Microscope and Specimen Preparation- Ch. 2

Question 1

light microscopy

Answer 1

microscope that uses visible light to observe specimens

Question 2

resolution

Answer 2

the distance by which two objects can be separated and still be distinguished

Question 3

properties of a Compound light microscope

Answer 3

• cannot resolve structures smaller than 0.2 micrometers, therefore, cannot be used to observe viruses
• commonly used to observe various stained (killed) specimens and to count microbes
• the specimen appears colored against a bright background (where as negative staining uses dark background)
• maximum magnification is about 2000X (200x oil immersion)

Question 4

Why use immersion oil?

Answer 4

• refractive index: measure of light-bending ability
• objective lens for oil immersion is small
• immersion oil has same refractive index as glass

Question 5

darkfield microscopy

Answer 5

• used for examining live microorganisms that do not stain easily, are distorted by staining, or are invisible in brightfield microscopy
• uses a special condenser with an opaque disc that blocks light from entering the objective directly
• specimen appears light against a black background
• can be used to detect Treponema pallidum, causative agent for syphilis (STI)

Question 6

fluorescence microscopy

Answer 6

• uses an ultraviolet source of illumination that causes fluorescent compounds to emit light
• some organisms fluoresce naturally (disadvantage)
• other organisms can be stained with fluorescent dyes called fluorochromes
• specimens appear as luminescent objects against a dark background

Question 7

Technique for preparing specimen to use fluorescence microscopy

Answer 7

1. Combine a fluorochrome to a specific antibody for a specific antigen
2. Allow the antibody to combine with the specific antigen
3. Wash to remove non-specific binding
4. the fluorochrome-antibody-antigen fluoresces under UV light

Question 8

electron microscopy

Answer 8

• uses a beam of electrons (shorter wavelength) instead of light
• structures smaller than 0.2 micrometers can be resolved (viruses, cellular structures like DNA)

Question 9

2 types of electron microscopy

Answer 9

1. transmission electron microscope (TEM)

2. Scanning electron microscope (SEM)

Question 10

transmission electron microscopy

Answer 10

-can resolve objects as close as 2.5 nm
-magnification is from 10,000X to 100,000X (total)
-only a very thin section (about 100nm) of a specimen can be studied effectively
can’t obtain 3D image
-Specimens must be fixed, dehydrated, and viewed under vacuum; therefore, shrinkage and distortion may occur; may get artifacts

Question 11

scanning electron microscopy

Answer 11

• the image is 3D
• it can study surface structures of intact cells and viruses
• resolves objects as close as 20 nm
• magnification is 1000X to 10,000X
• can only see external structure

Question 12

preparation of specimens

Answer 12

• most microorganisms appear colorless when viewed through a light microscope
• staining microorganisms with a dye makes them more visible
• prior to staining, organism must be fixed (attached) to a microscope slide (smear of microorganism is air dried and the heat fixed to denature protein to attach to glass slide

Question 13

Stains

Answer 13

• salts composed of a positive and a negative ion
• colored one is the chromophore
• bacteria are slightly negatively charged at pH 7.0

Question 14

2 types of dyes

Answer 14

1. basic dyes- have positive colored ions so binds to negatively charged bacteria (binds to the actual organism)
   
   • crystal violet (purple)
   • methylene blue (blue)
   • safranin (pink)
2. acidic dyes- have a negative colored ion so binds ot positively charged bacteria (binds to actual slide/background)

Question 15

Examples of acidic dyes

Answer 15

1. eosin
2. nigrosin (will use in lab)
3. India ink

Question 16

Simple stain

Answer 16

• an aqueous or alcohol solution of a single basic dye
• used to highlight the entire microorganism so that cellular shapes and structures are visible
• a mordant may be added to the simple stain to :
  
  1. increase the affinity of a stain for the specimen
  2. coat a structure (such as a flagellum) to make it thicker-so it assists in binding

Question 17

Examples of simple stains (4)

Answer 17

1. methylene blue (blue)
2. carbolfuchsin (red)
3. crystal violet (purple)
4. safranin (pink)

Question 18

Differential stains

Answer 18

react differently with different bacteria–> can be used to distinguish them

Question 19

types of differential stains (2)

Answer 19

1. Gram stain –> (+) vs. (-)

2. Acid-Fast stain –> acid-fast vs. non-acid fast

Question 20

Gram Stain

Answer 20

developed by Hans Christian Gram to differentiate/ classify bacteria into 2 large groups

Question 21

Gram-positive

Answer 21

-bacteria have a thicker cell wall composed of peptidoglycan than gram (-) bacteria

Question 22

Gram-negative

Answer 22

• bacteria have a thin layer of peptidoglycan and outside of that, a layer of lipopolysaccharide
  
  • outer layer gives cell extra protection and not easy for things to go into the cell– more resistant to antibiotics

Question 23

Gram stain procedure

Answer 23

• crystal violet and iodine go into the thick cell wall, but once inside it forms a complex (CV-I) which can’t get out
• gram (+) bact. stain purple b/c of the trapped CV-I complex
• gram (-) bacteria will be colorless b/c the alcohol wash disrupts the lipopolysaccharide and allow tthe CV-I complex to be washed out of cell
• gram (-) bact. are pinkb/c of counterstain with safranin
  
  • *Gram stain is most consistent when used on young growing bact (they make more peptidoglycan
• results provide better info on how to treat disease and generally gram (+) are easily killed with penicillin and sulfonamide drugs

Question 24

Acid-Fast Stain procedure

Answer 24

• stain binds to bacteria that have a waxy (mycolic acid) material in their cell wall
• used to identify all bacteria in the genus Mycobacterium
  (ex) . Mycobacterium tuberculosis cause