Wooden applicator stick
PROCEDURE FOR MAKING SMEARS.To
prepare smears for microscopic examination, follow
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
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
5. Let the slide cool. Once the slide is cooled, it is
ready to be stained.
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.
Grams 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
Grams iodine acts as a mordant, a substance that
causes the crystal violet to penetrate and adhere to the
mixture acts as the decolorizer that washes the stain
away from everything in the smear except the
The safranin is the
counter-stain that stains everything in the smear that
has been decolorized:
pus cells, mucus, and
organisms will stain a much deeper pink than the pus
cells and mucus will stain even lighter pink than the
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)
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
4. Remove mordant by gently washing with tap
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
Reading and Reporting Smears
Place a drop of oil on the slide and, using the oil
immersion objective of the microscope, read the