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.