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Principles of Microscopy and Staining

Microbial Stains

Purpose of Microbial Stains

Stains are used to colorize and contrast different parts of microbes; simple stains use a single color, while differential stains use more than one color.

Stains—or dyes—are used to add color and differentially emphasize structures in specimens. Staining is especially useful since many biological structures and components lack contrast or color and cannot be discriminated when visualized under a microscope. Different stains take advantage of different properties of chemicals and how they interact with parts of the microbe in order to provide information about the microbe. A stain may colorize an entire microbe or a specific component of a cell. A simple stain uses a single dye color and typically stains all microbes in a sample. A differential stain uses multiple dye colors—typically two—and can stain microbes in the same sample differently depending on certain properties, such as the composition of the bacterial cell wall or the way the microbe processes the chemicals used in the stain. The color of the specimen after being stained reveals properties of the microbe that would not be visualized without staining.

One simple stain is the negative stain, in which a dark stain is used to cover the background of the slide while the microbes on the slide remain clear. The stains used for negative staining do not permeabilize the microbes and instead are used to create a dark background in contrast with the unstained microbes. The images produced from a negative stain look like a black-and-white photograph. This technique can be used with light microscopes or electron microscopes.
Negative staining is a type of simple stain technique in which dark stain is used to cover the background of the slide and the specimen is left clear. This allows the specimen to stand out for observation. Negative staining is best for samples that may be too delicate to remain intact throughout the heat fixing required by other staining methods. (transmission electron microscope)
Credit: CDC/F. A. Murphy

Gram Staining

Gram-positive bacteria have a thick peptidoglycan layer and retain the crystal violet stain after being set with iodine. Gram-negative bacteria have a thinner peptidoglycan layer, which is dissolved with alcohol, and do not retain crystal violet but rather stain red with safranin.

One very common differential stain is Gram stain, a method of staining used to differentiate types of bacteria based on the thickness of the primary cell wall material peptidoglycan. After the sample has been stained, the bacteria will appear either red (gram-negative) or purple (gram-positive).

First, bacteria are fixed onto a glass slide using a smear where the sample is wiped on the slide and allowing it to dry. Crystal violet (purple dye) is added, and this stains all the bacteria on the slide. Next, iodine is added. Iodine binds to the crystal violet inside the bacteria, making a large complex. The slide is then decolorized, or washed with alcohol that removes the crystal violet complex from some (but not all) bacteria. The crystal violet plus iodine complex is too big to be washed out of bacteria with thick peptidoglycan cell walls, and these bacteria remain purple. However, it is able to be washed out of other bacteria. Finally, safranin (the counterstain) is added. The bacteria that were decolorized in the previous step are instead stained by safranin now, and they appear red. Looking under a microscope, the purple and red colors can be distinguished. The purple bacteria are gram-positive, while the red bacteria are gram-negative.

Gram Staining

Gram staining differentiates between gram-positive bacteria, which are stained with crystal violet (purple dye), and gram-negative bacteria, which are stained with safranin (the counterstain). After the steps of Gram's stain are complete, any gram-positive bacteria in the sample are left with a purple appearance, and any gram-negative bacteria are left with a pink or red appearance.
Gram-positive bacteria have a strong, thick cell wall, while gram-negative bacteria have a thin cell wall that is dissolved during the staining process. The component that differs between the cell wall of gram-positive and gram-negative bacteria is called peptidoglycan. Gram-positive bacteria have a cell wall that is made up of 90 percent peptidoglycan, which is organized into a thick layer. Because of this, the crystal violet and iodine complex remains inside the cells during the decolorization step. Gram-negative bacteria have a cell wall that is made up of only 10 percent peptidoglycan, arranged into a thin layer. The rest of the cell wall is made up of lipids, or fat molecules. During the decolorization step, the cell wall of a gram-negative bacteria dissolves, and the crystal violet can escape. These bacteria can be counterstained with safranin, turning them red so they can be seen under the microscope.

