.Microscopes are optical instruments that can observe microbes.Our magnifying lenses produce magnified images that help us to see the form and structure of these tiniest of living creatures.

You should understand what a microscope does before you begin using it.We always refer to terms such as "ocular lenses" and "condensers" when discussing ways to improve microscopic images, so it is essential to know the names and functions of each component.

Based on the picture of the binocular, compound light microscope in Figure 1, match the name of the major part (listed below) with its location on the microscope, and give a very brief description of what each is used for:

Ocular lenses_________________________________Locate the parts on the microscope that allow you to:Move the stage (stage adjustment knobs)Adjust the condenser lensAdjust the light intensityAdjust the iris diaphragmAdjust the distance between the ocular lenses

Objective lenses_________________________________
(Revolving) Nosepiece_________________________________
Stage and stage clips_________________________________
Course and fine focus knobs_________________________________
Condenser lens_________________________________
Iris diaphragm_________________________________

Making Images

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Waves that travel through and interact with an object may vary in speed, speed, or direction as they travel through "media" (such as air, water, oil, cytoplasm, etc.) of different densities.If light passes through a thick or dense part of a specimen (such as the nucleus of a cell), the light is reflected or refracted (changed in speed or direction) more than wave passing through a thinner part.Therefore, the thicker parts appear darker in the image, while the thin parts appear lighter.

For a compound microscope, the optical path leading to a detectable image involves two lenses – the objective lens and the ocular lens. The objective lens magnifies the object and creates a real image, which will appear to be 4, 10, 40, or 100 times larger than the object actually is, depending on the lens used. The ocular lens further magnifies the real image by an additional factor of 10, to produce a vastly larger virtual image of the object when viewed by you.

Condenser lens, which is located just below the stage and adjustable with condenser adjustment knob, provides a focus for light coming from an illuminator (light source) below the stage.Figure 2 illustrates how the condenser focuses light through the specimen to match the aperture of the objective lens above.


A high-quality image is dependent upon the appropriate use of the condenser, which is usually equipped with an iris diaphragm on most microscopes.With increased magnification lenses with small apertures, raising the condenser to just below the stage produces a spotlight effect on the specimen.While using scanning lenses or low power lenses, the condenser should be lowered since the apertures are much larger, and a lot of light can blind the subject.To create a great contrast in the image, the iris diaphragm can be opened to make the image brighter and closed to dim the light.It is subjective and the adjustments should be made to suit the audience's preferences.


When the light waves that have interacted with the specimen are collected by the lenses and eventually get to your eye, the information is processed into dark and light and color, and the object becomes an image that you can see and think more about.

Magnification

In lab, you'll use a microscope with a compound system of lenses. .Accordingly, you can determine the total magnification, or how much bigger the object appears to you when you view it, by multiplying the magnification of the objective lens by 10.

The magnifying power of each lens is engraved on its surface, followed by an “X.” In the table below, find the magnification, and then calculate the total magnification for each of the four lenses on your microscope.

Magnification of objective lensTotal magnification of viewed object
Scanning Lens
Low Power Lens
High Power Lens
Oil Immersion Lens

Take, for instance, a look at Bacillus cereus cells, which are rod-shaped and about 4 m long.Looking at B.cereus with a high power lens, how big would the cells appear to you?

Resolution Limits Magnification

Therefore, in a microscope, small cells appear enormous.But why can't we just use different or more lenses to magnify the image until it is big and easy to read?

The answer is resolution. Consider what happens when you try to magnify the fine print from a book with a magnifying glass. As you move the lens away from the print, it gets larger, right? But as you keep moving the lens, you notice that while the letters are still getting larger, they are becoming blurry and hard to read. This is referred to as “empty magnification” because the image is larger, but not clear enough to read. Empty magnification occurs when you exceed the resolving power of the lens.

An image's resolution is usually regarded as how clearly it shows details in the image.By definition, a resolution is the minimum distance between two objects that can be perceived as two separate entities.One can also think of it as the smallest object that we can see clearly.

The ability of a lens to resolve detail is ultimately limited by diffraction of light waves, and therefore, the practical limit of resolution for most microscopes is about 0.2 µm. Therefore, it would not be practical to try to observe objects smaller than 0.2 µm with a standard optical microscope. In addition, cells of all types of organisms lack contrast because many cellular components refract light to a similar extent. This is especially true of bacteria. To overcome this problem and increase contrast, biological specimens may be stained with selective dyes.

