Microscopic Techniques Dyes and Staining

It can be difficult with the bright-field microscope to observe living microorganisms. Most are nearly transparent and often move rapidly about the slide. Consequently, cells are frequently immobilized and stained with dyes. These dyes may be attracted to one or more cellular components. As a result, an entire organism, or its specific parts, can be made to contrast with the unstained background. Many different dyes and staining procedures can be used; each has specific applications (table 3.2).

Basic dyes, which carry a positive charge, are more commonly used for staining than are negatively charged acidic dyes. Because opposite charges attract, basic dyes stain the many negatively charged components of cells, including nucleic acid and many proteins, whereas acidic dyes are repelled. Common basic dyes include methylene blue, crystal violet, safranin, and malachite green. Simple staining employs one of these basic dyes to stain the cells. Acidic dyes are sometimes used to stain backgrounds against which colorless cells can be seen, a technique called negative staining.

To stain microorganisms, a drop of a liquid containing the microbe is placed on a glass microscope slide and allowed to air dry. The resulting specimen forms a film and is called a smear. The organisms are then attached, or fixed, to the slide, usually by passing the slide over a flame (figure 3.13). Dye is then applied and washed off with water. Heat fixing and subsequent staining steps kill the microorganisms and may distort their shape.

Differential Stains

Differential staining techniques are used to distinguish one group of bacteria from another. They take advantage of the fact that certain bacteria have distinctly different chemical structures in some of their components. These lead to differences in their staining properties. The two most frequently used differential staining techniques are the Gram stain and the acid-fast stain.

Gram Stain

The Gram stain is by far the most widely used procedure for staining bacteria. The basis for it was developed over a century ago by Dr. Hans Christian Gram (see Glimpse of History). His observations led to procedures by which bacteria can be separated

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Microbiology, A Human Microorganisms Structure Companies, 2003

Perspective, Fourth Edition

46 Chapter 3 Microscopy and Cell Structure

Table 3.2 A Summary of Stains and Their Characteristics



Simple Stains

Differential Stains

Gram stain

Acid-fast stain

Special Stains Capsule stain

Endospore stain

Flagella stain Fluorescent Dyes and Tags

Fluorescent dyes Fluorescent tags

Employ a basic dye to impart a color to a cell. Easy way to increase the contrast between otherwise colorless cells and a transparent background.

Distinguish one group of microorganisms from another.

Used to separate bacteria into two major groups, Gram-positive and Gram-negative.The staining characteristics of these groups reflect a fundamental difference in the chemical structure of their cell walls.This is by far the most widely used staining procedure.

Used to detect members of the genus Mycobacterium in a specimen. Due to the lipid composition of their cell walls, these organisms do not readily take up stains.

Stain specific structures inside or outside of a cell.

Because the viscous capsule does not readily take up stains, it stands out against a stained background.This is an example of a negative stain.

Stains endospores, a type of dormant cell that does not readily take up stains.These are produced by the genera Bacillus and Clostridium.

The staining agent adheres to and coats the otherwise thin flagella, enabling them to be seen with the light microscope.

Fluorescent dyes and tags absorb ultraviolet light and then emit light of a longer wavelength.They are used in conjunction with a fluorescence microscope.

Some fluorescent dyes bind to compounds found in all cells; others bind to compounds specific to only certain types of cells. Antibodies to which a fluorescent molecule has been attached are used to tag specific molecules.

into two major groups: Gram-positive and Gram-negative. We now know that the difference in the staining properties of these two groups reflects a fundamental difference in the chemical structure of their cell walls.

The Gram stain procedure involves four basic steps (figure 3.14):

1. The smear is first flooded with the primary stain, crystal violet in this case. The primary stain is the first dye applied in any multistep staining procedure and generally stains all of the cells.

2. The smear is rinsed to remove excess crystal violet and then is flooded with a dilute solution of iodine, called Gram's iodine. Iodine is a mordant, a substance that increases the affinity of cellular components for a dye. The iodine combines with the crystal violet to form a dye-iodine complex, thereby decreasing the solubility of the dye within the cell.

3. The stained smear is rinsed again, and then 95% alcohol or a mixture of alcohol and acetone is briefly added. These solvents act as decolorizing agents and readily remove the dye-iodine complex from Gram-negative, but not Gram-positive, bacteria.

4. A counterstain is then applied to impart a contrasting color to the now colorless Gram-negative bacteria. For this purpose, the red dye safTanin is used. This dye stains Gramnegative as well as Gram-positive bacteria, but because the latter are already stained purple, it imparts little difference.

