Here is the fungus I am trying to isolate!

With the help of another intern (Amanda) this week I was able to actually find and identify the fungal endophyte growing in my grass. Neotyphodium is the genes of the endophytic fungi i am finding in the festuca grass samples I am testing on. Now the hard part is to isolate them. In some further research some scientist are using antibiotic enriched media to prevent bacterial growth but not effect fungal growth. Some antibiotics have no effect on a fungus because fungi lack the proper chemical receptors for the antibiotics to target - much like viruses. This may be the next step in finding a media to grow what I want. In the mean time here are some of the pictures of endophytes I took this week. If you look at last weeks post the blue lines are fungus growing between the plant cells and they are very prevalent in these pictures as well. 

Rought draght was rough

One of my agar's has continued to grow and is looking like it could be an Endophtye but I am not going to hold my breath. It looks like just colonies of fungi have grown and no other microbes seem to be present on the plate, which is the hardest part so far this projects, getting an isolated microbe to test on. The rough draft due this week was just that very very rough and incomplete. I am feeling the stress of not having enough conclusive data by the end of the semester to conclude my research and answer the question for my experiment. I think I have had many successes in the last few weeks but still nothing in the way of fungal growth. I am almost positive that one of the bacteria I was able to isolate is an endophyte but I was not trying to isolate a bacterial endophyte to begin with. Fungal endophytes are much more selective and hard to grow. I am going back to the beginning to see if the grass i am using in fact has fungal endophytes in it. I will use a process of staining extremely thin slices of grass with Anil blue to identify if the grass is in fact infected with fungus. If all goes correctly it should look something like this.
     

granola troll hole

Last week the pressure of the semester really started to weigh on me and then before I realized it the week was over and it was time for a Friday fun day. Field trip with the other interns to Dreamy Draw park for a little lesson on navigation, latitude, longitude, and coordinates. There was a very interesting talk on ephemeral drainage, and the conspiracy theory that alien crash sites have been covered up in those hills. I went to hide a geocache for the first time and think we found a great spot.

The project is going slow and steady but at least it is going. I am still trying to isolate and identify the microbes I keep inoculating from the grass. I am at the same time adjusting my sterilization technique and trying to get a smaller number of microbes per slide. Unfortunately the dichotomous key Josh and I found for MEA is not for the type of fungus I am trying to isolate. MEA is still a good media to use for the fungus I am isolating I just need to find another dichotomous key. 

Malt shakes any one?

To all of the hard working mentors and students in the program, have a great and happy holiday. This week in lab I have made yet another media to try and isolate these fungal cells. In my research I found an interesting article talking about endophytes actually giving plants tolerance to pollutants in the air, like from automobiles. I am amazed at how symbiotic these relationships are between endophytes and plants and just how much each one can gain from each other. The malt extract I am using is going to require 20 grams of malt extract and 9 grams of agar solution as a standard from the dichotomous key I am using.  
 

Here is the video from last week sorry guys.

I will have to admit even with all the stresses of life weighing me down, the time spent in lab and with the great minds that encompass that area has been a centering point for thought, laughter, and of course some science work. I am thankful for all the joy this class brings me. Now with that said I made corn meal agar and will make malt extract agar this week to go along with the new dicotomist key we found. That way I can see if the microbes I have isolated are in fact endophytes. These media should help differentiate and help isolate the fungus I am looking for. Untill next week here is a video for your enjoyment.

This is why endophytes are important!

Things seem to be taking turns very quickly. One day I am excited that I got something isolated and a pure enough sample to test on, and then the next day back at square one. I am going to try using different media's designed for fungus growth to see if that makes any difference. With the help of Josh (thanks again by the way) I was able to find a dichotomous key that will help me in identifying the microbes I am finding. They used MEA (malt extract agar) to inoculate there microbes as well as corn meal agar. Josh will be ordering these media for me so I can make some new plates and try again. Even though things are going slow, I feel I am making strides and improvements in the right direction. This video shows how endophytes have already helped the world and the problems of agriculturist everywhere.    

Lots of things growing and new media to inoculate!

