For Paramecium cultures that are ordered from a biological supply company:
When the cultures arrive, immediately open the shipping container and remove the culture jars. Loosen the lids on the jars and aerate the cultures using disposable transfer pipettes. Use a different pipet for each culture and write the name of the culture on each pipet to avoid cross-contamination. To aerate, place the tip of a pipet into the culture water and squeeze the bulb, bubbling air into the water. Withdraw the pipet and release the bulb, allowing it to refill with air. Repeat about 4 times.
5 Wheat Berries
5 Clean IO Containers w/top
1 Donor Culture
~200 mL Sterile dH2O
Boil wheat berries in 50 mL sterile dH2O for 15 minutes. Add 24 mL of sterile dH2O to each Paramecium container. Drop 1 boiled wheat berry into each container using sterile forceps. Take a well developed (confirm there are living Paramecium inside using inverted scope) donor culture and homogenize the solution by pipetting up and down. Inoculate each container with 6 mL of homogenized donor culture solution. Cover loosely with lid. Place at room temperature. Check ~72 hours later to confirm successful subculture. Cultures last ~5 weeks. Always maintain extra cultures in case of contamination.
Cultures typically contain 3 organisms, bacteria, Chilomonas and Paramecium. Bacteria consume the wheat berry, Chilomonas consume the bacteria, and Paramecium consume the Chilomonas. At lower magnification the bacteria appear to be a murky substance typically surrounding the wheat berry, Chilomonas appear to be spiraling bell shaped organisms and Paramacium appear to be cruising flip-flop soles. Culture organisms normally maintain logistic growth (lag, log, stationary & death phases). At 1-2 weeks cultures will be dominated largely by Chilomonas. At 3-4 weeks Paramecium numbers begin to grow as Chilomonas numbers diminish. During weeks 4-5 the number of each organism stays relatively even before dropping off quickly near the end of 5 weeks.
Preparation for lab:
To ensure student success, we concentrate our Paramecial cultures as follows:
o Aerate Paramecia as usual
o Pipette about 7mL into each of six 15mL conical centrifuge tubes (need 1 tube per group; 6 tubes per room). Make sure the tubes are balanced prior to centrifugation.
o Centrifuge on 3 in clinical centrifuge for about 3 minutes.
o Decant the supernatant (pellet is the bottom most layer, supernatant is the remaining liquid above) in one swift movement into an empty or "used" paramecium jar leaving about 1mL in the centrifuge tube. (You can also use a plastic transfer pipette to remove supernatant from the top). DO NOT DISTURB THE PELLET.
o Once all 6 tubes are down to about 1mL, shake them up to re-suspend the pellet and take a look under the inverted microscope. If satisfied with the concentration of Paramecium, empty contents of conical centrifuge tube into labeled test tube. Once there are 6/rack, send them out to the rooms for students to use. If the concentration at this point is still too dilute, just add more Paramecial culture to the tube up to 7mL and centrifuge again.
Date Modified: 12/16/2010