What is Cystine Glucose Blood Agar?

• Cystine Glucose Blood Agar, also known as Cystine Heart Agar, is a specialized medium used for cultivating Francisella tularensis and other microorganisms. It was originally developed by Edward Francis and further modified by Rhamy for optimal growth of F. tularensis.
• Cystine Glucose Blood Agar is the historical medium of choice for isolating F. tularensis. It is an artificial medium supplemented with sulfhydryl compounds, specifically cystine, as F. tularensis is a fastidious organism that requires cysteine for its growth. The addition of cystine to the agar allows for the cultivation of F. tularensis.
• The medium is also enriched with hemoglobin, which further enhances the growth of F. tularensis. Hemoglobin provides additional nutrients and supports the optimal growth of the organism on the agar.
• The use of Cystine Glucose Blood Agar is not limited to F. tularensis. It can also support excellent growth of gram-negative cocci and other pathogenic organisms. Without the enrichment of hemoglobin, the medium can be used for the cultivation of various gram-negative cocci and other microorganisms.
• The name Cystine Glucose Blood Agar is sometimes used interchangeably with Cystine Heart Agar to refer to the same medium. It recognizes the importance of cystine and hemoglobin in the formulation of the agar and their roles in supporting the growth of F. tularensis and other organisms.
• Overall, Cystine Glucose Blood Agar or Cystine Heart Agar is a specialized medium that serves as a valuable tool in the isolation and cultivation of Francisella tularensis and supports the growth of other gram-negative cocci and pathogenic organisms.

Principle of Cystine Glucose Blood Agar

The principle of Cystine Glucose Blood Agar lies in its nutrient-rich composition and selective properties that facilitate the isolation and cultivation of Francisella tularensis and other challenging organisms.

The medium contains infusions derived from beef heart, which provide essential nutrients and growth factors necessary for bacterial growth. Proteose peptone, a protein digest, supplies nitrogen and vitamins that support microbial metabolism and proliferation.

L-Cystine, an amino acid, serves as a source of sulfur-containing compounds required for the growth of F. tularensis and other microorganisms. Dextrose acts as a carbon energy source, providing the necessary substrate for bacterial metabolism.

Sodium chloride is added to maintain the osmotic balance and provide essential ions for bacterial growth. Agar, a solidifying agent, helps to solidify the medium, allowing for the growth of microorganisms on its surface.

Enrichment with 2% hemoglobin further enhances the growth of F. tularensis by providing additional nutrients and growth factors. Hemoglobin, derived from blood, supports the optimal growth of the organism on the agar.

Without the enrichment of hemoglobin, Cystine Glucose Blood Agar still supports excellent growth of gram-negative cocci and other pathogenic microorganisms. This makes the medium versatile and suitable for cultivating a range of challenging organisms.

In summary, the principle of Cystine Glucose Blood Agar lies in its nutrient-rich composition, selective properties, and the enrichment of hemoglobin, which collectively support the growth of Francisella tularensis and other difficult-to-cultivate organisms.

Composition of Cystine Glucose Blood Agar

Ingredients	Gms/L
Infusion from Beef heart	500.0
Proteose peptone	10.0
Dextrose	10.0
Sodium chloride	5.0
L-Cystine	1.0
Agar	15.0

Final pH (at 25°C) 6.8±0.2

Preparation of Cystine Glucose Blood Agar

Here are the instructions for the preparation of Cystine Glucose Blood Agar:

1. Suspend 51 grams of the medium in 1000 ml of distilled water.
2. Heat the suspension to boiling, ensuring that the medium is completely dissolved.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. If enrichment with hemoglobin (2%) is desired, suspend 10.2 grams of the medium in 100 ml of distilled water.
5. Sterilize the hemoglobin suspension by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
6. Allow the medium to cool to 50°C.
7. Aseptically add 100 ml of 2% sterile hemoglobin solution to the cooled medium.
8. Mix the medium well to ensure thorough distribution of the hemoglobin.
9. Pour the prepared medium into sterile Petri plates.

Following these steps will result in the preparation of Cystine Glucose Blood Agar, ready to be used for the cultivation of various microorganisms, including Francisella tularensis and gram-negative cocci.

Results on Cystine Glucose Blood Agar

On Cystine Glucose Blood Agar, the interpretation of results for F. tularensis colonies can be as follows:

• At 24 hours of incubation, F. tularensis colonies may be very small and difficult to observe. They might appear as tiny pinpoint colonies or might not be visible to the naked eye.
• At 48 hours of incubation, characteristic F. tularensis colonies can be observed. They typically have a diameter of 1-2mm and appear white to grey to bluish-grey in color. The colonies are opaque, flat, and have an entire edge. They exhibit a smooth and shiny surface.

It’s important to note that the colony characteristics described above are specific to F. tularensis and can aid in the presumptive identification of this pathogen. However, further confirmatory tests, such as biochemical or molecular tests, are necessary to confirm the presence of F. tularensis in the colonies obtained on Cystine Glucose Blood Agar.

Uses of Cystine Glucose Blood Agar

Cystine Glucose Blood Agar, or Cystine Heart Agar, has specific uses in the cultivation of Francisella tularensis and other pathogenic microorganisms.

1. Cultivation of Francisella tularensis: Cystine Glucose Blood Agar is primarily used for the qualitative cultivation of Francisella tularensis. The medium provides the necessary nutrients and growth factors required for the growth of this fastidious pathogen. It allows for the isolation and identification of Francisella tularensis from clinical specimens.
2. Growth of gram-negative cocci and other microorganisms: In its non-enriched form, Heart Infusion Agar supplemented with 1% dextrose and 0.1% cystine can support the growth of gram-negative cocci and other pathogenic microorganisms. This makes it a useful medium for the cultivation and isolation of various gram-negative cocci, allowing for their identification and further characterization.

The versatility of Cystine Glucose Blood Agar in supporting the growth of Francisella tularensis and other pathogenic microorganisms makes it a valuable tool in the laboratory for the detection and study of these organisms. It provides a suitable environment for their growth and facilitates their identification through subsequent tests and analyses.

Limitations of Cystine Glucose Blood Aga

Cystine Glucose Blood Agar has certain limitations that need to be considered in its usage:

1. Identification limitations: While Cystine Glucose Blood Agar provides a suitable environment for the cultivation of Francisella tularensis and other microorganisms, it is important to note that further confirmatory tests are required for complete identification. Biochemical, immunological, molecular, or mass spectrometry testing should be performed on colonies from pure culture to accurately identify the microorganism present.
2. Biosafety considerations: F. tularensis is a highly virulent pathogen, and laboratory infections can occur through aerosols or droplets. Therefore, caution must be exercised when handling clinical specimens suspected to contain F. tularensis. It is essential to follow appropriate biosafety procedures, such as Biological Safety Level-2 (BSL-2) guidelines, for handling suspected specimens. Additionally, BSL-3 conditions are recommended for all culture manipulations as soon as F. tularensis is suspected.

These limitations highlight the importance of conducting further tests for accurate identification and the need for strict adherence to biosafety measures when working with F. tularensis or suspected samples. By acknowledging and addressing these limitations, the appropriate precautions can be taken to ensure safe and accurate laboratory practices.

FAQ

References

• Handbook of Media for Clinical and Public Health Microbiology ByRonald M. Atlas, James W. Snyder
• https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/211874.pdf
• https://www.jstor.org/stable/4585327
• https://doh.sd.gov/lab/resources/bt/Fancisella/colony.aspx
• https://exodocientifica.com.br/_technical-data/M172.pdf