GRAM STAINING

• Inoculating quadrant #1


  1. Sterilize inoculating loop

  2. Allow loop to cool

  3. Tease cells out of quadrant #1 into quadrant #2
• Streaking quadrant #2


  1. Sterilize loop

  2. Streak from quadrant #1 into quadrant #2
• Streaking quadrant #3


  1. Sterilize loop

  2. Streak from quadrant #2 into quadrant #3 without touching quadrant #1
• Streaking quadrant #4


  1. Sterilize loop

  2. Streak from quadrant #3 into quadrant #4 without touching any other quadrant
• Label plates properly
• Incubate plate upside down at 37C for 24 hours
• Preparing bacterial smear

  1. Draw 3 circles on slide

  2. Transfer unknown bacterial colony to middle circle

  3. Transfer Staphylococcus epidermidis to left circle

  4. Transfer Escherichia coli to right circle

  5. Heat fix slide
• Performing Gram stain


  1. Flood slide with crystal violet for 1 minute

  2. Cover with Gram's iodine for 1 minute

  3. Flood with 95% acetone alcohol for 15-20 seconds

  4. Flood with safranin for 1 minute

  5. Examine under oil immersion
• Viewing bacteria under oil immersion

  • Start at low power (100x)
  • Use coarse adjustment to focus
  • Switch to oil immersion objective (100x)
  • Place drop of immersion oil on slide
  • Use fine focus to sharpen image
• Caution when using oil immersion objectives and immersion oil
• Take micrographs of specimens viewed at 1000x
• Wash down bench with disinfectant at end of lab
• Gram positive control


  Microorganism seen, scientific name, color of stain retained, cell morphology and Gram stain reaction
• Gram negative control


  Microorganism seen, scientific name, color of stain retained, cell morphology and Gram stain reaction
• Unknown specimen


  Microorganism seen, scientific name, color of stain retained, cell morphology and Gram stain reaction
• Why is Gram staining important?
• What does aseptic transfer mean and why was it important?
• What is the purpose of preparing an isolation streak plate?