Isolating protozoa into monocultures:
For field collection
- many sterile 50ml tubes
- autoclaved pipette tips or sterile disposable pasteur pipettes with the tips cut off (using a scissor that has been wiped with ethanol)
At the field site, in order to obtain the largest diversity of protozoans, collect water from as many leaves as possible. Place the contents of one leaf into one 50mL tube.
**Do not pool the samples into one large container, as this will make isolating the protist species more difficult. Each leaf will contain a different diversity/abundance of species**
For laboratory
- Autoclaved deionized water – place in one sterile 50mL tube
- Autoclaved 200µl pipette tips
- Autoclaved microcentrifuge tubes
- Autoclaved fish food solution or autoclaved ants or Drosophila
- Ethanol
- Microscope plate
- 200µl pipette
- Cover slip
Initial Isolation
*An inverted compound microscope is best for this stage
1. Take a 50µl aliquot sample from one of the tubes containing Sarracenia water from the field.
2. Pipette onto the microscope slide and cover with the cover slip. This will allow you to have the first initial look at the diversity of protists in the sample. The cover slip at this stage really helps with visualizing which species and how many species are in the sample. This will be the only time that you will use the cover slip during the initial isolation stage.
3. Once you know what species are there, take another 50µl aliquot sample from the same tube.
4. Pipette out a row of small drops onto the ethanol-wiped microscope slide. Do not place a cover slip on these drops.
*It is not important to use the entire volume of aliquot, but just to have a row of drops*
5. Look at each drop under the microscope and find a drop that has a target species.
6. Pipette a drop of sterile deionized water onto the Sarracenia drop of water containing that target species. Do another row of drops of water with this dilution.
7. Look again through the diluted drops for the target species.
8. Once the target species is found, continue the process of diluting with sterile deionized water, creating rows of water drops, and searching for the drop containing the target species.
9. Repeat this process until you have a very clean sample with only the target species, and clear water (no detritus should be visible in the water).
Incubation stage
1. Take an autoclaved microcentrifuge tube filled halfway with a small amount of autoclaved fish food solution (or 1 ant/Drosophila) and 0.5mL of water
2. Pipette a small volume of this liquid onto the drop containing the clean sample of your target species.
3. Pipette the drop containing the target species into the autoclaved tube.
4. Close the tube and label it with species name, date, field site, your initials. Place it in the incubator that corresponds to the temperature of the field site where the protist originated from.
***To aid in the success of the isolation, it is helpful to have a population of at least 2-5 individuals within the autoclaved microcentrifuge tube. For flagellates, this is usually easy, as they are in high numbers and thus are easy to isolate. For ciliates, you may have to do the dilution process for several drops containing individuals of the target species. This means that you will probably place 2-5 very clean drops of water + target species into one 1.5mL autoclaved tube.***
Checking your isolations and possible reisolation
Wait 3-4 days, and then check your isolation by taking a 50µl aliquot from the microcentrifuge tube containing the target species. Place a coverslip over this aliquot and view it under the microscope.
***It is likely that at this stage, there will be some contamination. If this is the case, use the volume within the microcentrifuge tube and repeat the dilution/isolation process as stated above.
Transferring the target species to a larger volume
Once you have a clean sample of the target species within your microcentrifuge tube, and this species is in a high density:
Take a large, sterile 50ml macrocentrifuge tube. Place 5mL of sterile water and 5 autoclaved ants/Drosophila into it. Then pipette 50µl of the target species into the macrocentrifuge tube. Make several replicates. Place the tubes in the incubators corresponding to the correct field site.
If you use fish food instead, add 4.5ml of autoclaved deionized water to the macrocentrifuge tube and 0.5mL of 3mg/mL fish food solution.
Maintaining the Protist Monocultures:
The protist monocultures are typically fed every two weeks with 1 ant/Drosophila. One way to assess if the resource has been decomposed is if the ant/Drosophila has sunk to the bottom of the tube.
If fish food is used instead, it is 200µl of a 3mg/mL dilution every 1.5 weeks (or every 2-3 weeks for Cyclidium sp.).
***If you have monocultures of Rotifers, place several small, autoclaved beads at the bottom of the 50mL tube. This provides habitat heterogeneity and a structure for the Rotifers to hold onto***
Cultures can be transferred to new tubes/fresh media every few months.
PDF of the protocol can be found here.