U.S. patent application number 10/559797 was filed with the patent office on 2007-04-26 for test system for the determination of the presence of an antibiotic in a fluid.
Invention is credited to Cornelis Jacobus Bouwknecht, Angelina Dekker, Stark Jacobus, Pieter Cornelis Langeveld, Johannes Theodorus Arie Van Pelt, Angelique De Rijk.
Application Number | 20070092929 10/559797 |
Document ID | / |
Family ID | 34066502 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070092929 |
Kind Code |
A1 |
Dekker; Angelina ; et
al. |
April 26, 2007 |
Test system for the determination of the presence of an antibiotic
in a fluid
Abstract
Provided is a test system, a test method and a test kit based on
a test medium comprising Bromothymol Blue or a structurally related
indicator.
Inventors: |
Dekker; Angelina; (Delft,
NL) ; Bouwknecht; Cornelis Jacobus; (Delft, NL)
; Pelt; Johannes Theodorus Arie Van; (Delft, NL) ;
Rijk; Angelique De; (Ridderkerk, NL) ; Jacobus;
Stark; (Rotterdam, NL) ; Langeveld; Pieter
Cornelis; (Delft, DE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
34066502 |
Appl. No.: |
10/559797 |
Filed: |
July 1, 2004 |
PCT Filed: |
July 1, 2004 |
PCT NO: |
PCT/EP04/07288 |
371 Date: |
December 8, 2005 |
Current U.S.
Class: |
435/32 |
Current CPC
Class: |
C12Q 1/18 20130101 |
Class at
Publication: |
435/032 |
International
Class: |
C12Q 1/18 20060101
C12Q001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 2, 2003 |
EP |
03077073.9 |
Nov 24, 2003 |
EP |
03078707.1 |
Claims
1. Test system for the determination of the presence of an
antibiotic in a fluid comprising a test medium comprising a
microorganism, at least one substance that provides a solid state
and an indicator suitable for the detection of penicillin G,
characterized in that said indicator is a compound having the
general formula: ##STR3## wherein R.sub.1, R.sub.2, R.sub.3 and
R.sub.4 are independently of each other alkyl, halogen or hydrogen,
X=C or S, n=1 if X.dbd.C and n=0, 1 or 2 if X.dbd.S, and R.sub.5
and R.sub.6 are independently of each other: ##STR4## wherein
R.sub.7, R.sub.8 and R.sub.9 are, independently of each other
alkyl, branched alkyl, hydrogen or halogen and R.sub.10 is alkyl or
branched alkyl, or salts thereof.
2. Test system according to claim 1 wherein R.sub.10 is methyl.
3. Test system according to claim 2 wherein said indicator is
Bromothymol Blue.
4. Method for the determination of the presence of an antibiotic in
a fluid comprising the steps of: a) contacting a sample of said
fluid with a test medium comprising a micro-organism, at least one
substance that provides a solid state and an indicator; (b)
incubating the microorganism for a period of time to grow the
microorganism in case no antibiotic is present in the fluid sample;
and (C) detecting growth or inhibition of growth of the
microorganism with the indicator, characterized in that said test
system is a test system according to claim 1.
5. Method according to claim 4 wherein the antibiotic to be
determined is a .beta.-lactam antibiotic.
6. Method according to claim 4 wherein the fluid in which
antibiotics are to be determined is a fluid obtainable from an
animal or human body.
7. Method according to claim 4 wherein the ratio of the volume of
said fluid to the volume of test medium exceeds 0.68:1.
8. A method according to claim 4, wherein the ratio of the volume
of liquid sample to the volume of test medium exceeds 20:27
(0.74:1) (v/v), 25:27 (0.93:1) (v/v) or 2:1 (v/v).
9. A method according to claim 4, wherein the volume of liquid
sample is greater than the volume of test medium.
10. Kit suitable for the determination of an antibiotic in a fluid
comprising a container partially filled with a test medium
comprising a microorganism, a gelling agent and an indicator,
characterized in that said indicator is a compound with the general
formula (I).
