M. J. (1960). J. uppl. Rmt. 23 ( I ) , 37-52.

THORNLEY,

THE DIFFERENTIATION O F PSEUDOMONAS FROM

OTHER GRAM-NEGATIVE BACTERIA ON THE
BASIS O F ARGININE METABOLISM
BY MARGARET J. THORNLEY
Low Temperature Research Station, Cambridge

SUMMARY: Of 391 Gram-negative bacteria isolated from chicken meat spoiled at a

low temperature and claasified by the commonly used methods, 156 were considered
to be P 8 e u d m n a s and 188 Achromobacter, and 47 others belonged to the coli-asrogenes
group or remained unclassified. A test for the production of alkaline conditions in an
arginine medium incubated under a vaseline seal gave positive results for 155 of the
P8ewEomona.e isolates, and negative results for 1 Psewiomonas and all the 188 Achromo-
bacter strains. When named strains from culture collections were tested under these
conditions, 63 Pseudumoms strains produced alkalinity while two plant pathogenic
P~eudonzonasspecies and two non-pigmented strains did not. These last two, which
produced no acid from glucose, could not be regarded as typical P8eudomonm. All the
Achromobacter strains gave negative results, as did four Alcaligenea, but one species,
Alcaligenea bookeri, produced slightly alkaline Conditions. One strain of Chromobaeterium
and three of Vibrw were also positive. These could be distinguished from Pseudomom
by their metabolism of glucose.

THEMICRO-ORGANISMS which commonly spoil food such as meat, fish, eggs and milk
in cold storage are psychrophilic Gram-negative bacteria, and are often referred to
as the Pseudomom-Achromobacter group (Mossel & Ingram, 1955). Experience with
meats suggests that many of these bacteria have polar flagella but lack ability t o
form pigment (Brown & Weidemann, 1958) and, as already noted (Ayres, 1955;
Brown & Weidemann, 1958) they have been classified as Achromobacter or Pseudo-
mom-9 according to the criteria adopted. Early workers limited Pseudomom t o
strains producing a water soluble green pigment, while non-pigmented motile strains
were called Achromobacter whether the flagella were polar or peritrichous. This system
is still used by many French workers (Brisou, 1957). The definition was changed
in the 6th edition of Bergey’s Manual of Determinative Bacteriology (Breed, Murray &
Hitchens, 1948), where Pseudmnom was described as a genus of polarly flagellated
straight rods which might or might not produce water soluble pigments, while the
organisms in the genus Achromobacter were non-pigmented and either non-motile or
peritrichous. I n the 7th edition of Bergey’s Manual (Breed, Murray & Smith, 1957)
the previous definitions were retained except that the genus Pseudomow was limited
to strains which were known or thought to attack glucose by an oxidative pathway
only. I n this paper the criteria of Bergey (7th edition) will be adopted when ‘Pseudo-
m o m ’ and ‘Achromobacter’are referred to. The oxidative attack on glucose, whioh
is characteristic of Pseudomom, is possessed also by some Achromobacter strains
(Shewan, pers. comm.) ; but it is useful in helping t o distinguish between Pseudomonas
and members of the Enterobacteriaceae. The oxidase test of Kovacs (1956) was
also devised for this purpose, using clinical material, a positive result signifying
38 Margaret J.' 'Thornley
P s e u d o m o w . This observation was confirmed by Shewan for P s e u d o m o m strains
isolated from fish, while Achrombacter strains from the same source could be either
positive or negative. When tested against penicillin, Pseicdomonas strains were
resistant to 2.5 i.u., whereas Achromobacter were sensitive (Shewan, Hodgkiss &
Liston, 1954). Sensitivity to a vibriostatic agent was used t o separate out vibrios.
In these reports the only characters distinguishing P s e u d o m o m from Achromo-
bacter are flagellation, if the strains are motile, and penicillin sensitivity. In addition,
failure to produce acid from glucose and a negative oxidase test are likely to occur
in Achrombacter rather than P s e u d o m o m . The difficulties encountered in practice
arise from the time consumed in flagella staining. which may render that procedure
impracticable for large numbers of isolates and, even when it is used, the problem
of classifying non-motile organisms remains. This is well illustrated by the work of
Brown t Weidemann (1958).
I n the present work Gram-negative bacteria from chicken meat spoiled at low
temperature were classified by the tests outlined above, with flagella staining applied
only t o a selection of strains. A new test was developed and tried out on these
bacteria and on named strains from culture collections.

METHODSAND MATERIALS
In the preliminary classification of the cultures the following techniques were used.
Incubation. All tests were done a t 20".
Motility. Overnight cultures in yeast extract broth were examined under low
power dark ground illumination.
Glucose metabolism. The mode of action on glucose was studied by means of the
semisolid medium of Hugh & Leifson (1953) (see Table 1).
Fluorescence. Cultures were streaked on the medium recommended for maximum
fluorescin production by King, Ward & Raney (1954) and examined under ultra-
violet light after one day's incubation.
Antibiotic sensitivity. The method of Shewan, Hodgkiss & Liston (1954) was
employed with penicillin (2.5 i.u.) and terramycin (10 pg) Sentest tablets (Evans
Medical Supplies, Ltd.).
Oxidase test. Kovacs' (1 956) method was used, with cultures grown on solid medium
for either 24 or 48 hr.
Flagella stain. The modification of Fontana's method described by Rhodes (1958)
was used.
During classification by means of these tests it was observed (as will be detailed
later) that strains grouped as P s e u d o m o m because they produced no acid from
glucose in Hugh & Leifson's medium incubated anaerobically were not inert but
shifted the p H towards the alkaline side. This led to a study of the production of
alkaline substances from the peptone, from a simplified medium, and from known
amino acids in experiments with washed cells. The following methods were used.
Change of reaction i n peptone medium. An adaptation of Hugh & Leifson's medium
resulted in medium 1, whose composition is shown in Table 1. As will be seen, the
carbohydrate was omitted, the peptone concentration was increaeed and the indicator
 Arginine test for Pseudomonas 39
was changed to phenol red. To obtain the results to be described it wm essential
t o use Difco Bactio-peptone, as three other brands of peptone gave different results.
This medium was filled into 25 ml bottles with screw caps and rubber washers in
amounts of 15 ml and sterilized by autoclaving.
To perform the test, duplicate bottles were inoculated by stabbing. Sterile melted
vaseline was poured into one bottle t o a depth of 5 mm ; the screw cap of the other
bottle was left loose. Changes in reaction, shown by colour changes of the indicator,
were noted a t intervals during 21 days incubation a t 20".

