BOT/MICRO/ZOOL 5364

HANDLING OF SPECIMENS DURING PREPARATION FOR TEM

I.  Fixation

A.     Immersion Fixation

Most fixation procedures require the immersion of specimens in solutions.  A number of methods have been devised for handling specimens.  Some of these are described below.

1.      Blocks

If tissue is large, cut tissue into small blocks or strips with a very sharp razor blade.  Blocks should be no larger than 0.5 mm³ if 0s0₄ is used as the primary fixative.  Blocks may be several mm if the primary fixative is an aldehyde.

Small specimens which are hard to handle may be placed on a millipore filter and covered with a layer of 1-2% agar at a temperature just warm enough to keep the agar liquid.  The millipore filter is handled as a tissue block through the fixation and dehydration solutions until acetone.  Acetone will dissolve the millipore filter leaving specimens attached to the agar.  The millipore filter may also be cut and embedded with the specimen.

2.      Cell Monolayers

Monolayers of cells may be grown on a support of glass (coverslip), plastic, mica or millipore filters and handled as tissue blocks.

3.      Single Cells

Single cells may be centrifuged to form a pellet.  The pellet may be treated as a block if it remains intact, or the cells may be suspended and centrifuged during each step.

Concentrated cells may be collected on a millipore filter and covered with a layer of agar (see above).

Cells may also be embedded in agar.

a.      A 1% agar solution is prepared by heating agar in water, buffer, or culture medium.

A small amount of warm agar is placed in a centrifuge tube with a pellet of cells (usually fixed) in a water bath at 45 C.  The tube is shaken to suspend the cells.

The agar is immediately poured out onto a cool microscope slide so that the agar forms a drop.

The solidified agar is cut into small cubes with a sharp razor blade.

b.      Cells ar suspended in warm (50 C) 2% agar and centrifuged with the agar.  The carrier for the centrifuge tube is filled 1/2 full with hot tap water to keep the agar liquid.

The centrifuge tube is cooled in ice.  70% ethanol is added and the tube allowed to stand for 1 hr. or longer keeping the tube on ice.

The agar block may then be carefully displaced from the tube and cut into small blocks with a small razor blade.

c.      Cells embedded in agar by either of the above methods  may be removed from the centrifuge tube by using a disposable plastic centrifuge tube and cutting the tube.

Single cells may be embedded in bovine serum albumin using a 2% solution of albumin which has been millipore filtered.  A drop of 25% glutaraldehyde is added to cells suspended in 0.25 ml of albumin and the suspension centrifuged immediately.  The albumin will gel and become opaque about 5 min. after the addition of the glutaraldehyde.  The gel may be removed from the centrifuge tube by carefully displacing it or by cutting a disposable centrifuge tube.

Single cells may be encapsulated by placing them in an indentation made in a slightly dried 2% agar block.  The agar block will absorb the water.  The concentrated cells are covered with 2% agar to make a block.

B. Perfusion Fixation

Fixation of animal tissue critically dependent on a continuous blood flow may be fixed by perfusion.

1.      Whole Body Perfusion

A midline incision is made in an animal which has been anaesthetized or undergone cervical dislocation.

A needle connected to a tube from a reservoir is inserted into the left ventricle.  The needle may be secured with a ligature or clamp.  The right atrium is cut to allow the perfusate and blood to escape.

The blood may be washed out with an oxygenated isotonic saline solution maintained at body temperature.  When the perfusate solution is clear, the perfusate is changed to a fixation solution.  The abdominal aorta may be clamped if tissues from the head are desired in order to conserve fixative.

2.      Organ Perfusion

If the desired organ and a portion of the connecting artery are easily removed (such as the heart), a cannula may be placed in the artery of the removed organ for perfusion.

II. Embedding

Specimens may be embedded in a number of ways.

A.     Tissue Blocks

1.      Samples handled as blocks may be embedded by placing them in the bottom of BEEM capsules or gelatin capsules.

2.      Blocks may be placed in a commercially manufactured embedding mold.

3.      Aluminum or polyethylene weighing dishes, boats made from aluminum foil and coated with mold release (or PAM), or flexible plastic bottle caps may be used as embedding dishes.

Blocks are placed in embedding dishes containing 100% plastic and the plastic is polymerized.  The polymerized plastic with the blocks is removed from the embedding dish.

Individual blocks are cut from the plastic mold with a jeweler's saw or by warming the plastic on a hot plate and cutting the plastic with a razor blade.  The size of the cut plastic block will depend on the method used to hold the block in the microtome.

Individual blocks may be placed in a flat chuck for the microtome, or glued to the face of a cylindrical plastic block or small cylinder of plexiglass which will fit the cylindrical chuck for the microtome.

B.     Cell Monolayers

Monolayers of cells may be embedded on the support if the support can be sectioned (such as millipore filter or thin plastic).  If the support cannot be sectioned (glass), the cells must be removed from the support.

Glass coverslips may be removed from the cells if the cells are grown on coverslips coated with carbon, silicone, collodion (Parlodion), Formvar or teflon.  Cells on collodion or Formvar layers can be floated onto the surface of water.  Collodion also detaches from the glass in 100% ethanol or propylene oxide.

C.     Single Cells

Single cells may be placed in conical BEEM capsules and centrifuged in 100% plastic to form a pellet.

If individual cells are to be examined, they may be suspended in 100% plastic and placed in an embedding dish.  Individual cells may be selected and plastic blocks cut by the methods described for tissue blocks.  They may be mounted on the face of a plastic block or plexiglass rod, or placed in a flat or vise-type chuck.

WARNING: Blocks formed by centrifugation of cells in conical BEEM capsules often contain fragments of glass from Pasteur pipettes.  All glass must be removed from the face and sides of the block during trimming.  Glass fragments destroy a knife.

Be sure to label all blocks.  It is impossible to tell the method of fixation or the type of material in the block by looking at the block itself. Blocks may be labeled with small pieces of paper embedded along with the specimen.

WHEN MAKING LABELS USE PENCIL OR INDIA INK.  REGULAR INK OR BALL POINT PEN WILL FADE IN THE UNPOLYMERIZED PLASTIC LEAVING A BLANK PIECE OF PAPER.