JOURNAL OF BACTERIOLOGY, July 1972, p. 242-247 Vol. 111, No.

1
Copyright © 1972 American Society for Microbiology Printed in U.S.A.

Axenic Culture of Myxamoebae of the

Myxomycete Physarum polycephalum
E. M. GOODMAN
University of Wisconsin-Parkside, Kenosha, Wisconsin 53140
Received for publication 25 February 1972

Myxamoebae of the acellular slime mold Physarum polycephalum have been

cultured axenically in a soluble medium. The growth medium contains bovine
serum albumin, embryo extracts, liver infusion broth, peptone, and glucose.
Cell densities ranging from 3 x 106 to 5 x 106 cells/ml have been obtained with
this medium. To date, myxamoebae have been serially transferred more than
100 times without deleterious effect.

The myxomycete Physarum polycephalum is
an ideal organism for comparative biochemical
studies of both cellular growth and differentia-
tion. Previous investigators have shown that
fusion of compatible haploid myxamoebae re-
sults in formation of the diploid, multinucleate
plasmodium, a state concerning which exten-
sive biochemical and morphological data exist
(9). Future studies of the metabolism and re-
lated biochemical pathways in myxamoebae
should provide greater insight into the events
and control mechanisms associated with syn-
gamy and the resultant shift from the haploid
to the diploid state. Myxamoebae of P. polyce-
phalum divide by a true cell division, with
karyokinesis followed by cytokinesis
(Goodman and Haugli, unpublished data),
whereas only karyokinesis occurs in the plas-
modium (9). These contrasting states in the
same organism offer the investigator a new
approach for studying comparative controls
involved in cell division.
To maximize the experimental usefulness of
myxamoebae it is essential that they be cul-
tured under axenic conditions. This paper re-
ports the axenic culture of P. polycephalum
myxamoebae in a semidefined soluble me-
dium.
MATERIALS AND METHODS
A modification of Band's (1) basal salt solution
was found to be osmotically compatible with myx-
amoebae and was used in all subsequent attempts to
replace bacteria as a principal source of nutrient.
The basal salt solution (BSS) was prepared as fol-
lows: 120 mg of NaCl; 3 mg of MgCl2 6H20; 3 mg
of FeSO4.7H20; 3 mg of CaCl2; 1.24 g of Na2HPO4;
and 354 mg of KH2PO4; distilled water to 1,100 ml
(final pH, 6.9).
242
For experimental studies, 6-ml portions of BSS
were autoclaved separately in 125-ml siliconized Er-
lenmeyer flasks with stainless-steel caps (Bellco
Glass, Vineland, N.J.). Flasks were siliconized in
dimethyldichlorosilane (Alfa Inorganics, Beverly,
Mass.)-concentrated hydrochloric acid (HCl), 50:50
(v/v). Excess silicone and HCl were removed by
rinsing in ethanol-ether, 50:50 (v/v), followed by
heating at 160 C overnight.
Component preparation. (i) BSS, glucose (0.5 M),
peptone (Difco; 10%, w/v), and liver infusion broth
(Difco; 10%, w/v) were autoclaved separately at 121
C for 20 min. (ii) Lyophilized bovine serum albumin
(Sigma Chemical, St. Louis) was reconstituted in
distilled water (10%, w/v) and sterilized by filtration
through 0.45-tm membrane filters (Millipore Corp.,
Bedford, Mass.). (iii) Lyophilized chick embryo ex-
tract and beef embryo extract (Grand Island Biologi-
cal, Grand Island, N.Y.) were reconstituted in 10 ml
of sterile distilled water.
Individual components of the medium, in addition
to stock cultures, were checked routinely for contam-
ination by: (i) streaking on plates containing either
tryptone agar (1%, w/v) or Sabouraud dextrose agar;
(ii) inoculation in a nutrient broth; and (iii) micro-
scopic examination using phase optics.
Maintenance of stock cultures. Myxamoebae of
strain RSD4, mating type mt1, of P. polycephalum
were kindly supplied by F. Haugli. During the initial
search for an axenic medium, myxamoebae were
maintained by the methods of Haugli (F. Haugli,
Ph.D. thesis, Univ. of Wisconsin, 1971) on 2% agar
(w/v) supplemented with 0.05% (w/v) liver infusion
broth. Escherichia coli strain K was used as the pri-
mary nutrient source. Prior to use, bacteria in log
growth were harvested by centrifugation and sus-
pended in 0.01 M tris(hydroxymethyl)aminomethane
buffer, pH 8.0, containing 0.01 M MgSO4.7H20
(T-M buffer) and 2.5% Formalin (v/v). Cells were al-
lowed to remain in this solution for 24 hr followed by
two 24-hr washes in T-M buffer to remove residual
Formalin.
VOL. 111, 1972 CULTURE OF P. POLYCEPHALUM MYXAMOEBAE 243
medium for longer than 96 hr resulted in the
Axenic stock cultures were maintained in a sol-
uble medium consisting of: BSS (final volume after
appearance of spherical cysts ranging in size
autoclaving, 5 ml); 0.1 ml of chick embryo extract;
from 8.0 to 12.0 Am (Fig. lc). Flagellated
0.1 ml of beef embryo extract; 0.25 ml of glucose;
swarm cells were rarely seen in media that
0.25 ml of liver infusion broth; 0.25 ml of peptone;
contained growing cells. However, when myx-
