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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.

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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.


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 below how the background remains generally clear, while the bacterial cells are a deep blue.

Methylene blue stains all cells blue.
India ink turns the background a dark brown to black color.
slide with negative bacterial staining

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 different kinds of bacteria would look different.Enter the differential staining technique, a procedure that allows the observer to visually distinguish between different types of bacterial cells based on the idea that not all cell types stain equally.

This technique takes advantage of the different physical properties that different bacteria have evolved. The best way to understand this concept is to look at the most famous differential staining technique, the Gram stain.The Gram stain is a differential staining technique that can detect two different types of bacteria based on differences 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 different 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.
slide with gram positive and gram negative

Lesson Summary

Let’s review.Individual bacterial cells are nearly colorless, making them difficult to see under the light microscope. To overcome this problem, bacteria are stained to enhance visibility. There are many different 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 differential staining technique is a procedure that allows the observer to visually distinguish between different 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 specific bacteria. The resulting difference in color can be used to distinguish different cell types. The Gram stain is a differential 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
  • Differentiate between simple and negative staining techniques and identify the common dye used in each
  • Indicate how differential staining techniques work
  • Understand what is meant by Gram-negative and Gram-positive

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