Cutting Problems
Cut on an angle
It is important to make sectioning cuts that are perpendicular to the longitudinal axis of the root. This seems basic, however it is much more difficult to perform and to sustain throughout the sectioning of an entire segment. When one cut is made an angle, all subsequent sections will be at an angle, until a cut is made to renew the perpendicular surface.
Angled cuts can be identified in the following ways:
- the section or cells within it are oval in shape
- one can focus through several cell layers in one area of the section
- part of the section appears to be "smeared"
Cut too thin
This problem can be identified if a section has a part missing, and/or it is not completely round.
Cut too thick
This problem can be identified if boundaries within the section appear exceptionally thick, or if you are able to focus through several cell layers across the whole section.
Dull Razorblade
Cutting resistance from the blade may cause the researcher to exert extra pressure on the segment or to use a sawing motion with the blade, both without realizing it. This results in sections that appear "smeared" or misshapen, usually in an oval shape.
Sections cut in half
The problem here is that there are too many sections in the drop of water, and convection is bringing them back towards the razor where they are being cut in half.
Tissue Related Problems
Exposure to Excessive Fixative
Within a fixative mixture, the use of a high percentage of a strong fixative, such as an aldehyde, may result in break down or digestion of the tissue. This problem can be identified by tissue with a "frayed" appearance. The same problem may result if the tissue is left for a long period of time in the initial killing fixative (e.g. FAA), before being moved to storage in ethanol. Remedying this problem is relatively easy, if the traditional recipe for FAA is followed (see "Tips on Preserving Tissue"). Also, the length of time of initial fixation is usually relatively short, lasting only a few hours to a few days. This time must be optimized for your particular tissue.
Ethanol percentages exceeding 75% may make the tissue tough, rubbery and resistant to sectioning.
Lack of Exposure to Fixative
Using a low percentage of ethanol (<50%) for long-term storage may introduce the possibility of pathogen infection. Storage in water is not recommended for the same reasons. Chances of pathogen infection may be decresed if a brief and strong fixative used at harvest, and before storage. Whether using FAA, ethanol or both, some time is required for fixative penetration into tissue. As such, sectioning will be optimal if adequate time is allowed for fixative penetration.
Tissue Too Young
Young plant tissues are tender, and are much more difficult to handle and section than older tissues. Young tissue can be identified by premature vascular development and, occasionally by diameter. If young tissues are required, the researcher may consider sectioning with a microtome following embedding in agar, other plant tissue or paraffin.
