Nutrition and Cultivation of Bacteria – Page 1

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.

I. Nutritional Classification of Microorganisms (based on energy and carbon requirements)

The various life forms may be categorized as being either chemotrophs or phototrophs regarding the source of the energy which is used in ATP generation. Chemotrophs obtain their energy purely from the oxidation of chemical compounds. Phototrophs use light as the ultimate source of energy. Phototrophs include plants, algae, cyanobacteria, and the purple and green anoxygenic bacteria.

When it comes to describing any specific organism regarding its growth characteristics and means of obtaining energy, the terms "strictly aerobic," "facultatively anaerobic" and "strictly anaerobic" can be incomplete and misleading – as discussed here. In place of these terms – or in addition to them – one or more of the more-clearly-definable methods of energy generation of which the organism is capable should be indicated:

• aerobic respiration
  
• anaerobic respiration
  
• fermentation
  
• anoxygenic phototrophy
  
• oxygenic phototrophy

The common laboratory test for "oxygen relationships" determines if an organism (able to grow under the conditions given) can respire (aerobically) or ferment and is generally used to describe and compare chemoheterotrophic bacteria.

Another method of classifying organisms nutritionally is by the source of reducing power utilized. All organisms need reducing power in the form of electrons for biosynthesis. Organisms that oxidize organic compounds are called organotrophs and those that oxidize inorganic compounds are called lithotrophs (which literally means "stone eaters"). Some authors apply these terms exclusively to chemotrophs, using the terms "chemoorganotrophs" and "chemolithotrophs." In the generation of reducing power and energy, phototrophs may also oxidize either organic or inorganic compounds, and we may thus have the categories of "photoorganotrophs" and "photolithotrophs." Examples of inorganic substances oxidized by the latter include sulfur, sulfides, thiosulfates and hydrogen gas.

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 in one of the descriptive categories listed below. 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.

• CHEMOHETEROTROPHS. As these organisms are generally organotrophic, they may also be termed chemoorganotrophs. These organisms may use a variety of organic compounds as both carbon and energy sources. A common sugar so used is glucose. ATP is generated by either substrate-level or oxidative phosphorylation.
  
  
• CHEMOAUTOTROPHS. As these organisms are generally lithotrophic, they may also be termed chemolithotrophs. ATP is usually generated by oxidative phosphorylation.
  
  
• MYXOTROPHS do not follow the correlations noted for the above two groups. These organisms are actually "chemolithotrophic heterotrophs" and include the genus Beggiatoa.
  
  
• PHOTOTROPHS. Traditionally, these organisms are thought of as being autotrophs and lithotrophs, taking in carbon dioxide and generating reducing power by the oxidation of water with the release of oxygen. The classic "photosynthetic equation" we grew up with and applied to trees and other plants is based on this. In place of water, the purple and green sulfur bacteria substitute hydrogen sulfide (H₂S) or hydrogen (H₂). In Bacteriology 102, we study the purple non-sulfur bacteria, all of which are heterotrophs and organotrophs – obtaining carbon and reducing power at the expense of organic compounds. (Autotrophic/lithotrophic growth similar to that of the purple sulfur bacteria is an alternative for some bacteria in this group.) For phototrophs in general, ATP is generated by photophosphorylation.

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.

II. Other Nutritional and Physical Requirements

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.

III. Putting Together a Culture Medium

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:

1. A broth (i.e., liquid) medium which is designed to detect acid produced by an organism from glucose fermentation (i.e., Glucose Fermentation Broth) may include the following ingredients:
   
   
   • Distilled water
     
   • Glucose:  Supplied for carbon and energy. Without glucose, a "facultative anaerobe" (as "officially" defined here) would not be able to grow anaerobically, and an "aerotolerant anaerobe" (also as defined) would probably not be able to grow at all.
     
   • Peptone:  Supplied for reasons given above.
     
   • pH indicator:  Supplied to detect the change to an acidic condition.

   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.

2. Here we have an example of a medium which supplies the basic needs for prototrophic strains (i.e., strains typical of their species regarding their biosynthetic capabilities and requirements) of a common intestinal bacterium, Escherichia coli. Such a medium formulated with nutritional requirements of a given species in mind is called a "minimal medium" as discussed below. Any required element not seen in this list of ingredients is still assumed to be part of the actual medium – having come into the medium as a trace element in one or more of the individual ingredients.
   
   
   • Distilled water
     
   • Glucose:  Supplied for carbon and energy.
     
   • Ammonium sulfate ((NH₄)₂SO₄):  Supplied as the nitrogen source.
     
   • Magnesium sulfate (MgSO₄)
     
   • Di- and monopotassium phosphate (K₂HPO₄ and KH₂PO₄):  In a given combination of amounts, these can provide a certain pH and, as "pH buffers," assist in preventing the pH from varying widely due to an organism's formation of metabolic products, such as acids from the fermentation of the glucose.

   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.