How To Culture Amoeba? – (Preparation of Amoeba Culture)
Introduction to culture
The science of culture plays an important role in the biophysical and biochemical studies. Various methods are known to culture invertebrates in artificial media.
Invertebrate species like Amoeba has been used for many years in the laboratory and teaching environment. Various culturing techniques are used to study these species with detail and practical analysis under an artificial environment in the laboratories.
Researchers can create a short food chain, enabling the few collected amoeboid specimens to feed and readily reproduce under their guidance and observance. This introduces much more information about these collected microorganisms.
Culture media can be purchased commercially from science supply companies or easily prepared in the classroom. Practical culture studies in classrooms are usually done by students who want to learn more about the culture media.
The simple culture of amoeboid species involves one or many easy procedures, which are subject to many variables.
Something as simple as the type of glass used for the culture container, use of glass-stoppered dishes, or use of petri dish can highly influence the culture of the specimens.
Background of Amoeba
Amoeba is the most popular and free-living available protozoan. And, among other species inhabiting soil and water, Amoeba proteus is the largest fresh-water form that is mostly used in the various culture methods.
Amoeba actually is regarded as the lowest and simplest form of animal in the whole Animal Kingdom that belongs to Phylum Protozoa, Sub-phylum Sarcomastigophora, and Class Rhizopedia.
The body of Amoeba is really very simple consisting of a small spot of protoplasm resembling like that of a tiny drop of jelly that has all the cell organelles to perform movement, reproduction, excretion, respiration, etc.
Amoeba actually is the best option as an interesting and suitable microorganism for laboratory studies by the students because it is a large protozoan, very slow in locomotion, and easy to obtain.
Although there are several species of Amoeba, but the most commonly studied one is the large freshwater species Amoeba proteus which is an easy to find and useful experimental animal.
Where do you find Amoeba?
Here, in this post, we will specifically talk about the culture method of Amoeba proteus which is actually the most common, best, and easy to find Amoeba for laboratory culture.
Amoeba proteus is widely distributed and is commonly found on the bottom mud or on the underside of aquatic vegetation in freshwater ponds, ditches, lakes, springs, pools, and in slow-running streams as well.
Amoeba proteus is rarely found in free-running water as it requires a substratum to glide on from place to place.
Amoeba proteus is mostly found in abundance in only those water bodies where there’s already the presence of excessive bacteria and organic substances such as aquatic vegetation, leaves, and twigs. They can also be found among other decaying vegetable matters and aquatic plants.
They can also be seen in and near the water of lotus ponds, and also in the artificial watering point intended to provide drinking water to animals, and livestock.
Other Amoeba species can be found in hot springs, warm water coming from industrial plants, poorly maintained swimming pools, and water heaters kept at temperatures below 117°F (47°C). Some Amoeba species can also be found in soil.
Preparation of Amoeba Culture: How to culture Amoeba?
Amoeba proteus can be cultured using any of the 4 different methods:
- By Simple Collection Method
- By Hay Infusion
- By Nutrient Agar
- By Chalkley’s Stock Solution 10x
Let’s know about each of these in detail…
➜ By Simple Collection Method
1. Collect few Amoeba proteus and keep them on the bottom of a clean and sterilized petri dish by a micropipette.
2. Next, add some amount of rain or tap or well water to the petri dish. Next, add a few grains of wheat and cover the dish.
3. The petri dish should be kept at a dark place in a cool room with 18–22 °C (64–72 °F) and placed out of direct sunlight for their proper growth as they avoid light due to being negative phototaxic.
4. Observe sample cultures regularly using a dimly lit stereoscope. It’s better to place a piece of black paper under the culture dish to make Amoeba easier to view.
5. Amoeba proteus species start to stick to wheat grains and after sometime multiplies and will give rise to numerous daughter cells.
6. It is usually seen that the Amoeba population will increase to its maximum level in a dish within a period of 20 to 21 days and may then require additional wheat seeds.
