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Propagation methods for anaerobes

By Allison Faust

For anaerobic cultures, the exclusion of oxygen from the environment is essential for growth. Presently, there are a number of methods that can be used to ensure an oxygen-free environment. Here, we will discuss several common methods for the cultivation of anaerobic bacteria.

Roll tube

The roll tube method was developed by Hungate in 1950 for the isolation of oxygen intolerant anaerobes from rumen fluid and sewage. It was later modified and improved throughout the 1960s and 1970s. To use this method, 4.5 mL of pre-reduced agar medium is placed in a hungate or roll tube and kept melted in a 46°C water bath. Oxygen-free gas is then fed through sterile leads, of which at least one is bent and attached to a luer-lok tip syringe. The ambient air in the syringe is replaced with oxygen-free gas through a lead attached to a modified Pasteur pipet. The syringe is placed into the growing anaerobic culture, and the inoculum is removed and placed into the prepared roll tube of melted agar. The tube is then placed on a rolling device to spin the medium until it produces a solid layer on the wall of the tube. Historically, this method is not widely used as it is hard to perfect the technique.

Cannula System

Jars in ventilation boothThe cannula system is a modified and updated version of the roll tube method. Cannulas are sterilized with a Bunsen burner and attached to an anaerobic gas mixture. The gas mixture is turned on and set at the appropriate PSI to a gentle stream of gas so that the broth media does not bubble or splatter. The cannulas are then inserted into test tubes of media and/or growing cultures, and the anaerobic gas is supplied in a constant stream to maintain the cultures in an anaerobic environment; this work can be performed in a biological safety cabinet using pipettes for subcultures and inoculations. Please note, all media should be pre-reduced with a reducing agent and oxygen-free gas before use in this propagation method, and superior aseptic technique is critical to this method. Further, cannula systems are not readily available commercially and would most likely have to be built in-house.

Needle and Syringe

The needle and syringe method is the simplest and cheapest way to culture anaerobes. This method is useful for pre-reducing media and performing inoculations. To reduce media, first sterilize the rubber stoppers of the test tubes. Then, add a reducing agent and out-gas the tubes with a line of oxygen-free gas, displacing the air inside the tube through a syringe vent. Removing the vent first ensures no backflow of ambient air into the test tube. Subsequently, to perform inoculations, insert a needle and syringe through the rubber stopper of the test tube and invert the tube to withdraw an inoculum. Insert the needle and syringe into another tube and dispense the inoculum. Plates can be inoculated if the organism is facultative or aerotolerant and placed in jars or bags with a catalyst immediately after inoculation. Aseptic technique and the ability to work quickly and safely is critical to this method. Organisms that need to be re-pressurized or re-gassed would be performed in this manner.

Jars and Catalysts

There are various types of jars available for anaerobe growth. One example is the McIntosh Filde’s jar, which was first described in 1916. Originally, it was a metal jar with an air-tight lid and a screw to hold the lid into place. It had a pressure gauge to read internal pressure. After partial evacuation of the jar, an electric current was passed through a catalyst to heat it, which caused the residual oxygen to combine with hydrogen gas to produce water. The manometer measured this as a fall in internal pressure of the jar, thus leaving an anaerobic atmosphere within the jar. Hydrogen was then pumped into the jar so that the pressure of the jar equaled atmospheric pressure. McIntosh and Filde also developed a methylene blue indicator to ensure anaerobiosis.

Over time, anaerobe jars have evolved. Brewer’s Jars were developed in the 1950s and 1960s and featured a self-contained disposable hydrogen/carbon dioxide generation system. Hydrogen was produced when water activated a sodium borohydride tablet. The hydrogen then reacted with oxygen and the palladium catalyst present in the lid of the jar. Carbon dioxide was generated and produced a suitable atmosphere for anaerobic growth in about 90 minutes. Newer versions of this method feature a “cold catalyst” and involve a simple sachet, without the need for water and a catalyst. These produce an anaerobic environment in less than 30 minutes. Anaerobic indicators are useful in these systems to ensure the conversion to an anaerobic environment is successful.

Anoxomat™

The Anoxomat™ (Mart Microbiology B.V.) is an economic alternative to an anaerobe chamber. It is a small, self-contained unit that doesn’t use a lot of bench space. You can attach more than one gas tank, allowing for the customization of gas mixtures. It automatically evacuates and seals the jar after which the desired gas mixture is pumped into the jar. The anaerobic atmosphere is immediate as opposed to the jar and sachet method. The Anoxomat™ can also be used for microaerophilic organisms.

Anaerobe Chambers

Scientist scraping petri dishAnaerobe chambers are the ideal propagation method for anaerobes. Two examples of anaerobe chambers are the Bug Box Plus (Baker) and the Whitley MG500 Anaerobic Workstation (Don Whitley Scientific). Both use foot pedals to gas out the sleeves prior to entering. Items are brought in and out of the chamber through a pass box that is out-gassed to prevent oxygen from entering the chamber. Vials can be thawed or rehydrated in the chamber to prevent any oxygen exposure. Both of these models allow the user to set the interior temperature, so they can also be used as incubators. Using an anaerobe chamber allows you to perform all work in an oxygen-free environment.

Another anaerobe chamber is the AS-580 Gloveless Anaerobe Chamber (Anaerobe Systems). We ordered ours with the built-in incubator that we keep at 37°C. Having the incubator separate from the work space is much more comfortable for the user, and the incubator accommodates a large number of plates. Cultures that need another temperature are inoculated in the chamber and then placed in a jar with a gas pack. The jar is taken out through the pass-box and placed in the appropriate incubator.

Anaerobe Systems recommends using the 90% N- 5% H- 5% CO2 gas mixture. When working with an organism that requires a different mixture, we out-gas the test tubes using the needle and syringe method. The chamber uses positive pressure to push gas out through the sleeves when you enter and exit, eliminating the need for successively gassing out the sleeves. It also gasses out the pass box faster than other models, thus, using less gas. Moreover, this anaerobe chamber is economically priced and easy to maintain. Most importantly, using an anaerobe chamber keeps your anaerobes in a completely oxygen-free environment, which increases your odds for success.

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