Bridging the Gap Between Developmental Genetics and Paleontology


Alizarin Bone Staining Protocol:

Introduction: Staining with Alizarin Red S colors bones red, increasing both the speed and accuracy with which osteological traits can be scored. We use it on formalin-fixed specimens that have been stored in alcohol (50% isopropyl or 70% ethanol) to facilitate scoring of lateral plate, pelvic, gill raker, and other phenotypes in threespine and ninespine stickleback. Formalin fixation is mandatory before staining, and fish appear to stain better if they have been held in alcohol for at least a few days.

Preparing Staining Solution*:
*We use Fisher brand potasium hydroxide pellets and Sigma brand alizarin red S (alizarin sodium sulfonate)

1. Use 4 L flask, fill with a bit of water (<1/4 volume).
2. Add one large Potassium Hydroxide (KOH) pellet (approximately 400 mg), mix until it dissolves.
3. Add two large scoops of Alizarin Red (approximately 1.7 g), mix well.
4. Fill vial with water and mix again.

Preparing Destaining Solution:

1.Add one large potasium hydroxide pellet to 4 L of H20 and mix until the pellet is completely dissolved.

Staining Fish:

1. Remove isopropanol from jars and store for later use. Wash fish with water.
2. Place jars in plastic dishes. Remove labels from jars and place each label in the dish the jar it came from is in (labels should always remain with jars).
3. Add staining solution such that all fish are completely covered, turn over fish or place cheese cloth on top if necessary (if specimens are not completely submerged, the part of the fish that is not in contact with the solution will not stain). Leave specimens in the staining solution for 3-6 hours (samples are ready when fin rays look dark purple against a light background).
4. Once samples are stained remove staining solution and place it in appropriate repository in fume hood.
5. Add destaining solution and rinse samples in it well, cover jars and shake vigorously (the destaining solution will be heavily stained). Remove this solution and place in appropriate repository in fume hood.
6. Add new destaining solution and let samples sit in it for one hour. Remove destaining solution and dispose of it.
7. Repeat step 6 at least two more times.
8. Add water and rinse at least twice. Place labels back in jars. Add water and let rinse over night.
9. The next day, change water and let rinse all day. Change water several times during the day. Add 50% isopropanol and store samples.

Procedures for Shipping Live Threespine Stickleback or Fertilized Eggs

INTRODUCTION: We frequently ship fertilized eggs or one-year old threespine stickleback (Gasterosteus aculeatus) from Alaska to New York. We may ship them as baggage when somebody is flying back to Stony Brook, but we often ship by overnight courier. In Anchorage, Alaska, shipments may be brought directly to the main terminals of FedEx or UPS near the International Airport. Be sure to deliver the fish before the shippers?closing times (previously 2:00 pm) for overnight shipments and to determine that the carriers are shipping and delivering on both the day you plan to ship and to receive the shipment. We have found that FedEx often fails to deliver shipments in one day from Anchorage to New York, and we have switched to UPS for transcontinental shipments.

If you plan to air-ship stickleback or stickleback eggs and have never done it before, read this entire document because some of the steps require lead time. Time sensitive steps are highlighted in red. Preparing fish or eggs for shipment always takes longer than I anticipate, and I am always surprised how close we are to the deadline. Missing a shipping deadline means holding the fish or eggs an extra day, which may violate your protocol, if you are trying to treat all the animals the same way, and may cause stress due to fasting or mortality. If you miss a shipping deadline on a Friday, you may have to hold the shipment until Monday, which may result in eggs hatching. At the very least, you will have to use several hours to unpack and repack the animals that missed the shipping deadline. You do not want to deal with this!

MATERIALS ?/B> AC-powered aquarium pump, battery-powered aquarium pump, gang valves, tygon tubing, air stones, thermometers, duct tape, strapping tape (with fiber glass strands in it), and freezer packs (e.g., blue ice). For adult and juvenile fish: ice chests of appropriate size, one-gallon water bottles, two-liter or smaller juice and soft drink bottles. For fertilized eggs: small ice chest or Styrofoam box, 50 or 35 ml centrifuge tubes, refractometer calibrated to measure salinity (optional). Write your name and contact numbers on the inside and outside of the ice chest or box using a water-proof felt-tip marker or enclose a label with the address and contact information on it..

