How to Do a Serial Dilution?

Serial dilution is a laboratory technique used to systematically reduce the concentration of a solution. This allows accurate measurement and experimental procedures. It is a common technique widely applied in microbiology, chemistry and biochemistry for a variety of applications, including quantification of bacterial cultures, preparing standard solutions, and performing assays.

What is Serial Dilution?

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Serial dilution is a process of sequential dilution of a concentrated solution to achieve lower and more usable concentration. The method is commonly used in microbiology to decrease the concentration of cells in a culture. This leads to simplified operations such as plating and counting of bacterial colonies. The dilution is achieved by mixing a sample with a sterile diluent in a stepwise manner, applying a consistent dilution factor at each stage. When combined with standard plate count method, serial dilution ensures reliable enumeration of viable bacteria by producing well-distributed and countable colonies. Serial dilution is a fundamental tool in quantitative microbiological analysis and laboratory research.

Objectives of Serial Dilution

Simplifies the calculation of the concentration of cells in the original solution by using the total dilution factor.

Prevents the need to pipette extremely small volumes.

Produces incubated culture plates with an easily countable number of colonies.

Why is Serial Dilution Necessary in the Standard Plate Count Method?

The standard plate count method is designed to determine the number of viable bacteria. By counting colony-forming units (CFUs) that develop on agar plate, the method aids in determining the origin of the sample. Many samples, however, contain high concentration of microbes, hindering accurate observations. Therefore, the serial dilution is necessary to:

• Reduce the concentration of bacteria in the sample.
• Ensure the development of countable colonies on agar plates.
• Increase the accuracy of the bacterial count by promoting well-distributed colonies.
• Achieve isolated bacterial colonies that can be reliably counted.
• Maintain a contaminant-free culture environment.

Performing serial dilution enables easier count of isolated colonies. The process involves diluting the original sample with known dilution factor at each step. This results in manageable bacterial population for accurate enumeration.

Serial Dilution Method

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1. Prepare six test tubes with 9 ml of sterile diluent.
2. Draw 1 ml of the sample using a sterile micropipette and add it to the first test tube.
3. Mix thoroughly by pipetting up and down several times.
4. Discard the pipette tip and attach a new one.
5. Take 1 ml from the first tube and add it to the second tube.
6. Repeat the process for the remaining tubes to achieve final dilution of 1:1,000,000.

Formula and Calculation

Serial dilutions work by using a fixed dilution factor at each step. The dilution factor is the ratio by which the solution concentration is being reduced at each step. The key benefit being that scientists can work with lower concentrations whilst still maintaining a proportional relationship between original and diluted solutions. The diluted volumes are then used for spread, pour or streak plate methods. The extent of the dilution is determined by estimating the concentration of cells within the sample. Common dilution methods are 2-fold or 10-fold dilutions. With a 10-fold dilution, 1 ml of sample is mixed with 9 ml of diluent. The total dilution factor is then calculated by multiplying the dilution factors of each step in the series.

Tube number	Volume taken (ml)	Diluent (ml)	Dilution factor	Cumulative dilution
1	1	9	1/10 (10-1)	1/10 (10-1)
2	1	9	1/10 (10-1)	1/100 (10-2)
3	1	9	1/10 (10-1)	1/1000 (10-3)
4	1	9	1/10 (10-1)	1/10,000 (10-4)
5	1	9	1/10 (10-1)	1/100,000 (10-5)

Types of Serial Dilution Methods

10-fold serial dilution - This method is commonly used to reduce very high bacterial concentrations by rapidly diluting 1 ml of the sample into 9 ml of diluent.

2-fold serial dilution - This type of dilution is often used to determine the minimum inhibitory concentration (MIC) of antimicrobial agents. Performing this method offers precise MIC values than 10-fold dilution.

	Beginning	After first dilution	After second dilution	After third dilution	After fourth dilution	After fifth dilution
10-fold serial dilution	200 µg/ml	20 µg/ml	2 µg/ml	0.2 µg/ml	0.02 µg/ml	0.002 µg/ml
2-fold serial dilution	200 µg/ml	100 µg/ml	50 µg/ml	25 µg/ml	12.5 µg/ml	6.25 µg/ml

Applications of Serial Dilution

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In microbiology, serial dilution is commonly used to determine the number of microorganisms in a sample with an unknown concentration. An example would be having a bacterial culture and needing to estimate how many bacteria are present. If you were to count them individually without using serial dilution, it would be almost impossible due to the extremely high numbers. So instead, a small portion of the culture is diluted until the concentration is at a more manageable level, this allows for more accurate and manageable counting. The primary purpose of serial dilutions in microbiology is to systematically reduce the concentration of bacteria, viruses or other microorganisms to a reasonable level which allows for precise counting and analysis.

Serial dilutions are also useful in analytical chemistry, they can be used to create standard curves for quantitative analysis. Techniques such as spectrophotometry and ELISA rely on these dilutions to measure the concentration of substances which are unknown. It is also a technique used in pharmaceuticals, essentially it allows for determining the minimum inhibitory concentration (MIC) of antibiotics and drugs, this allows researchers to evaluate their effectiveness.

In a biochemistry laboratory, serial dilutions are very helpful in understanding the microbial concentration. They are applicable in the study of growth patterns, assessing enzyme production and testing antimicrobial compounds. A common use of serial dilutions in biochemistry is determining protein concentration, this is usually done through colorimetric assays like the Bradford assay, BCA assay and Lowry assay, all of which measure the amount of light a sample absorbs. An example of this includes starting with a known protein solution, such as 1 mg/mL of Bovine Serum Albumin (BSA) which is diluted in increments (0.5 mg/mL, 0.25 mg/mL and 0.125 mg/mL), creating a series of concentrations which are easier to measure. In the Bradford assay, Coomassie dye is mixed with each dilution, and the dye binds to the protein which causes a colour change. The intensity of the colour is directly related to protein concentration (dark blue correlating to higher concentrations). The next step involves the use of a spectrophotometer to measure the absorbance of each dilution, this is 595 nm for the Bradford assay. Absorbance readings are then plotted onto a graph to create a calibration curve, with concentration (X-axis) against absorbance (Y-axis). This curve then allows you to determine concentrations of unknown samples. Serial dilutions ensure that measurements are precise, but also reproducible as they cover a wide range of concentrations.

Limitations of Serial Dilution

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It is important that a serial dilution is conducted with precision and accuracy, as small errors during the dilution steps could lead to substantial errors in the counting. A small mistake such as a pipetting error or miscalculation can change the result by huge margins. This is because as the method goes on, you could essentially be accumulating the error, therefore this can be seen as a downside to serial dilutions as human error can never be ruled out. There is also a risk of contamination because there is a transfer between each tube. Furthermore, equipment not being properly sterilised can have a knock-on effect which will hinder the result. The method is time-consuming and has limited efficiency, as it does not separate the cells, only reduces concentration. However, by addressing these sources of possible error, it can allow you to take the necessary precautions to ensure your results are as accurate as possible when using a serial dilution.

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