Transcript Bacteria Oxygen Requirements

General Microbiology Laboratory
Bacteria Oxygen Requirements
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
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• Oxygen
• Both useful and harmful
• Useful in respiration
• Harmful because is a strong oxidizing
agent
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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.
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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
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.
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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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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 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 dismutasethey die in the presence of oxygen
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Oxygen detoxification
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The requirements for growth
SOD

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CATALASE
Obligate aerobes
Facultative anaerobes
Aerotolerant anaerobes
(Not obligate
anaerobes)
 (Usually not in
microaerophiles)
 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 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)
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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.
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Anaerobic 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 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 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
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Anaerobic Jar
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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.
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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.
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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.
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Growing Microaerophilic Microbes
CO2 Generating Packet
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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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Anaerobic Bacteria Of Clinical Importance
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End of lecture
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