Microbes are too small to be seen with the naked eye. It wasn’t until the invention of the microscope that scientists could peer directly into the unseen world around us. Microscopes make the microscopic world visible by enlarging the image of an object, and making the details more clear. These two features are called magnification and resolution, respectively. Without good resolution, a microscope could make an image larger, but you would not be able to see all of the fine features of the object.
There are two basic types of microscopes: light and electron. Light microscopes pass light through, or bounce light off, a sample. This light then goes through a series of lenses that magnify and focus the image. Other techniques are used with light microscopes to view samples more clearly, to identify microbes, or to see larger parts of the microbes. To view smaller objects — such as viruses — an electron microscope must be used.
Early Microscopes
The earliest microscopes relied on light to magnify images. In the beginning, drops of water were used like a magnifying lens. You can try this at home by placing a drop of water on a printed page, and viewing the text through it. Later, single lenses were made that enlarged — magnified — an image up to 20 times the object’s actual size. The earliest use of this type of single lens was in 1267.
It was not until 1595 that the first true microscope was built. This consisted of two lenses that could be adjusted to change the magnification. Later a third lens was added to the eyepiece of microscopes to provide improved focusing. One of the first people to peer at microbes through a microscope was the Dutch scientist Anton von Leeuwenhoek in the late 1600s. Using microscopes that he built himself, van Leeuwenhoek observed microbes living in lake water, and bacteria scraped from his teeth.
Light Microscopes
Light — or optical — microscopes are the most widely used. They are found in high schools and colleges, as well as in many hospitals and scientific laboratories. Microscopes with only a single eyepiece still exist, although the binocular microscope — having an eyepiece for each eye — is becoming more common. A binocular microscope allows you to view the image through both eyes at the same time. Some of these microscopes even allow you to see the image in three dimensions, although this is not as important when viewing microbes, which are extremely small.
Not all samples can be viewed well with bright light — called bright-field illumination. Scientists use another type of light microscope — phase-contrast — to change the light patterns of the image: some parts appear brighter and others darker. This allows smaller details to be seen more easily. A dark-field microscope, on the other hand, keeps light from shining directly on the specimen. This improves the contrast of the image. Another type is the polarizing light microscope, which enables scientists to view images in color.
Staining Microbes
Even with the magnifying power of a microscope, many microbes are colorless and transparent. In order to see them, scientists use a variety of stains and dyes that make parts of the microbes visible under the microscope. Early stains were natural products, such as saffron. The first synthetic stain was made in 1856. This sparked the search for other colored compounds that could be used to view cells and structures under the microscope.
Scientists quickly discovered that different microbes could be stained with different dyes. This provided another method of identification. One of the methods used on bacteria consisted of an iodine stain followed by an alcohol wash. Some bacteria kept the dye while others lost the color during the wash. Based upon this staining, bacteria were grouped into gram-positive and gram-negative, meaning they kept or lost their color, respectively. This technique — gram staining — was named after the scientist who discovered it, Hans Christian Joachim Gram.
FISHing for Microbes
Scientists use a technique called fluorescent in situ hybridization — FISH — to detect specific RNA or DNA sequences in tissues, cells and microbes. DNA contains the genetic instructions for an organism. RNA is used by the cell to turn the information stored in DNA into proteins that make up the cell. During FISH, molecules that bind to specific types of DNA or RNA are added to a sample. These molecules are also tagged so they give off a color when exposed to a certain type of light. This is called fluorescence. Using a special light microscope, scientists can identify cells that contain the specific DNA or RNA.
In microbiology, FISH is used to find the position of genes, identify certain types of microbes, and study the structure and function of cells. Unknown samples of microbes — such as from an infection — can be identified more quickly using FISH. Rather than growing the microbes, scientists can test them against known segments of DNA. This type of microscope can also be automated. Slides are moved and scanned by a robotic system, with the images captured on a computer. Image-recognition software — like the kind used to identify people in a crowd — can look for specific shapes of cells and fluorescent tags.
Electron Microscope
Because they are so small, viruses and many structures of microbes cannot be seen using an optical microscope. Electron microscopes, first built in 1931, can visualize objects less than one-billionth of a meter. This enables scientists to view molecules and even some atoms. Instead of light, they use a beam of electrons to view objects. As with light microscopes, several types of electron microscopes exist.
A transmission electron microscope (TEM) produces black-and-white images by passing electrons through an object. Some of the electrons are absorbed by the sample, while others are captured on a special photographic plate to produce the image. A scanning electron microscope (SEM) captures a three-dimensional image of an object by scanning its surface with electrons. Another type of microscope, the electron-probe microanalyzer, can analyze the materials that make up a sample.
