Agricultural and Biological Chemistry

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Turbidimetric Microbiological Assay of Amino

Acids

Tomoyuki Ishikura, Tadashi Sakamoto, Ichiya Kawasaki, Toshinao Tsunoda &

Kikuko Narui

To cite this article: Tomoyuki Ishikura, Tadashi Sakamoto, Ichiya Kawasaki, Toshinao Tsunoda &
Kikuko Narui (1964) Turbidimetric Microbiological Assay of Amino Acids, Agricultural and Biological
Chemistry, 28:10, 700-709, DOI: 10.1080/00021369.1964.10858295

To link to this article: https://doi.org/10.1080/00021369.1964.10858295

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[Agr. BioI. Chern., Vol. 28, No. 10, p. 700-709, 1964J

Turbidimetric Microbiological Assay of Amino Acids*

By Tomoyuki ISHIKURA, Tadashi SAKAMOTO, Ichiya KAWASAKI,

Toshinao TSUNODA* and Kikuko NARUI*
The Central Research Laboratories of Sanraku-Ocean Co., Ltd.
*The Central Research Laboratory of Ajinomoto Co., Inc.
Received March 30, 1964

To shorten the time necessary for the determination of L-glutamic acid a turbidimetric micro-
biological assay method with Lactobacillus arabinosus 17-5 has been studied. It was found that the
assay can be completed within 30 hours including the incubation period for the inoculum, the assay
and the time for medium preparation. Turbidimetric assay methods of other amino acids such as
DL-alanine with Leuconostoc citrovorum 8081, L-aspartic acid and L-lysine with Leuc. mesenteroides P-60
have also been studied.

and Schiaffino et a!.4,5) reported the use of a

INTRODUCTION
In the course of study on the production of
L-glutamic acid by fermentation processes, we
wished to present a simple method to determine
amounts of the acid in the culture media. It is
important that the assay be specific for L-glutamic
acid, precise, as well as not time-consuming.
The assay method should be adaptable to a large
number of samples. Comparing enzymic and
microbiological methods, one can deal with a
larger number of samples in one run by
the microbial method than by the enzymatic
methods. Nevertheless, the titrimetric micro-
biological method has a drawback in that it re-
quires a relatively long culture time (72 hrs).
Standardized techniques of turbidimetric assay
have been described in the U. S. PY and A. O.
A. CY refferring to the microbiological method
for cyanocobalamin. Gavin 3 ) recently reviewed
the status of the turbidimetric method in his
series of papers on microbiological process reports
* This work was presented at the annual meeling of this society in
April of 1959.
1) U.S. Pharmacopeia XV, 885 (1955).
2) Official Aft/hods oj' Anar)'sis oj the Assoc. Offic. Agr. Chemist,
8th editinn, 835 (1955).
3) Gavin, Appl. Microbiol. 4, 323 (1956). 5, 25 (1957), 5,
235 , 6, 80 (1958), 7, 180 (1959).
turbidimetric technique for the microbiological
determination of amino acids except L-glutamic
acid.
METHOD
The procedure used for turbidimetric assay of L-glutamic
acid with L. arabinosus 17-5 based on the titrimetric pro-
cedure has been reported by Tsunoda 6 ) and was conducted
with necessary modifications.
An outline of the procedure is as follows. The stock
culture was maintained on a culture medium shown in
Table I by transferring in to a new medium once a week.
It wa~ stored in a refrigerator. To prepare the inoculum,
a quantity of cells was transferred from" newly prepared
stab culture that had been grown over night onto a
preculture.
The composition of this medium is shown in Table II
and it was grown for 7 to 18 hours at 37°C. The main
culture was carried out 2 ml of liquid medium in test
tubes (150 X 15±0.5 mm). The basal medium used for
the assay of L-glutamic acid with L. arbinosus 17-5 contained
the components shown in Table III. After 16 to 18 hours
incubation the reaction was stopped by heating at lOOoe
for 10 minutes. Each tubes was diluted by the addition
of 2 ml of water with hand shaking. The turbidity was
4) S.S. Schiaffino, ].J. McGuire and H.W. Loy, J. Assoc. Olfie.
Age. Chemist, 4.1, 420 (1958).
5) S.S. Schiaffino, ibid., 42, 525 (1959).
6) T. Tsunoda, H Tanpakushitsu~Kagaku (Protein Chemistry) '.
Kyoritsu Shuppan, I, 1954, p. 282.
 Turbidimetric Microbiological Assay of Amino Acids 70t

