Staining Microscopic Specimens

In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. Indeed, numerous methods have been developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope. Here, we will focus on the most clinically relevant techniques.

Preparing Specimens for Light Microscopy

In clinical settings, light microscopes are the most commonly used microscopes. There are two basic types of preparation used to view specimens with a light microscope: wet mounts and fixed specimens.

The simplest type of preparation is the wet mount, in which the specimen is placed on the slide in a drop of liquid. Some specimens, such as a drop of urine, are already in a liquid form and can be deposited on the slide using a dropper. Solid specimens, such as a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid used is simply water, but often stains are added to enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on top and the specimen is ready for examination under the microscope.

The second method of preparing specimens for light microscopy is fixation. The “fixing” of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either by heating (heat fixing) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.

To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear), and the slide is then briefly heated over a heat source (shown below). Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples.

In addition to fixation, staining is almost always applied to color certain features of a specimen before examining it under a light microscope. Stains, or dyes, contain salts made up of a positive ion and a negative ion. Depending on the type of dye, the positive or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is called the counterion. If the chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye.

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 (shown below).

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. The table below provides more detail.

Simple stains

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. The table below provides more detail on these differential staining techniques.

Differential stains

Check Your Understanding

Check Your Understanding

Gram Staining

The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 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 the figure below.

1. First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells a purple color.
2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that complexes with the crystal violet, making the crystal violet–iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls.
3. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution. Cells that have thick peptidoglycan layers in their cell walls are much less affected by the decolorizing agent; they generally retain the crystal violet dye and remain purple. However, the decolorizing agent more easily washes the dye out of cells with thinner peptidoglycan layers, making them again colorless.
4. Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized cells pink and is less noticeable in the cells that still contain the crystal violet dye.

Extended description

Step 1, crystal violet, the primary stain added to the specimen smear, stains cells purple or blue; the drawings show both the round gram-positive cells and the rod-shaped gram-negative cells purple. Step 2, iodine, the mordant, makes the dye less soluble so it adheres to the cell walls; the cells remain purple or blue in both drawings. Step 3, alcohol, the decolorizer, washes stain away from gram-negative cell walls; the gram-positive cells remain purple or blue while the gram-negative cells turn colorless in the drawing. Step 4, safranin, the counterstain, allows dye to adhere to the gram-negative cells; the gram-positive cells remain purple or blue while the gram-negative cells appear pink or red.

The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative (shown in the micrograph below). 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 the decolorizer on for too long can affect the results. For example, if the decolorizer is left on a slide of pure gram-positive bacterial cells for too long, some of the gram-positive cells will be decolorized and stained red with safranin, suggesting a mixed culture. Therefore, if a mixed culture of gram-positive and gram-negative cells is evaluated with the Gram stain and the decolorizer is left on too long, it will be difficult to accurately interpret the results.

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.

Check Your Understanding

Acid-Fast Stains

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.

The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria (AFB). If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells (shown below).

Check Your Understanding

Capsule Staining

Certain bacteria and yeasts have a protective outer structure called a capsule. Since the presence of a capsule is directly related to a microbe’s virulence (its ability to cause disease), the ability to determine whether cells in a sample have capsules is an important diagnostic tool. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining. The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell. The specimen does not need to be heat-fixed prior to negative staining.

One common negative staining technique for identifying encapsulated yeast and bacteria is to add a few drops of India ink or nigrosin to a specimen. Other capsular stains can also be used to negatively stain encapsulated cells (shown below). Alternatively, positive and negative staining techniques can be combined to visualize capsules: the positive stain colors the body of the cell, and the negative stain colors the background but not the capsule, leaving a halo around each cell.

Check Your Understanding

Endospore Staining

Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most commonly used endospore-staining technique) uses heat to push the primary stain (malachite green) into the endospore. Washing with water decolorizes the cell, but the endospore retains the green stain. The cell is then counterstained pink with safranin. The resulting image reveals the shape and location of endospores, if they are present. The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible (shown below).

Endospore-staining techniques are important for identifying Bacillus, Clostridium, and Clostridioides, three genera of endospore-producing bacteria that contain clinically significant species. Among others, B. anthracis (which causes anthrax) has been of particular interest because of concern that its spores could be used as a bioterrorism agent. Clostridioides difficile is a particularly important species responsible for the typically hospital-acquired infection known as “C. diff.”

Check Your Understanding

Flagella Staining

Flagella (singular: flagellum) are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. Because they are so thin, flagella typically cannot be seen under a light microscope without a specialized flagella staining technique. Flagella staining thickens the flagella by first applying mordant (generally tannic acid, but sometimes potassium alum), which coats the flagella; then the specimen is stained with pararosaniline (most commonly) or basic fuchsin (shown below).

