Gram Stain

1. Prepare heat-fixed smears of E. coli, S. aureus, and the mixture of E. coli and S. aureus (see figure 7.1).
2. Place the slides on the staining rack.
3. Flood the smears with crystal violet and let stand for 30 seconds (figure a).
4. Rinse with water for 5 seconds (figure b).
5. Cover with Gram’s iodine mordant and let stand for 1 minute (figure c).
6. Rinse with water for 5 seconds (figure d).
7. 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).
8. Rinse with water for 5 seconds (figure f ).
9. Counterstain with safranin for about 60 to 80 seconds (figure g). Safranin preparations vary considerably in strength, and different staining
10. times may be required for each batch of stain. (If you are color-blind, use Bismark brown stain rather than safranin.)
11. Rinse with water for 5 seconds (figure h).
12. 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.

Smear Preparation and Simple Staining

Bacterial smear and staining preparation
Smear procedure:

1. With the pen (permanent ink)/pencil, mark the name of the bacterial culture in the far left corner on each slides.
2. For the broth culture, shake the culture tube and, with an inoculating loop, aseptically (see figure bellow) transfer 1 to 2 loopfuls of bacteria to the center of the slide. Spread this out to about a 1/2-inch area. When preparing a smear from a slant or plate, place a loopful of water in the center of the slide. With the inoculating needle, aseptically pick up a very small amount of culture and mix into the drop of water. Spread this out as above.
   
3. Allow the slide to air dry, or place it on a slide warmer (see figure bellow).
4. Pass the slide through a Bunsen burner flame three times to heat-fix and kill the bacteria.

Simple Staining:

1. Place the fixed smears on a staining loop or rack over a sink or other suitable receptacle
2. Stain one slide with alkaline methylene blue for 1 to 1,5 minutes, or carbolfuchsin for 5 to 10 seconds, or with crystal violet for 20 to 30 seconds.
3. Wash stain off slide with water for a few seconds (see figure bellow).
4. Blot slide dry with bibulous paper.Be careful not to rub the smear when drying the slide because this will remove the stained bacteria.
5. Examine under the oil immersion lens.