THE NUTRITIONAL REQUIREMENTS OF THE
PREDOMINANT BACTERIAL FLORA OF THE SOIL
BY C. B. TAYLOR
Imperial Chemical Industries Limited,
.Butterwick Research Laboratories, Welwyn, Herts
SUMMARY : Several aspects of techniques for nutritional classification of soil bacteria
have been critically investigated. The methods used by recent Canadian workers
are found to be unsatisfactory ; (a) because their seven standard media fail in several
respects t o give a n unequivocal grouping ; (b) because some of the media used contain
inadequate concentrations of essential nutrients ; (c) because recommended incubation
times are in some cases too short.
A simpler method of classification into five nutritional groups, using longer periods
of incubation and more satisfactory differential media, has been used to compare soils
receiving different manurial treatments. Results indicate that other factors, such as
the well-known rapid fluctuation in bacterial numbers which occurs in all soils, may
obscure any effects of soil treatment unless a very elaborate system of controls is
adopted. It is suggested that an even simpler method of nutritional grouping into
three classes may be of value : (a) organisms capable of using ammonia nitrogen :
(b) organisms requiring amino-acid nitrogen, with or without vitamin suppleinent,s :
(c) organisms requiring more complex nitrogenous materials.
The possibility of developing a grouping technique by direct plating on differential
solid media has been studied. It is considered that this is not possible; association
effects on plates with mixed colonies suffice to obscure differences between a series of
media of increasing nutritional complexity.
The growth-promoting properties of soil-extract are believed to be due in part to
complex nitrogenous substances, which can be replaced by certain concentrations of
suitable peptones, and in part to such cations as calcium, magnesium and strontium.
The concentration of such cations needed to allow growth of exacting strains is related
to the concentration of peptone in the medium and it is suggested that they may act
by antagonising in some way the inhibitive effects of certain peptone constituents.
NEITHER
microscopic study, nor the classical biochemical tests, are of much value
for grouping the more abundant kinds of bacteria found in soil or for the qualitative
comparison of the bacterial flora of different soils. The physiological instability of
many soil and water bacteria is well known (Taylor & Lochhead, 1938 : Taylor,
1942). Considerable, interest has therefore been aroused by a new approach to the
physiological differentiation of bacterial groups developed in Canada by Lochhead
and his colleagues. This involves determining the ability of isolated cultures to
grow in a number of media of differing nutritional complexity (West & Lochhead,
1940 ; Lochhead & Chase, 1943). The technique has been used to study the influence
of cropping on the bacterial flora of soil (Wallace & Lochhead, 1948), the rhizosphere
effect (West & Lochhead, 1940 ; Lochhead & Thexton, 1947), microbiological changes
in soil in relation to season and manurial treatment (Katzneleon & Chase, 1944)
and root-rot problems (Hildebrand & West, 1941).
I n this technique suitable dilutions of soil are plated on an agar medium containing
aqueous soil extract but no added energy-supplying material or salts. The plates
are incubated a t 25-28' for 10-12 days, all the colonies (100-200) from one or more
I02
C. B . Taylor
plates are picked off and subcultured in a semi-solid soil extract medium containing
in addition 0-0270potassium dihydrogen phosphate, 0.01 yeast extract and 0.37,
agar. From tubes showing growth, subcultures are made on a series of differential
media which enable the organisms to be grouped as follows :Group 1. Bacteria with simple requirements. These can grow in a basal medium
containing salts (potassium nitrate as nitrogen source) and glucose only.
Group 2. Bacteria requiring one or more amino-acids. These grow in the basal
medium if a mixture of ten amino-acids (cysteine, alanine, proline, asparagine,
glutamic acid, aspartic acid, arginine, leucine, glycine and lysine) is added.
Group 8. Bacteria which require growfh factors. These grow in the basal medium
if the following growth factors are added-cysteine, thiamin, biotin, pyridoxin,
pantothenic acid, nicotinic acid, riboflavin and inositol.
