Nutrition and Cultivation of Bacteria
In the late 20th Century, numerous authors contributed to an essay on bacterial nutrition and cultivation which became an essential appendix in lab manuals. This web page expands on that material, and much of the original authors' wordage remains in Sections II-VI. Included are links to related web material.
After the introduction, this page is arranged as follows:
ON THIS PAGE (after introduction):
The survival of microorganisms in the laboratory, as well as in nature, depends on their ability to grow under certain chemical and physical conditions. An understanding of these conditions enables us to characterize isolates and differentiate between different types of bacteria. Such knowledge can also be applied to control the growth of microorganisms in practical situations.
Media used in the laboratory for the cultivation of bacteria must supply all of the necessary nutrients required for cellular growth and maintenance of the organisms. A wide variety of culture media is employed by the bacteriologist for the isolation, growth and maintenance of pure cultures and also for the identification of bacteria according to their biochemical and physiological properties.
A culture medium must supply suitable carbon and energy sources and other nutrients, sometimes including "growth factors" (defined below). It is important to note that no one medium will support the growth of all microorganisms. Accordingly, the elements required for the maintenance, growth and reproduction of all organisms will be used by different organisms in different ways.
When one prepares a medium for the cultivation of microorganisms, one dissolves various organic and/or inorganic compounds sequentially in pure, distilled water. The importance of water cannot be overestimated. Water is the universal solvent in which all nutrients must be dissolved and all chemical reactions will take place. It can supply some hydrogen and oxygen in certain chemical reactions. Water containing a significant amount of solutes may not be osmotically compatible or "available" for use by microorganisms, so the concept of water availability needs to be considered.
The various life forms may be categorized as being either chemotrophs or phototrophs regarding the generation of the energy which is used in ATP generation. Generally speaking, energy production by chemotrophs is purely associated with the oxidation of chemical compounds; with phototrophs, light is essential. Phototrophs include organisms that oxidize water with the release of oxygen (such as plants, algae and cyanobacteria) and also those which do not (such as the purple and green phototrophic bacteria).
|Type of Organism||General Process and Major Features|
|Chemotroph||Respiration||"Derive energy by oxidative phosphorylation.
"Generally, respirers use molecular oxygen as the external, terminal electron acceptor; this is aerobic respiration. Respirers may also use nitrate or some other "oxygen substitute" in the process of anaerobic respiration.
"Certain organisms can only perform anaerobic respiration – for example, the methane producers and many sulfate reducers.
|Fermentation||"Derive energy by substrate-level phosphorylation.|
|Phototroph||Phototrophy||"Derive energy by photophosphorylation.
"Phototrophs may be oxygenic (oxygen-evolving) or anoxygenic (not oxygen-evolving).
Results of the common (and generally ill-taught) laboratory test for "oxygen relationships" are based on whether an organism can respire (aerobically) and/or ferment. However, this test is limited to those chemotrophic organisms that are able to utilize the test medium under the conditions given, and it can in no way be applied to the classification of organisms in general. In place of such terms as "strictly aerobic," "facultatively anaerobic", etc. (which can be interpreted in a number of different and conflicting ways), one or more of the following clearly-definable catabolic processes should be indicated for any given organism according to its capabilities:
For example, instead of referring to Escherichia coli as a "facultative anaerobe," one can indicate its ability to perform aerobic and anaerobic respiration and also fermentation. As for the ability of an organism to grow anaerobically, the middle three processes itemized just above are relevant as discussed here.
Associated with energy production is "reducing power." For biosynthesis, all organisms need reducing power in the form of electrons. Organisms that obtain their electrons from organic compounds are called organotrophs, and those that obtain their reducing power from inorganic compounds are called lithotrophs.
