Nutrition and Cultivation of Bacteria

Adapted from Appendices D.1 and E.1 in the lab manual by J. A. Lindquist (1999):
General microbiology: a laboratory manual, 3rd edition, McGraw-Hill, ISBN 0-07-235906-4.

This page is subdivided as follows: (Click on the X.)

X  Nutritional Classification of Microorganisms (based on energy and carbon requirements)
X  Other Nutritional and Physical Requirements
X  Putting Together a Culture Medium
X  Solid Media
X  Classification of Culture Media
X  Summary of Commonly-Used Constituents in Microbiological Media

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. Water can supply some hydrogen and oxygen in certain chemical reactions.

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

Regarding the source of energy which becomes trapped in an organism's ATP, the various life forms may be categorized as either chemotrophs or phototrophs. 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. That which is summarized in the following table is given further treatment here.

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 obtain their electrons from organic compounds are called organotrophs. Those that obtain their reducing power from inorganic chemicals are called lithotrophs ("stone eaters").

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 described above, nearly all living things can be placed in one of the following categories:

• CHEMOHETEROTROPHS. As these organisms are generally organotrophic, they may also be called 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 called 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. 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 below.

Given below is an example of a broth (i.e., liquid) 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 (see above).

• Distilled water
  
• Glucose – supplied for carbon and energy
  
• Ammonium sulfate ((NH₄)₂SO₄) – 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 the above medium is chemically defined or complex. 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.

As far as the growth of E. coli is concerned, a peptone can substitute for all of the compounds listed above and also supply needed trace elements, as it is a relatively crudely-prepared material.

IV. Solid Media

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.

V. Classification of Culture Media

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.

• A MINIMAL MEDIUM is one which supplies only the minimal nutritional requirements of a particular organism. As an example, a typical, prototrophic strain of E. coli is able to synthesize all of its cell components from a simple solution containing several "mineral salts" plus glucose as the source of carbon and energy – such as the medium given above. Minimal media vary in composition according to the minimal nutritional requirements of the particular species under study.
  
• An ALL-PURPOSE MEDIUM is rich in a wide variety of nutrients (including many growth factors) and will, therefore, support the growth of a wide range of bacteria. All-purpose media listed in Appendix E include Nutrient Agar, APT Agar, Plate Count Agar, Heart Infusion Agar, Brain Heart Infusion Agar and Penassay Agar.
  
• A SELECTIVE MEDIUM supports the growth of desired organisms while inhibiting the growth of many or most of the unwanted ones – either by purposely adding one or more selective agents which "poison" certain types of organisms or by including or deleting certain nutrients such that the desired organisms and few others are able to grow. Examples on how these things may be accomplished are as follows:
  
  • MacConkey Agar. This is an example of a medium where selective agents are added which directly suppress the growth of undesired organisms as much as possible. The particular selective agents chosen for this medium – bile salts and crystal violet – inhibit gram-positive bacteria, allowing the near-exclusive growth of gram-negative bacteria.
    
  • Nitrogen-Free Broth. Here the medium is made selective by the deletion of a required element; no nitrogen compounds are present. Therefore, the only organisms which can grow after inoculation into this medium are those which can utilize gaseous nitrogen (N₂) which diffuses in from the atmosphere. These are the nitrogen-fixing bacteria. While this medium does not utilize selective agents, it is still restrictive to an extensive number of various organisms.
    
  • Succinate Broth. In this example, a particular nutrient utilized by the desired organism – and few others – is included as the only carbon source. This medium is used for the enrichment of the purple non-sulfur photosynthetic bacteria; most other organisms tend not to metabolize succinate under the anaerobic conditions utilized. This is another example of a restrictive medium not utilizing selective agents.
• A DIFFERENTIAL MEDIUM is one which allows two or more different organisms to grow, but it contains dyes and/or other components upon which different organisms act in various ways to produce a variety of end products or effects, often detected by variations in color. These differences are often very apparent among colonies of a mixed culture growing in a petri dish. Pure cultures, growing in separate tubes of the same differential medium, may also be characterized and differentiated from one another according to a particular biochemical characteristic. Examples:
  
  • MacConkey Agar. This medium is used in plates. Organisms which ferment the lactose in the medium will lower the pH due to the production of acids. The pH indicator (neutral red) will turn red, and the colonies will consequently have a reddish appearance. Other colonies on the same plate which do not contain lactose-fermenting cells should appear whitish. (As this medium also appears in the above category, it is termed a selective-differential medium.)
    
