Fungal Biotechnology

Fungal Biotechnology

  • 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

  • Amirreza Amirmijani 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

    Amirreza Amirmijani · University of Jiroft, Kerman, Iran.

    Dear M. haider,

    Hope you are doing well,

    There are many differences between VAM fungi and Saprobs! As you know, VAM fungi are oblige and  produce Chlamydospore-like spores, but soprobs are not VAM. For saprobs, you can use from Atlas of soil Ascomycetes, This book has key, description and illostration.

    I think this can help you about some isolates of saprobs that you have.

    All the best,

    Amir

  • Shraddha A Sane asked a question:
    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.

  • 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?

  • Ricardo Delgado Santander 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!

    Ricardo Delgado Santander · University of Valencia

    I'm not sure this is helpful for you, but I'm substituting cycloheximide by Natamycin (also known as pimaricin), a very cheap and not toxic fungicide that is actually employed in food industry. You can buy it as Nataproq (50% Natamycin 50% glucose). I'm adding it to my culture media and it works perfectly. The only issue is that you need to dissolve it in DMSO prior to add it to culture media.   

  • 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.

  • Marwa Hosni asked a question:
    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?

  • 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.

  • Michaela Conrad added an answer:
    Does the accumulating CO2 levels become toxic to yeast during fermentation?

    What will happen to the yeasts if the vessel carrying out yeast fermentation is tightly sealed? Will the rising level of CO2 inside the vessel be toxic to the growing yeasts or the yeasts have a strategy to overcome this situation?

    Michaela Conrad · Vlaams Instituut voor Biotechnologie

    You will also need a very stable vessel otherwise it might just blow up. Not a direct problem for your yeast, but not nice for your bench neighbours ;)

  • Martua Manullang added an answer:
    How to prepare samples for fungal hypahe/spores for environmental scanning electron microscopy?
    For Alternaria alternata. Any suggestions?
    Martua Manullang · Universitas Riau

    How to prepare dry mycelium for SEM analysis ??
    I want to SEM (Scanning Electron Microscop ) analyzes on samples that had been dried mycelium with a freeze drier.. Thanks

  • Dhinakarasamy Inbakandan added an answer:
    Can someone explain the effects of nanoparticles on plant or fungi gene expression, please?

    Manganese peroxidase is a ligninolytic enzyme encoded by MnP genes in fungi. Expression of this gene is increased by Mn ions as substrate of MnP enzyme. Do manganese nanoparticles have any effect on mnp gene expression as well?

    Dhinakarasamy Inbakandan · Sathyabama University

    Dear Mojtaba Lotfi,

    Normally Metal Nanoparticles will have impact on oxidative stress genes, in particular metallothionein genes. In your case, hope Manganese peroxidase kinetics in the presence of manganese nanoparticles itself is a good work. Further you can confirm it in your fungal model with enzyme production followed by gene expression studies in the presence and absence of NP.

  • Ervín Jankura added an answer:
    How to control fusarium in microbiology QC lab?
    There are number of fungicide which can be used to control different type of fungi but when you test a particular product for presence of fungi you got to avoid such fungicide, what you will do in this situation if you are having problem of fusarium contamination in your media you used for yeast and mold analysis?
    Ervín Jankura · Plant Production Research Center Piešťany
    Formalin alternative:

    https://www.researchgate.net/publication/230182896_Evaluation_of_a_Peroxyacetic_Acid_Disinfectant_in_Hotwater_Treatment_for_the_Control_of_Basal_Rot_%28Fusarium_oxysporum_f._sp._narcissi%29_and_Stem_Nematode_%28Ditylenchus_dipsaci%29_in_Narcissus
  • Deepti Agrawal added an answer:
    How can I determine inulinase activity?
    I am working on inulinase production from fungal sources. I followed DNS method for inulinase assay. However, I have a problem. How can I calculate inulinase activity in Units per mL/min?
    Deepti Agrawal · Indian Institute of Petroleum (IIP)
    You are most welcome Aftab
  • Ali Vaiz Garipoglu asked a question:
    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?
  • Senthil Sankar added an answer:
    What is the best way to induce zoospores in a Phytophthora palmivora sample?
    I have a culture of P palmivore on V8 and agar oats, the culture is 10 days old, but the zoospores production is to low only 2 or 3 zoosporores. Could anyone help me with a better protocol?
    Senthil Sankar · Aix-Marseille Université
    V8 is used for zoospore production in wide Phytophtora species but i did not get enough zoospores of P. palmivora in V8 agar media. Even the host material can also be tried.
  • Andrea Lessing asked a question:
    Has a complete pathway for methoxypyrazine production in either plants or microorganisms specifically fungi been described?
    I have recently read a publication which describes the last enzyme in the pathway but I need information regarding enzymes which function in earlier steps.
  • Muhammad Faraz Bhatti added an answer:
    Does someone have a reliable protocol for transformation of Botrytis cinerea (or other fungi with melanised cell wall)?
    I have tried a couple of published protoplast/PEG mediated methods and tried a homebrew electroporation (similar to a method which has been very successful for Neurospora), none of which have been successful.
    I know that my protoplasting is working as the protoplasts look big and round and recover well before the antibiotic is added. I have a tried and tested positive control for insert DNA so it must be my transformation step that is not working.
    I heard from other Botrytis workers that transformation is tricky and needs optimizing, any hot tips would be appreciated.
    Muhammad Faraz Bhatti · National University of Science and Technology
    I have adapted this method of protoplast fusion / fungal transformation (Please see attached file). It worked really well for me. Please find the paper as an attachment. I hope that will help. Regards
  • Ayat AL-Laaeiby added an answer:
    What is the best protocol for transforming fungal cell?
    Is electroporation of conidia more efficient than peg mediated transformation of protoplasts? Does the protoplast suspension in peg plated on large sized petri plate have a layer of soft agar? How long does it take for regeneration of the protoplast?
    Ayat AL-Laaeiby · University of Exeter
    I want to knock out gene, I used protoplast it is succeed for one gene and I want to KO another gene with electroporation. Can someone share protocol for KO gene with electroporation technique?

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