Microbiology for the Health Sciences

Chapter 2. Microscopy

Chapter 2 Outline
Introduction
Using the metric system to express the sizes of Microorganisms Microscopes

Simple microscopes
Compound microscopes Electron microscopes

Using the Metric System to Express the Sizes of Microorganisms

Metric units are used to express the sizes of microorganisms. 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 (m). The sizes of microorganisms are measured using an ocular micrometer.

Representations of Metric Units of Measure and Numbers

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; those that cannot be seen with the unaided human eye.

Each optical instrument has a limit as to what can be seen

The limit is referred to as the resolving power or resolution of the instrument.

Simple Microscopes
A simple microscope is one that contains only one magnifying lens. A magnifying glass could be considered a simple microscope. With a magnifying glass, images appear 3-20 times larger than the objects actual size. Leeuwenhoeks simple microscopes had a maximum magnifying power of about 300X or 300 times.

Early Microscopes

Compound Microscopes
A compound microscope contains more than one magnifying lens. Because visible light is the source of illumination, the compound microscope is also referred to as a compound light microscope . Compound light microscopes usually magnify objects about 1000 times!

Photographs taken through the lens system of a compound microscope are called photomicrographs.

Compound Microscopes (continued)

It is the wavelength of visible light (~0.45 m) 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. Todays laboratory microscope contains two magnifying lens systems: The eyepiece lens or ocular (usually X10) The objective (usually 4 - X4, X10, X40, and X100)

A Modern Compound Microscope

Compound Microscopes (continued)

For optimal observation of the specimen, the light must be properly adjusted. This is accomplished using the condenser which is beneath the stage. Image clarity depends on the microscopes resolving power or the the ability of the lens system to distinguish between two adjacent objects. The resolving power of the compound light microscope is ~ 1000 times better than that of the unaided human eye. Increasing magnification without increasing the resolving power is called empty magnification.

Compound Microscopes (continued)

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 now a darkfield microscope. Other types of microscopes include:
Phase contrast microscopes Fluorescence microscopes

Darkfield Microscopy of Treponema pallidum

Phase Contrast and Fluorescent 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.

Fluorescent microscope contains a built-in ultraviolet (UV) light source

When UV light strikes certain dyes and pigments these substances emit a longer wavelength light causing them to glow against a dark background.

Electron Microscopes
Electron microscopes enable us to see extremely small infectious agents such as rabies and smallpox viruses.

Living organisms cannot be seen with an electron microscope the processing procedures kill the organisms.
An electron beam is used as a source of illumination and magnets are used to focus the beam. Electron microscopes have a much higher resolving power than the compound light microscope. There are 2 types of electron microscopes - transmission and scanning.

The Transmission Electron Microscope

Uses an electron gun to fire a beam of electrons through an extremely thin specimen (<1 m thick).

An image of the specimen is produced on a phosphorcoated screen.

Magnification is 1000 times greater than the compound light microscope.

The Scanning Electron Microscope

Electrons are bounced off the surface of a specimen and the image appears on a monitor. Used to observe the outer surfaces of specimens. Resolving power not as high as the transmission electron microscope. Scanning and transmission electron micrographs are black and white images.

S. aureus

S. aureus and red blood cells as seen by light microscopy

S. aureus as seen by transmission electron microscopy (TEM)

Scanning electron microscopy reveals the corkscrew shape of the spirochete, Treponema pallidum; note the illusion of depth.

Review of Key Points

A meter (m) can be divided into 10 decimeters, 100 centimeters, 1000 millimeters, 1 million micrometers, or 1 billion nanometers The metric system is used to describe the sizes of microorganisms. The development of simple and compound microscopes enabled the discovery and visualization of microorganisms. The limiting factor of compound light microscopes is the type of illumination being used.

Review of Key Points (continued)

Because visible light is the source of illumination in the light microscope, objects smaller than half the wavelength of visible light cannot be seen. The resolving power of the compound light microscope is 0.2 m. Electron microscopes allow the visualization of smaller objects because electrons are used as the source of illumination. The wavelength of electrons is much shorter than that of visible light.

Review of Key Points (continued)

Transmission electron microscopes enable scientists to see inside of cells. Their resolving power is 0.2 nm The scanning electron microscope enables scientists to study surface details. Their resolving power is 20 nm. Photographs taken with a compound light microscope are called photomicrographs, whereas those taken with electron microscopes are called transmission or scanning electron micrographs.