Week 5

This was a very productive week for me in lab. Last week I finally got the dyes I was waiting for and I made agar for the first time in my life so I'm finally ready to start plating plant tissues in an attempt to isolate endophytic fungi. I plan on using three species of plants: lantana, oleander and bougainville, and three tissue types: leaves flowers and stems. 

Endophytes are slow growing fungi and if the surface of the leaves or flowers I'm using have any sort of fungal spore or bacteria on it then those organisms will grow much faster than the endophyte, take over the agar and eat all the food before the endophyte has a chance to appear. To ensure that the only thing that is growing in my agar is an endophyte, I have to sterilize the surface of the tissues. 

Monday I came in early to design the sterilization method I will be using on the plants for my project. The materials are first rinsed in running tap water for 5 min to remove any dirt or debris and then cut into 5mm lengths. The sections are agitated in 100% isopropyl alcohol for 2 minutes and then placed in a 1.25% bleach solution. At the intervals of 0,1,5, and 10 min four sections of tissue are removed, triple rinsed in sucessive sterile water baths and placed on an agar plate. It will take more than three weeks to see any results. 

Week 4

This week was a short week because of the holiday. All Hail the Presidents!

Anyway, Wednesday I was waiting for Rose Bengal and coomassie blue stains to come in so I didn't have much to do. I put some grass seeds in a 5% solution of sodium hydroxie to soak for eight hours to soften their cell walls enough that a stain can penetrate it. As I've read some endophytic fungi infections can be passed from the mother plant to her seeds, I was hoping to be able to see if I could find some mycelium there. Alas I forgot to take them out of solution before leaving yesterday and, after soaking all night in such a caustic solution, the seeds were nothing but mush today. I had to throw them all away and will have to try again later next week. 

In better news the stains finally came in! Tooday I got to weigh out, mix and autoclave a special agar for my project this semester. I added the Rose Bengal dye to my plates as well as dilute ampicillin to select against fast growing fungi and bacteria, respectively. In addition to giving endophytic fungi a fighting chance in my agar plates, the rose bengal will be picked up by the fungal colonies making identification of any species easier. 

I'm really loving this program. 

Week 3

Most of this week has been spent doing research for my semester project. I've decided I would like to study plant endophytic fungi this semester. Plants often have symbiotic relationships with many different organisms. Jeremy's project is looking at bacterial endophytes in roots while I will be forcusing my attention on fungal endophytes in stems, leaves and flowers. 

The definition of an endophyte is a little hazy but most people agree that it is any organism, usually bacteria or fungi, that can live inside another without causing pathogenic symptoms. The endophytic organism can be a symbiote that benefits the host, like our gut bacteria that helps us absorb nutrients, or it can just be something that happens to live in the host. In some cases an organism that is a normally an endophyte can become pathogenic, such as when Staph aureus gets into a human's blood stream or if a plant begins to die.  

The first plants it was proven had endophytic fungi living between their cell walls were certain species of grasses and so most of the literature is about grasses, The gentleman that was working on a similar project last semester left behind a sack full of grass seeds known to harbor endophytes and I planted some of them earlier this week. Here is a photo: 

And here is one of the microtome I will be using to slice my plant samples for staining to see if I can find mycelium between the plant's cell walls. The potatoe is used to hold the sample in place so I can slice extremely thin slices off sample.

Week 2

The entirety of my second week in S-STEM has been used trying to identify my unknown bacterial sample. Even though I took Microbiology a year ago it took me three attempts to gram stain my unknown. It could have been because I wasn't heat setting the sample long enough, and the bacteria was just washing off into the sink, or it could be that I just couldn't find them under the microscope. Either way it was a little frustrating.

Finally on the third attempt I was able to get a sample to stick and, with a little assistance from Josh, was able to locate the sample under the microscope. It was a beautifully stained gram negative bacillus bacterium. I spent the rest of the week doing all the metabolic tests necessary to discover the identity of my sample.

Here are the results of my tests:

Lactose fermentation- negative, color remained red and there was no gas produced. 

Glucose fermentation- positive, the color of the phenol indicator changed from red to yellow and a small amount of gas was produced.

Tryptone indole test-positive, the media got a red ring on the top when the indole reagent was added.

Citrate test- positive, after inoculation the media changed color from green to blue. 

Sulfur indole motility test- positive for both motility and sulfur reduction, the inoculation site had feathered edges and changed the media around it to a black color. 

Catalase test- positive for catalase production, the sample began to bubble after adding a few drops of peroxide. 

Gelatin test- negative, the gelatin became solid again after leaving it in the fridge proving the organism cannot hydrolysis gelatin. 

With all this new information at my disposal I can now identify my unknown using the flow chart available. After following the chart it has been determined that my specimen is, in fact, Providencia stuartii. 

Here are some pictures of the tests I ran. Now I can move on to my semester project!