Anaerobic culture methods

Introduction

• Anaerobic bacteria require incubation without oxygen.
• However, some anaerobes (e.g. Clostridium histolyticum) are aero-tolerant which may produce some growth on the surface of aerobic plates.
• There are some bacteria which are strict anaerobes (Clostridium tetani).
• Anaerobiosis can be established by the following methods.

Methods of Anaerobiosis

1) By displacement of oxygen

i) Cultivation in vacuum in a vacuum dessicator has been tried but proved to be unsatisfactory.

ii) Displacement of oxygen by inert gas like hydrogen or nitrogen is done from a sealed jar loaded with inoculated media.

• The drawbacks in this method include repeated evacuation and refilling.
• Moreover, oxygen can never be removed completely.
• An effective but widely tried method is the use of candle.
• A lighted candle is kept in an airtight container loaded with inoculated plates.
• Although it is expected that the burning candle will use all the available oxygen inside before it gets extinguished.
• But practically, some amount of oxygen is always left behind.

2) Absorption of oxygen by chemicals

a) Pyrogallic acid

• Buchner (1888) first introduced alkaline pyragallol for anaerobiosis which absorbs oxygen.
• This method has subsequently been used with different modifications.
• In a large tube (Buchner’s tube) containing solution of sodium hydroxide, pyrogallic acid is added.
• The Buchner’s tube is then placed inside an air tight jar loaded with inoculated plates and tubes.

b) Mixture of powdered chromium and sulphuric acid

• Instead of alkaline pyrogallol, a mixture of chromium and sulphuric acid can also be used for producing anaerobiosis.
• The two chemicals react in presence of available oxygen and produce chromous sulphate.

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c) Gas pack

• A commercial product is available nowadays.
• It is in the form of a disposable packet of aluminium foil containing pellets of sodium borohydride and cobalt chloride and of citric acid and sodium bicarbonate, is now widely used for preparing anaerobic jars.
• The chemicals generate hydrogen and carbon dioxide inside the jar when water is added.
• Hydrogen combines with oxygen in the presence of catalyst (alumina pellets coated with palladium) present in the under surface of the lid of the jar.
• After the inoculated plates are placed inside a large air-tight jar, it is incubated at 37⁰C.
• This technique is simple and effective.

3) By displacement and combustion of oxygen

• Anaerobiosis is obtained by Mclntosh and Flide’s anaerobic jar.
• It is the most dependable and widely used method.

Principle

• Spongy palladium or platinum kept inside jar acts as a catalyzing agent.
• It causes slow combination of hydrogen and oxygen to form water.

  Procedure

• Mclntosh Filde’s jar consists of a 8×5 inches jar of stout glass or metal with a tight fitting metal lid.
• A metal jar is preferable because there is no risk of explosion.
• The lid can be clamped air-tight with a screw and is fitted with two tubes with taps.
• One tap is for introduction of gas inside and the other as an outlet for vacuum valve.
• A capsule containing alumina pellets coated with palladium (palladinized alumina) is suspended under the lid.
• Catalyst active at room temperature is used.
• Culture plates inoculated with specimens are kept inside the jar along with an indicator.
• The lid is screwed tight.
• The inlet tube is closed and the outlet tube is connected to the vacuum pump and at least three quarters of the air of the jar is removed.
• The pressure is reduced to 100 mm Hg which is noted on vacuum gauze.
• The outlet tap is now tightly closed and the inlet tap is connected to a hydrogen supply and then opened.
• Hydrogen is passed through a small wash bottle.
• The reduced pressure is brought up to 760 mm Hg which is monitored on the vacuum gauze as 0.
• The catalyst helps the combination of hydrogen and residual oxygen to form water.

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Indicator

• Reduced methylene blue is generally used as indicator.
• It is mixture of NaOH, methylene blue and glucose.
• It becomes colorless anaerobically but regains blue color on exposure to oxygen.

4) Biological methods

• Aerobic organisms are incubated along with the anaerobic bacteria in order to gain anaerobiosis.
• But this method is slow and ineffective.
• Two blood agar plates of 9cm diameter are taken where one is inoculated with Pseudomonas aeruginosa or Serratia marcescens and the other with specimen of anaerobic organism.
• Two plates are placed one over other and sealed along the rims and incubated.

5) By incorporating reducing agents in media

• Oxygen in culture media can be reduced by 1% glucose, 0.1% thio-glycollate, 0.05% cysteine, 0.1% ascorbic acid and cooked meat pieces.
• The two most widely used anaerobic liquid culture media are:

a) Thio-glycollate broth: it contains nutrient broth and 1% thioglycollate.

b) Robertson’s cooked meat medium

• It consists of nutrient broth and pieces of fat- free minced cooked meat of ox heart.
• It permits the growth of strict anaerobes and preserves delicate organisms.
• Unsaturated fatty acids present in meat utilize oxygen for autooxidation, the reaction is being catalyzed by haematin in the meat.
• Certain reducing substances such as glutathione and cysteine present in meat also utilize oxygen.
• With growth of saccharolytic anaerobes, colour of meat pieces turns red while it becomes black in case of proteolytic anaerobes.
• Before inoculation, the media are boiled in water bath at 80⁰C for half hour to drive out oxygen.
• For strict anaerobiosis, the surface of the medium may be covered with a 1 cm layer of sterile liquid paraffin.

References: 

i) https://www.sciencedirect.com/topics/earth-and-planetary-sciences/anaerobiosis

ii) https://www.slideshare.net/doctorrao/anaerobic-culture-methods

Anaerobic culture methods