Microscopy and Cytology

p 73

Compound Light Microscope

• Review proper use and care of microscope by watching video.

• Principles of microscopy p 78

A.Magnification

• Factor by which specimen is enlarged

B. Parfocal and parcentric

• Image will remain in focus and centered as you switch from one objective to the next.

C. Size of microscopic field

• Using a ruler, you will measure the diameter of the field using 4X scanning objective.

• Your measurement will be somewhere near 4.2 mm

• Ruler is in mm. Convert to micrometers

• 4200 micrometers (um)

• Formula to determine unknown field of view if field of view of scanning objective is known.

40 X X 4200 um

Total magnification of unknown field

Field of view of scanning objective

Total mag with scanning objective

40 X 4200 um

100Total magnification with low power objective

Field of view of scanning objective

Total mag with scanning objective

Answer: 1680 um

Calculating the field of view

Objective mag

Ocular mag

Total mag Diameter (mm)

Diameter (um)

Scan 4X 10X 40X 4.2 4200

Low 10X 10X 100X N/A 1680

High 40X 10X 400X N/A 420

Oil 100X 10X 1000X N/A 168

• If the size of bacterial cells is in the range of 1-10 micrometers, which objective should be used for viewing bacterial cells?

D. Depth of focus

Remember to focus on each individual thread to determine which is on top.

Defined as the vertical distance that an object remains in focus at one time.

E. Image orientation

• View the letter E slide

If you place the slide face up and right side up on the stage, how does the “e” appear when viewed through the microscope?

F. Resolving Power

• Degree at which 2 points on a specimen are seen as separate images

G. Contrast

• The degree to which details of a specimen stand out against the background

• Omit section “V. Other types of microscopes”

Microscopy

• Answer questions on microscopy on p 84-86.

• Label parts of microscope.

• You will need to know these concepts for the lab practical.

Part II:The Cell p 89

• Review information on cellular structures and organelles. This material will be a review of what you have covered in lecture.

• For the lab practical, I will only ask questions about the structures that we view in the lab.

Exercise 7.5 p 95

• Look over drawing and models of plant and animal cells.

• Concentrate on identifying these structures:– Nucleus, nucleolus, plasma membrane,

cytoplasm, chloroplast (in plant cells), cell wall (in plants), vacuole, cilia, flagella

Exercise 7.6 p 96

• A. Cyanobacteria-omit• B. Elodea leaf- prepare a wet mount, view and

sketch (use a drop of water, then add safranin)• C. Onion leaf- prepare wet mount using iodine.

Use a very thin section. Draw and label.• D. Stained cheek cell- prepare wet mount

stained with methylene blue. Draw and label.• E. Ear swab- omit

Cheek cells at 10X

Cheek cell at 1000X (using 100 X oil objective)

http://iws.ccccd.edu/jbeck/Cellsweb/Elodeacells10x.jpg

All photos by Jeff Beck, CCCCD unless otherwise noted.

http://commons.wikimedia.org/wiki/Image:Microphoto-cells-onion2.jpg

Onion cell

Exercise 7.7 Bacterial slides p.102

• You must use OIL IMMERSION objective to view bacteria.

• If you are not sure how to use oil, ask!• Find the area to view using scanning, 10X and 40X

objectives, then add oil and move to 100X objective.

• Always find the area with scanning objective first, then move to higher magnifications

• Detailed instructions for using the oil immersion objective http://biology.clc.uc.edu/fankhauser/Labs/Microscope/Oil_Immersion.htm

• View prepared slides of bacterial cells.

• Gram-stain– Purple- gram positive– Pink to red- gram negative

• Cell shapes– Coccus– Bacillus– Spirillum

Rods- bacillus

Spherical-coccus

Corkscrew- spirillum

p103

• Study drawing of bacterial cell

• Answer lab questions #2 and #3 on p 104-105.