Ashdown’s Agar- Composition, Principle, Preparation, Results, Uses

Ashdown’s agar is a selective and differential medium for the isolation and identification of Burkholderia pseudomallei, the causative agent of melioidosis. The medium was developed by Ashdown and colleagues in 1979 and has been widely used in areas where melioidosis is endemic.

The composition of Ashdown’s agar is as follows:

• Ingredient: Peptone
• Amount (g/L): 10.0

• Ingredient: Yeast extract
• Amount (g/L): 3.0

• Ingredient: Sodium chloride
• Amount (g/L): 5.0

• Ingredient: Agar
• Amount (g/L): 15.0

• Ingredient: Glycerol
• Amount (g/L): 40.0

• Ingredient: Neutral red
• Amount (g/L): 0.05

• Ingredient: Crystal violet
• Amount (g/L): 0.002

• Ingredient: Gentamicin sulfate
• Amount (g/L): 0.004

The medium contains crystal violet and gentamicin as selective agents to suppress the growth of other bacteria. Colonies of B. pseudomallei also take up neutral red which is present in the medium, and this further helps to distinguish it from other bacteria.

The medium is also enriched with 4% glycerol, which is required by some strains of B. pseudomallei to grow.

Source: Ashdown LR, Guard RW, Pitt TL. A selective medium for the isolation of Pseudomonas pseudomallei from clinical specimens. Pathology. 1979;11(4):293-300. Ashdown LR. An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens. Pathology. 1985;17(4):599-601.

Ashdown’s agar is a selective and differential medium that is designed to isolate and identify Burkholderia pseudomallei, the causative agent of melioidosis. Melioidosis is an infectious disease that can affect humans and animals, especially in tropical and subtropical regions. B. pseudomallei is a gram-negative, aerobic, motile bacillus that can be found in soil and water.

The medium contains two main selective agents: crystal violet and gentamicin. Crystal violet is a dye that inhibits the growth of most gram-positive bacteria and some gram-negative bacteria. Gentamicin is an antibiotic that inhibits the growth of most gram-negative bacteria except for B. pseudomallei and some other Burkholderia species. These two agents help to suppress the growth of other bacteria that may be present in the clinical specimens, such as sputum, urine, blood, or pus.

The medium also contains a differential agent: neutral red. Neutral red is a pH indicator that changes color from yellow to red in acidic conditions. B. pseudomallei produces acid from glycerol fermentation, which lowers the pH of the medium around the colonies. As a result, the colonies of B. pseudomallei appear pinkish-red on Ashdown’s agar, while other bacteria remain colorless or pale pink.

Another characteristic feature of B. pseudomallei on Ashdown’s agar is its metallic sheen, which is visible under reflected light. The metallic sheen is due to the production of exopolysaccharide by B. pseudomallei, which forms a capsule around the cells. The capsule helps the bacteria to evade the host immune system and to survive in harsh environments.

The medium also contains 4% glycerol as a carbon source for B. pseudomallei. Some strains of B. pseudomallei require glycerol for growth, while others can use other substrates such as glucose or lactose. The presence of glycerol in the medium ensures that all strains of B. pseudomallei can grow on Ashdown’s agar.

The principle of Ashdown’s agar is based on the selective inhibition of other bacteria by crystal violet and gentamicin, and the differential identification of B. pseudomallei by neutral red and metallic sheen.

To prepare Ashdown’s agar, you will need the following ingredients:

• Peptone: 10 g
• Yeast extract: 2 g
• Sodium chloride: 5 g
• Glycerol: 40 ml
• Agar: 15 g
• Distilled water: 1000 ml
• Crystal violet solution (0.1%): 1 ml
• Neutral red solution (0.5%): 2 ml
• Gentamicin: 4 mg

The steps to prepare and use Ashdown’s agar are as follows:

1. Mix peptone, yeast extract, sodium chloride, glycerol and agar in a bottle and dissolve them in distilled water.
2. Autoclave the mixture at 121°C for 15 minutes.
3. Cool the mixture to 50°C and add crystal violet solution and neutral red solution. Mix well.
4. Add gentamicin to a final concentration of 4 mg/l and mix well.
5. Dispense the agar into sterile petri dishes and let them solidify.
6. Store the plates at 4°C until use.

Note: The crystal violet solution should be incubated at 37°C for two weeks before being used in order to ensure optimal coloration.

To use Ashdown’s agar for isolation and identification of B. pseudomallei, you will need the following materials:

• Clinical specimens taken from non-sterile sites (e.g., sputum, urine, pus, etc.)
• Threonine-basal salt solution plus colistin 50 mg/L medium
• Ashdown’s agar plates
• Sterile inoculating loops or swabs
• Incubator
• Biosafety cabinet
• Latex agglutination test kit

The steps to use Ashdown’s agar are as follows:

1. Inoculate the clinical specimens into threonine-basal salt solution plus colistin 50 mg/L medium and incubate at 40°C for 24 hours.
2. Subculture 10 ml from the upper layer of the medium to an Ashdown’s agar plate in a biosafety cabinet, streaking to achieve single colonies.
3. Incubate the Ashdown’s agar plate at 40°C in air for up to 7 days.
4. Examine the Ashdown’s agar plate daily for colonies suspected to be of B. pseudomallei. They are usually pinkish-purple, flat, slightly dry and have a metallic sheen. They may also become dry and wrinkled after 96 hours.
5. Perform a latex agglutination test on the suspected colonies by following the manufacturer’s instructions. If positive, presumptively identify them as B. pseudomallei.
6. Confirm the identification of B. pseudomallei by performing further tests such as biochemical tests, PCR or MALDI-TOF MS.

