APPLIED MICROBIOLOGY, OCt. 1971, p. 666-670 Vol. 22, No.

4
Copyright ( 1971 American Society for Microbiology Printed int U.S.A.

Disc Plate Method of Microbiological

Antibiotic Assay
II. Novel Procedure Offering Improved Accuracy'
W. W. DAVIS AND T. R. STOUT
The Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46206

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Received for publication 7 June 1971
A detailed disc plate procedure is introduced for assay of antibiotics. The pro-
cedure is based on a previous study by the authors and deviates from conventional
procedures in several respects: selected plastic petri dishes are employed; critical
temperature control is simply provided at all stages of the test with refrigeration
of the plates never used; all dilution is done with displacement microburettes;
six pads (6.3 mm diameter) per dish are employed, all filled with the same unknown
or reference solution; the sequence of all plates handled on 1 day is made a part
of the protocol which allows accounting for the influence of the order of pouring
and setting the plates; external reference plates are set at specified locations in the
sequence; and, by averaging the diameters of all zones on a plate, most of the con-
sequence of wedge shape of agar in plates, which is common and almost unavoidable,
is removed. The present method is economical, uses simple facilities, and provides
good accuracy of test results. Bacillus subtilis was most commonly employed, but
other organisms may be employed in the present procedure.

We demonstrated a number of critical factors unconventional procedure. They are poured at a

