Hello and welcome back to the second installment of Dylan and Devin Discovering Dirt! In the past week Devin and I took a crucial step on the journey of characterizing our unknown microbe: choosing and isolating our microbe into individual colonies!


Now, without further adieu, I give you our plates after one week of growth:

The picture to the left shows the result of a full week of growth of each of the colonies cultivated from our 10 fold serial dilutions (10^-3 ug/ml through 10^-7 ug/ml). In this picture the clear tryptic soil agar plates are in order from highest concentration of microbes to lowest starting with the top right gel and ending with the bottom right gel. If you look closely, you can even visually tell the gradient in concentration by the varying amounts of growth from right to left! The pink Rose-Bengal gel (10^-4 ug/ml) is less conducive to the growth of bacteria, and was used to help us see the differences in fungal fungal and bacterial colonies.

As you can see, our plates are full of microbe colonies with different boundary shapes, textures, elevations, and colors. This is visual proof that our collected sample was quite biodiverse, despite having no fungi growth on our Rose-Bengal plate. We will touch on this more later.




You may be wondering how we went about isolating our microbes. Well, just sit back and relax as we break it down for you.


Last week(2-10-15) : Devin and I gathered a soil sample, and preformed a 10 fold serial dilution to a  lowest concentration of 10^-7 g soil/ml. Each concentration was then smeared on a plate, and left to incubate at room temperature for a week.


This week(2-17-15) :
As a team Devin and I examined the growth on each of our plates and counted the number of colonies on each plate, and used this information to estimate the number of microbes in our soil sample per gram of soil! To do this, we counted the number of individual colonies on our 10^-3 ug/ml plate and multiplied this number by 10,000 considering our dilution. Giving us:

We counted 44 colonies on our plate x 1,000= 44,000 microbes per gram of our original soil sample! This means our sample was teeming with life and diversity!

Next, we chose our favorite microbe from the plates.

Devin's microbe (top)- picked from our 10^-4 dilution plate was...
Border- circular
Color-Mucus
Texture-Smooth
Elevation-Flat

Dylan's Microbe (bottom)- picked from 10^-5 dilution plate was...
Border- Round
Color -Mucus with a brown circular center
Texture- Smooth
Elevation- Flat


We then individually created a t-streak of a colony of our favorite bacteria.

What is a T-streak you ask?
A t-streak is essentially the method of dipping a small gauged "looped" wire into a desired microbe and proceeding to smear this sample with said loop onto a portion of media. This is followed by sterilizing the loop by heating it in a bunsen burner, allowing the loop to cool, then dragging your loop through the previous "smearing" into a smaller portion of the media. This process is repeated from your second "smearing" into a new untouched portion of the media. The theory behind this practice is that with continued "smearing" eventually microbes will detach from the loop one organism at a time. After they fall off, a single colony of the desired microbe should proliferate from a single cell of the desired microbe. The reason it is called a T-streak, is because it the plate is divided with a "T" as seen above.

Here is a link to a Youtube video for a more in-depth description:

 http://youtu.be/DAm21yPGMRo

Food For Thought: Why Discover Dirt?

As Devin and I progress with our research we thought it may be helpful to discuss the importance of our dirty discoveries. I know what you're thinking: "Come on, it's just some small bacteria in dirt, how important could it really be?"

Well, believe it or not small bacteria in dumb ole dirt are actually responsible for many of the chemical processes that make Earth habitable by humans!  For starters, one of the most played-out examples of bacteria supporting human life is through nitrogen-fixation.

What is nitrogen fixation, you ask?

Well for starters, humans require Nitrogen as a source for many biomolecules (DNA, RNA, Proteins, and more!). The only source of useable nitrogen for these processes comes from symbiotic rhizobium. These symbiotic rhizobium convert Nitrogen Diatoms from the atmosphere, into a plant-usable form, ammonium (NH3 + H+ → NH4+). This now useable nitrogen is traded to legumes(think beans) in return for carbohydrates, proteins, and oxygen. As you may have guessed it, humans eat the legumes, or an animal who has eaten them and receive their useable nitrogen. If the nitrogen filled legumes are not consumed, they die and contribute their useable nitrogen into the soil, creating a fertile environment for plant growth.