FISH AGE ?/B> Alaskan stickleback usually breed at the end of their third summer (age 2+), and we prefer to ship 1+ fish because older fish are in reproductive condition and are not very resilient. In addition, 1+ fish are large enough to ship well but do not take up as much space; we can ship more small fish. However, young-of-the-year fish (0+) are too delicate in early summer to ship, and we are rarely in Alaska during the late summer. We do not have any experience shipping 0+ fish in late summer, but it might work well.

EGG AGE AND TREATMENT ?/B> We hold fertilized eggs in Petri dishes with 3 ppt (10%) sea water (use sea salt and deionized water) at 20 C for three days. Day 0: fertilize. Day 1: remove dead eggs and separate the adhering eggs from each other. Day 2: Check again to remove dead eggs. The embryos should be obvious. We dip our eggs for 30 seconds in Kordon Acriflavine (Kordon division of Novalek, Inc., 2242 Davis Ct., Hayward, CA 94545, USA) followed immediately by a 30-second dip in Methylene Blue, following the manufacturers?instructions for dilution, using 10% sea water. Using this procedure, the fry grow up free of Glugea anomala, which is a serious pathogen that can infect an entire fish colony if the lab is contaminated. Our developing eggs never grow water mould when they are treated this way (Check the fry when they hatch for infection when they hatch at the destination lab, and be prepared to discard 5 ?10% of the families.)

CONTAINERS ?JUVENILES AND ADULTS ?/B> Several days before it is time to ship the fish, obtain the right-size ice chest for your needs, plastic one-gallon water or milk bottles, and two-liter or smaller juice or soft drink bottles. We try to buy bottles of fluids that we want to drink and accumulate them until we need them for shipping. Select the right combination of bottles to fill your ice chest. We commonly stand one-gallon water bottles up in the ice chest and they fit tightly across the bottom of most chests in a double row. We lay two-liter or smaller soft drink or juice bottles on top of the gallon bottles to fill the space above them and keep the bottles from moving around in transit. Last summer we discovered that rectangular two-liter Ocean Spray cranberry drinks pack extremely well into medium-size ice chests. Be creative. Well before using the milk, juice, or soft drink bottles, fill them several times with hot water and allow them to soak all day with the cap off. Be sure to save the caps. Air-dry the bottles for a day or two with the cap off to allow odors to escape. Some bottles may still smell from their original contents after this process, but the fish seem to tolerate the odor.

CENTRAFUGE TUBES FOR EGGS ?/B> We use 35 ml tubes for small clutches of eggs (<40) and 50 ml tubes for full clutches of about 150 eggs. Clutches of a few hundred eggs from anadromous or large freshwater females may be divided up among a few tubes.

CAPTURE, HOLDING, AND INITIAL PROCESSING ?/B> It takes several hours to transfer, chill, and pack eggs or juvenile stickleback for shipping. FedEx and UPS accept overnight packages until early afternoon, so you must get an early start to make their closing times. If you take the shipment with you on an early flight, plan to stay up all night.

Set traps for live fish two full days before shipment. Bring the live fish back to the motel or lab the day before shipment in as much lake or stream water as possible. Make sure the water is as clean as possible, with a minimum amount of floating debris or mud. Be sure to put a water-proof label into each container of fish, if they come from more than one source. Fish can be transported from the field in large buckets or in ice chests with a hole drilled in the top for an air line. Your vehicle is going to get wet if you carry water in buckets, and it is always good to buy a small plastic tarp to put down to keep the vehicle dry. Aerate constantly from the time of capture until you seal up the fish for shipment. It is preferable to ship the fish in clean, aerated water that has never had fish in it or in water from a large holding tank that has had fish at low density, but this may not be possible. Keep the fish cool (<20 C) and do not feed them before shipment. The first day after capture, they will defecate most of their gut contents, and this will minimize feces in the shipping water. However, if you must ship them in the water in which they were held, be sure to use water from the top of the container so feces from the bottom is not poured into the shipping bottles.

