LAB 13 – ANAEROBIC BACTERIOLOGY Flashcards
What factors contribute to O2 sensitivity of anaerobes? How do they do this?
- Direct toxic effects of oxygen
- Toxic products of oxygen metabolism
- Strict anaerobes lack superoxide dismutase (SOD), which converts superoxide anion (O2-) to H2O2
- Some anaerobes lack catalase, which neutralizes H2O2 - Oxidation-reduction potential (Eh) must be low
- Oxidized organic constituents of media form in media stored in O2:
- C. perfringens can grow on BAP stored for weeks
- C. haemolyticum cannot grow if media exposed for 1-2 hrs
What clues may signal that a patient has an anaerobic infection?
- foul odor
- location of infection in proximity to mucosal surface
- infections secondary to human or animal bite
- gas
- previous therapy with aminoglycoside antibiotics (gentamicin, tobramycin, amikacin)
- black discoloration of blood-containing exudates (may fluoresce red)
- sulfur granules in discharges (Actinomyces)
- failure to grow, aerobically, organisms seen on Gram stain of original exudate
- growth anaerobically
- characteristic colonies
How long should primary anaerobic plates be incubated for?
at least 2 days/ 48 hours
Why are transport systems used for anaerobic specimens? Name one.
designed to avoid exposure of specimen to oxygen (Cary-Blair transport)
What media is used to isolate Clostridium difficile? What type of media is it?
Cycloserine-Cefoxitin-egg yolk-Fructose agar (CCFA):
- selective for flat, gray to white rhizoid colonies (flt gry/ wh rhz)
- yellowish under regular light
- yellow-green fluorescence under UV
BAP* or PEA: flt gry/ wh rhz
PEA - Phenyl Ethyl Alcohol agar
Why are reducing agents used for anaerobic media? What is the most commonly used reducing agent?
- Reducing agents prevent formation of oxidized constituents in media
Eg. cysteine
What are 2 indicators used to detect anaerobiosis?
- Methylene blue
- colorless when reduced
- blue when oxidized - Resazurin
- colorless when reduced
- pink when oxidized
Describe EYA agar and the 2 things it detects.
Egg-Yolk Agar detects enzymes produced by Clostridium sp.:
1. Lipase
2. Lecithinase
What are 2 conditions caused by Clostridium difficile? What causes them?
- Antibiotic-associated diarrhea
- Pseudomembrane colitis
- due to Toxins A and B
What are some diseases/conditions that Clostridium species cause?
C. botulinum = Botulism
C. tetani = Tetanus
C. perfringens = Food poisoning, gas gangrene
C. difficile = Antibiotic-associated diarrhea and pseudomembrane colitis (toxins A and B)
How would you work up a Gram positive bacilli, sporulating anaerobe?
- suspected Clostridium sp.
1. Aerotolerance test
2. Colony morph
3. Gram stain
4. EYA lipase + lecithinase
5. Reverse CAMP
6. GLC
Why do we do aerotolerance?
To confirm that a suspect isolate is actually an anaerobe
Describe the Nagler inhibition test.
- neutralization of C. perfringens alpha toxin (lecithinase) by an antitoxin
- 3-4 drops antitoxin is spread on half of EYA
- C. perfringens is inoculated heavily on both halves
Result: C. perfringens will grow on both halves, however lecithin will be precipitated (wide zone of opacity) only on the half without antitoxin - alpha toxin (lecithinase) acitivty is neutralized on the other half
What is the identification scheme for an anaerobic Gram negative bacilli?
- Aerotolerance test
2.Colony morph - Gram stain
- Antibiotic susceptibility
- Biochemical tests
- GLC
B. fragilis group ID
- g-b, pleo with capsule
- shy gy
- most frequently isolated anaerobes
- found in mixed infections of abdominal cavity
- Bile esculin agar (+)
- penicillin (B-lactamase), kanamycin, vancomycin, and colistin resistant
