General Laboratory Techniques

General Laboratory Techniques

Cleaning glassware and plastic buckets

Any buckets, beakers, components, etc. that are used with the rotifer culture should only be rinsed and scrubbed using hot tap water (no detergent). Glassware that is used with rotifers or zebrafish can be cleaned with detergent but must be thoroughly rinsed with hot tap water to remove any remaining detergent. Any lingering detergent will harm the rotifers and/or fish on contact and/or ingestion.

Maintaining water quality for the rotifer culture

In our continous rotifer culture, it is important to maintain the proper levels of several indicators of water quality to maximize rotifer production and larval zebrafish health. These indicators include: nitrite, nitrate, ammonia, pH, and dissolved oxygen. All of these indicators must be monitored and adjusted to properly maintain optimal water quality in the culture bucket.

  1. pH: optimal is 6.75 - 8.0. May be lowered by algae or chlor-am-x application. Add sodium hydroxide to increase pH and hydrochloric acid to decrease pH. pH probe should be cleaned and calibrated frequently.
  2. Ammonia: optimal is 0.0, frequently 1.0 - 8.0 though. May be lowered with Chlor-Am-X (100 ml, every 3 days or so). Caution: Chlor-Am-X will decrease pH drastically.
  3. Nitrite:
  4. Nitrate:
  5. Dissolved oxygen: Make sure the air pump and tubing are operating properly and supplying airflow to the culture bucket.

Cleaning and calibrating a pH meter

The pH probe from the rotifer culture bucket should be cleaned once per week, ideally at the same time that the bucket is cleaned. Over time, algal buildup will hinder proper pH testing. Be careful with the glass end of the probe; the glass will break easily if you knock it against a hard surface. The glass end should always remain wet. To clean the pH probe:

  1. Remove the pH probe from the rotifer culture bucket and disconnect the cable from the pH meter body.
  2. Rinse the pH probe under a stream of warm tap water in the sink.
  3. Use a scrub brush to carefully remove debris from the glass end and body of the probe.
  4. Rinse clean under tap water.
  5. Reconnect the probe wire to the pH meter body.
  6. To calibrate, place the probe into the pH7 standard (yellow). Adjust the corresponding screw on the meter body until the screen displays 7.0.
  7. Rinse the probe and place it into the pH10 standard (blue). Adjust the other screw until the screen displays 10.0
  8. Rinse the probe and place it back into the pH7 standard (yellow). Adjust the first screw again if necessary.
  9. Replace the probe in the rotifer culture bucket and record the new pH reading on the log sheet.

Mixing 15 ppt water

Rotifers thrive best in slightly salty water, optimized at 15 parts of salt per 1,000 parts of water (parts per thousand, ppt). Our laboratory uses ~20 L of 15 ppt water per week in the rotifer culture bucket. Our large carboy holds ~52 L of 15 ppt water. To mix new 15 ppt water:

  1. Using a clean white bucket and magnetic stirrer bar, add 210 g of Instant Ocean salt to the bucket.
  2. Take the bucket to the freezer room of the 5th floor BRI and add 14 L of purified (RO) water, creating 15 g/L (ppt) salinity.
  3. Bring the bucket back to the lab and place it on the magnetic stirrer to dissolve the salt. Add air tubing to incorporate oxygen and aerate for at least 24 hours.
  4. Titrate pH to ~7.00, adding hydrochloric acid as necessary (3-4 mls in total is typical).
  5. Once pH stabilizes, pour the 15 ppt water into carboy.

Measuring the salinity of water samples

Our laboratory animals prefer slightly salty water. Rotifers thrive at a salinity of ~15 ppt but can survive in much less (>2 ppt water). Zebrafish larvae find anything greater than 7 ppt salinity too toxic to survive but are typically housed in 0.8 ppt water as adults. The larval polyculture tank water that combines zebrafish larvae with rotifers should be ~5 ppt. The rotifer culture bucket water should be ~15 ppt. To test the salinity of a water sample:

  1. Open the cover and place a few drops of the water sample on the prism of the refractometer.
  2. Close the cover carefully, assuring the water makes a film across the prism without bubbles.
  3. Hold the refractometer up so that the prism is parallel to a light source (i.e., underneath one of the fluorescent fixtures in the ceiling).
  4. Look through the eyepiece and record the salinity reading from the right-hand scale (ppt), the line where the blue area stops.
  5. Wipe off the prism carefully to remove any water.