TROUBLESHOOTING: Cry-Ac

Problem: Trigger handle sticks open, won't stop spraying.
Solution: Rotate the trigger to the side until the next time you need to use it. If this doesn't work, depressurize it by unscrewing the top.

Problem: Excessive leaking/hissing from the relief valve.
Solution: The exterior of the Cry-Ac has too much frost built up. Try pushing down on the relief valve and twisting it back and forth to break up the ice. You can also clean the frost with alcohol and gauze.

Problem: Sprays only intermittently.

Solution: The liquid nitrogen supply may be contaminated with water. Check for cloudy droplets of ice.

Problem: Does not spray

Solution A) Trigger is too close. Twist the round silver part to adjust the trigger position.

Solution B) The Cry-Ac has been over-filled and there is insufficient air space inside the bottle to create an adequate build up of pressure required to spray. Do not fill past this line.

Solution C) Check that the rubber gasket is in place inside the cover and is not split or missing.

Solution D) The position of the trigger should be directly to the left or right of the delivery tube.

Slide Review: Water in the slide

At the end of staining, tissue sections must be fully dehydrated by alcohol before applying Cytoseal. If you rush the process and leave water mixed with the alcohol and glue, the tissue will have strange dark flecks in it. Notice how the tissue in the far lower right corner of this picture is not contaminated as much. The cytoseal was probably dropped directly on that area of tissue.

You will also notice what looks like streams of bubbles. Since cytoseal is toluene-based and toluene is insoluble in water, any water left on the slide will create this cloudy mess of bubbles.

Solution: Recoverslip. Slide off the coverslip. Give the slide a few good swishes in the alcohol, a few drops of glue, and a brand new coverslip. 

TROUBLESHOOTING: Loose Knife Clamp Arm

Rolling the cryostat over bumpy cobblestone or tactile pavement can loosen the pins that hold the knife clamp arms. The microtome will probably look something like this:

Assuming everything was working just fine at the last job, all the parts should be rolling around somewhere at the bottom. Put the knife clamp arm back where it belongs. 

Now find the pin and insert it into the hole.

Use your heat sink to tap the pin all the way in. 

All fixed! 

The next time you need to go over bumpy pavement, ROLL THE CRYO SLOWLY. 

TROUBLESHOOTING: OCT mount falls off the disc

Sad mount misses his disc.

Sometimes, when you're making rough cuts or even just regular cuts, the OCT mount gets knocked off the disc. This can be caused by several things, the most common reason being:

-You took too much time putting the mount together.
Once you get the disc onto the cold bar, you need to apply OCT immediately, before the disc has a chance to get too cold. If you take too long, the pre-chilled disc will freeze the OCT before it has a chance to ooze into those tiny nooks, crannies, and holes.

You can preheat your discs on the hotplate to give yourself a few seconds leeway and some burned fingers.

The most reliable way to make sure your mounts stay on the disc is to touch the tip of the OCT bottle to one of the holes and give it a good squeeze to force the OCT into the hole.

In figure A, the OCT froze before getting into the holes, making it the type that's likely to fall off the disc. You want the OCT to freeze inside the holes as shown in B, C, or D.

Other reasons why your mounts are getting knocked off the discs:

-The surface of the disc needs to be roughed up. Refer to the post below. 
-There are loose screws.  Check the knife screws, blade screws, etc. Check everything!
-Your cryo has been off for several hours and is too warm to cut on.  The OCT block isn't frozen solid enough to hold on to the disc. Bend a Freeze-It straw so that you can freeze the backside of the disc as well as the front.
-Your rough cuts are too aggressive.  When you're advancing the wheel by hand (e.g. cutting through the safety layer), avoid cutting too deeply with each turn.

TROUBLESHOOTING: How to cool a warm cryo

It's a Mohs Gnome morning.

You forgot to plug in your cryo before you went to bed, or the staff forgot to turn on the in-office cryo.  Maybe there was a power outage in the middle of the night.  For whatever reason, it's morning, your cryo is warm, and you don't have the luxury of waiting 3hrs for it to get cold.

Take a deep breath.  Here's what you need to do.

[optional] The Ice-Bag Method - MN

If your job is ≥30min away, you may want to consider using the Ice-Bag Method to start cooling your cryo while you drive.

Place bag directly on microtome.

STEP 1: Break the block of dry ice into smaller chunks. 

STEP 2: Dump everything into a plastic bag and double-knot the handles.

STEP 3: Place the bag directly on the microtome.

STEP 4: Close the lid, cover the cryo, strap it in, and get to that office!  (drive safely, of course)

30min of driving will give you a -13°C headstart on cooling, and the plastic bag will have prevented most of the frost from building up on your microtome.

The Standard In-Office Method: Dry Ice and LN₂

Most of the time, you’ll live close enough to your job that it’s best to just take your dry ice directly to the office and plug in your cryo ASAP. 

STEP 1: Get your knife into the machine so it can start cooling right away.

STEP 2: Balance the chunks of dry ice on the microtome.  Some well-placed damp paper towels will prevent the chunks from shifting and will act as ice cubes once they freeze up.  –EM, TR, CD

Caution: Make sure the dry ice doesn’t rest against the walls of the cryo or

else the Freon in the tubes will freeze solid and ruin your machine.  –BP

STEP 3: Slowly pour LN₂ over the microtome and knife.  Close the lid.  You will have to repeat this step several times before the machine is cold enough. 

The chunks are usually bigger. These have been sitting in a bag for a few hours.

Pro Tips:

Fill the "bowl" and ball joint socket with LN2.

-Use a damp paper towel to form a cup next to the knife and fill it with LN_2.  Also, keep pouring LN₂ into the socket for the ball joint. –GS

-Place the knife on the cold bar while the machine cools.  If you have a small knife, you can dunk it into the cry-ac to rapidly cool it.  You can cut with a warm machine as long as your knife is cold, but you have to be fast and use freeze spray between each cut. –GG

-Use the Quick Freeze by turning the dial clockwise from 0, where it’s usually at,
to 3. –BP, EM

-Cover up the thermostat as much as possible with paper towels/gauze so that when you pour in the LN_2, the thermostat doesn’t read the temp and shut off the machine. Not all machines have working thermostats though! –KM

-Use magic. –CS (Don’t bother asking her for the spell.  I’ve already tried.)

-Test for warm spots with dabs of OCT. (should freeze in less than 5 sec) –GG

-You can double stack two Styrofoam cups in the cryo under the blade or near the machine.  The double layer reduces the rate of evaporation of the LN₂  and allows for a cooler in-machine ambient temp. –GG

[experimental] The LN₂ Geyser Cup - GG

When you cut in a warm machine, the air makes the block sticky.  Get a urine cup, fill it with LN_2, and poke a hole in the lid.  The LN₂ will rise from the hole in a slow geyser.  Place the cup under the blade and direct the geyser towards the block.  This keeps you from needing to spray between cuts.