These are my lab notes on Gram Staining from Microbiology Lab 9/1/09
Gram Staining: Done twice one with the broth and one with the one of our plates
1. Get two microscope slides
2. Turn on the bunsen burner by first opening the gas valve and then using a striker to ignite the flame
3. Heat the inoculating loop until the entire wire tip is red
4. Allow the loop to cool for a few seconds
5. Open your bottle of broth and run its top through the flame to sterilize it
6. Dip your inoculating loop into the broth cover the broth up and smear the loop on one of your slides (be sure to label which slide has which one it)
7. Repeat steps 3-6 on the same slide in order to get enough broth on the slide
8. Let the slide cool by setting it underneath the bunsen burner
9. One the next microscope slide obtain bacteria from your plate
10. Put a small drop of water on your slide using one of the bottles provided by Dr. Robson
11. Heat your inoculating loop again until red
12. Allow it to cool
13. Touch your inoculating loop to an isolated colony of bacteria on your streak plate
14. Smear the inoculating loop on the water drop on your slide
15. Allow the slide to air dry under the bunsen burner
The following steps are performed on both of the slides:
1. Heat/Methanol fix
2. Cover bacteria with crystal violet and let sit for 1 or more minutes
3. Rinse gently and blot
4. Cover bacteria with gram iodine for 30 sec
5. Rinse gently and blot
6. Decolorize bacteria with 5-12 drops of 95% E & OH
7. Rinse gently and blot
8. Cover with safrinin for more than 1 minute
9. Rinse gently and blot
10. Once slide is air dry view with microscope if time is available
Amy Blass