Table 1. The relationship between the compositions* of

H q h t Leifson's medium and media 1, 2 and 2A
Ingredient Composition (yow/v)
h
7
Hugh & Medium Medium Medium
Leifson 1 2 2A
Peptone 0.2 o-5-f 0.1 0.I
NaCl 0.5 0.5 0-5 0-5
K,HPO, 0.03 0-03 0.03 0.03
Agar 0.3 0.3 0.3 0.3
Bromothymol blue 0-003 - - -
Phenol red - 0.001 0.001 0*001
Cerbohydrate 1.0 - - -
Arginine HCl - - - 1.0
* The pH of Hugh & Leifson's medium is 7*1,8ndthat of media 1, 2 and 2A
wa4 7.2. tDifco Bscto-peptone must be used.

Change of reaction in arginine medium. The basic medium 2 was similar t o

medium 1 but had less peptone (Table 1 ) . It was used alone or with the addition
of 1 % of L(+)-arginine monohydrochloride, when it was called medium 2A. The p H
in both cases was adjusted t o 7.2. All ingredients were dissolved and autoclaved
together in 5 ml bijou bottles which were filled t o a depth of about 16 mm. They
were inoculated in duplicate and one bottle was sealed with vaseline as with medium 1.

Techniques in experiments with washed suspensions

Washed suspensions were incubated at 25" under strictly anaerobic conditions in
Thunberg tubes with various substrates, including peptone solutions and amino acids
used singly and combined in groups.
Prepamtion of suspensions. The organisms were inoculated into 10 ml amounts
of medium 1 (without the agar) in T-tubes (Kay & Fildes, 1050) and incubated a t 25"
with shaking for 2 days. The cells were centrifuged, washed twice in quarter-strength
Ringer's solution and resuspended in the same solution to a standard opacity as
measured by the EEL nephelometer. These suspensions were stored at 1". N o
difference in activity was observed whether they were used the day after preparation
or u p t o a week later. a

Substrates. Medium 1 without the agar was compared with amino acids, mainly
combined in groups as in the experiments of Hills (1940). Table 2 shows the con-
stituents of the groups with the concentrations a t which they werc initially present
40 Margaret J . Thornley

in the reaction mixtures. Phenol red was added t o give a final concentration of
0.001%, as in medium 1.

Table 2. Amino acids, grouped after Hills (1940), as used

in experiments with washed suspensions
Group and Concn. Group and Concn.
composition (M x I 0-4) composition (Mx 10-4)
Glycine 40 DL-serine 40
DL-threonine 40

{
40

{
DL-AUI~~
I L(-)-proline 40 IV DL-methionbe 40
DL-v8line 40 L( -)-cystbe Trace
D L - I ~ U C ~ ~ 40 L-cysteine HCI Tram
DL-aspartic acid 40

i
DL-isoleucine 40
DL-norleucine 40 L( +)-glutemic acid 40
L( -)-phenylalanine 40 DL-asparagine 40
I1 L( -)-tyrosine 2 L( + )-glutamine 40
L( -)-hietidine 40 40
DL-tryptophene 40
26
L( + )-ornithinet 40
+
L( )-citrulline 40
* Monohydrochloride. t Dihydrochloride.

Nethod. I n a typical experiment, 4.5 ml of the substrate solution were put into
each tube and 0.5 ml of the bacterial suspension in the stopper. The tubes were
evacuated and filled with oxygen-free nitrogen four times, placed in a water bath
a t 25", and the contents mixed, The colour changes were recorded every hour and,
when the experiment was stopped, pH measurements were made with a glass electrode.

RESULTS
This work resulted from an investigation of the psychrophilic spoilage flora of chicken
carcases which had been treated with ionizing radiation, with chlortetracycline, or
had received both treatments. Two experiments were performed, in which samples
were taken from control and treated birds initially and after they had spoiled during
storage a t 1" and 3", and the bacteria enumerated on heart infusion agar (Difco)
plates incubated a t 1" and 20". From these plates colonies were picked, and 391
cultures of Gram-negative bacteria, were obtained. The tests used to claasify these
organisms were those recommended by Shewan (pers. comm.), and on the results
obtained they were placed in five groups, A-E, described below. Some of the groups
were subdivided, and the charactma of the organisms, separated into classes according
to their action on glucose, are set out in Table 3.
Group A, gas-producing #fermenters. These were recognized by their behaviour in
the Hugh & Leifson test. They were penicillin resistant, motile or non-motile and,
with one exception, oxidase negative. Further study of some of these cultures
(marked A, in Table 3) showed them to belong t o the genera Aerobader (Cloaca)and
Hafnia, while the one oxidase positive strain (marked A,) was a n Aeronzonas (Eddy &
Kitchell, 1959).
 Arginine test f o r Pseudomonas 4'
Table 3. The characters and a grouping of 391 Gram-negative organisms
isolated from chicken carcases
Teat

Motility
Fluorescence
-c--

A,
Reect,ions* of organisms classed on type of glucose metabolism as:
Fermenters,
groupt
A1 A,
-- +- +-
B C
Oxidizers,

+ + - - -
+ - - - -
GOUP

E,
t
D, D, D,
Inert,
groupt
D, I), Ds D, E,
_ _- -- -_ - _ _+
+
penicillin R R R R R R S S S S S R R R
Oxidaae
No. of
- - + + + + + -
16 1 1 0 19 0
+3 46+ 18- +4 -14 +I
{EEg:1
ieoletes
8 17 1
4 6 0 31 98 2 8 5 1 2 7 3 4 5 6 0
Total isolates 12 23 1 47 109 2 %I 5 4 1 3 5% 9 20 I
* +, present; -, absent; R, resistant; S, sensitive.
t &oups: A, ges-producing fermenters of glucose; B, pigmented Psewlolnonaa; C, non-pigmented
Paeudomonas; D, Achromobacter; E, unclassified.