Though flagella staining is uncommon in clinical settings, the technique is commonly used by microbiologists, since the location and number of flagella can be useful in classifying and identifying bacteria in a sample. When using this technique, it is important to handle the specimen with great care; flagella are delicate structures that can easily be damaged or pulled off, compromising attempts to accurately locate and count the number of flagella.

Preparing Specimens for Electron Microscopy

Samples to be analyzed using a TEM must have very thin sections. But cells are too soft to cut thinly, even with diamond knives. To cut cells without damage, the cells must be embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions (50%, 60%, 70%, and so on). The ethanol replaces the water in the cells, and the resin dissolves in ethanol and enters the cell, where it solidifies. Next, thin sections are cut using a specialized device called an ultramicrotome (shown below). Finally, samples are fixed to fine copper wire or carbon-fiber grids and stained—not with colored dyes, but with substances like uranyl acetate or osmium tetroxide, which contain electron-dense heavy metal atoms.

When samples are prepared for viewing using an SEM, they must also be dehydrated using an ethanol series. However, they must be even drier than is necessary for a TEM. Critical point drying with inert liquid carbon dioxide under pressure is used to displace the water from the specimen. After drying, the specimens are sputter-coated with metal by knocking atoms off of a palladium target, with energetic particles. Sputter-coating prevents specimens from becoming charged by the SEM’s electron beam.

Check Your Understanding

In clinical settings, indirect immunofluorescence is often used to identify Treponema. A primary, unstained antibody attaches directly to the pathogen surface, and secondary antibodies “tagged” with a fluorescent stain attach to the primary antibody. Multiple secondary antibodies can attach to each primary antibody, amplifying the amount of stain attached to each Treponema cell, making them easier to spot (shown below).

Extended description

Reading the diagram: the T. pallidum cell (blue) carries an unstained primary antibody (black, Y-shaped) bound to its surface. Two unstained secondary antibodies (green, Y-shaped), each carrying a yellow starburst fluorochrome, bind to the primary antibody, so several fluorescent tags cluster at the one site where the primary antibody attached.

Preparation and Staining for Other Microscopes

Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells (immunofluorescence); others may attach to DNA molecules in a process called fluorescence in situ hybridization (FISH), causing cells to be stained based on whether they have a specific DNA sequence.

Sample preparation for two-photon microscopy is similar to fluorescence microscopy, except for the use of infrared dyes. Specimens for STM need to be on a very clean and atomically smooth surface. They are often mica coated with Au(111). Toluene vapor is a common fixative.

Check Your Understanding

Summary

• Samples must be properly prepared for microscopy. This may involve staining, fixation, and/or cutting thin sections.
• A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining, capsule staining, endospore staining, and flagella staining.
• Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.
• Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.

Key terms

• wet mount — a slide preparation technique in which a specimen is placed on the slide in a drop of liquid.
• fixation — the process by which cells are killed and attached to a slide.
• smear — a thin layer of a specimen on a slide.
• staining — the addition of stains or dyes to a microscopic specimen for the purpose of enhancing contrast.
• basic dye — a chromophore with a positive charge that attaches to negatively charged structures.
• acidic dye — a chromophore with a negative charge that attaches to positively charged structures.
• positive stain — a stain that colors the structure of interest.
• negative stain — a stain that produces color around the structure of interest while not coloring the structure itself.
• simple staining — a staining technique that uses a single dye.
• differential staining — staining that uses multiple dyes to differentiate between structures or organisms.
• Gram stain procedure — a differential staining technique that distinguishes bacteria based upon their cell wall structure.
• primary stain — refers, in differential staining techniques, to the first dye added to the specimen.
• mordant — a chemical added to a specimen that sets a stain.
• decolorizing agent — a substance that removes a stain, usually from some parts of the specimen.
• counterstain — a secondary stain that adds contrasting color to cells from which the primary stain has been washed out by a decolorizing agent.
• acid-fast stain — a stain that differentiates cells that have waxy mycolic acids in their gram-positive cell walls.
• Ziehl-Neelsen technique — a method of acid-fast staining that uses heat to infuse the primary stain, carbolfuchsin, into acid-fast cells.
• Kinyoun technique — a method of acid-fast staining that does not use heat to infuse the primary stain, carbolfuchsin, into acid-fast cells.
• capsule staining — a negative staining technique that stains around a bacterial capsule while leaving the capsule clear.
• endospore staining — a differential staining technique that uses two stains to make bacterial endospores appear distinct from the rest of the cell.
• flagella staining — a staining protocol that uses a mordant to coat the flagella with stain until they are thick enough to be seen.
• thin sections — thin slices of tissue for examination under a TEM.
• ultramicrotome — a device that cuts thin sections for electron microscopy.

