EXPERIMENT

Microscopic Examination

Of Stained Cell Preparations

Learning Objectives

Once you have completed this experiment, you should be familiar with the

1. Theoretical principles of brightfleld microscopy.

2. Component parts of the compound microscope.

3. Use and care of the compound microscope.

4. Practical use of the compound microscope for visualization of cellular morphology from stained slide preparations.

Principle

Microbiology is a science that studies living or​ganisms that are too small to be seen with the naked eye. Needless to say, such a study must in​volve the use of a good compound microscope. Although there are many types and variations, they all fundamentally consist of a two-lens sys​tem, a variable but controllable light source, and mechanical adjustable parts for determining focal length between the lenses and specimen (Figure 4.1).

Components of the Microscope

Stage A fixed platform with an opening in the center allows the passage of light from an illumi​nating source below to the lens system above the stage. This platform provides a surface for the placement of a slide with its specimen over the central opening. In addition to the fixed stage, most microscopes have a mechanical stage that can be moved vertically or horizontally by means of adjustment controls. Less sophisticated micro​scopes have clips on the fixed stage, and the slide must be positioned manually over the central opening.

Illumination The light source is positioned in the base of the instrument. Some microscopes are equipped with a built-in light source to provide direct illumination. Others are provided with a

 

reversible mirror that has one side flat and the other concave. An external light source, such as a lamp, is placed in front of the mirror to direct the light upward into the lens system. The flat side of the mirror is used for artificial light, and the con​cave side for sunlight.

Abbe Condenser This component is found di​rectly under the stage and contains two sets of lenses that collect and concentrate light as it passes upward from the light source into the lens systems. The condenser is equipped with an iris diaphragm, a shutter controlled by a lever that is used to regulate the amount of light entering the lens system.

Body Tube Above the stage and attached to the arm of the microscope is the body tube. This structure houses the lens system that magnifies the specimen. The upper end of the tube contains the ocular or eyepiece lens. The lower portion consists of a movable nosepiece containing the objective lenses. Rotation of the nosepiece po​sitions objectives above the stage opening. The body tube may be raised or lowered with the aid of coarse-adjustment and flue-adjustment knobs that are located above or below the stage-depending on the type and make of the instrument.

Theoretical Principles of Microscopy

To use the microscope efficiently and with mini​mal frustration, you should understand the basic principles of microscopy: magnification, resolu​tion, numerical aperture, illumination, and focusing.

Magnification Enlargement, or magnification, of a specimen is the function of a two-lens system; the ocular lens is found in the eyepiece, and the objective lens is situated in a revolving nose​piece. These lenses are separated by the body tube. The objective lens is nearer the specimen and magnifies it, producing the real image that is projected up into the focal plane and then magni​fied by the ocular lens to produce the final image.

Ocular

(eyepiece)

lenses

Body tube lock screw

Head-

Arm

Mechanical stage

Coarse-

adjustment knob

Fine-

adjustment knob

Objective lenses

Diaphragm lever Condenser

Iris diaphragm lever

Substage light

Condenser adjustment knob

Light control

Base

Power switch

Figure 4.1 Leica ATC 2000 compound microscope

The most commonly used microscopes are equipped with a revolving nosepiece containing four objective lenses, each possessing a different degree of magnification. When these are com​bined with the magnification of the ocular lens, the total or overall linear magnification of the specimen is obtained. This is shown in Table 4.1.

Resolving Power or Resolution Although mag​nification is important, you must be aware that unlimited enlargement is not possible by merely increasing the magnifying power of the lenses or by using additional lenses, because lenses are limited by a property called resolving power. By definition, resolving power is how far apart two adjacent objects must be before a given lens shows them as discrete entities. When a lens

cannot discriminate, that is, when the two ob​jects appear as one, it has lost resolution. In​creased magnification will not rectify the loss and will, in fact, blur the object. The resolving power of a lens is dependent on the wavelength of light used and the numerical aperture, which is a characteristic of each lens and imprinted on each objective. The numerical aperture is defined as a function of the diameter of the objective lens in relation to its focal length. It is doubled by use of the substage condenser, which illuminates the ob​ject with rays of light that pass through the speci​men obliquely as well as directly. Thus, resolving power is expressed mathematically as follows:

