- Biology Required Practicals
RP1 – effect of named variable on rate of enzyme-controlled reactions
different influences on rate of reaction of enzyme-controlled reaction? temperature, pH,
concentration (enzyme or substrate)
equipment list? powdered milk suspension, trypsin solution, distilled water, HCl
(0.1M), 5cm syringes, flat bottomed tubes, water bath, timer
3
method: step 1? make two control samples
what are controlled samples made in? bottom flat tubes
what is in one control sample? 5cm3 milk suspension, 5cm3 distilled water
reason for control sample with distilled water? absence of enzyme activity
what is in another control sample? 5cm3 milk suspension, 5cm3 HCl
reason for control sample with hydrochloric acid? colour of completely hydrolysed
sample
method: step 2? 5cm3 milk in three test tubes
method: step 3? tubes in water bath at 10 for 5 mins
why do you place the test tubes in water bath? equilibrate
method: step 4? 5cm3 trypsin to each tube simultaneously and start timer
method: step 5? record time taken for milk samples to completely hydrolyse and
become colourless
method: step 6? repeat steps 2-3 at temperatures 20, 30, 40, 50
method: step 7? mean time at each temperature
rate calculation? 1 / mean time
hazard 1? broken glass
hazard 2? HCl
hazard 2: risk? cause harm/irritation
hazard 3? hot liquids
hazard 4? enzymes
graph plot? rate of reaction against temperature
what causes milk to turn colourless? protein in milk called casein, colourless when
broken down
what causes casein to be broken down? trypsin, protease enzyme, hydrolyses
why does rate of reaction increase up to optimum temperature? kinetic energy increases,
more E-S complexes
effect after optimum temperature? vibrations break hydrogen/ionic bonds in tertiary
structure, active site alters, no E-S complexes
how does active site of enzyme cause high rate of reaction? lowers Ea, induced fit
causes active site to change shape, E-S complex causes bonds to form/break
RP2 – preparation of stained squashes of cells from meristems; set-up
and use optical microscope to identify stages of mitosis; calculation of
mitotic index
what do plants undergo at shoot and root tips? mitosis
where do plant cells undergo mitosis at shoot and root tips? meristems
type of cells in meristems? totipotent
what is the mitotic index? ratio of cells undergoing mitosis to total number of cells in
sample
equipment list? optical microscope, microscope slides, cover slips, water bath, HCl,
toluidine blue O stain, distilled water, scalpel, forceps, 100 ml beaker, root tip
method: step 1? heat 1 mol dm-3 HCl at 60 in water bath
method: step 2? cut small sample of meristem with scalpel
method: step 3? transfer sample to HCl
, method: step 4? incubate for 5 mins
method: step 5? remove sample from HCl
method: step 6? wash sample in cold distilled water
method: step 7? remove very tip (~5 mm) using scalpel
why do you remove very tip? where mitosis occurs
why is it only ~5 mm? length that will fit under cover slip
method: step 8? place tip on microscope slide
method: step 9? add few drops of stain
stain used? toluidine blue O
what colour does toluidine blue stain chromosomes? blue
other possible stain? acetic orcein
what colour does acetic orcein stain chromosomes? purple-red
why is a stain added? makes chromosomes visible
why do you need the chromosomes to be visible? show which cells are undergoing
mitosis
method: step 10? lower cover slip down carefully on slide, press down firmly
ensure what when putting cover slip down? no air bubbles, cover slip doesn’t slide
sideways
why no air bubbles? may distort image
why ensure cover slip doesn’t slide sideways? could damage chromosomes
why do you press down firmly? thin layer of cells so light passes through
method: step 11? place under microscope
what do you set the objective lens on microscope to? lowest magnification
how do you re-adjust the focus? fine adjustment knob
how to make image clearer? higher magnification
mitotic index calculation? number of cells with visible chromosomes / total number of
cells in sample
describe how to calculate mitotic index? examine large number of cells, repeat count,
only count whole cells
why is a large number of cells examined for mitotic index? ensure representative
sample
why do you repeat the count of cells for mitotic index? ensure figures are correct
why are only whole cells calculated for mitotic index? standardise counting
how can you tell which cells are not dividing using an optical microscope? longer,
nuclei not in centre of cell
how can you tell which cells are dividing using an optical microscope? small and square,
nucleus in centre of cell
briefly describe how to prepare a root tip squash? squash stained meristem pressing
firmly on coverslip, produces single layer of cells
eyepiece graticule must be what before use for each magnification? calibrated
calibrating eyepiece graticule: step 1? place stage micrometre on microscope
calibrating eyepiece graticule: step 2? focus scale on micrometre using low-power
objective lens
calibrating eyepiece graticule: step 3? align scale of eyepiece graticule and
micrometre
calibrating eyepiece graticule: step 4? count number of divisions on eyepiece
graticule equal to 100 micrometres on micrometre
calibrating eyepiece graticule: step 5? length of one eyepiece division
calibrating eyepiece graticule: step 6? work out actual length of cell with calibrated
values
hazard 1? HCl
hazard 2? toluidine blue O stain
hazard 3? scalpel
