FAQ Leica CM1850

There are five different knife holders and four specimen clamps available. Each one has been developed to suit the different types of specimens and to allow you a great varety of applications - whether for use in the histopathology or research lab or for sample preparation in the industrial quality assurance lab. Please refer to the chart by clicking on the below hyperlink.

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1. Advantages in the application of disposable blades over conventional knives:
   - Substantial cost reduction
   - Consistent section reproducibility
   - Consistent section quality
   - Support of all common microtome systems
   - For all paraffin embedded samples
   - For all cryosectioning applications
   - Expensive resharpening no longer required
   - No longer dependent on quality of selected resharpening service
   - No downtime while knives are at the resharpening service
2. Standard re-usable steel knives and disposable blades in comparison with standard microtome blades: In typical histology laboratories, 50-100 blocks on average per day and person will be handled. Depending on the type of specimen and the way of trimming, up to 50 specimens and more can be worked on. Assuming the laboratory is handling all the different types of tissues that are typically processed in a histopathologyl lab, up to 20 blocks can be cut with high quality microtome blades retaining the same sectioning quality.


3. Conventional steel knife 16 cm c-profile: As a rule, no more than 80 to 100 blocks will be cut by a steel knife with consistent section quality. Thereafter, the steel knife has to be sharpened regularly to regain the required knife performance. Due to the design of microtome knife holders, only 70% to 80% of the total knife edge can be utilized for cutting. The remaining 20% to 30% are not accessible because of the clamping principle commonly used.

Unfixed, or fixed tissue can be frozen and sectioned using a microtome enclosed in a cold cabinet more commonly known as a cryostat. The sections that are prepared are referred to as “frozen” or “quick” sections; the latter term is used to denote the speed with which the section can be prepared. Techniques for low temperature sectioning are referred to as cryotechniques.

1. Rapid interoperative consultations and diagnosis.
2. Preservation of enzyme activity for enzyme histochemistry and antigenicity for immunohistochemistry and in situ hybridization.
Retention of substances that are soluble in routine processing solutions, such as lipids.
3. Preservation of cell morphology without exposure to chemicals and/or heat.
4. Can be performed on fixed and unfixed tissue specimens.

1. Selecting the optimum temperature for the specific tissue being sectioned.
2. Making certain that the cryostat maintains the set temperature for the cabinet and specimen.
3. Trimming the specimen to a size and shape that will optimize sectioning.
4. Eliminating ice crystal artifacts in the specimen by using an appropriate, rapid freezing technique.
5. Attaching the specimen to the specimen holder (chuck) in a manner that will facilitate artifact free sections (minimizing amount and avoiding creating layers of cryocompound).
6. Keeping the cryo-microtome in good working order by keeping it clean, defrosting on a regular basis and lubricating according to manufacturer’s recommendations.
7. Applying the appropriate tension to the clamping system for the knife or blade.
8. Selecting the correct knife angle.
9. Using a sharp, nick-free knife or disposable blade.
10. Properly adjusting the anti-roll plate and making sure it is damage free.
11. Collecting the sections in a manner that preserves the specimens integrity and morphology.

There are a variety of methods that can be successfully used for freezing various types of specimens and the best techniques are often determined by trial and error. Here are two basic methods that can be used to obtain good specimen freezing:

1. Using cryogens such as isopentane and solid carbon dioxide. An excellent freezing technique is using a combination of these two cryogens. Place 50 mL of Isopentane in a glass (or polypropylene) beaker and place it into a mound of solid carbon dioxide, CO2 pellets, so that the pellets surround and are in direct contact with the beaker. Allow the Isopentane to stabilize, the resulting temperature will be approximately -80°C.
   Trim your sample to a suitable size for sectioning, try to use samples that are not thicker than 4.0 mm, (a recommended sample size for cryosectioning is 20 mm x 20 mm x 4.0 mm) and put the sample into the Isopentane and allow to freeze.
   While the sample is freezing place a clean specimen disc on the freezing shelf of the cryostat. Remove the sample from the Isopentane and mount onto the specimen disc with a small volume of cryocompound. Allow the specimen to equilibrate to chamber temperature, as it will be too cold to section immediately.
2. Using a rapid freezing system mounted in the cryostat.
   The freezing shelf of the cryostat is an alternate method of freezing especially if the freezing shelf is fitted with a Peltier element. A Peltier element is a thermoelectric device that allows “heat” from the sample to be reduced at a very fast rate and thus freeze rapidly.
   To use the Peltier element, or freezing shelf, trim the specimen to a suitable size, with a maximum thickness of 4.0 mm. Turn on the Peltier element. Select a suitable specimen disc and mount the specimen onto the disc with a small volume of cryocompound. Allow the specimen to equilibrate to chamber temperature as it will be too cold to section immediately.
   Another excellent method of freezing is using the Precision Cryoembedding System. Please visit http://www.pathologyinnovations.com/ for details.

