What is Salmonella Shigella Agar (SS Agar)?

• Salmonella Shigella Agar (SS Agar) is a specialized medium used for the isolation and identification of Salmonella and Shigella species, particularly from stool samples, food samples, and clinical materials. It is a moderately selective and differential medium that helps in distinguishing between different types of bacteria based on their ability to ferment lactose and produce hydrogen sulfide (H2S).
• The principle behind SS Agar lies in its composition, which includes various components that inhibit the growth of gram-positive bacteria while allowing the growth of target organisms. Bile salts, brilliant green, and sodium citrate present in the agar act as inhibitory agents against gram-positive bacteria. These components prevent the overgrowth of unwanted bacteria, thereby facilitating the isolation of Salmonella and Shigella species.
• The medium contains essential growth nutrients like proteose peptone and beef extract, which provide the necessary elements for bacterial growth. Lactose is the fermentable carbohydrate present in SS Agar. When lactose-fermenting bacteria metabolize lactose, they produce acid as a byproduct. This acid production is indicated by a change in color from yellow to red due to the presence of a pH indicator called neutral red. As a result, lactose-fermenting organisms appear as red-pigmented colonies on the agar.
• On the other hand, non-lactose-fermenting organisms do not produce acid during lactose fermentation. These organisms grow as translucent, colorless colonies on SS Agar, with or without black centers. The black centers indicate the production of H2S gas. Certain species of enteric organisms possess thiosulfate reductase, an enzyme that reduces sodium thiosulfate present in the agar to produce H2S gas. The H2S gas reacts with ferric ions or ferric citrate in the medium, resulting in the formation of an insoluble black precipitate of ferrous sulfide. This black precipitate is observed in the center of colonies of organisms that produce H2S gas, such as Salmonella species.
• The SS Agar medium’s high selectivity allows for the direct inoculation of large quantities of specimens suspected to contain pathogenic enteric bacilli, such as feces or rectal swabs. However, because SS Agar can be inhibitory to some strains of Shigella, it is recommended to inoculate plates of less inhibitory media, such as Hektoen Enteric Agar or Deoxycholate Citrate Agar, in parallel with SS Agar for the easier isolation of Shigella species.
• In summary, Salmonella Shigella Agar is a selective and differential medium that aids in the isolation and identification of Salmonella and Shigella species. It selectively inhibits gram-positive bacteria while allowing the growth of target organisms. By differentiating lactose-fermenting and non-lactose-fermenting bacteria and detecting the production of H2S gas, the agar helps in the identification and characterization of these pathogenic enteric bacilli.

Principle of Salmonella Shigella Agar (SS Agar)

The principle of Salmonella Shigella Agar (SS Agar) is based on its composition and the specific interactions between its components and the targeted organisms. The agar contains various ingredients that inhibit the growth of certain bacteria, promote the growth of Salmonella species, and facilitate the differentiation of different types of enteric organisms.

Bile salts, sodium citrate, and brilliant green are included in SS Agar to inhibit the growth of gram-positive bacteria, coliform organisms, and swarming Proteus species. This selective action allows for the preferential growth of Salmonella species. These inhibitory components help create an environment that is suitable for the isolation and detection of Salmonella and Shigella species.

Beef extract, enzymatic digest of casein, and enzymatic digest of animal tissue provide essential nutrients such as nitrogen, carbon, and vitamins required for the growth of the targeted organisms. These nutrients support the metabolic activity and reproduction of Salmonella and Shigella species.

Lactose, a fermentable carbohydrate, is incorporated into the medium to differentiate between lactose-fermenting and non-lactose-fermenting bacteria. When lactose-fermenting organisms metabolize lactose, they produce acid as a byproduct. The presence of neutral red, a pH indicator, causes the colonies of lactose-fermenting bacteria to appear red or pink. This color change indicates acid production, which is characteristic of certain enteric organisms.

Non-lactose-fermenting bacteria, on the other hand, do not produce acid during lactose fermentation. As a result, their colonies remain colorless on the agar.

