Sporulation of Physarum

[Vicente Lopes]

I've tried quite a bit of sporulation induction for my Physarum culture since I need spores for experiments that I plan to do.

However, every time I try inducing the sporulation it appears to die while sporulating, leading to immature sporangia.

After crushing the sporangia some spheres are visible (maybe spores) but they don't come out of the sporangia naturally (no dust) and they don't germinate.

Here is a micro photo of the crushed sporangia with spore like spheres, and deformed sporangia in the right (looks like they couldn't finish developing).

Is this normal? Should I just keep doing it until they form properly? Tips are welcomed.

[Kenneth Ramos]

What is it that you mean by "induced sporulation?"  Sporulation is a touchy process.  As you probably already know, temperature, humidity, and light among the most prominent are major players in getting a plasmodium to sporulate.  I have never tried induced sporulation but allow it to occur naturally.  Even then it is a lengthy process ranging from 21 days to a month, sometimes longer depending on species.  Your photographs are quite good and the deformed sporangia are evidence of a disturbance of sorts, maybe? IMO that is, though I could be wrong.  It doesn't take much of any outside interference to cause this to happen from what I have read.  It will be quite interesting as to what you find out.

[Vicente Lopes]

By "induced sporulation" i mean i basically tried to slowly starve it (less food than needed) in light conditions, after different setups this seems to be the best at causing sporulation (within 7 days) however it never seems to finish properly... These images are from the best batch most of them stopped at just brown/black blobs.

You say your method takes close to a month? What do you do differently? Could the size of the plasmodium be a factor (bigger = better success %)?

[Kenneth Ramos]

I keep the plasmodia in the dark, with the exception of removal for observation with the dissecting microscope.  Using moist chamber cultures, petri dishes (plastic or borosilicate) with dampened paper towel and oat flakes.  I don't have great success but some success all the same and keeping viable plasmodia for some months in culture.  The plasmodia stay damp, not wet, fed, and kept in the dark.  Much like the American citizen when it comes to the matters of politics.  Personally, I believe the time taken for sporulation, may rely on the pH of the substrate from which I germinate my plasmodia, though I have yet to test that idea.  The substrate I employ is usually rough Flowering dogwood tree bark.  Thanks for explaining what you meant by "induced sporulation."  I understand what you mean, and that too had crossed my mind on occasion but I never had much success at it and ended up with the entire plasmodium dying before any resemblance of sporulation could occur.  I have done better by letting nature take its course.  As I mentioned, I haven't had a high percentage of success but enough to keep a keen interest in my cultures and observations.

[Vicente Lopes]

and in your cultures do your sporangia release spores into the air, like a dust cloud?

I assume this is a hallmark to know if the sporangia are well formed and viable for germination...

[Kenneth Ramos]

The sporangia are well formed as are the rest of the fruitbody components and spores are released but a dust cloud is seldom noticed but only on occasion and during observation through the dissecting microscope and slide preparation for observing spore characteristics and the capillitium, utilizing the light microscope.  There usually isn't a great many of them but what there are, are quite useful.  However, I oftentimes get a surprise by finding other fruitbodies outside of that which I was hoping for usually resulting from aphaneroplasmodia, which are difficult to observe at best even with the microscope.  During the warmer months, I scour pine forest deadfall and needles for other fruit bodies and try to culture from them in test tube or petri dish preparations with distilled water and substrate taken from the area of collection, hoping for amoeboflagellates or swarm cells and then move the developing plasmodia to culture with substrate also from the collection sites.  I also bag and label my substrate samples so as not to get them mixed up, hoping also to maintain the pH from which all samples were taken.  I also quite frequently scour remnants of decaying hardwoods and do the same.  In both instances a notebook, a Hastings 10x hand lens, and a camera with a macro lens or a lens utilizing a 36mm extension tube are pretty much a necessity and I also photograph the immediate surrounding environment of where my samples are taken for later reference should I require it.  Here the trusty "smart phone" camera helps to reduce the load in the field.