1. Bacteria oxygen requirements
& Anaerobic Bacteria

2. Introduction
Bacteria can exist in a wide variety of environments, including environments that lack oxygen.
In fact, oxygen is toxic to some bacteria. This toxicity is usually due to the accidental formation of superoxides (O2-) and hydrogen peroxide (H2O2), which react with other cell components.
Oxygen Species is harmful if :
Bacteria have no enzyme system to eliminate it.
Oxygen Species concentration is very high in media or air.

3. Toxic Forms of Oxygen Singlet oxygen (O2) : Peroxide anion (O2-2) :
is in high energy state & extremely reactive, exists in phagocytes.
Superoxide free radical (O2-) & hydrogen peroxide (H2O2):
is so toxic to cell , formed in small amount in cellular respiration.
Peroxide anion (O2-2) :
used as antimicrobial agent.
Hydroxyl radical (OH-) :
the most active form, form in the cytoplasm from respiration.

4. Oxygen Detoxification
Toxic forms of oxygen need to be neutralized by Enzyme systems.
Superoxide dismutase
Catalase
Peroxidase
If microbe does not produce these enzymes, must have anaerobic conditions.
Cells that live in oxygen-containing environments have the enzymes superoxide dismutase and catalase (among others) that convert superoxides and peroxides to oxygen and water..

5. Enzyme systems
Super oxide dismutase : Converts the superoxide ion to hydrogen peroxide in the existence of hydrogen ion.
Catalase : breaks up hydrogen peroxide to water and oxygen.
Peroxidase : converts hydrogen peroxide to water in presence of hydrogen ions (NADH).

6. Oxygen Requirements Classifications
Bacteria classified based on their oxygen requirements into 5 main classifications:
Obligate aerobes :
Require oxygen to live.
Their metabolic pathways require oxygen as the final electron acceptor (aerobic respiration).
They have no alternate means of producing ATP.
Facultative anaerobes :
grows in best where most oxygen is presented.
Utilize oxygen and aerobic respiration whenever possible.
They can undergo anaerobic respiration and/or fermentation to survive without oxygen.

7. Note : Capnophilic bacteria Require carbon dioxide.
Obligate anaerobes :
Growth occurs only when there is no oxygen.
Use anaerobic respiration and/or fermentation to
generate ATP.
Lack superoxide dismutase , catalase & peroxidases
they die in the presence of oxygen.
Aerotolerant anaerobes :
Growth occurs evenly, oxygen has no effect.
Utilize anaerobic respiration and/or fermentation to produce ATP.
They possess superoxide dismutase, and so are not affected by the toxic byproducts of oxygen.
Microaerophiles :
Growth occurs only where a law concentration of oxygen has diffused into medium.
Note : Capnophilic bacteria Require carbon dioxide.

9. Brain-Heart Infusion Agar (BHIA)
Components:
Peptone mixture, Beef Heart Infusion, Calf Brain Infusion, Sodium Chloride, Dipotassium Phosphate, Dextrose, Agar 1.5%.
Bacteria oxygen requirements Test:
Materials
3 Brain-heart infusion agar deeps.
1 Hot plate with water bath.
1 Ice water bath.
1 Thermometer.

10. Lab supplies: Procedure
Ice.
Nutrient broth cultures of Pseudomonas aeruginosa and Staphylococcus aureus( hrs.).
Thioglycolate broth culture of Clostridium sporogenesor C. butyricum ( hrs.).
Procedure
Place the brain-heart infusion (BHI) agar deeps into a boiling water bath.
After the BHI agar melts, allow the water to boil for at least five more minutes. Turn the heat off, and place a thermometer in the bath. Monitor the temperature of the bath.
When the temperature reaches 45°C, remove the tubes from the bath. Be careful not to shake, stir or otherwise disturb the agar.

11. Inoculate each culture into a separate agar deep by carefully stabbing with an inoculating needle . Avoid shaking, stirring or getting bubbles into the medium. Immediately after inoculation, place the deeps upright in an ice-water bath until they are completely solid.
Incubate the deeps at 37°C. Check the growth at 24, 48 and 72 hours.
After incubation, note the pattern of growth in each tube. Compare your results to Figure at the end of the exercise.

12. Obligate aerobe--growth only at the surface.
Facultative anaerobe--growth throughout, often with heavier growth
toward the top.
Obligate anaerobe--growth only at the bottom.
Aerotolerant anaerobe--relatively low growth throughout.
Microaerophile--growth just below the surface.

13. Anaerobic Bacteria
Bacteria are often categorized according to their growth responses to atmospheric oxygen. This may vary from species that can grow only in the presence of oxygen to those that can grow only in the absence of oxygen.

15. Fluid Thioglycolate Broth
Fluid Thioglycolate broth is a reducing medium & supports aerobic & anaerobic bacteria, it contains:
Sodium Thioglycolate removes O2 from the medium
Resazurin as an oxidation reduction indicator that turn to pink in presence of O2
Small amount of agar presented to retard gases

16. Gas Pack System : Anaerobic Jar
Anaerobic jars such as the Gas Pak are vessels in which an anaerobic environment is generated after inoculated media are sealed into the chamber. Anaerobiasis is achieved by adding water to commercially available gas generator envelopes that are placed in the jar just prior to sealing. Chemicals in the envelope produce hydrogen gas and carbon dioxide. The hydrogen combines with free oxygen in the chamber to produce water. The carbon dioxide is required for the growth of certain organisms. A methylene blue indicator strip is usually placed in the jar. It turns colorless when the oxygen has been removed.

18. Gas Pack System : Gas Pak Pouch
Procedure:-
Dispense GasPak liquid activating reagent into channel of pouch.
Place plates inside the pouch.
Lock in anaerobic environment with sealing bar and incubate.

19. Candle Jar
The candle jar is used to create Microaerophile conditions. It is a large screw-capped container into which the medium is placed along with a candle. The candle is lit and the jar is sealed. The candle will burn and reduce the oxygen concentration.

20. CO2 Generating Packet

21. Anaerobic Chambers
The Vinyl Anaerobic Chambers provide a strict anaerobic atmosphere using a palladium catalyst and hydrogen gas mix of 5%. 

22. END OF LECTURE

23. Yasser M. Aldahdoh Zeina A. Shubair Dr. Abdelraouf A. Elmanama
Prepared by :
Yussuf M. Abd Elal Hanan I. Abu Hasira
Yasser M. Aldahdoh Zeina A. Shubair
Islam F. Hasouna
Supervisor :
Dr. Abdelraouf A. Elmanama