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Intro to Microtomy: Preparing & Sectioning Paraffin Embedded Tissue

Microtomy and Paraffin Section Preparation

Creating great paraffin sections using a rotary microtome takes a great deal of skill and experience. "Microtomy and Paraffin Section Preparation" is a great training aid for new microtomists and is an excellent refresher for experienced operators. All of the essential aspects of cutting paraffin sections are covered, including:

  • Safety
  • Microtome setup
  • Microtome blades
  • Trimming, facing and roughing blocks
  • Techniques for consistent paraffin sections
  • Microtome maintenance
  • Common microtomy faults

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Microtomy and Paraffin Section Preparation


Steps to Better Microtomy + Flotation + Section Drying

From patient to pathologist, preparing tissue specimens for histological examination requires care, skill and sound procedures. This guide provides practical advice on best-practice techniques and simple ways to avoid common errors.

Tips for better microtomy + flotation + section drying are highlighted in this week's guide. We hope each step provides a valuable reminder of good histology practice and also helps with troubleshooting when unacceptable results do occur.

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Step 31 - Use High Quality Blades

 High quality, sharp blades are always used for cutting.

Blades are used for as long as possible–a few “train lines” are considered acceptable.

A: A section of spleen (H&E) showing many fine lines due to a defective blade.
Use High Quality Blades
B: Skin sections undergoing flotation. A severe knife line can be seen running right through the tissue. Defects such as this can be easily seen during flotation.
Use High Quality Blades

Step 32 - Optimize Knife Tilt Angle

 Knife tilt angle is always optimized for each microtome and blade type.

 Knife tilt angle is never adjusted when conditions are changed (different microtome, new blade type, different wax etc.).

This short ribbon of sections that was cut from a cold block shows considerable compression (30–40%). In this case re-setting the knife tilt angle overcame the problem.
Optimize Knife Tilt Angle

Step 33 - Carefully Trim Blocks

Blocks are carefully trimmed to expose tissue. The last few sections are always cut at what will be the final thickness to polish the block face.

Blocks are roughly trimmed to save time. The surface is not polished before taking final sections. This often produces a  “moth-eaten” appearance in the final section which is full of small, ragged holes.

Initial exposure of the tissue (roughing) in this block has pulled fragments from the block surface which has resulted in numerous holes in the final section (H&E).
Carefully Trim Blocks

Step 34 - Avoid Freezing Damage

Blocks are chilled on a cold wet surface and are always cold when cut (the surface of melting ice is excellent).

Blocks are frozen before cutting. This sometimes causes the blocks to crack.

This block face has cracked because it was frozen to –15°C in a freezer prior to cutting. The cracks may make sectioning and flotation difficult because the wax is no longer bound to the tissue.
Avoid Freezing Damage

Step 35 - Use Cold Blocks

Blocks are always cold when cut.

 There is sometimes a delay before final sections are cut from a block. The block may be warm and this may result in excessive compression of sections.

A The distortion of the glomeruli in this kidney section is due to excessive compression when the section was cut (H&E).
B Sections from the same block undergoing flotation. The sections on the left were cut without chilling the block while those on the right were cut when the block was cold.
Use Cold Blocks

Step 36 - Cut Sections Slowly

 The final sections from each block are cut gently with a uniform, slow rotation.

 Sections are cut as quickly as possible with a rapid rotation, in the belief that “any section compression will be overcome on the flotation bath.”

A Rodent liver (reticulin stain) showing fine chatter due to cutting a cold block of brittle tissue too fast. In this case the problem was overcome by allowing the block to warm slightly then cutting very slowly. Chatter can also result if the paraffin block or blade is poorly secured in the microtome.
B This H&E stained section of mucosal tissue shows fine chatter due to cutting a very cold block very quickly.

Step 37 - Use Clean Water

The water in the flotation bath is replaced regularly.

 The water in the flotation bath is topped up regularly but replaced only occasionally. Any contaminants in the bath may end upon the slide under the section (fungi, molds, etc.).

