Fungal Biotechnology

Fungal Biotechnology

  • Aadarsh Madhu asked a question:
    How to check the fungal spore germination?

    Currently I am working on the penicillium fungus strain and facing problem in spore germination. When I inoculate the production medium with spores 1X107 spores/ml, I did not get the desired concentration of cellulase protein but when I inoculated  by  disk of 1 cm diameter from plate I got the desired concentration of protein. I want to check the viability and germination of spores. Is it possible to see the germinating spores in microscope? If yes, Please do let me know. Please let me know any other relevant information regarding this. I am enclosing SDS gel pic for your convenience.

  • Olívio Fernandes Galão added an answer:
    What cell disruption method is recommended for freeze-dried fungal mycelium for the subsequent solvent extraction and fatty acid analysis by GC-FID?

    I have to determine total fatty acid content (mg/g dry biomass) and fatty acid composition from oleaginous filamentous fungi samples. 

    I have several freeze-dried fungal mycelium sample (low amount: 2-3 extraction possible with 50mg/extraction). I have tried so far grinding in morter with the addition of solvent. I did for at least 1 minute until complete homogenization, but still after extraction and transesterification (direct transesterification method by Lewis) I saw some intact hypha and lipid bodies inside the hypha. I'm working with Mucor, Mortierella and Penicillium at the moment. Does bed beating work without the addition of water? I need dry biomass for the extraction..

    Olívio Fernandes Galão

    Extraction of Lipids in Solution by the Method of Bligh & Dyer
    (Bligh,E.G. and Dyer,W.J. 1959. A rapid method for total lipid extraction and purification.
    Can.J.Biochem.Physiol. 37:911-917.)

  • Sufi Hanna added an answer:
    How long can a sample for mycology (infected plant) prior to screening be stored under 4 degree Celsius?

    I took sample from infected stem of tropical tree in the forest. I read from some sources that screening should be done as soon as possible to get accurate result. It is optional to store sample under 4 degree but the duration limit of fungi viability or potential of contamination is not stated.Is there any other way to lengthen the viability of fungi in the infected sampel?

    Sufi Hanna

    Dear D.M Hunupolagama,

    Thank you for your reply. I will try your method.

  • Alexander Vlassov added an answer:
    Fungal DNA extraction kit
    Does anyone have good recommendations for kits for fungal DNA extraction from solid medium? I have many colonies to identify.
    Alexander Vlassov

    Try PureLink Microbiome DNA Purification Kit –

  • Tim Alexander Dahlmann added an answer:
    How can I purify fungal isolate from unknown contaminant?

    I have a culture seems to be Alternaria alternata . Culture is looking pure having single type but very very less spores. In sequencing showing mixed peaks, what reason could be if it is contaminated how can i purify it?  

    Tim Alexander Dahlmann

    I'm glad to hear that you solved your problem. Good luck with your studies.

  • Damion Neath 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?

    Damion Neath

    Thank you all for your responses. 

  • Jegan Sekar added an answer:
    Any advice on the microbes (apart from human pathogen) involved in rotting of pulp and paper?

    name of the species responsible for rotting of pulp and paper specially bacteria and fungi.

    Jegan Sekar

    Fungal community will be best source, it can produce laccase, Mnp and Lip which degrade the complex material easily. In bacteria Clostridium, pseudomonas and bacillus

  • Agu K C added an answer:
    Does anyone know the protocol for investigation of fungal induced deterioration of mineral pigments?

    I wish to test the ability of wall painting fungal isolates to degrade mineral pigments (eg. Red Ochre, Green Earth...) often used during the painting of murals, but I am having problem finding the suitable method.

    Any idea or protocol on how to prove that degradation took place, or even better quantify the amount of degraded pigment, would be really helpful.

    Thank you in advance.

    Agu K C

    The vapour-Phase transfer method of Okpokwasili et al., will be helpful with subsequent mathematical models of Freudlich, Lagmuir etc

  • Tim Jenkins added an answer:
    Does anyone know what is the name of the Enzyme involved in Alcoholic sugar biosynthesis in Candida albicans?

    Candida albicans groups Does anyone know what is the name of the Enzyme involved in Alcoholic sugar biosynthesis in Candida albicans?

    Tim Jenkins

    Sugar Alcohol Biosynthesis: Potential enzymes can include xylose reductase for xylitol synthesis and aldose reductase for L-arabitol synthesis - see attached link. Mannose-6-phosphate isomerase has been reported in Candida albicans (this could be followed by mannose reductase as identified in high mannitol producing Candida magnoliae HH-01 but not sure if it is present in C. albicans).

  • 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

    Generally, yes. But you will need amplify or to re-amplifiy the sliced band using DGGE primers i.e. one of them should have GC-clamp.  For  100bp you should use 8% acrylamide gel. For first time use high denaturant range, 30-70% and according to bands position you can use narow range in next run.

  • Ephrem John Ora Agorob 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.

    Ephrem John Ora Agorob

    Thank You sir C.P. Chin-pao Huang and David N Ogbonna.

  • Prithvi Shirahatti added an answer:
    How to prepare the spore suspension of Magnaporthe oryzae?

