Differential Staining Techniques

Viewing bacterial Cells

The microscopic lense is a very important device in microbiology, however there are restrictions when it involves using one to observe cells in general and bacterial cell in particular. 2 of the many important involves are resolution and contrast. Resolution is a limitation that us can’t do lot about, because most bacterial cells are currently near the resolution limit of many light microscopes. Contrast, however, deserve to be enhanced by either utilizing a different kind of optical system, such as phase comparison or a differential interference comparison microscope, or through staining the cells (or the background) through a chromogenic dye the not just adds contrast, yet gives castle a shade as well.

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There are plenty of different stains and also staining measures used in microbiology. Some involve a single stain and just a couple of steps, if others usage multiple stains and a more complicated procedure. Prior to you can start the staining procedure, the cells need to be mounted (smeared) and also fixed onto a glass slide.

A bacterial smear is simply that—a little amount of society spread in a an extremely thin film on the surface of the slide. To prevent the bacteria native washing away during the staining steps, the smear may be chemically or physically “fixed” come the surface ar of the slide. Warmth fixing is straightforward and reliable method, and is completed by passing the slide briefly through the flame of a Bunsen burner, which causes the organic material come become an ext or much less permanently affixed come the glass surface.

Heat addressed smears are prepared for staining. In a an easy stain, dyes that room either attracted by fee (a cationic dye such as methylene blue or decision violet) or repelled by charge (an anionic dye such together eosin or India ink) are included to the smear. Cationic dyes bind the bacterial cell which deserve to be quickly observed against the glowing background. Anionic dyes are repelled by the cells, and also therefore the cells room bright versus the stained background. See figures 1 and also 2 for examples of both.

Figure 1. An unfavorable stain of Cyptococcus neoformans, one encapsulated yeast
Figure 2. Positive stain of Staphylococcus aureus.

Probably the most crucial feature made apparent when friend stain bacterial cell is your cellular morphology (not to be puzzled with early american morphology, which is the illustration of bacterial swarms on an agar plate). Many heterotrophic and also culturable bacteria come in a couple of basic shapes: spherical cell (coccus/cocci), rod-shaped cell (bacillus/bacilli), or rod-shaped cells through bends or twists (vibrios and also spirilla, respectively). Over there is better diversity of shapes among Archaea and also other bacteria found in ecosystems other than the human body.

Often bacteria create specific arrangements that cells, which type as a result of binary fission through the bacteria as they reproduce. Kinds are particularly obvious with non-motile bacteria, because the cells tend to continue to be together after the fission procedure is complete. Both the shape and arrangement of cells are features that have the right to be offered to distinguish among bacteria. The most commonly encountered bacterial forms (cocci and also bacilli) and also their possible arrangements are displayed in numbers 3 and 4.

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

Differential Staining Techniques

In microbiology, differential staining approaches are used an ext often than an easy stains as a means of collection information around bacteria. Differential staining methods, which generally require much more than one stain and several steps, space referred to thus because they allow the differentiation of cell varieties or cell structures. The most important of these is the Gram stain. Other differential staining methods incorporate the endospore stain (to recognize endospore-forming bacteria), the acid-fast stain (to discriminate Mycobacterium types from various other bacteria), a metachromatic stain to recognize phosphate storage granules, and also the capsule stain (to identify encapsulated bacteria). We will be performing the Gram stain and endospore staining steps in lab, and also view prepared slides the highlight some of the other cellular structures existing in some bacteria.

Gram Stain

Figure 5. Bacteria stained v Gram stain.

In 1884, medical professional Hans Christian Gram was studying the etiology (cause) the respiratory conditions such as pneumonia. He occurred a staining procedure that enabled him to determine a bacterium in lung organization taken indigenous deceased patients as the etiologic agent of a fatal kind of pneumonia. Although the did little in the way of treatment for the disease, the Gram stain an approach made it much less complicated to diagnose the cause of a person’s death at autopsy. This particular day we usage Gram’s staining approaches to help in the to know of bacteria, start with a preliminary group into among two groups: Gram positive or Gram negative.

The differential nature of the Gram stain is based upon the capability of some bacterial cell to retain a major stain (crystal violet) by resisting a decolorization process. Gram staining entails four steps. Very first cells are stained with crystal violet, followed by the enhancement of a setting agent because that the stain (iodine). Climate alcohol is applied, i m sorry selectively clears the stain from only the Gram an unfavorable cells. Finally, a second stain, safranin, is added, i beg your pardon counterstains the decolorized cell pink.