Choup B, pigmented Pseudomonas. Forty -seven cultures produced fluorescent

pigment on the medium of King et al. (1954) and also were penicillin resistant and
oxidase positive. All were motile rods, straight or slightly curved, and all produced
acid oxidatively from glucose.
Group C , rwn-pigmented Pseudomonas. Among the non-fluorescent, penicillin
resistant, oxidase positive cultures, 101) were motile and oxidized glucose. Flagella
stains were done on about one-fifth of these. They had polar flagella, and were
classified as non-pigmented Pseudomonas. Two similar but non-motile cultures were
left unclassified (group El, Table 3).
Group D, Achromobacter. Strains which were sensitive to penicillin and either
oxidative or inert with glucose were a t first regarded as Achromobactar. They were
either positive or negative in the oxidase test. Only 4 strains in this group were
motile (D1,Table 3) and 2 of these showed polar flagella. which excluded them from
Achrmobacter. They were finally left unclassified. The flagellation of the other
2 strains could not be determined; a heavy deposit of stain surrounded each cell,
probably due t o the presence of mucilaginous material. These cultures were provision-
ally retained in this group. The non-motile penicillin sensitive organisms (D, Table 3 )
showed a tendency for some cells to stain Graq-positive, especially when freshly
isolated, and were either coccoid rods, often in pairs, or even cocci, some resembling
Neissericc. This very characteristic morphology corresponded with that described by
Shewan and his colleagues for Achromobacter from fish, and it also occurred in two
groups of organisms resistant to penicillin and inert towards glucose (Da and D,,
Table 3). Group D, contained 9 oxidase positive and group D, 20 oxidase negative
strains. Although similar penicillin resistant groups had not been found by Shewan,
groups D, and D, were included in Achromobacter on the morphological grounds.
Excluding the 2 polarly flagellated penicillin sensitive D, cultures mentioned above,
this made a total of 188 Achromobacter strains. The 2 non-motile glucose oxidizing
organisms previously mentioned under group C (El, Table 3) did not fit into this
category, as they were slender rods of medium length.
42 Margaret J . Thornley
Group E, other organisms. In addition t o the 2 polarly flagellated organisms in
group D,, and the 2 non-motile organisms similar t o Pseudomom (group El), 7 peni-
cillin resistant, oxidase positive, motile organisms which were inert on glucose
remained unclassified (group E,, Table 3). They formed yellow pigment on milk
agar and in gelatin stabs. Flagella stains were done on 4 of these strains and all
proved t o have a single polar flagellum. They were excluded from Pseudomow
because of the failure to produce acid from glucose; from Flavobacterium by their
polar flagella; and from the genus Lophomonas of Galarneault & Leifson (1956) by
the fact that they were monotrichous.
As will be seen from this description the borderline between Achromobacter and
Pseudomow is doubtful, and the separation made here was based mainly on morpho-
logical grounds. The remainder of this paper concerns the development of a n
additional method of distinguishing these two genera.

Change of reaction in peptone medium

It was observed that when glucose oxidizing organisms were incubated in Hugh &
Leifson’s medium a n alkaline reaction sometimes appeared in the closed bottle.
This, which seemed to happen mainly with Pseudomom strains, was attributed
to the metabolism of peptone, and in order to find out more clearly what was
happening tests were done in the modified Hugh & Leifson medium (medium 1,
Table 1).

-
Table 4. The reactions produced in medium 1 by 380 Grarn-negative
bacteria isolated from chicken meat
Reaction*, in bottle Group Proportion
of showing
Open Closed organisms reaction
Alk. Alk. {B. Pigmented Peeudomaas
C. Non-pigmented PeeudomonaR
46/47
lOSjl09
B. Pigmented Peeudwrtonas 1/41

Alk.
Alk.
slight
acidOr
NC
Acid
{D. Achrmobacter
A. Gas producing fermentem
A. Gas producing fermenters
188/188
3/36
33/36
* Alk., elkaline reaction; NC, growth with no change in reaction.

Both open and closed bottles of this medium were inoculated, and in the open
bottles almost all the organisms produced an alkaline layer near the surface, but a t
differing rates. The reactions in the deeper layers and in the closed bottles varied,
as shown in Table 4. Of the 47 pigmented Pseudomonas strains described above,
46 produced alkali in closed bottles and 1 gave no change. The 109 non-pigmented
P s e d m o n a s all produced alkali, as did the two similar but non-motile organisms.
The 188 organisms classified as Achromobacter gave no change in closed bottles, or
a very slight shift to the acid side. Three gas-producing fermenters (1 Aeromonas,
1 Hafnia, 1 not studied in detail) behaved in the same way, while the other 33 gave
definite acidity in closed bottles together with strong surface alkali production in
open bottles. Thus the Pseudomom and Achromobacter strains gave different
 Arginine test for Pseudomonas 43
reactions when incubated in closed bottles of medium 1, and the results were
sufficiently promising for further investigation of the nature of the reaction concerned.
The conditions in the closed bottles were not strictly anaerobic, since the medium
had not been boiled to remove dissolved oxygen and contained no reducing sub-
stances. It was in fact shown, by inoculating 2 Pseudomow and 2 Achromobacter
cultures on t o plates of the same medium solidified with 1.5 yo of agar and incubating
in a McIntosh and Fildes jar, that in strictly anaerobic conditions no growth took
place, a t least with the strains selected. It was therefore possible that the Pseudomow
cultures possessed a greater capacity than the Achromobacter for growth at low
oxygen tensions and hence the difference might be quantitative. To check this
possibility some experiments were made with washed suspensions incubated in
oxygen-free nitrogen in Thunberg tubes.

Experiments with zomhed suspensions and peptone

In the first experiment two strains, one of Pseudomonas and one of Achromobacter,
were grown under two sets of conditions: (a) in 10 ml portions of medium 1 (agar
omitted) in 25 ml bottles and (b) in 10 ml portions of medium 1 (agar omitted) in
T-tubes, with aeration. Washed suspensions in Ringer’s solution were prepared and
adjusted to approximately the mme opacity. When these suspensions were added
to medium 1 in Thunberg tubes and incubated under nitrogen the reaction in the
tubes containing Pseudomow cells became alkaline, no matter whether they had
been grown in aerated or non-aerated conditions. No change in reaction took place
with the Achromobacter cells. Therefore all later experimenh were done with cells
grown in aerated culture.

Table 5 . The pH chnnges induced in medium. 1 after incubation

anaerobically for 22 hr with wnshed swpensiona* of Pseudomonas
and Achromobacter cells
Culture pH changes
7--in
\
Group no. duplicate tubes
B. Pigmented + 0.79 +on70
+0*31 +0.28
+0.72
C. Non-pigmented
P.sewiomonas { !EA ++64
+0*73
+0-64
-0.03 +0.01
D. Achronwbacter - 0.04 -0.06
+ 0.01 0.00
* Suspensions all of equal turbidity.