hazard 4? broken glass
RP1 – effect of named variable on rate of enzyme-controlled reactions
different influences on rate of reaction of enzyme-controlled reaction? temperature, pH,
concentration (enzyme or substrate)
equipment list? powdered milk suspension, trypsin solution, distilled water, HCl
(0.1M), 5cm syringes, flat bottomed tubes, water bath, timer
3
method: step 1? make two control samples
what are controlled samples made in? bottom flat tubes
what is in one control sample? 5cm3 milk suspension, 5cm3 distilled water
reason for control sample with distilled water? absence of enzyme activity
what is in another control sample? 5cm3 milk suspension, 5cm3 HCl
reason for control sample with hydrochloric acid? colour of completely hydrolysed
sample
method: step 2? 5cm3 milk in three test tubes
method: step 3? tubes in water bath at 10 for 5 mins
why do you place the test tubes in water bath? equilibrate
method: step 4? 5cm3 trypsin to each tube simultaneously and start timer
method: step 5? record time taken for milk samples to completely hydrolyse and
become colourless
method: step 6? repeat steps 2-3 at temperatures 20, 30, 40, 50
method: step 7? mean time at each temperature
rate calculation? 1 / mean time
hazard 1? broken glass
hazard 2? HCl
hazard 2: risk? cause harm/irritation
hazard 3? hot liquids
hazard 4? enzymes
graph plot? rate of reaction against temperature
what causes milk to turn colourless? protein in milk called casein, colourless when
broken down
what causes casein to be broken down? trypsin, protease enzyme, hydrolyses
why does rate of reaction increase up to optimum temperature? kinetic energy increases,
more E-S complexes
effect after optimum temperature? vibrations break hydrogen/ionic bonds in tertiary
structure, active site alters, no E-S complexes
how does active site of enzyme cause high rate of reaction? lowers Ea, induced fit
causes active site to change shape, E-S complex causes bonds to form/break
RP2 – preparation of stained squashes of cells from meristems; set-up
and use optical microscope to identify stages of mitosis; calculation of
mitotic index
what do plants undergo at shoot and root tips? mitosis
where do plant cells undergo mitosis at shoot and root tips? meristems
type of cells in meristems? totipotent
what is the mitotic index? ratio of cells undergoing mitosis to total number of cells in
sample
equipment list? optical microscope, microscope slides, cover slips, water bath, HCl,
toluidine blue O stain, distilled water, scalpel, forceps, 100 ml beaker, root tip
method: step 1? heat 1 mol dm-3 HCl at 60 in water bath
method: step 2? cut small sample of meristem with scalpel
method: step 3? transfer sample to HCl
, method: step 4? incubate for 5 mins
method: step 5? remove sample from HCl
method: step 6? wash sample in cold distilled water
method: step 7? remove very tip (~5 mm) using scalpel
why do you remove very tip? where mitosis occurs
why is it only ~5 mm? length that will fit under cover slip
method: step 8? place tip on microscope slide
method: step 9? add few drops of stain
stain used? toluidine blue O
what colour does toluidine blue stain chromosomes? blue
other possible stain? acetic orcein
what colour does acetic orcein stain chromosomes? purple-red
why is a stain added? makes chromosomes visible
why do you need the chromosomes to be visible? show which cells are undergoing
mitosis
method: step 10? lower cover slip down carefully on slide, press down firmly
ensure what when putting cover slip down? no air bubbles, cover slip doesn’t slide
sideways
why no air bubbles? may distort image
why ensure cover slip doesn’t slide sideways? could damage chromosomes
why do you press down firmly? thin layer of cells so light passes through
method: step 11? place under microscope
what do you set the objective lens on microscope to? lowest magnification
how do you re-adjust the focus? fine adjustment knob
how to make image clearer? higher magnification
mitotic index calculation? number of cells with visible chromosomes / total number of
cells in sample
describe how to calculate mitotic index? examine large number of cells, repeat count,
only count whole cells
why is a large number of cells examined for mitotic index? ensure representative
sample
why do you repeat the count of cells for mitotic index? ensure figures are correct
why are only whole cells calculated for mitotic index? standardise counting
how can you tell which cells are not dividing using an optical microscope? longer,
nuclei not in centre of cell
how can you tell which cells are dividing using an optical microscope? small and square,
nucleus in centre of cell
briefly describe how to prepare a root tip squash? squash stained meristem pressing
firmly on coverslip, produces single layer of cells
eyepiece graticule must be what before use for each magnification? calibrated
calibrating eyepiece graticule: step 1? place stage micrometre on microscope
calibrating eyepiece graticule: step 2? focus scale on micrometre using low-power
objective lens
calibrating eyepiece graticule: step 3? align scale of eyepiece graticule and
micrometre
calibrating eyepiece graticule: step 4? count number of divisions on eyepiece
graticule equal to 100 micrometres on micrometre
calibrating eyepiece graticule: step 5? length of one eyepiece division
calibrating eyepiece graticule: step 6? work out actual length of cell with calibrated
values
hazard 1? HCl
hazard 2? toluidine blue O stain
hazard 3? scalpel
hazard 4? broken glass
