Differential Staining Techniques

Viewing bacter Cells

The microscopic lense is a very important device in microbiology, however there are restrictions when it pertains to using one to watch cells in general and bacterial cells in particular. Two of the many important comes to are resolution and contrast. Resolution is a limitation that we can’t do lot about, because most bacterial cells are already near the resolution border of many light microscopes. Contrast, however, have the right to be boosted by either making use of a different form 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 the not just adds contrast, however gives castle a shade as well.

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There are many different stains and also staining steps used in microbiology. Part involve a single stain and also just a couple of steps, while others use multiple stains and a more complicated procedure. Before you can start the staining procedure, the cells need to be placed (smeared) and fixed top top a glass slide.

A bacterial smear is simply that—a small amount of culture spread in a really thin film on the surface of the slide. To stop the bacteria from washing away throughout the staining steps, the smear may be chemically or physically “fixed” come the surface ar of the slide. Warmth fixing is simple and efficient method, and is achieved by happen the on slide briefly with the fire of a Bunsen burner, which reasons the organic material to become much more or less permanently affixed come the glass surface.

Heat solved smears are all set for staining. In a basic stain, water that are either attracted by fee (a cationic dye such together methylene blue or decision violet) or repelled by charge (an anionic dye such as eosin or India ink) are added to the smear. Cationic dyes bind the bacterial cell which can be quickly observed against the shining background. Anionic dyes space repelled through the cells, and therefore the cells space bright versus the stained background. See numbers 1 and 2 for examples of both.

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

Probably the most essential feature made noticeable when friend stain bacterial cells is their cellular morphology (not come be puzzled with colonial morphology, i beg your pardon is the illustration of bacterial colonies on an agar plate). Most heterotrophic and also culturable bacteria come in a couple of basic shapes: spherical cell (coccus/cocci), rod-shaped cells (bacillus/bacilli), or rod-shaped cells v bends or twists (vibrios and spirilla, respectively). Over there is higher diversity the shapes among Archaea and other bacteria discovered in ecosystems other than the human body.

Often bacteria create specific arrangements the cells, which form as a result of binary fission by the bacteria as they reproduce. Species are particularly obvious with non-motile bacteria, since the cells often tend to remain together after the fission procedure is complete. Both the shape and arrangement of cell are attributes that can be supplied to distinguish among bacteria. The most commonly encountered bacterial forms (cocci and bacilli) and their possible arrangements are presented in figures 3 and 4.

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

Differential Staining Techniques

In microbiology, differential staining techniques are used more often than basic stains as a method of gathering information around bacteria. Differential staining methods, which generally require an ext than one stain and several steps, space referred to therefore because they permit the differentiation that cell types or cabinet structures. The most essential of these is the Gram stain. Various other differential staining methods encompass the endospore stain (to recognize endospore-forming bacteria), the acid-fast stain (to discriminate Mycobacterium varieties from various other bacteria), a metachromatic stain to identify phosphate warehouse granules, and the capsule stain (to identify encapsulated bacteria). We will be performing the Gram stain and also endospore staining procedures in lab, and also view ready slides that highlight several of the various other cellular structures existing in part bacteria.

Gram Stain

Figure 5. Bacteria stained with Gram stain.

In 1884, medical professional Hans Christian Gram was examining the etiology (cause) of respiratory diseases such together pneumonia. He occurred a staining procedure that allowed him to determine a bacterium in lung tissue taken indigenous deceased patients as the etiologic certified dealer of a fatal kind of pneumonia. Although it did little in the method of therapy for the disease, the Gram stain an approach made that much much easier to diagnose the cause of a person’s fatality at autopsy. Now we usage Gram’s staining techniques to assist in the to know of bacteria, beginning with a preliminary group into one of two groups: Gram positive or Gram negative.

The differential nature that the Gram stain is based upon the capacity of part bacterial cell to maintain a major stain (crystal violet) by resisting a decolorization process. Gram staining requires four steps. An initial cells room stained with crystal violet, complied with by the addition of a setting agent for the stain (iodine). Climate alcohol is applied, i beg your pardon selectively removes the stain from only the Gram negative cells. Finally, a second stain, safranin, is added, which counterstains the decolorized cells pink.

Although Gram didn’t know it at the time, the key difference in between these two species of bacterial cells is their cell walls. Gram an adverse cell walls have an outer membrane (also called the envelope) the dissolves during the alcohol wash. This patent the decision violet dye come escape. Just the decolorized cells take it up the pink dye safranin, which defines the difference in color in between the two species of cells. At the conclusion of the Gram stain procedure, Gram hopeful cells appear purple, and also Gram an adverse cells appear pink.