 In this weeks lab I got to do more testing on my unknown bacteria from the grass seeds I broke up. It is a pure sample to test on. I found it is Gram positive bacillus bacteria, microaerophilic, triptone negative and fermented glucose, fructose, and lactose while producing gas in all three of them. I will also try to recreate the inoculation of a fungus I got to grow from sterile grass with the root and the tips of the blades cut after sterilization. I found many articles that showed scientist inoculation endophytes at room temperature instead of the 37 degrees I am currently inoculating at, so I am adding this variable to my process and going to see what happens at a different temp.   

Endophytes can clean up polluted and contaminated soil from explosives like TNT.

Well it is something.

I started today with little hope, seeing as i am still having trouble getting anything to grow with the techniques I found online. I was pleasantly surprised to find that one of the agars from a grass seed inoculation I had done last week had a nice colony on it, and only the one! After doing a gram stain I found that the unknown in question is a gram positive bacillus microbe. After making a TSB today I will do more testing to further identify the microbe tomorrow.

Not getting the results I need

This week Matt and I have decided to just try and isolate the endophytic fungus itself and not try and name all the other microbes we are finding in the grass samples which are mostly bacteria. I have started to do more research on other people isolating endophytes and the process in which they used. The photo bellow is showing enodphytic bacteria harbor within alfalfa. Every light spots on the photo represent a endophytic bacterial group.

Grass is growing

My grass is growing great already and next week I will start doing some of the process to find and isolate any microbes within the plants microbiome. Last semester I simply tried rubbing the blades of grass on a TSA plate and waited to see what microbes grew. This semester I found an article on an extraction method for endophytes that used ethanol to sterilize the surface of the plant and then with a mortar and pistol ground the matter in to fine particles and started streaking from there on to a selected media.

The grass seed that I ground up and started streaking last week only produced microbes on one of four of the agars I tested. The TSA plate #2 grew 7 different colonies of microbes for re-isolation and testing. I am first going to grow the unknown microbes in a TSB before attempting to isolate any further. I plan on starting the gram stains and re-streaks of the unknown microbes this week.

     

Lets do this!

The first week back in the lab and I already feel more comfortable and less like I am going to mess up everything I touch. I have started making different media's for isolating the microbes I will find on the  endophytic rich grass I have planted this week. I am using a different media from last semester called the Sabouraud agar as well as a TSA or soy media. The Sorbouraud agar or SDA is going to help isolate and define the microbes I find. I plan on using different isolation techniques this semester as well to reduce the sample size.  

(Sabouraud on top, TSA bottom)

Back to School!

I am so happy to be back in the lab and in the program to continue my research on plant microbiomes. Over the summer I continued to do research and experiments on my own about plant environments and how to adapt them manually and monitor those changes. Over the summer I started to grow various types of mushrooms and have had some success. This could be a possible research project for the semester if plant microbiomes are already being researched by someone.    

The last and final week as been nothing short of crazy. I found the research conference to be an absolute joy and got most of the work done just in the nick of time. It was great seeing and hearing peoples ideas and overall enthusiasm for the research I was doing. I have been given a new vigor on science and the answers that I could possible unlock with hard work and some luck.

Week 13 has proven to be a tough week for me. I was not able to get as much done with my new unknowns from the desert Creosote plant as I wanted to but was able to finish identifying the other two unknowns from my drip irrigation plant here on campus. The wild grown Creosote plant had a total of 9 unkowns where I only had four with the plant grown on campus. I think this is a great sign and a start to answer my question of which plant would have more microbes in there microbiome.

Cool stuff I found about microbiomes

In Week twelve I was able to start on the first stages of inoculating my new creosote sample to find what is growing on it. I was able to identify a streptococcus and micrococcus genus on the drip irrigation plant here on campus and I only have two more unknowns to identify. This image is an MSA plate for further metabolic testing

In week 11 I was able to grab another creosote plant from the Papago mountains in Tempe Arizona while out mountain biking. I found that this plant is everywhere in the wild and has a stronger smell then the plant grown on the drip irrigation system. I am wondering how many different types of microbes I will be able to find on a wild plant from a drip irrigation plant.

In week 10 of the internship I have started working on metabolic test for the creosote plant I pulled off of campus and I have started to inoculate colonies from a wild grown creosote plant that I found in the Papago mountains in Tempe, AZ. I am getting close to determining the different types of microbes found on a drip irrigation plant. The most important is the FTM test to determine aerobic or anaerobic.

