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Amoeba Lore

Prepared by Keith Brocklehurst and Arranged by Mick Chaplin

Amoeba - the animal almost everyone knows by name, but few have seen either live or dead, is more correctly known as Amoeba proteus Leidy, 1878 (see figure 1). It was Leidy who in that year first gave an adequate recognisable description of this small freshwater protozoan. It is one of the larger species of the genus, reaching 0.4 mm (400 µm) in length when actively moving. Because it has only a single nucleus it is often said to be unicellular and it is this characteristic which is largely responsible for its name being so widely known from early biology lessons in schools.

Fig. 1

Amoeba proteus is difficult to ‘collect’ in the wild; it is generally found by chance. Normally one has to make cultures from freshwater samples and then search in order to find them. Success usually comes with persistence, after which one can easily maintain a live supply of this beautiful and remarkable little animal. A first encounter with Amoeba proteus under ideal microscopical viewing is a sight one is never likely to forget.

In this short web presentation we are looking to familiarise viewers with Amoeba’s more obvious characters through labelled diagrams and photomicrographs at first, and later add animations and short videoclips. Hopefully, we shall gradually build up this topic over a few months. Suggestions or offers of assistance would always be welcome. Contact either Mick Chaplin or Keith Brocklehurst.

A dark background is most rewarding when viewing cultures under low power stereomicroscopes but for higher magnification work phase contrast optics are ideal. It is, however, quite possible to achieve acceptable images using bright-field techniques.

Fig. 2

Fig. 2 shows the appearance of a ‘clean’ culture of six actively moving polypodial Amoeba proteus with ciliate and flagellate food organisms and very little organic debris.

Fig. 3a

Fig. 3b

Figs.3a and 3b. Compare the labelled line drawing of this amoeba with the photomicrograph from which it was made. Notice the longitudinal ridges which are characteristic of this species (proteus), and work through the named parts methodically. It was actively moving to the right when photographed and is monopodial unlike those in Fig.2.

Fig. 4

Fig.4 This shows a rapidly advancing pseudopodium; you can tell by the blurred central granules – exposure was 1/15 s. Notice the clear area at the advancing tip.


The body layers in Amoeba have gone through a confusing number of different names largely as a result of the chronic controversy about their mechanism of movement. We have adopted the following: Plasmalemma - the outermost membrane, much thicker than normal trilaminar plasma membranes of cells which are usually about 7.5 nm. (1 nanometre = 0.001 µm) thick : Amoeba’s outer layer has two extra outer coats and appears to be about 30 times thicker than normal plasma membranes.

Hyaloplasm - clear cytoplasm without crystals and other inclusions. It used to be called plasmagel or ectoplasm, but it can be in either the gel or sol state. Mitochondria (see later), the smallest dark spheres under phase contrast, are frequently visible in the hyaloplasm.

Granuloplasm - the main mass of cytoplasm containing crystals, food vacuoles and all other inclusions including the nucleus. Used to be called endoplasm or plasmasol.

Fig. 5

Fig.5 This shows the uroid, not invariably distinguishable, is dark under phase contrast, has numerous short, blunt pseudopodia (arrowed above) and thus appears wrinkled. The small dark spheres clustered around the water expulsion vesicle (WEV) or contractile vacuole are mitochondria. They are more clearly seen in Fig.6, below. Contractile proteins in the cytoplasm, using energy released from the mitochondria, cause the vesicle to collapse and expel fluid which has entered osmotically. Focussing above or below the centre of a full WEV usually shows mitochondria clearly with phase-contrast. They are also detectable using bright-field optics.

Fig. 6

Fig. 7

Fig.7 Nucleus and cytoplasm in a temporary preparation thinned by evaporation and without vaseline support for coverglass.


The nucleus appears distinctly granular and, when it is being carried in a moving stream of granuloplasm can be seen to be disc-shaped sometimes with a slightly concave side. It has many small nucleoli under the surface and is covered by a fine honeycomb lattice structure (not visible in optical microscopes), which is thought to give the nucleus protection and support. Notice the crystals, especially just below the nucleus, are enclosed in vacuoles.

