Section B: Culturing the Vegetative Stages

P. polycephalum is easy to maintain in culture in either the amoebal or the plasmodial stage. In appropriate culture conditions, the cells are prevented from proceeding through the life-cycle, and can be kept in active growth by frequent subculturing. As in other micro-organisms or cultured cells, however, long periods of proliferation can result in several types of changes in the genetic constitution of the population. It is therefore essential to re-initiate cultures frequently, using stocks that have been stored in conditions in which such changes cannot occur. Culture methods for plasmodia The choice of culture method will of course depend mainly on the type of experiments to be carried out. For biochemical analysis, plasmodia have usually been maintained in liquid, shaken cultures (Aldrich & Daniel, 1982). For studies related to the nuclear division cycle, microplasmodia are harvested from shaken culture and allowed to fuse on a surface, where they readily form large plasmodia in which the nuclei soon become synchronous. Events can then be followed through several successive cycles of nuclear division and DNA synthesis (Burland et al, 1993a). Surface cultures are also preferable to liquid cultures for studies of plasmodial myosin, for example, since it is produced in larger quantities in surface plasmodia, presumably because the 'vein' structure is more highly-developed than in microplasmodia. For genetic analysis, plasmodia are normally cultured on axenic agar media, since these provide the simplest conditions for testing characteristics such as nutritional requirements or plasmodial fusion behaviour (see Section D). For genetic analysis, sporulation is most easily obtained from such cultures also, though biochemical studies of the changes accompanying sporulation have been done using microplasmodia harvested from liquid medium (Schreckenbach & Werenskiold, 1986). Culture methods for amoebae For biochemical analysis of amoebae, it is probably most convenient to use liquid cultures of Axe strains since large quantities of amoebae can be easily harvested. It is also essential to use such cultures for DNA transformation (see Section E). Axe amoebae can also be cultured axenically on the surface of filters moistened with liquid axenic medium. In both types of culture, however, the doubling-time is much longer (16 - 18h) than for amoebae cultured on bacterial lawns (6-8h). Axe amoebae can also grow very slowly on axenic agar media but this method of culture has so far proved impracticable. Because of the slow growth rate, it has been found difficult to label amoebal proteins in axenic cultures, and this has been achieved more easily by culturing amoebae on labelled bacteria (Turnock et al, 1981). For many stages of genetic analysis, amoebae are cultured on lawns of live bacteria, where they can be plated to form single colonies, re-cloned, and tested for many inherited characteristics (see Section D). Stock cultures of amoebal clones, including Axe strains, are also maintained on bacterial lawns; these cultures can be easily stored for short periods in a refrigerator until they are required, unlike axenic liquid cultures, which must be subcultured frequently and kept in active growth (Dee et al, 1989, 1997). Cultures on live bacteria are also used to provide amoebae for the long-term storage procedures described below. Formalin-killed bacteria can be used as an alternative food supply but these support slower growth. For studies of individual cell growth and cell behaviour, and for analysis of cell pedigrees by time-lapse cinematography, an amoebal culture is set up on a bacterial lawn, spread on a thin agar layer and sealed into a cavity slide; such a culture continues in active growth for several days (Bailey et al, 1987). Formalin-killed bacteria are sometimes used in slide-cultures, instead of live bacteria, since this enables the food supply to be more precisely controlled. For particular purposes, amoebae have also been cultured on bacteria in other conditions. For example, lawns of bacteria spread on filters, supported over liquid medium, are more suitable than lawns on agar when amoebae are cultured for some mutagenic procedures; the filters, bearing the amoebae, can easily be transferred to different media or reagents. Axe amoebae can be cultured either in liquid axenic medium (SDM) or on lawns of bacteria, depending on the type of analysis required. The most recent recipe for SDM is given in Dee et al. (1997). Transferring the amoebae from liquid to agar presents no great difficulty, but must be carried out with a little care, following the recommended procedures (Dee et al., 1989). The reverse transfer is sometimes a little more difficult. The growth of amoebae inoculated into liquid axenic medium from bacterial lawns is inhibited by the presence of bacteria and by the presence of cysts in the amoebal culture. It is useful to induce excystment prior to inoculation since this process does not readily occur in the liquid medium (Dee et al., 1997) Washing the cell suspension to