Why gram staining is classified as differential staining

Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria with a thicker peptidoglycan layer retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. The process involves three steps:. Your Account. Gram Staining Created by Monica Z. Negative staining produces an outline or silhouette of the organisms against a colorful background Figure 2.

Figure 2. Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin , crystal violet , malachite green , methylene blue , and safranin typically serve as positive stains.

On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin , eosin , and rose bengal. Table 2 provides more detail. Some staining techniques involve the application of only one dye to the sample; others require more than one dye. In simple staining , a single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism.

In contrast, differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms in a differentially stained sample may appear to be different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. Table 3 provides more detail on these differential staining techniques. The Gram stain procedure is a differential staining procedure that involves multiple steps.

It was developed by Danish microbiologist Hans Christian Gram in as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.

The steps of the Gram stain procedure are listed below and illustrated in Table 1. Gram-staining is a differential staining technique that uses a primary stain and a secondary counterstain to distinguish between gram-positive and gram-negative bacteria. Step 2: Iodine. Cells remain purple or blue. Step 3: Alcohol.

Step 4: Safranin. Gram-negative cells appear pink or red. Figure 3. In this specimen, the gram-positive bacterium Staphylococcus aureus retains crystal violet dye even after the decolorizing agent is added. Gram-negative Escherichia coli, the most common Gram stain quality-control bacterium, is decolorized, and is only visible after the addition of the pink counterstain safranin. The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative Figure 3.

However, there are several important considerations in interpreting the results of a Gram stain. First, older bacterial cells may have damage to their cell walls that causes them to appear gram-negative even if the species is gram-positive.

Thus, it is best to use fresh bacterial cultures for Gram staining. Second, errors such as leaving on decolorizer too long can affect the results. In some cases, most cells will appear gram-positive while a few appear gram-negative as in Figure 3. This suggests damage to the individual cells or that decolorizer was left on for too long; the cells should still be classified as gram-positive if they are all the same species rather than a mixed culture.

Besides their differing interactions with dyes and decolorizing agents, the chemical differences between gram-positive and gram-negative cells have other implications with clinical relevance. For example, Gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. Gram-negative bacteria tend to be more resistant to certain antibiotics than gram-positive bacteria.

We will discuss this and other applications of Gram staining in more detail in later chapters. Figure 4. However, more information is needed to make a conclusive diagnosis. The technician decides to make a Gram stain of the specimen.

This technique is commonly used as an early step in identifying pathogenic bacteria. After completing the Gram stain procedure , the technician views the slide under the brightfield microscope and sees purple, grape-like clusters of spherical cells Figure 4. Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not.

Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique. Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing agent an acid-alcohol solution is applied. A secondary counterstain, methylene blue, is then applied, which renders non—acid-fast cells blue. The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process.

The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria AFB.

If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells Figure 5.

Figure 5. Ziehl-Neelsen staining has rendered these Mycobacterium tuberculosis cells red and the surrounding growth indicator medium blue. Mycobacterium tuberculosis , the bacterium that causes tuberculosis , can be detected in specimens based on the presence of acid-fast bacilli. If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification.

Variations of this approach can be used as a first step in determining whether M. An alternative approach for determining the presence of M. In this technique, fluorochrome-labeled antibodies bind to M. Antibody-specific fluorescent dyes can be used to view the mycobacteria with a fluorescence microscope. Certain bacteria and yeasts have a protective outer structure called a capsule. Capsules do not absorb most basic dyes; therefore, a negative staining technique staining around the cells is typically used for capsule staining.

The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell. The specimen does not need to be heat-fixed prior to negative staining.

One common negative staining technique for identifying encapsulated yeast and bacteria is to add a few drops of India ink or nigrosin to a specimen. Other capsular stains can also be used to negatively stain encapsulated cells Figure 6. Alternatively, positive and negative staining techniques can be combined to visualize capsules: The positive stain colors the body of the cell, and the negative stain colors the background but not the capsule, leaving halo around each cell.

Figure 6. Endospores are dormant forms of living bacteria and should not be confused with reproductive spores produced by fungi. These structures are produced by a few genera of Gram-positive bacteria, almost all bacilli, in response to adverse environmental conditions. Two common bacteria that produce endospores are Bacillus or Clostridum. Both live primarily in soil and as symbionts of plants and animals, and produce endospores to survive in an environment that change rapidly and often.

The process of endosporulation the formation of endospores involves several stages. After the bacterial cell replicates its DNA, layers of peptidoglycan and protein are produced to surround the genetic material.

Once fully formed, the endospore is released from the cell and may sit dormant for days, weeks, or years. When more favorable environmental conditions prevail, endospores germinate and return to active duty as vegetative cells. Mature endospores are highly resistant to environmental conditions such as heat and chemicals and this permits survival of the bacterial species for very long periods. Endospores formed millions of years ago have been successfully brought back to life, simply by providing them with water and food.

Because the endospore coat is highly resistant to staining, a special method was developed to make them easier to see with a brightfield microscope. This method, called the endospore stain , uses either heat or long exposure time to entice the endospores to take up the primary stain, usually a water soluble dye such as malachite green since endospores are permeable to water.

Following a decolorization step which removes the dye from the vegetative cells in the smear, the counterstain safranin is applied to provide color and contrast. When stained by this method, the endospores are green, and the vegetative cells stain pink, as shown in Figure 7. Although endospores themselves are resistant to the Gram stain technique, bacterial cells captured in the process of creating these structures can be stained. In this case, the endospores are seen as clear oval or spherical areas within the stained cell.

Endospores can also be directly observed in cells by using phase contrast microscopy, as shown in Figure 8. Because many differential staining methods require several steps and take a long time to complete, we will not be performing all of the differential staining methods discussed above.

Pre-stained slides will be used to visualize bacterial capsules, metachromatic granules, and acid-fast bacilli. Obtain one slide of each of the three bacteria listed in the table below. Your environmental isolate may have one or more of these cellular features, and learning to recognize them will aid in identification. These should all be viewed using the oil immersion objective lens. All staining procedures should be done over a sink.

The Gram stain procedure will be demonstrated, and an overview is provided in Table 1. A volunteer from your lab bench should obtain cultures of the bacteria you will be using in this lab, as directed by your instructor. One of the cultures will be a Gram positive bacterium, and the other will be Gram negative.

Below, write the names of the bacteria you will be using, along with the BSL for each culture:. Obtain two glass slides, and prepare a smear of each of the two bacterial cultures, one per slide, as demonstrated. Stain both smears using the Gram stain method. Observe the slides with a light microscope at 1,X and record your observations in the table below.

Heat fix the smear and Gram stain it. You should be able to determine the Gram stain reaction, cellular morphology and arrangement of BOTH bacteria in this mixed smear. Your instructor may ask to see this slide and offer constructive commentary. Only a few genera of bacteria produce endospores and nearly all of them are Gram-positive bacilli.

Most notable are Bacillus and Clostridium species, which naturally live in soil and are common contaminants on surfaces. The growth of Clostridium spp. Endospore-forming bacteria are distinct from other groups of Gram positive bacilli and distinguishable by their endospores.

After staining, endospores typically appear as light green oval or spherical structures, which may be seen either within or outside of the vegetative cells, which appear pink. The shape and location of the endospores inside the bacterial cells, along with whether the sporangium is either distending D or not distending ND the sides of the cell, are important characteristics that aid in differentiating among species see Figure 9.

A volunteer from your lab bench should obtain bacterial cultures for endospore staining, as directed by your instructor. Note that these will all be species of Bacillus. Prepare smears and stain each using the endospore staining technique.