Introduction & Review
First, a quick review of respiration vs. fermentation (from the Bacteriology 102 lab manual): In our introductory laboratory courses, we deal mostly with chemotrophic bacteria – primarily the chemoheterotrophs. Depending on the abilities of any specific chemotrophic organism and the environment in which it is found, the catabolic pathway is involved with either oxidative or substrate-level phosphorylation. If the former, the organism is obtaining energy by respiration; if the latter, the process is fermentation. Relative comparisons are made between respiration and fermentation in the following outline. (The three kinds of "phosphorylation" are diagrammed under respiration, fermentation and phototrophy on the catabolism page.)
- There is a greater variety of potential substrates (amino acids, sugars, etc.). The substrate is more completely broken down than by fermentation.
- A relatively smaller variety of end products is produced. A small amount of acidic intermediates can accumulate when respiring organisms catabolize sugars (such as for Pseudomonas species which utilize the Entner-Douderoff pathway).
- ATP is generated by oxidative phosphorylation wherein relatively more ATP is generated than by substrate-level phosphorylation, and oxygen is utilized as the terminal electron acceptor. Certain respiring organisms can use an alternate terminal electron acceptor such as nitrate under anaerobic conditions; this situation is termed anaerobic respiration.
- Relatively more cell mass is generated.
- A smaller variety of substrates can be fermented. Many organisms which can ferment sugars will not ferment amino acids. The substrate is less completely broken down.
- A relatively larger variety of end products is produced. Much acid (and possibly gas) is produced when sugars are fermented.
- ATP is generated by substrate-level phosphorylation wherein relatively less ATP is generated than by oxidative phosphorylation. Oxygen is not involved in the process.
- Relatively less cell mass is generated.
Both Glucose Fermentation Broth (as used in our lab courses) and Glucose O/F Medium include the following major ingredients:
- Glucose – a sugar from which most common chemoheterotrophic bacteria can obtain energy – by fermentation and/or respiration. Glucose can also be utilized as a source of carbon, but these media include a large number of potential carbon sources (amino acids as well as glucose), and whether or not glucose is used as a carbon source cannot be directly determined from the reactions seen in these media.
- Peptone – a commonly-used medium ingredient which mainly supplies amino acids (sources of nitrogen, carbon, sulfur and energy for many bacteria). It is a crude preparation of a partially-digested protein, and a peptone solution can serve as a complete medium for a number of organisms such as E. coli. If too much peptone (relative to glucose) is incorporated in the medium, detection of acidic products of fermentation or respiration may not be possible, as overabundance of ammonium (which is alkaline) released from the breakdown of amino acids can neutralize the acids.
- pH indicator – the pH indicators employed in these media turn yellow under acidic conditions. Brom-cresol purple is in Glucose Fermentation Broth (which also contains the Durham tube), and brom-thymol blue is in Glucose O/F Medium.
Glucose Fermentation Broth
Testing whether an organism can ferment glucose is one of the basic, primary tests in the identification of chemoheterotrophic bacteria. For this test we routinely use a "Glucose Fermentation Broth."
- Fermentation of glucose results in the abundant production of acidic end products, the presence of which can be detected by the pH indicator in the medium.
- Many organisms produce gas – either CO2 alone or a mixture of H2 and CO2. H2 is insoluble and is detected by bubble formation in a Durham tube placed in the medium.
Note the examples shown below.
- Tube 1: No fermentation. The pH indicator remains purple. There can still be growth due to the use of amino acids as sources of energy (usually by respiration).
- Tubes 2A and 2B: Fermentation with the production of acid (yellow color) but no gas. A slight amount of acid is seen in tube 2A, but fermentation is still recorded for this tube.
- Tubes 3A and 3B: Fermentation with the production of acid (yellow color) and insoluble gas (bubble in Durham tube). Tube 3B shows an alkaline reaction on top; this is simply due to deamination of amino acids whose alkaline reaction has not been over-neutralized by the acid diffusing through the tube from fermentation.
Furthermore, with any of the reactions shown here, amino acids and/or glucose can be used as sources of carbon, but determination of what is or is not used as a carbon source cannot be made with this medium.
