What is Sorbitol MacConkey Agar?

• Sorbitol MacConkey Agar, also known as MacConkey Sorbitol Agar, is a specialized type of agar used in microbiology for the selective and differential detection of a specific strain of Escherichia coli called serotype O157:H7. This strain is associated with a severe form of foodborne illness known as hemorrhagic colitis, which is caused by the production of a Shiga-like toxin.
• The traditional MacConkey Agar is a commonly used medium for the cultivation of Enterobacteriaceae, including E. coli. It contains lactose as a fermentable carbohydrate, allowing lactose-fermenting bacteria to produce acid, which leads to the development of pink or red colonies on the agar plate. However, E. coli O157:H7, unlike most E. coli strains, exhibits a unique characteristic – it ferments sorbitol slowly or not at all.
• Sorbitol MacConkey Agar is formulated to address this distinguishing feature of E. coli O157:H7. It incorporates sorbitol as the fermentable carbohydrate instead of lactose. This change in the composition of the agar allows for the specific identification of E. coli O157:H7 in stool cultures. When E. coli O157:H7 is grown on Sorbitol MacConkey Agar, it appears as colorless or pale colonies since it does not ferment sorbitol efficiently.
• In addition to the sorbitol component, Sorbitol MacConkey Agar also contains cefixime and tellurite. These antimicrobial agents are added to the medium to inhibit the growth of Proteus mirabilis, non-O157 E. coli strains, and other sorbitol-nonfermenting strains that may be present in the sample. By selectively inhibiting these bacteria, the agar facilitates the isolation and identification of E. coli O157:H7.
• The selective and differential properties of Sorbitol MacConkey Agar make it a valuable tool in clinical and food microbiology laboratories for the detection and isolation of E. coli O157:H7. Its use helps in the early identification of this pathogenic strain, enabling timely intervention and prevention of the spread of the associated illnesses.

Composition of Sorbitol MacConkey Agar

Ingredients	Gm/L
Peptone	17.0g
Proteose peptone	3.0g
D-Sorbitol	10.0g
Sodium Chloride	5.0g
Bile Salts Mixture	1.5g
Neutral Red	0.03g
Crystal Violet	0.001g
Agar	13.5g

Final pH 7.1 +/- 0.2 at 25ºC

Preparation Procedure of Sorbitol MacConkey Agar

he preparation of Sorbitol MacConkey Agar involves several steps to ensure its proper formulation and sterility. Here is a step-by-step guide on how to prepare this medium:

1. Start by measuring out 50.03 g of Sorbitol MacConkey Agar powder. This quantity is suitable for preparing 1000 ml of the medium.
2. Suspend the measured amount of powder in 1000 ml of distilled water. It is important to use distilled water to avoid any contamination that may interfere with the growth of desired bacteria.
3. Mix the suspension thoroughly to ensure even distribution of the agar powder in the water.
4. Heat the suspension while agitating frequently to aid in dissolving the agar powder completely. It is essential to dissolve the powder thoroughly to avoid any clumps or uneven agar distribution in the final medium.
5. Boil the suspension for 1 minute to ensure complete dissolution of the agar powder and to sterilize the medium.
6. After boiling, the next step is autoclaving the medium. Transfer the suspension to appropriate containers, such as glass bottles or flasks, that can withstand autoclave conditions.
7. Autoclave the containers at 121°C for 15 minutes. Autoclaving is a crucial step as it ensures the sterility of the medium by eliminating any potential contaminants.
8. After autoclaving, allow the containers to cool down to approximately 45-50°C. It is important to cool the medium to this temperature range to prevent any heat-related damage to the Petri plates and to avoid killing the desired bacteria during the pouring process.
9. Once the medium has cooled, it is ready for pouring. Sterile Petri plates should be prepared beforehand and placed in a sterile environment, such as a laminar flow hood or a clean bench.
10. Pour the Sorbitol MacConkey Agar into the sterile Petri plates, ensuring that the plates are evenly filled. It is important to work quickly and smoothly to prevent any contamination during the pouring process.
11. Allow the poured plates to solidify at room temperature. Once solidified, the plates can be stored in a refrigerator at 2-8°C until they are ready to be used.

