Staining Techniques

INTRODUCTION

Bacteria have almost the same refractive index as water. This means when you try to view them
using a microscope they appear as faint, gray shapes and are difficult to visualize. Staining is one method
for making microbial cells easier to visualize.

Simple stains use only one dye that stains the cell wall of bacteria much like dying eggs at Easter.
Differential stains use two or more stains and categorize cells into groups. Both staining techniques allow
the detection of cell morphology, or shape, but the differential stain provides additional information
concerning the cell. The most common differential stain used in microbiology is the Gram stain.

The Gram stain uses four different reagents and the results are based on differences in the cell
wall of bacteria. Some bacteria have relatively thick cell walls composed primarily of a carbohydrate
known as peptidoglycan. Other bacterial cells have thinner cell walls composed of peptidoglycan and
lipopolysaccharides. Peptidoglycan is not soluble in organic solvents such as alcohol or acetone, but
lipopolysaccharides are nonpolar and will dissolve in nonpolar organic solvents.

Crystal violet acts as the primary stain. This stain can also be used as a simple stain because it
colors the cell wall of any bacteria. Gram’s iodine acts as a mordant. This reagent reacts with the crystal
violet to make a large crystal that is not easily washed out of the cell. At this point all cells will be the
same color. The difference in the cell walls is displayed by the use of the decolorizer. A solution of
acetone and alcohol is used on the cells. The decolorizer does not affect those cell walls composed
primarily of peptidoglycan but those with the lipid component will have large holes develop in the cell
wall where the lipid is dissolved away by the acetone and alcohol. These large holes will allow the crystal
violet-iodine complex to be washed out of the cell leaving the cell colorless. A counterstain, safranin, is
applied to the cells which will dye the colorless cells.

The cells that retain the primary stain will appear blue or purple and are known as Gram positive.
Cells that stain with the counterstain will appear pink or red and are known as Gram negative. The
lipopolysaccharide of the Gram negative cell not only accounts for the staining reaction of the cell but
also acts as an endotoxin. This endotoxin is released when the cell dies and is responsible for the fever
and general feeling of malaise that accompanies a Gram negative infection.

When reporting a Gram stain you must indicate the stain used, the reaction, and the morphology
of the cell. Round, purple (blue) cells would be reported as Gram positive cocci and rod-shaped, purple
(blue) cells would be reported as Gram positive bacilli.

In order to survive some bacteria produce endospores that are highly resistant to harsh
environmental conditions. The malachite green staining procedure is a differential staining that is used to
distinguish between vegetative cells and endospores.

Stains attach to something because of charge differences between the object and the stain.
Different stains can appear as a different color because they contain different chromophore groups, which
vary in the wavelength of light they absorb. In general there are two main stain types. Positively charged
stains have a positive chromophore. The second type, negatively charged stains, has a chromophore that
carries a negative charge. Positively charged stains are excellent in binding negatively charged structures
such as bacterial cell walls and, if they can enter the cell, many macromolecular structures such as DNA
and proteins.

Cationic (basic) stains have a positive charge associated with them while anionic (acidic) stains
carry a negative charge. Examples of cationic stains include crystal violet, safranin, basic fuschin, &
methylene blue. Examples of anionic stains include eosin, nigrosin, & congo red. Acid dyes are often
used to stain the slide background, which leaves the microbe transparent. Thus, in the field of view the
microbe will appear as clear dots against an opaque background. Stains require a short exposure time to
their target followed by a brief, light rinse with deionized (DI) water. This removes any excess stain and
allows better viewing of the cells that carry the stain.

Stains are used to recognize bacteria in clinical specimens and in cultures. Simple stains are used
to demonstrate the presence of organisms as well as the nature of the cellular contents in exudates. This
exercise is useful to practice staining and observing bacteria before doing more complicated stains.
Examples of simple stains include Loeffler’s Methylene Blue, Polychrome Methylene Blue and Dilute
Carbol Fuchsin. The typical bacteria will be visualized as about 0.5 – 1.0 micrometer (m) in width to 2-7
m long and are usually rods, cocci or spiral shaped.

Gram Staining

One of the most important stains performed by both the fledgling microbiologist and professional
microbiologist is the Gram stain. This stain is named after Hans Christian Gram who was the first to
implement the technique. Although Hans did not know it at the time, the Gram stain allows differentiation
between Gram negatives and Gram positives. This is largely due to the structure of the cell wall and the
presence or absence of an outer membrane that occurs in Gram negative bacteria.

After heat-fixing a loop-full of overnight culture, the cells are ready to be stained. First a primary
stain, called crystal violet, is used as a primary stain. This cationic stain will adhere to all organisms,
since cells carry an overall negative charge. After rinsing, a mordant is applied called Gram's iodine,
which promotes retention of the primary stain.

The second rinse is performed with EtOH. The EtOH functions to do several things one of which
is to shrink the pores in the peptidoglycan layer. This traps the crystal violet-iodine complex in the Gram-
positive cell. The larger pores and thinner peptidoglycan in Gram-negative organisms is not changed to
such an extent compared to the Gram-positive organisms. However, the EtOH also removes the outer
membrane of the Gram-negative organisms. In essence, the EtOH rinse effectively leaves the Gram-
negative cells colorless.

While the Gram positives are stained purple, a counterstain is applied to stain the colorless Gram
negatives. These will appear reddish in color.

What would a simple drawing of a Gram-positive and Gram-negative cell look like? What are the
different parts (cell wall, outer membrane, periplasmic space, etc.) and their relative sizes?

Name:______________________________ Date Completed: _____________________________

Class:____________ Lab Minutes:______________ Teacher: _______________________________

ACTIVITY No. 3

Gram Stain

Materials:

• Overnight cultures of S. aureus, E. coli, & an unknown

• Crystal violet

• Gram's iodine

• Safranin

• DI H2O
Methods:

1. Prepare two smears of each of the cultured organisms. Heat fix the air dried smear by carefully passing
the slide through a Bunsen burner/alcohol lamp three times. Let cool.

2. Add the primary stain (crystal violet) to the smear/slide and stand for 1 minute. Rinse slide with a
gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet.

3. Add Gram's iodine for 1 minute.

4. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water.
The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the
alcohol remains on the sample for too long, it may also decolorize Gram positive cells.

5. Add the secondary stain, safranin, to the slide and stand for 1 minute. Wash with a gentle stream of
water for a maximum of 5 seconds. If the bacteria is Gram positive, it will retain the primary stain (crystal
violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If
the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to
appear red when viewed under a microscope.

Questions:

1. What is the function of the Lugol’s iodine solution in the Gram stain? If it was omitted, how
would stain results be affected?

2. What is the purpose of the iodine acetone solution in the Gram stain?

3. What is the advantage of the Gram stain over the simple stain?
4. Draw the following Gram Stain results for the following:
a. Staphylococcus aureus
b. Neisseria gonorrhoeae
c. Escherichia coli
d. Listeria Monocytogenes
e. Haemophilus influenzae