Section from serosa of gastrointestinal tract (H&E). Clusters of weakly staining micro organisms are present within the tissue but could also be seen on the slide, outside the section. The likely source of these contaminating organisms was the flotation bath.
Use Clean Water

Step 38 - Ensure Slides are Clean

The cleanliness of slides is always checked before they are used. Handling of slides is kept to a minimum to avoid contamination with squamous cells prior to flotation.

Slides are not checked for cleanliness: “As long as the sections stay on the slide during staining we consider them to be satisfactory.” Dust, organisms and other contaminants can spoil an otherwise good slide.

A This section of kidney is spoilt by a black contaminant that was present on the slide prior to use.The deposit could be seen under the section in other parts of the slide.
B A section of lung containing stained adhesive “pools” which have been formed as the section dried. The adhesive (probably gelatin based) was present in the flotation bath. The protein content of the adhesive has been concentrated as the water evaporated. Proper draining of the section prior to drying may have avoided the problem.
Ensure Slides are Clean

Step 39 - Avoid Cross-contamination

The water surface is always skimmed between specimens to avoid contamination of one section with cells from another.

The water surface is not skimmed between every block. This can result in specimen-to-specimen contamination which can cause confusion and even an inaccurate diagnosis.

A section of cardiac muscle has been contaminated with a fragment of thyroid from another case. This example of specimen-to-specimen transfer occurred on the flotation bath.
Avoid Cross-contamination

Step 40 - Avoid Contamination with Squames

Care is taken not to brush hair or hands whilst floating-out sections (squames can contaminate sections).

 “Some of our staff produce slides containing many squames. They seem unaware that this can be avoided.”

A kidney section containing many extraneous squames that were deposited on the surface of the section while it was on the flotation bath. They adhere firmly and are subsequently stained with eosin.
Avoid Contamination with Squames

Step 41 - Don’t Float from Multiple Blocks

Sections from more than one block (case) are never simultaneously floated on the water bath.

Sometimes sections from two or more blocks (cases) are left floating-out simultaneously. This is a dangerous practice that can lead to inaccurate identification of specimens. There is a particular risk when the sections happen to be from the same type of specimen.

Here sections from two different cases are being floated-out simultaneously. This practice can result in confusion and lead to inaccurate identification of sections.
Don’t Float from Multiple Blocks

Step 42 - Check Water Temperature

Flotation bath temperature is carefully checked. A temperature 4–5°C below the melting point of the wax is optimal. Sections should readily flatten but the wax should not melt.

 If sections are left on the flotation bath for more than 15 seconds the wax melts. Although this may seem to make the process faster it can rapidly cause over-expansion and tissue and cell damage.

These sections of skin clearly show cracks and excessive separation of layers, the typical effects of over-expansion. Poorly processed tissue is very prone to this problem.
Check Water Temperature

Step 43 - Avoid Wrinkles in Sections

Sections flatten readily on the flotation bath.

 Sections never quite flatten on the flotation bath. The bath may be too cold and the sections may remain wrinkled when picked up on the slide.

In this case flotation has not overcome the wrinkles produced during the cutting of these sections. Better cutting technique and slightly warmer water would overcome this problem.
Avoid Wrinkles in Sections

Step 44 - Avoid Over-expanding Sections

Sections are left on the flotation bath for just long enough to flatten then promptly picked up on a slide.

For convenience, some sections are left for extended periods on the flotation bath. This can cause over-expansion and tissue damage (particularly to delicate specimens such as lymphoid tissue).

A section of intestinal mucosa stained with PAS shows a laminapropria that is over-expanded (shows excessive separation from the intestinal glands). In this case the section was floated for too long on a bath that was too hot.
B A section of lymphoid tissue that has cracked due to over-expansion on the flotation bath. Lymphoid and hematopoietic tissues are particularly prone to damage in this way.
Avoid Over-expanding Sections

Step 45 - Don’t Damage Floating Sections

Extreme care is taken to avoid damaging floating sections when mechanically removing wrinkles with a brush or forceps.

