incubation of fungi

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stopherlogic
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incubation of fungi

Post by stopherlogic » Sun Feb 15, 2009 5:10 pm

Hi, I have been doing a project on sampling fungi but I have been incubating samples 37 degrees. The project is related to human health so i suppose i could say that i was just attempting to isolate pathogens, does anyone have any helpful advice, can it be rescued, can i reincubate at a lower temperature or will i have killed the spores off?
Last edited by stopherlogic on Sun Mar 08, 2009 9:46 am, edited 2 times in total.

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Post by biohazard » Sun Feb 15, 2009 8:44 pm

If I recall correctly, +37C is bit too high for most human pathogen fungi, because they are mainly adapted to live outside the human body (on skin, nails, mucous membranes), where the temperature is generally lower. However, I don't think that +37C kills them either, it just isn't optimal.

Fungi often take longer to grow (especially from spores) than most bacteria, so you may also have to wait a while before you see anything. And make sure the culture medium is something where your fungus likes to grow :)

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Post by stopherlogic » Mon Feb 16, 2009 4:34 am

So do you think I will be able to get away with reincubating at 25C. as I have already incubated at 37 for 48 hours?

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Post by biohazard » Mon Feb 16, 2009 6:57 am

It depends on the type of fungus and medium, but is well possible that you can do it. For human pathogens 48 hours may be too short time for you to see colonies anyway - in medical laboratories fungi are often incubated as long as 2 to 4 weeks, and the temperatures commonly used vary from +28 to +35 C.

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Post by miles500 » Mon Feb 16, 2009 12:14 pm

We incubate 20-25'C and ID when growth is the size of a 10pence piece. This can take between 8-30 days. Be watchful for mites with long incubations.

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Post by miles500 » Mon Feb 16, 2009 12:16 pm

We incubate 20-25'C and ID when growth is the size of a 10pence piece. This can take between 8-30 days. Be watchful for mites with long incubations.

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Re: incubation of fungi

Post by JorgeLobo » Mon Feb 16, 2009 1:59 pm

It would help if you'd add some details - identify the specimen that serves as inoculum and the medium you're using.

Generally and as biohazard said, incubation conventionally is at a lower temp for fungi but rarely do medical labs incubate 2 to 4 weeks. If you're unsure - incubatre separate plates at two temps - ~35C and ~ 28C and be aware that some fungi grow in different morphologies based on temp of incubation.

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Post by biohazard » Mon Feb 16, 2009 3:34 pm

In the university hospital lab I used to do fungal diagnostics the lab manual states that the most common incubation time is 2 to 4 weeks. In reality the time frame is larger: many samples can be identified after one week's incubation, but some species require as long as 6 weeks.

And if the fungus is not a human pathogen, then the incubation temperatures can be considerably lower, often around +20-25C like miles500 wrote.

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Re: incubation of fungi

Post by JorgeLobo » Mon Feb 16, 2009 3:52 pm

It may say that but it should not be practice. This is a hangover from the old days and has litle technical justification. The medium will be dried out at 4 -6 weeks - whatever the precautions to keep it hydrated. Changes in Aw will diminish any recover and growth at this point is more apt to be contamination. Clinically, it's unwise to extend the time of reported exclusion of fungal etiology so long - delaying pursuit of alternative etiologies and therapy.
Results of studies at Duke Univ and UCLA (e.g. http://www.pubmedcentral.nih.gov/articl ... tid=105263)
have clearly shown de minimus benefit to extended incubation and the FDA's BAM recommends 5 days with 48 more hours if no growth.

Further, one should consider the fungi that might be present - which would require that an extended incubation?

I am a mycologist and have written and validated sop's for recovery of fungi from clinical environmental sources. The extended incubation is nonproductive.
For recovery of fungi from an environmental source - one should attempt to reproduce the conditions of that source - temperature, pH, soluble growth factors etc. Extended incubation is similarly limited in value. Slow growth is due to a limiting factor - often as not Aw. If there's a chance for osmophiles, culture for them. Still, there are no hard and fast rules.

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Post by biohazard » Mon Feb 16, 2009 5:12 pm

Fair enough, you seem to know what you are talking about. It's been a few years since I last did diagnostics on fungi, so maybe the methods have changed since that.

Conveniently, though, I'm supposed to keep a microbiology course for some physicians-to-be, which includes mycology, so maybe I get to see what the up-to-date practices are around here nowadays.

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Re: incubation of fungi

Post by JorgeLobo » Mon Feb 16, 2009 5:28 pm

may I help?

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Post by stopherlogic » Mon Feb 16, 2009 5:35 pm

The samples are indoor environmental samples. I have decided to lower incubation to 30C as I don't have the time left to retake the samples they have only been in at 37 for 48 hours and stored in a cold room for a week or so, so hopefully the ones requiring incubation will not be killed off. I know this is far from ideal but it is probably the best option that I have left open to me, thank you for your help everyone. I think the spores of the fungi that have lower optimal growth can survive the 37C incubation from what I gather/wishful thinking (any one who has any other information on this or knows differently I would appreciate any advice on this).

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