Ashdown’s Agar 50 FAQs and 20 MCQs
Master the use of Ashdown’s Agar, a selective medium for isolating Burkholderia pseudomallei (the causative agent of melioidosis), with our 50 frequently asked questions (FAQs) covering its composition, preparation, interpretation, and clinical significance. Additionally, test your knowledge with 20 multiple-choice questions (MCQs) designed for medical lab technicians, microbiologists, and students. Whether you’re preparing for exams or enhancing your lab skills, this resource on LabTestsGuide.com provides in-depth insights into this critical culture medium.
Ashdown’s Agar 50 FAQs
What is Ashdown’s Agar?
A selective culture medium for isolating Burkholderia pseudomallei (causes melioidosis).
Who developed Ashdown’s Agar?
LR Ashdown in 1979.
What is the primary purpose of Ashdown’s Agar?
To selectively isolate and identify B. pseudomallei from clinical samples.
What disease is B. pseudomallei associated with?
Melioidosis, a potentially fatal infectious disease.
Is Ashdown’s Agar selective or differential?
Both (selective due to inhibitors, differential due to colony morphology and neutral red uptake).
What are the key ingredients in Ashdown’s Agar?
Tryptone soya broth, agar, glycerol, gentamicin, crystal violet, neutral red, and distilled water.
Why is glycerol added to Ashdown’s Agar?
Some B. pseudomallei strains require it for growth.
What is the role of crystal violet in Ashdown’s Agar?
A selective agent that inhibits Gram-positive bacteria
Why is gentamicin included?
Suppresses other bacteria but slightly inhibits B. pseudomallei.
What is the function of neutral red?
Helps differentiate B. pseudomallei (uptakes the dye, turning colonies purple).
How is Ashdown’s Agar prepared?
Mix ingredients, autoclave (121°C, 15 min), cool to 50°C, add gentamicin, and pour into plates.
Why is crystal violet pre-incubated before use?
Ensures optimal coloration (incubate at 37°C for 2 weeks).
What is the final concentration of gentamicin?
4 mg/L.
How much glycerol is added?
40 mL per liter (4% final concentration).
How does Ashdown’s Agar select for B. pseudomallei?
Crystal violet and gentamicin inhibit other bacteria.
Why does B. pseudomallei grow despite gentamicin?
It is naturally resistant but slightly inhibited (needs 96h incubation).
How does neutral red help identify B. pseudomallei?
Colonies absorb the dye, turning purple/pinkish.
What is the typical colony morphology of B. pseudomallei on Ashdown’s Agar?
Wrinkled, dry, purple colonies with a metallic sheen by 96h.
How does B. pseudomallei appear at 24h vs. 96h?
24h: Pinpoint, pale pink → 96h: Wrinkled, purple, dry.
What distinguishes B. pseudomallei from B. thailandensis on Ashdown’s Agar?
B. pseudomallei: Wrinkled colonies; B. thailandensis: Smooth purple colonies.
How long should Ashdown’s Agar be incubated?
Minimum 96 hours (4 days), up to 7 days.
Why is incubation longer than standard agar (48h)?
Gentamicin slightly slows B. pseudomallei growth.
At what temperature is Ashdown’s Agar incubated?
40°C (enhances selectivity).
How should specimens be streaked on Ashdown’s Agar?
Subculture from enrichment broth and streak for single colonies.
What type of specimens is Ashdown’s Agar used for?
Non-sterile sites (e.g., sputum, wounds).
Should Ashdown’s Agar be examined daily?
Yes, for up to 7 days to detect slow-growing colonies.
What indicates a positive result for B. pseudomallei?
Wrinkled, purple, metallic-sheen colonies by 48–96h.
Can Ashdown’s Agar definitively identify B. pseudomallei?
No, it’s presumptive; confirm with latex agglutination/biochemical tests.
What other bacteria grow on Ashdown’s Agar?
B. thailandensis (smooth purple colonies).
How is B. pseudomallei confirmed after growth on Ashdown’s Agar?
Colistin resistance + co-amoxiclav susceptibility + latex agglutination.
Where is Ashdown’s Agar most commonly used?
Endemic melioidosis regions (e.g., Southeast Asia, Australia).
Why is Ashdown’s Agar preferred for melioidosis diagnosis?
High selectivity for B. pseudomallei in mixed flora samples.
Can Ashdown’s Agar be used for sterile sites (e.g., blood)?
Less common (preferable for sputum, pus, non-sterile samples).
Is Ashdown’s Agar used in environmental sampling?
Yes, for detecting B. pseudomallei in soil/water in endemic areas.
What are the limitations of Ashdown’s Agar?
Some B. pseudomallei strains are inhibited.
Low sensitivity in sputum with high commensal flora.
Requires additional confirmatory tests.Why might Ashdown’s Agar fail to detect B. pseudomallei?
Low bacterial load or inhibition by competing flora.
Does Ashdown’s Agar work for all B. pseudomallei strains?
No, some strains may not grow well.
What is a major challenge in sputum testing with Ashdown’s Agar?
Overgrowth of normal respiratory bacteria masking B. pseudomallei.
How does Ashdown’s Agar compare to blood agar for B. pseudomallei?
More selective (blood agar allows more contaminants).
Are there alternative media for B. pseudomallei?
Threonine-basal salt + colistin, Burkholderia cepacia selective agar.
Why is Ashdown’s Agar better than MacConkey agar for B. pseudomallei?
MacConkey lacks glycerol/gentamicin, reducing selectivity.
What if no growth is seen after 96h?
Extend incubation to 7 days; consider retesting with enrichment.
How to avoid contamination on Ashdown’s Agar?
