 |
 |
 |
 |
The investigator has Salmonella that are either kanamycin-resistant or
ampicillin-resistant. The ampicillin-resistant bacteria also have a
plasmid with the gene encoding green fluorescent protein (I remember
seeing some querys on the network concerning this protein, but this is
the first time I have been exposed to it).
The experiment was to mix these two populations so that one mixture
contained 10 amp-resistant GFP (bright) cells per 1 x 10E6 kanamycin
resistant (dark) cells. This mixture was then run on our FACS 440 and we
sorted the bright cells (which could be nicely distinguished). A second
mixture contained 10 dark (kanamycin-resistant) cells per 1 x 10E6 bright
(amp-resistant) cells and in this sort we sorted for the dark cells. In
each sort we collected approximately 1,000 cells and these were put back
into LB medium - the amp-resistant sorted cells into LB medium containing
ampicillin and the kanamycin-resistant sorted cells into kanamycin-containing
LB medium. After a period of time sufficient for them to grom up (2 to 3
days) there was no growth in either culture.
The cells were sorted using 400 mwatts of the 488 nm line. Isoton II was
used as sheath fluid and the cells sorted into buffer supplied by the
investigator. Because so few cells were sorted, the recovery (or even
the presence) of cells was not checked. Sort conditions (drop delay, etc.)
were determined using 1 u beads.
When it was evident the cells were not
going to grow, I did go back and check to see if it was possible to sort
them. For this, I did a sort on another culture of cells (dark
ones) and centrifuged the sorted cells to concentrate them to a point where
I could look at them with a microscope. And there were sorted cells. I
did not, however, put these sorted cells back into culture to see if the
sort affected them in a way that precluded their growth subsequently.
Can anyone shed some light on this. Are there difficulties in getting
sorted bacteria to grow? Could the GFP affect their growth? After being
exposed to that much laser light? Any responses would be helpful.
Thanks.
Ray
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Raymond B. Hester, Ph.D. Someday, after we have mastered the
Flow Cytometry Lab winds, the waves, the tides and
The University of South Alabama gravity, we shall harness the energies
Mobile, AL 36688-0002 of love. Then, for the second time
Email: rhester@jaguar1.usouthal.edu in the history of the world,
Voice: (334) 460-6029 man will have discovered fire.
FAX: (334) 460-6073 -Teilhard de Chardin
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
|
|
|
|