2Anaerobic microorganisms are widespread and very important Do not require oxygen for growth - often extremely toxic
3Facultative anaerobes - can grow in the presence or absence of oxygen Obtain energy by both respiration and fermentationOxygen not toxic, some use nitrate (NO3-) or sulphate (SO42-) as a terminal electron acceptor under anaerobic conditions
4Obligate (strict) anaerobes - oxygen is toxic to these organisms, do not use oxygen as terminal electron acceptorArchaea such as methanogens and Bacteria, e.g Clostridia, Bacteriodes etc. etc.
5Microaerophilic organisms - require low levels of oxygen for growth, but cannot tolerate the levels present in the atmosphereAerotolerant Anaerobes: Metabolism is anaerobic but they are unaffected by the presence of oxygen.
6Oxygen ToxicityOxygen is used by aerobic and facultatively anaerobic organisms as its strong oxidising ability makes it an excellent electron acceptorDuring the stepwise reduction of oxygen, which takes place in respiration toxic and highly reactive intermediates are produced reactive oxygen species (ROS).
8ROS production during respiration O2 + e- => O superoxide anionO2- + e- + 2H+ => H2O hydrogen peroxideH2O2 + e- + H+ => H2O + OH. Hydroxyl radicalOH. + e- + H+ => H2O water
9Organisms that use O2 have developed defence mechanisms to protect themselves from these toxic forms of oxygen - enymesCatalase: H2O2 + H2O2 => 2H2O + O2Peroxidase: H2O2 + NADH + H+ => 2H2O NAD+Superoxide dismutase: O2- + O2- + 2H+ => H2O O2
10Oxygen tolerance of bacteria is dependent on which of these enzymes they possess.
11Anaerobic 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 aquifersAnaerobes also important in some infections, e.g. C. tetanii and C. perfringens important in deep puncture wound infections
14Also microaerophiles like Campylobacter jejuni very important in medial terms For proper diagnosis and for studies of anaerobic environments culture techniques are desirable
15Culture of anaerobes is extremely difficult due to the need to exclude oxygen, slow growth and complex growth requirementsMolecular 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
17Culture methodsAnaerobes 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 anaerobesVessels filled to the top with culture medium can be used for organisms not too sensitive
18Most 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, cysteineActs 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
19Deep culture tubes can be used to test whether an unknown organism is anaerobic/facultative or aerobicThioglycollate added to culture medium, oxygen only found near top where it can diffuse from air -pattern of colony formation characteristic of organisms
21Pyrogallic 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 agentAnaerobic jars (GasPak System) are sued to incubate plates in an anaerobic atmosphere, useful if brief exposure to oxygen is not lethal
24P. aeruginosa Strict aerobe Enterococcus FacultativeGrows aerobic or anaerobic.Bacteriodes fragilis
25Culture 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 manipulationsMethanogenic archaea from rumen and sewage treatment plants killed by even a brief exposure to O2Medium usually boiled during preparation and reducing agent added, stored under O2-free atmosphere
26Manipulations 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
271.Exclude oxygen by flushing the tube with the desired gas 2. Place 4.5ml of pre-reduced anaerobic agar medium into tube3. Seal the tube with the butyl rubber stopper and screw cap4.Autoclave the tube5.Inoculate with a syringe6.Prepare on roll tube spinner7.Incubate in water bath
28Use of anaerobic cabinet/glove box allows conventional bacteriological techniques e.g. replica plating, antibiotic sensitivity testing etc. to be carried out anaerobically