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

What is Ashdown’s Agar?

• Ashdown’s Agar is a specialized culture medium that was developed by LR Ashdown in 1979. It is primarily used for the selective isolation and characterization of Burkholderia pseudomallei, the bacterium responsible for causing melioidosis, a potentially fatal infectious disease.
• The purpose of Ashdown’s Agar is to provide optimal conditions for the growth of B. pseudomallei while inhibiting the growth of other bacteria present in clinical specimens. This medium contains specific components that contribute to its selectivity and differential characteristics.
• To achieve selectivity, Ashdown’s Agar incorporates crystal violet and gentamicin as selective agents. These substances help suppress the growth of competing bacteria, allowing B. pseudomallei to thrive and form colonies. The inclusion of gentamicin in the medium slightly inhibits the growth of B. pseudomallei, which necessitates a longer incubation period of a minimum of 96 hours, compared to the typical 48 hours required for other bacterial cultures.
• Additionally, Ashdown’s Agar is enriched with 4% glycerol. This addition provides a necessary nutrient source for the growth of certain strains of B. pseudomallei. The presence of glycerol enables these strains to develop and flourish on the medium.
• The distinctive feature of B. pseudomallei colonies on Ashdown’s Agar is their characteristic morphology. The colonies typically appear as flat, wrinkled, and purple in color. Another distinguishing factor is the uptake of neutral red by B. pseudomallei, which is present in the medium. This uptake further aids in the differentiation of B. pseudomallei from other bacteria during the isolation and characterization process.
• In summary, Ashdown’s Agar is a selective culture medium designed specifically for the isolation and characterization of Burkholderia pseudomallei. It utilizes crystal violet, gentamicin, and glycerol to promote the growth of B. pseudomallei while inhibiting the growth of other bacteria. The distinctive morphology and the uptake of neutral red by B. pseudomallei colonies on this medium contribute to its effectiveness in identifying this bacterium.

Ashdown’s Agar Principle

The principle of Ashdown’s Agar lies in its selective and differential components, which facilitate the isolation and characterization of Burkholderia pseudomallei, the causative bacterium of melioidosis.

To achieve selectivity, the medium incorporates crystal violet and gentamicin as selective agents. These substances effectively inhibit the growth of other bacteria, allowing B. pseudomallei to thrive and form colonies on the agar. However, it is worth noting that gentamicin also slightly inhibits the growth of B. pseudomallei itself. As a result, specimens inoculated onto Ashdown’s Agar require a longer incubation period of at least 96 hours, in contrast to the standard 48-hour incubation period for many other bacterial cultures.

One of the distinguishing characteristics of B. pseudomallei colonies on Ashdown’s Agar is their ability to take up neutral red, which is present in the medium. This uptake of neutral red by B. pseudomallei colonies further aids in differentiating them from other bacteria, enhancing the specificity of the medium.

Furthermore, Ashdown’s Agar is enriched with 4% glycerol. This addition is essential as certain strains of B. pseudomallei require glycerol as a nutrient source to grow and develop on the medium.

During the incubation period, the appearance of B. pseudomallei colonies on Ashdown’s Agar undergoes characteristic changes. Initially, after 24 hours of incubation, the colonies are often pinpoint in size, clear, and pale pink in color. Over the following two days, the colonies transition to a pinkish-purple hue, becoming flat and slightly dry. One of the most distinct features of B. pseudomallei colonies is their metallic sheen. Finally, at 96 hours of incubation, the colonies typically become dry and wrinkled, exhibiting their characteristic appearance on Ashdown’s Agar.

In summary, the principle of Ashdown’s Agar revolves around its selective agents, crystal violet and gentamicin, which suppress the growth of other bacteria, while allowing B. pseudomallei to thrive. The uptake of neutral red by B. pseudomallei colonies helps differentiate them from other bacteria. The inclusion of 4% glycerol enriches the medium to support the growth of specific strains of B. pseudomallei. The progressive changes in colony appearance, from pinpoint and pale pink to pinkish-purple, flat, and dry with a metallic sheen, further aid in the identification and characterization of B. pseudomallei on Ashdown’s Agar.

