Fungal Biotechnology

Fungal Biotechnology

  • Gemma Assante added an answer:
    How can I extract secondary metabolites from fungus mycelium without homogenization?

    i would like to to extract the secondary metabolites trapped in the fungus mycelium without homogenizing them ?

    Any ideas 

    Gemma Assante · University of Milan

    Just overlay the agar culture of the fungus with solvent (Ethylacetate + Methanol 100:1). Leave the solvent for 24 h, collect it and re-extract after 24 h. Pay attention: your culture container must be glass or plastic resistant to the solvent you use. Use anhydrous Na2SO4 to adsorb water, evaporate the solvent and purify the extract. 

  • Elena Shersher added an answer:
    What factors might cause Aspergillus niger fungus to turn bright yellow during growth in a liquid culture?

    I am expressing a heme protein in Aspergillus niger fungus. The spores I work with are brown, and when I inoculate the liquid culture, most of the growing fungus has a light brown color. However, I usually get at least one flask where the fungus turns bright yellow and the culture itself becomes yellow-brown. I noticed that those are the cultures that have my protein expressed in much larger quantities than light brown cultures. I also noticed that the fungus turns yellow if I add fructose instead of glucose to the culture and grow it in a flask that has indentations on the bottom of the flask instead of flat-bottomed flask. I would like to have all my cultures yellow and now grow small cultures first, then peak yellow cultures and transfer to larger cultures. However, I never see consistency in the fungus turning yellow even though the growing conditions are the same. Does anyone have an explanation for those observations? What factors might contribute to the color change and larger protein expression?  If you work with Aspergillus niger, have you ever had a similar experience? 

    Elena Shersher · Florida International University

    Thank you, Ghanshyam and Matthias!

    I do use spores from the same colony, and my promoter is constitutive. The reason I am interested in yellow color cultures is that if I have more protein expressed,  I don't have to grow that many liters of culture. In order for me to get enough protein for characterization I need to grow about 50 liters of culture. It is a lot of work! If all my cultures were yellow I would probably need to grow a half of that. 

  • Abrory Agus Cahya Pramana added an answer:
    What is the second layer of this extract?

    Last week, I extracted my fungal culture in Wickerham media by using ethyl acetate. I disrupted the cells by using the Ultraturax machine. After that I macerated my extract. Then, three layers formed. I am still confused with the second layer which is located between the medium and ethyl acetate.    

    Abrory Agus Cahya Pramana · Gadjah Mada University

    I would like to say thank you for the answering of my question...
    And I hope we can discuss again...
    Now, I have evaporated the first layer and then I add methanol in my last extraction.

  • Jai Ghosh added an answer:
    How can I differentiate bacterial contamination in fungal wet mount microscopically?

    Basically I work on solid state fermentation of fungus and find it really difficult to confirm if my seed cultures are pure.

    Jai Ghosh · Shivaji University, Kolhapur

    simply stain with cotton blue instead of lactophenol blue and you should be able to differentiate the two. The best method is if you have a phase contrast microscope you can see the difference very well.

  • Jun-Seob Kim asked a question:
    What is the highest OD number of Pichia pastoris GS115 in MMH medium?

    Hi guys, what is the maxium cell number (OD) of P.pastoris GS115 in MMH medium in batch culture (e.g. 300 rpm, 30 degree)?

  • Priyanka Kudalkar added an answer:
    Has anyone tried using Lyse and Go PCR reagents for their fungal cultures ?

    I have always used the DNeasy Plant Mini Kit for extracting fungal DNA. This is a rather long protocol and did not seem to work for one of my cultures. I was wondering if anyone has used Lyse and Go on their fungal cultures? I wanted to give it a shot. According to the protocol, it eliminates the lengthy DNA protocols and excessive sample manipulations. 

    Priyanka Kudalkar · Montana State University

    Thank you for the help! I will be certainly be trying some of these suggestions. 

  • Partha Pratim Dhar added an answer:
    What is the protocol for DNA extraction, testing for identification of Arbuscular Mycorrhizal Fungi?

    Molecular identification of AM fungi is important. I need the protocol for DNA analysis.