necessary to secure constancy of response of zone
diameters (1). Control of the critical factors and
the introduction of a schedule of operations pro-
duced very uniform test response. The present
paper describes such a schedule of operations
usable for routine testing. The precise control of
several factors has proven to be necessary. How-
ever, the necessary procedures are little if any
more difficult than less rigidly controlled pro-
cedures.
Equally as important as the new procedures is
the new treatment of data. The conventional in-
corporation of internal reference zones in each
plate was necessary in the past for control of the
large plate-to-plate variation which occurred due
to variables which are now recognized and either
controllable or accountable. The major plate-to-
plate variation in the present system is a sys-
tematic, regularly decreasing zone diameter for a
constant concentration of antibiotic as the plate
sequence number and time of setting increases.
This regular variation is controlled and accounted
for in the present procedures. Also, the subtle
and very serious effects of swings of temperature
in the handling of plates are minimized by an
1 Presented at Analytical Microbiology Section, A and IM Divi-
sion of the American Society for Microbiology at Detroit, 6
May 1968.
666
precisely controlled temperature, the poured
plates are cooled for a full 20 min before stacking,
and all subsequent operations are carried out
without refrigeration. Incubation is at room tem-
perature or, if much better growth dictates, at
30 C.
MATERIALS AND METHODS
Seeded nutrient agar is poured into disposable
plastic petri dishes.
No mixture of dishes of different shapes or design
should be used in any one set of plates since even a
small variation of diameter results in a large change of
area and, therefore, of the average thickness of agar.
Plates with tilt are acceptable but not plates with any
form of nonplanar bottom. This is a characteristic of
mold design.
A reciprocating, syringe-type, automatic pouring
machine may be employed. This machine draws the
seeded, melted agar into a syringe and discharges it
through a tube into a petri dish. The speed is variable
and can be set to fill successive dishes at about 5-
sec intervals. The standard volume of seeded agar
employed is 8.0 ml. The delivered quantity in all
dishes must be very constant, as thickness of agar is a
critical factor. As each plate is poured, it is picked up,
rotated, and replaced on a leveled surface to assure a
flat and uniform agar layer.
The temperature at which agar is delivered into
VOL. 22, 1971 MICROBIOLOGICAL ANTIBIOTIC ASSAY. I6667
petri dishes must remain constant throughout the
pouring procedure. Accordingly, a special provision
is suggested to prevent a gradual drift in pouring tem-
perature from beginning to end of the series. The
flask with melted agar is placed in a water bath at 50 C
beside the pump along with a flask of sterile distilled
water at the same temperature. Before beginning the
delivery of agar, the distilled water is pumped and dis-
carded for a period of perhaps 20 deliveries to warm
the syringe and valve assembly to 50 C. Then, the in-
take tube of the pump is switched to the agar flask
and after discarding 5 to 10 more pours the pouring
into dishes is begun. This assures that the temperature
will be up to the constant 50 C value from the be-
ginning.
After pouring all plates in the series, which takes
generally less than 10 min, plates are allowed to re-
main on the bench top for a full 20 min before they are
inverted and stacked in the same order as that in
which they were poured. They are preferably stacked
in two or three stacks of 34 plates each.
These stacks are held together with a lengthwise
ring of masking tape. They are placed in a steel tube
just large enough to accommodate them and closed at
top and bottom with large rubber stoppers. The en-
closed stacks are held at room temperature until
needed for setting. They may be held for a period of
up to 3 hr between pouring and setting. Longer hold-
ing will result in smaller zones and poor zone develop-
ment. When test organisms other than B. subtilis are
employed, it may be advantageous, after setting, to
incubate the enclosed stacks at somewhat higher tem-
perature.
A parent antibiotic solution to be used for diluting
to make reference solutions is made in a neutral buffer
in which the antibiotic has good stability at refrigera-
tor temperature. This more concentrated solution is
kept refrigerated or frozen and serves as parent solu-
tion for making standard dilutions each day for a
standard curve. This parent solution is made to an ac-
curacy of concentration of better than 0.5%. This
limits the absolute accuracy to 0.5%, but the activity
of the dry reference sample of antibiotic is unlikely to
be known to be better than this accuracy. The solution
is made 50 ,ug per ml in a neutral buffer. It may be di-
vided into approximately 5-ml portions, frozen or re-
frigerated, and held until a portion is needed for
making up reference solutions for the tests of 1 day.
This practice avoids repeated opening of the same
supply bottle. Experience must dictate how long such
solutions may be kept and used.
The second step in making reference solutions is the
addition of a very small volume of the 50 ,ug per ml
parent antibiotic solution to small volumes of the same
diluting fluid as that in which the unknown will be
presented for test. This is done employing a micro-
burette of 0.2 ml total capacity. An amount of the 50
jig per ml solution was measured into a small vial to
produce, when diluted to 1 ml, a series of concentra-
tions of 0.35, 0.5, 1.0, 2.0, 4.0, and 6.0,ug/ml. These
are the standard solutions and are only needed in
small amounts in the subsequent protocol, never more
than 1.0 ml.
Setting each plate in turn involves removing it from
the stack in which it has been held and putting it up-
right on a mechanical guide for the plate. This guide
shows clearly six circles of approximately Y inzh
(ca. 0.95 cm) diameter over which the pads are to be
centered, equally spaced at equal distance from the
center. One pad at a time is placed on the agar, and
immediately a full capillary pipette of antibiotic solu-
tion is applied to it. The capillary is not rinsed but is
again filled automatically from the supply bottle and
is delivered into the next pad which is placed on the
agar after the pipette is full. A fine needle is convenient
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for picking up the pads.
This is a rapid operation and continues until 12
pads (two plates) have been set with one solution. The
pipette is then rinsed twice with distilled water by
automatic ifiling and emptying by withdrawal onto a
clean cloth. The pipette is then ready for setting plates
with the next solution.
In this way, the setting of plates can be done easily
on a schedule of 0.5 min per plate. A regular time
schedule of setting all plates in each stack is important
because the time of setting has a direct effect upon the
resulting zone diameter. A few moments interruption
between stacks is necessary and is accounted for in
treatment of data.
As plates are set, they are inverted and stacked.
Each stack has 30 plates with pads and in addition
four poured plates without pads, two at the bottom
and two at the top to minimize end effects. The stack
when completed may be held together by a strip of
masking tape extending around the stack from top
to bottom, as it is during storing.
The favored arrangement for incubating plates is
to place the stacks in the same close-fitting steel cylin-
ders as used for storing the plates before setting. These
are incubated at room temperature in a location pro-
tected from irregular drafts, as in a large glass jar.
This room temperature incubation is always con-
tinued from setting in the afternoon to reading the fol-
lowing morning, generally approximately 16 hr. No
consideration has been given to small fluctuations of
room temperature during incubation. In those cases
in which incubation is done at higher than room tem-
perature, the steel cylinders are placed in equivalent
positions in a constant temperature incubator.
A unique feature of the test procedure is the ar-
rangement of plates set with standard reference solu-
tions and with 2.0 jAg of solution per ml, only in "ex-
ternal" relation to the plates set with unknowns. The
one regular variable, namely, time of setting plates
for any sample, is accounted for by reference to the
zone diameters of 2.0 jig of solutions per ml set at
regular intervals throughout the sequence of a day's
protocol. Two pairs are located in each stack, one near
the bottom and one near the top, namely at 4 and 14
pairs in each stack. The six standard solutions of con-
centrations 0.35, 0.50, 1.0, 2.0, 4.0, and 6.0 ;&g/ml are
set at pairs 6 through 11, respectively, in the first stack.
The 1st and 17th pairs of plates in each stack are left
blank to avoid end effects. The complete order of
setting of three stacks of 34 plates each (17 pairs) is
shown in Table 1. Three stacks contain positions for 33
pairs of plates assignable to unknowns. If between 20
and 33 samples are presented for assay, three stacks
668 DAVIS AND STOUT APPL. MICROBIOL.