A second experiment showed that an alkaline reaction was obtained, as before.

with 4 other Pseudomonas cultures, 2 pigmented and 2 non-pigmented. whereas
3 further strains of Achromobacter failed t o produce any change (Table 5). No change
took place when boiled Pseudomonm suspensions were used, and growth did not
occur during the experiment. The Thunberg tubes were made t o fit into an EEL
nephelometer. and readings were madc on a duplic&e set of tubes containing no
44 Margaret J . Thornley

indicator: these showed that there was no increase in turbidity during the period
of incubation.
These results showed that Pseudomoms cells, even when grown with aeration,
contained enzymes which would lead to the production of an alkaline reaction in
medium 1 under anaerobic conditions, without growth occurring, whereas the
Achromobacter cells did not. The difference therefore lay in the enzymic constitution
of the bacteria and not merely in their ability to grow a t reduced oxygen tensions.

Experiments with washed suspensions and amino acids

An attempt was made to identify the ingredient in the Difco peptone which gave
rise t o the alkaline reaction, and initially 21 amino acids in groups (Table 2) were
used as substrates. As well as 2 pigmented (nos. 11 and 145A) and 3 non-pigmented
(nos. 155A, 141 and 167) Pseudomonas cultures from chicken meat, 1 pigmented
culture from water (VGC/l)and 1 from soil (D/418) were tested. So also was a culture
of Alcaligenes bookeri (NCIB 8165), another organism which had been found to give
alkaline conditions in the peptone medium. The incubation periods varied between
18 and 20 hr, and slight pH changes of less than 0.2 units were disregarded. To
summarize the results, all the cultures produced alkaline conditions only with
medium 1 and arginine as substrates. Of the p H changes in medium 1 the smallest
was recorded for Alcaligenes bookeri (+0-26 units), while the Pseudomorn strains, in
suspensions of the same turbidity, produced changes from + 0 5 5 to $0.74 units.
With arginine as substrate, no buffer being present, the amount of change wm +1.3
units with Alcaligenes bookeri, and with the Pseudomonas strains it varied from
4-1.15 to +1.65 units.
It was thought that arginine might be decomposed by the arginine dihydrolase
enzyme system (see Table 8 and Discussion). It was shown by Knivett ( 1 9 5 2 ~that
)
with Streptococcus faeculis cells treated with cetyltrimethylammonium bromide
(CTAB), the reaction proceeded only as far as the intermediate product, citrulline.
This could be estimated by the colorimetric method of Archibald (1944), used in a
modified form by Knivett (19523). Accordingly, CTAB treated suspensions of the
pigmented Pseudomonas strain 145A and the non-pigmented strains 155A, 141 and
167 were incubated with an arginine-buffer mixture, as described by Knivett (19523).
After 1 hr a t 25" the colour test showed the presence of citrulline in the mixture
with CTAB treated cells, and its absence in that with untreated cells. This effect
was the same in tubes incubated aerobically and anaerobically. It therefore appeared
that the arginine dihydrolase enzymes were present in the pigmented and the non-
pigmented organisms tested.

Experiments with arginine media

An arginine containing medium was required, to provide a simple test for large numbers
of cultures. Medium 2 (Table 1 ) was derived from medium 1 by reducing the peptone
concentration to the very low value of 0*1%,and medium 2A was derived from
medium 2 by adding 1 % of arginine. The reduction in peptone concentration wtbs
designed to minimize any interference with reactions due to breakdown of arginine.
 Arginine test for Pseudomonas 45
In spite of the low concentration of nutrients in medium 2 , almost all the strains
inoculated produced visible growth in it.
Open and closed bottles of media 2 and 2A were inoculated with a variety of
organisms, and some of the results obtained are shown in Table 6. I n medium 2 all
the Pseudomom cultures formed alkali in the open bottles, with no change or a
very slight alkaline shift in the closed bottles. Its buffer action was presumably
sufficient to cope with any alkali produced from the small amount of peptone present.
I n the arginine medium a strongly alkaline reaction appeared in both the open and
sealed bottles after 2-3 days incubation. One strain o f Ps. formicans and 3 of
Aeronzonas gave the same reaction. With 10 of the Pseudomow cultures from
chicken, and with Ps. formicans and the Aeromonas cultures, tests were done with
Nessler's reagent on the closed bottles of media 2 and 2A. These showed that
ammonia was present after incubation only in the arginine containing medium.

Table 6. A comparison of the reactions produced by various &am-negative

organisms in medium 2 (without arginine) and medium 2A
(with added arginine)
Organism Source
or

no.*

Chicken
No. of
cultures
collection tested

8
r
2, with bottle
&
Open

1
Closed
-
Reactionst in medium:
,
2A. with bottle
Open Closed

Slight
(non-pigmented) Chicken 18 ' :A
: alk. Alk.+ Alk. +
PS.f O T 6 & n S Shewan 1 slight or
Aerontonaa hydrophila NCTC 7810 1 NC
Aero. hydrophila NCTC 7812 1
A ero . liquefaciens L417 (Shewan) 1
Achromobacter Chicken
Ahligenea viscosw NCIB 8154 1
Alc. viacoaua NCIB 8506 1 Alk. NC Alk.
Alc. faeculis NCIB 8156 1 or or or NC
Alc. faemlid NCTC 415 1 slight acid slight
Fhvibacteriurn ark. alk.
aurantiacma NCIB 8204 1
Flaw. d e n i t h h n s NCIB 8205 1
Rchronzobucier lacticum NCIB 8208
F b .Jlavescens NCIB 8187
Flav. esterourornuticum NCIB 8188
Flav.quatile NCIB 8748
1
1
1
1
} Alk. NC Or
acid
Alk.+ NC or
acid

Gaa producing NC Or Alk. NC or

fermenters Chicken 4 Alk. acid acid
* NCIB, National Collection of Industrial Bacteria.; NCTC, National Collection of
Type Cultures. t Alk., alkaline reaction; NC, growth with no change in reaction;
Alk.+, a stronger reaction with arginine present than in the corresponding bottle
without arginine. Medium 2 had only 0.1 % of peptone; medium BA contained
1% of arginine in addition.

