5-1 Bacterial nutrition

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The growth of any microorganism, whether in its natural niche or in a laboratory, is dependent upon the presence of certain essential compounds in its environment. In the lab all these components must be provided for in the culture medium for the organisms to grow. The potential composition of media is as diverse as the number of microorganisms under study. This section discusses the basic requirements for all organisms and the components of culture media. If you want to know more about microbial requirements for growth, read the chapter on bacterial nutrition in your microbiology textbook

A fundamental and often ignored ingredient in all growth media is water. Water is an amazing compound. It is the only natural substance found in all three states; liquid, solid and gas at temperatures commonly found on earth. Water has a high specific heat index and can hold a large amount of heat. Water is the universal solvent, dissolving many more substances than any other liquid. All biological reactions take place in water. Its unique properties allowed the development of life on this planet. When humans discover life on other planets, life forms will likely be water-based. Besides being the solvent of choice, water can also donate H or O to certain reactions, but this contribution is minimal. In the preparation of most culture media, the first ingredient added is water.

The second most important ingredient added to media is the carbon source. For many of the media we will use in this course, an organic compound, such as glucose, will serve as the carbon source. However, this is not always the case. Autotrophs will utilize CO2 for cell carbon and the medium used for the growth of some autotrophs consists of just a few salts. In many cases the carbon source will also serve as the source of energy. E. coli, growing in a minimal glucose medium utilizes the glucose both as carbon and energy source. In other cases, another source will be used to generate energy, including inorganic compounds and light.

Carbon is not the only element needed in relatively large amounts by microorganisms. Hydrogen, oxygen, nitrogen, phosphorous, sulfur, and potassium are usually provided in a culture medium. Some microbes can assimilate these elements in their most simple form (i.e. O2, H2,or N2), while in other cases they have to be provided as a part of a larger molecule (KNO3, amino acids, MgSO4). Other elements that are also essential, but are not added in as large a quantity include, magnesium, iron, calcium, and potassium. Whatever the source, all these elements must be provided to support growth.

Components of Media and its Classification

Since the list of growth requirements is quite extensive, providing all of them to a microorganism in a culture medium would seem complicated. In practice, most media is easy to make, but some formulations can be tremendously difficult. The ingredients added to culture media can range from pure chemical compounds to extracts and digests of plant or animal tissues. If all the components of a medium are known both qualitatively and quantitatively, it is referred to as chemically defined medium. This type of medium is often used to study the nutritional requirements of an organism or is necessary when elucidating various metabolic processes. Complex medium contains components that are extracts or digests whose exact chemical composition is impossible to determine and often varies from lot to lot. Therefore the exact amount of ingredients in complex medium is generally unknown. Common extracts and digests used in the preparation of microbial medium include; brain heart infusion (boiled, concentrated cow brains and hearts), yeast extract (killed and purified, dehydrated yeast) and various peptones (a digest of certain plant and animal proteins). These complex materials can provide carbon and energy sources, all necessary minerals, and growth factors (known and unknown) to an organism. Complex medium is often used in diagnostic tests, since they often provide all necessary components for growth of many different microorganisms.

Media used in the cultivation of microorganisms can also be classified according to the way in which it is used.

  1. A medium that contains only the minimal components necessary for growth of a microorganism is termed a minimal medium. This type of medium can be simple, containing a few salts ((NH)4SO4, KH2PO4, MgSO4) and a carbon source (glucose). Minimal medium can also be extremely complex. A medium formulated for the growth of Leuconostoc mesenteroides, a fastidious organism, contains glucose, 7 salts, 19 amino acids, 4 nucleotides, and 10 vitamins.
  2. All purpose media are able to support the growth of a wide variety of microorganisms. These media are usually complex. Some examples of all purpose media include; Brain Heart Infusion Broth, All Purpose Tween (APT), Penassay agar and Luria Broth.
  3. Some media contain ingredients that inhibit the growth of a certain class of microorganisms. For example, MacConkey's Agar contains bile salts and crystal violet to inhibit most gram positive microorganisms. This type of medium is termed selective medium, since it selects for a certain class of microorganisms. Other examples include Eosin Methylene Blue (EMB) Agar and Lactose Lauryl Tryptose Broth.
  4. Differential media help us distinguish between different groups of microorganisms by some biochemical or physiological criteria. This type of medium is useful in identifying the genera of microorganisms under study. Examples of this type include MacConkey's agar sugar fermentation broths, and Kliger's Iron Agar.

If a solid medium is necessary, agar is usually added as the solidifying agent. For plates or slants, a 1.5% concentration of agar is typical. For semi-solid medium < 0.5% agar is employed. Agar is a complex, long chain polysaccharide derived from certain marine algae and has several useful properties. When added to a solution, it melts at 100 °C forming a slightly viscous liquid that solidifies at ~43 °C. After solidification, the agar will not melt unless the temperature is again raised to 100 °C. This is a tremendously useful property as you will discover later in the semester. Some other useful properties of agar include its resistance to microbial degradation and its translucence for easy viewing of colonies embedded in the agar. One important disadvantage of agar is its tendency to harbor impurities, which are virtually impossible to completely extract. With certain organisms, these impurities can sometimes interfere with nutritional studies, and even inhibit growth. Chemically defined medium that contains agar must technically be considered complex. If agar presents a problem in certain studies, silica based solidifying agents are usually used as a substitute.

We owe the adoption of agar as a solidifying agent in microbiology to Fanny Eilshemius Hesse. Robert Koch and her husband Walter Hesse were searching for a method of culturing microorganisms on solid surfaces. Walter had tried using potato slices and solidified gelatin, both with unsatisfactory results. In 1881, Fanny suggested he try agar (a thickener she often used in cooking). With this new solid medium, Walter was able to develop pure culture techniques and discover the causes of many diseases, including tuberculosis.

Sterilization of Media

In almost all cases, once a medium is made, it must be treated to eliminate any microorganisms contaminating containers, media ingredients, weighing papers, or other surfaces that come in contact with the medium. If this is not performed correctly, contaminates arise during incubation, making microbiological investigations impossible. Sterilization is defined as the inactivation (or removal) of all life forms (including the pseudo-life forms, viruses) in a specific area. Culture media must be made sterile without inactivating nutrients necessary for growth of the microorganism. Equipment and media used in the microbiology laboratory are most often sterilized using one of the methods listed in Figure 5-14.

Figure 5-14 Sterilization of media

Method Mode of action Materials
Autoclaving - moist heat (121 C) under pressure (15-17 lb/in2). coagulates proteins heat stable items such as most culture media, glass and metal, but not plastic.
Oven - dry heat (160 C for several hours). coagulates proteins glass and metals but not liquids nor plastics.
Filtration - 0.22 to 0.45 m pore size. prevents organisms from passing through the filter, but does allow viruses to pass so therefore not sterilization in true sense. Solutions of heat sensitive compounds such as some amino acids, vitamins, some sugars, antibiotics.
Radiation - ultraviolet light or gamma rays. damages nucleic acids heat sensitive solids such as plastics, however effective on surfaces only.
Gas-ethylene oxide. inactivates enzymes. heat sensitive solids such as some plastics.

Some common methods for the sterilization of media and instruments. The methods used depends upon the material and its intended use.

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