Preparation of Infectious CJD Standard for Transmission and Biochemical Studies

Equipment :

Eppendorf EDOS 5222 Liquid dipensing system

CamLab Omni-Mixer ES Homogeniser

Class 2 Microbiological Safety Cabinet

Solution

0.25M-sucrose (MW 342.30) : 85.56g/L in pyrogen-free water.
Pre-chilled to ~4 degrees centigrade

Procedure:

1. Weigh frozen brain. Thaw enough to allow slicing into 4-5g chunks and transfer to homogenisation container.
   
2. Homogenise approx. 20g and 180 mL sucrose solution in sealed 250 mLcontainer using programmable Omni-Mixer. Setting at 4000rpm, 6 x 30seconds.*
   
3. Transfer homogenate to 2L beaker on ice/salt. Stir on magnetic stirrer.
   
4. Repeat homogenisation : 4 x 20g brain to create a pool of 1L of 10%-brain homogenate.
   
5. Dispense 100 x 0.5 mL into pre-labelled, Nalgene 1.2mL cryovials, immerse in bleach (10%-Chloros) for >15 minutes at 4^oC.
   
6. Rinse in water to remove surface bleach. "Flash Freeze " by immersing in liquid nitrogen for 5 minutes.
   
7. Transfer onto dry ice for transport to storage facility at NIBSC (-80 ^oC).

* Shown to be adequate by facsimile homogenisation of frozen sheep brain