Cystine Glucose Blood Agar- Composition, Principle, Preparation, Results, Uses

Cystine Glucose Blood Agar (CGBA) is a selective and differential medium that is used for the isolation and identification of Francisella tularensis, the causative agent of tularemia. Tularemia is a zoonotic disease that can affect humans and animals, and is transmitted by various vectors such as ticks, flies, rodents, and rabbits. The symptoms of tularemia vary depending on the route of infection, but they may include fever, headache, lymphadenopathy, ulceration, pneumonia, and septicemia. F. tularensis is a gram-negative coccobacillus that requires special media and conditions for growth, as it is fastidious and highly virulent. CGBA is one of the recommended media for the cultivation of F. tularensis from clinical specimens such as blood, sputum, lymph node aspirates, and ulcer swabs.

Cystine Glucose Blood Agar is a complex and enriched medium that contains the following ingredients:

• Beef heart infusion: This provides a rich source of organic nitrogen, carbon, vitamins, and minerals for the growth of fastidious bacteria.
• Proteose peptone: This is a mixture of peptides and amino acids that supplies additional nitrogen and vitamins for bacterial metabolism.
• L-Cystine: This is an essential amino acid that is required by some bacteria, such as Francisella tularensis, for their growth and survival.
• Dextrose: This is a simple sugar that serves as a fermentable carbohydrate and an energy source for bacteria.
• Sodium chloride: This maintains the osmotic balance and the isotonicity of the medium.
• Agar: This is a polysaccharide extracted from seaweed that acts as a solidifying agent and provides a firm surface for bacterial growth.
• Hemoglobin: This is an optional enrichment that can be added to the medium to provide additional nutrients and growth factors, such as iron and heme. Hemoglobin also enhances the hemolytic reactions of some bacteria.

The final pH of the medium is 6.8±0.2 at 25°C. The medium has a light amber color when prepared without hemoglobin and a dark brown color when prepared with hemoglobin.

Cystine Glucose Blood Agar is a selective and differential medium that allows the growth and identification of Francisella tularensis, the causative agent of tularemia. F. tularensis is a fastidious and highly virulent gram-negative coccobacillus that requires cystine and other amino acids for its growth. Cystine Glucose Blood Agar provides these essential nutrients, as well as glucose as a carbon and energy source, proteose peptone as a nitrogen and vitamin source, sodium chloride as an osmotic stabilizer, and agar as a solidifying agent. The medium is enriched with 2% hemoglobin, which provides additional growth factors such as heme and iron. Hemoglobin also serves as an indicator of hemolysis, which is a characteristic of some strains of F. tularensis.

Cystine Glucose Blood Agar allows the differentiation of F. tularensis from other bacteria based on colony morphology and hemolysis. F. tularensis produces small, white to grey to bluish-grey, opaque, flat, smooth, and shiny colonies with an entire edge. Some strains may exhibit a greenish-white color or an opalescent sheen. F. tularensis does not produce hemolysis on Cystine Glucose Blood Agar, whereas some other bacteria may produce alpha (partial) or beta (complete) hemolysis. The absence of hemolysis is a presumptive identification of F. tularensis, but further tests are required for confirmation.

To prepare Cystine Glucose Blood Agar, you will need the following ingredients and equipment:

• Cystine Glucose Blood Agar powder (51 grams)
• Distilled water (1000 ml)
• Hemoglobin solution (2%, 100 ml)
• Autoclave
• Water bath
• Sterile Petri plates
• Measuring cylinder
• Balance
• Flask
• Stirring rod
• Pipette

Follow these steps to prepare the medium:

1. Weigh 51 grams of Cystine Glucose Blood Agar powder and transfer it to a flask.
2. Add 1000 ml of distilled water to the flask and mix well with a stirring rod until the powder dissolves completely.
3. Loosely cap the flask and place it in the autoclave. Sterilize the medium at 15 lbs pressure (121°C) for 15 minutes.
4. Remove the flask from the autoclave and cool it to 50°C in a water bath.
5. If you want to enrich the medium with hemoglobin, prepare a separate flask with 10.2 grams of Cystine Glucose Blood Agar powder and 100 ml of distilled water. Sterilize this flask in the same way as the previous one.
6. Cool the hemoglobin-enriched medium to 50°C in a water bath and aseptically add 100 ml of 2% sterile hemoglobin solution. Mix well with a pipette.
7. Pour the medium into sterile Petri plates, either plain or hemoglobin-enriched, depending on your needs. Allow the medium to solidify and store it in a refrigerator until use.

You have now prepared Cystine Glucose Blood Agar for your microbiological experiments. 😊

Cystine Glucose Blood Agar is a selective and differential medium that allows the identification of F. tularensis based on its colony morphology and hemolysis pattern. Other microorganisms may also grow on this medium, but they can be distinguished by their different characteristics.

