Lab Manual 1 - Lab safety and Microscopy PDF

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UNIVERSITI SAINS MALAYSIA BOI 207: GENERAL MICROBIOLOGY LAB PRACTICAL 1: LAB SAFETY AND MICROSCOPY Name: Matric number: Group:

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JERRY BEN

Section 1: Study the cartoon and answer the following questions: 1. List 3 unsafe activities shown in the illustration and explain why each is unsafe.

2. List 3 correct lab procedures depicted in the illustration.

3. What Sue should have done to avoid an accident?

4. What are three things shown in the lab that should not be there?

5. Compare Joe and Carl's lab techiques. Who is doing it the correct way?

6. Compare Luke and Duke's lab techniques. Who is following the rules?

7. What is Jerry doing wrong?

8. What is correct procedure that Ben should be following?

Section 2: Label the parts of the light micoscope:

Section 3: Procedure for looking at a slide. Each group will be provided with four bacteria slides (Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Herbaspirillum seropedicae). Look at the slides under microscope using procedures laid out below and draw the images of these bacteria according to the respective magnification. Always use this procedure when you look at a new microscope slide: 1. Turn the 4X objective lens into place. 2. Put the slide into the holder on the stage center the object to be seen over the opening in the stage. 3. Use the coarse adjustment knob to move the stage as close to the lens as it will go (a brake will prevent the slide from actually hitting the lens). 4. While looking,through the ocular, use the coarse adjustment knob to move the stage away from the objective lens until the image is in focus. 5. Adjust the iris diaphragm for the proper amount of light. 6. To increase magnification, slowly turn the 10X objective lens into place. 7. Use the fine focus knob to bring it into proper focus. Note: This microscope is parfocal. Parfocal means that when the image is in focus with one objective lens, it will be almost in focus at the next higher magnification. You should only need to make slight adjustments to bring it into perfect forcus. 8. Adjust the iris diaphragm for the proper amount of light. 9. To increase magnification further, slowly turn the 40X objective into place. Note: We sometimes use or make slides that have a thick specimen mounted on them. This may prevent you from using a higher power objective. You should watch from the side as you move the objectives into place to avoid hitting the lens on the specimen. This may damage the lens. 10. Use only the fine focus knob to focus. You should adjust the amount of light entering your specimen in order to form the best image possible. You will probably need to increase the amount of light as you increase magnification and decrease the amount of light as you decrease magnification. If the field is dark or the image very grainy, then increase the amount of light. If the image appears "washed out", reduce the amount of light reaching the specimen.

Section 4: Learn to calculate magnification. Total magnification is calculated by multiplying the magnification of the objective lens by the magnification of the ocular. Why does it work this way? A microscope requires two sets of magnifiers. On low power (10X), for example, the objective forms a primary image inside the tube that is 10X larger than the viewed object. The ocular (10X) then magnifies the primary image another 10 times. Thus, the image that finally reaches your eye has been enlarged to 100X the size of the object. Calculate the Total Magnification for each objective lens (4X, 10X, 40X and 100X) for a microscope with a 10X ocular lens. Record in the table. Ocular Magnification 10X

Objective Magnification 4X

10X

10X

10X

40X

10X

100X

Total Magnification

Diameter of Field (mm)

Working Distance (mm)

The following procedure shows you how to use the microscope and introduces you to some related concepts and terminology. 1. Take any slide that is provided to you. 2. Place the slide on the stage so that the “sample” is upright. Hold it in place with the clip on the microdrive. 3. Turn the 4X objective into place. Use the coarse focus knob to focus on the “sample”. 4. Adjust the lighting with the iris diaphragm. Note any change in the image. 5. Observe the space between the objective lens and the microscope slide. This is the Working Distance. Use a plastic ruler to measure this distance. Record in the table. Note: An object must be at this specific distance from the objective lens in order for its image to be in focus. 6. Draw the image of the “sample” that you see in the appropriate space below. Show its size relative to the size of the field. Note: The field is the area of the microscope slide that is visible through the microscope.

7. Question 1: Compare the image of the “sample” under microscope to the “sample” on the slide. How do they differ?

8. Use the stage microdrive to move the slide to the right and then left. Use the stage microdrive to move the slide away from you and then closer to you. 9. Question 2: Compare the movement of the image to the movement of the slide. Do they move in the same direction?

10. Re-center the “sample” in the field. Focus. Adjust the light. 11. Slowly turn the 10X objective lens into position. Take your eyes away from the ocular lens and look at the microscope from the side as you do this. 12. Focus carefully. The lenses are parfocal so little adjustment is needed. 13. Adjust the light. 14. Measure the Working Distance. Record in the table. 15. Question 3: Draw the image that you see.

16. Question 4: How does it compare to the first image that you drew? Is it bigger? Does it fill a larger portion of the field?

17. Re-center the “sample” in the field. Focus. Adjust the light. 18. Slowly turn the 40X objective lens into position. Take your eyes away from the ocular lens and look at the microscope from the side as you do this. 19. Note the tiny Working Distance. Make an estimate of this distance and record in the table. 20. Focus using the fine focus knob. 21. Adjust the light. 22. Question 5: Do you have to increase or decrease the light level?

23. Question 6: Draw the image that you see.

24. Question 7: How is the image of the “sample” under the microscope different from the actual “sample” mounted on the slide?

25. Let’s look at how light levels change with magnification. Question 8: Turn the 10X objective lens back into place. Does the field get brighter or darker?

Adjust the light so that the “sample” is visible but it is as dark as possible. Question 9: Turn the 40X objective lens back into place. Does the field get brighter or darker?

Question 10: How does the requirement for light change as total magnification increases?

Section 5: Measure the diameter of the microscope field. The Field is the circle of light that you see through the ocular. It is the portion of the microscope slide that you can see at one moment. It is useful to know the diameter of field because you can then estimate the size of objects you are viewing. Let’s measure the diameter of this field. 1. Lay a clear plastic ruler on the stage and measure the diameter of field at 40X and 100X total magnification. Record the values in the table to the nearest 0.1 mm. Use the data that you recorded in the table to summarize the trends that you observed. Answer the following questions. Question 11: Based on your observations, at which magnification (4X or 40X) will field diameter be largest?

Question 12: As you increase magnification, what happens to the working distance?

Question 13: At which magnification will you be able to see the largest part of your specimen?

Question 14: Draw the images of the remaining samples according to their respective magnification (4X, 10X, 40X and 100X)....