Chapter 4: Microscopy Staining and Classification

Question 1

centimeter

Answer 1

1 * 10^-2 (0.01)

Question 2

milimeter

Answer 2

1*10^-3 (0.001)

Question 3

decimeter

Answer 3

1*10^-1 (0.1)

Question 4

micrometer

Answer 4

1*10^-6 (0.000001)

Question 5

nanometer

Answer 5

1*10^-9 (0.000000001)

Question 6

wavelength of radiation

Answer 6

lambda

Question 7

magnification

Answer 7

-x factor
-the apparent increase in size

Question 8

resolution

Answer 8

-the ability to distinguish between two separate points that are close together

Question 9

contrast

Answer 9

-differences in intensity between two objects, or between an object and a background
-is important in determining resolution, staining increases contrast

Question 10

What type of microscope is used in school?

Answer 10

compound bright field

Question 11

Simple microscope

Answer 11

-microscope with only one lens for magnifying

Question 12

compound microscope

Answer 12

-microscope with two lenses for magnifying

Question 13

Ocular lenses

Answer 13

-lenses that are the eyepieces to see the specimen usually 10X

Question 14

objective lenses

Answer 14

-lenses that are optical elements closest to the specimen, comes in 4X, 10X, 40X, and 100X

Question 15

How do you determine total magnification using a compound microscope?

Answer 15

• you multiple ocular lens and the objective lens together

Question 16

Bright-field compound scope characteristics

Answer 16

-light rays pass through the specimen and into the objective lens
-specimen appears dark against light background
-oil immersion lens increases resolution b/c oil minimizes bending of light (refraction) and prevents damage to lens
-a condenser is used to direct light through the specimen and our scopes have an iris diaphragm to adjust the aperture
-light microscopes cannot resolve structures smaller or closer together than 200 nm b/c shortest wavelength of visible light is 400 nm

Question 17

Dark-field microscope

Answer 17

-best for observing pale objects
-only those light rays scattered by specimen enter objective lens
-specimen appears light against dark background

Question 18

Phase microscope

Answer 18

-treat one set of light rays differently from one another b/c light waves are 1/2 wavelengths “out of phase” which increases contrast = better resolution
-they re use to examine living organisms or specimens that would be damaged or altered by attaching them to slide or staining
-light rays in phase produce brighter image, while light rays out of phase produce darker
-two types are phase contrast-microscope and differential interference contrast microscope (Nomarski)

Question 19

Fluorescent microscope

Answer 19

-direct UV light at a specimen which radiates energy back as a longer, visible wavelength to increase resolution and contrast
-some cells and molecules are naturally fluorescent while others may be stained
-immunofluorescence is used to identify pathogens and locate and make a variety of proteins visible
-confocal scopes use fluorescent dyes but also lasers to take laser thin “slice images” of specimen

Question 20

Electron microscope

Answer 20

-electrons have wavelengths of 0.01 nm to 0.001nm, which provides greater resolving power and greater magnification
-electron microscope magnify objects up to 1,000,000X
-they provide detailed views of ultrastructure of bacteria, viruses, internal cellular structures, molecules, and large atoms
-electron microscopy is preformed in a vacuum so its only good for fixed (dead) specimens or non living objects
-two types Transmission electron microscope (TEM) and scanning electron microscope (SEM)

Question 21

Transmission electron microscope (TEM)

Answer 21

-internal details of cell or object

Question 22

Scanning electron microscope (SEM)

Answer 22

-external details of cell or object

Question 23

Simple stains

Answer 23

-only have 1 primary stain

Question 24

Gram stain procedure from a smear

Answer 24

1. Slide is flooded with crystal violet for 1 minute, then rinsed with water
   (all cells are purple)
   2.Slide is flooded with iodine (mordant) for 1 minute and rinsed with water
   (iodine acts as a mordant and all cells remain purple)
2. Slide is flooded with alcohol/acetone for 10-30 sec and rinsed with water
   (gram positive cells are purple, and gram negative are colorless)
   4.Slide is flooded with safranin for 1 minute, then rinsed with water
   (gram positive cells now purple, gram negative cells now pink)

