Materials Included In Kit

Additional Materials Required

Prelab Preparation

At least two weeks prior to the lab, start the protozoan culture by filling a small jar, microaquarium or culture bowl with spring, mineral or aged tap water. Note: Tap water may be aged by allowing it to sit out in an open container for at least 48 hours. Add the packet of protozoa mix to the water and place in a location where it can sit undisturbed overnight while receiving normal room light and temperatures in the range of 65–75 °F. Boil the wheat grains by adding them to a beaker of boiling water for 10 minutes. After boiling, allow the wheat grains to sit in the water at room temperature overnight and add the seeds to the protozoan culture approximately 24 hours later. The culture population will change over time. Exact species and population vary.

Safety Precautions

Serious injury may occur if a microscope is dropped. Remind students to always carry microscopes with two hands—one hand on the arm and one hand underneath the base of the microscope. Have students wash their hands after completing the lab.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Slides and coverslips should be placed in a proper glass disposal container. Sodium chloride and sucrose may be disposed of in the trash according to Flinn Suggested Disposal Method #26a. The protozoan culture may be disposed of down the drain or saved for future activities.

Lab Hints

• Enough materials are provided in this kit for five classes of 30 students working in pairs, or for 75 groups of students (each prepared cork slide must be shared by three lab groups). This laboratory activity is intended to be reasonably completed in a 50-minute class period, however, Activity 4 may require additional time, dependent on the level of the class. The pre-laboratory assignment should be completed before coming to lab and the Microscope Worksheet should be completed during the lab.
• Never let students with eye infections use a microscope.
• To sterilize a microscope eyepiece, use a cotton swab with 70% isopropyl alcohol. Do not oversaturate the lens or allow alcohol to drip on other areas of the microscope. Wipe eyepiece with clean lens paper.
• If a stereoscope is available, have students locate protozoans under a stereoscope while pipetting a sample to view under the compound microscope. This helps ensure the presence of an organism in the sample and will allow students to view the differences between a compound microscope and a stereoscope. Keep the culture in a dim location, if possible.
• Methyl cellulose solution may be used to slow down protozoans, such as paramecium, and ease viewing under the compound microscope; a 3% solution works well.
• Provide glass disposal containers for slides and cover slips.
• Never touch microscope lenses with anything except lens paper.
• For information on microscope maintenance, please contact Flinn Scientific to request Publication No. 10194 or go to the teacher resources section at www.flinnsci.com.
• http://biodidac.bio.uottawa.ca/ is a great online reference for more information and drawings of protists (Accessed May 2018).

Teacher Tips

• Additional activities involving the protozoan culture may include making wet mounts using samples from different locations in the culture container for comparison. Have students investigate which types of organisms tend to congregate in the corners, on the bottom, along the sides or in the center of the culture.
• Activity 2 may be done without the ruled stickers as a follow-up activity, requiring calculation of the size of the field of view (FOV) rather than using the mm squares as a guide. This is done by first dividing the magnification of the objective in use by four. This number obtained is referred to as the magnification factor (MF). The FOV diameter, which is a standard 4.5 mm under 4X, is then divided by the magnification factor (MF) to obtain the FOV for each objective lens.
  
  Example: An objective of 10X—(a) divide 10/4 = 2.5 (MF), (b) divide 4.5 mm/2.5 = 1.8 mm, this is the FOV for 10X objective.
  
  For an objective of 40X—(a) –40/4 = 10 (MF); (b) divide 4.5 mm/10 = 0.45 mm, this is the FOV for 40X objective.
  
  The formula for estimating the size of objects under a microscope for a particular objective is: d = D/X where
  

  d = size of the object in mm
  D = diameter of the FOV for the objective in use
  X = estimated number of granules that “fit” across the FOV diameter.

  Example: Sodium chloride under 10X—D = 1.8 mm and X = 40
  

  d = 1.8 mm/40 = 0.045 mm. The size of a salt crystal is then 0.045 mm or 45 μm (microns)

  A micron is 1000X smaller than a millimeter (mm). An object with the size just calculated—45 μm—is just slightly larger than the size of a single human cheek cell.

Answers to Prelab Questions

1. Define magnification.
   

   Magnification is defined in the Background section as the ratio of the size of the image of the object viewed through the lens to that of the actual size of the object. The total magnification of a sample may be found by multiplying the magnification of the ocular by the magnification of the objective in use. Accept all reasonable variations of this definition.

2. In what ways is the compound microscope used in this activity different from a stereomicroscope?
   

   Stereoscopes are primarily used to scan three-dimensional opaque objects in which light will not filter through, whereas the compound microscope requires light to pass through the sample in order for it to be visible.

