Lab 2

Microscopy

Introduction: Connecting Your Learning

This lab exercise will introduce the microscope and microscopy (microscope techniques). The microscope is an important tool in the microbiology lab. The use of microscopes greatly enhances the study of microorganisms. This lab exercise will familiarize students with the compound microscope and how to use it to view bacteria and other microorganisms too small to be seen by the unaided eye.

Readings, Resources, and Assignments

Multimedia Resources	Microscopy Video
Required Assignments	Lab 2 Assignment

Focusing Your Learning

Course Competency

1. Utilize compound microscope to visualize microbial organisms.

As you watch the assigned Microscopy Video from above and work through this lab, consider the objectives below.

Lab Objectives

By the end of this lab, you should be able to:

1. Describe the difference between a compound, darkfield, and phase contrast microscope.
2. Identify the components, use, and care of the compound brightfield microscope.
3. Use the microscope for observation of microorganisms.

Background Information

Microscopy Video

Please watch and take notes on this video: Microscopy Video

Three types of light microscopes

The compound brightfield microscope is the most commonly used microscope in the laboratory. This type of microscope uses two lenses between the eye and the object to magnify the object. It also uses an illumination system to ensure adequate light is available for viewing dark objects in a bright field.

The beam of light focused on it by a condenser illuminates the specimen; the result is a specimen that appears dark against a bright background. Compound brightfield microscopes require a light source. The light is usually a built-in illuminator in the base of the microscope. The intensity of the light (how bright the light is) can often be adjusted. In brightfield microscopy, objects are dark, and the background is light. This type of microscopy can be used to view unstained living microorganisms in addition to killed, stained microorganisms on prepared slides. This type of microscope is used to study the size, shape, and arrangement of microbial cells, but it provides little information about internal cell structure.

A major limitation of this system is the absence of contrast between a living specimen and its surroundings, which makes it difficult to observe living cells. Therefore, most brightfield observations are performed on nonviable, stained preparations.

A brightfield image of unstained simple squamous epithelial cells. Notice that the cells are difficult to see against the background.

Creative Commons https://openstax.org/books/microbiology/pages/2-3-… is licensed under CC BY 4.0.

In addition to the brightfield microscope, there are several other microscopes used in the microbiology lab. In darkfield microscopy, the objects are light and the background is dark. Darkfield microscopy is similar to the light microscope; however, the condenser system is modified so that the specimen is not illuminated directly. The condenser directs the light so that the light is deflected or scattered from the specimen, which then appears bright against a dark background. This enables one to observe the shape and motility of unstained live organisms. Living specimens may be observed more readily with darkfield than with brightfield microscopy. Darkfield microscopy is the method of choice for viewing the bacterium that causes syphilis (Treponema pallidum), as this bacterium is not stainable with most conventional stains but can be observed in darkfield microscopy.

Creative Commons https://openstax.org/books/microbiology/pages/2-3-… is licensed under CC BY 4.0.

A darkfield microscope allows us to view living samples of the spirochete Treponema pallidum. Similar to a photographic negative, the spirochetes appear bright against a dark background. (credit: Centers for Disease Control and Prevention)

Creative Commons https://openstax.org/books/microbiology/pages/2-3-… is licensed under CC BY 4.0.

In phase-contrast microscopy, the organisms appear as degrees of brightness against a darker background. Its optics include special objectives and a condenser that makes visible cellular components that differ only slightly. As light is transmitted through a specimen, a portion of the light is bent due to slight variations in density and thickness of the cellular components. The special optics convert the difference between transmitted light and refracted rays resulting in a significant variation in the intensity of light and producing an image of the structure being studied. The image appears dark against a light background. The advantage of phase-contrast microscopy is that structural detail within live cells can be studied. Observation of microorganisms in an unstained state is possible with this microscope.

This figure compares a brightfield image (left) with a phase-contrast image (right) of the same unstained simple squamous epithelial cells.

Creative Commons https://openstax.org/books/microbiology/pages/2-3-… is licensed under CC BY 4.0.

Please review the flashcards below which summarize three types of light microscopes.

