Note: Descriptions are shown in the official language in which they were submitted.
~V~ 91/On9n3 2 ~ ~ 2 ,~ ~ 1 PCT/GB90/0l067
"Appaxatus for microbloloqical testinq"
The invention relates to apparatus for
microbiological testing, e.g. for assaying
microbial sensitivity to one or more antibiotics.
It is known to test antibiotic sensitivity
by measuring zones of inhibition of microbial
growth on an agar plate. In this known method,
agar, or other nutrient medium, is poured into a
container such as a Petri dish and allowed to
solidify, to produce an uninterrupted plate of
agar. The surface of the agar is then inoculated
with the microorganism under test, and paper
discs, each impregnated with an antibiotic are
applied to the surface. The plate of agar is
incubated, whilst keeping the surface of the agar
horizontal, until zones of inhibition of microbial
growth form. During the incubation period, the
antibiotic diffuses through the medium so that a
concentration gradient, which is a function of the
amount of antibiotic on the paper disc, is
established in the medium. Zones of inhibition
form in regions where the antibiotic is present in
sufficient concentration to prevent microbial
growth. In these regions, the medium remains
clear, whereas microbia~ growth produces turbidity
in or on the medium.
With conventional methods for testing
antibiotic sensitivity, zone sizes are measured
and compared to sizes obtained for known sensitive
and resistant strains and zone size limits are set
enabling classification of the organism as
"sensitive", "resistant" or "intermediate".
However, if more than one test is carried
out on the same plate nf agar, interference can
occur between the separate tests. Unless
inconveniently large plates of agar are used,
antibiotics in separate tests can diffuse from
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different paper discs into the same region and
produce overlapping zones of inhibition. Only six
antibiotics may conveniently be tested on the
standard 9 cm diameter Petri plate.
A further disadvantage of this method is the
need for measurement and comparison or calculation
before a result can be obtained. Such extra
handling is time-consuming, open to error and
increases the risk of the operator coming into
contact with dangerous pathogens.
Alternative methods of testing antibiotic
sensitivity currently in use include measurement
of the electrical conductance or impedance of
cultures grown in the presence of the drug, or of
the turbidity generated in such cultures. These
values are then compared with those of a culture
which has already been classified. Again, these
methods require a certain ~mount of calculation
and comparison usually requiring continuous
read-out by expensive equipment.
Such methods may also involve tests on a
multiplicity of media containing different
concentrations or combinations of the antibiotic,
and are thus time-consuming to set up and prone to
errors.
According to the invention, there is `
provided apparatus for performing a plurality of
microbiological tests, said apparatus comprising a
plurality of channels which communicate with a
common region from which the apparatus may ~e
charged with liquid agar or other nutrient gel
prior to use; characterised in that indicia are
provided adjacent said channels to permit a direct
visual measurement of the effect of a test
substance on the growth of a microorganism on said
agar, when said test substance has been allowed to
diffuse along said channel from a determined point
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of application, to form a concentration gradient
of said substance along said channel.
It is evident that the apparatus may be used
for any kind of assay or test which requires the
S measurement of a zone of inhibition or activation
of microbial growth. Thus, the substance under
test may either inhibit growth (e.g. antibiotics),
or promote it (e.g. vitamins). For use in assays,
the concentration of an antibiotic in a sample,
for example, may be determined by comparing the
zone of inhibition produced by a known amount of
the solution of unknown concentration with those
produced by known amounts of standard solutions of
the antibiotic, or by the dehydrated solids from
these volumes and concentrations.
In a preferred embodiment, the channels are
parallel, communicating with a common region at
one or both ends. Said common region may
communicate with parallel channels on either side
thereof, thereby permitting a greater number of
tests to be performed, e.g. at two different
concentrations of antibiotics, to determine
sensitivity break-point values. If desired, said
common region may be open-sided, whereby the
apparatus may be charged with liquid agar after
closing over said test regions with a remova~le
cover and tilting the apparatus so that said
common region is uppermost.
Other configurations, e.g. channels
extending radially from a central common region,
are also feasible.
The substance or substances under test can
be applied to the channels using conventional
methods e.g. by applying to the surface paper
3~ discs impregnated with the substance. Preferred
forms of apparatus, however, possess means for
facilitating the application of test substances to
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t~e channels, e.g. by locating a carrier on which
the test substance or substances are impregnated.
In one such preferred form of apparatus,
each channel comprises a depressed portion forming
a well. The wells, which conveniently are
centrally disposed in the test regions, may
be of any convenient shape or size, but they are
preferably provided with means adapted to retain a
disc or other carrier impregnated with the
substance under test. Such means may e.g. be an
overhanging rim, lip or projection beneath which
the carrier may be trapped, or a protrusion from
the bottom of the well on which the carrier may be
impaled, e.g. a conical peg designed to locate in
a suitably placed aperture in the carrier. It is
also possible to retain the carrier in the well by
adhesive means.