NUMBER OF FISH PER BOTTLE ?/B> About fifteen 2.5 cm standard length (SL) stickleback can fit into a one-gallon bottle and seven or eight in a two-liter bottle. Fewer large stickleback can be shipped in the same volumes, and we ship two anadromous stickleback (~ 6.5 cm SL) in each two-liter bottle. It is hard to know if more fish per unit volume would fit, but this works, and if you exceed a density threshold, you will lose the entire bottle of fish. Don’t get greedy or you may lose them all! Some people like to use zip-lock bags or special fish shipping bags. The former may pop open from depressurization and then collapse, suffocating the fish. I have not used special fish shipping bags. Nor have I used oxygen, but this may be an option to extend shipping time and possibly fish density.

COOLENT ?/B> The carriers do not ship ice. If you use ice, do not volunteer that information and be sure to seal up your ice as well as you seal up your fish. I recommend that you use freezer packs or the coolant from freezer packs that you have repacked in zip lock sandwich bags. You can cut open the freezer packs (it is not generally toxic) and distribute the contents to smaller sandwich-size zip lock bags to avoid over-cooling one area in an ice chest . Be sure to tape the bags of coolant closed with duct tape if you do this. We always buy larger, less expensive freezer packs and divide the contents into about half-full sandwich bags about 1 cm thick before freezing for shipment of eggs in Styrofoam boxes. Be sure to prepare your sandwich bags in advance and to freeze them or freezer packs the night before you ship. They will not freeze in a few hours the day you use them.

PREPARING FISH FOR SHIPMENT ?/B> Be sure you know the combination of bottles and number of fish per bottle before you prepare to pack the fish for shipment. Do a dry run with the bottles you plan to use. Of course, water bottles do not need to be washed, but bottles that had other liquids in them should have been thoroughly rinsed and dried by this time (see above). Otherwise, you may discover that you cannot ship all your fish or that you have unused bottles or space in the cooler. Again, be sure to freeze your freezer packs the night before shipping.

Packing the fish is going to take a few hours regardless of how large your shipment is because you must cool the water with the fish in it; larger shipments take substantial additional time because packing is time consuming. Never move fish between water with sharply contrasting temperatures.

Put a label with the name of the population inside the bottle and label the outside of the bottle with a water-proof felt-tip marker. Fill shipping bottles to were the neck begins to narrow. If you are using water in which the fish have been held, be careful to avoid pouring feces or other debris into the shipping bottle. Pouring the water through an aquarium net may help. Once the bottle is filled, catch the fish with an aquarium net and carefully transfer them to the bottles. Your hands should be wet before you touch a fish. Once the appropriate number of stickleback is in a bottle, put it into a sink or tub full of ice so the ice comes almost to the top of the bottle. Put an air stone in the bottle, and monitor the temperature. The air stone should be bubbling vigorously but hung high enough in the bottle so it is in the top few cm of water. If the air stone is near the bottom of the bottle and bubbling vigorously will tumble, batter, and stress the fish. The gallon bottles will take more than an hour to cool down to 5 ?8 C, and smaller bottles cool slightly faster. Do not let the water chill to 0 C, or the fish may die. As the bottles reach the desired temperature, move them into the ice chest, while continuing to aerate the water in them. If you do not have enough air stones to aerate all the bottles, move the air stones among the bottles from time to time.

You can use about 300 ml of coolant from freezer packs per gallon bottle, if the coolant was frozen in a conventional freezer. Distribute the freezer packs among the bottles. A large ice chest (e.g., one with 8 ?10 one-gallon bottles and several two-liter bottles) with the water in the bottles chilled to about 5 C can actually be shipped over night without any additional cooling, but it could be risky, and we have done this only when a particularly uncooperative airline (Alaska Airlines) made us remove the ice from an ice chest. The freezer packs provide a margin of error, in case there is a delay.

If it will be a long trip to the shipper or airport, take the bottles of fish as is. They should be kept open and aerated en route, but minimize warming during this time. (Some plastic bags of ice may be packed around the bottles to keep them cold.) If you are laying smaller bottles on their sides on top of the larger ones, it will be necessary to keep these bottles in a bucket or box with ice. However, if the point of departure is nearby, go directly to the next step.

