It will of course depend on the fungus you are working with........

What are the liquid cultures for? For DNA it may be best to isolate a lot af spores and start at high density and grow short rather than growing a few spores long time

This is our protocol for Botrytis, it also works with other fungi that make conidiophores

COLLECTING SPORES (ASEXUAL MACROCONIDIA)

Grow Botrytis on MEYA plates for 2 weeks, with white light and/or one night near UV

Material (per plate)

Sterile 10-15 ml in 0.05% Tween 80

water

15 ml centrifuge tube (blue cap)

5 ml tips

5 ml tips with a bit of glasswool in the tip

yellow + blue tips

closed + bend pasteur pipette

tube rack

centrifuge (Heraeus, centrifuge room)

20 + 200 + 1000 + 5000 pipettes

Flood plate with circa 5-10 ml 0.05% Tween 80 and carefully scrape of conidia with bend pasteur pipette (2X)

With 5 ml pipette transfer to homemade glasswool filters (in 5 ml tips) placed in 12-15 ml centrifuge tube to collect spores and catch away mycelium.

Spin 5 min at 800 rpm/ 114g.

Discard supernatant, wash pelleted spores in sterile water, resuspend in 10 ml

(If needed take sample for counting in haemocytometer).

Spin 5 min at 800 rpm/ 114g.

Discard supernatant, take up pelleted spores in sterile water, calculate volume from count, best is 10^8 per ml.

High concentrations (more then 10^7 per ml) can be stored for several days at 4 degrees in the dark.

For long storage mix moitié-moitié with sterile 30% glycerol, store 0.5 ml in screwcaps in liquid nitrogen or -80 C, make 4 tubes, store in duplo boxes (2x2).

May you tell us what the microorganism and the aim of this job are?

I would like to draw your attention that as soon as you collect spores as an aqueous suspension, dormancy can be broken and germination initiated. Therefore it is important to determine for which purpose suspensions are prepared. Otherwise the key is to add Tween 80 (2 droplets per litre) to collect spores.

I am not keen on using centrifugation or filtering as usually very few fragments of mycelium are removed while using bend Pasteur pipette. Another method consisted in harvesting dry conidia by turning over the dish rolled with Parafilm, tapping the bottom to recover spores in the lid, then discarding the bottow and substituting for a sterile one in such a way that you obtained dry conidia such as they are disseminated into the atmosphere.

Conclusion, all depends on for what purpose you are preparing fungal spores.

The organism is Trichoderma and I want to inoculate into liquid medium to investigate secondary metabolites. Previous inoculations with blocks of agar leads to ''clumped" growth in the form of cotton like balls distributed through the media. If one was to inoculate with conidia/spores would one get more homogenous growth?

Thank you all for your replies.

Cenek Novotny Institute of Microbiology ASCR, Prague

I use a similar protocol as described by Henk-Jan above for Alternarias. Tween can be replaced by 0.01% Triton X-100 that might be more stable in the autoclave, compared to Tween.

For fungal culture take 6 ml blank (autoclaved Dwater with tween 80) and tranfer the whole 6 ml water in one fungal slant and make a spore suspension of that after that use that spore suspension ( if u are using 50 ml of liquid medium then add 2 ml of spore suspension in it) it is the simple method

If you inoculate a liquid medium with colonized agar plugs or with conidia suspension, and incubate the culture with shaking, you will obtain Trichoderma colonies as you observed (‘cotton balls’). However, you can take carefully the liquid medium with a pipette without Trichoderma colonies. If colonies are too many or too much big, incubate for a shorter time. Alternatively, use a stable culturing instead of a shaking one, and Trichoderma will grow on the surface of liquid medium, making easier the taking of liquid medium without colonies. I suggest to inoculate the liquid medium with a conidia suspension:

1) Inoculate agar plates (e.g. PDA) with a agar plug or conidia suspension obtained from collection tubes, and incubate in the dark at 20-25°C

2) After 7 days, pour about 20 mL distilled sterile water (Tween 80, Tween 20 or other surfactants are note mandatory) in the plate and swirl handily and gently to favor detachment of conidia

