Differential Staining Techniques

Viewing Bacterial Cells

The microscope is an extremely vital tool in microbiology, but there are constraints when it concerns making use of one to observe cells in general and bacterial cells in specific. Two of the the majority of vital involves are resolution and contrast. Resolution is a limitation that we can’t perform a lot around, considering that most bacterial cells are already near the resolution limit of many light microscopes. Contrast, yet, have the right to be enhanced by either making use of a different form of optical mechanism, such as phase contrast or a differential interference contrast microscopic lense, or by staining the cells (or the background) via a chromogenic dye that not just adds contrast, however offers them a shade as well.

You are watching: The gram stain works because of differences in the ________ of bacteria.

Tbelow are many kind of different stains and also staining procedures offered in microbiology. Some involve a solitary stain and also just a couple of procedures, while others usage multiple stains and a more facility procedure. Before you deserve to begin the staining procedure, the cells need to be mounted (smeared) and also solved onto a glass slide.

A bacterial smear is sindicate that—a small amount of society spreview in an extremely thin film on the surchallenge of the slide. To proccasion the bacteria from washing ameans throughout the staining measures, the smear may be chemically or physically “fixed” to the surface of the slide. Heat solving is an easy and also efficient method, and also is achieved by passing the slide briefly through the flame of a Bunsen burner, which causes the organic material to become more or less permanently afresolved to the glass surchallenge.

Heat solved smears are all set for staining. In a straightforward 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 have the right to be conveniently observed against the bright background. Anionic dyes are repelled by the cells, and also therefore the cells are bbest versus the stained background. See Figures 1 and also 2 for examples of both.

Figure 1. Negative stain of Cyptococcus neoformans, an encapsulated yeast
Figure 2. Hopeful stain of Staphylococcus aureus.

Probably the a lot of necessary feature made evident when you stain bacterial cells is their cellular morphology (not to be puzzled via early american morphology, which is the appearance of bacterial colonies on an agar plate). Many heterotrophic and also culturable bacteria come in a couple of standard shapes: spherical cells (coccus/cocci), rod-shaped cells (bacillus/bacilli), or rod-shaped cells with bends or twists (vibrios and spirilla, respectively). Tbelow is greater diversity of shapes among Archaea and also various other bacteria uncovered in ecosystems other than the humale body.

Often bacteria produce particular arrangements of cells, which create as an outcome of binary fission by the bacteria as they recreate. Arrangements are particularly obvious through non-motile bacteria, because the cells tend to continue to be together after the fission procedure is finish. Both the form and also setup of cells are qualities that deserve to be used to differentiate among bacteria. The the majority of typically encountered bacterial shapes (cocci and bacilli) and their feasible arrangements are shown in Figures 3 and 4.

Figure 3. Possible bacterial cell arrangements for cocci
Figure 4. Possible bacteria cell arrangements for bacilli

Differential Staining Techniques

In microbiology, differential staining approaches are used even more often than straightforward stains as a means of gathering indevelopment about bacteria. Differential staining approaches, which typically call for more than one stain and a number of procedures, are described as such bereason they permit the differentiation of cell kinds or cell frameworks. The most important of these is the Gram stain. Other differential staining approaches encompass the endospore stain (to recognize endospore-forming bacteria), the acid-fast stain (to discriminate Mycobacterium species from various other bacteria), a metachromatic stain to identify phosphate storage granules, and the capsule stain (to determine encapsulated bacteria). We will be perdeveloping the Gram stain and also endospore staining steps in lab, and see all set slides that highlight some of the other cellular structures present in some bacteria.

Gram Stain

Figure 5. Bacteria stained with Gram stain.

In 1884, medical professional Hans Christian Gram was researching the etiology (cause) of respiratory illness such as pneumonia. He occurred a staining procedure that allowed him to recognize a bacterium in lung tproblem taken from deceased patients as the etiologic agent of a fatal kind of pneumonia. Although it did bit in the way of therapy for the disease, the Gram stain technique made it much much easier to diagnose the cause of a person’s death at autopsy. Today we usage Gram’s staining approaches to help in the identification of bacteria, start through a preliminary classification right 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 entails 4 procedures. First cells are stained with crystal violet, complied with by the enhancement of a setting agent for the stain (iodine). Then alcohol is applied, which selectively gets rid of the stain from just the Gram negative cells. Finally, a second stain, safranin, is added, which counterstains the decolorized cells pink.

Although Gram didn’t understand it at the time, the primary distinction in between these two types of bacterial cells is their cell wall surfaces. Gram negative cell walls have actually an external membrane (additionally referred to as the envelope) that dissolves throughout the alcohol wash. This permits the crystal violet dye to escape. Only the decolorized cells take up the pink dye safranin, which describes the difference in color in between the two kinds of cells. At the conclusion of the Gram stain procedure, Gram positive cells appear purple, and Gram negative cells show up pink.

