Salmonella Shigella (SS) Agar- Composition, Principle, Preparation, Results, Uses

Salmonella Shigella (SS) Agar is a type of culture medium that is used in microbiology to isolate and differentiate bacteria of the genera Salmonella and Shigella from other enteric bacteria. These bacteria are responsible for causing various gastrointestinal infections in humans and animals, such as typhoid fever, salmonellosis, shigellosis, and food poisoning. SS Agar was developed by Leifson in 1951 as a modification of the original Salmonella Agar devised by Kauffmann in 1935. SS Agar is also known as Brilliant Green Bile Agar or Kauffmann`s Modification of Brilliant Green Agar.

SS agar is a complex medium that contains various ingredients to selectively inhibit or promote the growth and differentiation of enteric bacteria. The main components of SS agar are:

• Bile salts: These are natural detergents that disrupt the cell membranes of gram-positive bacteria and some gram-negative bacteria, such as coliforms. Bile salts also inhibit the swarming of Proteus species, which can obscure the growth of other organisms on the agar surface.
• Sodium citrate: This is a salt that chelates metal ions and reduces the pH of the medium. Sodium citrate also inhibits the growth of gram-positive bacteria and some gram-negative bacteria, such as enterococci and Klebsiella species.
• Brilliant green: This is a synthetic dye that acts as a selective agent against most gram-positive bacteria and some gram-negative bacteria, such as Escherichia coli and Pseudomonas species. Brilliant green also imparts a green color to the medium, which helps to distinguish it from other agar types.
• Lactose: This is a disaccharide sugar that serves as a carbohydrate source for some enteric bacteria. Lactose fermentation produces acid, which lowers the pH of the medium and changes the color of the neutral red indicator from yellow to red or pink. Lactose-fermenting bacteria form red or pink colonies on SS agar, while lactose-nonfermenting bacteria form colorless or transparent colonies.
• Sodium thiosulfate and ferric citrate: These are inorganic salts that enable the detection of hydrogen sulfide (H2S) production by some enteric bacteria. H2S is a gas that reacts with ferric ions to form ferrous sulfide, a black precipitate. H2S-producing bacteria form colonies with black centers on SS agar, while non-H2S-producing bacteria form colonies without blackening.
• Neutral red: This is a pH indicator that changes color depending on the acidity or alkalinity of the medium. Neutral red is yellow at neutral or alkaline pH and red or pink at acidic pH. Neutral red helps to differentiate lactose-fermenting bacteria from lactose-nonfermenting bacteria on SS agar based on their acid production.
• Beef extract, enzymatic digest of casein, and enzymatic digest of animal tissue: These are organic substances that provide nitrogen, carbon, vitamins, and other nutrients for the growth of enteric bacteria. They also enhance the color contrast between colonies and the medium.
• Agar: This is a polysaccharide derived from seaweed that solidifies the medium and provides a surface for bacterial growth.

The final pH of SS agar is 7.0 +/- 0.2 at 25°C. The composition of SS agar per liter of deionized or distilled water is:

SS agar is a selective and differential medium that allows the growth and differentiation of Salmonella and some Shigella species from other enteric bacteria. The principle of SS agar is based on the following features:

• Bile salts, sodium citrate, and brilliant green: These are the selective agents that inhibit the growth of gram-positive bacteria and most coliforms, while allowing Salmonella and some Shigella to grow. These agents also prevent the swarming of Proteus species, which can interfere with the isolation of the target organisms.
• Lactose: This is the differential agent that distinguishes between lactose-fermenting and non-lactose-fermenting bacteria. Lactose-fermenting bacteria produce acid from lactose, which lowers the pH of the medium and turns the neutral red indicator red or pink. Non-lactose-fermenting bacteria do not produce acid and remain colorless or transparent on the medium. Most Salmonella and Shigella are non-lactose-fermenters, except for some rare strains such as Salmonella arizonae and Shigella sonnei.
• Sodium thiosulfate and ferric citrate: These are the indicators for hydrogen sulfide (H2S) production, which is a characteristic of some Salmonella species. H2S is produced by the reduction of thiosulfate in the medium by certain bacteria. Ferric citrate reacts with H2S to form ferric sulfide, which appears as a black precipitate in the center of the colonies. H2S-producing bacteria form black-centered colonies on SS agar, while non-H2S-producing bacteria form colorless or red/pink colonies without black centers.

By using these features, SS agar can help isolate and identify Salmonella and some Shigella species from clinical and non-clinical specimens. However, this medium is not recommended for the primary isolation of Shigella, as some strains may be inhibited by the high concentration of bile salts and brilliant green. Moreover, SS agar cannot differentiate between all species of Salmonella and Shigella, as some may share similar characteristics such as lactose fermentation or H2S production. Therefore, further biochemical and serological tests are required to confirm the identification of these organisms.