Use aseptic techniques, work in a BSC (Biosafety Cabinet).
Why might colonies not show the expected purple color?
Neutral red uptake issues (check dye preparation/storage).
Can Ashdown’s Agar expire?
Yes, store properly and check for dehydration/contamination.
Why is 40°C incubation used?
Enhances selectivity by inhibiting some competitors.
How does B. pseudomallei’s resistance to gentamicin help detection?
Most bacteria are inhibited, allowing B. pseudomallei to dominate.
What is the role of colistin in B. pseudomallei identification?
B. pseudomallei is resistant, helping differentiate from other species.
Can PCR replace Ashdown’s Agar for B. pseudomallei detection?
PCR is faster but culture (Ashdown’s) is still gold standard for viability.
Is Ashdown’s Agar used in research beyond diagnostics?
Yes, for antibiotic susceptibility testing and bacterial studies.
Ashdown’s Agar 20 MCQs
- What is the primary purpose of Ashdown’s Agar?
a) Isolate Escherichia coli
b) Detect Staphylococcus aureus
c) Selectively grow Burkholderia pseudomallei
d) Culture Mycobacterium tuberculosis - Who developed Ashdown’s Agar?
a) Robert Koch
b) Louis Pasteur
c) LR Ashdown
d) Alexander Fleming - Which disease is caused by Burkholderia pseudomallei?
a) Tuberculosis
b) Melioidosis
c) Cholera
d) Leprosy - Ashdown’s Agar is a:
a) Selective medium
b) Differential medium
c) Both selective and differential
d) Enriched medium
- Which selective agent inhibits Gram-positive bacteria in Ashdown’s Agar?
a) Gentamicin
b) Crystal violet
c) Neutral red
d) Glycerol - Why is glycerol added to Ashdown’s Agar?
a) To inhibit B. pseudomallei
b) As a nutrient for some B. pseudomallei strains
c) To enhance pigment production
d) To neutralize pH - What is the concentration of gentamicin in Ashdown’s Agar?
a) 1 mg/L
b) 4 mg/L
c) 10 mg/L
d) 50 mg/L - How should crystal violet be prepared before use?
a) Autoclave immediately
b) Incubate at 37°C for 2 weeks
c) Freeze at -20°C
d) Mix with distilled water only
- What is the key feature of B. pseudomallei colonies on Ashdown’s Agar at 96 hours?
a) Shiny blue colonies
b) Wrinkled, purple, dry colonies
c) Yellow mucoid colonies
d) Transparent colonies - Which dye uptake helps differentiate B. pseudomallei?
a) Methylene blue
b) Neutral red
c) Safranin
d) Malachite green - How does B. thailandensis differ from B. pseudomallei on Ashdown’s Agar?
a) Forms green colonies
b) Smooth purple colonies (non-wrinkled)
c) No growth
d) Yellow pigment - Why is incubation longer (96h) for Ashdown’s Agar?
a) Gentamicin slightly inhibits B. pseudomallei
b) Slow autoclaving process
c) Neutral red takes time to stain
d) Glycerol delays growth
- At what temperature should Ashdown’s Agar be incubated?
a) 25°C
b) 37°C
c) 40°C
d) 55°C - How long should Ashdown’s Agar be observed for growth?
a) 24 hours
b) 48 hours
c) 96 hours to 7 days
d) 2 weeks - Which specimen is NOT ideal for Ashdown’s Agar?
a) Sputum
b) Blood (sterile site)
c) Wound swabs
d) Urine - What confirms B. pseudomallei after Ashdown’s Agar growth?
a) Catalase test
b) Latex agglutination
c) Oxidase test
d) Indole test
- A major limitation of Ashdown’s Agar is:
a) Overgrowth of Candida
b) Inhibition of some B. pseudomallei strains
c) Turns black after 24h
d) Only works at 25°C - Ashdown’s Agar is least effective for:
a) Sputum with high commensals
b) Pure cultures
c) Soil samples
d) Sterile fluids - Which antibiotic resistance helps identify B. pseudomallei?
a) Penicillin
b) Colistin
c) Vancomycin
d) Metronidazole - PCR vs. Ashdown’s Agar for B. pseudomallei detection:
a) PCR is faster but culture confirms viability
b) Ashdown’s Agar is obsolete
c) PCR cannot detect DNA
d) Both are equally slow
Ashdown’s Agar: A Selective Medium for Burkholderia pseudomallei
Ashdown’s agar is a specialized selective medium used for the isolation and identification of Burkholderia pseudomallei, the causative agent of melioidosis. Developed by Dr. T.W. Ashdown in 1979, this agar contains crystal violet and gentamicin, which inhibit the growth of most other bacteria while allowing B. pseudomallei to thrive. The medium is available from various suppliers, including HiMedia and Oxoid, each offering slightly different formulations. Researchers and microbiologists often refer to the Ashdown agar recipe, which typically includes tryptone, glycerol, and neutral red as key components, aiding in colony morphology differentiation. Due to its selectivity, Ashdown’s selective agar remains a gold standard in clinical and environmental microbiology for detecting this pathogenic bacterium.
For those studying microbiology, resources like Ashdown’s agar MCQs, FAQs, and mock tests are valuable tools for understanding its applications and preparation. These assessments often cover topics such as the medium’s composition, its inhibitory agents, and the distinctive colony characteristics of B. pseudomallei. The Ashdown test is particularly crucial in regions where melioidosis is endemic, helping ensure accurate diagnosis. Whether using Ashdown agar Oxoid or HiMedia’s version, consistency in preparation and interpretation is key for reliable results. By mastering the use of this medium, laboratories can significantly improve the detection and management of melioidosis cases worldwide.
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