Anaerobe Introduction Flashcards
A bacterium that is able to replicate w/o O2
Anaerobe
Anaerobes often require a ____ redox potential
Low
3 types of anaerobes
- Strict obligate anaerobes
- Moderate obligate anaerobes
- Aerotolerant anaerobes
3 types of aerobes
- Facultative anaerobe
- Microaerophile
- Obligate aerobe
Strict obligate anaerobes
- 2 facts about O2
- 2 examples
- Extremely sensitive; killed by O2
- Clostridium novyi, C. haemolyticum
Tolerate moderate exposure to O2; requires an anaerobic environment for growth
Moderate obligate anaerobes
Example of a moderate obligate anaerobe
Bacteroides fragilis
Able to tolerate exposure to O2; able to grow more slowly in presence of O2, grows best in anaerobic environment
Aerotolerant anaerobes
3 examples of aerotolerant anaerobes
- Clostridium tertium
- Clostridium carnis
- Clostridium histolyticum
Grows under aerobic and anaerobic conditions; classified as an aerobe
Facultative anaerobes
2 examples of facultative anaerobes
- E. coli
- Staphylococcus sp.
Need reduced O2 (5%) and increased CO2 for growth
Microaerophiles
Example of a microaerophile
Campylobacter sp
Requires O2 and usually don’t grow under anaerobic conditions
Obligate aerobes
2 examples of obligate aerobes
- Pseudomonas spp
- Micrococcus spp
Where can anaerobes be found as normal flora?
- Nose
- Mouth
- Oropharynx
- Vagina
- Gastrointestinal tract
- Skin
- Colon
- Soil
- Freshwater/saltwater sediments
In the colon, ____ outnumber facultative anaerobes 1000:1
Anaerobes
Most anaerobic infections are ____ infections caused by trauma to kin or mucus membranes
Endogenous
____ infections are usually by spore forming GPRs and often from soil or ingetsion of contaminated food
Exogenous
Infections are most often ____; containing multiple anaerobes
Polymicrobial
Factors that predispose a person to anaerobic infections
- Vascular stasis
- Human/animal bite wounds
- Aspiration of oral contents into lungs after vomiting
- Tooth extraction
- Oral surgery
- Puncture of oral cavity
- GI tract surgery or traumatic puncture of bowel
- Genital tract surgery or traumatic puncture of genital tract
- Introduction of soil into a wound
List acceptable specimens for anaerobic cultures
- Needle aspirates at active site of infection, tissue, blood (anaerobic blood culture bottle)
- Lower respiratory specimens
- Normally sterile body fluids
- Stool (only for C. difficle)
List unacceptable specimens for anaerobic culture
- Voided urine
- Upper respiratory specimens
- Stool (except for C. difficle)
- Vaginal or cervical
- Surface wounds
- Swabs are NOT appropriate (usually)
Direct smear microscopic evaluation may present w/ what two signs?
Polymicrobial and pleomorphic
Characteristics of myonecrosis (gangrene)
- Absence of inflammatory cells (WBCs)
- Large GPRs
- Gas
- Possible spores
All primary plates should have these 3 requirements for anaerobes
- Vitamin K
- Hemin
- Yeast extract
Prior to inoculation what should be done to media?
- Stored at room temperature
- Pre-reduced (ensures redox potential stays low)
After inoculation, media should be stored in a holding chamber for less than ____ _____ or placed in the anaerobic chamber immediately
1 hour
List 5 reducing agents that can be added to media to maintain an anaerobic environment
- Enriched thioglycollate (THIO)
- Chopped meat
- Serum
- Cysteine
- Rumen fluid
What does PRAS stand for?
