Serial dilution is a foundational laboratory technique used to systematically reduce the concentration of a substance in a solution. The method involves a sequence of dilution steps, where a small, measured volume of the initial sample is transferred into a larger volume of a sterile diluent, such as water or a nutrient broth. This process is then repeated using the newly diluted solution as the starting material for the next step, ensuring a constant and precise reduction factor at each stage. This step-by-step reduction allows scientists to achieve extremely high dilutions with accuracy, which is far more reliable than attempting a single, massive dilution.
The Necessity of Gradual Concentration Change
The systematic nature of serial dilution addresses the practical impossibility of accurately measuring substances in their original, highly concentrated state. When a sample is too dense, such as a thick bacterial culture or a deeply colored chemical solution, direct measurement is often inaccurate or impossible. A spectrophotometer cannot distinguish between light absorbances that are too high, and a petri dish covered in a dense film of bacteria is impossible to count.
The gradual, step-wise dilution ensures that at least one of the resulting solutions falls within a “measurable range” for the chosen instrument or technique. This range represents the concentration where measurement is possible, but the substance is not virtually undetectable. Attempting to achieve a high dilution, such as a 1:1,000,000 ratio, in a single step would require pipetting volumes too small to measure accurately with standard laboratory equipment. The sequential process avoids this limitation by compounding smaller, highly accurate dilution steps to reach the final dilution.
Quantifying Microorganisms and Viruses
One of the most frequent uses for serial dilution is in microbiology to determine the number of microorganisms in an unknown sample. Since microbial cultures often contain millions or even billions of cells per milliliter, direct plating would result in a solid, unreadable smear of growth. Serial dilution systematically lowers this count until a small portion of a dilution can be spread onto an agar plate, a process known as the Plate Count Method.
The goal is to produce a plate where the colonies are distinct and numerous enough for statistical accuracy, a range generally accepted to be between 30 and 300 colonies per plate. Plates with fewer than 30 colonies are statistically unreliable due to sampling errors, while plates with more than 300 have colonies that merge, leading to underestimation. By counting the colonies on a plate and multiplying that number by the total dilution factor, scientists calculate the concentration of viable organisms in the original sample.
This final concentration is expressed as Colony-Forming Units per milliliter (CFU/mL), which represents the number of bacteria or fungi in the sample capable of growing into a visible colony. A related application is in virology, where serial dilution is used to determine the concentration of a virus, known as its titer. The viral titer is calculated by observing the effects of various dilutions on host cells, which provides a quantitative measure of the infectious particles present in the starting material.
Creating Measurement Standards for Assays
Serial dilution is fundamental to quantitative chemistry and biochemistry, where it is used to create a set of known standards for analytical assays. This is accomplished by taking a stock solution of a substance, such as a protein, drug, or antibody, and performing a series of dilutions to create a range of solutions with precisely known concentrations. These known concentrations are then measured using a specific assay, such as an enzyme-linked immunosorbent assay (ELISA) or a spectrophotometer, which records a corresponding signal, like color intensity or light absorbance.
When plotted on a graph, the relationship between the known concentration and the measured signal generates a straight line called a standard curve or calibration curve. This curve acts as a baseline against which unknown samples can be measured. When an unknown sample is run through the same assay, its measured signal can be located on the standard curve, allowing scientists to interpolate its precise concentration. This application of serial dilution is less about counting physical particles and more about establishing a proportional relationship between concentration and a measurable chemical response.