    Partha Pratim Dhar · King Saud University

    Thank you very very much, Alvaro.

  • Rolf Henrik Nilsson added an answer:
    Which is the simplest method to identify the fungi whether it is saprophytic or mycorrhizal?

    i currently isolating different fungi from the soil by soil-plate and serial dilution method. i am interesting to know that is there method to identify the mycorrhizal or saprophitic fungi from these soil-isolated fungi. please give your valuable suggestions in this regard. Thanks in advance

    Rolf Henrik Nilsson · University of Gothenburg

    UNITE (unite.ut.ee) is for all groups of fungi, not only mycorrhizal. It focuses on ITS sequences, the formal fungal barcode. In my experience it works well for most groups of fungi.

  • Senthilarasu Gunasekaran added an answer:
    How can I find or obtain Lentinula edodes and Bacillus pumilus cultures to be used in my study?
    How can I find or obtain Lentinula edodes and Bacillus pumilus cultures to be used in my study?
    Senthilarasu Gunasekaran · SRM University

    Lentinula edodes is a common, edible mushroom and a vernacular name is shiitake mushroom. You can get the mushroom at any grocery shop. Bring the mushroom to the lab. Cut it into two half and transfer inner tissue into PDA medium amended with an antibiotic. Incubate the plate at 28 degree C. the mycelium growing out from the tissue can be transferred to  another plate. Or simply obtain the pure culture from any fungus culture collection center near your place.

  • Matthias Brock added an answer:
    How do you extract intracellular siderophores from the mycelium in Aspergillus fumigatus?

    I have the freeze-dried mycelium of Aspergillus fumigatus - what are the next steps?

    Matthias Brock · Friedrich-Schiller University Jena and Leibniz Institute for Natural Product Research and Infection Biology

    You could have directly used the frozen mycelium that can be ground to a fine powder under liquid nitrogen. However, the procedure should also work with lyophilised samples that also need to be ground to a fine powder to disrupt the cells. Subsequently, follow the protocol described here:

    Oberegger et al., Mol Microbiol. 2001 Sep;41(5):1077-89; PMID: 11555288

    Standards should be used to verify chromatographic results.

  • Sara Tomiolo added an answer:
    Is there a fungicide similar to Benomyl that is not illegal or out of production in Germany?

    I am planning to conduct experiments on the effect of mycorrhiza on desert plants and I don't seem to find a single fungicide that is not illegal or out of production in Germany. I tried with Benomyl, Benlate and Carbendazim. All of them are unavailable.

    Do you have any suggestions?

    Thanks!

    Sara Tomiolo · University of Tuebingen

    Thanks a lot Beatriz! Your suggestion was very useful, and I just found it in Germany. :)

  • Luis Monteagudo added an answer:
    What should the primer concentration be in a "multiplex PCR"?

    I am using a mixture of 2 forward primers and 4 reverse primers in a single pcr-mix for the detection of Arbuscular Mycorrhiza Fungi and I was wondering in what concentration should I use them. The reference article I am using is Kruger et al. (2009) where they describe the mixture of 2-4 primers respectively as "one primer" and the concentration they use of this "each primer" is 0.5 uM. I doubt whether the concentration of the 2 or 4 primers together would be 0.5 uM or each single primer's concentration should be 0.5 uM.

    Luis Monteagudo · University of Zaragoza

    When preparing multiplex PCR reactions you can be obliged to test different proportions of the different couples of primers. Besides the final concentration, the relation among different couples must be tested, starting by 1:1 and followig by 1.5:1 then 2:1 and even 3:1. The higher the number of PCR reactions you are multiplexing, the more important the proportion testing. Good luck!

  • Christian Q. Scheckhuber asked a question:
    Is anyone familiar with the use of CMAC-based vacuole markers in filamentous fungi?

    I recently tested the components of the "Yeast vacuole staining kit" from Life technologies for staining vacuoles in Penicillium chrysogenum. Carboxy-DCFDA and MDY-64 (both use the GFP filter) gave acceptable results. However, none of the CMAC-based dyes (CellTracker Blue CMAC, CMAC-Arg, CMAC-Ala-Pro) worked... These would be especially useful to me as I would prefer to use the DAPI filter installed in my microscope.

