Microscopy - 2.1 - BASIC COMPONENTS OF LIVING SYSTEMS (3)

Question 1

In basic light microscopy, where is the sample illuminated?

Answer 1

from below with white light and observed from above (brightfield microscopy)

Question 2

What happens to the sample after it is illuminated from below?

Answer 2

the whole sample is illuminated at once (wide-field microscopy)

Question 3

What do the images tend to have?

Answer 3

low contrast as most cells do not absorb a lot of light

Question 4

What is resolution limited by?

Answer 4

the wavelength and diffraction of light as it passes through the sample

Question 5

What is diffraction?

Answer 5

it is the bending of light as it passes close to the edge of an object

Question 6

What is the cytosol?

Answer 6

the liquid found inside of cells

Question 7

What is often transparent?

Answer 7

the cytosol of cells and other cell structures

Question 8

What do stains increase?

Answer 8

they increase contrast as different components within a cell take up stains to different degrees

Question 9

What does the increase in contrast allow?

Answer 9

it allows components to become visible so they can be identified

Question 10

What do you do in order to prepare a sample for staining?

Answer 10

the sample is first placed on a slide and allowed to air dry

Question 11

What happens after the sample is allowed to air dry?

Answer 11

it is then heat-fixed by passing through a flame

Question 12

What happens to the sample after it is heat-fixed?

Answer 12

the specimen will adhere to the microscope slide and will then take up stains

Question 13

What is crystal violet and methylene blue?

Answer 13

they are postively charged dyes

Question 14

What is crystal violet and methylene attracted to?

Answer 14

negatively charged materials in cytoplasm leading to staining of cell components

Question 15

Which dyes are negatively charged?

Answer 15

nigrosin and Congo red

Question 16

What are nigrosin and Congo red repelled by?

Answer 16

negatively charged cytosol

Question 17

Where does the nigrosin and Congo red dye stay?

Answer 17

they stay outside cells

Question 18

What happens when dyes such as nigrosin and Congo red stay outside of the cell?

Answer 18

this leaves the cells unstained, which then stand out against the stained background -> this is a negative stain technique

Question 19

What can differential staining distinguish?

Answer 19

it can distinguish between two types of organisms that would be hard to identify

Question 20

What can differential staining also differentiate between?

Answer 20

different organelles of a single organism within a tissue sample

Question 21

What is the Gram stain technique used for?

Answer 21

it is used to seperate bacteria into two groups

Question 22

What are the names of the two groups where Gram stain technique is used?

Answer 22

gram-positive bacteria and gram-negative bacteria

Question 23

Gram stain technique - What happens first in the gram stain technique?

Answer 23

crystal violet is first applied to a bacterial specimen on a slide, then iodine, which fixes the dye

Question 24

Gram stain technique - What happens after the dye is fixed by iodine?

Answer 24

the slide is then washed with alcohol

Question 25

Gram stain technique - what happens after the slide is washed with alcohol?

Answer 25

the gram-positive bacteria will keep the crystal violet stain and will appear blue or purple under a microscope

Question 26

Gram stain technique - what happens to the gram-negative bacteria?

Answer 26

as gram-negative bacteria have thinner cell walls, they will lose the stain

Question 27

Gram stain technique - what happens after the gram-negative bacteria loses the stain?

Answer 27

they are then strained with safranin dye, which is called a counter stain

Question 28

Gram stain technique - what colour will the gram-negative bacteria appear after being stained with safranin dye?

Answer 28

red

Question 29

Gram stain technique - what is the gram-positive bacteria susceptible to?

Answer 29

antibiotic penicillin, which prevents the formation of cell walls

Question 30

Gram stain technique - as gram-negative bacteria have much thinner cell walls, are they susceptible to penicilin or not?

Answer 30

they are not susceptible to penicillin

Question 31

Acid fast technique - what is this used for?

Answer 31

to differentiate species of Mycobacterium from other bacteria

Question 32

Acid fast technique - what is a lipid solvent used for?

Answer 32

to carry carbolfuchsin dye into the cells being studied

Question 33

Acid fast technique - what happens after the lipid solvent is used?

Answer 33

the cells are then washed with a dilute acid-alcohol solution

Question 34

Acid fast technique - what happens after the cells are then washed with a dilute acid-alcohol solution?

Answer 34

mycobacterium are not affected by the acid-alcohol and retain the carbolfuchsin stain, which is bright red

Question 35

Acid fast technique - what happens to the other bacteria?

Answer 35

other bacteria lose the stain and are exposed to a methylene blue stain, which is blue