How to do a serial dilution?

Serial dilution is an important method used in microbiology, biochemistry, and pharmaceutical research. The technique involves dilution of a concentrated sample to achieve lower concentration. This allows accurate measurement and experimental procedures. When it comes to standard plate count method, serial dilution is an important step in estimating the number of viable microbes within the sample by reducing the concentration.

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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Serial dilution method

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

During serial dilution, samples are diluted through a series of standard volumes of sterile diluent. The 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.

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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• Microbiology: To estimate the concentration of cells/organisms and obtain countable colonies.
• Biochemistry: To achieve desired reagent concentrations from higher stock solutions.
• Pharmaceuticals: To prepare specific concentrations of chemicals and compounds.
  

Limitations of serial dilution

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• Potential for errors in sample propagation and transfer, leading to inaccuracies in higher dilutions.
• Time-consuming, limiting efficiency.
• Does not separate bacteria/cells, only reduces concentration.
• Requires expertise in aseptic techniques and microbiological practices.

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