Sorbitol MacConkey Agar is now ready to be used for the selective and differential isolation of E. coli O157:H7. The agar’s composition inhibits the growth of non-pathogenic E. coli strains while allowing for the detection and differentiation of EHEC strains based on their ability to ferment sorbitol.

Principle of Sorbitol MacConkey Agar

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The principle behind Sorbitol MacConkey Agar (SMAC) lies in its ability to selectively inhibit the growth of certain microorganisms while allowing for the differentiation of specific bacterial strains based on their ability to ferment sorbitol.

The medium contains various components that contribute to its selective and differential properties. Although it is only slightly selective compared to other enteric plating media, the concentration of bile salts present in SMAC is sufficient to inhibit the growth of most gram-positive microorganisms.

Peptone and proteose peptone in the medium provide essential nutrients like nitrogenous and carbonaceous compounds, long-chain amino acids, minerals, vitamins, and trace ingredients necessary for bacterial growth. These components support the growth of both gram-negative and gram-positive bacteria.

Crystal violet, another component of SMAC, plays a role in inhibiting the growth of gram-positive bacteria, particularly enterococci and staphylococci. By suppressing the growth of these organisms, the medium ensures a more favorable environment for the growth of gram-negative bacteria.

Sodium chloride is included in the medium to maintain osmotic equilibrium, which is crucial for the optimal growth of bacteria.

The neutral red indicator present in the medium acts as a differentiating agent. It is a pH indicator that changes color depending on the fermentation of sorbitol.

D-Sorbitol, a fermentable carbohydrate, is the key component of SMAC. Most fecal flora can ferment sorbitol, resulting in the production of acid byproducts. However, E. coli O157:H7, a specific strain of E. coli, is unable to ferment sorbitol due to the absence of the necessary enzymes. As a result, when this strain is grown on SMAC, it forms colorless colonies.

The combination of sorbitol and the neutral red indicator allows for the differentiation of enteric microorganisms. Most fecal flora that can ferment sorbitol will appear as pink colonies due to the acid production and subsequent pH change. In contrast, E. coli O157:H7, which does not ferment sorbitol, will appear as colorless colonies.

In summary, the principle of Sorbitol MacConkey Agar involves the selective inhibition of gram-positive bacteria through the presence of bile salts and crystal violet. The differential aspect relies on the ability of the neutral red indicator to distinguish between sorbitol-fermenting bacteria (pink colonies) and E. coli O157:H7, which does not ferment sorbitol (colorless colonies). This allows for the detection and differentiation of specific bacterial strains, particularly E. coli O157:H7, in clinical and research settings.

Intended Use

The recommended method for identifying and isolating of strains of Enteropathogenic Escherichia Coli that are associated with diarrhea in infants.

Type of specimen Used

This medium is employed for the isolating of Escherichia coli O157 (and other serotypes that are sorbitol-negative) from stool samples of patients who are suspected to have been infected by this pathogen, as well as for food, veterinary and environmental samples.

Technique for working with Sorbitol MacConkey Agar (SMAC)

The technique for working with Sorbitol MacConkey Agar (SMAC) involves the following steps:

