Anaerobic Culture Techniques

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Anaerobic Culture Techniques

• 3rd Science Practical Seminar 1
• Vincent OFlaherty

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• Anaerobic microorganisms are widespread and very
  important
• Do not require oxygen for growth - often
  extremely toxic

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• 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

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• Obligate (strict) anaerobes - oxygen is toxic to
  these organisms, do not use oxygen as terminal
  electron acceptor
• Archaea such as methanogens and Bacteria, e.g
  Clostridia, Bacteriodes etc. etc.

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• 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.

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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).

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Anaerobic and Aerobic Respiration
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ROS production during respiration

• O2 e- gt O2- superoxide
  anion
• O2- e- 2H gt H2O2 hydrogen
  peroxide
• H2O2 e- H gt H2O OH. Hydroxyl
  radical
• OH. e- H gt H2O water

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• Organisms that use O2 have developed defence
  mechanisms to protect themselves from these toxic
  forms of oxygen - enymes
• Catalase H2O2 H2O2 gt 2H2O O2
• Peroxidase H2O2 NADH H gt 2H2O
  NAD
• Superoxide dismutase O2- O2- 2H gt H2O2
  O2

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Oxygen tolerance of bacteria is dependent on
which of these enzymes they possess.
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Anaerobic environments

• Anaerobic environments (low reduction potential)
  include
• Sediments of lakes, rivers and oceans bogs,
  marshes, flooded soils, intestinal tract of
  animals oral cavity of animals, deep undrground
  areas, e.g. oil packets and some aquifers
• Anaerobes also important in some infections, e.g.
  C. tetanii and C. perfringens important in deep
  puncture wound infections

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• Also microaerophiles like Campylobacter jejuni
  very important in medial terms
• For proper diagnosis and for studies of anaerobic
  environments culture techniques are desirable

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• Culture of anaerobes is extremely difficult due
  to the need to exclude oxygen, slow growth and
  complex growth requirements
• Molecular methods based on DNA analysis and
  direct microscopy have shown that we are largely
  ignorant of the microbial world and previously
  unknown diversity has been discovered

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Microbial Numbers in Natural Environments
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Culture methods

• Anaerobes differ in their sensitivity to oxygen
  and the culture methods employed reflect this -
  some are simple and suitable for less sensitive
  organisms, others more complex but necessary for
  fastidious anaerobes
• Vessels filled to the top with culture medium can
  be used for organisms not too sensitive

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• 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

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• Deep culture tubes can be used to test whether an
  unknown organism is anaerobic/facultative or
  aerobic
• Thioglycollate added to culture medium, oxygen
  only found near top where it can diffuse from air
  -pattern of colony formation characteristic of
  organisms

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Redox potential
500 mV
- 300 mV
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• Pyrogallic acid-sodium hydroxide method can be
  used, again relies on a chemical reaction to
  generate an anaerobic environment, but a catalyst
  rather than a reducing agent
• Anaerobic jars (GasPak System) are sued to
  incubate plates in an anaerobic atmosphere,
  useful if brief exposure to oxygen is not lethal

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P. aeruginosa Strict aerobe
Enterococcus Facultative Grows aerobic or
anaerobic.
Bacteriodes fragilis
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Culture of strict anaerobes

• For culture of strict anaerobes all traces of
  oxygen must be removed from medium and for many
  organisms sample must be kept entirely anaerobic
  during manipulations
• Methanogenic archaea from rumen and sewage
  treatment plants killed by even a brief exposure
  to O2
• Medium usually boiled during preparation and
  reducing agent added, stored under O2-free
  atmosphere

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• Manipulations usually carried out under a jet of
  O2-free N2 or N2/CO2 to exclude O2
• Roll-tube (Hungate) method often used instead of
  conventional plates for isolation and culture of
  strict anaerobes

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1.Exclude oxygen by flushing the tube with the
desired gas 2. Place 4.5ml of pre-reduced
anaerobic agar medium into tube 3. Seal the tube
with the butyl rubber stopper and screw
cap 4.Autoclave the tube 5.Inoculate with a
syringe 6.Prepare on roll tube
spinner 7.Incubate in water bath
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• Use of anaerobic cabinet/glove box allows
  conventional bacteriological techniques e.g.
  replica plating, antibiotic sensitivity testing
  etc. to be carried out anaerobically

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