How to Prepare Mycelium Culture of Mushroom (Part 2/4)

[@valvetann]

Thank you Dr Rakib, glad to look back at this video

[@uzmapathan6853]

Thank you for ur hard work i can do great in my exam now

[@playwithtoys9055]

Very informative video

[@ritwikbasak4960]

That animation helped a lot. Thank you sir.

[@ropenedu]

You're welcome

[@sonotuttotuo7782]

Thanks for this video. Please, do you have video on how the mycelium culture can be stored to be use later.

[@ropenedu]

Unfortunately there's no such video of mine. Mycelium can be stored in slanted agar tube, at 4-8 degree Celsius for long term storage (~1 year)

[@sonotuttotuo7782]

@@ropenedu Thank you immensely for your reply. Am so happy that you replied to my comment. Am just a beginner in mushroom farming and I really want to know so many things about it. Thanks once again. Have a nice day.

[@sonotuttotuo7782]

Thanks so much. Your video is very nice.

[@khairsrar1134]

Please please complete the mushroom series. (Loading mushroom spores on wheat grains)

[@ropenedu]

will upload a video on production of spawn using grains later. However, we usually use pure mycelia culture, instead of spore.

[@khairsrar1134]

@@ropenedu Thank you very much and appreciate and respect We are waiting for you to support you in the field of mushrooms.

[@lilit_ghazaryan]

thank you for the great video!!! what should I do with the rest of the master culture, after cutting a part for the subculture? I assume throwing in a bin will be wrong

[@ropenedu]

The proper way to dispose is to autoclave them before throwing them into a bin.

[@BettaVibesAquatic]

Awesome Video👌👌. Can you please make a video on liquid culture please.

[@ropenedu]

sure. thanks for the advice.

[@khairsrar1134]

All thanks, appreciation and respect. Please want to supplement the production of mushroom spores loaded on wheat grains

[@steveraman4562]

excellent keep it going

[@fabilizboa]

Is it not necessary any kind of antibiotics, or acid to prevnt bacteral growth in the PDA with the fungi?

[@youtubeshinobi8932]

Hi, Currently I've made two sample - one of Wood ear and another one of Oyester. The wood ear Sample got all contaminated, while the oyester mushroom is in it's 3rd day and still not showing any mycelium growth. Please explain.

[@ropenedu]

I'm not familiar with wood ear mushroom. For oyster mushroom. Try to use as fresh as possible mushroom.

[@ejamafricamovies]

I love this video but I want to ask, did you add any liquid content into the peltri before you putting the mushroom tissue inside?

[@ropenedu]

thank you for your comment. There's no liquid added.

[@gardyzafridi1]

Good work

[@hadishidayattullah891]

after cutting the mycilia square from the edge with the inoculation needle then put it in an empty petri dish or put it in a petri dish that contains potato dextrose, please answer

[@ropenedu]

put onto a fresh/new potato dextrose agar in a petri dish.

[@mr.punk___6693]

Can i ask if did you used pda in the first innoculation ? I mean when you cut the tissue and placed it into another pertri dish where u said it was a PDA, i just want to ask if when starting the process, did u alreasy used PDA or another type of agar ? Our thesis is culminating enoki mushrooms, this video helped alot in our process, especially in spawner and substrate preparation. My questio again is did you used another type of agar or it was just a PDA at all ?

[@mr.punk___6693]

Hoping for your response🥺

[@ropenedu]

We used PDA only throughout the process. It's a general agar media for fungi, and it's cheaper compared to other agar like malt extract agar (MEA). thank you for your support.

[@nusantaraenthusiast5595]

Buat lah video lagi bang, saya suka utube abang

[@ropenedu]

terima kasih di atas sokongan dan semangat yg diberikan. Insyaallah akan buat video baru. cuma sekarang ni agak sebok dengan tugas hakiki.

[@bobanristic9047]

Great video! Thanks thanks thanks!

[@pedrotenorio6327]

What is the difference between the first culture and the sub-culture?

[@ropenedu]

The first culture is the original culture directly from the basidiocarp (fruiting body) of a mushroom. It may contain impurity. Thus, sub-culturing them once or twice will result in purified mycelium culture. Genetically, they should be the same. Thanks for your comment.

[@torvaldchan7641]

Well explained video do you plan to make another video on how to do liquid culture?

[@ropenedu]

will consider it. thanks for your suggestion.

[@davka0318]

Hi . Incubation °C first and second can u tell me . Thank u

[@ropenedu]

room temperature, it can be within 25-30.

[@holahesmia8194]

Can i do the same procédure with agaricus bisporus? incubation; sub-culture, but it grows differently, not like cotton, like oysters, how can I do the sub-culture?Why is the tissuse removed from the top? The body tissue cannot be used(oyster or agaricus?

[@weiping9970]

Hello, may i know why do we need to place the culture inversely during 2nd culture? Does 1st culture need to be placed inversely too?

[@ropenedu]

It's advisable to place the plates inversely during incubation. The main reason is to avoid accumulation of water near the mycelia, because sometime there's water vapor in the plates. Thus, this will improve the successfulness of isolation of the culture.

