Biology - Unit 3: Core Practicals 1-9

1.3 CORE PRACTICAL 1 (BENEDICT’S TESTING)

1. set up standard solutions with known concentrations of reducing sugar via serial dilution

2. add same volume of Benedict’s reagent + heat in water bath for set time = ensure excess of Benedict's solution used

3. same procedure repeated with sample of unknown concentration = compared to stock solution colours

4. avoid issues with human interpretation of colour = colourimeter used

• sample of known solution added to cuvettes = inserted in colourimeter to measure absorbance/transmission of light = establish range of values form a calibration curve

1.3 CORE PRACTICAL 1 (IODINE TESTING)

1. set up standard solutions with known concentrations of starch via serial dilution

2. add same volume of iodine's solution + heat in water bath for set time 

3. same procedure repeated with sample of unknown concentration = compared to stock solution colours

4. avoid issues with human interpretation of colour = colourimeter used

• sample of known solution added to cuvettes = inserted in colourimeter to measure absorbance/transmission of light = establish range of values form a calibration curve

• sample with unknown concentration is compared against calibration curve to estimate concentration of reducing sugar present

what is a colourimeter

colourimeter: instrument that beams specific wavelength (colour) of light through sample + measures light is absorbed by sample

how does the colourimeter work

colour filters used to control light wavelength emitted
- colour used in contrast to colour of solution = assesses solution's colour intensity (Benedict's) = uses blue light filter via shining blue light through sample
- blue light absorbed by orange solution = orange light reflected to show orange appearance
- extent of blue light absorbed differs depending on intensity of orange colour

how are colourimeters calibrated

insert empty cuvette in colorimeter (reference) = should be 0 (no light absorbed)
- results used to plot a calibration/standard curve = absorbance against known concentrations

where can vitamin C be found in

vitamin C found in green vegetables + fruits
- ascorbic acid is a reducing agent = easily oxidised
- detection of vitamin C involve titrating it against an oxidising agent called DCPIP
- DCPIP is a blue dye, turns colourless in presence of vitamin C

what is the safety risk assessment for DCPIP

DCPIP is an irritant 
- avoid contact with skin 
- wear eye protection + gloves

1.14 CORE PRACTICAL 2

1. make up a series of known vitamin C concentrations by serial dilution

2. use measuring cylinder = measure 1 cm3 of DCPIP in test tube

3. add 1 of vitamin C solutions to DCPIP solution via pipette/burette

4. shake tube for set period of time via stop watch = keep shaking time same (control variable)

5. solution turns colourless record volume (number of drops of vitamin C solution added)

6. repeat steps 2-5 of same concentration twice more + calculate average = repeat steps 2-6 per known concentrations

7. results plotted as line of best fit = average volume of vitamin C needed to decolourise DCPIP

results of vitamin C solution and DCPIP

volume of vitamin C solution needed to decolourise DCPIP decreases + concentration of vitamin C solution increases
- results plotted on graph of volume of vitamin C to decolourise DCPIP against concentration of vitamin C
- line of best fit = calibration curve = estimate concentration of vitamin C in fruit juices 

what are the factors that affect permability of cell membranes

permeability of cell membranes affected by several factors = temperature + pH = investigated using beetroot
- beetroot cells contain a dark purple-red pigment
- higher permeability of beetroot cell membrane = more pigment leaks out of cell

2.3 CORE PRACTICAL 3

1. using cork borer + scalpel = cut 5 equal-sized sections of beetroot

   • pieces must have same dimensions = equal surface areas + volumes

2. rinse beetroot pieces = remove loose pigment

3. add beetroot pieces to 5 different test tubes containing same volume of water

4. put test tube in water bath at different temperatures for same length of time

5. remove beetroot pieces= transfer samples of coloured liquid to colorimeter cuvettes via pipettes

6. colorimeter = measure how much light is absorbed as it passes through 5 samples of coloured liquid

how does temperature increase affect

temperature increases = membrane permeability increases
- phospholipids in cell membrane move more = more kinetic energy = loosely packed together
- temperature affects proteins’ 3D shape
- volume of water in cells expands = puts pressure on membrane + damages membrane

temperature decrease = membrane permability increases
- temperatures below 0 ℃ = membrane permeability may increased after cells have thawed
- ice crystals form = pierce cell membrane = highly permeable

what are the limitations in core practical 3

cuvettes may differ in thickness = thicker cuvette will absorb more light than thinner cuvette
- scratched cuvettes have impact on absorbance as thickness
- solution = use same cuvette for every reading/repeat investigation + calculate a mean

beetroot pieces may not be identical in size/shape = some test tubes contain more beetroot tissue
- solution: cut discs accurately as possible via scalpel + ruler = repeat investigation several times to find a mean