and 2.5 ml of bovine serum albumin. The final pH of
the growth medium was 6.35. Cultures were main- amoebae were placed in a medium which could
tained at 24 C on a reciprocating shaker (100 not support growth (i.e., substituting hemin for
embryo extracts), the proportion of swarm
strokes/min) and transferred to fresh medium every
cells dramatically increased.
72 to 96 hr. Myxamoebae were serially transferred as
follows. (i) Cells were collected in 50-ml conical The results obtained when cultures were
tubes and centrifuged at 250 x g for 10 min, and all
transferred to fresh medium indicate a dou-
but 0.5 to 1.0 ml of the old medium was decanted.bling in the original cell number approxi-
(ii) Myxamoebae were resuspended by brief agitation
mately 32 hr after inoculation (Fig. 2). At 72 hr
on a Vortex mixer, and 0.1 ml of this suspension the number of cells increased about 3.5-fold
(approximately 7 x 106 cells) was inoculated into 8.45
over the initial inoculum, or the equivalent of
ml of soluble growth medium. Comparing cultures of
myxamoebae maintained in stationary flasks with 1.75 doublings. The pH of the medium in-
creased 0.2 pH unit (from 6.35 to 6.55) during
other cultures grown on a shaker revealed approxi-
the first 48 hr of growth (Fig. 3). During the
mately one-third more growth under the latter con-
ditions. next 48-hr period, there was a less marked in-
Analytical procedures: analysis of growth. crease resulting in a pH of 6.65 at 96 hr
Growth curves were obtained by adding 0.05 ml of a
postinoculation. Data obtained from the incor-
l 4C-reconstituted protein hydrolysate mixture poration of 'IC-protein hydrolysate also show
(Schwarz/Mann, Orangeburg, N.Y.) to cultures fol-that the period of maximum growth occurs
lowing their transfer to fresh medium. Duplicate 0.5-
during the first 24 to 48 hr after transfer to
ml samples were removed at 24-hr intervals and cen-
new medium (Fig. 3). In the ensuing 48-hr pe-
trifuged at 250 x g for 5 min at 4 C in an Interna-
riod a substantial decrease in growth rate was
tional PR-J centrifuge. Pellets were resuspended and
washed twice with BSS, followed by homogenizationobserved.
at 500 rev/min in 0.5 ml of BSS with a Potter-Elve- Several experiments were performed to as-
jehm homogenizer. The homogenate was extracted certain the absolute requirements for various
three times in trichloroacetic acid (10%, w/v), and
constituents found in the soluble medium. The
the final pellet was dissolved in 0.5 ml of 0.4 Ndata obtained when either individual or combi-
NaOH. Protein was analyzed by the Lowry method nations of components were deleted from the
(8) and radioactivity was determined by counting
0.1-ml samples in a mixture of PPO-POPOP (4 g of growth medium are summarized in Table 1.
2,5-diphenyloxazole and 50 mg of 1,4-bis-2-[5- Deleting beef or chick embryo extract, glucose,
or peptone as individual components had a
phenyloxazolyl]-benzene in a liter of toluene) and
moderate effect, decreasing growth from 12 to
95% alcohol (3:1) by using a Nuclear-Chicago Unilux
11-A spectrometer. Counts were corrected to 100%28% relative to control cultures. In contrast,
efficiency by the Nuclear-Chicago CURVFIT pro- removing liver infusion broth or bovine serum
gram. albumin resulted in a 48% and total inhibition
Growth rates of myxamoebae were also deter- of growth relative to their respective controls.
mined by counting cells at 24-hr intervals in a Cell growth was completely inhibited when
Spencer-Neubauer hemacytometer. Microscopic ob- both liver infusion and peptone were removed,
servations and photographs were made using a Wild
M-2 phase-contrast Photoautomat system. or when chick and beef embryo extracts were
deleted from the medium.
Doubling the concentration of bovine serum
RESULTS albumin, glucose, peptone, or both embryo
Comparisons of myxamoebae grown axen- extracts had a limited effect, increasing the
ically in synthetic soluble medium (Fig. la) and final yield of myxamoebae about 5 to 10%.
those grown with bacteria on an agar substrate However, when the concentration of liver infu-
(Fig. lb) revealed no discernable morphological sion was doubled, a 16% increase in cell num-
differences, with both presenting the incon- bers relative to controls was observed (Table 1).
stant shape characteristic of amoebae. Myx- Increasing the concentration of liver infusion
amoebae ranged in size from 14 to 18 ,um. The fourfold produced no further enhancement of
nucleus varied from 2.0 to 4.0 ,um and con- growth.
tained a single distinct nucleolus surrounded To determine whether both embryo extracts
by numerous chromocenters. Myxamoebae were essential or whether it was only necessary
with two nuclei were occasionally observed. to maintain a certain extract concentration,
Allowing myxamoebae to remain in growth one of the extracts was removed and the con-
244 GOODMAN J. BACTERIOL.