[@weiping9970]

@@ropenedu owhh i see, thank you so much! Appreciate this informative video :)

[@Filmafamily93]

Assalamu'alaikum prof. Bisakah prof. Membuatkan vidio pembibitan jamur kancing/ champignon? Trimakasih ( Indonesia)

[@malinatezycka]

Is the darkness necessary? Or no sun exposure should be enough? Great video!

[@ropenedu]

dark condition is the best for mycelial growth. mycelia will grow when exposed to light, but at slower growth rate.

[@Akhirra]

Hi it's a great video 🙏🏻 I wonder if it is possible to transfer "Example: Trichoderma Sp(Harzianum)inoculated Mycelium into a Liquid Culture?" A method like mushroom farmer's do, culturing a mushrooms tissue into Liquid Culture. I really wish you can make the video about it.. That would be very awesome.

[@礼晟]

Yes u can

[@Akhirra]

@@礼晟 how to do it? What is the best liquid composition and the liquid ratio for Trichoderma into liquid culture ?

[@ropenedu]

yes, can use similar procedure. PDB media will work as well.

[@ayeshashaikh9729]

Thanks a lot 👍😀😊💗

[@holahesmia8194]

Please how you prepare spawn and substrat?

[@ropenedu]

coming in future video. stay tuned.

[@cobbylexis1598]

Please i don't have an incubator So what is the alternative

[@ropenedu]

room temperature 25-30 degree Celsius should be ok

[@cobbylexis1598]

What should be the temperature of the incubator before incubation

[@mahmudulhussain7552]

Sir can we make banana culture like mushroom culture or pda

[@ropenedu]

The technique of culturing banana and mushroom on agar media are similar, but it uses different type of agar media, and different part of the plant (explant).

[@PetaniDKurt]

Can we directly transfer mushroom tissue to sterilize grain? I saw videos they skip the PDA making part

[@ropenedu]

Yes. However, the purity of the culture is not ensured.

[@PetaniDKurt]

@@ropenedu what do u mean by purity? The spawn is not strong?

[@ropenedu]

@@PetaniDKurt yes, purity of the spawn. no issue with the strength of spawn. there is higher possibility the mushroom tissue contain other microorganism. Using mycelia culture is better as it usually reduce the possibility of having contaminants.

[@PetaniDKurt]

@@ropenedu i see. What about grain to grain transfer? For example, f1 grains transfer to new sterilize grains. Is it true some people say the spawn is getting weaker after too many transfer f1...f5..hope u understand my question. Thanks for your response.

[@ropenedu]

@@PetaniDKurt yes, it's true. Culture getting weaker after sub-cultured for many times. they become "lazy" when we provide them all the necessary nutrients without their effort to use their natural mechanisms to breakdown lignin.

[@ritwikbasak4960]

Can we obtain the first necessary piece from any part of the mushroom?

[@ropenedu]

it can be from the stipe (stem), or pileus (cap). The most important thing is from the internal unexposed tissue. Avoid culturing mushroom from the spore or gill (may contain spore), because culture from spores may cause genetic variation.

[@ritwikbasak4960]

@@ropenedu thanks

[@vencieregalado9215]

Mycelium is that a glass .if you try to culture mushroom refregerated is ok to process. Reps pls

[@ropenedu]

the culture can be kept refrigerated not below 4 degree celsius for storage purposes. Let me know if I get your question wrong.

[@darmansaul136]

How to take/slice the tissue from split gill fruit body.. since its very small compared with other mushroom?

[@礼晟]

Use precision tweezer and tear a small piece.

[@malshasamarasiri3779]

Hello sir, is it necessary to do sub-culturing after 3 days? How about after 5-7 days?

[@ropenedu]

It depends on the growth of the mycelia. usually good to subculture when the radius of the mycelia achieved 1-2 cm

[@malshasamarasiri3779]

@@ropenedu Thank you very much sir!

[@holahesmia8194]

Sub-culture is 100% pure ? It's not the seconde generation? How long we can storage at 6°c?

[@ropenedu]

the culture usually 100% pure, which mean no other microorganisms associated with the culture. We be more assure about the purity in 2nd culture. I usually suggest approximately 1 year to ensure high chance of survival.

[@holahesmia8194]

@@ropenedu in this period, we restore the culture in the same way, meaning a culture and sub-culture or we grow it(culture) on a media culture(7 days at 28°c) and use it directly. Why it's considered a clone and that it is another generation that is not similar to the first in characteristics? Why it's incubated at 28°c not at 22.5°c . What about PDA and Malt extract agar who is the best one for the good results?

[@holahesmia8194]

@@ropenedu thank you

[@mr.purrfect6500]

Hello sir, does this method also apply in isolating beauvaria bassiana mycelium? I hope you can help me.

[@ropenedu]

Beauveria bassiana has no fruiting body. the method of isolation is different.

[@mr.purrfect6500]

Okay sir thank you so much!

