SIMPLE AND DIFFERENTIAL STAINING OF BACTERIA

In previous exercises you have observed live bacteria via compound brightfield
microscopy. This approach allows one to observe bacteria in terms of their motility and
provides some insight on the organisms overall morphology. However, since the
bacterial cell is transparent and motile and therefore somewhat difficult to observe when
using the compound brightfield microscope, cells are often fixed and stained to make
them more visible. Staining provides a reliable means for observing bacteria in terms of
their relative size, morphology and cellular arrangement.
Stains are solutions of a dye that has been dissolved in water or alcohol. The dye
is a salt (like Na+Cl-). One component of the dye consists of a negatively charged ion,
while the other component of the cell consists of a positively charged ion. The ionic
component of the dye that imparts color to the cell is called a chromophore. In the case
of basic dyes the chromophore is positively charged. The positively charged
component of a basic dye is able to interact strongly with a bacterial cell, because most
cells under normal conditions have an overall negative charge. The stains that we will
be using in the following exercises, methylene blue, crystal violet, malachite green and
safranin (red) are all examples of basic dyes.
You will be observing fixed bacteria subjected to simple staining in the first
exercise. In a simple stain a basic dye is added to a population of bacteria that has been
fixed to the surface of a microscope slide such that all cells in the population take on the
color of the dye used. In the second set of exercises you will be performing a Gram
stain (a type of differential stain). Gram staining involves the use of two different basic
dyes, one that stains a sub-population of the cells and the other that stains another
subpopulation. While the simple and differential stains both provide information with
respect to size, morphology and bacterial arrangement, the Gram stain is used to
distinguish between bacteria with different cell wall structures and compositions.
Bacteria can be divided into two major subclasses with respect to how they react to the
basic dyes employed in a Gram stainGram positive or Gram negative. Gram positive
and Gram negative bacteria both have cell walls that are comprised of peptidoglycan.
The cell wall of Gram positive bacteria are very thick (up to 80 nm thick); the
peptidoglycan that make up the cell wall of Gram positive bacteria is highly crosslinked
in the second dimension and also crosslinked in the third dimension. The cell walls of
Gram-negative bacteria are thinner (10 nm or less); the peptidoglycan is not as fully

Figure1 Principles behind Gram staining

and observations after staining with crystal
violet, treating with iodine decolorizing with
acetone and counterstaining with safranin.

crosslinked in the second dimension and not crosslinked at all in the third dimension. In
Gram staining one first stains the fixed bacteria with a primary dye (crystal violet) and
adds a mordant (iodine) to generate a crystal violet-iodine complex within the cell. The
crystal violet-iodine complex is larger than the crystal violet alone and therefore more
difficult to remove in subsequent steps. Acetone is added to remove the crystal violetiodine complex from Gram negative bacteria. The fact that the peptidoglycan layer of
Gram-negative bacteria is thin and not fully crosslinked makes it easy to remove the
primary dye, conversely, the peptidoglycan layer of Gram positive cells are dehydrated in
the presence of acetone such that the crystal violet-iodine complex is trapped within the
cell. As the Gram negative cells are decolorized by the acetone, one then counterstains
with safranin such that those cells can be easily observed (Figure 1.)
Simple staining: In the first exercise, you will observe bacteria subjected to simple
staining in order to analyze different bacteria with respect to their size, shape and
arrangement. To this end you will observe, Escherichia coli, Bacillus subtilis and
Vibrio spp. as well as Staphylococcus epidermidis and Streptococcus lactis.
Bacteria have three basic characteristic morphologies. Those are the coccus
(cocci pl), the bacillus (bacilli pl) and the spiral shaped bacteria such as the spirillum,
spirochetes and Vibrio spp. The cocci are spherical shaped bacteria while the bacilli are
rod shaped bacteria. There is a great deal of variety with respect to the morphologies that
spiral shaped bacteria assume; the spirillum and spirochetes appear different under
observation because of the manner in which they move. When spirillum move the long
axis of their bodies remain rigid such that they appear to bend at the ends, when the
spirochetes move, the long axis of their bodies are not fixed such that they take on a
wavy, permed hair appearance. Vibrio spp. are rod shaped bacteria that are bent near
the middle such that they are often called the comma shaped bacilli. When bacteria
grow and divide they can stick together in a manner that aids in their characterization
with respect to their genus and species. For example if cocci shaped bacteria divide and
stick together to form pairs, they are called diplococci; if they stick together to form long
chains they are called streptococci and if they adhere to each other to form grape like
clusters they are called staphylococci. Certain bacillus shaped species such as Bacillus
subtilis can adhere to each other in a chain like formation known as streptobacilli.
Describe in the spaces below how the bacteria look in terms of size, shape and
bacterial arrangement at a total magnification of 1000X !!!
Escherichia coli

