Nicotiana tabacum (BY-2) Suspension cells contaminated! Help! - (Sep/15/2011 )

Hi there,

I have a range of BY-2 Nicotiana tabacum suspension culture cells expressing a variety of constructs which were on a 2 week sub-culture cycle and growing perfectly fine to a thick yellow consistency by the end of the two weeks. Then our shaker broke and I had to rescue them from old backups, some of which were looking almost dead/unhleathy.

Since then I have been struggling to get some of them to grow happily and healthy again. I thought it was just due to the shock of them having to be resurrected from the old cultures but now some which seemed to regrow OK for a while are having problems too.

Some cultures die/begin to look unwell much quicker then they were previously - turning brown (usually they turn more grey) after a week of growth. Others don't seem to get growing at all and end up looking cloudy and white. Now I think I may have infection in some of these lines (perhaps different types - some bacterial, some yeast).

I use hygromycin as my antibiotic (and kanamycin in some double lines). I have not used any fungicides and do not know how to get rid of a fungal infection if this is the problem.

Any advice would be appreciated, I can't afford to lose them!

Thank you,

Petra

Can you look at the cultures under a microscope - that should tell you if you have yeast or bacteria. Yeast will appear as small spheres-ovals with buds or chains coming off the side. Bacteria will just be a shimmer in the medium. Yeast tend to have a characteristic odour, but it varies according to the type as to what the odour is.

It is really tricky to get rid of yeast and other fungal infections, but you can try treating with drugs such as amphotericin B. Be aware that these will also affect your cultures as yeast etc. are also eukaryotes so the drugs have some affect on the plant cells too.