When you interpret a Gram stained smear, you need to likewise define the morphology (shape) of the cells, and their arrangement. In Figure 5, tright here are two unique types of bacteria, distinguishable by Gram stain reaction, and likewise by their form and also plan. Below, describe these attributes for both bacteria:

Acid Fast Stain

Some bacteria develop the waxy substance mycolic acid when they construct their cell walls. Mycolic acid acts as a obstacle, protecting the cells from dehydrating, and also from phagocytosis by immune system cells in a organize. This waxy barrier also avoids stains from penetrating the cell, which is why the Gram stain does not work-related through mycobacteria such as Mycobacterium, which are pathogens of people and animals. For these bacteria, the acidfast staining technique is supplied.

Figure 6. Acid-fast bacilli in sputum

To perdevelop the acid-rapid stain, a heat-fixed smear is flooded through the main stain carbol fuchsin, while the slide is heated over a steaming water bath. The heat “melts” the waxy cell wall and also permits the absorption of the dye by the cells. Then the slide is allowed to cool and a solution of acid and alcohol is added as a decolorizer. Cells that are “acid-fast” because of the mycolic acid in their cell wall stand up to decolorization and also retain the major stain. All various other cell kinds will certainly be decolorized. Methylene blue is then provided as a counterstain. In the end, acid-fast bacteria (AFB) will be stained a bbest pink color, and also all various other cell kinds will certainly appear blue.

Staining Methods to Highlight Specific Cell Structures

Capsule: The polysaccharide goo that surrounds some species of bacteria and also a couple of kinds of eukaryotic microbes is finest visualized when the cells are negative stained. In this strategy, the bacteria are initially mixed through the stain, and also then a drop of the mixture is spread across the surconfront of a slide in the thin film. With this strategy, capsules show up as a clear layer roughly the bacterial cells, with the background stained dark.

Metachromatic granules or other intracytoplasmic bodies: Some bacteria may contain storage bodies that deserve to be stained. One example is the Gram positive bacilli Corynebacterium, which stores phosphate in frameworks called “volutin” or metachromatic granules that are hooffered within the cell membrane. Various staining methods are offered to visualize intracytoplasmic bodies in bacteria, which frequently provide an identification clue as soon as oboffered in cells.

Endospore Stain

Endospores are dormant develops of living bacteria and should not be puzzled with reproductive spores created by fungi. These structures are created by a couple of genera of Gram-positive bacteria, nearly all bacilli, in response to adverse eco-friendly conditions. Two prevalent bacteria that develop endospores are Bacillus or Clostridum. Both live primarily in soil and as symbionts of plants and animals, and also develop endospores to survive in an setting that adjust quickly and also regularly.

The procedure of endosporulation (the development of endospores) involves a number of steras. After the bacterial cell replicates its DNA, layers of peptidoglydeserve to and protein are created to surround the hereditary material. Once fully formed, the endospore is released from the cell and also might sit dormant for days, weeks, or years. When even more favorable ecological problems prevail, endospores germinate and also go back to active duty as vegetative cells.

Mature endospores are highly resistant to eco-friendly problems such as warm and chemicals and also this permits survival of the bacterial species for incredibly long periods. Endospores created millions of years earlier have actually been successfully carried ago to life, ssuggest by providing them with water and also food.

Since the endospore coat is highly resistant to staining, a one-of-a-kind technique was developed to make them easier to view via a brightarea microscope. This technique, dubbed the endospore stain, supplies either heat or long exposure time to tempt the endospores to take up the primary stain, normally 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 used to administer shade and also comparison. When stained by this technique, the endospores are green, and also the vegetative cells stain pink, as displayed in Figure 7.

Figure 7. Bacterial cells via endospores, stained via the endospore stain.
Figure 8. Bacilli with endospores viewed by phase-comparison microscopy.

Although endospores themselves are resistant to the Gram stain strategy, bacterial cells caught in the procedure of developing these structures can be stained. In this instance, the endospores are viewed as clear oval or spherical locations within the stained cell. Endospores have the right to likewise be straight oboffered in cells by using phase comparison microscopy, as displayed in Figure 8.


Because many type of differential staining techniques need a number of actions and also take a lengthy time to finish, we will certainly not be performing all of the differential staining methods debated above.

Pre-stained slides will certainly be offered to visualize bacterial capsules, metachromatic granules, and acid-rapid bacilli. Obtain one slide of each of the three bacteria provided in the table listed below. As you watch these slides, make note of the “highlighted” frameworks. Your environmental isolate might have actually one or even more of these cellular functions, and learning to identify them will assist in identification. These have to all be perceived utilizing the oil immersion objective lens.

BacteriumStainDescription or sketch of cells with the stated feature
Flavobacterium capsulatumCapsule stain
Corynebacterium diphtheriaeMethylene blue(metachromatic granules)
Mycobacterium tuberculosisAcid quick stain

Gram Stain

All staining steps have to be done over a sink. The Gram stain procedure will be demonstrated, and an overview is offered in Table 1.

