Viewing Bacterial Cells
The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Two of the most important concerns are resolution and contrast. Resolution is a limitation that we can’t do much about, since most bacterial cells are already near the resolution limit of most light microscopes. Contrast, however, can be improved by either using a different type of optical system, such as phase contrast or a differential interference contrast microscope, or by staining the cells (or the background) with a chromogenic dye that not only adds contrast, but gives them a color as well.
There are many different stains and staining procedures used in microbiology. Some involve a single stain and just a few steps, while others use multiple stains and a more complicated procedure. Before you can begin the staining procedure, the cells have to be mounted (smeared) and fixed onto a glass slide.
A bacterial smear is simply that—a small amount of culture spread in a very thin film on the surface of the slide. To prevent the bacteria from washing away during the staining steps, the smear may be chemically or physically “fixed” to the surface of the slide. Heat fixing is an easy and efficient method, and is accomplished by passing the slide briefly through the flame of a Bunsen burner, which causes the biological material to become more or less permanently affixed to the glass surface.
Heat fixed smears are ready for staining. In a simple stain, dyes that are either attracted by charge (a cationic dye such as methylene blue or crystal violet) or repelled by charge (an anionic dye such as eosin or India ink) are added to the smear. Cationic dyes bind the bacterial cells which can be easily observed against the bright background. Anionic dyes are repelled by the cells, and therefore the cells are bright against the stained background. See Figures 1 and 2 for examples of both.
. .Archaea and other bacteria living outside the human body exhibit greater differences in shapes than those found in our bodies. When the Rgroup is alkyl, diazonium ions readily decompose via the SN1mechanism, with dinitrogen as the leaving group.
Differential Staining Techniques
Differential staining techniques are used more frequently than simple stains when studying bacteria in microbiology.Methods of differential staining, which often involve more than one stain and several steps, are known as such because they allow the differentiation of cell types or structures.Among these, Gram stain is of the most importance. .In lab, we will perform the Gram stain and endospore staining procedures and view slides illustrating some other cellular structures found in bacteria.
In 1884, physician Hans Christian Gram was studying the etiology (cause) of respiratory diseases such as pneumonia. He developed a staining procedure that allowed him to identify a bacterium in lung tissue taken from deceased patients as the etiologic agent of a fatal type of pneumonia. Although it did little in the way of treatment for the disease, the Gram stain method made it much easier to diagnose the cause of a person’s death at autopsy. Today we use Gram’s staining techniques to aid in the identification of bacteria, beginning with a preliminary classification into one of two groups: Gram positive or Gram negative.
The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process. Gram staining involves four steps. First cells are stained with crystal violet, followed by the addition of a setting agent for the stain (iodine). Then alcohol is applied, which selectively removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is added, which counterstains the decolorized cells pink.
Although Gram didn’t know it at the time, the main difference between these two types of bacterial cells is their cell walls. Gram negative cell walls have an outer membrane (also called the envelope) that dissolves during the alcohol wash. This permits the crystal violet dye to escape. 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:
Acid Fast Stain
During the assembly of their cell walls, some bacteria produce a waxy substance known as mycolic acid.A layer of mycolic acid protects cells from dehydration and from being phagocytosed by an immune system cell in the host.A waxy barrier also prevents stains from penetrating the cell, which is why Gram stains don't work on pathogenic bacteria, such as Mycobacterium, which are pathogens of humans and animals.Staining with acid-fast is used on these bacteria.
.Heat melts the waxy cell wall, which allows the dye to be absorbed by the cells.In the next step, the slide is allowed to cool and a solution of acid and alcohol is added as a decolorizer.As a consequence, acid-fast cells are resistant to decolorization and maintain the primary stain.All other types of cells lose their color. .As a result, acid-fast bacteria (AFB) will appear bright pink, and all other types of cells will appear blue.
Staining Methods to Highlight Specific Cell Structures
In eukaryotes and some bacteria, the polysaccharide goo surrounding the cells is best visualized using negative staining.Using this method, the stain is mixed with the bacteria before a drop is applied to the slide surface in a thin film.A clear layer surrounds the bacterial cells in this method, while the background is stained dark. A few bacteria contain metachromatic granules or other intracytoplasmic bodies that may be stained. .Several methods are available for visualizing intracytoplasmic bodies in bacteria, which provide essential clues for identification 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. When stained by this method, the endospores are green, and the vegetative cells stain pink, as shown in Figure 7.