[@vitaly5209]

What is master mycelium then or G1 and how to get it? As I know you showed us cloning process

[@ropenedu]

The first culture from the mushroom fruiting body is considered as master culture. Then, u have to subculture at least once to ensure the purity of the mycelium culture. Keep the culture as stock for further use or cloning to multiply them. There should be no genetic variation between the master culture and the cloned cultures as it involves asexual reproduction, and not sexual reproduction.

[@vitaly5209]

@@ropenedu thanks a lot! That way I can get mother culture from mushroom tissue, right? i'm asking cause many growers say that power of mycelium degradets from generations to gen.. and it's needed to refresh culture by taking it from master dish, my point was to find out what is actually "mother culture", " generation 1“ or "master dish mycelium"!

[@ropenedu]

@@vitaly5209 Yes, it's true. Mycelium culture will degrade if sub-sub-sub cultured over and over again... it's better to clone them from the initial culture. Thanks.

[@darmansaul136]

Same procedure for Schizophyllum commune? any specific temperature , duration etc?

[@ropenedu]

frankly, I have no experience with Schizophyllum commune. However, I think the procedure should be similar... the duration and temperature are similar as well since both fungi can grow in tropical climate.

[@vencieregalado9215]

Its ok to culture grow to a glass? Reps pls

[@ropenedu]

do u mean glass petri dish?... If that is your question, the answer is yes.

[@amsalaron8316]

Tahan berapa lama bibit jamur tiram disimpan, bagaimana cara penyimpanan bibit jamur tiram. Thanks

[@ropenedu]

1 tahun, simpan di chiller 4-6 darjah celsius

[@malshasamarasiri3779]

Hello sir, I followed a similar procedure in a biosafety cabinet. But most of the time, my petri dishes were contaminated with green mould (Trichoderma harzianum). Is there any specific reason where it went wrong and a method to prevent it?

[@ropenedu]

there are many factors that possibly cause contamination. 1. agar media prepared properly? to troubleshoot this. Prepare a few media plates without any culture and make observations. If no microbial growth is observed, that means the agar media is clean and good. 2. the laminar airflow or biosafety cabinet working well?... expose a sterile cotton bud to the air inside of the biosafety cabinet, then swab on agar media. see if any microbe grows. If no microbe grows, this means the biosafety cabinet is working as well. 3. stage of the cabinet, hands, glove, apparatus properly sanitized with 70% ethanol? 4, the original culture probably was contaminated.

[@Polarcupcheck]

Wonderful

[@kanakakumarkarri7758]

Sir what is the name of sealing tape

[@ropenedu]

it's a parafilm... also can use electrical tape which is easier to get.

[@kanakakumarkarri7758]

@@ropenedu thank you sir

[@kanakakumarkarri7758]

@@ropenedu sir for milky mushroom tissue culture tissue has to take from stem or mushroom cap sir

[@ropenedu]

@@kanakakumarkarri7758 either one should be fine.

[@kanakakumarkarri7758]

@@ropenedu thank you sir

[@pyarafrommm702]

thanks

[@muhammadarslan1291]

sir how much quantity of agar was used on Petri dishes?

[@ropenedu]

do you mean how much PDA poured into each petri dish?... if yes, the answer is 12-15 mL per plate. This is a standard 90mm diameter disposable petri dish.

[@HelaYak]

Thank you sir

[@sriyaniamarasinghe5880]

Tanks sir

[@holahesmia8194]

The seconde step is?

[@ropenedu]

Hai Haha Ch. sorry for the late respond. May I know what's you're referring to?

[@holahesmia8194]

@@ropeneduthe difference between the mother spawn and the spawn commercial and how it made ? what about the grain preparation (the best preparation)thank you so much for your answer.

[@ropenedu]

@@holahesmia8194 I will publish a video on mushroom spawn preparation later. In that example, I will use rice husk as the substrate. stay tune.

[@holahesmia8194]

@@ropenedu ok thank you but why not the wheat?

[@holahesmia8194]

@@ropenedu Why is not wheat (it is considered one of the hardest grains which is used because the mycelium grows very slowly on wheat incubated ( the temperature range from twenty- two to twenty-five degrees Celsius )because it contains starch from the type of albumin amylase and cellulose of the type of hemicelilulose), which takes a long time to grow despite the modification of the pH (with 15 g Calcium carbonate and 15 g calcium sulfate / kg) It cannot accelerate the growth, neither by changing the temperature nor by adding something else, and even by adding more than one petri dishe to accelerate the growth(one petri dish/kg grain), making two hundred grams of mother's spawn and transferred it to two liters of wheat( it's mean a cloning )meaning a different and second generation with the first, and therefore in the copmost the growth is not uniform and dense and the fruits are few.

[@ShinobiVuDU]

Poor sterile procedure, never hold something "sanitized" over top of a sterile surface or the tissue you are trying to cultivate IE the sanitized work gloves over top of the exposed mushroom tissue.

[@treebeard8475]

99% isopropyl would be better for this?

[@NotoriousHighBP]

@@treebeard8475 no, it evaporates too fast.

[@kartalbaba981]

👍👌👏

[@حجيمسلم-م5ظ]

🌷🌷🌷👍

[@thunderedsun203]

I love how we gone to eating real vegetable and real meat to mushroom