TABLE 1. MEDIUM FOR MAINTANING L. arabinosus TABLE III, BASAL MEDIUM OF L. arabinosus 17-5
17-5 CULTURES
FOR TURBIDIMETRIC METHOD OF L-GLUTAMIC
Baker's yeast extrate* IOml ACID ASSAY (SINGLE STRENGTH II)
Glucose I g Glycine 0.1 g
Na-acetate I g DL-Alanine O. 2 g
Polypeptone I g L-Valine 0.1 g
Agar (powder) 2g L-Leucine 0.1 g
pH 6.0 L-Isoleucine O. I g
* Barker's yeast was heated with 1O·fold volume water for 1 hour DL-Serine O. I g
on boiling wate-r bath and boiled for 5 min on a burner. Solid
L-Threonine O. I g
matter was removed by filtration. Filtrate was employed.
L-Cystine 0, I g
TABLE II. MEDIUM FOR INOCULUM CULTURE
L-Methionine O. I g
OF L. arabinosus 17-5
DL-Asparagine 0.4 g
Baker's yeast extrate lOami L-Proline 0.1 g
Glucose Ig L-Phenylalanine 0.1 g
Na-acetate Ig L-Tyrosine O. I g
Polypeptone I g L-Tryptophan O. I g
Salt A* 2.5ml L-Lysine·HCl 0.2 g
Salt B+ 0.5ml L-Arginine·HCI 0.2 g
Tween 80 0.1 g L-Histidine·HCI 0.1 g
pH 6.0 Adenine·H 2SO, 0.01 g
*- KH 2 PO.. 1 g., K 2 HPO. 1 g.) per 100 m!' Guanine·HCI 0.01 g
MgSO•• 7H,O 2 g., FeSO.,7H,O 0.1 g., MnSO.·4H,O 0.1 g.•
X.Cl 0.1 g., per 100 mi. Uracil 0.01 g
Xanthine 0.01 g
measured by Hitachi Spectrophotometer Type EPB-II
Thiamine· HCI I. mg
with 10 mrn cell. The value obtained by the use of
Riboflavin 2. mg
distilled water was taken as a control. Standard con-
p-Aminobenzoic acid I . mg
centration response curves were prepared each time and
Pyridoxine·HCI I. mg
the amino acid content in samples was calculated by
Pyridoxal· HCl O. 02 mg
interpolation.
Ca-Pantothenate I. mg
The procedures used for turbidimetric assay of other
Niacin I. mg
amino acids were designed on the basis described above.
Biotin 0.01 mg
EXPERIMENTAL RESULTS AND DISCUSSION Folic acid 0.01 mg
1. Assay of L-Glutamic Acid with L. arabinosus KH 2 PO, 0.5 g
17-5. K,HPO, 0.5 g
(A) Assay range MgSO,·7H 2 0 0.2 g
FeSO,· 7H 20 0.01 g
Though the assay range by the titrimetric
MnSO,,4H,O 0.01 g
method with L. arabinosus 17-5 was 1 to 10 micro-
NaCI 0.01 g
grams of L-glutamic acid per tube (2 ml), that
Glucose (anhydrous) 20. g
of the turbidimetric method was more than 10 Na-Acetate (anhydrous) 20. g
micrograms per tube. Turbidimetric response NH,CI 3. g
recorded with the degree of optical density (0. D.) Tween (10 I. g
after 18 hours incubation period is shown in pH 6.0
Fig. 1. These results indicate that the O. D.
values between 10 to 100 microgram per tube which plot of data was more meaningful, a
(2 ml) are readable but values above an O. D. variety of plots with arithmetic dose-arithmetic
of 0.5 are not. response, log dose-arithmetic response and log
To determine which reading was more practical, dose-log response was examined. It was found
optical density or transmission per cent, and that an arithmetic plot of optical density versus.
702 Tomoyuki ISHIKURA, Tadashi SAKAMOTo, Ichiya KAWASAKI, Toshinao TSUNODA and Kikuko NARUI

.8
0.7 30

.6
h
b/) 40
..£ .4
I

.2 50

·°0 50 100 150 200 250 300

L-Glutamic acid (ig/tube (2 ml) 60 c/.
f,
FIG. I. Standard Response Curve of Turbidimetric
L-Glutamic Acid Assay with L. arabinosus 17-5.
70

