Recipes for Voucher Preservation

PVLG | Melzer’s reagent | Sodium Azide | Distilled water

Polyvinyl-Lacto-Glycerol (PVLG)

PVLG is used to permanently mount whole or broken spores on glass slides. For best results, mounted specimens should not be studied for 2-3 days after they were mounted to give time for spore contents to clear. Whole spores will change color, generally darkening to varying degrees, and shrink or collapse with plasmolysis of spore contents. Discrete layers of the spore wall or flexible inner walls of broken spores will swell to varying degrees and appear fused after long storage in some instances.

Ingredient Quantity
Distilled water 100 ml
Lactic acid 100 ml
Glycerol 10 ml
Polyvinyl alcohol (PVA) 16.6 g

It is most important to mix all ingredients in a dark bottle BEFORE adding the polyvinyl alcohol. The PVA should have the following properties: 99-100% hydrolyzed, and a viscosity of 24 – 32 centipoise in a 4% aqueous solution at 20°C. The PVA is added as a powder to the other liquid ingredients. The PVA dissolves slowly, and then only when placed in a hot water bath (70 – 80°C). The solution will be clear in 4-6 hours; we usually prepare the mix in the evening and letting it incubate in the water bath overnight. PVLG stores well in dark bottles for approximately one year.

Rick Koske writes that the PVA powder can be added to the water, followed by autoclaving for 15 minutes. The lactic acid and glycerin are added, and the solution then is stored at room temperature for at least 24 hr before using.

Melzer’s Reagent

Melzer’s reagent has become an important diagnostic tool in morphology-based diagnoses. Iodine staining reactions will vary from pale pink (weak reaction) to dark red-brown (moderate reaction) to dark reddish-purple (intense dextrinoid reaction). The iodine binds to hydrophobic regions of macromolecules (in spore or germinal walls) and intensity of the reaction is related in part to the length of carbohydrate chains. In most instances to date, the intensity of the reaction is directly correlated to plasticity of the structure in acidic mountants.

Ingredient Quantity
Chloral hydrate 100 g
Distilled water 100 ml
Iodine 1.5 g
Potassium iodide 5.0 g

When Melzer’s reagent is used according to the recipe above, staining reactions will be most intense. However, mounts are temporary even when a coverslip is sealed, and often dries out within 1-2 years of storage. More permanent mounts are made by mixing Melzer’s reagent with PVLG in a volume ratio of 1:1 (and storing the mixture in a dark bottle). The staining reaction is diminished slightly, but not enough to cause any confusion as to the intensity of the reaction. In structures staining weakly, the color reaction fades within a year or two of storage.

Sodium azide for voucher specimens

Sodium azide is a respiratory inhibitor and therefore should be handled with care (wearing gloves) in the preparation of stock solutions (2.5 g in 50 ml of distilled water). A one ml aliquot of the stock is added to 90 ml of distilled water for a 0.05% working solution.

For vial vouchers, spores are collected and added to 2 ml vials in a minimum of water. The vial is then filled with the sodium azide working solution and labelled. Solutions and vials are stored at 4°C as an added precaution to optimize safety of the workplace.

Spores die in the sodium azide and therefore will begin to degrade naturally over time. They will begin to float, contents often darken or lose their integrity (appearing either cloudy or vacant). However, subcellular structures largely retain their integrity. Other preservative solutions such as FAA (Formalin + Acetic Acid + Alcohol) and lactophenol (lactic acid + phenol) have been used extensively in the past, but evidence from type specimens indicates they can cause major changes or degradation of subcellular structure of spores.

Distilled water and sand for voucher specimens

Serendipitously, on one occasion we stored washed spores of Gigaspora gigantea in distilled water at 4°C and were surprised to discover that the spores did not change color after 30 days. In contrast, spores stored in sodium azide or any other preservative turn red-brown to brown within 10 days. We have since tried this with other fungi, and obtain similar results. When the spores are killed by the preservative, some degradation of structure occurs. The only drawback with storing in water is that any parasitized spores (often not detectable at the time of extraction) will be a source of spread of colonizing fungal saprophytes or actinomycetes to adjacent spores. The approach we have taken to minimize this problem is similar to that used to prepare spore samples slated for molecular studies (viewed here). Fungal species with small (< 150 Ám) light-colored spores tolerate these conditions best (large darkly pigmented spores tend to degrade no matter what), and we have stored some of them for over a year this way. Avoid Ringer’s and other solutions that provide an isotonic environment; buffering appears to serve no useful purpose and in some cases, causes plasmolysis.