Brucella Agar- Composition, Principle, Preparation, Results, Uses

Brucella Agar is a type of culture medium that is used for the isolation and cultivation of Brucella spp. and other fastidious microorganisms. Brucella spp. are gram-negative, facultative intracellular bacteria that cause brucellosis, a zoonotic disease that affects humans and animals. Brucella Agar was developed by Brewer in 1940 as a modification of the serum dextrose agar used by Huddleson.

The composition of Brucella Agar is as follows:

• Peptones: 10 g/L
• Yeast extract: 2 g/L
• Dextrose: 1 g/L
• Sodium chloride: 5 g/L
• Sodium bisulfite: 0.1 g/L
• Agar: 15 g/L

The peptones provide organic nitrogen and amino acids for the growth of microorganisms. The yeast extract is a source of B-complex vitamins and other growth factors. The dextrose serves as an energy source. The sodium bisulfite acts as a reducing agent to create a low-oxygen environment. The sodium chloride maintains the osmotic balance of the medium. The agar is the solidifying agent that allows the formation of discrete colonies.

Brucella Agar has a final pH of 7.0 ± 0.2 at 25°C, which is suitable for the growth of most bacteria. It is a non-selective and non-differential medium, meaning that it does not inhibit or differentiate between different types of bacteria. However, it can be supplemented with blood, antibiotics, or other additives to enhance its performance for specific purposes.

Brucella Agar is widely used in clinical and veterinary microbiology, as well as in food and environmental testing. It is especially useful for the isolation of Brucella spp., which are highly fastidious and require special growth conditions. Brucella Agar can also support the growth of other bacteria, such as Streptococcus pneumoniae, Streptococcus viridans, Neisseria meningitidis, and Campylobacter spp.

Brucella Agar is a selective and differential medium that supports the growth of fastidious microorganisms, such as Brucella spp., Streptococcus spp., Neisseria spp., and Campylobacter spp. The medium contains peptones, dextrose, yeast extract, sodium bisulfite, sodium chloride, and agar as its main ingredients.

The peptones provide organic nitrogen and amino acids for the growth of microorganisms. The yeast extract is a rich source of B-complex vitamins, which are essential for many metabolic processes. Dextrose is a fermentable carbohydrate that serves as an energy source. Sodium bisulfite acts as a reducing agent that lowers the oxidation-reduction potential of the medium and creates an anaerobic environment. Sodium chloride maintains the osmotic balance of the medium. Agar is a polysaccharide that solidifies the medium and allows the formation of discrete colonies.

The principle behind Brucella Agar is based on the ability of different microorganisms to grow and produce characteristic changes on the medium. For example, Brucella spp. grow well on Brucella Agar and produce small, smooth, convex, translucent colonies that are non-hemolytic and non-pigmented. Streptococcus pneumoniae grows as small, round, alpha-hemolytic colonies that have a greenish discoloration around them. Streptococcus viridans grows as small, round, non-hemolytic colonies that are white or grayish in color. Neisseria meningitidis grows as small, round, grayish-white colonies that are surrounded by a narrow zone of beta-hemolysis. Campylobacter spp. grow as small, flat, irregular colonies that are grayish-white or pinkish in color and have a moist appearance.

By observing the growth and appearance of different microorganisms on Brucella Agar, one can identify and differentiate them from each other. However, further biochemical and serological tests are required to confirm the identification of the isolates. Brucella Agar is a useful medium for the isolation and cultivation of fastidious microorganisms from various clinical and nonclinical specimens.

Brucella Agar is a ready-to-use medium that can be purchased from various suppliers. However, if you want to prepare it from scratch, you will need the following ingredients:

• Peptone: 10 g
• Proteose peptone: 10 g
• Yeast extract: 2 g
• Dextrose: 1 g
• Sodium chloride: 5 g
• Sodium bisulfite: 0.1 g
• Agar: 15 g
• Distilled water: 1 L

To prepare Brucella Agar, follow these steps:

1. Weigh the ingredients and dissolve them in distilled water in a suitable container. You may need to heat the solution and stir it frequently to ensure complete dissolution.
2. Adjust the pH of the solution to 7.0 ± 0.2 using a pH meter and a suitable buffer solution. If needed, add more distilled water to make up the final volume of 1 L.
3. Sterilize the medium by autoclaving at 121°C for 15 minutes. Make sure the container is loosely capped to allow steam escape.
4. Cool the medium to 45-50°C and pour it into sterile Petri plates. Alternatively, you can store the medium in sterile screw-cap bottles at 2-8°C until use.
5. Label the plates or bottles with the name of the medium, the date of preparation, and the expiry date.

