Streptococcus Selective Agar – Composition, Preparation, and Principle

What is Streptococcus Selective Agar?

• Streptococcus Selective Agar is a specialized medium developed by Roantree et al for the isolation of group A beta-hemolytic streptococci, particularly Streptococcus pyogenes (S. pyogenes). This agar is recommended for primary isolation from oral culture and respiratory specimens. It has selective properties that inhibit the growth of gram-negative bacilli and staphylococci, allowing for the isolation, subculture, and identification of pathogenic streptococci, including beta-hemolytic streptococci and S. pneumoniae.
• The majority of infections caused by beta-hemolytic streptococci in humans are attributed to group A streptococci, specifically Streptococcus pyogenes. This pathogen is responsible for various inflammatory and suppurative conditions such as sore throat, scarlet fever, cellulitis, wound infections, erysipelas, impetigo, puerperal fever, otitis media, septicemia, and necrotizing fasciitis. It is commonly found in the throat or nasal cavity.
• The medium used for selective isolation of beta-hemolytic streptococci, including Streptococcus pyogenes, is known as ß-Streptococcus Selective Agar Base. This medium, described by Liebermeister and Braveny, restricts the growth of accompanying flora by providing limited nutrients. As a result, the growth of other bacteria is significantly reduced. Beta-hemolytic streptococci exhibit reduced colony size but distinct beta-hemolysis, which is enhanced on this medium.
• ß-Streptococcus Selective Agar contains yeast extract, lysine, meat extract, and meat peptone as sources of carbon, nitrogen, and essential growth factors. These components promote the hemolytic action of beta-hemolytic streptococci and result in higher yields compared to standard blood agar. Sodium chloride helps maintain the osmotic equilibrium of the medium, while disodium hydrogen phosphate acts as a buffer.
• Group A beta-hemolytic Streptococcus, also known as Streptococcus pyogenes, is a type of beta-hemolytic Streptococcus bacteria. It is a gram-positive bacterium responsible for a wide range of both invasive and non-invasive infections in humans.
• Group A Streptococci Selective Agar is specifically designed for the selective isolation of group A Streptococcus from throat cultures and skin specimens. The addition of blood to the medium provides additional growth nutrients and aids in the formation of well-defined hemolytic zones. The sodium chloride content of the agar prevents the lysis of erythrocytes.
• The medium contains a combination of Tryptone and Soya peptone, which provide nitrogenous compounds, carbon, sulfur, trace elements, and vitamin B complex essential for the growth of Streptococci. Sodium chloride helps maintain the osmotic balance. The selective supplement in the agar inhibits the growth of gram-negative bacteria and most gram-positive bacteria, further enhancing the selectivity of the medium for group A Streptococcus.

Composition of Streptococcus Selective Agar

Ingredients	Grams/L
Casein Peptone	15.0 g
Peptic Digest of Soybean Meal	5.0 g
Sodium Chloride	5.0 g
Nucleic Acid	3.0 g
Maltose	0.5 g
Neomycin	10.0 mg
Polymyxin B	10.0 mg
Sheep Blood	50.0 ml (5%)
Agar	15.0 g

Final pH 7.3 +/- 0.2 at 25ºC.

Principle of Streptococcus Selective Agar

The principle of Streptococcus Selective Agar lies in its composition and the inclusion of specific ingredients that promote the growth and identification of Streptococcus species, particularly group A beta-hemolytic streptococci (Streptococcus pyogenes), while inhibiting the growth of other bacteria.

The medium contains casein and soy peptones, which serve as a rich source of organic nitrogen, including amino acids and long-chained peptides necessary for bacterial growth. This combination makes the medium highly nutritious and supports the growth of streptococci.

Sodium chloride is included in the agar to maintain the osmotic equilibrium of the medium. This helps create an optimal environment for streptococcal growth.

