Index >> Mycorrhizae >> Isolation of AM Fungal Spores

The residue containing the rootlets in the sieve is set aside. The filtrate in the cylinder is stirred constantly and allowed to pass through a 250 μm sieve and the filtrate collected in a second 1 litre measuring cylinder until the filtrate reaches the high mark. The residue in the sieve is set aside. This procedure is 'again repeated twice, once with 105 μm sieve and the other with a 53 μm mesh sieve and the filtrates collected in separate 1 litre measuring cylinders. The objective of stirring, sieving and washing so many times is to facilitate quantitative

retrieval of spores depending on their size. Large circular sieves can also be used over glass troughs instead of small sieves in a funnel and the filtrates collected in a serial fashion.

The finer roots are washed gently in tap water and simmered in 10 percent KOH at 90°C for 1-2 hours, rinsed in tap water, immersed in 2 per cent HCI and stained with 0.05 per cent trypan blue in lactophenol. This is done by boiling roots in the stain for 3 minutes, draining the excess stain and immersing the stained roots in lactophenol overnight to destain the cortical cells. Later the stained specimens may be examined under a microscope in lactic acid-glycerol medium (1:1) when only the hyphal strands and spores retain the stain. Due to the corrosive nature of the materials used in these studies, the procedures of staining and destaining are done in a fumehood.