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- Prepare heat-fixed smears of E. coli, S. aureus, and the mixture of E. coli and S. aureus (see figure 7.1).
- Place the slides on the staining rack.
- Flood the smears with crystal violet and let stand for 30 seconds (figure a).
- Rinse with water for 5 seconds (figure b).
- Cover with Gram’s iodine mordant and let stand for 1 minute (figure c).
- Rinse with water for 5 seconds (figure d).
- Decolorize with 95% ethanol for 15 to 30 seconds. Do not decolorize too long. Add the decolorizer drop by drop until the crystal violet fails to wash from the slide (figure e). Alternatively, the smears may be decolorized for 30 to 60 seconds with a mixture of isopropanol-acetone (3:1 v/v).
- Rinse with water for 5 seconds (figure f ).
- Counterstain with safranin for about 60 to 80 seconds (figure g). Safranin preparations vary considerably in strength, and different staining
- times may be required for each batch of stain. (If you are color-blind, use Bismark brown stain rather than safranin.)
- Rinse with water for 5 seconds (figure h).
- Blot dry with bibulous paper (figure i) and examine under oil immersion. Gram-positive organisms stain blue to purple; gram-negative organisms stain pink to red. There is no need to place a coverslip on the stained smear. See figure bellow for an example of gram-positive and gram negative bacteria.
