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

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

3. 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

4. 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.

5. 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.

6. 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).

7. 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

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

9. 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+ => H2O O2

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

11. 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

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

15. 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

16. Microbial Numbers in Natural Environments
Habitat
Typical counts
Cultivable (%)
Soil
cells/g
Lakes/rivers
cells/ml
Ground water
cells/ml
<1
Marine (surface)
cells/ml
Marine (depth) ND
Sediments
cells/g

17. 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

18. 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

19. 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

20. Redox potential
+500 mV
- 300 mV

21. 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

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

25. 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

26. 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

27. 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

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