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http://www.practicalbiology.org/areas/advanced/control-and-communication/control-of-heart-rate/investigating-factors-affecting-the-heart-rate-of-daphnia,92,EXP.html
http://www.aquarticles.com/articles/management/Childers_Raising_Daphnia.html\
http://en.wikipedia.org/wiki/Daphnia#Reproduction
Caffeine
While Reading up on Daphnia we learned that many pet owners use Daphnia as fish food. We thought that it would be a practical experiment to see how extreme the living conditions a Daphnia fish can handle to improve or reduce shipping and storage cost. Daphnia females can reproduce parthenogenesis (asexually) during ideal conditions about every ten days. In the fall and winter the Daphnia reproduce sexually, and create what is called winter eggs. These winter eggs have an extra layer to them so that they can survive the winter and begin the cycle in the spring. We began to wonder if harsher living conditions would be better to cultivate Daphnia because it seemed to produce eggs that could survive more strenuous living conditions than eggs cultivated in ideal living conditions. After reading we learned that Daphnia’s heart rate increases when caffeine is introduced into their environment, and that we could count the Daphnia’ heart rate by watching the heart beat through a microscope. This gave us the idea to use change the amount of caffeine in the water and monitor the effects this difference has on the heart rate of the Daphnia. The higher the heart rate we are assuming the greater level of stress, which is the aspect that we will be testing. If the Daphnia are stressed they will being to reproduce sexual. When they are not stressed they produce asexual in a reproduction process that takes approximately ten days. The Daphnia eggs produced during higher stress levels should be the winter eggs (sexually produced eggs that have a harder outer shall) but if the Daphnia eggs are produced asexually they will not have the harder outer shall and therefore the eggs will die easier after being placed in harsh living conditions. After the Daphnia have had their heart beats reordered and assumed stress levels recorded for a period of ten day we will put the all of the colonies into the refrigerator to put all of the variables under the same strenuous living conditions. After a short period in the refrigerator (which supposable Daphnia can survive) we will take all of the colonies out and put them all into ideal living conditions with out the caffeine present. This period of time we will call the ideal period or spring period of time. During this spring time period we will observe the eggs that were frozen and record which ones colonies had the most eggs that hatched and from the eggs that hatched which of those Daphnia did not miserly hatch but lasting survival.

Materials
1 Shrimp net
2 Shallow tank
3 Water
4 Light
5 Thermometer
6 Microscope – low power, transmission
7 Petri dish
8 Small piece of cotton wool
9 Stop watch
10 Pasteur pipette
11 Caffeine powder
12 yeast
13 soy flour
14 hard-boiled egg

Maintenance of the Daphnia
Food (document how often each tank is feed)
1 package of dry active yeast w/ one cup of soy flour
¼ teaspoon of this dry mixture into a 1 cup of warm water 1 or 2X daily
Note: when water is clear again it is time to feed
Blending 1 hard-boiled egg in 500 cm3 of water
Caffeine powder
Harvest (document how often each tank is needed to be harvested)
Pour old water down the drain while using a shrimp net to filter out Daphnia
Refill tank with clean water
Water temperature
Keep water at room temperature; preferably 72 degrees F (if tank is close to floor)
Use a temperature tank; if not available to maintain water temperature put Daphnia tank in a larger tank that will help to slow the loss of heat. We will allow the water to vary slightly in degree, and will have the specific water temperature in the data and results.
Light
Give them some light 24 hours a day
Oxygen
Shallow tank to ensure enough oxygen
Cleaning (document how often each tank is needed to be cleaned)
Take scum off surface of the water
Leave debris at the bottom – it is probably Daphnia eggs
Taking Heart Rate
1 Take cotton wool in Petri dish.
2 Transfer Daphnia onto cotton
3 Cover Daphnia in water
4 Observe Daphnia under microscope on low power. Locate heart on the dorsal side just
5 above the gut but in front of the brood pouch.
Note: Don’t count the flapping of the gills/movement of gut. The heart can only be seen with transmitted light.
6 Count heart beats in 3 periods of 20 seconds
Note: to count the heat beats the best tap a pencil for every heat beat during the 20 seconds and then count the number of pencil dots after the 20 seconds are up
7 Return Daphnia