AS Practicals Flashcards
Calibrating an eyepiece graticule
Calibrating an eyepiece graticule:
1). Set up microscope to required magnification
2). Place a stage graticule on the stage
3). Line up the two scales (the stage and eyepiece graticule)
4). Count the number of divisions on the eyepiece graticule equivalent to each division on the stage micrometer
Purpose of staining
Staining provides contrast between different structures within a sample
Differential staining
Differential staining is when multiple stains are used, and each stain binds to a specific cell structure, staining each structure differently so the structures can be easily identified
Aecetic orcein stain
Aecetic orcein binds to DNA and stains chromosomes dark red
Eosin stain
Eosin stains cytoplasm dark red or pink
Iodine stain
Iodine stains starch blue/black
Iodine in potassium iodide solution as a stain
Iodine in potassium iodide solution stains cellulose yellow
Haematoxylin stain
Haematoxylin stains RNA/DNA a purple/blue colour
Methylene blue
Methylene blue is an all-purpose stain, used often to stain DNA blue
Wet mount technique
Wet mount:
-Use pipette to add a drop of water on the slide
-Use tweezers to place specimen in water
-Put cover slip on
-Add stain (drop on the edge of the cover slip)
-Put paper towel on the opposite edge to absorb stain, drawing it under the coerslip and staining the specimen
Dry mount technique
Dry mount:
-Slice specimen thinly
-Use tweezers to pick up and put on the middle of the slide
-Put cover slip ontop of it
How to use a light microscope
Using a light microscope:
1). Clip the slide onto the stage
2). Select the lowest-powered objective lens
3). Use the coarse adjustment knob to move the objective lens to just above the slide
4). Look down eyepiece, adjust focus by moving the lens away from the slide using the fine adjustment knob until clear image appears
How to measure size of structures with a microscope
Measuring size:
Structures can be measured by counting the number of eyepiece divisions (once eyepiece graticule has been calibrated)
-To find actual length, the number of divisions should be multiplied by the length of one division
Why is resolution limited by diffraction?
Resolution is limited by diffraction of light as it passes through samples (and lenses). Diffraction is the tendency of light waves to spread as they pass close to physical structures such as those present in the specimens being studied.
Heart dissection method (external examination)
Heart dissection method: (external examination)
1). Feel the differences in thickness of the walls using fingers - identify left and right atria + ventricles (left = thicker, ventricles = thicker)
2). Identify 4 main vessels attached to heart (arteries = thick + rubbery, veins = thinner)
3). Identify coronary artery on external surface and where it connects to the aorta
Heart dissection method (internal examination)
Heart dissection method (internal examination):
1). Cut along left ventricle and through left atrium wall
2). Open up atrium and ventricles, look for tendinous chords and observe how they area attached to the atrioventricular valve
3). Look for the AV alve and Sl valves
4). Use ruler to measure thickness of walls
Plant dissection method
Plant dissection method:
1). Cut cross-section of stem thinly (transverse + longitudinal)
2). Place transverse section in water for 2 mins (stops them from drying out)
3). Lift sections of stem onto watch glass containing a stain (Eg tuolodine blue)
4). Rinse off excess stain
5). Place under microscope on slide with drop of water and coverlip, on lowest mag
Effect toludine blue on plant tissue
Effect toludine blue on plant tissue: (stains non-lignified and lignified tissue to allow contrast between xylem and phloem)
Phloem - red
Xylem - green/blue
Parenchyma = red/purple
mm to micrometer
1mm = 1000 micrometeres
Micrometeres to nanometers
1 micrometer = 1000 nanometers
Typical stage micrometer sizes
Stage micrometeres are typically 2mm long with subdivisions that are 10 micrometers apart
External examination of a heart dissection
External examination of a heart dissection:
-Place fingers inside 4 chambers to feel the differences in thickness of walls -> left should be thicker than right, ventricles thicker than atria
-Identify main vessels -> arteries are thick and rubbery, veins are thinner
-Identify coronary artery on external surface -> see where it connects to the aorta
Internal examination of a heart dissection
Internal examination of a heart dissection:
-Cut along left atrium, cut down apex to left ventricle
-Look for tendinous heart strings that are attached to the AV valve
-Look for AV and SL valves -> AV has two flaps, SL has half-moon shape
-Examine wall and internal structures of right atrium
