Flashcards in Unit 3 - Practical Deck (60):
What must you ensure when marking using the capture-mark-recapture technique?
Don't use a toxic substance as you mustn't cause them harm, only mark on underside to ensure you don't make them vulnerable to predators. Mark must be permanent to endure for rest of investigation. Collect a large sample.
What is the equation for population estimation? What is it called?
(s1 x s2) / r
s1 = total num captured first time
s2 = total num captured second time
r = total num recaptured
Lincoln Index/Peterson Estimate
What is the method for the capture recapture technique?
Capture num of organisms. Record total caught + mark each. Release + allow to reintegrate for long enough, depending on typical movements. Recapture some. Record total recaptured + total of those marked. Use equation to estimate.
What assumptions are made when using capture mark recapture technique?
No significant gains/losses through immigration/emigration or births/deaths (closed conditions), trapping doesn't affect animal so it becomes more wary and less likely to be trapped, marked animals have remixed.
What is a haemocytometer?
Instrument for counting cell nums (density). (Yeast/blood cells) Modified microscope slide with grid of squares slightly lower giving depth of 0.1mm so liquid has known volume.
What are the volumes of these squares:
Type A, B + C.
A = 0.1mm3
B = 0.004mm3
C = 0.00025mm3
How do you determine the number to multiply average num of cells by to get the number/mm3?
How do you count the cells in a haemocytometer?
First, choose which size square to count from. Then, count using the North West Rule (only count touching cells if touching the top or left side of each square)
How do you set up a haemocytometer?
Clean haemocytometer + cover slip with lens tissue + ethanol. Mix sample well. Use pipette (sample at same depth in solution) to add few drops onto grooves of haemocytometer. Cover slip on. Place on stage + focus. Don't get solution on top of cover slip or haemocytometer grooves. Ensure to replicate for reliability. Hard to tell between dead + alive cells so not good for decline + stationary phase.
What should you do if there are too many cells to count, even in a type C square?
Serial dilution. 1 part cell suspension to 9 parts isotonic buffer + multiply raw count by dilution factor (10)
What should you do if sampling from natural environment (phytoplankton from lake)?
Retrieve samples from same depth at same time of day.
How do you do the haemocytometer + yeast practical?
Use 3 flasks of diff sizes (150,250,500), pour 50ml apple juice into each + 1 drop yeast suspension. Cover each with muslin (keep out dust + allow air), leave flasks in warm place (25-30c, 3-4days). Set up haemocytometer + estimate population.
What is aseptic technique used for?
To prevent contamination when working with microorganisms, including preventing contamination of microbe culture + individuals working with microbes.
What can be used to culture microorganisms?
Solid agar or liquid broth. Care needed when transferring microorganisms to Petri dishes/other containers for investigation.
What is the procedure for transferring microorganisms?
Metal inoculating/disposable plastic loops can be used. When transferring from culture to petri, lid should be held in same hand as culture bottle + not allowed to touch bench with other hand holding inoc loo. After opening culture bottle, neck should be passed through Bunsen flame to sterilise lid region. Repeat before replacing lid. Lid of petri only raised enough to add microorganisms to agar.
What must you do with a metal inoculating loop?
Flame it in the hottest part of a Bunsen flame until it becomes red hot to sterilise it. Must then allow it to air cool as it would kill any microorganisms it came into contact with. After transfer, must resterilise. Don't create microorganism-rich aerosol when doing this for safety reasons.
What must you do with disposable plastic loops?
Discard into a solution of disinfectant after use.
What are the 2 methods for transferring cultures to petri dishes?
Inoculating loop or spread plate method.
What is the spread plate method?
Used when cells in suspension used in inoculation. L shaped spreader used to spread inoculating bacteria over agar surface (plastic alt to glass which require repeated sterilisation)
What must you do after transferring culture to petri dish?
Label petri dish on outside of base. Incubate upside down in incubator at appropriate temp for it.
What are 2 different types of antibiotic applications?
Antibiotic discs or e-strips (more accurate)
How do you complete an experiment using antibiotic discs?
Place antibiotic disc on agar in petri dish. Inoculate agar with bacteria.
What are e-strips?
Prepared strips which contain concentration gradient of antibiotic running length of strip. More sophisticated way of measuring effect of diff concs of antibiotic on bacteria. Contain markings which show antibiotic concs at certain points on strip so can work out min antibiotic conc to inhibit bacterial growth.
How do you complete an experiment using e-strips?
Place e-strip on agar of petri inoculated with bacteria.
What would you use a streak plate for?
Isolate single colonies of microbial cells (pure culture of cells) which all share same parental cell.
Describe the procedure of a streak plate to isolate single colonies.
Spread microorganisms over agar in petri dish using inoculating loop. Sterilise loop. Streak lines of microorganism across agar at angle (not overlap lines), Repeat x2.
Make final streak. Incubate dish at suitable temp, should be possible to identify after 24 hours.
Describe the procedure to investigate antimicrobial properties of plants.
Grind up plant leaves + add little water/ethanol in separate mortars. Prepare agar plates. Spread fungi/bacteria over plate using aseptic techniques. Soak small disk of filter paper with plant extract + place on agar. Incubate 24-48 hours + compare results.
What are some things which can be measured about plants in terms of immunity?
Compare antimicrobial properties, effect of extract conc, temp or incubation + other variables.
Describe pour plates?
They allow microorganisms to grow on surface + within medium. Most of colonies grow within medium + are small in size + may be confluent. Colonies on surface are same size + appearance as streak plate.
What is the purpose of pour plates?
Can be used to test anti microbial effects of various substances.
What is the method for creating a pour plate?