Wrinkles are vigorously removed from floating sections with a brush or forceps. Macroscopic and microscopic damage can easily be caused by this procedure.

A section of cardiac muscle shows mechanical damage (gouging), caused when attempting to remove a fold in the section (heart, H&E).
Don’t Damage Floating Sections

Step 46 - Carefully Choose Sections

The first one or two sections in a ribbon are never picked up on slides.

The first and second sections in a ribbon are selected for mounting because they look better than the later sections. They look better because they are invariably thicker due to the expansion of the cold block during the first couple of passes across the knife.

The sections in the ribbon prepared from this block are numbered in the order in which they were cut. Note that the first couple of sections are widest (least compressed) but as the block warmed the sections got narrower (more compressed). Although the microtome was set on 3 µm the first couple of sections would be 4–5 µm thick due to thermal expansion.
Carefully Choose Sections

Step 47 - Prevent Bubbles Under Sections

Care is taken to avoid the formation of air bubbles in the flotation bath. Any visible bubbles are dislodged before the sections are laid on the water.

Small air bubbles in the flotation bath are ignored. Any bubbles that are trapped under the section apparently disappear as the section dries. Although the bubbles may apparently disappear, the areas in the section above the bubble are often distorted and are likely to float off during staining.

A liver section stained H&E, showing a circular, cracked area where the section has lifted. The cause was a bubble that lodged under the section during flotation and prevented proper flattening and adhesion.
Prevent Bubbles Under Sections

Step 48 - Prevent Section Lifting

The use of “sticky” (charged) slides or section adhesives such as AAS is considered and used appropriately.

Sometimes sections float off during staining (particularly during antigen retrieval for IHC, or when methods require the use of heat). Charged slides or section adhesives are required in these circumstances.

This slide shows an area where the section has lifted and been deposited on adjacent tissue (lung, H&E).
Prevent Section Lifting

Step 49 - Drain Before Drying

Sections are drained briefly before being placed in the slide drier or onto a hotplate.

Sections are not drained properly before being dried horizontally. Sections move on the slide and sometimes do not dry flat.

A This section was dried horizontally without effective preliminary draining. As a result, a raised out-of-focus area is visible in the center of the field.
B This section has just been picked up from the flotation bath and will be drained vertically for a brief time before being placed in the slide drier. This will avoid the problem shown in A.
Drain Before Drying

Step 50 - Monitor Drying Temperature

The temperature of the slide drier is carefully monitored.

Sometimes the slide drier is very hot. Excessive heat can produce hot-spots in sections and cause uneven staining.

This section of prostate shows the features of “nuclear meltdown”, one possible cause being excessive heat when drying slides. Faulty tissue processing can produce a similar effect. Nuclear meltdown is typically seen at the perimeter of specimens and usually affects epithelial tissue. Nuclei show uneven staining, sometimes appearing pink or very blue and completely lacking detail.
Monitor Drying Temperature

Step 51 - Dry for Appropriate Time

The minimum and maximum slide drying time is monitored.

Slide drying times vary considerably. Extended drying at higher temperatures may be detrimental to sections.

A section of lymphnode H&E that shows extensive cracking due to prolonged drying at too high temperature. Cracking like this can also be caused by other factors including over-processing.
Dry for Appropriate Time

Click here to download the full 101 Steps to Better Histology

This reference document is presented as a service to health care professionals by Leica Biosystems and has been compiled from available literature. Although every effort has been made to report faithfully the information, Leia Biosystems cannot be held responsible for the correctness. This document is not intended to be, and should not be construed as medical advice. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Leica Biosystems and the editors disclaim any liability arising directly or indirectly from the use of drugs, devices, techniques or procedures described in this reference document.

Copyright © 2016 Leica Biosystems, a division of Leica Microsystems Inc.
All rights reserved. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.

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