• At 24 hours, colonies of F. tularensis are very small and may not be visible to the naked eye.
• At 48 hours, colonies of F. tularensis appear as 1-2 mm in diameter, white to grey to bluish-grey, opaque, flat, with an entire edge, smooth and have a shiny surface. They may also produce a greenish-white opalescent sheen.
• F. tularensis produces weak beta-hemolysis on Cystine Glucose Blood Agar. This means that it partially breaks down the red blood cells in the medium, causing a clear zone around the colonies.
• Other gram-negative cocci, such as Neisseria spp., may also grow on Cystine Glucose Blood Agar without hemoglobin enrichment. They produce larger, rounder and more mucoid colonies than F. tularensis. They may also produce different types of hemolysis, such as alpha-hemolysis (greenish discoloration) or gamma-hemolysis (no change).
• Other pathogenic microorganisms, such as Brucella spp., Yersinia pestis, Pasteurella multocida and Haemophilus influenzae, may also grow on Cystine Glucose Blood Agar with hemoglobin enrichment. They can be differentiated from F. tularensis by their colony size, shape, color and hemolysis pattern.

The following figure shows an example of F. tularensis colonies on Cystine Glucose Blood Agar with hemoglobin enrichment:

Figure: F. tularensis on Cysteine heart agar with blood (CHAB) – Colonies are 2 to 4 mm, smooth, entire, greenish-white, and butyrous with opalescent sheen at 48 to 72 hours. Source: © 2012 South Dakota Department of Health.

Cystine Glucose Blood Agar (CGBA) is a useful medium for the isolation and cultivation of various microorganisms, especially those that are fastidious or difficult to grow. Some of the main uses of CGBA are:

• It is the most suitable medium for isolating Francisella tularensis, the causative agent of tularemia, a zoonotic disease that can infect humans and animals. F. tularensis is a highly virulent and infectious bacterium that requires special nutrients and growth factors to grow in vitro. CGBA provides these nutrients and factors by incorporating beef heart infusion, proteose peptone, L-cystine, dextrose, sodium chloride, agar, and hemoglobin. F. tularensis forms characteristic colonies on CGBA that are small, white to grey to bluish-grey, opaque, flat, smooth, shiny, and entire. CGBA is also useful for subculturing and maintaining F. tularensis strains.
• Without hemoglobin enrichment, CGBA can also support the growth of gram-negative cocci and other pathogenic microorganisms that may be present in clinical specimens or environmental samples. Gram-negative cocci are a group of bacteria that have a spherical shape and a negative reaction to the Gram stain. Some examples of gram-negative cocci are Neisseria spp., Moraxella spp., Acinetobacter spp., and Veillonella spp. These bacteria may cause infections such as meningitis, gonorrhea, pneumonia, otitis media, endocarditis, and periodontitis. CGBA provides a rich and balanced medium for these bacteria to grow and produce visible colonies that can be further identified by biochemical or molecular tests.
• CGBA can also be used for the cultivation of other fastidious or rare bacteria that may not grow well on other media. For example, CGBA can be used to isolate and grow Bartonella quintana, the causative agent of trench fever, a louse-borne disease that affects homeless people and soldiers in war zones. B. quintana is a slow-growing bacterium that requires hemin and L-cystine for growth. CGBA provides these factors along with other nutrients that allow B. quintana to form small, smooth, convex, and grey colonies after 5 to 7 days of incubation. Another example is Brucella abortus, the causative agent of brucellosis, a zoonotic disease that can cause abortion in cattle and undulant fever in humans. B. abortus is a facultative intracellular bacterium that requires CO2 and amino acids for growth. CGBA provides these conditions along with other nutrients that enable B. abortus to form small, smooth, convex, and translucent colonies after 2 to 3 days of incubation.

CGBA is a versatile and valuable medium for the isolation and cultivation of various microorganisms that are fastidious or difficult to grow. It is especially useful for detecting F. tularensis, the causative agent of tularemia, which is a highly virulent and infectious bacterium that poses a serious threat to public health and biodefense.

• Cystine Glucose Blood Agar is not a selective medium and may support the growth of other bacteria besides F. tularensis. Therefore, it is important to use other methods to confirm the identity of the isolate, such as biochemical tests, immunological tests, molecular tests, or mass spectrometry.
• Cystine Glucose Blood Agar may not be suitable for the cultivation of some strains of F. tularensis that have different nutritional requirements or are more fastidious. For example, some strains may require additional supplements such as thiamine or cysteine, or may grow better on chocolate agar or buffered charcoal yeast extract agar.
• Cystine Glucose Blood Agar may not detect low numbers of F. tularensis in clinical specimens, especially if they are mixed with other bacteria or cellular debris. Therefore, it is recommended to use enrichment broth or selective media to increase the recovery rate of F. tularensis from specimens.
• Cystine Glucose Blood Agar may not differentiate F. tularensis from other closely related bacteria, such as Francisella novicida, Francisella philomiragia, or Francisella hispaniensis. These bacteria may also produce similar colonies on Cystine Glucose Blood Agar and may cause confusion in diagnosis. Therefore, it is necessary to use specific tests to distinguish them from F. tularensis, such as serological tests, PCR tests, or MALDI-TOF MS.
• Cystine Glucose Blood Agar requires careful handling and disposal, as F. tularensis is a highly virulent and infectious agent that can cause serious disease in humans and animals. Laboratory personnel must follow strict biosafety precautions and wear appropriate personal protective equipment when working with F. tularensis cultures. All culture manipulations should be performed in a biosafety level 3 (BSL-3) facility or a biosafety cabinet with HEPA filters. All waste materials should be autoclaved or incinerated before disposal.

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