required practicals Flashcards
RP1 - light microscope - preparing a slide
If you want to look at a specimen (e.g. plant or animal cells) under a light microscope, you need to put it on a microscope slide first.
For example, here’s how to prepare a slide to view onion cells:
1. Add a drop of water to the middle of a clean slide.
2. Cut up an onion and separate it out into layers. Use tweezers to peel off some epidermal tissue from the bottom of one of the layers.
3. Using the tweezers, place the epidermal tissue into the water on the slide.
4. Add a drop of iodine solution. Iodine solution is a stain. Stains are used to highlight objects in a cell by adding colour to them.
5. Place a cover slip (a square of thin, transparent plastic or glass) on top. To do this, stand the cover slip upright on the slide, next to the water droplet. Then carefully tilt and lower it so it covers the specimen. Try not to get any air bubbles under there -they’ll obstruct your view of the specimen.
RP1 - light microscope - Observing the specimen
You need to know how to set up and use a light microscope (see Figure 2) to observe your prepared slide:
1. Start by clipping the slide you’ve prepared onto the stage.
2. Select the lowest-powered objective lens (i.e. the
one that produces the lowest magnification).
3. Use the coarse adjustment knob to move the stage up to just below the objective lens. Don’t use the eyepiece yet while you’re doing this, as you could cause the lens and stage to collide.
4. Look down the eyepiece. Use the coarse adjustment knob to move the stage downwards until the image is roughly in focus.
5. Adjust the focus with the fine adjustment knob, until you get a clear image of what’s on the slide.
6. If you need to see the slide with greater magnification,
swap to a higher-powered objective lens and refocus.
RP1 - light microscope - drawing observations
Drawing rules:
- use a pencil with a sharp point
- draw with clear, unbroken lines - no colouring or shading.
- If you are drawing cells, the subcellular structures should be drawn in proportion (the correct sizes relative to each other).
- drawing needs to take up at least half of the space available.
- title of what you were observing
- label your drawing (e.g. nucleus, chloroplasts) using straight, uncrossed lines.
- you need to work out and write down the magnification of your drawing. You can work this out using this formula: magnification = length of drawing of cell ÷ real length of cell.
For example, in Figure 4, 40 mm ÷ 0.3 mm = 133.
RP2 - How are microorganisms cultured in the lab?
Bacteria (and some other microorganisms) are grown (cultured) in a “culture medium”, which contains the carbohydrates, minerals, proteins and vitamins they need to grow. The culture medium used can be a nutrient broth solution or solid agar jelly. Bacteria grown on agar plates’ will form visible colonies on the surface of the jelly, or will spread out to give an even covering of bacteria.
RP2 - Growing microorganisms on an agar plate
To make an agar plate, hot agar jelly is poured into shallow round plastic dishes called Petri dishes. When the jelly’s cooled and set, inoculating loops (wire loops) can be used to transfer microorganisms to the culture medium. Alternatively, a sterile dropping pipette and spreader can be used to get an even covering of bacteria. The microorganisms then multiply
RP2 - Investigating the effect of antibiotics on bacterial growth
- Place paper discs soaked in different types (or different concentrations) of antibiotics on an agar plate that has an even covering of bacteria. Leave some space between the discs.
- The antibiotic should diffuse (soak) into the agar jelly. Antibiotic-resistant bacteria will continue to grow on the agar around the paper discs, but non-resistant strains will die. A clear area will be left where the bacteria have died - this is called an inhibition zone
- Make sure you use a control. This is a paper disc that has not been soaked in an antibiotic. Instead, soak it in sterile water. The control is not expected to have any effect on the bacteria, so you can then be sure that any difference between the growth of the bacteria around the control disc and around one of the antibiotic discs is due to the effect of the antibiotic alone (and not something weird in the paper).
- Leave the plate for 48 hours at 25 °C.
- The more effective the antibiotic is against the bacteria, the larger the inhibition zone will be
RP2 - Preparing an uncontaminated culture
Contamination by unwanted microorganisms will affect your results and can potentially result in the growth of pathogens. To avoid this, follow these steps:
- The Petri dishes and culture medium must be sterilised before use (e.g. by heating to a high temperature), to kill any unwanted microorganisms that may be lurking on them.