.8 80

.6
~ 90
.s .4
I

.2
. 0 L4c":o-'-O--;5oc!O;O==:::;60S0=:~7:'::OO::==;82;o""o
Wavelength (mp)
FIG. 2. Wavelength and Optical Density of L. ara-
binosus 17-5 Culture Solution.
A: Turbid solution after incubation with L~glutamic acid lOOllg/
tube. B: Almost clear solution after incubation without L-gluta-
mie acid.
·dose was more linear and the most satisfactory.
(B) Wavelength used for turbidity measurement
Changes in optical density measured at wave-
lengths between 400 and 1000 mp. are shown in
Fig. 2. Absorbance by the media was too high
at shorter wavelengths while on the other hand
at longer wavelengths, absorbance was too low
to measure accurately.
The wavelengths from 600 to 700 mp. gave
more accurate values and red light of 625 mp.
was selected as the best. The gaps in absorption
curves at 600 m/~ in Fig. 2 were caused by the
use of different phototubes. Probably the scat-
tering of light by cell suspensions is the cause
of this difficulty since when the instrument is
used as colorimeter, no break of curve takes place.
0.0 O':---O~.';;2---;0"".4-;---;;:O'r.6---;O~.';;'8---;-1";;.O-;-'
100
B Cell concentration, dry matter mg/ml
==9""O"';O~-:1-:O~OO FIG. 3. Relation between Turbidity and Cell Con-
centration.
(C) Relation between turbidity and cell concentration
In general, the strength of light transmitted
through a turbid solution does not always obey
Beer's Jaw. With the instrument employed, if
the optical density is less than about 0.5, the
relation between the bacterial cell concentration
and the transmitted light follows this law.
Relation between optical density or per cent
transmission at 625 mp. of the turbid cell suspen-
sion and bacterial cell concentration is shown in
Fig. 3.
(D) Change in turbidity during incubation periods
A previous report 6l suggested that the turbidi-
metric bioassay was less reliabile than the acido-
metric because turbidimetry is a measure of the
speed of growth of the test organism. On the
other hand a test in which the change in turbidi-
metric response during incubation periods was
measured was carried out to make certain of
this point (Fig. 4). In the earlier incubation
periods, the speed of growth seemed to depend
 Turbidimetric Microbiological Assay of Amino Acids 703:

TABLE IV. FACTORS AND LEVELS OF THE EXPERI-

.5 MENTS DESIGNED WITH FACTORIAL REPLI-

hrs CATION IN 2' FACTORIAL IN 16 UNITS
16
20 Factor Low level High level
40
12 Inoculum incubation period 7 hours 18 hours
Inoculum ratio I: 100 1:5
pH 6.0 6,8
Tween 80 None 0,1%
Temperature 30°C 3rC

