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.