What is the purpose for fixing the specimen to the slide?

Created by Monica Z. Bruckner, Montana State University, Bozeman

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This Giemsa stained micrograph depicts an example of a slightly acidic slide that yielded a pink colored resultant stain. The micrograph shows malarial cells. Photo courtesy of the Public Health Image Library.

What is Cellular Staining?

Cell staining is a technique that can be used to better visualize cells and cell components under a microscope. By using different stains, one can preferentially stain certain cell components, such as a nucleus or a cell wall, or the entire cell. Most stains can be used on fixed, or non-living cells, while only some can be used on living cells; some stains can be used on either living or non-living cells.

Why Stain Cells?

The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample. Cells may also be enumerated by staining cells to determine biomass in an environment of interest.

How Are Cells Stained and Slides Prepared?

Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the following procedures may be required to prepare a sample:

• Permeabilization - treatment of cells, generally with a mild surfactant, which dissolves cell membranes in order to allow larger dye molecules to enter inside the cell.
• Fixation - serves to "fix" or preserve cell or tissue morphology through the preparation process. This process may involve several steps, but most fixation procedures involve adding a chemical fixative that creates chemical bonds between proteins to increase their rigidity. Common fixatives include formaldehyde, ethanol, methanol, and/or picric acid.
• Mounting - involves attaching samples to a glass microscope slide for observation and analysis. Cells may either be grown directly to the slide or loose cells can be applied to a slide using a sterile technique. Thin sections (slices) of material such as tissue may also be applied to a microscope slide for observation.
• Staining - application of stain to a sample to color cells, tissues, components, or metabolic processes. This process may involve immersing the sample (before or after fixation or mounting) in a dye solution and then rinsing and observing the sample under a microscope. Some dyes require the use of a mordant, which is a chemical compound that reacts with the stain to form an insoluble, colored precipitate. The mordanted stain will remain on/in the sample when excess dye solution is washed away.

What Are Some Common Stains?

There are several types of staining media, each can be used for a different purpose. Commonly used stains and how they work are listed below. All these stains may be used on fixed, or non-living, cells and those that can be used on living cells are noted.

• Bismarck Brown - colors acid mucins, a type of protein, yellow and may be used to stain live cells
• Carmine - colors glycogen, or animal starch, red
• Coomassie blue - stains proteins a brilliant blue, and is often used in gel electrophoresis
• Crystal violet - stains cell walls purple when combined with a mordant. This stain is used in Gram staining
• DAPI - a fluorescent nuclear stain that is excited by ultraviolet light, showing blue fluorescence when bound to DNA. DAPI can be used in living or fixed cells
• Eosin - a counterstain to haematoxylin, this stain colors red blood cells, cytoplasmic material, cell membranes, and extracellular structures pink or red.
• Ethidium bromide - this stain colors unhealthy cells in the final stages of apoptosis, or deliberate cell death, fluorescent red-orange.
• Fuchsin - this stain is used to stain collagen, smooth muscle, or mitochondria.
• Hematoxylin - a nuclear stain that, with a mordant, stains nuclei blue-violet or brown.
• Hoechst stains - two types of fluorescent stains, 33258 and 33342, these are used to stain DNA in living cells.
• Iodine - used as a starch indicator. When in solution, starch and iodine turn a dark blue color.
• Malachite green - a blue-green counterstain to safranin in Gimenez staining for bacteria. This stain can also be used to stain spores.
• Methylene blue - stains animal cells to make nuclei more visible.
• Neutral/Toluylene red - stains nuclei red and may be used on living cells.
• Nile blue - stains nuclei blue and may be used on living cells.
• Nile red/Nile blue oxazone - this stain is made by boiling Nile blue with sulfuric acid, which creates a mix of Nile red and Nile blue. The red accumulates in intracellular lipid globules, staining them red. This stain may be used on living cells.
• Osmium tetroxide - used in optical microscopy to stain lipids black.
• Rhodamine - a protein-specific fluorescent stain used in fluorescence microscopy.
• Safranin - a nuclear stain used as a counterstain or to color collagen yellow.

After staining cells and preparing slides, they may be stored in the dark and possibly refrigerated to preserve the stained slide, and then observed with a microscope.

Related Links and Teaching Activities

• Core Microscopy Skills: Instructional Scaffolding for the Gram Stain
  This teaching activity uses a step-wise process to aid the student in familiarity with the use of the microscope as well as to increase the success rate with which they are able to stain and view microorganisms.
• Science Learning Network Cell Staining Activity
• University of Maryland Laboratory Protocol for Gram Staining
• Gram Stain Process Animation - this page from North Carolina University contains a link to an animation depicting how Gram staining (a technique that distinguishes between two groups of bacteria) works.
• Case Studies in Microscopy - this resource was accessed through BioSciEd Net (BEN) digital resources collection, which is the National Science Digital Library (NSDL) Pathway for biological sciences education. The resource itself requires a subscription or purchase of the activity from MicrobeLibrary.org. This website has three auto-tutorial case studies (about biofilms, microbes in a watershed, and an outbreak) that ask students to analyze and interpret microscopic images.
• MicrobeLibrary Atlas CD - The American Society for Microbiology (ASM), a member of the BEN Collaborative who are contributors to the NSDL Biological Sciences Pathway, has just released the MicrobeLibrary Atlas CD. This CD provides 329 images that portray results from the use of standard microbiology protocols and media such as Gram Stain, Blood Agar, MacConkey Agar, Triple Sugar Iron Agar, and more. The MicrobeLibrary Atlas CD is available for $18.00 plus shipping and handling. To order, visit the ASM estore.

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 (Figure \(\PageIndex{1}\)). 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 (Figure \(\PageIndex{1}\)).

Figure \(\PageIndex{1}\): (a) A specimen can be heat-fixed by using a slide warmer like this one. (b) Another method for heat-fixing a specimen is to hold a slide with a smear over a microincinerator. (c) This tissue sample is being fixed in a solution of formalin (also known as formaldehyde). Chemical fixation kills microorganisms in the specimen, stopping degradation of the tissues and preserving their structure so that they can be examined later under the microscope. (credit a: modification of work by Nina Parker; credit b: modification of work by Nina Parker; credit c: modification of work by “University of Bristol”/YouTube)

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 (Figure \(\PageIndex{2}\)).

Figure \(\PageIndex{2}\): (a) These Bacillus anthracis cells have absorbed crystal violet, a basic positive stain. (b) This specimen of Spinoloricus, a microscopic marine organism, has been stained with rose bengal, a positive acidic stain. (c) These B. megaterium appear to be white because they have not absorbed the negative red stain applied to the slide. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by Roberto Danovaro, Antonio Pusceddu, Cristina Gambi, Iben Heiner, Reinhardt Mobjerg Kristensen; credit c: modification of work by Anh-Hue Tu)

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. Figure \(\PageIndex{10}\) provides more detail.

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 stainingdistinguishes 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. Figure \(\PageIndex{11}\) provides more detail on these differential staining techniques.

Exercise \(\PageIndex{1}\)

1. Explain why it is important to fix a specimen before viewing it under a light microscope.
2. What types of specimens should be chemically fixed as opposed to heat-fixed?
3. Why might an acidic dye react differently with a given specimen than a basic dye?
4. Explain the difference between a positive stain and a negative stain.
5. Explain the difference between simple and differential staining.