Collecting and culturing protozoans

COLLECTING WATER SAMPLES AND PREPARING NUTRIENT RICH SOLUTIONS

Microscopic life can be found in ponds, lakes, streams, rivers, ocean backwater estuaries, and, surprisingly, rain puddles that have been in existence for a few days. Water samples can easily be collected using wide-mouth glass jars with tight-fitting lids. The collection jars you use should be completely clean and detergent free.  A great place to find protozoans and tiny water animals is in a fresh water fish tank.  Take samples right near or on the filter material (where the pump is).  You will be surprised at what you find!

Collections from one site could include surface or bottom samples. When sampling from the bottom, use a kitchen basting tool and include some of the bottom solid material with your sample. When sampling from the surface, include any floating material or scum that you might find.

Upon close examination of your sample with a microscope, you may see very few living things, perhaps only one protozoan per drop. Culturing techniques can now be employed to set up a short food chain, enabling the few protozoans to feed and readily reproduce. In effect, you will be upsetting the ecological balance in your sample jar!

Culture media can be purchased commercially from science supply companies or easily prepared in the classroom. Six different solutions that can be made easily are listed below:

Solution A:

The "hay infusion" is perhaps the most well known culturing technique. Boil one liter of pond, spring or rain water. As the water comes to a boil, add a small handful of hay (ideally, timothy hay) and boil for ten additional minutes. The boiling will break down the hay and set up an ideal medium for the growth of bacteria. Allow this mixture to stand for two to three days. Add 25-50 milliliters (2 to 4 T.) of your sample (this is called "inoculation"). In a few days, small protozoans such as Chilomonas will populate your culture. If Paramecium are present, they will feed on the Chilomonas and eventually increase in number (in 10 to 14 days). The organism at the top of the food chain will become the most common but will quickly die off as the food supply is exhausted. You may be successful in maintaining one organism for long periods of time by sub-culturing into newly prepared media.

Solution B:

Boil 100 milliliters of pond or spring water for ten minutes. Add five grains of wheat to the cooled water. Let this mixture stand in open dishes for one to two days, then inoculate this culture media with one or two tablespoons of your sample.

Solution C:

Follow the instructions for Solution B, but use five grains of uncooked rice instead of the wheat.

Solution D:

Hard boil an egg and grind a pinch (1/4 gram) of the yolk in a bowl with a small amount of water to form a paste. Add the paste to 1 liter of boiled pond or spring water and let stand for two days before inoculation.

Solution E:

Boil 250 ml of pond or spring water for ten minutes. Cool and add a pinch of powdered skim milk. Mix thoroughly and immediately inoculate your sample.

Solution F:

Boil 250 ml of pond or spring water for ten minutes. Cool and add 1/4 package (2 grams) of dehydrated yeast. Mix well and let stand in open containers for a few hours before inoculation. A rich culture should develop within one week.

 Different microorganisms thrive better in different culture media. If you are transferring Amoeba, use Solution C. For ciliates, try any of the above solutions. Paramecium are best cultivated in Solutions A, D and E. For Paramecium bursaria, use Solution C. For Blepharisma, try Solutions B or C.

Vorticella are cultured best in Solution D. After the solution has set for two days, pour off some of the clear top liquid and add the Vorticella. Sub-culture every two weeks by transferring Vorticella from the parent culture to a new, clear, egg yolk solution.

Stentor are best cultured with Solution C, but will also thrive in Solutions B and D.

Flagellates can be successfully cultivated in Solutions B and D. A modified Solution B can be prepared that will save time. Boil four wheat grains in 80 milliliters of pond water. Cool and add a few milliliters of sample containing the organisms that are to be cultured.

For Eudorina, Pandorina or Volvox, add 1 gram of 4-10-4 or 5-10-5 fertilizer to one liter of pond, spring or rain water. Heat to 30 degrees C for twenty minutes. Filter the mixture while still hot, allow it to cool and inoculate with the microorganism. Store in an area that is moderately lit.

Experiment with these yourself! As a class project, you may try the effectiveness of one solution versus another. Try a combination of Solutions A and C (hay and rice), or invent your own. Perhaps you might use two crushed pellets of rabbit food in one liter of water.

When culturing microorganisms, choose suitable water. Because of trace metals, tap water is usually not recommended. Keep your culture lightly covered to exclude dust but not air. Use only clean glassware. Traces of soap or other chemicals may be toxic to the organisms. Store your cultures in areas of dim to moderate light at temperatures of 20 - 21 degrees C (68 - 70 degrees F).

Finally, only sub-culture when the parent culture reaches its maximum and heavily inoculate the new culture media. 

Daily observations of your culture may provide a valuable lesson in population dynamics.  The best type of microscope to use for observation of protozoa is a compound microscope with 3 powers (10X, 40X and 400X).  You can use depression slides at the two lower powers but must use a plain slide and coverslip at 400X as the objective will be very close to the specimen when in focus. 

More Good Stuff!   See our illustrated guide to protozoans.

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