TABLE 1. Protocol assignment of sequential pairs of plates

Stack 1 Stack 2 Stack 3

Pair sequence Assignment Pair sequence Assignmnent Pair sequence Assignment

1 18 35
2 Unknown 19 Unknown 36 Unknown
3 Unknown 20 Unknown 37 Unknown
4 2.00 tie 21 2.00 tie 38 2.00 tie
5 Unknown 22 Unknown 39 Unknown

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6 0.35a 23 Unknown 40 Unknown
7 0. 50a 24 Unknown 41 Unknown
8 1 . Ooa 25 Unknown 42 Unknown
9 2.00 tieh 26 2.00 tieb 43 2.00 tie'
10 4.OOa 27 Unknown 44 Unknown
11 6.00a 28 Unknown 45 Unknown
12 Unknown 29 Unknown 46 Unknown
13 Unknown 30 Unknown 47 Unknown
14 2.00 tie 31 2.00 tie 48 2.00 tie
15 Unknown 32 Unknown 49 Unknown
16 Unknown 33 Unknown 50 Unknown
17 34 51
a Standard reference curve.
b These are optional extra ties.

would be employed. If less than 20 samples are pre- ZONE

sented, the third would be omitted. DIAMETER
(millimeters)
RESULTS
This protocol was employed to illustrate the 20-j mcg/ml
e 6.0
test procedure and the results represented in Fig. 19-
1. In this experiment the arrangement of 2.0 Ag
of reference solutions of cephalothin per ml was 18-
employed as shown in Table 1. In addition, how- 17- 0 4.0
ever, the same solutions from 0.35 to 6.0 ,ug per
ml employed for the standard curve in stack 1 16-
were placed in the corresponding positions in 15-
stacks 2 and 3 and regarded as unknowns. Also all
other positions designated for unknowns were set 144
with solutions of 2 ,g per ml, and regarded as 13-
unknowns.
The data which result from this experiment, 12-
shown in Fig. 1, clearly indicate that the time of 11-
setting produces a regular decrease in resulting
zone diameters for any constant concentration of 10- 1.0

antibiotic. Note that all other operations with the

plates, pouring, stacking, etc., are carried out in 9_
this same order, and, if they do contribute to a 8-
progressive variation in zone diameter, their con- ~~~~~~0.5
sequence would also be cancelled by the external
reference procedure employing the plates set with I I
2.0 Mg of solution per ml near the bottom and 15 10 15 20 25 30 35 40 45 50
top of each stack. PAIR No.
The sequence of values for 2 Mg of "tie" refer- FIG. 1. Decline of zone diameter with setting zwnber.
ence solutions per ml, set in the indicated posi- Each point represents the average zone diameter for 12
tions, permits drawing a line that serves as a zonies in two plates. Starred values are pairs which are
reference within each stack. These lines may also used as 2.0 ,ug of "ties" per ml or external reference
approximate a single straight line for the six 2 zonies.
VOL. 22, 1971 MICROBIOLOGICAL ANTIBIOTIC ASSAY. II 669
TABLE 2. Two-plate test performance in six experiments
Concn (ug/ml)
Expt no. Stack no.a
0.35 0.50 1.00 2.00 2.00 6.00

1 2 0.50 1.04 2.04 4.00 5.80

3 0.47 1.03 1.99 4.00 5.80
2 2 0.40 0.51 1.02 1.93 4.02 6.10
3 0.41 0.55 1.05 2.06 4.03 6.00

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3 2 0.35 0.50 0.99 2.06 4.02 6.25
3 0.32 0.48 0.91 1.99 3.80 6.02
4 2 0.48 0.96 1.99 4.00 6.10
3 0.49 0.98 1.99 4.03 6.20
5 2 0.48 0.91 1.95 3.92 6.00
3 0.97 2.03 4.10 6.15
6 2 0.34 0.48 0.99 1.95 4.10 6.08
3 0.35 0.48 1.00 2.02 4.22 6.42
Avg value 0.36 0.49 0.99 2.00 4.02 6.08
Avg per cent error from
stated value in listed
two-plate tests 7.45 3.91 3.62 1.75 1.67 2.38
Avg per cent error from
2.0 ;g/ml in 160 two-
plate tests included in
six expt 1.62
a Stack 1 contains the standard reference curve and only2.0,ug of plates per ml considered " unknowns."

