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Media Making

Microbial Requirements for Growth

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.

A fundamental and often ignored ingredient in all growth media is water. Water is an amazing compound. Its unique properties allowed the development of life on this planet. It is the universal solvent in which all biological reactions take place. If (or 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. An All purpose medium is 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. A differential media distinguishes 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 MacConkeys 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 100C forming a slightly viscous liquid that solidifies at ~43C. After solidification, the agar will not melt unless the temperature is again raised to 100C. 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.

Period 2

Materials

Media Making

To become familiar with what goes into mixing up a batch of medium, 4 plates of a minimal medium and 4 plates of a complex medium will be prepared.

  1. Go to one of the designated measuring areas and weigh out each component required for the Minimal Medium - Part A. Use the recipe listed above. Note: The above recipe is in grams/liter and you are making 100 ml not 1 liter of each medium.
  2. Carry the measured amounts back to your lab bench. Use the graduated cylinder to measure 50 ml of distilled water (dH2O) and pour this into one 250 ml flask.
  3. Place a magnetic stir bar in the minimal medium flask, place it on the magnetic stirrer and turn it on. Add the first ingredient on the list and let it dissolve completely. Then add the next ingredient. Continue until all ingredients have been added. When finished, place a foam plug in the flask. (The stir bar can remain in the flask).
  4. Fill the other 250 ml flask with 100 ml of dH2O and place the stir bar in the flask. In this case we are using a dehydrated ready-made medium. Weigh out an appropriate amount of Nutrient Agar powder, add it to the flask, and stir. That's it! Many types of media come in dehydrated, premixed form. They are convenient and cheap. Which brings up a rule to remember, scientists always try to make necessary chores, like media making, as quick and convenient as possible, thus freeing their time for more fruitful pursuits.
  5. Place the finished medium in the trays provided. When everyone is finished, attend the autoclaving demonstration. Autoclaving will take about 45 minutes total. During the waiting period, perform the other experiments scheduled for today.
  6. When the medium is sterile, place it in the 50C water bath to cool down. The minimal medium needs some additions (a carbon source and agar). Once the minimal medium is cool, add 0.4 ml of 50% glucose (Minimal Medium Part C) to the medium and 50 ml of Minimal Medium Part B (This is pre-made and is located in the 50C water bath). Swirl the flask and pour the now complete Minimal Medium into 4 plates. Also, swirl the Nutrient Agar flask and pour 4 plates.
  7. Let the plates cool and when completely solidified place them in your lab bench drawer, upside down, until next period
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