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Media preparation
and training

Experimental Biosciences Resources

Training Sessions

Session 1: Lab tour and introduction to media preparation

  • An instructor will take you on a tour of the laboratory. Here's is what you should learn from the tour:
  • Locations of supplies, media bench, autoclave, sterile cabinet for pouring plates, incubators, gas shutoff valve, fire extinguisher, fire alarms, glass waste container, "death row" for disposing of agar plates
  • General considerations for preparing media, including
    • Measuring and rehydrating prepared media
    • Loading and starting an autoclave using a liquid cycle
    • Safely removing materials from an autoclave
    • Cooling media and pouring agar plates using a laminar flow hood
    • How to prepare agar slant tubes
    • How to prepare broth tubes with Durham inserts
  • Sterilizing and/or cleaning contaminated materials, including broth tubes, inserts, closures, media bottles, and contaminated agar plates

Session 2: Aseptic technique, serial dilution and spread plate techniques

As the teams prepare agar plates for the projects, we will introduce you to the procedures for processing samples for Project 1.

Session 3: Culture methods and Gram staining

Step by step, an instructor will demonstrate the technique, explain the rationale, and then you will carry out the step individually. After your introduction to the technique you will then carry it out again for practice. If you are to successfully carry out Project 1 you must become proficient at preparing smears, Gram stains, dilution streak plates, and spread plates.

  1. From the agar plate provided, aseptically prepare three bacterial smears on a single marked slide.
  2. While the smears dry, prepare two dilution streak plates, each from a different colony, and incubate inverted at RT.
  3. Prepare Gram stains from the dried smears, to be examined during session 3.

Session 4: Microscopic observation of Gram stains and living bacteria

  1. Using the Gram stains from Session 3, review use of the Nikon Labophot microscope, including dark field and phase contrast optics.
  2. Learn to use the oil immersion method, observe each Gram stain at 1000x, and record observations.
  3. Examine your streak plates, correct mistakes if necessary
  4. Prepare wet mounts of living bacteria from your streak and/or spread plates and view them in dark field and in phase contrast, observing motility and endospores if possible.

Created by David R. Caprette (caprette@rice.edu), Rice University 27 Dec 10
Updated 15 Mar 16
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