1. BIO244 Lab 1 Microscopy Students will need: microscope and cord (sign out number, use all semester) 2 sterile agar plates, stack and tape to keep lids shut 4 sterile swabs 1 vial sterile water 1 “e” slide 1 stage micrometer immersion oil: share one bottle per student pair lens tissue & methanol: share per student pair laboratory supplemental packet lab manual or photocopied pages BIO244 Microscope Worksheet Chapter 3 Notes outline

2. Microscopy Text book Chapter 3 Lab Notes

4. Light Microscope

6. http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/intro/illu/illu.html BrightfieldDarkfield Phase contrastD.I.C.

7. Fluorescence Microscopy

8. D.I.C. Confocal

9. Electron Microscopy

10. DNABacterial Surface Protein Scanned-Probe Microscopy

11. Positive StainingNegative Staining

12. Gram Stain Acid Fast Stain

13. Capsule Stain Special Stains

14. Lab 1 Goals and Objectives: Microscopy lecture: Text Chapter 3 Exercise 1: Brightfield Microscopy Learn how to use the microscope (guide in suppl. packet p.51) Diopter and interocular adjustments Light adjustments (iris diaphragm) Oil immersion Exercise 5: Microscopic Measurements Calibrate the ocular micrometer (suppl. packet p.52) Microscope Worksheet Measure the provided specimens Complete the microscope worksheet and turn it in Microscope Measurement Concept Practice Homework: complete measurement practice sheet in supplemental packet, self check: answers in packet (p. 53-54) Exercise 7: Ubiquity of Bacteria (Replacement activity in packet p. 55) Homework: “Testing Household Surface” handout, Take home plates, swabs, and sterile water READ DIRECTIONS CAREFULLY! then do experiment

15. Exercise 7: Ubiquity of Bacteria (Replacement activity in packet p. 55) Homework: “Testing Household Surface” handout, Take home plates, swabs, and sterile water READ DIRECTIONS CAREFULLY! then do experiment

16. Exercise 1: Brightfield Microscopy Learn how to use the microscope (guide in suppl. packet p.51) Diopter and interocular adjustments Light adjustments (iris diaphragm) Oil immersion

17. Exercise 5: Microscopic Measurements Calibrate the ocular micrometer (suppl. packet p.52)

18. 1.Make sure left ocular is turned fully counter-clockwise 2.Start with 10X objective 3.Get stage micrometer (2mm ruler on slide) in focus: you will see the ocular micrometer with labels in multiples of 10 (0, 10, 20, 30) and the stage micrometer with labels in multiples of 0.1 (0.0, 0.1, 0.2) stage micrometer ocular micrometer

19. 3. Line up the zero lines of the two micrometers (you can turn the top of the ocular to position the ocular micrometer and the mechanical stage controls to move the stage micrometer) At 100X total magnification they should line up as shown below “0.1” on stage micrometer is 0.1mm on that actual 2mm ruler. This distance corresponds to 10 “units” (spaces between lines) on the ocular micrometer (0-10): the 0.1 line of the stage ruler lines up with the 10 line on the ocular one. How long in millimeters is one unit on the ocular micrometer with the 10X objective in place?

20. 0.1mm = 10 “units” 1 unit = 0.1mm ÷ 10 1 unit = 0.01mm to convert mm to µm, multiply by 1000 When the microscope is set to 100X total magnification, one unit on the ocular micrometer is worth ________µm

21. 4. Repeat for the 40X objective:

22. 0.1mm = 40 “units” 1 unit = 0.1mm ÷ 40 1 unit = 0.0025mm to convert mm to µm, multiply by 1000 When the microscope is set to 400X total magnification, one unit on the ocular micrometer is worth ________µm

23. When the microscope is set to 100X total magnification, one unit on the ocular micrometer is worth ____10____µm When the microscope is set to 400X total magnification, one unit on the ocular micrometer is worth ____2.5____µm When the microscope is set to 1000X total magnification, one unit on the ocular micrometer is worth ____1____µm

24. Dinoflagellate 4Xobjective (40X) 10Xobjective (100X) 40Xobjective (400X) Czura 2005

25. Lilium Pollen 4Xobjective (40X) 10Xobjective (100X) 40Xobjective (400X) Czura 2005

26. Frog Blood 4Xobjective (40X) 10Xobjective (100X) 40Xobjective (400X) Czura 2005

27. 100X objective (1000X) 40X objective (400X) Streptococcus/Enterococcus faecalis Czura 2005

28. Dinoflagellate Frog Blood Lilium Pollen Streptococcus faecalis

29. Lab 1 Goals and Objectives: Microscopy lecture: Text Chapter 3 Exercise 1: Brightfield Microscopy Learn how to use the microscope (guide in suppl. packet p.51) Diopter and interocular adjustments Light adjustments (iris diaphragm) Oil immersion Exercise 5: Microscopic Measurements Calibrate the ocular micrometer (suppl. packet p.52) Microscope Worksheet Measure the provided specimens Complete the microscope worksheet and turn it in Microscope Measurement Concept Practice Homework: complete measurement practice sheet in supplemental packet, self check: answers in packet (p. 53-54) Exercise 7: Ubiquity of Bacteria (Replacement activity in packet p. 55) Homework: “Testing Household Surface” handout, Take home plates, swabs, and sterile water READ DIRECTIONS CAREFULLY! then do experiment