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 •CHO •NO3- •CHO + NO3

- + H+ ==> CO2+ N2+ H2O

•649

•Sulfate Reduction •CHO •SO42- •2CHO + SO4

2-+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 •0.01-0.1

•Ground water •104-105 cells/ml •<1

•Marine (surface)

•104-106 cells/ml •0.001-0.1

•Marine (depth) •104-105 cells/ml •ND

•Sediments •106-109 cells/g •<1

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.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

+500 mV

- 300 mV

Redox potential

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 FacultativeGrows 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