Although Gram didn’t understand it in ~ the time, the main difference in between these two varieties of bacterial cells is your cell walls. Gram negative cell walls have an external membrane (also called the envelope) the dissolves during the alcohol wash. This patent the decision violet dye to escape. Only the decolorized cells take up the pink dye safranin, which defines the difference in color between the two types of cells. In ~ the conclusion that the Gram stain procedure, Gram positive cells show up purple, and Gram an adverse cells appear pink.

When you translate a Gram stained smear, friend should likewise describe the morphology (shape) of the cells, and their arrangement. In number 5, there space two distinct types of bacteria, distinguishable by Gram stain reaction, and likewise by their shape and arrangement. Below, define these qualities for both bacteria:

Acid quick Stain

Some bacteria develop the waxy problem mycolic acid once they build their cell walls. Mycolic mountain acts as a barrier, protecting the cell from dehydrating, and also from phagocytosis through immune device cells in a host. This waxy barrier likewise prevents stains from penetrating the cell, i m sorry is why the Gram stain go not occupational with mycobacteria such together Mycobacterium, which are pathogens of humans and also animals. Because that these bacteria, the acidfast staining an approach is used.

Figure 6. Acid-fast bacilli in sputum

To execute the acid-fast stain, a heat-fixed smear is flooded v the primary stain carbol fuchsin, while the on slide is heated end a steaming water bath. The warm “melts” the waxy cell wall and patent the absorb of the dye by the cells. Climate the on slide is allowed to cool and a systems of acid and also alcohol is included as a decolorizer. Cell that space “acid-fast” because of the mycolic acid in your cell wall resist decolorization and also retain the major stain. All various 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 all various other cell types will show up blue.

Staining methods to Highlight particular Cell Structures

Capsule: The polysaccharide goo the surrounds some varieties of bacteria and also a couple of types of eukaryotic microbes is best visualized as soon as the cells are an unfavorable stained. In this method, the bacteria are first mixed with the stain, and then a fall of the mixture is spread across the surface of a slide in the thin film. V this method, capsules appear as a clean layer roughly the bacter cells, through the lift stained dark.

Metachromatic granules or other intracytoplasmic bodies: some bacteria may contain storage bodies that can be stained. One example is the Gram optimistic bacilli Corynebacterium, which shop phosphate in structures dubbed “volutin” or metachromatic granules that are housed in ~ the cabinet membrane. Various staining approaches are offered to visualize intracytoplasmic body in bacteria, i m sorry often carry out an identification clue when observed in cells.

Endospore Stain

Endospores space dormant develops of life bacteria and should not be perplexed with reproductive spores produced by fungi. These frameworks are created by a couple of genera the Gram-positive bacteria, practically all bacilli, in an answer to adverse environmental conditions. Two usual bacteria that create endospores are Bacillus or Clostridum. Both live mainly in soil and also as symbionts that plants and also animals, and also produce endospores to survive in an environment that readjust rapidly and often.

The procedure of endosporulation (the development of endospores) involves several stages. After the bacterial cabinet replicates that is DNA, class of peptidoglycan and protein are produced to surround the genetic material. Once fully formed, the endospore is exit from the cell and may sit dormant for days, weeks, or years. When more favorable environmental conditions prevail, endospores germinate and also return to energetic duty as vegetative cells.

Mature endospores are extremely resistant come environmental problems such together heat and also chemicals and also this permits survive of the bacterial species for very long periods. Endospores developed millions that years ago have been successfully brought back to life, simply by providing them through water and also food.

Because the endospore cloak is highly resistant to staining, a special technique was emerged to make them much easier to see with a brightfield microscope. This method, called the endospore stain, uses either heat or long exposure time to lure the endospores to take up the primary stain, normally a water soluble dye such together malachite green since endospores are permeable to water. Complying with a decolorization step which gets rid of the dye indigenous the vegetative cells in the smear, the counterstain safranin is applied to administer color and also contrast. As soon as stained through this method, the endospores are green, and the vegetative cells stain pink, as shown in number 7.

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

Although endospores themselves room resistant to the Gram stain technique, bacterial cells caught in the process of developing these structures can be stained. In this case, the endospores are viewed as clear oval or spherical locations within the stained cell. Endospores can also be straight observed in cells by making use of phase comparison microscopy, as presented in figure 8.


Because plenty of differential staining techniques require number of steps and also take a long time come complete, we will certainly not be performing every one of the differential staining methods debated above.