  • Carbohydrate Fermentation Broth. This medium is used in tubes, usually with Durham tubes. Organisms which ferment the particular carbohydrate in the medium (e.g., glucose, lactose, sucrose) will cause the pH indicator to change color. Also, if insoluble gas (H₂) is produced during fermentation, a bubble will be seen in the inverted Durham tube.
    
  • Other examples of differential media include Motility Medium (exploiting a morphological characteristic – production of flagella), Nutrient Gelatin, Starch Agar, Kligler Iron Agar and Blood Agar.

VI. Summary of Commonly-Used Constituents in Microbiological Media

• AGAR. Agar is used as a solidifying agent in media. It is an impure polysaccharide gum obtained from certain marine algae. Agar dissolves and melts around 100°C and solidifies around 43°C. Generally agar itself is not used as a nutrient by microorganisms.
  
• BODY FLUIDS. Whole or defibrinated blood, plasma, serum or other body fluids are frequently added to culture media for the isolation and cultivation of many pathogens. Body fluids contribute many growth factors and/or substances which detoxify certain inhibitors.
  
• BUFFERS. These compounds are incorporated to maintain the optimum pH range of the organism. Substances like sodium and potassium phosphates and calcium carbonate prevent marked changes in pH which otherwise would result from microbial production of organic acids or bases. Crude organic preparations such as peptones (see below) also act as buffers.
  
• EXTRACTS. Eucaryotic tissues (yeast, beef muscle, liver, brain, heart, etc.) are extracted by boiling and then concentrated to a paste or dried to a powder. These extracts are frequently used as a source of amino acids, vitamins and coenzymes, including many needed as growth factors by fastidious organisms. Trace elements and other minerals and usually some sugar are also present. (The term infusion refers to the aqueous extracts of these materials used for these purposes without being dried or otherwise concentrated, although "infusion" is sometimes used synonymously with "extract.")
  
• PEPTONES. These complex mixtures of organic and inorganic compounds are obtained by digestion of protein-containing tissues of animals and plants such as meat scraps, beef muscle, gelatin, milk protein (casein) and soybean meal. These materials are then dried down to a powder and made commercially available to microbiology laboratories. Peptones primarily contain peptides and single amino acids. Being crude digests of complex materials, they contain a great variety of other organic and inorganic materials, but they may be deficient in certain minerals and vitamins. Three examples of brand names of peptones are Tryptone (or Trypticase; a pancreatic digest of casein), Phytone (or Soytone; a papaic digest of soybean meal) and simply Peptone (a digest of beef muscle). Peptones are used frequently in conjunction with extracts for the cultivation of fastidious organisms, and a simple peptone solution will support the growth of many organisms. Peptone in a concentration of 0.1% is often used as a diluent.
  
• pH INDICATORS. An acid-base indicator is often added to differential media to detect changes in hydrogen ion concentration during the growth of an organism such as in Carbohydrate Fermentation Broth, Kligler Iron Agar, Simmons Citrate Agar, MacConkey Agar and Glucose O/F Medium. Brom-cresol purple, brom-thymol blue and phenol red are commonly used; for each of these, an acidic pH turns the indicator a yellow color.
  
• REDUCING AGENTS. Certain chemicals may stimulate growth by reducing the oxidation-reduction potential in the environment. Cystine and thioglycollate are reducing agents often used for the cultivation of anaerobes.
  
• SELECTIVE AGENTS. Antimicrobial agents such as crystal violet, bile salts, brilliant green, potassium tellurite, sodium azide and antibiotics can be employed in selective media to suppress or inhibit the growth of certain groups of microorganisms while allowing growth of desired organisms. These agents are usually bacteriostatic.