3) Transfer with a sterile pipette the conidia suspension obtained to a sterile vial, and add sterile distilled water to reach a given volume (e.g. 50 mL)

4) Take an aliquot of this suspension and count conidia with a haematocytometer under the microscope. Once you know the conidia concentration, you can adjust all your Trichoderma isolates suspension to the same conidia concentration by dilutions (quantity of metabolites also depends on the medium colonization level)

5) Inoculate the liquid medium (e.g. 100 or 250 mL vials or others, or Roux Culture Bottles) with the conidia suspension (e.g. 1-2 mL or others) and stop vials with cotton plug or parafilm, at 20-25°C

6) Incubate under shaking for a rapid growth or under stable culturing for a slower but superficial growth, for 2-4 days

7) Take carefully the liquid medium with a pipette without Trichoderma colonies

Micro dilution drop tail method:

The method employs the use of flame sterilized slides for cleaning ascomata or fungal spore’s cluster, a micropipette for making dilutions and trailing of a drop to spread ascospores on nutrient medium to obtain clean isolates.

All steps will be performed in a laminar air flow.

1. A fine flamed sterilized needle will be dipped into sterile distilled water and an ascoma pick from soil or other part.

2. The ascoma place on a flamed sterilized slide containing a drop of sterilized distilled water. It retransfer on a new water drop to remove soil particles and contaminating spores adhering to the ascoma. The ascoma handle gently in order to reduce the loss of ascospores.

3. The ascoma crush and pipetting in and out several times with a micropipette to make an ascospore suspension.

4. Five hundred µ ltr of basal salt solution (1.5gm-KH2PO4, 25mg-MgSO4.7H2O, 25mg-CaCl2, 15mg-FeSO4.7H2O, 5mg-ZnSO4.7H2O, 5gm-glucose, 1 litre distilled water) will be taken containing 2mg chloramphenicol ml-1 to prevent bacterial contamination. The ascospore suspension will be added and mixed thoroughly with the help of the micropipette.

5. The suspension incubate for 2-3 hrs. at 28˚C.

6. Two hundred µ liter of spore suspension take and place in the form of a drop on nutrient medium plate near plate margin. Depending on the size of the ascoma, dilutions will be prepared at this point.

7. The lid cover up and the plate tilt so that the drop flows and leaves a trail behind, thus spread ascospores on the nutrient plate.

8. The plates incubate at 28˚C for a week and monitored for the development of colonies. Subcultures then prepare once pure colonies developed.

After spore formation collect mycelia along with spore add Tween 20 shake vigorously spores settle at the bottom

can Tween 20 be used in place of Tween 80 for making saline soln of spore suspension? what will be the recipe...

I used 1% Tween 20. 0.1% Tween 80 also can be used. right?

Grow the fungus over cellophane in the PDA media, use needle collect mycelium and take it in tube with Tween 20 and spin at low speed in centrifuge spores settle at the bottom.

Or Use slide culture  harvest the mycelium and agitate in the sterile water  spores settle at the bottom.

0.1% v/v Tween 80 can be poured over petri dish.

Anyone know how to prepare Rhizopus oryzae spore suspension from plate/slant /broth culture of  Rhizopus oryzae ? How to count there spore?

Grow the fungus over  the 3.9% PDA+0.1% tryptophan  plate agar. After that, pour over petri dish with dH2O and use cotton bud to scrape the fungus colony. Then, the mixing of spores, agar,mycelium and dH2O  were filtrated with fritted filer funnel. The agar and mycelium were stuck on the fritted filter funnel. The solution of spore was drop into the centrifuge tube. Spin at low speed in centrifuge spores settle at the bottom of the centrifuge tube. Discard the supernatant and keep the solid with the 20% glycerol.

Spores can be harvested from lawn cultures of

the organism on potato dextrose agar by flooding the culture

with sterile saline containing 0.01% (v/v) Tween (BDH), and

dislodging spores from the hyphae with the aid of a sterile glass

spreader. The solution with spores then filtered through

4 consecutive sterile absorbent cotton wool plugs to remove

any hyphal fragments present. The number of spores can be

counted using a hemocytometer, diluted to ... spores/mL as a

stock spore solution, and kept at 4 C until use.

