Fungal Biotechnology

Fungal Biotechnology

  • Rainer W Bussmann added an answer:
    using herbarium material for extraction?

    I would like to know how old can the fungal fruiting body be for detection and screening of antioxidants. Has anyone tried this with herbarium material? Does the preservation method affect the contents?

    Rainer W Bussmann · Missouri Botanical Garden

    It really depends in the preservation method. In vascular plants and pteridophytes many samples are collected and preserved in ethanol before drying (we do that mostly in tropical regions). That greatly alters the chemical composition. Drying of course (if too hot or too slow) will also have an effect. In case of fungi that are air-dried or lyophilized I would think the effect will be minimal.

  • David N Ogbonna added an answer:
    Good Day! What is the ratio for the amount of fungi to be included in the waste-water to the BOD/COD of the untreated waste-water?

    Good Day! I'm a chemical engineering student and I am doing my final year research on waste-water treatment using fungal strain (A. oryzeae). I just want to ask what is the ratio for the fungi to be placed in the waste-water (COB*:fungi or BOD*:fungi)

    *data fron the untreated waste-water to be used in the study.

    David N Ogbonna · Rivers State University of Science and Technology

    refer You to the article on: wastewater treatment with microalgae by Karin Larsdotter, Vatten 62: 31-38,. Lund 2006

  • Sara Salcedo added an answer:
    Is there a specific protocol for disrupting fungal tissue using the TissueLyser?

    Has anyone developed a specific protocol for the disruption of fungal tissue using a TissueLyser, or is a protocol for plant tissue described in the TissueLyser handbook fully applicable?

    Sara Salcedo · Universidade Federal de Viçosa (UFV)

    Monosporic colonies grown for 4 days in liquid medium BD (potato dextrose), dried overnight in sterile filter paper.

    place a small amount of dry mycelium 2ml microtube with two spheres of 6 mm in the TissueLyser II - QIAGEN for 2 minutes at 30 Hz, repeat it takes more disruption (you can work with up to 48 samples).

    Then we use the modified protocol :Wizard® DNA Purification System, PromegaTM

    Lyse Cells

     1. Add 600µl Nuclei Lysis Solution.

    2. Incubate for 15 minutes at 65°C. Vortex repeated every 5 min.

    Protein Precipitation

    3. Add 200µl of Protein Precipitation Solution. Vortex.

    4. Centrifuge at 14,000 RPM for 10 minutes.

    5. Transfer the supernatant to a clean tube containing 600µl of chloroform:isoamyl alcohol (24:1, Sigma-Aldrich®) Vortex  20s.

    6. Centrifuge at 14,000 RPM for 10 minutes.

    DNA Precipitation and Rehydration

    7. Transfer the supernatant to a clean tube containing 600µl of containing room temperature isopropanol. Gently mix by inversion and place in the freezer for 15 min.

    8. Centrifuge at 14,000 RPM for 10 minutes and discard the supernatant carefully for the pellet.

    9. Add 600µl of room temperature 70% ethanol. Repeat twice.

     10. Centrifuge for 5 minutes at 14,000 RPM.

    11. Aspirate the ethanol and air-dry the pellet for 10–15 minutes.

     12. Rehydrate the DNA pellet in 50µl of Rehydration Solution for 1 hour at 65°C or overnight at 4°C.

  • Christian Q. Scheckhuber added an answer:
    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!

    Christian Q. Scheckhuber · Senckenberg Research Institute

    Hello Arman!

    There is a nice overview on OD 660 nm values and cell numbers here:

    http://www.pangloss.com/seidel/Protocols/ODvsCells.html

    Please keep in mind that this table shows data for S. cerevisiae. But I think it should serve well as a template for H. polymorpha cells. 

    In the attachment you will find the manual for this kit. You will need 10^6 cells/mL so a 1/10 dilution of cells at OD 0.680 (corresponding to 1*10^7 cells/mL) should work fine. Hint: use 50 mM sodium citrate buffer, pH 5, for the dilution.

    Success with your experiments!

    Best regards, Christian

  • Vasileios Bitas added an answer:
    How can I cryo-preserve my magnaporthe spore solution in -80 without making fresh spore suspension each time?

