Fall 2015 Week 11

This week in lab I haven't done much on my endophyte project. I transferred very small squares of mycelium from the original test plates to plates that have fewer biological controls so I can characterize them.

The rest of the week I worked with Matt on the allelopathy project we have been doing. Some plants have compounds in their leaves that, when they fall to the ground, prevent seeds in the neighboring area to not germinate to lower the competition for resources. We are composting leaves that we know to have allelopathic traits and seeing if the chemicals prevent various seeds to germinate. So I spent all week counting seeds into petri plates in preparation of this experiment.

Fall 2015 Week 10

I ran my last sterilization experiment of the semester this week in lab. The grass plates I made weeks ago are as sterile as the day I put them in my cabinet and so I decided to go ahead with the procedure I used on the grass. This was a summary of my procedure:

1. Rinse leaves in running tap water for 5 minutes to remove any debris or dirt
2. Soak leaves in 75% ethanol for 30 seconds
3. Soak leaves in 4% bleach for 5 minutes
4. Soak leaves in 75% ethanol for 30 seconds to rinse any residual bleach from the leaves
5. Cut the leaves in to small squares and put pieces in a petri plate 

This time I varied the times I soaked the silver leaved nightshade leaves in bleach to see if it had any affect. I soaked the leaves in bleach for 5 minutes, 6 minutes, 8 minutes, 10 minutes, and 15 minutes. Things began to grow on most of the plates by Thursday and by today the growth was dense enough to see without squinting. WHAT DOES THIS MEAN? I have no clue, Cori has no clue, Matt has no clue. So I'm just going to characterize this fungi for my project. Its everywhere on these plants.

To characterize the fungi I will use their distinctive characteristics when growing on petri plates, lumpy green with white mycelium on the fringes:

What the mycelium looks like stained and put under a microscope:

And what the spores look like under a microscope: 

I will also look up any biochemical tests I could possibly do to narrow the search to a genus of fungi I'm working with. 

Fall 2015 Week 9

This week in lab there has been no growth on my grass species plates, indicating the sterilization procedure is working for the grass. Growth of the gray mold is significantly quicker than the week I've given it, with noticeable mycelium by day 3 or 4. Next week I plan on using the same concentrations of alcohol and bleach but letting the silver leaved nightshade leaves sit longer than 5 minutes so the bleach penetrates the trichomes.

While waiting to see results from my grass plates I have been working more with my unknown bacteria. To even begin to classify a bacterium species one must know the cell shape, whether it is Gram positive or negative, and the organism's oxygen requirements. The first two pieces of information can be deciphered using Gram staining but the last is a requires other tests.

To test if a bacterium is capable of growing in oxygen free environments, two test chambers are prepared. The first is in a giant pickle jar with a burning candle that will use up the oxygen in the container. Only a very small amount of oxygen can get in through the edges of the lid making an environment most suitable to microaerophiles: bacteria that like a very specific, low level of oxygen.

The other test is a box with a pouch of chemicals that use up the all the oxygen in the box, while producing carbon dioxide. This creates an environment that only strict anaerobes or facultative anaerobes can live in. Strict anaerobes are bacteria that can only live in oxygen free environments, which I know I don't have because my bacteria was growing fine on my plates in open air. Facultative anaerobes are bacteria that can switch from oxygen rich environments to oxygen poor environments. 

And as a bonus a photo of my bacterium Gram stain under the microscope: