Page 229 - Algae Anatomy, Biochemistry, and Biotechnology

212 Algae: Anatomy, Biochemistry, and Biotechnology

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. Alterna-
tively, 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 21 ) of germanium dioxide, GeO 2, 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 uni-
algal 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 exper-
imental work, and may lead to the extinction of the desired algal species in culture through out-
competition 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 con-
tamination 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 21 ultra-
sonic water bath for varying lengths of time (a few seconds to tens of minutes) can sometimes phys-
ically 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, tetra-
cycline, and bacitracin. Antibiotic solutions should be made with distilled water and filter-sterilized
(0.2 mm 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 micro-
algal 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 hand-
ling 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
2
of about 1.5 W m and a temperature of 16–198C. Constant illumination is suitable for the