-Measure thickness of walls and compare
Plant dissection method
Plant dissection method:
-Cut cross-section of stem, perpendicularly and thinly
-Place transverse sections in water for 2 mins
-Set up microscope
-Lift stem onto stain (toludine blue) + leave for 1 min
-Wash off excess stain
-Place on slide, add drop of water and coverslip (repeat for three thinnest sections)
-Use lowest mag and increase until find clearest view of cascular bundle
Effect of toluidine blue on vascular bundles
Toluidine blue:
-Non-lignified tissue/phloem = pink/purple
-Lignified tissue/xylem = green/blue
Types of sampling
Types of sampling: random and non-random
Non-random sampling techniques
Non-random sampling techniques:
-Stratified
-Opportunistic
-Systematic
Stratified sampling: explanation, positives, limitations
Stratified sampling: explanation, positives, limitations:
-Explanation: habitat divided into groups, each area sampled separately, each group mutually exclusive (each population can only be in one group) and collectively exhaustive (no population left out)
-Positives: All areas are sampled, non missed out, areas of different population levels in same habitat sampled equally
Opportunistic sampling: explanation, positives negatives
Opportunistic sampling: explanation, positives negatives:
-Explanation = Areas are chosen to sample by reseacher using prior knowledge, or what is just readily available
-Positives =Easier and quicker than random sampling, and produces more data
-Negatives = potential for bias, as species more noticeable may be included more often, leading to overestimation of importance and inaccurate estimate of biodiversity
Systematic sampling: explanation, positives and negatives
Systematic sampling: explanation, positives and negatives:
-Explanation = This is when samples are taken at set distances, eg transects
-Positives = Good to view a change in biodiversity along a line
-Negatives = risk of missing species as only small area is sampled
Species diversity definition
Species diversity is a measure of the number of different species in a given community
Method on calculating species diversity
Method on calculating species diversity:
1). Place quadrats at random coordinates
2). Identify the different species in the quadrat, and count the number of each present
3). Calculate percentage cover
4). Use simpsons index of diversity to calculate species diversity
Method on calculating species abundance
Method on calculating species abundance:
2). Identify the different species in the quadrat, and count the number of each present
3). Calculate percentage cover
4). Population size of each species can be estimated by multiplying up the sample size
5). Produce graph of species abundance
Investigating the effect of temperature on catalase activity
Investigating the effect of temperature on catalase activity:
1). Suggest hypothesis
2). Water baths of 40, 60, 80 degrees + ice bath of a constant temp
3). 4 boiling tubes with same volume and concentration of hydrogen peroxide solution
4). Keep pH constant by adding equal volumes of suitable bugger
5). Put each tube in ice bath of a different temp, allow apparatus to equilibriate
6). Add same volume and conc of catalase to each boiling tube
7). Put bung on and start timer
8. Measure oxygen produced every 30 secs for 3 mins
9). Repeat and find mean
10). Calculate rate of gas producing for each temperature
Investigating the effect of enzyme (amylase) concentration on the rate of reaction
Investigating the effect of enzyme (amylase) concentration on the rate of reaction:
1). Drop of iodine in potassium iodine solution into each well
2). Boiling tubes with different conc of amylase using serial dilutions method
3). Add same conc and volume of starch to first boiling tube and start timer
4). Put drop of this mixture into one of the wells containing the iodine iodine solution at regular intervals
5). Observe colour change
6). Time how long it takes for colour to no longer turn blue-black
7). Repeat and find mean
8). Calculate rate of reaction by dividing conc of starch by time taken
Investigating the effect of substrate concentration on the rate of an enzyme-controlled reaction
Investigating the effect of substrate concentration on the rate of an enzyme-controlled reaction:
1). Set up 5 beakers of varying conc of hydrogen peroxide using serial dilutions technique. Transfer each solution to conical flask
2). Use syringe to add the same volume and conc of catalase to first conical flask
3). Put bong on and start timer
4). Record how much oxygen is produced every 30 seconds for 3 minutes
5). Repeat for each conc
6). Calculate mean and standard deviation for each concentration
7). Calculate rate of gas production for each conc of hydrogen peroxide
How to carry out serial dilutions?
