ANTIGEN PREPARATION LABORATORY: Methods
I. Purpose: To prepare E. coli for use as an antigen in immunological procedures.
II. Cell Growth: Cells were grown in nutrient broth under aerobic conditions
overnight at 37 degrees C.
III. Preparation of Cells
1. Centrifuge cells in sterile tubes at 3500 x g for 10 minutes. (Follow directions
for centrifuge using handouts distributed by instructor)
2. Remove supernatant and discard. Add 3-5 ml of buffer, resuspend cells.
3. Pool cells from tubes into one small tube and centrifuge again using table top
centrifuge.
4. Remove supernatant, add 3-5 ml of PBS (phosphate buffered saline),
resuspend and centrifuge again if necessary (see instructor).
5. Discard supernatant.
6. Resuspend cells in PBS in a 50-100 ml stereile beaker. Add enough buffer to
give a 10% cell suspension (approximately 5 to 9 ml.).
Prepare a simple stain (crystal violet) to observe cells at this point. Draw and
record results.
IV. Cell Disruption
The cell suspension will be sonicated while immersed in an ice bath in order to
avoid heat denaturation of the antigenic determinants during sonication.
Check the cells microscopically ( simple stain) before and after sonication to
assure complete disruption.
 Set the Ultrasonic Dismembrator as directed by instructor.
 With the probe held stationary on a clamp, raise the cells to the probe for
10 - 20 second bursts. Keep the ice bath around the cells to keep them
cool.
 Store the sonicated preparations in a screw-capped tube in the freezer.
V. Plate disrupted cells on nutrient agar and incubate at room temperature until
next week to check for loss of viability.
CFM2013