Transcript Lab-13-Bacterial-oxygen-requirement-anearobic

Introduction
Bacteria can exist in a wide variety of environments, including
environments that lack oxygen.
In fact, oxygen is toxic to some bacteria. This toxicity is usually
due to the accidental formation of superoxides (O2-) and
hydrogen peroxide (H2O2), which react with other cell
components.
Oxygen Species is harmful if :
1) Bacteria have no enzyme system to eliminate it.
2) Oxygen Species concentration is very high in media or air.
Toxic Forms of Oxygen
 Singlet oxygen (O2) :
is in high energy state & extremely reactive, exists in phagocytes.
 Superoxide free radical (O2-) & hydrogen peroxide (H2O2):
is so toxic to cell , formed in small amount in cellular respiration.
 Peroxide anion (O2-2) :
used as antimicrobial agent.
 Hydroxyl radical (OH-) :
the most active form, form in the cytoplasm from respiration.
Oxygen Detoxification
Toxic forms of oxygen need to be neutralized by Enzyme
systems.
 Superoxide dismutase
 Catalase
 Peroxidase
If microbe does not produce these enzymes, must have
anaerobic conditions.
Cells that live in oxygen-containing environments have the
enzymes superoxide dismutase and catalase (among others)
that convert superoxides and peroxides to oxygen and water..
Enzyme systems
1) Super oxide dismutase : Converts the superoxide ion to
hydrogen peroxide in the existence of hydrogen ion.
2)Catalase : breaks up hydrogen peroxide to water and oxygen.
3) Peroxidase : converts hydrogen peroxide to water in
presence of hydrogen ions (NADH).
Oxygen Requirements
Classifications
Bacteria classified based on their oxygen requirements into 5
main classifications:
1) Obligate aerobes :
Require oxygen to live.
Their metabolic pathways require oxygen as the final electron
acceptor (aerobic respiration).
They have no alternate means of producing ATP.
2) Facultative anaerobes :
grows in best where most oxygen is presented.
Utilize oxygen and aerobic respiration whenever possible.
They can undergo anaerobic respiration and/or fermentation to
survive without oxygen.
3) Obligate anaerobes :
Growth occurs only when there is no oxygen.
Use anaerobic respiration and/or fermentation to
generate ATP.
Lack superoxide dismutase , catalase & peroxidases
they die in the presence of oxygen.
4) Aerotolerant anaerobes :
 Tolerates the presence of oxygen and can survive in air but does not
require it for growth.
 Utilize anaerobic respiration and/or fermentation to produce ATP.
 They possess superoxide dismutase, and so are not affected by the
toxic byproducts of oxygen.
5) Microaerophiles :
 Growth occurs only where a law concentration of oxygen has
diffused into medium.
Note : Capnophilic bacteria Require carbon dioxide.
Brain-Heart Infusion Agar (BHIA)
Components:
Peptone mixture, Beef Heart Infusion, Calf Brain Infusion,
Sodium Chloride, Dipotassium Phosphate, Dextrose, Agar 1.5%.
Bacteria oxygen requirements Test:
 Materials
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3 Brain-heart infusion agar deeps.
1 Hot plate with water bath.
1 Ice water bath.
1 Thermometer.
 Lab supplies:
 Ice.
 Nutrient broth cultures of Pseudomonas aeruginosa
and Staphylococcus aureus(24 - 48 hrs.).
 Thioglycolate broth culture of Clostridium
sporogenesor C. butyricum (48 - 72 hrs.).
 Procedure
1) Place the brain-heart infusion (BHI) agar deeps into a
boiling water bath.
2) After the BHI agar melts, allow the water to boil for at
least five more minutes. Turn the heat off, and place a
thermometer in the bath. Monitor the temperature of the
bath.
3) When the temperature reaches 45°C, remove the tubes
from the bath. Be careful not to shake, stir or otherwise
disturb the agar.
4) Inoculate each culture into a separate agar deep by
carefully stabbing with an inoculating needle .
Avoid shaking, stirring or getting bubbles into the
medium. Immediately after inoculation, place the
deeps upright in an ice-water bath until they are
completely solid.
5) Incubate the deeps at 37°C. Check the growth at 24, 48 and
72 hours.
6) After incubation, note the pattern of growth in each tube.
Compare your results to Figure at the end of the exercise.
1) Obligate aerobe--growth only at the surface.
2) Facultative anaerobe--growth throughout, often with heavier growth
toward the top.
3) Obligate anaerobe--growth only at the bottom.
4) Aerotolerant anaerobe--relatively low growth throughout.
5) Microaerophile--growth just below the surface.
Anaerobic Bacteria
Bacteria are often categorized
according to their growth responses to
atmospheric oxygen. This may vary
from species that can grow only in the
presence of oxygen to those that can
grow only in the absence of oxygen.
Fluid Thioglycolate Broth
Fluid Thioglycolate broth is a reducing
medium & supports aerobic & anaerobic
bacteria, it contains:
 Sodium Thioglycolate removes O2 from
the medium
 Resazurin as an oxidation reduction
indicator that turn to pink in presence
of O2
 Small amount of agar presented to
retard gases
Gas Pack System : Anaerobic Jar
Anaerobic jars such as the Gas Pak are vessels in which an
anaerobic environment is generated after inoculated media are
sealed into the chamber. Anaerobiasis is achieved by adding
water to commercially available gas generator envelopes that
are placed in the jar just prior to sealing. Chemicals in the
envelope produce hydrogen gas and carbon dioxide. The
hydrogen combines with free oxygen in the chamber to
produce water. The carbon dioxide is required for the growth
of certain organisms. A methylene blue indicator strip is
usually placed in the jar. It turns colorless when the oxygen has
been removed.
Gas Pack System : Gas Pak Pouch
Procedure:1) Dispense GasPak liquid activating reagent into channel of pouch.
2) Place plates inside the pouch.
3) Lock in anaerobic environment with sealing bar and incubate.
Candle Jar
The candle jar is used to create
Microaerophile conditions. It is
a large screw-capped container
into which the medium is
placed along with a candle. The
candle is lit and the jar is
sealed. The candle will burn
and reduce the oxygen
concentration.
CO2 Generating Packet
Anaerobic Chambers
The Vinyl Anaerobic Chambers provide a strict anaerobic atmosphere
using a palladium catalyst and hydrogen gas mix of 5%.
END OF LECTURE