remove bacteria before inoculation is also desirable, even though antibiotics should be added to the initial cultures. Even with these precautions, the growth rate of amoebae in axenic liquid medium is often initially slower than that observed in established cultures, and the reasons for this are not yet clear (see section below on changes during culture). Storage of stock cultures Microplasmodia and surface plasmodia can be stored in dormant state as 'spherules' and sclerotia, respectively (Mohberg, 1982). Repeated use of such storage procedures is not recommended, because of the genetic changes that may be propagated, but they may be useful occasionally and are preferable to maintaining the same plasmodia in active culture for long periods. Since the plasmodial stage is multinucleate, however, even in liquid, shaken cultures, it is not possible to re-clone a plasmodium from a single nucleus. It is therefore preferable to store stocks in the form of amoebae, which can be re-cloned if necessary, and to derive plasmodia from these when required; all strains used for genetic work are maintained in this way. Several methods for preserving amoebae (or cysts derived from them) have been described and most of them are very simple to use. All laboratories using P. polycephalum should keep their own collection of strains stored by one of these methods. When amoebal strains are received from another laboratory, they should be cultured for the shortest time possible before a stock is preserved for future use, so that minimal genetic change is allowed. To avoid maintaining amoebae or plasmodia for long periods in active culture, the stored stocks can then be used at intervals to re-initiate the cultures. Plasmodia can easily be re-constructed by selfing or crossing amoebae on bacterial lawns. The genetic characteristics of strains should also be checked at intervals and amoebae should be re-cloned by plating if necessary to eliminate cells that have changed. In our experience, stocks of amoebae stored in the freezer or with silica gel (Anderson et al., 1983) have remained viable for several years. We have not made any careful measurements of viability or longevity, however; our criterion of success has been simply that we could obtain an active culture from a sample of amoebae stored at high cell density. It is likely that some cells die during storage and that the viability of a stored culture gradually decreases. Another area of uncertainty is the state of the cells at the time of storage. It is usually assumed that most amoebae have formed cysts before they are prepared for storage, but we do not usually check this; it is possible that the proportion of encysted cells varies widely between different cultures and strains. If only cysts remain viable during long- term storage, the viability of stored cultures may also vary widely. It is therefore advisable for each laboratory to standardise its own methods and check the longevity of stored strains. Amoebal stocks to be stored are usually grown to high cell density on lawns of live bacteria on agar. As a confluent lawn of amoebae is formed, the cells become immobile, rounded, and phase- bright, and they are prepared for storage at this stage. The proportion of cysts can be checked by tests for triton-resistant cells (Gorman et al., 1977; Dee et al., 1997), although this is not normally done. Lawns of formalin-killed bacteria should probably not be used to produce amoebae for storage, since we have found that the amoebae grow more slowly in these conditions and have poor viability when incubated for more than a week or when stored in a cold-room. Axe strains of amoebae have usually been stored after culture on live bacteria, but a method for inducing encystment and freezing cysts on axenic agar medium is now available (Dee et al., 1997). The frozen axenic cells can be induced to excyst when required and used to re-inoculate axenic liquid cultures. It is hoped that this method will avoid the initial stage of slow growth caused by transferring stored amoebae into liquid medium from bacterial lawns. Incubation temperatures The optimum temperature for growth of P. polycephalum amoebae and plasmodia probably lies between 25oC and 30oC. Strains of amoebae capable of apogamic development must be cultured at the upper end of this range (29-30oC) if plasmodium formation is to be prevented. Other strains may be found to grow better at slightly lower temperatures (25-27oC). It is not possible to be more precise because we have found by experience that optimum temperatures vary between laboratories, probably due to other factors affecting conditions in the incubator or hot room. Humidity of the atmosphere may be particularly important for surface cultures, and good aeration is essential for liquid cultures; these conditions may differ in different laboratories, and are likely to be affected by temperature. It will therefore be necessary to define the best conditions by trial and error in each laboratory. The most suitable temperature for apogamic strains is the most difficult to determine, since amoebal