I have successfully used the following staining procedure for arbuscular mycorrhizal (AM) fungi in plant roots of many types. Note that the hydrogen peroxide step is added if the roots are still quite dark after the KOH treatment. This is based on the protocol used by Koske & Gemma published in a Mycological Research paper (1998), with a few modifications made for use by undergraduates in my Fungal Biology class with the equipment we have available.

Reagents

1. 2.5% KOH (25 g in 975 ml H20)

2. alkaline H202 (6 ml 20% NH4OH in 660 ml 3% hydrogen peroxide)

3. 1% HCl (30 ml concentrated HCl (38%) in 1170 ml of water)

4. acidic glycerol (500 ml glycerol; 450 ml H20; 50 ml 1%HCl)

5. trypan blue in acidic glycerol (to the same volume of acidic glycerol above, add 0.50 g trypan blue)

Materials

root capsules (plastic tissue culture capsules)

2 hot plates OR a moderately large water bath set at 90 C.

2 thermometers

2 500 ml beakers

25 scintillation vials

Method

1. Put the roots in labeled capsules and place up to 3 capsules in a 500 ml beaker with 2.5% KOH (+/- 200 ml). Heat for 15 minutes while holding temperature steady at 90 C.

2. Rinse the roots well in 5-8 changes of water in the beaker.

3. If roots are already white - yellow proceed directly to step 5. If pigment is still present in roots, bleach with alkaline hydrogen peroxide at room temperature for 10 - 30 minutes until roots lose color. (Don’t go longer than 30 minutes as roots will disintegrate). Use sufficient volume to cover the capsules (+/- 200 mls).

4. Rinse well in water.

5. Soak the roots in 200 ml of 1% HCl for 1-24 hours (overnight is fine), at room temperature (roots should be submerged in the acid).

6. Stain the roots in trypan blue/acidic glycerol for 20 minutes at 90 C.

7. Pour off trypan blue solution into the “used” bottle. Place the stained roots in labeled scintillation vials containing acidic glycerol, for long-term storage at room temperature. Used tb solution can be re-used once.

80 c is best for heating (for 15 mnts)

You can also try heating the roots for longer - overnight at 60 C, but the problem probably is related to non-alkali soluble pigments, and the colour will probably need to be bleached away (if hydrogen peroxide doesn't do the job, try sodium hypochlorite (just commercial bleach at 10% with water (v/v). You can watch the roots clear so that you don't bleach for too long. Another trick used by the folks in Louvan-la-Neuve, is to change the KOH one or more times until there is no further clearing (i.e., until the KOH doesn't discolour). Also, trypan blue is a carcinogen. Try using something else (ink is fine, or methyl blue is supposedly less dangerous). Other methods include autoclaving the roots in KOH. I haven't tried it, but I have had reports that it works well (I'm sure you can find reference in an internet search). The key to all these things is really to treat the recipes in journals as a starting point only, and then fiddle around a bit. Incidentally, a method I find useful is to simply bring the KOH (1M) to the boil (or heat to near-boiling) and then just leave to cool overnight. You need to try different times or temperatures with the KOH as if you overdo it, the roots will disintegrate. The acidification is almost instant, though there is no harm in leaving in acid for a longer period. It is there just because the stains used are acid stains See my little paper on staining

Walker, C. (2005). A simple blue staining technique for arbuscular mycorrhizal and other root-inhabiting fung. Inoculum, 56(4), 68–69 which is on ResearchGate as full text.

Thank you all for help.