Charcoal Selective Medium 50 FAQs and 30 MCQs
Charcoal Selective Medium (CCDA), also known as Charcoal Cefoperazone Desoxycholate Agar, revolutionized the isolation of Campylobacter species from clinical and food specimens. Developed by Bolton et al. in 1984, this blood-free medium uses charcoal to replace traditional blood components while enhancing aerotolerance and selectivity for Campylobacter . Its optimized formulation with antibiotics like cefoperazone, vancomycin, and cycloheximide suppresses commensal flora, making it indispensable in diagnostic microbiology .
Key Features of Charcoal Selective Medium
1. Composition & Mechanism
CCDA combines:
- Nutrient Base: Beef extract, gelatin/casein peptones (nitrogen/vitamin sources).
- Aerotolerance Enhancers: Charcoal, hematin, sodium pyruvate, ferrous sulfate (quench toxic oxygen radicals) .
- Selective Agents:
- Cefoperazone (inhibits Gram-negative bacilli).
- Vancomycin (suppresses Gram-positive bacteria).
- Cycloheximide (blocks fungi) .
Table: Core Components of CCDA
| Component | Concentration | Function |
|---|---|---|
| Charcoal | 4.0 g/L | Detoxifier, reduces oxygen tension |
| Sodium desoxycholate | 1.0 g/L | Inhibits non-target bacteria |
| Cefoperazone | 32 mg/L | Targets Gram-negative enterics |
| Sodium pyruvate | 0.25 g/L | Enhances Campylobacter recovery |
2. Historical Significance
- 1984: Bolton’s team proved charcoal’s efficacy over blood-based media .
- 1986: Karmali added vancomycin/cycloheximide, boosting selectivity vs. Skirrow’s medium .
- 1991: Endtz confirmed superior Campylobacter recovery rates .
3. How It Works
- Charcoal absorbs toxins and reduces oxygen tension, mimicking microaerophilic conditions.
- Selective antibiotics inhibit contaminants (e.g., E. coli, Proteus), while sodium pyruvate supports Campylobacter growth .
4. Lab Applications
- Specimen Types: Stool (liquid/formed), rectal swabs, food samples .
- Incubation: 42°C for 48–72 hours in microaerophilic atmosphere (5% O₂, 10% CO₂, 85% N₂) .
- Colony ID: Two morphotypes:
- Type 1: Small, convex, grayish-brown, glistening.
- Type 2: Flat, mucoid, spreading colonies .
Best Practices for Lab Use
- Specimen Preparation:
- Emulsify formed stools in saline before streaking.
- Use fresh media for optimal colony morphology .
- Quality Control:
- Test with C. jejuni ATCC 33291 (growth) and E. coli ATCC 25922 (inhibition) .
- Limitations:
- Some C. coli and C. fetus strains are inhibited by cefoperazone.
- Confirm results with biochemical/MALDI-TOF testing .
Charcoal Selective Medium 50 FAQs
What is Charcoal Selective Medium used for?
It is used for selective isolation and presumptive identification of Campylobacter species from food and human fecal specimens.
Who first proposed charcoal as a substitute for blood in Campylobacter culture?
Bolton et al. in 1984 suggested that charcoal could efficiently replace blood in culture media for Campylobacter isolation.
Does charcoal improve Campylobacter isolation rates?
Yes, Endtz et al. confirmed that charcoal selective medium increases Campylobacter isolation rates.
What is the pH of Charcoal Selective Medium?
The pH is 7.4 ± 0.2 at 25°C.
What type of organisms does Charcoal Selective Medium target?
It selectively isolates Campylobacter species, particularly C. jejuni and C. coli.
What are the key ingredients in Charcoal Selective Medium?
Beef extract, gelatin peptone, sodium chloride, casein peptone, charcoal, sodium desoxycholate, ferrous sulfate, sodium pyruvate, cycloheximide, cefoperazone, hematin, vancomycin, and agar.
Why is beef extract included in the medium?
It supplies nitrogen, carbohydrates, vitamins, and other nutrients for Campylobacter growth.
What role does charcoal play in the medium?
Charcoal acts as a detoxifying agent, reduces oxygen tension, and improves aerotolerance.
Why is hematin added to the medium?
Hematin, along with sodium pyruvate and ferrous sulfate, helps neutralize toxic oxygen derivatives, aiding Campylobacter survival.
What is the purpose of sodium desoxycholate?
It inhibits some bacteria, making the medium selective for Campylobacter.
Why are antibiotics like cefoperazone and vancomycin included?
Cefoperazone suppresses Gram-negative enteric bacilli, while vancomycin inhibits Gram-positive bacteria.
What is the role of cycloheximide?
It acts as an antifungal agent, preventing yeast and mold contamination.
How is Charcoal Selective Medium prepared?
Components are dissolved in distilled water, heated to boiling, autoclaved (121°C, 15 psi, 15 min), cooled to 45-50°C, and poured into Petri dishes.
Why must the medium be shaken while dispensing?
