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Why is oxygen toxic to anaerobes?
- Oxygen is a good reducer (O2 acceptor)
- Creates free radicals (O2-, H2O2, OH-)
- Anaerobes lack (or have low levels) of protective enzymes (superoxide dismutase, catalase)
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What are the two outcomes of O2 exposure to anaerobes?
- Bacteriostatic: for aerotolerant anaerobes
- e- for metabolic functions diverted to O, decreased growth
- reversible
- Bacteriocidal: strict anaerobes
- damanged in presence of O2 and radicals, cell damage/death
- irreversible
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What are some unacceptable specimens for anaerobic culture?
- Sputum
- feces
- gastric contents (shouldn't be any bacteria present)
- all swabs (if abs. necessary use w/ anaerobic transport media)
- urine
- *don't take samples exposed to O2
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Why must anaerobe specimens remain at RT?
refrigeration can oxygenate the specimen via condensation
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What are the 3 kinds of anaerobic transport systems?
- Rubber-stoppered collection vial for liquid specimens
- O2-free collection tube for swab specimens
- Self-contained anaerobic bag for tissue specimens
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What is PRAS media?
- Pre Reduced Anaerobically Sterilized
- Pre-packaged "ready-to-go" anaerobic media
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Brucella Agar
- Nonselective
- Vit K and Hemin help w/ Prevotella fluorescence
- Sheeps blood for hemolysis
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PEA agar
- Phenyl Ethyl Alcohol
- inhibits facultative anaerobic GNR (eg enteros)
- Inhibits swarming (Clostridia, Proteus)
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LKV agar
- Laked Kanamycin Vancomycin agar
- Helps detect Prevotella pigmentation (black)
- Vanc inhibits G+
- Kan inhibits aerobic GNR
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BBE agar
- Bacteroides Bile Esculin agar
- Detects esculin hydrolysis of Bacteroides sp
- Bile inhibits most other organisms
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Egg Yolk Agar
- Detects presence of lecithinase and lipase (Clostridium)
- lechithinase: opaque ppt around colonies
- lipase: iridescent sheen on colony surface
- proteolysis: clearing around colonies
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Chopped Meat Carbohydrate
- Enrichment broth that will "grow anything"
- Backup media, used for low # organisms
- Turbidity = growth
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Thioglycolate broth
Used to determine O2 usage based on where growth appears
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What are the general ways to speciate an anaerobe?
- Colony morphology
- Gram stain
- Antibiotic sensitivity
- handful of biochem tests
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What are the incubation conditions for anaerobic boxes and jars?
- 90% N2
- 5% CO2
- 5% H2 (too reactive)
- 35-37C for 48 hours
- *anaerobes are very fastidious, and very slow
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Describe the production of an anaerobic atmosphere in an anaerobic box
- Catalyst chamber: palladium pellets convert free O2 to H2O
- gas generator envelope: chemicals react using H2O or O2 and creating CO2 and H2
- Anaerobic indicator: indicator varies
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What is a roll tube? How is it used?
- Prepared tube with a thin coat of agar inside (agar prepared anaerobically)
- suspected anaerobic specimens are streaked along the walls of this tube and incubated
- Used as a quick visual check to see if any anaerobes are present
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What is unique about anaerobic gram stains?
- G- anaerobes stain poorly with safranin (leave on for 3-10 mins)
- Some G+ anaerobes will stain pink
- *THERE ARE NO SPORE FORMING GNRs
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Describe aerotolerance testing
- Two plates (anaBAP and choc) are divided into sections
- Colonies of interest are streaked into the corresponding section of each plate
- Plates are incubated overnight (anaBAP anaerobic and choc in capnophillic)
- growth on plates is compared to determine aerotolerance
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Describe the methods of presumptive ID and definitive ID for anaerobes
- presumptive: colony morph, spot tests, gram stains, antibiotic disc paterns
- definitive: catalase, spot indole, kanamycin, vancomycin, colistin, lipase, NH3- reduction
- *often kits
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What are KVC discs and what are the important patterns?
- An anaerobic isolate is streaked onto one Brucella plate w/ KVC discs
- Kanamcyin, Vancomycin, Colistin
- **PATTERNS GIVE PRELIM ID (KVC order)
- RRR: Bacteroides
- RRV: Prevotella
- SRS: Fusobacterium, Veillonella
- RSR: Porphyromonas
- SSR: G+ rod/cocci (except Peptostreptococcus anaerobius = RSR)
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What is unique about the anaerobic catalase test?
15% H2O2 rather than 3%
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What is unique about the anaerobic spot indole test?
- DMACA is used in place of Kovac's reagant
- A positive result is robin's egg blue, NOT PINK
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What is unique about the anaerobic CAMP test?
Staphylococcus aureus is replaced by Streptococcus agalactiae (group B strep) as 'center line'
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How are anaerobic biochemicals usually tested?
- Old: conventional methods (slow, dependant on growth, expensive)
- new: enzyme kits (no growth req, O2 incubation, rapid results)
- newest: PCR instead
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