Here is something to note: When we ask for these terms to be defined on quizzes and exams, take care that you define them according to their original, intended meanings. Additional descriptions, analogies and examples generally do not help in defining terms. Even though we may learn that the word "lithotroph" is derived from a Greek word meaning "stone-eater," we would never define the term that way any more than we would find acceptable the definition of an autotroph as an organism that "makes its own food" – an unfortunate statement that makes no sense, as all organisms assimilate and process all of their nutrients, appropriate to their kind. Following the strict definitions, one would not treat autotroph and lithotroph as synonyms – nor heterotroph and organotroph, for that matter.
Details about phosphorylation and examples of energy-yielding pathways can be found in most modern textbooks, and a very general overview of catabolism is given here.
As carbon is a major and essential element in all living things, organisms may also be classified according to the nature of their source of carbon. Organisms which assimilate organic compounds for their carbon needs are termed heterotrophs. Those which utilize carbon dioxide are called autotrophs.
Considering the various requirements for carbon and energy with the above-defined terms, nearly all living things can be placed (hopefully readily) in one of the descriptive categories listed below. The discussion of each category is more descriptive of the organisms than defining of the terminology; the actual definitions of the root words have been given just above.
The supply of carbon and energy for a particular organism may be relatively simple such as (1) providing light and an atmosphere containing carbon dioxide for photoautotrophs, or (2) providing glucose for the majority of the chemoheterotrophs.
Besides carbon, other required major elements include hydrogen, oxygen, nitrogen, sulfur, phosphorus, potassium, and – to a lesser extent – magnesium, iron, calcium, chlorine and sodium. Other elements, generally required at relatively very low levels, include manganese, cobalt, zinc, molybdenum and copper. (Attempting to group elements according to importance is somewhat arbitrary.) Certain organisms may use one or more of the first four elements in this listing (H, O, N, S) in their simplest, pure molecular forms. Otherwise, elements are always taken in as part of compounds with other elements. For example, organisms which are termed aerobic and facultatively anaerobic regularly use molecular oxygen (O2) in respiration; see our oxygen relationships page. Also, nitrogen-fixing bacteria can obtain their nitrogen from the reduction of atmospheric nitrogen (N2) to ammonium; the nitrogen becomes incorporated into amino acids and ultimately proteins.
Many of the latter elements in the above listing are required in such small amounts that one can depend on their compounds to be present as inorganic chemical contaminants in the various ingredients used to make media. Such elements not individually added are termed trace elements.
To a greater or lesser degree, various organisms may require pre-formed organic compounds which these organisms are incapable of synthesizing. Depending on a particular organism's capabilities of producing the essential organic compounds it needs for structure or metabolism, certain amino acids, fatty acids, nucleic acids, vitamins or other compounds may have to be supplied to that organism. A growth factor is therefore defined as a specific organic compound that is required – generally in a very small amount – by a particular organism as it cannot be synthesized by that organism. Organisms termed fastidious tend to require a variety of growth factors.
Each organism has its range of growth and its optimum pH value. Organisms themselves may change the pH of their immediate environment. For example, the pH of a medium tends to decrease when microbial fermentations take place, producing acidic products. Buffers, such as phosphates and calcium carbonate, are often utilized to help stabilize the pH during the growth of the cultures studied.
Incubation conditions must be appropriate for the organism under study. Considerations include the provision of a suitable atmosphere, a suitable temperature, and anything else which may be required such as a light source for the cultivation of phototrophs.
The ingredients in culture media range from pure chemical compounds to complex materials such as extracts or digests of plant and animal tissues. If all the ingredients of a culture medium are known, both qualitatively and quantitatively, the medium is called a chemically-defined medium. These media are of great value in studying the nutritional requirements of microorganisms or in studying a great variety of their metabolic activities. In a complex medium, the exact chemical composition is not known, and such a medium is often prepared from very complex materials, e.g., body fluids, tissue extracts and infusions, and peptones. A peptone is a commercially-available digest of a particular plant or animal protein, made available to organisms as peptides and amino acids to help satisfy requirements for nitrogen, sulfur, carbon and energy. Peptones also contain small amounts of various organic and inorganic compounds, and a peptone solution can serve as a complete medium for many organisms including E. coli. Complex media often contain all nutrients, known and unknown, which may be required for optimal growth of a wide assortment of bacteria. Commonly-used constituents of microbiological culture media are summarized in Section VI.
