This protocol describes the process to use the ball mill grind to homogenize samples (muscle, liver, etc) to prep for analyses (e.g. bulk C/N). Samples are generally freeze dried beforehand.
Ethanol is considered an acutely toxic chemical. Ethanol can induce dizziness and shortness of breath. Ethanol is flammable. Always wear the proper personal protective equipment, including gloves and eye protection. Familiarize yourself with the MSDS and SOPs for all chemicals prior to starting this protocol.
- Ball mill grind machine (FSH 333)
- Ball mill canister components (in drawer below machine- labeled “Ballmill capsules- Please return to drawer when done”)
- Two Canister bases
- Grinding balls
- Cap with o-ring (lid)
- Screw on top (threaded band top)
- OR: (preferred)
- If using 4×4 capsules -> contains 4 base pieces, 8 capsules comprised of 16 end caps (with black o-rings), 8 metal tubes, and 8 small grinding balls
- Ethanol in squeeze bottle
Be sure there is sufficient ethanol.
Checking out the ball mill grinder
- In FSH 333 reserve ball mill grind in notebook by machine
Ball mill usage
- Gather samples to be ground
- Turn on machine by pressing the switch on the back-right corner of the machine
- Before beginning, be sure all metal canisters, grinding balls, and caps are clean and free of all previous sample residue. If cleaning is needed, use gloves, kim wipes and ethanol to wipe all exposed areas to sample.
- Use metal spatula to place all unground samples into separate metal canisters (can grind up to two samples at a time- be sure to keep track which sample is in which capsule OR with 4×4 capsules can grind up to eight samples -> refer to page 6 of protocol for specific tips for 4×4 capsules )
- Place two of the same size metal grinding balls in canister with sample
- Place cap with black O-ring side down
- Screw top to close canister, make sure it is screwed tight enough to keep closed but not too tight where cap cannot be unscrewed
- Secure canisters in each fork using the nut, secure with the secondary nut
- Set timer on control panel to desired time of grinding (most tissue samples homogenized in 1 minute). Press start
- Once grinding is complete, unscrew fork and remove canister.
- Remove cap, check if sample is a fine powder (some fattier samples may need more than one grinding session)
- If sample is fine enough, use metal spatula to scrape out sample and place into desired vial.
- Use ethanol and kim wipes to clean all components of metal capsule. Make sure they are clean of all sample reside before moving on to next sample
- Repeat steps 3-13 for all subsequent samples
- Clean up station by wiping down metal canister components are counter area for the next user
- Return supplies to appropriate drawers
Tips for Using 4×4 Capsules
General steps of this protocol still apply, but this section has tips specific to 4×4 capsules. Instead of grinding up to two samples at a time, you can grind up to eight samples at a time with the 4×4 capsules, but be sure to keep track of which sample is associated with which capsule. We recommend labeling a paper towel and coordinating samples.
When inserting the capsules into the fork, support the bottom, and make sure the capsules are straight. Tighten the fork and nut until it is hand-tight.
When finished with grinding, to remove capsules from forks, grip the two base pieces together and support bottom of capsules with your other hand.
When unloading samples from the base pieces, to prevent any spillage, invert base holding the capsules to hand to release all four at one time.
When cleaning beware of o-rings by not using any sharp objects (forceps) to clean. O-rings may pop out, this is okay, just be sure to re-insert them fully into caps.
Even when only processing one sample, include both metal canisters (one can be empty) to ensure the ball mill is balanced. Likewise with the 4×4 capsule set up, include all capsules and base pieces in both forks of the ball mill.