Media Choice and Preparation

The correct maintenance of algal strains is dependent on the choice of growth media and cultureparameters. Two approaches are possible for selection of media composition:

• In theory it is best to work on the principle that if the alga does not need the addition of any particular chemical substance to the culture media (i.e., if it has no observable positive effect on growth rate), do not add it.
• In practice it is often easier to follow well-known (and presumably, therefore, well tried) media recipes, and safer to add substances “just in case” (providing they have no observable detrimental effect on algal growth).

When choosing a culture medium, the natural habitat of the species in question should be considered in order to determine its environmental requirements. It is important to know whether the environment is eutrophic, hence nutrient rich, or oligotrophic, hence nutrient poor, and whether the algae belong to a r-selected or a k-selected species. r-selected species are characterized by a rapid growth rate, autotrophic metabolism, and a wide environmental plasticity, whereas k-selected species shows a slow growth rate, mixotrophic or photoheterotrophic metabolism, and a low environmental tolerance.

The media recipes currently available are not always adequate for many species, and the exact choice for a particular species therefore is dependent on trial and error. It must be remembered that in culturing in general there are (within limits) no right and wrong methods; culture media have only developed trying out various additions, usually based on theoretical considerations. Refinement of media composition for laboratory-maintained algal cultures have been the object of research for several decades, resulting in many different media recipes being reported in the literature and being used in different laboratories.

Media can be classified as being defined or undefined. Defined media, which are often essential for nutritional studies, have constituents that are all known and can be assigned a chemical formula. Undefined media, on the other hand, contain one or more natural or complex ingredients, for example, agar or liver extract and seawater, the composition of which is unknown and may vary. Defined and undefined media may be further subdivided into freshwater or marine media.

In choosing or formulating a medium, it may be important to decide whether it is likely to promote heavy bacterial growth. Richly organic media should be avoided unless the algae being cultivated are axenic. For contaminated cultures, mineral media should be used. These may contain small amount of organic constituents, such as vitamins or humic acids and, in either case, provide insufficient carbon for contaminating organisms to outgrow the algae.

Culture collections have attempted to rationalize the number of media recipes, and to standardize recipes for algal strain maintenance. In particular, the use of undefined biphasic media (soil/water mixture) is declining, due to lack of reproducibility in media batches and occasional contamination of the media from soil samples.

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 magnetic 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 30° 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 126°C (20 min, 1 atm pressure), or by filtration, using 0.2 µm 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.