1. Prepare the agar: Follow the provided directions to prepare the SMAC agar. This typically involves suspending the agar powder in water, heating to dissolve it, and autoclaving for sterilization. After autoclaving, the agar can be poured into sterile plates.
2. Drying the surface (if required): Depending on the specific requirements of the experiment or analysis, it may be necessary to dry out the surface of the agar in the plates before inoculation. This can be done by allowing the plates to dry in a sterile environment, such as a laminar flow hood or a clean bench.
3. Inoculation: Once the plates are ready, inoculate them with the test substances, such as food samples or fecal specimens, to introduce the bacteria for analysis. This can be done by streaking or spot inoculation methods. It is important to create isolated colonies by ensuring proper separation of the inoculated areas.
4. Incubation: Place the inoculated plates in an incubator set at the appropriate temperature. The recommended temperature for the growth of Escherichia coli O157 is typically around 35°C. Incubate the plates for 24 hours to allow bacterial growth and colony formation. It is important to follow the specified incubation time, as delaying the reading of plates beyond 24 hours may result in decreased intensity of sorbitol fermenting colonies.
5. Colony interpretation: After the incubation period, examine the colonies on the SMAC plates. Sorbitol fermenting colonies will appear as pink to red, while sorbitol non-fermenting colonies will be colorless. Other Gram-negative organisms, such as Pseudomonas, Proteus, and Klebsiella species, may also grow on SMAC but can be differentiated based on their colony appearance.
6. Confirmation tests: To ensure accurate identification and confirmation of suspect colonies, it is recommended to perform additional tests. One such test is the Escherichia coli O157 test (DR0620), which is a diagnostic reagent readily available for immediate confirmation tests on suspect colonies. This test can provide further confirmation of the presence of Escherichia coli O157.

By following these techniques, researchers and laboratory personnel can effectively work with Sorbitol MacConkey Agar for the detection and differentiation of bacterial strains, particularly Escherichia coli O157:H7.

Result and Interpretation of Sorbitol MacConkey Agar

The interpretation of results on Sorbitol MacConkey Agar (SMAC) is based on the ability of bacteria to ferment sorbitol. After 18-24 hours of incubation, the following observations can be made:

1. Sorbitol fermenters: Bacterial strains that can ferment sorbitol will appear as pink to red colonies on SMAC. These colonies may vary in intensity of color. Some sorbitol fermenters may exhibit zones of precipitated bile around the colonies, creating a halo-like appearance. This precipitation occurs due to the acid production resulting from sorbitol fermentation.
2. Sorbitol non-fermenters: Bacterial strains that are unable to ferment sorbitol, such as E. coli O157:H7, will appear as colorless colonies on SMAC. These colonies do not produce acid as a byproduct of sorbitol fermentation and, therefore, do not exhibit any color change.

It is important to note the colony morphology and growth pattern on the SMAC plates. Isolated colonies may be observed in areas where the inoculum was streaked thinly, while areas of heavy inoculation may show confluent growth.

The presence of pink to red colonies surrounded by zones of precipitated bile is indicative of sorbitol-fermenting bacteria, which includes most fecal flora. These organisms are typically considered normal intestinal microbiota.

On the other hand, the presence of colorless colonies indicates the potential presence of sorbitol non-fermenting bacteria, particularly E. coli O157:H7. This strain is an important pathogen associated with foodborne illnesses and has been implicated in outbreaks of severe gastrointestinal infections.

Therefore, the interpretation of SMAC results involves identifying and distinguishing between sorbitol-fermenting (pink to red colonies) and sorbitol non-fermenting (colorless colonies) bacteria. Further confirmatory tests, such as biochemical or serological assays, may be required to definitively identify specific bacterial strains, particularly if E. coli O157:H7 is suspected.

Storage and Shelf Life of Sorbitol MacConkey Agar

Storage temperature is between 10 and 30 degrees Celsius in a tightly sealed container. The prepared medium at 20-30 degrees Celsius. Utilize before the expiry date is listed in the bottle. After opening, the product must be stored dry after sealing the bottle tightly to avoid the formation of lumps as a result of the hygroscopic character of the product. Incorrect storage of the product can result in the formation of lumps. Store in a dry and ventilated location shielded from extreme temperatures and ignition sources. The container should be sealed tightly following use. Make sure to use it before the expiry date is in the container’s label.

