4 Lab 4: Microscopy
Lab Objectives
After completing this lab, the student should be able to:
- Calculate total magnification of a specimen.
- Relate changes in field of view, working distance, amount of light needed, and magnification to image size and clarity.
- Examine microscopic specimens to determine cell shape and arrangement.
- Summarize the major differences between prokaryotes and eukaryotes and between fungi, algae, and protozoa.
Introduction
Microscopy is one of the key concepts in the field of microbiology. In this lab we will review the basic parts of a microscope, how to produce a good image, and how to proper use a microscope, include the oil immersion lens.
The Microscope and its Parts: Most microscopes that are used in teaching labs today are compound light microscopes which indicates that the microscope contains at least two or three lens systems including the ocular lens, objective lenses, and the condenser lens. Below is a list of common parts found on most compound microscopes and their functions. Be sure to refer to the “Labeled Microscope” diagram provided on Canvas.
- Ocular lens (eyepiece) – the lens that the user looks through to view the specimen; this lens magnifies the sample 10x
- Microscope arm (body tube) – to properly carry a microscope, place one hand on the arm and a second underneath the microscope.
- Slide holder – the is a lever that acts to bracket the slide and allow for smooth, easy movement of the slide across the stage by the stage control (X-Y stage control) knobs.
- Coarse focus knob (coarse adjustment knob) – controls movement of the stage up and down such that large height changes are produced quickly. The course adjustment knob should only be used with the 4x objective lens.
- Fine focus knob (fine adjustment knob) – controls movement of the stage up and down such that small height changes are produced. The fine adjustment knob can be used with any objective lens though is favored for use with the 10x, 40x, and 100x objectives.
- Power switch (on-off switch) – the switch that turns on the microscope and may be combined with the rheostat which increases the amount of light produced by the lamp. Power switches are located at the base or the lower center back of most microscopes.
- Illumination (lamp) source – the light source for a microscope. Light intensity is controlled by the rheostat and the Iris Diaphragm.
- Iris Diaphragm – the lever located underneath the Condenser that regulates the amount of light that reaches the condenser lens and thus the specimen.
- (Abbe) Condenser – a lens that focuses light on the specimen. The condenser is located just underneath the stage.
- Objective lenses – a series of lenses (usually 4) that provide the majority of specimen magnification.
Table 1: Lens name, color and magnification for common used objective lenses.
|
Objective Lens
|
Color
|
Magnification
|
|
Scanning
|
Red |
4x |
|
Low power
|
Yellow |
10x |
|
High dry
|
Blue |
40x |
|
Oil immersion
|
White |
100x |
- (Revolving) Nosepiece – the part of the microscope that holds the objective lenses.
Microscopic Images: When viewing a specimen under the microscope, it is important to remember four terms that will help to produce a good image – light, resolution, magnification, and working distance.
- The circle where the specimen is seen through a microscope is termed the field of view and it changes as magnification and working distance change.
- Light is going to be key in producing a good image. As a specimen is viewed through the field of view, the light should not be too bright (it can cause damage to the eyes) or too dark (poor resolution and contrast). Adjust the rheostat to provide sufficient light for create contrast between the specimen and the background. The amount of light necessary to create contrast will vary between slides, especially when chromophores (charged ions that make stains) are used which further help to create contrast.
- Resolution is the ability to distinguish two cells that are spatially close to each other as separate objects. Each type of microscopy will have limits to resolution based on the illumination source. Light microscopes have lower resolution that electron microscopes. Resolution can be increased by creating contrast through the manipulation of light and staining specimens.
- Magnification refers to the visual increase in size of an image and is presented by the unit “x” (or “X”). Each lens contributes to image magnification. When a microscope uses more than one lens system, each lens’s contribution to image size must be considered using the equation:
Total Magnification = Ocular Magnification * Objective Magnification
- For the microscopes that are used in our lab, the ocular provides 10x magnification and the objective magnification will range from 4x to 100x.
- Working distance describes the space between the stage and the objective lens. The higher the magnification of the objective lens, the longer the objective lens will be thus the shorter the working distance. Care must be taken when working with the longer 40x and 100x objectives.