11. Kit according to claim 8 further comprising nutrients suitable
for allowing the microorganism to grow.
12. Kit according to claim 10, further comprising a thermostatic
device, with the aid of which test samples can be kept at a pre-set
temperature.
13. Kit according to claim 10, further comprising a data carrier
loaded with a computer program suitable for instructing a computer
to analyze digital data obtained from a sample-reading device.
14. Use of a compound having the general formula (I) as indicator
in a test system for an antibiotic.
15. Use according to claim 14 wherein the antibiotic is a
.beta.-lactam antibiotic.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an improved novel
microbiological test system and a new method for the rapid
determination of the presence of antibacterial compounds in fluids
such as milk, meat juice, serum and urine using said test
system.
BACKGROUND OF THE INVENTION
[0002] Microbiological test methods for the determination of
antibacterial compounds, particularly residues of antibiotics such
as cephalosporin, penicillin, tetracycline and derivatives thereof
and chemotherapeutics such as sulfa's, in fluids such as milk, meat
juice, serum and urine have been known for a long time. Examples of
such tests have been described in CA 2056581, DE 3613794, EP
0005891, EP 0285792, EP 0611001, GB A 1467439 and U.S. Pat. No.
4,946,777. These descriptions all deal with ready to use tests that
make use of a microorganism and will give a result by the change
indicated by an indicator molecule added to the test system. The
principle is that when an antibacterial compound is present in the
fluid in a concentration sufficient to inhibit the growth of the
microorganism the color of the indicator will stay the same, while,
when no inhibition occurs, the growth of the microorganism is
accompanied by the formation of acid or reduced metabolites or
other phenomena that will induce an indicator signal.
[0003] The test systems mentioned above include a test medium, such
as an agar medium, inoculated with a microorganism, preferably a
strain of Bacillus, Escherichia coli or Streptococcus, and a pH
indicator and/or a redox indicator. The microorganism and the
indicator are introduced into an optionally buffered agar solution,
optionally nutrients are added to the solution and optionally
substances to change the sensitivity to certain antimicrobial
compounds are added to the solution. Finally the agar solution is
allowed to solidify to form the test medium such that the
microorganisms stay alive but cannot multiply because of lack of
nutrients and/or low temperature. A suitable test should have the
desired sensitivity with regard to the compounds to be tested
for.
[0004] The problem with the test systems currently distributed on
the market and/or described in literature is that they have a
limited sensitivity towards certain antibiotics. One of the
consequences of this problem is that for certain applications, for
instance when threshold requirements are changed, an adequate test
system cannot be made available with the current technology. There
is thus a need for an improved test method that does not have this
problem.
SUMMARY OF THE INVENTION
[0005] It is an object of the present invention to provide an
improved method for the determination of antibiotics in fluids.
Surprisingly, we have found that there is a positive effect
attainable when applying the indicator according to the
invention.
[0006] By applying the indicator of the present invention in
microbiological test systems, an advantage in sensitivity towards
antibiotics, such as for instance .beta.-lactams and
aminoglycosides can be achieved. By applying said indicator in a
method for the determination of antibiotics in fluids, increases in
sensitivity can be achieved. Said increases can amount up to 25%
and even up to 100% depending on the antibiotic in question.
Additionally, it has been found that the use of said indicator also
results in a test system showing an improved visual contrast when
comparing positive and negative samples. This latter phenomenon
greatly facilitates accurate visual evaluation of test results.
[0007] The present invention provides a test system for the
determination of the presence of an antibiotic in a fluid which
comprises a test medium comprising a microorganism, a substance
that provides a solid state and an indicator suitable for the
detection of penicillin G, characterized in that said indicator is
a compound having the general formula: ##STR1## wherein R.sub.1,
R.sub.2, R.sub.3 and R.sub.4 are independently of each other alkyl,
halogen or hydrogen, X.dbd.C or S, n=1 if X.dbd.C and n=0, 1 or 2
if X.dbd.S, and R.sub.5 and R.sub.6 are independently of each
other: ##STR2##
[0008] wherein R.sub.7, R.sub.8 and R.sub.9 are, independently of
each other alkyl, branched alkyl, hydrogen or halogen and R.sub.10
is alkyl or branched alkyl, or salts thereof.