growth will be poor if the temperature is too high and plasmodium formation may occur if it is too low; the amount of fluctuation in temperature is also important, since apogamic amoebae may need only a short time (less than an hour) at low temperature to become committed to plasmodium develoment. Since amoebal strains of genotype matA2 gadAh npfC5 (e.g. CLd, LU352) form plasmodia only when revertant mutations occur in npfC, it is possible to culture amoebae of these strains for short periods at temperatures permissive for development; this is not recommended, however, since plasmodia may form, and, particularly in liquid cultures, they may not be detected until they have reached a large size. Incubation temperature is critical also when amoebal cultures are re-initiated from stored stocks. In Leicester (but not in Sheffield) it is normally found that amoebal growth cannot be obtained from stocks stored in a cold room or freezer unless cultures are incubated initially at a temperature below 29-30oC. If the amoebae are of an apogamic strain, they could be shifted to the higher temperature after about 24 h, or when active amoebae are visible at low density in the culture. Plasmodium formation will not begin, even at low temperature, until the amoebae reach a critical cell density. The initial incubation at low temperature necessary for growth from stored stocks may be due to the need for excystment. However, as discussed above, it is not clear whether amoebae survive storage only in the form of cysts, and there is evidence that low temperature may also be required when cultures are inoculated with amoebae at very low density. For example, it has been found possible to grow Axe amoebal cultures from single cells in liquid medium incubated at 26oC but not at 30oC (Dee et al. 1989). Incubation at the lower temperature has also proved helpful when Axe amoebae are first inoculated in liquid medium from cultures on bacteria, and when subcultures are made from liquid cultures that have reached stationary phase, when the cells begin to appear rounded and to form clumps. Changes in genetic constitution of amoebae during culture Several types of change have been observed during long-term culture of amoebae and it is important to be aware of these in order to avoid adverse changes or to detect them quickly if they occur. Clones derived after re-cloning amoebae produced by spores usually consist of haploid cells, but diploid and aneuploid clones also occur, presumably as the result of aberrant events during sporulation (see Section D). Haploid amoebae maintained in culture on bacteria for long periods have also been found to give rise to cells of higher ploidy (Adler & Holt, 1974). In some cases, strains which were initially haploid have later been found to consist almost entirely of diploid amoebae, though it is not clear whether these have arisen during culture or by some accident of differential survival during storage. (See for example LU381 in Dee et al. 1989). Diploid or aneuploid amoebae are sometimes detected during re-cloning because they form slow- growing colonies of irregular form; this is not always the case, however, and some diploid amoebae grow well and form good colonies. Diploid and aneuploid strains can cause great problems if they are used for genetic analysis. The best method for checking the ploidy of amoebal cultures is probably flow cytometry of cells stained with a DNA-specific fluorochrome; this gives a very precise analysis of the distribution of cells with different DNA contents in the population. If this method is not available, an indication of ploidy can be obtained by microscopic measurements of cyst diameter (Dee & Anderson, 1984). Using flow cytometry, it was found that some strains of Axe amoebae, maintained in liquid culture, still consisted almost entirely of haploid cells after being repeatedly subcultured during active growth for a period of several months (Dee et al. 1989). In contrast, the same strains cultured on bacterial lawns contained diploid and aneuploid cells, sometimes at high frequency. Earlier reports from several laboratories had suggested that the strain CLd-AXE, which was originally haploid when it was isolated in Leicester, had become diploid during culture in liquid medium. It is not clear, however, whether such changes actually occurred in liquid medium, or during periods of storage. Changes in genetic constitution resulting from mutation and inadvertent selection during culture of amoebae have also been observed, and should indeed be expected in certain circumstances. Firstly, amoebae which revert to wild-type in a drug-resistant strain may grow faster than the mutant type when cultured in the absence of the drug and will therefore become more common during repeated subcultures in such conditions. Secondly, amoebae with mutations delaying or reducing their ability to undergo plasmodium development are expected to increase in frequency during repeated subculture of an apogamic srain in conditions permissive for development. Cells that become committed to development will fail to multiply