step 1 - sterilization -spray membrane & Petri dish
with 70% EtOH
-leave membrane in Petri dish*
-sterilize for 1 minute
step
2 - drying -remove excess of EtOH,
allow membrane in Petri dish
to dry completely -make sure shiny side is up*
step 3a - adding media -pipette in media, keeping pipette away from membrane
at all times
step
3b - floating membrane -continue to add media until
membrane is floating (~10 ml.)
-it is critical that the upper
surface of the membrane is not
wetted*
step 4 - plating on raft -add cells in drops to top
surface of membrane
(recommended drop volumes
5ul-250ul)
filter paper/gel plating -place filter paper or gel in
Petri dish
-place dry membrane shiny
side up on dry filter paper or gel
-wet filter paper wish PBS or
media*
-plate cells in drops on top of
membrane
step 5 - media change -siphon out supporting media and replace with fresh media without disturbing membrane
-to change media in drops,
pipette off & replace media,
each drop separately*
step
6 - passing -transfer cultures by placing membrane, face down, in new Petri dish
-add enough media to cover membrane*
-remove membrane 48hrs. later
-trypsinization is also possible*
"culture flexibility equals responsiveness to science"
