2.8@
Removal of contaminants
Introduction
Contamination
is a serious problem but you can recover the sterile culture with the following
methods. However, it takes time to remove contaminants and the best way is to
clean your bench and laboratory not to contaminate.
(Trial 1)
Solution required
E Sterilized water
E Jiffy7 (see section 2.3 Induction of antheridia, archegonia and
sporophytes). @
E Forceps (autoclave)
E Solutions necessary for spore germination (see section 2.1 Germination
of spores)
Procedure
1.
Cultivate
contaminated tissue on a Jiffy7 pot and induce sporangia (see section 2.3 Induction
of antheridia, archegonia and sporophytes).
2.
Sterilize
outer surface of sporangia with sodium hypocholorite and sow spores on the
germination medium (see section 2.1 Germination of spores in detail). Usually
spores in the sporangia are sterile and germinated protonemata should be appropriate
for sterile culture.
(Trial 2)
Solution required
E Forceps (autoclave)
E Medium (see section 2.2 Culture and storage of protonemata and
gametophores)
Procedure
1. Pick up a tip of protonemata with a sterile forceps
under a dissection microscope and move to a new sterile medium. When your line
contains an antibiotic-resistant gene, you should use the antibiotics for
protonema cultivation. For strains without any introduced antibiotic-resistant
genes, you should use medium containing ampicillin or kanamycin, to which P. patens is resistant.
2. Incubate the inoculated plate for 7 days at 25‹C and
then inoculate a tip of protonemata on new sterile medium again.
3. Repeat these steps until you can get a sterile
protonemata.
(Trial 3)
Solution required
E Sterilized water
E PPM (PLANT PRESERVATIVE MIXTURE, Plant Cell Technology, Inc.; http://www.ppm4plant-tc.com/)
E Forceps (autoclave)
E Eppen tubes (autoclave)
E Sterile medium (see section 2.2 Culture and storage of protonemata and
gametophores)
Procedure
1. Pick up gametophores with more than 15 leaves from colonies
into a 1.5 ml sterile eppen tube containing 0.1% PPM solution and keep them for
7 days at 25˚C.
2. Move gametophores to a sterile plate and keep under
the regular cultivation conditions. Most of gametophore cells die, but a few
cells inside of a gametophore stem survive and regenerate after several days.