Faculty of Medicine and Health Sciences

General Microbiology Lab (7105404)

Lab-13
Culturing of Anaerobic Bacteria

Second Semester 2020-2021

1
Introduction:

Oxygen is so important and indispensible for survival of many living organisms. However, some of
the living organisms can be killed by Oxygen or any of its derivatives.

For example, anaerobic bacteria are either killed of their growth is inhibited upon exposure to
Oxygen.

Why Oxygen can be toxic for some living organisms?

Utilization of O2 (and/or exposure to O2), results in the generation Superoxide (O2-) and Hydrogen
peroxide (H2O2), which are known as free-radicals.

Free-radicals, are highly-reactive molecules that can oxidize various cellular molecules and enzymes,
resulting in their inactivation. This consequently may lead to cell death or inhibition of various
important cellular processes such as cell division .

Bacteria that utilize O2 (( or withstand its presence in the surrounding environment )) have certain
enzymes (such as Superoxide dismutase and Catalase) that can neutralize free-radicals and thus
avoiding their lethal or harmful effects.
Classification of Bacteria Based on Oxygen Requirement
Based on Oxygen requirement, bacteria may be classified into:

Obligate aerobes: they require O2 for growth. They use O2 as a final electron acceptor in
aerobic respiration.

Obligate anaerobes: they do not need or use O2 . In fact, O2 is a toxic substance for them,
which either kills or inhibits their growth.

Facultative anaerobes: these organisms can grow in the presence or absence of O2, but
they grow better when Oxygen is present.

Aerotolerant anaerobes: these are exclusively anaerobic. However, they are insensitive
(not killed) to the presence of O2. They live by fermentation alone whether or not O2 is
present in their environment.

Microaerophilic: these bacteria need O2 in low concentration (1-10%).

Aerobic bacteria, facultative anaerobic bacteria, aerotolerant and microaerophilic
bacteria, possess the enzymes that can neutralize free-radicals and thus can avoid their
harmful effects.

However, because of the fact that Obligate anaerobic bacteria lack such enzymes, they
either get killed or have their growth inhibited due to the accumulation of free radicals
upon the presence of O2 in their surrounding environment
Medically-Important Anaerobic Bacterial Species:
These species belong to different anaerobic genera, such as:

1- The genus Clostridium (such as C. tetani, C. perfringens, C. difficile & C. botulinum)

2- The genus Bacteroides (such as B. fragilis)

3- The genus Actinomyces (such as Actinomycetes israelii and Nocardia asteroides)

4-The genus Fusobacterium  (such as F. necrophorum)

5- Anaerobic Streptococci (such as Enterococcus fecalis)

Methods Used in Culturing Obligate Anaerobes

1-Candle Jar:

Any a sealable, non-flammable jar large enough to hold Petri-

dishes can be used as a candle jar.
After placing the cultured plates in the jar, a candle is lighted
inside it and then, hen the jar is closed firmly. The candle flame
will consume most of the Oxygen in the jar and generating an
elevated level of carbon dioxide.
Then the jar is placed in the incubator
Candle jar is ideal for the growth of microaerophilic organisms.

2- Anaerobic Incubators:

Carbon dioxide incubators are frequently used to culture

anaerobes.

After the cultures are placed in the incubator, the air is

evacuated and replaced by carbon dioxide gas and other gases
such as nitrogen.
3- GasPak Jar

The GasPak is a commercially-produced

disposable Hydrogen and Carbon dioxide
generator envelope.

While culturing anaerobic bacteria using

this method, the cultured plates are placed
in a GasPak jar, then water is added to the
GasPak and then the lid of the jar is
sealed firmly.

After that, the jar is placed in an incubator

Note-1: when water is added to the GasPak, Hydrogen and Carbon dioxide are produced. In
the presence of palladium catalyst, Hydrogen reacts with Oxygen to form water, thus creating
an anaerobic atmosphere within the jar.

Note-2: Methylene blue impregnated paper may be used as an indicator for the generation of
an anaerobic atmosphere within the jar. In the absence of O2, Methylene blue becomes
colorless.
Culturing Anaerobic Bacteria In A Broth Medium
Water contains soluble Oxygen, which must be removed when a broth is intended to
be used for culturing anaerobic bacteria.

It is possible to remove soluble O2 from water by using a reducing-chemical agent,

such as Sodium Thioglycolate, which reduces O2 to H2O.

After that, the broth can be cultured and then the tubes are incubated under anaerobic
conditions.