Lab 1:

The Laboratory Microscope

Information gathered by:
Dr. Chuks Ogbonnaya

 

Introduction

Microorganisms, as their name implies, cannot be seen with the naked eye.  Although they were observed as early as 1674 by Anton Van Leeuwenhoek using a simple, single-lens microscope, it was not until the development of the modern compound microscope that the real diversity of microorganisms became apparent.

There are two basic categories of microscopes: light microscopes and electron microscopes.  Light, or optical, microscopes require light waves to provide the illumination while electron microscopes use electrons to provide the illumination.  Light microscopes are used for most general laboratory work, while electron microscopes are used to view extremely small objects such as sub-cellular components or viruses. 

In each basic category of microscope, there are a variety of sub-types.  Light microscopes may be bright field, dark field, phase contrast, or fluorescence, while electron microscopes can be either transmission or scanning.  The most commonly used laboratory microscope is the bright-field microscope, so this lab will be concerned exclusively with bright-field microscopes. 

For this lab, you will need to use a microscope, which should be available at most wastewater treatment plants.  Read all of the introductory information carefully before beginning the exercises.  The first exercise is meant to familiarize you with the parts of the microscope.  Then you will learn to focus the microscope while viewing a sample of pond water.


 

 

Parts of a Microscope

The main parts of a bright-field microscope are shown by the diagram and table below.

 

 

 


Diagram of a microscope
PART
FUNCTION
Base Supports the microscope
Light Source Illuminates the object to be studied 
Iris Diaphragm Controls the light intensity on the object 
Condenser Concentrates light on the object 
Stage Platform which supports the slide containing the object to be studied 
Arm Carrying handle 
Coarse Adjustment Knob
Large outer knob which brings the image into rough focus 
Fine Adjustment Knob
Small inner knob which brings the image into clear focus 
Low Power Objective Magnifies the object ten times (10×
High Power Objective Magnifies the object 43 times (43×
Ocular Lens Magnifies the image produced by the objective lens ten times (10×
Revolving Nosepiece Holds the objective lenses and allows you to change directly from one objective to another without having to refocus 

Basically, the microscope consists of a support system, a light system, a lens system, and a focusing system.  Each of these systems works together to produce a magnified image of the specimen. 


Support System

The support system consists of the base, arm, and stage.  The base and arm are structural elements which hold the other parts of the microscope in place while the stage holds the slide.  Depending on the microscope, the slide can be positioned under two spring clips and moved by the fingers, or it can be held in place by a mechanical stage and moved by means of two control knobs.


Light System

The light system passes light through the specimen using the light source, the condenser, and the iris diaphragm.  In a bright-field microscope, an incandescent bulb is usually used as the source of illumination.  Light from the light source then passes through the condenser which focuses the light on the specimen.  An iris diaphragm is used to control the intensity, or brightness, of light which passes through the specimen, thus allowing the operator to adjust the intensity and achieve an optimum viewing contrast. 


Lens System

The lens system forms the actual image which you will see when you look through a microscope.  A typical compound microscope has two lenses - an objective lens near the specimen and an ocular lens at the top - each of which magnifies the image of the specimen by a certain amount. 

The ocular lens on most microscopes magnifies 10x (meaning that the image produced by the ocular lens is ten times as large as the specimen).  In contrast, the typical microscope has at least three objective lenses mounted on a revolving nosepiece to allow for different magnifications.  The low-power objective is the shortest and generally magnifies 10x; the middle-sized lens is the high-dry objective which usually magnifies between 40x and 45x; and the longest lens is the oil-immersion lens which usually magnifies between 97x and 100x. 

To determine the total magnification of the image, simply multiply the magnification power of the ocular lens by the magnification power of the objective lens which is being used.  For example, if you are using a high-dry objective with a magnification power of 40x, then the total magnification will be 10 × 40 = 400x.  Alternatively, using the 10x low-power objective, the total magnification would be simply 10 × 10 = 100x.  As you can see, you would use the higher power objective lenses to magnify smaller objects and the low-power objective lens to magnify large objects. 

There is a limit to the amount of useful magnification one can achieve with a light microscope.  The highest magnification which can be achieved without producing a poorly resolved image is known as the resolving power of the lens.  The resolving power is the shortest distance between two closely adjacent points which can be seen and is based on the wavelength of light used for illumination and on the nature of the lens.  Because of these constraints, the maximum resolving power normally possible with a light microscope is about 0.2 micrometers (um), or 1/100,000 inch.  Smaller objects can be viewed using an electron microscope. 


Focusing System

The final system at work in the microscope is the focusing system.  So far, we have learned how all of the components of the microscope are held together by the support system, how the light system sends light through the specimen, and how the lens system uses that light to magnify the specimen's image and transmit it to our eyes.  The focusing system adjusts the distance between the slide and the objective lens so that the image comes into focus. 

The focusing system consists of two knobs - the coarse adjustment knob and the fine adjustment knob.  When focusing, the operator first turns the coarse adjustment knob (which is the larger focus knob) in order to move the stage a large distance and bring the image into the focal plane of the objective lens.  At this stage, the image will be visible but fuzzy.  Then the operator turns the smaller knob, known as the fine focus knob, to fine tune the focus and to make the image sharply focussed. 



