BASHKIR STATE MEDICAL UNIVERSITY
The department of Microbiology, Virology
Associate professor
Farzana Faritovna Musyrgalina
Breathing is a set of chemical
reactions, as a result of which the
energy necessary for growth and
development,
the
formation
of
spores and capsules is released.
Microorganisms receive energy due
to
the
oxidation
of
organic
substances (glucose, alcohols, fats).
The
bacterial
metabolism
is
dependent on whether they are aerobic
or anaerobic.
➢Aerobic bacteria utilize glucose by
oxidation
➢The anaerobes utilize glucose by
fermentation.
AEROBIC RESPIRATION
is a process that can completely
catabolize a reduced organic energy
source to CO2 using the glycolytic
pathways
and
TCA
cycle
(the
tricarboxylic acid cycle) with O2 as
the terminal electron acceptor for an
electron transport chain.
The catabolism of glucose can be
divided into three steps.
It begins with the formation of
pyruvate (1).
AEROBIC RESPIRATION
Next pyruvate is fed
into the TCA cycle (2)
and
oxidized
completely to CO2 with
the production of some
GTP or ATP, NADH,
and FADH2.
Finally, the NADH and FADH2 formed by
glycolysis and the TCA cycle are oxidized by an
electron transport chain, using O2 as the
terminal electron acceptor (3).
It is the activity of the electron transport chain that
conserves most of the energy used to make ATP during
aerobic respiration.
AEROBIC RESPIRATION
Aerobic respiration is represented by the
following equation.
C6H12O2 → 6CO2 + 6H2O + 686
k/calories (energy in the form of ATP)
In biochemical reactions involving
oxygen, highly reactive molecules are
formed such as hydrogen peroxide and
superoxide free radicals which cause
harm to the organism. In order to
combat the effects of these molecules,
bacteria have enzymes that convert free
radicals into safer forms of oxygen
compounds such as water.
Aerobes possess enzymes such as catalase,
peroxidase and superoxide dismutase used in
oxygen metabolism.
Catalase breaks down hydrogen peroxide
(H2O2) to water and oxygen.
Peroxidase, by which 1NADH + H2O2 are
converted to 2NAD and O2.
Superoxide dismutase, by which superoxide,
O2 is converted to H2O2.
These enzymes detoxify peroxide and oxygen
free radicals produced during metabolism in the
presence of oxygen.
The anaerobes, do not possess enzymes to
convert these free radicals thus they cannot
survive in the presence of oxygen in the
surroundings.
Anaerobic respiration includes glycolysis and
fermentation.
During the latter stages of this process NADH
(generated during glycolysis) is converted back
to NAD by losing a hydrogen. The hydrogen is
added to pyruvate and, depending on the
bacterial species, a variety of metabolic endproducts are produced.
ANAEROBIC RESPIRATION
The second possible outcomes for pyruvate
are fermentation.
In fermentation, the only energy
extraction pathway is glycolysis.
Fermentation generates some energy
for the cell, but it also generates toxic
products like alcohol, acetic acid (or
vinegar), and lactic acid that have to be
cleaned up, or removed from the cell.
In
Alcohol
fermentation
NADH donates its electrons to a
derivative
of
pyruvate,
producing ethanol.
In lactic acid fermentation, NADH
transfers its electrons directly to
pyruvate, generating lactate as a
byproduct.
Butyric acid fermentation is performed with the
anaerobes Clostridium perfringens or Clostridium
butyricum resulting in the production of butyric acid
Propionic acid fermentation is demonstrated by
anaerobes from genus Propionibacterium spp.
End-Products of Fermentation
Anaerobes are normally found within certain
areas of the body but result in serious infection
when they have access to a normally sterile body
fluid or deep tissue that is poorly oxygenated.
Some anaerobes normally live in the crevices of
the skin, in the nose, mouth, throat, intestine,
and vagina.
Injury to these tissues (cuts, puncture wounds,
or trauma) especially at or adjacent to the
mucous membranes allows anaerobes entry into
otherwise sterile areas of the body and is the
primary cause of anaerobic infection.
A second source of anaerobic infection occurs
from the introduction of spores into a normally
sterile site.
Spore-producing anaerobes live in the soil and
water, and spores may be introduced via
wounds, especially punctures.
Anaerobic infections are most likely to be
found in persons who are immunosuppressed,
those treated recently with broad-spectrum
antibiotics, and persons who have a decaying
tissue injury on or near a mucous membrane,
especially if the site is foul-smelling.
On the basis of oxygen requirements, bacteria can
be divided into following different categories:
Aerobes: Grow in ambient air, which contains 21%
oxygen
and
small
amount
of
(0,03%)
of
carbondioxide (Bacillus cereus).
Obligate aerobes: They have absolute requirement
for oxygen in order to grow. (Psuedomonas
aeruginosa, Mycobacterium tuberculosis).
Obligate anaerobes: These bacteria grow only
under condition of high reducing intensity and for
which oxygen is toxic (Clostridium perfringens,
Clostridium botulinum).
Facultative anaerobes. They are capable of
growh under both aerobic and anaerobic conditions
(Enterobacteriaceae group, Staphylococcus aureus).