    Does anybody have some experience for using these vacuole markers in filamentous fungi? Help would be greatly appreciated!

  • Shraddha A Sane added an answer:
    Why does high quality rock phosphate solubilization by fungi differ much in shaking and static condition?

    I have compared different types of rock phosphates for their solubilisation using a fungal strain in both static and shaking (150rpm) conditions (rest all growth parameters are identical for both sets). Most of the rock phosphates of poor grade exhibit almost comparable solubilisation in both conditions except the purest one. For this high quality RP, P solubilises is 4 times less in static than shaking. Growth was good in both the flasks. Please suggest some possible reason.

    Shraddha A Sane · Gujarat State Fertilizers and Chemicals (GSFC)

    Thanks for response.

    All the growth conditions were identical. My point is does the impurities of rock phosphate has anything to do with its solubilisation. As the low grade RP shows almost equal solubilisation in both the conditions. But the purest form show almost four times more solubilisation in shaking than static. I agree that oxygen is the driving force but then why the  impure RP are not showing such difference.

  • Eman H F Abd El-Zaher added an answer:
    How do we prevent condensation from happening on the inner top of a petri dish?

    Currently, I am culturing fungus in petri dishes. Frequently, condensation of water occurred on the inner top of the petri dish. Although the water droplets didn't fall down it still blocks the view. Does anyone know why and how to prevent this?

    Eman H F Abd El-Zaher · Tanta University

    your culture temperature must be between (40-45°)  C not more  when you pour it in petri dishes .

    Best wishes

  • Jing Zhang added an answer:
    Why did the primers AML1/AML2 and NS31/AML2 amplify so many Dikarya Ascomycota?

    We tried to amplify  AM fungal DNA from root or soil samples from Tibetan Plateau with the primer combination AML1/AML2 or NS31/AML2. But the result is not good. There are so many sequences belonging to Dikarya ,Ascomycota. Only 20% was AM fungi. It was like winning the lottery. I do not know whether you had similar results. How to solve it ?

    Jing Zhang · Chinese Academy of Sciences

    Thanks for your answer. We know these mixed-primers but we have not tried for various consideration, for example the cost. And we are also not sure now whether the mixed-primers can weaken the probability of non-AMF,especially for samples from Tibetan Plateau.Do you have any related research?

  • Jai Ghosh added an answer:
    How can i purify a fungal metabolite from lipids during liquid-liquid extraction using methylene chloride?

    During extraction of fungal secondary metabolites using methylene chloride from culture filtrate, the methylene chloride layer forms lipids and fats that contaminate the extraction process. I need to know how to purify the filtrate from these lipids prior to extraction. I have already used n-hexane as a defatting agent three times but I still found the fats.

    Jai Ghosh · Shivaji University, Kolhapur

    First of all try to identify the lipids using MS. Once you know the lipids, try to identify the mixture of solvents (one of which has to be methylene chloride) which will show you the difference of partition coefficient of those lipids. Also know the partition coefficients of the metabolites too and then by using logic select a combination of liquid which will suit you best. Alternatively, freeze the mixture of metabolites and lipids in methylene chloride and slowly thaw it so that the lipids separate out  leaving you with very little contamination. Lastly if you can get a hold of Preparative HPLC, nothing like it. good luck.

  • Dominique Liger added an answer:
    Is it possible to grow Pichia using McIlvaine buffer (phosphate/citrate)?

    Hi everybody.

    I want to try to grow Pichia at various pH in flask to see what will be the best for their growth AND my protein. I want to try range about 3.5-4.5 and 6.5-7.5, because my protein has pI 5.5. In the neutral-basic range we usually use K-phosphate buffer to buffer the culture, as we do not have control over the pH because it's only in flasks, but in the range 3.5-4.5 the phosphate does not buffer.

    I'd prefer to use the same buffer to prevent some effect due to different medium composition, so I was thinking about McIlvaine buffer. But the question is, whether the yeast won't eat the citrate as carbon source (I'll be growing it on MeOH).