Care of the Microscope

Microscopes are delicate pieces of equipment, so you should follow a few basic rules to prevent damage to the microscope.  These rules are meant to prevent you from dropping the microscope, from damaging the lenses, or from storing the microscope improperly. 

Dropping a microscope can break the lenses or can alter the alignment of the lenses.  To prevent this damage, you should always carry the microscope with two hands - one hand under the base and the other hand on the arm of the microscope.  When using the microscope, keep the instrument at least six inches from the edge of the lab table and keep any excess electrical cord on the table top to keep the microscope from being pushed or pulled off the table. 

The microscope's lenses are very delicate and can easily be scratched or damaged by oils.  Lenses should be cleaned before and after each use with special lens paper.  (Cleaning with paper towels or cloth can damage the lenses.)  In addition, you should refrain from touching the glass lens with your finger to avoid depositing oils or scratching the glass. 

When using the microscope to view a specimen, you should follow common sense rules of behavior.  Do not tamper with any part of the microscope unless you understand its purpose.  A common mistake is to focus quickly while looking through the eyepiece of the microscope so that the objective lens bumps into the slide.  To prevent damage to the lens, you should always make large focus changes slowly while observing the movement of the objective lens from the side of the microscope.

Finally, the microscope should be stored carefully.  Unplug the electrical cord by pulling on the plug instead of the cord.  Remove oil from the oil-immersion objective using lens paper, then turn the nosepiece so that the low-power objective is in place.  Carefully lower the objective to its lowest position by turning the coarse adjustment knob.  Then store the microscope under a dust cloth.    


Exercise 1: Parts of the Microscope

Examine your microscope carefully.  Find the location of each of the parts listed below:


Exercise 2: Viewing Specimens

The purpose of this exercise is to view microorganisms found in pond water while learning to operate a microscope. 

Equipment
Procedure

1. Collect a jar of pond water containing microorganisms.  To ensure that you capture the largest number of microorganisms, do not simply scoop a jar of water from the center of a pond.  Instead, fill the jar partway with pond water and then squeeze water into the container from water plants or pond scum. 

2. Prepare a specimen of pond water.

Place a droplet on the slide.
a. Using the dropper, place a few drops of pond water onto the center of a clean, dry slide. 
Coverslip
b. Hold the side edges of the coverslip and place the bottom edge on the slide near the drop of pond water. 

Finished slide
c. Slowly lower the coverslip into place.  The water should spread out beneath the coverslip without leaving any air bubbles.  If air bubbles are present, you can press gently on the coverslip to move the air bubbles to the sides. 

3. Set up the microscope.

a. Remove the dust cover from the microscope.

b. Plug in the microscope. 

c.Turn on the microscope's light source. 

d. Place the condenser at its highest position and close the iris diaphragm somewhat to prevent overbrightness. 

e. If the microscope has two ocular lenses, adjust the width between the lenses to your eyes.  You will know the width is correct when you see one bright circle of light rather than two.

4. View the specimen with the low-power objective. 

a. Turn the nosepiece until the low-power objective locks into place.

b. Place the slide on the stage and center it over the condenser.

c. Looking from the side, turn the coarse adjustment knob until the low-power objective is in its lowest possible position. 

d. Looking through the ocular lens, slowly turn the coarse adjustment knob in the other direction.  This raises the low-power objective away from the slide.  Continue until a clear image appears. 

e. Slowly turn the fine adjustment knob until the object comes into sharp focus. 

f. Adjust the light for maximum contrast using the iris diaphragm. 

g. Move the slide around on the stage using your fingers or the control knobs until you find a microorganism. 

5. View the microorganism with the high-power objective. 

a. Center the microorganism exactly in the center of the field of view. 

b. Turn the nosepiece until the high-power objective locks into place.  At this point, the microorganism should still be roughly in focus.

c. Turn the fine adjustment knob slowly to focus the image.  (Never use the coarse adjustment when the high power objective is in place!)

d. Open the iris diaphragm until optimum contrast is achieved. 

e. Sketch a picture of the microorganism. 

6. If the microscope has an oil immersion objective, then view the microorganism using this lens. 

a. Turn the nosepiece so that no objective lens is directly over the slide.  Then place a small drop of immersion oil on top of the coverslip. 

b. Turn the nosepiece so that the oil immersion objective is locked into place over the specimen, making sure that the lens is immersed into the oil.

c. Slowly turn the fine adjustment knob until the image comes into focus.

d. Adjust the iris diaphragm to achieve maximum contrast. 

e. Sketch a picture of the microorganism. 

f. When you are finished, remove the oil from the oil immersion lens using lens paper. 

g. Remove the oil from the slide by wiping it gently with a paper towel.

7. Repeat steps 4, 5, and 6 until you have sketched at least five different microorganisms.

8. Turn off the microscope. 

a. Turn the nosepiece of the microscope until the low-power objective locks into place. 

b. Carefully lower the objective to its lowest position by turning the coarse adjustment knob.

c. Turn off the light source.

d. Remove your slide.  Clean the slide and coverslip with water. 

d. Unplug the microscope and store it under a dustcloth.


9. Identify each of your microorganisms using the Pond Identification Kit.