MOUNTING MEDIA, REAGENTS, AND STAINS


Polyvinyl alcohol-lactic acid-glycerol

Polyvinyl alcohol-lactic acid-glycerol (PVLG) is used to prepare permanent slides with unbroken and crushed spores, as well as with fragments of mycorrhizal roots. Its viscosity enables to manipulate the position of the specimen examined and, hence, accurately determine their properties. To stabilize the mounted specimens, slides have to dry for ca. 2 weeks at a room temperature or at 65oC for 12-24 h. The latter procedure is recommended, because it clears the contents of spores and their surroundings from oil droplets and air bubbles. Such permanently mounted specimens remain unchanged even after 20 years of their storage.

Polyvinyl alcohol-lactic acid-glycerol used in studies of arbuscular fungi consists of the ingredients listed below.

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

To obtain a high quality PVLG, one needs to:

a) add polyvinyl alcohol as a powder to distilled water and dissolve it in a hot water bath at 70-80oC,

b) mix the solution with lactic acid and glycerol,

c) pour PVLG into a dark bottle.

Melzer's reagent

Melzer's reagent shows dextrinoid reactions of components of the spore wall and inner germination walls of arbuscular fungi. In freshly extracted spores, intensity of the staining reaction mainly depends on the length of carbohydrate chains present in the components. In stored spores, the reactivity of their subcellular structures usually decreases or completely disappears.

Melzer's reagent consists of the following ingredients.

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

Melzer's reagent may be used alone or as a mixture with PVLG. However, spores mounted in a pure Melzer's reagent quickly dry out even when a coverslip is sealed and, thereby, usually are unsuitable for further studies. A mixture of Melzer's reagent and PVLG in a volume ratio of 1:1 enables to prepare permanent slides of advantages similar to those with PVLG alone. The mixture should be stored in a dark bottle.

Trypan blue

Ingredient
Quantity
Lactic acid
100 ml
Trypan blue
1 g

Trypan blue may be replaced with other stains, e. g., with aniline blue.