Table 1. Gram stain procedural procedures.
Primary stain(crystal violet)Add a number of drops of crystal violet to the smear and permit it to sit for 1 minute. Rinse the slide with water.Both Gram-positive and Gram-negative cells will certainly be stained purple by the crystal violet dye.
Mordant (iodine)Add numerous drops of iodine to the smear and also enable it to sit for 1 minute. Rinse the slide through water.Iodine “sets” the crystal violet, so both forms of bacteria will certainly remain purple.
Decolorization (ethanol)Add drops of ethanol one at a time until the runoff is clear. Rinse the slide through water.Gram-positive cells stand up to decolorization and remain purple. The dye is released from Gram-negative cells.
Counterstain(safranin)Add numerous drops of safranin to the smear and permit it to sit for one minute. Rinse the slide through water and blot dry.Gram-negative cells will certainly be stained pink by the safranin. This dye has no result on Gram-positive cells, which reprimary purple.

A volunteer from your lab bench should attain cultures of the bacteria you will be making use of in this lab, as directed by your instructor. One of the cultures will certainly be a Gram positive bacterium, and the various other will be Gram negative. Below, create the names of the bacteria you will certainly be using, along with the BSL for each culture:


Obtain 2 glass slides, and also prepare a smear of each of the two bacterial cultures, one per slide, as demonstrated. Allow to COMPLETELY air dry and warmth deal with. Stain both smears making use of the Gram stain approach. Observe the slides via a light microscope at 1,000X and record your monitorings in the table listed below.

Name of cultureGram stain reactionCellular morphologyArrangement

Gram Stain “Final Exam”: prepare a smear that has a mixture of the Gram-positive AND Gram-negative bacteria by including a tiny amount of each bacterium to a solitary drop of water on a slide. Heat resolve the smear and also Gram stain it. You need to have the ability to identify the Gram stain reaction, cellular morphology and plan of BOTH bacteria in this combined smear. Your instructor might ask to watch this slide and also market constructive commentary.

Endospore Stain

Only a few genera of bacteria create endospores and practically all of them are Gram-positive bacilli. Many notable are Bacillus and Clostridium species, which naturally live in soil and are common contaminants on surencounters. The development of Clostridium spp. is generally restricted to anaerobic environments; Bacillus spp. may flourish aerobically and also anaerobically. Endospore-developing bacteria are unique from various other teams of Gram positive bacilli and also distinguishable by their endospores.

An overcheck out of the endospore stain procedure is provided in Table 2.

Table 2. Endospore stain procedural actions.
Main stain(malachite green)Add a number of drops of malachite green to the smear and enable it to sit for 10 minutes. If the stain starts to dry out, include added drops.Vegetative cells will automatically take up the primary stain. Endospores are resistant to staining yet eventually take up the dye.
Decolorization(water)Rinse the slide under a gentle stream of water for 10-15 secs.Once the endospores are stained, they remajor green. A thostormy rinse through water will certainly decolorize the vegetative cells.
Counterstain(safranin)Add numerous drops of safranin to the smear and permit it to sit for 1 minute. Rinse the slide and also blot dry.Decolorized vegetative cells take up the counterstain and also show up pink; endospores are light green.

After staining, endospores frequently appear as light green oval or spherical structures, which might be viewed either within or outside of the vegetative cells, which show up pink.

The shape and location of the endospores inside the bacterial cells, together with whether the sporangium is either distending (D) or not distending (ND) the sides of the cell, are important characteristics that help in distinguishing among species (view Figure 9).

Figure 9Oval, central, not distended (ND)Oval, terminal, ND (and paraspdental crystal)Oval, terminal, distended (D)Oval, central, DSpherical, terminal, DOval, lateral, D

Endospores are quite resistant to many staining procedures; yet, in a frequently stained smear, they might be visible as “outlines” with clear room within. If you observe “outlines” or what show up to be “ghosts” of cells in a Gram stained smear of a Gram-positive bacilli, then the endospore stain have to additionally be perdeveloped to confirm the presence or absence of endospores.

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A volunteer from your lab bench should achieve bacterial societies for endospore staining, as directed by your instructor. Note that these will all be species of Bacillus. Prepare smears and also stain each making use of the endospore staining strategy. Observe the slides and also note the form and place of the endospore and also the appearance of the sporangium (swollen or not swollen) in the table below:

Name of cultureEndospore ShapeLocationSporangium

In enhancement, pick ONE of the cultures from above and Gram stain it. Record your outcomes listed below in the spaces provided:

Name of Gram stained culture: __________________________________________________

Gram stain reactivity and also cellular morphology: ______________________________________

Are endospores visible in the Gram stained smear? _________________ If you see endospores, define how they appear in the Gram stained preparation, and exactly how this is similar to and various from what you see in the endospore stained preparation.