Plant Material
This procedure must be optimized to suit the particular root tissue you are using, and will be affected by the age, growing environment, general condition and fixation method (if any) of your roots. It is central to the success of this staining procedure that the tissue be fully cleared by KOH before staining.
Some things to bear in mind:
- If starting with roots stored in a fixative, it is necessary to rinse them in tap water a couple of times.
- Older or field-grown roots will tend to resist clearing, due to accumulation of tannins and other compounds.
Procedure
- Place roots in holders so that your sample fits in the lower 50% of the container. Commonly used containers are scintillation vials and test tubes of various sizes. Aside from the size, the only other consideration is that the container is made of a material that is autoclavable. If you are processing many samples, you may want to cover the containers with snug-fitting mesh so solutions can be easily changed out. Open lids (with mesh in them) may be purchased for 40mL scintillation vials.
- Add 10% KOH (w/v) to each container, making sure that the sample is completely covered and that the fluid does not fill more than half of the container. For field grown roots, you may soak the tissue overnight, making sure to replace with fresh KOH before the heat treatment. You will find that the KOH turns yellow overnight, indicating that tannins have left your tissue and gone into solution. The overnight soak may be done prior to or following heat treatment. In some tissues two periods of soaking may be necessary.
- The heat treatment may be via boiling in a waterbath, or by autoclaving. This is the part of the procedure that most needs optimizing. For greenhouse-grown maize roots 2-3 weeks old, only 15 minutes of autoclaving (with no prior KOH soaking) is necessary. Field grown adventitious roots of maize that are 1cm in diameter and 2-3 months old require overnight soaking and 1 hour of autoclaving. Some tissues may require multiple soaking and more than an hour in the autoclave. Boiling time is generally longer than corresponding autoclave time.
Roots that have been well cleared resemble cooked rice noodles. They are both translucent and transparent, and are white (not even remotely yellowish!). Remember that clearing with KOH removes many compounds in the cell wall, so after this process, your roots will be mushy and flimsy. Lateral roots may partially or completely disintegrate. Caution should be taken not to dissolve parts of the ground tissue (the cortex usually goes first). It is difficult to ascertain if roots have been completely cleared without staining, but examination of cleared and unstained roots under 4x magnification of a light microscopy will reveal white tissue composed of cells surrounded by thin grey cell walls. Also, you should be able to focus through the sample. - Remove samples from autoclave and rinse in tap water three times. Add 5% HCl and let it sit for about a minute. Pour out the HCl and add trypan blue stain. Let it sit overnight (or longer is fine as well), and examine the roots under a dissecting scope.
If the roots are very dark blue, they did not clear long enough in KOH and further optimization is necessary.
Stain:
1 L glycerol
950 ml distilled water
50 ml acetic acid
0.2 g trypan blue - The typical method of quantifying colonization of the root is by the gridline intersect method (McGonigle et al, 1990). This method has been adapted many times, and each investigator should consider optimizing these examination and quantification procedures to suit desired data collection. Below are a few possible approaches.
Methods of Examination
Light Microscope
Roots may be examined in small segments between two microscope slides (a version of the "squash"method), and quantification may be done using either an ocular or stage grid graticule or micrometer. Several advantages exist with this method. First, tissue is being examined under a light microscope which allows for better imaging than with a stereomicroscope. Further, tissue is easily smashed and spread out here, allowing for visualization of more fungal structures. One disadvantage here is an increased expenditure of time
Stereo (Dissecting) Scope
Whole roots may be examined using a Petri dish that has been fitted with a gridded piece of plexiglass. Roots are placed in the dish with water, and the grid is placed over them. The investigator moves the dish in a back and forth motion so that all intercepts of the grid are examined. An advantage here is that a larger amount of tissue can be examined. A disadvantage is that it may be more difficult to recognize or identify fungal structures with a dissecting scope.
Methods of Quantification
Quantification is done by calculating the percent colonization of your sample. You may use only two categories ("mycorrhizal" or "non-mycorrhizal") or you may choose to subdivide into various categories of fungal structures (i.e. vesicles, arbuscules, hyphae). In either case, you will need to keep track of numbers. If two categories are used, you can keep track with hand-held click counters, having one in each hand. If you only have one counter, you can keep track of the other category in your head. Tallying is necessary with more than two categories.