Fig. 8

Fig. 8 Nucleus and food vacuoles (numbered 1 – 4) in a thinned preparation without coverglass support. Five flagellates of genus Chilomonas are have been captured in vacuole numbered 4. The characteristic notch of Chilomonas is clear in one of the specimens (2 o’clock, lower end).

Amoeba is a difficult subject for stained squash preparations which show chromosomes, because its chromosomes, being very numerous and small, are commonly obscured by food vacuoles, crystals and other cytoplasmic inclusions. Making such preparations is an exacting task! Thanks are due to Dr L.G.E.Bell who made the preparation and micrograph (Fig.9 below) which originally appeared in 'The Biology of Amoeba', Academic Press 1973.

Fig. 9

Fig.9. A lactopropionic squash preparation of an A.proteus division sphere at late metaphase plate stage. Most of the nuclear region is included; the large number of small chromosomes suggests that the species is polyploid.

Before nuclear division A. proteus withdraws its pseudopodia, assumes a spherical shape covered by numerous very short pseudopodia and ceases active movement. In this motionless state, when the nucleus is undergoing mitotic division, the animal is spherical and covered with very short motionless pseudopodia. Such division spheres have no attachment to the substrate, will roll gently on the bottom of a culture dish and can be picked up readily in a fine micropipette. If then mounted under a coverglass with vaseline support, the process of cytoplasmic division, which follows the nuclear division can be observed and recorded.

Simple fission is the only fully substantiated method of reproduction in A. proteus. It involves division of the nucleus followed by a separation of the cytoplasm into two. The whole process lasts less than an hour at 18 – 20ºC. The onset of mitotic nuclear division is marked by the withdrawal of long locomotory pseudopodia and formation of the division sphere (see Fig.10). Cytoplasmic division begins with a lengthening of the division sphere, in which two terminal masses become progressively obvious (see Fig.11) as movement in them recommences. A connecting strand of cytoplasm, at first thick becomes noticeable, then it gradually thins as each terminal mass makes attachment and moves stretching the strand until it finally breaks with an elastic recoil. This cytoplasmic division is simple to observe and record (see fig.12) once one has a culture containing division spheres, and a fine micropipette with which they can be picked out and mounted for examination under a microscope. Bright illumination in the early stages may prevent completion of fission. A heat filter in the microscope's light path is recommended.

Fig. 10

Fig. 11

Fig. 12

Feeding or phagocytosis

Amoeba proteus is a carnivore – see Fig.8, vacuole 4 with 5 flagellates. It also feeds on ciliates, bacteria, rotifers, and other small freshwater animals. In simple terms, it senses the food organisms and surrounds them with pseudopodia thus enclosing them in a food vacuole membrane. Stages in this food capture are shown in Figs.13 and 14 below. When in a rich food culture they eventually stop feeding, probably because they cannot make cytomembrane fast enough to supply the food vacuoles! The food in these cases are once again the flagellate Chilomonas paramecium which is about 16µm in length. Digestion occurs in the vacuoles and undigested remains of the food are voided near the uroid during active movement.

Fig. 13

Fig. 14

Drinking or pinocytosis

Foods in solution may be ingested by pinocytosis (cell drinking).If mounted in the more liquid albumen of a chick egg, Amoeba proteus responds in a remarkable way. It becomes immobilised and rapidly covered all over with about 80 short pseudopodia, each with a fine channel leading in from the external medium (see Fig. 15). The inner ends of these pinocytic channels are difficult to follow into the granuloplasm, but there is no doubt vacuoles containing albumen are pinched off there, and presumably digestion of this almost pure protein takes place. Careful inspection of the channels at high magnifications also reveals waves of constriction passing down the channels, which promotes albumen uptake. Plasma proteins and amino-acid solutions also elicit this response which can last for a half hour.
Notice that the bright phase halo is reversed to dark with phase contrast viewing in albumen.

Fig. 15

Locomotion

Examine Fig.16 which shows 3 still images taken at 30 second intervals. The fine strand of glass wool acts as a static marker. Look at images 1 and 2; try to judge which pseudopodia have been advancing and which have been withdrawn. Repeat for images 2 and 3. A videoclip will be added soon. Watching pseudopodia formation and withdrawal has fascinated observers for a long time. There has been much experimental investigation of the biochemical mechanisms involved and considerable controversy. However, there is evidence of an ATP-powered actin/myosin system at work as in muscular systems of locomotion in higher animals.