3. Explain what is meant by a “field of view.”
   

   An area seen through the lens system—the circle in view through the eyepiece. The actual size of a field of view varies with the magnification setting.

4. You see a classmate carrying a microscope across the room holding it by the arm with one hand and swinging it back and forth. The teacher asks you to explain to the student how to properly transport a microscope and why proper handling is so important. Write what you would tell the student.
   

   Microscopes should always be carried with two hands, with one hand on the arm and one hand underneath the base of the microscope. It should be held comfortably at chest height so the microscope does not hit tabletops, chairs, or other students. The microscope should never be swinging; it must be held tightly to your body.

Sample Data

Activity 1. Becoming Familiar with the Microscope

Sketch the cork sample under 10X and 40X.

Activity 2. Estimating Crystal Size Salt


Activity 3. Preparing a Wet Mount with Newsprint


Activity 4. Finding and Identifying Microorganisms
(Student answers will vary—refer to the Protozoa Identification Key.)

Sketch and name three microorganisms located under the microscope.

Introduction

Microscopes are tools which allow the human eye to view tiny objects that would otherwise not be observable. Become familiar with the functional parts of the compound microscope and some common applications involved in slide preparation with this four-part activity.

Concepts

• Microscopy
• Slide preparation
• Basic usage and care of the microscope
• Observation

Background

Throughout recent history, microscopes have proven to be a vital instrument in scientific advancements. It is not clear who invented the microscope, however Robert Hooke is usually recognized as the creator of the first compound microscope in the 1600s. Hooke’s valuable discovery of cells in cork can be attributed to his makeshift compact microscope. His invention of the microscope was driven by a desire for a closer look at cork to figure out what properties made it so light, buoyant, and compressible. Under microscopic investigation, he found that cork, in fact, contained small chambers filled with air, which he called “cells” as they reminded him of the small confines of monasteries. The term microscope is derived from the Greek words micron, meaning small, and scopos, meaning aim. Microscopes allow scientists to observe samples in detail far exceeding the limits of the human eye. The study of this microscopic world is called microscopy. Organisms, such as bacteria, are not visible at all without the use of a microscope. There are many different varieties of microscopes exist ranging from simple magnifiers to very complex electron microscopes with the capacity to view objects at up to two million times their original size.

The most common laboratory scopes, the compound microscope (also referred to as light or compact microscope) and the stereomicroscope (also referred to as a dissecting microscope), have different uses. Compound microscopes have a glass lens contained in the ocular (the eyepiece typically has 10X magnification), and a lens in each objective (4X, 10X, 40X and 100X are the common objectives found in microscopes used in education, although not all microscopes have a 100X objective). Magnification is simply the ratio of the object size viewed through the lens to that of the actual size of the object. The total magnification of a sample is found by multiplying the magnification of the ocular by the magnification of the objective in use (see Figure 1).

Stereoscopes are primarily used to scan three-dimensional opaque objects in which light will not filter through. This activity focuses on the compound microscope only.

Microscopes always need to be carried with two hands—one hand on the arm and one hand underneath the base of the microscope. Hold it comfortably at chest height so the microscope does not hit tabletops, chairs or other students. Never swing the microscope—hold it to your body securely. Microscopes should be used on uncluttered laboratory tables or lab benches. The microscopes should be placed squarely on the flat surface, away from the edge of the table. Leave room for individuals to sit or stand comfortably around the microscope for viewing. After use, microscopes should be returned to their storage location.

All microscopes are slightly different in terms of objectives, mechanical stages, light regulators, and parfocal capabilities. It is important to become familiar with the particular microscope that you are working with. See Figures 2 and 3 to familiarize yourself with common terminology related to the compound microscope and definitions of the working parts.

Materials

Prelab Questions

1. Define magnification in your own words.

2. In what ways is the compound microscope different from a steromicroscope?

 

3. Explain what is meant by a “field of view.”

 

4. You see a classmate carrying a microscope across the room, holding it by the arm with one hand and swinging it back and forth. The teacher asks you to explain to the student how to properly transport a microscope and why proper handling is so important. Write what you would tell the student.

Safety Precautions

Serious injury and microscope damage may occur if a microscope is dropped. Microscopes should always be carried with two hands—one hand on the arm and one hand underneath the base of the microscope. Wash hands with soap and water after completion of the lab.

Procedure

Activity 1. Becoming Familiar with the Microscope

As mentioned in the Background section, Robert Hooke first used a compound microscope to investigate the unusual properties of cork. In this activity, prepared slides containing a thin cork sample will be used to practice focusing and to become familiar with the use of a compound microscope.

