Isolation of Amoebae

Isolation of Amoebae
Page updated 23/8/02
Amoeba are essentially everywhere and can be isolated from almost any kind of sample; soil, freshwater or salt water. Most methods for isolating amoeba are based on the "walk out" method in which a mono-layer of bacteria (usually E.coli) is deposited on an agar plate, and any amoeba present inoculum crawl as they consume the bacteria, to produce characteristic haloes of growth. Of course, just as 95% of bacteria cannot be easily cultured, by simple inoculative methods, so we can be certain that a proportion of the amoeba initially present cannot be cultured by this simple method and are rapidly overgrown by the usual amoebal "weeds"! This makes it very important to consider what type of organism we would wish to culture. The source of sample is a very important influence over which type of amoeba predominates in the culture. For example, if one is interested in obtaining Naegleria, then the best source would be freshwater. If Acanthamoeba are desired then soil is the best bet especially if the soil is rich in organic content. Flattened or flabellate amoeba (Vannella, Platyamoeba etc) are most likely to be found in marine samples Several different approaches can be taken for the isolation of amoebae depending on which type of amoebae is required. The main determinant in the choice of method is size. Three methods are outlined below in order of the size of the amoebae the isolator is interested in.
Small Amoebae (2-30 microns spherical diameter) Acanthamoeba, Naegleria, Vannella This basis of this method was published in the late 50's (Harrison, 1957; Neff, 1958). If amoeba are being sampled from water, this is poured over a petri dish in which 2% agar has been deposited. For freshwater, the 2% agar should be in Neff's saline (see below). If the water is seawater, then the plates should contain 2% agar in 75% seawater (for no clear reason many amoeba from fully marine environments do better in this than 100% sewater (Page, 19). If soils/sediments are being sampled a small amount of the material should be deposited on the plates with suitable water to cover. Amoebae should be given a few days to gather on the plates, after which, small cubes (A, figure 1) should be excised and placed upside down on fresh plate into which bacteria (usually E.coli) have been spread and allowed to dry. Ciliates and other non crawling protists together with metazoans are left under this block (sometimes nematodes wriggle out if the plates are too wet), while amoebae are free to crawl out from under the block consuming the bacteria and growing as they move out. After a few days characteristic growth rings appear on the surface (Figure 1, left). Amoeba are often very numerous at the leading edge (red), but for some reason Acanthamoeba does not do this much. Being this concentrated region, amoebae are present as small groups and individuals, but without much bacteria on which to feed (blue), further back toward the original block, amoebae especially from soil and freshwater will have formed cysts as a result of descication and lack of bacteria (green). The cysts often flocculate. The amoeba from this culture turned out to be Naegleria gruberi.
Figure 1. Culturing of amoeba by the "walk out" method.
Small blocks of agar from this plate should then be excised and the amoebae shaken off the block by swirling in media (such as sterile Neff's or 75% seawater), and deposited on fresh agar plates. Small blocks from these plates are now excised and inverted on fresh bacterial lawn plates and the process repeated. By now the culture should be clonal, but the only way to be sure is to isolate a single amoeba by carefully excising a block on which only one amoeba is resting. Axenic culture Many of these small amoeba can be cultured axenically, that is without any other organism present. This is often desirable if the amoeba is to be characterised at the molecular level or grown in large amounts as a source of a particular molecule. The walk out method can be used for axenification by creating lawns of heat killed (autoclaved bacteria) and the use of antibiotics (such as penicillin) in the agar. Blocks of agar containigndead bacteria and living amoeba can then be transferred to sterile axenic media. (see Culturing amoebae).
WARNING.As some of this group are known to be pathogenic in humans and animals, care should be taken with the resulting cultures. Workers wearing contact lenses should be especially cautious with Acanthamoeba strains as some cause keratitis, a potentially sight threatening condition. The pathogenicity problem can be reduced by discarding any cultures in which amoeba can grow at 37^oC.
*Medium Amoebae (30-150 microns) Mayorella, Saccamoeba,* The walk out method described above can be used for these larger species, but the small amoeba tend to out grow them, obscuring there presence and out competing them due to their more limited requirement for surface water films. Instead the following dilution method may be better. Pond, sea or soil is plated out on suitable agar plates as above and amoeba are allowed to settle. It is also worth doing without the agar. A single interesting larger amoeba can be scooped up by the careful use of a pipette and this transferred to a well in a 24 well plate. Let everything settle down and then look for the successfully transferred amoeba. If it is there and there are no other similarly sized amoeba present, growth can be encouraged by adding food organisms such as bacteria or Tetrahymena. Larger amoeba tend to require more than just bacteria to grow, many need ciliates or even other amoeba.
Giant Amoebae (150-4000 microns) Amoeba, Chaos, Pelomyxa Giant amoeba such as those of the genera Amoeba, Chaos and Pelomyxa are not the most numerous and can be difficult to find. They are to be found most often on the muddy surfaces of well established ponds and lake sediment. One method to collect them is to invert a clear glass jar into the water so that air is retained until the lip reaches the silty pond bottom. Air is then let out so that the inrushing water sucks in the top layer of silt. Move the vessel (jar) around to suck up a large surface of the pond bottom. Tall jars are particularly useful as the next step is to settle the contents of the jar in diffuse (not direct sunlight) with the bottom of the jar covered in foil. Amoeba and Chaos are phototactic (I don't know if Peloxyma is). The amoeba will concentrate on the surface of the "pond" within the jar. Once the amoeba is discovered it can be transferred into a petri dish with a pipette and filtered pond water added. Boil a few grains of rice let them cool and transfer one to the petri dish, organisms that will have inevitably come along with the amoeba will grow and provide food. Alternatively, ciliates such as Tetrahymena can be grown and fed to the amoeba. The amoeba to the right ( A. proteus) was isolated from the pond above.	Amoeba proteus
References:- Harrison, D. (1957) A technique for obtaining clone cultures of soil amoebae in sterile liquid medium. Nature. 180, 1301-1302. Neff, R. J. (1958) Mechanisms of purifying amoebae by mirgation on agar surfaces. J.Protozool. 5, 226-231.