When you analyze a Gram stained smear, you should likewise describe the morphology (shape) the the cells, and also their arrangement. In figure 5, there are two distinct species of bacteria, distinguishable by Gram stain reaction, and additionally by your shape and arrangement. Below, describe these features for both bacteria:

Acid fast Stain

Some bacteria produce the waxy substance mycolic acid as soon as they build their cell walls. Mycolic mountain acts together a barrier, protecting the cells from dehydrating, as well as from phagocytosis by immune mechanism cells in a host. This waxy barrier additionally prevents stains indigenous penetrating the cell, i beg your pardon is why the Gram stain does not work with mycobacteria such together Mycobacterium, which are pathogens the humans and animals. For these bacteria, the acidfast staining method is used.

Figure 6. Acid-fast bacilli in sputum

To perform the acid-fast stain, a heat-fixed smear is flooded v the major stain carbol fuchsin, while the slide is heated over a steaming water bath. The warmth “melts” the waxy cell wall surface and permits the absorption of the dye through the cells. Then the slide is enabled to cool and also a systems of acid and also alcohol is included as a decolorizer. Cells that space “acid-fast” since of the mycolic acid in your cell wall resist decolorization and retain the major stain. All other cell varieties will it is in decolorized. Methylene blue is then offered as a counterstain. In the end, acid-fast bacteria (AFB) will be stained a glowing pink color, and also all various other cell varieties will appear blue.

Staining techniques to Highlight specific Cell Structures

Capsule: The polysaccharide goo the surrounds some varieties of bacteria and a few types of eukaryotic microbes is best visualized when the cells are an unfavorable stained. In this method, the bacteria are very first mixed with the stain, and also then a autumn of the mixture is spread throughout the surface of a on slide in the slim film. V this method, capsules appear as a clear layer about the bacterial cells, through the lift stained dark.

Metachromatic granules or other intracytoplasmic bodies: some bacteria may contain storage bodies that have the right to be stained. One instance is the Gram optimistic bacilli Corynebacterium, which shop phosphate in structures referred to as “volutin” or metachromatic granules that room housed within the cabinet membrane. Various staining techniques are offered to visualize intracytoplasmic body in bacteria, which often provide an to know clue when observed in cells.

Endospore Stain

Endospores room dormant creates of living bacteria and also should no be confused with reproductive spores created by fungi. These frameworks are developed by a couple of genera the Gram-positive bacteria, almost all bacilli, in an answer to adverse eco-friendly conditions. Two common bacteria that develop endospores space Bacillus or Clostridum. Both live primarily in soil and also as symbionts the plants and also animals, and produce endospores to survive in an environment that readjust rapidly and also often.

The process of endosporulation (the formation of endospores) involves several stages. ~ the bacterial cabinet replicates the DNA, layers of peptidoglycan and also protein are developed to surround the hereditary material. Once totally formed, the endospore is released from the cell and also may sit dormant for days, weeks, or years. When much more favorable environmental problems prevail, endospores germinate and return to energetic duty as vegetative cells.

Mature endospores are very resistant come environmental problems such together heat and chemicals and also this permits survival of the bacterial species for really long periods. Endospores developed millions the years earlier have been properly brought ago to life, simply by offering them through water and also food.

Because the endospore cloak is highly resistant to staining, a special an approach was arisen to make them simpler to see with a brightfield microscope. This method, referred to as the endospore stain, supplies either heat or lengthy exposure time to attract the endospores to take it up the primary stain, usually a water soluble dye such together malachite green due to the fact that endospores are permeable to water. Complying with a decolorization action which clears the dye from the vegetative cell in the smear, the counterstain safranin is applied to carry out color and also contrast. When stained through this method, the endospores space green, and the vegetative cells stain pink, as displayed in number 7.

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

Although endospores themselves are resistant to the Gram stain technique, bacterial cells caught in the process of developing these structures have the right to be stained. In this case, the endospores are seen as clear oval or spherical locations within the stained cell. Endospores can additionally be directly observed in cells by using phase contrast microscopy, as displayed in figure 8.


Because numerous differential staining methods require numerous steps and take a lengthy time come complete, we will not be performing every one of the differential staining methods questioned above.

Pre-stained slides will be supplied to visualize bacter capsules, metachromatic granules, and acid-fast bacilli. Attain one slide of every of the three bacteria listed in the table below. As you watch these slides, make keep in mind of the “highlighted” structures. Your eco-friendly isolate may have actually one or much more of this cellular features, and also learning to identify them will help in identification. These must all be regarded using the oil immersion objective lens.