I n medium 2, as would be expected, the Achromobacter cultures tested gave alkaline

shifts, often only slight, in the open bottles, and no change or slight acidity in the
closed bottles (Table 6). I n many cases, the results in the arginine medium were
exactly the same. It would seem that these strains did not utilize arginine, or not
46 Margaret J . Thornley
in such a way as to cause a marked pH change. As well as Achronwbacter, strains of
some species of Alw~ligeuesand Phvobacteriuni fell into this group. A slight variation
of this behaviour occurred with dchromobacter hcticurn and several other E'lavo-
bacterium species Here, the open bottles of the arginine containing medium 2A
developed a strongly alkaline reaction, much in excess of that in the open bottles
without arginine, and ammonia was present in the arginine medium. No alkaline
shift took place in the closed bottles, however.
A few gas producing fermenters of the coli-aerogenes group were also tested; they
all produced a strongly alkaline reaction under aerobic conditions, and no change
or acid conditions in the sealed bottles.
It seemed of little interest, from these results, to continue to compare the results
in open and closed bottles of media with and without arginine, as the aerobic con-
ditions of the open bottles allowed the possibility of too many alkaline reactions.
In the closed bottles, an alkaline reaction from the peptone alone had not been
observed without a corresponding arginine reaction ; therefore, for screening a large
number of cultures, an alkaline pH shift in the sealed bottle of the arginine medium 2A
was considered sufficient evidence of arginine breakdown of the type brought about
by Pseudomonas strains.

Tests on &am-negative bacteria from other sources i n arginine,

peptone and glucose media
Tests in the arginine medium, the peptone containing medium 1 (closed bottle) and
the Hugh & Leifson glucose medium were applied to a large number of Gram-negative
bacteria from culture collections, with particular attention to strains of P s e u d o m o w
end Achromobacter. These results are summarized in Table 7, where the organisms
are grouped according to their reactions in the arginine medium 2A in closed bottles,
group I forming alkali and group I1 giving no change or slight acidity. It can be
seen that the production of alkali separates most P s e u d o m o w species and strains
from nearly all the rest. An exception was Alcaligenes bookeri NCIB 8155, which
gave an alkaline reaction after 14 days. However, most P s e u d o m o m strains gave
that reaction in 3 days or less. Also, this organism could easily be distinguished
from a P s e u d o m o m by its failure to oxidize glucose. Three glucose-fermenting Vibrio
species also fell into group I, while other Vibrio species occurred in group I1 (section !

B). Two halophilic Vibrio strains, G220 and RS368, supplied by Dr. Shewan and
5 Vibrio paracholerae strains (NCTC 30,47 11,4715,4716and 8012) also fell in group I1
but their behaviour in the other conditions was not tested. Chrombacteriurn violaceum
NCTC 9757 also produced the alkaline reaction, but differed from P s e u d o m o w in
pigmentation and in fermenting glucose. A number of other Chronwbacterium
violaceurn strains which oxidized glucose failed to give the alkaline reaction (group 11,
section A, Table 7). One strain of P s . f o m i c u n s and 10 Aeromorm cultures gave
strongly alkaline reactions in the arginine medium, but slight acid and no change
respectively in the peptone medium 1 in closed bottles. As the results obtained
with 2 strains of Cloaca cloacae were the same as with the Aeromonas cultures this
test is evidently of no use for distinguishing the fermentative polarly flagellated
group from members of the Enterobacteriaceae.
 Arginine test for Pseudomonas 47
Table 7 . The association between the reactionsproduced ,inclosed bottles of medium 2A
by Gum-negutivebacteria from culture wllections, and those produced in
Hugh & Leifson’s glucose medium and in closed bottles of medium 1
Organisni No. of Designation. Reaction produced**
cultures and u - 7
tested source Glucose, Glucose, Medium 1
open closed closed
Grouu I. Ornanisms uroducine alkali in closed bottles of medium 2A
P&udom&
Ps. jlumesccns
aednosa - 3 NCTC 6750 8060 5083
4 NCIB 3756,’ 8251,’ 8248, NCTC 4755
7
Ps. walw 1 NCIB8296
Ps. fragi 1 NCIB8542
9 Human plasma (Rhodes)
Acid Alk. Alk.

i
10 Sew (Rhodes)

’
10 S o i l x o d e s )
Psew&nnmas strains, 10 Milk (Rhodes I
pigmented 1 Fish 1005 (Shewan)
1 Soi1,’C 123B (Paton)
1 P6009-2 (Brennerl
( 10 Lake Wat;?r(Collins) Acid Acid: Alk.
Pseudomonas s 1 NCTC7452 Alk. ’ Alk. Alk.2
tA&a{igenes boo,!.& 1 NCIB 8155 Alk. S1. alk. Alk.:
V i h ichthyodermis 1 SH1 (Shewan) Acid Acid No change
V . anquillarum 1 (Shewan) Acid Acid No change
V i b r b sp. Smith & Krueger 1 (Shewan) Acid Acid Alk.
Chromobactm‘wm violaceuw 1 NCTC975i Acid Acid Alk.
PsPwlmnmtas formicans 1 (Shewan) Acid .4cid 91. acid
Aeromnaa hydrophila
A m . liquefoeisns
Aeromonas strains
Cloaca cloacae
2 NCTC 7810, 7812
1 L417 (Shewan)
7 (Barnes)
2 NCTC 8155, 8168
] Acid
and
gas
Acid
w
and No
change

Group 11. Organisms producing no change or acid in closed bottles of mcdium 2A.
A. Oreanisms which oxidize elucose to acid.
PaeGiomonaa mmu-p-unowk 1 330 (Dowaon) S1. acid
Ps. phaseolicola 1 52 (Dowson) 91. acid
Achrowwbacter anitratus 1 NCTC 8102 No change
Achr. lacticum 1 NCIB 8208 No change
Achr. hartbbii 1 NCIB 8129 NO Change
Achromobacter strains 17 Lake water (Collins) No No change
Flavobacterium suavolem 1 NCIB 8188 Acid No change
Flav. jlavescens 1 NCIB 8187 change No change
$ Flav. estmoarmticunl 1 NCIB 8186 No change
Flav. dcnih.ificans 1 NCIB 8205 51. acid
Flav. aquafils 1 NCIB 8748 51. acid
SFlavobacierium strain 1 F31 (Windle Ta lor, Shewan) No change
Chrmbactekum violaceurn 5 Lake water (Codins) No change