- If an inoculating loop is used to transfer the bacteria to the culture medium, it should be sterilised first by passing it through a hot flame.
- After transferring the bacteria, the lid of the Petri dish should be lightly taped on - to stop microorganisms from the air getting in.
- The Petri dish should be stored upside down-to stop drops of condensation falling onto the agar surface
RP2 - Measuring inhibition zones
You can compare the effectiveness of different antibiotics (or antiseptics) on bacteria by looking at the relative sizes of the inhibition zones. The larger the inhibition zone around a disc, the more effective the antibiotic is against the bacteria.
You can do this by eye if there are large differences in size. But to get more accurate results it’s a good idea to calculate the area of the inhibition zones using their diameter (the distance across). You can measure this with a ruler - see Figure 5. Don’t open the Petri dish to measure the inhibition zones they should be visible through the bottom of the dish.
RP2 - calculating the area of an inhibition zone -
The diagram below shows the inhibition zones produced by antibiotics A and B. Use the areas of the inhibition zones to compare the effectiveness of the antibiotics.
To calculate the area of an inhibition zone, you need to use this equation:
Аrea = πг^2
e.g:
The diagram below shows the inhibition zones produced by antibiotics A and B. Use the areas of the inhibition zones to compare the effectiveness of the antibiotics.
- Divide the diameter of zone A by two to find the radius:
Radius of A = 14 ÷ 2 = 7 mm - Stick the radius value into the equation, area = πг²:
Area of А = π x 7² = 154 mm² - Repeat steps 1 and 2 for zone B: Radius of B = 20 ÷ 2 = 10 mm Area of В = π × 10² = 314 mm²
- Compare the sizes of the areas
314 mm² is just over twice 154 mm², so you could say that:
The inhibition zone of antibiotic B is roughly twice the size of the inhibition zone of antibiotic A.
RP2 - Finding the area of a colony
The equation on the previous page can also be used to calculate the area of a bacterial colony. You just need to measure the diameter of the colony you are interested in first.
RP3 - effect of sugar solutions on plant cells
- Cut up a potato into identical cylinders and measure their masses. Make sure there’s no skin left on your potato cylinders, as this could affect your results.
- Get some beakers with different sugar solutions in them. One should be pure water and another should be a very concentrated sugar solution (e.g. 1.0 mol/dm³). Then you can have a few others with concentrations in between (e.g. 0.2 mol/dm³, 0.4 mol/dm³, 0.6 mol/dm³, etc.)
- Place one potato cylinder in each beaker, as shown in Figure 1. Leave them in the beakers for 24 hours
- Take the cylinders out, dry them with a paper towel and measure their masses again.
- If the cylinders have drawn in water by osmosis, they’ll have increased in mass. If water has been drawn out, they’ll have decreased in mass. You can calculate the percentage change in mass (see p. 399), then plot a few graphs and things (see below).
RP3 - Variables
The dependent variable in this experiment is the cylinder mass and the independent variable is the concentration of the sugar solution. All other variables (volume of solution, temperature, time, type of sugar used, etc.) must be kept the same in each case or the experiment won’t be a fair test.
RP3 - errors
Sometimes errors may occur when carrying out the method, e.g. if some potato cylinders were not fully dried, the excess water would give a higher mass, or if water evaporated from the beakers, the concentrations of the sugar solutions would change. You can reduce the effect of these errors by repeating the experiment and calculating a mean percentage change at each concentration
RP3 - Producing a graph of your results
You can create a graph of your results by plotting the percentage change in mass against the concentration of sugar solution. You can then draw a line of best fit, which you can use to determine the concentration of the solution in the potato cells.
RP5 - Investigating the effect of pH on amylase
The enzyme amylase catalyses the breakdown of starch to maltose. It’s easy to detect starch using iodine solution. This is how you can investigate how pH affects amylase activity:
- Put a drop of iodine solution into every well of a spotting tile.