Ribonucleic acid and maltose are added to enhance the production of hemolysis. Streptococci are known for their ability to cause hemolysis of red blood cells, resulting in distinct zones around the bacterial colonies. The presence of ribonucleic acid and maltose in the medium promotes increased colony size and enhances the clarity and sharpness of the hemolytic zones produced by streptococci.

Supplementation with sheep blood at a concentration of 5% is done to provide additional growth factors for fastidious microorganisms and aid in determining hemolytic reactions. The blood contains nutrients that support the growth of streptococci and helps differentiate between different types of hemolysis (e.g., alpha, beta, gamma).

To selectively inhibit the growth of commensal oral microflora, including coliforms, staphylococci, Micrococcus, Haemophilus, and Neisseria species, selective agents such as Neomycin and Polymyxin B are added to the agar. These agents have antimicrobial properties that specifically target and suppress the growth of these bacteria, allowing for the isolation and identification of streptococci.

In summary, the principle of Streptococcus Selective Agar involves providing a nutritious medium with organic nitrogen sources, maintaining osmotic equilibrium, promoting hemolysis, supplementing with sheep blood for growth factors, and incorporating selective agents to inhibit the growth of unwanted bacteria, thereby facilitating the isolation and identification of Streptococcus species, particularly group A beta-hemolytic streptococci.

Purpose

Selective Streptococcus Agar was designed to aid in the isolating of streptococci from group A of respiratory origin.

Physical Properties of Streptococcus Selective Agar Media

• Appearance: Cream to yellow homogeneous free flowing powder
• Gelling: Firm, comparable with 1.5% Agar gel
• Colour and Clarity of prepared medium:  Light to medium amber-coloured clear, to slightly translucent gel forms within Petri plates
• Reaction: Reaction of 4.56% w/v aqueous solution at 25°C. pH : 7.4±0.2
• pH: 7.20-7.60

Preparation of Streptococcus Selective Agar

The preparation of Streptococcus Selective Agar involves the following steps:

1. Add all the components of the agar, except for sheep blood, maltose solution, and antibiotics, to distilled or deionized water. Make sure the volume reaches 930.0 mL.
2. Mix the components thoroughly to ensure uniform distribution.
3. Gently heat the mixture and bring it to a boiling point.
4. Autoclave the medium at 121°C and 15 psi pressure for 15 minutes. Autoclaving helps sterilize the agar and eliminate any potential contaminants.
5. Allow the agar to cool down to a temperature of 45°–50°C. It is important to cool it down sufficiently to avoid denaturation of the added blood and antibiotics.
6. Aseptically add sterile sheep blood, sterile maltose solution, and sterile antibiotic inhibitors to the cooled agar. This step ensures the addition of necessary growth factors, enhancers of hemolysis, and selective agents.
7. Mix the agar thoroughly to ensure even distribution of the added components.
8. Finally, pour the prepared agar into sterile Petri dishes or distribute it into sterile tubes, depending on the desired format for further use. The agar-filled Petri dishes or tubes should be sealed or covered to prevent contamination.

Following these steps ensures the proper preparation of Streptococcus Selective Agar, creating a suitable medium for the isolation and identification of Streptococcus species, particularly group A beta-hemolytic streptococci, while inhibiting the growth of other unwanted bacteria.

Result and Interpretation of Streptococcus Selective Agar

The result and interpretation of Streptococcus Selective Agar can be described as follows:

After an incubation period of 18 to 48 hours in a carbon dioxide-enriched atmosphere, the plates will exhibit specific characteristics indicative of different bacterial species.