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Phase contrast microscope

Phase contrast microscopeStructures in the specimen create refraction and interference which results in high contrast. High resolution views can be obtained without staining. Useful to view living samples and structures like endospores and organelles.

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Brightfield microscope

Brightfield microscopeThe standard light microscope, most commonly used in labs. It produces a lighter background and a darker specimen. It is most useful for stained, prepared slides. Can be difficult to use for viewing living samples due to lack of contrast.

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Darkfield microscope

Darkfield microscopeStaining is not required, as contrast is increased to produce a bright image on a darker background. Very useful for viewing live specimens, and those that do not stain well.

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Phase contrast microscope

Phase contrast microscopeStructures in the specimen create refraction and interference which results in high contrast. High resolution views can be obtained without staining. Useful to view living samples and structures like endospores and organelles.

Brightfield microscope

Brightfield microscopeThe standard light microscope, most commonly used in labs. It produces a lighter background and a darker specimen. It is most useful for stained, prepared slides. Can be difficult to use for viewing living samples due to lack of contrast.

Flashcard 1 of 3

Flashcard Transcript

A tour of a light microscope

The basic frame of the microscope consists of a base, a stage to hold the slide, an arm for carrying the microscope, and the body tube for transmitting the magnified image. The stage is a fixed platform with an opening in the center that allows the passage of light from the illuminating 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. The stage will have clips attached to it to hold the slide in place for easy viewing and moving of the slide. The light source is located at the base of the microscope. Above the light source is a condenser, which consists of several lenses to collect and concentrate the light onto the slide by focusing the light into a small area. The condenser has an iris diaphragm, a shutter which controls the angle and amount of light concentrated onto the slide. Above the stage, is a revolving nosepiece that holds three or four objective lenses. Rotation of the nosepiece positions objectives above the stage opening. At the upper end of the body tube is an ocular or eyepiece lens. A microscope may be monocular, having one eyepiece, or binocular, having two eyepieces. When using a binocular light microscope, the user will need to adjust the two oculars closer together or farther apart as is appropriate to match the individual’s own interpupillary distance, which is the distance from the center of one pupil to the center of the other pupil. When the oculars have been correctly set to the user’s interpupillary distance, then the left and right fields of view will become one view when looking through the eyepieces.

By moving the stage closer to the objective lens using the adjustment knobs, one can focus the image first into relative focus and then into fine focus. The larger knobs, the coarse adjustment, are used to relatively focus the microscope starting with one of the lower magnification objectives (4x or 10x). The small knobs, the fine adjustment, are then used for fine focusing with the high-power objectives and the oil-immersion lens.

Image Hotspots Transcript

The magnification of the microscope depends on the type of objective lens used with the ocular lens. Compound microscopes usually have three or four objective lenses mounted on a nosepiece. The magnifications of the objectives are: 4x used for scanning; 10x for low power; 40x for high-dry power; and 100x for oil-immersion. The magnification of each objective lens is provided on the objective lens itself.

The total magnification of the microscope is calculated by multiplying the magnification of the objective lens with the magnification of the ocular lens. The magnification of the ocular lens is almost always 10x. The lens most often used in the microbiology lab is the oil-immersion lens. It has the highest magnification and must be used with immersion oil. Immersion oil is required when using the 100x objective because it reduces light refraction which increases the resolving power at this high level of light magnification. One common mistake encountered with first time microscopy users is selecting an incorrect objective lens for use with oil immersion, which can damage the lens. The photo below displays the damage caused to the 40x objective from immersion in oil.

A 40x objective damaged by immersion oil.

The microscope is a useful and important tool in the microbiology lab. Proper use and care of the microscope is very important. The following are general guidelines for use of the microscope and instructions for focusing objects on a microscope slide.

General Guidelines

1. Carry the microscope with both hands, making sure to support the base of the microscope.
2. Observe the slide with both eyes open to avoid eyestrain.
3. Always focus by moving the lens away from the slide. This will prevent both the lens and the slide from breaking.
4. When using the low-power objectives, the diaphragm should be barely open so that a small amount of light is used to create contrast. More light is needed with higher magnification.
5. Always focus with low power (4x or 10x) first.
6. Keep all objective lenses except the oil-immersion lens free of oil (reference the above photo).

