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