1. Obtain four clean glass slides.
2. Using sterile technique, prepare a smear of each of the three organisms and on the remaining slide prepare a smear consisting of a mixture of S. aureus and E. coli. Do this by placing a drop of water on the slide and then transferring each organism separately to the drop of water on the slide with a sterile, cooled loop. Mix and spread both organisms by means of a circular motion of the inoculating loop.
3. Allow smears to dry air and then heat fix in the usual manner.
4. Flood smears with crystal violet and let stand for 1 minute.
5. Wash with tap water.
6. Flood smears with the Gram’s iodine mordant and let stand for 1 minute.
7. Wash with tap water.
8. Decolorize with 95% ethyl alcohol. Caution: Do not over-decolorize. Add reagent drop by drop until crystal violet fails to wash from smear.
9. Wash with tap water.
10. Couterstain with safranin for 45 seconds.
11. Wash with tap water for a maximum of 5 seconds
12. Blot dry with bibulous paper and examine under oil immersion.