Materials

• Colonies of bacteria from Exercise 12.2
• Toothpicks
• Crystal violet
• Gram''s iodine
• 95 ethanol
• Safranin
• Microscopes with oil immersion

Procedure

1. Before staining the individual colonies, you should first practice the technique by observation of the gram positive micro- organisms normally found in the gum linings of your mouth.

2. Use a clean toothpick to rub along the gingival crevices (area between tooth surface and gums) of your mouth.

   Rub lightly!

3. Mix the scrapings with a drop of water previously placed on a clean slide, spread in a thin film over the center of the slide and allow to air dry.

4. Fix the smear to the slide by passing the slide (smear side up) quickly through a flame three times. If the slide is held directly in the flame, it will heat up too rapidly and break. The trick is to gently dry the smear without overheating the slide.

5. Place the slide on a staining rack. Apply the stains on the fixed smear as follows:
   • Flood the slide with crystal violet for 30 sec.
   • Rinse with water.
   • Flood with Gram''s iodine for 60 sec.
   • Rinse with water.
   • Decolorize with 95 ethanol.
   • Rinse with water.
   • Counterstain with safranin for 60 sec.
   • Rinse with water and blot dry (no rubbing!).
   • Examine under oil-immersion objective lens. 3

   Gram-positive bacteria retain crystal violet after washing with 95 ethanol, while gram-negative bacteria lose the purple dye after washing with 95 ethanol. The positive or negative reaction is a measure of the presence or absence of specific polysaccharide components of their cell walls. Safranin is used as a pink counterstain, so that Gram - cells can be visualized. In practice then, the distinction is made between purple cells (+) and pink cells(-).

6. Determine the basic cell shape of the bacteria.

7. Add the information on Gram stain and cell shape to the work done in Exercise 12.2.