storage of bacterial plates

Marieke R. Koedood Zhao rkoedood at bu.edu
Fri Jan 30 16:35:46 EST 1998


Here's a protocol I have and have used once long ago :-)

Make 2 replicas of you plate on nylon filters. Put the replica filters with 
bacteria face up on fresh agar-plates and grow overnight. 
Use one filter for screening, the other for freezing:

Freezing: 
Put filter,bacteria face up, for 10 min. on 3MM paper soaked in 1x HMFM:
10x HMFM:	6.3 g K2HPO4
		1.8 g KH2PO4
		0.45 g Na citrate
		0.09 g MgSO4.7H20
		0.9 g (NH4)2SO4
		44 g glycerol
		H2O to 100 ml
Put filter, bacteria face up on some support - we used plastic 'sheets',
about 0.5mm thick, firm.
Put an overhead with a 1 mm grid xeroxed onto it on the filter (the 1 mm 
grid is to make it easier to find your postitives later).
Press on the overhead, and rub lightly with a Kleenex so the overhead sticks to the colonies.
Mark the orientation spots on the filter (mark on the overhead)
put @ -70degreesC for 30 min., then stack the plates with filters on top of
each other, cover with aluy foil and store at -70degreesC.

To pick colonies from forzen master plates:
Put the master plate on a layer of dry ice so it remains frozen
match the orientation marks on the film with the grid on the plate (overhead)
Look where the positive is on the grid with respect to the orientation marks
Cut out a square with a scalpel sterilized with EtOh and a flame. Cut
through grid and filter.
Put both grid and filter in 1 ml LB with 20% glycerol
Vortex very well andmake dilutios for plating out
Freeze rest in case another screens lights upa colony in the same area.

Hope this helps 

Marieke

Tulene McCabe (tcmccabe at cyllene.uwa.edu.au) wrote:
: Hi everyone,

: I am performing colony blots with E.coli and I have heard there is a
: method of storing master plates by freezing.  Unfortunately my attempts to
: actually find this method have been unsuccessful.  If anyone knows of
: such a method, or has advice about long-term storage of the colonies I am
: screening, I'd be so pleased!

: Thanks,

: Tulene



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