Gram Staining

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Block C3 - Childhood


Difference between Gram positive and Gram negative[edit]

Gram Positive[edit]

  • Violet dye retained by cells.
  • Darker, denser, and larger appearing cells under the microscope.
  • Thick, multi-layered peptidoglycan layer.
  • Teichoic acids present.

Gram Negative[edit]

  • Pink or red dye retained by cells.
  • Thin single-layered peptidoglycan layer.
  • Outer layer of lipopolysaccharides.
  • Periplasmic space present.
  • High lipid and lipoprotein content due to existence of outer membrane.

Regents[edit]

  1. Gram A - Crystal violet (primary stain)
  2. Gram B - Iodine (mordant)
  3. Gram C - Ethanol-acetone (decolorizer)
  4. Gram D - Safranin (counterstain)

Procedure[edit]

  1. Put bacteria on film:
    1. Aseptically pull out the loop (attached to the cap) on the bacteria sample test-tube.
    2. Gently rub the loop onto the thin film.
  2. Dry the film:
    1. Air dry it by waving the thin film gently in the air.
    2. Once dry, drop a couple of drops of 1% formalin onto the film. Air dry again.
    3. Quickly pass the film over a flame three times to heat fix. Heat fixing allows remaining water to evaporate, kills cells so they're safe to handle, and allows cells to absorb stains easier.
  3. Gram staining:
    1. Add 5 drops of crystal violet stain (Gram A). Wait 1 to 3 minutes, and then pour it off into chemical waste container.
    2. Add 5 drops of iodine solution (Gram B). Wait 1 minute, pour it off, and then rinse the slide with water.
    3. Add a few drops of decolorizer (Gram C). Wait 5 seconds, and then rinse it off.
    4. Add 5 drops of safranin (Gram D). Wait 10 seconds, and then rinse it off.
    5. Air dry or use filter paper to absorb and remove water from the slide.
  4. Examine under microscope:
    1. Start examining at 10x and then move to 40x.
    2. Once it is in focus at 40x, put a drop of immersion oil on the film and switch to the 100x lens.

Exam[edit]

For batch 2011, the exam consisted of a pre-test and an open-book practical. The pre-test asked one question only: "Describe how to properly collect the sample." The practical exam required students to create their own films using a bacterial sample and then stain them correctly. The instructor then asked questions personally to each student, such as: "What did you see in the microscope?", "What type of bacteria do you think is present?", "Describe the distribution of the bacteria", and "Which bacteria is gram-positive and which is gram-negative? How do you know?"