Practical 7 ST
anaerobic cultivation
• Bacteria in relation to oxygen
• Anaerobic infections - sampling and
transport
• Cultivation
- establishing of anaerobic environment
- anaerobic culture media,
- procedure: inoculation, isolation
Anaerobes
• are the predominant components of the
skin’s and mucous membranes normal flora
- cause infections of endogenous origin
• mixed infections caused by numerous
aerobic and anaerobic bacteria are often
observed in clinical situations.
Anaerobic infections
• Usually endogenous bacteria - disturbance of the
balance of normal flora (ATB and colon microflora)
or unusual anatomic sites (perforation of colon -
anaerobic peritonitis)
• Exogenous bacteria - wound contamination
• Exudate, pus, abscess
• Sampling and transport in anaerobic conditions -
aspirate in the syringe with the needle capped or
protected with rubber. Surgical sampling can be
required. Never use the dry swab!
Anaerobic bacteria
can be divided into:
• strict anaerobes that can not grow in the
presence of more than 0.5% oxygen
• moderate anaerobic bacteria that are able of
growing between 2 to 8% oxygen.
• Anaerobic bacteria usually do not possess
catalase
• can generate superoxide dismutase which
protects them from oxygen.
• Facultative anaerobes - can grow in the presence
or absence of oxygen
• Obtain energy by both respiration and
fermentation
• Oxygen not toxic, some use nitrate (NO3-) or
sulphate (SO42-) as a terminal electron acceptor
under anaerobic conditions
• Obligate (strict) anaerobes - oxygen is toxic
to these organisms, do not use oxygen as
terminal electron acceptor
• e.g Clostridia, Bacteriodes etc....
• Microaerophilic organisms - require low levels of
oxygen for growth, but cannot tolerate the levels
present in the atmosphere
• Aerotolerant Anaerobes: Metabolism is anaerobic but
they are unaffected by the presence of oxygen.
Bacteria in relation to oxygen
• Obligate aerobic -require gaseous oxygen,
cannot grow without oxygen
• Obligate anaerobic - oxygen is toxic for
them. Methabolic pathway used for gaining
energy is fermentation with production of
foul-smelling end products.
• Facultative anaerobic - able to adapt to
aerobic or anaerobic
• Microaerophilic - require reduced oxygen
tension
Oxygen Toxicity
• Oxygen is used by aerobic and facultatively
anaerobic organisms as its strong oxidising ability
makes it an excellent electron acceptor
• During the stepwise reduction of oxygen, which
takes place in respiration toxic and highly reactive
intermediates are produced reactive oxygen species
(ROS).
The clinically important
anaerobes
• 1. Gram-negative rods (Bacteroides, Prevotella,
Porphyromonas, Fusobacterium,
• 2. Gram-positive cocci (primarily
Peptostreptococcus spp.);
• 3. Gram-positive spore-forming (Clostridium spp.)
and nonspore-forming bacilli (Actinomyces,
Propionibacterium, Eubacterium, Lactobacillus
and Bifidobacterium spp.);
• 4. Gram-negative cocci (mainly Veillonella)
Anaerobic environments
• Anaerobic environments (low reduction potential)
include:
• Sediments of lakes, rivers and oceans; flooded
soils, intestinal tract of animals; oral cavity of
animals, ...
• Anaerobes also important in some infections, e.g.
C. tetanii and C. perfringens important in deep
puncture wound infections
• Culture of anaerobes is extremely difficult due to
the need to exclude oxygen, slow growth and
complex growth requirements
Culture methods
• Most common adaptation of media is the addition
of a reducing agent, e.g. thioglycollate, cysteine
• Acts to reduce the oxygen to water, brings down
the redox potential -300mV or less.
• Can add a redox indicator such as rezazurin, pink
in the presence of oyxgen - colourless in its
absence
Redox potential
+500 mV
- 300 mV
Culture media
• Blood agar
• Blood agar - enriched with growths factors
and antibiotics (selective, kanamycin,
neomycin, aminoglycosides - to inhibit
most aerobic and facultative anaerobic
bacteria)
• VL agar, VF bouillon
• Should be stored in refrigerator in plastic
bag - decrease the solution of oxygen in the
agar - use freshly prepared media
Establishing of anaerobic conditions
• Candle jar - microaerophilic, facultative anaerobic
• Anaerobic jar with atmosphere generator (foil envelopes): release
hydrogen and carbon dioxide -after addition of water.
• Biological method - cultivation with Serratia marcescens
• Tubes of broth cultures (thioglycolate, cooked meat) - their
formulations contain reducing substances - anaerobic conditions at the
lower part - regeneration - 15 minutes in boiling water bath
Innoculation, isolation
• Innoculation without delay
• Innoculation of anaerobic and aerobic culture (anaerobic jar, candle jar or anaerobic
incubator)
• 48hrs
• Often mixed infection
• Comparison of aerobic and anaerobic culture
• Gram staining: principal is the presence of anaerobic infection G+coccus, G+rod, G-
coccus, G- rod, sporulation
Algorythm
• anaerobic jar aerobic incubator
• anaerobic
• facultatively
anaerobic
• aerobic
P. aeruginosa Strict aerobe
Enterococcus Facultative
Grows aerobic or anaerobic.
Bacteriodes fragilis
Anaerobic jar
Biological method
• Half of medium in the Petri´s dish is innoculated
with the tested sample, the other with Serratia
marcescens - bacteria able to
Biological sample
produce -------------------
anaerobic ----------------------- environment
by the Serratia consumption of
oxygen. marcescens Petri dishes is
sealed with the wax or parafin
and introduced to the not oxygen - free incubator