To obtain reliable results, the Gram stain must be done properly. One of the most common mistakes is to decolorize a smear for too long a time period. Even Gram-positive cells can

Steps Gram Stain Technique
Figure 3.13 Staining Bacteria for Microscopic Observation

Steps in Staining

State of Bacteria

Step 1: Crystal violet Cells stain purple. (primary stain)

Step 2: Iodine (mordant)

Step 3: Alcohol (decolorizer)

Step 4: Safranin (counterstain)

Cells remain purple.

Gram-positive cells remain purple; Gram-negative cells become colorless.

Gram-positive cells remain purple; Gram-negative cells appear red.

3.2 Microscopic Techniques: Dyes and Staining 47

3.2 Microscopic Techniques: Dyes and Staining 47

Endospore Staining Aureus

Figure 3.14 Gram Stain (a) Steps in the Gram stain procedure. (b) Results of a Gram stain.The Gram-positive cells (purple) are Staphylococcus aureus; the Gram-negative cells (reddish-pink) are Escherichia coli.

lose the crystal violet-iodine complex during prolonged decol-orization. An over-decolorized Gram-positive cell will appear pink after counterstaining. Another important consideration is the age of the culture. As bacterial cells age, they lose their ability to retain the crystal violet-iodine dye complex, presumably because of changes in their cell wall. This also causes the cells to appear pink. In the case of certain types of Gram-positive bacteria, this aging may occur within 24 hours. Thus, the Gram stain results of fresh cultures are more reliable.

Acid-Fast Stain

The acid-fast stain is a procedure used to stain a small group of organisms that do not readily take up stains. Among these are members of the genus Mycobacterium, including a species that causes tuberculosis and one that causes Hansen's disease (leprosy). The cell wall of these acid-fast bacteria contains high concentrations of lipid, preventing the uptake of dyes, including those used in the Gram stain. Therefore, harsh methods are needed to stain these organisms. Once stained, however, these same cells are very resistant to decolorization. Because mycobac-teria are among the few organisms that retain the stain in this procedure, the acid-fast stain can be used to presumptively identify them in clinical specimens that might contain a variety of different bacteria. ■ tuberculosis, p. 580 ■ Hansen's disease, p. 670

The acid-fast stain, like the Gram stain, requires multiple steps. The primary stain in this procedure is carbol fuchsin, a red dye. In the classic procedure, the stain-flooded slide is heated over boiling water, which facilitates the staining. A current variation does not use heat, instead using a prolonged application of a more concentrated solution of dye. The slide is then rinsed briefly to remove the residual stain before being flooded with acid-alcohol, a potent decolorizing agent. This step removes the carbol fuchsin from nearly all cells, including tissue cells and most bacteria. Those few unusual organisms that retain the dye are called acid-fast. Methylene blue is then used as a counter-stain to make the non-acid-fast cells visible. Acid-fast organisms, which do not take up the methylene blue, appear a bright reddish-pink. They stand out against a background of the other cells, which are stained blue (figure 3.15).

Special Stains to Observe Cell Structures

Dyes can also be used to stain specific structures inside or outside the cell. The staining procedure for each component of the cell is different, being geared to the chemical composition and properties of that structure. The function of each of these structures will be discussed in more depth later in the chapter.

Capsule Stain

A capsule is a viscous layer that envelops a cell and is sometimes correlated with an organism's ability to cause disease. Capsules stain poorly, a characteristic exploited with a capsule stain, an example of a negative stain. It colors the background, allowing the capsule to stand out as a halo around an organism (figure 3.16).

To observe capsules, a liquid specimen is placed on a slide next to a drop of India ink. A thin glass coverslip is then placed over the two drops, causing them to flow together. This creates a gradient of India ink concentration across the specimen. Unlike the stains discussed previously, the capsule stain is done as a wet mount—a drop of liquid on which a coverslip has been placed—rather than as a smear. At the optimum concentration of India ink, the fine dark particles of the stain color the background enough to allow the capsule to be visible.

Chapter 3 Microscopy and Cell Structure

Chapter 3 Microscopy and Cell Structure

Carbol Fuchsin Fluorescence Spectrum

Figure 3.15 Acid-Fast Stain Mycobacterium species retain the red primary stain, carbol fuchsin. Counterstaining with methylene blue imparts a blue color to cells that are not acid-fast.

