LABORATORY ACTIVITY FOR MEDICAL BIOLOGY STUDENTS
Palawan State University
Activity No. 3: Gram Staining
I. Materials
• Dropper/disposable pipet • Cultured bacteria
• Lab gown • Tissue paper
• Gloves • Alcohol lamp
• Glass slides • Petri dish
• Cover slip • Inoculating loop
• Board marker • Stains:
• Distilled water ✓ Crystal violet
• Wash bottle ✓ Grams iodine
• Wooden stick ✓ Grams decolorizer
• Microscope ✓ Safranin
, II. Procedure
1. Preparation of the glass microscopic slide
Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil
from the fingers on the slides is removed by washing the slides with soap and water. Wipe the
slides with alcohol. After cleaning, dry the slides and place them on laboratory towels until
ready for use.
2. Labeling of the slides
Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful
to clearly designate the area in which you will prepare the smear. You may also label the slide
with the initials of the name of the organism on the edge of the slide. Care should be taken that
the label should not be in contact with the staining reagents.
3. Preparation of the smear
• Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth culture
on the slide. Spread by means of circular motion of the inoculating loop to about one
centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement.
A satisfactory smear will allow examination of the typical cellular arrangement and isolated
cells.
• Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or saline
solution on the slide. Sterilize and cool the loop again and pick up a very small sample of a
bacterial colony and gently stir into the drop of water/saline on the slide to create an
emulsion.
• Swab Samples: Roll the swab over the cleaned surface of a glass slide
4. Heat Fixing
• Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows
the sample to more readily take up stains.
• Allow the smear to air dry.
• After the smear has air-dried, hold the slide at one end and pass the entire slide through the
flame of a Bunsen burner or alcohol lamp two to three times with the smear-side up.
• Now the smear is ready to be stained. Please Note: Take care to prevent overheating the
slide because proteins in the specimen can coagulate causing cellular morphology to appear
distorted.
5. Gram Stain Procedure
a) Place slide with heat fixed smear on staining tray.
b) Gently flood smear with crystal violet and let stand for 10 seconds.
c) Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
d) Gently flood the smear with Gram’s iodine and let stand for 10 seconds.
e) Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
The smear will appear as a purple circle on the slide.
Palawan State University
Activity No. 3: Gram Staining
I. Materials
• Dropper/disposable pipet • Cultured bacteria
• Lab gown • Tissue paper
• Gloves • Alcohol lamp
• Glass slides • Petri dish
• Cover slip • Inoculating loop
• Board marker • Stains:
• Distilled water ✓ Crystal violet
• Wash bottle ✓ Grams iodine
• Wooden stick ✓ Grams decolorizer
• Microscope ✓ Safranin
, II. Procedure
1. Preparation of the glass microscopic slide
Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil
from the fingers on the slides is removed by washing the slides with soap and water. Wipe the
slides with alcohol. After cleaning, dry the slides and place them on laboratory towels until
ready for use.
2. Labeling of the slides
Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful
to clearly designate the area in which you will prepare the smear. You may also label the slide
with the initials of the name of the organism on the edge of the slide. Care should be taken that
the label should not be in contact with the staining reagents.
3. Preparation of the smear
• Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth culture
on the slide. Spread by means of circular motion of the inoculating loop to about one
centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement.
A satisfactory smear will allow examination of the typical cellular arrangement and isolated
cells.
• Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or saline
solution on the slide. Sterilize and cool the loop again and pick up a very small sample of a
bacterial colony and gently stir into the drop of water/saline on the slide to create an
emulsion.
• Swab Samples: Roll the swab over the cleaned surface of a glass slide
4. Heat Fixing
• Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows
the sample to more readily take up stains.
• Allow the smear to air dry.
• After the smear has air-dried, hold the slide at one end and pass the entire slide through the
flame of a Bunsen burner or alcohol lamp two to three times with the smear-side up.
• Now the smear is ready to be stained. Please Note: Take care to prevent overheating the
slide because proteins in the specimen can coagulate causing cellular morphology to appear
distorted.
5. Gram Stain Procedure
a) Place slide with heat fixed smear on staining tray.
b) Gently flood smear with crystal violet and let stand for 10 seconds.
c) Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
d) Gently flood the smear with Gram’s iodine and let stand for 10 seconds.
e) Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
The smear will appear as a purple circle on the slide.