To use Brucella Agar for culturing microorganisms, follow these steps:

1. Obtain a specimen from a clinical or nonclinical source and inoculate it onto the surface of Brucella Agar using a sterile loop or swab. You can use different methods of inoculation, such as streaking, spreading, or spot inoculation, depending on your purpose and preference.
2. Incubate the plates at 35 ± 2°C for 24-72 hours in an aerobic atmosphere supplemented with carbon dioxide. You can use a candle jar, a CO2 incubator, or a CO2 generator system to provide the required CO2 level.
3. Examine the plates for growth and colony morphology after incubation. You may need to use a microscope or other tools to identify the microorganisms based on their appearance and characteristics.

These are the basic steps for preparing and using Brucella Agar in a laboratory setting. However, you may need to modify them according to your specific needs and protocols. Always follow good laboratory practices and safety precautions when handling biological specimens and media.

After incubation, most plates will show an area of confluent growth. This means that the microorganisms have grown densely and uniformly over the surface of the agar. However, this does not indicate the identity or purity of the culture. To obtain isolated colonies of the organisms contained in the specimen, you need to examine the streaked areas of the plate.

The streaking procedure is a technique that reduces the number of microorganisms deposited on each successive area of the agar. This allows for the separation of different species or strains of bacteria that may be present in the specimen. By observing the morphology, color, size, and hemolysis of the colonies on each streaked area, you can identify and differentiate the microorganisms.

For example, if you are using Brucella Agar with blood to determine bacterial hemolytic reactions, you can look for zones of clearing around the colonies. These zones indicate that the bacteria have lysed (broken down) the red blood cells in the agar. There are three types of hemolysis:

• Alpha-hemolysis: partial lysis of red blood cells, resulting in a greenish discoloration around the colonies.
• Beta-hemolysis: complete lysis of red blood cells, resulting in a clear zone around the colonies.
• Gamma-hemolysis: no lysis of red blood cells, resulting in no change in the agar color around the colonies.

Some examples of bacteria that show different types of hemolysis on Brucella Agar with blood are:

• Streptococcus pneumoniae: alpha-hemolytic
• Streptococcus pyogenes: beta-hemolytic
• Streptococcus viridans: alpha-hemolytic or gamma-hemolytic
• Neisseria meningitidis: gamma-hemolytic

You can also use Brucella Agar as a base for the isolation of Campylobacter species, which are fastidious microaerophilic bacteria that cause gastroenteritis in humans and animals. To enhance their growth, you need to add antibiotics and incubate them at 42°C in a microaerobic atmosphere (5% oxygen, 10% carbon dioxide, and 85% nitrogen). Campylobacter colonies on Brucella Agar are small, flat, gray-white, and have a moist appearance.

Finally, you can use Brucella Agar to isolate Brucella species, which are gram-negative coccobacilli that cause brucellosis in humans and animals. Brucella species are very slow-growing and require enriched media and carbon dioxide for optimal growth. They also produce tiny colonies that are difficult to see with the naked eye. Therefore, you need to use a magnifying lens or a dark-field microscope to observe them. Brucella colonies on Brucella Agar are smooth, convex, translucent, and non-hemolytic.

In summary, Brucella Agar is a versatile medium that can support the growth of various fastidious microorganisms. By modifying its composition or incubation conditions, you can enhance its selectivity or specificity for different bacteria. By examining the colony characteristics on different streaked areas of the plate, you can interpret the results and identify the microorganisms. However, you should always confirm your identification by performing additional biochemical or serological tests.

Brucella Agar is a versatile medium that can be used for different purposes in a microbiology laboratory. Some of the common uses are:

