1. The CCA phage hunt 2013 Phage

2. Lab outline Isolate phage visualize phage Soil samples

3. The host: Mycobacterium smegmatis Soil bacterium Mycobacteria and their phage are found in rich organic soil and mulch piles. Aerobic Hardy, resistant to: toxic compounds desication Harmless relative of the bacterium that causes tuberculosis

4. Bacteriophage = bacteria eater

5. Interesting facts about phage 10 31 phage on earth 10 25 infections/second Largest unexplored reservoir of genetic diversity Transfer genes between bacteria (transduction)

6. A gallery of mycobacteriophage

7. Genomics reveals that these 60 phage group into 9 major clusters, plus 5 singletons (phage that do not have sequenced relatives):

8. Sterile/Aseptic Technique Sterile: An environment (solution, tool) that is free of living organisms and viruses Aseptic: A process that is performed under sterile conditions

9. 1. To grow only the desired phage and bacteria, rather anything that can find its way to your petri dishes! The goals of sterile technique: 2. To prevent cross contamination of your phage samples 3. To minimize exposure to the phage and bacteria we work with

10. Things to remember Always know where your hands are. Never pass your hands or fingers over the top of a sterile field (open bottles, flasks, tubes and bottle caps, and agar plates). Never have more than one tube, bottle, or flask open on the bench at a time. Never set a bottle or tube cap or Petri dish lid on a bench top. Never turn the caps or lids UP.

11. More things to remember Always work with an open flame when opening sterile tubes or bottles. Never go into a sterile solution with a used pipette or tip. Change tips every time! The shaft of a pipetman is not sterile. Do not introduce it into a tube or bottle!

12. Start the phage hunt

13. Enrichment A method that favors the growth of certain organisms from a complex mixture, such as soil or water. The Gulf oil spill Enriched for oil- degrading bacteria that used the oil (as carbon & energy source)

14. Mycobacterium culture Our enrichment protocol Environmental sample Environmental sample Clear solution Filter Provides mycobacteria for mycobacteriophage to infect, so they will outcompete other phage

15. Enrichment protocol (part 1) Transfer ~1 gram of each sample to your flask Pour culture solution and broth into flask Label the flask with your initials, date, period, and sample name Cover top with aluminum foil and place on shaker overnight