No change
51. acid
Achrmbactcr strains 2 A 1011 NC80 (Shewan) 51. acid
Alcaligsnes viscosus 2 NCIB 8154 8596 No change
Ale, faecalia 2 NCIB 8156’ NCTC 415 No change
A&. dsnitrificans 1 NCTC858d No change
Ale. hlarrms 1 NCIB 8552 > Alk. No Acid
Flavobachrium aurantiacum 1 NCIB 8204 change No c-e
5 F h . acErphureum 1 NCIB 8207 Acid
IIFlovobactdwn strains 3 L 624, MacLeod A 18, G 1566(Shewan) Acid
vibrio yclositcs 1 NCIB 2581 91. acid
v. ne&ies 1 NCIB 2582 S1. acid
v. p e w o h 1 NCIB 8193 91. acid
Acid

Acid
Acid

and

NCTC, NCIB, National Collections of T pe Cultures and Industrial Bacteria respectively; LTRS, Low Tem-
perature Research Station, Cambridge. &her designations are those of the workers listed, who supplied them.
* * Alk., alkaline; Sl., slight. :These reactions developed slowly.
t Formed alkali slowly and to a relatively small extent in medium 28.
5 These organisms produced only sli$ht acidity in closed bottles of Hugh k Leibon’s glucose medium.
II T h e strains produced no change in open bottles of Hugh & Leifson’s glucose medium.
48 Margaret J . Thornley
The cultures which did not give rise to alkali in medium BA (group 11) were
further subdivided, for convenience, according t o their action on glucose. Those
which oxidized it to acid were placed in group IIA, those which failed to give acid
from it in group I I B and those which fermented it in group IIC.
Two Pseudomonas species were unusual in not giving alkali from arginine, though
they produced a fluorescent pigment and oxidized glucose. These were the plant
pathogens Ps. mors-prunorum and Ps. phaseolicola, which fell into group IIA. Two
other Pseudomonas cultures, Ps. diminuta NCTC 8545 and Ps. 816, which produced no
fluorescent pigment also gave no alkaline reaction in the arginine medium. They
did not attack glucose and were placed in group IIB. As the genus Pseudomonas is
now defined in Bergey’s Manual (7th ed., Breed, Murray & Smith, 1957), only
organisms which attack glucose can be included, and therefore these must be regarded
as belonging elsewhere. This has already been suggested by Rhodes (1958) for Ps.
diminuta, which she considers might be more closely related t o Vibrio or Acetobacter.
Other organisms which gave either no change or a very slight acid reaction in
medium 2A and attacked glucose by an oxidative mechanism (group IIA) included
many strains and species of Achromobacter and some of Flavobacteriurn and
Chromobacterium violaceurn. Organisms placed in group IIB, inert with glucose,
included many cultures of Achromobacter from chicken meat (these are not shown
in Table 7) and some species and strains of Alcaligenes and Flavobacterium, and also
4 Vibrio species. Only a few fermentative organisms (group IIC) were tested. All
except the Cloaca cloacae strains already mentioned and placed in group I gave
no change or acid in the arginine medium 2A. To sum up the results in Table 7, the
Pseudomonas strains and species which oxidized glucose, whether pigmented or not,
usually also showed the alkaline reaction in the arginine medium in closed bottles.
Exceptions so far discovered are 1 pigmented culture from chicken meat, and 2 pig-
mented plant pathogenic species. Some organisms classed as Pseudomom, but not
able to produce acid from glucose, failed to give the alkaline reaction, as did the
seven polarly flagellated unclassified organisms from chicken (Table 3, group E,).
An exception t o this was Pseudomonas sp. NCTC 7452, which gave no acid from
glucose and a slow alkaline reaction in medium 1 and the arginine medium, in
closed bottles in both cases.

DISCUSSION
Some of the theoretically possible mechanisms of anaerobic arginine metabolism are
shown by Table 8. Arginine decarboxylase produces agmatine which may be broken
down to putrescine and urea by agmatinase; and if urease is present, ammonia and
carbon dioxide are formed. Breakdown as far as putrescine has been demonstrated
in the Enterobacteriaceae by Moeller (1955),but has not been recorded in Pseudomow.
The arginine dihydrolase enzyme system consists of arginine desimidase, which
breaks down arginine to citrulline and ammonia, and citrulline ureidaae, which gives
ornithine, ammonia and CO, from citrulline, with the formation of a high energy
phosphate bond. The overall reaction, shown in Table 8, was demonstrated in strepto-
cocci by H ills (1940) and the system has since been studied intensively in the
streptococci (Knivett, 1952a, b, 1953; Oginsky & Gehrig, 1952; Korzenovsky &
 Arginine test for Pseudomonas 49
Table 8. Possible methods of breakdown of arginine (after Moeller, 1955)

-r hr
&matine+ CO,
I
P e t r e s c1i n e + r

2NH,+COe
ArE%oxylase
Agmatinase

Urease
Citrulline+ NH,

1
Ornithine+CO,+ NH,
*Ezdase
Citrulline
urnidase
1 ihydrolase

Overall arginine dihydrolase reaction:

