3.2: Staining of microscope slides and descriptions (2023)

Learning objectives

• Distinguish between simple and differential points
• Describe the unique characteristics of commonly used dyes
• Explain procedures and name clinical applications of Gram stain, spores, acids, negative envelopes and flagella
• What to describe about cells in the microscope

Clinical Focus: Part 2

Wound infections such as Cindy can be caused by many different types of bacteria, some of which can spread rapidly with serious complications. Identifying the specific cause is very important to choose a drug that can kill or stop the growth of the bacteria.

After calling the local doctor about Cindy, the camp nurse sends a wound sample to the nearest medical lab. Unfortunately, because the camp is in a remote area, the nearest laboratory is small and poorly equipped. A more modern lab would probably use other methods to culture, grow, and identify the bacteria, but in this case the technician decides to mount the sample and view it under a bright-field microscope. In a wet holder, a small drop of water is added to the slide and a coverslip is applied to the slide to hold it in place before being placed under the objective.

Under a bright field microscope, the technician can barely see the bacterial cells because they are almost transparent against the light background. To increase the contrast, the technician places an opaque light barrier over the device. The resulting dark-field image clearly shows that the bacterial cells are spherical and clustered, like grapes.

Sample preparation for light microscopy

In clinical settings, light microscopes are the most commonly used microscopes. There are two basic types of slides used to view slides under a light microscope: liquid mounts and fixed slides.

The simplest type of preparation is the liquid preparation, in which the sample is placed on a slide in a drop of liquid. Some samples, such as a drop of urine, are already in liquid form and can be placed on a slide using a dropper. Solid samples, such as scraped skin, can be placed on a slide before adding a drop of liquid to prepare a liquid mount. Sometimes the liquid used is simply water, but stains are often added to increase the contrast. After adding the liquid to the slide, a coverslip is placed on it and the slide is ready for examination under the microscope.

The second method of preparing samples for light microscopy is fixation. "Fixing" a sample refers to the process of attaching cells to a slide. Fixation is usually achieved by heating (thermal setting) or chemical treatment of the sample. In addition to securing the slide to the slide, fixation also kills the microorganisms in the slide, stopping their movement and metabolism, preserving the integrity of their cellular components for observation.

To fix the slide with heat, a thin layer of the slide is spread on the slide (called a smear) and then the slide is briefly heated over a heat source (Figure \(\PageIndex{1}\)). Chemical fixatives are often better than heating tissue samples. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, disrupt biochemical reactions, and stabilize cellular structures in tissue samples (Figure \(\PageIndex{1} \)).

In addition to fixation, staining is almost always used to color certain characteristics of the sample prior to light microscopic examination. Stains or dyes contain salts consisting of a positive and a negative ion. Depending on the type of dye, the positive or negative ion can be a chromophore (color ion). the other colorless ion is called the counterion. If the chromophore is a positively charged ion, the dye is classified as a basic dye. If the negative ion is a chromophore, the stain is considered an acid dye.

Dyes are selected for staining based on the chemical properties of the dye and the observed sample, which determine how the dye will interact with the sample. In most cases, it is preferable to use a positive dye, a dye that will be absorbed by the cells or organisms being observed, adding color to objects of interest to make them stand out from the background. However, there are cases where it is beneficial to use a negative dye that is absorbed by the background but not by the cells or organisms in the sample. Negative staining creates an outline or silhouette of organisms on a colored background (Figure \(\PageIndex{2}\)).

Since cells normally have negatively charged cell walls, positive chromophores in basic dyes tend to attach to cell walls, causing them to stain positively. Thus, commonly used basic dyes such as basic magenta, crystal violet, malachite green, methylene blue, and safranin usually serve as positive dyes. On the other hand, the negatively charged chromophores in acid dyes are repelled by the negatively charged cell walls, making them negative colors. Commonly used acid stains include acid fuchsin, eosin, and rose bengal.