To keep charcoal in suspension and ensure even distribution.
What sterilization method is used for the medium?
Autoclaving at 121°C for 15 minutes under 15 psi pressure.
At what temperature should the medium be cooled before pouring?
45-50°C to prevent solidification and ensure proper mixing.
At what temperature should Campylobacter jejuni be incubated?
42°C, as it is thermophilic and higher temperatures inhibit competing flora.
Why is 42°C incubation preferred for C. jejuni?
It enhances selectivity by inhibiting other microorganisms while promoting Campylobacter growth.
How long should incubation be extended to improve isolation rates?
Up to 72 hours may increase Campylobacter recovery.
What are the two types of colonies produced by C. jejuni?
Type 1: Small, raised, grayish-brown, smooth, glistening, with entire edges.
Type 2: Flat, mucoid, translucent, grayish, with irregular edges.Do Campylobacter colonies change appearance over time?
Yes, fresh medium may show flat/spreading colonies, while older medium forms 1-2 mm round, convex, glistening colonies.
Are Campylobacter colonies hemolytic?
No, they are non-hemolytic and may appear yellowish, gray, or pinkish.
What are the limitations of Charcoal Selective Medium?
Some Campylobacter strains (e.g., C. coli, C. fetus) may be inhibited by cephalosporins.
False negatives can occur if competing bacteria overgrow.Why should nonselective media also be used alongside selective media?
Some Campylobacter strains may be inhibited by selective agents, so nonselective media help ensure recovery.
Which Campylobacter species are inhibited by cephalosporins?
C. coli, C. fetus, and some C. jejuni strains.
Can Charcoal Selective Medium inhibit desired Campylobacter strains?
Yes, selective agents may sometimes inhibit target strains, especially if present in low numbers.
What additional tests are needed for complete Campylobacter identification?
Biochemical, immunological, molecular (PCR), or mass spectrometry (MALDI-TOF) testing.
How does charcoal improve Campylobacter aerotolerance?
It quenches toxic oxygen derivatives, creating a microaerophilic environment.
Why is sodium pyruvate included in the medium?
It helps neutralize reactive oxygen species, aiding Campylobacter survival.
What is the role of ferrous sulfate?
It supports bacterial metabolism and helps counteract oxidative stress.
How does sodium chloride contribute to the medium?
It maintains osmotic balance and cell integrity.
Can charcoal fully replace blood in Campylobacter culture media?
Yes, studies show it is an effective substitute for blood.
What are alternative selective agents for Campylobacter isolation?
Skirrow’s medium, Preston agar, and CCDA (Charcoal Cefoperazone Deoxycholate Agar).
How does Charcoal Selective Medium compare to blood-based media?
It provides similar or better isolation rates without requiring blood.
Why might Campylobacter fail to grow on this medium?
Overgrowth of competing bacteria, incorrect incubation temperature, or antibiotic inhibition.
What should be done if no growth is observed after 48 hours?
Extend incubation to 72 hours and check for slow-growing strains.
Extend incubation to 72 hours and check for slow-growing strains.
Ensure proper sterilization, aseptic techniques, and correct antibiotic concentrations.
Is Charcoal Selective Medium used in clinical diagnostics?
Yes, for isolating Campylobacter from human fecal specimens.
Can this medium be used for food testing?
Yes, it is used for detecting Campylobacter in food samples.
What specimens are suitable for culture on this medium?
Stool samples, food homogenates, and environmental swabs.
How should Charcoal Selective Medium be stored?
At 2-8°C, protected from light and moisture.
What is the shelf life of prepared plates?
Typically 4-6 weeks when stored properly.
Does Campylobacter require microaerophilic conditions?
Yes, it thrives in 5-10% oxygen, making charcoal beneficial for reducing oxygen tension.
Can this medium detect antibiotic-resistant Campylobacter strains?
Resistance to cefoperazone or vancomycin may affect growth, requiring alternative media.
Are there automated alternatives to charcoal-based culture?
PCR and ELISA tests offer rapid detection but culture remains the gold standard.
Have newer formulations improved Campylobacter isolation?
Modified charcoal media (e.g., CCDA) enhance recovery rates further.
What future improvements are needed for Campylobacter culture media?
Reducing antibiotic inhibition and improving fastidious strain recovery.
Can this medium be used for veterinary samples?
Yes, it is effective for isolating Campylobacter from animal feces.
Are there colorimetric indicators in this medium?
No, but colonies may appear grayish, pinkish, or yellowish.
What is the most critical factor for successful Campylobacter isolation?
Proper incubation conditions (42°C, microaerophilic) and extended incubation (up to 72h).