Pre-stained slides will be used to visualize bacter capsules, metachromatic granules, and acid-fast bacilli. Obtain one slide of each of the 3 bacteria listed in the table below. Together you watch these slides, make note of the “highlighted” structures. Your environmental isolate may have actually one or an ext of these cellular features, and learning to acknowledge them will aid in identification. These need to all be viewed using the oil immersion objective lens.

BacteriumStainDescription or map out of cells through the stated feature
Flavobacterium capsulatumCapsule stain
Corynebacterium diphtheriaeMethylene blue(metachromatic granules)
Mycobacterium tuberculosisAcid rapid stain

Gram Stain

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

Table 1. Gram stain procedural steps.
Primary stain(crystal violet)Add number of drops of crystal violet to the smear and allow it to sit for 1 minute. To wash the slide v water.Both Gram-positive and also Gram-negative cells will be stained purple by the decision violet dye.
Mordant (iodine)Add several drops that iodine to the smear and permit it come sit because that 1 minute. To wash the slide through water.Iodine “sets” the decision violet, therefore both species of bacteria will stay purple.
Decolorization (ethanol)Add autumn of ethanol one at a time till the runoff is clear. Rinse the slide v water.Gram-positive cells stand up to decolorization and also remain purple. The dye is exit from Gram-negative cells.
Counterstain(safranin)Add numerous drops of safranin to the smear and allow it come sit because that one minute. To wash the slide with water and blot dry.Gram-negative cells will be stained pink through the safranin. This dye has actually no effect on Gram-positive cells, which remain purple.

A volunteer from your lab bench should achieve cultures the the bacteria you will be utilizing in this lab, together directed by her instructor. One of the cultures will it is in a Gram hopeful bacterium, and the various other will be Gram negative. Below, create the names of the bacteria you will be using, together with the BSL for each culture:


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

Name the cultureGram stain reactionCellular morphologyArrangement

Gram Stain “Final Exam”: prepare a smear that has a mixture the the Gram-positive and also Gram-negative bacteria by including a tiny amount of every bacterium come a solitary drop the water top top a slide. Warm fix the smear and Gram stain it. Friend should have the ability to determine the Gram stain reaction, moving morphology and arrangement the BOTH bacteria in this mixed smear. Her instructor might ask to see this slide and also offer constructive commentary.

Endospore Stain

Only a couple of genera the bacteria create endospores and also nearly all of them space Gram-positive bacilli. Most notable space Bacillus and Clostridium species, which normally live in soil and are usual contaminants ~ above surfaces. The expansion of Clostridium spp. Is typically limited to anaerobic environments; Bacillus spp. May thrive aerobically and anaerobically. Endospore-forming bacteria are distinctive from other groups of Gram positive bacilli and distinguishable by your endospores.

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

Table 2. Endospore stain procedural steps.
Primary stain(malachite green)Add numerous drops of malachite green to the smear and permit it to sit because that 10 minutes. If the stain beginning to dried out, include additional drops.Vegetative cells will immediately take increase the primary stain. Endospores room resistant come staining but eventually take it up the dye.
Decolorization(water)Rinse the on slide under a gentle stream that water because that 10-15 seconds.Once the endospores are stained, they stay green. A thorough rinse through water will decolorize the vegetative cells.
Counterstain(safranin)Add numerous drops the safranin come the smear and allow it come sit for 1 minute. Wash the slide and also blot dry.Decolorized vegetative cells take it up the counterstain and also appear pink; endospores are light green.

After staining, endospores typically appear as light green oval or spherical structures, which may be checked out either in ~ or exterior of the vegetative cells, which appear pink.

The shape and also location of the endospores within the bacter cells, together with whether the sporangium is either distending (D) or not distending (ND) the political parties of the cell, space important attributes that aid in separating among types (see number 9).

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

Endospores are quite resistant to most staining procedures; however, in a regularly stained smear, they may be visible as “outlines” through clear an are within. If you watch “outlines” or what appear to it is in “ghosts” of cells in a Gram stained smear of a Gram-positive bacilli, climate the endospore stain should additionally be perform to check the existence or absence of endospores.

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A volunteer from your lab bench should achieve bacterial cultures for endospore staining, together directed by your instructor. Keep in mind that these will all be varieties of Bacillus. Prepare smears and stain each using the endospore staining technique. Watch the slides and also note the shape and location that the endospore and the appearance of the sporangium (swollen or no swollen) in the table below:

Name that cultureEndospore ShapeLocationSporangium

In addition, pick ONE of the societies from over 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 visible in the Gram stained smear? _________________ If you see endospores, define how they show up in the Gram stained preparation, and how this is comparable to and different indigenous what you check out in the endospore stained preparation.