Laboratory Standard Operating Procedure for:

CRYOSTAT SECTIONING (non-hazardous)

This SOP requires a Designated Trainer!
Description
This SOP describes the process of cutting non-hazardous frozen tissue on the Leica CM3050-S Cryostat.

Potential Hazards
Blades are extremely sharp. If a cut occurs, immediately remove your gloves, wash thoroughly with soap and
water and flush for 5 minutes. Inform the Histologist and your Principal Investigator as soon as possible.

Work Practice Controls

Always lock the hand wheel to immobilize the block when not actively cutting tissue and before insertion or
removal of the blade.
Never walk away from an exposed blade; use the anti-roll plate as a blade guard (if there is one available) and
leave a note saying “Caution! Sharp blade in use”.
Do not leave motorized microtomes running unattended.
Wash hands when done.

Personal Protective Equipment (PPE)

Gloves, lab coat, and safety glasses/goggles.

Storage
When finished sectioning, please set the cryostat temperature to -15°C.

Sectioning Procedure – please refer to Leica’s Instructions for Use manual for this cryostat:
https://www.igb.illinois.edu/sites/default/files/upload/core/PDF/Leica_CM3050_S_Manual_EN.pdf
Section 5.3.1 Key Functions in Control Panel 1 and Section 5.4 Control Panel 2. Hard copies are attached.
Steps for basic sectioning:
1. If the cryostat is locked, press and hold the key function with the key symbol until the cryostat unlocks.
2. Turn on the light by pushing the key function for cryochamber illumination (looks like a light bulb).
3. Set the temperature by pressing the key function for setting/changing instrument parameters (looks
like 2 arrows making a circle). Use the up/down arrows to adjust temperature as needed. It will take a
few seconds for the parameters to be accepted and the screen reverted to normal display.
4. Place your blocks inside the cryostat and insert a blade into the holder so they have time to equilibrate
to the temperature of the cryochamber. The blade is secured by the pressure lever on the left side of
the holder. Note: Hard tissue needs a tungsten carbide blade and appropriate blade holder, which is
kept in a freezer. This blade is secured in the holder by allen head screws instead of a pressure lever.
5. Retract the specimen holder to its’ starting position by pressing and releasing the double-arrow retract
key.
6. Optional: Lay gauze or aluminum foil in the waste tray behind the blade to make cleanup easier.
7. Set the cutting thickness using the +/- key function.

1 Release date: 4-22-2020

 8. Attach a specimen to a specimen disc using freezing medium (typically OCT compound). This only takes
a couple minutes for this to set; the clear embedding medium should turn completely white. If there
isn’t a bottle stored with the cryostat supplies retrieve another bottle from the fridge.
9. Place the specimen disc into the specimen head and tighten the screw. Adjust the specimen head by
releasing the lever and turning either screw for both horizontal and vertical alignment. Secure lever
completely into a horizontal position.
10. Release the clamping mechanism of the Blade Holder Base and move the base forward or backward to
position the blade near, but in front of, the block face. Secure the clamping lever. Do NOT overtighten!
11. While looking at the block from above, use the Slow Coarse Feed Forward button (button with single
arrow) to move the block close enough to the blade to cut a section. Rotate the hand wheel slowly to
gauge how close the block is to the blade. If the block is stopped by the blade you have gone too far
and must use the Backward button to get the block behind the blade.
12. Unlock and rotate the hand wheel to take sections from the block until the tissue of interest (TOI) has
been exposed; this is called “facing” the block. Selecting the Trim function will allow you to choose a
pre-set number of microns as shown by the lighted red dot underneath the numbers.
13. Switch to section mode using the TRIM/SECT button and rotate the hand wheel a few more time to
smooth out the block face, and then lock the hand wheel.
14. Brush all debris up and away from the blade to clean the area. Move the blade to a sharper area, if
needed and check the block for orientation again. Take one section of your TOI (keep the block below
the blade and out of the way for the next step).
15. Orient a charged slide above the section and quickly touch the slide to the section. The section will
melt onto the slide.
16. Place the slide in a chilled slide box for storage.
17. If using the anti-roll plate, be sure that before it is used for the first time you test its’ orientation in
relation to the blade; rotate the hand wheel to move the specimen below the blade, flip the anti-roll
plate into place over the blade, and rotate the hand wheel up. If the block hits the plate, the plate is
too far forward and can be cracked if used for sectioning before it is adjusted backwards by turning the
knurl to the right (‘righty tighty’ pulls the plate towards you and away from the blade).
Automated sectioning:
18. The automated sectioning feature can be used by pushing one of the 3 automated sectioning keys. The
bottom automation key will cause the block to be raised and lowered (cut) and then stop behind the
blade.
19. Use the foot pedal to start/stop the motor. If the pedal is pressed too hard, everything will stop, and
the red emergency light will turn on. The red emergency Stop button needs to be released by turning it
to the
20. When finished sectioning, remove all blocks and slides and follow the Cleaning Procedure before
leaving the instrument.

