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Preparation of S9 Mix: A Step-by-Step Guide

Preparation of S9 Mix: The S9 mix is a crucial component in various biological and biochemical experiments, particularly in the Ames test for mutagenicity. It is derived from the liver of rats (typically induced with Aroclor 1254), providing a rich source of mammalian enzymes, including those involved in the metabolic activation of many compounds. This article will guide you through the process of preparing the S9 mix.

Preparation of S9 Mix

Preparation of S9 Mix: A Step-by-Step Guide

Components of S9 Mix

The S9 mix is composed of the S9 fraction, which contains the enzymes, and a cofactor solution that provides the necessary elements for enzymatic activity. The cofactor solution typically contains glucose-6-phosphate, NADP (Nicotinamide adenine dinucleotide phosphate), and magnesium chloride (MgCl2).

Preparation of S9 Mix

Step 1: Preparation of S9 Fraction

The S9 fraction is prepared from the liver of rats treated with Aroclor 1254. The liver is homogenized, and the homogenate is centrifuged at 9000 g for 20 minutes. The supernatant is then centrifuged at 105,000 g for 60 minutes to obtain the S9 fraction.

Step 2: Preparation of Cofactor Solution

The cofactor solution is prepared by dissolving the following components in distilled water:

Glucose-6-phosphate33 mM
MgCl28 mM

Step 3: Mixing the S9 Fraction and Cofactor Solution

The S9 mix is prepared by combining the S9 fraction and the cofactor solution. The typical ratio is 1 part S9 fraction to 9 parts cofactor solution. For example, to prepare 10 ml of S9 mix, combine 1 ml of S9 fraction with 9 ml of cofactor solution.

Storage of S9 Mix

The S9 mix should be stored in aliquots at -80°C to maintain enzyme activity. It should be thawed just before use and kept on ice during the experiment.

Applications of S9 Mix: A Broad Spectrum of Uses

The S9 mix, with its rich content of metabolic enzymes, finds extensive application in various fields of biological and biochemical research. Here are some of the key applications:

1. Mutagenicity Testing

The most common use of the S9 mix is in the Ames test, a widely used method for detecting chemical substances that can cause mutations in the DNA of organisms. The S9 mix provides the metabolic enzymes necessary to mimic the conditions in a living organism, allowing for the detection of mutagens that require metabolic activation.

2. Metabolic Studies

The S9 mix is used in in vitro metabolic studies to investigate the metabolism of various compounds, including drugs, environmental pollutants, and natural products. It can help researchers understand how these compounds are metabolized in the body and identify their metabolic products.

3. Drug Development

In the field of drug development, the S9 mix is used to study the metabolic stability of new drug candidates. It can help predict the drug’s behavior in the body, including its potential for interactions with other drugs.

4. Toxicology

The S9 mix is used in toxicological studies to assess the potential toxicity of various substances. It can help identify substances that are not inherently toxic but can be converted into toxic compounds through metabolic processes.

5. Environmental Monitoring

In environmental monitoring, the S9 mix can be used to assess the potential health risks of environmental pollutants. By studying how these pollutants are metabolized, researchers can gain insights into their potential effects on human health.

In conclusion, the S9 mix is a versatile tool in biological and biochemical research. Its ability to mimic the metabolic processes in living organisms makes it invaluable in a wide range of applications, from drug development to environmental monitoring.


The preparation of the S9 mix is a straightforward process, but it requires careful handling to maintain the activity of the enzymes. With this guide, you should be able to prepare the S9 mix for your experiments. Remember, the quality of your S9 mix can significantly impact the results of your experiment, so always ensure to use fresh and high-quality components.

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