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 finally 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
magnification, oil immersion, and a digital camera. Bacteria are everywhere, and, hopefully, you finally 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 find
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 flooded across a sample, adding color to the cells. Before we move on, let's
define 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 off 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 fixed to the slide. During heat fixation,
a glass slide is waved over an open flame. This kills the bacteria, attaches the cells to the slide, and enhances the stain uptake. This
process makes staining more effective 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 how the background remains generally clear, while the bacterial cells are a deep
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 unaffected by direct contact
with the dye and distortion from heat fixing, 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. These are cells of the fungal pathogen Cryptococcus. The India ink has colored the background brown,
leaving the cells their natural color.