"32 C.NH(CHI)8CH(NH,).COOH+2H,0---2NH,+C0,+ NH,(CH,)&H(NH,)COOH

Werkman, 1952, 1953; Slade & Slamp, 1952). I n streptococci, the incidence of
arginine dihydrolase is used as a test for differentiating species (Niven, Smiley &
Sherman, 1942). I n the lactobacilli, it is usually present only in heterofermentative
species (Briggs, 1953). Ammonia production from arginine by Staphylococcus aureus
was noted by Hills (1940), who thought that the enzyme concerned was arginine
dihydrolase. Soru (1958) detected urea as well as ornithine among the products of
arginine metabolism, and concluded that arginase was responsible. Soru also stated
that among staphylococci only the pathogenic strains contained arginase. An enzyme
system attacking the guanidine group of arginine is present in Bacillus subtilis
(Tomota, 1941), and enzymes of the arginine dihydrolase system have been shown in
several species of Clostridium (e.g. Woods, 1936; Schmidt, Logan & Tytell, 1952). Some
Gram-positive bacteria were tested in the peptone containing medium 1 (Table 9),
and the development of alkalinity in the closed bottles showed a close correlation
with the known occurrence of the arginine-decomposing enzymes described above.
Strep. faecalis var. liquefaciem gave negative results, but tests on the arginine medium
were positive for all the streptococci tested.
Among Gram-negative bacteria, the attack on the guanidine group of arginine
was demonstrated as early as 1924 by Hino with Pseudomom aeruginosa and Ps.
fluorescens. The arginine desimidase was first described by Horn (1933) in Ps. aeru-
ginosa (B. pyocyaneus). Arginine dihydrolase was shown by Roche, Lacombe &
Girard (1950) t o occur in Ps. ovalis, where ornithine was the end product, and in
Ps. eisenbergii, which decomposed ornithine with the liberation of an extra molecule
of ammonia. Cell free extracts of Ps. aeruginosa have been used by Oginsky (1955)
t o study the citrulline to ornithine reaction. Attack on arginine, which in some
cases proved to be by the dihydrolase enzymes, has therefore been demonstrated
in four species of Pseudomonm. It was also shown by Neilson & Eagles (1947) to
occur in Proteus ichthyosmiw, also known as Ps. ichthyomius (Breed, Murray &
Hitchens, 1948) and now regarded as Aeronzonm hydrophila (Miles & Miles, 1951).
I n the Enterobacteriaceae, Tomota (1940)demonstrated an attack on the guanidine
group of arginine by Salmonella enteritidis, and Moeller (1955) showed that the
products of arginine dihydrolase were formed by some strains of E . coli, Cloaca and
Salmonella. It was usually absent in Klebsiella and the use of a n arginine test for
distinguishing between Klebsiella and Cloaca has been suggested (Moeller, 1955 ;
Hormaeche & Munilla, 1957).
 Margaret J . Thornley
The present work has shown the prevalence in the genus Pseudomow of an
anaerobic enzyme system which attacks arginine. In the 4 cultures tested, 1 pig-
mented and 3 non-pigmented, the accumulation of citrulline by CTAB treated cells
indicates the action of arginine dihydrolase, already known to occur in several
pigmented species. It seems probable that this enzyme system is responsible for the

Table 9. The associdion between the reactions produced in medium 1 by Qram-

positive bacteria from culture collections and those produced in Hugh &

-
Levson's glucose medium
Organism No. of Designation. Reaction producedt
cultures and r L \
tested source Medium 1 Glucose
open Closed
Staphylococcus aurew 2 NCTC 7447, 8532 Alk. Acid Acid
Staph. sapmphyiicus 1 NCTC 7292 Acid Acid Acid

1
Staph. laetia 1 NCTC7584 Alk. 91. acid Acid Acid
Staph. +oseua 1 NCTC75U) 91. acid NC NC
Staph. ofemLentam 1 NCTC2665 91. acid NC NC

1
. .
Streplococcusj a e c i m 2 9748 R/298 (Barnea)
Sirep. jaecalia 1 N/8d(Barnes) 91. alk.
var. zymogenes 1 C1 (Barn@ 91. alk. Acid Acid
var. liquefaciena 1 EID76 (Barnea) ' Alk.
Btrep. dumna 2 C3 H2 (Barnes) Alk.
Streplocoem sp. 1 R/h14, oup D unclassified Alk.
type ?(Barnes)
lactobm'Uw bra&
t L.buchneri
L.P l a n h M
1
1
1
NCDO473
NCDO110
NCDO83
fk g:}
Acid Acid
Acid Acid

Microbacterium 2 CRlPA, WR7 (Niven) Acid Acid

Acid Acid
thmmosphacium
Coynebactmiurn EB/F/37/5 31 170 (Barnes) Alk. Acid Acid NC
strains
Bacterium 1Lm 1
{
EB/F/37/3b (dames)
739 (Garvie)
Alk. 91. acid
Alk. NC '
Alk.
Alk.
NC
NC
Kurthia zopfii 2 NCTC404,q Alk. NC Alk. NC
Bacillus solbtilia 2 LTRS collection A&. 91. acid Acid 91. acid
NCTC, NCDO, National Collections of Type Cultures and Dairy Organisms renpectively; LTRS, Low
Tem erature Research Stacion, Cambridge. Other denignatiom are those of the workers listed, who
su pfied them.
t A!.,alkaline reaction; 91., 81' ht. NC owth with no cha in reaction.
t Not tested in open bottles of%& id f%faonvs glucose m%m.

rapid development of alkalinity in the arginine medium by members of the genus

Pseudomow.
The anaerobic production of alkalinity from arginine was also effected by the
10 strains of Aeronww which were tested, and by several Vibrio species, a mesophilic
strain of Chromobacterium violaceum and one species of Alcaligenes, where it has not
hitherto been described.
Of the various methods used in this work, the test recommended for screening
a large number of cultures employs the arginine medium 2A (Table I), autoclavedy
in approximately 3 ml amounts in 5 ml bottles. Each organism is inoculated by
stabbing into one bottle, which is then sealed with sterile melted vaseline and
incubated. Colour changes of the indicator in inoculated bottles, compared with a
control, are noted, and a pH change towards the alkaline side is regarded as a positive
result. Nearly all Pseudomow strains gave a positive result after 3 days at 20",
and the slowest alkaline change (for Alealigenes bookeri) was recorded after 14 days.
 Arginine test for Pseudomonas 5’
The incubation temperature of 20” was chosen for the psychrophilic organisms
studied here, but this could be varied according to the nature of the organisms.
It is suggested that this test may prove useful for distinguishing Pseudormnus
from other Gram-negative glucose-oxidizingbacteria, particularly the Achromobacter
group. The occurrence of the same reaction in several groups with a different type
of glucose metabolism makes it imperative to combine this test with Hugh & Leifson’s
test; and since exceptions to the arginine test have already been shown to occur in
undoubted Pseudomom species, it remains desirable to use these two tests in
conjunction with as many others as possible.