Fig. 16

Culturing

Amoeba proteus has been called a 'domesticated' animal, and with some justification, since indoor enrichment cultures normally have to be made from fresh sphagnum moss collections if a ready supply of live specimens is to be made available. Enrichment involves sterilising clean (unsprayed) ripe wheat grains in water from the moss collections using medical 'flats' and a pressure cooker. Full details of this procedure are available in Balsam Post No.23, April 1994, p.28. Copies are still available from the librarian. Obtaining the right water may be the key to success. Recently Volvic bottled water has been used successfully, and in the 1950s Monica Taylor of Glasgow Zoology Dept. maintained her magnificent cultures in water from Loch Katrine in the Trossachs!
Convenient culture dishes are glass, stackable, with loose-fitting lids and 13cm. x 13cm. x 5cm in size. Petri dishes and lids 9cm. diameter will serve. Adding wheat grains, now burst open, and sterile water promotes growth of bacteria, flagellates and other food organisms. Bacterial films and fungal growth can be removed from the water surface with fine clean tissues. Examine regularly until you find an established culture, then sub-culture at intervals to maintain it. Perhaps a less tedious way to obtain live amoebae could be to contact Sciento., Algae and Protozoa cultures, tel:0161 773 6338 or e-mail bob@sciento.co.uk . I understand they can supply live cultures.
Several other rhizopods, both free-living and shelled (testate) are also common in these mixed enriched cultures.

Fig. 17

Fig. 18

Other Amoebae

Once you have got to know Amoeba proteus you may like to extend your acquaintance with rhizopods (Protozoa which possess pseudopodia). It is important to allow specimens time to settle to their characteristic locomotory form before attempting an identification.

Thecamoeba terricola (fig.19) surface layer showing irregular curved or long ridges, sometimes called a pellicle. Normally moves slowly by eruptive waves but develops long ridges when moving fast. Herbivorous, feeding on algae, especially Scenedesmus. A vesicular nucleus with a central smooth nucleolus. Length 120 – 150µm.

Fig. 19

Mayorella viridis (fig.20) green because of numerous zoochlorellae in the granuloplasm. Nucleus vesicular, several hyaline conical pseudopodia produced from a broad zone. This gives a 'web-footed' appearance to the advancing region. Some authors call these advancing tips sub-pseudopodia. Length 90 – 140µm.

Fig. 20

Mayorella bigemma (fig.21) no zoochlorellae, vesicular nucleus, web-footed appearance as in M.viridis ,but usually larger. Under phase contrast bright hour-glass shaped crystals each attached to a dark hemispherical body are characteristic and readily noticeable during active movement in the granuloplasm.

Fig. 21

Saccamoeba limax (fig.22) monopodial and slender, up to 100µm. in length. Single contractile vacuole (better called a water expulsion vesicle) near a posterior dark rough uroid. About 20 – 30 bipyramidal crystals, relatively large vesicular nucleus, short hyaline cap at advancing end.

Fig. 22

Vexillifera sp. (fig.23) often stellate in free-floating state, settles down with many short digitate pseudopodia at first. Can also produce long thin pseudopodia which bend and wave. Nucleus vesicular.

Fig. 23

Nebela flabellulum (fig.24) a squat, bilaterally flattened, pear-shaped amoeba with a shell supported by silica plates. Blunt pseudopodia issue from the ovoid aperture of the shell, make attachments and pull the animal along. A testate rhizopod from Cwm Idwal, N.Wales. Length 90µm., the only species of Nebela which is broader than long.

Fig. 24

Hyalosphenia papilio (fig.25) the most amenable testate rhizopod for live study because its flask-shaped test is transparent and without any siliceous plates or attached opaque particles. Its cytoplasm can be viewed in near perfect optical conditions. Length 110 – 140µm. Test flask-shaped and mildly flattened bilaterally. Numerous zoochlorellae, Large nucleus aborally with numerous nucleoli. Pseudopodia blunt, never more than two at one time. Rapid locomotion observed in vaseline supported mounts. Specimens from sphagnum pools on Heron Crag above Grasmere, Lake District.

Fig. 25

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