1. Slow, poor freezing produces large hexagonal ice-crystals within the tissue that can damage insoluble structural elements and cause displacement of water-soluble components.
2. Melting ice from mounting the frozen section on a room temperature slide causes water-soluble components of the cell to be displaced by the flow.
3. Water-soluble substances may wash out of the sections when unfixed wet or dry sections are placed in aqueous solutions (even aqueous fixatives).

1. Poor air circulation around cryostat.
2. Sliding window seal is defective. Replace seal.
3. Locating the cryostat in an area where it is exposed to air currents (open windows or doors, overhead air vents, etc). A more suitable area should be found.
4. Leaving the sliding window of the cryostat open for an extended period. This can permit warm air from the room to enter the cryochamber and cause frost to form on the microtome and knife holder system. Ensure that the sliding window is closed after terminating work.

Ensure that all tools, brushes, forceps, wipes etc. are pre-cooled in the chamber before use. Store all tools on storage shelf in the cryochamber.

1. Room temperature and humidity might be too high, check the installation requirements, and adjust as required.
2. Verify that the heater in the sliding window is operating.

1. The specimen is too warm. Select a lower temperature, wait for a few minutes for the temperature to stabilize and try sectioning again.
2. Either the blade or anti-roll plate is too warm. Allow the blade and anti-roll plate to stabilize at the selected temperature (the anti-roll plate should be in position touching the pressure plate).

1. The specimen is too cold. Select a higher temperature, give specimens time to adapt to the selected cutting temperature or gently warm the specimen surface with the Delrin portion of the warm/cold block, if available.
   a) Specimens coming directly from the freezing shelf, Peltier element or freezing outside the cryostat in isopentane/dry ice or liquid nitrogen mixture are too cold.
   b) Specimens that have been stored in a deep freeze (e.g. -80 °C) are too cold.
2. The specimen surface is rather large: trim the specimen parallel and increase the section thickness.
3. The anti-roll plate is not correctly adjusted: adjust the anti-roll plate correctly.
4. You might be using a dull blade: use another area of the blade or a new blade.
5. A knife angle of 2-5° for disposable blades is recommended.

1. Try to avoid air currents entering the cryochamber which can cause static electricity.
2. The anti-roll plate is too warm; allow the anti-roll plate to stabilize at selected sectioning temperature.

1. The anti-roll plate and/or the knife are dirty, clean with dry cloth or brush.
2. You might be using a dull blade, use another area of the blade or a new blade.

1. The anti-roll plate does not protrude far enough beyond the knife edge, re-adjust correctly.
2. Use vacuum assisted section stretching aid.

1. The blade edge is damaged; use different part of the cutting edge.
2. The edge of the anti-roll plate is damaged, replace the anti-roll plate.

1. The specimen might be improperly frozen onto the specimen disc. Remove the specimen; ensure that the disc is clean and that the grooves are cleared of any build up of cryocompound. Remount the specimen on the disc using a minimum amount of cryocompound.
2. The specimen disc is not secured properly. Ensure that the disc is properly inserted into the specimen head.
3. The specimen is very hard, or not homogeneous. Reduce the specimen surface area if necessary; cut at the temperature necessary for the most difficult to freeze component of the tissue; if possible, trim the block into a V shape and orient the block so that the smallest surface will contact the blade first.
4. Rigorous trimming of the block has caused the specimen to detach from the disc. Re-mount the specimen onto the disc.
5. The blade is not clamped properly. Check the clamping. It may be overtightened. (Position the locking lever so it is in the same plane as the disposable blade front plate.)
6. The cutting edge is dull. Use different parts of the cutting edge or replace the blade.
7. The knife profile is inappropriate for the specimen to be cut. Use knife with different profile that can withstand the cutting force.
8. The clearance angle is incorrect. Set correct angle.
9. Cutting speed is too fast and should be slowed down.

