Page 252 - Algae Anatomy, Biochemistry, and Biotechnology

Algal Culturing 235

. CSIRO (CSIRO Collection of living microalgae, Tasmania, http://www.marine.csiro.au/
microalgae/collection.html)
. PCC (Pasteur Culture Collection of Cyanobacteria http://www.pasteur.fr/recherche/
banques/PCC/)

STERILIZATION OF CULTURE MATERIALS

All vessels used for culture purpose should be scrubbed (abrasive brushes not appropriate for most
plastics) and soaked with warm detergent (not domestic detergents, which leave a residual film on
culture ware, but phosphate-free laboratory detergent), then rinsed extensively with tap water. After
soaking in 10% HCl for 1 day–1 week (not routinely necessary, but particularly important for new
glass and polycarbonate material), vessels should be rinsed extensively with distilled and finally
bidistilled water, and left inverted to dry in a clean, dust-free place, or in an oven.
Sterilization can be defined as a process which ensures total inactivation of microbial life, and
should not be confused with disinfection, which is defined as an arbitrary reduction of bacterial
numbers. The primary purpose of sterilization is to prevent contamination by unwanted organisms,
but it may also serve to eliminate unwanted chemicals. Sterilization can be obtained by means of
several methods, the choice depending on the purpose and material used, either empty glassware/
plasticware or medium-containing vessels, but also on the facilities available in a laboratory.
Several methods are available for sterilization of material, some of which can be used also for
growth media:

. Gas such as ethylene oxide, EtO, finds the best application on heat sensitive equipment on
which steam autoclaving (sterilization with heat) cannot be performed. EtO sterilizes by
alkylation, it substitutes for hydrogen atoms on molecules such as proteins and DNA,
and, by attaching to these molecules and disrupting them, EtO stops these molecules’
normal life-supporting functions. This method is widely used for the sterilization of
medical devices, but it is not a routinely available technique for algology laboratories.
Moreover, EtO is a potent carcinogen.
. Dry heat: some laboratories use dry heat to sterilize empty vessels, putting the material in
an oven for at least 3–4 h at 1608C; however, only higher temperature (200–2508C)
guarantees an effective result. Vessels are covered with aluminum foil to maintain sterility
on removal from oven. This procedure is suitable only for few materials that stand high
temperatures, such as glass, teflon, silicone, metal, and cotton.
. Autoclaving (moist heat) is the most widely used technique for sterilizing culture media and
vessels, and is the ultimate guarantee of sterility (including the destruction of viruses). A
commercial autoclave is the best, but pressure cookers of various sizes are also suitable.
Sterility requires 15 min at 1–2 Bar pressure and a temperature of 1218C in the entire
volume of the liquid (i.e., longer times for larger volumes of liquid; approximately
10 min for 100 ml, 20 min for 2 l, 35 min for 5 l). Flasks containing media should not be
more than half full, and should be left partially open or plugged with cotton wool or
covered with aluminium foil or paper, because for sterility the steam must penetrate the
material. Autoclave steam may introduce chemical contaminants; empty glass and polycar-
bonate vessels should be autoclaved containing a small amount of bidistilled water which is
poured out (thus diluting contaminants) under sterile conditions immediately prior to use.
Vessels should never be closed, because of the risk of implosion, by using cotton wool
bungs, or by leaving screw caps slightly open. Ensure that heating elements are covered
with distilled water, and the escape valve should not be closed until a steady stream of
steam is observed. After autoclaving, the pressure release valve should not be opened
until the temperature has cooled to below 808C. Autoclave steam may contaminate the
media (i.e., with trace metals from the autoclave tubing). Autoclaving also produces