1. Isolated colonies in streaked areas and confluent growth in heavy inoculation areas: The agar will show isolated colonies in areas where the sample was streaked, indicating the presence of individual bacterial colonies. In heavily inoculated areas, the growth will be confluent, with colonies merging together due to the high concentration of bacteria.
2. Group A Streptococcus (S. pyogenes): Group A streptococci will appear as small (1 to 2 mm), translucent, or opaque colonies. These colonies will be white to gray in color and surrounded by a zone of beta hemolysis. Beta hemolysis refers to the complete lysis of red blood cells around the colony, resulting in a clear zone around it.
3. Pinpoint or very small colonies of other streptococci: Other streptococci, such as alpha-hemolytic, nonhemolytic, or other beta-hemolytic streptococci, may grow as pinpoint or very small colonies in small numbers. These colonies may exhibit different hemolysis patterns or no hemolysis at all.
4. Inhibition of certain bacteria: Streptococcus Selective Agar is designed to inhibit the growth of specific bacteria. Neisseria species, viridans streptococci, staphylococci, gram-negative rods, and most beta-hemolytic streptococci other than groups A and B will be inhibited on the medium. This selective property allows for the isolation and identification of group A streptococci while suppressing the growth of unwanted bacteria.

Interpreting the results on Streptococcus Selective Agar involves identifying the characteristic colonies of group A streptococci, which display small, translucent to opaque, white to gray colonies surrounded by a zone of beta hemolysis. Other bacterial colonies may also be observed but in smaller numbers or with different hemolysis patterns. The inhibition of certain bacteria provides further confirmation of the selective nature of the medium.

Organism	Growth	Recovery	Hemolysis
Streptococcus agalactiae	50-100	Good	>= 50%
Staphylococcus aureus	>= 10^4	Inhibited	–
Streptococcus pneumoniae	>= 10^4	Inhibited	–
Streptococcus pyogenes	50-100	Good-luxuriant	>= 70% beta
Escherichia coli	>= 10^4	Inhibited	–
Bacillus cereus	Fair-good	Positive	–
Pseudomonas aeruginosa	Fair-good	Positive	–
Enterococcus hirae	Fair-good	Negative	–
Streptococcus agalactiae	Fair-good	Negative	–
Staphylococcus aureus	Fair-good	Negative	–
Streptococcus pyogenes	Fair-good	Positive	–
Enterococcus faecalis	Fair-good	Negative	–

Uses of Streptococcus Selective Agar

Streptococcus Selective Agar has several important uses in the field of microbiology. Some of these uses include:

1. Primary isolation of group A streptococci from respiratory sources: The agar is specifically designed for the primary isolation of group A streptococci, particularly Streptococcus pyogenes, from respiratory specimens. It provides a selective environment that inhibits the growth of unwanted bacteria, allowing for the isolation and identification of group A streptococci.
2. Isolation of various Streptococcus species: Streptococcus Selective Agar can also be used for the isolation of other Streptococcus species besides group A streptococci. This includes streptococcal groups B (S. agalactiae), C, D, F, G, and S. pneumoniae. The medium’s selective properties, combined with appropriate incubation conditions, enable the growth and identification of a wide range of Streptococcus species, particularly those found in respiratory specimens.
3. Identification of streptococci from respiratory specimens: The agar is particularly useful for the identification of streptococci present in respiratory specimens. These specimens may include throat cultures, sputum samples, or other respiratory tract samples. Streptococcus Selective Agar provides a suitable medium to support the growth of streptococci while inhibiting the growth of unwanted bacteria commonly found in respiratory sources.

By facilitating the selective isolation and identification of streptococci, including group A streptococci, Streptococcus Selective Agar plays a crucial role in diagnosing and studying various streptococcal infections. It helps microbiologists and healthcare professionals in accurately identifying the causative agents of respiratory tract infections and other diseases caused by streptococci, thereby guiding appropriate treatment decisions and infection control measures.

Limitations of Streptococcus Selective Agar

Streptococcus Selective Agar, like any diagnostic medium, has its limitations. Here are some important limitations to consider:

