Serial dilution is a foundational laboratory technique used to systematically reduce the concentration of a substance in a controlled, step-wise manner. This process is particularly important in microbiology because samples, such as bacterial cultures, often contain an extremely high number of microorganisms. Since direct counting of these high concentrations is impossible, serial dilution creates a series of solutions with progressively lower concentrations. The primary goal is to achieve a concentration low enough for accurate quantification of the original sample’s microbial load.
The Physical Steps of Serial Dilution
The process begins with a high-concentration sample and a set of sterile tubes, each containing a fixed volume of an inert liquid called the diluent. For microbiological work, this diluent is typically sterile saline or a nutrient broth, often at a volume of nine milliliters (9 mL) per tube. A small, fixed volume of the original sample, commonly one milliliter (1 mL), is accurately measured and transferred into the first diluent tube. This action creates a 1:10 dilution, meaning the concentration is now one-tenth of the starting material.
Thorough mixing of this first tube is performed to ensure the microorganisms are evenly suspended throughout the total ten-milliliter volume. Next, a fresh, sterile pipette tip is used to transfer one milliliter from this newly mixed tube into the second tube containing nine milliliters of diluent. This transfer repeats the 1:10 dilution process, making the second tube one-hundredth as concentrated as the original sample.
This sequence of transferring a small volume into the next tube of diluent and mixing is repeated multiple times until the desired level of reduction is achieved. Using a fresh pipette tip for each transfer is crucial to prevent “carry-over” contamination, which would compromise the accuracy of the dilution series. The systematic nature of this process allows for massive reductions in concentration using only small, manageable volumes of liquid.
Calculating the Dilution Factor
The physical steps of transferring liquid create a quantitative reduction in concentration that must be tracked precisely using the dilution factor (DF). The DF for a single step is calculated by dividing the final volume by the volume of sample added; for example, adding 1 mL of sample to 9 mL of diluent results in a total volume of 10 mL, giving a single-step DF of 10. The resulting concentration is often expressed as a fraction or in scientific notation, such as 10^-1 or 1/10.
The power of serial dilution lies in its geometric progression, where the total dilution factor for the entire series is found by multiplying the individual dilution factors of each step. For instance, the second tube, which is a 10^-1 dilution of the first 10^-1 tube, has a total dilution factor of 100 (10^-2). The third tube would be 1000 (10^-3), and so on.
This multiplicative effect means that a highly concentrated sample can be reduced by a factor of one million (10^-6) in just six simple steps. Attempting to achieve such a massive dilution in a single step would be impractical, requiring the addition of one milliliter of sample to 999,999 milliliters of diluent. Serial dilution avoids the need for impractically large volumes, providing a mathematically precise and manageable way to reduce concentration over several orders of magnitude.
Estimating Colony Forming Units (CFU)
The ultimate purpose of performing serial dilution in microbiology is to estimate the concentration of viable microorganisms in the original sample, a value expressed as Colony Forming Units per milliliter (CFU/mL). After the dilution series is prepared, a small volume, typically 0.1 or 1.0 mL, is taken from several of the final tubes and spread onto a sterile agar plate. The plates are then incubated, allowing each single, viable microorganism to multiply and form a visible cluster known as a colony.
The goal is to select a plate that contains a statistically significant and countable number of colonies, typically considered to be between 30 and 300 colonies. Plates with fewer than 30 colonies are considered too sparse to be representative, while plates with more than 300 colonies are designated “Too Numerous To Count” (TNTC) because the colonies merge, leading to an inaccurate count. By selecting a plate within the countable range, scientists can be confident in the representation of the original sample’s population.
The final CFU/mL concentration of the original sample is calculated by using the number of colonies counted on the selected plate and working backward through the known dilution steps. The formula used is: CFU/mL = (Number of Colonies) / (Volume Plated in mL x Total Dilution Factor of the Plate). This calculation essentially reverses the process of dilution, scaling the small, countable number of colonies back up to determine the concentration of viable microbes present in the initial sample.