Ashdown’s Agar Composition

Ingredients	Gram/Litre
Tryptone soya broth	10 g
Agar	15 g
Glycerol	40 ml
Gentamicin	4 mg
Crystal violet 0.1%	5 ml
Neutral Red 1%	5 ml
Distilled water	950ml

Preparation of Ashdown’s Agar

The preparation and method of use of Ashdown’s Agar involve several steps to ensure optimal growth and identification of Burkholderia pseudomallei colonies:

1. Prepare the medium by mixing all the required ingredients in a bottle.
2. Autoclave the medium at 121°C for 15 minutes to sterilize it.
3. Allow the medium to cool down to approximately 50°C.
4. Add gentamicin to the medium, achieving a final concentration of 4 mg/L. This step helps in suppressing the growth of other bacteria, enhancing selectivity for B. pseudomallei.
5. Dispense the prepared Ashdown’s Agar into petri dishes, following aseptic techniques to maintain sterility.
6. Before use, ensure that the crystal violet solution of 0.1% has been incubated at 37°C for two weeks. This incubation period allows for optimal coloration of the solution, which is important for the differentiation of B. pseudomallei colonies.

To isolate B. pseudomallei using Ashdown’s Agar, the following method can be followed:

1. In a biological safety cabinet (BSC), subculture 10 mL of the upper layer of the threonine-basal salt solution plus colistin (50 mg/L) medium.
2. Transfer the subculture onto an Ashdown’s Agar plate, using streaking techniques to achieve single colonies.
3. Incubate the Ashdown’s Agar plate at 40°C in an ambient air environment.
4. Examine the Ashdown’s Agar plate daily for a period of 7 days within the biological safety cabinet, specifically looking for colonies suspected to be Burkholderia pseudomallei.

By following these steps, the selective and differential properties of Ashdown’s Agar can be utilized to isolate and identify B. pseudomallei colonies effectively.

Result Interpretation of Ashdown’s Agar

The result interpretation of Ashdown’s Agar is based on the growth and appearance of colonies, specifically for Burkholderia pseudomallei and Burkholderia thailandensis. Here is the interpretation:

1. Burkholderia pseudomallei:
   • Growth: Burkholderia pseudomallei exhibits highly wrinkled circular purple colonies on Ashdown’s Agar.
   • Timing: These characteristic colonies can typically be observed by 48 hours of incubation on Ashdown’s Agar.
2. Burkholderia thailandensis:
   • Growth: Burkholderia thailandensis colonies on Ashdown’s Agar appear as circular purple colonies.
   • Appearance: The colonies of Burkholderia thailandensis lack the highly wrinkled morphology observed in Burkholderia pseudomallei colonies.

It is important to note that Ashdown’s Agar is primarily used for the selective isolation and characterization of Burkholderia pseudomallei, the causative bacterium of melioidosis. While Burkholderia thailandensis colonies may also grow on this medium, their morphology differs from Burkholderia pseudomallei colonies. Therefore, the focus of result interpretation on Ashdown’s Agar is primarily on the presence and characteristics of Burkholderia pseudomallei colonies, such as their highly wrinkled, circular, and purple appearance after 48 hours of incubation.

