Microscopy and Specimen Preparation

Question 1

What is light microscopy?

Answer 1

compound light microscopes use visible light to illuminate specimens

Question 2

What are the types of light microscopy?

Answer 2

• bright field
• dark field
• phase contrast
• fluorescence

Question 3

What is the refractive index?

Answer 3

• describes how much light is refracted (bent) when passing from one medium to another
• light rays bend towards the normal once they pass through the lens

Question 4

What focal length (f) gives you greater magnification?

Answer 4

shorter focal length (distance between the normal point and the focal point (F))

Question 5

How are specimens visualized in Bright-Field microscopy?

Answer 5

due to differences in contrast (density) between specimen and surroundings

Question 6

How is total magnification calculated?

Answer 6

magnification of ocular lens x magnification of objective lens

upper limit for Bright-Field= 1000x

Question 7

What is resolution (d)?

Answer 7

the ability to distinguish between 2 objects as distinct and separate when viewed under a microscope

d= 0.5(λ)/n(sinθ)
d= 0.5(λ)/NA

upper limit for Bright-Field= 0.2 micrometers

Question 8

What are ways to achieve a better resolution?

Answer 8

• use a shorter wavelength
• decrease the working distance (bring objective lens closer to specimen)
• increase refractive index (n)

Question 9

What is the numerical aperture (NA)?

Answer 9

• a measure of light gathering ability

- larger=higher resolution

Question 10

What does fixation do?

Answer 10

• preserves specimens and fixes them in position

- organisms are usually killed and firmly attached to the microscope slide

Question 11

What are the two types of fixation?

Answer 11

• heat fixation: routinely used for bacteria and archaeons

- chemical fixation: used with larger, more delicate organisms

Question 12

What does staining do?

Answer 12

makes internal and external structures of a cell more visible my increasing contrast with the background

Question 13

What are the two common features of stains?

Answer 13

• chromophore groups: chemical groups with conjugated double bonds, gives color
• the ability to bind cells

Question 14

What are basic stains and what do they bind to?

Answer 14

• dyes with positive charges

- bind to negatively charged structures: nucleic acids, proteins, surfaces of bacteria and archaea

Question 15

What are acidic stains and what do they bind to?

Answer 15

• dyes with negative charges

- bind to positively charged structures

Question 16

What is simple staining?

Answer 16

• the use of a single stain

- for visualization of morphological shape and arrangement

Question 17

What is differential staining?

Answer 17

• the use of two contrasting strains separated by a decolorizing agent
• can be used for identification: gram stain, acid fast stain
• -presence or absence of structures
• can be used for visualization of structure: spore stain, capsule stain (can be simple or differential)

Question 18

What are the steps of the Gram stain?

Answer 18

1. Flood the heat-fixed smear with crystal violet for 1 min
2. Add iodine solution for 1 min
3. Decolorize with alcohol for 20 sec
4. Counterstain with safranin for 1-2 min

Question 19

What color are gram-negative and gram-positive cells after flooding with crystal violet?

Answer 19

both purple

Question 20

What color are gram-negative and gram-positive cells after being treated with iodine?

Answer 20

both purple

Question 21

What color are gram-negative and gram-positive cells after decolorizing with alcohol?

Answer 21

• gram-negative is colorless

- gram-positive is purple

Question 22

What color are gram-negative and gram-positive cells after counterstaining with safranin?

Answer 22

• gram-negative is pink

- gram-positive is purple

Question 23

What does the Gram stain divide bacteria based on?

Answer 23

• differences in cell wall structure
• gram-positive cells have a thick layer of peptidoglycan and no outer membrane
• gram-negative cells have a thin layer of peptidoglycan with an outer membrane

Question 24

What is the acid-fast stain used for?

Answer 24

• useful for staining members of the genus Mycobacterium that don’t stain well with the Gram stain
• acid-fast cells have high lipid (mycolic acid) content in their cells walls which is responsible for their staining characteristics
• wax-like, impermeable cell walls

Question 25

What do the colors represent in acid-fast staining?

Answer 25

• pink=acid-fast cells

- blue=non acid-fast cells

Question 26

What is endospore staining?

Answer 26

• heated, double staining technique
• bacterial endospores are stained green (malachite green) and vegetative cells are stained pink (safranin)
• the heat is used as the mordant
• bacteria with endospores are harder to stain because of their layers of protein

Question 27

What is capsule staining?

Answer 27

• uses a negative stain (colors the background)
• the capsules are colorless against a stained background (look like a white halo)
• capsules are made out of sugar and therefore do not pick up the stain

Question 28

What is flagella staining?

Answer 28

• mordant is applied to increase the thickness of the flagella so that it can be viewed under the microscope
• flagella are fragile and difficult to stain
• uses basic stain

Question 29

How are specimens prepared and stained?

Answer 29

1. Spread culture in thin film over slide
2. Dry in air
3. Pass slide through flame to heat fix
4. Flood slide with stain; rinse and dry
5. Place drop of oil on slide and examine with 100x objective lens