Cryostat Sectioning (non-hazardous) 2 Release date: 4-22-2020

 Cleaning Procedure
1. Lock the hand wheel.
2. Release the lever, carefully remove the blade and:
a. discard a steel blade in the Biohazard Waste sharps container, or
b. if a steel blade still has a lot of unused area, it can be left in the machine if the blade guard is in
place and a large note saying “Caution, Sharp Blade!” is placed on the cryostat window.
c. carefully clean a tungsten carbide blade with gauze wetted with 70% ethanol by only wiping up
and away from the blade. Rinse the blade with 100% ethanol to facilitate drying. Return it to
the Histologist.
3. Brush sectioning debris from around the chamber into the waste tray behind the blade or into a pile
elsewhere. Empty the waste tray by picking it (or just the gauze/aluminum foil lining it) up and
disposing of it in a trash bin.
4. Wipe all sectioning debris and exposed surfaces of the cryochamber with gauze wetted with 100%
ethanol.
5. Using a fresh wetted gauze, wipe all sectioning brushes (including the bristles)/forceps/tools/etc.
6. Wipe the outer surfaces of the cryostat, including the hand wheel and window, with a paper towel
wetted with dH20 only. Also wipe embedding medium from anything else that you may have touched
– the microscope, freezing compound container, boxes of slides, etc.
7. Leave any gauze wetted with ethanol in the hood to evaporate.
8. Leave the window partially open for 10 minutes to allow the vapors to dissipate.
9. Turn off the light and set the temperature to -15°C unless another user is working immediately after
you.
10. Turn off and cover the microscope. Be sure that the media bottle is closed. Waste glass can be
disposed of in the white Uncontaminated Glass Waste bucket.

Trouble Shooting
A. Sections that curl – There are several reasons why a section will curl:
a. New blades have a thin coating of oil on them from the manufacturing process. Remove most
of this oil by lightly pinching a piece of gauze around it from the bottom and swiping across the
entire blade. Turn the blade around and repeat from the other side.
b. The blade is actually too sharp and needs to be conditioned. If a new part of a blade is to be
used next, try to use it first by doing the final trimming of a block in this area.
c. At the start of a curling section, place a small brush above the curled beginning and lightly hold
it there. Move the brush with the block while taking the rest of the section.
d. Adjust the anti-roll plate so that the section slides under the plate with minimal wrinkling. Use
quarter-turns of the knurl to move the plate closer to or farther from the blade as needed.
Always turn the hand wheel slowly when testing out the position of the plate. If the block is
hitting the plate on the upstroke, it is too close to the blade.

B. Tissue crumbling out of the section:

a. Try increasing or decreasing the thickness then try the same with the speed and temperature.
A good temperature for bone is usually around -20-22°C. Undecalcified bone should be cut 2-5°
cooler.

Cryostat Sectioning (non-hazardous) 3 Release date: 4-22-2020

 b. The sample may contain hard tissue of some sort. A tungsten carbide blade may be necessary.
c. Use the Kawamoto tape transfer technique (see Histologist for training).

Waste Disposal
Gloves and any material used to work in or clean this equipment can be disposed in a regular trash bin.
Steel blades must be disposed into a Biohazardous Waste Sharps container.

Exposures/Unintended Contact

If the employee needs emergency medical attention, call 911 immediately.

In case of skin or eye exposure, remove contaminated clothing and/or PPE, and flush eyes and/or skin for at
least 15 minutes. Contact the Histologist and your supervisor as soon as possible for follow-up care and
documentation.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure
assessment.

TREATMENT FACILITIES:
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021

University Health Services -- University students (non-life threatening conditions)

Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320

UMHS Emergency Department -- after clinic hours or on weekends

1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

Report all emergencies, suspicious activity, injuries, spills, and fires to UMPD by calling 911 or texting 377911.
Register with the University of Michigan Emergency Alert System via Wolverine Access.
Training of personnel
All personnel are required to complete the General Laboratory Safety session (BLS025w or equivalent) and
Bloodborne Pathogen Training (BLS100w) via MyLINC. Furthermore, all personnel shall read and fully adhere to
this SOP when handling the chemical.

Cryostat Sectioning (non-hazardous) 4 Release date: 4-22-2020

Certification
I have read and understand the above SOP. I agree to contact my Supervisor or Lab Manager if I plan to modify
this procedure.

Name Signature Trainer’s signature Date

Cryostat Sectioning (non-hazardous) 5 Release date: 4-22-2020

 Name Signature Trainer’s signature Date

Principal Investigator / Safety Officer_____________________________ Date ___________

(signature)

Printed name: Carol Whitinger

Designated Trainer ___________________________________________ Date_________________

(signature)

Printed name: Carol Whitinger

Cryostat Sectioning (non-hazardous) 6 Release date: 4-22-2020