Simple and Differential Stains: Definition and Examples

Observing tiny bacteria under the microscope is not as easy as it sounds. In this lesson, we will examine several
staining techniques used to color bacteria, enhancing their visibility.

A Microscope for Christmas

It is nally Christmas morning! This year you know you're going to get what you asked for. You
didn't go ludicrous and ask for a pony to share your New York City apartment. You didn't go
techno-shallow and ask for a new smart phone 12, now complete with a tiny microwave for nuking
pizza rolls while texting. This year, you asked for a compound light microscope - a good one, with
1000X magni cation, oil immersion, and a digital camera. Bacteria are everywhere, and, hopefully,
you nally get to look at them!

Success! You are now the proud owner of a shiny new microscope. Forgetting about the rest of
your presents, you swab the bottom of your 2-year-old running shoes, roll the grime across a new
glass slide, and quickly start scanning for microbes. But, you can't nd anything. There must be
bacteria on your shoes. After all, bacteria are everywhere - everywhere except your shoes,
apparently.

Stains
In actuality, your slide is likely teeming with bacteria. In colonies made up of millions of cells,
bacteria can be a wide range of colors and textures. But, the vast majority of individual bacterial
cells are nearly completely colorless. The cell wall and cytoplasm of one individual bacterium is
simply not going to be visible enough for you to see, even with your expensive light microscope.
What you need is something to enhance the visibility of that one bacterium. What you need is a
stain.

A stain is a chemical compound used to enhance the visibility of a microscopic object or organism.
But, not all stains are created equal. The type of stain and the technique you use depends on what
you're looking at, what structure you're looking for, and what you want the staining procedure to
accomplish. Let's take a quick look at a few of the more common categories of staining techniques.

Simple Staining Technique

In a simple staining technique, a basic, cationic dye is ooded across a sample, adding color to
the cells. Before we move on, let's de ne the word cationic. A cation is simply a positively charged
ion. The molecules that make up basic dyes have a positive charge. This is important because the
cell wall and cytoplasm of bacterial cells have a negative charge. The positively charged dye is
attracted to the negatively charged cells, enhancing the ability of the stain to stick to and color the
cells. Now, those nearly colorless cells should pop o the slide in any number of colors.

It is important to note that before a sample can be stained with a simple stain, it must be heat xed
to the slide. During heat xation, a glass slide is waved over an open ame. This kills the bacteria,
attaches the cells to the slide, and enhances the stain uptake. This process makes staining more
e ective but can damage or distort the cells, changing their appearance from a truly natural, free-
living state.

Methylene blue is a classic example of a simple stain. This blue stain will color all cells blue, making
them stand out against the bright background of the light microscope. Notice below how the
background remains generally clear, while the bacterial cells are a deep blue.

Methylene blue stains all cells blue.

Negative Staining Technique

In a negative staining technique, an acidic, anionic dye is mixed with a cell sample. The dye
changes the color of the background, not the cells, causing the cells to stand out. This process can
be considered the opposite of simple staining. An anion is a negatively charged ion, therefore an
anionic dye has a negative charge. When the negatively charged dye is added to the negatively
charged cells, the two repel each other, meaning they push apart. When the mixture is placed on a
slide and air dried, what results is a darkly dyed background, surrounding clear, unstained cells.
The transparent cells are now highly visible but are una ected by direct contact with the dye and
distortion from heat xing, which is not needed in a negative stain.

India ink is the classic example of a negative stain. It will turn the background a dark brown to
black, leaving the clear, bright cells unstained and highly visible. Below are cells of the fungal
pathogen Cryptococcus. The India ink has colored the background brown, leaving the cells their
natural color.
 India ink turns the background a dark brown to black color.

Differential Staining Technique

Simple stains and negative stains are great for looking at cells, but they will stain nearly all cells
equally. What if you have a mixed sample, meaning more than one type of bacteria is present, or
suspect your pure culture is contaminated? It would be nice if you could stain some cells, but not
others, or if di erent kinds of bacteria would look di erent.

Enter the di erential staining technique, a procedure that allows the observer to visually
distinguish between di erent types of bacterial cells based on the idea that not all cell types stain
equally. This technique takes advantage of the di erent physical properties that di erent bacteria
have evolved. The best way to understand this concept is to look at the most famous di erential
staining technique, the Gram stain.

The Gram stain is a di erential staining technique that can detect two di erent types of bacteria
based on di erences in the cell wall structure. There are two major types of cell walls, named after
how they appear after Gram staining. Gram-positive cell walls have a thick layer of peptidoglycan, a
mesh-like compound that adds strength and rigidity to the cell wall. Gram-negative cell walls have a
thin layer of peptidoglycan that is covered by an outer membrane.

During a Gram stain, the primary stain, crystal violet, turns all cells purple but can be easily washed
out of the thin peptidoglycan layer of Gram-negative cell walls, leaving them transparent. The thick
peptidoglycan layer traps the crystal violet in the Gram-positive cells, preventing it from being
washed out.

Adding a second stain, safranin, will stain the transparent, Gram-negative cells, a red color. The
Gram-positive cells will remain purple. You can see below how this will distinguish the Gram-
positive cells from the Gram-negative cells based on the di erent ways the cell walls take up the
stain. Bacteria with cell walls composed of a thick peptidoglycan layer turn purple or are Gram-
positive. Bacteria with cell walls composed of a thin peptidoglycan layer and an outer membrane
turn red or are Gram-negative.
 Purple-stained bacteria are Gram-positive, while red-stained bacteria are Gram-negative.

Lesson Summary
Let's review.

Individual bacterial cells are nearly colorless, making them di cult to see under the light
microscope. To overcome this problem, bacteria are stained to enhance visibility. There are many
di erent staining techniques.

In a simple staining technique, a positively charged stain colors the negatively charged cells,
making them stand out against the light background. Methylene blue is a simple stain that colors
cells blue.

In a negative staining technique, a negatively charged stain colors the background, leaving the
cells light colored and unstained. The bright cells are easily visible against the dark background.
India ink is a negative stain that colors the background brown, leaving the cells bright and visible.

A di erential staining technique is a procedure that allows the observer to visually distinguish
between di erent types of bacterial cells based on the idea that not all cell types stain equally.
Some physical characteristic leads to unequal uptake of a stain, depending on the speci c bacteria.
The resulting di erence in color can be used to distinguish di erent cell types. The Gram stain is a
di erential technique that colors Gram-positive cells with a thick peptidoglycan cell wall purple
while coloring Gram-negative cells with a thin peptidoglycan cell wall red.

Learning Outcomes
When you've gone through this lesson, you could be able to:

Realize why it is necessary to stain bacteria to view them under a microscope

Di erentiate between simple and negative staining techniques and identify the common dye used in each

Indicate how di erential staining techniques work

Understand what is meant by Gram-negative and Gram-positive