FEMS Microbiology Ecology 31 (1985) 103-109
Published by Elsevier
103
FEC 0014
A method for the enumeration of myxomycetes in soils and its
application to a wide range of soils
(Myxogastrids; slime moulds; most-probable-number method)
A. Feest and M.F. Madelin
Department of Botany, The Unioersityof Bristol, BristolBS8 1UG, U.K.
Received 8 February 1985
Accepted 22 February 1985
1. SUMMARY
A standardised most-probable-number technique for estimating the number of myxomycete
plasmodium-forming units (PFUs) in soils has been
developed and tested experimentally. Application
of the method to a local soil showed that the
uppermost layers contained the most PFUs. Its
application to 44 surface softs from various parts
of the world detected myxomycetes in 36, of which
4 were desert soils. In general, highest numbers of
PFUs (up to 9000. g-1 soil) were recorded in
grassland and agricultural softs. The nature of the
PFUs has not been definitely resolved but they are
probably myxamoebae, myxoflageUates and
microcysts.
2. INTRODUCTION
The literature relating to the geographical distribution of myxomycetes (myxogastrids or acellular slime moulds) suggests that they are nearly
ubiquitous throughout the vegetated land masses
of the world. Little is known about their actual
ecological role, because the literature on the ecology of these organisms is almost wholly concerned
with records of the natural occurrence of their
reproductive structures, rather than with their
trophic phases [1,2]. There is likewise little information on the natural occurrence of myxomycetes in soils, though there are reports that indicate
that soft is a habitat for some [3-7]. In the course
of investigating the ecological role of myxomycetes
in soft, we had need of a technique for their
selective isolation and quantification. We here describe such a technique, based on the most-probable-number method, and report the numbers of
myxomycetes detected in a variety of soils from
diverse locations.
3. METHODS
Half-strength corn meal agar (CMA/2) was
prepared by dissolving 8.5 g of Oxoid Corn Meal
Agar (Oxoid CM 103) and 7.5 g of Oxoid Purified
Agar (Oxoid L 28) in 1 1 distilled water. Saccharomyces cerevisiae (Culture No. Y37 from the
culture collection of the University of Bristol, Department of Botany), used as food for myxomycetes, was grown in Sabouraud's dextrose broth
(Oxoid CM 147) for 4 days at 30°C in shake
culture, harvested by centrifugation, washed 3
times by repeated suspension in sterile distilled
water and centrifugation, and finally prepared as a
0168-6496/85/$03.30 © 1985 Federation of European Microbiological Societies
104
standard suspension containing 1% packed cell
volume. To prepare C M A / 2 plus yeast, 0.5 cm 3
standard yeast suspension was added to each 9-cm
diameter petri dish containing approximately 20
cm 3 of solidified sterile C M A / 2 . Media were sterilised in an autoclave at 121°C for 20 min.
4. RESULTS A N D DISCUSSION
4.1. Standard method for enumerating myxomycetes
in a soil sample
In the light of experimentation and extensive
practical experience gained over numerous trials, a
standard procedure based on the most-probablenumber technique [8-12] was developed for enumerating myxomycetes in a given soil sample. The
unit which we have adopted for the expression of
myxomycete abundance
in soil is the
'plasmodium-forming unit' (PFU) analogous to
the colony-forming unit of filamentous fungi. The
nature of PFUs and the interpretation of their
abundance are discussed below. It was necessary
that the medium adopted for the most-probablenumber technique should promote the appearance
of myxomycete plasmodia in mixed cultures, since
it was by the production of plasmodia that
myxomycetes in the soil inoculum were recognised
unequivocally. Since plasmodia appear able to live
wholly phagotrophically [2], weak culture media
with an overlay of washed cells of S. cerevisiae
were used to discourage osmotrophic competitors.
S. cerevisiae is suitable for the nourishment of
plasmodia of numerous myxomycetes [13]. To
satisfy the needs of the preceding phagotrophic
unicellular phases, reliance was placed on bacteria
derived from those inevitably introduced in the
soil inoculum. Weak underlying agar media tested
included 1.5% plain agar, corn meal agar, halfstrength corn meal agar and apple bark extract
agar, singly and in combination, but none of these
gave greater recoveries from soil than C M A / 2
with yeast overlay.
