incubation of fungi

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    • #10909
      stopherlogic
      Participant

      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?

    • #89123
      biohazard
      Participant

      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 🙂

    • #89137
      stopherlogic
      Participant

      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?

    • #89138
      biohazard
      Participant

      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.

    • #89145
      miles500
      Participant

      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.

    • #89146
      miles500
      Participant

      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.

    • #89147
      JorgeLobo
      Participant

      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.

    • #89155
      biohazard
      Participant

      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.

    • #89157
      JorgeLobo
      Participant

      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.

    • #89158
      biohazard
      Participant

      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.

    • #89160
      JorgeLobo
      Participant

      may I help?

    • #89162
      stopherlogic
      Participant

      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).

    • #89165
      JorgeLobo
      Participant

      Indoor air – dust will have osmophiles. Air from the exterior will carry Asperigillus, Penicillium and Cladosproium spore.

      As biohazard suggested, the temp is prob still to high. What medium are you using?

    • #89166
      stopherlogic
      Participant

      Malt extract agar

    • #89171
      JorgeLobo
      Participant

      Good medium for that purpose. Assume you’re not that interested in osmophiles?

    • #89196
      stopherlogic
      Participant

      Just interested in possible pathogens really thought it was the best general purpose media. Didn’t want to be too selective.

    • #89197
      JorgeLobo
      Participant

      No worries for osmophiles if you’re looking for "pathogens." Are you also looking for toxigenic fungi?

    • #89204
      stopherlogic
      Participant

      Yes, anything which you would say has an effect on human health but it will be necessary to identify whatever I find.

    • #89222
      JorgeLobo
      Participant

      malt extract will prob be adequate. How do you plan to id – and th what extent (genus, species?)

    • #89231
      stopherlogic
      Participant

      I was planning to just id using colony morphology, and use the adhesive tape method to observe microscopically, not sure whether i will need to stain as yet. Just going to categorise by genus.

    • #89240
      JorgeLobo
      Participant

      Understand you may have limitations in what’s available but colony morphology is not going to serve well, even if you’re an experienced mycologist, and no way will you be able to adequately id "pathogens" even fumigatus. Scotch tape is messy and it’s real hard to get decent conidiophores.

      Can you set up slides culture? That would help seeing mode of spore production. Establishing higher temp incubation would help with fumigatus and a black fungus or two.

      In terms of "pathogenic fungi" what do you think you’d need to id?

    • #89261
      stopherlogic
      Participant

      Pathogenic fungi is of particular interest which is probably why I made the mistake of incubating too high in the first place. So at 37 all I think I am seeing is a few colonies of penicillium up to now. I plan incubate the same samples at 25 on monday and hopefully I will get some more growth if everything hasn’t died off. I realise pathogens are an unlikely prospect but human health was the original angle which I was taking with my project. I haven’t thought about slide cultures but I will look into it. I am running low on time though.

    • #89263
      JorgeLobo
      Participant

      short of marneffii, what penicillium, would you have considered pathogenic?

    • #89265
      stopherlogic
      Participant

      There are no others as far as I am aware and the chances of it being marneffii are unlikely. Finding pathogens is extremely unlikely unless I count opportunistic pathogens such as some aspergillus (?) which I would have to incubate at a lower temperature. I was looking for pathogens or anything affecting human health but have to identify anything I find.

    • #89272
      JorgeLobo
      Participant

      agree re, marnefii. Not sure whatu will see =- good luck.

    • #89273
      stopherlogic
      Participant

      Thanks you’ve been a great help.

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