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NUTRITION OF CELLULAR SLIME MOLDS
III. SPECIFIC GROWTH REQUIREMENTS OF POLYSPHONDYLIUM PALLIDUM
HANS-RUDOLF HOHL' AND KENNETH B. RAPER
Departments of Bacteriology and Botany, University of Wisconsin, Madison, Wisconsin
Received for publication 21 August 1963
ABSTRACT Sussman (1963) described the axenic cultivation
HOHL, HANS-RUDOLF (University ofWisconsin, of Polysphondylium pallidum (Hohl and Raper,
Madison), AND KENNETH B. RAPER. Nutrition strain WS-320) in a medium containing milk
of cellular slime molds. III. Specific growth re- solids, proteose peptone, and lecithin, the latter
quirements of Polysphondylium pallidum. J. Bac- being reported as an absolute requirement. More
teriol. 86:1314-1320. 1963.-Soluble media for recently, Allen et al. (1963) reported "moderate
the axenic growth of certain strains of Poly- growth through at least two serial transfers" on
sphondylium pallidum are described. One of the defined media, and suggested that fatty acids are
media, referred to as the complex medium, was required. Gezelius (1962) obtained equal growth
composed of 2% tryptose and 4% serum albumin of Dictyostelium discoideum in liquid media on
in an inorganic salt solution. Cell densities of up either living or dead bacteria, and found that the
to 107 myxamoebae per ml were obtained. Sup- rather slow growth rate of the myxamoebae on
plementing this medium with amino acids, vita- dead bacteria (see also Hohl and Raper, 1963a)
mins, nucleic acid bases, and glucose resulted in could be improved by supplementing the suspen-
growth of strain Pan-17 up to 3 X 107 cells per sion with glucose, amino acids, and vitamins, and
ml with a generation time during the log phase bylowering the initial pH of the medium. A study
of about 4.5 hr. A defined medium was subse- of the food spectrum of Acrasis rosea by Weitz-
quently developed that allowed growth of strain man (1962) showed that in some cases mixtures
Fr-47 up to 2 X 106 to 5.5 X 106 myxamoebae of two organisms (e.g., Hansenula anomala plus a
per ml in the presence of 1% serum albumin. rod-shaped bacterium, D7) supported growth,
The base medium contained the following essen- although neither of the two was able to do so
tial nutrients: six amino acids (glycine, methio- alone. Furthermore, colonies of Hansenula upon
nine, lysine, isoleucine, tryptophan, and tyrosine), which A. rosea failed to survive supported growth
riboflavine, purines and pyrimidines, and a carbo- and fructification after the addition of a Seitz-
hydrate (dextrose) dissolved in an inorganic salt filtered, nondialyzable, and heat-stable extracel-
solution containing trace elements. lular factor from Rhodotorula, the yeast employed
by Olive and Stoianovich (1960) as a host organ-
ism. Media for several other amoeboid organisms,
In an earlier paper, Hohl and Raper (1963b) such as Hartmanella rhysodes (Band, 1962) or
described the successful cultivation of strains of Acanthamoeba sp. (Adam, 1959; Band, 1961),
Polysphondylium pallidum in a liquid-soluble have already been devised. Thus, the prospects
medium containing embryo extract, serum al- of obtaining additional cellular slime molds in
bumin, tryptose, dextrose, vitamins, and inor- axenic culture are encouraging.