The standard procedure was as follows. The
collected soil sample was thoroughly mixed in a
sterile plastic bag by kneading and shaking, and a
sub-sample of 50 cm 3 was removed. The packing
of the measured sub-sample was made to ap-
proximate to the density of the soil in its natural
state. It was weighed and added to 450 cm 3 sterile
0.1% Brij 35 solution (a wetting agent; polyoxyethylene lauryl ether; B.D.H. Ltd., Poole, U.K.)
in a conical flask and agitated thoroughly for 20
min on a wrist-action shaker to yield a 10 -1
dilution. These initial operations were scaled down
for smaller soil samples. A further small sub-sample of soil was oven-dried and weighed so that
results could be expressed in relation to volume,
fresh weight or dry weight. The 10 -1 suspension
was serially diluted in 0.1% Brij 35 solution to give
a range of tenfold dilutions to suit the expected
numbers of PFUs in the soil. Usually the dilution
range used was from 10 -1 to 10 -3 , but where high
numbers were expected, from 10 -2 to 10 -4 . One
cm 3 of each of the 3 consecutive dilutions was
placed on each of 5 plates of C M A / 2 plus yeast.
The 15 plates were placed in an unsealed polythene bag in a 20°C incubator for 1 week and then
examined with a binocular dissecting microscope
( x 10) and a phase-contrast microscope ( x 200).
The former was usually adequate for observing
phaneroplasmodia, but the latter was necessary for
observing aphanoplasmodia and protoplasmodia.
Incubation was continued for a further 4 weeks in
natural daylight on the laboratory bench
(18-22°C) with the plates enclosed in a clear
plastic seedling-propagating box to minimise drying and risk of contamination, and examined
weekly for plasmodia and sporangia. If the plates
lost their surface moisture film and appeared dull,
sterile distilled water was sprayed onto the agar
surface, sufficient to restore the film but not
enough to flow freely across the surface. The number of PFUs in the original soil was calculated
from the numbers of plasmodium-containing plates
at each dilution step with the aid of most-probable-number tables [8,9].
4.2. Tests of the reproducibility of the standard
method
2 Experiments were conducted to test the consistency of the standard method in practice. In the
first, 9 independent estimations were conducted on
a pooled sample of soil. Red marl soil from 4
rectangular cores, 5 x 8 x 3 cm deep, was collected
from a field site in grass turf at the edge of an
105
Table 1
Independent estimates by the most-probable-number method
of the number of plasmodium-forming units (PFUs) in 9
subsamples of a pooled sample of soil from an apple orchard
Sample No.
PFUs. c m - 3 soil
PFUs. g - ] dry soil
1
2
3
4
5
6
7
8
9
800
350
350
1700
450
500
200
1300
500
1720
754
580
2369
698
786
305
2088
841
683
1127
Mean
unsprayed apple orchard at Long Ashton Research Station, Bristol, England (O.S. map reference, ST 535705) and mixed thoroughly. Nine
50-cm3 samples of the mixed soil were separately
assayed by the standard procedure, using dilutions
of 10 -2 , 10 -3 and 10 -4 . The results are presented
in Table 1. The individual estimates ranged from
200-1700 P F U . cm -3 soil, with a mean value of
683 cm -3. The upper and lower 95% confidence
limits of a single estimate based on 5 samples from
inocula diluted in tenfold steps are 3.3 and 0.303
times the estimate, respectively [10]. The best
available estimate of the number of PFUs in this
soil is the mean of the 9 separate assays. This lies
within the 95% confidence limits of 8 of the 9
individual estimates, and is only fractionally beyond the limit for the single low estimate of 200.
The same is true for the data expressed per g dry
weight. The method thus furnished essentially reproducible estimates.
The second test of consistency was made with
soil gathered on a different occasion from the
same field site, but instead of a well-mixed fresh
collection of soil being divided into sub-samples,
an aqueous suspension of soil was divided. 50 cm 3
Soil was suspended in 450 cm 3 sterile 0.1% Brij 35
and 10 samples were taken for independent assays
of the number of PFUs present. Because the culture medium and incubation conditions allowed
the numbers of certain other organisms to be
estimated simultaneously, by the most-probable-
number method, these too were recorded, so that
the reproducibility of the estimates of PFUs could
be compared with that of estimates of other
organisms. 'Presumptive myxoflagellates' were
organisms with the distinctive morphology and
locomotory behaviour of myxomycete swarm cells.
