GROWING AND MOUNTING MEDIA, REAGENTS, AND STAINS


Czapek-Dox Agar

Ingredient
Quantity (g)
NaNO3
2.00
K2HPO4
1.00
MgSO4 7H2O
0.50
KCl
0.50
FeSO4
0.01
Sucrose
30.00
Agar
20.00
Distilled water
1 L

If glass distilled water is used, add 1 ml of 1% ZnSO4 and 0.5% CuSO4. Heat the full chemical solution without sucrose in a water bath for 15 min. After cooling add sucrose and agar. Melt the agar and autoclave. Do not filter (Dhingra and Sinclair 1985).

Malt extract agar

Ingredient
Quantity (g)
Malt extract
20.00
Agar
20.00
Distilled water
1 L

Heat 20 g malt extract in 1 L of water until dissolved, then add 20 g agar and heat until the agar melts (Dhingra and Sinclair 1985).

Potato Dextrose Agar

Ingredient
Quantity (g)
Potato peptone
4.00
Glucose
20.00
Agar
15.00
Distilled water
1 L

Sabouraud Dextrose Agar

Ingredient
Quantity (g)
Peptone, Difco
10.00
Glusoce
40.00
Bacto-Yeast Extract
3.00
Agar
15.00
Distilled water
1 L

Polyvinyl alcohol-lactic acid-glycerol

Polyvinyl alcohol-lactic acid-glycerol (PVLG) is used to prepare permanent slides with unbroken and crushed fungal specimens, 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 the specimens 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 consists of the ingredients listed below:

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

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 fungal structures examined. In freshly extracted fungal structures, intensity of the staining reaction mainly depends on the length of carbohydrate chains present in the components. In stored specimens, the reactivity of the 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, specimens 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.


REFERENCES

Dhingra O. D., Sinclair J. B. 1985. Basic plant pathology methods. CRS Press, Inc. Boca raton, Florida.