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AS BIOLOGY
CORE PRACTICALS
PRACTICAL ONE
INVESTIGATING FACTORS AFFECTING THE HEART RATE OF DAPHNIA
Preparation a Take a small piece of cotton wool, tease it out and place it in the middle of a small Petri dish. b Select a large Daphnia and use a pipette to transfer it onto the cotton wool fibres. c Immediately add pond water to the Petri dish until the animal is just covered by the water. d Place the Petri dish on the stage of a microscope and observe the animal under low power. The beating heart is located on the dorsal side just above the gut and in front of the brood pouch (see diagram). Make sure that you are counting the heart beats, and not the flapping of the gills or movements of the gut. The heart must be observed with transmitted light if it is to be properly visible.
e Use a stopwatch to time 20 seconds, and count the number of heart beats in several periods of 20 seconds. The heart beat of Daphnia is very rapid, so count the beats by making dots on a piece of paper. Count the dots and express heart rate as number of beats per minute.
f At the end of the investigation, return the Daphnia to the stock culture.
General Notes 1.
Daphnia, the single, small heart is easily visible when viewed under transmitted light under a low power microscope. The heart rate (which can be up to 300 beats per minute) can be monitored and counted in different conditions for example changing water temperature, or changing the type and concentration of chemicals added to the water. A change in Daphnia heart rate may not be a predictor of a similar change in human (or vertebrate) heart rate under the same conditions, but the procedure provides an interesting technique for investigating the effects of different chemicals on a metabolic process. Daphnia is poikilothermic, which means that its body temperature and therefore its metabolic rate are affected directly by the temperature of the environment. The change in metabolic rate is reflected in the rate at which the heart beats (cardiac frequency). Within a range of 10 C above and below normal environmental temperatures, the rate of a metabolic process is expected to double for every 10 C rise in temperature. There will be considerable variation in the data gathered. Class results for the heart beat at any temperature should be recorded and mean results calculated.
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Ethical issues
In the UK it is considered ethical to use invertebrates, such as Daphnia in scientific studies, for the following reasons: Daphnia has reduced awareness of pain because of the lack of a well-developed nervous system. It is transparent and its heart is visible without the need for dissection. Daphnia is abundant in nature and there is no threat to it or its dependent species (food chains). Daphnia can reproduce asexually and may be clones, therefore there is no loss of genetic variation.
Factors to be controlled
Size of daphnia Habitat from which daphnia is obtained Temperature of the surrounding Oxygen concentration of the water surrounding the daphnia
If daphnia is treated with a chemical, The volume and concentration of the chemical should be controlled The duration of exposure to the chemical should be controlled Time should be allowed for acclimatisation
Instead of heating water in a water bath, you could surround the Daphnia in the Petri dish with a circular heating coil connected to a 6V battery. This will gradually heat the water in the dish, and the cardiac frequency can be estimated at 5 C or 10 C intervals. An additional, larger dish outside the small one could also be filled with water at the appropriate temperature to help reduce heat loss from the experimental chamber. Ethanol is highly flammable and harmful because of the presence of methanol. Once diluted to 10% and 1%, this is low hazard for the students. Physiologically-active compounds: Each compound will have different hazards and associated risk control measures. Acetylcholine is an irritant (to eyes, respiratory system and skin). L-adrenaline (epinephrine) is toxic by inhalation, in contact with the skin and if swallowed. Aspirin is harmful if swallowed, but a soluble tablet dissolved according to the manufacturers instructions would give a suitable concentration to use in the investigation at low hazard to the students. Heating due to the microscope lamp: When working with organisms under a microscope, the effects of heating due to the microscope lamp itself can be significant. Turning the lamp on only when observing the Daphnia will help, and LED microscopes produce less heat than those with incandescent lamps.