In the absence of evidence that they were able to
give rise to plasmodia, we have, for the present,
hesitated to use them as a basis for enumerating
the abundance of myxomycetes in the soil.
Myxobacteria were recognised by their characteristic fruit bodies. The results are presented in Table
2.
The 95% confidence limits of 7 of the 10 estimates of PFUs embraced the mean. However, for
the other groups of organisms this occurred for all
10 estimates of ciliates, 9 of presumptive
myxoflagellates, and 9 of myxobacteria (some of
the estimates of amoebae and nematodes were
outside the quantifiable range). The results suggest
that sometimes the standard method may underestimate the numbers of PFUs present in the soil
and there may be a biological reason for this.
Firstly, the estimates whose 95% confidence limits
did not embrace the mean were the 3 lowest values
(170, 250, 250). Secondly, there were 2 results
(Tests 2 and 3) for which improbable score sequences were recorded, i.e., 3-3-0 and 4-4-0. When
a given dilution of inoculum yields 2 or more
positives, the next tenfold higher concentration
should yield appreciably more. The occurrence of
unlikely most-probable-number results has been
discussed by Taylor [12]. Because the results for
the other organisms in Table 2 lacked such sequences, the most likely explanation of anomalous
results for PFUs is that competition from other
organisms in the most concentrated series sometimes led to failure of potential PFUs to give rise
to detectable plasmodia. Microscopic examination
of isolation plates suggested that ciliates and
nematodes were among the deleterious competitors. It may be significant that the 2 anomalous
sequences were ones in which the numbers of
either ciliates or nematodes were the highest recorded. There is other supporting evidence. In
assays by the authors of the abundance of PFUs in
artificial microcosms in which only myxomycetes,
bacteria and angiosperm roots were present,
106
Table 2
Results of 10 independent estimates by the most-probable-number method of the numbers of plasmodium-forming units (PFUs),
presumptive myxoflagellates, amoebae, ciliates, myxobacteria and nematodes in a single sample of soil from an apple orchard
Score-sequences a and most-probable-numbers of organisms per cm3 soil
Test
No.
PFUs
1
2
3
4
5
6
7
8
9
10
5-4-1
3-3-0
4-4-0
4-3-0
5-4-0
5-4-0
5-4-0
4-3-0
5-3-0
5-5-1
Mean
1700
170
350
250
1300
1300
1300
250
800
3500
1092
Presumptive
myxoflagellates
Amoebae
5-4-2
5-5-1
5-5-2
5-5-3
5-5-2
5-5-3
5-5-3
5-5-4
5-5-3
5-5-3
5-5-2
5-5-5
5-5-5
5-5-4
5-5-5
5-5-4
5-5-5
5-5-4
5-5-5
5-5-5
2250
3500
5500
9000
5500
9000
9000
16000
9000
9000
Ciliates
5500
> 18000
> 18000
16000
> 18000
16000
> 18000
16000
> 18000
> 18000
7775
5-2-0
5-4-0
5-2-0
5-2-0
5-3-1
5-2-0
5-2-1
5-2-0
5-2-0
4-2-0
-
500
1300
500
500
1100
500
700
500
500
200
Myxobacteria
Nematodes
5-4-2
5-5-2
5-5-1
5-4-3
5-5-3
5-5-1
5-4-2
5-5-4
5-5-3
5-4-0
2-0-0
0-0-0
2-0-0
1-0-0
1-0-0
0-0-0
1-0-0
0-0-0
1-0-0
0-0-0
630
2250
5500
3500
2750
9000
3500
2250
16000
9000
1300
50
< 20
50
20
20
< 20
20
< 20
20
< 20
5505
a The 'score sequence' is the number of positive plates out of 5 at each of 3 dilutions of the inoculum differing by factors of 10,
highest concentration first.
