Tiny Earth Process

Part A Biology- TEPIs isolate antibiotic-producing bacteria from the soil

Soil is a great place to look for antibiotic- producing bacteria!

Productive soil has 100,00 million to 1 billion bacteria per teaspoon. So in 2 liters of rich soil with 100,000 million bacteria per teaspoon, there would be more bacteria than starts in the Milky Way Galaxy! ( High estimates of stars is 400 billion)*

About 75% of the antibiotic in clinical use were discovered in soil bacteria.**

At Tiny Earth’s Partner Schools…

1. Students take soil samples. Data about the soils is recorded in our database for later usage.

Photo by Bryce Richter / UW-Madison

2. Dilute soil with saltwater.  We only want to focus on a few of the bacteria.

3.Place dilutions on petri dishes, incubate, and grow bacteria on dilution plates

Photo by Celia Puleo

 

 

 

 

 

 

4.Transfer bacteria onto master plates to allow colonies to grow more

Photo Property of Wisconsin Institute for Discovery
Photo by Celia Puleo

 

 

 

 

 

 

 

5. Transfer to screening plate with a safe relative of an ESKAPE pathogen and look for Zones of Inhibitions  (the clear area around the bacteria where it is producing an antibiotic that kills the pathogen!)

Photo by Celia Puleo

6. Once antibiotic producing bacteria are found, they are either sent to the Chemistry hub, or students might run further protocols and experiments on them

Part B Chemistry- Extract chemicals and identify the species of antibiotic

At the Tiny Earth Chemistry Hub (TECH) at the Wisconsin Institute for Discovery at UW Madison we have 8 Undergraduate interns and a few post-docs working to process these samples. They…

7. Freeze bacteria

Photo by Celia Puleo
8. Test for tagalong bacteria

Photo by Celia Puleo

Some times there are more than just 1 bacteria in one isolate shipped into the chemistry hub. Doing a 3 way street can test for these tagalongs.

9. Repeat steps 4 and 5 to screen more (additional screening of actinomycetes may occur)

Photo by Celia Puleo

10. Identify species of antibiotic-producing bacteria

a. Amplify (make lots of) copies of the 16S gene with a PCR reaction.

b. Run the product on a gel to determine if amplification occurred by detecting a 1500 nucleotide band.

c. Submit product of successful PCR reactions to Functional Biosciences for sequencing

Photo by Celia Puleo
This is a PCR Machine that is making copies of a 16S a part of the bacteria’s DNA.

11. Test molecular compound without bacteria in assay

Photo by Celia Puleo
12. Chromatography

Photo by Celia Puleo

13. Isolate bacteria from molecular compound→Future Mass Spectrometry to identify what the antibiotic molecule is

Once we test the most promising bacteria’s molecular compounds through the Mass Spectrometry we will be able to see if we have discovered a new antibiotic! And when we can figure out what species the bacteria is, we can trace the antibiotic back to the species producing it!

Want to learn more and be part of this amazing process? See if your college has a Tiny Earth class on the Our Network page under the Community tab!

*Fact paraphrased from  Ingham, Elaine R. “Natural Resources Conservation Service.” Soil Bacteria | NRCS Soils, NRCS, www.nrcs.usda.gov/wps/portal/nrcs/detailfull/soils/health/biology/?cid=nrcs142p2_053862.

High estimate of starts in Milky Way galaxy from Masetti, Maggie. “How Many Stars in the Milky Way?” NASA, NASA, 22 July 2015, asd.gsfc.nasa.gov/blueshift/index.php/2015/07/22/how-many-stars-in-the-milky-way/. Comparison of bacteria in soil to Milky Way calculated by Celia Puleo

**Fact from Tiny Earth’s Brochure

Special thanks to Martel DenHartog, Pat Pointer, Amanda Hurley, and Sarah Miller who helped review this process and edit this page.