some parts of beetroot tissue have more pigment in cells than others
-solution: conduct several repeats, using different parts of beetroot + find mean

what is the impact of alcohol on membrane permability

alcohol concentration increases permeability of membrane increases
- dissolves lipids in cell surface membrane = membrane loses structure + beetroot pigment leaks out

how can temperature affect rate of reactions

higher temperatures speed up reactions
- molecules move more quickly
- higher frequency successful collisions between substrate + active site
- more frequent enzyme-substrate complex = likely for bonds to be form/break

lower temperatures slows down reaction
- molecules move slow
- lower frequency of successful collisions between substrate + active site
- less frequent enzyme-substrate complex = unlikely for bonds to be form/break

what happens when temperature passes over optimum temperature

if temperatures continue to increase = rate enzyme catalyses a reaction drops = enzyme denature
- bonds holding enzyme molecule into shape break
- causes tertiary structure of the protein change
- damages active site = prevents substrate from binding

how can pH cause enzymes to denature

enzymes denature at extremes of pH
- hydrogen + ionic bonds hold tertiary structure of protein together
- below/above optimum pH of enzyme, acidic/alkaline solutions can cause bonds to break
- alters shape of active site = harder for enzyme-substrate complexes to form

why are buffer solutions used

buffer solutions used to measure rate of reaction at different pH values
- buffer solutions have specific pH + maintain specific pH
- measured same volume of buffer solution added to reaction mixture + add to all pH investigated

how does enzyme concentration affect the rate of reaction

increase enzyme concentration = more active sites available = higher likelihood of enzyme-substrate complex formation
- sufficient substrate available = initial rate of reaction increases linearly with enzyme concentration
- amount of substrate is limited = reaction rate won't increase as amount of substrate = limiting factor

how does substrate concentration affect the rate of reaction

higher substrate concentration = faster rate of reaction
- more substrate molecules = more collision between enzyme + substrate
- saturation point reached where all active sites occupied = increasing substrate concentration won’t affect rate of reaction

2.8 CORE PRACTICAL 4 (TEMPERATURE)

1. use 5 test tubes + add 2cm³ of 1% trypsin solution + add into water baths with different temperatures

2. take another 5 test tubes + add 2cm³ of milk + add into water baths with different temperatures for 5 minutes

3. add 2cm³ trypsin solution + distilled water in cuvette (reference cuvette)

4. pour 2cm³ of milk suspension into 1st temperature into another cuvette

5. add 2cm³ of trypsin of same temperature to milk in cuvette = mix trypsin solution + milk via shaking

6. place cuvette into colorimeter = measure absorbance in 15 seconds intervals

7. repeat steps 4-6 for other temperatures

2.8 CORE PRACTICAL 4 (pH)

1. add 1cm³ of trypsin solution + buffer solution + 2cm³ of distilled water into cuvette (reference cuvette)

2. add 1cm² of trypsin solution + buffer solution in cuvette

3. measure 2cm² of milk suspension in another cuvette

4. mix mixture in step 2 to milk in cuvette via shaking cuvette + place into colorimeter

5. repeat steps 2-5 for other pHs

2.8 CORE PRACTICAL 4 (ENZYME CONCENTRATION)

1. work dilution calculations via serial dilutions

2. add 2cm³ of trypsin + distilled water in cuvette (reference cuvette)

3. measure 2cm² of milk suspension

4. mix solution in step 2-3 + measure in colorimeter in 15-second intervals

5. repeat steps 2-5 for other concentratiosn

2.8 CORE PRACTICAL 4 (SUBSTRATE CONCENTRATION)

1. work dilution calculations via serial dilutions

2. add 2cm³ of 1% trypsin + distilled water in cuvette (reference cuvette)

3. measure 2cm³ of 0.2% milk suspension

4. add 2cm³ trypsin to milk in cuvette = mix trypsin solution + measure in colorimeter

5. measure absorbance in 15-second intervals

6. repeat steps 3-5 for other milk concentrations

methods of preparing a microscope slide using solid specimen

take care using sharp objects + wear gloves to prevent stain from dying your skin
- use scissors/scalpel cut small sample of tissue
- use forceps to peel thin layer of cells from sample to place on slide = microscope's light pass through
- apply stain to make cells more visible + place coverslip on top + press down to remove any air bubbles
- some tissue samples need to be treated with chemicals to kill cells/make tissue rigid

method of preparing a slide using a liquid specimen

add few drops of sample to slide via pipette
- cover liquid with coverslip + press down to remove air bubbles
- wear gloves to ensure no cross-contamination of foreign cells

3.8 CORE PRACTICAL 5 (CALIBRATION)

1. place micrometer slide on stage of microscope + focus micrometer scale using low-power objective

