Nutrition and Cultivation of Bacteria – Page 2

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 on the previous page. Minimal media vary in composition according to the minimal nutritional requirements of the particular species under study. A minimal medium for a "fastidious" organism such as Lactobacillus may contain many growth factors such as vitamins and amino acids.
  
  
• 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 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 which does not contain selective agents.
    
    
• A DIFFERENTIAL MEDIUM is one which allows two or more different types of 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 of differential media include the following:
  
  
  • 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.)
    
    
  • Glucose Fermentation Broth. This medium is used in tubes, usually with Durham tubes. As discussed on the previous page, organisms which ferment the sugar (glucose) will cause the pH indicator to change color upon production of acidic products. Additionally, if insoluble gas (H₂) is produced during fermentation, a bubble will be seen in the inverted Durham tube.
    
    
  • Examples of differential media which do not involve pH-related reactions include Motility Medium (exploiting a morphological characteristic – i.e., production of flagella), Nutrient Gelatin, Starch Agar, Kligler Iron Agar (for H₂S production) 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. A list of pH indicators is given here.
  
  
• 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 and are found in the Thioglycollate Medium utilized to determine oxygen relationships.
  
  
• 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. A list of selective agents often used in media to inhibit gram-positive bacteria is given here.