.2
I was preferable to that of a low inoculum ratio,
,OO':---;:2':c----:ej';:0---I+iC:-)--c:!c:-- - 7 "jl:":-olOc------1
O SO
L-Glutamic acid ltg/tube
FIG, 4. Change in Turbidity During Incubation
Period.
upon the size of inoculum and the response curve
was not linear. Nevertheless at the stage of
maximum growth (sixteen hours) the response
curve became a fine straight line. For at this
stage, the substrate concentration must have been
a limiting factor for the growth of the organism
and the responses seemed to be independent of
the size of inoculum. At later incubation stages
bacterial cells autolysed themselves and a small
decrease in the optical density was observed.
These results indicate that a maximum growth
response instead of speed of growth plays and
important part in the measurement by this
turbidimetric method and the previous suggestion
should be altered. In practice, turbidity response
should be measured after a 16 to 18 hour in-
cubation period.
(E) Size and incubation period of inoculum, addition
of Tween 80 to medium and pH and incubation
temperature
As times the response curve of L. arabinosus
17-5 to L-glutamic acid was abnormal and a
phenomenon called "lag in response n at lower
dose concentrations was observed. To prevent
this trouble, use of a large inoculum ratio (1 : 5)
(1 : 100) even in the titrimetric method. S )The
effect of pH on the utilization of glutamic acid
by this organism was reported by Sondheimer
et al. 7l and the effect of oleic acid on the cell
permiability of the same organism was described
by Traub. S ) Our preliminary work suggested
that a younger inoculum such as incubated for
seven hours was more effective in preventing
the trouble than an inoculum incubated for the
usual eighteen hours. Growth at an initial pH
6.0 seemed to be better than at pH 6.8 and the
addition of Tween 80 to 0.1 % final concentra-
tion in the media to suspend the bacterial cell
stimulates the growth.
In regard to the four above-mentioned factors
and incubation temperature, an experiment de-
signed 9 ) with partial fractional design in 2'
factorial in 16 units was carried out. The
factors and experimented levels are shown in
Table IV. The plan and the results of response
curves in each unit are demonstrated in Fig. 5.
The values in vertical scale were sums of dupli-
cated optical density values.
Since all three or higher order interactions
were assumed negligible, all main effects and
two-factors' interaction could be detected by the
use of this design. An experiment with 32 units-
was considered to be too large to obtain reliable
results. To easily find each main effect, the
sums of optical densities against each dose
concentration in eight units of low levels and
sums of these in high levels regarding each
7) E. Sondheimer and D.C. 'Nilson, Arch. Biochem. Biophys., 61.-
313, (1956).
8) A, Traub, ibid., 62. 222 (1956).
9) rv1. t-.-Jasuyama, "Jikken Keikaku Ho (Experimental Designs), ",
Iwanami Shoten, 1956.
704 Tomoyuki ISHIKURA, Tadashi SAKAMOTO, Iehiya KAWASAKI, Toshinao TSUNODA and Kikuko NARUI

Inoculum size 1 :5
1:100

Tween - 0.1% 0.1 %

'C
c
.~
~
e-
o
..c
::::
CJ)

co

':kLl/k/
':Llil/l/
0.

"
.8
;;
.c CD
3 f=:
.9 b£
8, s:
.0

"~':k l/l/[/
§
.!J

CJ)

co

'n
e-
~
0
.0
..c

0
'.0

:r: ':l/[Cl/,l/
.0 0 50 1000 50 1000
L-Glutamic acid J.!g/tube
50 1000 50 100

FIG. 5. The Plan Designed with Factorial Replication in 2 5 Factorial in 16 Units and Each
Response Curve

factor were plotted against doses, which is shown
in Fig. 6. Care had to be taken that only the
height difference between two curves could be
the subject of this discussion whereas the height
of curves in itself could not. It was found that
advantageous conditions were seven hours in-
cubation period of inoculum, 1: 5 ratio of
inoculum, pH 6.0 of basal medium, addition of
Tween 80 to medium and 37°C incubation
temperature, against another level of each factor.
Furthermore, in order to obtain more detailed
information about these effects, results of an
experiment of 16 units were divided into two
groups with respect to two conditions (l: 5 and
1: 100) of inoculum ratio. In each group, sums
of optical densities of response for each concentra-
tion of dose in four units of low levels and sums
of those in four units of high levels in regard
to each factor were plotted against doses. Results
are shown in Fig. 7. In a group of 1: 100
inoculum, the advantage of seven hours incuba-
tion, pH 6.0, addition of Tween 80 and 37°C
incubation against different level of each factor
were apparent. In the group of 1: 5 inoculum,
however, there were no significant differences
in response between high and low levels of each
factor except that an incubation temperature
of 30°C was better than 37°C. The analysis of
variance from the data of these experiments
proved these main effects and their interactions
 Turbidimetric Microbiological Assay of Amino Acids 705

Inoculum incubation Inocu lum size

S period
7 hrs. 1 :5
E:;' 6
bIJ
..9
I 4
~-
:;[-.1 1 : Ill')
2

0
pH
Tween SO Temperature
S

6.0 0.1%

100 0 20 40 60 80 100 60 80 100

L-Glutamic acid pg/tube
FIG. 6. Main Effects of Each Factor.
lnoc. inc. period pH Tween SO Temp.
4
37'C
3 7 hrs
6.0 0.1%

2
6.S None
30'C

E:;' 0
bIJ
..9
I
~~ Inoe. inc. Tween 80 Temp.
'" period 7 hrs
0.1%
.~ 3
None
,6 IS hrs
:; 2
g
..s

00 20 40 60 SO 1000 20 40 60 SO 100 0 20 40 60 SO 1000 20 40 60 SO 100

L-Gluramic acid pg/tube

FIG. 7. Effects of Each Factor in 8 Units in Which Inoculum Size is Same.