Aerotolerant anaerobes are anaerobic bacteria
that are not killed by exposure to oxygen.
Capnophiles. Capnophilic bacteria require
increased concentration of carbondioxide (5% to
10%) and approximately 15% oxygen. This
condition can be achieved by a candle jar (3%
carbondioxide) or carbondioxide incubator, jar or
bags
(Haemophilus
influenzae,
Neisseria
gonorrhoeae).
Microaerophiles.
Microaerophiles are
those groups of bacteria that can grow
under reduced oxygen (5% to 10%) and
increased carbondioxide (8% to 10%).
Higher oxygen tensions may be inhibitory
to them. This environment can be obtained
in
specially
designed
jars
or
bags
(Campylobacter jejuni, Helicobacter pylori).
Different categories of bacteria on the basis of oxygen requirements
A culture tube containing nutrient broth
ANAEROBIC CULTURING
The identification of anaerobes is highly complex, and
laboratories may use different identification systems.
Organisms are identified by their:
1) colonial and microscopic morphology,
2) growth on selective media,
3) oxygen tolerance,
4) biochemical characteristics:
➢ sugar fermentation,
➢ bile solubility,
➢ esculin, starch, and gelatin hydrolysis
➢ casein and gelatin digestion,
➢ catalase, lipase, lecithinase and indole production
➢ nitrate reduction
5) volatile fatty acids (determined by gas chromatography)
6) susceptibility to antibiotics.
The methods of obtaining specimens for
anaerobic culture and the culturing procedure
are performed to ensure that the organisms are
protected from oxygen.
The specimen must be protected from oxygen
during collection and transport and must be
transported to the laboratory immediately.
Cultures should be placed in an environment
that is free of oxygen, at 35°C for at least 48
hours before the plates are examined for growth.
Anaerobic Culture Methods
Anaerobic condition can be achieved by
➢ Cultivation in vacuum
➢ Displacement of oxygen with other gases
➢ Chemical or biological methods
➢ By displacement and combustion of
oxygen
➢ By reducing agents
➢ Anaerobic chamber
For
anaerobic
culture
transportation the thioglycolate
broth is widely used.
It contains sodium
thioglycolate (the salt
of
mercaptoacetic
acid) that is a strong
reducing agent for
elimination
of
dissolved oxygen.
After primary inoculation the medium-containing test tubes
with specimens should be tightly closed.
Iron sulphite agar
(MPA, glucose, iron citrate or chloride, and NaHSO3)
It is prepared as high butt of
agar covered with liquid vaseline.
Microbial inoculation is made
like deep agar culture. The
growth of anaerobes (e.g.,
Clostridium
perfringens)
is
followed by the blackening of the
medium from the production of
hydrogen sulfide.
A Kitt-Tarozzi medium
(broth, 0.5% of glucose and pieces of animal
organs (as liver) or minced meat for oxygen
absorption)
The top of medium is
covered with liquid
vaseline.
Deep agar cultures are performed by stab
inoculation of the specimen into long tubes
filled with solidified anaerobic nutrient medium,
covered with mineral oil.
Special culture techniques for anaerobic bacteria
Anaerobic
jar
(or
anaerostat), where the
air oxygen has been
pumped out. A number
of Petri dishes with
inoculated
anaerobic
media are placed inside
the jars for incubation.
Candle Jar
A candle jar is a container into
which a lit candle is introduced
before sealing the container's
airtight lid
The candle's flame burns until extinguished by oxygen
deprivation, which creates a carbon dioxide-rich, oxygen-poor
atmosphere in the jar.
Candle jars are used to grow bacteria requiring an increased
CO2 concentration (capnophiles). Candle jars increase CO2
concentrations and still leave some O2 for aerobic
capnophiles
The chemical reagent placed into the jar as a
disposable sachet (gas-pack or anaeropack).
This reagent removes oxygen from closed jar
volume. For instance, in some techniques the
chemical reagent produces hydrogen that
(under the action of catalyst) binds to any free
oxygen inside the jar to form water.
Anoxomat Mark II
➢ Fast anaerobes and micro-aerophilic
condition
➢ Low gas consumption
➢ Fully automatic self control
➢ Flexible
Chemical Methods
✓ Alkaline
pyrogallol
(pyrogallic
acid in NaOH) absorbs O2
➢ Biological Methods
Biological method can be use
to
establish
anaerobic
conditions. One half of the solid
medium in the Petri´s dish is
innoculated with the tested
sample, the second half is
inoculated
with
Serratia
marcescens – aerobic bacteria
able
to
produce
anaerobic
environment by the consumption
of oxygen. Petri dish is sealed
with the wax or parafin and
cultured in aerobic environment.
Other Anaerobic Culture System
ANAEROBIC CHAMBER
It is an anaerobic incubation system
It provides oxygen free atmosphere for
inoculating culture media and for incubation
It is fitted with airtight rubber gloves to insert
hands for working with specimens
The anaerobic chamber contains catalyst,
desiccant, hydrogen gas, carbon dioxide gas,
nitrogen gas and an indicator