1. The temperature is incorrect for the tissue to be cut. Select correct temperature.
2. The cryocompound and specimen have become detached from disc after freezing. Remove the specimen and ensure that the disc is clean and that the grooves are cleared of any build up of cryocompound. Remount the specimen on the disc using a minimum amount of cryocompound.
3. The specimen disc is not clamped tightly enough. Check disc clamping.
4. The specimen arm is fully extended and the knife holder is too far back to be clamped tightly onto the microtome base. Move the specimen arm all the way to the home position and reposition that knife holder squarely over the clamping device.
5. The knife profile is inappropriate for the specimen to be cut. Use a knife with different profile (c or d) or possibly switch to a disposable blade system.
6. The knife may have ice or debris built-up on the back side. It should be cleaned.
7. The knife/blade is clamped using either too much or not enough pressure. Check knife clamping.
8. You may be using a blunt knife. Use a different area of the knife edge or replace the knife.
9. The cutting speed is irregular or too fast. Turn the handwheel in a moderate, hesitation-free manner. Automate if possible.
10. The clearance angle may be incorrect. Set correct angle.
11. The section thickness may be inappropriate. Select correct section thickness.

1. The anti-roll plate is too warm or incorrectly positioned. Cool down the anti-roll plate or reposition the plate.
2. Specimen is too warm. Select lower temperature.
3. There is adipose tissue, other tissue fragments or debris on the edge of the anti-roll plate or blade edge. Clean with alcohol or acetone.
4. There is rust on the knife. Remove rust.

1. Static electricity or air currents: Remove static electricity.
2. The anti-roll plate is too warm: Cool down the anti-roll plate.

1. The blade/knife edge is blunt or there is dirt, dust, frost or rust on the edge. Remove the cause or replace the blade.
2. The leading edge of the anti-roll plate is damaged. Replace the plate.
3. There are hard particles in the tissue. Use tape transfer technique or select another specimen.
4. The back of the blade back is dirty. Clean, or replace the blade.
5. The temperature is too low for the tissue to be cut: increase (raise) the temperature, wait for a few minutes and try sectioning again.

1. The anti-roll plate projects too far beyond the cutting edge and is scraping on the specimen surface. Re-adjust anti-roll plate correctly.


2. Check knife angle and increase if necessary.
3. If there is a singing noise, the specimen might be too hard for the type of blade/knife. Select a blade/knife that is appropriate for the specimen.

1. The plate is extended too far past the cutting edge.
2. The adjustment of the plate was carried out in direction of the cutting edge with the glass on the sharp surface.
3. If the plate is damaged, it must be replaced or another portion of the plate must be used.

The cold air inside a cryostat is very dry and will rapidly dehydrate the specimen.

• Specimens may be stored during the day when the automatic defrost cycle is not activated but they must be covered by a layer of cryocompound or wrapped in aluminum foil to prevent dehydration.
• Not only is the specimen dehydrated, but also the cryocompound surrounding the specimen. The cryocompound sectioning properties are negatively influenced (feels like sectioning chewing gum).
• When sectioning of a specimen is completed, remove the remaining sample from the specimen holder while still frozen and wrap in a double layer of aluminum foil, label and store in a sealed container in a -80 °C freezer.

1. Check that the mains lead is connected properly to the outlet and the instrument.
2. Check to see if the over-current (GFCI) or circuit breaker has been activated. Reset and switch the cryostat on again with the mains power switch after approximately 5 minutes.
3. Check the mains fuse.
4. If all of the above are OK – call Technical Service.

1. Ensure that there is a minimum clearance distance of 10 cm on all sides of the instrument.
2. Check that the compressor ventilation grids are not dirty or covered. Remove any obstruction or remove dust with brush or vacuum cleaner.
3. If the instrument has been switched off overnight and the automatic defrost cycle did not take place, activate a manual defrost cycle.
4. Select a more suitable location for the cryostat if it is exposed to air currents (open windows or doors, overhead air vents).
5. Call Technical Service if there might be a leak in the cooling system or if the compressor might be defective.