It deserve to be an overwhelming with the bright-field microscope to watch living microorganisms. Many are virtually transparent and also often relocate rapidly about the slide. Consequently, cells are frequently immobilized and stained v dyes. This dyes may be attracted to one or an ext cellular components. As a result, an entire organism, or its certain parts, deserve to be do to comparison with the unstained background. Many different dyes and staining procedures can be used; every has certain applications (table 3.2).

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Basic dyes, which bring a hopeful charge, are much more commonly offered for staining than space negatively charged acidic dyes. Since opposite dues attract, straightforward dyes stain the plenty of negatively charged contents of cells, including nucleic acid and many proteins, whereas acidic dyes are repelled. Common basic dyes include methylene blue, decision violet, safranin, and also malachite green. An easy staining employs one of these basic dyes come stain the cells. Acidic dyes are periodically used come stain backgrounds against which colorless cells deserve to be seen, a an approach called an unfavorable staining.

To stain microorganisms, a fall of a liquid containing the microbe is placed on a glass microscopic lense slide and permitted to waiting dry. The resulting specimen develops a film and is referred to as a smear. The organisms space then attached, or fixed, to the slide, normally by passing the slide end a flame (figure 3.13). Dye is then applied and also washed off through water. Heat fixing and also subsequent staining steps kill the microorganisms and may distort their shape.

Differential Stains

Differential staining techniques are provided to differentiate one group of bacteria native another. Castle take advantage of the reality that specific bacteria have distinctly various chemical structures in several of their components. These lead to distinctions in your staining properties. The two most typically used differential staining approaches are the Gram stain and also the acid-fast stain.

Gram Stain

The Gram stain is by far the most widely offered procedure because that staining bacteria. The basis for it was arisen over a century earlier by Dr. Hans Christian Gram (see Glimpse that History). His observations resulted in procedures whereby bacteria deserve to be separated

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Perspective, 4th Edition

46 chapter 3 Microscopy and also Cell Structure

Table 3.2 A an overview of Stains and also Their Characteristics



Simple Stains

Differential Stains

Gram stain

Acid-fast stain

Special Stains Capsule stain

Endospore stain

Flagella stain Fluorescent Dyes and Tags

Fluorescent water Fluorescent tags

Employ a straightforward dye to send a color to a cell. Easy method to boost the contrast between otherwise colorless cells and also a transparent background.

Distinguish one group of microbe from another.

Used to separate bacteria into two significant groups, Gram-positive and also Gram-negative.The staining characteristics of these groups reflect a basic difference in the chemical structure of their cell walls.This is by far the many widely offered staining procedure.

Used come detect members that the genus Mycobacterium in a specimen. Due to the lipid ingredient of your cell walls, these organisms execute not easily take up stains.

Stain certain structures within or exterior of a cell.

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

Stains endospores, a form of dormant cell the does not conveniently take increase stains.These are developed by the genera Bacillus and Clostridium.

The staining agent adheres to and also coats the otherwise thin flagella, enabling them to it is in seen v the light microscope.

Fluorescent dyes and tags absorb ultraviolet light and then emit irradiate of a much longer wavelength.They are provided in conjunction v a fluorescence microscope.

Some fluorescent dyes tie to compounds discovered in every cells; others bind to compounds certain to only certain species of cells. Antitoxin to i m sorry a fluorescent molecule has been attached are supplied to tag specific molecules.

into two major groups: Gram-positive and also Gram-negative. We now know that the distinction in the staining properties of this two groups reflects a an essential difference in the chemical framework of their cell walls.

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

1. The smear is very first flooded v the major stain, decision violet in this case. The major stain is the very first dye applied in any type of multistep staining procedure and generally stains all of the cells.

2. The smear is rinsed to remove excess crystal violet and then is flooded through a dilute systems of iodine, dubbed Gram"s iodine. Iodine is a mordant, a problem that rises the affinity that cellular components for a dye. The iodine combines through the decision violet to kind a dye-iodine complex, in order to decreasing the solubility that the dye in ~ the cell.

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

4. A counterstain is then used 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 and also Gram-positive bacteria, but since the last are already stained purple, it imparts little difference.

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

Figure 3.13 Staining Bacteria because that Microscopic Observation

Steps in Staining

State the Bacteria

Step 1: decision 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 stay purple; Gram-negative cells appear red.

3.2 microscopic Techniques: Dyes and Staining 47

3.2 microscopic Techniques: Dyes and Staining 47


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

lose the decision violet-iodine facility during prolonged decol-orization. One over-decolorized Gram-positive cell will show up pink after ~ counterstaining. One more important factor to consider is the age of the culture. As bacterial cell age, they shed their ability to retain the crystal violet-iodine dye complex, presumably because of changes in their cell wall. This likewise causes the cells to show up pink. In the situation of certain varieties of Gram-positive bacteria, this aging may take place within 24 hours. Thus, the Gram stain outcomes of fresh societies are an ext reliable.