Question 25

Acid-Fast stain (Ziehl-Neelsen) from a smear

Answer 25

-This is used to stain mycobacterium and norcardia species
1. Cover smear with tissue paper (kim wipe)
2. Flood the tissue with the primary stain of carbolfuchsin for (3-5 min) while warming with steam to drive stain through waxy layer into cell
3. Remove tissue with forceps, cool, the decolorize with concentrated HCL and alcohol for 30 sec to bleach background and non-acid cells
(acid cannot penetrate wax layer if you let it cool before applying)
4. Counterstain with methylene blue for 45 sec rinse with water
* pink cells have mycolic acid
*if you don’t let it cool the decolorizer will get rid of all the color

Question 26

Endospore stain

Answer 26

-used to stain bacillus and clostridium species
-Schaeffer- Fulton endospore stain
1. Flood tissue with malachite green and heat (3-5 min) to drive stain into the endospore
(heat is the mordant important to drive dye into the endospore)
2. Cool then decolorize with water
3. counterstain with safranin for 45 sec then rinse with water

Question 27

Negative stain and capsule stain

Answer 27

-acidic dyes like Eosin or Nigrosin stain the background but leave cells colorless b/c they are repelled by the negative charges on the cell’s surface
-the cells are often counterstained with a 2 primary basic stain to stain the cells when looking for bacterial capsule

Question 28

Flagellar stain

Answer 28

-flagella are difficult to stain so different than other staining
-stains most commonly used are pararosaniline and carbolfuchsin
-mordants like tannic acid and potassium alum are used
-these stains and mordants are used b/c they also increase the diameter of the small flagella

Question 29

Taxonomy

Answer 29

-consists of classification, nomenclature and identification

Question 30

Why do we need classification

Answer 30

-taxonomy enables scientists to organize large amounts of information about organisms and make predictions based on knowledge of similar organisms

Question 31

who provided systems that standardize the naming and classification of organisms based on characteristics they have in common?

Answer 31

Carl Linnaeus
-developed binomial nomenclature
-grouped similar organisms that can successfully interbreed into categories called species
-linnaeus goal was to classify and name organisms as a means of cataloging them while the major goal of modern taxonomy is to reflect phylogenetic hierarchy
-more recently greater emphasis placed on comparisons of an organisms genetic material led to add super taxons (DOMAINS) to the whitaker taxonomic hierarchy (Woese)

Question 32

What are the two kingdoms Linnaeus proposed

Answer 32

-plantae and animale

Question 33

How many kingdoms did Whitaker propose

Answer 33

• 5 kingdoms
  1. plantae
  2. animale
  3. protists
  4. fungi
  5. Bacteria

Question 34

What is phylogenetic hierarchy

Answer 34

tree that arranges species into groups based on their evolutionary origin and relationships. It uses hierarchy which smaller groups are placed within larger groups

Question 35

Domain review

Answer 35

-Carol Woese compared nucleotide sequences of small rRNA subunits b/c they rarely change
-proposed three domains (Archaea) (Eukarya) (Bacteria)
-cells in these three domains also differ in respect to many other characteristics than a nucleus vs nucleoid
1. Physical characteristics (size, shape)
2. biochemical test (carb usage, fatty acid composition, enzymes present)
3. serological test (antigen/antibody interaction)
4. phage testing (phage bacteria interaction by species)
5. analysis of nucleic acid (genome sequencing, GC ratio)

Question 36

King Philip Came Over For Great Spaghetti

Answer 36

DOMAIN
Kingdom
Phyla
Class
Order
Family
Genus
Species

Question 37

Dichotomous keys

Answer 37

-set of paired statements worded so that only one of two either/or, yes/no choices applies to any particular organisms
-a key directs a user to another pair of statements and eventually provides the name of organism by genus and species