Glucose O/F Medium
Original Paper: Rudolph Hugh and Einar Leifson. 1953. "The Taxonomic Significance of Fermentative versus Oxidative Metabolism of Carbohydrates by Various Gram Negative Bacteria." J. Bacteriol. 66 (1): 24-26. You can download this paper as a .pdf file from the link provided here. The original intention of this medium was to be able to differentiate between gram-negative bacteria (1) that can ferment, (2) that only catabolize glucose by respiration and (3) that do not catabolize glucose at all. This differentiation is not as important in the identification of gram-positive bacteria, and it so happens that gram-positive bacteria do not grow in Glucose O/F Medium well (if at all) anyway – probably because of some sensitivity to the pH indicator. It is a waste of time and money to use this medium to characterize gram-positive cultures.
Whether an organism can respire or ferment glucose can be tested with Glucose O/F Medium. A small amount of acid production can be associated with glucose respiration. The original paper which describes the medium gives the example of the Entner-Douderoff pathway that is utilized by a variety of generally gram-negative bacteria (including Pseudomonas) to convert glucose to pyruvate – an alternative method of pyruvate formation to that of the Embden-Meyeroff pathway. (Pyruvate is further oxidized to CO2 in the aerobic respiration process.) Among the intermediates in the Entner-Douderoff pathway are forms of gluconic acid. So, where a strictly aerobic organism producing this acid is growing – i.e., at the top of the medium where O2 is available – a net acidic reaction will be seen. However, this acidic reaction would be rendered indistinguishable if the organism were a facultative anaerobe – in which case the large amount of acid (produced by fermentation in the anaerobic environment of the tube) would be diffusing throughout the entire medium.
Duplicate tubes are inoculated for each organism, and the medium in one of the tubes is overlayed with mineral oil. Mineral oil does not in itself cause anaerobic conditions but rather prevents oxygen from continuing to diffuse into the medium. After incubation, one looks for the presence and location of growth and acid.
It is important to emphasize that this medium contains relatively less peptone and more glucose than Glucose Fermentation Broth, so the acid associated with respiration can be detected in the aerobic part of the non-overlayed tube – if it is not made indistinguishable by acid production from fermentation which turns both tubes yellow throughout.
Note the examples shown below. For each pair of tubes, the tube on the right was overlayed with mineral oil after inoculation.
- First pair of tubes: Tubes were inoculated with a strict aerobe which neither respires nor ferments glucose – therefore no acidic reaction. The blue alkaline reaction shows up where there is growth at the top of the "aerobic" tube. This is the negative reaction.
- Second pair of tubes: Tubes were inoculated with a strict aerobe which respires but does not ferment glucose. The small amount of acid associated with respiration shows up where there is growth at the top of the "aerobic" tube. This is the "O" reaction, typical for most species of Pseudomonas. (The alkaline reaction from amino acid deamination is overneutralized.)
- Third pair of tubes: Tubes were inoculated with a facultative anaerobe – i.e., one which can respire (with O2) and ferment. Acid from the fermentation of glucose diffuses throughout both the "aerobic" and "anaerobic" tubes. This is the "F" reaction, typical of the enterics. (The alkaline reaction from amino acid deamination is overneutralized. Also, one cannot discern any acid production that might be associated with respiration.)
|Pair of Tubes
|Aerobic deamination of amino acids – not relevant to glucose catabolism
(This occurs in upper part of open tube.)
(Blue alkaline reaction seen; not over-neutralized by any acid production.)
(Alkaline reaction overneutralized by acid.)
(Alkaline reaction overneutralized by acid.)
|Respiration of glucose
(Acid seen in upper part of open tube.)
|This cannot be discerned due to the high amount of acid produced from fermentation.
|Fermentation of glucose
(Acid diffuses throughout both tubes.)
Additional comparisons between these two media are illustrated and discussed on "Page 2." A discussion of these media with regard to their formulation is given here.
For certain groups of bacteria, different formulations of Glucose Fermentation Broth are employed which satisfy special growth requirements. For example, clinical streptococci and dairy lactobacilli require a much richer basal medium than that provided by peptone. Likewise, a variation of Glucose O/F Medium is used for the characterization of Staphylococcus and Micrococcus. Bergey's Manual and other reference books give more specific information.