The method used for staining roots at CICG was proposed by Koske & Gemma (1989). According to the authors, this method has been extensively and successfully used in 150 plant species from 65 botanical families of Angiosperms, ferns, lycopods, psilophytes and bryophytes.
Regardless of the origin of the root samples (from the field or plants in the greenhouse), it is important to select fine roots to detect mycorrhizal colonization. Older, thicker roots may even contain mycorrhizal colonization, usually in the form of external hyphae over the root surface. Pigmented roots, such as those of some pteridophytes (ferns) or even tree species, may require an additional immersion step in alkaline H2O2.
It is important to wash the roots well to remove organic debris or soil particles that can interfere with the process of visualization of colonization. Thus, before starting the staining process, the roots must be washed and then placed in beakers or inside plastic cassettes. In CICG we use rectangular cassettes with 0.9 mm pores or 100 ml beakers. Cassettes with larger pore sizes can lead to root loss during the staining process. The roots must be accommodated inside the cassettes (0.1 to 0.2 g maximum) in order to allow maximum infiltration of the solutions during staining. Cassettes containing too many roots can lead to uneven discoloration.
The roots are bleached by immersion in a 10% KOH (potassium hydroxide) solution and placed in a water bath (90o C) for 60 minutes. The original protocol by Koske & Gemma (1989) recommends using a 2.5% KOH solution only. This step is important to remove the cytoplasmic content from the cells and at the end of it, the solution has a brown color. In cases where the root is very pigmented, it can be left immersed in cold 10% KOH overnight and then placed in a water barth. In more extreme cases, the 10% KOH solution must be changed at least once.
Pour the KOH into a container for appropriate discarding (avoid pouring it into the sink) and rinse the roots under running water to remove excess KOH. If the roots are still dark, place them in an alkaline H2O2 solution (30 ml of a H2O2solution and 3 ml of a NH4OH solution) and leave for 10 minutes (depends on how dark the root is after KOH).
IMPORTANT: Alkaline H2O2 solution must be prepared minutes before use and root must be washed under running water after this step.
Afterwards, cover the roots with a 1% HCl solution (hydrochloric acid) for 5-10 minutes. It is important that the roots are well acidified for proper staining as the dyes used are basic. So, if you use the above step with alkaline H2O2, you can leave a few more minutes in the HCl solution. Roots from soils with high pH (basic soils), the immersion time in HCl can be longer.
Remove HCl properly and DO NOT wash roots. Place them in an acidified glycerol solution (500 ml Glycerin, 450 ml distilled water, 50 ml 1% HCL) containing 0.05% trypan blue (0.5 g in 1 L of solution). The roots are left in a water bath (90o C) for 50-60 minutes. Afterwards, the dye solution is placed in amber vials and can be used once or twice more (it is important to filter it through a piece of gauze to remove eventual pieces of roots), or it can be properly discarded. The stained roots can be kept in a refrigerator at 4o C covered with distilled water (to prevent further growth of saprophytic fungi). A greater contrast of the fungal structures against plant cells is obtained after the samples have been stored for a week, as excess of dye is removed from the roots.
To store the roots longer, they can be placed in screw capped glass tubes containing a mixture of water:glycerin (2:1, vol/vol) with 1-2 drops of 0.05% sodium azide. The dye is retained in fungal tissues for over a year. This procedure is adopted at CICG to observe the colonization pattern of fungal isolates.
Root staining procedure:
1) Arrange root samples in beakers or cassettes
2) Properly identify samples
3) Cover the samples with 10% KOH so that no part of the roots are uncovered
4) Place the samples in a water bath previously heated to a temperature of 90o C for 50-60 minutes
5) Rinse roots under running water (2-3 times) to remove excess KOH
6) If necessary, bleach the roots in alkaline H2O2 (3 ml 20% NH4OH solution and 30 ml 3% H2O2 solution) for 5 minutes. Wash root under running water
Step 6 is optional and depends on root pigmentation
7) Cover samples with 1% HCL for 10 minutes
8) Discard 1% HCL
DO NOT WASH THE ROOTS AFTER THIS STEP
9) Cover samples with acidified glycerol solution (500 ml Glycerin, 450 ml distilled water, 50 ml 1% HCL) containing 0.05% trypan blue (0.5 g in 1 L acidified glycerol solution) and place them in water bath (90o C) for 50-60 minutes.
10) Dispose of the dye solution properly and wash roots under running water to remove excess of dye.
11) Store the roots in distilled water in a 4o C refrigerator for at least one week before analyzing.
Reference:
Koske RE & Gemma JN (1989) A modified procedure for staining roots to detect VA mycorrhizas. Mycological Research 92(4):486-505.
PERCENTAGE OF ROOT COLONIZATION
The measurement of mycorrhizal colonization is one of the assessments commonly performed on roots collected from plants in the field or from plants from experiments (in a greenhouse or in the field). Mycorrhizal colonization estimates the growth of a fungal isolate or an AMF community within the root cortex.
The most used method to measure mycorrhizal colonization is the grid line method proposed by Giovannetti & Mosse (1980). For this method, a Petri dish with a checkered grid of 1.1 x 1.1 cm at the base is required. Using this grid size, both the percentage of mycorrhizal colonization and the length of colonized root can be obtained. Initially, the roots must be colored according to the different methods proposed in the literature or in an acidified glycerol solution with 0.05% trypan blue (Koske & Gemma 1989).
Procedure
1) Spread the stained roots evenly in the Petri dish
2) Under the dissecting microscope, observe the horizontal and vertical lines of the grid and record:
a) the total number of intersections between grid lines and roots (R1)
b) the number of intersections with mycorrhizal roots (R2)
3) The percentage of mycorrhizal colonization (%MC) is obtained by the formula:
%MC = (R2/R1) * 100
Example:
After homogeneously spreading the roots on the checkered 1.1 x 1.1 cm Petri dish, the number of intersections between a piece of root and the vertical and horizontal lines are as follows:
- 25 intersections (horizontal lines)
- 18 intersections (vertical lines)
- 12 intersections showing mycorrhizal colonization
Thus:
a) Total Root Length = 43 cm (25 + 18)
b) Length of Colonized Root = 12 cm
c) Mycorrhizal Colonization Percentage = (12/43) * 100 = 27.9%
Therefore, the total number of intersections between roots and lines represents the total length of roots if the entire root system of a plant is spread out in the Petri dish. It is also possible to calculate the total root length if only one sample of roots is obtained from the root system (in cases where the plant produces too much root that makes it difficult to evaluate the entire root system). For more details on how to proceed in this case, see explanation on the website of INVAM: http://invam.caf.wvu.edu/methods/mycorrhizae/rootlengths.htm
Giovannetti M & Mosse B (1980) An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytologist 84:489-500.