2. move slide + rotate eyepiece to align scales of eyepiece graticule + stage micrometer

3. count number of divisions (eyepiece units) on eyepiece graticule equal to known length on micrometer

4. repeat steps 1-3 with medium/high-power objectives

3.8 CORE PRACTICAL 5 (MAKING OBSERVATIONS)

1. wash hands with soap + water

2. take cotton bud + rub inside cheek + rub cotton bud in circle in centre of glass slide = place cotton bud in disinfectant solution

3. add drops of methylene blue to sample + cover with cover slip

4. turn objective lens to low power + examine stained slide under microscope = bring lens close to slide + use coarse focusing knob to focus

5. sketch cells + use eyepiece graticule to measure cell’s diameter = add scale bar

6. turn objective disc to medium/high-power lens + focus via fine-focusing knob till cells are clear = draw + label cells

7. measure length of 2 cells = place glass slide in beaker of disinfectant solution

core practical 5 considerations with microscope when viewing

dehydration of tissue
- thin layers of material placed on slides can dry up rapidly
- add drop of water to specimen beneath coverslip = prevent cells from damaged by dehydration

unclear/blurry images
- switch to lower power objective lens + use coarse focus
- consider if specimen sample is thin enough for light to pass through to see structures clearly
- could be cross-contamination with foreign cells

what are the limitations for core practical 5

size of cells/tissue structures may appear inconsistent
- cell structures are 3D = different tissue samples will be cut at different planes = inconsistencies viewed on 2D slide
- optical microscopes have weaker magnification power = some structures cannot be seen
- treatment of specimens could alter cellular structure

3.15 CORE PRACTICAL 6

1. garlic/onion root tips commonly used

2. prepare boiling tube of 1M hydrochloric acid + place in water bath at 60C for 10 minutes

3. remove tips of roots (about 1cm) + place in warmed hydrochloric acid for 5 minutes

4. rinse tips in cold water + dry with paper towel 

5. cut 2mm off tip + place on microscope slide

6. add drop of stain (eg. acetic orcein = stains chromosomes purple)

7. stained root tip squashed on glass slide using blunt instrument

8. view slide under microscope + draw cells in mitosis

4.6 CORE PRACTICAL 7

1. cut thin cross-section of stem via scalpel

2. transfer each section into dish containing a stain + leave for 1 minute

   • toluidine blue O (TBO) = xylem + sclerenchyma appear blue-green + phloem appear pink-purple

3. rinse section in water + place on microscope slide add cover slip (lower coverslip slowly over sample to avoid trapping air bubbles = mistaken for plant tissues/structures)

4. view under microscope + adjust focus to form a clear image

5. make labelled drawing of positions of xylem vessels, phloem sieve tubes + sclerenchyma

how to draw low/high-power images

If drawing from a low-power image:

• do not draw individual cells

• read question carefully = may only have to draw portion of image

• include magnification on drawing

If drawing from a high-power image:

• draw only few of the required cells

• draw cell wall of plant cells 

• include magnification on drawing

4.9 CORE PRACTICAL 8

1. fibre should be attached to clamp stand

2. attach weight on other end of plant fibre

3. carefully add 1 weight at a time until fibre breaks

4. record mass at which fibre broke = represents tensile strength

5. increase accuracy of results = repeated with more samples of same plant fibre

   • can calculate mean tensile strength for fibre

   • ensure fibres are all of same length + all variables kept constant

4.12 CORE PRACTICAL 9

1. bacteria grown in mixture of distilled water + nutrients in specific bacterial culture (broth)

2. transfer bacteria from broth to agar plate via sterile pipette

3. spread out bacteria via sterile spreader

   • open lid of agar plate + place lid back on of agar plate immediately = prevent contamination

   • prepare plant extracts = must be dried + ground finely

4. soak in ethanol to extract antimicrobial substances + filtered

5. equal sized discs cut from sterile absorbent paper dipped in plant extract via sterile forceps

6. leave discs in extract for set time = ensure to absorb similar amount of plant extract = controlled disc soaked in ethanol

7. space discs out on agar plate, close lid on, invert plate + incubate it 25°C = incubate for 24-48 hours

what is the clear zone

clear zone = area around disc where bacteria cannot grow
- larger clear zone = more effective antimicrobial properties of plant extract
- size of clear zone determined by measuring diameter/area
- repeat experiment thrice + calculate mean of results

aseptic techniques used

aseptic techniques = prevent bacterial cultures being contaminated 
- contamination = negative impact on growth of bacteria
- keep windows/doors closed = prevent air movement
- disinfect surfaces + utensils = prevent contamination
- ensure use of sterile equipment + discard
- work near Bunsen flame when transferring bacteria = ensure microbes in air drawn away
- hold flame close to neck of glass container of broth when opened/closed