Bunsen burner for convection current. Flame neck of inoculum test tube, take pipette worth of it into petri dish barely open. Don't squeeze teat bulb of pipette after it's in broth as it could cause bubbles or aerosols. Reflame bottle neck + replace cap. Same method for
sterile molten agar from water bath but no pipette, entire bottle. Swirl dish so well mixed and even. Allow to solidify. Ensure aspetic techniques.
What is the method for using a pour plate to investigate the antimicrobial effects of certain plants?
Crush 3g plant material + add 10cm3 ethanol + shake to make plant extract. Soak paper disks in these + use sterile forceps to drop onto pour plates (in 4 sections labelled). Incubate inverted. Aseptic techniques.
What is the method for creating a streak plate?
Aseptic techniques. Fill petri with agar. Flame inoculation loop + bacterial tube neck. Get bacteria on loop + smear on agar. Rub horizontally in section A, turn petri + repeat for zones B - D. Flame loop + bottle again. Invert petri + place in incubator.
How do you calculate oxygen uptake using a respirometer?
Add KOH to respirometer. CO2 produced by resp absorbed by KOH, reduces pressure + causes liquid to move towards respiring object. Distance moved reps O2 used in resp.
How do calculate CO2 production using a respirometer?
Add water to respirometer. CO2 produced by resp. If no change in liquid, vol of CO2 produced is same as O2 absorbed. Leave for same time + in same conditions as for KOH. If liquid moves away from respiring object, more CO2 produced than O2. Otherwise, less is produced.
How do you calculate RQ?
Vol of CO2 released/vol of O2 consumed.
RQ less than 1 =
RQ of more than 1 =
RQ of infinity
Some carb respired with lipids.
Anaerobic respiration with aerobic.
Just anaerobic respiration.
What measures should be taken to ensure the experiment is reliable and valid?
Use same apparatus, same organisms (age/metabolism), temperature (water bath), time period. Temp as affects resp rate and changes in gas vol.
How can we demonstrate that dehydrogenation is occurring?
Use redox indicators (change colour when reduced). Take up H2 + become reduced.
Give a method for demonstrating dehydrogenation?
Crush soaked peas to paste with pestle + mortar, place in boiling tube, repeat with boiled peas. Add 2cm3 methylene blue to each tube + place in 35c water bath. Observe colour changes.
How do you prepare the chromatogram?
Chromatography paper cut to fit tank, long enough to be slightly in solvent which will first saturate atmosphere. Pencil line drawn above base above solvent level. Spot solutions onto paper with micropipette onto origin line. Avoid contaminating, touch at edges.
How do you run the chromatogram?
Paper must be straight. Stop experiment before solvent reaches top of paper. Dry paper and mark solvent front with pencil. Calculate Rf values.
How do you calculate Rf values?
Distance moved by solute (pigment)/distance moved by solvent front. Always less than 1.
What should you be aware of when doing redox indicator photosynthesis experiment?
Use isolated chloroplasts by homogenising + centrifuging leaves to max electron production and avoid contamination with mitochondrial reactions which can also reduce DCPIP.
How do you demonstrate hydrogen acceptors in photosynthesis using redox indicators?
5g chopped lettuce in mortal + 10cm3 buffer solution, grind with pestle. Pour through muslin into beaker. Pour into centrifuge tube, ensure to counterbalance. Centrifuge at high speed 10-12 mins, discard supernatant. Add chloroplasts to 2 test tubes with buffer. Cover one with foil. Another tube with just buffer. Add DCPIP to each + place tubes in beaker of crushed ice. Place under light + check colour every 5 for 20 mins.
What are the results of DCPIP test on photosynthesis experiment?
Blue-green to green if positive or stays blue if no chloroplasts. If in darkness, stays blue-green as chloroplasts are green. Changes due to light dependent reactions so if in darkness, no change.
Using a haemocytometer:
Calculate num of yeast cells per mm3. There are 10 cells in a type C square (0.0025mm2) and the haemocytometer is 0.1mm deep.
0.0025 x 0.1 = 0.00025mm3
0.00025 x 4000 = 1mm3
10 x 4000 = 40,000 cells/mm3
1 x 10^-3
1 x 10^-6
1 x 10^-9
What is the yeast experiment for respiration to demonstrate hydrogen acceptors?
2cm3 boiled yeast in 1 tube, fresh yeast in other 2. Add 2cm3 distilled water to boiled and 1 fresh. Add 2cm3 glucose to final tube. Acclimatise in water bath for 5 mins then add 2cm3 methylene blue to each. Record how fast they change from blue to colourless.
What respiratory substrate would you expect to find in seeds?
Lipids. These provide more energy in a smaller mass, allowing for dispersal.
What would you expect to be the RQ of a growing yeast culture?
Very high as growing quickly and respiring anaerobically so takes in very little oxygen.
How do you extract DNA?
Add salt + cold water to plant, grind with pestle + mortar, strain off big pieces + mix rest with detergent. Leave 5-10 mins. Add protease. Add layer of cold ethanol. DNA precipitates out in series of white strands.
Why do you add salt to DNA for extraction?
Reduces solubility of DNA in water so easier to precipitate out.
Why do you add detergent to DNA for extraction?
Breaks down cell membrane.
Why do you add protease to DNA for extraction?
Separates DNA from chromosomes.
Why do smaller fragments of DNA travel further in gel electrophoresis?
Agarose gel has tiny pores which allow smaller DNA through easier than larger.
Why does DNA move towards anode (+) during electrophoresis?
DNA has small negative charge due to phosphate presence.
Describe how gel electrophoresis works.
Small electric current flows in buffer solution through agarose gel between cathode + anode connected to battery. Travel diff speeds + distances depending on size + charge. Wells in gel formed by placing comb in situ in gel + allowing molten agarose to set around it. DNA into wells. When DNA has moved, electric current stopped + dye may be added to make visible. UV light used to pick out fluorescing DNA bands.