• Cultivation of Brucella spp. and other fastidious microorganisms: Brucella Agar is originally designed to support the growth of Brucella spp., which are the causative agents of brucellosis, a zoonotic disease that affects humans and animals. Brucella spp. are facultative intracellular bacteria that require enriched media and carbon dioxide for optimal growth. Brucella Agar provides the necessary nutrients and reducing agents for their cultivation. Other fastidious microorganisms, such as Francisella tularensis, Haemophilus influenzae, and Bordetella pertussis, can also grow on Brucella Agar with or without blood supplementation.
• General purpose medium for the cultivation of Streptococcus pneumoniae, Streptococcus viridans, and Neisseria meningitidis: These bacteria are common pathogens that cause respiratory and meningitis infections in humans. They are also fastidious and require enriched media and carbon dioxide for growth. Brucella Agar can serve as a general purpose medium for their isolation and identification from clinical specimens, such as sputum, blood, and cerebrospinal fluid.
• Determination of bacterial hemolytic reactions: Hemolysis is the lysis of red blood cells by bacterial enzymes or toxins. It is an important characteristic that helps to differentiate bacteria based on their hemolytic patterns. Brucella Agar can be used as a base for the addition of blood, usually sheep or horse blood, to observe the hemolytic reactions of bacteria. There are three types of hemolysis: alpha (partial), beta (complete), and gamma (none). For example, Streptococcus pyogenes produces beta-hemolysis, while Streptococcus pneumoniae produces alpha-hemolysis on blood agar.
• Isolation of Campylobacter spp.: Campylobacter spp. are gram-negative bacteria that cause gastroenteritis in humans and animals. They are microaerophilic, meaning they require low oxygen and high carbon dioxide for growth. They also require a selective medium to inhibit the growth of other bacteria. Brucella Agar can be used as a base for the isolation of Campylobacter spp. by adding antibiotics, such as cefoperazone, amphotericin B, and trimethoprim, to suppress the growth of competing flora. The plates are incubated at 42°C in a microaerobic environment for 48 hours.
• Isolation of Brucella species from foods: Brucellosis can also be transmitted through the consumption of contaminated foods, such as milk, cheese, meat, and vegetables. The detection of Brucella spp. in foods is important for food safety and public health. Brucella Agar is recommended by APHA (American Public Health Association) for the isolation of Brucella spp. from foods. The food samples are enriched in tryptic soy broth with antibiotics and then streaked onto Brucella Agar plates. The plates are incubated at 35°C in an aerobic atmosphere supplemented with carbon dioxide for 24-72 hours.
• Isolation and cultivation of non-fastidious and fastidious microorganisms from a variety of clinical and nonclinical specimens: Brucella Agar can also be used as a general purpose medium for the isolation and cultivation of various microorganisms from different types of specimens, such as urine, feces, wound swabs, water samples, etc. By adding 5% horse blood to the medium, it enhances the growth of both non-fastidious and fastidious microorganisms. The plates are incubated at 35°C in an aerobic atmosphere supplemented with carbon dioxide for 24-72 hours.

These are some of the common uses of Brucella Agar in a laboratory setting. However, there may be other applications depending on the specific needs and objectives of the laboratory.

Brucella Agar is a useful medium for the cultivation of various fastidious microorganisms, but it also has some limitations that should be considered:

• Brucella Agar is not selective or differential for any specific organism. Therefore, it may support the growth of unwanted contaminants or commensals that may interfere with the isolation and identification of the target organism. For example, some strains of Staphylococcus aureus may grow on Brucella Agar and produce hemolysis that may mask the hemolytic reactions of other organisms. To overcome this limitation, some selective or differential agents may be added to Brucella Agar, such as antibiotics, dyes, or indicators.
• Brucella Agar may not support the growth of some organisms that have very specific nutritional or environmental requirements. For example, some strains of Haemophilus influenzae may not grow on Brucella Agar unless supplemented with hemin and NAD. Similarly, some strains of Campylobacter jejuni may not grow on Brucella Agar unless incubated at 42°C in a microaerophilic atmosphere. To overcome this limitation, some specialized media may be used for the cultivation of these organisms, such as chocolate agar for Haemophilus influenzae or Campylobacter agar for Campylobacter jejuni.
• Brucella Agar may not provide enough information for the definitive identification of some organisms. For example, some strains of Brucella spp. may not show any distinctive characteristics on Brucella Agar, such as colony morphology, pigmentation, or hemolysis. Similarly, some strains of Streptococcus pneumoniae may not show their typical alpha-hemolysis on Brucella Agar due to the presence of sodium bisulfite. To overcome this limitation, some additional tests may be performed on the isolates from Brucella Agar, such as biochemical tests, serological tests, or molecular tests.

These are some of the limitations of Brucella Agar that should be taken into account when using this medium in a laboratory setting. However, despite these limitations, Brucella Agar remains a valuable and versatile medium for the cultivation of many fastidious microorganisms.

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