Using this form of apparatus, a known
quantity of test substance may be applied to the
disc using conventional methods. The disc is then
inserted into the well before the molten medium is
charged to the apparatus.
It is preferred that the discs or other
carriers bearing the test substances or standard
samples for use in assays be inserted in the
apparatus prior to hermetically packaging said
apparatus e.g. in plastics film or foil. The
whole apparatus may then be sterilised, e.g. by
X-rays or ga~ma radiation, and stored in a sterile
condition until required for use.
In another embodiment of the invention, the
wells are not provided with carriers for the test
substances. Instead, the aforementioned cover
plate, or a separate well-forming member, carries
projecting portions, preferably of t;-1ncated
pyramidal or truncated conical form, which align
with the wells and cause a void to be left therein
when the apparatus is charged with nutrient
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medium. On removal of the cover plate or well-
forming member, the voids in each well may be
filled with solutions of test substances by means
of a micropipette or the like.
Alternatively, a test substance applicator
may comprise projections, e.g. analogous to the
teeth of a comb, dimensioned to align with the
wells previously formed in the nutrient medium.
Each tooth is provided with an inlet e.g. in the
form of a bore or slot designed to draw up a
standard volume of liquid by capillarity. The
applicator may then be charged from reservoirs of
standard concentrations of test substances, and
may be used to transfer determined volumes to the
respective wells.
In another embodiment, the test substance or
substances are applied from a carrier strip placed
across said channels, means being provided to
locate such strip in a determined position, e.g.
by lugs, projections, groove or slot.
It is preferred to apply the test substances
to a central point in each channel so that they
cannot diffuse through said common region and also
so that large zones of inhibition are able to
form.
The size of the zone of inhibition or
activation may be estimated visually by inspection
against the indicia provided adjacent said
channel. It is preferred to provide a fixed scale
marked on the apparatus itself. Each channel may
be calibrated so that e.g. the sensitivity of a
microorganism to the antibiotic under test can be
read directly from the apparatus. Thus, in
contrast with conventional methods, once the
apparatus has been calibrated-there is no need for
any measurement, calculation or comparison.
Alternatively, the indicia may be prov~ded
on a carrier, e.g. of paper or other sheet
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material, to which the test substance or
substances have been applied.
Generally the apparatus will be made of a
transparent or translucent material, e.g. glass or
synthetic resin, so that it may be trans-
illuminated for assessment of the tests.
The invention will now be described with
reference to the accompanying drawings, which are
by way of example only.
Figure 1 shows a partial perspective view of
one embodiment of our invention.
Figure 2 shows a cross-section through the
apparatus of Figure 1, together with the cover (8)
in position. The cross section is taken through
the line X-X shown in Figure 1.
Figure 3 shows a cross-section through one
of the wells (1) shown in Figure 1. The section
is taken through the line Y-Y.
Fi~ures 4, 5 and 6 show partial perspective
views of three further embodiments of our
invention.
Figure 7 is a perspective view of a test
substance carrier for use with the embodiment of
Figure 6.
Figure 8 is a perspective view of a well-
forming member for use with the apparatus of
Figure 5; and
Figure 9 is a perspective view of a test
substance applicator for use with the apparatus of
Figure 5.
The apparatus of Fiyure 1 possesses a
plurality of test regions (2) in the form of
parallel channels, communicating with common
regions (3) and (4). Each test region is provided
with a well (l) and a scale (7) on the base of
each channel. This scale may alternatively be on
the surface of the separating walls (5). The test
regions are separated by walls (5), and a
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~)91/~)90~ PCT/GB90/01067
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containing wall (6) retains nutrient medium (not
shown) within the apparatus. The apparatus is
moulded from glass or synthetic resin.
The cover (8) shown in Figure 2 has a planar
lower surface and rests in intimate contact with
the top surface of the container and separating
walls. The apparatus is open-sided and part of
the communicating channel (4) is uncovered so that
the apparatus may be charged with liquid agar when
lo the cover is in position. The cover may be
provided with narrow grooves aligned with the
walls (5) so that air can escape from the
apparatu~. Using this form of apparatus, each
test region is completely filled to the height of
the separating and container walls, without the
inclusion of air.
The well shown in cross-section in Figure 3
houses a paper (or other porous) disc (9) retained
by projections (10). If desired the closure of
the well may be performed by the disc (9) and the
wall (13) may be omitted.
The apparatus shown in Figure 4 possesses
all four container walls. After charging with
liquid agar into common region (3), the apparatus
25 must be held horizontally so that the agar is --
uniform in depth when set.