Vibrio anquillarum

Staphylococcus epidermidis

Streptococcus lactis

Bacillus subtilis

Gram staining: The next part of the exercise involves using a differential staining
procedure called Gram staining to distinguish between Gram positive and Gram
negative bacteria. To this end you will stain and observe, Escherichia coli and/or
Staphylococcus epidermidis and/or Bacillus subtilis in a mixed culture.
Preparing a smear from a liquid culture:
Place a small amount of the culture on a microscope slide as shown by your
instructor and heat fix the sample.
Using a wax pen (not Sharpie, Why?) label your slide with your initials. This
is the top side of your slide and the side of the slide that will be subjected to all
of the staining steps
You will make a smear of the bacterial species, by aseptically transferring
bacteria from a culture tube onto the glass slide. To this end, obtain a sterile
cotton swab and swab into the test tube containing the mixture of bacteria.
Wring out the swab on the inner surface of the test tube. Make a thin smear
on the surface of your microscope slide. It is important that you make a thin
smear such that it is easier to observe individual bacteria.
Allow the smears to dry completely; the smears should become somewhat
cloudy as they dry, it is important that the smears are completely dry as the
following heat fixation step will distort the morphology of organisms in a
smear that is not completely dry.
Heat fix the bacteria in the smear by quickly passing the microscope slide
through a flame 8-10 times.

2. Crystal violetprimary stain: Place the slide containing heat fixed smears on a
test tube rack in the sink with the smear facing up. Cover the smear with crystal violet
and leave the stain on for 1 minute. (What color will the cells be?)
3. Grams Iodinemordant: Use a wash bottle containing WATER to gently wash
BOTH sides of the microscope slide, tip off the extra water and IMMEDIATELY cover
the slide with Grams iodine. Allow the iodine to remain on the slide for one minute.
(What color will the cells be?)
4. Acetone wash--Decolorizing step: Use a wash bottle containing WATER to gently
wash BOTH sides of the microscope slide.
5. Rinse the slides with acetone for 3 seconds only. Immediately rinse both sides of
the microscope slide with water to remove the acetone and to halt the decolorizing
process. It is important that you do not allow the acetone to remain in contact with the

specimen for longer than 3 seconds because longer contact will remove the crystal violetiodine complex from the Gram positive cells as well. (What color will the cells be?)
6. Safranincounterstain: Place the slide on the test tube rack in the sink (smear side
up) and cover the slide with safranin; allow the safranin to remain on the slides for 90
seconds. Use a wash bottle containing WATER to gently wash BOTH sides of the
microscope slide and gently blot the slides with bibulous paper as described by your
instructor.
7. Save your slides until the next lab period where you will observe your stained bacteria
using a microscope.
Describe in the space below how the bacteria look in terms of size, shape and
bacterial arrangement AND color at a total magnification of 1000X !!!

Which of the bacteria are Gram positive/Gram negative?

Caveats:
It is important to note that not all bacilli are Gram negative and not all cocci are Gram
positive!! Also some bacteria can be Gram variable. A good example of this is Bacillus
subtilis that is a Gram positive rod that forms endospores. Older cultures of Bacillus
may react to the dyes in the Gram stain such that one may observe what look like Gram
positive bacilli and Gram negative bacilli in the same population. When observing
bacteria in terms of whether they are Gram positive or negative it is important to work
with young cultures.
The instructor has placed a demonstration slide of a Gram stain of a four day old
culture of Bacillus subtilis (the south side of the lab). Note how some of the bacilli
appear pink (or Gram negative).