Mug of ties per ml employed in the entire sequence
of three stacks. A procedure is described below by
which this variable is treated in the conversion of
sample zone diameters into antibiotic concentra-
tions.
When all plates have been read, zone diameters
for the readable zones in each plate are averaged.
If an individual zone is unreadable or distorted,
the zone directly opposite in the plate should also
not be read or should be dropped from the plate
average. This prevents the inadvertent use of a
high or low zone due to wedge shape of agar
without the compensation by the opposite zone
to average out the effect. The averages for pairs of
plates set with the same sample are combined to
give an average for the sample, and the results
are tabulated.
For conversion of zone diameter data to con-
centrations, the usual graphic method may be
employed as follows. Semilog paper is employed
to produce a standard curve, the zone diameter
averages for the standard concentrations being
plotted on the abscissa. The concentration in
micrograms per milliliter of the standard dilu-
tions are plotted on the ordinate. The values are
connected point to point and generally produce a
reasonably straight line. This is the conventional
presentation of the standard reference curve.
A simple graphic procedure has been devised by
which to read the concentration of an unknown
corresponding to any zone diameter, for that
sample in the time sequence of plates. A vertical
line is drawn through the standard curve at the
value of diameter corresponding to 2 ,g/ml in
the standard curve. A laterally movable scale
like the zone diameter scale is used and is offset
progressively by an amount indicated by the 2.0-
,ug ties in the sequence. From this scale, concen-
tration of the unknown is read where the meas-
ured zone diameter coincides with the standard
curve. This correction assumes that the zone
diameter decreases equally with setting number
for all concentrations of antibiotic. Figure 1
indicates that this is approximately true.
An alternative procedure for making the cor-
rection for position of a sample in the setting
sequence is to adjust the tabulated, averaged zone
diameters by a progressive correction calculated
670 DAVIS AND STOUT
by interpolation between the tie points and then
to use the adjusted, tabulated zone diameters in
the usual way, by using the standard curve. The
tie plates have the same use regardless of the pro-
cedure. A third procedure, a simple computer
program, has been devised by John Quay in
these laboratories and is not yet published. It is
used in routine testing by the assay procedure
described here.
Since the standard curve is spread over six
pairs of plates, the interpolative correction should
be applied to the resulting diameters as for all
other samples.
DISCUSSION
The quantitative test of performance of this
assay procedure can be presented by the results of
repeated assay of solutions of known concentra-
tion. Also, a regular series of standard dilutions
was made and employed for making the standard
curve and provided samples set in the second and
third stacks to be also treated as unknowns. The
results of three such test runs are summarized in
Table 2. The variability is creditable for a micro-
biological test.
In evaluating a test procedure, it is, of course,
also desirable to consider the relation of sample
tests performed per man-day of effort. In this
respect, the described procedure appears to be at
least as economical of operator time as alterna-
APPL. MICROBIOL.
tives known to the authors. The operation of the
test procedure on a routine basis, as it was em-
ployed for periods of several days at a time, was
by one man. The supporting operations, per-
formed by others, were the preparation of an
original parent spore suspension in distilled water
and the preparation and autoclaving of solutions
of nutrient agar. All other operations in the
assay of 33 unknowns per day were performed by
the single operator. These operations included
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melting of nutrient agar and admixture with test
organisms, pouring of plates, all handling and
setting of plates, and the reading of plates on the
next day. They included also the preparation of
standard solutions, the handling of all unknowns,
and finally the calculation and presentation of
results.
This protocol appears to be applicable to
testing programs over a wide range of complexity,
from clinical or research testing to industrial
assay. It is obvious that in large operations some
rearrangement of individual responsibilities from
the conventional division of labor would be neces-
sary to assure the regular sequence of operations
with one set of plates, which is essential in the
present protocol.
LIERATURE CITED
1. Davis, W. W., and T. R. Stout. Disc plate method of micro-
biological antibiotic assay. I. Factors influencing vari-
ability and error. Appl. Microbiol. 22:659-665.