SEALING FISH FOR SHIPPING ?/B> This step will take a person working alone several minutes per bottle because fish tend to escape and it is easy to spill out too much water if you rush. Pour about two thirds of the water out of the bottle. Once you seal the bottle, oxygen is going to be a more seriously limiting factor than water for the chilled fish. You may wish to press an aquarium net against the mouth of the bottle to prevent fish from spilling. Pour at least some of the discarded water into a clean container so you can add some water to a bottle, if you pour out too much. Once the volume is adjusted, squeeze the bottle slightly so that when you replace the cap tightly, the bottle will have a slight indentation. This is intended to prevent the bottle from popping open due to depressurization during a flight. With the bottle still slightly squeezed, screw or snap the cap on securely. Tape the bottle cap securely to the bottle using duct tape. Check the bottle to be sure it has a slight depression to allow expansion. Repeat this until all the bottles are sealed.

Distribute the freezer packs among the bottles. If you are forced to use a few larger freezer packs, separate them from the adjacent bottle with a little Styrofoam or bubble wrap. Alternatively, tape them in place across the top of the gallon bottles so that they will not come into contact with the wall of a bottle with water on the opposite side. This is intended to prevent any single bottle from chilling too much.

Latch the cooler closed and tape it shut with strapping tape around each end perpendicular to the hinged edge. Also tape it length wise along the free edge (i.e., away from the hinges; taping it near the hinge adds no strength). Remember that a large cooler is not going to be lifted, but dragged, so be sure it is taped well enough not to come open. You can use duct tape to seal the ice chest if you wish, but I prefer strapping tape. Finally, try to label the outside of the ice chest with "perishable" stickers. This is crucial if you are sending the fish using an overnight shipper because they sometimes choose to leave some freight behind if their shipment is too large. Packages marked perishable should not be left behind (though FedEx left one of our shipments of stickleback eggs behind in Anchorage, ruining the shipment for an experiment).

PREPARING FERTILIZED EGGS FOR SHIPMENT ?/B> If possible, check the eggs to remove dead eggs immediately before shipping. The three-day post-fertilization eggs we ship still do not have a lot of living tissue and do not consume much oxygen after they are chilled.

Transfer the eggs to 35 or 50 ml tubes. Fill the tubes with fresh 10% sea water that has been kept at about the same temperature as the eggs and aerated. Cap the tubes and put them into ice to chill. Monitor the tubes periodically so that they do not chill below about 5 C. Chilling only takes about 15 minutes. As the tubes reach 5 C, we briefly aerate them with an air line attached to a Pasteur pipette, but this is probably not necessary. Top off the tubes with chilled, aerated 10% sea water, but leave a small bubble of air. We try not to leave a large enough air bubble to agitate the eggs if it moves back and forth through the tube when it is on its side and tilted slightly.

We usually recycle Styrofoam boxes from reagent shipments, which may be available in biology departments. If they are not available, small ice chests that are designed to hold a six-pack of drinks are suitable for shipping eggs. Use about 300 ml of coolant from a freezer pack that has been transferred into a plastic bag and pressed flat. Lay this bag across the bottom of the box. Cover the coolant bag with a 1 cm layer of Styrofoam cut so it covers the coolant bag but does not seal it away completely from the tubes. Alternatively, cover the coolant bag with a double layer of small-bubble bubble wrap. It is important to be sure none of the tubes is in direct contact with the coolant. The tubes in each layer should face in alternate directions for better packing. Add another layer above the first, and repeat until the box is nearly full. Then put down a layer of Styrofoam or bubble wrap as before, and add another 300 ml bag of freezer pack coolant. We ship 40 to 60 35-ml tubes in Styrofoam reagent boxes this way. If there is any space left, you can fill it with additional bubble wrap, Styrofoam, or paper toweling to absorb water. Put on the top, and tape it shut with strapping tape all the way around the box in two parallel applications so you tape the tape to itself.

Three-day old eggs shipped this way generally arrive 20 hours later by overnight courier at < 15 C. However, one shipment of eggs that had been delayed by FedEx for 24 hours (44 hour shipment) was still alive. Another shipment that took 70 hours to arrive was warm and dead.

