Microscope  Wooden applicator stick  Saline solution  Forceps  Bunsen burner PROCEDURE FOR MAKING SMEARS.—To prepare smears for microscopic examination, follow these steps: 1. Spread the specimen with a wood applicator stick across a slide that has been cleaned with alcohol or acetone and polished with lens paper. The smear should be thin and uniformly spread. If the smear is opaque, it is too thick and should be emulsified with a drop or two of saline. 2. Label the smear and circle the material to be stained with a diamond point pen for easier identification and location of the material after staining. 3. Let the smear air dry. Do not use forced heat drying; forced drying will distort bacterial cells and other materials. 4. Hold the smear with forceps and fix the smear by passing it through a flame (smear side up) three or four times. Avoid overheating the smear; overheating will cause cellular wall destruction. 5. Let the slide cool. Once the slide is cooled, it is ready to be stained. Gram’s Stain As previously explained, the most common staining procedure used in bacteriologic work is the Gram stain. This method yields valuable information and should be used on all smears that require staining. Gram’s stain is also used for examining cultures to determine purity and for identification purposes. PRINCIPLE OF GRAM STAINING.—As touched on previously, the crystal violet stain, the primary stain, stains everything in the smear blue. The Gram’s iodine acts as a mordant, a substance that causes the crystal violet to penetrate and adhere to the gram-positive organisms. The acetone-alcohol mixture acts as the decolorizer that washes the stain away from everything in the smear except the gram-positive organisms. The safranin is the counter-stain that stains everything in the smear that has been decolorized: pus cells, mucus, and gram-negative organisms. The gram-negative organisms will stain a much deeper pink than the pus cells and mucus will stain even lighter pink than the pus cells. MATERIALS REQUIRED FOR GRAM STAINING.—To Gram stain a smear, the following materials are required:  Gram stain kit, which consists of: —Crystal violet stain —Iodine or stabilized iodine (mordant) —Acetone-alcohol decolorizer —Safranin stain  Staining rack  Blotting paper or paper towel PROCEDURE FOR GRAM STAINING S M E A R S . — A f t e r s m e a r s h a v e b e e n d r i e d , heat-fixed, and cooled, proceed as follows: 1. Place the slide on a staining rack. Then flood slide with primary stain (crystal violet). Let stand 1 minute. 2. Remove the primary stain by gently washing with cold tap water. 3. Flood the slide with mordant (iodine or stabilized iodine) and retain on slide for 1 minute. 4. Remove mordant by gently washing with tap water. 5. Tilt slide at a 45-degree angle and decolorize with the acetone-alcohol solution until the solvent that runs from the slide is colorless (30 to 60 seconds). 6. Wash the slide gently in cold tap water. 7. Flood the slide with counter-stain (safranin) and let stand for 30 to 60 seconds. 8. Wash slide with cold tap water. 9. Blot with blotting paper or paper towel or allow to air dry. 10. Examine the smear under an oil immersion objective. Reading and Reporting Smears Place a drop of oil on the slide and, using the oil immersion objective of the microscope, read the 7-28