KMH's+Milestone+Map

__**KMH’s Milestone Map**:__ This Milestone Map is a compilation of my work with zebrafish, including my work this semester and my work over the summer at Notre Dame’s Friemann Animal Research Center. I am sorry if it sounds repetitive at times, as some sections overlap. This Milestone Map tells you everything that you need to know about the care and development of zebrafish. I hope that you enjoy your holiday season and if you have any questions feel free to email either Lesley or myself. Lesley's Map and mine overlap on most of our information. For a more detailed synopsis of background information look to her milestone report (a written record of the powerpoint slides) and look to mine for a more detailed version of setups. Thanks for everything and have a very merry christmas!!!!!!!!!!!!!!!!


 * __Fundamentals__:** What you need to Know/Have to Get Started

-Learn background information on the species. See PowerPoint, located on the Zebrafish main page. -Learn background information about brine shrimp. General information: live in salt water, only hatch if the water is heated and is on constant motion, and once they have been immersed in filtered tank water, they will live for up to 3 hours. They are also attracted to light.
 * Background Information on Zebrafish:**


 * How to Tell the Difference Between a Male and Female Zebrafish:**

It is very difficult to tell the difference between a male and a female. Females’ stomachs protrude more than the males do and they have a golden tint. Males’ bodies are more stream-lined and have a silver tint to them.


 * Supplies Needed to Setup Your Own Aquarium:**

-Glass tanks -Lids for the tanks -Gravel or pebbles to line the bottom of the tanks -An air pump to circulate the water in the tanks through the filters. -Filters and filter pads, one for every tank -water heaters for every tank -thermometers for every tank -Fish (obviously), wild type and albino is preferred, so one can cross breed them in experiments. Also, a normal 2.5 gallon tank should not house more than 15 fish. In conclusion, the males and females should be housed in separate tanks. -Marine Salt Mix: to salinize the water to be used to hatch the brine shrimp. -Reverse Osmosis tank: provides clean, filtered water for the fish tanks -with this, availability to tap water is required -Power strips: to connect the heaters and air pump to an outlet. -25 ft package of air tubing -Air tubing holder (non-corrosive aquarium gang valve with anti-siphon capabilities works best) -Labeling tape (in multiple colors), I have found that color-coding all of the wires with the tank it connects to, helps everything run much more smoothly. -Pipettes -Petri dishes: to house the newly-formed fry -Many plastic, 1000mL containers: these are to hold hatched fry, transport water, etc. -Tetramin: fish flake food, is to be used when the Brine shrimp have not yet hatched. -2 L plastic bottle (pop bottle): to use as a brine shrimp hatchery - Fry diet: Powder 0&1 for days 4 to 7, Powder 1&2 for days 9 to 14, and finely crushed Tetramin for the third week. -Egg water: to put in Petri dishes with the collected eggs -Brine shrimp eggs -Breeding tanks and Tupperware bins -Lamp with a bendable neck: to attract brine shrimp to the side. -Graduated cylinder: to measure the amount of brine shrimp eggs to put into hatchery. -Funnel: to add salt to the water to salinize it. -Marbles: if fish are breed in a tank instead of a Tupperware bin. -Bleach: to clean the tanks, heaters, and other containers. -Green nets to catch the zebrafish. -White nets to catch the brine shrimp.


