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| When sampling roots to detect and/or measure the amount of mycorrhizal colonization, it is important to select finer, more fibrous roots. Older roots or those from plants with taproots or other coarse roots, may have some mycorrhizae, but it ususally is sparse, and consists only of hyphae that often is most visible outside the roots. For plants with a fibrous root system, then a random sampling suffices. Darkly pigmented roots should be avoided -- to use them means going through an additional bleaching step (not covered here). |
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| After thorough washing to remove all particulates, roots are placed in plastic cassettes used in medicine for processing biopsy tissues. Different types of cassettes are marketed (Fischer, Ted Pella, etc.), but the best for root samples are rectangular with 0.9 mm holes (photo at right). Cassettes with larger holes result in escape of roots, especially when roots are cut into small fragments. Pack roots loosely in cassettes (0.1-0.2 g max) for maximum infiltration of solutions. | 
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| The roots are cleared (removing cytoplasmic contents from cells) using hot 10% KOH. Different approaches are used, from autoclaving cassettes for 5-10 minutes to boiling them in some container on the lab bench. Much depends on number of samples to be processed simultaneously. We usually process only 10-20 samples at a time. To minimize agitation, we preboil the KOH in a large beaker over a bunsen burner, and immediately add cassettes when the burner is shut off. Incubation time varies with thickness and fragility of roots, but 10 minutes usually is sufficient. Older thicker roots require longer incubation times. Browning of the solution is an indication of the clearing process. |  |
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| Because most of the histological stains used for this procedure are acidic, it is critical that roots be acidified. So we wash roots (4 -5 x), and then immerse cassettes in 2% HCl for 15-20 minutes. For roots from highly alkaline soils, longer incubation times are recommended. If roots are uniformly dark and mycorrhizae are hard to see, then inadequate clearing often is the cause. | ||
| The same procedure described above to clear roots is carried out again, only with 0.05% direct blue or some other suitable stain (acid fuchsin, chlorazol black E). The stain is prepared by mixing with water, glycerin, and lactic acid in proportions of 1:1:1 (v/v/v). Incubation time varies, but 3-4 minutes works best for us with greenhouse-grown plants. In the photo at right the cassettes were added to the boiling solution and then the burner shut off immediately thereafter. This step can be done without application of heat, but the incubation period then needs to be extended to 12 hr. |  |
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| The stain is poured off into another container (it can be used again 1-2 times after filtration through cheesecloth) or discarded in a wastebottle. Rinse the cassettes with 4-5 changes of water and store at 4C in water. More contrast between fungal tissue and background plant cells is obtained when the samples have been stored for a week or longer (excess stain leaches from roots). | ||
| For long term storage of stain roots, we place them in screw-top glass tubes containing a water-glycerin mix (2:1 v/v) with 1-2 drops of 0.1% sodium azide. Stain is retained in fungal tissue for > one year. Roots can be stored in fixative solutions as well (e.g. formalin), but only if you want to contract allergies, cancer, or other possible health problems. |  |
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| Mycorrhiza stained with trypan blue | Mycorrhiza stained with 0.05% acid fuchsin (alternative stain) | Mycorrhiza stained with 0.05% acid fuchsin (alternative stain) |