with the factor of inoculum ratio to be significant. From these results, it can be concluded that
The effect of temperature on nutritional require- routine assays should be carried out under
ment of L. arabinosus was already described by conditions as follows; seven hour incubated
Borek et al. IO ) inoculum, pH 6.0 of media and 0.1 % addition
10) E. Borek and H. Waelsch, J. BioI. Chern., 190, 191 (1951). of Tween 80, since these conditions were ap-
706 Tomoyuki ISHlKURA, Tadashi SAKAMOTO, Ichiya KAWASAKI Toshinao TSUNODA and Kikuko NARUI

.5 .5

L -Glu.
.4
.4

h .3
.3 bD
h ..s
I
~
I .2 IJ-Glu .
.2

.0 OL.---c:2:':oO--4-;';O:---~60;;----c:S'c-O---o1.".Or"...)
c-1,-_G"'Ic-u-.fJ-g-,J/tube-
o 40 St) 120 160 21ln D-Glu. fJg/tube-
. 00;,----"I20"..---4""0--...."6'::-0---:S,,,,0,,----,1..,07"0----...J FIG. 9. Activity of D-Glutamic Acid .
L-Glutamic acid Ilg/tube

FIG. 8. Influence of Sterilization Condition.

:-\: 10 Lbs/inch 2, 10 min. .5
B: 20 Lbs/inch 2 , 15 min.
L-Glu.
parently more effective in the case of a low .4
inoculum ratio and did not depress the response
even with a larger inoculum ratio.
(F) Sterilization of culture medium h .3
bD
Autoclaving of culture medium is known to 0

I
cause color formation and the color influences
.2
turbidity measurements. The results of heating
carried out under a pressure of 20lbs/inch 2 for
15 minutes showed that the reading of optical .1 ~-PCA

density value in lower range of dose were raised

as shown in Fig. 8. The conditions whereby
sterilization is effected in the shortest length of .00 20 40 60 SO 100 L Glu. fJg/tube

time is most desirable. It was found that a pres- 0 .5 1.0 1.5 2.0 2.'5 L-PCA rng/tube-

sure of 10 Ibs/inch 2 for ten minutes was sufficient. FIG. 10. Activity of L-Pyrrolidone Carboxylic Acid.

(G) Influences of D-glutamic acid and L-pyrolidone

L-glutamic acid assay with L. arabinosus 17-5 were
carboxylic acid
tested. It was found that D-glutamic acid had
Studies on utilization of D-glutamic acid by
about one tenth the activity of the L-form (Fig.
this organism were reported by Ayengar et al.ll)
9) and L-pyrrolidone carboxylic acid (not more
and Carnien et a1. 12 )
than 25 mg per tube) had no activity (Fig. 10).
D-Glutamic acid and L-pyrrolidone carboxylic
These results are similar to those by the titri·
acid which might interfere with this turbidimetric
metric method for this amino acid with the
II) P. Aycngar and E. Roberts,]. Bioi., Chern., 197,453 (1952).
same organism.
12) lvLN. Carnien and :\LS. Ounn, ibid., 217,125 (1955).
 Turbidimetric Microbiological Assay of Amino Acids 707

(H) Response of Leuconostoc mesenteroides P-60.

