Culturing Amoeba

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**Background**

Amoeba can be found among decaying vegetable matter and aquatic plants in ponds, puddles, and eddy areas of streams. They are not found in high quantities in waters where crustaceans are present, since many crustaceans feed on Amoeba. Their abundance in natural water sources is not as high as one might guess. Those who have tried to locate Amoeba in the wild will agree that they are difficult to find. For this reason, it is advantageous to culture Amoeba under lab conditions. In culture, Amoeba are whitish-gray in appearance and are approximately the size of a pinhead to the naked eye. When healthy, they tend to be very active, although disturbances may cause them to stop moving and retract their pseudopods to “hide.” Fortunately, Amoeba, are relatively simple to culture from purchased stock cultures and easy to locate using a dimly lit stereoscope. Amoeba, a popular and often remembered protist, move around by means of pseudopodia (flowing cytoplasm) for locomotion. (Movements can best be observed under 400 X.)

**Culturing/Media**

Upon arrival of Amoeba stock cultures, loosen the caps and immediately aerate the cultures by forcing air into the liquid
using a clean pipet. Cultures should be kept at **18–22 °C** (64–72 °F) and placed out of direct sunlight as they avoid light (negative phototaxic). Avoid moving the culture! A certain amount of experimentation may be necessary to find a suitable place to house stock cultures. It is wise to initially place multiple subcultures in a variety of sites while trying to determine the best culturing environment in a particular classroom. Observe sample cultures regularly using a dimly lit stereoscope. Placing black paper under the culture dish makes Amoeba easier to view. The **Amoeba population will increase for 21 days** and may then require **additional wheat seeds**. Split a culture when 50 or more Amoeba are seen in one field of view on low power of a stereoscope. Stir the media in the dish and divide the culture evenly into three clean culture dishes. Add enough liquid media solution to each dish to restore the volume to that of the original culture (approximately 2/3 full). Add two new boiled wheat seeds to each subculture. Dishes should be covered loosely and stacked to **limit evaporation and contamination**. Fresh medium should be prepared monthly if the Amoeba will be maintained long-term.

If Amoebae are to be cultured for extended periods of time, the following recipes and procedures should be followed to insure that cultures remain viable.

**Chalkley’s Stock Solution \*Recommended\***

**Timothy** **Hay Infusion**

**Tips**

I always keep an old culture until the new one is well established.

• To assure positive transfers of Amoeba to student slides, do collection transfers while looking through a stereoscope. Amoeba tend to be on the bottom of the culture dish and can be viewed as they are being drawn into the transferring pipet. The transferred Amoeba should be obvious on the student slide.

• Students skilled in microscope use should view Amoeba in a depression slide. Looking for Amoeba should not be a
first microscope practice exercise. Do not allow students to randomly take pipets full of water from an Amoeba culture. Continual agitation of the culture will cause Amoeba to “ball up” and become inactive. Slides of Amoeba can be kept viable for hours if slide gel is used and the sample is kept sealed around the coverslip.

• Allow students to view prepared microscope slides of Amoeba before working with the live specimens. This will eliminate
some initial frustration.

• Monitor the culture regularly using a stereoscope. Have extra media already mixed and ready when sub culturing is necessary. If ~50 specimens can be seen in one field of view, it is time to split the culture.

• For long term culturing, fresh media should be prepared and changed monthly.

**Disposal**

 Amoeba cultures may be disposed of according to Flinn Suggested Biological Waste Disposal Method Type IV. Please consult your current Flinn Scientific Catalog/Reference Manual for proper disposal procedures.