Guide to the Reichert-Jung Ultra-Microtome
Magnification
Coarse Advance
Knob
Focus
Binoculars
Light Buttons (2)
On/Off
Auto Advance:
Fine Adjust
Presets
Reset
Green button-motor
start/stop
Handwheel
Block Holder
Block angle adjust
Knife Holder with
tightener
Knife rotator
Knife lock front to back
Knife - Side to Side
Knife base position triangle
Trimming the block
Use a single edged razor blade, to shave off thin pieces of excess resin.
Shape the block so that you have 4, ~45 sloped sides.
The blade will dull, so move to a sharp part every few cuts.
A trapezoid or square are good shapes for the block face, remember to keep the
top and bottom parallel to each other.
Before Trimming
After Trimming
Sample
Top View
Angled Side View
Side View
Setting up the Microtome
Press and hold the reset button of the microtome’s control box. It will beep when it is
done resetting. Check the “Knife base position triangle” Turn the coarse advance knob
until the bottom line of the triangle is barely visible.
Place trimmed block into Block base, tighten side screw.
Place block base in block holder on the arm of microtome, (long edge of the trapezoid
down), tighten side screw.
Water level
Place Knife in knife holder, tighten side screw.
Fill knife boat with water. Water tension should pull the water’s edge up to the knife
edge. The water level should then dip, away from the knife edge.
Unlock the knife and push it towards the block, about 2mm apart.
Using the knife side-to-side adjuster, align the block to the side of the knife for thick
sections, at the first place where the edge is flat.
Using the block angle adjust, roughly tilt the block so the the face is parallel with the
back of the knife.
Before
After
Block
knife
knife
Smoothing a rough faced block
(if you do not see a reflection)
The block face surface needs to be smooth and shiny to see the
reflection.
Move the block down below the knife edge (with handwheel), and
move the block forward (with the coarse advance knob) until you
see the block touch the flat face (back) of the knife.
Move the block back slightly so that it no longer touches the knife
(<10 turn of the coarse advance wheel).
Set the auto advance to 1000 nm, and press the green button. This
will start sectioning the block, 1.0m at a time. Press the green
button again to stop when the face has been cut smooth. Or turn
handwheel to manually smooth blockface.
Alignment of Block face to the Knife (1of 3)
Look in the binoculars. Zoom out all the way, and focus on
the knife edge.
Rotate the block holder in the arm so that the face is level.
Rotate the base of the knife holder so that the bottom edge of
the sample is parallel to the knife edge.
With your right hand, turn the handwheel to move your
block up and down behind the knife edge. Look for a
reflection of the knife edge on the block face. (If the face is
rough, this will be hard to see.)
The distance from the reflection to the knife edge is equal to
the distance of the knife to the block face.
If you see the reflection, skip the next slide.
Note: if the control box starts beeping, make sure the dot
next to the handwheel is within the wheel’s row of dots. If it
is, press and hold the reset button.
Reflection
Alignment of Block face to the Knife (2 of 3)
Using the reflection, you can see the angle of the block face relative to the knife.
Move the handwheel up and down and watch the reflection. If the block is angled, the reflection/knife
distance will vary as the block moves up and down.
In this example, the knife is farther away at the bottom of the block than it is at the top of the block.
Turn the block angle adjust knob, so the reflection is a consistent distance when you move the block up and
down.
Final Alignment of Block face to the Knife (3of 3)
Zoom in all the way, and refocus on the knife edge.
Move the block towards the knife,
Check all angles between knife and block
Repeat the previous steps if you see anything out of alignment. Move the block closer to the knife.
Here is a block that is properly lined up and very close to the knife. The black reflection is barely visible,
about the thickness of the red lines.
Thick sections
Set the auto advance to 500 nm, and press the green button to begin cutting sections - If you are too far away
from the block no sections will be cut
Incomplete sections indicate that block/knife alignment may not be correct - Either re-align or continue
cutting until an entire block face is sectioned.
Prior to collecting sections clean boat with piece of tape, then cut desired sections.
Fill the knife boat and adjust the reflection.
Press start/stop, section until 4-5 nice sections float away on the water. Press start/stop button to stop
sectioning.
Put 2-3 drops of water on a gel-coated slide. Use a bamboo stick or “glass ball on a pipette” to pick up
sections. Dip the sections into the water on the slide, rotate until they float off.
Place the slide on the slide warmer at 60 until all the water evaporates.
Stain sections then observe with a light microscope. Check the quality of your sections; complete sections
with the target cells, even staining, dark enough stain to see clearly.
Once satisfied with the sections either continue sectioning or move to a new part of the knife, realign and thin
section (see next page).
If not satisfied with your location in the tissue, continue sectioning.
The knife is dull if the sections look wrinkled, torn or have gouges, move the block to a new part of the knife.
Thin Sections
Once you have cut to your area of interest, and are cutting along your desired angle, it is time to cut ultrathin
sections for the TEM.
Depending on your tissue, you may be able to only cut as few as 3-4 sections on your cutting surface before
you need to move the knife over to a fresh spot. Thin sections require a very sharp knife.
Set the auto advance to 70nm, and press the reset button.
To cut thin sections, move to a fresh spot on the knife. Approach the knife, taking extra care not to hit the
knife edge with the block. If you do take a chunk while approaching, the knife will be too dull to cut thins,
move farther over.
Move the block as close to the knife as you possibly can. Zoomed in all the way, the reflection should look
about the width of a piece of paper.
Start the motor with the green button. Since these sections are about a 1/10 th of before, the cuts will take 10
times more passes to start.
Once the blade starts cutting, wait till it is about to make full face sections. (It will probably start off by
shaving thin spaghetti-like pieces.) Stop the cutting, clean out the boat with tape, then resume cutting. Note
the color of the sections and refer to the interference color guide on the wall. The ideal interference colors are
light gold or silver. If the sections are blue/purple/green, and they are not making sections every pass,
ensure that all the set screws are tight, if they are, the knife is most likely dull already.
Try to get as many silver sections as you can, or until the boat is about 3/4 th full.
Pick up Thin sections with a grid
Use a hair-stick (or 2) to arrange the sections in the boat. If the sections are large (grid size) isolate them. If
the sections are smaller, group them into clumps smaller than a grid. The goal is to get as many sections as
possible onto each grid.
Use self-closing forceps to scratch a fine line around a formvar coated grid to tear the excess formvar. Pick
up the grid by the rim, and dip it into the boat away from any sections at a 45 angle. Move the grid so it is
underneath your clump of sections, lift the grid up so that it catches an edge of the section then slowly lift it
up out of the water at about 15. Use the hair stick to encourage the sections onto the grid
Place the grid section-side up on a piece of filter paper or set the forceps and grid under a lamp to dry the
water for a couple minutes, then place the grid in a gridbox. Do not forget to write down what sample is on
the grid either in a notebook or on the grid box index card.
One large section per grid
A Ribbon of Serial Sections
As many as possible