Quality Control of Sorbitol MacConkey Agar

• Appearance Color: Light pink to yellow homogeneous free-flowing powder
• Gelling: Firm, comparable with 1.35% Agar gel.
• Colour and Clarity of prepared medium: A purplish red colour clear to lightly translucent gel forms inside Petri plates
• Reaction: Reaction of 5.0% w/v aqueous solution at 25°C. pH : 7.1±0.2
• pH: 6.90-7.30
• Cultural Response: Culture traits that are observed following an incubation period of 35-37°C for 18-24 hrs.

Unsupplemented

Positive control:	Expected results
Escherichia coli O157:H7 Non-toxigenic NCTC12900 *	Good growth; 1-2mm straw colonies
Negative control:
Escherichia coli ATCC® 25922 *	Good growth; 1-2mm pink colonies

Supplemented with CT Supplement SR0172 and tested in accordance with ISO 11133;2014¹².

Positive control:	Expected results
Escherichia coli O157:H7 Non-toxigenic NCTC12900 * WDCM00014	Good growth; 1-2mm straw colonies
Negative controls:
Escherichia coli ATCC® 25922 * WDCM00013	No growth/pinpoint-0.25mm pink colonies
Escherichia coli ATCC® 8739* WDCM00012	No growth/pinpoint-0.25mm pink colonies
Staphylococcus aureus ATCC®25923* WDCM00034	No growth
Staphylococcus aureus ATCC®6538* WDCM00034	No growth

Precautions

When working with Sorbitol MacConkey Agar (SMAC), it is important to take certain precautions to ensure accurate and reliable results. Here are some precautions to consider:

1. Limitations of SMAC: Recognize that while SMAC is commonly used for the detection and differentiation of Escherichia coli O157 strains, it may not be sufficient as the sole method to identify all varieties of Verotoxigenic Escherichia coli (VTEC). Some non-toxic strains of Escherichia coli may not ferment sorbitol, leading to potential false-negative results. Therefore, it is essential to use additional confirmatory tests and procedures, such as serological assays and biochemical tests, for accurate identification.
2. Expiry date and product integrity: Ensure that the SMAC medium and any other related diagnostic products, such as Cefixime Tellurite Sorbitol MacConkey Agar, are used before their expiry date. Do not use the medium or product if it shows any signs of degradation, contamination, or damage. This precaution is crucial to maintain the quality and reliability of the medium.
3. Sterilization: Prioritize proper sterilization of all equipment, specimens, and media used in the process. Sterilization can be achieved through appropriate techniques, such as autoclaving, to ensure the elimination of any potential contaminants that may interfere with the accuracy of the results. Timely sterilization after use is important to prevent cross-contamination and maintain a sterile working environment.
4. Proper training and qualified microbiologists: The use of SMAC and related diagnostic media should be performed by qualified microbiologists who have received proper training in handling and interpreting the results. This ensures that the procedures are conducted correctly and that the results are accurately interpreted, minimizing the risk of errors and misdiagnosis.

By adhering to these precautions, laboratories and microbiologists can enhance the reliability and accuracy of their results when working with Sorbitol MacConkey Agar for the detection and differentiation of Escherichia coli strains, particularly Escherichia coli O157.

Uses of Sorbitol MacConkey Agar

Sorbitol MacConkey Agar (SMAC) has specific uses in microbiology, primarily for the isolation, detection, and differentiation of enteropathogenic Escherichia coli O157:H7, which is the primary serovar associated with hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Here are the key uses of SMAC:

