After reading about the interesting endophyte in Fescue Arundinacea I looked for some of the grass.
I believe I found the grass growing in a few places so I took various samples from the different plants I had found. I stripped the outer layer of grass from the main stem and rinsed the stem in 3% H2O2 and rubbed with a cotton ball soaked in the H2O2 as well. The stem was than cut into smaller pieces with finger nail clippers that had been flame sterilized and than soaked in 3% H2O2 as well.
The smaller pieces of the grass were allowed to soak in the H2O2 for around a minute ~ -/+ 30 seconds. The ends of them were bubbling tiny O2 bubbles and the sides of the grass had small bubbles forming. The sterilized grass stems while still dripping wet with the store bought 3% H2O2 were shoved in 8oz polypropylene deli cups that have about 1cm of PDA agar in them.
I was pretty sure I had killed everything since the H2O2 treatment seemed pretty extreme. I am at least sure that nothing came from the outside of the grass.
4 of these deli cups/jimmy-rigged petri dishes sat in a dark drawer at room temperature for about 3-4 days with varying results. All sad and questionable.
They appear to ALL have bacterial contamination severely that has grown rapidly and nearly colonized half of the deli cup.
A few samples have, along side with what appears to be bacterial contamination, extremely long and fibrous mycelium (at least more fibrous than what I have ever noticed growing on PDA in the past)coming out of the ends of the stem and some, but less from the sides. The growth is WAY to fast to be the endophyte that I was hoping to get. It grew way faster than even shiitake mycelium on PDA at RT. Obviously though, it is an endophyte of some sort.
So, I need to try this over again obviously. From this attempt though, it seems like it is absolutely needed to use antibiotics/antibacterial PDA agar - the growth of the bacteria is way to fast and ruins everything.
In the thumbnail uploaded, hopefully you can see the ring of bacteria and than the fluffy clump of mycelium growing out of the stick - which is hidden in the mycelium, however the slightly dark part is the stick/stem.
Not to sure if this is worthy of a new post or a topic but I figured I would share anyways. This is not as easy as getting a clean culture from a sample of a stem from a store bought mushroom such as shiitake.
I believe I found the grass growing in a few places so I took various samples from the different plants I had found. I stripped the outer layer of grass from the main stem and rinsed the stem in 3% H2O2 and rubbed with a cotton ball soaked in the H2O2 as well. The stem was than cut into smaller pieces with finger nail clippers that had been flame sterilized and than soaked in 3% H2O2 as well.
The smaller pieces of the grass were allowed to soak in the H2O2 for around a minute ~ -/+ 30 seconds. The ends of them were bubbling tiny O2 bubbles and the sides of the grass had small bubbles forming. The sterilized grass stems while still dripping wet with the store bought 3% H2O2 were shoved in 8oz polypropylene deli cups that have about 1cm of PDA agar in them.
I was pretty sure I had killed everything since the H2O2 treatment seemed pretty extreme. I am at least sure that nothing came from the outside of the grass.
4 of these deli cups/jimmy-rigged petri dishes sat in a dark drawer at room temperature for about 3-4 days with varying results. All sad and questionable.
They appear to ALL have bacterial contamination severely that has grown rapidly and nearly colonized half of the deli cup.
A few samples have, along side with what appears to be bacterial contamination, extremely long and fibrous mycelium (at least more fibrous than what I have ever noticed growing on PDA in the past)coming out of the ends of the stem and some, but less from the sides. The growth is WAY to fast to be the endophyte that I was hoping to get. It grew way faster than even shiitake mycelium on PDA at RT. Obviously though, it is an endophyte of some sort.
So, I need to try this over again obviously. From this attempt though, it seems like it is absolutely needed to use antibiotics/antibacterial PDA agar - the growth of the bacteria is way to fast and ruins everything.
In the thumbnail uploaded, hopefully you can see the ring of bacteria and than the fluffy clump of mycelium growing out of the stick - which is hidden in the mycelium, however the slightly dark part is the stick/stem.
Not to sure if this is worthy of a new post or a topic but I figured I would share anyways. This is not as easy as getting a clean culture from a sample of a stem from a store bought mushroom such as shiitake.