Group 4. Bacteria requiring amino-acids and growth fcctors. These grow in the
basal medium if both amino-acid and growth factor mixtures are added.
Group 5 . Bacteria requiring unidentified substances i n yeast extract. These will
not grow in the basal medium plus amino-acids and growth factors unless Difco
yeast extract (O.lyo)is also added.
Group 6. Bacteria requiring unidentified substances in soil extract. These will not
grow in the basal medium plus amino-acids and growth factors unless soil extract
is also added.
Group 7. Bacteria requiring unidentified substances in both yeast and soil extract.
These will only grow when yeast extract, soil extract, amino-acids and growth factors
have all been added to the basal medium.
This paper describes an investigation of the reliability of the technique ancl an
attempt to develop a simpler procedure for nutritional grouping.
MATERIALSAND METHODS
Soils. Samples of soil were collected from the Broadbalk plots (farmyard manure,
complete minerals, nitrogen, phosphorus and potash, and unmanured plots) a t
Rothamsted Experimental Station and from the Barnfield plots of similar treatment.
Isolation medium. Although more colonies usually develop on an agar medium
containing soil extract only than on morn conventional bacteriological media, it was
considered that the concentration of vitamins and other nutrients in soil extract
might be insufficient for the development of some soil bacteria. After various trials
a medium was developed containing agar (1.5), salts (K,HPO,, 0.04 ; MgS0,.7H20,
0.005 ; NaC1,O.Ol ; FeCl,, 0.001), Difco ' Tryptone ' (0.05),Difco yeast extract (0.05),
all as yo w/v, and soil extract (2.5% viv). The pH was adjusted to 7.0 before autoclaving. The soil extract was prepared by autoclaving equal weights of soil and
water a t 15 1b./ix2 for 1 hr. and filtering through paper pulp. This medium gives
consistently higher numbers of colonies than plain soil extract agar. The results
of a comparison are given in Table 1 ; i t must be noted, too, that in this experiment
the soil extract gave counts little higher than a simple glucose mineral salts ammonium
phosphate medium.
Nutritional grouping
of soil
bacteria
103
Table 1 . d comparison of bacterial numbers in the variously manured
Broadbalk plots, determined by plate counts on three media
(38 April, 1949)
Bacteria ( 106/g.moist soil)
Medium
7
Salts, ammonium phosphate, glucose
Soil extract
Soil extract, yeast extract, tryptone, salts
Farmyard
manure
41
55
111
A-
Complete
minerals
31
33
95
7
No
manure
42
49
120
Culture medium. A semi-solid medium of the same composition as the isolation
medium, but with the agar concentration reduced to 0.3 yo,proved very satisfactory
for the growth and maintenance of isolated cultures.
Vitamin-free casein hydrolysate. The preparation used was ' Difco ' Vitamin-Free
Casamino Acids (Difco Laboratories Inc., Detroit). The makers claim this to be
vitamin-free but no quantitative information on vitamin content is given.
Preparation of silica gel media,. The method described by Taylor (1950) was used.
RESULTS
The nutritional groups
Some 300 cultures have been tested by the technique proposed by Lochhead and
his collaborators (West & Lochhead, 1940 ; Lochhead & Chase, 1943). This experience has suggested certain improvements in the procedure, and these are dealt with
below, as they concern the various groups.
Group 1. Bacteria with simple requirements. Preliminary heating of the basal
medium before adjustment of pH (i.e. a t about pH 7.9) as recommended by
Lochhead & Chase (1943) results in the production of a substantial precipitate,
which may well remove much of the calcium and magnesium. The second precipitate
produced on autoclaving after adjustment to pH 6.8 is still sufficient to interfere
with the reading of tubes with light growths. Less precipitate is obtained if the
p H is adjusted to 6-8 before the first heating ; the medium proposed later in this
paper gives less trouble with precipitates.