A much more in-depth, technical, and ecological source of information can be found at www.Nature.com :

 http://www.nature.com/scitable/knowledge/library/biological-nitrogen-fixation-23570419

The Circle of life and Agriculture
Another type of bacteria is also responsible for the degradation of dead organic matter. Sure, we often attribute this to larger animals, but many soil microbes are vital in the carbon cycle and for distributing the nutrients of organic matter as it dies. These bacteria specifically are specialized in decomposing chitin and cellulose which are molecules of plant cells that are not easily digested by humans. This redistribution of resources via Nitrogen fixation and Carbon cycling provides nutrient rich soil for plant life. So you can see how these microbes could be useful in agriculture.

More in depth information can be found at the FAO corporate document repository:

http://www.fao.org/docrep/009/a0100e/a0100e0d.htm

Midnight Snack for thought: If we have rhizobium, why do we need Nitrogen fertilizers?

With your new appreciation for rhizobium and the job they do for us, you may be wondering why we need nitrogenous fertilizers for agriculture or even for just planting a small garden. Do you think the addition of fixed nitrogen to soil could effect the actions of nitrogen fixing bacteria? I'll leave you with this thought until next week.


Tune in next week to see if our microbes fair in a Gram-stain, and learn what the heck a Gram stain is! I'm Gram positive you'll love the results.

The What: To kick off our second lab of the semester we (DYLAN and DEVIN) are going to be DISCOVERING DIRT.
The Why: Dylan and I along with the rest of the Birmingham-Southern College BI 304 students will be trying to determine an unknown microbe in soil samples taken in random locations across the Birmingham-Southern campus.
The Where: The sample that Dylan and I will be handling was taken outside of the Stephens Science Center first floor entrance.

Our sample was taken from the surrounding soil at the base of  an Oak Tree outside the Stephens Science Center first floor entrance. In order to collect an adequate sample Dylan and I had to brush away all leaves, twigs, and other possible debris that had collected around the base of the tree. Doing this ensured that our sample was purely soil with no additional materials.

The soil that we collected was dense and somewhat moist, but not wet too wet to inhibit the experiment. The sample was surrounded by a few patches of grass and a few groups of dandelions. The fact that this sample is surrounded by plant life will provide more of a variety of bacteria because it will contain the roots of the nearby plants. This soil that contains plant roots is called rhizosphere, and luckily the location we picked had an abundant amount of rhizosphere.

The How: In order to collect our sample we had to obtain:
1.) 1 trowel.
2.) 1 scoopula.
3.) 2 alcohol wipes.
4.) 15 mL conical tube.
5.) 1 pair of latex gloves.
6.) 2-3 paper towels.
Collecting the Soil: to collect our soil we took our trowel and dug approximately 5 inches into the ground, making sure not to surpass a six inch depth. Once we had created our hole and collected our sample with our scoopula we placed the soil into our sanitized 15mL conical tube, then headed back to the lab!
Back in the Lab...
Sample Preparation: once back in the lab Dylan and I weighed out approximately 0.5g of soil in out balance and mixed it with 50mL of sterile water in a 50mL conical tube. The tube was then shaken to mix up the soil particles with the water and inverted to ensure a thorough mixing. Once all of the mixing was complete the sample was set aside so the soil could collect at the bottom of the conical tube...and now we wait...
Assessing Cultural Microbes: Dylan and I took 5 different microfuge tubes to carry out our dilutions (ranging from 10^-3 to 10^-7). Once we had completed all of our dilutions we set up 5 Tryptic Soil Agar Plates with corresponding soil dilution concentrations. Each plate received one drop (100 microliters) of its corresponding solution. Once the drop had been applied to the plate it was spread out with a freshly sterilized spreader, and was then closed and ready for incubation at room temperature.
      Once all of the Tryptic Soy Agar Plates had been covered and set aside it was time for the beautiful Rose-Bengal! The Rose-Bengal plates suppress bacterial growth, which was used in order to control the fast-growing bacteria colonies from taking over the plate. This single Rose-Bengal Plate was covered in the 10^-4 soil solution and covered for incubation.

Two Types of Agar Plates Directly After Addition of Soil Dilution Solutions

One of five Tryptic Soy Agar Destination Plates. This
plate contains the 10^-3 soil dilution (the most concentrated
of all of the dilutions). 

One of one Rose-Bengal Agar Plates. This plate
contains the 10^-4, which is the second most
concentrated dilution.

Check us out next week! Will there be growth...?