T2: Anaerobic Bacteriology Introduction Flashcards
a bacterium that is able to replicate without oxygen
An anaerobe
Anaerobes often require a ______ redox potential
low
Three types of Anaerobes
- Strict obligate anaerobes
- Moderage obligate anaerobes
- Aerotolerant anaerobes
Three types of Aerobes
- Facultative anaerobe
- Microaerophile
- Obligate aerobe
Strict obligate anaerobes
- 2 facts about oxygen
- Extremely oxygen sensitive
- Killed by oxygen
Strict obligate anaerobes
- 2 examples
Clostridium novyi and C. haemolyticum
Tolerate moderate exposure to Oxygen, Require an anaerobic environment for growth
Moderate Obligate Anaerobes
Example of a moderate obligate anaerobe
Bacteroides fragilis
Able to tolerate exposure to oxygen; able to grow slowly in presence of oxygen, grows best in anaerobic environment
Aerotolerant anaerobes
Three examples of Aerotolerant anaerobes
Clostridium tertium, C. carnis, and C. histolyticum
grow under aerobic or anaerobic conditions, classified as an aerobe
Facultative anaerobes
two examples of facultative anaerobes
E. coli and Staphylococcus spp
Need reduced oxygen (5%) and increased CO2 for growth
microaerophiles
Example of a microaerophile
Campylobacter spp
Require oxygen and usually don’t grow under anaerobic conditions
Obligate aerobes
two examples of obligate aerobes
Pseudomonas spp and Micrococcus spp
Where can anaerobes be found as normal flora?
Nose, Mouth, oropharynx, vagina, gastrointestinal tract, skin, colon, soil, and freshwater/saltwater sediments
In the colon ______ outnumber facultative anaerobes 1000:1
anaerobes
Most anaerobic infections are _________ infections caused by trauma to skin or mucous membranes
Endogenous
_______ infections are usually by spore forming GPRs and often from soil or ingestion of contaminated food
Exogenous
Infections are most often ________ ; containing multipe anaerobes
Polymicrobic
Factors that predispose a person to anaerobic infection
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 culture
Needle aspirates at active site of infection, tissue, blood (anaerobic blood cult. bottle), lower respiratory specimens, normally sterile body fluids, Stool (only for C. diff)
Unacceptable specimens for anaerobic culture
voided urine, upper respiratory specimens, stool (except for C. diff), vaginal or cervical, surface wounds; swabs are NOT appropriate (usually)
Direct smear microscopic evaluation may present with what two signs?
polymicrobial and pleomorphic
Characteristics of Myonecrosis (gangrene)
absence of inflammatory cells (WBCs), Large GPRs, gas, and possible spores
All primary plates should have these 3 requirements for anaerobes
1 vitamin K
2 hemin
3 yeast extract
Prior to inoculation what should be done to media?
1 Stored at room temp
2 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, and rumen fluid
What does PRAS stand for?
Pre-Reduced Anaerobically Sterilized; made with no oxygen exposure and sealed in oxygen 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 with esculin hydrolysis (differential)
Incubation Conditions
-Ambient Air
21% O2, 0.03% CO2
Incubation Conditions
-CO2
15-21% O2, 5-10% CO2
Incubation Conditions
-Microaerophilic
5% O2, 10% CO2
Incubation Conditions
-Anaerobic
0% O2, 5-10% CO2
List 3 anaerobic systems used in lab to maintain anaerobic conditions
1 Anaerobic Chamber (glove box)
2 Anaerobic Jars
3 Anaerobic Bags and Pouches
Advantages of using an anaerobe chamber (5)
Specimens never exposed to oxygen during set up, plate observed as often as desired, incubator option, large volume cultures, gloves allow for less Oxygen introduction
Disadvantages of using an anaerobe chamber (4)
Cumbersome, airlock, space, contamination
Anaerobe Chamber requirements (4)
Catalyst, desiccant, oxidation reduction indicator, and anaerobic gas (5% H2, 5-10% CO2, and 85-95% N2)
removes residual O2, often composed of palladium coated aluminum pellets
catalyst
used to absorb water
desiccant
CO2 needed for the growth of many anaerobes and inert ____ is used as filler for remaining % of anaerobic atmosphoere
N2
used to verify daily that anaerobic conditions are maintained, can use indicator such as methylene blue or resazurin
Oxidation Reduction Indicator
useful for small labs with SMALL anaerobe VOLUME
Anaerobic jars
Anaerobic jars must be incubated for ______ hours because 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
Anaerobic bags and pouches advantages
Transport of organisms, plates can be examined without removal from bag
Gas Liquid Chromatography 2 forms of extraction
1 Ether Extraction (Most common); short chain volatile acids
2 Chloroform Extraction; organic acids, non-volatile
Media used to grow anaerobes for gas liquid chromatography
PYG Broth (peptone yeast extract-glucose)
To detect fermentation large tubes contain ______ or ________ biochemical test media; reactions measure the _____ with a _______
PRAS or Non-PRAS; pH; Probe
List 3 tests for Proteolysis
1 Gelatin Hydrolysis
2 Milk Liquefaction
3 Meat Digestion
Two newer technologies that are very reliable for ID of bacteria including anaerobes
16S rRNA sequencing and MALDI-TOF