• Poor growth of certain strains: Some strains of group A streptococci (S. pyogenes) may exhibit poor growth on Streptococcus Selective Agar. Factors such as the nature of the specimens and the physiological state of the organisms can influence the recovery of the desired species and affect the inhibitory characteristics of the medium. To overcome this limitation, it is recommended to perform nonselective control examinations and compare them to the selective medium to gather additional information and ensure optimal recovery of potential pathogens.
• Additional confirmatory tests required: While Streptococcus Selective Agar provides a selective environment for the growth of streptococci, additional biochemical tests and serological procedures are often necessary for final confirmation of the bacterial species. These supplementary tests help validate the identity of the isolates and ensure accurate diagnosis.
• Variable inhibition of susceptible organisms: Organisms that are generally susceptible to the antimicrobial agent present in a selective medium may be completely or only partially inhibited. The inhibitory effect can vary based on the concentration of the agent, characteristics of the microbial strain, and the inoculum size. Conversely, organisms that are generally resistant to the antimicrobial agent will not be inhibited. It is essential to compare cultures grown on selective media with those grown on nonselective media to obtain additional information and aid in the recovery of potential pathogens.
• Hemolytic reactions of group D streptococci: Some strains of group D streptococci may exhibit altered hemolytic reactions depending on the type of animal blood used. They may exhibit beta-hemolysis on horse, human, and rabbit blood agar, but display alpha-hemolysis on sheep blood agar. This variation in hemolysis can affect the interpretation of hemolytic patterns on Streptococcus Selective Agar.
• Influence of incubation atmosphere: The atmosphere of incubation can influence hemolytic reactions of beta-hemolytic streptococci. To achieve optimal performance, it is recommended to incubate blood agar base media under increased levels of carbon dioxide (5-10%) as per established laboratory procedures. This controlled incubation environment helps maintain the appropriate conditions for accurate interpretation of hemolytic reactions.

Quality Control

Quality control measures for Streptococcus Selective Agar involve several steps to ensure the reliability and performance of the medium. Here are the key aspects of quality control:

1. Examination for product deterioration: Before using the agar, examine the plates according to the provided instructions for any signs of product deterioration. Look for physical imperfections, such as cracks, uneven surfaces, or contamination, that may affect the usability of the plates.
2. Visual inspection: Perform a visual inspection of the plates to ensure their overall quality. Check for any abnormalities or visual defects that could impact the results or the growth of bacteria.
3. pH measurement: Potentiometrically measure the pH of the agar at room temperature. The pH should be within the standard range of 7.3 ± 0.2. This ensures that the pH is suitable for the growth of streptococci and the performance of the selective properties of the medium.
4. Plate firmness: Assess the firmness of the plates during the inoculation process. They should maintain their structural integrity and not be excessively soft or brittle. Firm plates provide a suitable surface for bacterial growth and facilitate accurate interpretation of results.
5. Incubation of representative plates: Set aside a representative sample of plates that have not been inoculated. Incubate these plates at the appropriate temperature, typically 35 ± 2°C, for a specified duration, such as 72 hours. After incubation, check for any contamination by microbial species. The absence of microbial growth on these plates ensures that they are free from contaminants that could interfere with the identification of streptococci.

These quality control measures help ensure the reliability, consistency, and performance of Streptococcus Selective Agar. By verifying the physical condition, pH, firmness, and absence of contamination, laboratories can have confidence in the medium’s ability to support the selective growth and identification of streptococci.

Storage Instructions

When you receive the plates, keep them in the dark for 2-8 degrees Celsius. Avoid overheating and freezing. Don’t open the appliance until it is you are ready to use. Reduce exposure to sunlight. The plates that are kept in the original sleeve wrapper at temperatures between 2 and 8 degC for a few hours the time of use could be inoculated until the expiration date, and incubated according to the recommended time of incubation. Let the medium cool to room temperature prior to inoculation.

FAQ

References

• https://exodocientifica.com.br/_technical-data/M1608.pdf
• https://bioactiva.com/pub/media/sebwite/productdownloads//s/e/selective-strep-agar-manual.pdf
• https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU1857.pdf
• https://www.bd.com/resource.aspx?IDX=8988
• https://www.growinglabs.com/products/selective-strep-agar-coba-medium-for-the-selective-isolation-of-all-species-of-strep-15x100mm-plate-hardy-diagnostics
• https://alphabiosciences.com/selective-streptococcus-agar-s19-106/