OPENING THE PACKAGE ?/B> If you are flying with the package, be sure to bring a battery-powered aquarium pump. Also be sure to be picked up by a colleague; a shuttle may delay your arrival in the lab inordinately. Open the ice chest as soon as possible to take the temperature of the contents and the condition of fish. If they are above 18 C, ice the contents down and start aerating immediately. (You may have to buy a very expensive soft drink with ice [hold the drink].) It is prudent to have the person who picks you up bring a cooler full of ice to dump into your cooler of bottles upon arrival. If the contents are cold but the fish are at the surface, swimming on their side, or on the bottom of the bottle motionless, aerate immediately, moving the air stone from bottle to bottle. Again, avoid placing the air stone on the bottom of the bottle. Of course if any sample has a pathogen, moving the air stone among bottles of fish from different sites may spread the pathogen. You may wish to have labeled air lines with separate stones for different populations.

If you ship by courier, be sure to email the tracking number to the recipient, and the recipient should know when to expect the package. The procedures described above should be used by the recipient.

Either way, the fish will have to be moved to aquaria in the lab. Again, avoid moving fish between water with contrasting temperature or chemistry, though we find that they easily tolerate 2 C changes and transfer from lake water to 10% sea water without distress. You can float bottles in aquaria and dilute the contents of the bottle with aquarium water to make the change more gradual.

If you are unpacking eggs, many of the same procedures should be followed. Be sure the eggs are still cool. Transferred them back to Petri dishes or incubator tubes with mesh bottoms or sides and suspended in an aquarium near an air stone.

You are done.

Protocol for Egg Washing
Written by Kaitlyn E. Ellis and Peter J. Park

Introduction: We perform in vitro fertilizations to produce progeny for our work in genetics, behavior, and development of threespine and occasionally ninespine stickleback. After the eggs have been fertilized and cleaned, we hold them in fish water in 10 cm diameter Petri dishes without aeration in an incubator at 20 C. We wash the eggs on post-fertilization day 3 (day 1 = fertilization day).

Using this method, we prevent post-hatching infections (usually fungus) that often kill embryos. We have also found that in 5% of our families, conspicuous infections (usually bulbous, transparent skin lesions all over the body) occur prior to hatching. If infections are found, we sacrifice these families and sterilize all tools that have come in contact with the infected water in a weak bleach solution. This is done to avoid contaminating other families.


  • Fish Water: add 6 g of dry sea salt (e.g., Instant Ocean) to 2 L of distilled or deionized water to make up 3 ppt sea water (~10% of full sea water). The salt concentration can be measured using a refractometer. Bring the water to a boil and cool to room temperature. An ice bath can be used to accelerate the cooling process. Aerate the fish water with a clean air stone for several minutes before use.
  • Acriflavine: add 0.9 ml Acriflavine to 2 L of fish water. We use item no. 37244, Trypflavine 3.84% solution, manufactured by the Kordon Division of NovalekTM, 2242 Davis Ct., Haywood, CA 94545, USA
  • Methylene Blue: add 1 drop of methylene blue (e.g., Aqauatrol, Inc. stock # 641) to 2 L of fish water. This product is available in pet stores from various manufacturers. Follow the manufacturer’s instructions for dilutions.

Discard and replace the Acriflavine and Methylene Blue solutions every second day. Kordon states that Acriflavine can be discarded down the drain into domestic sewage. Be careful when handling Acriflavine because it will stain skin bright yellow.

Acriflavine egg wash: Decant the old fish water from the Petri dish with embryos. Add the Acriflavine solution and swirl gently for 30 sec (This includes time to decant Acriflavine solution).

Methyline Blue egg wash: Add the Methylene Blue solution to the Petri dish immediately after decanting the Acriflavine and swirl for 30 seconds (This includes time to decant Methylene Blue).

Rinse: Rinse embryos with fish water four times.

Discard dead embryos: Observe the embryos under a dissecting microscope. Live embryos will have large, conspicuous eyes and have undergone somatic segmentation by day 3. Remove any embryos that have abnormal or undeveloped morphology.

Final rinse: Rinse embryos with fish water once more and decant. Add fish water and return the Petri dish with embryos to the incubator.