The inclusion of sodium thiosulfate and ferric citrate in the medium allows for the detection of hydrogen sulfide (H2S) production by certain enteric organisms. Some species possess thiosulfate reductase, an enzyme that can reduce sodium thiosulfate to produce H2S gas. When H2S gas reacts with ferric ions or ferric citrate in the agar, it forms an insoluble black precipitate of ferrous sulfide. This results in the formation of colonies with black centers, indicating the production of H2S gas.

Additionally, the pH indicator neutral red, which turns red in the presence of an acidic pH, helps to confirm the fermentation of lactose by the organisms. The red color observed in the colonies of lactose-fermenting bacteria indicates that they have undergone fermentation.

In summary, the principle of Salmonella Shigella Agar relies on the selective and differential properties of its components. The medium selectively inhibits gram-positive and certain coliform bacteria while allowing the growth of Salmonella and Shigella species. The inclusion of lactose, pH indicators, and H2S detection components enables the differentiation of lactose-fermenting and non-lactose-fermenting organisms, as well as the identification of H2S-producing bacteria.

Salmonella Shigella Agar (SS Agar) Composition

Ingredients	Gms/litre
Lactose	10.0
Bile salts no.3	8.5
Sodium citrate	8.5
Sodium thiosulfate	8.5
Beef extract	5.0
Proteose peptone	5.0
Ferric Citrate	1.0
Brilliant green	0.00033
Neutral red	0.025
Agar	13.5

Final pH 7.0 +/- 0.2 at 25ºC.

Step by Step Preparation of Salmonella Shigella Agar (SS Agar)

To prepare Salmonella Shigella (SS) Agar, follow these steps:

1. Suspend 60 grams of the SS Agar medium in one liter of deionized or distilled water. Ensure that the water used is of high quality to avoid any contaminants that could interfere with the growth and differentiation of the target organisms.
2. Mix the suspension thoroughly to ensure even distribution of the agar powder in the water. Agitate the mixture to break up any clumps and achieve a homogenous solution.
3. Heat the mixture while continuously agitating it to facilitate the dissolution of the agar and other components. It is important to maintain a gentle heat and avoid boiling the mixture vigorously.
4. Allow the mixture to come to a gentle boil and continue boiling for one minute. This step ensures proper sterilization of the medium and helps in activating its components.
5. Do not autoclave the SS Agar medium. Autoclaving can lead to the breakdown of certain components and compromise the selective and differential properties of the agar.
6. After boiling, the medium is ready for pouring into plates. Use sterile techniques and appropriate precautions to avoid any contamination during this step.
7. Pour the SS Agar mixture into sterile petri dishes. Ensure that the plates are evenly filled, leaving enough space at the top for proper solidification of the agar.
8. Allow the agar to cool and solidify in the plates. This process typically takes some time, depending on the room temperature and the depth of the agar in the plates.
9. Once the agar has solidified, store the plates in a refrigerator. It is important to avoid freezing the plates, as it can lead to the alteration of the agar’s physical properties and the subsequent growth of ice crystals that may affect bacterial growth.
10. Prepared SS Agar plates can be kept in the refrigerator for at least a week. Proper labeling and storage practices should be followed to ensure the integrity and quality of the agar during storage.

By following these steps, you can prepare Salmonella Shigella (SS) Agar, which will provide a suitable medium for the isolation, cultivation, and differentiation of Salmonella and Shigella species from various samples.

Culturing the sample on Salmonella Shigella Agar (SS Agar)

Culturing the sample on Salmonella Shigella (SS) Agar involves a series of steps to ensure proper inoculation and incubation for the growth and differentiation of target organisms. Here’s the process:

1. Allow the plates to warm to room temperature: Before inoculating the agar plates, it is important to let them reach room temperature. This step helps prevent any condensation on the agar surface, which can interfere with the growth of bacteria. Additionally, allowing the agar surface to dry helps ensure an even distribution of the sample.
2. Heavily inoculate and streak the specimen: As soon as possible after collection, the specimen should be heavily inoculated onto the agar plate. If the specimen is on a swab, gently roll the swab over a small area of the agar surface. This process transfers the bacteria onto the agar for growth and subsequent analysis.
3. Streak for isolation: Using a sterile loop, streak the inoculated specimen onto the agar surface. This streaking technique helps to separate individual bacteria from the original sample and encourage their isolated growth. Streaking for isolation involves spreading the sample in a pattern that thins out the bacterial concentration to obtain single colonies.
4. Incubate plates aerobically: After streaking the specimen, the agar plates should be incubated aerobically at a temperature of 35-37°C (95-98.6°F). The optimal temperature facilitates the growth of the target organisms while inhibiting the growth of unwanted bacteria. Incubation time usually ranges from 18 to 24 hours, but it may vary depending on the specific bacterial species being targeted.
5. Examine colonial morphology: After the incubation period, the agar plates should be examined for colonial morphology. This examination involves observing the appearance, color, size, shape, and any other distinguishing characteristics of the colonies that have grown on the agar. These observations provide initial clues for identification and further analysis of the bacterial isolates.

