Method: Removal of Yeast Contamination from Lymphoblast
Cultures
May 30, 1990
Rosalie Veile
Purpose:
This method is advantageous for saving the occasional cultures that
become contaminated. Yeast contaminated cultures will appear cloudy
when slightly shaken and lymphocytes will not cluster together as much
as normal. If cultures are suspect, a drop of culture can be streaked
on a YPD media plate to check for growth of yeast colonies, or a 5 ml
sample can be taken to Barnes Diagnostic Center for identification of
yeast strain.
Procedure:
- Pipet 5 ml histopaque into a 15 ml centrifuge tube.
- Carefully layer on top of histopaque 10 ml of contaminated culture
(or concentrated cells/yeast resuspended in growth media).
- Centrifuge tube for 25 minutes at 2500 rpm (no brake) at room
temperature, using the TJ-6 centrifuge.
- The yeast cells will pellet to the bottom of the histopaque
gradient and the lymphoblast cells will be located on top of
histopaque gradient. Remove the lymphoblast cells with a 10 ml
disposable pipet, and transfer to a 15 ml centrifuge tube.
- Wash cells by adding 10 ml of wash media to cells. Centrifuge 10
minutes at 1200 RPM, no brake, at room temperature. Aspirate off the
wash media and resuspend in RPMI-growth media containing 1X
antimycotic/antibiotic. This will remove the rest of the yeast cells.
- Transfer the cells to a 25cm2 Tissue Culture Flask and feed the
culture twice a week with 1X antimycotic/antibiotic media until all
traces of contamination are gone. This will depend on the severity of
the contamination (usually for cultures moderately contaminated, 2
weeks or 4 feedings will suffice). After contamination is no longer
visible, feed the cultures with growth media containing only antibiotic
and not the antimycotic.
Solutions:
Growth media: (1 liter)
To 1 liter of strerile RPMI 1640 with 2mM L-glutamine, add:
165.0 ml fetal bovine serum, heat inactivated at 50-60 degrees C for
one half hour
12.0 ml 200mM (100 X) L-glutamine
1.2 ml 50 mg/ml gentamicin reagent
Filter sterilize through a 0.22 µm cellulose acetate filter and store
up to 2 weeks at 4 degrees C.
1X Cyclosporin media: (100ml)
To 100 ml of growth media add:
1.0 ml 100X cyclosporin A
100X Cyclosporin A: (100ml)
Dissolve 1 mg CSA in 0.1 ml ethanol in a sterile 15 ml centrifuge tube
with a small magnetic stirrer. Add 0.02 ml (or 20µl) of Tween 80 and
mix well. While continually stirring, add 1 ml RPMI drop by drop.
Quanitate to a final volume of 100 ml with RPMI. Filter sterilize with
a 0.22 µm filter. Store at 4 degrees C for up to 4 months.
Antimycotic/antibiotic media:
To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add:
165.0 ml fetal bovine serum, heat inactivated
12.0 ml 200mM (100X) L-glutamine
12.0 ml antimycotic/antibiotic (100X), liquid, Gibco, Cat. No.
600-5240AG
Filter sterilize through a 0.22 µm cellulose acetate filter and store
up to 2 weeks at 4 degrees C.
References:
Biotechniques, Overhauser, Joan, et al. Department of Biochemistry and
Molecular Biology, Thomas Jefferson University, Philadelphia, PA.
