I am novice in culturing fungi, Rhizopus spp.
I would like to make the filamentous Rhizopus into pellet form. I had followed a few journals but it doesn't seem to be successful.
Could you please to share your experience here?
1. Sub-culturing of fungi from master stock to PDA plate, PDA plate to PDA plate.
1.1 How frequent does sub-culturing + condition need to be to store the PDA plate after Rhizopus has been grown under optimal circumstances?
2. How do I calculate the fungi spore cells, in 10x2 per ml? Any simple method to follow?
3. Transfer PDA Rhizopus into liquid medium to form pellet fungi. Any simple method?
Do I need a control pH using CaCO3?
Thank you.
Hope can hear and share your experience here.
Hi Jacky,
I haven't worked with Rhizopus but I have had a lot of experience with other filamentous fungi. Routine culture tranfers between agar plates can be done with a pointed blade scalpel. Flame the blade to sterilize then quench in alcohol, burn off the alcohol before applying to the culture.
Cultures on agar slopes should remain viable for a year or more if stored at 5 degrees. Check that they have formed plenty of spores before you store them. Sub-culture 'working' stocks once a month.
Spore counts can be done microscopically with a 'Cell-Vu' counting chamber. These are designed for doing sperm counts but are ideal for fungal spores.
To produce pellets you need shake flasks inoculated either with spore suspension or hyphal fragments. Spore suspensions an be prepared by washing a plate culture with a 0.1% solution of Tween 20. Hyphal fragments can be prepared from a static liquid culture by shaking for 20 minutes with 5mm diameter glass beads.
I hope that's of some help.
Steve
Dear Stephen,
Great day.
I had tried to grow the Rhizopus pellet using various media.
The Media that used were
1. PDB (Potatoes soap + glucose)
2. glucose,
3. glucose + CaCO3,
3. Glucose +CaCO3 + peptone (meat)
pH was adjusted to 5-5.1 using Acetic acid.
conditions: 180-200 rpm
Temp. 37C.
Innnoculum: 5-6ml sterile dH2O washed the 3-5days Rhizopus agar plate (incubate at 37C) to get spore suspension.
innoculumm added to shake flask was 2%, 4, 6, 8, and 10%.
However, most tested media produced clump cell and growing in pellet cell was considered less growth...
Based on your experience....
1. Is the temperature too high?
2. Rpm to slow/fast?
3.pH?
4. Innoculum should further dilute at least 10x, 100x?
Thank you.
Jacky
Dear Jacky,
I'm affraid that some mycelial growth in the form of 'clumps' is unavoidable (clumps tend to form when the mycelium sticks to the glass). However, you can probably minimallize the clumping by optimising the shaking speed, you could also try variations in the volume of culture medium.
The shape of the flasks is another factor, those with baffles produce higher shearing forces than plain ones
Your other suggestion of further diluting the inoculum might help but I doubt if other environmental factors will have much influence.
If you have the facilities, a small stirred and aerated fermenter will also produce pelleted growth.
As you have obtained some pellets at the first attempt, some improvement should be acheivable.
Best Regards
Steve
Dear Jacky,
1. You should use well-homogenized mycelium.
2. Try to vary the shaker speed.
3. Try to vary the volume of medium.
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