 * __Comprehensive Guide to Get Started:__**


 * How to Setup the Lab:**

-Tanks: Fill up the Tanks with filtered water and place them side by side. Make sure you label each tank numerically and with label telling the sex and date of birth of the fish that occupy it. In each tank, there should be gravel, heaters, filters, and thermometers. (Unless it is a breeding tank). -Shelves: The shelves are adjustable and are able to slide up and down. To do so, pull the shelf straight up and readjust the black clips where you want them. -incubator -Heaters: After being plugged in to your power strip, insert one in every Tank. There will be a lot of cords so it is helpful to color code the cords to the appropriate tank with colored tape. Slide the suction cups on over the base of the heater and insert it onto the wall of the tank. Make sure that it is fully submerged and only the head is protruding or it will not turn on. -Air tubing: Cut the air tubing to the appropriate length, making sure that it can comfortably reach from the tank to the gang valve. Place the gang valve on the middle of the shelf and connect a piece of tubing to each valve. Next, connect a piece of tubing from the gang valve to the air pump setup on the shelf below. Have the air pump on all the way and have each gang valve that is connected to the tank filters on half way. Air will now run from the air pump, to your filters to circulate the water in your tanks. Again, it is very helpful to color code the tanks to the gang valves so you are quickly able to tell what tube goes to what filter.
 * The fish have a specific light schedule that they must follow: Every school day turn the lights on in the lab and leave them on throughout the day, turning them off when you leave at the end of the day. During the weekend, just leave the blinds cracked so natural light will be able to get in.
 * Color coding is the best way to keep your lab organized!
 * Make sure to label the date of birth, sex and phenotype of your fish when you place them in a tank because it is very easy to loose track of them!


 * How to Setup a Brine Shrimp Hatchery:**

- First, you need to make salt water. (See above instructions) - Supplies needed for your brine shrimp hatchery: shrimp eggs, water heater, lamp, a 2 L pop bottle, air tubing and one hard, clear pipe with a diameter of 1 cm. - Next, cut off the top of the pop bottle and insert the heater, making sure that it does not touch the sides of the bottle. - Fill up the pop bottle (with ½ a gallon of salt water). Make sure to leave at least five inches exposed at the top of the bottle. - Measure 7 mL of brine shrimp eggs into a graduated cylinder. - Pour the brine shrimp into the pop bottle - Finally, connect the plastic pipe to air tubing that is connected to an air pump and insert it into the bottle; put the air on about half way. - The brine shrimp take 18 hours to hatch. You will see best results after 2 days. - After about four days, or when you are no longer able to collect the brine shrimp, empty the bottle (pour it down the drain) and make another.


 * How Best to Care for the Fry:**

-Incubator -Diet -Egg Water -Once the eggs have been collected, place them into a Petri dish that is filled half way with egg water. -The eggs will take three days to hatch. During this time, they need to be labeled and put into an incubator. -The incubator should be on level 3 (about 27 degrees Celsius) -When the eggs are placed into the incubator, take a plastic 1000mL container, fill it with filtered water and place it into the incubator. -Once the eggs have all hatched, they are ready to be transferred into the 1000 mL container. -With a pipette, place them in the new container. They are now fed once a day with the appropriate powdered diet. Dab the top of your finger on the powder and sprinkle it over the top. Make sure not to feed them too much because it will put a coating over the surface of the water and they will be unable to get oxygen. -We have found it best to keep these containers in the incubator so the temperature never fluctuates. They are very sensitive to drafts and often die before they become adults.
 * It is important to label them when you first breed them, so you know what type of fish they are and so you can distinguish which powder diet you ought to be feeding them.


 * How to Clean Tanks and Filters:**

__Cleaning Filters__: - Filter pads should be changed about every 3 weeks (It depends on the amount of fish in your tank). - To change your pad, pull the blue filter out of the tank and pop the front cover off. - Pull out the used filter pad and put another one in its place. - Then, pop the cover back on and place it back into the tank. __Cleaning Tanks and Gravel__: - Remove the fish from the tank and place them in a holding tank. - Unplug the heater and the filter. - Remove the heater, thermometer, and filter from the fish tank. - Next, slowly pour the dirty water into the sink, making sure not to let any rocks go down the sink. - Then, pour the gravel/marbles into a strainer or mesh bin. - Rinse the stones thoroughly, using very little bleach. - Next, fill the empty tank up with normal tap water. - Add bleach and let it soak for 24 hours. - Then, scrub the sides with a sponge, pour the bleached water out and rinse the tank repeatedly with tap water, making sure that there is no scent of bleach left over. - After there is no trace of bleach left, fill the tank up with water from the reverse osmosis tank and reinsert the gravel, thermometer, water heater, filter, and of course the fish. - *It is really important that there is no bleach left, they are very sensitive to it and the littlest amounts will kill them.


