Culture Types

A culture can be defined as an artificial environment in which the algae grow. In theory, culture conditions should resemble the alga’s natural environment as far as possible; in reality many significant differences exist, most of which are deliberately imposed. In fact, following isolation from the natural environment, algal strains are maintained under largely artificial conditions of media composition, light, and temperature. The imposition of an artificial environment on a cell population previously surviving under complex, fluctuating conditions and following a seasonal life cycle inevitably causes a period of physiological adaptation or selection, during which population growth will not occur or is very slow.

While contaminated algal cultures have previously been satisfactory for certain application and experiments, modern experimental methods and application demand that contaminants are not generally present, and that the taxonomy and growth characteristics of strains are defined. Hence, for most purposes, algal cultures are maintained as unialgal, contaminant-free or axenic stocks. “Unialgal” cultures contain only one kind of alga, usually a clonal population (but which may contain bacteria, fungi, or protozoa), whereas “axenic” cultures should contain only one alga and no bacteria, fungi, or protozoa.

To obtain a unialgal culture one species must be isolated from all the rest; three major techniques borrowed from microbiology are available for obtaining unialgal isolates: streaking and successive plating on agar media, serial dilution, and single-cell isolations using capillary pipettes. Streaking is useful for single-celled, colonial, or filamentous algae that will grow on an agar surface. Filaments can be grabbed with a slightly curved pipette tip and dragged through soft agar (less than 1%) to remove contaminants. It is best to begin with young branches or filament tips that have not yet been extensively epiphytized.

Many flagellates, however, as well as other types of algae must be isolated by single-organism isolations or serial-dilution techniques. A particularly effective means of obtaining unialgal cultures is isolation of zoospores immediately after they have been released from parental cell walls, but before they stop swimming and attached to a surface. Recently released zoospores are devoid of contaminants, unlike the surfaces of most algal cells, but catching zoospores requires a steady hand and experience.

Sterile cultures of microalgae may be obtained from specialized culture collections. Alternatively, axenic cultures can be obtained by treating isolated algae to an extensive washing procedure, or with one or more antibiotics. Resistant stages such as zygotes or akinetes can be treated with bleach to kill epiphytes, then planted on agar for germination. It is usually necessary to try several different concentrations of bleach and times of exposure to find a treatment that will kill epiphytes without harming the alga. When diatoms represent the contaminating species, addition of low concentrations (5 mg l^-1) of germanium dioxide, GeO₂, to a culture medium can inhibit diatom growth, because it disrupts silica deposition.

“Cleaning” previously contaminated cultures is a skilful and time-consuming process, and could take several years in sizeable collections. Extensive measures must be taken to keep pure unialgal cultures chemically and biologically clean. Chemical contamination may have unquantifiable, often deleterious, and therefore undesirable effects on algal growth. Biological contamination of pure algal cultures by other eukaryotes and prokaryotic organisms in most cases invalidates experimental work, and may lead to the extinction of the desired algal species in culture through outcompetition or grazing. In practice, it is very difficult to obtain bacteria-free (axenic) cultures, and although measures should be taken to minimize bacterial numbers, a degree of bacterial contamination is often acceptable.

If biological contaminants appear in a culture, the best remedy is to isolate a single cell from the culture with a micropipette, and try to establish a new, clean clonal culture. Alternatively the culture can be streaked on an agar plate in the hope of attaining a colony free of contaminants. Neither of these methods works well, however, for eliminating bacteria that attach firmly to the surface of microalgae. Placing a test-tube of microalgal culture in a low-intensity 90 kilocycles sec^-1 ultrasonic water bath for varying lengths of time (a few seconds to tens of minutes) can sometimes physically separate bacteria without killing the algae, making it easier to obtain an axenic culture by micropipette isolation. Often, however, to achieve an axenic culture, antibiotics must be added to the growth medium to discourage growth of contaminating cyanobacteria and other bacteria. Best results appear to occur when an actively growing culture of algae is exposed to a mixture of penicillin, streptomycin, and gentamycin for around 24 h. This drastically reduces the growth of bacteria while allowing the microalgae to continue to grow, increasing the chances of obtaining an axenic cell when using micropipette or agar streaking isolation. Different algal species tolerate different concentrations of antibiotics, so a range of concentrations should be used (generally 50–500% w/v). Other antibiotics that can be used include chloramphenicol, tetracycline, and bacitracin. Antibiotic solutions should be made with distilled water and filter-sterilized (0.2 µm filter units) into sterile tubes, and should be stored frozen until use. Another approach is to add a range of antibiotic concentrations to a number of subcultures and then select the culture that has surviving algal cells but no surviving bacteria or other contaminants. Sterility of cultures should be checked by microscopic examination (phase contrast) and by adding a small amount of sterile bacterial culture medium (e.g., 0.1% peptone) to a microalgal culture and observing regularly for bacterial growth. Absence of bacterial growth does not, however, ensure that the microalgal culture is axenic, because the majority of bacteria do not respond to standard enrichments. In reality there is no way of demonstrating that a microalgal culture is completely axenic. In practice, therefore, axenic usually means “without demonstrable unwanted prokaryotes or eukaryotes.” Some microalgal cultures may die when made axenic, probably due to the termination of obligate symbiotic relationships with bacteria.

The collection of algal strains should be carefully protected against contamination during handling and poor temperature regulation. To reduce risks, two series of stocks are often retained, one which supplies the starter cultures for the production system and the other which is only subjected to the handling necessary for maintenance. Stock cultures are kept in test-tubes at a light intensity of about 1.5 Wm² and a temperature of 16–19°C. Constant illumination is suitable for the maintenance of flagellates, but may result in decreased cell size in diatom stock cultures. Stock cultures are maintained for about a month and then transferred to create a new culture line.