Charcoal Selective Medium 30 MCQs
- What is the primary purpose of Charcoal Selective Medium?
a) Isolate Salmonella
b) Isolate Campylobacter✔
c) Culture fungi
d) Detect viruses - Who first proposed charcoal as a substitute for blood in Campylobacter culture?
a) Endtz et al.
b) Bolton et al.✔
c) Skirrow
d) Preston - What is the pH of Charcoal Selective Medium?
a) 6.5 ± 0.2
b) 7.0 ± 0.2
c) 7.4 ± 0.2✔
d) 8.0 ± 0.2 - Which organism is primarily targeted by Charcoal Selective Medium?
a) Escherichia coli
b) Campylobacter jejuni✔
c) Staphylococcus aureus
d) Candida albicans - What does charcoal in the medium help neutralize?
a) Toxins
b) Oxygen radicals✔
c) Acids
d) Alkalis
- Which ingredient provides nitrogen and vitamins for Campylobacter growth?
a) Sodium chloride
b) Beef extract✔
c) Charcoal
d) Cycloheximide - What is the role of sodium desoxycholate in the medium?
a) Nutrient source
b) Selective inhibitor✔
c) pH buffer
d) Antifungal agent - Which antibiotic inhibits Gram-positive bacteria in the medium?
a) Cefoperazone
b) Vancomycin✔
c) Cycloheximide
d) Ampicillin - What is the function of ferrous sulfate in the medium?
a) Acts as an antifungal
b) Neutralizes oxygen toxicity✔
c) Provides electrolytes
d) Enhances pigment production - Which component prevents fungal contamination?
a) Cefoperazone
b) Cycloheximide✔
c) Hematin
d) Sodium pyruvate
- What is the autoclaving condition for Charcoal Selective Medium?
a) 100°C for 10 minutes
b) 121°C for 15 minutes✔
c) 115°C for 20 minutes
d) 130°C for 5 minutes - Why must the medium be shaken while dispensing?
a) To prevent contamination
b) To keep charcoal suspended✔
c) To enhance antibiotic activity
d) To speed up solidification - At what temperature should the medium be cooled before pouring?
a) 25-30°C
b) 37°C
c) 45-50°C✔
d) 60°C - What happens if the medium is poured too hot?
a) Charcoal settles unevenly✔
b) Antibiotics degrade
c) Agar solidifies too quickly
d) pH becomes alkaline - How should prepared plates be stored?
a) At room temperature
b) Frozen at -20°C
c) Refrigerated (2-8°C)✔
d) Exposed to light
- At what temperature should Campylobacter jejuni be incubated?
a) 25°C
b) 37°C
c) 42°C✔
d) 55°C - Why is 42°C incubation used for C. jejuni?
a) Kills other bacteria
b) Enhances pigment production
c) Selects for thermophilic Campylobacter✔
d) Prevents fungal growth - How long should incubation be extended to improve isolation rates?
a) 24 hours
b) 48 hours
c) 72 hours✔
d) 1 week - Which type of colony is raised and grayish-brown?
a) Type 1✔
b) Type 2
c) Type 3
d) Type 4 - Are Campylobacter colonies hemolytic?
a) Yes, always
b) No, never✔
c) Sometimes
d) Only on blood agar
- Which Campylobacter species may be inhibited by cephalosporins?
a) Only C. jejuni
b) C. coli and C. fetus✔
c) Only C. lari
d) None - Why should nonselective media be used alongside selective media?
a) To detect antibiotic resistance
b) To recover inhibited strains✔
c) To enhance pigment production
d) To speed up growth - What additional test is needed for definitive Campylobacter identification?
a) Gram staining
b) Biochemical tests✔
c) Blood culture
d) None - What happens if incubation is too short (<48 hours)?
a) No colonies form
b) Only contaminants grow
c) Slow-growing strains are missed✔
d) Medium dries out - Which factor is most critical for successful Campylobacter isolation?
a) High oxygen levels
b) Proper incubation temperature (42°C)✔
c) Low pH
d) Bright light exposure
- What is the role of sodium pyruvate in the medium?
a) Acts as an antibiotic
b) Neutralizes reactive oxygen species✔
c) Enhances fungal growth
d) Increases pH - Which alternative medium is used for Campylobacter isolation?
a) MacConkey agar
b) Skirrow’s medium✔
c) Sabouraud agar
d) Mannitol salt agar - Can Charcoal Selective Medium detect antibiotic-resistant Campylobacter?
a) Yes, always
b) No, never
c) Only if resistant to cefoperazone✔
d) Only if resistant to vancomycin - Which oxygen requirement does Campylobacter have?
a) Strict aerobe
b) Strict anaerobe
c) Microaerophilic✔
d) Facultative anaerobe - What is the shelf life of prepared Charcoal Selective Medium plates?
a) 1 week
b) 2 weeks
c) 4-6 weeks✔
d) 6 months
Conclusion
Charcoal Selective Medium remains a gold standard for Campylobacter isolation due to its cost-effectiveness, high selectivity, and compliance with CLSI protocols. Its unique composition addresses the oxygen sensitivity of Campylobacter while suppressing background flora, making it critical for outbreak investigations and diagnostic labs. For deeper insights into colony interpretation and troubleshooting, explore the 50 FAQs and 30 MCQs sections below.
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