Some staining techniques involve applying only one dye per sample. others require more than one painting. In single staining, a single dye is used to highlight specific structures in a sample. A single stain usually makes all 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 different dyes. In other words, two organisms in a different colored sample may have different colors. Differential staining techniques commonly used in the clinical setting include Gram stain, acid-fast stain, endospore stain, flagella stain, and capsule stain.

Stains in grams

The Gram stain procedure is a differential staining procedure that involves several steps. It was developed by the Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different cell wall types and remains one of the most widely used staining techniques to this day. The steps of the Gram staining procedure are shown in the figure \(\PageIndex{3}\) and listed below.

1. First, crystal violet, the primary dye, is applied to the heat-fixed smear, giving all cells a purple color.
2. Gram's iodine, a flavoring agent, is then added. A mordant is a substance used to set or fix stains or dyes. In this case, Gram's iodine acts as a scavenging agent that forms complexes with crystal violet, causing the crystal violet-iodine complex to accumulate and be trapped in thick layers of peptidoglycan in the cell walls.
3. A bleaching agent, usually ethanol or an acetone/ethanol solution, is then added. Cells that have thick layers of peptidoglycan in their cell walls are much less susceptible to the bleaching agent. they usually retain the crystal violet dye and remain purple. However, the decolorizing agent more easily removes the dye from cells with thinner layers of peptidoglycan, rendering them colorless again.
4. Finally, an additional remedy, usually safranin, is added. This stains discolored cells pink and is less noticeable in cells that still contain crystal violet pigment.

Violet cells stained with crystal violet are referred to as Gram-positive cells, while red cells stained with safranin are Gram-negative (Figure \(\PageIndex{4}\)). However, there are several important considerations regarding the interpretation of Gram stain results. First, older bacterial cells may have damaged cell walls, making them appear Gram-negative, even though the species is Gram-positive. Therefore, it is best to use fresh bacterial cultures for Gram staining. Secondly, errors such as prolonged whitening time can affect the results. In some cases, most cells will appear Gram-positive while some will appear Gram-negative (as shown in \(\PageIndex{4}\)). This indicates that individual cells have been damaged or that the bleach has been left on for too long. Cells should be classified as Gram-positive if they are all from the same species and not from a mixed culture.

In addition to various interactions with pigments and decolorants, the chemical differences between Gram-positive and Gram-negative cells have other clinically significant implications. For example, Gram staining can help clinicians classify bacterial pathogens in a sample into categories related to specific properties. Gram-negative bacteria are usually more resistant to some antibiotics than gram-positive bacteria. We will discuss this and other applications of Gram staining in more detail in later chapters.

Acid-resistant dyes

Rapid acid staining is another commonly used differential staining technique that can be an important diagnostic tool. Acid-fast staining is able to distinguish between two types of Gram-positive cells: those that have waxy mycolic acids in their cell walls and those that do not. Two different acid staining methods are the Ziehl-Neelsen technique and the Kinyoun technique. Both use carbolic fuchsin as the primary dye. The acid-resistant wax cells retain carbo-fuchsin even after the use of a bleaching agent (acid-alcohol solution). A secondary counterstain, methylene blue, is then applied to turn the anaerobic cells blue.

The main difference between the two methods based on carbolic fuchsin is whether or not heat is used during the main dyeing process. The Ziehl-Neelsen method uses heat to inject carbolic fuchsin into acidic cells while the Kinyoun method does not use heat. Both techniques are important diagnostic tools because several specific diseases are caused by acid-fast bacteria (AFB). If AFB is present in the tissue sample, its red or pink color is clearly visible against the blue background of the surrounding tissue cells (Figure \(\PageIndex{6}\)).

The use of microscopy in the diagnosis of tuberculosis

Mycobacterium tuberculosis, the bacteria that causes tuberculosis, can be detected in samples by the presence of acid-alcohol-fast bacilli. Often, a smear is prepared from the patient's sputum sample and then stained with the Ziehl-Neelsen technique (Figure \(\PageIndex{6}\)). If acid bacteria are confirmed, they are usually cultured for positive identification. Variations of this approach can be used as a first step to determine whethertuberculosisor other acidic bacteria, although samples from other parts of the body (such as urine) may contain othersmycobacteriaPolite.

