Madhadron:PreparingCellsForMicroscope

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m
Current revision (06:15, 19 October 2007) (view source)
(Removing all content from page)
 
Line 1: Line 1:
-
===Materials===
 
-
* 1mL sterile syringe (BD 1mL tuberculin slip tip)
 
-
* 5μm filter (Millex-SV, fits onto end of syringe)
 
-
===Directions===
 
-
# Take a culture of ''M. smegmatis'' in exponential phase.  Pipette 1mL into each of two Eppendorf tubes.
 
-
# Centrifuge the tubes for 5 minutes at 5,000rpm.
 
-
# Pipette off the supernatant, and resuspend in 100μL 7H9 medium.
 
-
# Place a clean Eppendorf in a rack.  Put the filter on the end of the syringe, and remove plunger from syringe.  Place it upside down in another slot of the rack.
 
-
# Load the contents of both Eppendorf tubes into the syringe with a pipette, replace the plunger, and use it to force the liquid through the filter into the clean Eppendorf.  The cells are ready for microscopy.
 
-
 
-
===Notes===
 
-
Mycobacteria clump horribly, so to do microscopy sensibly, you have to break up or remove these clumps.  All of this should be done in a hood, as with any other manipulation of such slow growing bacteria.
 

Current revision

Personal tools