a n o m a l o u s sequences n e v e r arose. Also, in assays
in w h i ch soil samples were frozen to destroy the
vegetatively active stages o f m y x o m y c e t e s , a n o m a lous sequences were e n c o u n t e r e d only once, possibly because the c o m p e t i n g p o p u l a t i o n s h a d b e e n
d e p l e t e d or eliminated. In the course o f a p p l y i n g
the s t a n d a r d p r o c e d u r e in studies which will be
r e p o r t e d elsewhere, a n o m a l o u s results have p r o v e n
to b e u n c o m m o n . In a survey of 38 w h e a t f i e l d
soils, only o n e a n o m a l o u s sequence was encountered, a n d in a survey of m o r e t h a n 200 o t h e r
soils of diverse types a n d usage there were 4; 3 of
t h e m for the same o r c h a r d soil used above. T h e
m e t h o d is e v i d e n t l y satisfactory in the m a j o r i t y of
soils. Th e o r c h a r d soil used in the a b o v e experim e n t s was the p ri n c ip a l exception, t h o u g h on the
o c c a s i o n of the s a m p l i n g date on which the d a t a in
T a b l e 1 are based, n o n e of the score sequences was
anomalous.
4. 3. Application of the standard enumeration procedure to a wide range of soils
T h e foregoing e x p e r i m e n t s a n d similar w o r k
i n d i c a t e d that, in local soils, n u m b e r s of P F U s of
the o r d er o f 1 0 0 - 1 0 0 0 . c m -3 or g - 1 fresh weight
were c o m m o n . T o d e t e r m i n e w h e t h e r these values
were representative of soils generally, a n u m b e r of
soils collected f r o m a w i d e r an g e o f geographical
situations was assayed. T o ensure s o m e c o m p a r a bility of soil-sampling p r o c e d u r e s in these differen t locations, it was necessary investigate first
w h e t h e r the d e p t h f r o m which the sample was
t ak en i n f l u e n c e d the result.
A 6 - c m - d i a m e t e r core was r e m o v e d in early
s u m m e r f r o m the top 8 c m of the soil in the
o r c h a r d site specified above. T h e core was d i v i d ed
i n t o 2-cm lengths, an d the n u m b e r s of P F U s assayed in each b y the s t a n d a r d procedure. T h e
results p r e s e n t e d in T a b l e 3 show that the ab u n d a n c e of P F U s in the s t r a t u m b e t w e e n 6 an d 8 c m
Table 3
Numbers of plasmodium-forming units (PFUs) recovered from
each 2-cm length of an 8-cm-long core of soil from an apple
orchard
Depth of sample (cm)
PFUs. cm-3 of soil *
0-2
2-4
4-6
6-8
5500 a
3500 a
1300 a
250 b
• Estimates with the same letter are not significantly different
at the P = 5% level.
107
Table 4
Abundance of plasmodium-forming units in soil samples from geographically diverse locations
Location
Habitat
AFRICA
Zimbabwe
Harare, Nat. Bot. Gdns.
Harare, Greenwood Pk.
Sabi-Limpopo Valley
Kopje area
Lawns
Lowveld tree savanna
20
110
110
Acacia confusa woodland
Mud
Regenerating slope after
felling
Rubber plantation
Rubber plantation
Slope understorey
Slope understorey
<2*
17
4
110
80
4
11
Hong Kong
New Territories
New Territories
New Territories
Broadleaf evergreen forest
Herbaceous cover by path
Pine wood
130
< 20
< 20
AUSTRALASIA
Australia
Gawler Ranges, S. Aust.
Gawler Ranges, S. Aust.
Gawler Ranges, S. Aust.
Hummock grassland hillside
Open Acacia sowdeni woodland
Barren plain
EUROPE
Albania
Butrint
Fier
Lezhe
Saranda
Saranda
Shkodra
Low grass
Low grass hill vegetation
Woodland litter
Lush meadow
Lush meadow
Garden soil
ASIA
China
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Hainan
Germany
Mosbach, Baden-Wfirttemberg
Mosbach, Baden-Wiirttemberg
Mosbach, Baden-Wilrttemberg
Mosbach, Baden-Wiirttemberg
Mosbach, Baden-Wiirttemberg
Mosbaeh, Baden-Wiirttemberg
Mosbach, Baden-Wtirttemberg
Walldurn, Baden-WOrttemberg
Walldurn, Baden-Wiirttemberg
Walldurn, Baden-Wilrttemberg
Walldurn, Baden-W~ttemberg
Switzerland
Gstaad
Rinderberg Peak
NORTH AMERICA
U.S.A.