    I want to preserve Magnaporthe spores in a stock solution (e.g. skim milk-Glycerol, or trehalose  ). Could someone give me some suggestion?

    Vasileios Bitas · Pennsylvania State University

    You collect the spores by filtering the culture suspension with a cheese cloth and centrifuge them. Put them in a 20% glycerol in water solution and store them at -80C.
    In addition you can modify the spore suspension concentration by using a hemocymeter so you can have spore suspension of a known concentration.

  • Xabier Vázquez Campos added an answer:
    What does the growth morphology of a fungus in a shaken liquid medium say about the medium?

    I want to achieve maximum cell biomass over 72 h in a shaken flask inoculated with a fungus. Some grow really well but others don't particularly Curvularia lunata and Rhizopus Stolonifer. Are there any studies dealing primarily with fungal growth in shaken Liquid media?

    Xabier Vázquez Campos · University of New South Wales

    Sometimes even different isolates of the same species have different growth rates.

    In addition, pO2 and shearing, which will be partially determined for the shaking speed and many other paramenters, including the age and development stage of the inoculum will affect to the growth.

    Take a look to the linked paper.

  • Virgil Tudor asked a question:
    How to extract ahcc from shiitake mushroom?

    I am trying to find some information on how the extraction of Active Hexose Correlated Compound (AHCC) from Shiitake mushroom can be done. I can't seem to find anything online. 

    Thank you for the help. 

  • Beng Fye Lau added an answer:
    Is it wrong if I extracted mycelium fungi using ethyl acetate?

    I am doing my research about secondary metabolite from Sponge Associated Fungi. I have cultivated my fungi in broth medium, then I separated the mycelium from the media. Then, I extracted the mycelium using Ethy Acetat after disrupted it. Someone said for me, it is uncommon for the extraction.
    Thank you.

  • Saurabh Guleri added an answer:
    How can I perform pathogenicity test for fungi using petri plate method ?

    i am working on pathogenic diseases of some medicinal plants and need to carry out pathogenicity test using petri plate method.

    Saurabh Guleri · Hemwati Nandan Bahuguna Garhwal University

    thanks a lot Nirmal Sahay Sir.

  • Michael Seegers added an answer:
    Where can I get spores of Cordyceps sinensis and C. militaris?

    Can anyone suggest me how to buy the fresh spores of Cordyceps sinensis and C. militaris fungi?Or please let me know any lab which is working with these fungi?

    I am very interested in these species, since they are high value for medicine.

    Thank you in advanced.

    Michael Seegers

    Taken from their site is this quote,

    "For all of you formulators - Aloha Medicinals also offers bulk powder ingredients for use in your formulations, which are 100% USDA and EU Certified Organic. Call our customer service representative at (775) 886-6300 for more information and to place your order today!"

    Might be worth a phone call.

  • Barry Preuett added an answer:
    How long are fungal spore cultures stable in physiological salt solution stored in a fridge (+4C)?

    How long are fungal spore cultures stable in physiological salt solution stored in a fridge (+4C)?

    Barry Preuett · Children's Mercy Hospitals and Clinics

    As Johannes has mentioned, it depends on the species.  My particular spore suspensions are quite happy even after 6 months in sterile saline stored at 4C.  Sure, there is some viability loss, but it's never been an issue to sub-culture from.  

    If you store your solid media preparations at 4C, sealing them with a gas permeable shrink seal helps slow down the evaporation from the media (for plates that is).  

    I've also had great success with Christine's method of filter paper preservation.  Works great by just placing the disk saturated with spores onto a plate, and then you can sub-culture from there once it grows out.  

  • Marwan Al-Maqtoofi added an answer:
    Could anyone suggest why I found no bands in my gel although I extracted DNA with high purity (as shown by nanodrop)?

    I did extract by CTAB and I tried phenol. DNA from fungi.

    Marwan Al-Maqtoofi · University of Exeter

    Thanks for all who tried to help me figure out this case.

  • Muinat Olanike Kazeem added an answer:
    How can we calculate CFU/ g- 1 (Colony forming unit) of tissue burden candidasis for experimental study in mice?