Procedure: Label the Parts of a Brightfield Microscope Activity

Refer to the background reading above to complete the Brightfield Microscope Labeling activity. Select the “Check Latest Move” button to check your answers.

1. Ocular lens
2. Body tube
3. Objective lenses
4. Stage
5. Condenser
6. Iris diaphragm lever
7. Illuminator/light source
8. Base
9. Coarse focus knob
10. Fine focusing knob
11. Arm

Choices

CHECK LATEST MOVE CHECK WHOLE QUESTION RESET

Correct!

Record the correct names of Parts A through K in your Lab Notebook.

A.

B.

C.

D.

E.

F.

G.

H.

I.

J.

K.

How to Use a Brightfield Microscope

1. Place the microscope evenly on a benchtop.
2. Plug the microscope into an electrical outlet and turn it on. The power switch is usually located at the base of the microscope.
3. Place the slide with the specimen within the stage clips on the stage. Move the slide with the mechanical stage adjustment knob to center the specimen over the opening of the stage directly over the light source. Make sure the specimen on the slide is right side up.
4. Rotate the nosepiece until the 10x objective clicks (locks) into place straight up and down.
5. Adjust the eyepieces.
   • Look through the eyepieces and adjust the interpupillary distance between the eyepieces until one circle of light appears.
6. Raise the condenser up to the stage.
7. Adjust the coarse focus until the objects on the slide appear focused.
8. When an image has been brought into focus with low power, rotate the nosepiece to the next high magnification objective lens.
   • All of the objective lenses are parfocal. This means that when an image is in focus with one objective lens, it will focus with all of the other lenses with minimal adjustment from the fine focus knob when rotating from the in-focus objective to the next higher power objective.
9. When using the oil-immersion objective, be sure to keep the other objective lenses away from the immersion oil.
   • Move the 40x high-dry objective out of position, and place a drop of immersion oil on the area of the slide being observed. Place the oil-immersion objective into position.
10. When done, move the nosepiece to bring a low power objective into position, taking care not to rotate any of the objective lenses through the oil on the slide. Clean off the oil on the objective with lens paper. Remove the slide.

Procedure: Practice Using the Virtual Microscope

Go to the Virtual Microscope to learn more about using the Brightfield microscope and what objects look like when viewed through the microscope.

1. Utilize the VIRTUAL MICROSCOPE to view the letter “e,” and observe how the orientation of a specimen appears under light microscopy. When using the virtual microscope, one must complete the steps in the correct order. Failure to properly perform the steps in the necessary order will result in failure to complete subsequent steps and properly view the slide.
   • Turn on the light switch.
   • Get the slide tray.
   • Select the desired slide and place it on the mechanical stage.
   • Click on the stage clip to fasten the slide onto the microscope stage.
   • Adjust the interpupillary distance to ensure the specimen is viewed as one image.
   • Adjust the slide position so the student has a clear view of the desired specimen. Both the x-axis position and the y-axis position should be adjusted to proceed.
   • Adjust the iris diaphragm to regulate the amount of light that strikes the specimen and control the clarity and depth of field of the image.
   • Adjust the diopter until a clear image is obtained by compensating for differences between the vision of the left and right eyes.
   • Adjust the coarse focus knob to bring the specimen into relative focus.
   • Adjust the fine focus knob to bring the image into sharp, clear focus.
   • Adjust the objective magnification by clicking on the objective numbers on the microscope. Objective selections must proceed sequentially in either direction, without skipping any objectives. Note the total magnification when viewing through each different objective lens.
   • Once you’ve reached your final desired total magnification, click the ‘ok’ button. This will take you back to the main VM page and will reveal the ‘Slide Info’ button on the upper right. Please review the slide info to learn more about each assigned specimen.

1. Utilize the VIRTUAL MICROSCOPE to view a slide of colored threads. Be sure to view the slide under all available levels of magnification. Observe how the depth of field, the field of view, resolution, and contrast change at the different levels of magnification.

Assessing Your Learning

Warning: You are expected to submit your own, individual work. Using work completed by anyone other than yourself is plagiarism. This includes resources found on Internet sites. Posting assessments on an unauthorized website, soliciting assessment answers or the acquisition of assessments, assessment answers, and other academic material is cheating. Cheating and/or plagiarism will result in a failing grade for the course.

