Gram Staining a Heat Fixed Smear Slide

Ingredients

• heat fixed smear mount
• paper towel
• pipettes
• distilled water
• small non corrosive glass or ceramic bowl
• ethanol
• lint-free cloth or tissue
• microscope
• hucker's crystal violet stain
• gram's iodine stain
• safranin O stain

Procedure for Gram Staining

1. Place a paper towel on a clean, flat surface, then place the heat fixed smear mount onto the towel.
2. Use a clean pipette to place a drop or two of Hucker's crystal violet stain onto the smear, then use the tip of the pipette (don't touch the smear) to spread the stain until it covers the entire smear.
3. Allow the Hucker's crystal violet stain to remain in contact with the smear for one minute.
4. Then, using a pipette filled with distilled water, hold the slide at an angle over the bowl and drip water above the stain so the slide floods with water and washes away any excess stain. 
5. Drain the slide and place it flat on the paper towel.
6. Use a clean pipette to place a drop or two of Gram's iodine stain onto the smear. Again, use the tip of the pipette to spread the stain over the entire smear.
7. Allow the Gram's iodine stain to remain in contact with the smear for one minute.
8. Fill a clean pipette with ethanol (drugstore 70% ethanol is fine). Hold the slide at an angle over the bowl and drip the ethanol above the smear so it floods the smear. Continue until the ethanol runs colorless.
9. Then repeat step 4 to rinse all of the ethanol from the slide. Drain the slide and place it flat on the paper towel.
10. Use a clean pipette to place a drop or two of safranin O stain onto the smear, and again use the tip of the pipette to spread the stain until it covers the entire smear.
11. Allow the safranin O stain to remain in contact with the smear for one minute.
12. Repeat step 4 to rinse excess safranin O stain from the slide.
13. Allow the slide to air dry. If you'r in a hurry, you can gently pat the slide dry with a lint free cloth or tissue. Do not rub the smear area.
14. Observe the slide under your microscope. You do not need a coverslip.
15. The Gram-negative bacteria should appear pink or red, and the Gram-positive bacteria should appear violet.

Observations

The bacteria stained only with
Hucker's crystal violet

After staining the smear with Hucker's crystal violet stain, I looked at it under the microscope but the bacteria was to dark to really identify anything except the blobs of bacteria.

 

The bacteria stained with Hucker's
crystal violet and Gram's iodine

After having stained the smear with Gram's iodine stain, the blobs of bacteria were considerably more transparent. I could also see that some parts of the blobs were lighter in color than the others, which could indicate that those spots were made up of Gram-negative bacteria.

After staining the smear with safranin O, I could clearly see the bacteria at 100x with the different colorations of blue and pink. I also noticed that there is more of the Gram-negative bacteria than the Gram-positive bacteria.