Serial dilutions:
-Set up 5 boiling tubes
-Start with 2% enzyme solution, add 4cm^3 to first boiling tube
-Use syring to add 2cm^3 from first tube to next tube, which makes a 1% enzyme solution
-Repeat this to make 0.5% solution, 0.25% solution and 0.13% solution
Using a colorimeter
Using a colorimeter:
1). Switch on and leave to stabilise for 5 minutes
2). Select red filter (complementary to blue benedicts solution)
3). Set colorimeter to 0 using cuvette 3/4 filled with distilled water to callibrate
4).Ensure cuvette is placed so that light passes through clear sides
5). Ensure slides are clean and no bubbles in solution
6). Use pipette to fill cuvette 3/4 with sample
7). Place in colorimeter and read absorbance of light
8). Less light absorbed by solution in a paler solution, so there is greater transmission
How to produce a callibration curve in order to measure the concentration of a solution (commonly reducing sugars) using a colorimeter
Callibration curve:
1). Standard solutions of reducing sugar should be used
2). Carry out benedicts test on each sample
3). With colorimeter, measure percentage transmission for each sample
4). Plot graph of transmission against reducing sugar concentration
What do the results mean when testing for reducing sugars?
Testing for reducing sugars:
More transmission/less absorbance = more sugar present/greater concentration
Why is benedicts test semi-quantitative?
Benedicts test is semi-quantitative as it is only an pproximate results
Why is benedicts solution less blue after testing?
Benedicts solution will be less blue after testing because some copper ions have reacted and are no longer in the solution
Purpose of a centrifuge
Centrifuge seperates solution from precipitate
Purpose of a potometer
Potometers are used to estimate the rate of transpiration of a plant. It measures water uptake, but this is assumed be associated with rate of water loss in plants
Limitations of potometers
Limitations of potometers:
-Not all the water taken up by the plant is used to transpiration - eg some used in cells to maintain turgidity
-Some water used in photosynthesis
-The plant is dying once roots are cut off
How to use a potometer?
Potometer method:
1). Cut shoot underwater to prevent air entering xylem, cut it at a slant to increase surface area available for water uptake
2). Asssemble potometer in water and insert shoot under water
3). Remove apparatus from water but kepe end of capillary tube submerged in water
4). Check it is watertight and airtight by using screws or petroleum jelly
5). Dry leaves
6). Allow plant to acclimatise
7). Remove capillary tube end from water until one air bubble formed then put back under water
8) Record distance moved by bubble at regular intervals
How to calculate rate of air bubble movement after using a potometer?
Calculate rate of air bubble movement by dividing the distance travelled by time - estimation of transpiration rate
What is chromatography?
Chromatography is an analytic method used to seperate a mixture into the different biological molecules
-> two components: stationery phase, mobile phase
Adsorption meaning
Adsorption = where molecules bond to the surface of a substance
Limitations of chromatography
Limitations of chromatography: similar molecules may have similar Rf values, meaning that it may be difficult to distinguish between them
Using chromatography to separate a mixture of amino acids
Using chromatography to separate a mixture of amino acids:
1). Set up chromatography paper so that there is a dot of stained mixture with unknown amino acids, and known standard solution of amino acids
2). Lower paper into beaker with solvent, and wait for solvent to travel up paper
3). Dry chromatography paper and spray with ninhydrin solution
What does the time/distance amino acids move in the mobile phase depend on?
The time/distance that amino acids move in the mobile phase is dependant on their charge and size
How to identify an unknown amino acid using the chromatography method?
The unknown amino acid can be identified by comparing them with the chromatograms of the known solutions,
-If the spot is at the same distance as a standard solution, it contains the same amino acid
Why is ninhydrin solution used in chromatography?
Ninhydrin solution is a chemical that reacts with amino acids, producing an easily visible blue-violet colour
What determines how far each component can travel in chromatography?
Differences in solubility of each components in the mobile phase affects how far each component can travel
Potato osmosis practical procedure
Potato osmosis practical procedure:
1). 7 beakers with varying concentrations of sucrose (0.0M-0.6M)
2). Cut potato sylinders to equal lengths, each to one beaker, also dilute with distilled water
3). Weight each and record masses
4). Time
5). Remove each and dry excess, then record new masses and percentage changes
Sources of error for the potato osmosis practical
Sources of error for the potato osmosis practical:
-Potato cylinders taken from different parts of the plant may have different water potentials
-Equilibrium may not have been fully reached when cylinders weighed second time
Conclusion of potato osmosis practical
Conclusion of potato osmosis practical:
-Potatoes in lower concentration of glucose conc will increase in mass
-Potatoes in higher concentation of glucose conc will decrease in mass
-In the dilute glucose concentrations, solution has higher water potential than potato so water moves passibely by osmosis - potatoes in crease in mass
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