Dear Adnan,In some cases it's possible overcome blending.If you grow your mycelium in shaking culture you may get better inoculum for some species. Some basidiomysetes can form arthrospores, or similar structures that can be good inoculum. If you work with such species you may collect the spores from liquid culture by filtering. When I worked with Agaricus mycelium I used so called  Waring blender, which can carefully homogenize the mycelium. I can type more detailed info within a week when I get the access to my documentation.Anyway, every species differ from each other. Exact optimal protocol may vary for different fungi.

When preparing an inoculum suspension of Pythium aphanidermatum for a drench, I used a small Waring blender. 

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I am also agreed with Maria. Liquid culture will be good. I worked with Penicillium so my experience is also that for homogenize culture liquid culture is good.

Dear Adnan,I hoped that you tell us about the fungus you want to prepare its inoculum. If the fungus has great ability to produce spores, then you have to be used solid culture. But if your fungus has not this ability, you can prepare your inoculum on sterilized sand amended with suitable medium. In case of non-sporulated Alternaria you can culture it in liquid culture and use homogenizer  at very low speed for about 2 min, and you have to examine this inoculum under light microscope. 

Dear Adnan,

It is important to know which substrate you want to inoculate. The use of plates with agar can be used as inoculum if you need to replicate the mycelia. In this case, you use plugs of agar. You can prepare liquid inoculum, using flask and a shaker. In this way, you will be able to obtain beads of agar with mycelia (the diameter of the beads depends on the rpm of the shaker). You can also prepare spawn, as it is used for mushroom cultivation. The spawn is usually produced on sterilized cereal grains or on sawdust.

Thank you Maria, Mike Ibatsan, Mostafa and Edgardo for your comments/suggestions. Here are some answers to questions. I am working with Dichomitus squalens and I want to inoculate it onto wood. I am already growing it on sterilized cereal grain (to check if the fungus if producing chlamydospores and if they can be detached and be used as inoculum). I am also growing it in shake cultures (125 rpm at 25C) and there  am seeing nice big beads of mycelia forming in the nutrient broth after 5 days of incubation. I will check tomorrow if there are arthrospores forming. The next stage will be either filtration (if there are plenty of arthrospores) or the homogenisation part to shatter mycelium into small propagules that can be used in a sprayer. Maria I would be very much interested to hear the details about your Waring blender specifics to gently homogenise the mycelium.  I will also check your paper Mike. In past  we used powerfull Dupont Sorvall Omni mixer for 10 sec using its 16000 RPM. Based on some of your comments that may be an overkill. Perhaps I should just buy one of food blenders  as they may be more gentle and where speed can be controlled?

Mostafa's method for non sporulation fungi on sterile sand sounds interesting  and it  would be nice to hear more details about it.

Dear Dr. Adnan Uzunovic

Thank you for your reply. Concerning the preparation of inoculum in sand I suggest the following protocol:

1- Chose fine sand,wash it in 0.1N HCl then in distilled water for several times( you have to check the pH). then leave it to dry.

2-Pour this washed sand in glass Petri dishes then wet this sand with appropriate liquid medium.

3- Strilize the plates by autoclaving at 121C for 15 min.

4- After cooling you can infest the plates by pieces of your inculum.

5- Incubate the plates at suitable Temp. for at least 10 days, then you will get very good inoculum.

With my best wishes.

Dear Adnan,

I also agree with Maria. As regards the preparation of inoculum of basidiomycetes little or non-sporulating, you can start from a liquid culture incubated in static conditions. Put mycelium and soil in the waring Blendor with sharp blades and use it at medium speed for progressive time (e. g. 1, 2, 3 minutes). For each time pick up few milliliters, do progressive dilution and inoculate 5 Petri dishes filled with a conducive medium for the fungus you're working with (I usually use 100 microliters for each Plate). The plates where the highest number of colonies will grow, will tell you which is the best time of blending. Of course you can repeat the process with different speeds, as well as with the different times.

Best wishesAdd your answer

Thanks Alessandra. Doing a pilot study on homogenisation for one's particular fungus certainly makes sense to avoid guessing game  and I am already starting a small pilot study.

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... 

Hi,I'm currently working with submerged shaked cultures (liquid medium of course)  of filamentous fungi and I do often need to homogenize them. I use a UltraTurrax device with stainless steel dispersing element ( so similar to a food blender) , it homogenizes mycelium but fungi don't die , I think is a easy , fast and ready to use system especially if you need to disperse extremly "gummy" mycelium growth on liquid... bye 

 Hi, I am interested in homogenize my fungal biomass but is there a way to tell if the mycelium is from the aerial hyphae or the submerged hyphae? 

Here is additional discussion on enumerating mycelial fragments that are relevant to the question on fragmenting mycelium for inoculum, that I also inlcude here. If someone else could shed more light/share thought on this issue that would be great.

Maizan Ismail: Good afternoon Dr. Adnan. I'm Maizan from Malaysia. I would like to ask you about the mycelia/mycelium concentration. How do you count a concentration of your inoculum using mycelium base? Lot of literature reviews teach how to do the inoculum concentration base on spore and the concentration will be like 1x10^7 conidial/ml. But I have no idea how to count the same concentration using mycelium. I hope to get your answer as it can really helps me to produce a right inoculum with a right method of concentration. Best regards and thank you so much.

Adnan Uzunovic : Dear Maizan It is tricky to count mycelia/mycelium concentration. I did try to use regular counting methods like those used for spores (e.g. hemocytometer) but that gives very rough estimate and tells only if you have some mycelial fragments and provides very rough count. Whichever method you use to generate mycelial propagules each time you will get different size of these and you can never be sure if these fragmented particles will grow into live colonies or the damage is to big to produce a live colony. These fragments also mix and tangle on the hemacytometer so are not easy to accurately count uless they are really small which then raise further qeustion on their vitality. Even if you count them somehow the final results when these are used in inoculation studies may differ as some of mycelia may further untangle during inoculation procedure creating new propagules. If you want to count them the best is to chop them to be as small yet viablel and use some sort of dilution until you come up to known volumes with not dense population of fragments that you can observe under microscope where you can count and then estimate for the whole liquid inoculum amount. However the most useful and meaningful way to count these propagules really is to do a dilution series and spreading known volumes on nutrient plates to count colony forming units after 2-3 days of incubation. This is the most relevant count as this give you the amount of propagules that will create new colonies however this unfortunately cannot be done ahead of time and before your inoculation experiment but rather to confirm the fragment concentration you used. You can perhaps in preexperiment phaze play with comparing your counts under hemacytometer and counts of colony forming units from the same liquid suspension of mycelial fragments and see how they compare. You can then use some sort of calculating factor to estimate live colony forming propagules based on your hemacytometer counts. I guess you will need to do this each time you prepare a different inoculum and for each experiment. I did get quite a different number when I tried it on very small scale with not much repetition. Hope this helps.

Dear Adnan

Your question is fairly wide .. It is better to determine which fungi you working with to get homogenized inoculum, as you know there are a significant difference between the fungi in propagules production ….

Good luck

could you guide me with how to produce a homogenized mycelial inoculum from Ganoderma lucidum grown on PDB broth with shaking for five days. I urgently need completely homogenous broth because i am going to take inocula for experiment with results as dry weight so i dentical inocula are very important to obtain before starting my experiment. given that this mycelial broth without spores as Ganoderma lucidum does not for spores in this mycelial stage.