Anaerobic Culture Techniques 3rd Science Practical Seminar 1 Vincent O’Flaherty

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

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 Archaea such as methanogens and Bacteria, e.g Clostridia, Bacteriodes etc. 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.

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).

Anaerobic and Aerobic Respiration Reaction name Reduct. Oxid. Reaction Stoichiometry kcal/mol Aerobic Respiration CHO O2 C6H12O6 + 6O2 ==> 6CO2 + 6H2O 686 Nitrate Reduction NO3- CHO + NO3- + H+ ==> CO2+ N2+ H2O 649 Sulfate Reduction SO42- 2CHO + SO42-+2H+ => 2CO2+ H+ 2H2O 190 Methanogenesis CHO or H2 CO2 4H2 + CO2 ==> CH4 + 2H2O 8.3

ROS production during respiration O2 + e- => O2- superoxide anion O2- + e- + 2H+ => H2O2 hydrogen peroxide H2O2 + e- + H+ => H2O + OH. Hydroxyl radical OH. + e- + H+ => H2O water

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

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

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

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

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

Microbial Numbers in Natural Environments Habitat Typical counts Cultivable (%) Soil 109-1010 cells/g 0.01-0.1 Lakes/rivers 106-107 cells/ml Ground water 104-105 cells/ml <1 Marine (surface) 104-106 cells/ml 0.001-0.1 Marine (depth) ND Sediments 106-109 cells/g

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

Most common adaptation of media is the addition of a reducing agent, e 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

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

Redox potential +500 mV - 300 mV

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

P. aeruginosa Strict aerobe Enterococcus Facultative Grows aerobic or anaerobic. Bacteriodes fragilis

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

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

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

Use of anaerobic cabinet/glove box allows conventional bacteriological techniques e.g. replica plating, antibiotic sensitivity testing etc. to be carried out anaerobically