    This is method use for this study:
    Lungs, brains, spleens, livers and kidneys were aseptically removed and approximately half of each organ was weighed and homogenized in 1 ml of sterile saline. Serial 10-fold dilutions of the homogenates were inoculated on SDA, incubated at 35 C and examined daily for 3 days. The number of CFU/g-1 of tissue was calculated for each sample.
    May I help me to answer these questions?
    1. Is it necessary we weighed organ?
    2. What is exactly meaning of Serial 10-fold dilutions?
    3. How can we calculate CFU? Any protocol or methods for this?

    Muinat Olanike Kazeem · University of Ilorin

    All your comments were absolutely right. Thanks

  • Maria V. Kozlova added an answer:
    How do I homogenise fungal mycelium from plates or liquid cultures to make an inoculum?

    Often in literature it is only mentioned that fungal cultures are homogenized but rarely it mentions the  time, type of homogenizer etc. There surely must be optimal choices as some bad choices as different homogenizers have different rpm power, blades can be of different quality and can be blunt of sharp. How long to homogenize to get best  propagules (mucelial fragments, blastospores, other spores..)  and the least of dead shattered mycelial fragments. I am working with basidiomycetes (wood decay fungi) . Any suggestions and personal experimences?

    Maria V. Kozlova · State Oceanographic Institute

    Dear Adnan,

    I lost your question and found it just now....so I can answer.

    For homogenization we used a handle device similar to that on attached photo.

    My colleagues and I obtained good results with such homogenizer, although in your case an automatic blender is better I guess. Nevertheless, the handle blender might be useful for some purposes... 

  • Ramona Pezzotta added an answer:
    Is the E-test on SAB agar for the Scedosporium apiospermum reliable?

    I had to perform a MIC test on Scedosporium apiospermum last week (isolated from a BAL). Unfortunatly I couldn't use the specific and required media I was suppose to (potato's agar to select the conidia and R.P.M.I. for the E-test). I was in a sort of rush and then I decided to perform the E-test on Sabourad agar. I know that it's not correct, but I need to know if the results I obtained are reliable or not. Can I use them as an indication for the therapy?

    I tested Voriconazole (which looks Sensible) Itraconazole (which has a strange MIC of 0.5 but I think is dose-related) Ketoconazole (sensible as VOR) and Caspofungin (that's surely resistant).

    Any suggestion? Can I confirm to the clinical doctor this results?

    (He already started a VOR therapy, and the patient seems to feel, after few days, better. But the doctor wants a confirm anyway).

    Ramona Pezzotta · Spedali Civili di Brescia

    I definitely will .

    Thank you.

  • Heba Shawky Mohamed added an answer:
    What is the best way of starting to look for commercially important metabolites in a newly characterized fungus?

    1. We can not order standards for all important metabolites.

    2. What type of Bioassays should be conducted to know if the fungi are producing any important metabolites?

    3. From Chemical or molecular approach, which one should be selected first? As both have their advantages and disadvantages. 

    Heba Shawky Mohamed · Egyptian Atomic Energy Authority

    I suggest to start with the chemical characterization firstly to know the major metabolites produced by this fungus. This is carried out by cultivating the fungus in a liguid rich medium as PDB or YSB media,then extract using different solvent systems to obtain the crude extract using rotary evaporator. this extravt can be then characterized through different techniques of chromatography such as column chromatography or GC/MS for recognition of these compounds. this can help you for chracterization.

  • Miss Biology added an answer:
    How can I read the OD from minimal salt solution plus with oil and fungus, for biodegradation of petroleum by using studies of isolated fungi?

    The 10ml minimal salt solution + 2ml of petrol(oil) and selected fungi was put in the test tube and being incubated in the incubator shaker(120rpm) for 20 days with 28 degree celsius, and the reading of Optical Density will be taken in 5 days interval at wavelength 540nm..in order to know the degradation rate of oil using fungi. Is this the best way to study the biodegradation rate of oil using isolated fungi?

    Miss Biology · Universiti Teknologi MARA

    Thank  you very much Eleni Xenofontos for answering my questions with the useful suggestions. I really appreciate it.

  • Dyoni Matias de Oliveira added an answer:
    Can fungi utilize the degradative products of lignin as their carbon sources?

