I am working on Arbuscular Mycorrhizal Fungi (AMF) with emphasizing on Cucumber productivity in the presence or co-inoculation with organic substrate, Could someone guide how to check the effective AMF inoculation% ?or suitable staining techniques to visualize the AMF roots interaction with substrate .
best staining procedure.
Procedure
AMF infection can be detected in the roots of plants by Trypan blue staining. The fungal spores and hyphae appear blue after staining and observing it under light microscope.
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
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.
@ Ahmad, in addition to above answers; to observe structure within roots, it is necessary to clear cortical cells of cytoplasm and phenolic compounds, and then to deferentially stain the fungal tissues. Phillips and Hayman (1970) developed the method to visualize AM fungi in roots using trypan blue in lactophenol, but the use of phenol is not discouraged. The KOH is used as cleaning agent for non-pigmented roots, but H2O2 or NaOCl may be used for pigmented roots. In general, colonized root length has been estimated by Grindline-Intersect method given by Giovannetti and Mosse (1980). The spores in the soil can be recovered by Wet Sieving and Decanting technique or Density gradient centrifugation (Hyman, 1984; Schenck and Perez, 1990).
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining