Recipes
Polyvinyl-Lacto-Glycerol (PVLG)
PVLG is used to permanently mount whole or broken AMF spore on slides. Mounted spores should not be studied for 2-5 days after mounting to allow the spore content to clear.
Ingredients - Quantity
Distilled water - 100 mL
Lactic acid - 100 mL
Glycerol - 10 mL
Polyvinyl Alcohol (PVA) - 16.6 g
Important – mix all ingredients in a dark amber bottle before adding polyvinyl alcohol. PVA dissolves slowly and only when placed in a water bath (70-80o C). The solution will clear in 4-6 hours and we usually leave the mixture for 1-2 days in a water bath until the PVA is completely dissolved. You can store PVLG in tightly sealed dark bottles for 1 year.
Melzer's Reagent
Melzer's reagent has become an important tool for the morphological identification of AMF species. The reaction to iodine varies from faint pink (weak reaction) to dark reddish-brown (moderate reaction) to dark reddish-purple (intense dextrinoid reaction). Iodine binds to the hydrophobic regions of macromolecules making up the spore and germinal walls) and the intensity of the reaction is related in part to the length of the carbohydrate chains. In most cases, the intensity of the reaction is directly correlated with the plasticity of the structure in acidic mounting media.
Ingredient - Quantity
Chloral Hydrate - 100 g
Distilled water - 100 mL
Iodine - 1.5 g
Potssium Iodide - 5.0 g
When Melzer's reagent is used according to the recipe above, the reactions will be more intense. However, mounts will be not last long, even when the cover slip is sealed and often dries out after 1-2 years of storage. More permanent mounting medium can be achieved by mixing Melzer's reagent with PVLG (1:1, vol/vol) and storing the mixture in a dark bottle. The reaction is diminished a little, but not to the point of misinterpreting the intensity of the reaction. In structures that stain weakly, the reaction color disappears within a year or two after storage.
Sodium azide for “vouchers”
Sodium azide is an inhibitor of cell respiration and so must be handled with great care (wearing gloves) when stock solutions are prepared (2.5 g in 50 ml distilled water). A 1 ml aliquot of the stock solution is added to 90 ml of distilled water to obtain a 0.05% solution, which is used to store AMF spores.
For vouchers kept in eppendorf tubes, spores are collected and placed in 5 ml eppendorfs with a minimum of water. The eppendorf is then filled with 0.05% sodium azide and identified with a tag (containing the species name, code and date). Spores in eppendorfs with sodium azide are stored at 4oC.
Spores die in sodium azide and begin to naturally degrade over time. They start to float, the content often turns dark or loses its integrity (becoming condensed). However, the subcellular structures maintain their integrity for the most part. Other solutions to preserve spores have been used such as FAA (Formaldehyde, Acetic Acid and Alcohol) and lactophenol (lactic acid and phenol), but evidence from AMF-type specimens shows that they cause major changes or degradation of the subcellular structure of the spores and should be avoided.