Cryostat Protocol

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Contents

CRYOSTAT USERS PROTOCOL

Generally any Cryostat should be appropriate for 10 – 50μm sectioning of frozen soft tissue onto glass slides at temperatures between -10 and -40 degrees Celsius. These guideline parameters can be exceeded with discretion. Read the users manual before using the cryostat!

Before You Begin

Before you begin sectioning, you should check the following:

  • you have an adequate supply of embedding compound (e.g. OCT)
  • there is a clean brush inside the chamber
  • there are (4) unoccupied chucks in the chamber
  • you have the appropriate low profile disposable sectioning blades
  • that the chamber temperature is correct (explained below)
  • the chamber is clean! (also explained below)

All materials (besides the glass slides and OCT compound) should be in put in the cryostat for at least 2 hours before starting to cut. In particular, any tissue to be cut, chucks to be mounted upon, blades to be cut with, and brushes for the sweepin.

You can usually turn on the instrument by pressing any of the buttons or opening the chamber window (the latter is preferred), the display shows might show something like:

  • the configuration of the instrument (e.g. HM 550 MVP)
  • the section thickness selection
  • chamber temps with set and actual values
  • the symbol for the cutting window (region where cut speed slows down)


Typical Button Functions

BUTTON FUNCTIONS For additional info please refer to the users manual.

UP/DOWN buttons These two buttons are used to change pre-selected values of different kinds in connection with the chosen function.

  • section thickness
  • trimming thickness
  • specimen temperature
  • chamber temperature

Object temperature button This button allows you to set the object quick freeze for the unused chucks

Chamber temperature button This button allows you to set the chamber temperature using the UP/DOWN buttons.

Fine and Trim thickness button This button highlights and toggles between the fine thickness (the thickness you actually cut sections at) and trim thickness (the thickness a section is cut after pressing the trim button 1 time). These values are changed by small preset intervals using the UP/DOWN buttons.

Reset button This button is used to reset added values (e.g. number of sections, sum of section thicknesses) to zero.

Menu button This button is used to get into the submenus. Basic settings, times, service functions and options are set and/or called.

Enter/scroll button Within the submenu, basic settings are carried out, further submenus are called and other functions are activated via this button. During operation of the instrument, e.g. while cutting, this button is used to choose between the indication of the section thickness, section sum, real time and indication of the distance to the end position of the specimen.

SPECIMEN COARSE FEED For the fast forward and backward travel between knife and specimen, the cryostat microtome has a motorized coarse feed system. The approach between specimen and knife can also be carried out by means of an automatic function

Chuck Return travel button

  • Pressing the button briefly leads to short return travels only.
  • Press the button for more than approx. 2 seconds, the function is carried out continuously. The specimen clamping will entirely be moved backwards.
  • To stop this function, briefly press button once more.
  • When the rear end position of the specimen clamping is reached, the coarse feed turns off. At this point an LED in light blinks, which means that the specimen clamping is in its rear end position.

Chuck Feed Travel Button

  • Briefly press this button, to carry out a forward travel of that trimming section thickness which was chosen via the control panel and shown on the display.
  • The continuous forward travel is only carried out as long as the button is being pressed.
  • Warning: With the function coarse feed forwards specimen and knife are adjusted very quickly. To avoid damage to specimen and knife, BE CAREFULL!
  • Carefully observe the narrowing gap between specimen and knife edge to stop the function coarse feed forwards in time before the specimen touches the knife. Take note that the specimen and knife edge do not come in contact with each other.
  • When front end position of the specimen clamping is reached, the coarse feed turns off.
  • The LED in button lights up and shows that the specimen clamping has reached its front end position.

Cutting Window Button To activate an automatic approach process, it is necessary to set a cutting window first. The size of this window is then shown in the symbol cutting window. When passing the cutting window this symbol is shown inverted on the display. Manual setting:

  • The cutting window can be determined by manual entries.
  • For this, press the Reset Button and while this button is still being pressed, press the Cutting Window Button.
  • Turn the handwheel so that the lower edge of the specimen is positioned slightly above the knife edge.
  • Press Cutting Window Button to set the upper limit of the cutting window.
  • Continue turning the handwheel clockwise to place the upper edge of the specimen just below the knife edge.
  • Press Cutting Window Button to set the lower limit of the cutting window.
  • The LED in lights up during each further passing through of the cutting window. The length of the cutting window is shown in mm within the cutting window symbol on the display.

Note: A cutting window should only be set while the specimen is moved downwards. If, by mistake, a cutting window limit is set during return travel of the specimen, the set limits are applied to the cutting movement accordingly. Note: In case the cutting window limits are set incorrectly, please repeat above-mentioned process again. Note: If no cutting window is needed, briefly press the Cutting Window Button twice. The automatic approach system can now be released in any position.

Automatic Chuck Approach Button

  • Do not use unless you consult the users manual.


Cutting Modes

SELECTION CUTTING MODES For the motorized cutting movement of the microtome, the following operating modes are available: - continuous stroke - single stroke - interval stroke

Cutting Mode Button

  • If the cutting drive is turned off, choose one of the above-mentioned operating modes by pressing the button MODE once or twice.
  • The selected operating mode is shown on the display as CONTIN, SINGLE and INTERV and its LED in button lights up.