 * How to Make Salt Water:**

- Fill a gallon container (with a lid) half way with filtered water from the reverse osmosis system. - Next, measure out one cup of Marine Salt Mix and pour it into the funnel. - Place the funnel on top of the gallon container. - Next, let water run through the funnel into the container, this helps to dissolve the salt quickly and it allows it to pass through the funnel opening. - Then, finish filling the gallon container up to the top. - Finally, screw the lid onto the container and shake the mixture up until the salt is completely dissolved. - Now it is ready to use in your brine shrimp hatchery!


 * How to Breed the Zebrafish:**

-Although, we have had limited success at St. Joe with this endeavor, we did however have the technique mastered over the summer. - It is best to use three females and two males (always have more females than males in the breeding tank) -They breed in the mornings (probably the reason for our unsuccessful attempts) -They breed asexually. -Make sure that you do not feed them when they are in a breeding tank because it causes them to swim to the bottom of the tank to look for any sunken food and they will stumble upon their eggs, and eat them! -There are two different methods that we have done. -1st method: A glass tank, with no filtration system, that is filled with marbles. -Place the fish into the tank and the next day collect the eggs that are embedded in the marbles. (See How to Collect Eggs) -2nd method: A plastic breeding tank, with holes in the bottom, placed into a Tupperware bin filled with filtered water. -Fill the Tupperware bin with filtered water. - Next, suspend the plastic breeding tank a ½ in. from the bottom of the bin with string. Make sure that the tank stays in the middle! -Place the fish into the tank and tape the lid down. Also, tape the lids to the walls o the Tupperware so that the tank is not disturbed. -The next morning, collect the eggs. (See How to Collect Eggs)


 * How to Collect Eggs:**

-It depends on the method of breeding that you used. -If you used the 1st breeding method, egg collection is much easier. -First, remove the fish and place them back into their original tanks. -Next, remove the small plastic breeding tank. -Then, slowly pour the water out into the sink. Carefully, so as not to disturb the eggs that are settled on the bottom of the Tupperware bin. -Slowly pour the remaining water into Petri dishes. -Fill up a Petri dish with egg water and make a label with the fish’s phenotype and date of birth on it to place on the lid. -Using a pipette, suck out the fertilized eggs and place them in the dish. Make sure to differentiate between the fertilized and unfertilized eggs. The unfertilized eggs will be ruptured-looking or be a milky-white color. The fertilized eggs are transparent and have a visible nucleus. -If you are unsure if the egg is fertilized or not, place in the Petri dish, it will not hurt anything! -If you used the 2nd breeding method, egg collection will be a bit more time consuming. - For this collection method, you will need a siphon, bulb, and container. - This method works best with two people. -First, place the tip of the siphon tubing on the bottom of the tank in the marbles. -Next, connect the bulb to the end of the tubing and contract it in and out until suction is created. -Then, remove the bulb from the end of the tubing and place the exposed end in a container placed on the floor. -Run the tip through the marbles like a vacuum, sucking up all of the eggs. -The collected water from the tank will run through the tubing into the container below. -Once you have collected all of the eggs, lift the tip out and tip it up so all of the water in the tubing runs into the container located on the floor. -Then follow the same steps as before to gather the eggs out of the collected water and place them in a labeled Petri dish. -* If you have trouble locating the eggs in the tank, it is helpful to look from underneath and then they are much easier to locate.


