Cultivation of Aerobic and Anaerobic Bacteria: Methods, Principles, and Equipment

A surgeon debrides a deep wound and sends tissue for culture. The Gram stain shows gram-negative rods in the specimen. After 48 hours, the aerobic blood agar and MacConkey plates show no growth. The organism visible on the smear has failed to grow in ambient air because it is an obligate anaerobe that dies on oxygen exposure. The anaerobic culture set up in parallel grows Bacteroides fragilis.

This discrepancy (organisms seen on Gram stain but no growth on routine aerobic plates) is one of the most important clues to anaerobic infection in clinical microbiology. Recognizing it requires knowing that cultivation of anaerobes demands completely different conditions from routine culture.

Understanding oxygen requirements is the starting point for choosing the right cultivation method. Bacteria are classified by oxygen tolerance: obligate aerobes, facultative anaerobes, microaerophiles, aerotolerant anaerobes, and obligate anaerobes and each category requires different cultivation conditions. For the complete classification with clinical examples and the thioglycollate tube growth pattern, see: Oxygen Requirements of Pathogenic Bacteria.

A. Aerobic Bacteria

Main Principle: Provide Oxygen

Atmospheric condition is generally satisfactory for the culture of aerobes or facultative anaerobes. However, for the growth of many aerobes, it is necessary to provide extensive aeration. Forced aeration of cultures is therefore frequently desirable. It is achieved either by vigorously shaking the flask/tube on a shaker or by bubbling sterilized air into the medium. When aerobic organisms are to be grown in large quantities, it is advantageous to increase the exposure of the medium to the atmosphere. This is  accomplished by dispensing the medium in shallow layers or by providing aeration by constantly shaking the inoculated liquid cultures.

B. Cultivation of Anaerobic Bacteria

Main Principle: reduce the O₂ content of the culture medium and remove any oxygen already present inside the system or in the medium.

Oxygen is ubiquitous in the air so culture of anaerobic microorganisms require special methods. A number of procedures are available for reducing the O₂ content of cultures. Some are simple but suitable mainly for less sensitive organisms. Whereas others are more complex but necessary for the growth of strict anaerobes.

• Bottles or tubes filled completely to the top with culture medium and provided with a tightly fitting stopper. Suitable for organisms not too sensitive to small amounts of oxygen.
• Addition of a reducing agent that reacts with oxygen and reduces it to water e.g., Thioglycolate in thioglycolate broth. After thioglycolate reacts with oxygen throughout the tube, oxygen can penetrate only near the top of the tube where the medium contacts air.
  • Obligate aerobes grow only at the top of such tubes.
  • Facultative organisms grow throughout the tube but best near the top.
  • Microaerophiles grow near the top but not right at the top.
  • Anaerobes grow only near the bottom of the tube, where oxygen cannot penetrate.

A redox indicator dye called resazurin is added to the medium because the dye changes color in the presence of oxygen. Thereby indicating the degree of penetration of oxygen into the medium.

Strict anaerobes, such as methanogenic bacteria can be killed by even brief exposure to O₂. In these cases, a culture medium is first boiled to render it oxygen- free. Then a reducing agent such as H2S is added and the mixture is sealed under an oxygen-free gas. All manipulations occurs under a tiny jet of oxygen-free hydrogen or nitrogen gas, directed into the culture vessel when it is open. Thus driving out any O₂ that might enter. For extensive research on anaerobes, special boxes fitted with gloves, anaerobic glove boxes, permit work with open cultures in completely anoxic atmospheres.

Methods of cultivating Anaerobic Bacteria

Stringent anaerobes can be grown only by taking special precautions to exclude all atmospheric oxygen from the medium. Such an environment can be established by using one of the following methods:

1. Pre-reduced media
   During preparation, the culture medium is boiled for several minutes to drive off most of the dissolved oxygen.  A reducing agent e.g., cysteine, is added to further lower the oxygen content. Oxygen-free N2 is bubbled through the medium to keep it anaerobic. The medium is then dispensed into tubes which are flushed with oxygen-free nitrogen, stoppered tightly, and sterilized by autoclaving. Such tubes are continuously flushed with oxygen-free CO₂ by means of a cannula, restoppered, and incubated.

