- Slides: 25
Bacteria oxygen requirements & Anaerobic Bacteria
Introduction v. Bacteria can exist in a wide variety of environments, including environments that lack oxygen. v. In fact, oxygen is toxic to some bacteria. This toxicity is usually due to the accidental formation of superoxides (O 2 -) and hydrogen peroxide (H 2 O 2), which react with other cell components. v. Oxygen Species is harmful if : 1) Bacteria have no enzyme system to eliminate it. 2) Oxygen Species concentration is very high in media or air.
Toxic Forms of Oxygen § Singlet oxygen (O 2) : is in high energy state & extremely reactive, exists in phagocytes. § Superoxide free radical (O 2 -) & hydrogen peroxide (H 2 O 2): is so toxic to cell , formed in small amount in cellular respiration. § Peroxide anion (O 2 -2) : used as antimicrobial agent. § Hydroxyl radical (OH-) : the most active form, form in the cytoplasm from respiration.
Oxygen Detoxification v. Toxic forms of oxygen need to be neutralized by Enzyme systems. § Superoxide dismutase § Catalase § Peroxidase v. If microbe does not produce these enzymes, must have anaerobic conditions. v. 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. .
Enzyme systems 1) Super oxide dismutase : Converts the superoxide ion to hydrogen peroxide in the existence of hydrogen ion. 2) Catalase : breaks up hydrogen peroxide to water and oxygen. 3) Peroxidase : converts hydrogen peroxide to water in presence of hydrogen ions (NADH).
Oxygen Requirements Classifications v. Bacteria classified based on their oxygen requirements into 5 main classifications: 1) Obligate aerobes : v. Require oxygen to live. v. Their metabolic pathways require oxygen as the final electron acceptor (aerobic respiration). v. They have no alternate means of producing ATP. 2) Facultative anaerobes : vgrows in best where most oxygen is presented. v. Utilize oxygen and aerobic respiration whenever possible. v. They can undergo anaerobic respiration and/or fermentation to survive without oxygen.
3) 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. 4) Aerotolerant anaerobes : § Growth occurs evenly, oxygen has no effect. § The bacteria don’t use oxygen, but oxygen doesn’t harm them § Utilize anaerobic respiration and/or fermentation to produce ATP. § They possess superoxide dismutase, and so are not affected by the toxic byproducts of oxygen. 5) Microaerophiles : § Growth occurs only where a law concentration of oxygen has diffused into medium. v. Note : Capnophilic bacteria Require carbon dioxide.
Brain-Heart Infusion Agar (BHIA) v. Components: Peptone mixture, Beef Heart Infusion, Calf Brain Infusion, Sodium Chloride, Dipotassium Phosphate, Dextrose, Agar 1. 5%. v. Bacteria oxygen requirements Test: ü Materials § § 3 Brain-heart infusion agar deeps. 1 Hot plate with water bath. 1 Ice water bath. 1 Thermometer.
ü Lab supplies: § Ice. § Nutrient broth cultures of Pseudomonas aeruginosa and Staphylococcus aureus(24 - 48 hrs. ). § Thioglycolate broth culture of Clostridium sporogenesor C. butyricum (48 - 72 hrs. ). v Procedure 1) Place the brain-heart infusion (BHI) agar deeps into a boiling water bath. 2) 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. 3) When the temperature reaches 45°C, remove the tubes from the bath. Be careful not to shake, stir or otherwise disturb the agar.
4) 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. 5) Incubate the deeps at 37°C. Check the growth at 24, 48 and 72 hours. 6) After incubation, note the pattern of growth in each tube. Compare your results to Figure at the end of the exercise.
1) Obligate aerobe--growth only at the surface. 2) Facultative anaerobe--growth throughout, often with heavier growth toward the top. 3) Obligate anaerobe--growth only at the bottom. 4) Aerotolerant anaerobe--relatively low growth throughout. 5) Microaerophile--growth just below the surface.
Anaerobic Bacteria v. 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.
Fluid Thioglycolate Broth v. Fluid Thioglycolate broth is a reducing medium & supports aerobic & anaerobic bacteria, it contains: § Sodium Thioglycolate removes O 2 from the medium § Resazurin as an oxidation reduction indicator that turn to pink in presence of O 2 § Small amount of agar presented to retard gases
Gas Pack System : Anaerobic Jar v. 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.
Gas Pack System : Gas Pak Pouch v. Procedure: 1) Dispense Gas. Pak liquid activating reagent into channel of pouch. 2) Place plates inside the pouch. 3) Lock in anaerobic environment with sealing bar and incubate.
Candle Jar v. 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.
CO 2 Generating Packet
Anaerobic Chambers v. The Vinyl Anaerobic Chambers provide a strict anaerobic atmosphere using a palladium catalyst and hydrogen gas mix of 5%.
END OF LECTURE
Prepared by : Supervisor : Dr. Abdelraouf A. Elmanama