    Instead of the spore spread all over, I see lumps while using the haemocytometer for spore counting. 

    Prithvi Shirahatti

    The simplest way is to overlay tween 20/tween 80 to the culture and tease the ooze a bit with the needle and then collect the liquid using a pipette. Once you have this suspension, you can filter it to remove the impurities and then you are good to set your spore concentration.

  • Heba Shawky Mohamed added an answer:
    I want to know how to calculate the diameter of fungal spores and the length of the hyphae through scanning electron microscope?

    I make analysis using scanning electron microscope on fungal spores and I want to measure their diameters.

    Heba Shawky Mohamed

    I install it but can not use it

  • Virgil Tudor added an answer:
    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. 

    Virgil Tudor

    Hi Woo-Sik Jo,

    Thank you for your troubles.

  • Oadi Matny added an answer:
    How do I extract spore of Rhizoctonia solani?

    I want to extract spore of Rhizoctonia solani, so please anyone suggest me how can i extract the same and is their any specific stain for spore staining...?


    Oadi Matny

    you can grow Rhizoctonia on millet sorghum seed after soaking in water for 3-6 h after that pour out the water and sterilized in autoclave. after the seed be could inoculated with 3-4 disc of Rhizoctonia and incubated in 25 C for 10 day with shaking every 3 days. You can use this seed as inoculation to apply the soil with the fungus, 

  • Hero M. Ismael added an answer:
    How to exract fungul DNA from grains?

    I am trying to exract DNA of fungi out of grains. How do I extract DNA of fungi which are on a plant, grains and silage, since the DNA is dameged from enzymes of the host? How can I exract the DNA without damage?


    Hero M. Ismael

    as mentioned above CTAB is the best, also you can use kits as bioneer kit or GenAid kit

  • Raoni Gwinner added an answer:
    Can anyone recommend methods to quantify the oxalic acid production in Botrytis cinerea isolates?

    What is the best way to directly quantify oxalic acid production in fungi?

    Does anyone suggest an appropriate protocol to incubate the fungi before the quantification ?

    Thank you so far

    Raoni Gwinner

    Thanks Robin, I found a paper where they optimize the Sclerotinia protocol to Botrytis.

    I really appreciate your help

  • Arun Babu Vathsalan added an answer:
    What is the best method for the biosorption through freeze-dried (physical treatment) of fungal biomass?

    Actually I want to know that what is the procedure for freeze-dried pretreatment of fungal biomass using biosorption.

    Arun Babu Vathsalan

    Yes, you can use liquid nitrogen for drying your sample. But different drying methods may have different effect on the biosorption process.

  • Sandip Chakraborty added an answer:
    What is the best fungi to grow and harvest cellobiose dehydrogenase from?

    Looking for a easy to grow fungus that I can purify the enzyme cellobiose dehydrogenase from. 

    Sandip Chakraborty

    Fungi belonging to the genus Cladosporium can act as important source of cellobiose dehydrogenase.  White rot fungus Termitomyces sp. OE147 can also be used for this purpose. You can further refer to the following links to get certain valuable details:-



  • A. K. Vijayan added an answer:
    How can I increase fungal growth in PDB medium?

    I am doing mass culture of fungus but i am getting very less amount like dry weight of fungus is 0.50-1.60 gm only because of this am getting very less amount of extract.

    A. K. Vijayan

     Both PDA broth  in a shaker and   stationary condition for culturing  various  specimens  of fungi   vary under  a particular condition. Fungal mat yield / spore load  also varies under a particular condition and also depending on the  fungal strain .  Metabolite content  varies depending on the duration of growth, media type and groth conditions etc.  More  extracts of metabolites up to 10-15 days duration in a  liguid culture under a particular condition.

  • Kenneth Jones 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.

    Kenneth Jones

    An account of the first successful culture of Hirsutella sinensis, the true anamorph of Ophiocordyceps sinensis, was related to me in confidence many years ago. What I can reveal today is that the first attempts to culture the anamorph after transporting the fungus from its natural habitat located at high altitude to the laboratory at sea level resulted in failure. A second attempt using a portable refrigeration unit was also a failure. Only when both the temperature and atmospheric pressure of the transportation unit matched those of the area in which the fungus was harvested were researchers able to successfully isolate and culture H. sinensis.

  • Maria V. Kozlova 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?

    Maria V. Kozlova

    I think for Magnaporthe  it will be ok

  • 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

    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.

  • 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

    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

    Hello Arman!

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

    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

  • 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

    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.

    • [Show abstract] [Hide abstract]
      ABSTRACT: Filamentous fungi are important organisms industrially and continue to attract research interest as microbiologists attempt to overcome the problems associated with their behavior in submerged culture. This review critically examines the literature describing these problems and where available suggests possible solutions to them. The influence of the chemical and physical environment on culture morphology, the process engineering challenges presented by different fungal morphologies, and the relationship between fungal morphology and metabolite production are all discussed.
      Critical Reviews in Biotechnology 02/2000; 20(1):17-48. DOI:10.1080/07388550091144177
  • 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

    thanks a lot Nirmal Sahay Sir.

  • 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

    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

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

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