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Working with bacteria

Table of contents
  1. Practice pipetting and serial dilutions
  2. Handprint Culture

Practice pipetting and serial dilutions

Serial dilution is a method used to manipulate the amount of something in a sample. In the future, you will be working with phage stocks of unknown concentrations. You will use serial dilutions to purify, amplify, and titer your phage. This protocol uses 10-fold serial dilutions, meaning that the concentration of dye in each tube is 10 times less than the previous tube, allowing for easy mathematical calculations.

Materials

  • Buffer
  • Blue dye requiring dilution
  • Microcentrifuge tubes

Procedure:

  1. Prepare your bench for aseptic work and assemble your supplies.
  2. ๐Ÿ–Š๏ธ๐Ÿงช Arrange the 7 microcentrifuge tubes in a rack and label them 10-1, 10-2, 10-3,โ€ฆ.10-7.
  3. โž• Add 90 ฮผl of buffer to each of the tubes.

    Perform 10-fold serial dilutions like in the picture below.

  4. โž• Add 10 ฮผl of your undiluted dye sample to the โ€œ10-1โ€ tube and vortex well.
  5. The solution in this โ€œ10-1โ€ tube contains 1/10th the concentration of dye as your undiluted sample. It is also referred to as a 1:10 dilution.

    Make sure to use a clean pipette tip for each transfer and pipette carefully, vortexing your sample well before making each dilution. Otherwise, you will not make accurate 10-fold dilutions.

  6. Transfer 10 ฮผl of the โ€œ10 -1โ€ sample to the โ€œ10-2โ€ tube and vortex well.
  7. This solution contains 1/100th as much dye as your undiluted sample. It can also be referred to as your 1:100 dilution.
  8. Continue each successive dilution until you get to your last tube. This tube is a 1:10 million dilution of your original dye!


Handprint Culture

Your skin is covered with bacteria. All of them are awesome, and youโ€™ve probably never seen them before. We are going to press our hands (gently) into an agar gel made of bacterial food, with or without antibiotics. If there are extra plates you can use something besides your hands. Then we are going to see what grows! PLEASE FEEL FREE TO DESIGN YOUR OWN SIMPLE EXPERIMENT! For example, if you wanted to see how the antibiotic Kanamycin impacts what grows, how could you test that?

Materials

  • Hands (unwashed!)
  • Anything else you want to stick in the agar
  • Agar plates (3 different kinds)
    • LB agar - made from digested milk proteins, yeast, and salt. TWO BLACK STRIPES
    • TSA - made from digested milk proteins, soybean, and salt. ONE BLACK STRIPE
    • TSA Kan - TSA plus kanamycin, an antibiotic that inhibits protein translation. ONE GREEN STRIPE

Procedure

  1. ๐Ÿงซ Grab some agar plates. There are enough to get one of each and a few extra.
  2. ๐Ÿ–Š๏ธ Label the bottom of your plate with your name, the type of plate, the date, and what you are putting on the plate (e.g., hand โ€“ filthy, hand โ€“ rinsed, hand โ€“ gloved, dollar bill, etc.) If you want to put more than one thing on the plate, make sure you label what went where.
  3. โœ‹ GENTLY press your unwashed hand into the agar. Try not to break it or smear your hand at all. Imagine you are trying to press your hand onto a sheet of JELLO without destroying it.
  4. ๐Ÿค‘ If you are curious, repeat Step 3 again but try a freshly washed hand, or put a glove on first. Or, try putting something dirty on the plate, like money. Or use the same object on a different kind of
  5. ๐ŸŒก๏ธ Incubate plates at either 30โ„ƒ or 37โ„ƒ overnight.

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