Ammonium phosphate was compared with potassium nitrate as nitrogen source.
I n one experiment 38 cultures grew well in the ammonium phosphate medium, only
7 in the nitrate medium ; in another, 29 grew in the ammonium phosphate medium
but only 9 in the nitrate medium. Previous workers have found that all bacteria
capable of using nitrate as a nitrogen source can also use ammonia, while many which
use ammonia easily are unable to assimilate nitrate. There can be no doubt that
many cultures capable of using ammonia nitrogen, but unable to use nitrate, have
been classified under the Canadian system as requiring more complex sources of
nitrogen. I n the work described later in this paper, nitrate was replaced by 0.05%
ammonium phosphate.
Group 2. Bacteria requiring one or more amino-acids. Many cultures grew very
poorly in the recommended amino-acid medium ; to a great extent this may have
‘04
C . R . Taylor
been due to the low nitrogen content of only 0.09%. Much speculation is involved
in choosing the constituent amino-acids and their concentrations for a purely
synthetic medium. A medium containing instead 0-4% vitamin -free acid hydrolysed
casein supported good growth of many organisms that grew poorly in the synthetic
medium, and such a medium was used in further work described below.
Group 3. Bacteria requiring one or more growth factors. Lochhead & Thexton (1947)
presented data which indicate that many of the organisms which would fall in
group 2 require only the amino-acid cysteine. This amino-acid is also included in
their growth factor medium so that many organisms would be placed by these tests
in both groups 3 and 4. Lochhead & Thexton’s results also suggest that in some
instances the growth factors accelerate growth but are not essential.
Group 4. Bacteria requiring amino-acids and growth factors. Results obtained in
the present study indicate that this is a well-founded group, but it appeared that
incubation for 5 days was often insufficient. Incubation €or 12 days in some
instances produced easily visible growth where none was apparent after 5 days ;
the unsatisfactory criterion ‘ submaximal growth ’ used by Lochhead and his
collaborators can thus be avoided.
Group 5 . Bacteria requiring substances in yeast extract. I n many experiments, with
several hundred cultures, no organisms have been found which will grow in the yeast
extract medium of Lochhead & Chase (1943) but which are unable to grow in a
medium containing vitamin-free casein hydrolysate and a growth factor mixture.
On the contrary, many organisms growing in the latter medium will not grow in a
0.1 yo yeast extract medium, though they will do so if the yeast extract concentration
is increased to 0.3%.
Croup 6. Bacteria requiring substances in soiE extract. Many of the organisms
which by Lochhead & Chase’s media would be placed in this group will grow in a
casein hydrolysate growth factor medium or in a similar medium in which the casein
hydrolysate is replaced by tryptone. This suggests that the effect of soil extract
observed by Lochhead and his collaborators was in part due to substances (probably
protein) also present in some peptones. Nevertheless, there remain some organisms
which require soil extract ; the growth promoting properties of soil extract have
been investigated and the results are presented in a later section of this paper.
Group 7. Bacteria requiring substances in both yeast extract and soil extract. Results
of several experiments suggest that the main difference between groups 6 and 7 is
due to the deficiency in nitrogen and possibly in growth factors of the medium used
for group 6, organisms requiring greater concentrations of these substances thus
appearing in group 7. All organisms which would fall into this group will grow in
a casein hydrolysate growth factor soil extract medium or a tryptone growth factor
soil extract medium.
Modified nutritional grouping
On the basis of the above observations and criticisms i t was concluded that the
grouping resuIting from use of the media developed by Lochhead & Chase (1943)
is not based entirely on simple nutritional differences. The arguments and evidence
Nutritional grouping of soil bacteria
105
suggest that a more accurate classification into the following five nutritional groups
can be achieved by the use of the media listed :Group I. Bacteria using inorganic nitrogen, differentiated in a basal medium
containing 0.04% K,HPO,, 0.00596 MgS0,.7H20, 0.01'$b NaC1, 0.001 % FeCI,,
0.05% (NH,),HPO, and 0.17, glucose.
