Oxygen Requirement

Exercise 11 | MBC 101 G-1L | Palis, Maria Camille D.

Oxygen
Is molecular oxygen (O2) always needed to support life?
Great Oxygenation Event / Oxygen Revolution

Earliest signs of life are dated to a period when free

oxygen gas was nonexistent.

Cyanobacteria started releasing oxygen as a

byproduct of photosynthesis.

Oxygen levels increase in the atmosphere.

Great Oxygenation Event / Oxygen Revolution

It caused a massive extinction.

Most organisms could not survive the powerful

oxidative properties of oxygen (ROS)

The organisms that were able to detoxify reactive

oxygen species thrived in the new environment.
Oxygen

• Universal • Elemental • Highly

component constituent reactive
of cells of water gas
• Almost all and organic • Constitutes
plants and compounds 20% of the
animals earth’s
need it to atmosphere
live.
Oxygen

• Many • Despite the • Depending

ecosystems lack of upon the
/places are oxygen, oxygen
still free of some requirement
molecular organisms of bacteria,
oxygen can still they are
thrive. classified
into 5
groups
Oxygen Requirement of Bacteria

Source:https://courses.lumenlearning.com/microbiology/chapter/oxygen-requirements-for-microbial-growth/
Oxygen Requirement of Bacteria
Obligate Aerobe
• Require O2 as their final electron acceptor (cellular respiration)
• Live in atmospheric oxygen level (20%)

Microaerophiles
• Also require O2 as their final electron acceptor
• Can only live in environments with only 2-10% atmospheric oxygen
level

Facultative Anaerobe
• Use O2 as their final electron acceptor in aerobic environments
• Can also use alternate electron acceptors in anaerobic environments
• Thrive better in aerobic environments
Oxygen Requirement of Bacteria
Aerotolerant Anaerobe
• Never use O2 as their final electron acceptor
• Presence of oxygen does not affect their growth
• Fermentation of organic substrates
• May grow in both aerobic and anaerobic environments

Strict Anaerobe
• Never use O2 as their final electron acceptor
• Harmed by molecular oxygen
• Can only live in anaerobic environment
Exercise 8: Objective & Expected Outcomes

The students should be able to:

• determine the oxygen requirement of the unknown bacterium based on growth

pattern in fluid thioglycollate medium, catalase and oxidase test.

• discuss the principle behind the differences in oxygen requirement of bacteria

• explain the principle of the use of fluid thioglycollate medium

• describe the principles of catalase and oxidase tests in relation to the oxygen
requirement of bacteria
Procedures

A. Growth in Anaerobic Agar

B. Catalase Test
C. Oxidase Test
Fluid Thioglycollate Medium (FTM)

Components Quantity

Sodium Thioglycollate 0.5 g

Resazurin 0.001 g

Agar 7.5 g

Sodium chloride 2.5 g

Cysteine 0.5 g

Yeast extract 5g

Glucose (dextrose) 5.5 g

Casitone 15 g
Fluid Thioglycollate Medium (FTM)

Sodium Thioglycollate (Thioglycollate acid)

• Prevents the oxygen from entering the entire medium

Resazurin

• Indicator for the amount or presence of oxygen / redox potential indicator

• Colorless, but when oxidized, changes to red/pink

Agar

• Low percentage of agar allow motile bacteria to move throughout the

medium
• Higher viscosity of medium prevents rapid intake of oxygen
Fluid Thioglycollate Medium (FTM)

Sodium Thioglycollate and Cysteine

• Reducing agents responsible for anaerobiosis by binding with

free oxygen

Dextrose, peptone, cysteine, and yeast extract

• Provide growth factors necessary for growth

pH

• Neutral
A. Growth in Anaerobic Agar

Boil FTM tubes Inoculate Rapidly cool Incubate

• To remove • Tolerable • Dipping in • At 30°C for

excess temperature water 24-48h
oxygen • Molten
• Red color medium
shoud not
be greater
than 1/3 of
the medium
A. Growth in Anaerobic Agar

Source:https://courses.lumenlearning.com/microbiology/chapter/oxygen-requirements-for-microbial-growth/
Results

• Facultative • Obligate aerobe • Strict anaerobe

anaerobe • Growth only on top • Growth only at the
• More growth on top of the medium bottom of the
• Growth present medium
throughout

Escherichia Pseudomonas Clostridium

coli fluorescens sporogenes
Results

• Aerotolerant • Obligate • Microaerophile

anaerobe aerobe • Growth only
• Even growth • Growth only near, but not
throughout the on top of the on the surface
tube medium
Lactobacillus Pseudomonas Campylobacter
bulgaricus cerevisiae jejuni
Procedures

A. Growth in Anaerobic Agar

B. Catalase Test
C. Oxidase Test
B. Catalase Test

Reactive oxygen species (ROS)

