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Amal Hulangamuwa Intec Asia Campus


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.

Investigating the effect of temperature

g Record the temperature of the water in the Petri dish. h Add pond water at a different temperature to the Petri dish. Allow the Daphnia some time to acclimatise, but keep a check on the temperature of the water in the dish and add more hot or cool pond water if necessary to adjust the temperature. i Record the heart rate again as in step e. j Plot a graph of mean frequency of heart beats per minute against temperature.

Investigating the effect of chemicals

k Take a large Daphnia from the stock culture and record its heart beat at room temperature in pond water. l Add one drop of 1% ethanol to 5 cm3 of pond water in a beaker. Mix well. Draw the pond water off the Daphnia with a pipette and replace it with 2 or 3 cm3 of the water containing ethanol. Record the rate of heart beat again. m Repeat step l using 10% ethanol in place of 1%. n Repeat with other chemicals such as acetylcholine, L-adrenaline, caffeine or aspirin.

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

Health, Safety and Technical notes

1. Keeping live cultures of Daphnia:

Daphnia are crustaceans, commonly found in ponds and lakes and widely sold as live fish food.They feed by filtering minute particles such as bacteria and algae, from the fresh water in which they live. Daphnia can be kept in any watertight container containing tap water that has been allowed to stand for a few days. Keeping a few Daphnia is not difficult, but cultivating a vigorous, dense colony requires some care. A good supply of oxygen is necessary, either by aeration or by using a large shallow tank to ensure that a large surface area of water is exposed to the air. Warming the water to about 15 C also ensures rapid growth of the colony. You can purchase live cultures from suppliers, including local aquarists. Alternatively, you can collect adult Daphnia by pond dipping. In this case you must observe strict hygiene procedures. Stock purchased from aquarists is usually free from this hazard. The safest, most hygienic and most convenient ways to provide the necessary food for a colony of Daphnia is to feed them on a few drops of a suspension of fresh yeast or of egg-yolk medium (made by blending a hard-boiled egg in 500 cm 3 of water). Small, regular supplies of food are required.

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.