The apparatus shown in Figure 5, may be
charged with test substance in a number of
differentlways. In one embodiment, the test
substancé is applied to the surface of the agar.
A paper strip (not shown) is first impregnated
with the test substances and then laid across the
apparatus, at right angles to the test zones. The
paper is held in position by the projections (11)~
To prever.' diffusion of the test substances
through the paper, the regions containing the test
substances may be separated by hydrophobic regions
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~VO91/Q090~ 2 0 fi ~. ~ I 1 PCT/GB90/01067
impregnated with an impenetrable substance such as
PVC or silicone or other suitable compound.
The lugs (12) are arranged to cooperate with
a suitably-shaped end portion of the paper strip
so that only a correctly-shaped and impregnated
strip is usable with the apparatus. It is
envisaged that a number of types of apparatus be
provided, e.g. for testing antibiotics against
different main classes of pathogen - e.g. Gram
positive, Gram negative or urological organisms.
Each apparatus could be designed to accommodate a
uniquely-shaped paper strip impregnated with
suitable antibiotics for testing the organisms
concerned and may be marked with the appropriate
inhibition zone sizes to enable the grading
"sensitive", "resista~t" or "intermediate" to be
made, or to enable minimum inhibitory
concentrations (MIC values) to be calculated.
For a particular drug the MIC value
20 corresponding to any particular zone size can be ~ -
derived from a regression line analysis of the
plots of log2 MIC, obtained by a standard tube
dilution method, versus zone diameter in
millimeters, derived by an agar diffusion method
under equivalent conditions, performed for a large
number (several hundred) of different bacterial
cultures. Such regression line data already exist
for a large number of antibiotics in common use
and form the basis of the decisions of disc drug
potencies for the performance of standard tests.
Alternatively, the device itself could be used to
obtain the zone diameters appropriate to the MIC
values performed at the same time.
In a preferred embodiment for the perform- -
ance of microbiological assays, a plate of tnetype shown in Fig.5 is charged with molten agar
containing the assay organism and a well-forming
"comb" (Fig.8) comprising frusto-pyramidal "teeth"
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is inserted into the plate such that the "teeth"
of the com~ are inserted into the mid-positisns of
the channels by locating the spine of the well-
former exactly between the lugs (11, Fig.5), the
width of the spine being the size of the inter-lug
space.
When the agar has set the well-former is
removed carefully. To assist easy removal the
protrusions which form the wells may be of
truncated pyramidal shape, or of truncated conical
shape.
Each well so formed is then charged with a
standard volume of solution of the antibiotic or
other substance under test so as to form a
suitably graduated concentration series in the
majority of the wells. One or more wells are
reserved for the test samples, being unknown
concentrations of the antibiotic.
After incubation of the plate the sizes of
the zones of inhibition for each known
concentration may be read directly from the plate
and used to plot a standard curve of log
concentration vs. zone diameter. The
concentration of the unknown solution may be
obtained by interpolation of its zone diameter
into the standard curve.
Alternatively it may be sufficient simply to
read directly the zone diameter of the unknown,
and to compare this directly with the zone
diameter of standard concentration nearest to the
unknown. Such an estimate, using the appropriate
graduated series of ctandards (e.g. 2mg/1
concentration differences for gentamicin), would
give adequate evaluations of serum concentration
for the monitoring of ~ost clinical dosag~
regimes.
A simplified method of establishing the
concentration gradient series of drug standards
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W09l/0090~ PCr/GB90/01067
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may be effected by means of a capill~ry co~b,
Fig.9. In this comb there is a slot in the centre
of each "tooth" (14) of such dimension as to draw
into each "tooth" a standard volume of liquid by
S capillary absorption. Such a comb could be
charged from reservoirs of appropriate standard
concentrations and used directly (by hanging the
comb in the plate wells each of which has a
standard volume of diluent already charged), or
the comb may be dehydrated and stored (under
appropriate conditions) for future use.
Similar methodology could be used for the
assay of vitamins, e.g. folic acid in samples
except that there would be growth only in the
areas proximate to the vitamin source, related in
zone size to the concentration of the vitamin
source.
The teeth of a capillary comb could be of
round or rectangular shape, the prime
consideration being that each lumen should draw up
the same volume of liquid.
An alternative to a capillary comb would be
an absorbent paper comb but with an impervious
hydrophobic spine. Each tooth would contain an
absorbed amount of dehydrated active agent such
that on immersion in standard volu~es of suitable
liquid in the wells, the required concentration of
agent would be established.
In an alternative application of the method
the lugs (11, 12) in Fig. 5 may be omitted from
the design and the drug-bearing paper strips may
be centrally located in a plate of the type shown
in Fig. 6, using a paper template as shown in Fig.