The oil immersion lens represents a special case in combatting the problems associated with microscopy. First the lens itself is so long and has such as small opening that not much light can actually pass through the sample and be collected by the lens thus oil immersion lenses often require an increase in the amount of light used to view the specimen and create the necessary contrast to produce a good image. Second, the glass of the microscope will bend light from the illuminator differently than the air which causes problems with light scattering. To decrease light scatter and thereby increase the likelihood of light reaching the oil immersion lens, a drop of immersion oil is added to the specimen before moving the oil immersion objective into position. This creates a continual “lens system” from the glass of the microscope slide, the oil, through the oil immersion lens so that most of the light is bent into the oil immersion objective. Some microscope may be use a blue or green filter over their illuminators to help reduce light refraction but the use of such filters is not always necessary.
In this lab we will be viewing images of both eukaryotes and prokaryotes. Instructions for how to use the microscope are found in the Methods section of the lab. Be sure to read through the protocol and ask your lab instructor if you have any questions.
Method (Lab@Home)
For this lab you will need:
Compound light microscope
Colored pencils
Prepared slides
Blank slides
Toothpicks
Bacterial colony
Instructions for Microscope Observation of Prepared Slides
Before beginning this lab exercise, you should thoroughly wash your hands with warm soap and water and wipe down your bench using the provided disinfectant.
1. Prepare the lab work space by taking out the materials used in today’s lab
___ Prepared slides ___ Microscope
___ Colored pencils
2. Turn on the Microscope
- Depending on your microscope, this may be different than instructions described here. These instructions are for the IQCrew Microscope.
- Add batteries (provided with microscope kit) to the base of the microscope. You may need a small star end screwdriver to remove the base plate. Once batteries have been added, return the base plate and tighten the screws.
- Add one of the prepared slides to the stage of the microscope.
- Flip the microscope mirror up to where the light shine through the condenser.
3. Observe Eukaryotic Slides
Begin by viewing the eukaryotic slides as the specimens are easier to find and can be clearly seen on either at 300x or 600x total magnification (30x or 60x objective lenses).
- Take a slide from the prepared slide case. Be sure that the stage clips rest on top of the slide.
- Begin by making sure the 30x objective is in position on the revolving nosepiece. Raise the stage with the coarse adjustment knob as far up as possible. Slowly rotate the course adjustment knob in the opposite direction while looking through the ocular lens until the image comes into view.
- Rotate the 60x objective lens into position using the coarse adjustment knob to make the image clear.
- Record your field of view in the provided circles on the Results page. Be sure to label which specimen you are drawing in each circle.
- View these slides:
- Broad bean leaf
- Cotton stem
- Goldfish scale
- Apple
- Onion bulb epidermis
- Note: If you are using a different microscope, then other prepared slides may have been included. It is fine to substitute the prepared slides in your microscope kit but please update your Results section to reflect what you are drawing.
4. Observe Prokaryotic Slides
Prokaryotic slides often require the use of the 100x oil immersion objective lens and immersion oil. Because our microscope kits do not have an oil objective, we will be using the 120x provided objective and a condenser filter to provide contrast.
- Take a blank slide from the slide box.
- Add a small drop of water to the slide.
- Using a clean toothpick, mix a small amount of bacteria from one of your petri plates into the drop of water.
- Set the slide aside until it has completely air dried.
- Begin by making sure the 30x objective is in position on the revolving nosepiece. Raise the stage with the coarse adjustment knob as far up as possible. Slowly rotate the course adjustment knob in the opposite direction while looking through the ocular lens until the image comes into view.
- Rotate the 60x objective lens into position using the coarse adjustment knob to make the image clear.
- Rotate the 120x objective lens into position and carefully adjust the image using the coarse adjustment knob to make the image clear. Note that you may need to adjust the condenser filters (located under the stage) to create greater contrast with the microscope.
- Record your field of view in the provided circles on the Results page. Be sure to match the correct organism to its circle. Note that all bacterial specimens with the exception of “Algae Blue Green” or “Cyanobacteria” will be viewed with the 100x objective.
- View these slides:
- Prepared bacterial slide
Lab clean up
Once you have completed the lab be sure to:
- Clean your bacterial slide with warm soapy water
- Return the slides to the slide box and put away slide box, color pencils, and microscope
- Disinfect your bench.
- Wash your hands.
Results
In the section below, draw example organisms in your field of view. You do not have to fill up the field of view, only communicate what you are observing. Be sure to include total magnification, any necessary labels, and match the correct organism to its location on the Results sheet.
Eukaryotic Cells
Slide: Broad bean leaf Cotton stem Goldfish scale
Total Magnification ________ ________ ________
Organism: Apple Onion bulb epidermis Bacterial colony [Prokaryotic]
Total Magnification ________ ________ _______