Section 3.1: Units of Measurement

1. List the units used to measure microorganisms.

• Microorganisms are measured in even smaller units, such as micrometers and nanometers. A micrometer (μm) equals 0.000001 m (10−6 m). The prefix micro indicates the unit following it should be divided by 1 million, or 106. A nanometer (nm) equals 0.000000001 m (10−9 m). Angstrom (Å) was previously used for 10−10 m, or 0.1 nm, but the term has been replaced with the appropriate nanometers.

Section 3.2: Microscopy - The Instruments

Light Microscopy - the use any kind of microscope that uses visible light to observe specimens.

2. Diagram the path of light through a compound microscope.

   

   • For an electron microscope, magnification is achieved when light rays from an illuminator, the light source, pass through a condenser, which has lenses that direct the light rays through the specimen. From here, light rays pass into the objective lenses, the lenses closest to the specimen. The image of the specimen is magnified again by the ocular lens, or eyepiece.

3. Define total magnification and resolution.

   • Total Magnification: the magnification of a microscope specimen, determined by multiplying the ocular lens magnification by the objective lens magnification.

     Most microscopes used in microbiology have several objective lenses, including 4× (scanning power) 10× (low power), 40× (high power), and 100× (oil immersion). Most ocular lenses magnify specimens by a factor of 10.

   • Resolution (resolving power): the ability of the lenses to distinguish fine detail and structure. For example, if a microscope has a resolving power of 0.4 nm, it can distinguish two points if they are at least 0.4 nm apart.

To obtain a clear image under a compound light microscope, specimens must have a sharp contrast with their medium, achieved by altering their refractive index through staining. The refractive index indicates how much light bends when passing through a medium. By staining the specimen, the differing refractive indexes enhance contrast as light rays refract at the boundary between the specimen and its medium, allowing for better visibility of details.

For high magnification and resolution, it's crucial to maintain the direction of light rays after they pass through the stained specimen. This is done using immersion oil, which is placed between the glass slide and the oil immersion objective lens. The immersion oil matches the refractive index of glass, preventing light rays from being refracted as they enter the air. This allows for finer resolving power and avoids image fuzziness that can occur without it.

4. Identify a use for darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, two-photon, and scanning acoustic microscopy, and compare each with brightfield illumination.

   • Brightfield illumination: a microscope that uses visible light for illumination; the specimens are viewed against a white background.

   • Compared to a brightfield illumination, darkfield microscopes use a darkfield condenser that contains an opaque disk that blocks light that would normally enter directly. Only light reflected off the specimen enters the objective lens, making the specimen appear light against a black background. It is used to examine live microorganisms that either are invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics are obscured. They are used to examine very thin spirochetes like Treponema pallidum.

   • Phase-contrast microscopes: Phase-contrast microscopy enhances the observation of microorganisms by sharply defining internal structures, allowing for detailed examinations of living specimens without the need for fixation or staining, which could distort or kill the cells. In this microscope, light rays are divided into two sets: one set comes directly from the light source, while the other set includes light reflected, refracted, or diffracted from the specimen's structures. Diffraction occurs when light scatters as it interacts with the specimen's edge, bending the rays away from the direct path. The combination of these light rays creates an image of the specimen on the ocular lens, displaying various contrasts from light areas (in phase) to dark (out of phase).

   • Differential Interference Contrast Microscopy - similar to phase-contrast microscopy in that differences in refractive indexes are used. However, it uses two beams of light with prisms to split light beam, adding contrasting colors. It often has a higher resolution and seems nearly three-dimensional.

   • Fluorescence microscopy - Fluorescence microscopy utilizes fluorescence, allowing substances to absorb short wavelengths of light (ultraviolet) and emit light at longer wavelengths (visible). Some organisms naturally fluoresce under ultraviolet light, while others require staining with fluorochromes to achieve fluorescence. When stained microorganisms are viewed under a fluorescence microscope with an ultraviolet light source, they appear as bright objects against a dark background. Fluorochromes, such as auramine O and fluorescein isothiocyanate (FITC), have specific attractions to different microorganisms. For instance, Mycobacterium tuberculosis glows yellow when stained with auramine O, while Bacillus anthracis appears apple green when stained with FITC.