Group 11. Bacteria requiring amino-acids, requiring the basal medium to be
supplemented with 0.4% vitamin-free casein hydrolysate.
Group 111. Bacteria requiring amino-acids and growth factors. These need the
group I1 medium plus the following quantities of growth factors/litre :-200 pg.
riboflavine, 400 pg. calcium pantothenate, 10 pg. biotin, 200 pg. aneurin, 15 mg.
uracil, 15 mg. adenine, 1.2 mg. pyridoxin hydrochloride, 600 pg. nicotinic acid,
50 pg. p-aminobenzoic acid, 7.5 mg. choline chloride and 0-2 pg. folic acid.
Group IV. Bacteria requiring substances in tryptone. For these, the basal medium
needs to be supplemented with 0*50,/,tryptone and 0.3% yeast extract, but in fact
the yeast extract seems either unnecessary or replaceable by the growth factors
added for group 111.
Group V. Bacteria requiring soil extract. These grow in the group IV medium
t o which has been added 35q6 (v/v) of soil extract.
One criticism of the suggested media may be anticipated here. This is, that it is
difficult to obtain really vitamin-free casein hydrolysates, so it is possible for there
to be some overlap between groups I1 and 111. It is thought that this would be
small; we have in fact isolated numerous organisms able to grow in medium I11
though unable to grow in medium 11.
The incidence of the modified nutritional groups i n Rothnrnsfed soils
Using these five media, the distribution of nutritional groups in manured and
unmanured plots a t Rothamsted was determined (Table 2 ) . The soil from the
Broadbalk plots was taken from between rows of wheat. Organisms using inorganic
nitrogen (group I) were dominant in the plot receiving complete minerals ;
organisms with complex requirements (groups IV and V) were most abundant in
the unmanured soil ; in the farmyard manured plot no one group was really outstanding but growth factor requiring organisms (group 111) were more common
than in the other two soils.
Table 2 . The incidence, in variously manured Rothamsted plots, of bacteria
falling into five nutritional groups (14 March, 1950)
Numbers of bacteria (percentage of all organisms isolated)
Broadbalk plots
Nutritional
group
I
11
I11
IV
V
A
Farmyard
manure
30
14
27
20
9
Barnfield plots
7
Complete
minerals
51
10
J
24
10
No
manure
14
15
14
42
15
Farmyard
manure
53
8
10
21
8
Complete
minerals
11
6
31
26
26
No
manure
36
9
28
20
7
I06
C. B . Taylor
The Barnfield soils gave a different result. These plots are cropped annually with
mangolds and the tops are ploughed in after harvesting : a t the time of sampling
the plots had just been ploughed. Organisms using inorganic nitrogen (group I)
were dominant in the farmyard manured plots ; these organisms were also numerous
in the unmanured plots, where groups I11 and IV were also common. The plots
receiving complete minerals showed a predominance of the more exacting types.
These data are insufficient to warrant drawing any definite conclusions but i t does
seem possible, judging by the very different results obtained in the Broadbalk and
Barnfield plots, that such a group analysis reflects a state of affairs existing a t the
moment of sampling and which might change radically in a short time,
Deterrnination of nulritional groups by direct plating on
silica gel media
The techniques for nutritional grouping described in the preceding paragraphs are
very laborious. Moreover, many isolated cultures become less exacting on subculture.
Direct plating of soils on to solid media differing in nutritional complexity was
therefore investigated.
I n a preliminary experiment the media of Lochhead & Chase (1943) were used,
solidified with agar and modified by replacement of nitrate in the basal medium
with diammonium hydrogen phosphate, for the examination of three local soils.