.As can be seen here, the endospores appear as spherical or oval areas within the stained cells.Endospores can also be observed directly in cells using phase contrast microscopy, as shown in Figure 8. Often, bacteria produce specific arrangements of cells, which form as the bacteria divide. Non-motile bacteria display arrangement particularly prominently, since the cells tend to stay together after the fission process is complete.On pre-stained slides, we will observe bacterial capsules, metachromatic granules, and acid-fast bacilli.Take one slide of each of the three bacteria listed below.As you view these slides, pay attention to the "highlighted" structures.There are several features of your environmental isolate that may be distinctive, so learning to recognize them will be helpful in identifying it.
|Bacterium||Stain||Description or sketch of cells with the specified feature|
|Flavobacterium capsulatum||Capsule stain|
|Corynebacterium diphtheriae||Methylene blue(metachromatic granules)|
|Mycobacterium tuberculosis||Acid fast stain|
All staining procedures should be done over a sink. The Gram stain procedure will be demonstrated, and an overview is provided in Table 1.
|Table 1. Gram stain procedural steps.|
|Primary stain(crystal violet)||Add several drops of crystal violet to the smear and allow it to sit for 1 minute. Rinse the slide with water.||Both Gram-positive and Gram-negative cells will be stained purple by the crystal violet dye.|
|Mordant (iodine)||Add several drops of iodine to the smear and allow it to sit for 1 minute. Rinse the slide with water.||Iodine “sets” the crystal violet, so both types of bacteria will remain purple.|
|Decolorization (ethanol)||Add drops of ethanol one at a time until the runoff is clear. Rinse the slide with water.||Gram-positive cells resist decolorization and remain purple. The dye is released from Gram-negative cells.|
|Counterstain(safranin)||Add several drops of safranin to the smear and allow it to sit for one minute. Rinse the slide with water and blot dry.||Gram-negative cells will be stained pink by the safranin. This dye has no effect on Gram-positive cells, which remain purple.|
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. Allow to COMPLETELY air dry and heat fix. Stain both smears using the Gram stain method. Observe the slides with a light microscope at 1,000X and record your observations in the table below.
|Name of culture||Gram stain reaction||Cellular morphology||Arrangement|
Gram Stain “Final Exam”: prepare a smear that contains a mixture of the Gram-positive AND Gram-negative bacteria by adding a small amount of each bacterium to a single drop of water on a slide. 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. is typically limited to anaerobic environments; Bacillus spp. may grow aerobically and anaerobically. Endospore-forming bacteria are distinct from other groups of Gram positive bacilli and distinguishable by their endospores.
An overview of the endospore stain procedure is provided in Table 2.
|Table 2. Endospore stain procedural steps.|
|Primary stain(malachite green)||Add several drops of malachite green to the smear and allow it to sit for 10 minutes. If the stain starts to dry out, add additional drops.||Vegetative cells will immediately take up the primary stain. Endospores are resistant to staining but eventually take up the dye.|
|Decolorization(water)||Rinse the slide under a gentle stream of water for 10-15 seconds.||Once the endospores are stained, they remain green. A thorough rinse with water will decolorize the vegetative cells.|
|Counterstain(safranin)||Add several drops of safranin to the smear and allow it to sit for 1 minute. Rinse the slide and blot dry.||Decolorized vegetative cells take up the counterstain and appear pink; endospores are light green.|
Differentiation among species is greatly assisted by observing how the endospores were formed and where they were located inside the bacterial cells. If the sporangium was distending (D) or not (ND) the sides of the cell is another characteristic that can be observed each species has (see Figure 9).
Endospores are quite resistant to most staining procedures; however, in a routinely stained smear, they may be visible as “outlines” with clear space within. If you observe “outlines” or what appear to be “ghosts” of cells in a Gram stained smear of a Gram-positive bacilli, then the endospore stain should also be performed to confirm the presence or absence of endospores.
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. Observe the slides and note the shape and location of the endospore and the appearance of the sporangium (swollen or not swollen) in the table below:
|Name of culture||Endospore Shape||Location||Sporangium|
In addition, choose ONE of the cultures from above and Gram stain it. Record your results below in the spaces provided:
Name of Gram stained culture: __________________________________________________
Gram stain reaction and cellular morphology: ______________________________________
Are endospores visible in the Gram stained smear? _________________ If you see endospores, describe how they appear in the Gram stained preparation, and how this is similar to and different from what you see in the endospore stained preparation.