Gram staining is a method developed by the Danish Scientist Hans Christian Gram in 1884 to distinguish two types of bacteria based on the structure of their cell wall. Gram-positive bacteria stain purple because of its thick peptidoglycan layer in the cell wall. Gram-negative bacteria will stain pink due to having a very thin peptidoglycan layer. The gram staining characteristic of the bacteria will not only tell us about differences in the cell wall but also differences in the behavior of the two types of bacteria.

Function of the gram staining chemicals in this procedure

  1. Crystal Violet: Penetrate and stain cell wall and membrane
  2. Iodine: Binds with crystal violet and traps it in the cell to give a purple color
  3. Alcohol: A decolorizer to remove purple stain from cell wall. Gram positive bacteria will retain purple stain due to thick wall while gram negative will lose purple stain due to thin wall
  4. Safranin: A pink counter-stain. This will have no effect on the crystal violet in the gram positive bacteria. However since gram negative bacteria is decolorized, it will stain pink with the safranin

Gram negative bacteria

Gram positive bacteria

Culture Interpretation From Previous Class

In the previous class you cultured bacteria from a washed hand and an unwashed hand. The red-violet colonies that you now see on the bacteria plate are either S. aureus, S. hyicus or S. intermedius. These are all gram-positive bacteria so they will stain the same (purple). Further differentiation procedure can be done to isolate S. aureus but we will not go to that step in this lab. Just count all red-violet colonies as S. aureus. Each red colony represents millions of bacteria. Count the number of colonies you have. The recommended limit on 3M Petri plate Staph Express Count Plate is 150 colonies. If you have more than this, just label it as TNTC (too numerous to count). For easy count, you can look for a square that has a number of colonies that are representative of the rest. Count the number of colonies in that square and multiply it by 30 since the area of the inoculated area is 30 cm2.

Transferring Bacteria from Culture

Caution: Remember that S. aureus is a pathogen. Make sure to handle it carefully and wear gloves. Although you will wear gloves, please still wash your hands thoroughly at the end of the lab.

  1. Collect a clean microscope slide
  2. To the far left of the slide, add your initial using a wax pencil
  3. On that same side of the slide, use the wax pencil to draw a circle in the center of the slide. This is where you will be putting your bacteria. This step is important in helping you to find the position of the bacteria when it comes time to view it under the microscope
  4. Using a sterile loop, add a drop of water on the center of the side (directly opposite to the side where you drew the circle)
  5. Using the same sterile loop, touch a colony of the bacteria and transfer it by mixing the tip of the loop in the water
  6. Use a board clothespin as a handle to hold the slide and then pass the slide gently through a Bunsen burner flame until all the water is dehydrated. This will fix the bacteria on the slide. Now that your bacteria is fixed, you are ready to stain it

Gram Staining Bacteria

Note: Make sure that in this step you are staining the side that has the bacteria and not the opposite side with the wax and label. 

  1. Cover the bacteria with crystal violet and hold for 60 seconds
  2. Rinse with distilled water
  3. Cover the bacteria with iodine for 60 seconds
  4. Rinse with distilled water
  5. Decolorize the bacteria by washing with alcohol and promptly rinse with distilled water to avoid excessive decolorization
  6. Add safranin to counter-stain and hold for 60 seconds
  7. Rinse with distilled water
  8. Gently dab slide between Kim wipes to dry
  9. Observe under microscope using 100X oil immersion objective (note the guidelines below)

Using the Microscope

  1. Focus the slide on 10X first and then 40X
  2. Once you can see clearly on the 40X, move the lens away from the slide and hold between the 40X and 100X. Now drop a small drop of oil on the slide and move to the 100X
  3. At this point, use the fine adjustment ONLY to focus on the bacteria
  4. If you cannot find the bacteria, go back to the 10X to refocus. DO NOT PASS THE LENS THROUGH THE 40X SINCE YOU WILL GET OIL ON IT. THIS WILL DAMAGE THE LENS!
  5. Once have a sharp image on the 10X, go back to 100X. DO NOT PASS THROUGH THE 40X!
  6. If this does not work, lower the stage of the microscope, remove the slide, and clean off oil by dapping with Kim wipe and start over
  7. If by accident the 40X gets contaminated with oil (hope this does not happen), clean quickly by dabbing with lens paper. DO NOT USE KIM WIPE OR ANY OTHER KIND OF PAPER ON THE LENS AS THIS WILL SCRATCH IT!
  8. When you are done with the lab, clean the oil immersion lens by dabbing with lens paper
  9. Shut down the microscope:
    • Turn the lens to the lowest power (10X)
    • Turn off microscope power
    • Completely lower the stage
    • Center the slide arm
    • Unplug and wrap the cord around back
    • Replace the cover on microscope
    • Put back in the microscope cabinet carrying it with both arms (one arm holding the arm of the microscope and the other holding the base¬†¬†

Lab Questions (Total points = 20)

  1. Label the parts of the microscope below (7 points)
  2. Calculate the magnification of the most powerful lens on the microscope you used (2 point)
  3. Draw the gram stain that you see in the microscope (2 points)
  4. Based on your gram statin results, is Staphylococcus aureus gram-negative or positive? (1 point)
  5. Draw a table showing the bacteria count on the plate for students who washed their hands and those who did not (include your results). (4 points)
  6. What conclusion can you make about the importance of handwashing in food handling? (2 points)
  7. For students who washed their hands, what conclusion can you make about how effective the handwashing was? (2 points)

Courtney Simons
Courtney Simons is a food science professor. He holds a BS degree in food science and a Ph.D. in cereal science from North Dakota State University.
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