It is also the stain of choice for identifying the metachromatic granules in Corynebacterium diphtheriae. The granules will stain a distinctly deeper blue than the surrounding blue bacteria.
Other species of Corynebacterium do not have the metachromatic granules. Any basic dyes, such as methylene blue, crystal violet, malachite green, or safranin work well.
Basic cationic or positively charged dyes bind to negatively charged components in the cell membrane and cytoplasm. Staining is part art and part science. There are no hard and fast rules for staining and rinsing times. The times listed are suggestions that usually work well. You will need to experiment with what works for the bacteria you have and the techniques you use.
It is essential that you record exactly what you do and the results you observe in your lab book. It would be useful for each lab bench member to pick a different stain so you can see what they all look like. Negative stains are even simpler than simple stains because you do not have to make a smear. A drop of cells is spread on a slide and viewed without fixation. The stain is a suspension of carbon, found in India ink or nigrosin.
The carbon particles are negatively-charged, as is the cell membrane. The background looks black or sepia colored and the cells remain clear, since they repel the dye. Some positively charged inclusion bodies, such as sulfur, may stain. This stain gives accurate information on cell morphology and capsule presence because the cells are not fixed. Cell size appears slightly larger because any extracellular coatings or secretions on the outside of the cell membrane also do not stain.
Negative stains are useful for rapid determination of the presence of Cryptococcus neformans , the causative agent of cryptococcisis, in cerebral spinal fluid. This technique is also used when you stain for endospores and capsules. Just as in preparing a smear, you only need a small amount of organism. It is also important not use too much nigrosin. If it is too thick, the background will have a cracked appearance similar to mud puddles drying in the sun.
You want to get a light film. Your instructor will demonstrate this technique for you. Nigrosin comes off the slide and onto your oil immersion lens very easily. Be sure to thoroughly clean your oil lens when you are finished. Then clean it again. Once it dries on the lens it is very difficult to remove and will impair your ability and the other micro students using that scope to see clearly out of the lens.
The Gram stain is the most common differential stain used in microbiology. Differential stains use more than one dye. The unique cellular components of the bacteria will determine how they will react to the different dyes.
The Gram stain procedure has been basically unchanged since it was first developed in Almost all bacteria can be divided into two groups, Gram negative or Gram positive. A few bacteria are gram variable. Trichomonas , Strongyloides , some fungi, and some protozoa cysts also have a Gram reaction. Very small bacteria or bacteria without a cell wall, such as Treponema , Mycoplasma , Chlamydia , or Rickettsia do not have a gram reaction. The characterization of any new bacteria must include their gram reaction.
Typically a differential stain has four components; the primary stain, a mordant that sets the stain, a decolorizing agent to remove the primary stain, and a counter stain.
Dyes are selected for staining based on the chemical properties of the dye and the specimen being observed, which determine how the dye will interact with the specimen. In most cases, it is preferable to use a positive stain , a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out against the background.
However, there are scenarios in which it is advantageous to use a negative stain , which is absorbed by the background but not by the cells or organisms in the specimen. 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. True to its name, the simple stain is a very simple staining procedure involving a single solution of stain. Any basic dye such as methylene blue, safranin, or crystal violet can be used to color the bacterial cells. These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged.
Since the surface of most bacterial cells and cytoplasm is negatively charged, these positively charged stains adhere readily to the cell surface. Only the decolorized cells take up the pink dye safranin, which explains the difference in color between the two types of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram negative cells appear pink.
When you interpret a Gram stained smear, you should also describe the morphology shape of the cells, and their arrangement. In Figure 5, there are two distinct types of bacteria, distinguishable by Gram stain reaction, and also by their shape and arrangement.
Below, describe these characteristics for both bacteria:. Some bacteria produce the waxy substance mycolic acid when they construct their cell walls. Mycolic acid acts as a barrier, protecting the cells from dehydrating, as well as from phagocytosis by immune system cells in a host. This waxy barrier also prevents stains from penetrating the cell, which is why the Gram stain does not work with mycobacteria such as Mycobacterium , which are pathogens of humans and animals.
For these bacteria, the acid — fast staining technique is used. To perform the acid-fast stain, a heat-fixed smear is flooded with the primary stain carbol fuchsin, while the slide is heated over a steaming water bath. Then the slide is allowed to cool and a solution of acid and alcohol is added as a decolorizer. All other cell types will be decolorized.
Methylene blue is then used as a counterstain. In the end, acid-fast bacteria AFB will be stained a bright pink color, and all other cell types will appear blue. Capsule : The polysaccharide goo that surrounds some species of bacteria and a few types of eukaryotic microbes is best visualized when the cells are negative stained.
In this method, the bacteria are first mixed with the stain, and then a drop of the mixture is spread across the surface of a slide in the thin film. With this method, capsules appear as a clear layer around the bacterial cells, with the background stained dark. Metachromatic granules or other intracytoplasmic bodies : Some bacteria may contain storage bodies that can be stained.
Various staining methods are used to visualize intracytoplasmic bodies in bacteria, which often provide an identification clue when observed in cells. 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.
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