The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
STAINING BACTERIA AND USE OF THE MICROSCOPE
Background
There are many different ways to stain bacteria so that they can be more easily visualized under the microscope.
Some stains can also be used to identify and classify bacteria. The Gram stain is a differential stain that allows you
to classify bacteria as either Gram-positive or Gram-negative. The staining technique consists of four steps:
1) A primary stain (crystal violet). All bacteria are stained purple by this basic dye.
2) A mordant (Gram’s iodine). The iodine combines with the crystal violet in the cell to form a violet-iodine
complex.
3) A decolorizer (ethyl-alcohol). The primary stain is washed out of some bacteria, while others are unaffected.
4) A secondary or counterstain (safranin). This basic dye stains bacteria that have been decolorized red.
Bacteria that decolorize easily are called Gram-negative (red) and those that retain the primary stain are called
Gram-positive (purple). Bacteria stain differently because of differences in their cell walls. Gram-positive cell walls
consist of many layers of peptidoglycan. The crystal violet-iodine complex is larger than either the crystal violet or
iodine molecules that entered the cell and the complex cannot pass through this thick cell wall. Gram-negative
bacteria have a thin layer of peptidoglycan and an outer lipopolysaccharide layer. The alcohol dissolves the
lipopolysaccharides so that the crystal violet-iodine complex can wash out of the cell.
Before bacteria can be stained, a smear of bacteria must be made on a slide and heat fixed. A smear is made by
spreading a bacterial suspension on a clean slide and letting it air dry. The dry smear is heated on a hot plate or
passed through a flame several times to heat fix it. Heat fixing denatures bacterial enzymes, preventing them
from digesting cell parts, which causes the cell to break, a process called autolysis. The heat also enhances the
adherence of bacterial cells to the slide.
Each group will be provided with:
– Gram staining reagents:
 Crystal violet
 Gram’s iodine
 Decolorizer
 Safranin
– Wash bottle of distilled water
– Slides
– Sterile inoculating loops
– Forceps
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
THE GRAM STAIN
1) Prepare a bacterial smear.
a) Place 10 μl of sterile water in the center of a clean glass slide.
b) Remove lid from culture plate with free hand. Do not place lid on bench. Hold lid face
down just above culture plate to help prevent contaminations from falling on plate or lid.
c) Use a sterile inoculating loop to scrape up a visible cell mass from a colony. Do not gouge
agar. Replace culture lid.
d) Mix bacteria with water drop on slide. Spread the water-bacteria mixture over an area of
about 1 inch square.
2) Allow the smear to air dry.
3) Hold the slides with forceps and heat fix the smears by heating on the hot plate for several
minutes.
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
4) Staining
a) Cover the smear with a few drops of crystal violet (1) and leave for 1 minute.
b) Turn the slide over so the smear is facing down. Wash the slide carefully over the top
with distilled water from a wash bottle or the sink until no large amounts of color wash
off. Do not squirt water directly onto the smear.
c) Cover the smear with Gram’s iodine (2) and leave for 1 minute.
d) Without washing, decolorize with 95% ethyl alcohol (Decolorizer (3)). Wash the slide
carefully over the top with distilled water from a wash bottle or the sink until no large
amounts of color wash off.
e) Immediately rinse with distilled water.
f) Cover the smear with safranin (4) and leave for 1 minute.
g) Gently rinse with distilled water.
h) Blot dry with a paper towel or absorbent paper. Do not rub slide, as the bacterial smear
can be rubbed off.
5) Observe under the microscope.
6) Record your observations from the microscope.
Is it Gram-positive or Gram-negative?
What shape are the bacteria?
How are the cells arranged relative to one another?
Sketch a few bacteria as you see them in the microscope in the circle below.
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
What are some other things, besides staining, that might be affected by the properties of the
cell wall that differentiate Gram-positive and Gram-negative bacteria?
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
GRAM STAINING OF ORAL SAMPLES
Background
Microorganisms are found everywhere in the environment. One common place where bacteria
can be found is in the mouth and throat. We will be collecting samples from the mouth and
throat and doing a Gram stain on the samples we collect and examining them under the
microscope. There are three basic shapes of bacteria:
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
Each group will be provided with:
– Gram staining reagents:
 Crystal violet
 Gram’s iodine
 Decolorizer
 Safranin
– Wash bottle of distilled water
– Slides
– Sterile swabs
– Clothes pins
Protocol
1) Place 10 μl of sterile water in the center of a clean glass slide.
2) Open individually wrapped sterile swab, and collect sample from mouth or have someone else
collect sample from back of throat.
3) Mix sample with water on slide.
4) Follow instructions from Gram staining exercise starting with step 2.
5) Record your observations from the microscope.
Is it Gram-positive or Gram-negative?
What shape are the bacteria?
How are the cells arranged relative to one another?
Are there cells that don’t look like bacteria in size or shape? What are they?
The Young Scientist Program
Microbiology Teaching Team
http://medicine.wustl.edu/ysp
Funding by Pfizer Inc.
Sketch a few bacteria as you see them in the microscope in the circle below.