     The principal use of fluorescence microscopy is the fluorescent-antibody (FA) technique, also known as immunofluorescence. This diagnostic method involves injecting an animal with a specific antigen (such as a bacterium), prompting the animal to produce antibodies against that antigen. After a period of time, these antibodies are extracted from the animal's serum and combined with a fluorochrome to create fluorescent antibodies. When these fluorescent antibodies are added to a microscope slide containing an unknown bacterium, they will bind to the corresponding antigens on the bacterium's surface if it is the same as the one injected, resulting in fluorescence. This can be used to find microorganisms that even within cells, tissues, or other clinical specimens. It can be used to find a microbe in minutes.

   • Confocal Microscopy - Confocal microscopy is a light microscopy technique that reconstructs three-dimensional images by illuminating one plane of a specimen at a time. Specimens are stained with fluorochromes to emit light, and short-wavelength (blue) light is used for illumination. The returned light passes through a pinhole aperture aligned with the illuminated region, which eliminates blurring and enhances clarity. Successive planes are scanned, corresponding to fine slices of the specimen, leading to exceptionally clear two-dimensional images and improved resolution. Most confocal microscopes are linked to computers, allowing for digital data conversion to create three-dimensional representations that can be rotated for different views. This technique is effective for imaging entire cells and evaluating cellular physiology by monitoring substances like ATP and calcium ions.

   • Two-Photon Microscopy - Two-Photon Microscopy (TPM) uses fluorescence staining similar to confocal microscopy, but it employs long-wavelength (far red) light, requiring two photons to excite the fluorochrome. This allows TPM to image living cells in tissues at greater depths. Unlike confocal microscopy, which is limited to imaging at shallow depths, TPM minimizes damage to cells by generating less singlet oxygen and enables real-time tracking of cellular activity, such as immune cells responding to antigens.

   • Scanning acoustic microscopy - Scanning acoustic microscopy (SAM) involves interpreting the action of sound waves transmitted through a specimen. A specific frequency sound wave travels through the specimen, reflecting back every time it encounters an interface within the material. SAM is utilized to study living cells attached to surfaces, including cancer cells, arterial plaque, and bacterial biofilms.

5. Explain how electron microscopy differs from light microscopy.

   • Electron microscopes are essential for examining objects smaller than about 0.1 micrometers, such as viruses and cellular internal structures, as they utilize a beam of electrons rather than light. The resolving power of electron microscopes is significantly greater than that of light microscopes due to the shorter wavelengths of electrons compared to visible light. Images from electron microscopes are typically black and white but can be artificially colored to highlight specific features. There are two main types of electron microscopes: transmission electron microscopes (TEM) and scanning electron microscopes (SEM).

6. Identify uses for the transmission electron microscope (TEM), scanning electron microscope (SEM), and scanned-probe microscopes.

• Transmission electron microscope - Transmission electron microscopy has high resolution and is extremely valuable for examining different layers of specimens. The transmission electron microscope can resolve objects as close together as 10 pm, and objects are generally magnified 10,000 to 10,000,000×. Because most microscopic specimens are so thin, the contrast between their ultrastructures and the background is weak.

• Scanning electron microscope - overcomes the sectioning problems associated with a transmission electron microscope. overcomes the sectioning problems associated with a transmission electron microscope. The SEM is especially useful in studying the surface structures of intact cells and viruses. In practice, it can resolve objects as close together as 0.5 nm, and objects are generally magnified 1000 to 500,000×.

• Scanned-probe microscopes - They use various kinds of probes to examine the surface of a specimen using electric current, which does not modify the specimen or expose it to damaging, high-energy radiation. Such microscopes can be used to map atomic and molecular shapes, to characterize magnetic and chemical properties, and to determine temperature variations inside cells.

