Lectures 5, 6, 7 (Chapter 3)

Question 1

The smallest object the human eye can see is about

Answer 1

0.2mm (half a grain of salt)

Question 2

Light microscopes can visualize a range of sizes from

Answer 2

200nm or .2um to 10mm

Question 3

1,000nm = ______ um

Answer 3

1

Question 4

1nm = _____ um

Answer 4

.001

Question 5

1 bacterium length

Answer 5

about 1 um long

Question 6

10mm = ______ cm

Answer 6

1

Question 7

Microscope with only one lens who was it used by?

Answer 7

simple microscope Anton Van Leeuwenhoek

Question 8

Light Microscopy and types?

Answer 8

-use of any kind of microscope that uses visible light to see specimens -brightfield -darkfield -phase-contrast -fluorescence -confocal

Question 9

Compound light microscope

Answer 9

-First used by Hooke -has 2 lenses

Question 10

Compound microscope parts

Answer 10

….add pic

Question 11

In a compound microscope, the image from the ________ lens is magnified again by the ________ lens

Answer 11

objective, ocular

Question 12

Total magnification =

Answer 12

objective lens x ocular lens

Question 13

resolution compound light microscope resolution?

Answer 13

ability to distinguish two adjacent objects as separate and distance -0.2um

Question 14

resolving power limited by wavelength. to improve resolving power a ______ wavelength of light is needed

Answer 14

shorter

Question 15

refraction in compound light microscope

Answer 15

the bending of light as it travels through substances with different densities

Question 16

what does immersion oil do

Answer 16

a drop of immersion oil that is placed on the slide, which has the same refractive index as the glass slide allows the light to continue in the correct pathway (prevents refraction) -oil immersion lenses (usually 100x) are used to minimize refraction -light rays are collected to increase clarity

Question 17

Bright Field Microscopy

Answer 17

•Dark objects are visible against a bright background •Light reflected off the specimen does not enter the objective lens •Best for fixed, stained specimens, specimens that absorb light •Image is dark and/or highly colored against a bright, often light gray/white background •Specimens are visualized because of differences in contrast (density) between specimen and surroundings •Compound microscope used

Question 18

Darkfield illumination

Answer 18

uses darkfield condenser that contains an opaque disk to examine unstained live specimens

Question 19

Phase contrast

Answer 19

way to observe live specimens, permits more detail to view internal structures

Question 20

Fluorescence

Answer 20

takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light (UV) and give off a light at a longer wavelength(visible).

Question 21

Confocal

Answer 21

microscopy illuminates each plane in a specimen to produce a three-dimensional image up to 100 µm deep and also uses fluorescence.

Question 22

Staining improves ______

Answer 22

contrast

Question 23

Positively charged dyes bind to negatively charged cell components:

Answer 23

membranes, nucleic acids (ribosomes), proteins, cell wall

Question 24

Examples of common stains are:

Answer 24

methylene blue, safranin, and crystal violet

Question 25

Types of stains:

Answer 25

simple, differential, special

Question 26

What do you use for a simple stain and Steps for simple stain:

Answer 26

-Use a single basic dye -A mordant may be used to hold the stain or coat the specimen to enlarge it I. preparing a smear - spread culture on thin film over slide, and allow to air dry II. Heat fix and III. then stain - flood slide with stain; rinse and dry III. view

Question 27

Separate bacteria into groups and examples

Answer 27

differential stain -acid fast stain -gram stain

Question 28

Differential Stain: The Gram Stain

Answer 28

Gram (+) vs Gram (-) Gram rxn can provide valuable info for treatment downsides: kills cells, distorts features

Question 29

Because peptidoglycan wall is negatively charged - gram positive stains what color

Answer 29

Gram positive: pepidoglycan wall stains blue/purple (dye will bind to all negatively charged areas in the cell)

Question 30

Differential Acid Fast Stain

Answer 30

-acid fast stain – for identification of mycobacteria and pathogenic Nocardia, which have a waxy lipid cell wall (mycolic acid). ♣Acid fast stains retain red/pink ♣Non-acid bacteria cells will be colorless – counterstain stains them blue

Question 31

Special Stains

Answer 31

♣Special stains – are used to color and isolate a specific part of a microbe, such as endospores or flagella. Stain special structures -Flagella: carbolfuschin -Capsules: Colloids (India Ink) – presence of a capsule is a means of determing an organisms’ virulence (note colorless capsules that surround the purple stained rods) -Endospores: Malachite Green

Question 32

How do unstained endospores appear?

Answer 32

refractive under the light (white hollow hole)

Question 33

Addition of alcohol is the critical step in gram staining b/c

Answer 33

-alcohol will cause the outer part of cell membrane of gram - bacteria to pull off (dehydrate) -gram (+) it will cause cell wall to shrink and will hold purple dye a lot tighter

Question 34

EM can be used to examine

Answer 34

objects smaller than about 0.2 um (which is the limit of resolution for a light microscope), such as viruses and macromolecules which are in the nm range.

Question 35

EM vs. Light Microscope

Answer 35

The source of illumination is visible light for a light microscope and an electron beam for an EM. Instead of using glass lenses, an EM uses electromagnetic lenses to focus a bean of electrons onto a specimen. Like light, free electrons travel in waves, however in EM, the waves travel in a vacuum as opposed to air.

Question 36

TEM

Answer 36

Transmission Electron Microscope (TEM) was the first type of Electron Microscope to be developed and is patterned exactly on the Light Microscope except that a focused beam of electrons is used instead of light to “see through” the specimen. Specimen must be prepared in ultra thin slices since electrons must pass through the sample (electron transparency). Incredible internal detail. Images produced by EM are always black and white, but they may be colored artificially to accentuate certain details.

Question 37

SEM

Answer 37

Scanning the beam of electrons across the sample. The SEM overcomes the sectioning/slicing problem associated with the TEM and produces a 3D-image surface of the specimen, and especially useful in studying the surface structures of intact cells and viruses. Best for viewing external structures.

Question 38

We saw in lecture 5 that the compound light microscope will increase out ability to see by about _______ fold. An EM gives us approximately another ______ fold magnification.

Answer 38

1,000, 1,000 - EM’s show us viruses and macromolecular structures

Question 39

EM vs. Light Microscopes

Answer 39