resolving power =

wavelength of light

2 x numerical aperture

Overall Linear Magnification

MAGNIFICATION

TOTAL MAGNIFICATION

OCULAR LENS

OBJECTIVE LENSES

OBJECTIVE MULTIPLIED BY OCULAR

40x 100x 400x 1000X

Scanning 4x Low-power 10x High-power 40X Oil-immersion 100x

10x 10X 10X 10X

Based on this formula, the shorter the wave​length, the greater the resolving power of the lens. Thus, for the same numerical aperture, short wavelengths of the electromagnetic spec​trum are better suited for higher resolution than are longer wavelengths.

However, as with magnification, resolving power also has limits. You might rationalize that merely decreasing the wavelength will automati​cally increase the resolving power of a lens. Such is not the case, because the visible portion of the electromagnetic spectrum is very narrow and borders on the very short wavelengths found in the ultraviolet portion of the spectrum.

The relationship between wavelength and numerical aperture is valid only for increased re​solving power when light rays are parallel. There​fore, the resolving power is also dependent on another factor, the refractive index. This is the bending power of light passing through air from the glass slide to the objective lens. The refrac​tive index of air is lower than that of glass; as light rays pass from the glass slide into the air, they are bent or refracted so that they do not pass into the objective lens. This would cause a loss of light, which would reduce the numerical aperture and diminish the resolving power of the objective lens. Loss of refracted light can be compensated for by interposing mineral oil, which has the same refractive index as glass, be​tween the slide and the objective lens. In this way, decreased light refraction occurs and more light rays enter directly into the objective lens, producing a vivid image with high resolution (Figure 4.2).

Illumination Effective illumination is required for efficient magnification and resolving power. Since the intensity of daylight is an uncontrolled variable, artificial light from a tungsten lamp is

the most commonly used light source in mi​croscopy. The light is passed through the con​denser located beneath the stage. The condenser contains two lenses that are necessary to produce a maximum numerical aperture. The height of the condenser can be adjusted with the condenser knob. Always keep the condenser close to the stage, especially when using the oil-immersion objective.

Objective lens

Refracted (lost)

Slide —[

Condenser

Light source

Figure 4.2 Refractive index in air and in mineral oil

Between the light source and the condenser is the iris diaphragm, which can be opened and closed by means of a lever, thereby regulating the

amount of light entering the condenser. Exces-

sive illumination may actually obscure the speci​men because of lack of contrast. The amount of light entering the microscope differs with each objective lens used. A rule of thumb is that as the magnification of the lens increases, the distance between the objective lens and slide, called working distance, decreases, whereas the nu​merical aperture of the objective lens increases

(Figure 4.3).

Use and Care of the Microscope

You will be responsible for the proper care and use of microscopes. Since microscopes are ex​pensive, you must observe the following regula​tions and procedures.

The instruments are housed in special cabi​nets and must be moved by users to their laboratory benches. The correct and only acceptable way to do this is to grip the microscope arm firmly with the right hand and the base with the left hand, and lift the instrument from the cabinet shelf. Carry it close to the body and gently place it on the laboratory bench. This will prevent colli​sion with furniture or coworkers and will protect the instrument against damage.

Once the microscope is placed on the labora​tory bench, observe the following rules:

1. Remove all unnecessary materials (such as books, papers, purses, and hats) from the lab​oratory bench.

2. Uncoil the microscope’s electric cord and plug it into an electrical outlet.

3. Clean all lens systems; the smallest bit of dust, oil, lint, or eyelash will decrease the efficiency of the microscope. The ocular, scanning, low-power, and high-power lenses may be cleaned by wiping several times with acceptable lens tissue. Never use paper toweling or cloth on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened with xylol is used to wipe it clean. The xylol is immedi​ately removed with a tissue moistened with 95% alcohol, and the lens is wiped dry with lens paper. Note: This xylol cleansing pro​cedure should be performed only by the in​structor and only if necessary; consistent use of xylol may loosen the lens.

The following routine procedures must be followed to ensure correct and efficient use of the microscope.