7. So, after 21 days, if you want the Amoeba to be cultured for extended period of time, you can split the culture into the other three petri dishes. To do so, stir the media in the dish, and divide the culture evenly into three different clean culture dishes.
8. Next, add enough liquid media solution to each dish to restore the volume to that of the original culture.
9. Next, add few grains of wheat to each subculture and cover the dishes and keep the dishes in dark and cool room again with 18–22 °C (64–72 °F) room temperature.
10. If Amoeba species are to be cultured for extended periods of time, the following recipes and procedures should be followed to create sub-cultures to ensure that cultures remain viable. You can use these prepared cultures for laboratory studies.
➜ By Hay Infusion Method – I
1. Boil about 12 gm of hay stems in a large flask in 100 cubic centimetre of distilled water for about 45 minutes.
2. Gradually pour the liquid into another flask and add a few drops of Sodium hydrooxide (NaOH) to make it alkaline.
3. Next, sterilize the flask in an autoclave by subjecting them to pressurized saturated steam at 60 °C for around 10–15 minutes.
4. Next, take the flask out of the autoclave and after cooling it, pour the liquid into a smaller glass-stoppered dish.
5. Now it’s time to inoculate the liquid in the glass-stoppered dish with 500 mg of any soil containing pure Amoeba proteus.
6. After a day or two if everything is perfect, then numerous Amoeba proteus will multiply and develop rapidly.
➜ By Hay Infusion Method – II
1. First boil 100 cc of distilled water along with a few grains of wheat for about 10 minutes and then let it cool.
2. Next after cooling, gradually transfer the liquid in a petri dish and add the Amoeba culture to the dish and let it stand uncovered for 24 hours in a cool and dark place.
3. After 24 hours or so, you will see that Amoeba tend to congregate on the bottom and around the edges where the sidewalls and bottom come together. It will now multiply to large number.
4. The culture is now prepared and should be okay, as long as, it is kept aerated to prevent surface scum from forming due to excessive bacterial growth.
➜ By Nutrient Agar Method
1. First prepare agar in a flask by dissolving 1.5 grams of nutrient agar in 100 cc of distilled hot water.
2. Next, while it is hot, pour the solution in a sterilized bowl by passing it through a funnel of filter paper.
3. The amount of agar should be in sufficient quantity to form a thin layer of 0.2 cm thick on the bottom of the bowl.
4. Now, as soon as possible, drop several grains of ordinary polished white rice on the layer inside the bowl before the agar has set. The agar serves not only to anchor the starch grains, but also offers a good surface for the Amoebae to feed and crawl upon.
5. Next, when the agar has hardened, add about 10 cc to 15 cc of culture water containing a large amount of Amoebae as possible, into the bowl. Next add almost an equal quantity of distilled water.
6. Make sure that each day you add about 5 cc of distilled water into the bowl until the bowl contains about 50 cc of water. In adding the water, the contents of the bowl should be agitated as little as possible, so as not to disturb the Amoebae, which will tend to gather around the rice starch grains.
7. Make sure that the bowl is kep at a dark and shady room in at least 17°C to 18°C, somewhat below the usual room temperature.
8. To maintain a constant temperature, the bowls may be stored in any kind of water bath, with water from the tap constantly circulating around it.
9. One biggest advantage of this culture technique is that, the cultures prepared in this particular way described above will, within two to three weeks, will develop thousands of Amoeba species per bowl. And, they will often last for several months without sub-culturing.
10. Also the presence and growth of Chilomonas will be seen in the culture bowl. The presence of large numbers of Chilomonas is very favorable for the cultures, as they also serve as food for the Amoebea.
➜ By Chalkley’s Stock Solution 10x
1. First, boil and cool about 1L of spring water.
2. Next, add 0.06g CaCl2, 1.00g of NaCl, and 0.04g KCl, and mix thoroughly.
3. This prepared 1L solution of spring water mixed with CaCl2, NaCl, and KCl is the 1x stock solution. Store this 1x solution in a media bottle until ready to culture or subculture the Amoeba.
