2.8@ Removal of contaminants

 

Yuji Hiwatashi and Mitsuyasu Hasebe

 

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.