General Microbiology LaboratoryGeneral Microbiology Laboratory

Bacteria Oxygen RequirementsBacteria Oxygen Requirements

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Bacterial requirements for growthBacterial 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

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• Oxygen• Both useful and harmful

• Useful in respiration• Harmful because is a strong oxidizing

agent

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Oxygen RequirementsOxygen 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.

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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)

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3- Aerotolerant anaerobes• the bacteria don’t use oxygen, but oxygen do

esn’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.

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5- Microaerophilic bacteria• grow well in low oxygen concentrations and higher car

bon 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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Why is oxygen is harmful?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 ( O2-) - 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

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Oxygen detoxificationOxygen 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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Oxygen detoxificationOxygen detoxification

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The requirements for growthThe 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)

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Anaerobic Bacteria

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Categories Based Upon Oxygen RequirementsCategories Based Upon Oxygen Requirements

• Aerobic bacteriaRequire oxygen as electron acceptor• Microaerophilic bacteria Require oxygen in reduced quantity (2-10%) • Capnophilic bacteriaRequire carbon dioxide• Facultative bacteriaGrow either with or without oxygen• Anaerobic bacteriaBoth obligate and aerotolerant (Aerotolerant do not use aerobic metabolism but have

some enzymes that detoxify toxic forms of oxygen)

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Physiology And Growth ConditionsPhysiology 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.

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Anaerobic bacteriaAnaerobic bacteria

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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.

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Methods for excluding oxygenMethods 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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Gas Pack SystemGas Pack System

2- A: Anaerobic JarInoculate 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 jarIncubate overnight at 37C

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Anaerobic JarAnaerobic Jar

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Anaerobic JarAnaerobic 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.

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2-B: Gas Pak Pouch2-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.

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2-C: Candle jars2-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.

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Growing Microaerophilic MicrobesGrowing Microaerophilic Microbes

CO2 Generating Packet

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3: Anaerobic Chambers3: 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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Anaerobic Bacteria Of Clinical ImportanceAnaerobic Bacteria Of Clinical Importance

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End of lecture