as amoebae and will therefore be lost from the population. Cultures maintained at high temperature are less likely to undergo such changes, but since the temperature sensitivity of development is not 'tight' in most apogamic strains (see Section A), inadvertent selection is still possible in these conditions. This type of change has been recorded frequently by research workers culturing apogamic amoebae, and it can occur during a few subcultures; great care is therefore required to maintain such strains without losing their potential to develop. Mutations in the opposite direction, giving rise to npfC+ revertants in a strain such as CLd or LU352, for example, are less likely to cause difficulties because they will rapidly become obvious when plasmodia are formed in the culture. Care must be taken to detect such mutations during studies of stage-specific gene expression, however (see Section C). Loss of ability to grow in axenic media has been recorded in a number of amoebal clones after culture on bacteria but the basis of this change has not been elucidated. Axe amoebae are often cultured on bacteria and returned to axenic medium without difficulty but it is advisable to re-test strains for their Axe phenotype if they have been cultured or stored in non-axenic conditions for long periods. In liquid axenic culture, Axe amoebae are often observed to increase in growth rate. During the first ten subcultures, after transfer from a bacterial lawn, the change is probably due to the gradual elimination of bacteria and encysted cells and perhaps to gradual conditioning of the medium. Up to about 30 subcultures, further slight increases in growth rate continue unaccompanied by any adverse changes (Dee et al., 1989). After about 60 subcultures, however, dramatic increases in growth rate have been observed and it has been found that various normal cellular properties have been lost at about the same time. For example, the Axe strain LU352 was found to lose the ability to undergo the amoeba-flagellate transformation on several occasions when the amoebae had been cultured in liquid axenic medium for a period of several months (Glyn, 1989). It has also been repeatedly observed that the proportion of cells able to undergo the amoebal- plasmodial transition and the proportion able to encyst when transferred to bacterial lawns have declined after 60-70 subcultures in axenic medium. The genetic basis of all these changes is currently under investigation. Meanwhile, we recommend that fast-growing sublines of Axe amoebae should be treated with caution and that axenic cultures should be re-initiated from stored stocks at intervals. Changes in plasmodia during culture Little is known about mutational changes during plasmodial culture. Since plasmodia are multinucleate, new mutations are not likely to be expressed, though they are expected to occur during long periods of culture, and to accumulate as a mutational load', particularly in diploid plasmodia. When plasmodia are stored in the form of sclerotia or spherules, these dormant phases are again multinucleate and thus mutations present in only some of the nuclei are likely to remain. Selection cannot operate to eliminate mutant alleles that are not expressed. Plasmodia have frequently been found to be heterogeneous in nuclear DNA content, and some progressive changes in the proportions of nuclei with different contents have also been recorded. Plasmodia derived from crosses between heterothallic amoebal strains have sometimes been found to contain a mixture of nuclei of haploid and diploid DNA content. During prolonged culture in liquid medium, the proportion of haploid nuclei in such plasmodia was found to increase (Mohberg et al, 1973). Similar changes were observed in heterokaryons constructed by fusing haploid and diploid plasmodia; changes in the expression and transmission of genetic markers indicated that the diploid nuclei were always lost when the heterokaryons were cultured on agar medium (Dee & Anderson, 1984). In plasmodia derived from apogamic amoebae, the majority of nuclei are haploid, but a small proportion of diploid nuclei is usually present also: it is not known whether this proportion shows consistent changes during culture. When maintained in surface culture on agar medium, plasmodia eventually show senescence: this is associated with particular morphological changes, decreased growth rate and, ultimately, death. The time course of these changes was found to be repeatable in several sublines of each strain studied, but differed between strains (Poulter, 1969). There is evidence that the final stages of senescence, at least, are associated with large increases in nuclear DNA content (McCullough et al, 1973). Plasmodial cultures maintained in liquid medium were not observed to senesce, perhaps because competition between microplasmodia was possible in these conditions, leading to the survival of those that had not suffered the degenerative changes observed in surface-grown plasmodia. The mechanism of senescence is still unknown.

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