4. Now, when you are ready to culture the Amoeba, this 1x stock solution should be diluted to a 10x solution. For doing so, add 100 mL of this 1x stock solution to a newly collected 1L of spring water. This will make the Chalkley’s Stock Solution 10x.
5. Next, to prepare the culture of Amoeba, add 2 rice or wheat grains for every 50 mL of 10x solution. This will encourage the growth of bacteria.
6. Next, you will find that various ciliates and Amoeba species started to grow on the culture media. It’s because small ciliates feed on the bacteria, and the Amoeba will start to grow feeding on the ciliates.
How do you keep an Amoeba alive?
Culture practices means to maintain the Amoeba species in laboratory conditions suitable for growth.
The culture practices of Ameoba proteus done by simple collection method, or by hay infusion method, or by nutrient agar method, or by preparing Chalkley’s Stock Solution 10x method, are all the best practices to increase the growth of Amoeba for about a period of 20 to 21 days maximum.
After this, if you want to prolong the culture and growth for extended periods of time then you must prepare many sub-cultures with the same conditions and media as that of the main culture.
The food nutrients that you will be providing to the Amoeba in the form of rice or wheat grains is very necessary. This will ensure the proper gowth and reproduction of the amoeaba in the culture.
Ameoba proteus feed on a variety of organisms, such as bacteria , algae , other protozoans, and even other smaller amoebae. Most members of the order of Amoebida are free-living in fresh or salt water or soil , and ingest bacteria.
It is always a best practice to create a sub-culture as soon you see as many as 50 Amoeba individuals in a quantity of about 100 cc of culture water in a petri dish or bowl.
Can we see Amoeba with naked eyes?
Some species of Ameoeba can be blurrily seen with naked eyes, while some species of Amoeba are very microscopic and cannot be seen so easily with a naked eye and so needs a stereoscope. Or, to see some extremely smallest ones needs a microscope.
Some species of Amoeba are called Xenophyophores which lives in the deep oceans can be seen easily with our open eyes. They has a size of about at 4 inches (10 cm) long and is one of the largest single-celled organisms in the world.
Species of amoeba like Chaos carolinense (700-200 micrometers), Pelomyxa palustris (up to 5000 micrometers), and Syringammina fragilissima (up to 200,000 micrometers), etc. are really very large kind of amoeba species that can be seen under naked eyes.
The average size of an Amoeba is about 700 micrometers or 0.7 millimeters. Amoeba proteus ranges in size between 250 and 750 microns and it can almost be seen with the naked eyes as very fine jelly dots in water.
And, in a culture with 50 or more Amoeba proteus species, they can be easily seen in one field of view on a low power stereoscope as well.
Amoeboid movements of Amoeba proteus are very best seen under a microscope with 400x lense power or more. Amoebas are a type of microscopic, unicellular protist and therefore will be best to see under a compound light microscope if possible.
What do Amoebas look like?
In culture, Amoeba are whitish-gray in appearance and are approximately the size of a pinhead to the naked eye.
Amoeba depicts body organization of a protoplasmic mass or a single cell into a complete organism.
If seen under a microscope, they look like a tiny blob of colorless or transparent jelly with a dark speck inside it. The colorless jelly is cytoplasm, and the dark speck is the nucleus.
You will find them moving as with their false feet ‘pseudopodia’. They have the ability to form temporary cytoplasmic extensions called pseudopodia, or false feet, by means of which they move about. This is called the amoeboid movement.
You will also see that they reproduce very fast asexually through binary fission or multiple mission. Amoeba proteus are nearly “immortal”, as it does not die due to natural death on its own, due to fission.
Amoeba also has the great power of regeneration. If an individual is cut into pieces, accidently or in laboratory, you’ll eventually find that the every piece containing a part of nucleus grows into a complete Amoeba.