FIG. 1. (a) Phase-contrast micrograph of myxamoebae grown on bacteria. Note metaphase plate at arrow.
x 1,800. (b) Phase-contrast micrograph of myxamoebae grown in soluble mediufn. x 1,800. (c) Phase-contrast
micrograph of encysted myxamoebae. x 1,800.

centration of the remaining one was doubled.
The results (Table 1) show that doubling the
concentration of chick embryo extract and de-
leting beef embryo extract depressed the
growth approximately 12% relative to the con-
trol cultures. Essentially no change in growth
was obtained when the concentration of beef
embryo extract was doubled and chick embryo
extract was deleted from the medium (Table 1).
If the concentration of bovine serum al-
bumin was decreased from 3 to 1%, growth was
inhibited approximately 28% (Table 1). To
ascertain whether the apparent growth-pro-
moting effects of bovine serum albumin could
be attributed merely to increased viscosity,
0.2% (w/v) Methocel (a high-molecular-weight,
inert methylated cellulose polymer, 4,000 cen-
tipoise) was introduced as an albumin substi-
tute. The experimental data indicate that in-
creasing medium viscosity alone is not suffi-
cient to sustain growth.
Substituting hemin for chick and beef em-
bryo extracts at either 6.0 or 0.6 jig per ml re-
sulted in total cessation of growth (Table 1).
At both hemin concentrations, numerous en-
cysted myxamoebae were observed within 72
VOL. 111, 1972 CULTURE OF P. POLYCEPHALUM MYXAMOEBAE 245
100 6.7

50 6.6

.C

6.5 a
E
-E
6.4
0
I 10
E
E-5 ( 6.3
0 0 24 48 72 96
Time (hr)
FIG. 3. Incorporation of "IC-protein hydrolysate,
left ordinate (0); and changes in pH, right ordinate
(0). Data based on an average of four experiments.

dium. It is interesting to note that these re-

0 24 48 72 96 quirements were also described by Hohl and
Time (hr) Raper (6, 7) for axenically culturing the cel-
FIG. 2. Typical growth curve of myxamoebae on lular slime mold Polysphondylium pallidum.
soluble medium. Standard deviation based on 10 However, this need apparently varies among
replicate counts per sample. slime molds since the acellular slime mold
Echinostelium minutum (5) and the cellular
hr after inoculation. slime mold Dictyostelium discoideum (10)
Prepared tissue culture media (Gibco; have both been grown axenically without bo-
NCTC-135, RPM1-1634 with glutamine, vine serum albumin or chick and beef embryo
Puck's N-16 with glutamine, Scherer's mainte- extract.
nance solution, and McCoy's 5a medium) used
Although the function of bovine albumin is
at concentrations that were osmotically com- not known, our data indicate a role other than
patible with myxamoebae were also unable to that of merely increasing the viscosity of the
sustain growth. Introduction of either the de- medium. It has been suggested that the
fined or semidefined media developed by growth-promoting effects observed with bovine
Daniel et al. (2, 4) for culturing Physarum serum albumin might be a result of its ability
plasmodia resulted in immediate lysis of myx- either to adsorb toxic metabolites (7), or to act
amoebae. Substitution of BSS for the salts as a carrier for some critical growth factor (1 1).
used by Daniel prevented cell lysis, but the In the original semidefined medium devel-
modified medium still failed to support oped for the axenic culture of Physarum plas-
growth. modia, chick embryo extract proved to be an
essential ingredient (4) although hemin is now
DISCUSSION commonly substituted for this component (3).
As reported herein, myxamoebae demonstrate
This study has demonstrated that myxam- a similar requirement for embryo extract;
oebae of P. polycephalum can be grown free of however, unlike the plasmodia, hemin cannot
bacteria in submerged liquid culture. To date, be used as a substitute for embryo extract in
myxamoebae have been serially subcultured culturing myxamoebae. These findings are
over 100 times without exhibiting any morpho- similar to those of Hohl and Raper (6, 7) who
logical variations from myxamoebae grown on found that bovine embryo extract used in
media containing bacteria. axenic cultivation of Polysphondylium pallidum
To maintain viable myxamoebae, embryo could not be replaced by hemin. The decreased
extracts and bovine serum albumin were found growth that resulted when one embryo extract
to be indispensable components in the me- was removed and the concentration of the
246 GOODMAN J. BACTERIOL.
TABLE 1. Effect on growth when components of the medium are altereda
Components" Controlc Experimental

