You could harvest some spores from your fungus, serially dilute the spore suspension and plate out the dilutions. You could then harvest a young fungal colony that has grown far from any yeast colony after several days and transfer that to a clean plate.

If your fungus does not sporulate you could use homogenised mycelium instead.

Oh Patricia I feel your pain. Yeasts are difficult to get rid of. I suppose you don't have a clean stock to go back to?

What kind of fungus are you trying to keep clean? If your filamentous fungus does sporulate you can use the good advice of Greg, and make sure you dilute carefully, maybe even sonicate lightly to detach yeastcells that were clinging to your spores or mycelial fragments. For some filamentous fungi it may be posiible to isolate clean hyphal tips with steril scalpel from the edge of the colony before they are covered with yeast. And sometimes less is more, eg make weaker strength nutrient agar so that hyphae grow out further than yeast. Goodluck and keep us posted.

In addition to Greg suggestion, in case your filamentous fungus does not develop conidiophores with conidia you can also take a portion (1.0 mm X 1.0 mm) of your slant or culture plate containing both yeast and the filamentous fungus. Place it in a conical 50 ml plastic tube and add 40 ml sterile distilled water, vortex (low speed) for 2 min and discard the water. Repeat this procedure 10 times. Then prepare a 2% water agar plates and Corn meal agar plates. Take a portion of the washed agar containing your contaminated sample and incubate at room temperature. In water agar and Corn meal agar (poor media) filamentous fungi tend to emerge faster. Look the inoculated plates under an inverted microscope (if you do not have one, use magnifier glasses) and cut the advancing hyphae from the plate and transfer to Sabouraud plate. The rate of getting your filamentous fungus in pure culture with this technique is about 80%.

@Leonel

So you basically try to wash all the yeast cells off? Would a tiny bit of tween help, eg 0.01-0.05%? I supposse that the cornmeal agar can be replaced by other poor media that the fungus in question grows on.

@Patricia

what fungus are you trying to rescue?

Thank you all for your answers. I will try then and post the results!

@Henk

I'm trying to isolate a non identified filamentous fungus from a bone sample (actually is a relic of saint undertaking a conservation process). It grows slowly and produces no spores in the conditions I'm using.

Henk-jan, yes that is purpose of the technique. Of course some yeast cells will remain, but the numbers are low. I presume the addition of tween may help. You can use any poor media, water agar is the best.

I agree, your best bet is to sub culture until you feel confident that you have a clean culture.

That sounds like a nice challenging isolation. Of course the yeast may also limit the sporulation, I have seen that happen.

Maybe you can use agar that doesn't support yeast as good as Sabouraud? maybe just collagen.?

And since it is non-identified, could it be a dimorphic fungus, eg the same specimen growing both yeast-like and hyphal? Some fungi I worked with completely change depending on light, food, temperature, aeration, anything really. sometimes you have boths stages in the same prep.

I would second Gregor's dilution and the sub-culture ideas In addition there are some differential antifungal activities you can exploit - for example fluconazole is active against most yeasts but is less commonly active against filamentous moulds - so there is a chance that combining this with the dilution might help. Test your filamentous fungus first with FLU levels between 1 and 16 mg/l to get an idea if this might work.

In addition to what have been suggested so far, you may would like to try the following medium: Sabouraud dextrose agar medium (Oxoid) (SDA) amended with, per liter, 50-mg chloramphenicol, gentamicine sulfate (100 mg/L), and 5-flurocytosine (50 mg/L) (5-FC medium).

Please so also the attached paper.