Practice

Differentiate between simple and differential stains

Describe the unique features of commonly used stains

Explain the procedures and name clinical applications for Gram, endospore, acid-fast, negative capsule, and flagella staining

This section is adapted from Microbiology, Section 2.4: Staining Microscopic Specimens by Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister, Brian M. Forster, and OpenStax, © OpenStax, licensed under CC BY-NC-SA 4.0. Access the original for free at openstax.org. Changes: figures re-encoded as WebP, with kind="photo" set explicitly on twelve photographic figures and kind="diagram" on the two drawn/labeled figures (the Gram stain process table and the indirect-immunofluorescence schematic), overriding the media manifest’s JPEG-based guess; alts rewritten for all fourteen figures to describe what is visibly drawn or photographed, since this chapter’s source alts contain defects (the Gram-stain-process alt misspells “purple” as “pruple,” and the two stain-table alts misspell “fuchsin” as “fuschsin,” “India ink” as “india in k,” and contain a 1,000-character run-on of cells) rather than reused; a longdesc added for the Gram stain process figure (required) and the indirect-immunofluorescence diagram, walking each in reading order; the two summary tables printed as images in the source (Simple Stains, Differential Stains) are transcribed as Markdown tables from the image, checked against the printed page, because the source alt for both misspells several dye names (the Differential Stains image itself prints “pararosaline” in its flagella-stain row; the transcribed table prints pararosaniline, the spelling this section’s own text uses, and the image’s spelling is reported as a source defect) — the source images are kept immediately after their tables for their sample micrographs only, with alts limited to the micrograph column and not re-transcribing the table cells; both tables are placed at the point in the text where the module’s own sentence introduces them (“provides more detail” / “provides more detail on these differential staining techniques”), not at their later position in the source’s print layout, where they are typeset at the end of the Flagella Staining section for page-fit reasons that do not apply to a continuously scrolling page; feature boxes rendered as callouts; of the fourteen Check Your Understanding bullets, eleven are now graded — eight multiplechoice, two sortbins (one built from the Simple Stains table, one classifying named techniques as simple or differential), and one textin — from this section’s own sentences and tables (11 Check Your Understanding questions graded from the module’s own sentences or tables rather than answered in prose; the source prints no key for them), and three (why acid-fast stains are useful; why cells must be dehydrated before electron microscopy; and the difference between a positive and a negative stain) remain body self-checks with model answers and rubrics assembled from this section’s own preceding text, because each combines more than one module sentence rather than resting on a single one; the source’s single five-bullet box split into two labeled “Check Your Understanding” runs of three and two so no more than three self-checks run back to back without intervening prose or a figure; a second sortbins for the Differential Stains table was considered and skipped, since four of its five rows’ distinguishing facts (Gram stain’s mordant/decolorizer/counterstain roles and color outcome; acid-fast stain’s mycolic-acid identification; endospore staining’s classification; capsule staining’s background-only mechanism) are already asked by converted items elsewhere on the page, leaving only the flagella-stain row untested — not enough for a second 4-item sortbins without repeating a fact; the Clinical Focus box’s Part 3 now names all three earlier parts of the case — it began in Section 2.1, continued in Section 2.3, and continues in the Resolution below — replacing the source’s “Jump to the next / Go back to the previous Clinical Focus box” links, and the Resolution names where the case began, since it is not accurate to call Part 2 “where the case began” once a case runs across three sections; the Link to Learning sentence’s title, which the source printed twice in a row (once as plain text, once repeated as the link text), is printed once as the Markdown link text; the end-of-section Multiple Choice and Fill in the Blank questions and the unkeyed Short Answer and Critical Thinking questions are adapted into the closing interactive Practice block, sorted under the objective each supports; of the two, the Short Answer question (identifying mycolic-acid-rich cell walls) is now a multiplechoice graded from this section’s own sentence, among the differential-stain names this section’s tables print (1 of the source’s unkeyed Short Answer questions is graded from the module’s own sentence rather than answered in prose; the source prints no key for it), and the model answer for the remaining Critical Thinking question is written from this section’s own text, because the source prints no answer key for it; five filler items (one multiple choice and one self-check for the first objective, one text-recall and one self-check for the second, both from this section’s own body sentences) fill out the first two objective groups, which the source’s own exercise set does not reach on its own — the first objective’s filler self-check (whether two species look different under a simple stain) is now a multiplechoice, and the second objective’s filler self-check (why capsule staining needs no heat-fixing) remains a self-check, because the module states only the one reason and no alternative module-printed mechanism exists to build defensible wrong options from; key terms compiled from the module’s twenty-three defined terms and the book’s Glossary appendix (all twenty-three taken from the glossary; none from a defining sentence); the Treponema pallidum figure’s caption is printed verbatim, including the source’s “used to visualized” (reported as a source defect rather than corrected, because the caption rule keeps the source’s wording); the µm figure in the syphilis Micro Connection is set with the micro sign (µ), not the Greek letter mu, per the book’s notation rule; cross-references to Sections 2.1 and 2.3 rendered as absolute links, since both pages exist.