    Or would you recommend another buffer to use?

    Dominique Liger · Université Paris-Sud 11

    Hi there,

    I think the best would be to use a non metabolisable weak acid with pK around 4 for buffering. But I don't have any to suggest right now...

  • Sambhaji Chavan added an answer:
    Can anybody tell me about haploidization ?

    I want to do protoplast fusion, for that I need to do haploidization.

    Can anybody tell me the protocol of fungal haploidization?

    Sambhaji Chavan · PRAJ Industries Ltd

    haploidization : after protoplast fusion fusants can be transfer on regenation media containing benomyl this is one of  the very common  method to do haploidization .

  • Ivan Schlembach added an answer:
    How do you produce high cellulase titers with T. reesei RutC30?

    It it widely reported that Trichoderma reesei RutC30 produces around 2 FPU/ml cellulase, when cultivated in shake flask and far more in stirred tank fermenters. I'm not able to reproduce this value using the classical Mandels and Weber Basal Medium. Best values I've reached so far is 0,7 FPU/ml in shake flasks.

    Does anyone have experience in cellulase fermentation and know how to reach the reported 2 FPU/ml values?

    Ivan Schlembach · RWTH Aachen University

    Hi,

    I´m not using any sugar as substrate, only mandels medium with avicel. Addition of peptone was clearly enhancing cellulase production.

    It is weird, in literature you can find anything you want. Some people say, peptone is good, other say its not. The one says avicel is better, an other one says alpha cellulose is better, the third one says PASC is the best substrate. I´ve found yeast extract to be clearly inhibiting cellulase production whereas there are publications, that clearly show its improving cellulases production.

    The only thing you can do as a researcher is trying everything yourselve.  There is nothing you can really rely on. Cellulase research is so prone to methodolocigal errors, only small changes in the filter paper assay, can have drastic effects on the results.

    Is there anyone out there, who personally has achieved high FPU titers in shake flasks? Can you please explain me the exact procedure for your filter paper assay?

  • Khaled Alkassem added an answer:
    is there any method to isolated and purified ganoderic acids from Ganoderma lucidum without HPLC ?
    I only have this file that ganoderic acids from Ganoderma lucidum isolated by HPLC.
    Khaled Alkassem · Martin Luther University of Halle-Wittenberg

    It is not possible to obtain pure ganoderic acids in good amounts without a final run of preparative HPLC (following column chromatography to exclude any other compounds)

  • Ulrike Jung added an answer:
    Is there any possibility for us to use RNA in fungus in-vitro experiments?

    Hello everyone,

    If dsRNA or siRNA is used in the solution by the in-vitro experiments, what should we do to save or protect the RNAs?

    What should we do when using in-vitro solutions which contain RNA?

    Ulrike Jung · City of Hope National Medical Center

    What do you mean with in vitro experiments? Test tube solution only? Transfected into the fungus cells?

    I would not add ribonuclease inhibitors if you transfect into cells, in the later case I would definitely look into chemical modifications of your RNAs. There is a huge literature spectrum out there if that is what you want to look into.

    In any case make sure you use barrier tips to prevent contamination from your pipettes and make sure never to move with hands/wrists over open tubes/bottles, don't sneeze or cough while something is open and don't talk with open tubes. The main contamination is through aerosol RNAse and in my experience you don't need RNase Zap or masks or anything if your air (avoid open windows, air condition vents etc) "space" is clean.

  • Marko Lõoke added an answer:
    What is the method to extract fungal DNA from infected human clotted blood from a gel separator tube?

    As most of the serum-gel separator tubes end-up in the biological hazardous waste bin after serum has been taken for serology test, I am currently experimenting the usefulness of the clotted blood from candidaemia samples (result known by serology test) that may contain Candida sp. cells and its DNA could be extracted and applied as a template in PCR. Please advice other methods than Steven et al. 2007 (attached) as this method to obtain human DNA, and not yeast DNA, which the cells are lesser and seems to stuck with gel separator and fibrin during fragmentation process. 