By following these steps, culturing the sample on Salmonella Shigella (SS) Agar allows for the growth and isolation of the target organisms, which can be further analyzed for identification and characterization. It is essential to maintain sterile techniques throughout the process to prevent contamination and ensure accurate results.

Result Interpretation on Salmonella Shigella Agar (SS Agar)

The interpretation of results on Salmonella Shigella (SS) Agar is based on the colony characteristics and color changes observed on the medium. Here is the interpretation for different types of organisms:

1. Lactose fermenter: If an organism is capable of fermenting lactose, it will produce acid as a byproduct, leading to a decrease in pH. This acid production is indicated by a change in color of the medium from its original yellow color to red. Examples of lactose fermenters include Escherichia coli and Klebsiella pneumoniae, which will form red colonies on SS Agar.
2. Non-lactose fermenter: Salmonella, Shigella, and other non-lactose fermenting organisms will not produce acid during lactose fermentation. Therefore, their colonies will appear as transparent or translucent, colorless colonies on SS Agar. Salmonella species may exhibit colonies with or without black centers, depending on the specific species isolated.
3. Other organism characteristics:

• Enterobacter/Klebsiella: These organisms may show slight growth on SS Agar and form pink colonies.
• Proteus: Proteus species typically appear as colorless colonies on SS Agar, often with black centers. The black centers indicate the production of hydrogen sulfide (H2S) gas.
• Shigella: Shigella species appear as colorless colonies on SS Agar.
• Pseudomonas: Pseudomonas may show irregular and slight growth on SS Agar.
• Gram-positive bacteria: SS Agar is moderately selective against gram-positive bacteria, so they will not grow on this medium. Therefore, no growth is expected for gram-positive bacteria.

Interpreting the colony characteristics on Salmonella Shigella Agar allows for the differentiation of lactose fermenting and non-lactose fermenting bacteria, as well as the identification of specific pathogens such as Salmonella and Shigella species based on their characteristic colony appearance.

Organisms	Result
Shigella	Clear, colorless, transparent
Escherichia coli	Small, pink to red
Enterobacter, Klebsiella	Larger than E.coli, mucoid, pale, opaque cream to pink
Salmonella	Colorless, transparent, with a black center if H2S is produced
Proteus	Colorless, with black center

Quality Control

Quality control is an essential aspect of using Salmonella Shigella (SS) Agar to ensure the reliability and accuracy of the medium. Here is the information related to quality control for SS Agar:

1. Appearance: The SS Agar should appear as a light yellow to pink homogeneous free-flowing powder. Any deviation from this appearance may indicate a quality issue.
2. Gelling: The medium should gel firmly and be comparable to a 1.35% Agar gel. This gelling property ensures the solidification of the agar for bacterial growth.
3. Colour and Clarity of prepared medium: When prepared, the SS Agar should form a reddish-orange-colored, clear to slightly opalescent gel in Petri plates. The medium’s color and clarity contribute to the proper visualization and differentiation of bacterial colonies.
4. Reaction: A 6.0% w/v aqueous solution of SS Agar should exhibit a pH of 7.0 ± 0.2 at 25°C. This pH range is critical for the growth and differentiation of target organisms.
5. pH: The pH of the prepared SS Agar should fall within the range of 6.80 to 7.20. Maintaining the appropriate pH range is essential for optimal bacterial growth and colony characteristics.
6. Cultural Response: The cultural response of SS Agar can be evaluated by inoculating specific organisms and observing their growth and colony characteristics after incubation at 35-37°C for 18-24 hours. The expected growth and recovery, as well as the color and characteristics of the colonies, should align with the specified criteria for each tested organism.

