Oxygen Requirements of Bacteria

An experiment to determine the oxygen needs of bacterium.

Various genera of bacteria have differing needs for oxygen. Some, like Clostridium, find oxygen toxic and usually don't grow in air. Others can grow only when oxygen is present. There are variations in between. The drawings below show the location of growth of some common bacteria in Glucose Shake Tubes. CAUTION: do not shake the tubes because presence of air (oxygen) will ruin the assay. Directions for the experiment are given below.

The possible oxygen relationships are as follows:

AA = strict anaerobes: Clostridium, Sarcina, and many genera from the rumen of cattle, intestines and similar sites. Strict anaerobes grow only were oxygen is absent. Some are more sensitive to oxygen than others. Some species, especially those from the rumen and intestines, die rapidly when exposed to oxygen. Most Clostridium are not killed by such brief exposure but can't grow in oxygen. Some species of Clostridium can grow slowly in the presence of air. No species of Clostridium is able to produce spores when free (uncombined) oxygen is present.

FA = facultative anaerobes: Escherichia, Citrobacter, Enterobacter, and Proteus grow best in oxygen but can grow in the absence of oxygen by stealing oxygen from foods such as nitrate, sugars, and other "honorary oxygens". The result of this is production of nitrite, organic acids (lactic acid, formic acid, etc.), and other substances which are often foul-smelling. Tubes containing facultative anaerobes contain growth throughout when the bacteria are evenly distributed, but there is usually a heavier growth on the surface of the agar because they grow best in air.

MA = microaerephilic: Azospirillum, Aquaspirillum, Cytophaga require oxygen but grow best just below the surface of the agar where oxygen is reduced. This type of bacteria is relatively uncommon in laboratories because some won't grow on GST and other common media. It is difficult to find a species which will grow in a well defined band a millimeter or so below the surface to produce a mice band below the surfarce as seen here. Often the band is so near the surface that you can confuse these for aerobic species, however, they do not grow profusely on the surface of the agar like aerobic species.

A = strict aerobes: Acetobacter, Arthobacter, Azomonas, Bacillus, Micrococcus, Pseudomonas, Xanthomonas grow only on the surface of the agar where they get plenty of oxygen. Most produce a heavy growth above the agar which may be a liquid. Some bacteria produce slimes or capsules and these produce exceptional profuse growth above the agar and may burrow into the agar slightly. The liguid containing cells may run down between the walls of the tube and the agar plug, but you should not confuse this with growth.

I = indifferent: Lactobaccilus, some Streptococcus, and most other milk organisms grow equally well on the surface and within the agar because they are indifferent to the oxygen level. Notice that the bacteria grow uniformly on the surface and to the bottom of tube provided the cells are distributed uniformly. The growth is similar to that of facultative anaerobes except FAs have a heavy growth of bacteria at the surface. At the surface indifferent bacteria may have barely noticeable growth of cells. We know they can grow on the surface because they do so when spread on a pertri plate. Actually, many of these bacteria require vitamines, amino acids, and other growth factors and the colonies may be tiny even on the media most suitable for them.

Gas Fracturing

Gas fracturing may or may not be seen in tubes containing the growth of bacteria deep within the medium. When a bacterium is growing deep within a medium and produces any gas rapidly enough the agar will be fractured and the gas escapes. This is easily seen at 37C. At room temperature, gas is produced more slowly and may diffuse out of the agar without any fracture. Sometimes bubbles of gas are seen. Some gases produced by bacteria include one or more of these: carbon dioxide, hydrogen, methane, and hydrogen sulfide. Gas fracturing is also seen in slants and other media when abundant gas is produced. The agar plug may be driven out of the tube pushing the cotton plug out.

GST Glucose Shake Tubes

Prepare this medium in advance, stopper or cap the tubes, and autoclave 15 minutes at 15 pounds pressure. Cool to 45C (agar gels at 38C) for inoculation. See our media preparation pages.

Inoculation of GST tubes (maintain pure culture conditions).

  1. Melt the agar (this heating also drives out oxygen so anaerobes can grow)
  2. Cool to 44 C. in water bath (cooling to avoid killing bacteria)
  3. Add 3 to 5 drops of a 24-hour liquid culture or bacteria from a slant
  4. Mix tube by rollling between hands. Don't get air into medium.
  5. Cool tubes in an upright position until agar is solid. If using screw cap tubes, loosen caps 1/4 turn so air can get into the culture.
  6. Incubate at a temperature which permits growth of the organism. Incubate in light if you are testing for photosynthesis. Except for the blue-green bacteria (called blue-green algae in old books), bacterial photosynthesis occurs only in anaerobic conditions.
  7. After incubation (24 hours or as needed), determine location of bacterial growth by trying to view newspaper print through the tube.

This info on catalase may be moved to another page.

Like most organisms, bacteria generate hydrogen peroxide in the presence of oxygen, but Clostridia lack catalase and can't break hydrogen peroxide down. Humans have catalase. Drops of hydrogen peroxide antiseptic on glass just lie there. Touch the drops with human blood or put the antiseptic on an open wound and you get bubbles of free oxygen. If human saliva contains catalase, it will also cause bubbles. Test saliva on a drop of hydrogen peroxide solution. Similarly, hydrogen peroxide produces bubbles when dropped onto colonies of bacteria species which produce catalase.

Hydrogen peroxide ------> Oxygen + water (when catalase is present)

H202 --------------------> O2 + H2O

A suggested experiment.

This experiment is harmless, unless you accidentally use pathogenic bacteria. You may use bacteria you have isolated or species which are normally safe. See safety notes at bottom.

During any experiment such as this you must maintain pure culture conditions. If the culture is contaminated by other organims. The observed results are for the mixture of bacteria not just for the species you intended to put into the tube. You do not have to limit this test to bacteria. You may use algae, protozoa, yeasts, or fungi. However, many of these may not grow on the QST medium and some will be killed by the temperature of the melted agar. Also not all bacteria will grow on this medium. In fact, this medium was choosen because many (most?) human pathogens will not grow on it. For example, some pathogens don't grow well unless blood is included in the medium

Expected Observations

Methylene blue will be colorless in oxygen-free regions of the tube and blue where oxygen is present. Phonel red will be yellow in acidic regions of the tube and red in alkaline regions of the tube. Some organisms produce gas by fermentation and the gas ruptures the agar. All these traits will help you identify unknown bacteria.

Add a discussion of oxidative vs fermentive metabolism.

This experiment is one of a series testing the ability of bacteria to grow in various environments. We plan other experiments in this series. It is interesting to accumulate a set of bacteria and test the response of each in the different experiments. In fact, this is one of the ways we identify bacteria. The experiments in the series are:

add detailed discussion of how anaerobes live how hey get oxygen. link to eleecton pathwsy

cover aerobes as above; discuss lack of microaerophilic.cover catalase.

Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 4716

Last modified 1999 Mar 18.
Suggestions, corrections, and comments are appreciated: Contact Harold Eddleman (indbio@disknet.com).