No. of cells
per ml No. of cells per ml Growth
relative
Beef Chick Bovine serum Liver Pep-
infu- tPe
x (5 x 104)
Glu-
u(5
X (5 x 104)
1 to
embryo embryo albumin
extract extract (BSA) sion Initial Final Initial Final c(ntr)o

+ + + + + - 17.8 1.3 61.9 3.4 19 ± 1.6 56 ± 3.2 88

+ + + + - + 30 ±3.0 102.5 ±2.5 26.9 ±2.7 67.5 ±4.2 72
+ + + - + + 20.1 ± 1.3 65.2 ±2.8 25.6 ±3.42 49.5 ± 1.79 52
+ + - + + + 28.5 2.4 71.6 2.1 23.9 1.8 16.8 1.4 _d
+ - + + + + 16.6 1.8 48.2 2.8 20.9 1.5 49.6 1.9 81
_ + + + + + 23.5 ± 1.5 81.6 ±3.5 19.8 ± 1.2 51.6 ± 1.9 77
+ + + - - + 29.6 ± 1.99 77.3 ±3.6 26.9 ±3.1 29.8 ± 1.86 _d
_ _ + + + + 25.6 ± 2.5 61.2 ±4.7 22.9 ± 1.3 18.2 ± 2.6 -d
+ + + 2x + + 21.3 ± 1.2 72.1 ±2.5 17.6 ± 1.7 72.4 ±2.2 116
2x - + + + + 14.0 ± 1.6 48.8 ± 1.9 15.0 ± 1.1 50.3 ±2.2 100
_ 2x + + + + 11.9 ± 1.2 41.1 ±2.7 16.3 2.0 47.3 ±3.9 88
+ + 0.2% Methocel + + + 26.4 ± 2.8 71 ± 4.2 23.5 ± .75 22.7 ± 1.8 _d
+ + 1% BSA + + + 25.2 ±2.1 88.6 ±3.9 26.4 ±2.7 51.5 ±2.5 72
6.0 jg of hemin/ml + + + + 29.1 ± 2.9 92.8 ± 6.7 32.1 ± 3.2 26.4 ± 2.7 _d
0.6 ,ug of hemin/ml + + + + 30.3 ± 1.5 71.6 ± 4.0 32.5 ± 1.3 27.8 ± 1.3 - d
a Table is based on samples counted between 62 and 72 hr post-inoculation. Each experiment has been
repeated at least twice and each deletion represents one experiment. Standard deviation of the mean is
based on 10 replicate counts per sample. In all experiments the observed difference between the control and
experimental is significant at the 95% confidence interval.
'Composition of experimental culture medium indicated as follows: normal concentration of component
present (+), component deleted (-), component concentration doubled (2x), component replaced (as indi-
cated).
c Grown in "complete medium" with no deletions.
d No growth observed.

remaining one doubled suggests that both ex-
tracts essential to maintain optimum con-
are
ditions, although beef embryo extract seems to
have more of the required growth factor. How-
ever, it remains to be determined what partic-
ular components of the embryo extracts are
actually needed by the myxamoebae.
Data obtained from both the growth curve
and incorporation of "C-protein hydrolysates
indicate that following the first doubling,
some factor(s) apparently become rate-lim-
iting. Since increasing the concentration of
medium components had limited effects on
growth, an alternative suggestion is that met-
abolic by-products may induce pH changes
and thereby exert an inhibitory effect. The
latter suggestion appears to be a distinct possi-
bility since the pH was observed to increase
0.2 unit during the first 48 hr of growth. In
addition, the time at which the growth rate of
myxamoebae begins to decline coincides with
the time period during which the pH of the
medium displays its most rapid increase.
The time required to complete one doubling
in axenic culture (approximately 32 hr) is sub-
stantially longer than the 8 hr required when
myxamoebae are grown on bacteria (Haugli,
personal communication) and suggests that the
present medium, although able to sustain pro-
liferating myxamoebae, probably does not pro-
vide optimal growth conditions.
ACKNOWLEDGMENTS
I wish to thank Joyce Mohberg and Thomas Evans for
helpful discussion during the preparation of this paper.
The work was supported by the National Science Foun-
dation (GB-20074) and the Research Committee of the grad-
uate school from funds provided by the Wisconsin Alumni
Research Foundation.
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