  • Scanning tunneling microscopy - Scanning tunneling microscopy (STM) utilizes a thin tungsten probe to scan specimens, creating images that reveal atomic bumps and depressions on the surface. It has a resolving power greater than that of electron microscopes, capable of resolving features as small as an atom, and does not require special specimen preparation. STMs are particularly valuable for providing detailed views of molecules like DNA.

  • Atomic force microscopy - Atomic Force Microscopy (AFM) utilizes a metal-and-diamond probe that is gently applied to a specimen's surface. As the probe scans the surface, its movements are recorded to produce a three-dimensional image. Unlike Scanning Tunneling Microscopy (STM), AFM does not necessitate special specimen preparation. AFM is capable of imaging biological substances at nearly atomic resolution and molecular processes, such as the assembly of fibrin, which is integral to blood clot formation.

Section 3.3 - Preparation of Specimens for Light Microscopy

7. Differentiate an acidic dye from a basic dye.

   • Basic dyes are colored by cations and attract slightly negatively charged bacteria, making them more commonly used for staining. Examples of basic dyes include crystal violet, methylene blue, malachite green, and safranin. In contrast, acidic dyes are colored by anions and are generally repelled by the negatively charged bacterial surface, resulting in background staining rather than the bacteria themselves. This method is called negative staining, useful for examining overall cell shape, size, and capsules against a dark background.

8. Explain the purpose of simple staining.

   • Simple Staining:

     • A simple stain is an aqueous or alcohol solution of a single basic dye.

     • Purpose: The primary purpose of a simple stain is to highlight the entire microorganism, allowing cellular shapes and basic structures to be visible.

     • Procedure:

       • The stain is applied to the fixed smear for a specific length of time.

       • After staining, the specimen is washed off, dried, and examined.

     • Mordants: Occasionally, an additive called a mordant is used to intensify the stain.

       • Functions of a mordant include:

         • Increasing the affinity of a stain for a biological specimen.

         • Coating structures (e.g., flagellum) to make them thicker and more visible after staining.

     • Common Dyes Used: Some commonly used simple stains in the laboratory include methylene blue, carbolfuchsin, crystal violet, and safranin.

9. List Gram stain steps, and describe the appearance of gram-positive and gram-negative cells after each step.

   • A heat-fixed smear is covered with a basic purple dye, usually crystal violet. Because the purple stain imparts its color to all cells, it is referred to as a primary stain.

     ➋ After a short time, the purple dye is washed off, and the smear is covered with iodine, a mordant. When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple.

     ➌ Next, the slide is washed with alcohol or an alcohol-acetone solution. This solution is a decolorizing agent, which removes the purple from the cells of some species but not from others.

     ➍ The alcohol is rinsed off, and the slide is then stained with safranin, a basic red dye, which functions as a counterstain. The smear is washed again, blotted dry, and examined microscopically.

   • Bacteria that retain this color after the alcohol has attempted to decolorize them are classified as gram-positive bacteria; bacteria that lose the dark violet or purple color after decolorization are classified as gram-negative bacteria

10. Compare and contrast the Gram stain and the acid-fast stain.

    • Acid-fast stain is used to bind to cells walls that have a waxy material. This is to identify microbes and bacteria that have a waxy cell wall and differentiate them from other microbes. Acid-fast staining can identify bacterial agents responsible for tuberculosis and leprosy.

11. Explain why each of the following is used: capsule stain, endospore stain, flagella stain.

    • Special stains are used to color parts of microbes like endospores, flagella, and capsules.

    • Capsule staining can be used to determine an organism’s virulence. Because capsules do not accept most biological dyes, they appear as halos around the bacterial cell.

    • Endospore staining can help identify the relatively few bacteria that have endospores like Clostridium, which includes agents that cause botulism and tetanus. Because endospores are highly refractive, they can be detected under the light microscope when unstained, but without a special stain, they cannot be differentiated from areas of stored material (inclusions) in cells. They are green after a staining.

    • Flagella Staining enables scientists to ensure the flagella are visible under a microscope.