This document discusses various methods for achieving anaerobic culture conditions necessary for growing obligate anaerobic bacteria. These include: 1) Excluding or displacing oxygen using vacuum, gases like hydrogen or carbon dioxide, or chemical/biological oxygen absorbers; 2) Absorbing oxygen using chemicals like pyrogallic acid or biological methods like GasPak envelopes; 3) Reducing residual oxygen using reducing agents in culture media like glucose or meat extracts. Achieving strict anaerobic conditions is important for cultivating bacteria that cannot tolerate even small amounts of oxygen.
2. methods of Anaerobic culture
āObligate anaerobes grow only in absence of free oxygen
āThese bacteria lack mechanism of oxidation through respiratory
enzymes like cytochrome oxidase, catalase and peroxidase resulting in
H2O2 accumulation
āThis H2O2 is toxic for the growth of anaerobic bacteria.
3. methods of Anaerobic culture
āClostridium tetani & Cl. Perfringens are strictly anaerobic
4. Principles followed to achieve Anaerobiosis
āExclusion of oxygen
āProduction of vacuum
āDisplacement of oxygen with other Gases
āAbsorption of oxygen by chemical or biological means
āReduction of oxygen
5. A. Cultivation in Vacuum
āTried by incubating culture in vacuum desiccator
āSome oxygen is always left behind
āFluid culture may boil over and media get detached
6. B. Displacement of oxygen with gases
āHydrogen, nitrogen, helium, or carbon dioxide
āInoculated plates are placed, inside airtight container and lighted
candle is kept before lid is sealed
āBurning candle use all the oxygen before it get extinguished
7. C. Absorption of oxygen by chemical or biological mean
1. Pyrogallic acid and sodium
hydroxide
2. Chromium and sulfuric acid
3. GASPAK METHOD
āEnvelop contains 3 tablets
ā Citric acid
ā sodium carbonate
ā Sodium borohydride
8. Absorption of oxygen by chemical or biological mean
āOn addition of water it generates
hydrogen and carbon dioxide
ā hydrogen combines with oxygen to
produce water , thus create
anaerobiosis.
9. Absorption of oxygen by chemical or biological mean
4. McIntosh and fildesā aerobic Jar
āIt consist of glass or metal jar with
metal lid which can be clamped
airtight with screw
āThe lid has two tubes, one act as a gas
inlet and the other one as outlet
ā two terminals which can be
connected to electric supply
10. Absorption of oxygen by chemical or biological mean
āInnoculated culture plates are
placed inside jar
āOutlet tube is connected to vacuum
pump and jar inside is evacuated
āThe outlet tap is closed & inlet tube
is connected with hydrogen gas
cylinder
11. Absorption of oxygen by chemical or biological mean
āAfter filling jar with hydrogen,
electric terminals are connected so
that palladinised asbestos is
heated
āThis acts as a catalyst for
combination of hydrogen and
residual oxygen
āIt ensures complete anaerobiosis
12. Absorption of oxygen by chemical or biological mean
āCarries risk of explosion
āReduced methylene blue is used as
Indicator for verifying anaerobic
condition
āColorless anaerobically
āRegains blue color on an exposure to
oxygen
13.
14. D. Anerobic Glove box
āSelf contained anerobic system with
provisions of circulation hydrogen, nitrogen,
and carbon dioxide
āCatalytic conversion of residual oxygen to
water
āRecommended for total anerobic gut flora
studies
āIt is expensive
15. E. Reduction of oxygen
Used reducing agents :
1. 1% glucose
2. 0.1% thioglycolate
3. 0.1% ascorbic acid
4. 0.05% cystine
16. E. Reduction of oxygen
Used reducing agents :
5. Broth containing of iron pieces flamed red hot
6. Broth containing of fresh animal tissue e.g., rabbit
kidney, spleen, etc
17. E. Reduction of oxygen
Used reducing agents :
7. Robertson cooked meat medium produces anaerobiosis as
under:
āUnsaturated fatty acids present in meat utilize oxygen for
autooxidation
āGlutathione and cysteine are reducing agents of meat and use
oxygen
āSulfahydryl compound of cysteine also precipitate for reduced
oxidation reduction
18. E. Reduction of oxygen
ā¢ It has fat free minced meat in broth
ā¢ It permits growth of even strict anaerobes
ā¢ The meat itself contains reducing substances, particularly
glutathione which helps in the growth of anerobes
ā¢ Fresh entry of oxygen is prevented into the medium by
layering the top with sterile liquid paraffin
Editor's Notes
H2O2: Hydrogen peroxide
Anaerobiosis :Ā life in the absence of air or free oxygen.
catalyst, in chemistry,Ā any substance that increases the rate of a reaction without itself being consumed. Enzymes are naturally occurring catalysts responsible for many essential biochemical reactions.
catalyst, in chemistry,Ā any substance that increases the rate of a reaction without itself being consumed. Enzymes are naturally occurring catalysts responsible for many essential biochemical reactions.
An oxidation-reduction (redox) reaction isĀ a type of chemical reaction that involves aĀ transfer of electronsĀ between two species. An oxidation-reduction reaction is any chemical reaction in which the oxidation number of a molecule, atom, or ion changes by gaining or losing an electron.
An oxidation-reduction (redox) reaction isĀ a type of chemical reaction that involves aĀ transfer of electronsĀ between two species. An oxidation-reduction reaction is any chemical reaction in which the oxidation number of a molecule, atom, or ion changes by gaining or losing an electron.
