Chapter 2: Viewing the Microbial World

Using the Metric System to Express the Sizes of Microbes Metric units are used to express the sizes of microbes. The basic unit of length in the metric system is the meter (m); it is equivalent to 39.4 inches. The sizes of bacteria and protozoa are usually expressed in terms of micrometers (µm). A micrometer is one millionth of a meter. A typical spherical bacterium (coccus) is approximately 1 µm in diameter. A typical rod-shaped bacterium (bacillus) is approximately 1 µm wide  3 µm long.

Representations of Metric Units of Measure and Numbers

Relative Sizes of Staphylococcus and Chlamydia Bacteria and Several Viruses

Using the Metric System to Express the Sizes of Microbes (cont.) The sizes of viruses are expressed in terms of nanometers (nm). A nanometer is equal to one billionth of a meter. Most of the viruses that cause human diseases range in size from 10 to 300 nm. One exception is Ebola virus, a cause of viral hemorrhagic fever. Ebola viruses can be as long as 1,000 nm (1 µm). When using a microscope, the sizes of microorganisms are measured using an ocular micrometer. This must be calibrated using a stage micrometer for each microscope objective Stage micrometer acts as a scale of measurement

Microscopes The human eye, a telescope, a pair of binoculars, a magnifying glass, and a microscope are various types of optical instruments. A microscope is an optical instrument that is used to observe tiny objects, objects so small that they cannot be seen with the unaided human eye. Each optical instrument has a limit as to what can be seen using that instrument; this limit is referred to as the resolving power or resolution of the instrument. The resolving power of the unaided human eye is approximately 0.2 mm.

Early Microscopes

Simple Microscopes A simple microscope is one that contains only one magnifying lens. A magnifying glass could be considered a simple microscope when using a magnifying glass, images appear 3 to 20 times larger than the object’s actual size. Leeuwenhoek’s simple microscopes had a maximum magnifying power of about 300 (about 300 times) Not as widely used in research and labs Often uses external light source

Simple Microscopes

Compound Microscopes A compound microscope contains more than one magnifying lens. Because visible light is the source of illumination, a compound microscope is also referred to as a compound light microscope. Compound light microscopes usually magnify objects about 1,000 times. The resolving power of a compound light microscope is approximately 0.2 µm about 1,000 times better than the resolving power of the unaided human eye.

Compound Microscopes (cont.) It is the wavelength of visible light (~0.45 µm) that limits the size of objects that can be seen. Objects cannot be seen if they are smaller than half of the wavelength of visible light. Today’s laboratory microscope contains two magnifying lens systems: The eyepiece or ocular lens (usually 10) The objective lens (4, 10, 40, and 100 are the four most commonly used objective lenses)

A Modern Compound Light Microscope

Compound Microscopes (cont.) Total magnification is calculated by multiplying the magnifying power of the ocular lens by the magnifying power of the objective lens being used. 10 ocular  4 objective = 40 total magnification 10 ocular  10 objective = 100 total magnification 10 ocular  40 objective = 400 total magnification 10 ocular  100 objective = 1,000 total magnification Photographs taken through the lens system of the compound light microscope are called photomicrographs.

Compound Microscopes (cont.) Because objects are observed against a bright background or “bright field,” the compound light microscope is sometimes referred to as a brightfield microscope. If the condenser is replaced with what is known as a darkfield condenser, illuminated objects are seen against a dark background or “dark field”; the microscope is then called a darkfield microscope. Other types of compound microscopes include Phase-contrast microscopes Fluorescence microscopes

Darkfield and Fluorescence Micrographs

Phase-Contrast and Fluorescence Microscopes Phase-contrast microscopes are used to observe unstained living microorganisms. Organisms are more easily seen because the light refracted by living cells is different from the light refracted by the surrounding medium. Fluorescence microscopes contain a built-in ultraviolet (UV) light source. When the UV light strikes certain dyes and pigments, these substances emit a longer-wavelength light, causing them to glow against a dark background. Different parts of the same cell can be stained with different dyes to allow for complex analysis of proteins within the cell

Electron Microscopes Electron microscopes enable us to see extremely small microbes such as rabies and smallpox viruses. Living organisms cannot be observed using an electron microscopethe processing procedures kill the organisms. Microscope uses a vacuum An electron beam is used as the source of illumination, and magnets are used to focus the beam. Electron microscopes have a much higher resolving power than compound light microscopes. Wavelength of the beam is 100,000 times shorter than visible light There are two types of electron microscopes  transmission and scanning.

Transmission Electron Microscope (TEMs) Electron beam produced at the top of a tall column This microscope uses an electron gun to fire a beam of electrons through an extremely thin specimen (<1 µm thick). Some electrons transmit through the specimen while others are blocked creating an image An image of the specimen is produced on a phosphor-coated screen. Magnification is approximately 1,000 times greater than with the compound light microscope. Resolving power is approximately 0.2 nm. Can visualize intermal cell structures

A Transmission Electron Micrograph of Influenza Virus A

Scanning Electron Microscope (SEM) Composed of a shorter column Electrons are bounced off the surface of a specimen and captured by detectors that create an image that appears on a monitor. This is used to observe the outer surfaces of specimens. Resolving power of this microscope is about 100 times less than that of transmission electron microscope.

Electron microscopes Micrographs collected are black and white images If color is used in photograph, it was artificially enhanced 2-dimensional (2-D) micrographs obtained Micrographs collected are black and white images If they are seen in color it is because they are artificially enhanced Two dimensional image of the specimen is obtained

Staphylococcus aureus (Blue) and Red Blood Cells as Seen by Light Microscopy

S. aureus in the Process of Binary Fission, as Seen by Transmission Electron Microscopy

Scanning Electron Micrograph of S. aureus

Atomic force microscopes Provides a 3-dimensional (3-D) image Silicon or silicon nitride cantilever with a sharp tip scans surface of specimen When tip and cantilever are in close proximity, deflection is caused due to atomic forces between the two Deflection is measured using a laser reflected off the cantilever onto a photodiode creating an image Atomic force microscopes Figure 2-14 Provides a 3-D surface profile of the specimen Silicon or silicon nitride cantilever with a sharp tip scans the surface of the specimen When the top and sample are in close proximity, it leads to a deflection due to atomic forces between the two This deflection is measured using a laser reflected off the cantilever onto a photodiode to create an image