Culture Media

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

Ashdown's medium can be described as a selective culture medium that allows for the identification and characterisation of Burkholderia pseudomallei (the bacteria...

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This article writter by MN Editors on January 23, 2022

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Ashdown’s medium is a selective culture medium for the isolation and characterisation of Burkholderia pseudomallei (the bacterium that causes melioidosis). Ashdown’s medium was first described by LR Ashdown in 1979.It is used for the selective isolation of B. pseudomallei from clinical specimens taken from non-sterile sites (e.g., sputum) 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. Gentamicin slightly inhibits the growth of B. pseudomallei and so specimens inoculated onto Ashdown’s agar needs to be incubated for a minimum of 96 hours instead of 48 hours. The medium is also enriched with 4% glycerol, which is required by some strains of B. pseudomallei to grow. B. pseudomallei usually produces flat wrinkled purple colonies on Ashdown’s agar.

Ashdown’s Agar Principle

The medium has crystal violet as well as gentamicin, which are selective agents to stop some other bacteria’s growth. The colonies of B. pseudomallei soak the neutral red present in the medium which helps identify the bacteria from others. Gentamicin is a mild inhibitor of development of B. pseudomallei, and as such samples inoculated on Ashdown’s agar require incubation for at least 72 hours instead of the 48 hours. In addition, the medium has been enhanced with 4percent glycerol which is essential for certain varieties from B. pseudomallei to develop. After 24 hours after incubation, the medium colonies, they tend to be small, clear and pale pink. They then change to pinkish-purple flat and dry over the following two days. The most distinctive characteristic that distinguishes B. pseudomallei is its metallic sheen, as well as the typical progression from dry to wrinkled colonies at 96 hours.

Ashdown’s Agar Composition

IngredientsGram/Litre
Tryptone soya broth10 g
Agar                           15 g
Glycerol40 ml
Gentamicin4 mg
Crystal violet 0.1%5 ml
Neutral Red 1%5 ml
Distilled water             950ml

Preparation and Method of Use of Ashdown’s Agar

  1. Mix all ingredients together in one bottle.
  2. Autoclave at 121°C for 15 minutes.
  3. Cool to about 50 degrees, then add gentamicin until the final concentration of 4 mg/l
  4. Dispense the agar in petri-dishes.
  5. Subculture 10ml of the upper layer of salt solution containing threonine and colistin 50 mg/L medium into an Ashdown Agar Plate in the form of a BSC streaking process to produce single colonies.
  6. Incubate the Ashdown Agar Plate at 40°C in air.
  7. Examine the Ashdown agar plate every day for seven days in the BSC for colonies you suspect to be from B. pseudomallei.

Note that the solutions of crystal violet of 0.1 percent should be incubated at 37°C for 2 weeks prior to being used to get the most optimal color.

Result Interpretation of Ashdown’s Agar

OrganismGrowth
Burkholderia pseudomallei  Highly wrinkled circular purple colonies on ASA by 48 h
Burkholderia thailandensisCircular purple colonies

Uses of Ashdown’s Agar

  • It is employed for the specific isolation from B. pseudomallei from clinical specimens collected from non-sterile locations (e.g. the sputum) and to generate the characteristic appearance of B. pseudomallei.
  • The Ashdown’s Selective Agar (ASA) is currently the preferred method for the isolation and presumed detection of B. pseudomallei in locations that have melioidosis as a prevalent.

Limitations of Ashdown’s Agar

  • It was noted in local clinical lab use that ASA inhibits certain species of B. pseudomallei.
  • Additional tests should be conducted to determine the presence as B. pseudomallei. Any organism with the appearance of a colonial B. pseudomallei, on Ashdown agar that is not resistant to colistin, and susceptible to co-amoxiclav may be presumed to be B. pseudomallei when the test results are positive for lax Agglutination.
  • It is especially difficult when a small amount of B. pseudomallei are found and there is an abundance of and abundant flora of the upper respiratory tract that is common to.

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://dbpedia.org/page/Ashdown’s_medium
  • https://en.wikipedia.org/wiki/Ashdown%27s_medium
  • https://microbenotes.com/ashdowns-agar/
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