REQUIRED PRACTICAL 2 - SETUP AND USE OF A MICROSCOPE

Question 1

what is the aim of this practical

Answer 1

to identify the stages of mitosis in the stained squashes and calculation of a mitotic index

Question 2

discuss mitosis and its relevance in this practical

Answer 2

• plant cells undergo mitosis at the SHOOT and ROOT TIPS in areas called the MERISTEMS
• cells in the MERISTEMS are TOTIPOTENT and retain the ability to DIFFERENTIATE

Question 3

what is the mitotic index

Answer 3

• the ratio of cells undergoing mitosis to the total number of cells in a sample

Question 4

how do you find the mitotic index

Answer 4

cells from the meristem must be viewed under a optical microscope

Question 5

what is the equipment needed for this practical

Answer 5

• optical microscope
• microscope slides
• cover slips
• HCL acid
• toluidine blue O stain
• distilled water
• scalpel
• forceps
• 100 ml beaker
• root tip

Question 6

what is the method for this practical

Answer 6

• heat 1 mol/dm3 at 60 degrees C in a water bath
• cut a small sample of the root tip using a scalpel
• transfer the root tip to HCL and incubate for 5 mins
• remove the root tip from HCL and wash sample in cold distilled water and remove the tip using a scalpel
• place the tip on a microscope slide and add a few drops of stain, toluidine blue O = makes chromosomes visible and will therefore show which cells are undergoing mitosis
• lower the cover slip down carefully onto the slide
• ensure there are no air bubbles in the slide as this may distort the image
• ensure the coverslip doesn’t slide sideways which could damage the chromosomes
• place the coverslip under a microscope and set the objective lens on the LOWEST magnification
• use the coarse adjustment knob to move the lens down to just above the slide
• use the fine adjustment knob to carefully re-adjust the focus until the image is clear
• calculate the mitotic index

Question 7

how do you calculate the mitotic index

Answer 7

the number of cells with visible chromosomes/total number of cells in the sample

Question 8

name the potential risks in the practical

Answer 8

• HCL acid
• toluidine blue O stain
• scalpel
• broken glass

Question 9

what is the risk of HCL acid

Answer 9

may cause harm/irritation to eyes or in cuts

Question 10

what is the safety precaution of HCL acid

Answer 10

• wear eye protection
• avoid contact with the skin
• tie up long hair

Question 11

what is the risk of toluidine blue O stain

Answer 11

may cause harm/irritation to eyes or in cuts

Question 12

what is the safety precaution for toluidine blue O stain

Answer 12

• wear eye protection
• avoid contact with skin

Question 13

what is the risk of using a scalpel

Answer 13

cuts from sharp objects

Question 14

what is the safety precaution for using a scalpel

Answer 14

• cut away from fingers
• use forceps to hold sample whilst cutting
• keep away from the edge of the desk

Question 15

what is the risk for broken glass

Answer 15

cuts from sharp objects

Question 16

what is the safety precaution for broken glass

Answer 16

• take care when handling slides and coverslips
• keep glassware away from the edge of the desk

Question 17

why are root tips used

Answer 17

as this is where dividing cells are found and mitosis occurs

Question 18

why is a stain used

Answer 18

• to distinguish chromosomes
• chromosomes are not visible without a stain

Question 19

why is it necessary to squash/press down on the cover slip

Answer 19

• it spreads out cells to create a single layer of cells
• allows light to pass through to make chromosomes visible

Question 20

why dont you push the coverslip sideways

Answer 20

to avid rolling cells together/ breaking chromosomes

Question 21

why do you soak the roots in acid

Answer 21

• separate cells/cell walls
• to allow the stain to diffuse into the cells
• to allow the cells to be more easily squashed
• to stop mitosis

Question 22

describe how to set up and use an optical microscope

Answer 22

• clip slide onto stage and turn on the light
• select the lowest power objective, usually x4
• use the coarse functioning dial to move stage close to lens
• OR turn the coarse functioning dial to move the stage away from the lens until the image comes into focus
• adjust the fine focusing dial to get a clearer image
• swap to a higher power objective lens, then refocus

Question 23

are chromosomes visible in interphase

Answer 23

• interphase is not mitosis
• chromosomes aren’t visible BUT nuclei are
• in mitosis, chromosomes are visible

Question 24

how can prophase be identified

Answer 24

• chromosomes are visible/ distinct due to CONDENSING
• chromosomes are randomly arranges, as they are not attached to their spindle fibre

Question 25

how can metaphase be identified

Answer 25

• chromosomes are lined up on the equator, as they are attaching to the spindle

Question 26

how can anaphase be identified

Answer 26

• chromatids are at the poles of spindle
• chromatids are V SHAPED, as they are being pulled apart via their centromeres by the spindle fibres

Question 27

how can telophase be identified

Answer 27

• chromosomes are in 2 sets, one at each pole

Question 28

how do you determine a reliable mitotic index from observed squashed

Answer 28

• count cells in mitosis in the field of the view
• count only WHOLE cells/ cells on top and right edges, this standardises counting
• divide this ^ by the total number of cells in the field of the view
• repeat with many/ at least 5 fields of the view selected RANDOMLY, makes the representative sample
• calculate a reliable mean

Question 29

suggest how to calculate the time that cells are in a CERTAIN phase of mitosis

Answer 29

• identify a proportion of cells in the named phase at any one time
• ^ through number of cells in that phase/total number of cells observed
• multiply this by the length of the cell cycle