Bryce Canyon, UT
Death Valley (Furnace
Creek), CA
Beech wood
Grass ley
Kale field
Lawn wormcasts
Mangold field
Ploughed stubble
Stubble
Beech wood
Conifer wood
Hornbeam wood
Oak wood
Meadowland
Meadow above treeline, 2500 m
PFUs. g - 1 fresh wt.
14
5
5
170
11
> 1800
550
65
25
< 20
5500
200
50
9000
350
140
< 20
< 20
50
40
13
17
Scattered pines on sandy soil
2
Almost no vegetation
2
108
Table 4 (continued)
Location
Grand Canyon (North Rim), AZ
Grand Canyon (South Rim), AZ
Inyo National Forest, CA
Las Vegas, NV
Painted Desert, AZ
Sonoran Desert, hr. Phoenix, AZ
Yosemite National Park, CA
Habitat
Pines on brown earth
Pines on sandy soil
Coniferous forest
Roadside desert
Desert flora
Desert flora
Coniferous forest
P F U s . g - ~ fresh wt.
8
14
5
2
< 2
35
< 2
* The symbol < signifies that if myxomycetes were present they were below the specified limit of detection, none being recovered.
deep was significantly less ( P ~< 5%) than in the
upper layers of the soil. Though differences between the estimates for the 3 upper layers were not
statistically significant, the numbers suggest a
steady decline with depth. (The ratios of 2 separate
estimates obtained by the described method, which
may be judged as significantly different at different critical values of probability for 2-tailed tests,
can be calculated [10]; these are 3.73 for P = 10%,
4.80 for P = 5%, 7.87 for P = 1% and 13.95 for
P = 0.1%). The observed vertical distribution of
myxomycetes resembles that of protozoa in temperate soils [15,16]. In view of these results, those
who agreed to collect soil samples were therefore
asked to take them from the first 4 cm of the soil
profile. The samples of soil in field condition were
placed in clean plastic containers and assayed
within 4 weeks of collection, usually within 2. In
other experiments, samples were assayed on the
day that they were collected, but in order to establish a general indication of the abundance of
myxomycetes in soils worldwide, this practice was
waived. The results of applying the standard enumeration procedure to these samples are given in
Table 4.
The data suggest that myxomycetes are of
widespread distribution and that their abundance
is sometimes high. In only 8 of the 44 soils assayed
were no plasmodia recovered, and even these may
have been soils with numbers of PFUs below the
limits of detection of the standard method. It may
be ecologically significant that 6 of these 8 nonyielding soils were from woodland. On the other
hand, one assay of an Albanian woodland soil
revealed a high abundance. Four of the desert soils
yielded myxomycetes, a result which accords with
floristic studies which show that myxomycetes can
be found in desert regions [17]. In general, higher
numbers were recovered from grassland and agricultural soils than from woodland soils. In view of
these data and those in the preceding tables it
appears that the role of myxomycetes in terrestrial
ecology has been underestimated in the past.
4.4. General discussion
The question of the nature of the enumerated
PFUs has yet to be resolved. A PFU might be a
spore, swarm cell, myxamoeba, microcyst, or part
of a plasmodium or sclerotium. The evidence we
have is compatible with PFUs in the soil being
uninucleate stages rather than plasmodia, but the
whole question of the form of activity of
myxomycetes in the soil requires further examination. Chuang and Ko [18] present an equation
which relates propagule size to the maximum
population density of the microorganisms within
the soil, and which may be used predictively. If it
is assumed that swarm cells, myxamoebae and
microcysts are the likely form in which myxomycetes occur in the soil, then, on the supposition,
based on observation, that their volumes will be
mostly between 100-400 /~m3, the corresponding
population densities can be calculated as being
between 4800-2270 g-1 of soil, respectively. Such
predictions are of the order of the numbers of
PFUs reported here in a number of soils, and
suggest that such uninucleate forms may be the
dominant state in which myxomycetes live in soil,
at least when present in numbers in the order of
thousands per g.
109
It is likely that the enumeration m e t h o d described underestimates the numbers of P F U s present in the soil, because in at least some m y x o m y cetes, single spores, amoebae or microcysts are
incapable of yielding plasmodia. Instead, 2 compatible cells are required for plasmodium formation. This situation could lead to a several-fold
underestimation. It m a y therefore be significant
that the 'presumptive myxoflagellates' in Table 2,
whose m o r p h o l o g y and m o v e m e n t closely resembled myxomycete swarm cells, were present in
numbers about sevenfold higher than the PFUs. It
is possible that all or most of these presumptive
myxoflagellaes genuinely were myxomycete cells.
