Chapter #2: The Study of Microbial Structure

Question 1

Compound Microscopes

Answer 1

Microscopes having two sets of lenses. All modern microscopes.

Question 2

Objective Lens

Answer 2

Lens closest to the specimen. It forms a magnified image that is further enlarged by one or more additional lenses.

Question 3

Resolution

Answer 3

The ability of a lens to separate or distinguish between small objects that are close together.

Question 4

Working Distance

Answer 4

Numerical aperture is related to another characteristic of an objective lens, the working distance. It is the distance between the front surface of the lens and the surface of the cover glass (if one is used) or the specimen when it is in sharp focus. Objectives with large numerical apertures and great resolving power have short working distances.

Question 5

Fixation

Answer 5

The process by which the internal and external structures of specimens are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation. A microbe is usually killed and attached firmly to the microscope slide.

Question 6

Heat Fixation

Answer 6

Used to observe bacteria and archaea. Typically, a film of cells is gently heated as a slide is passed through a flame. Heat fixation preserves overall morphology. Although heat fixation inactivates enzymes, it also destroys proteins that may be part of subcellular structures.

Question 7

Chemical Fixation

Answer 7

Used to protect fine cellular substructure as well as the morphology or larger, more delicate microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, to render them inactive, insoluble, and immobile. Common fixative mixtures contain such components as ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.

Question 8

Chromophore Groups

Answer 8

Groups with conjugated double bonds that give the dye its color, and they can bind cells with ionic, covalent, or hydrophobic bonding.

Question 9

Negative Staining

Answer 9

Where the background but not the cell is stained.

Question 10

Dyes Binding Cells By Ionic Interaction

Answer 10

Most commonly used dyes. These ionizable dyes divided into two general classes, basic and acidic.

Question 11

Basic Dyes

Answer 11

Basic dyes bind to negatively charged molecules.

Question 12

Acidic Dyes

Answer 12

Acidic dyes, in their ionized form, have a negative charge and bind to positively charged cell structures.

Question 13

Simple Staining

Answer 13

A single dye is used. It’s simple and ease of use. The fixed smear is covered with stain for a short period, excess stain is washed off with water, and the slide is blotted dry.

Basic dyes such as crystal violet, methylene blue, and carbolfuchsin are frequently used in simple staining to determine size, shape, and arrangement of bacterial and archaeal cells.

Question 14

Gram Stain

Answer 14

Developed in 1884 by Danish physician Christian Gram. It’s the most widely employed staining method in bacteiology. An example of differential staining. Divides most bacteria but not archaea) into two groups, gram negative and gram positive.

1) The smear is stained with the basic dye crystal violet, the primary stain.
2) Followed by treatment with an iodine solution functioning as a mordant. Iodine increases the interaction between the cell and the dye so that the cell is stained more strongly.
3) Smear is next decolorized by washing with ethanol or acetone. This step generates the differential aspect of the gram stain; gram positive bacteria retain the crystal violet, whereas gram-negative bacteria lose the crystal violet and become colorless.
4) Finally, the smear is counterstained with a simple basic dye different in color from crystal violet. Safranin, the most common counterstain, colors gram-negative bacteria pink to red and leaves gram-positive bacteria dark purple.

Question 15

Differential Staining

Answer 15

Procedures used to distinguish organisms based on their staining properties.

Question 16

Mordant

Answer 16

Substance that helps bind the dye to a target molecule.

Question 17

Acid-Fast Staining

Answer 17

Another important differential staining procedure. It’s most commonly used to identify Mycobacterium tuberculosis and M. leprae, the pathogens responsible for tuberculosis and leprosy, respectively.

These bacteria have cell walls containing lipids constructed from mycolic acids, a group of branched-chain hydroxy fatty acids, which prevent dyes from readily binding to the cells.

However, M. tuberculosis and M. leprae can be stained by harsh procedures such as the Ziehl-Neelsen method, which uses heat and phenol to drive basic fuchsin into cells. Once basic fuchsin has penetrated, M. tuberculosis and M. leprae are not easily decolorized by acidified alcohol (acid-alcohol) and thus are said to be acid-fast.

Non-acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain.

Question 18

Endospore Staining

Answer 18

Requires heat to drive dye into a target, in this case an endospore. Endospore morphology and location with species and often are valuable in identification; endospores may be spherical to elliptical and either smaller or larger than the diameter of the parent bacterium. Endospores aren’t stained well by most dyes, but once stained, they strongly resist decolorization. This property is the basis of most endospore staining methods.

In the Schaeffer-Fulton procedure, endospores are first stained with malachite green in the presence of heat. After malachite green treatment, the rest of the cell is washed free of dye with water and is counterstained with safranin. This technique stains endospores bluish green and the vegetative cells pink to red.

Question 19

Capsule Staining

Answer 19

Simple staining procedure. A technique that reveals the presence of capsules, a network usually made of polysaccharides that surrounds many bacteria and some fungi.

Cells are mixed with India ink or nigrosin dye and spread out in a thin film on a slide.

After air-drying, the cells appear as lighter bodies in the midst of a blue-black background because ink and dye particles can’t penetrate either the cell or its capsule. Thus capsule staining is a kind of negative staining.

The extent of the light region determined by size of capsule and cell itself. There is little distortion of cell shape, and the cell can be counterstained for even greater visibility.

Question 20

Flagella Staining

Answer 20

Provides taxonomically valuable info about the presence and distribution pattern of flagella on prokaryotic cells. Bacterial and archael flagella are so slender they can only be seen directly using the electron microscope.

To observe bacterial flagella with the light microscope, their thickness is increased by coating them with mordants such as tannic acid and potassium alum, and then staining with paraosaniline (Leifson method) or basic fuchsin (Gray method).

Question 21

Electron vs. Light Microscopes

Answer 21

Resolution of a light microscope increases with a decrease in the wavelength. In electron ms, electrons replace light as the illuminating beam. The electron beam can be focused with a wavelength about 100,000 times shorter than that of light. Therefore, electron microscopes have a resolution 1000 times better.

Question 22

Transmission Electron Microscope (TEM)

Answer 22

A heated tungsten filament in the electron gun generates a beam of electrons that’s focused on the specimen by the condenser.

Since electrons can’t pass through a glass lens, doughnut-shaped electromagnets called magnetic lens are used to focus the beam. The column containing the lenses and specimen must be under high vacuum to obtain a clear image because electrons are deflected by collisions with air molecules.

The specimen scatters some electrons, but those that pass through are used to form an enlarged image of the specimen on a fluorescent screen. A denser region in the specimen scatters more electrons and therefore appears darker in the image since fewer electrons strike that area of the screen; these regions are said to be “electron dense.” In contrast, electron-transparent regions are brighter. The image can be captured digitally or on photographic film.

Question 23

Scanning Electron Microscope (SEM)

Answer 23

Produces an image from electrons released from atoms on an object’s surface. The SEM has been used to examine surfaces of microbes in great detail; mainy have a resolution of 7nm or less.