Incorporation of amino-acids, growth factors or soil extract in the medium had
little or no effect on the number of colonies developing. I n an investigation of the
effect of medium composition on plate counts of bacteria in water (Foot & Taylor,
1948) it was found that the nutritional effects of substances added to an agar medium
in low concentration are frequently masked by nutrient impurities in the agar itself.
.In order to avoid such interference a further experiment was carried out with silica
gel media. I n this experiment five selective media were used, similar to, but not
quite identical with, those used for separating groups I to V in liquid media. The
media comprised respectively, (a) Basal inorganic medium plus glucose (as for group
I) ; (b) Basal medium with vitamin-free casein hydrolysate ; (c) Medium (b) with
growth factors added ; (d) Medium (c) with 25'7; soil extract added ; (e) Medium (d)
with casein hydrolysate replaced by tryptone. These media were used with soils
from the Broadbalk plots ; the soil suspensions used were those also used for
preliminary isolation of the cultures for the grouping in liquid media (Table 2 ) . The
results are presented in Table 3. The figures shown for each medium are obtained
by subtracting from the appropriate count the value obtained from the immediately
less exacting group. Three points should be noted : first, there is no difference of
grouping between plots and the grouping is unrelated to that obtained on liquid
media (Table 2 ) ; second, there is no response to growth factors and factors in
tryptone ; third, there is a response to amino-acids (casein hydrolysate) and a larger
one to soil extract. The lack of response to growth factors is considered further
in the next section.
Nutritional grouping of soil bacteria
107
Table 3. The incidence, in the variously manured Broadbalk plots, of bacteria
capable of growth on five dqferent media (14 March, 1950)
Medium*
Bacterial population, as
Bacterial numbers in
percentage of all isolations
millions/g. moist soil
u--7
No
Farmyard Complete
Farmyard Complete
No
manure
manure
minerals
manure
minerals
manure
17
10
18
9
3
6
33
31
41
17
9
14
0
0
0
0
0
0
50
59
41
26
17
14
0
0
0
0
0
0
I-
B
B+C
B+C+V
B+C+V+S
B+T+V+S
*
B, basal medium containing ammonia nitrogen ; C, casein hydrolysate ; V, growth factor
mixture ; S, soil extract ; T, tryptone.
Association effects of mixed cultures in plate counts
I n mixed cultures on solid media bacteria which require vitamins for growth can
obtain these from neighbouring colonies capable of synthesizing an excess by diffusion
through the agar or silica gels. This effect was demonstrated by streaking a layer
of vitamin-free casein medium with a culture capable of growth on that medium.
At increasing distances from the streaked culture drops of a washed suspension of a
growth factor requiring organism were placed. After incubation the exacting
organism was seen to have grown well near the streak of the non-exacting culture,
the amount of growth diminishing rapidly with increasing distance. The certain
prevalence of such association effects on dilution plates is sufficient t o explain the
observed lack of response to added growth factors.
Similarly, association effects may explain certain apparently contradictory results
obtained by Lochhead and his colleagues. These workers have described many
instances of organisms isolated on soil extract agar, with no other added nutrients,
showing amino-acid requirements when grown on the liquid media used for nutritional
grouping. Tests carried out in the course of the work described in this paper have
invariably failed to demonstrate any amino-acids in soil extract. There seem to be
two possible explanations for the appearance of amino-acid exacting organisms on
the soil extract plates ; they may be able to break down the proteins probably
present in soil extract, or amino-acids may diffuse outwards from colonies of organisms
able to break down protein or to synthesize amino-acids from inorganic nitrogen,
thus enabling neighbouring exacting organisms incapable of these activities to develop
into colonies.
The growfh-promoting properties of soil extract
The work of Lochhead and his colleagues on the nutritional grouping of soil bacteria
has given prominence to the fact that some constituent or constituents of soil extract
are essential for the growth of many soil bacteria. Soil extract cannot be replaced
by any known growth factors or amino-acids, though, as shown earlier in this paper,
I08
C . B . Taylor
it can be replaced by tryptone with some organisms. The report that unmanured
soil gives inactive extracts (Lochhead & Chase, 1943) adds interest to the problem.