1. Obtain a prepared cork slide.
2. Turn on the microscope light source and then place the 4X objective in line with the eyepiece by turning the revolving nosepiece to the appropriate locked position.
3. Carefully place the prepared slide so that the cork sample is directly under the objective lens and light is shining through the sample. Note: Microscopes will vary. Some may have stage clips that hold down the slide by placing the slide underneath the metal clips, whereas other microscopes may have a mechanical stage with a slide clip that rests on the side of the slide rather than on top of it. Consult your instructor for assistance if needed.
4. Use the coarse focusing knob to raise the stage as close to the objective as possible.
5. While looking through the ocular, use the coarse focusing knob with the 4X objective still in place, bring the image of the cork sample into focus. The diaphragm may also need to be adjusted to achieve the proper amount of light.
6. Switch to the 10X objective lens. You will feel it click indicating that the objective is properly in position.
7. Adjust the focus using the fine focusing knob only. Record your observations on the Microscope Worksheet.
8. Switch to the 40X objective lens.
9. Adjust the focus using the fine focusing knob only. Sketch the observations on the Microscope Worksheet.
10. Turn the revolving nosepiece back to the 4X objective. Lower the stage completely. Remove the slide from the stage.

Activity 2. Crystal Size Estimation

1. Obtain a glass microscope slide and a ruled microscope slide sticker.
2. Carefully peel the backing from the sticker and center it with the sticky side down on top of the slide. Place the sticker as evenly as possible onto the slide and smooth it down with your finger.
3. Using a scoop, place a few granules of sodium chloride onto the ruled section of the sticker.
4. Place the slide onto the microscope stage. With the 4X objective in place, taking care not to tip the slide and spill the salt.
5. Pick out a single salt crystal to focus using the 4X objective. Sketch the magnified granule on the Microscope Worksheet.
6. Keeping in mind that each square on the ruled slide sticker is one square millimeter, estimate the size of the salt crystal in mm². Record your estimate on the Microscope Worksheet under Trial 1, Activity 2.
7. Repeat step 6 for two more crystals of salt on the slide. Record the estimated sizes under Trial 2 and 3 on the Microscope Worksheet.
8. Carefully remove the slide with salt and pour the salt in the trash. Use care not to spill the granules on the lab bench or floor.
9. Repeat steps 3–8 using sucrose crystals.

Activity 3. Preparing a Wet Mount with Newsprint

1. Using scissors, carefully cut out a small letter “e” from newsprint. Note: Choose a small font size—do not use a letter from a title or headline.
2. Using a pipet, place a single drop of water in the center of the ruled glass slide. Note: Save the pipet for Activity 4.
3. Using forceps, place the newsprint e on the drop of water.
4. Carefully lower the coverslip at an angle over the wet sample to minimize air bubbles.
5. Sketch what the newsprint e looks like with the naked eye on the Microscope Worksheet.
6. Place the slide on the microscope stage so that the letter e is right side up from your perspective. Note: Remember to ALWAYS have the 4X objective in place and the stage lowered when putting a slide on or off the stage.
7. Raise the stage completely. While looking through the oculars, focus with the course focusing knob and adjust lighting using the diaphragm under the 4X objective.
8. Use the fine adjustment knob to bring the newsprint into sharp focus.
9. Sketch the image of the letter on the Microscope Worksheet.

Activity 4. Finding and Identifying Microorganisms Under the Microscope

1. Expel the air from a pipet and gently place the tip near the sides or bottom of the protozoan culture where the organisms tend to congregate. Release the bulb to draw a sample.
2. Add a drop of the protozoan culture from the pipet into the depression of a depression slide. Optional: Add a drop of methyl cellulose solution to the depression first if it is being used.
3. Carefully place the slide on the microscope stage.
4. Use the 4X objective to survey the protozoan sample.
5. Adjust the field of view to different depths in the sample—there are multiple fields of view since the sample is not flat as seen in previous activities.
6. Try to locate as many organisms as possible. Once and organism has been located, change the objective to 10X to get a closer look at the morphology of the protozoa.
7. Use the Protozoan Identification Key to identify the organism.
8. Sketch the organism and record the name on the Microscope Worksheet.
9. Find and sketch three different organisms. Three organisms may not be present in a single sample. If this occurs, rinse the depression slide and repeat steps 1–9.
10. Prepared cork slides and microscope slides may be stored or disposed of according to your teacher’s instructions. Excess sodium chloride and sucrose may be thrown away in the regular trash.

Student Worksheet PDF

10789_Student1.pdf