# 3.7: Gram Stain

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## Preparation

1. Have test tubes with sterile TSB on hand.
2. 12-18 hours before the laboratory, inoculate test tubes of sterile TSB with a mixture of Escherichia coli and Staphylococcus aureus and incubate at 37 °C .
##### Note

Remember that you will have multiple groups needing these cultures so make enough cultures so each group has their own or enough so each group only shares the culture with another group.

## Materials

• 12-18 hour cultures of E. coli and S. aureus mixture - see Preparation section above
• plain microscope slides (at least 1 per group)
• Bunsen burners (1 per group)
• inoculating loops (1 per group)
• (if using bacteria on solid medium rather than TSB) droppers with saline, DI water, or distilled water
• slide warmer
• test tube racks (1 per group plus one or more racks to hold cultures)
• wood test tube clamps/holders (1 per group)
• small chemical waste containers (1 per group)
• crystal violet in a dropper bottle (1 per group or enough for two groups to share one bottle)
• Gram's iodine in a dropper bottle (1 per group or enough for two groups to share one bottle)
• 95% ethanol in a dropper bottle (1 per group or enough for two groups to share one bottle)
• safranin in a dropper bottle (1 per group or enough for two groups to share one bottle)
• distilled or DI water in squirt bottles (1 per group)
• bibulous paper (enough for each groups to blot their slides)
• light microscopes (1 per group)
• colored pencils
• (optional, but recommended) Wax pencils (1 per group)
• (if using oil immersion) immersion oil dropper bottles
• (if using oil immersion) lens paper

This page titled 3.7: Gram Stain is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Rosanna Hartline.