2. Anaerobic Chambers

   This refers to a plastic anaerobic glove box that contains an atmosphere of H2, CO₂, and N2. Culture media are placed within the chamber by means of an airlock which can be evacuated and refilled with N2. Any oxygen in the media is slowly removed by reaction with hydrogen, forming water; this reaction is aided by a palladium catalyst.  After being rendered oxygen-free, the media are inoculated within the chamber (by means of the glove ports) and incubated (also within the chamber).

3. Anaerobic Jar

   

   Anaerobic jar is a heavy-walled jar with a gas-tight seal within which tubes, plates, or other containers to be incubated are placed along with H2 and CO₂ generating system (GasPak System). After the jar is sealed oxygen present in the atmosphere inside the jar and dissolved in the culture medium, is gradually used up through reaction with the hydrogen in the presence of a catalyst. The air in the jar is replaced with a mixture of H2 and CO₂, thus leading to anoxic conditions.

4. Candle Extinction Jar

A simple, inexpensive method suitable for microaerophilic organisms particularly Campylobacter and Helicobacter pylori. These organisms require reduced oxygen (5%) and increased CO₂ (10%) rather than complete anaerobiosis.

Method: Inoculated plates are placed inside a sealed jar or tin with a lit candle. The candle burns until it consumes sufficient oxygen (reducing O₂ to approximately 17%) and produces CO₂ (raising CO₂ to approximately 3%). When the candle extinguishes from CO₂ accumulation and O₂ depletion, the jar is sealed and incubated.

Limitations: Does not produce true anaerobiosis — only microaerophilic conditions. Not suitable for obligate anaerobes. CO₂ and O₂ levels are not precisely controlled. Residual O₂ (~17%) is too high for strict anaerobes.

Current use: Largely replaced by commercial CO₂ tablets and CO₂ incubators in well-equipped laboratories, but remains useful in resource-limited settings.

5. Chemical Methods: Pyrogallol and Alkaline

An older method for emergency or resource-limited anaerobic culture: pyrogallic acid (pyrogallol) absorbs oxygen when mixed with an alkaline solution (sodium hydroxide or sodium carbonate). The reaction vessel is sealed after inoculation to consume residual oxygen. Produces true anaerobic conditions but is less reliable and more hazardous than GasPak systems.

Anaerobic Indicators to Confirm Anaerobiosis

Simply sealing an anaerobic jar does not guarantee anaerobic conditions have been achieved. Two types of indicators are used:

Resazurin Indicator

Resazurin is a redox indicator dye added to pre-reduced media and anaerobic transport systems:

• Pink/red = oxidized = oxygen present = NOT anaerobic
• Colorless = reduced = oxygen absent = anaerobic conditions achieved

This is the same dye mentioned in the existing thioglycollate tube section — elevated here as a named principle.

Methylene Blue Indicator Strip

A strip of filter paper impregnated with methylene blue is placed inside the anaerobic jar before sealing:

• Blue = oxidized = oxygen still present — do not open, check seal
• Colorless/white = reduced = anaerobic conditions confirmed — safe to incubate

The indicator strip is read after 10–15 minutes. If still blue after 30 minutes, the jar has failed: check the catalyst, sachet, and seal before repeating.

Anaerobic Growth Indicator Plate

Some laboratories include a Clostridium species or strict anaerobe reference strain on a control plate inside every anaerobic jar run. If this control grows, anaerobiosis was achieved. If it fails to grow, the entire jar run is invalid.