Some preliminary work on the nature of the soil extract effect irreplaceable by
tryptone is now described. A brief account of this has already been given by Taylor
(1951). I n this work soils from plots a t Rothamsted receiving no manure, farmyard
manure and complete minerals have been compared. Several bacteria requiring soil
extract for growth were used as test organisms. The basal medium contained 0.5Ob
tryptone and 0.30/, yeast extract.
Methods of extracting soils. Soils from the Rothamsted plots were autoclaved with
water (equal weights of soil and water) at 15 Ib./in.2 for varying periods. Soils from
unmanured plots or plots receiving complete minerals gave inactive or partially
active extracts if the period of autoclaving were 30 min. or less but fully active
extracts if autoclaving were prolonged. Soils from the farmyard manure plots gave
active extracts with shorter periods of autoclaving. All soils gave extracts allowing
growth of the exacting organisms when added in 2% (v/v) concentration, if the
period of autoclaving was sufficient.
Extracts obtained by refluxing or autoclaving with dilute hydrochloric acid (1
v/v of conc. hydrochloric acid, 37OTw., in water) were always active. Four successive
hydrochloric acid extractions of soil from a farmyard manure plot each produced
active extracts. Extracts prepared with sodium hydroxide (0.1 or 0.01 N), ethyl
alcohol or acetone were inactive. Extraction with sulphuric acid was usually
ineffective.
Examination of extracts. The active substance in soil extract was removed from
solution by such cation-removing base-exchange substances as ' Soucol,' or ' ZeoKarb 215', or '216', but not by the anion-removing material ' De-acidite.' Contrary
bo the findings of Lochhead & Chase (1943), activity was not removed by filtration
through charcoal (B.D.H.). When an extract was heated to dryness, the active
substance could be extracted in water but not in ether. Water extracts of the ash
produced by ignition of the dry matter, or from a wet ash obtained by heating with
concentrated sulphuric acid, were inactive. These latter findings, together with the.
discovery that limited activity could be obtained from extracts of straw, and even
from certain batches of filter papers, considerably obscured the solution of the
problem. It was eventually found that when the dry matter was oxidized with
perchloric acid, the neutralized aqueous solution of the ash residue was active.
Experiments with likely ash constituents led to the discovery that following the
addition of calcium chloride to the autoclaved basal medium all the test cultures
would grow.
The effect of calcium. The calcium chloride requirements of different test organisms
were found to vary considerably. This explains many discrepancies encountered in
the early experimental work, because small amounts of calcium may well have
been present in some of the reagents or materials used. When the basal medium
containing 0.5 yo tryptone was used the minimum requirements of CaC1, varied
between 10 and 200 p.p.m. Strontium (as nitrate) was found to replace CaCl, :
magnesium (as chloride or sulphate) replaced CaCl, with some cultures only, in.
so
Nutritional grouping of soil bacteria
I09
.approximately equivalent amounts. Aluminium compounds were ineffective.
Comparison of anhydrous CaCI,, CaCI,.GH,O (A.R.), and extremely pure CaCI,
(' Specpure ') showed that all were equally effective and thus that the active
substance was not a contaminating trace element. Tests carried out with different
salts of calcium showed that their activity was related to solubility, thus : nitrate>
acetate>sulphate>citrate>carbonate (ineffective). The addition of the calcium
compound t o the medium in the commonly employed manner before autoclaving
led to the formation of precipitates during sterilization and usually to partial or
even complete inactivation of the medium. It was found best to sterilize the basal
medium and the calcium salt solution separately and to mix these under aseptic
conditions.
Table 4. T'he growth of three soil extract requiring bacteria in media
containing 0.3yo yeast extract and varying concentra,tions
of tryptone and calcium chloride
Culture
no.