• Highly unstable ions and molecules
 derived from partial reduction of oxygen

• Most organisms would not survive its powerful oxidative

properties due to extreme damage to cell.
steal an electron from neighboring molecules
the molecule then steals from another neighboring molecule
B. Catalase Test

Reactive oxygen species (ROS)

• Some of these toxic forms of oxygen (superoxide free
radicals, O2−)are formed in small amounts during
respiration
organisms that use oxygen as their final electron acceptor
In order for the organism to live, it must have the means to
neutralize it.
B. Catalase Test

Reactive oxygen species (ROS)

• Examples:
singlet oxygen (O2•)
superoxide (O−2)(O2−)
peroxides (H2O2)
hydroxyl radical (OH•)
hypochlorite ion (OCl−)
B. Catalase Test

• Obligate aerobes, facultative anaerobes, and aerotolerant

anaerobes
produce superoxide dismutase (SOD) to catalyze the
neutralization of O2− produced in respiration.

However, the produced H2O2 is also a reactive oxygen species.

B. Catalase Test

• To compensate for the production of H2O2 ,

the enzyme, catalase , is produced for the breakdown of hydrogen
peroxide into water and oxygen
B. Catalase Test

• Used to detect the presence of catalase enzyme

• Differentiates aerobic and obligate anaerobic bacteria
Anaerobes lack this enzyme, that’s why they cannot thrive in
aerobic environments (no means to neutralize ROS = cell damage)
B. Catalase Test

Deposit bacterial
+ 1-2 drops of Observe for
growth on a
3% H2O2 bubble formation
clean glass slide
B. Catalase Test

Source: https://courses.lumenlearning.com/microbiology/chapter/oxygen-requirements-for-microbial-growth/
Results

• Catalase- • Catalase-
positive negative
• Facultative • Obligate aerobe
anaerobe

Escherichia Lactobacillus
coli bulgaricus
Procedures

A. Growth in Anaerobic Agar

B. Catalase Test
C. Oxidase Test
C. Oxidase Test

Cytochrome System (hydrogen carrier system or electron transfer system)

• present in aerobic respiration

• most of the energy produced during respiration is made by this
system
C. Oxidase Test

Cytochrome System

Source: http://www.biology-online.org/1/3_respiration.htm
C. Oxidase Test

Cytochrome System

Source: http://osp.mans.edu.eg/medbiochem_mi/cources/biochemistry/2nd_year_medicine/Biological_Oxidation/files/Lecture_03.htm
C. Oxidase Test

• This test determines if a microorganism produces certain

cytochrome C oxidase enzymes.
• catalyze the transport of electrons from donor compounds to electron acceptors

• transfer of electrons to molecular oxygen to form water

• Use of reagent dyes such as Kovac’s reagent (oxidase rgt.)

• 0.5% N, N, N’, N’ -tetramethyl-p-phenylenediamine (TMPD)
• Acts as a redox indicator and as artificial electron donor.
C. Oxidase Test

cyt. C oxidase
2 reduced cyt. C + 2H+ + ½ O2 2 oxidized cyt. C + H2O
oxidized cyt. C + TMPD Wurster’s blue or Indophenol
C. Oxidase Test

Observe for
Moisten sterile Deposit a colony formation of blue
filter paper with on the filter violet color within
oxidase reagent paper 1 min
C. Oxidase Test

Source: https://microbiologyinpictures.com/bacteria-photos/staphylococcus-epidermidis-photos/staphylococcus-oxidase-test.html/
Results

• Oxidase-negative • Oxidase-positive
(OX-) (OX+)
• Facultative anaerobe • Obligate aerobe
• Enterobacteriaceae
family

Escherichia Pseudomonas
coli fluorescens
Can the presence of specific type of
enzyme that detoxify reactive
oxygen species distinguish bacteria?
Yes, it can.
Distinguishing Bacteria

Catalase Test Oxidase Test

Primarily used to distinguish among Used to distinguish among Gram-
Gram-positive cocci negative bacilli

Members of the genus Staphylococcus Pseudomonas, Neisseria, Campylobacter,

are catalase-positive Vibrio, Brucella and Pasteurella species

Members of the genera Streptococcus

and Enterococcus
References

• https://courses.lumenlearning.com/microbiology/chapter/oxygen-
requirements-for-microbial-growth/

• https://www.biotecharticles.com/Biology-Article/Oxygen-Requirements-of-
Different-Bacteria-952.html

• http://iws2.collin.edu/dcain/CCCCD%20Micro/catalase_test.htm

• http://iws2.collin.edu/dcain/oxygenclass.htm

• https://microbiologyinfo.com/oxidase-test-principle-uses-procedure-types-
result-interpretation-examples-and-limitations/