1. Place the microscope slide with the specimen within the stage clips on the fixed stage. Move the slide to center the specimen over the opening in the stage directly over the light source.

2. Raise the microscope stage up as far as it will go. Rotate the scanning lens or low-power lens into position. Lower the body tube with the coarse-adjustment knob to its lowest po​sition. Note: Never lower the body tube while looking through the ocular lens.

3. While looking through the ocular lens, use the fine-adjustment knob, rotating it back and forth slightly, to bring the specimen into sharp focus.

4. Adjust the substage condenser to achieve op​timal focus.

5. Routinely adjust the light source by means of the light-source transformer setting, and/or the iris diaphragm, for optimum illumination for each new slide and for each change in magnification.

6. Most microscopes are parfocal, which means that when one lens is in focus, other lenses will also have the same focal length and can be rotated into position without fur​ther major adjustment. In practice, however, usually a half-turn of the fine-adjustment knob in either direction is necessary for sharp focus.

7. Once you have brought the specimen into sharp focus with a low-powered lens, prepa​ration may be made for visualizing the speci​men under oil immersion. Place a drop of oil on the slide directly over the area to be viewed. Rotate the nosepiece until the oil-immersion objective locks into position. Note: Care should be taken not to allow the high-power objective to touch the drop of oil. The slide is observed from the side as the objective is ro​tated slowly into position. This will ensure that the objective will be properly immersed in the oil. The fine-adjustment knob is read​justed to bring the image into sharp focus.

8. During microscopic examination of microbial organisms, it is always necessary to observe several areas of the preparation. This is ac​complished by scanning the slide without the application of additional immersion oil. Note: This will require continuous, very fine ad​justments by the slow, back-and-forth rota​tion of the fine-adjustment knob only.

Diaphragm Opening

Objective

Working Distance

Scanning 4x

4x

Reduced

9-10 mm

Slide

Low power 10x

10x

Not fully opened

5-8 mm

Slide

High power 40x

40x

Not fully

opened

0.5-0.7 mm

J

Slide

Oil immersion 100x

100x

Fully opened

0.13-0.18 mm

Slide

Figure 4.3 Relationship between working distance, objective, and

Diaphragm opening

On completion of the laboratory exercise, re​turn the microscope to its cabinet in its original condition. The following steps are recommended:

1. Clean all lenses with dry, clean lens paper. Note: Use xylol to remove oil from the stage only.

2. Place the low-power objective in position and lower the body tube completely.

3. Center the mechanical stage.

4. Coil the electric cord around the body tube and the stage.

5. Carry the microscope to its position in its cabinet in the manner previously described.

AT THE BENCH

Materials

Slides

Commercially prepared slides of Staphylococcus aureus, Bacillus subtilis, Aquaspirillum iter-sonii, Saccharomyces cerevisiae, and a human blood smear.

Equipment

Compound microscope, lens paper, and immer​sion oil.

Procedure

1. Review the parts of the microscope, making sure you know the names and understand the function of each of these components.

2. Review instructions for the use of the micro​scope, giving special attention to the use of the oil-immersion objective.

3. Examine the prepared slides, noting the shapes and the relative sizes of the cells un​der the high-power (also called high-dry, be​cause it is the highest power that does not use oil) and oil-immersion objectives.

4. Record your observations in the Lab Report.

Observations and Results

Draw several cells from a typical microscopic field as viewed under each mag​nification, and give the total magnification for each objective.

Lab Report

High Power

Oil Immersion

S. aureus

Magnification

B. subtilis

Magnification

A. itersonii

Magnification

S. cerevisiae

Magnification

Blood smear

Magnification

Review Questions

1. Explain why the body tube of the microscope should not be lowered while you are looking through the ocular lens.

2. For what purpose would you adjust each of the following microscope components during a microscopy exercise?

a. Iris diaphragm:

b. Coarse-adjustment knob:

c. Fine-adjustment knob:

d. Condenser:

e. Mechanical stage control:

3. As a beginning student in the microbiology laboratory, you experience some difficulties in using the oil-immersion lens. Describe the steps you would take to correct the following problems:

a. Inability to bring the specimen into sharp focus.

b. Insufficient light while viewing the specimen.

c. Artifacts in the microscopic field.