7. In this template the cross connecting strips
~14) are printed with the scale (15) for measuring
the zone sizes. The said cross strips in this
application are water resistant and rest upon the
tops of the walls (5).
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In both latter embodiments the carrier for
the test substance or substances may be held in
contact with the agar in the channels by means of
projections of appropriate length descending from
s the underside of a lid which covers the entire
agar area of the plate.
In order to make the zone edge more obvious,
and to do so at an earlier time, an indicator may
be applied as a solution by rapidly flooding the
plate and removing the excess. The layer of cells
outside the zone would be stained, leaving the
inhibition zone unstained. This could be achieved
by the use of an appropriate oxidation-reduction
indicator since all growing bacterial cells lower
the redox potential. Alternatively a fluorescent
dye might be used, or any other suitable means.
For reading the MIC value from zone sizes,
nomographs may be provided in the form of a
printed transfer on the lid.
All the applications mentioned can be
readily automated by the use of an optical scanner
capable of detecting opacity differences and the
signals can then be processed to yield the
sensitivity classification of the organism for
25 each drug, the MIC value, the dosage required in ~ `
clinical use and the cost of treatment; all
controlled by means of suitable computer software,
and printed in a hard copy format most suited to
the users needs. Additionally the data stored
could be computer analysed at any time to give the
epidemiological progress of drug-resistant strains
in the local community, and by com~ination with
data from other sources, the national picture.
It is additionally convenient if the
specific test is also automatically identified.
This may be achieved by a bar code strip on the
panel (16) which was also scanned. The said panel
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may incorporate the relevant patient and specimen
data.
A device of the type shown in Fig. 6 could
be used in a number of additional different
applications. For example it could be used to
facilitate the phage-typing of bacteria. For this
purpose the cross strips of paper would be
designed to contact the agar surface in the
channels, but preferably only half of each channel
so that the other half would serve as a control of
normal growth. In use each paper arm would be
impregnated with dried phage particles comprising
the number used in a typing set, using one phage
strain per arm. The phage template would then be
impacted onto the inoculated plate, then removed
and the plate incubated to reveal the subsequent
formation of phage plaques. The pattern of
positive and negative effects within the typing
set determines the phage type.
In another application, we have designed a
system for the identification of bacterial species
based on theix biochemical reactions. The
substrates to be modified by the organism are
dried onto a paper template similar to the one
~5 used for phage-typing. Each substrate area is
isolated from the next by a hydrophobic barrier,
and the substrate-bearing arms are left in contact
with the inoculated agar surface once the strip
has been applied. The substrates diffuse along
the agar channels, and are converted by the
growing organisms. The strips also include where
possible an indicator which directly indicates the
nature of the change wrought by the organism; for
example, a lactose substrate including Andrade's
indicator would turn pink on fermentd~ion of the
lactose with formation of acid products. The
tests for identifying members of the
Enterobacteriaceae might include the following
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su~strates: glucose, lactose, mannitol, sorbitol,
inositol, melibiose, saccharose, rham~ose,
arabinose, amygdalin, arginine, ornithine, lysine
sodium citrate, urea and tryptophan. Other
S changes resulting from metabolism of the growing
bacteria may be tested by adding reagents to the
test strips after a period of growth. Such tests
may include addition of Kovacs reagent to show
indole production, alpha-naphthol and potassium
hydroxide for acetoin production, tetramethyl p-
phenylenediamine to indicate the presence of
cytochrome oxidase, Greiss reagent to indicate
nitrate reduction, hydrogen peroxide to
demonstrate catalase, etc.
It has been possible to establish the
dimensions required for the channels (2) in the
various applications. For sensitivity testing by
the paper strip method, experiments have been
performed to determine the zone size by a standard
method for single drugs and standard strains of
organism. The conditions required to produce the
same zone size with the same organism and the same
drug disc on agar strips are then established. As
an example, a standard method using Iso-sensitest
agar (Oxoid) with a 30 mcg Cephradine disc and
Escherichia coli NCTC 10418 gave a zone diameter
of 18 mm. Using the same batch of discs the zone ~ -
diameters obtained with the same organism
inoculated to the same extent on the same agar but
in strips of varying depths and widths was
determined. ~
By interpolation it was possible to state ~ `
the parameters for agar strip diffusion which will
yield the same size of zone. These are: 7 mm wide
by 5.4 mm deep, or 10 mm wide by 4.; mm deep.
Using apparatus which gives equivalent sized zones
for agar strip (channels) diffusion, means that
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the standards already established for determining
"resistant", "intermediate" and "sensitive"
classification will be directly applicable.
Similar experimentation can be used to determine
the parameters for a device to utilise the
standards established for the Kirby-Bauer
technique, as given by the National Committee for
Clinical Laboratory Standards for use in the
United States.
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