Introduction to the Microscope

Learning Objectives
Demonstrate and explain how to use a microscope Explain how different types of microscopes affect magnification and resolving power Identify parts of a microscope

Concept Animation: Introduction to the Microscope

Introduction

Microbiology is the study of microorganisms. Microorganisms are organisms that are too small to be seen with the human eye (Figure 1). This is a huge field for such small subjects. There are many times more microorganisms on Earth than all the other organisms put together. It is estimated, for example, that in the human body alone there are 10 microorganisms for every one human cell. That means that 90% of the cells found in the human body are not human!

There are many other objects besides microorganisms, such as biological structures, that cannot be seen with the human eye. What types of objects can you think of that cannot be seen without the aid of some magnification? Biological structures might include a valve found in a vein that prevents blood from moving backwards or the cells that make up a multicellular organism. The fact is that there are an unimaginable number of objects that we cannot see with the naked eye.

Various bacterial species of all shapes and sizes

Types of Microscopes

Microscopes are used for the visualization of objects too small for us to see. There are many types of microscopes that range from low-level magnification (e.g., hand-held magnification lens) to mid-level magnification (e.g., compound light microscopes) to very high-power magnification (e.g., an electron microscope). The type of microscope that is used depends on the application. For example, if you were trying to locate and remove a small splinter from your finger, a hand-help magnification lens (magnifying glass) would be extremely helpful. If you wanted to examine the structure of a tiny virus, you would need an electron microscope.

Magnification

Magnification is one characteristic to consider when deciding what type of microscope is needed for a particular application. The other key characteristic is the resolving power. The resolving power refers to the ability of a microscope to show detail. Having a device that can greatly magnify an object is helpful only if the magnified object is well defined (not fuzzy). The resolution is largely dependent on the wavelength of light used. Shorter wavelengths produce higher resolutions. Light microscopes use visible light and electron microscopes use a beam of electrons that travel in wavelike patterns. These waves are 100,000 times shorter than visible light waves! Electron microscopy can therefore be used to visualize extremely small structures.

The compound light microscope is the most commonly used type of microscope. The name compound refers to presence of more than one lens. The use of these microscopes can be traced to the late 1500s. The early compound microscopes could magnify objects up to about 10X (this means 10 times). In the 1600s a Dutch linen merchant, Antonie van Leeuwenhoek, greatly improved upon the production of lens and was able to create microscopes that could magnify objects 200X. He observed and recorded many types of microorganisms with his microscopes. Because of this, van Leeuwenhoek is frequently referred to as "the Father of Microbiology". Modern day compound light microscopes can typically magnify objects up to 1000X.

A representative compound light microscope is shown in Figure 2. Note the two sets of lenses:

The ocular lenses (close to your eyes)
The objective lenses (close to the "object" on the stage)

Along with a light source, these lenses work together to magnify the object being viewed. In the case of the compound light microscope, the total magnification is equal to the magnification power of the ocular lens multiplied by the magnification power of the objective lens. For example, if the ocular lens magnifies 10X and the objective lens magnifies 10X, the total magnification is 100X.

Figure illustrating the parts of a microscope, shown counterclockwise from the top: the Resolving Nosepiece, Objective Lenses, Stage, Diaphragm, Light, Base, Fine Adjustment Knob, Coarse Adjustment Knob, Stage clips, Arm, Head, and Ocular Lens

Microscope Components

Below is a list of the parts of a compound light microscope. Refer to Figure 2 (above) as you read through this list to familiarize yourself with these parts.

Base: The flat support of the microscope.
Light: Illuminates the object being viewed. This can be either in the form of a light source or a mirror that reflects ambient light onto the image. In the latter case, it is important to be working in an environment with adequate ambient light.
Stage: Supports the slide or other material to be viewed.
Diaphragm: Controls the amount of light allowed on the object.
Stage Clips: Secure the slide in place.
Revolving Nosepiece: Rotates the objective lenses of different magnifications and allows one of them to be positioned over the slide.
Arm: Connects the lower base and the upper head of the microscope (also used to carry the microscope).
Head: Supports both the ocular lens and the revolving nosepiece.
Ocular Lens (eyepiece): The lenses on the microscope typically have a magnification of 10X. If your microscope has a pointer, which is used to indicate a specific area of the specimen, it is attached here. Monocular Microscopes have a single ocular eyepiece while binocular microscopes have two ocular eyepieces.