Continuous stroke (1)

  • Press Cutting Mode Button until the operating mode continuous stroke CONTIN is shown on the display.
  • To start a continuous cutting cycle, use the foot pedal or press the two start buttons.
  • To stop the continuous stroke, press foot pedal or one of the two start buttons.
  • However, if a cutting cycle has been started, it will continue through and stop in the next lower reversal point.

Single stroke (2)

  • Press Cutting Mode Button until the operating mode single stroke SINGLE is shown on the display.
  • Press the two start buttons or the foot pedal once to release a single cutting cycle.
  • The movement stops in the lower reversal point.

Interval stroke (3)

  • Press Cutting Mode Button until the operating mode interval stroke INTERV is shown on the display.
  • The cutting drive only moves while the two start buttons or the foot pedal are pressed.



SETTING THE CUTTING SPEED

Cutting Speed Knob

  • The desired cutting speed is set on the operating knob.

Note: To save time, the return travel speed is enhanced in relation to the cutting speed, especially for slow cutting speeds.


START AND STOP OF THE CUTTING DRIVE

Cutting Start/Stop Buttons

  • Press the two buttons start buttons of the safety switch either at the same time or briefly one after the other.
  • As an option, the motorized cutting drive can also be started via the foot pedal.
  • The LED in button lights up while the motorized cutting drive is active.

Note: To start the motorized cutting drive, the functions handwheel brake and emergency stop must not be activated. The course function of the cutting drive results from the selected cutting window, the selected operating mode and the set cutting speed.

Stop in operating mode continuous stroke

  • Press one of the two buttons start/stop buttons or
  • press the foot pedal.
  • The LED goes off.

Handwheel Brake Button Unintended movements of the specimen holder can be avoided via the handwheel brake. This reduces the danger of being injured while adjusting specimen clamp and knife carrier! Warning: When the instrument is turned off, the handwheel brake is not operative. For your personal safety, the handwheel brake should be turned on when working on the specimen holder or knife carrier.


Optional Vacutome

SETTING THE STAGE VACUUM FOR DISPOSING OF SECTION WASTE

Setting the vacuum

  • To select the suction power or the required vacuum between 0 - 10, turn the operating knob. (0 = no suction power, 10 = highest suction power).

Note: The user sets the desired value depending on the desired function (stretching or suction process), section thickness and size of the specimen. Note: The applied vacuum for stretching sections and/or disposing of section waste is only active during the cutting window representing also the suction window. Outside the cutting window, the selected vacuum is turned off by means of a valve. Setting the suction window is carried out in the same way as the cutting window.

WHEN THE VACUUM FILTER IS FULL, THE CRYO WILL TELL YOU THAT IT NEEDS TO BE CHANGED.

INSERTING AND REPLACING THE FILTER UNIT When the suction power and suction window are set in a correct way, the section to be disposed of gets via the suction slot in the knife carrier into the filter unit, consisting of a coarse filter (fig. 93-2) for collecting section waste and a micro filter for filtering the air. Section waste is collected in the coarse filter. This coarse filter must be replaced depending on the volume of waste. The coarse filter is actively cooled. The micro filter must also be replaced regularly, however, at least every 6 months. Removing coarse filter:

  • Press the bow of the coarse filter downwards either manually or via the foot.
  • The coarse filter tilts forwards.
  • Remove the coarse filter by means of the attached clamp for filter replacement.

Replacing the coarse filter: The coarse filter must be replaced depending on the volume of the existing section waste. If the integrated counter detects that the sum of all sections exceeds 50,000 μm after the last resetting, the message "Change coarse filt." appears on the display.

  • For this, remove the coarse filter as described above.
  • Use lab gloves!!!
  • Remove the coarse filter from the metal carrier by using forceps or the like.
  • Dispose of the coarse filter together with the section waste according to the respective lab regulations.

Note: The coarse filter is to be understood as a disposable filter and must not be used again.

  • Insert new coarse filter and close the filter.


Clean Up

WHEN YOU ARE DONE CUTTING IT IS TIME TO CLEAN UP!

Clean the cryo chamber from all section waste after.

  • Remove all tissue specimens, brushes and chucks from the chamber.
  • Lock the handwheel. Carefully take the blade out of the knife holder.
  • Use the coarse feed button to completely retract specimen
  • Swivel the anti-roll guide upwards.
  • Remove the two section waste trays. Thoroughly clean and disinfect them.
  • Use the Vacutome to dispose of the section waste into the coarse filter.

Thoroughly Clean the Chamber!! Remove frozen section waste with a cold brush. Empty the section waste tray. Wipe down the storage shelves and brush shelf with 75% ethanol. The cryostat should be left immaculately clean after each use. Remove all specimens from the cryostat. Close the sliding window.


EMPTYING THE DEFROSTING LIQUID Note: The liquid from the daily defrosting process is collected in the container (fig. 62.1). Please note that this container is emptied in due time according to the safety precautions at the beginning of this instruction manual. We highly recommend to treat the container with a disinfection solution after having emptied it.

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    Dec 18 2011 4:24 am

    Good tip. I did it and it worked. Thanks.

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