ganic salts. The present report relates to further MATERILS AND METHODS
experiments performed to simplify the complex
medium, to increase the final cell yields, and to Strains Pan-17 and Fr47 were used exclusively
study the minimal nutritional requirements of in this study, since they do not require embryo
these organisms. were interest in the extract for continuous growth as is the case with
Since these studies begun, revived. strain WS-320, which was studied intensively and
nutrition of the cellular slime molds has illustrated in our earlier reports (Hohl and Raper,
I Present address: Pacific Biomedical Research 1963a, b). These strains are presently assigned to
Center, University of Hawaii, Honolulu. the species P. pallidum Olive (1901, 1902) but
1314
VOL. 86, 1963 GROWTH REQUIREMENTS OF P. PALLIDUM 1315
are morphologically distinct. In contrast to WS- cell density in the complete medium (Table 1)
320 with its typically erect sorocarps bearing with only 1% bovine serum albumin compared
regularly spaced whorls of side branches, strain with the 2 to 4% that is necessary for other strains
Pan-17 tends to produce long, rangy, and often investigated. This relatively low reqwrement
senaiprostrate fructifications, while Fr47 pro- rendered Fr47 especially suitable for the study
duces sorocarps in which the distal internode is of minimal nutritional requirements, since the
typically quite long and bears a very small termi- concentration of impurities from the serum al-
nal spore mass, or sorus. bumin could be cut to one-half or even one-fourth.
Pan-17 grows very rapidly and yields high It may be added that strain WS-320 does not
concentrations of cells when grown in the com- grow on either the complete or minimal medium
plex medium (Hohl and Raper, 1963a), whereas listed in Table 1 unless embryo extract is added
Fr47 grows rather slowly in the same medium (see earlier paper for details).
and yields only about half the number of cells. The preparation of the inoculum, methods of
However, this latter strain is able to reach a high cultivation, growth measurements, and sterility
TABLE 1. Media developedfor the cultivation of strains of Polysphondylium pallidum with
details of their composition
Complex medium Complete medium Minimal medium
(simplified)
Component Amt Component Amt Component Amt
Tryptose 2.0 Amino acid mixture 1 Amino acid mixture 2
Serum albumin 4.0 Vitamin mixture Riboflavine*
Inorganic salts Dextrose 1.0 Dextrose 1.0
Bases Bases
Trace elements Trace elements
Serum albumin 1.0 Serum albumin 1.0
Inorganic salts Inorganic salts
Amino acid mixture 1: Amino acid mixture 2: Vitamin mixture:
Glycine 75t Glycine 300t D-Biotin 0 35t
DL-Isoleucine 131 DL-Isoleucine 520 Choline chloride 2.10
L-Lysine 182 L-Lysine 730 Folic acid 0.20
DL-Methionine 149 DL-Methionine 595 Niacin 1.00
L-Tryptophan 204 L-Tryptophan 204 Ca-pantothenate 1.10
L-Tyrosine 36 L-Tyrosine 11 Pyridoxal-HCl 2.20
L-Arginine 210 Bases: Thiamine*HCl 0.70
L-Cysteine*HCI 175 Guanine*HCl 25 Riboflavine 1.10
L-Histidine-HCl 191 Adenine sulfate 25 Pyridoxine HCl 2.20
L-Leucine 131 Thymine 25 Inositol 2.80
DL-Valine 117 Uracil 25 p-Aminobenzoic acid 0.20
L-Alanine 89 Cytosine 25 B12 0.003
L-Asparagine 150 Xanthine 25 Ascorbic acid 0.30
L-Aspartic acid 133 Hypoxanthine 25 Trace elements:
L-Proline 115 Inorganic salts: FeSO4*7H20 10
L-Glutamic acid 147 NaCl 800 FeCl3 10
DL-Phenylalanine 165 KCI 400 MnCI2*4H20 1
DL-Serine 105 CaC12*2H20 100 H3BO3 1
DL-Threonine 119 MgSO4-7H20 100 (NH4)2A12(S04)4.24H20 1
L-Cystine 48 MgCl2 6H20 100 ZnSO4,7H20 1
DL-Norleucine 131 Na2HPO4*7H20 60 MnSO4.H20 1
KH2PO4 60 CuS04 1
CoCl2 1
* Riboflavine: 0.1 mg per liter.
t All amounts in the mixtures are expressed as milligrams per liter of the final medium.
