Hi, if you are able to cultivate the yeasts on agar medium you could try to identify it with MALDI-TOF MS technology. E.g the Bruker MALDI Biotyper.

Plate your contaminated culture medium on a yeast agar medium (like sabouraud + chlormaphenicol). Wait 24-48h at 37°C to see yeast colonies. Select some of them and use API like systems for identification. You have several on the market : API20C, Vitek ...

Alternatively you have chromogenic media for quick identification by plating like the chromID or ChromAgar

If you have access then MALDI-TOF MS is best choice, but if not u can go for ITS region sequencing of the culture.

For yeast identification I would recommend to isolate gDNA followed by amplification and sequencing of the D1/D2 domain of the 26S rRNA as described by Kurtzmann and Robnett (Antonie van Leeuwenhoek 73: 331–371, 1998) rather than with the internal transcribed spacer (ITS).

Hi,

For a quick result if you just want to know if you have different species in the sample I would go for a mtDNA-RFLP. It has been used mainly for industrial/wine yeasts but you could maybe try if it works for your samples. You will need to isolate single colonies, perform mtDNA-RFLP (Lopez et al Int J Food Microbiol. 2001 Aug 15;68(1-2):75-81) for each and compare the restriccion patterns. If you want to identify the species you will need to include reference strains and perhaps also use other methods like rRNA or ITS sequencing.

Regarding the spores I do not have other idea than concentrating the sample and checking in the microscope.

There are no short ways around it. Combinations of ITS or D1/D2 std PCR followed by RFLP against know species specific to your line of work. So detemine the substrate or medium from where isolated and select references accordingly.

PCR amplification and sequencing of rDNA.

Dear Michael:

Other way is to use yeast selective media for different species in order to detect which one control your media, this technique is easy to reproduce at any laboratory without too much stuff. Also sometimes by microscope observation you may distinguish among some genus such as Saccharomycodes, Schizosaccharomyces, Kloeckera, some Candida…

Best regards.

I agree there is no super-fast simple way. Chromo-agar can help ID Candida and other related yeast but if the colony is filamentous this methodology will not be all that useful. I think the best way to know would be to isolate the colony onto a new plate. Then do ITS/rDNA PCR. I do this all the time with environmental isolates. In fact you may not need to do a special DNA prep. I tend to do a crude colony DNA prep and this usually suffices for ITS/rDNA PCR. Basically I use 500ul autoclaved ddH20 then scrap off the newly grown purified colony into the water with a pipette tip or toothpick. Then heat 65C, vortex, freeze (-20) and thaw, then repeat again. 2-3ul for each PCR reaction works nicely increase the initial denaturing from 94 to 95 for 5min. Occasionally I'll add DMSO to the crude-prep to aid in the PCR amplification but only with difficult templates.

If, as said already, you will get isolated colonies. You may use universal primers to amplify the internal transcribed spacer region (ITS 1 and ITS2) (Med Mycol. 2007 Nov;45(7):619-2). If you sue RT-PCR you may see the melting curve of the product. If you get different meting temperatures you will have different yeast.

If after you want to know more you have to sequence the fragments and identify the contaminants.

This is the faster way that I may think and not so expensive.

I follow manuel Quiros, as ITS is not the perfect tool to segregate between yeasts, The D1/D2 region is better. To isolate easily yeast from moulds, I would suggest as an alternative to chloramphenicol, to add 5% v/v ethanol to Sabouraud agar medium.

In the case your study deal with a limited number of contaminants (species or strains), a fingerprinting technique might help to make rather quick identification. The method is based on visual or digital analyses of gels containing PCR products amplified with unspecific primer sets. A few "names" have been used for such techniques, e.g. MSP-PCR and RAPD.

Here are a few studies utilizing this method for strain grouping and identification:

https://www.researchgate.net/publication/6780629_Yeast_diversity_in_the_extreme_acidic_environments_of_the_Iberian_Pyrite_Belt?ev=srch_pub

https://www.researchgate.net/publication/9042355_Assessment_of_yeast_diversity_in_a_marine_environment_in_the_south_of_Portugal_by_microsatellite-primed_PCR?ev=srch_pub

https://www.researchgate.net/publication/225786643_Geographical_Races_of_Certain_Species_of_Ascomycetous_Yeasts_in_the_Moscow_and_Novosibirsk_Regions?ev=prf_pub

https://www.researchgate.net/publication/43099772_Description_of_Holtermanniella_gen._nov._including_Holtermanniella_takashimae_sp._nov._and_four_new_combinations_and_proposal_of_the_order_Holtermanniales_to_accommodate_tremellomycetous_yeasts_of_the_Holtermannia_clade?ev=srch_pub

Article Yeast Diversity in the Extreme Acidic Environments of the Ib...

Article Assessment of yeast diversity in a marine environment in the...

Article Geographical Races of Certain Species of Ascomycetous Yeasts...

Article Description of Holtermanniella takashimae sp. nov., Holterma...

We used PFGE to follow yeast populations during wine fermentation.

I think the easiest way is MALDI. See a review like this:

https://www.researchgate.net/publication/51809551_Applications_of_MALDI-TOF_mass_spectrometry_in_clinical_diagnostic_microbiology

Article Applications of MALDI-TOF MS mass spectrometry in clinical d...

For making differentiation between 2 or more yeasts in culture, it would be better primarily use a conventional method such as chromogenic media or CMA, also a PCR-RFLP using restriction enzyme MspI on ITS regions will be helpful. To avoid growth of contaminant spores on the culture, adding 0.5% of actidione (cyclohexamide) can be useful.

You can use the amplification and sequencing of the D2 region of the large-subunit rDNA.

For yeasts determination, you can use API tests (API 20C AUX or ID 32 C) from Biomérieux (after isolation of pure cultures):

http://www.biomerieux-usa.com/servlet/srt/bio/usa/dynPage?doc=USA_PRD_LST_G_PRD_USA_5&pubparams.sform=0

http://jcm.asm.org/content/36/11/3396.full

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC230154/pdf/353240.pdf