Turbidity response of Leuc. mesenteroides P-60 .5
to L-glutamic acid after 16 hours incubation are
.4
shown in Fig. 11. With increasing concentration h
of doses more than 60 microgram per tube, the bl> .3
..9
I
response reached a plateau, so that the assay .2
range was comparatively narrow. .1
In spite of continued incubation up to 14 hours, .0
the response of higher concentration doses did 10 15 20
Replication (days)
not rise. From these results, the turbidimetric
method of L-glutamic acid assay proved to be FIG. 12. Reproducibility of Response of L. arabinosus
17-5 to L-Glutamic Acid.
more convenient with L. arabinosus 17-5 than
with Leuc. mesenteroides P-60.
error in this method were as follows: When
a standard series was carried out in duplicate
.5 at ten levels and an unknown series in duplicate
A at one level, the limits of error (P=0.95) were
estimated to be 7.1 per cent. When a standard
.4 series was carried out in six tubes in parallel at
ten levels and an unknown series of six tubes
in parallel at five levels, the limits of error (P
B =0.95) were estimated to be 2.0 per cent.
. .3
"" The comparison of the assay value of the same
:tI sample between this method and titrimetric
method with the same organism and with the
.2
enzymic manometric method with Escherichia
coli Crooks is demonstrated in Fig. 13. In the
vertical scale, an index number of assay value
.1 of the turbidimetry was shown and in the
horizontal scale, an index number of assay value
of titrimetry in the left figure and that of the
.°0;---;:;2';;-0--4-;.,00;----;!-60~-r8!A0----,1*00;;----J manometric method in the right figure. It was
L-Glutamic acid I1g/tube found that all points fell with in the limits of
FIG. 11. Response of Leuc. mesenteroides P-60 to L- error and there was no bias between the assay
Glutamic Acid.
A: L. arabinosus 17-5, B: Leuc. mesenteroides P~60) Both are u 10
incubated for 16 hours.
E 8
'i3
(1) Reproducibility, precision and accuracy :eB
The results in Fig. 12 show that the re- .,., 6
•..,;0.':'
' .:i:
..!.
producibility of the standard response in this 4
method was satisfactory for routine work.
In Fig. 12, optical density values are shown in
.
~
"
~

"
~
~

the vertical scale, and in the horizontal scale the ...j 00 2 4 8 10 0 2 4 6 8 10

days of continuous running during routine work. E. coli crooks enzymic L. arabinosus ]7-5 titrimetric
Standard response curves were constructed for FIG. 13. Comparison Between Data of Turbidimetric
every assay and the potencies were calculated Assay and Those of Titrimetric Assay or Those
by interpolation from the curves. The limits of of Enzymic Assay for L-Glutamic Acid.
708 Tomoyuki ISHIKURA, Tadashi SAKAMOTO, Ichiya KAWASAKI, Toshinao TSUNODA and Kikuko NARUI

value of turbidimetric method and those of the

other two methods. .5
According to the results above mentioned, it
is noteworthy that the determination of gluta-
mic acid can be completed within 30 hours inc- .4
luding the incubation period for the inoculum,
the assay and the time for medium preparation.
II. Assay of Other Amino Acids by Turbidimetric
.3
Method
Some other amino acids can be determined ]'
I
within a comparatively short duration by the
.2
use of this same turbidimetric technique.
The standard curve of DL-alanine with Leuc.
citrovorum 8081 is shown in Fig. 14. It was found
that the assay range is 5 to 50 micrograms per .1
tube.
In Figs. 15 and 16, the standard curbes of L-
aspartic acid and L-lysine hydrochloride with . 0O~--;;'2OO;-----7c
4 OO;-----;6;!;O,------,S;!;O,---.,-;j0\-;;0-----'
Leuc. mesenteroides P-60 are shown. The range
LMAspartic acid ,ug/tube
of 5 to 50 micrograms was found per tube of
FIG. 15. Standard Response Curve for L-Aspartic
L-aspartic acid and 8 to 80 microgram per tube Acid with Leuc. mesenteroides P-60.
of L-lysine hydrochloride.
.6 , - - - - - - - - - --, .6.------------------,

.5 .5

.4 .4

E-o
E-o
~ .3 .3
"I .9"
I

.2 .2

.1
.1

. 0 ~O- - 71OO;-----;2,;,O,----;3;!;;O---}4AO-~5.frOc----J . 0 O~-~20:;----47:0,----;6;!;;O--~S~O---;-17;:00;;--~

DL-Alanine fig/tube L~Lysine. Hel Ilg/tube

FIG. 14. Standard Response Curve for DL-Alanine FIG. 16. Standard Response Curve for L-Lysine
with Leuc. citrol'orum 8081. Hydrochloride with Leuc. mesenteroides P-60.
 Turbidimetric Microbiological Assay of Amino Acids 709

Acknowledgements. The authors are indebted
to Mr. M. Suzuki, President of Sanraku-Ocean
Co., Ltd. and Mr. T. Domen, President of
Ajinomoto Co., Inc. for their encouragement
and to Dr. Y. Takeda, Director and Dr. A. Ozaki,
vice-Dicector of the Central Research Laboratory
of Sanraku Co. for their constant guidance
through the course of this work and Miss S. Imai,
Miss T. Kanno and Miss T. Furuya for their
assistance in carrying out the experiments.