Cultivation of Anaerobic Bacteria

Anaerobic Bacteria

An anaerobic organism or anaerobe is any organism that

does not require organism for growth. It may react
negatively or even die if oxygen is present.

Cultivation

A microbiological culture is a method of multiplying microbial

organisms by letting them reproduce in predetermined
culture media under controlled laboratory conditions.
Microbial cultures are used to determine type of organism,
its abundance in the sample being tested, or both.

Different kind of Anaerobes

OBLIGATE
They are harmed by the presence of Oxygen.
ANAEROBES
AEROTOLERANT ANAEROBES

They cannot use oxygen for growth, but tolerate its presence (20%).

FACULTATIVE ANAEROBES

They can grow without oxygen but use oxygen if it is present.

MICROAEROPHILES

They can tolerate Oxygen present in less amount (5%).

Growth related to oxygen

Anaerobic Environments Present in Nature

Sediments of lakes, rivers and oceans; bogs, marshes, flooded soils, intestinal
tract of animals; oral cavity of animals, deep underground areas, e.g. oil
pockets and some aquifers.

Anaerobes of Clinical Importance

CLOSTRIDIA
e.g- C tetani; C perfringens; C difficile; C botulinum

BACTEROIDES

B fragilis

Prevotella

Porphyronomas

ACTINOMYCES

FUSOBACTERIUM

ANAEROBIC STREPTOCOCCI

C. tetani

Porphyromonas
gingivalis
Prevotella intermedia

Actinomyces

Fusobacterium
Anaerobic
streptococci

Factors that inhibit the growth of

Anaerobes by Oxygens
Toxic

compounds are produced, e.g- Hydrogen

Peroxide (H2O2) , Superoxide Anions (O2-), O2- and
Hydroxyl radical (OH-)

Oxidation

of essential Sulfahydral groups in

enzymes without sufficient reducing power to
regenerate them.

Absence

of enzymes like Catalase, Proxidase &

Superoxide dismutase.

Requirements of Anaerobic Media

Mostly Anaerobes are inoculated into blood agar supplemented with Vitamin
K and Hemin.

Most common adaption of media is the addition of a reducing agent, e.gThioglycolate and L-cysteine.

These reducing agents convert oxygen to water, bringing down the redox
potential to -300 mV or less.

We can also add a redox indicator such as Rezazurin, pink in the presence of
oxygen, colourless in its absence.

Anaerobic Culture Methods

Anaerobes differ in their sensitivity to oxygen and the culture

methods employed reflect this- some are simple and suitable for less
sensible organisms, others more complex but necessary for fastidious
anaerobes.

Vessels filled up to the top with culture medium can be used for the
organisms which are not too sensitive.

Anaerobic Jar
1.

McIntosh and Fildes anaerobic jar

- CONSTRUCTION
The body is made of Metal.
.

The Lid

A screw, going through a curved metal strip.

A thermometer

A pressure Gauge

Another side tube.

A wire cage hanging from the lid

*.

CO2 gas packets can be used instead of candle.

Method of Use

The culture are placed inside the jar, stacked on one or the other.

Indicator system: Methylene Blue or Pseudomonas aeruginosa, inoculated on to a

nutrient agar plate is kept inside the jar along with the other plates. As these
bacteria need oxygen to grow . A growth free culture plate at the end of the
process indicates a successful anaerobiosis.

II

6/7 of the air inside is pumped out and replaced with either unmixed Hydrogen
or as a 10% CO2 + 90% H2 mixture.

Formation of H2O.

Manometer will measure this as a fall in internal pressure.

III

Hydrogen is pumped in to fill up the jar so that the internal pressure equals
atmospheric pressure. The jar is now incubated at desired temperature settings.

Candle Jar

Reduces O2 environment.

Only

CO2 tension.

2. Gas Pak Jar

a. Palladium Aluminium coated pellets

Catalyst

Chemically reduces O2

Reacts with residual O2 in the presence of H2 to form H2O

b. Gas Pak envelope

Generates CO2 & H2 gases

c. Methylene blue strip

Indicator

blue

(+)O2

white

(-)O2

3. Anaerobic Glove Chamber

Close system

Used for premature babies

E.g Incubator

Anaerobic Glove Chamber

3. Roll Tube
has a pedal through which gas (CO2 &H2)which would come out

Place test tube directly to the outlet.

Identification
Plates are checked at

18-24 hours for faster growing species like

Cl. perfringens & B. fragilis

5-7 days for slowly growing species like

Actinomyces, Eubacterium & Propionbacterium

Genus

determination

By gram stain, cellular morphology, gas- liquid chromatography.

Species

determination

Based on fermentation of sugars & other biochemical methods.