Hello Amit Ranjan 

I do have the same question. I want to make Rhizopus oryzae and A. niger spore suspension from liquid medium, make a spore count and use it for solid state fermentation. If you have already done this and got results, please forward me the protocol.

I also appreciate if anyone could help me here. Thanks!

Hello !

I would like to store spores of botrytis cinerea for long time , for future experiments. How can i process ! thank you !

someone have protocols?

You can either store  it in sterile distill water or store the spore suspension after freeze drying .

Hello every one, I have also one question

For how long specific concentration of fungal suspensions could be stored in freezer ? for the next use.

This is based on the method you are employing to preserve the fungal suspension.Generally, 2-3 months if it is properly stored in the freezer.Better you freeze dry your suspension that can last for even year .

How long can the stock spore solution be kept at 4C?

How long can phytophthora species causing cocoa black pod disease be stored and the media?

I store mine at -80C. I do a conidial count and make a 1x108 conidia concentration. To this I add equal volume of 50% glycerol to my suspensions then vortex and aliquot into one use volumes. I then freeze in liquid nitrogen and store at -80C. When I need that conidia at correct conidia concentration all I have to do is take out one vial from -80.

I am going to run an MIC with Aspergillus Niger and Fumigatus. I cultured them in broth, Sabouraud Dextrose Broth, overnight at 37 one time at and 25 the second time, but they form big cotton balls and I cannot get an accurate OD to run MIC using microdilution method. I also spin them down and did vortex to get them dissolved but it did not help. Is there anybody who knows how to fix this problem? Should I add Tween to the broth to make the cotton ball/droplets soluble?

hello Marjan M. Hashemi

i understand that your fungi is growing like a cotton ball. this is because of the mycelial growth of the fungi. When you need to do MIC for fungi, all try to make a matt growth on an agar plate and try to retrieve the spores from the plate by using saline buffer with 0.01% tween 20 or 80. spread with a sterile L-rod, and then collect the spores. you can confirm the spores by viewing it under a microscope.

Hope it was helpful.

thank you

Just to quickly answer Enoch's question: because the spores used for Phytophthora inoculum are nearly always zoospores (the most ephemeral life stage) you can't really save up Phytophthora inoculum to be used for later, like you can with these spore suspensions (I'm currently trying to make a Fusarium spore suspension in talc, which can theoretically be used for years. But when I make Phytophthora inoculum I make fresh sporangia and zoospores every single time I inoculate. .

I have also a question in this matter, i want to work with Bauveria bessiana! can i replace tween 20 or 80 with sterile water in harvesting and counting spores? will the water affects spores? what is the major function of tween 20 or 80 in harvesting fungi?

Dear All

Please answer this question too.

https://www.researchgate.net/post/How_exactly_fungal_spore_concentration_values_given_below_will_be_written_in_SPSS_under_conc_variable_for_probit_analysis_to_calculate_of_LC50

I have a question, how do you retrieve the fungal spores that are grown on rice and then adding to a mineral oil?

Franziske Coetzee you can wash the rice in distil autoclaved water or PBS (PBS is preferred) and collect spores using miracloth to get rid of the rice. I suppose you have to count the spores later just for your reference by using hemocytometer and microscopy. Then you can add a portion of your spore suspension into a mineral oil. I hope I was helpful enough.

Martin John Martin hi I mainly work with B. bassiana and I have to tell you that I have never used tween 20 or 80 to collect spores. I always use PBS and water but PBS is preferred, also I freeze the spores in PBS in -80c and they are still viable.

hai.. I mainly work in Alternaria solani. i used PDA medium..tha fugus no produced spore.. why

Thanks the responses are helpful

Does miracloth efficiently separate the spores from mycelium?

Tween 80 or 20 breaks the surface tension of the medium and homogenized the mixture, which cannot be achieved by using water.