An alternative approach to determining its presencetuberculosisis immunofluorescence. In this technique, antibodies labeled with a fluorochrome are attachedtuberculosis, if it exists. Antibody-specific fluorescent dyes can be used to visualize mycobacteria under a fluorescence microscope.

Capsule staining

Some bacteria and yeast have an outer protective structure called a capsule. Since the presence of the capsule is directly related to the virulence of the microbe (its ability to cause disease), being able to determine whether cells in a sample have capsules is an important diagnostic tool. The capsules do not absorb most basic dyes. Therefore, a negative staining technique (staining around the cells) is usually used to stain the capsules. The dye stains the background but does not penetrate into the capsules, which appear as a halo around the edge of the cell. The sample does not need to be heat fixed prior to negative staining.

A common negative staining technique for detecting encapsulated yeasts and bacteria is to add a few drops of Indian ink or nigrosin to the sample. Other capsular dyes can also be used to negatively stain capsular cells (Figure \(\PageIndex{7}\)). Alternatively, positive and negative staining techniques can be combined to visualize the capsules: positive staining stains the cell body and negative staining stains the background but not the capsule, leaving a shell around each cell.

endospore staining

Endospores are structures produced inside certain bacterial cells that allow them to survive in harsh environments. Gram stain alone cannot be used to visualize spores, which are clearly visible when Gram stained cells are visible. Endospore staining uses two dyes to distinguish endospores from the rest of the cell. The Schaeffer-Fulton method (the most commonly used technique for spore staining) uses heat to introduce the primary dye (malachite green) into the spore. Washing with water discolors the cell, but the endospore retains a green stain. The cell is then stained pink with safranin. The resulting image reveals the shape and location of the spores, if present. Green spores will appear inside the pink germ cells or will separate completely from the pink cells. If there are no spores, only pink germ cells will be visible (Figure \(\PageIndex{8}\)).

It is important to define endosperm dyeing techniquesBacillusMClostridium, two types of spore-forming bacteria containing clinically important species. Including,B. anthracite(causing anthrax) is of particular interest due to fears that its spores could be used as an agent of bioterrorism.It is hardis a particularly important species responsible for the common nosocomial infection known as "C. miscellaneous."

Eyelash coloring

Flagella (singular: flagella) are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. Because they are so thin, prokaryotic flagella cannot normally be seen under a light microscope without a specialized flagella staining technique. Flagella staining thickens the flagella by first applying a fragrance (usually tannic acid, but sometimes potassium alum) to coat the flagella. then the sample is stained with pararosaniline (most common) or basic fuchsin (Figure \(\PageIndex{9}\)).

Although flagella staining is rare in a clinical setting, the technique is commonly used by microbiologists because the location and number of flagella can be useful for classifying and identifying bacteria in a sample. It is important to handle the sample very carefully when using this technique. Flagella are delicate structures that can be easily damaged or severed, making it difficult to accurately locate and count the flagella.

Preparation of samples for electron microscopy

Samples for TEM analysis must have very thin sections. However, the cells are too soft to be finely cut even with diamond knives. To excise the cells intact, they must be embedded in a plastic resin and then dehydrated by a series of immersions in ethanol solutions (50%, 60%, 70%, etc.). Ethanol replaces water in the cells, and the resin dissolves in the ethanol and enters the cell where it solidifies. Thin sections are then cut using a specialized device called an ultramicrotome (picture \(\PageIndex{12}\)). Finally, the samples are attached to thin copper wires or carbon fiber mesh and painted - not with colored dyes, but with substances such as uranyl acetate or osmium tetroxide, which contain electrically condensed heavy metal atoms.

When samples are prepared for visualization by SEM, they should also be dehydrated with an ethanol series. However, they need to be even drier than needed for TEM. Critical point drying with inert liquid carbon dioxide under pressure is used to displace water from the sample. After drying, the samples are sputter coated, removing atoms from the palladium target with energetic particles. Sputter coating prevents samples from being charged by the SEM electron beam.