AND
1316 HOHL RAPER J. BACTERIOL.
tests were similar to those described in earlier could not only grow in the absence of embryo
papers (Hohl and Raper, 1963a, b). The myxa- extract, a component of the earlier medium, but
moebae were pregrown on dead bacteria, or on yielded cell numbers up to 107 per ml in a sub-
complete or minimal medium, then washed with strate containing only 2% tryptose and 4% serum
0.016 M phosphate buffer, counted, and diluted albumin in a salt solution. This simplified version
to 5 X 105 cells per ml. Portions (0.1 ml) of this of the medium is a clear solution and is very
suspension were then added to tubes containing simple to prepare (see Table 1). The highest cell
5 ml of the medium to be tested, thus giving a yields recorded were obtained in a combination
concentration of 104 cells per ml of medium. The of the simplified complex medium and the com-
tubes were incubated on a rotary shaker at 25 C. plete medium, with Pan-17 as the test organism.
The 'growth of the myxamoebae was measured By combining the defined part of the complete
by duplicate hemacytometer cell counts. medium with 2% tryptose and 4% serum albu-
The three media finally adopted, together with min, cell yields up to 3 X 107 per ml were ob-
their detailed compositions, are given in Table 1. tained with a generation time during the log
The salt solution (10 X concentration), the trace phase of about 4.5 hr.
elements (300 x concentration), and the Difco The interaction of tryptose and serum albumin
TCBovine SerumAlbumin (asterile 5% solution) in various concentrations and ratios is shown in
were stored in the refrigerator. The vitamins Fig. 1. Tryptose alone allows a final yield of up
(75 X concentration), the amino acids (75 mm to 6.3 X 106 myxamoebae per ml. Part of the
concentrations, except for tyrosine at a 15 mn tryptose can be replaced by serum albumin; for
concentration), and the nucleic acid bases (75 X example, the addition of 1.6% tryptose to the
concentration) were kept frozen, each substance defined medium allowed an increase of about
as a separate aqueous solution. Difco tryptose 500 times. If 0.3% serum albumin was added to
and dextrose were freshly dissolved for each ex- the medium, the same increase was obtained
periment. The pH was adjusted to 6.0 with NaOH with only 0.7% tryptose. On the other hand,
before sterilization. The media were autoclaved serum albumin alone allowed only a meager
for 20 min at 121 C prior to the aseptic addition growth of this strain (Fr-47, as mentioned above,
of the serum albumin. reacted more favorably in this respect), and the
addition of small amounts of tryptose was very
RESULTS beneficial. Thus, 2% serum albumin led to an
Theresults are presented in two sections: results increase of about 80 times; the addition of 0.1%
relating to the complex medium, and those ob- tryptose raised the increase to 380 times, i.e.,
tained with the more defined media. approximately five times the former value. The
Complex medium. It was noted earlier (Hohl effect of tryptose can be replaced by yeast ex-
and Raper, 1963b) that, of five strains tested, tract, yeast autolysate, or neopeptone.
Pan-17 grew best on the complex medium then More defined media. The simplified complex
employed. Further tests showed that this strain medium contains two chemically undefined com-
ponents, namely, tryptose and serum albumin.
