- Anaerobic bacteria require oxygen-free conditions to grow as oxygen is often toxic to them. There are different types including strict anaerobes, microaerophiles, aerotolerant anaerobes, and facultative anaerobes. - Oxygen toxicity occurs as it is used in respiration, producing reactive oxygen species that can damage enzymes and cell structures in anaerobic bacteria. Special culturing techniques are needed to exclude oxygen like using vacuum, inert gas displacement, or chemical oxygen absorbers. - Common culturing methods include the McIntosh jar using hydrogen gas and a catalyst to remove oxygen, or automated systems like the Anoxomat. Special reduced media and culturing

1. ARYA
1st MSc MICROBIOLOGY

2. INTRODUCTION
 Anaerobic bacteria grow only in the absence of oxygen –often extremely toxic.
 Anaerobic bacteria differ in their requirements of and sensitivity to oxygen.
 Some such as C.histolyticum are aerotolerant and may produce some growth on the
surface of aerobic plates ,while others such as C.tetani are strict anaerobes and form
surface growth only if the oxygen tension is less than 2mmHg.
 Facultative anaerobes:Can grow in the presence/absence of oxygen.
 Obtain energy by both respiration and fermentation
 Oxygen not toxic, some use nitrate or sulphate as a terminal electron acceptor under
anaerobic conditions.

3.  Obligate /strict anaerobes:Oxygen is toxic to these organisms, do not use oxygen as
terminal electron acceptor.
 Microaerophilic organism:Require low level of oxygen for growth ,but cannot
tolerate the levels present in the atmosphere.
eg: H pylori
 Aerotolerant anaerobes:Metabolism is anaerobic but they are unaffected by the
presence of oxygen
OXYGEN TOXIXITY :Oxygen is used by aerobic and facultatively anaerobic
organisms as its strong oxidizing ability makes it as excellent electron acceptor
 During the stepwise reduction of oxygen, which takes place in respiration toxic and
highly reactive intermediates are produced reactive oxygen species (ROS).

12. FACTORS THAT INHIBIT THE GROWTH OF
ANAEROBES BY OXYGEN
1.Toxic compounds are produced
Eg:H2O2 , Superoxides
2.Absence of catalase and superoxide dismutase
3.Oxidation of essential sulfhydryl groups in enzymes
without sufficient reducing power to regenerate them

13. CULTURING OF ANAEROBES
• Culture of anaerobes is extremely difficult due to the need to
exclude oxygen,slow growth and complex growth
requirements
• Molecular methods based on DNA analysis and direct
microscopy have shown that we are largely ignorent of the
microbial world and previously unknown diversity has been
discovered

14. ANAEROBIC CULTURE METHODS
Production of vacuum
Displacement of oxygen
Displacement and combustion of oxygen
Absorption of oxygen by chemical or biological methods
By readucing agents
Anaerobic chamber

15. PRODUCTION OF VACUUM
 This is achieved by incubating cultures in a vacuum dessicator.
 Is not an effective method .
 Not in use now
DISPLACEMENT OF OXYGEN
 Bt inert gases like hydrogen, nitrogen, carbon dioxide or helium.
 A lighted candle is kept in a large air tight container loaded with inoculated plates.
 It is expected that burning candle will use up all the oxygen inside before it is
extinguished but some amount of oxygen is always left behind.
 Unsatisfactory.

17. Displacement & combustion of
oxygen
 This involves evacuation of air from the jar and replacement with
inert gas like hydrogen followed by removal of residual oxygen by
use of a catalyst.
 McIntosh and filde’s anaerobic jar.
 Anoxomat instrument.
 McIntosh and filde’s anaerobic jar.
 This is the most reliable and widely used anerobic metod
.
 Consists of a stout glass or metal jar with a metal lid that
can be clamped air tight with a screw.
 Lid has 2 tubes with taps, one acting as gas inlet and
other as outlet.
 Lid has 2 terminals connected to an electric supply.

18.  A catalyst alumina pellets coated with palladium is suspended
under the lid by small wires which are connected with the terminals
to heat the catalyst for its activity.
 Now a days catalyst at room temperature is used.
 Culture plates inoculated with specimens are placed inside the anaerobic jar with an
indicator.
 Tight the lid, outlet tube connected to vacuum pump while inlet tube is closed.
 Air inside is evacated and outlet tube is closed and hydrogen gas is passed through inlet
tube till reduced atmospheric pressure is brought to normal atmospheric
pressure(760mmHg) which is monitored on vacuum gauze as zero
 Electric terminals are switched on to heat the catalyst and this helps to combine
hydrogen and residual oxygen to water.
 Reduced methylene blue is generally used as indicator of anaerobiosis in jar which
remains colourless in anaerobic conditions, but turns blue on exposure to oxygen

20. Anoxomat
• Is the new standard for culturing anaerobes and microaerobes in virtually any
laboratory , clinical or industrial.
• This is achieved according the evaluation – replacement method of Maclntosh
&Fildes , by means of a microprocessor controlled automatic valve system.
• Is an automatic equipment which evacuates the air from jar and replaces by
hydrogen gas from a cylinder.
• Same catalyst is used here to remove the traces of oxygen.
• More easier.
Advantages
 Is rapid :anaerobic condition in 70 sec and microaerobic in 20 sec
 Easy to operate
 Is space saving
 Is multifunctional
 Is cost saving
 Produces no environmental pollution, no chemical waste, no plastics
 Can be used continously: 365 days no start up time, maintenance and clearing is not
necessary
 Can handle both fluid as well as fixed media.