Figure 3.15 Acid-Fast Stain Mycobacterium species retain the red primary stain, carbol fuchsin. Counterstaining with methylene blue imparts a blue color to cells that are not acid-fast.

Staphylococcus Aureus Capsule Stain

Figure 3.16 Capsule Stain Capsules stain poorly, and so they stand out against the India ink-stained background as a halo around the organism.This photomicrograph shows an encapsulated yeast.

Figure 3.16 Capsule Stain Capsules stain poorly, and so they stand out against the India ink-stained background as a halo around the organism.This photomicrograph shows an encapsulated yeast.

Endospore Stain

Members of certain Gram-positive genera including Bacillus and Clostridium form a special type of dormant cell, an endospore, that is resistant to destruction and to staining. Although these structures do not stain with a Gram stain, they can often be seen as a clear smooth object within an otherwise purple-stained cell. To make endospores more readily noticeable, a spore stain is used. This stain, like the classic acid-fast staining procedure, uses heat to facilitate staining.

The endospore stain is a multistep procedure that employs a primary stain as well as a counterstain. Generally, malachite green is used as a primary stain. Its uptake by the endospore is facilitat

Escherichia Coli Safranin Stain

Figure 3.17 Endospore Stain Endospores retain the green primary stain, malachite green. Counterstaining with safranin imparts a red color to other cells.

Figure 3.17 Endospore Stain Endospores retain the green primary stain, malachite green. Counterstaining with safranin imparts a red color to other cells.

ed by gentle heat. When water is then used to rinse the smear, only endospores retain the malachite green. The smear is then coun-terstained, most often with the red dye safranin. The spores appear green amid a background of pink cells (figure 3.17).

Flagella Stain

Flagella are appendages that provide the most common mechanism of motility for bacterial cells. Because not all bacteria have flagella, and those that do can have them in different arrangements around a cell, the presence and location of these structures can be used to classify and identify bacteria. However, the diameter of a single flagellum is generally 30 nm or less and, ordinarily, is too thin to be seen with a light microscope. The flagella stain overcomes this limitation by using a mordant that allows the staining agent to adhere to and coat these thin structures, effectively increasing their diameter (figure 3.18). Unfortunately, this staining procedure is difficult and requires patience and expertise.

Staphylococcus Aureus Wet Mount Flagella

Figure 3.18 Flagella Stain The staining agent adheres to and coats the flagella.This increases their diameter so they can be seen with the light microscope.


Figure 3.18 Flagella Stain The staining agent adheres to and coats the flagella.This increases their diameter so they can be seen with the light microscope.