    Marko Lõoke · Massachusetts Institute of Technology

    HI,

    Could try this...

    http://www.ncbi.nlm.nih.gov/pubmed/21548894

    Marko

  • Suhas Ballal added an answer:
    Is anyone familiar with Methanol/Ethanol precipitation of a desired protein fraction from a small volume sample, such as 10 ml?

    I am in the process of concentrating a lectin from the crude extract of fungal mycelial mat. I want to approach this problem by organic solvent precipitation by sequential increase in % saturation.

    However, the volume of my sample is less ,approx 10 ml. Can somebody advise me how to approach this experiment? At each step of increase in % saturation, i will test the activity of the lectin.

    Suhas Ballal · Karnatak University, Dharwad

    Dear Ravi, 

    Thank you for the help.

  • Vartika Srivastava added an answer:
    Can any one explain the mechanism of nystatin action in blocking fungal growth?

    Can any one give me the reference about it?

    Vartika Srivastava · Netaji Subhas Institute of Technology

    Hello Wöstemeyer sir,  I am unable to access the paper you suggested. can you please send me the copy of the article

    "Jacques Bolard: Biochimica et Biophysica Acta 864 (1986) 257-304 257, "How do the polyene macroUde antibiotics affect the cellular membrane properties?""  

    Thanks

  • What are your sampling and DNA extraction methods for fungal communities?
    I have been trying to get a sample with a high concentration of fungal DNA. I tried using dry sterile swabs, swabs moistened with saline solution, sodium chloride and Tween 20 solution and water. However I wasn't able to get a concentration good enough for amplification (my kit calls for 5-20 ng/ul).
    I was hoping to get any advice from those who has already done that.
    Thank you!
    Mohammed saleem Ali-shtayeh · Biodiversity and Environmental Research Center

    You might like to see the attached file.

    Good luck

  • Which is the best Fungal DNA extraction Kit in your opinion?

    I need to extract DNA from spores from herbaria material. I've used the Phenol-Chloroform method with success, but I'm interested in a Kit.

    Eduardo Hernández-Navarro · Research Center for Food and Development A.C.

    Thank you so much, Dr. Baseia! I'll look for it.

  • Sangeetha Siva Sangu added an answer:
    What is the air humidity inside of a Petri dish?
    What is the air humidity inside of a Petri dish (94x16) with fresh PDA medium, inoculated with 0,5 ml of spore suspension, at 25 degrees Celsius, during a 32 hours incubation? It depends on the incubator humidity? I need saturated atmosphere for 32 hours. Thank you very much in advance!
    Sangeetha Siva Sangu · University Malaysia Sarawak

    thank you very much Mr. Vijay Kumar Sharma

  • Mansoor Hussain added an answer:
    What is the treatment condition for KT5720, PKA inhibitor for fibroblast cells?

    I would like to know the treatment condition for KT 5720 PKA inhibitor for fibroblast cells. Earlier i have used 5um concentration for 16 hours. 

    Mansoor Hussain · National Institute of Immunology

    thats informative Mr.Kaushik.. my only concern is the limitation of inhibitor that i have. i've used 5um concentration. i cant do time course expt as i mentioned above. But i think i should do that. And yeah i will also use the positive controls when i do my experiment next time. Thanks for your valuable suggestions!!! 

  • Helmut Brandl added an answer:
    What else can cause by-product formation in Aspergillus terreus fermentations besides low pH conditions?

    I am using a strain of Aspergillus terreus which mainly produces oxalic acid and a little of other organic acids, but it doesn't produce much of itaconic acid which is the metabolite of interest, even at pH 2. Considering the fact that low pH is one factor that minimizes undesirable by-product formation, what else can i do to ensure the strain produces more itaconic acid. The medium contains sufficient amounts of glucose and N-source

    Helmut Brandl · University of Zurich

    Perhaps this publication helps: Hevekert et al. (2014). Applied Microbiology and Biotechnology 98:6983.  I do not have experience with A. terreus, but with A. niger regarding the formation of citrate and gluconate (see Bosshard et al. (1996) Environmental Science and Technology 30:3066). In the absence of manganese citrate is formed, whereas in its presence gluconate is formed. Perhaps there is a similar regulation in A. terreus.

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