Serial dilution is a laboratory technique used to systematically reduce the concentration of a substance in a solution by fixed, proportional amounts. This process involves repeatedly diluting the solution in a sequence, creating a series of solutions with progressively lower concentrations. It is a foundational method employed across various scientific disciplines because it allows researchers to work with a range of concentrations that are more appropriate for accurate measurement. The technique is particularly valuable when the initial sample is so concentrated that direct analysis would be impossible or yield inaccurate results.
Purpose and Core Principles
The primary purpose of performing a serial dilution is to achieve a concentration of the sample that is within a measurable range for a specific assay or instrument. Many analytical methods accurately measure a substance only when it is present above a lower limit and below an upper limit, often called the saturation point. When a sample is too concentrated, it may exceed this upper limit, making it impossible to determine the true value. Serial dilution brings the concentration down to a level that can be reliably quantified.
The core principle governing this technique is the maintenance of a constant dilution factor, or ratio, at each step of the series. A common factor is a 1:10 dilution, where one part of the solution is mixed with nine parts of a diluent, resulting in a tenfold reduction in concentration. By applying this same factor sequentially, the concentration decreases geometrically (e.g., to 1/10th, 1/100th, and 1/1000th of the original stock solution). This geometric progression ensures that a broad spectrum of concentrations can be generated from a single stock solution with precision.
Step by Step Procedure Guide
Performing a serial dilution requires the concentrated stock solution, a sterile diluent (such as distilled water, saline, or a buffer), and multiple containers, like test tubes or microplate wells. Precision instruments, typically micropipettes, are necessary for accurately transferring small, measured volumes of liquid. The process begins with the preparation of the dilution containers.
The first step is to label each test tube or well with the final dilution factor it will represent, which helps track the concentration. Next, a set volume of the sterile diluent must be added to every container in the series. This ensures that each one receives the same amount to maintain the constant dilution factor. For a 1:10 dilution, this often involves adding 9 milliliters of diluent to each subsequent tube.
After the diluent is prepared, the concentrated stock solution is introduced into the first container. A small, measured volume of the stock—for example, 1 milliliter for a 1:10 dilution—is transferred into the first tube and thoroughly mixed. Adequate mixing is important to ensure the solute is evenly distributed throughout the new volume of diluent.
The process continues by taking the same small volume (e.g., 1 milliliter) from the first, now-diluted tube and transferring it into the second tube containing only the diluent. This action creates the next, lower concentration in the series, and the contents are mixed again. This transfer step is repeated for the entire series, with the sample for each new tube always being drawn from the tube immediately preceding it.
Maintaining accuracy depends heavily on preventing cross-contamination between the different concentration levels. To achieve this, a fresh pipette tip must be used for every transfer between tubes. Alternatively, if the same pipette is used, it must be thoroughly rinsed with the diluent before drawing the sample from the next tube. Finally, after the aliquot is removed from the final tube, that volume is often discarded so that all containers in the series have the same final volume.
Calculating Dilution Factors
Understanding the mathematical relationships between the tubes is necessary for accurately interpreting experimental results. The individual dilution factor for a single step is determined by the ratio of the volume of the sample transferred to the total volume in the new container.
The formula for the individual dilution factor is the volume of sample transferred divided by the total volume (sample volume + diluent volume). For instance, if 1 milliliter of solution is transferred into 9 milliliters of diluent, the total volume is 10 milliliters, making the individual dilution factor 1/10, or 10⁻¹. This indicates the sample’s concentration has been reduced to one-tenth of the concentration of the preceding tube.
To calculate the total or cumulative dilution factor for any tube in the entire series, you must multiply the individual dilution factors of all the preceding steps. Using the 1:10 example, the first tube has a total dilution factor of 10⁻¹. The second tube, created by diluting the first, has a total dilution factor of 10⁻² (or 1/100). The third tube would be 10⁻³ (1/1000), and so on, creating a predictable, logarithmic decrease in concentration.
Common Applications in Science
Serial dilution is a broadly applicable technique employed to prepare samples for quantitative analysis across numerous fields. One frequent application is in microbiology, where it determines the concentration of bacteria or other microorganisms in a culture. By diluting the culture until a small portion can be plated on agar, researchers achieve a “countable” plate (typically 30 to 300 colonies), which allows them to calculate the original cell density of the stock.
In biochemistry and analytical chemistry, serial dilutions are routinely used to prepare a set of known concentrations to generate a standard curve. A standard curve plots the concentration of a substance against its measured signal, such as absorbance in spectrophotometry, allowing the concentration of an unknown sample to be accurately interpolated. In immunology, the technique is used in assays like antibody titration, where researchers determine the lowest concentration of an antibody that still produces a measurable effect, such as binding to a target antigen.