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

Gram Stain

All staining steps should be excellent over a sink. The Gram stain procedure will certainly be demonstrated, and review is detailed in Table 1.

Table 1. Gram stain procedural steps.
Primary stain(crystal violet)Add several drops of crystal violet come the smear and enable it come sit because that 1 minute. To wash the slide with water.Both Gram-positive and also Gram-negative cells will be stained violet by the crystal violet dye.
Mordant (iodine)Add several drops of iodine come the smear and enable it to sit because that 1 minute. To wash the slide with water.Iodine “sets” the crystal violet, therefore both varieties of bacteria will remain purple.
Decolorization (ethanol)Add drops of ethanol one at a time till the runoff is clear. Wash 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 that safranin come the smear and allow it to sit for one minute. Rinse the slide through water and blot dry.Gram-negative cells will certainly be stained pink through the safranin. This dye has no result on Gram-positive cells, which continue to be purple.

A volunteer from her lab bench should obtain cultures of the bacteria you will be using in this lab, as directed by her instructor. One of the cultures will be a Gram positive bacterium, and the other will be Gram negative. Below, create the names of the bacteria you will certainly be using, together with the BSL because that each culture:


Obtain 2 glass slides, and also prepare a smear of every of the two bacterial cultures, one per slide, as demonstrated. Allow to fully air dry and also heat fix. Stain both smears utilizing the Gram stain method. Observe the slides with a light microscope at 1,000X and record your monitorings in the table below.

Name of cultureGram stain reactionCellular morphologyArrangement

Gram Stain “Final Exam”: prepare a smear that consists of a mixture of the Gram-positive and also Gram-negative bacteria by adding a tiny amount of every bacterium come a solitary drop the water ~ above a slide. Warm fix the smear and also Gram stain it. Girlfriend should be able to determine the Gram stain reaction, moving morphology and arrangement of BOTH bacteria in this combined smear. Her instructor may ask to see this slide and offer constructive commentary.

Endospore Stain

Only a couple of genera the bacteria create endospores and nearly every one of them space Gram-positive bacilli. Many notable room Bacillus and Clostridium species, which normally live in soil and also are usual contaminants on surfaces. The development of Clostridium spp. Is typically restricted to anaerobic environments; Bacillus spp. May prosper aerobically and anaerobically. Endospore-forming bacteria are distinctive from other teams of Gram confident bacilli and also distinguishable by their endospores.

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

Table 2. Endospore stain procedural steps.
Primary stain(malachite green)Add numerous drops of malachite environment-friendly to the smear and allow it to sit for 10 minutes. If the stain beginning to dried out, include additional drops.Vegetative cell will automatically take up the main stain. Endospores are resistant to staining however eventually take it up the dye.
Decolorization(water)Rinse the slide under a tenderness stream that water for 10-15 seconds.Once the endospores are stained, they continue to be green. A thoroughly rinse through water will decolorize the vegetative cells.
Counterstain(safranin)Add numerous drops the safranin to the smear and permit it come sit because that 1 minute. Wash the slide and blot dry.Decolorized vegetative cells take it up the counterstain and appear pink; endospores space light green.

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

The shape and location that the endospores within the bacter cells, together with whether the sporangium is one of two people distending (D) or no distending (ND) the political parties of the cell, are important characteristics that aid in differentiating among species (see number 9).

Figure 9Oval, central, no distended (ND)Oval, terminal, ND (and parasporal crystal)Oval, terminal, distended (D)Oval, central, DSpherical, terminal, DOval, lateral, D

Endospores are rather resistant to most staining procedures; however, in a regularly stained smear, they might be visible as “outlines” through clear an are within. If you observe “outlines” or what show up to be “ghosts” of cell in a Gram stained smear that a Gram-positive bacilli, climate the endospore stain should also be carry out to check the visibility or lack of endospores.

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A volunteer from her lab bench should attain bacterial societies for endospore staining, as directed by your instructor. Keep in mind that these will all be varieties of Bacillus. Prepare smears and also stain each making use of the endospore staining technique. Observe the slides and also note the shape and location that the endospore and the figure of the sporangium (swollen or no swollen) in the table below:

Name the cultureEndospore ShapeLocationSporangium

In addition, choose ONE the the societies from above and Gram stain it. Record your results below in the spaces provided:

Name that Gram stained culture: __________________________________________________

Gram stain reaction and cellular morphology: ______________________________________

Are endospores clearly shows in the Gram stained smear? _________________ If you see endospores, explain how they show up in the Gram stained preparation, and also how this is comparable to and different from what you watch in the endospore stained preparation.