This work has been carried out a8 part of the programme of the Low Temperature
Research Station, Cambridge.
REPERENOES
A R C ~ & DR. , M. (1944). Determinstion of citrulline and allantoin and demonstration of
citrulline in blood plasma. J . biol. C h m . 156, 121.
AYBES.J. C. (1955). Microbiological implications in handling, slaughtering and dressing of meat
&ah. Advanc. Food Rea. 6, 109.
BREED, R. S., MURRAY, E. 0. D. & HJTCHENB, A. P. (1948). Bergey’e Manud of Detemincrtive
Bacteriology. 6th ed. London: BailliBre, Tindall & Cox.
BREED, R. S., MURRAY, E. G. D. & Smm, N. R. (1957). Bergey’s Man& of Determinative
Bacteriology. 7th ed. London: BeilliBre, Tindall & Cox.
BBIQW, M. (1953). The classilkation of lactobacilli by means of phyaiological tests. J . gen.
Mkrobiol. 9, 234.
BRISOU,J. (1957). Contribution 8, 1’6tude de la syat6matique de Paeudmmnadaoeae. Ann. Inat.
Paatew 93,397.
BROWN,A. D. & WEIDEMA”, J. F. (1958). The taxonomy of the psychrophilic meat-spoilage
becteria: a reassessment. J . appl. Bact. 21, 11.
EDDY,B. P. & I(ITcmm, A. G. (1959). Cold-tolerant fermentative Gram-negative organisms
from meat and other sources. J . appl. Bact. 22, 57.
GAL~~M~AXJLT, T. P.& LEIIWON, E.(1956). Taxonomy of L o p h m n a e n. gen. C a d . J . MiCrobiol.
2, 102.
Ehm, G. M. (1940). Ammonia production by pathogenic becteria. Biochem. J . 34, 1057.
IEINo, 8. (1924). Des Vorkommen der Arginese in verschiedenen Bekterien. Hoppe-Seyl. 2.
133, 100.
HORWCEIE, E. & MUNILLA, M. (1957). Biochemical tests for the differentiation of Klebsieh
and Cloaca. Id. Bull. bact. Nomencl. Taxon. 7, 1.
Horn, F. (1933). n e r den Abbau des Arginins eu Citrullin durch Bacillus pyocyaneus.
2.phy8wl. Chem. 216, 244.
HUQE,R. & LEIBYION, E. (1953). The taxonomic si&cance of fermentative versu8 oxidative
metabolism of carbohydrates by verious Gr8m-negative b8CteI’ifb. J. Bact. 66, 24.
Kay, D. & FILDES, P. (1950). The calcium requirement of a typhoid bacteriophage. Brit. J .
exp. Path. 31, 338.
KINQ, E. O., WARD,M. K. & R ~ YD., E. (1954). Two simple media for the demonstration of
* pyocyenin and fluomin. J. Lab. clin. Med. 44, 301.
M n ,V. A. (19520). Citrulline 88 an intermediate in the breakdown of arginine by Strepto-
CODCUB foecaclis. Biochern. J . 50, xxx.
WTT, V. A. (1952b). Arginine metabolism in Streptococcusfaecalk Dissertation, Cambridge.
~ T T V. , A. (1953). Phosphorylation coupled with maerobic breakdown of arginine by
Streptococcw,faeedis. Biochem. J . 55, X.
KORZENOVSKY, M. & WERKMAN, C. H. (1952). Bacterial metabolism of erginine. Arch. Biochem.
Bbphya. 41, 233.
KORZENOVSKY, Ed. & WE-, C. H. (1953). Conversion of citrulline to ornithine by cell-fm
extrects of Streptococcw, lOct6. Arch. Biochm. Bwphys. 46, 174.
KOVACS, N. (1966). Identification of Paeudomonaa pyocyanm by the oxidwe reaction. Nature,
Lond.,178, 703.
52 Margaret J . Thornley
MILES, E. M. & Mnzs, A. A. (1951). The identity of Proteus hydrophilus Bergey et al. and Protew
melonovogenes Miles & H a h a n , and their relation t o the genus Aeromonas Kluyver &
van Niel. J. gen. Microbiol. 5 , 298.
MOELLER,V. (1955). Simplified tests for some amino acid decarboxylases and for the mginine
dihydrolase system. Acta path. rnicrobiol. scand. 36, 158.
MOSSEL,D. A. A. & INGRAM, M. (1955). The physiology of the microbial spoilage of foods. J. WpZ.
Boet. 18.232.
NEILSON, N. E. & EAGLES,B. A. (1947). The deaminase activities of Proteus ichthyosmius.
Proc. roy. SOC.Con. 41, series 111, section 5, 91.
NIVEN, C. F. (Jr.), SMILEY,K. L. & SHERMAN,J. M. (1942). The hydrolpis o f arginine by
streptococci. J. Bact. 43, 651.
OGINSKY,E. L. (1955). Mechanisms of arginine and citrulline breakdown in micro-organisms.
In Amino Acid Metabolisnt, ed. W. D. MCELROY & H. B. GLASS.Baltimore: Johns Hopkins
Press.
OGINSKY, E. L. & GEHRIG,R. F. (19524. The arginine dihydrolase system o f Streptococcusfaeculis.
I. Identification of citrulline as an intermediate. J . biol. Chem. 198, 791.
OQINSKY, E. L. & GEHRIO,E. F. (1952b). The arginine dihydrolase system ofStreptococcusfaecaZh.
11. Properties of arginine desimidase. J. biol. Chem. 198, 799.
RHODES,M. E. (1958). The cytology of Psedomonas spp. as revealed by a silver-plating staining
method. J. gen. Microbiol. 18, 639.
R o c m , J., LACOMBE, G. & GIRARD,H. (1950). Sur la sp6cScit6 de certaines d6guenideses
bacthriennes g6n6ratrices d’ur6e et sup l’arginine dihydrolase. Biochim. biophys. Acta 6 , 210.
SCHMIDT,G. C., LOGAN, M. A. & TYTELL, A. A. (1952). The degradation of arginine by CZostridium
perfringens (BP6K). J. biol. Chem. 198, 771.
SHEWAN,J. M., HODOKISS, W. & LISTON,J. (1954). A method for the rapid differentiation of
certain non-pathogenic asporogenous bacilli. Nature, Lond., 173, 208.
SLADE,H. D. & SLAMP,W. C. (1952). The formation of arginine dihydrolese by streptococci
and some properties of the enzvme system. J. Bact. 64, 455.
SORU,E. (1958). Application of circular paper chromatography t o the differentiation of bacteria
by enzyme tests. J. Chromatogr. 1, 380.
TOMOTA,S. (1940). Bacterial arginase. 11. Hydrolysis o f arginine by Salmonella enteritidis
Glrtneri. J. Biochem., Tokyo, 32, 401.
TOMOTA, S. (1941). Bacterial arginase. IV. Studies on subtilia arginase. J. Biochem., Tokyo,
33, 205.
WOODS,D. D. (1936). The metabolism of the strict anaerobes (genus Clostridiurn). V. Further
experiments on the coupled reactions between pairs of amino acids induced by C1. sporogenes.
Biochem. J . 30, 1934.

(Received 14 May, 1959)