Scale. A Microscope s job in life. The Light Microscope. The Compound Microscope 9/24/12. Compound Microscope Anatomy

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1 The Study of Microbial Structure: Microscopy and Specimen Preparation Scale A Microscope s job in life 1.Magnify 2. Resolve ability to separate or distinguish between two points 3. Contrast How much or little an object stands out against a background The Light Microscope Two types Compound Stereoscope The Compound Microscope Compound Microscope Anatomy Magnifies 40 X X image formed by action of 2 lenses < 0.2 um is not a clear image 1

Discovery of Microorganisms Stereoscope Anton

describe microorganisms accurately Looked at

dissecting microscope 7 X -30 X View 3D image

Size of Objectives Occular will always be 10 X

2 Discovery of Microorganisms Stereoscope Anton van Leeuwenhoek first person to observe and describe microorganisms accurately Looked at pond water and saw Animacules Also called dissecting microscope 7 X -30 X View 3D image How can you determine total magnification? Size of Objectives Occular will always be 10 X Occular x objective = total magnification Scanning = 4 X Total magnification? Low = 10 X Total magnification? High = 40 X Total magnification? 10 x 4 = 40 X 10 x 10 = 100 X 10 x 40 = 400 X Working Distance Distance between the objective and the stage Working distance 2

Focusing summary 1. Clean the microscope with lens tissue 2. Start on the shortest objective (4x or scan) 3. Center slide 4. Use coarse adjustment to focus. 5.

3 Focusing summary 1. Clean the microscope with lens tissue 2. Start on the shortest objective (4x or scan) 3. Center slide 4. Use coarse adjustment to focus. 5. After focus is achieved move to the low objective. Use fine adjustment to focus. 6. After focus is achieved, move to the high objective. Use fine to focus. * Adjust light using the iris diaphragm as necessary. Light microscope measurements are in micrometers, or µm. There are 1,000 µm in one millimeter, or mm µm Guestimate the size of an object The field of view is the circle of white light you see when you look through the ocular This can be measured with a microscopic ruler or micrometer. 5.5 mm or 5500 µm FIELD OF VIEW The scanning power field of view is about 4500 µm How many stars would it take to cover the diameter of the field of view? Seven stars would do it. Divide seven into 4500 µm The star is about 630 µm An answer between µm is reasonable Guestimate the size of an object The low power field of view is about 1700 µm How many wood chips would it take to cover the diameter of the field of view? Four chips would do it. Divide four into 1700 µm The chip is about 425 µm The high power field of view is about 450 µm How many cells would it take to cover the diameter of the field of view? About 14 or 16 cells would do it. Divide 14, 15, or 16 into 450 µm The cell is about µm 3

The oil immersion field of view is about 170 µm How many cells would it take to cover the diameter of the field of view?

Divide five into 170 µm The cell is about 34 µm Little Tricky Suppose you know the field of view under scanning is 2 mm.

How big is the field of view if you move the objective to low power? Remember Scan = 40 X Low = 100 X High = 400 X 2.

5 of the original image 1/2.5 x 2 = 0.8 mm 10 X as high as scan Suppose you know the field of view under low is 10 mm.

4 The oil immersion field of view is about 170 µm How many cells would it take to cover the diameter of the field of view? Five cells would do it. Divide five into 170 µm The cell is about 34 µm Little Tricky Suppose you know the field of view under scanning is 2 mm. How big is the field of view if you move the objective to low power? The Facts Suppose you know the field of view under scanning is 2 mm. How big is the field of view if you move the objective to low power? Remember Scan = 40 X Low = 100 X High = 400 X 2.5 X as high as scan 4 X as high as low You move from scan at 40 X to low at 100 X That s a leap of 2.5 Your image than is 1/2.5 of the original image 1/2.5 x 2 = 0.8 mm 10 X as high as scan Suppose you know the field of view under low is 10 mm. How big is the field of view if you move the objective to high power? You move from low at 100 X to high at 400 X That s a leap of 4 Your image than is 1/4 of the original image 1/4 x 10 = 2.5 mm Depth of field What ever is in focus looks like it is on top. To tell top from bottom, you must focus through the layers. USE FINE ADJUSTMENT! 4

5 Depth of field Bizzaro World Colored threads e Objective You move to the right label Focal point coverslip slide e Field of view moves to the left Electron Microscopy To study objects 0.2 µm Uses beams of electrons rather than light, to produce images. The Scanning Electron Microscope Uses electrons reflected from the surface of a specimen to create image Produces a 3-D image Flea Fly head 5

6 9/24/12 Virus and Erythrocytes Microscopic terlit The Transmission Electron Microscope Ebola virus Electrons scatter when they pass through thin sections of a specimen Transmitted electrons (those that do not scatter) are used to produce image Preparation and Staining of Specimens Yeast spore Increases visibility of specimen Accentuates specific morphological features Preserves specimens 6

7 Differential Staining divides microorganisms into groups based on their staining properties Gram stain Acid-fast stain Acid-fast staining particularly useful for staining members of the genus Mycobacterium Gram staining Most widely used Divides bacteria into two groups based on differences in cell wall structure Gram - = pink Gram + = purple Escherichia coli a gram-negative rod Dyes and Simple Staining Make internal and external structures of cell more visible by increasing contrast with background Fixation process by which internal and external structures are preserved and firmly attached to microscope slide 7

Laboratory Techniques Cell culture Place a

$Bending of Light light is refracted (bent)$ $refractive index a measure of how greatly$

8 Laboratory Techniques Cell culture Place a single cell into a dish containing nutrient solution. Single cell reproduces into a gigillion cells Laboratory Techniques Cell Fractionation Separates different cell parts working distance distance between the front surface of lens and surface of cover glass or specimen Lenses and the Bending of Light light is refracted (bent) when passing from one medium to another refractive index a measure of how greatly a substance slows the velocity of light Figure 2.6 8

Scanning Probe Microscopy Lenses scanning tunneling microscope steady current (tunneling current) maintained between microscope probe and specimen up and down movement of probe as it maintains

9 EM Field of view is the white circle of light you see when you look in the ocular. If you know the diameter of the field of view, you can guestimate the size of a cell Count how many cells could fit across the field of view, and divide the field of view by that number. Scanning Probe Microscopy Lenses scanning tunneling microscope steady current (tunneling current) maintained between microscope probe and specimen up and down movement of probe as it maintains current is detected and used to create image of surface of specimen focus light rays at a specific place called the focal point distance between center of lens and focal point is the focal length strength of lens related to focal length short focal length more magnification What to listen for. Anatomy of a microscope Resolving power Van Leeuwenhoek Compound microscope Lens strength When to use coarse/fine adjustment Bizzaro world Guesstimate Electronmicroscopy SEM TEM 9

Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation

Chapter 2 The Study of Microbial Structure: Microscopy and Specimen Preparation 1 Lenses and the Bending of Light light is refracted (bent) when passing from one medium to another refractive index a measure