SS Agar is a ready-to-use medium that can be purchased from various suppliers. However, if you want to prepare it from scratch, you will need the following ingredients:

• Bile salts
• Sodium citrate
• Brilliant green
• Enzymatic digest of casein
• Beef extract
• Enzymatic digest of animal tissue
• Lactose
• Sodium thiosulfate
• Ferric citrate
• Neutral red
• Agar
• Deionized or distilled water

The preparation steps are as follows:

1. Weigh 60 g of the dry ingredients and transfer them to a suitable container.
2. Add one liter of deionized or distilled water and mix well until the ingredients are dissolved.
3. Heat the mixture with frequent agitation and boil for one minute to sterilize the medium. Do not autoclave the medium as this may affect its performance.
4. Pour the medium into sterile Petri dishes and let it solidify at room temperature.
5. Store the plates in the refrigerator (2-8°C) and avoid freezing. The plates can be used for up to one week.

Alternatively, you can use a microwave oven to prepare the medium. In this case, you will need a microwave-safe container and a plastic wrap to cover it. The steps are as follows:

1. Weigh 60 g of the dry ingredients and transfer them to the microwave-safe container.
2. Add one liter of deionized or distilled water and mix well until the ingredients are dissolved.
3. Cover the container with plastic wrap and pierce it several times with a fork to allow steam to escape.
4. Microwave the mixture on high power for about 15 minutes or until it boils. Watch carefully and stop the microwave if the mixture overflows.
5. Carefully remove the container from the microwave and swirl it gently to mix the medium.
6. Pour the medium into sterile Petri dishes and let it solidify at room temperature.
7. Store the plates in the refrigerator (2-8°C) and avoid freezing. The plates can be used for up to one week.

To use SS Agar for isolation of Salmonella and Shigella, you will need to inoculate the plates with your samples and incubate them at 35°C for 18-24 hours. You can then observe the colony characteristics and perform further tests to confirm the identification of the organisms.

The appearance of colonies on SS agar depends on the type and characteristics of the bacteria growing on it. The following table summarizes the common results and interpretations for SS agar:

Some examples of colony morphology on SS agar are shown below:

[Include images of colony morphology]

It is important to note that some strains of Salmonella and Shigella may show atypical reactions on SS agar, such as lactose fermentation or no hydrogen sulfide production. Therefore, other biochemical tests and serological methods are required to confirm the identification of these bacteria.

SS Agar is a selective and differential medium that is used for the isolation and identification of Salmonella and some Shigella species from clinical and non-clinical specimens. These bacteria are important pathogens that cause gastroenteritis, typhoid fever, dysentery, and other infections in humans and animals. SS Agar helps to distinguish these organisms from other enteric bacteria based on their ability to ferment lactose and produce hydrogen sulfide.

Some of the common uses of SS Agar are:

• It is used for the isolation of Salmonella and Shigella from stool samples of patients with suspected enteric infections. The medium inhibits the growth of most gram-positive and coliform bacteria, while allowing Salmonella and Shigella to grow. The colonies of Salmonella and Shigella appear colorless or transparent on SS Agar, while lactose-fermenting bacteria produce red or pink colonies. Additionally, Salmonella colonies may have black centers due to hydrogen sulfide production.
• It is used for the isolation of Salmonella from food samples that may be contaminated with these bacteria. The medium can detect low levels of Salmonella in foods such as meat, poultry, eggs, dairy products, and spices. The food samples are enriched in a suitable broth before inoculating on SS Agar. The presence of Salmonella can be confirmed by biochemical and serological tests.
• It is used for the isolation of Shigella from environmental samples that may be sources of infection. The medium can isolate Shigella from water, soil, sewage, and other materials that may harbor these bacteria. The medium is not recommended for the primary isolation of Shigella, as some strains may be inhibited by the bile salts and brilliant green in the medium. Therefore, a pre-enrichment step in a non-selective broth may be necessary before inoculating on SS Agar.

SS Agar is a useful medium for the detection and differentiation of Salmonella and Shigella from various specimens. However, it has some limitations and should be used in conjunction with other methods for the confirmation and identification of these bacteria. Some of the limitations are:

• Some strains of Salmonella may not produce hydrogen sulfide or may produce it weakly, resulting in colorless colonies without black centers. These strains may be overlooked or confused with Shigella on SS Agar.
• Some strains of Shigella may produce hydrogen sulfide or ferment lactose, resulting in black or red colonies on SS Agar. These strains may be mistaken for Salmonella or coliforms on the medium.
• Some non-pathogenic bacteria such as Proteus, Citrobacter, Edwardsiella, and Hafnia may also grow on SS Agar and produce similar colonies as Salmonella or Shigella. These bacteria may interfere with the isolation and identification of the target organisms on the medium.

Therefore, SS Agar should be used as a screening tool rather than a definitive diagnostic medium for Salmonella and Shigella. The colonies that are suspected to be Salmonella or Shigella should be further tested by biochemical, serological, or molecular methods to confirm their identity and pathogenicity. SS Agar is a valuable medium for the isolation and differentiation of Salmonella and Shigella from various specimens. It helps to identify these bacteria based on their ability to ferment lactose and produce hydrogen sulfide.

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