Rotifer Culture

Rotifer culture

Rotifers (Brachionus plicatilis)

Rotifers are small invertebrate zooplankton, fast swimmers, and voracious eaters; they possess a high nutritional value when fed with algae concentrates, making them an excellent first-food for raising larval zebrafish. We keep a continuous culture system thriving to provide plenty of rotifers for larval polyculture and supplemental feeding to older zebrafish. In our setup, two pumps provide constant food and oxygen to support a dense rotifer culture. Maintaining a dense and healthy rotifer culture depends on good water quality and oxygenation plus a high rate of algae feeding.

Each culture bucket setup consists of: 5 gallon bucket, Compact Culture system (floss chimney, T-assembly), Air pump plus tubing, Sno-cap dosing pump (comes with tubing), Magnetic stirrer plus large stirrer bar, Refrigerator (poke algae tube through door seal), pH meter, ammonia test kit, Chlor-Am-X, RGcomplete, and Instant Ocean Salt.

Maintaining a dense rotifer culture

Daily tasks
  1. In the morning, visually inspect the culture bucket for proper function: the culture water should be dark green-brown, slightly translucent, with minimal foam/bubbles on the surface.
  2. Using a transfer pipette, collect a sample of rotifers for visual inspection. Hold the sample up to the ceiling light and look through the culture. It should be clear, not cloudy, with a greenish-brown tinge. You should be able to see many tiny rotifers swimming in it. If it looks normal, simply squirt it back into the culture bucket. If it looks abnormal, please inform Dr. Schmoutz immediately.
  3. Check the air pump and tubing to ensure proper bubbling (a medium boil).
  4. Check the pH meter and record pH on the log sheet.
  5. Check the ammonia and record it on the log sheet. Add Chlor-Am-X if necessary.
    • Pipette a small amount of the culture water into the glass tube until the 5 ml mark.
    • Add 8 drops of ammonia bottle #1 and 8 drops of ammonia bottle #2 to the glass tube.
    • Cap and shake, wait 5 minutes before reading by comparing to color chart.
    • For ammonia levels over 8.0 ppm, add 100 ml of Chlor-Am-X solution to the culture bucket.
    • For ammonia levels of 2.0 - 4.0 ppm, add 50 ml of Chlor-Am-X solution.
    • For ammonia levels of 0.0 - 2.0 ppm, add 25 ml of Chlor-Am-X solution.
    • Chlor-Am-X tends to decrease the pH. Titrate it back to pH 7.0 - 7.5 using the sodium hydroxide solution.
  6. Open the fridge and confirm that there is at least 100 mL of RGcomplete solution in the flask in the door of the fridge and that the tubing is completely full of the dark green solution.
  7. Confirm the dosing rate on the pump’s display (typically 4 mL/hr).
  8. Ensure that the carboy is more than half full of 15 ppt water.
  9. Harvest any rotifers that are needed for supplemental feeding (3 times per week) or larval polyculture.
In the afternoon
  1. Visually inspect the bucket for proper aeration and green water (indicates recent feeding) and note any foam buildup.
  2. Open the fridge and confirm that there is at least 200 mL of RGcomplete solution in the flask in the fridge and that the tubing is full of dark green RGcomplete solution.
Before an extended holiday break
  1. Reduce the feeding frequency by increasing the time to 2 hours between metered feedings.
  2. Harvest and save (refrigerate) a portion of concentrated rotifers as a backup.

Harvesting rotifers

The rotifer culture should be harvested 3-4 times per week to remove older rotifers that produce fewer eggs. These harvest events may be scheduled based on polyculture or supplemental feeding needs. Harvesting on back-to-back days is typically avoided to allow time for the culture to replenish.

  1. Make sure the ammonia levels are below 1.0 ppm. Add Chlor-Am-X if ammonia is too high.
  2. Remove the air tubing and pH probe. Place these in a pan to contain drips.
  3. Make sure the magnetic stir bar is operating to prevent settling.
  4. Harvest at least 25% of the culture by siphoning it through the 53 um sieve over a separate white bucket. Save a few drops of the harvested water for salinity testing
  5. Rinse these harvested rotifers with 15 ppt water from the squeeze bottle and wash them off the screen of the sieve into a smaller flask with a known volume (rotifers/mL).
  6. Dilute the concentrated rotifers with culture water to 500 mL and transfer these rotifers to the Fish Room for supplemental feeding.
  7. Clean the culture bucket.

Cleaning the culture bucket

The culture bucket should be cleaned at least once per week, typically directly after a harvest event. This prevents algal contamination, ammonia buildup, and helps to keep the rotifer population younger for better reproductive success. All components that come in contact with rotifers should not be cleaned with detergent, just hot water.

  1. Remove the air tubing and pH probe. Place these in a pan to contain drips.
  2. Disconnect the T-assembly and floss chimney, rinse it thoroughly under a strong jet of hot tap water in the sink.
  3. Brush the interior surfaces of the bucket to remove any adhering green waste.
  4. Replace the harvested volume with fresh 15 ppt water from the carboy.
  5. Re-install T-assembly, pH probe and air tubing. Restart magnetic stirrer.
  6. Once per week, swap the bucket, stirrer bar, T-assembly, and floss chimney for a clean set-up. Clean the air tubing and the pH probe.

Counting and calculating rotifer culture density

The density of the rotifer culture is largely dependent on the rate of algae feeding and the presence of oxygenated water. I have found that feeding 4.0 mL/hr (actually 0.8 mL/hr since the pump is calibrated down) will produce a density of 100-300 rotifers/ml in a 15L culture, depending on the timing of harvest events. To count and calculate the culture bucket density:

  1. Ensure that the culture bucket is well-stirred for a representative sample. Only take samples while the magnetic stirrer is operating.
  2. Using a plastic transfer pipette with the tip cut off, take a ~2 mL sample of rotifers from the middle of the culture bucket.
  3. Deposit into a glass vial containing 5 drops of distilled white vinegar. Mix gently and the vinegar should immobilize the rotifers within 30-60 seconds.
  4. Shake the vial to redistribute the immobilized rotifers and quickly remove ~1 mL with the transfer pipette.
  5. Fill a Sedgewick-Rafter counting slide with exactly 1 mL of the sample, placing it under the microscope to settle for 3-5 minutes before counting.
  6. Count the number of rotifers with and without attached eggs in at least two rows or five columns (>100 squares or >100 rotifers for good statistical accuracy).
  7. Record these values on the log sheet.
  8. The approximate density of the rotifer culture can be calculated by using the number of squares counted (ZZZ), the total number of rotifers counted (YYY), and the total number of squares (1,000): density = (1,000 / ZZZ) x YYY.