22. ABSORPTION OF OXYGEN BY CHEMICAL METHODS
1. PYROGALLOL
• Alkaline pyrogallol absorbs oxygen.
• A large tube containing solution of NaOH and pyrogallic acid placed inside air tight jar
produces anaerobic conditions.
2. CHROMIUM AND SULPHURIC ACID
• A mixture of chromium and sulphuric acid is used
• They two react in presence of oxygen and produce chromous sulphate
3. GAS PACK SYSTEM
• Most commonly used method
• Very simple to perform
• Uses a sachet containing sodium biocarbonate and sodium borohydride which react
chemically in presence of water to produce hydrogen and CO2.
• Cold catalyst –permits combination of hydrogen and oxygen
• Reduced methylene blue is used as indicator
In anaerobic condition it is colourless and becomes blue on exposure to oxygen

23. • Traces of O2 is removed by using same catalyst in McIntosh Fildes
method.Anaerobic indicator strips included to monitor the anaerobic condition.

24. BY REDUCING AGENTS
Oxygen in culture media can be reduced by various agents such as glucose
,thioglycollate ,cooked meat pieces ,cystein and ascorbic acid
Based on this principle 2 most commonly used liquid culture media are
 THIOGLYCOLLATE BROTH
 COOKED MEAT BROTH(RCM)

29. ANAEROBIC CHAMBER
• Is an anaerobic incubation system
• Also known as anaerobic glove boxes
• Provides oxygen free environment
• Is fitted with airtight rubber gloves to insert hands for working with specimens
• Contains catalyst, dessicant, H gas, CO2, N

31. PRE REDUCED ANAEROBICALLY STERILIZED (PRAS)
• Are prepared entirely under O2 free condition
• Media used in the anaerobic bacteriology can be freshly prepared or purchased
from commercial suppliers.
• Media for anaerobic culture prepared in the laboratory should be used within 2 weeks of
preparation as long storage degrades the quality of the media due to peroxides
accumulation and dehydration.
• Anaerobic culture media contains reducing agents such as cysteine.
• Pre-reduced, anaerobically sterilized media are produced
bydifferent commercial suppliers, which have extended shelf life of up to six months.
• The primary plating media for inoculating anaerobic specimen includes a non-selective
blood agar and one or all of the following mentioned selective media

32. Non selective media used in anaerobic bacteriology:
Cooked meat broth (e.g. Robertson’s Cooked Meat Medium): Non-selective for
cultivation of anaerobic organisms; with addition of glucose, can be used for gas-
liquid chromatography.
Anaerobic blood agar: It is a non-selective medium for isolation of anaerobes and
facultative anaerobes.
Egg-yolk agar (EYA): Non-selective for determination of lecithinase and lipase
production by Clostridia and Fusobacteria.
Peptone-yeast extract glucose broth (PYG): Non-selective for cultivation of
anaerobic bacteria for gas-liquid chromatography.
Selective and differential media used in anaerobic bacteriology:
Bacterioides bile esculin agar (BBE): It is selective and differential for
Bacteriodes fragilis group and good for presumptive identification.
Laked Kanamycin-vancomycin blood agar (LKV): It is selective for isolation of
Prevotella and Bacteriodes spp.
Anaerobic phenylethyl alcohol agar (PEA): Selective for inhibition of gram
negative rods and swarming by some Clostridia.
Cycloserine cefoxitin fructose agar (CCFA): selective for Clostridium difficile.
Thioglycollate broth: Non selective for cultivation of anaerobes; as well as
facultative anaerobes and aerobes.

33. HUNGATE TECHINIQUE
Hungate Type Anaerobic Culture Tube
• Gas tight hungate tubes designed for obtaining and maintaining anaerobic culture
conditions.
• Method of hungate et.al , utilizes syringe methods for anaerobiosis
• Anaerobic culture hungate tubes contain 3 autoclavable parts
a scerw cap with 9mm opening
a flange type non toxic rubber stopper that is gas permeable
a disposible screw cap culture tube
• The Hungate anaerobic culture tube was developed for growing and maintaining
anaerobic bacteria
• Technique is simple
• Exclude o2 by flushing the tube with desired gas
• Place 4.5ml of pre reduced anaerobic agar medium into tube
• Seal the tube with the butyl rubber stopper and screw cap
• Autoclave the tube
• Inoculate with syringe
• Prepare on roll tube spinner
• Incubate in water bath

34. • QUALITY ASSURANCE (Indicator of anaerobiosis)
Chemical indicator: reduced methelene blue remains colourless in
anaerobic condition and turns to blue on exposure to O2
Biological indicator: plate inoculated with pseudomonas is incubated
along with other inoculated plates for anaerobic culture.Absence of growth
indicates perfect anaerobiosis

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