Proper quality control measures should be followed to confirm that the SS Agar meets the specified standards. By ensuring the quality of the medium, reliable and consistent results can be obtained during bacterial culture and identification processes.

Uses of Salmonella Shigella Agar (SS Agar)

SS Agar has several important uses in microbiology, particularly in the isolation and differentiation of Salmonella and Shigella species. Here are the main applications of SS Agar:

1. Isolation of Salmonella and Shigella: SS Agar is primarily used as a selective and differential medium for the isolation of Salmonella and some Shigella species from various clinical and non-clinical specimens. The selective components in the agar, such as bile salts, sodium citrate, and brilliant green, inhibit the growth of gram-positive bacteria and certain coliform organisms, creating an environment that favors the growth of Salmonella and Shigella. The agar’s differential properties allow for the differentiation of lactose-fermenting and non-lactose-fermenting organisms, aiding in the identification of these target pathogens.
2. Differentiation of lactose and non-lactose fermenters: SS Agar was specifically designed to assist in the differentiation of lactose-fermenting and non-lactose-fermenting bacteria. Organisms that can ferment lactose produce acid as a byproduct, leading to a change in the pH of the medium and the appearance of red or pink colonies. On the other hand, non-lactose fermenters do not produce acid, resulting in the growth of colorless colonies. This differentiation is helpful in identifying different types of enteric bacteria in clinical specimens, suspected food samples, and other similar materials.
3. Secondary isolation of Shigella: Although SS Agar can support the growth of some Shigella species, it is not recommended for the primary isolation of Shigella. Instead, it is advised to use other agar media, such as Hektoen Enteric Agar or Deoxycholate Citrate Agar, in parallel with SS Agar for easier isolation of Shigella species. These additional media provide better selectivity for Shigella, enhancing their isolation and identification.

In summary, SS Agar is a valuable tool in the laboratory for the selective isolation and differentiation of Salmonella and Shigella species. It serves as a useful medium for the detection and characterization of these pathogens in clinical specimens, suspected food samples, and other materials. Additionally, it aids in the differentiation of lactose-fermenting and non-lactose-fermenting bacteria, providing valuable information for microbial identification.

Limitations of Salmonella Shigella Agar (SS Agar)

Salmonella Shigella (SS) Agar, despite its usefulness, has certain limitations that should be taken into consideration. Here are the main limitations associated with the use of SS Agar:

1. Inhibition of some Shigella strains: The presence of brilliant green in SS Agar, while contributing to the selectivity of the medium, has been found to inhibit the growth of certain Shigella strains. This limitation may result in the underrepresentation or failure to isolate certain Shigella species or strains.
2. Crystallization of bile salts: Over time, the bile salts in SS Agar may crystallize, leading to the formation of small spider-like puff balls within the medium. These crystals do not affect the performance of the medium but may cause visual interference or confusion during colony observation.
3. Slow lactose fermentation by some Shigella strains: Certain strains of Shigella, such as Shigella sonnei and S. dysenteriae serovar 1, may exhibit slow lactose fermentation. As a result, their colonies may initially appear as non-lactose fermenters but can change to lactose-fermenting after extended cultivation for two or more days. This delayed lactose fermentation can lead to ambiguous or misleading results if interpretation is done before sufficient incubation time.
4. Growth of non-pathogenic organisms: While SS Agar is designed to selectively promote the growth of target pathogens like Salmonella and Shigella, there is a possibility for non-pathogenic organisms to grow on the medium. This occurrence can complicate the interpretation and identification of true pathogens.
5. Further testing required for complete identification: SS Agar provides initial differentiation and identification of Salmonella and Shigella species based on their growth characteristics and lactose fermentation. However, for a comprehensive and accurate identification, it is recommended to perform additional tests such as biochemical, immunological, molecular, or mass spectrometry analysis on colonies obtained from pure cultures.

Considering these limitations, it is important to interpret SS Agar results cautiously, taking into account the possibility of inhibited growth, delayed lactose fermentation, and the need for further confirmatory testing to ensure accurate identification of the target organisms.

FAQ

References

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