The Oil Immersion Objective

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Among the lenses, the oil immersion lens achieves the highest magnification of 1000X with a resolution of 0.2 m.We need to pay attention to this lens because without it, we would spend a lot of time in the lab.

The resolving power of this lens is dependent on “immersing” it in a drop of oil, which prevents the loss of at least some of the image-forming light waves because of refraction. Refraction is a change in the direction of light waves due to an increase or decrease in the wave velocity, which typically occurs at the intersection between substances through which the light waves pass. This is a phenomenon you can see when you put a pencil in a glass of water. The pencil appears to “bend” at an angle where the air and water meet (see Figure 3). These two substances have different refractive indices, which means that light passing through the air reaches your eye before the light passing through the water. This makes the pencil appear “broken.”

The same thing happens as the light passes through the glass slide into the air space between the slide and the lens. The light will be refracted away from the lens aperture. To remedy this, we add a drop of oil to the slide and slip the oil immersion objective into it. Oil and glass have a similar refractive index, and therefore the light bends to a lesser degree and most of it enters the lens aperture to form the image.

It is important to remember that you must use a drop of oil whenever you use the oil immersion objective or you will not achieve maximum resolution with that lens. However, you should never use oil with any of the other objectives, and you should be diligent about wiping off the oil and cleaning all of your lenses each time you use your microscope, because the oil will damage the lenses and gum up other parts of the instrument if it is left in place.

Using the Microscope

If you are new to microscopy, you may initially feel challenged as you try to achieve high quality images of your specimens, particularly in the category of “Which lens should I use?” A simple rule is: the smaller the specimen, the higher the magnification. The smallest creatures we observe are bacteria, for which the average size is a few micrometers (μm). Other microscopic organisms such as fungi, algae, and protozoa are larger, and you may only need to use the high power objective to get a good view of these cells; in fact, using the oil immersion objective may provide you with less information because you will only be seeing a part of a cell.

This brings us to two additional concepts related to microscopy—working distance and parfocality. Working distance is how much space exists between the objective lens and the specimen on the slide. As you increase the magnification by changing to a higher power lens, the working distance decreases and you will see a much smaller slice of the specimen. Also, once you’ve focused on an object, you should not have to make any major adjustments when you switch lenses, because the lenses on your microscope are designed to be parfocal. This means that something you saw in focus with the low power objective should be nearly in focus when you switch to a high power objective, or vice versa. Thus, for viewing any object and regardless of what lens you will ultimately use to view it, the best practice is to first set the working distance with a lower power lens and adjust it to good focus using the coarse focus knob. From that point on, when you switch objectives, only a small amount of adjustment with the fine focus knob should be necessary.

Here is a final consideration related to objective lenses and magnification. Look at the lenses on your microscope, and note that as the magnification increases, the length of the lens increases and the lens aperture decreases in size. As a result, you will need to adjust your illumination to compensate for a darkening image. There are essentially three ways to vary the brightness; by increasing or decreasing the light intensity (using the on/off knob), by moving the condenser lens closer to or farther from the object using the condenser adjustment knob, and/or by opening/closing the iris diaphragm. Don’t be afraid to experiment to create the best image possible.

Guidelines for safe and effective use of a microscope:

1. Carry the microscope to your lab table using two hands, and set it down gently on the bench. Once placed on the bench, do not try to slide it around on its base, because this is extremely jarring to the optical system.

2. Clean all of the lenses with either lens paper or Kimwipes (NOT paper towels or nose tissues) BEFORE you use your microscope, AFTER you are done, and before you put it away.

3. When you are finished with the microscope, check the stage to make sure that you don’t leave a slide clipped in the stage. Make sure to switch the microscope OFF before you unplug it. Gently wrap the cord around the base and cover your microscope with its plastic cover.

4. Return the microscope to the cabinet before you leave the lab. Make sure that the ocular lenses are facing IN.

Together we will review how to effectively achieve an exceptional image using a standard optical microscope. This will include not only locating and focusing on the object, but also using the condenser lens and iris diaphragm to achieve a high degree of contrast and clarity.

Rectal Smear

We’ll start by looking at a prepared slide of a “rectal smear,” which is quite literally a smear of feces on a slide stained with a common method called the Gram stain. You will observe several different types of bacterial cells in this smear that will appear either pink or purple. While the main purpose of this is to develop proficiency in use of the oil immersion objective lens, it also provides the opportunity to look at bacteria, observe the differences in cell shapes and sizes, and note that when Gram stained they turn out to be either purple or pink.

When you have achieved an exceptional image of the fecal bacteria at 1000X, consider the following questions.