Ashdown’s agar is a selective and differential medium that allows the growth and identification of B. pseudomallei based on its characteristic morphology and color. The medium contains crystal violet and gentamicin as selective agents to inhibit the growth of most other bacteria. It also contains neutral red as a pH indicator that turns red in acidic conditions. B. pseudomallei produces acid from glycerol, which is present in the medium, and thus forms red or pink colonies. The colonies also have a metallic sheen and become dry and wrinkled after prolonged incubation.

To interpret the results of Ashdown’s agar, the following steps should be followed:

1. Examine the plates daily for up to 7 days at 40°C in air for the presence of suspected B. pseudomallei colonies.
2. Look for colonies that are red or pink, flat, slightly dry, and have a metallic sheen. These are typical of B. pseudomallei and can be presumptively identified as such.
3. Confirm the identity of B. pseudomallei by performing additional tests, such as latex agglutination, colistin resistance, co-amoxiclav susceptibility, and biochemical tests.
4. Report the results as positive, negative, or inconclusive for B. pseudomallei.

The following table summarizes the interpretation of Ashdown’s agar results:

Note: Some strains of B. pseudomallei may not grow well on Ashdown’s agar or may not produce the typical morphology or color. Therefore, negative results do not rule out the possibility of melioidosis and further testing is required.

Ashdown’s selective agar (ASA) is the currently favored medium for the isolation and presumptive identification of B. pseudomallei in areas where melioidosis is endemic. Melioidosis is a serious infectious disease caused by B. pseudomallei that can affect humans and animals. It is also known as Whitmore’s disease or soil-borne sepsis.

ASA is used for the selective isolation of B. pseudomallei from clinical specimens taken from non-sterile sites (e.g., sputum, urine, blood, pus, etc.) as well as to produce the characteristic morphology of B. pseudomallei. The medium contains crystal violet and gentamicin as selective agents to suppress the growth of other bacteria. Colonies of B. pseudomallei also take up neutral red which is present in the medium, and this further helps to distinguish it from other bacteria.

ASA is especially useful for detecting B. pseudomallei in specimens with low bacterial load or mixed flora, such as sputum samples from patients with pneumonia or chronic lung infections. The medium can also be used for subculturing B. pseudomallei from other media, such as threonine-basal salt solution plus colistin (TBSC), which is used for enrichment of B. pseudomallei from soil or water samples.

ASA can also be used for presumptive identification of B. pseudomallei based on its colonial appearance and biochemical properties. The most characteristic feature of B. pseudomallei on ASA is its metallic sheen, which is visible under oblique light. The colonies are usually pinkish-purple, flat, and slightly dry at 48 hours of incubation, and become dry and wrinkled at 96 hours. The colonies are also resistant to colistin and susceptible to co-amoxiclav, which are two antibiotics commonly used for treating melioidosis.

ASA can be further supplemented with latex agglutination test, which is a rapid and specific method for confirming the presence of B. pseudomallei in colonies grown on ASA. The test uses latex particles coated with monoclonal antibodies that react with the capsular polysaccharide antigen of B. pseudomallei. A positive result is indicated by visible agglutination of the latex particles within 2 minutes.

ASA is therefore a valuable tool for the diagnosis and surveillance of melioidosis, which is an emerging and neglected tropical disease that poses a significant public health threat in many parts of the world.

Ashdown’s agar is a useful medium for the isolation and presumptive identification of B. pseudomallei, but it also has some limitations that need to be considered.

• One limitation is that Ashdown’s agar does not allow the differentiation of B. pseudomallei from other closely related species, such as B. thailandensis, B. oklahomensis, and B. cepacia complex. These species can also grow on Ashdown’s agar and produce similar colonies with metallic sheen and neutral red uptake. Therefore, further tests must be carried out for confirmation of B. pseudomallei, such as latex agglutination, biochemical tests, or molecular methods.
• Another limitation is that Ashdown’s agar may be inhibitory to some strains of B. pseudomallei, especially those that are glycerol-negative or have low gentamicin susceptibility. These strains may fail to grow or produce atypical colonies on Ashdown’s agar, leading to false-negative results. Therefore, it is recommended to use Ashdown’s agar in conjunction with other non-selective media, such as blood agar or MacConkey agar, to increase the recovery rate of B. pseudomallei.
• A third limitation is that Ashdown’s agar may not be effective for the isolation of B. pseudomallei from sputum samples, especially when there is a low bacterial load and a high commensal flora. The selective agents in Ashdown’s agar may not be sufficient to suppress the growth of other respiratory bacteria, which may overgrow and mask the presence of B. pseudomallei. Therefore, it is advised to use a pre-enrichment step before inoculating sputum samples onto Ashdown’s agar, such as threonine-basal salt solution plus colistin (TBSSC) broth.

In summary, Ashdown’s agar is a valuable medium for the isolation and presumptive identification of B. pseudomallei from clinical specimens, but it also has some limitations that need to be addressed by using additional tests and methods.

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