Using microscopy to diagnose syphilis

The causative agent of syphilis ispale treponema, a flexible spiral cell (spirochete) that can be very thin (<0.15 μm) and correspond to the refractive index of the medium, making it difficult to visualize by bright-field microscopy. In addition, this species has not been successfully cultured in the laboratory on artificial media. Therefore, diagnosis depends on successful identification using microscopic techniques and serology (analysis of body fluids, often for antibodies to the pathogen). Because fixation and staining kill cells, dark-field microscopy is commonly used to study living specimens and visualize their movement. However, other approaches can be used. For example, cells can be coated with silver particles (in tissue sections) and observed under a light microscope. Fluorescence or electron microscopy can also be used for visualizationTrembling(Picture \(\PageIndex{13}\)).

In a clinical setting, indirect immunofluorescence is often used for identificationTrembling.The unstained primary antibody binds directly to the surface of the pathogen, and the secondary antibodies "labeled" with a fluorescent dye bind to the primary antibody. Multiple secondary antibodies can be attached to each primary antibody, increasing the amount of dye associated with each.Tremblingcells, making them easier to locate (Image \(\PageIndex{14}\)).

Preparation and staining for other microscopes

Fluorescence and confocal microscopy slides are prepared similarly to light microscopy slides, except that the dyes are fluorochrome. Stains are often diluted with liquid before being applied to the glass. Some dyes bind to the antibody, staining specific proteins in specific cell types (immunofluorescence). others can bind to DNA molecules in a process called fluorescent in situ hybridization (FISH), causing cells to stain depending on whether they have a particular DNA sequence. Sample preparation for two-photon microscopy is similar to fluorescence microscopy except that infrared dyes are used.

Cornella UniversityCase studies in microscopyoffers a series of clinical problems based on real events. Each case study guides you through a clinical problem using appropriate microscopic techniques at each stage.

Description of cells under the microscope

Once the cells have been stained, the next step is to use a microscope to characterize them. You'll always be looking for cell shape, grouping, and display colors. These descriptions of cell morphology (shape and structure) are his microscopic observations. This is most commonly done with prokaryotes, and the tables below contain the appropriate names of shapes and groups to use.

Image \(\PageIndex{15}\): ("Coccus" thumbnail: modified from Janice Haney Carr, Centers for Disease Control and Prevention; "Coccobacillus" thumbnail: modified from Janice Carr, Centers for Disease Control Centers for Control and disease prevention; thumbnail of "Spirochetes": edited by Centers for Disease Control and Prevention)

Eukaryotic cells exhibit a wide variety of different cell morphologies. Possible shapes include spheroidal, ovoid, cuboid, cylindrical, flat, lenticular, spindly, disc, crescent, star ring, and polygonal (Figure \(\PageIndex{17}\)). Some eukaryotic cells are irregular in shape and some are capable of changing shape. The shape of a particular type of eukaryotic cell can be influenced by factors such as its basic function, cytoskeletal organization, viscosity of the cytoplasm, stiffness of the cell membrane or cell wall (if present), and physical stress exerted on it from the surrounding environment and/or neighboring cells.

Main concepts and summary

• Samples must be properly prepared for microscopy. This may includecoloring,claspand/or cutthin sections.
• Various staining techniques can be used in light microscopy, includingGram staining, oxidative staining,capsule staining,endospore staining,Mflagella coloring.
• TEM samples require very thin sections while SEM samples require sputtering.
• Preparation for fluorescence microscopy is similar to preparation for light microscopy, except that fluorochromes are used.
• Cells have many unique characteristics when viewed under a microscope. When recording observations, describe the shape, grouping, and color(s) of the cells.

taxpayer

• Nina Parker (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph's University) with many tax-paying authors . Original content via Openstax (CC BY 4.0; free access athttps://openstax.org/books/microbiology/pages/1-introduction)