Page 253 - Algae

236 Algae: Anatomy, Biochemistry, and Biotechnology

leaching of chemicals from the medium receptacle into the medium (silica from glass
bottles, toxic chemicals from plastics). Autoclaving in well-used Teflon or polycarbonate
vessels reduces leaching of trace contaminants.
. Pasteurization (heating to 90–958C for 30 min) of media in Teflon or polycarbonate bottles
is a potential alternative, reducing the problems of trace metal contamination and alteration
of organic molecules inherent with autoclaving. Pasteurization does not, however, comple-
tely sterilize seawater containing media; it kills all eukaryotes and most bacteria, but some
bacterial spores probably survive. Heating to 90–958C for at least 30 min and cooling,
repeated on two or more successive days (“tyndallization”) may improve sterilization
efficiency; it is assumed that vegetative cells are killed by heat and heat resistant spores
will germinate in the following cool periods and be killed by subsequent heating.
. Ultraviolet radiation (240–280 nm) is not often used for culture materials, because very
high intensities are needed to kill everything in a medium such as seawater (1200 W
lamp, 2–4 h for culture media in quartz tubes). Such intense UV light necessarily alters
and destroys the organic molecules in seawater and generates many long lived free radicals
and other toxic reactive chemical species (Brand, 1986). Seawater exposed to intense UV
light must, therefore, be stored for several days prior to use to allow the level of these highly
reactive chemical species to decline.
. Sterile filtration is probably the best method of sterilizing certain media, especially
seawater-based media, without altering their chemistry, as long as care is taken not to con-
taminate the seawater with dirty filter apparatus. Sterilization efficiency is, however, to
some extent reduced compared with heat sterilization methods. Membrane filters of
0.2 mm porosity are generally considered to yield water free of bacteria, but not viruses.
0.1 mm filters can be used, but the time required for filtration of large volumes of culture
media may be excessively long. The filtration unit must be sterile: for small volumes
(,50 ml) pre-sterilized single use filter units for syringe filtration (e.g., Millipore Millex
GS) can be used; for volumes up to 1 l reusable autoclavable self-assembly filter units
(glass or polycarbonate) with 47 mm cellulose ester sterile membrane filters (e.g., Millipore
HA) can be used with suction provided by a vacuum pump; for larger volumes an inline
system with peristaltic pump and cartridge filters may be the best option. Filter units
(particularly disposable plastic systems) and the membrane filters can also leak toxic
compounds into the filtrate. The first portion of filtrate (e.g., 5% of the volume to be filtered)
should be discarded to alleviate this problem.
. Most stock solutions of culture medium additions can be sterilized separately by auto-
claving, although vitamin stock solutions are routinely filtered through 0.2 mm single
use filter units (e.g., Millipore Millex GS), because heat sterilization will denature
these organic compounds. Filter sterilization of all additions may reduce uncertainties
about stability of the chemical compounds and contamination from autoclave steam,
but absolute sterilization is not guaranteed. Stock solutions can be stored in ultraclean
sterile glass, polycarbonate, or Teflon tubes/bottles. In order to minimize effects of
any microbial contaminations, all stock solutions should be stored in a refrigerator at
48C, except vitamin stocks which are stored frozen at –208C and thawed immediately
prior to use.

CULTURE METHODS

Algae can be produced according to a great variety of methods, from closely-controlled laboratory
methods to less predictable methods in outdoor tanks. Indoor culture allows control over illumina-
tion, temperature, nutrient level, contamination with predators, and competing algae, whereas
outdoor algal systems, though cheaper, make it very difficult to grow specific algal cultures for