Pre-Reduced Anaerobically Sterilized
- Made w/ no O2 exposure and sealed in O2-free tubes
Purpose of ABAP
Anaerobe blood agar plate; non-selective
Purpose of PEA
Phenylethyl alcohol; anaerobe blood agar; selective; supports Gram positive
Purpose of PV, KV, and LKV
Paromomycin-Vancomycin; Kanamycin-Vancomycin; and Laked Blood Vancomycin; selective isolation of Gram negative anaerobes
Purpose of BBE
Bacteroides Bile Esculin; supports growth of bacteroides (selective); media turns black w/ esculin hydrolysis (differential)
Ambient air incubation conditions
- % O2 and CO2
21% O2, 0.03% CO2
CO2 incubation conditions
- % O2 and CO2
15-21% O2, 5-10% CO2
Microaerophilic incubation conditions
- % O2 and CO2
5% O2, 10% CO2
Anaerobic incubation conditions
- % O2 and CO2
0% O2, 5-10% CO2
List 3 anaerobic systems used in lab to maintain anaerobic conditions
- Anaerobic chamber (glove box)
- Anaerobic jars
- Anaerobic bags and pouches
5 advantages of using an anaerobe chamber
- Specimens are never exposed to O2 during set up
- Plate is observed as often as desired
- Incubator option
- Large volume cultures
- Gloves allow for O2 introduction
4 disadvantages of using an anaerobe chamber
- Cumbersome
- Airlock
- Space
- Contamination
4 anaerobe chamber requirements
- Catalyst
- Desiccant
- Oxidation reduction indicator
- Anaerobic gas (5% H2, 5-10% CO2, 85-95% N2)
Removes residual O2, often composed of palladium coated aluminum pellets
Catalyst
Used to absorb water; may be a silica gel
Desiccant
CO2 needed for the growth of many anaerobes and inert ____ is used as filler for remaining % of anaerobic atomosphere
N2
Used to verify daily that anaerobic conditions are maintained, can use indicator such as methylene blue or resazurin
Oxidation reduction indictator
Useful for small labs w/ SMALL anaerobe VOLUME
Anaerobic jars
Anaerobic jars must be incubated for ____ hours b/c especially susceptible
48 hours
Disadvantages of anaerobic jars
Cannot provide all advantages of a chamber, cost analysis over time shows chamber is better, plates must be removed from jar in order to examine and process
Advantages of anaerobic bags and pouches
Transport of organisms, plates can be examined w/o removal from bag
Gas liquid chromatography
- 2 forms of extraction
- Ether extraction (most common); short chain volatile acids
- Chloroform extraction; organic acids non-volatile
Media used to grow anaerobes for gas liquid chromatography
PYG broth (peptone yeast extract glucose)
Aerotolerance test
- Growth on SBA plate incubated aerobically in ambient air
- Growth on CHOC plate incubated anaerobically in CO2
- No growth on CDC BPA incubated anaerobically
Obligate aerobe
Aerotolerance test
- No growth on SBA plate incubated aerobically in ambient air
- Growth on CHOC plate incubated anaerobically in CO2
- No growth on CDC BPA incubated anaerobically
Capnophilic aerobe
Aerotolerance test
- Growth on SBA plate incubated aerobically in ambient air
- Growth on CHOC plate incubated anaerobically in CO2
- Growth on CDC BPA incubated anaerobically
Facultative anaerobe
Aerotolerance test
- No growth on SBA plate incubated aerobically in ambient air
- No growth on CHOC plate incubated anaerobically in CO2
- Growth on CDC BPA incubated anaerobically
Obligate anaerobe
Which form of GLC is most commonly used, volatile vs. non-volatile
Volatile
3 tests for proteolysis
- Gelatin hydrolysis
- Milk liquefaction
- Meat digestion
Gelatin hydrolysis
- Positive appearance
- Negative appearance
- Pos: liquid
- Neg: solid
Milk liquefaction
- Positive appearance
- Negative appearance
- Pos: curds or curdling/digestion
- Neg: no change
Meat digestion
- Positive appearance
- Negative appearance
- Strong pos: no individual particles
- Weak pos: some individual particles
- Neg: undigested particles
What media is used to anaerobic identification of biochemicals?
Egg yolk agar (EYA)
What reactions take place on EYA?
- Lecithinase
- Lipase
- Proteolysis
Lipase
- Positive test
- Pos: oil on water appearance
Lecithinase
- Positive test
- Pos: opaque appearance around colonies
Catalase
- Positive test
- Negative test
- Pos: bubbles
- Neg: no bubbles
Indole
- Positive test
- Negative test
- Pos: red
- Neg: no color change
Spot indole
- Positive test
- Negative test
- Pos: color change (blue/purple)
- Neg: no color change
Esculin
- Positive test
- Negative test
- Pos: black
- Neg: no color change
Growth in presence of bile (20%)
- Positive test
- Negative test
- Growth: orange and turbid
- Stimulated growth: opaque yellow
- No growth: no change
Urease
- Positive test
- Negative test
- Pos: pink
- Neg: yellow
2 newer technologies for identification of anaerobes
- 16S rRNA
- MALDI-TOF
