Culture contamination - yeast contamination (May/07/2007 )

Hello, I have a yeast contamination problem on my cell culture. I'm culturing hydridoma cells and I started working in an old cabinet, once they notice the problem they changed me to a new cabinet, where there is not contamination problems, I'm trying to get read of the yeast by washing the culture but it is almost impossible cause they proliferate very quickly. My hybridoma cells look fine, but the proliferation is limited because of the space the yeast are taking to grow. Any suggestions? I need to check on the antibody production on those hybridomas, but I guess I need to wait until I have more hybridoma cells proliferating per well in order to have enough antibody concentration in supernatant. Am I doing ok? What should I do?

there is a protocol that a colleague told me it's working in cell culture studies :

Decontamination of cells from the yeast
I Destroy yeast
1. Aspirate medium and wash cell in PBS.
2. Incubate cells at 37oC for 5 min in non-diluted antibiotic-antimycotic.
3. Incubate cells at 37oC for 5 min in trypsin. Spin them down (100g, 5 min). DO NOT EXCEED 100g.
II Secure cells from crosscontamination
4. Slowly and carefully aspirate supernatant. Resuspend cells in 0.5 ml of regular medium.
NB! We assume that your regular medium contains 1x antibiotic.
5 Transfer cells to a new tube and dilute them with regular medium to 6 ml.
III Prepare cell cultures for regular culturing
6. Seed cells on 30 mm Petri dishes: 1:1, 1:3, 1:7, 1:15, 1:31, 1:63 and 1:127 (cell suspension/regular medium).
7. Change the medium before you leave for home.
8. Next day: check for results