Transcript Culturing bacterial Pathogens

Culturing bacterial Pathogens
Pathogenic Bacteria
• Pathogenic bacteria are bacteria that cause bacterial infections in
human beings, animals and plants.
 Pathogenic bacteria contribute to globally important diseases, such
as pneumonia, which can be caused by bacteria such as Streptococcus
and Pseudomonas and
 Food borne illnesses, which can be caused by bacteria such as
Shigella, Campylobacter and Salmonella.
 Inoculated media should be incubated as soon as possible.
WHY?
 Delay in incubation can affect the viability of pathogens.
 Increase the risk of plates becoming contaminated.
Microorganisms require incubation at the
temperature, in the humidity and gaseous
atmosphere most suited to their metabolism.
Temperature of incubation
 Optimum temperature: The temperature at which a microorganism
grows best.
 The temperature below which growth stops is called the minimum
temperature and that above which growth stops is called the
maximum temperature.
 Growth of microorganisms is more affected by slight rises above their
optimum temperature than by reductions below it.

Most of pathogenic bacteria are Mesophiles i.e. Middle/Moderate
temperature loving (Neither too hot nor too cold).

Growth is in between 25 to 40oC.

Optimum temperature is commonly 37oC.

Many have adapted to live in the bodies of animals.
 In laboratory we incubate inoculated culture media in a
closed instrument with a temperature control, known as
an incubator.
Humidity during incubation

While incubating agar plates, make sure to keep an open beaker of
water in the incubator.

Periodically check that the beaker has water in it - do not let it run
dry.

The water will maintain a constant level of humidity in the incubator.

Most of modern incubators have build in water reservoirs by which
humidity in the chamber can be regulated.
Gonococci are rapidly killed in
dry conditions
Why humidity in incubator……….?
 Humid environment inside incubator will help in uniform transfer of
heat through out the incubator.
 Moist condition inside an incubator will also tend to minimize drying
out of agar media in plates, test tubes or screw cap bottles.
 Culture media tend to deteriorate more rapidly when undue drying
occurs.
 Most organisms grow maximally when humidity is 70% or higher e.g.
 H. pylori and N. gonorrhoeae require an atmosphere with high
humidity for their growth.
Presence of O2 and CO2 in incubator
According to the gaseous requirements microorganisms are:
Obligatory aerobe
Microaerophilic
• Requires free oxygen to survive.
• Pseudomonas aeruginosa.
• Grows best in the presence of only a trace of free oxygen.
• Campylobacter jejuni.
Obligatory anaerobe
• Survives only in the absence of oxygen.
• Clostridium tetani.
Facultative anaerobe
• Can live with or without free oxygen.
• Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli.
A carboxyphilic organism
• Requires an atmosphere which contains carbon dioxide.
• Neisseria meningitidis.
Aerotolerant Anaerobes
• Can’t use oxygen, but tolerate its presence e.g. Lactobacillus
Capnophiles
• Grow better at high CO2 levels and low O2 levels. Similar to
environment of intestinal tract e.g. Clostridium
The Requirements for Growth:
Related to Oxygen
 Oxygen (O2)
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Dr.T.V.Rao MD
Table 6.1
Culturing of anaerobes
Aim
1.
An anaerobic atmosphere is essential for the growth of strict
anaerobes such as Clostridium species.
2.
Anaerobic incubation also helps to differentiate pathogens and
to isolate facultative anaerobes from specimens containing
commensals, e.g. Streptococcus pyogenes from throat swabs.
3.
The haemolytic reactions of beta-haemolytic streptococci are also
more pronounced following anaerobic incubation.
Methods for obtaining anaerobic
conditions
1. Commercially produced kits containing oxygen removing
chemicals.
2. Reducing agents in culture media.
I. Commercially produced kits
The GasPak Anaerobic System is used to create an oxygen-free
environment for the growth of anaerobic microorganisms.

Inoculated plates or tubes are placed inside the chamber.

Anaerobic conditions are created by adding water to a gas generator
envelope that is placed in the jar just before sealing.

The envelope contains two chemical tablets, sodium
borohydride and sodium bicarbonate.
 Water reacts with these chemicals, producing hydrogen
gas and carbon dioxide.
 The hydrogen gas combines with free oxygen in the chamber to
produce water, removing all free oxygen from the chamber.
 This reaction is catalyzed by the element palladium, which is
attached to the underside of the lid of the jar.
 The carbon dioxide replaces the removed oxygen, creating a
completely anaerobic environment.
Requirements:
1. Anaerobic container.
2. Anaerobic gas generating kits.
3. Anaerobic indicators.
Technique:
 Aseptically inoculate the media.
 Place the inoculated plates into the plate carrier.
 Prepare the anaerobic indicator by cutting and exposing it. Use
forceps to insert it into the plate carrier ( it will ensure anaerobic
conditions by change in its color).
 Cut off the corner of the kit sachet as indicated by the broken line.
 Add 10 ml of water.
 Immediately place the sachet upright in the anaerobic jar.
 Close the lid of the jar according to instructions.
II. Use of reducing agents in culture media
 Cooked meat medium which is used to culture Clostridium and Bacteroides
species.
 The anaerobes grow at the bottom of the medium among the meat
particles which contain effective reducing substances.

Used to grow anaerobes that might be killed by oxygen.

Contain ingredients (reducing agents) that chemically
combine with oxygen and remove it from the medium.
Example: Sodium thioglycolate
Culturing in carbon dioxide
Aim
 Carbon dioxide enriched atmosphere is required for the growth of
Neisseria gonorrhoeae, Neisseria meningitidis, Brucella species, and
Streptococcus pneumoniae.
Methods for obtaining carbon dioxide conditions.
I. Commercially carbon dioxide gas-generating systems are
available
II. Simple ways of providing a carbon dioxide enriched
atmosphere:
A. Burning candle: Enclose the inoculated plates in an
airtight jar with a lighted candle. As the candle burns, the
oxygen content is reduced leaving a carbon dioxide content
of 3–5%.
Important: It is necessary to use a white wax smokeless candle to avoid the release of fumes
which may be bactericidal or interfere with the growth of bacteria.
B. Chemical method
 Carbon dioxide can be generated chemically in a jar by
reacting sodium bicarbonate with tartaric acid or citric acid.
Generation of carbon dioxide from chemicals:
To obtain a 10% carbon dioxide atmosphere in a jar of
about 3 liters capacity, mix 0.7 g sodium bicarbonate with
1.7 g tartaric acid (or 2.4 g citric acid). Immediately
before closing the jar, moisten the chemicals with water.