Organism	Growth
Burkholderia pseudomallei	Highly wrinkled circular purple colonies on ASA by 48 h
Burkholderia thailandensis	Circular purple colonies

Uses of Ashdown’s Agar

Ashdown’s Agar, also known as Ashdown’s selective agar (ASA), has several important uses in the field of microbiology, particularly in areas where melioidosis, caused by Burkholderia pseudomallei, is endemic. Here are the primary uses of Ashdown’s Agar:

• Selective Isolation: Ashdown’s Agar is the preferred medium for the selective isolation of Burkholderia pseudomallei from clinical specimens obtained from non-sterile sites, such as sputum. The selective agents, including crystal violet and gentamicin, incorporated into the medium suppress the growth of other bacteria, allowing for the preferential growth of B. pseudomallei.
• Presumptive Identification: Ashdown’s Agar aids in the presumptive identification of Burkholderia pseudomallei due to its ability to produce the characteristic morphology of the bacterium. The colonies of B. pseudomallei on Ashdown’s Agar exhibit specific traits, such as being highly wrinkled, circular, and typically purple in color. These colony characteristics, combined with other laboratory tests, provide valuable clues for the preliminary identification of B. pseudomallei.
• Endemic Areas: Ashdown’s Agar is particularly valuable in areas where melioidosis is endemic. It allows for the reliable isolation and identification of Burkholderia pseudomallei from clinical samples in regions where the disease is prevalent. This aids in diagnosing melioidosis and understanding the epidemiology of the disease in these specific geographical areas.

By utilizing Ashdown’s Agar, laboratories and healthcare providers can effectively isolate and identify Burkholderia pseudomallei from clinical specimens, contributing to the accurate diagnosis and management of melioidosis cases. It serves as a valuable tool in regions where melioidosis is endemic, helping to improve patient outcomes and facilitate appropriate epidemiological studies.

Limitations of Ashdown’s Agar

While Ashdown’s Agar is a valuable medium for the selective isolation and presumptive identification of Burkholderia pseudomallei, it does have certain limitations. Here are the limitations associated with Ashdown’s Agar:

• Additional Tests Required: While Ashdown’s Agar provides a presumptive identification of Burkholderia pseudomallei based on the characteristic colonial appearance, further tests are necessary for definitive identification. For conclusive identification, other laboratory tests, such as latex agglutination, need to be performed.
• Inhibitory Effect: Some strains of Burkholderia pseudomallei may be inhibited or have limited growth on Ashdown’s Agar. This limitation has been observed in local clinical laboratory use, suggesting that the medium may not support the growth of all strains of B. pseudomallei effectively.
• Low Sensitivity in Sputum Isolation: Isolating Burkholderia pseudomallei from sputum samples can be challenging, especially when the organism is present in low numbers and there is a significant presence of commensal bacteria from the upper respiratory tract. The abundant upper respiratory flora can hinder the isolation of B. pseudomallei, reducing the sensitivity of Ashdown’s Agar in these cases.

In summary, while Ashdown’s Agar is a useful tool for the selective isolation and presumptive identification of Burkholderia pseudomallei, it has limitations. Definitive identification requires additional tests, and the medium may be inhibitory to certain strains of B. pseudomallei. Moreover, its sensitivity in isolating B. pseudomallei from sputum samples may be compromised in the presence of a large commensal upper respiratory tract flora. Awareness of these limitations is essential to ensure accurate diagnosis and appropriate follow-up testing in cases of suspected melioidosis.

FAQ

References

• Comparison of Ashdown’s Medium, Burkholderia cepacia Medium, andBurkholderia pseudomallei Selective Agar for Clinical Isolation ofBurkholderia pseudomallei, https://www.researchgate.net/publication/7557561_Comparison_of_Ashdown%27s_Medium_Burkholderia_cepacia_Medium_and_Burkholderia_pseudomallei_Selective_Agar_for_Clinical_Isolation_of_Burkholderia_pseudomallei
• https://www.jcdr.net/articles/PDF/17135/57824_CE(AD)_F[SH]_PF1(SG_SHU_SS)_PFA(SG_SS)_PN(KM).pdf
• https://www.melioidosis.info/info.aspx?pageID=104&contentID=1040209
• https://dbpedia.org/page/Ashdown’s_medium
• https://en.wikipedia.org/wiki/Ashdown%27s_medium
• https://microbenotes.com/ashdowns-agar/