instrument. Unknown samples can then be measured, and their concentration is determined by interpolating the signal onto the established standard curve.