Acid-Fast Stain

The acid-fast stain is a procedure offered to stain a little group of biology that carry out not conveniently take up stains. Amongst these are members the the genus Mycobacterium, including a types that reasons tuberculosis and also one that causes Hansen"s an illness (leprosy). The cell wall of this acid-fast bacteria consists of high concentration of lipid, avoiding the uptake of dyes, consisting of those offered in the Gram stain. Therefore, harsh methods are needed to stain this organisms. As soon as stained, however, these same cells are very resistant to decolorization. Since mycobac-teria are amongst the couple of organisms that retain the stain in this procedure, the acid-fast stain have the right to be supplied to presumptively identify them in clinical specimens that could contain a selection of various bacteria. ■ tuberculosis, p. 580 ■ Hansen"s disease, p. 670

The acid-fast stain, choose the Gram stain, needs multiple steps. The main stain in this procedure is carbol fuchsin, a red dye. In the classic procedure, the stain-flooded slide is heated over boiling water, i m sorry facilitates the staining. A current variation does not use heat, rather using a an extensive application of a an ext concentrated equipment of dye. The slide is then rinsed summary to remove the residual stain before being flooded v acid-alcohol, a potent decolorizing agent. This step clears the carbol fuchsin from practically all cells, consisting of tissue cells and also most bacteria. Those couple of unusual organisms that retain the dye are referred to as acid-fast. Methylene blue is then used as a counter-stain to do the non-acid-fast cell visible. Acid-fast organisms, which execute not take it up the methylene blue, show up a shining reddish-pink. They stand out versus a background of the various other cells, which are stained blue (figure 3.15).

Special Stains to watch Cell Structures

Dyes can additionally be used to stain details structures within or exterior the cell. The staining procedure for each ingredient of the cell is different, being aligned to the chemistry composition and properties of that structure. The function of every of these frameworks will be disputed in much more depth later on in the chapter.

Capsule Stain

A capsule is a viscous layer that envelops a cell and also is sometimes correlated with one organism"s capacity to cause disease. Capsules stain poorly, a properties exploited v a capsule stain, an instance of a negative stain. That colors the background, allowing the capsule to stand out together a halo about an organism (figure 3.16).

To observe capsules, a fluid specimen is put on a slide next to a autumn of India ink. A thin glass coverslip is then put over the two drops, leading to them to flow together. This creates a gradient that India squid concentration throughout the specimen. Uneven the stains debated previously, the capsule stain is done together a wet mount—a autumn of fluid on i beg your pardon a coverslip has been placed—rather 보다 as a smear. At the optimum concentration that India ink, the well dark particles of the stain color the background sufficient to permit the capsule to it is in visible.

Chapter 3 Microscopy and also Cell Structure

Chapter 3 Microscopy and also Cell Structure


Figure 3.15 Acid-Fast Stain Mycobacterium varieties retain the red main stain, carbol fuchsin. Counterstaining through methylene blue imparts a blue shade to cell that space 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 shade to cells that are not acid-fast.


Figure 3.16 Capsule Stain Capsules stain poorly, and also so they was standing out versus the India ink-stained background together a halo about the organism.This photomicrograph mirrors an encapsulated yeast.

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

Endospore Stain

Members of details Gram-positive genera consisting of Bacillus and Clostridium type a special type of dormant cell, an endospore, the is resistant come destruction and to staining. Although these structures carry out not stain v a Gram stain, lock can often be viewed as a clean smooth object within an otherwise purple-stained cell. To do endospores much 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 the employs a main stain as well as a counterstain. Generally, malachite eco-friendly is used as a primary stain. Its absorb by the endospore is facilitat


Figure 3.17 Endospore Stain Endospores maintain the eco-friendly primary stain, malachite green. Counterstaining v safranin imparts a red shade to other cells.

Figure 3.17 Endospore Stain Endospores maintain the eco-friendly primary stain, malachite green. Counterstaining v safranin imparts a red color to other cells.

ed by gentle heat. As soon as water is then provided to rinse the smear, only endospores maintain the malachite green. The smear is climate coun-terstained, most often with the red dye safranin. The spores show up green between a background of pink cells (figure 3.17).

Flagella Stain

Flagella are appendages that administer the most typical mechanism that motility because that bacterial cells. Since not every bacteria have actually flagella, and those that do have the right to have lock in various arrangements approximately a cell, the presence and also location of this structures can be offered to classify and identify bacteria. However, the diameter the a solitary flagellum is usually 30 nm or much less and, ordinarily, is too thin to be seen v a light microscope. The flagella stain overcomes this limitation by utilizing a mordant that permits the staining agent to adhere to and also coat these thin structures, properly increasing their diameter (figure 3.18). Unfortunately, this staining procedure is difficult and needs patience and also expertise.


Figure 3.18 Flagella Stain The staining certified dealer adheres to and coats the flagella.This increases their diameter so they deserve to be seen v the light microscope.

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Figure 3.18 Flagella Stain The staining agent adheres to and also coats the flagella.This boosts their diameter for this reason they have the right to be seen with the light microscope.