I have so far read McGonigle et al. 1990: "A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi", as I am preparing to collect plant specimens from which I will take measurements of the % root colonization by AMF. Does anybody have any advice on the best way to take such measurements, any tips or tricks or important points to remember?
The per cent mycorrhizal colonization in plant roots was determined according to the method outlined by Phillips and Hayman (1970) and Giovannetti and Mosse (1980). The root materials were cut into bits of 1 cm length and fixed in FAA (Formalin: Acetic acid: Alcohol) in the ratio of 5:5:90 (v/v/v) respectively. The roots were cleared by autoclaving with 10 percent KOH at 15-lbs/inch2 pressures for 15 minutes. The alkalinity due to KOH was neutralized by 1% HCl for 5 minutes and the staining was done by simmering the roots in 0.05% tryphan blue in lacto glycerol (Lactic acid 40ml: Glycerol 50ml: Distilled water 10 ml) for 10 minutes. The stained root bits were arranged on the slide and observed under microscope to study the mycelia, vesicles and arbuscules. The stained root pieces of 1 cm length were selected randomly, arranged on a slide in groups of ten. The length of infection was assessed in mm or cm for each root piece and averaged for ten pieces, and expressed in percentage of colonization. The presence or absence of infection was also recorded in groups of ten each and results expressed in percentage (on the basis of slide method ±). 100 root bits were observed in each replicate sample. The percentage colonization was calculated by the following formula,
Per cent of root colonization = No. of root bits showing colonization x 100
____________________________
Total number of root bits observed
The per cent mycorrhizal colonization in plant roots was determined according to the method outlined by Phillips and Hayman (1970) and Giovannetti and Mosse (1980). The root materials were cut into bits of 1 cm length and fixed in FAA (Formalin: Acetic acid: Alcohol) in the ratio of 5:5:90 (v/v/v) respectively. The roots were cleared by autoclaving with 10 percent KOH at 15-lbs/inch2 pressures for 15 minutes. The alkalinity due to KOH was neutralized by 1% HCl for 5 minutes and the staining was done by simmering the roots in 0.05% tryphan blue in lacto glycerol (Lactic acid 40ml: Glycerol 50ml: Distilled water 10 ml) for 10 minutes. The stained root bits were arranged on the slide and observed under microscope to study the mycelia, vesicles and arbuscules. The stained root pieces of 1 cm length were selected randomly, arranged on a slide in groups of ten. The length of infection was assessed in mm or cm for each root piece and averaged for ten pieces, and expressed in percentage of colonization. The presence or absence of infection was also recorded in groups of ten each and results expressed in percentage (on the basis of slide method ±). 100 root bits were observed in each replicate sample. The percentage colonization was calculated by the following formula,
Per cent of root colonization = No. of root bits showing colonization x 100
____________________________
Total number of root bits observed
Giovannethi and Mosse (1980) method:
The percent root colonization was calculated by the formula:
Percent colonization = (Total numbre of infected roots interesecting griglines/total number of roots intersecting gridlines)x100
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