Difference between Gram positive and Gram negative
- Violet dye retained by cells.
- Darker, denser, and larger appearing cells under the microscope.
- Thick, multi-layered peptidoglycan layer.
- Teichoic acids present.
- 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.
- Gram A - Crystal violet (primary stain)
- Gram B - Iodine (mordant)
- Gram C - Ethanol-acetone (decolorizer)
- Gram D - Safranin (counterstain)
- Put bacteria on film:
- Aseptically pull out the loop (attached to the cap) on the bacteria sample test-tube.
- Gently rub the loop onto the thin film.
- Dry the film:
- Air dry it by waving the thin film gently in the air.
- Once dry, drop a couple of drops of 1% formalin onto the film. Air dry again.
- 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.
- Gram staining:
- Add 5 drops of crystal violet stain (Gram A). Wait 1 to 3 minutes, and then pour it off into chemical waste container.
- Add 5 drops of iodine solution (Gram B). Wait 1 minute, pour it off, and then rinse the slide with water.
- Add a few drops of decolorizer (Gram C). Wait 5 seconds, and then rinse it off.
- Add 5 drops of safranin (Gram D). Wait 10 seconds, and then rinse it off.
- Air dry or use filter paper to absorb and remove water from the slide.
- Examine under microscope:
- Start examining at 10x and then move to 40x.
- Once it is in focus at 40x, put a drop of immersion oil on the film and switch to the 100x lens.
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?"