Microscopy and Morphology

Question 1

4 types of light microscopes

Answer 1

bright-field
dark-field
phase-contrast
fluorescence

Question 2

What parts of the microscope are responsible?

Answer 2

ocular (eyepiece) and objective lens

Question 3

How to calculate magnification?

Answer 3

Total magnification = (objective) x (eyepiece)

Question 4

resolution

Answer 4

ability of the lens to distinguish between two objects that are close together (best light microscope resolution = 0.2 um)

Question 5

approximate size of viruses

Answer 5

0.01 - 0.1 um (need an electron microscope)

Question 6

bright-field microscopy

Answer 6

must heat-fix (kill) and stain bacteria
background appears light, while bacteria are the color of the stain

Question 7

dark-field microscopy

Answer 7

can look at live cells
light is refracted by the organism, then enters the objective lens
background appears dark, organism appears light

Question 8

phase contrast microscopy

Answer 8

usually live cells
phase contrast converts slight differences in refractive indexes into easily detected variations in light intensity –> helps differentiate organelles and motility structures (flagella)

Question 9

fluorescent microscopy

Answer 9

uses a UV light to excite fluorescent molecules
a) green fluorescent dye can be taken up by live cells and red dye by dead cells, allows you to see amounts of live/dead cells
b) fluorescent-labeled antibodies can bind to bacteria, allowing you to visualize the bacteria

Question 10

what to do before staining for bright-field microscopy

Answer 10

usually using heat, kill/fix the microbes to the slide (heat fixation)

Question 11

different dyes in fluorescence microscopy

Answer 11

basic dyes (methylene blue, crystal violet, etc) bind negatively charged structures like DNA, proteins, and phospholipid bilayer
acidic dyes (eosin, acid fuschin) bind positively charged structures like amino acids and other structures in the cytoplasm

Question 12

simple stain

Answer 12

one stain is used to increase the visibility of microbes

Question 13

differential stains

Answer 13

divides microbes into different categories depending on structural characteristics

Question 14

gram stain

Answer 14

type of differential stain that separates bacteria by the type of cell wall they have
gram + = thick wall = purple
gram - = thin wall = red

Question 15

acid fast stain

Answer 15

identifies bacteria that are neither gram + nor - that have a thick waxy layer around them (mycolic acid)
examples: mycobacterium, including TB and leprosy

Question 16

steps of acid-fast stain

Answer 16

heat fixation (carbol fuschin)
acid alcohol to wash - stain leaves the structures that do not have mycolic acid; only sticks to things with mycolic acid
counterstain with methylene blue

Question 17

endspore staining

Answer 17

identifies spore-producing bacteria (have to use heat to get spores to absorb the stain)
steps:
add heat and malachite green
wash with water
safranin counterstain

Question 18

flagella stain

Answer 18

identifies bacteria that have flagella, which are used for motility (do not need to know steps)