3.3 Morphology of Prokaryotic Cells

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  • anne bayer
    Is e coli endospore green?
    5 years ago
  • Jorma Hyypi
    Do I have to do a capsule stain on a negative Gram?
    5 years ago
  • hildifons tunnelly
    What agents in gram staining are colored dyes?
    5 years ago
  • michael
    How can an endospore stain be used to separate two indistinguishable species?
    5 years ago
  • ahti
    Can you make your own violet microscopic stain/dye at home?
    5 years ago
  • yonatan
    What structures can be seen with the gram stain?
    5 years ago
  • margaret woolbright
    How could an enspore stain be used to separate the two indistinguishable species?
    5 years ago
  • RALF
    What structure can be seen by gram staining?
    4 years ago
  • tiziano
    How can an endospore be used to seperate two indistinguishable species?
    4 years ago
  • Isabelle
    How could an endospore stain be used to seperate the two indistinguishable species?
    3 years ago
  • Pansy
    How can a endospore stain be used ro saperate the two indistinguishable species?
    3 years ago
  • luca
    What are the colored dyes applied in the gram staining techniques?
    3 years ago
  • barbara boothe
    How to perform heam and safranin staining for bacteria?
    3 years ago
    Can a dye repelled by microorganisms be used for staining?
    3 years ago
  • charlie mckay
    What dye is used for capsule staining?
    3 years ago
  • joseph
    What is primary dye and which part of flagella does it stain?
    3 years ago
  • Elaine
    Why is the dye used in staining called primary dye?
    3 years ago
  • bernd
    What is Microscopy techniques & Stain?
    3 years ago
    What are the physical and visual characteristics of a negative stain?
    2 years ago
  • Will
    How can these dyes and stains be applied in identifying bacteria cell?
    2 years ago
  • LISA
    Why do the cerevisiae and s.aureus remained unstained in the negative staining procedure?
    2 years ago
  • vittoria
    What are the acidic dye used for staining bacteria?
    2 years ago
  • gildo esposito
    Why the endospores do not take up the colour of safranin?
    2 years ago
  • Ines
    When is spore staining carried out?
    2 years ago
  • sinit adonay
    Why do we employ the use of dye and stain?
    2 years ago
  • Mezan
    Why some microorganisms retain primary colour and others does not?
    2 years ago
    What are the types of staining agent in microscope?
    2 years ago
  • thorsten
    What is the chemical nature of the dyes employed to stain microorganisms?
    2 years ago
    Why do we employ microscopic?
    2 years ago
  • Sebhat
    How could an endospore stain be used to separate the two indistinquishable species?
    2 years ago
  • liya fikru
    Why does crystal violet or methylene blue act as decolorizer to capsule stain?
    2 years ago
  • lavinia
    Why do bacteria cell stain more readily with basic dyes?
    2 years ago
  • Carmen
    How do endospor staining apply in medical industry?
    2 years ago
  • tuomo
    How are staining applied in medical industry?
    2 years ago
  • Dora
    Why is that basic dyes are used more frequently than acidic dyes in staining?
    2 years ago
  • dexter
    Why do cells readily take up basic dyes?
    1 year ago
  • Olga
    Why is acid alcohol used as a decolorizing agent?
    1 year ago
  • bertha
    What stain can show the cell walls of S. aureus in a microscope?
    12 months ago
  • emmi
    Why are endospores difficult to stain?
    11 months ago
  • marilyn
    What colour will Escherichia take when flooded with malachite green and counterstained with safanin?
    11 months ago
  • Christina Waechter
    Why is stain used in microscopic process why other dye are not uses?
    11 months ago
  • glenn mackay
    How many dyes in staining of bacteria?
    11 months ago
  • Scolastica
    Why is fresh culture used in flagella stain?
    10 months ago
    What will happen when acid dye is stain with a tissue cell?
    10 months ago
  • marcho
    Why yeast cell wall does not get stained with malachite green?
    9 months ago
  • jarmo
    What type of organism stains positive during spore stain and gram negative?
    9 months ago
  • dieter
    What causes the simple stain to dye the cells?
    9 months ago
  • aida
    Why are acidic dyes are not commonly used to colour bacteria?
    9 months ago
  • kaarlo myllyl
    How old culture is used for capsular staining procedure?
    9 months ago
  • erminio
    Can we use crystal violet instead of malachite green to stain bacterial spore?
    8 months ago
  • jemima
    What microscopic techniques are used for identification of spore,capsule,flagella,and cell wall?
    7 months ago
  • miranda
    What is the microscopic technic use for spore stain?
    7 months ago
  • Darcey
    What type of staining technique and microscopic technique is used for spores?
    7 months ago
  • roderic gawkroger
    Why are basic stain and not acidic usually employ in bacteria cell?
    5 months ago
    Why acidic dye are not used for bscteria staining?
    5 months ago
  • james scott
    Can staphylococcus be negatively stained?
    5 months ago
  • melissa
    Why are acidic dyes not commonly used to stay bacteria?
    5 months ago
    What is the function of each dye used during staining mycrobactrium tubacle?
    4 months ago
  • Iva
    Why are basic stains and not acidic stains usually employed in bacterial staining?
    4 months ago
  • Laura
    Why does bacterial cells retain basic stain and repel acid stain?
    3 months ago
  • ana
    Why was staphylococcus aureus smaller when stained with safranin?
    3 months ago
  • Berilac
    What is the result of staining with three dyes?
    2 months ago
  • juliane holtzmann
    What is the result of staning with three dyes?
    2 months ago
  • elias fikru
    How does microorganism looks when stained with three dyes?
    2 months ago
  • senja
    What is the result of staining a cell with three dyes?
    2 months ago
  • samsa
    What are the results of staining with three dyes?
    2 months ago
    What staining technique is used to stain clostridium?
    2 months ago
  • Stella
    Is transparency a reason why endospore hard to stain?
    1 month ago
  • alastair forbes
    Why is spore and acid fast staining referred to as special stain?
    1 month ago
  • Manuela Moench
    What is structural / special /specific stain technique?
    22 days ago
  • cynthia scales
    Why are basic dyes more successful in staining than acid dye's?
    13 days ago
  • ilse
    What is the type of staining technique whic employs more than one dye?
    12 days ago

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