Here are a couple examples of media, each formulated for a purpose:
An organism which is not fermenting glucose may still be able to grow in the medium by respiring the glucose and/or one or more of the amino acids in the peptone. In any event, an alkaline reaction will occur at the top of the medium if an organism deaminates amino acids aerobically, producing ammonium. A cautionary note: The alkaline reaction from this ammonium can overneutralize acid which permeates throughout the medium from glucose fermentation (an anaerobic process), and acid may not be detectable at all if the peptone concentration is too high. So, one is careful regarding the addition of the peptone and usually any acid from fermentation is detectable at least in the lower part of the tube. With Glucose O/F Medium, the formulation elevates the amount of glucose and decreases that of peptone such that even the very small amount of acid associated with glucose respiration is detectable for organisms which do not ferment. Glucose Fermentation Broth and O/F Medium are discussed more fully here. How competing acid and alkaline reactions in a differential medium can be used to advantage in bacterial identification is discussed here.
If one is studying an auxotrophic strain of E. coli – i.e., one which cannot produce (from the constituents of the E. coli minimal medium) a compound essential for its metabolic needs which prototrophic (typical) strains can so produce – that compound will have to be added specifically to the medium in which case it is then termed a growth factor.
One may ask the question as to whether this example is a chemically defined or complex medium. Given that trace elements may be present as chemical contaminants of the listed ingredients, which (furthermore) are not indicated as being provided in specific amounts, one would have to call this medium complex. Chemically-defined media – as strictly defined – are very exceptional, utilizing ingredients of extreme purity and including a long list of additional compounds to compensate for the lack of trace elements in those pure ingredients.
Agar is the major solidifying agent used in bacteriological media. It is an impure polysaccharide gum obtained from certain marine algae. It is added as a powder at a more or less standard concentration (1.5% for plates and slanted media, 0.5% or less for "semisolid" media), usually after the other medium components have been added and dissolved in the water. Agar dissolves at approximately 100°C, and an agar-containing medium thus heated will not solidify until the temperature is brought down to about 43°C. Once solidified, the medium will not melt until brought back up to about 100°C. Among the advantages of this interesting temperature-related property are the following: (1) The medium can be inoculated while in a liquid state at a low enough temperature (approx. 43-50°C) such that the cells will not die off, and (2) the medium, once solidified, will stay solid over a wide range of incubation conditions.
Two additional attributes of agar are its resistance to degradation by nearly all organisms and its relative clarity, permitting easy viewing of growth on or in the medium. One drawback to agar is the fact that it is very difficult, if not impossible, to purify it fully of trace impurities. Thus, when agar is added to a chemically-defined liquid medium, the medium must be considered complex. If an absolutely chemically-defined solid medium is required, silicon-based solidifying agents can be employed.
Previous to agar, potato slices and gelatin were utilized to form solid substrates upon which microbial colonies could be grown and studied. These materials were unacceptable for general use due to their ability to be broken down by a wide variety of microorganisms. Furthermore, gelatin liquefies in a warm room, and potato slices are opaque. In 1881, Fanny Eilshemius Hesse, a technician in the laboratory of Robert Koch in Germany, introduced the concept of agar to bacteriology, having used it for many years in the preparation of homemade jellies.
A classification of media based on their respective uses follows. Note that these categories can overlap. Furthermore, by now you should be using these terms correctly: Medium is always the singular form of the word, and media is always (and only!) the plural form.
|Selected General Microbiology Topics:
" General Overview of Catabolism
" Review of the Cycles of Elements
" Differential Media Site
Archived Bacteriology 102 site
Site Outline of related pages
These general microbiology pages found