1. Gram Staining Method Mr. Beyer

2. Individual Supplies Microscope Slide Clothes Pin Innoculating Loop Paper Towels

3. Shared Supplies Crystal Violet Stain Safranin Red Stain Gram’s Iodine Ethyl Alcohol (denatured) Water Bottle Rinsing Basin Alcohol Burner Wax Pencil Matches Microscope

4. Create a Staining Area On Your Microscope Slide Obtain a clean microscope slide Draw a circle on your slide with wax pencil Warming the slide will make the wax pencil work better when drawing your circle

5. Create a Suspension of Bacteria Place 1-2 drops of water inside the circle on your microscope slide Use the inoculating loop to obtain a tiny sample of bacteria from your bacteria culture Add the bacteria sample to the water on your microscope slide, mixing to create a suspension of bacteria

6. Heat Fixing Bacteria to the Microscope Slide Pass your microscope slide through the flame until all of the water evaporates This will leave your bacteria stuck or “fixed” to the microscope slide

7. Primary Staining of Gram Positive Bacteria Attach the clothes pin to one end of your slide Place 2-3 drops of Crystal Violet Primary Stain inside your circle (on your bacteria) Leave the stain on the bacteria sample for one minute Rinse stain off with water (gently)

8. Mordant (Sealer) Add the Gram’s Iodine to your bacteria sample inside the circle Leave Gram’s Iodine on your sample for one minute Rinse off with water

9. De-Colorizing Add ethyl alcohol to the bacteria Let the alcohol set, then rinse with water Continue de-colorizing until the crystal violet stain is no longer released from your sample. You should just be able to see a hint of purple at this point

10. Counter-Staining Gram Negative Bacteria Add 2-3 drops of Safranin Red Stain to your bacteria sample inside the circle on your slide Leave safranin red on your bacteria sample for one minute Rinse with water, blot or air dry

11. Viewing Bacteria View under oil immersion lens of microscope (1000x magnification) Sketch in color, Label bacteria as gram (+) or gram (-) Identify shape and arrangement of bacteria

12. Gram Positive Coccus

13. Gram Positive Streptococcus

14. Gram Positive Rods (Bacillus)

15. Gram Positive

16. Gram Positive Staphylococcus

17. Gram Negative Bacillus

18. Gram Negative

19. Gram Negative vs. Gram Positive NegativePositive

20. Gram (+) and (-) Cell Structure