LAB - Anaerobic Bacteriology Flashcards
aerotolerant anaerobes
can proliferate in the presence of trace oxygens
factors contributing to O2 sensitivity may include:
- direct toxic effect of molecular oxygen
- toxic products of oxygen metabolism
- oxidation-reduction potential (Eh in millivolts)
- oxidized organic constituents of culture media
toxic products of O2 metabolism
- superoxide anion (O2-): accumulates when bacteria don’t have superoxide dismutase (SOD) to convert superoxide to hydrogen peroxide; anaerobic bacteria do not have this
- formation and accumulation of hydrogen peroxide: due to lack of catalase
> formerly believed that H2O2 toxicity was an important factor in oxygen intolerance but some anaerobes do possess catalase activity and some facultative organisms capable of growth in air are devoid of catalase activity
media used for the growth of anaerobes must have a low __ which is usually achieved by the addition of ______ ____. the presence of molecular oxygen hinders the achievement of a low __
Eh
reducing agents
Eh
these form in media stored in air and may be inhibitory to certain anaerobes
oxidized organic constituents of culture media
- ex: C. haemolyticum will not grow on blood agar which is exposed to air for 1-2 hrs but C. perfringens will still grow on blood agar which has been stored in presence of air for several weeks
- optimal recovery = fresh media or media that have been stored anaerobically are required
major genera of anaerobes in clinical infection are …
Actinomyces, Bacteroides, Clostridium, Fusobacterium, and Peptostreptococcus (exception = Peptococcus niger)
Certain hints that a given specimen is likely to contain anaerobic bacteria:
- foul odor
- location of infection = 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)
- unique gram stain morph
- failure to grow, aerobically, organisms seen on Gram stain of original exudate
- growth anaerobically
- characteristic colonies
good specimen in anaerobic bacteriology
- aspirated pus
- tissue
- body fluids
- transtracheal aspirates
- direct lung aspirates
- ‘sulfur granules’ from patients with suspected actinomycosis
these specimens are NOT cultured for anaerobic bacteria
specimens contaminated with normal flora
use of swab is often not suitable for these organisms
anaerobes
transport systems for anaerobes
designed to avoid exposure of specimen to O2
- tubes containing oxygen-free gas
- swabs sterilized under O2 free CO2 in tubes containing a non-nutritive transport medium, for example, Cary-Blair transport
media for anaerobes
- must be freshly prepared OR pre-reduced supplemented media (fastidious anaerobes with low redox potential)
supplemented media
media for cultivation of anaerobes
- 5% sheep blood yeast extract, hemin and menadione (vit. K) added to a rich nutrient agar bse such as brain-heart infusion agar
- reducing agents
these are added to media to provide a low redox potential for the growth of anaerobes
reducing agents
- beef heart tissue in chopped meat medium
- glucose
- ascorbic acid
- sodium thioglycolate
- cysteine hydrochloride
most commonly used reducing agent in anaerobic media
cysteine
These are used in fluid media for the cultivation of anaerobes
Eh indicators such as resazurin
media for primary isolation of anaerobes
- liquid = small #s in OG; backup cultures
- plated agar media
> non-selective enriched = hemin, vit. K1, yeast extract, reducing agent (cysteine)
> selective media = to prevent overgrowth of facultative bacteria
menadione
vitamin K
examples of non-selective enriched media for anaerobes
Columbia agar
Brucella agar
BHI bases
examples of selective media for anaerobes
Phenyl ethyl alcohol blood agar (PEA)
kanamycin-vancomycin-laked blood agar (KVLB)