Tryptone
( % wlv)
173
4,r 0.2
0.5
1.0
Growth* with calcium chloride (p.p.m.)
r-
0
h
5
-
+++
+++
-
-
_.
12.0
352
50
+++
+++
+++
r 0.2
{ ;::
(2.0
4r, 0-2
0.5
1.0
I
362
-
7
20
10
~~
100
+++
+++
+++
+++
+++
+++
++
+++
+++
12.0
* + + +, vigorous growth ; + +, moderate growth ;
+, slight growth ;
-, no growth.
It appeared unlikely that such relatively large concentrations of calcium would
be required directly for the metabolism of the organisms. More information concerning the rBle of calcium was obtained by the use of different concentrations of
%aCl, in conjunction with different concentrations of various brands of peptone
in a peptone yeast extract medium, The results showed that for any effective peptone,
the amount of CaCl, required to provide growth had a direct relationship to the
,concentration of peptone in the medium ; also, that different peptones varied
considerably in their ability to support growth. Table 4 shows the results of
experiments of this kind with tryptone. It thus appears possible that the r61e of
calcium is to antagonise in some way an inhibiting material in the peptone. This
can only be finally proved by further experiment.
DISCUSSION
It is well established that the total number of bacteria in soils, including fallow soils
receiving no manurial treatment, fluctuates rapidly ; such fluctuations do not apply
II 0
C . R . Taylor
equally to all kinds of bacteria a t any time. Any effect 011 the microbiological
population produced by manuring or cropping can only be satisfactorily measured
by reference to a control receiving no treatment and to observations made on the
same soils over a period preceding the application of the manure or sowing of the
crop. These precautions have not always been observed in investigations of the
incidence of bacteria of different nutritional groups under different soil conditions.
Another source of error in such work, dealt with in some detail in this paper,
arises from incorrect formulation of the media used for nutritional grouping. Results
presented show that the differential media used by Lochhead and his colleagues are
susceptible of improvement. For instance, their medium for determining bacteria
with simple nutritional requirements (group 1 ) contains nitrogen as nitrate ;
consequently the many bacteria which cannot utilize nitrate but which can utilize
ammonia nitrogen are classified as amino-acid requiring bacteria. Again, their
results show that many of the organisms requiring one or more amino-acids (group 2 )
require the amino-acid cysteine only ; nevertheless this amino-acid is included in
the medium used to determine organisms which require one or more growth factors
but not requiring amino-acids (group 3), with 'the result that many organisms must
appear in both groups 2 and 3. Further, there are strong indications that the yeast
extract medium used for determining group 5 and the soil extract medium used to
determine group 6 are deficient in nitrogen and carbon so that many organisms
fail t o develop which would develop in media with higher concentrations.
For these reasons i t is suggested that nutritional classification of soil bacteria
as a means for studying qualitative changes in the soil flora has as yet yielded little
information of unequivocal value. A modified series of media, which will distinguish
five nutritional groups with somewhat greater accuracy, has been described in this
paper. It appears likely that an even greater simplification of the system of
classification might be advantageous. Three nutritional groups could be clearly
differentiated : organisms capable of utilising inorganic (ammonia) nitrogen ;
organisms requiring amino-acid nitrogen with or without growth factor supplements :
organisms requiring more complex nitrogenous substances.
Grateful acknowledgment is paid to Rothamsted Experimental Station for
facilities for soil sampling, to Mr. A. J: Woiwod and Dr. E. B. Vischer for chemicaL
assistance, and to Miss A. Stokes for the work on vitamins.
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KATZNELSON,
H. & CHASE, F. E. (1944). Qualitative studies of soil micro-organisms : VI.
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Nutritional grouping of soil bacteria
I11
LOCHHEAD,
A. G. & CHASE,F. E. (1943). Qualitative studies of soil micro-organisms. V.
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A. G . & THEXTON,R . H . (1947). Qualitative studies of soil micro-organisms : VII.
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(Received 9 April, 1951)