Microscopy lecture: Text Chapter 3 (supplement pg 39) Lab 2 Goals and Objectives: Microscopy lecture: Text Chapter 3 (supplement pg 39) Exercise 1: Brightfield Microscopy Learn how to use the microscope (guide in supplement pg 41) Diopter and interocular adjustments Light adjustments (iris diaphragm) Oil immersion Exercise 5: Microscopic Measurements Calibrate the ocular micrometer (supplement pg 42) Microscope Worksheet (supplement pg 43) 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 supplement pg 45-46

Microscopy Text book Chapter 3 Lab Notes Supplemental Packet page 39

Light Microscope

Brightfield Darkfield Phase contrast D.I.C. http://www.microscopy-uk.org.uk/index.html?http://www.microscopy-uk.org.uk/intro/illu/illu.html

Fluorescence Microscopy

D.I.C. Confocal

Electron Microscopy

Scanned-Probe Microscopy DNA Bacterial Surface Protein

Positive Staining Negative Staining

Gram Stain Acid Fast Stain

Capsule Stain Special Stains

BIO244 Microscopy Students will need: microscope and cord (sign out number, use all semester) 1 “e” slide 1 stage micrometer immersion oil: share one bottle per student pair lens tissue & methanol: share per student pair lab manual or photocopied pages (Exercises 1 & 5) laboratory supplemental packet: Guide to Using the Microscope (page 41) BIO244 Microscope Worksheet (page 43) Measurements using the Ocular Micrometer (page 42)

Exercise 1: Brightfield Microscopy Learn how to use the microscope (guide in supplement pg 41) Diopter and interocular adjustments Light adjustments (iris diaphragm) Oil immersion

Exercise 5: Microscopic Measurements Calibrate the ocular micrometer (supplement page 42)

Make sure left ocular is turned fully counter-clockwise Start with 10X objective (find it with 4X objective first!) 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

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?

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

4. Repeat for the 40X objective:

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

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

Dinoflagellate 4Xobjective (40X) 40Xobjective 10Xobjective (400X) Czura 2005

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

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

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

Lilium Pollen Dinoflagellate Coccus Bacteria Frog Blood

Microscopy lecture: Text Chapter 3 (supplement pg 39) Lab 2 Goals and Objectives: Microscopy lecture: Text Chapter 3 (supplement pg 39) Exercise 1: Brightfield Microscopy Learn how to use the microscope (guide in supplement pg 41) Diopter and interocular adjustments Light adjustments (iris diaphragm) Oil immersion Exercise 5: Microscopic Measurements Calibrate the ocular micrometer (supplement pg 42) Microscope Worksheet (supplement pg 43) 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 supplement pg 45-46