How to Use a Microscope

The following steps describe the proper use of a compound light microscope.

Always carry a microscope with one hand securely around the arm and the other underneath the base for support. Place the microscope on a table, plug it in, and turn on the light source (or adjust the mirror as necessary).

Note: When cleaning a microscope, do not use paper towels or cloths as this will scratch the lens. To preserve the microscope, use only lens paper that will not scratch the optics.

To prevent damage to the lens or slides, always start and end with the scanning power objective lens (the shortest one) above the light source.
Place your slide on the stage and secure it with the stage clips. It is helpful to visually orient the slide so the object to be viewed is directly in the middle of the opening in the stage where the light is directed up toward the slide.
Turn the course adjustment knob to bring the stage all the way up to the scanning power objective lens. While looking through the lens, use the course adjustment knob to slowly lower the stage until the specimen comes into focus.

Note: When using a binocular microscope, adjust the distance between the two oculars until only one object is seen when using a binocular microscope. Record this distance and set your microscope to this distance every time you use it. The lenses may be re-adjusted if someone else uses the microscope.

To adjust the light, open or close the diaphragm located over the light source. The specimen should not be gray or exceptionally bright when properly illuminated.
Rotate the revolving nosepiece to the low-power lens (the next longest) when the object is in general focus. Switch to the fine adjustment knob to obtain more precise and greater detail after focusing with the course adjustment knob. It may also be necessary to adjust the light because more light reduces contrast (sharpness).
To become familiar with the mechanical stage knobs around the base of the microscope (if present), turn one slowly to the right, noting that the image will be moving toward the left. This image inversion is caused by the lenses.
Slowly rotate the high-power lens into place (the next longest lens) if you need higher magnification. This will bring the tip of the lenses very close to the slide. Make sure the objective lens does not touch the slide.
Whenever you use the high-power lens, only use the fine adjustment knob. If the object was well focused while viewing with the low-power lens, very little adjustment should be necessary.
If you cannot bring the object into focus, return to the low-power lens, focus the object, and then return to the high-power lens. Move the revolving nosepiece to the scanning objective lens position before removing the slide when finished.

Specimen Preparation

Proper specimen preparation helps produce the best visualization possible with any microscope. Glass slides are used to contain the specimen. Live specimens are usually prepared as wet mounts. In wet mount preparation, the specimen is placed on the slide and a much thinner glass cover slip is placed on top of the specimen. Samples are frequently heated to simultaneously kill and fix (secure) the sample to the glass slide. Fixed samples are then stained to enhance the observable contract between the cellular features.

Did You Know? Different types of stains may be used depending on the desired outcome. For example, if a specific structure is being studied, one might select a dye that stains only the unique structure. Perhaps the most well-known strain is the Gram stain. The Gram stain was developed in 1884 by Hans Christian Gram. Gram staining is often used to categorize bacteria as Gram positive or Gram negative. The Gram stain distinguishes between bacteria with thick cell walls (Gram positive species) from those with thin cell walls (Gram negative species). Thick cell walls entrap the crystal violet, resulting in purple colored cells. Thin cell walls do not entrap the crystal violet dye. Instead, the safranin stain is accepted and the red colored cells result.

How to Prepare a Wet Mount Slide

To make a wet mount for a specimen that is not already in liquid, take a clean slide and place the specimen in the center.
Add one drop of water.
- Note: For cells that are transparent, it may be necessary to add a small drop of stain as opposed to water.
Carefully add a coverslip by placing one end down and slowly lowering the other end.
- Note: If the coverslip is added too quickly, large air bubbles may become trapped which can cause difficulty viewing the slide. If this happens, gently remove the coverslip, add another drop of water and try again.
Remove excess liquid on the bottom of the slide or around the edges before it is placed on the microscope to avoid damage to the lens. Just touch a tissue to the edge of the coverslip to draw away the water (this is an example of capillary action).
If the specimen is already in liquid, place a drop in the middle of the slide and add the coverslip as you did in Step 3.