 * How to Collect Brine Shrimp:**

-When the eggs are hatched and ready to be collected, the water will have an orange hue and the water will be very cloudy. - Next, pull the air tubing out of the pop bottle. -Place the lamp head on the side of the bottle near the top. -Turn the lamp on and wait several minutes. -The light attracts the brine shrimp and they swim towards it. -You will know that the shrimp are ready to collect when you see a clump of bright orange on the side of the transparent bottle. -Place your pipette in the middle of the orange blob and suck the brine shrimp out of the bottle. -*There is another way to collect the brine shrimp, but it is too time-consuming and inefficient and not worth explaining.


 * How to Feed to Fish:**

-How much and How often. -The fish can be fed either brine shrimp or Tetramin flakes. -They will eat everything you give them so make sure not to put in too much! -Feed them with brine shrimp, and if the shrimp has not hatched yet, feed them the Tetramin flakes. -They need to be fed three times a day, and if it’s the weekend, just make sure to feed them extra at every meal. -Amounts: brine shrimp: feed them two full pipettes for every 5 fish at each meal. Tetramin flakes: one large pinch for every 5 fish at each meal.


 * How to Setup a Reverse Osmosis Tank:**

-Locate Mr. Mankowski and hand him the instructions, the man’s a genius! -Notre Dame ordered it for us and he assembled it. -To install a reverse osmosis filtration system, you first need to have access to tap water. -The tank filters the tap water twice, purifying it, and then it places it in a 3 gallon holding tank (located under the sink) at a rate of 10 cc a minute. -There is a special faucet in the lab that allows us to take water out of the holding tank and it has a steady and consistent flow. -The tank, once drained, takes about five hours to refill.


 * How to Deal with the Fish (Getting your License):**

-mistreating -When Lesley and I worked at the Friemann Animal Research facility this summer, we were required to take a course on the proper care of lab animals. -Luckily, we passed and received certificates. -The course included the proper care of research specimen, especially aquatic creatures. -We learned the proper techniques and instruments used to euthanize the animals and the extremes that our experiments can reach.

- Most common ones -odd behavior, indications of problems with the fish
 * Fish Diseases:**

-The most common zebrafish disease, and the only one you will see, is called Ick. -It is highly contagious bacterial disease and it is characterized by bleeding around the gills. -The fish then need to be quarantined, so as not to expose any more fish, and given antibiotics. -If the fish are floating near the top or are swimming belly up, that is not a good sign. -Also, if they are not swimming around and are all huddled together near the bottom of the tank, something is not right. -If the fish are exhibiting any of these signs, the temperature could be unstable, the filter could need to be changed, they could be hungry, the tank could need to be cleaned or the water is contaminated. -If you feel like water could be contaminated, take the fish out of the tank immediately and clean it out.


 * __Future Aspirations:__**

-Stabilize our breeding techniques: our breeding techniques have yet to become 100 percent efficient. -Stabilize a colony of offspring: for some reason our 2nd generation fish die after several weeks and we have yet to come up with a solution as to why this occurs. -Get in contact with Dr. Hyde for some direction on research
 * have already emailed twice

-email the lady from Thomas Jefferson University (possibility) - Learn more about the fish and the studies that are already being done in research labs around the world. -Develop a program to be used for Mrs. Brunner’s Biology classes
 * Concerning development and phenotypes and genotypes

-put together some experiments for them, based off of the Thomas Jefferson University’s program. -Come up with our own research project to pursue in the 2nd semester. - Some possibilities that interest me are: - Looking at the Vitamin D receptors in the fish and possibly build off of Notre Dame graduate student, James Whitcomb’s work with Vitamin D receptors. -Looking at different elements that have affected human fetal development and duplicating it on the fertilized Zebrafish eggs. An example is alcohol. - Develop a way of highlighting certain body parts on the zebrafish, internally. I have been researching the harmless effects of highlighting fluids and we could somehow figure out a way of directing the concentration of these fluids to a certain part of the zebrafish’s body, scientists could be better able to pinpoint exact body parts (internal organs) on the fish.