Comparison of Anaerobic Cultivation Methods

Method	Anaerobiosis Level	Cost	Equipment	Best For
Pre-reduced media	Complete	Moderate	Anaerobic glove box ideal	Strict anaerobes; transport
Anaerobic jar (GasPak)	Complete	Low-moderate	Basic jar + sachets	Routine clinical anaerobic culture
McIntosh-Fildes jar	Complete	Low (reusable jar)	Vacuum pump + H₂ source	Resource-limited settings; research
Anaerobic chamber	Complete	High	Dedicated glove box	Reference labs; extensive anaerobic work
Candle extinction jar	Microaerophilic only	Very low	Any sealable jar	Campylobacter, H. pylori; field use
Pyrogallol method	Complete	Low	Basic chemicals	Emergency/resource-limited; not preferred

Detailed Guides for Each Method

This article provides the principles and overview. For complete procedures, compositions, and troubleshooting for each method, see the dedicated articles:

• GasPak Anaerobic System: the most widely used commercial anaerobic jar system: GasPak Anaerobic System: Principle, Procedure, and Uses
• McIntosh-Fildes Anaerobic Jar: the classical jar method using vacuum and hydrogen gas: McIntosh-Fildes Anaerobic Jar: Principle, Procedure, and Uses
• Anaerobic Culture Media: complete list of media used for anaerobic bacteriology including PEA blood agar, pre-reduced BHI, kanamycin-vancomycin blood agar: Commonly Used Anaerobic Culture Media
• Clinical Anaerobic Infections: when to suspect anaerobic infection, appropriate specimens, and clinical significance: Anaerobic Infections: Etiology, Characteristics, and Diagnosis

How to Remember: Cultivation Methods by Oxygen Requirement

The simple rule is to match the method to the organism:

• Obligate aerobe → forced aeration (shaking flask, bubbling air)
• Microaerophile → candle jar or CO₂ incubator (5% O₂, 10% CO₂)
• Facultative anaerobe → standard incubator (no special method needed)
• Obligate anaerobe → anaerobic jar / pre-reduced media / anaerobic chamber

The anaerobic jar sequence — "Seal, Generate, Confirm":

1. Seal: plates inside, catalyst active, sachet activated
2. Generate: H₂ + CO₂ produced; H₂ reacts with residual O₂ over catalyst → water
3. Confirm: indicator strip colorless = anaerobiosis achieved

The indicator color rule:

• Methylene blue blue = oxygen present = NOT anaerobic
• Methylene blue colorless = reduced = anaerobic ✓
• Resazurin pink = oxygen present = NOT anaerobic
• Resazurin colorless = reduced = anaerobic ✓

Key Exam Facts in One Table

Feature	Detail
Obligate aerobe cultivation	Forced aeration — shaking flask or bubbled sterilized air
Microaerophile cultivation	Candle jar or CO₂ incubator: 5% O₂, 10% CO₂
Obligate anaerobe cultivation	Anaerobic jar (GasPak/McIntosh-Fildes), pre-reduced media, or anaerobic chamber
Pre-reduced media principle	Boiled to remove O₂ + reducing agent (cysteine) + sealed under O₂-free N₂
GasPak sachet contents	Sodium borohydride + citric acid → H₂ + CO₂; H₂ + O₂ → H₂O (over palladium catalyst)
McIntosh-Fildes method	Jar evacuated by vacuum pump → filled with H₂ + CO₂ → H₂ reacts with residual O₂
Candle jar O₂ level	~17% O₂ (not true anaerobiosis — microaerophilic only)
Anaerobic indicator (methylene blue)	Colorless = reduced = anaerobic; Blue = oxidized = O₂ present
Anaerobic indicator (resazurin)	Colorless = reduced = anaerobic; Pink = oxidized = O₂ present
Thioglycollate tube — anaerobe position	Bottom of tube (oxygen cannot penetrate)
Thioglycollate tube — aerobe position	Top of tube only
Catalyst in anaerobic jar	Palladium pellets — must be active (regenerate by heating at 160°C for 2h if inhibited)

References

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