Assignments

Submit LAB 2.

Important information:

Copy and paste the list of Laboratory Exercise Questions into a Word document. Compose answers to these questions in the Word document and save the file as a backup copy in the event that a technical problem is encountered while attempting to submit the assignment. Make sure to run a spell check.

You will be submitting your answers to the lab assignment in two parts. The first part of the lab assignment consists of the laboratory exercise questions. The second part of the lab assignment is the application question. The first textbox on the submission page corresponds to the first part of the lab. Be sure to paste the laboratory exercise questions, with your answers, into this textbox. The second textbox on the submission page will be for your response to the application question.

NOTE: This lab assignment has questions related to binomial nomenclature. Please note that the assessment system does not accept formatted text, so you will need to indicate any italicized words by adding “IT” in parentheses after each word that is to be written in italics, (IT). For example, the common fruit fly is genus Drosphila and species melanogaster. When written, it is Drosophila melanogaster. From this example, the correct way to represent the italicized words is by typing, Drosphila (IT) melanogaster (IT).

Laboratory Exercise Questions

~~1. Identify the best choice of light microscope for viewing each of the following samples:

a. A preserved Gram-stained sample of Streptococcus (1 point)

b. Endospores of an unstained Bacillus sample (1 point)

c. A living sample where the microbiologist wants to observe the motility of the organism (1 point)

~~2. What is the purpose of each of the following parts of the microscope?

a. Condenser (1 point)

b. Objective lenses (1 point)

~~3. Name four important components of the microscope (besides those listed in Question #2). In your own words, describe the function of each component as covered in the video, and also in the lesson and lab. (4 points)

a.

b.

c.

d.

~~4. Why is immersion oil required when using the 100x objective in light microscopy? (1 point)

~~5. A Doctor suspects that her patient may have syphilis, caused by the spirochete Treponema pallidum. A specimen has been collected to confirm the diagnosis. Which type of microscope should be used to view this specimen? Describe why this choice is the best choice. (3 points)

~~6. If a light microscope is monocular, will it be necessary to adjust the interpupillary distance? Briefly justify your answer. (1 point)

~~7. From the “Procedure: Label the Parts of a Brightfield Microscope Activity”, located above, please identify the names of microscope parts that are labeled: B, C, E and I. (4 points)

a. Part B:

b. Part C:

c. Part E:

d. Part I:

~~8. If you are getting 200x magnification with a 25x power objective, what is the magnification of the ocular? Show how you derived your answer. (3 points)

~~9. What does parfocal mean, and why is it a useful and desirable feature of a microscope? (3 points)

Application Question

~~10. How might the information gained from this lab pertaining to microscopy be useful to you as a healthcare professional? (6 points)

Key components of critical thinking and application include the following:

1. Demonstrates application and comprehension of the scientific principles. (40%)
2. Displays competence in applying scientific knowledge to your professional life. Relevant content is supported by facts, data, and detailed examples. (40%)
3. The application paragraph is organized and structured. (10%)
4. Use of accurate scientific terminology. (10%)

Critical Thinking and Application of Information	0%	1-59%	60-89%	90-100%
Is your application a detailed description of how the lab content is relevant to your life?	Application did not adequately demonstrate application or comprehension of the scientific principles. Did not include detailed examples, facts or data. Or the application was not included.	A few areas of the application demonstrated some application and comprehension of the scientific principles by applying the knowledge to the student’s personal and professional life, but lacked detailed examples to support the content provided. Application demonstrated some organization and structure within the paragraph.	Most areas of the application demonstrated evidence of critical thinking and comprehension of the scientific principles. Displayed good competence and the relevant content was supported with good use of examples that apply the concepts and describe how the information will be relevant and useful to the student’s personal or professional life. The application paragraph is primarily presented in an organized and structured manner.	Application included a complete and detailed description of how the concepts are relevant and useful or applicable to the student’s personal or professional life. The application includes detailed examples and reveals insight into the scientific principles. The application paragraph maintains a strong sense of purpose and organization throughout.

Summarizing Your Learning