Gram staining is frequently used in a hospital setting to differentiate between different kinds of bacteria that cause infections. Gram-positive and gram-negative bacteria respond differently to medical treatment. Some kinds of gram-positive bacteria are Staphylococcus and Streptococcus. Staphylococcus can cause skin infections, and in clinical settings methicillin-resistant Staphylococcus aureus (MRSA) is a potential threat to patients. Certain strains of Streptococcus can cause strep throat. Some other types of organisms also stain gram-positive, including the infectious algae Prototheca wickerhamii. Some gram-negative bacteria include Salmonella and Yersinia enterocolitica, which can cause food poisoning, and Vibrio, which can cause cholera.

The difference in the composition of the cell wall between gram-positive and gram-negative bacteria means that the bacteria respond differently to antibiotic treatment. Gram-negative bacteria are more likely to become resistant to antibiotics—that is, unable to be killed by them. This is because many antibiotics target peptidoglycans. Gram-positive bacteria can be killed by antibiotics because their cell wall is composed of 90 percent peptidoglycans. In gram-positive bacteria, the thin peptidoglycan layer is coated with lipids, protecting these bacteria from the effects of antibiotics.
Prototheca wickerhamii is a gram-positive infectious algae and stains purple with Gram stain. Yersinia enterocolitica is a gram-negative bacteria and stains pink or red with Gram stain.
Credit: CDC/Dr. James Feeley (left), CDC (right)

Other Staining Techniques

The most common differential stains are Gram, acid-fast (Ziehl-Neelsen), and Schaeffer-Fulton.

In addition to Gram stain, many other differential stains are commonly used in the lab. While the color that bacteria turn following Gram stain provides information about the composition of the bacterial cell wall, other differential stains cause color changes based on other distinguishing properties of the bacterial cell, for example differentiating resistant cells called endospores.

The acid-fast stain (Ziehl-Neelsen stain) identifies acid-fast bacteria (those that are resistant to decolorization by acid alcohol because of their waxy or fatty cell walls) that are able to keep an acid-based stain inside their cells. Similar to Gram stain, the stain that may or may not wash out of bacterial cells is added first. In an acid-fast stain, the stain used is called carbolfuchsin and it stains cells pink because it is soluble in the lipids of the cell wall. The decolorization step in this stain uses acid, and the acid-fast bacteria remain pink while the bacteria that are not acid-fast will become clear. A counterstain of methylene blue or malachite green is then applied because they provide high contrast with the pink coloration of carbolfuchsin. Counterstained bacteria appear blue or green, respectively, under the microscope.

The acid-fast stain is used to identify Mycobacterium, the genus of bacteria responsible for tuberculosis and leprosy. These bacteria have a cell wall made of a thick layer of lipids. Despite the presence of peptidoglycan in their cell walls, Mycobacterium do not retain crystal violet well and stain neutral with a Gram stain, but the acid-fast stain readily identifies them as acid-fast bacteria.

An endospore stain is accomplished by using the Schaeffer-Fulton stain and distinguishes between endospores and bacteria. Some gram-positive bacteria form resistant cells, endospores, in order to survive dangerous changes in the environment and protect the genetic material of the bacterial cell. In the Schaeffer-Fulton stain, endospores stain green using malachite green, while the counterstain safranin stains bacteria red.

Common Differential Staining Techniques

Stain Dyes Uses Results Example
Gram stain Crystal violet, Gram's iodine, safranin Differentiates between bacteria based on peptidoglycan composition of cell walls Gram-positive bacteria stain purple; gram-negative bacteria stain red.
Credit: CDC/Dr. William A. Clark
Bacillus cereus
Acid-fast stain Carbolfuchsin, methylene blue Differentiates between bacteria with waxy external surfaces that are impenetrable to Gram staining Acid-fast bacteria stain red; non-acid-fast bacteria stain blue.
Credit: CDC/DPDx - Melanie Moser
Cyclospora cayetanensis (oocysts)
Endospore stain Malachite green, safranin Visualizes endospore structures within bacteria cells Endospores stain green; cells stain pink.
Credit: Suhail Ahmad et al.License: CC BY 3.0
Lodderomyces elongisporus

Staining of bacteria before microscopy enables determination of cellular structures that differentiate groups of bacteria based on their physical characteristics.