    Or it degrades lignin to reach cellulose or hemicellulose inside?

    Dyoni Matias de Oliveira · Universidade Estadual de Maringá

    Yes, lignin degradation products can be used as a carbon source for ligninolytic fungi.

  • Kuldeep Patel added an answer:
    What techniques are available to study the viability of arbuscular mycorrhizal spores?

    I am using Tetrazolium (INT) as a viability stain for AMF spores but I am finding some inconsistencies in the results I obtain. Any other technique that I could use to contrast my results with INT? Thanks for your answers.

    Kuldeep Patel · BACL

    We are using MTT staining of mycorrhiza spores and IP by root staining

  • Andrej Gregori added an answer:
    Is anyone willing to share Monascus purpureus cultures?

    We are conducting cultivation of Monascus purpureus on solid media in order to reduce citrinin and increase monakolin production. We are having quite some problems finding M. purpureus cultures. Is anyone willing to share/exchange? We have many basidiomicetes (especially lignivorous) - Ganoderma, Lentinula, Pleurotus, Grifola, Trametes etc. in our collection which we can offer to you.

    Andrej Gregori · Zavod za Naravoslovje

    Dear Ravi,

    Thank you for your reply. Is TISTR 3541 the strain you are willing to share?

  • Hani Antoun added an answer:
    Can I use PDA medium with ampicillin to isolate endophytic fungi from macerated roots?

    Sterile-surface roots were macerated. From this material I isolated dozens of endophytic bacteria. Now I want to take this material to isolate endophytic fungi. I plan to use PDA medium with the only antibiotic that I have, ampicillin. Would it be okay supplement the PDA with ampicillin for this purpose?

  • Afreen Haider added an answer:
    How can I get the ribosomes in a total nucleic acid extraction?

    I need to do total nucleic extraction from fungi out of clinical samples. At the moment we are using bead beating followed by a total nucleic extraction protocol on the Roche MagNA pure compact. Quantification of spores going into the extraction (can't really quantify mycelia but have tried those too) and subsequent PCR (with a ribosomal target) suggests we are getting a good yield of DNA from this procedure. However, when we do reverse transcription PCR to pick up the RNA in the ribosomes as a target too. We are not seeing much more of a PCR product. Is there a way to ensure ribosomal RNA gets extracted in total nucleic extraction protocol.?

    Afreen Haider · Central Drug Research Institute

    You should first check the integrity of your RNA i.e. on agarose gel you should obtain intact bands for rRNA (3 prominent bands where the top two should be more intense than the lower one). If you see smears in the gel then your RNA is probably degradng hence you are not getting RT-PCR. But If that is fine and you are also using the same primer and buffers for both your normal DNA PCR and RT-PCR, then the only thing is that the RT step is not working. Are you incorporating a 65 degrees melting step before you incubate with RT? this removes secondary structures that can inhibit your RT reaction.

  • Ulrike Martens added an answer:
    Do you know any recipe for cellulose agar media?

    I made up cellulose agar media to grow fungi but the only growth was bacteria, some gram negs. I thought the salts would inhibit the bacteria and select for fungi that broke down cellulose

    Cellulose  (cmc) 5.0g

    NaNO3 1.0g

    K2HPO4 1.8g

    MgSO4 7H2O 0.9g

    KCl 0.5g

    Agar 20g

    dH2O 1000ml

    Any suggestions on tweaks to be made?

    Ulrike Martens · University of Greifswald

    I also used the described method. To test the cellulolytic activity add CMC in a concentration of 0,1 %. After incubation period cover the plates with aqueous solution of Congo Red (1 mg/ml) for 15 minutes. Then float it off with a solution of NaCl (1 M). Cleared zones indicate a cellulolytic activity.

  • Emmanuel Manirafasha added an answer:
    Anyone know where I can conduct the supercritical fluid extraction in Saskatoon or in Saskatchewan?

