Question

You are using a microtome but are having trouble cutting the tissue into sections. Which troubleshooting strategy/strategies would you choose?

Answer 1

Steps to Better Microtomy

1. Use High Quality Blades

Do's - Use High quality, sharp blades always for cutting.

Don't Do - Dont use Blades that are used for as long as possible.

2. Optimize Knife Tilt Angle

Do's - Knife tilt angle is always optimized for each microtome and blade type for better use.

Don't Do - Knife tilt angle is never adjusted when conditions are changed (different microtome, new blade type, different wax etc.). In this case problems occur.

3. Carefully Trim Blocks

Do's - 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.

Don't Do - 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.

4. Avoid Freezing Damage

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

Don't Do - Blocks are frozen before cutting. This sometimes causes the blocks to crack.

5. Use Cold Blocks

Do's - Blocks are always cold when cut.

Don't Do - 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.

6. Cut Sections Slowly

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

Don't Do - Sections that are cut as quickly as possible with a rapid rotation, in the belief that “any section compression will be overcome on the flotation bath.” will trouble.

7. Use Clean Water

Do's - The water in the flotation bath is replaced regularly.

Don't Do - 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.).
And will cause trouble.

8. Ensure Slides are Clean

Do's - 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.

Don't Do - 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.

9. Avoid Cross-contamination

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

Don't Do - 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.

10. Avoid Contamination with Squames

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

Don't Do - “Some produce slides containing many squames. They seem unaware that this can be avoided.”

11. Don’t Float from Multiple Blocks

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

Don't Do - 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.

12. Check Water Temperature

Do's - 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.

Don't Do - 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.

13. Avoid Wrinkles in Sections

Do's - Sections flatten readily on the flotation bath.

Don't Do - 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.

14. Avoid Over-expanding Sections

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

Don't Do - 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).

15. Don’t Damage Floating Sections

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

Don't Do - Wrinkles are vigorously removed from floating sections with a brush or forceps. Macroscopic and microscopic damage can easily be caused by this procedure.

16. Carefully Choose Sections

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

Don't Do - 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.

17. Prevent Bubbles Under Sections

Do's - 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.

Don't Do - 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.

18. Prevent Section Lifting

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

Don't Do - 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.

19. Drain Before Drying

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

Don't Do - Sections are not drained properly before being dried horizontally. Sections move on the slide and sometimes do not dry flat.

20. Monitor Drying Temperature

Do's - The temperature of the slide drier is carefully monitored.

Don't Do - Sometimes the slide drier is very hot. Excessive heat can produce hot-spots in sections and cause uneven staining.

21. Dry for Appropriate Time

Do's - The minimum and maximum slide drying time is monitored.

Don't Do - Slide drying times vary considerably. Extended drying at higher temperatures may be detrimental to sections.