Back from spring break

Back from spring break and ready to start on the endophytes isolation. First I am going to grow the endophytic rich grass and then I am going to try and inoculate some agars and see what grows. Once I have grown some microbes I am then going to isolate those microbes and begin identifying them. Once identified I will grow each microbe with some soy beans at various watering levels and record the growth patters I observe. I may even use the grass grown in these for my soy bean grow if I can not isolate any microbes out of the leaves and vermiculite.

I have got the supplies

This week in my time in the lab I was able to finish some test on the plant I am working with in my abstract and I also got the necessary material to start my research project on endophytes. The grass I am planning to use has lots of fungi and bacteria to aid in drought tolerance and heat resistance of plants. This should give the plants I am going to grow a very healthy microbiome that I can test and isolate to find what is causing these beneficial factors in the growth cycle.

Finishing my abstract

This week I worked on and finished my abstract for the student conference. I am going to be finding the different microbes on a creosote plant, one grown from an irrigation drip system and one grown naturally in the desert. We happen to have a Larrea tridentata plant here on campus so I decided to grab some leaves and inoculate a TSA to see what grows. I am hoping to start isolating the microbes I find and start inoculations for a naturally grown plant next week. In my other research project I am waiting for some endophytic rich grass seeds to come from New York so I can start to plant soy beans and. I have decided that I will plant as many variables that I can control to get a more conclusive result for the experiment. 

The next step

With most of my time in the lab this week I finished researching my project and came up with a decisive plan on how I will conduct the different phases of the experiment. I completed an excel spread sheet of the test I did on my unknown and my unknown contaminant for quality purposes. My unknown is Staphylococcus aerus. In the lactose test my unknown showed positive for fermentation and the gas carbon dioxide.I am hoping to finish getting the materials I need to conduct the plant microbiome experiment and find out if they endophytes can make plants thermo-resistance.

Almost done with my unkown, ready to start the research project.

This week I made great progress in my unknown and unknown contaminant research. I concluded the msa test on both my microbe and the contaminant and found that they are different bacteria. I did notice that the unknown given to me fully turned the msa agar to yellow showing that it was positive for acid. However the unknown contaminant did not have the same results and did not show signs of acid fermentation. My unknown is defiantly Staphylococcus aureus and needs no further testing. The contaminant found on my original streak plate showed negative for acid in the msa test and needs to go through a tsi and lactose test. I believe the bacteria that contaminated my sample is Staphylococcus epidermidis but I will not know for sure until next week. As for my research project I have found the materials needed to complete the research and I hope to start setting up the experiment next week. Getting a control group of seeds planted is my first step then adding endophytes in different amounts will to each plant will show how the plant microbiome can have an effect on the overall health and production of the plant.

Week 2: Having some contamination.

In week one I made a culture of my first unknown microbe. There were however contaminants in the culture so I decided to test both bacteria from the culture in a gram stain. Both microbes stained positive and were both staphylococci in morphology. I did not have enough of the microbe contaminating my sample so am going to incubate a new TSB for this bacteria re-plate it on an augur trying to isolate the growth of microbe so I can get more precise testing done with out having both bacteria contaminate one another. When I come back next week I will be then conclude test on both the unknown microbe and the contaminant in my samples with definitive answers. I also hope to get results from the glucose fermentation test I started today to further test the microbes in my sample. 

Week 1 first day.

This is my week one blog post and for my first day as an intern I did lawn and split cultures of an unknown microbe. When I return to the lab on Tue. I will have a nice culture of my unknown and will begin the next process in Koch's postulates. I put the microbe in the incubator after using proper technique and do not expect any contaminants in my sample. The next step was to start researching microbiomes and how we could go about testing the concepts that through microbiomes one could make a plant more fruitful and healthy.

Works cited

Martin Grube . 2013. The plant microbiome and it's importance on plant and human health. Frontiers (M.     Schloter) [Internet]. [2013 November 8, cited 2014 Febuary 3] **Journal Info**. Available from:                 http://www.frontiersin.org/Journal/SpecialTopicDetail.aspx?name=plant-                     microbe_interaction&st=1543&sname=The_plant_microbiome_and_its_i