    I am trying to extract Fusarium mycotoxins from some grain samples using supercritical fluid extraction. Does anyone know any institution/facility have the capacity or experience for this process? I am a graduate student in University of Saskatchewan. If the facility close to Saskatoon or within Saskatchewan will be prefect. Any place in Canada will be great

    Emmanuel Manirafasha · Xiamen University

    I know one Institution in China, College of Chemistry and Chemical Engineering, Department of Chemical and Biochemical Engineering, Xiamen University 

  • Einar Martinez de la Parte added an answer:
    Can Hemileia vastatrix grow on agar?

    I want to know if Hamileia vastatrix can grow in the lab on artificial media. And if yes what type of agar?

    Einar Martinez de la Parte · Instituto de Investigaciones de Sanidad Vegeta

    I agree with D. Andrivon, most of the people that works with H. vastatrix maintain the pathogen on detached coffeee leaves. Despite the article of Bakala et al. (1975), recent works with this rust do not used culture media for maintain H. vastatrix inoculum in the lab.

    best regards

    Einar

  • Shivakumar Banakar added an answer:
    How can I purify and characterize the extracted natural pigments from the fungi?

    I am a Postdoctoral Fellow and having a few difficulties in purifying the natural fungal pigments.

    I extracted pigments (colors) in liquid medium (Smf),  and came to know that they are soluble in water. I tried a few solvents, but they are insoluble.

    I want to know the purity of the natural compound, either one compound or multiple compounds and I want to characterise all.

    Please help me regarding this.

    Shivakumar Banakar · North West University South Africa

    Thank you for your reply Prashanta Kumar pal.

    Can you mention any of method to try for the suitability of the solvents.

    Initial samples were sent for the PREP HPLC, but they were told that tailing was observed after the running, because I do not have the facility so.

  • Hideo Toyama added an answer:
    Do you know The Mega-mushroom?

    Are there someone who study about the Mega-mushroom?

    I have a interest in the lignin and cellulose degrading ability of this fungus.

    I hope to exchange informations about this fungus.

    Biofuel is quite easy for this fungus???

    45lb Mushroom found in Mexico

    http://www.supersizedmeals.com/food/article.php/20070715-Giant_Mexican_Mushroom/print

    Hideo Toyama · Minamikyushu University

    Dear Ricardo Valenzuela

    Thank you very much for your informations.

    I begin to study about this monster.

    Thank you very mucj again!

    Sincerely

    Hideo Toyama, Ph.D.

  • Phurpa Wangchuk added an answer:
    Is anyone working with the isolation and characterisation of molecules from worms and insect fungi?

    The sources of drug discoveries has been a synthetic chemical pools and the natural resources including microbes, proteins, plants and animals. The worms especially the intestinal helminths has been neglected and I am wondering if anyone has any experience in this area including isolation, characterisation and biological activies-protocols and findings would be appreciated?

    Phurpa Wangchuk · James Cook University/University of Wollongong, Australia

    Thank you all for your comments. Prof Russell, how have you been doing? I saw one of your paper on insect fungi. I have made a request and would appreciate if you could also give a link to related works of yours.  Thank you.

  • Juraj Medo added an answer:
    How can I design a primer with GC-clamp attached to ITS ?

    I have read the articel that is suggested by Kuldeep Patel.Thank you so much.

    I carried out DGGE to study microflora on 18S RNA using primer (NS1 and GC fung) based on Technical report from National Institute for Agro-Environmental Science japan (NIAES).

    Currently,I would like to identify spesies from DNA band of DGGE

    I would like to use ITS1 and ITS4 for DGGE to study microflora of fungi, I would like to ask how to attach GC clamp on the primer, and how long  the GC sequence?

    Anybody could help me please?

    Thank you

    Juraj Medo

    Beware on double bands, It is common if Muyzer`s GC clamp is attached to ITS primers. In my opinion IGS primers gave nicer gels

  • Hani Antoun added an answer:
    Is staining for mycorrhizae better with blue-black ink?

    I have been staining Celastrus orbiculatus roots and I have noticed that there are a variety of things that are being stained besides the mycorrhizae (hyphae, vesicles and arbuscules). They could either be dark septate endophtyes, microesclerotia, or the cell wall of the plant cells in the cortex being stained. I figured changing colors could help decipher the difference, but am reluctant to switch to blue ink because I have seen images of unclear samples of arbuscules when using blue ink. Does anyone have a particular preference or insight when using blue-black ink?

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