Page 233 - Algae

216 Algae: Anatomy, Biochemistry, and Biotechnology

Media may be prepared by combining concentrated stock solutions, which are not combined
before use, to avoid precipitation and contamination. Reagent grade chemicals and bidistilled (or
purer) water should be used to make stock solutions of enrichments. Gentle heating and/or mag-
netic stirring of stock solutions can be used to ensure complete dissolution. When preparing a
stock solution containing a mixture of compounds, each compound has to be dissolved individually
in a minimal volume of water before mixing, then combined with the other and the volume diluted
to the needed amount.
Another practical way of preparing artificial defined media is to mix the ingredients together
and dry them prior to long-term storage. Constituents are added sequentially to distilled water,
smallest quantities first, for a solution and finally a stiff slurry. Prior to the addition of the major
salts, the pH is adjusted between 4 and 5. The mixture is then transferred to a clean desiccator
of suitable size and the final additions are made. The well-stirred slurry is vacuum-dried, and the
dry mixture is then stored with calcium chloride as a desiccant. It can be stored for some years
if kept dry.
Solid media are usually prepared using 1.0–1.5% w/v agar, tubes being rested at an angle of
308 during agar gelation to form a slope that increases the surface area available for growth.
As described earlier, media are sterilized either by autoclaving at 1268C (20 min, 1 atm
pressure), or by filtration, using 0.2 mm pore nitrate cellulose sterile filters. Generally, subculturing
is performed using aseptic microbiological techniques; laminar flow cabinets, equipped with
Bunsen flame, microbiological loops, and glass or plastic sterile pipettes are required.

FRESHWATER MEDIA
Freshwater media are generally selected because they possess characteristics similar to the natural
environment or they differentially select for a specific algal component of the habitat. Media of an
artificial nature, with known chemical composition, are often employed as additives to natural
media with an unknown chemical composition, such as lake water, to enrich them. They are
often used to simulate diverse nutritional or physical requirements of a particular species or
groups of species, especially when the exact nutritional requirements are unknown.
Media are generally prepared from premixed stock solutions. Aliquots from these stocks are
measured and added to a given volume of water. Some, however, must be prepared by weighting
or measuring the desired components and adding them directly to a given volume of liquid.
Accuracy in measuring liquid aliquots from stock solution or water, and weighing of chemicals
is essential. Improper procedures may result in precipitation of one or more of the components
of the medium, such as nitrates and phosphates, or a failure of some of the constituents to go
into solutions.
Stock solution can be prepared and stored at low temperature in tightly sealed glassware,
because evaporation may alter initial concentrations.
Water generally employed for freshwater media should belong to one of the following types:
copper-distilled water; single glass-distilled water; double glass-distilled water; membrane filtered
water; and deionized water. In most laboratories single or double-distilled water is routinely used,
which can be deionized by passing it through a prepacked deionizing column.
As for the marine media, also freshwater media can be “defined” and “undefined.” Defined
medium such as Beijerinck or Bold Basal Medium have been proved successful for many algal
classes. Most of these defined media can be used for additional algal groups by adding a variety
of other components or modifying the amounts of certain reagents. These “undefined” media
often have the advantage of supporting growth of large number of different algal species, but,
when highly organic, they have also the disadvantage of encouraging more bacterial growth than
strictly inorganic media.
Some of the most commonly used freshwater media, defined and undefined, are listed in
Table 6.2–Table 6.9.