However, until the p o p u l a t i o n structure of
myxomycetes in soil samples has been studied, it is
prudent to base conclusions on the numbers of
PFUs, which are unequivocally m y x o m y c e t o u s
even if they are underestimates.
The majority of myxomycetes recovered from
British soils and induced to fruit proved to be
Didymium species. Whether this reflects selectivity
of the enumeration method, or the capacity of
these Didymium species for rapid growth, or truly
reflects the a b u n d a n c e of D i d y m i a in the soil
microflora, is the subject of continuing investigation. It should be noted that species of other
genera have been successfully isolated on the
m e d i u m used.
ACKNOWLEDGEMENTS
During the period in which most of the research
reported here was conducted, A.F. was supported
b y a Science and Engineering Research Council
studentship. Part of the investigation was supported by a grant to M.F.M. from the Natural
Environment Research Council. W e thank colleagues who furnished us with soil samples from
diverse localities.
REFERENCES
[1] Madelin, M.F. (1984) Myxomycetes, microorganisms and
animals: a model of diversity in animal-microbial interactions, in Invertebrate-Microbial Interactions (Anderson,
J.M., Rayner, A.D.M. and Walton, D.W.H., Eds) pp.
1-33, Cambridge University Press, London.
[2] Madelin, M.F. (1984) Myxomycete data of ecological significance, Trans. Br. Mycol. SOc. 83, 1-19
[3] Farquharson, C.O. and Lister, G. (1916) Notes on South
Nigerian Mycetozoa, J. Bot. 54, 121-133.
[4] Indira, P.U. (1968) Some slime moulds from Southern
India, IX, J. Indian Bot. 41, 792-799.
[5] Lister, G. (1918) The Mycetozoa: a short history of their
study in Britain; an account of their habits generally; and
a list of species recorded, Essex Field Club Memoirs 6,
1-54.
[6] Thorn, C. and Raper, K.B. (1930) Myxamoebae in soil and
decomposing crop residues, J. Wash. Acad. Sci. 20,
362-370.
[7] Warcup, J.H. (1950) The soil plate method for the isolation of fungi from soft, Nature 166, 117-118.
[8] Alexander, M. (1965) Most-probable-number method for
microbial populations, in Methods in Soil Analysis, Part 2.
Chemical and Microbial Properties (Black, C.A., Ed.) pp.
1467-1472, Am. SOc.Agronomy, Madison, WI.
[9] Ministry of Health; Ministry of Housing and Local
Government (1969) The bacteriological examination of
water supplies, in Reports on Public Health and Medical
Subjects No. 71, 4th ed. H.M. Stationery Office, London.
[10] Cochran, W.G. (1950) Estimation of bacterial densities by
means of the 'most probable number', Biometrics 6,
105-116.
[11] Maloy, O.C. and Alexander, M. (1958) The 'most probable number' method for estimating populations of plant
pathogenic organisms in the soil, Phytopathology 48,
126-128.
[12] Taylor, J. (1962) The estimation of numbers of bacteria by
tenfold dilution series, J. Appl. Bacteriol. 25, 54-61.
[13] Hok, K.A. (1954) Studies of the nutrition of myxomycete
plasmodia, Am. J. Bot. 41, 792-799.
[14] Gray, W.D. and Alexopoulos, C.J. (1968) The Biology of
Myxomycetes. Ronald Press, New York.
[15] Stout, J.D. and Heal, O.W. (1967) Protozoa, in Soil Biology (Burgess, A. and Raw, F., Eds.). pp. 149-195,
Academic Press, New York.
[16] Sleigh, M.A. (1973) The Biology of Protozoa, Edward
Arnold, London.
[17] Blackwell, M. and Gilbertson, R.L. (1980) Sonoran Desert
Myxomycetes, Myeotaxon 11, 139-149.
[18] Chuang, T.Y. and Ko, W.H. (1981) Propagule size: its
relation to population density of microorganisms in soil,
Soil Biol. Biochem. 13, 185-190.