1. Isolation of E. coli O157:H7: SMAC is a selective medium that inhibits the growth of most non-pathogenic coliform bacteria while allowing the growth of E. coli strains, including E. coli O157:H7. By providing a specific environment that favors the growth of this pathogen, SMAC facilitates its isolation from various samples, such as food, water, clinical specimens, or environmental sources.
2. Detection of E. coli O157:H7: SMAC contains sorbitol as the sole fermentable carbohydrate. E. coli O157:H7 is unable to ferment sorbitol due to the absence of the necessary enzymes, while most other E. coli strains can ferment sorbitol. This property allows for the differentiation and detection of E. coli O157:H7 based on its inability to ferment sorbitol, resulting in the formation of colorless colonies on SMAC.
3. Differentiation from other E. coli strains: SMAC aids in the differentiation of E. coli O157:H7 from other E. coli strains, particularly lactose fermenters. Most fecal flora and non-pathogenic E. coli strains can ferment sorbitol, leading to the production of acid and the appearance of pink to red colonies on SMAC. In contrast, E. coli O157:H7, being a sorbitol non-fermenter, forms colorless colonies on SMAC. This differentiation is crucial for identifying and distinguishing E. coli O157:H7, which is associated with severe clinical outcomes, from other strains.
4. Epidemiological surveillance: SMAC is utilized in surveillance and outbreak investigations to identify and track the prevalence of E. coli O157:H7 infections. It allows for the selective growth and detection of this pathogen, helping in the monitoring of its presence in various settings, such as food production facilities, hospitals, and community environments.

By employing Sorbitol MacConkey Agar, laboratories and researchers can specifically isolate, detect, and differentiate E. coli O157:H7 from other strains, aiding in the diagnosis, surveillance, and control of infections caused by this pathogen.

Limitations of Sorbitol MacConkey Agar

Sorbitol MacConkey Agar (SMAC) has certain limitations that should be considered when using this medium for bacterial analysis. These limitations include:

1. Inhibition in CO2-enriched atmosphere: It has been observed that some Enterobacteriaceae and Pseudomonas aeruginosa may be inhibited when incubated on MacConkey Agar in a CO2-enriched atmosphere. Therefore, the growth and detection of these organisms may be compromised under specific incubation conditions.
2. Loss of characteristic appearance with prolonged incubation: Prolonged incubation of E. coli serotype O157:H7 on SMAC can result in a loss of their characteristic colorless appearance. Additionally, the color of sorbitol-positive colonies may fade, making it challenging to distinguish them from sorbitol-negative colonies. This can lead to difficulties in interpretation and accurate identification of E. coli O157:H7.
3. Fading of pink-colored sorbitol-positive colonies: Prolonged incubation on SMAC can also result in the fading of pink-colored sorbitol-positive colonies. This fading can make the interpretation of results more difficult, potentially leading to misidentification or uncertainty in differentiating between sorbitol-positive and sorbitol-negative colonies.
4. Non-fermenting strains and other bacteria: Some strains of E. coli O157:H7 may acquire the ability to ferment sorbitol upon prolonged incubation, leading to the formation of pink-colored colonies. Additionally, there are other facultatively anaerobic gram-negative rods that do not ferment sorbitol, and they can appear similar to O157:H7 colonies. Confirmatory tests, such as biochemical tests and serological procedures, may be necessary to accurately identify and differentiate these strains.
5. Sorbitol-negative strains of other serotypes: There are sorbitol-negative strains of E. coli serotypes other than O157:H7, which may or may not produce toxins or cause clinical symptoms. MacConkey Agar with Sorbitol alone cannot differentiate between toxin-producing and non-producing strains of E. coli O157. Additional tests, such as serological assays, may be required to confirm the presence of specific toxins or pathogenicity.
6. Limitations in detecting E. coli O157:H7: It is important to note that SMAC should not be solely relied upon to detect pathogenic E. coli O157:H7 strains, as some non-toxic strains may also not ferment sorbitol. Gram staining, biochemical tests, and serological procedures should be performed to confirm the findings and accurately identify pathogenic strains.

Considering these limitations, it is essential to use SMAC in conjunction with other tests and procedures for accurate identification and confirmation of bacterial strains, particularly when detecting and characterizing pathogenic E. coli O157:H7.

FAQ

References

• https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/576/280/109207-smac-agar.pdf
• https://www.dalynn.com/dyn/ck_assets/files/tech/PM19.pdf
• http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0813&c=UK&lang=EN
• https://www.bd.com/resource.aspx?IDX=25485
• https://exodocientifica.com.br/_technical-data/M298.pdf
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