To study the two components separately, a
medium was developed in which only one of
2000 these was required. The medium finally adopted
(Table 1, complete medium) supported good
0 100/
-g growth of Pan-17 if tryptose was used instead
x 1500 of serum albumin, and it allowed strain Fr-47
0.30%S
c 1000 -020%
~~ ~ ~ ~ ~ to reach a cell density of up to 5.5 X 106 per ml
E within 8 to 10 days in the presence of only 1%
2D 500 ~~~~~~~~~~~- 0 serum albumin. It is called the complete medium,
Oz 0.1 05 10 15 2.0 because it contains 21 amino acids, 13 vitamins,
Concentration of tryptose (%) purines, pyrimidines, a carbon source (dextrose),
FIG. 1. Influence of different concentrations of trace elements, and inorganic salts besides the
tryptose and serum albumin (SA) on final cell yield serum albumin. The growth kinetics of strain
of Polysphondylium pallidum Pan-17. Fr-47 on the complete medium are presented in
VOL. 86, 1963 GROWTH REQUIREMENTS OF P. PALLIDUM 1317
Fig. 2. The lag phase was considerably longer 7
than on the complex medium, and no clear loga-
rithmic growth phase was obtained. The end of
the growth period was usually reached after 8 to
9 days. The slow growth rate was probably due 86
to the relatively high osmotic pressure of the 0
medium, resulting from the many osmotically E
0
active substances added toobtainoptimal growth. E
It was shown earlier (Hohl and Raper, 1963a) 0o5
that the myxamoebae of P. pallidum are very
sensitive to the osmotic pressure of the medium;
in these experiments, further decreasing the salt
concentration did not improve growth, while in- 4 3 5 7 9
creasing it depressed growth. Incubation time in days
The active factor(s) in tryptose was found to FIG. 2. Growth of Polysphondylium pallidum
be extractable with a 99% methanol-1% water Fr-47 on the complete medium.
mixture (v/v), dialyzable, and heat stable (121 C
for at least 40 min). It passed freely through a growth was not impaired cannot be considered
basic ion-exchange coluni (Amberlite CG45, nonessential since they might be present as im-
type 1, -OH form) but was adsorbed by an acid purities in the serum albumin fraction. This
column (Dowex 50-WXI, hydrogen form) from point will be discussed in more detail subse-
which it could not be extracted even with 1.0 N quently.
HCl. From the tryptose powder, the growth- Of the 13 vitamins tested, only riboflavine was
promoting activity could not be extracted with an absolute requirement. With riboflavine alone,
ethanol, n-butanol, acetone, chloroform, ether, the growth reached about 60% of that of the
or petroleum ether, and not by charcoal followed control. As little as 3.3 X 10-4 mg per liter had a
by pyridine extraction. Two-dimensional paper very pronounced effect, but in the medium finally
chromatography of the active fraction after pas- adopted this level was raised to 0.1 mg per liter.
sage through the basic ion-exchange column, with Repeated subculturing on the medium containing
n-butanol-acetic acid-water (120:30:50) and riboflavine as the only added vitamin did not
n-butanol-pyridine-water (60:60:60) solvent result in diminished cell yields.
systems, revealed at least 13 different ninhydrin- The amino acid requirement was more difficult
positive spots, demonstrating the complexity of to assess. In a first attempt, each of the 21 amino
the fraction. It was concluded that the active acids (Table 1) was omitted singly. Omission of
fraction is not a protein of high molecular weight isoleucine, methionine, and tryptophan depressed
(heat stability, dialysis), is hydrophilic, and is growth to a great degree. Cystine, norleucine,
probably basic in nature. serine, threonine, and phenylalanine had no ap-
The complete medium allowed a more detailed parent influence on growth. The others showed a
investigation of the specific nutritional require- moderate influence; i.e., their omission depressed
ments of strain Fr-47, even in the presence of growth to 40 to 80% of that of the control which
1% serum albumin. This was true because the contained all amino acids, and may have reflected
omission of any major part of the medium, amino the limitations of transaminating amino acids.
acids, vitamins, carbohydrate, or serum albumin, Subsequent deletion of amino acids by groups
prevented any growth of the slime mold. The eventually showed that six have a rather pro-
nucleic acid bases were not an absolute growth found effect on the growth of Fr-47. Table 2
requirement, but their omission from the medium summarizestheseeffects. Cell growthon the media
reduced growth to about 25% of that obtained in from each of which one of the six amino acids
the control. Of course, the presence cf the serum was deleted was measured after 5 and 9 days,
albumin imposed certain limits on the accuracy that is, at the middle and at the end of the growth
of our analysis. Those substances in the absence period. Glycine, methionine, and lysine were very
of which no growth occurred are definitely es- important; isoleucine and tryptophan were mod-
sential, but compounds in the absence of which erately so; and the absence of tyrosine depressed
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