2. General Microbiology Laboratory Bacteria Oxygen Requirements

3. Mohammed laqqan Bacterial requirements for growth  Source of energy  Source of "organic" carbon  sugars  fatty acids  Source of metal ions (e.g. iron)  Optimal temperature  Optimal pH  The need or lack of need for oxygen

4. Mohammed laqqan Oxygen Both useful and harmful Useful in respiration Harmful because is a strong oxidizing agent

5. Mohammed laqqan Oxygen Requirements  Bacteria classified based on their oxygen requirements into 5 main classifications: 1- Obligate or strict aerobes lack of oxygen kills the bacteria; ex. Bacillus & Pseudomonas they can not carry out fermentation they use O2 as a final electron acceptor in aerobic respiration.

6. Mohammed laqqan 2- Obligate or strict anaerobes do not carry out oxidative phosphorylation Obligate anaerobic may live by fermentation, anaerobic respiration, bacterial photosynthesis. oxygen kills the bacteria; ex. Clostridium tetani & Bacteroides lack certain enzymes superoxide dismutase O2-+2H+ to H2O2 catalase (H2O2 to H20 + O2) peroxidases (H2O2 to H20 using NAD to NADH)

7. Mohammed laqqan 3- Aerotolerant anaerobes the bacteria don’t use oxygen, but oxygen doesn’t harm them; ex. Lactobacillus They live by fermentation alone whether or not O2 is present in their environment. (respire anaerobically) 4- Facultative anaerobes can shift their metabolism (anaerobic if oxygen is absent or aerobic if oxygen is present); ex. E. coli, Staphylococcus Under anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but in the presence of O2 they switch to aerobic respiration.

8. Mohammed laqqan 5- Microaerophilic bacteria grow well in low oxygen concentrations and higher carbon dioxide concentrations; ex. Campylobacter & Heliobacter pylori killed by higher concentrations of oxygen mixture of 5 % O2, 10 % CO2 and 85 % N2

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10. Why is oxygen is harmful? Toxic forms of oxygen  Singlet oxygen-normal molecular oxygen that has been boosted into high energy state.  Superoxide free radicals ( O 2 - ) - formed by all living organisms during respiration.  Peroxide anion-found in hydrogen peroxide.  Toxic forms of oxygen It is a strong oxidizing agent It pulls electrons off other molecules toxic to cells must be neutralized

11. Mohammed laqqan Oxygen detoxification  Toxic forms of oxygen need to be neutralized by enzymes Superoxide dismutase Catalase Peroxidase  If microbe does not produce these enzymes, must have anaerobic conditions.  Superoxide dismutase (SOD) O2 + O2 + 2 H+ -- SOD --- H2O2 SOD- neutralizes oxygen free radicals *ok, but hydrogen peroxide also toxic…  Catalase 2 H2O2 ------Catalase---- 2 H2O + O2 – Obligate anaerobes lack catalase and superoxide dismutase- they die in the presence of oxygen

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13. Oxygen detoxification

14. Mohammed laqqan The requirements for growth  SOD  Obligate aerobes  Facultative anaerobes  Aerotolerant anaerobes  (Not obligate anaerobes)  (Usually not in microaerophiles)  CATALASE  Obligate aerobes  Facultative anaerobes  (not aerotolerant anaerobes)  (Not obligate anaerobes)  (Usually not in microaerophiles)

15. Mohammed laqqan Anaerobic Bacteria

16. Mohammed laqqan Categories Based Upon Oxygen Requirements Aerobic bacteria Require oxygen as electron acceptor Microaerophilic bacteria Require oxygen in reduced quantity (2-10%) Capnophilic bacteria Require carbon dioxide Facultative bacteria Grow either with or without oxygen Anaerobic bacteria Both obligate and aerotolerant (Aerotolerant do not use aerobic metabolism but have some enzymes that detoxify toxic forms of oxygen)

17. Mohammed laqqan Physiology And Growth Conditions  Anaerobic bacteria will not grow in the presence of oxygen.  Possible mechanisms: Lack of cytochrome systems for the metabolism of O2. Most lack superoxide dismutase (SOD) and catalase. Obligate anaerobe - lack cytochrome, SOD and catalase. Aerotolerant anaerobe - has some SOD and or catalase. Facultative anaerobe - grow equally well under either aerobic or anaerobic conditions.

18. Mohammed laqqan Anaerobic bacteria

19. Mohammed laqqan  Ability of anaerobes to tolerate oxygen or grow in its presence varies from species to species. Most anaerobic clinical isolates are moderately obligate anaerobes, and have small amount of both catalase and superoxide dismutase.

20. Mohammed laqqan Methods for excluding oxygen 1. Fluid Thioglycollate Media (FTM)  supports aerobic & anaerobic bacteria  – Sodium thioglycollate removes O2 from the medium  – Resazurin as an oxidation reduction indicator. When = dye that turns pink in presence of O2

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24. Gas Pack System 2- A: Anaerobic Jar  Inoculate FTM tube : B. subtilis or P. aeruginosa (strict aerobes) S. pyogenes (microaerophile) E. coli (facultative anaerobe) C. sporogenes (strict anaerobe)  Place in anaerobic jar with lighted candle & seal jar  Incubate overnight at 37C

25. Mohammed laqqan Anaerobic Jar

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27. Anaerobic Jar  For this exercise, we use the GasPak anaerobic system. This system consists of a polycarbonate jar, a lid with a gasket to prevent air flow, a strip infused with methylene blue, and a pouch containing sodium borohydride, sodium bicarbonate, citric acid, and a palladium catalyst.  When water is added to the pouch, the sodium borohydride, sodium bicarbonate, and citric acid react to form hydrogen and carbon dioxide. The palladium catalyzes a reaction between the hydrogen and the oxygen within the jar; this reaction creates water, which forms as condensation on the inside of the jar.  Methylene blue is blue in the presence of oxygen but is colorless in an anaerobic environment. When the oxygen is converted to water and condensation forms on the side of the jar, the indicator strip will turn from blue to white.

28. Mohammed laqqan 2-B: Gas Pak Pouch  Procedure for GasPak Pouch. (a) Dispense GasPak liquid activating reagent into channel of pouch. (b) Place plates inside the pouch. (c) Lock in anaerobic environment with sealing bar and incubate.

29. Mohammed laqqan 2-C: Candle jars  The candle flame will consume most of the oxygen in the jar.  Produce an elevated level of carbon dioxide.  These conditions are ideal for the growth of microaerophilic organisms.

30. Mohammed laqqan Growing Microaerophilic Microbes CO2 Generating Packet

31. Mohammed laqqan 3: Anaerobic Chambers  The Vinyl Anaerobic Chambers provide a strict anaerobic atmosphere of 0-5 parts per million (ppm) using a palladium catalyst and hydrogen gas mix of 5%.

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34. Anaerobic Bacteria Of Clinical Importance

35. Mohammed laqqan End of lecture