- Place a Bunsen burner on a heat-proof mat, and a tripod and gauze over the Bunsen burner. Put a beaker of water on top of the tripod and heat the water until it is 35 °C (use a thermometer to measure the temperature). Try to keep the temperature of the water constant throughout the experiment.
- Use a syringe to add 1 cm³ of amylase solution and 1 cm³ of a buffer solution with a pH of 5 to a boiling tube. Using test tube holders, put the boiling tube into the beaker of water and wait for five minutes.
- Next, use a different syringe to add 5 cm³ of a starch solution to the boiling tube.
- Immediately mix the contents of the boiling tube and start a stop clock.
- Use continuous sampling to record how long it takes for the amylase to break down all of the starch. To do this, use a dropping pipette to take a fresh sample from the boiling tube every thirty seconds and put a drop into a well. When the iodine solution remains browny-orange, starch is no longer present - note down how many seconds in this happens
- Repeat the whole experiment with buffer solutions of different pH values (e.g. 6, 7, 8, 9) to see how pH affects the time taken for the starch to be broken down.
- Remember to control any variables each time (e.g. concentration and volume of amylase solution) to make it a fair test.
https://www.youtube.com/watch?v=JyXXoevEWc8
RP6 - method
A source of white light is placed at a specific distance from the pondweed. You should then leave the pondweed for a couple of minutes to adjust to the new light intensity before starting the experiment. When you’re ready to start, the pondweed is left to photosynthesise for a set amount of time. As it photosynthesises, the oxygen released will collect in the capillary tube.
At the end of the experiment, the syringe is used to draw the gas bubble in the tube up alongside a ruler and the length of the gas bubble is measured. This is proportional to the volume of O2 produced.
The experiment is repeated twice with the light source at the same distance and the mean volume of O, produced is calculated. Then the whole experiment is repeated with the light source at different distances from the pondweed.
RP7 - Measuring reaction time
Caffeine is a drug that can speed up a person’s reaction time. The effect of caffeine on reaction time can be measured. You will need another person for this experiment - this is the person whose reaction time will be tested. You will provide the stimulus. Here’s what to do:
- The person being tested should sit with their arm resting on the edge of a table (this should stop them moving their arm up or down during the test).
- Hold a ruler vertically between their thumb and forefinger.
Make sure that the zero end of the ruler is level with their thumb
and finger. Then let go without giving any warning. - The person being tested should try to catch the ruler as quickly as they can as soon as they see it fall.
- Reaction time is measured by the number on the ruler where it’s caught, at the top of the thumb (see Figure 1) the further down it’s caught (i.e. the higher the number), the slower their reaction time.
- Repeat the test several times then calculate the mean distance that the ruler fell.
The person being tested should then have a caffeinated drink (e.g. 300 ml of cola). After ten minutes, repeat steps 1-5.
RP8 - Investigating the effect of light
You can investigate the effect of light on the growth of cress seeds like this:
1. Put 10 cress seeds into three different Petri dishes, each lined with
moist filter paper. (Remember to label your dishes, e.g. A, B, C.)
2. Shine a light onto one of the dishes from above and two of the dishes from different directions (see Figure 1).
3. Leave your cress seeds alone for one week until you can observe their responses - you’ll find the seedlings grow towards the light.
4. You know that the growth response of the cress seedlings is due to light only, if you control all other variables. Here are some examples:
Number of seeds -use the same number of seeds in each dish.
Type of seed -use seeds that all come from the same packet.
Temperature - keep your Petri dishes in a place where the temperature is stable (i.e. away from heat sources and draughts).
Water - use a measuring cylinder to add the same amount of water
to each dish.
Light intensity - keep the distance between the bulb and dish the
same for each dish.
RP8 - Investigating the effect of gravity
You can do another experiment to investigate the effect of gravity on the growth of cress seeds, like this:
1. Put four cress seedlings into a Petri dish that’s lined with damp cotton wool. The roots of each cress seedling should be pointing in a different direction (see Figure 2).
2. Store the Petri dish vertically for a few days in the dark, so you know your results won’t have been affected by light.
3. You should find that the roots of each seedling grow downwards (towards gravity) and the shoots grow upwards (away from gravity).
RP9 - quadrats
RP9 - Transects