[0009] Furthermore, there is provided a method for the
determination of the presence of an antibiotic in a fluid
comprising the steps of: [0010] (a) contacting a sample of said
fluid with a test medium comprising a micro-organism, at least one
substance that provides a solid state and an indicator; [0011] (b)
incubating the microorganism for a period of time to grow the
microorganism in case no antibiotic is present in the fluid sample;
and [0012] (c) detecting growth or inhibition of growth of the
microorganism with the indicator, characterized in that said
indicator is a compound having the general formula (I).
[0013] Furthermore, there is provided a kit suitable for the
determination of an antibiotic in a fluid comprising a container
partially filled with a test medium comprising a micro-organism, a
gelling agent and an indicator, characterized in that said
indicator is a compound with the general formula (I).
[0014] Finally there is provided the use of a compound having the
general formula (I) as indicator in a test system for an
antibiotic.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The terms and abbreviations given below are used throughout
this disclosure and are defined as follows.
[0016] The term `CFU` is an abbreviation of Colony Forming Units
and refers to the number of microorganisms, spores of
microorganisms, partially germinated spores of microorganisms or
vegetative cells capable of producing colonies of
microorganisms.
[0017] The term `fluid` refers to a substance (as a liquid) tending
to flow or conform to the outline of its container.
[0018] The term `gelling agent` refers to a compound that assists
in changing a mixture into or taking on the form of a gel.
[0019] The term `indicator` refers to a substance used to measure
(for example by change of color or fluorescence) the condition of a
test medium with respect to the presence of a particular material
(for example an acid, a base, oxidizing or reducing agents). For
instance, the term `indicator` may refer to one or more compounds
that are known as pH-indicators, but also to one or more compounds
that are known as redox-indicators. Also, the term `indicator` may
refer to mixtures of two or more different types of indicators,
such as a combination of a pH- and a redox-indicator. In general,
when two or more indicators are used, these indicators are
co-operating to increase the indicator effect of each of the
indicators when taken alone.
[0020] The term `nutrient` refers to one or more nutritive
substances or ingredients that promote and/or are required for the
growth of microorganisms as used in the method of the present
invention.
[0021] The term `sampling device` refers to a device with the aid
of which a sample of a fluid can be added to a test medium. Such a
device may be a container, optionally with volume markings. Such a
container may be a capillary, a syringe, a pipette or an automated
pipetting system. Such a syringe or pipette may be designed in such
a way that with only one mode of operation a predetermined volume
can be withdrawn from the fluid to be analyzed.
[0022] The term `sensitivity` refers to the degree of receptiveness
of a given system to sense a certain state. More, particularly, in
the present case `sensitivity` refers to the degree by which
concentrations of antibiotics in a sample can be determined.
[0023] The term `spore` refers to a primitive usually unicellular
often environmentally resistant dormant or reproductive body
produced by microorganisms and capable of development into a new
individual microorganism.
[0024] The term `test medium` refers to a composition such as a
solution, a solid or, preferably, in the form of a sol or a gel,
for instance comprising a gelling agent. Suitable examples of
gelling agents are agar, alginic acid and salts thereof,
carrageenan, gelatin, hydroxypropylguar and derivatives thereof,
locust bean gum (Carob gum), processed eucheuma seaweed and the
like. However, the person skilled in the art will understand that
other types of solid test media may be based on carrier materials
such as ceramics, cotton, glass, metal particles, paper, polymers
in any shape or form, silicates, sponges, wool and the like.
Usually, a test medium contains one or more indicators, however,
these compounds may also be added later when the test is being
performed. The test medium comprises one or more types of
microorganisms as detecting agents. Optionally, the test medium may
also contain one or more buffers, nutrients, stabilizers,
substances that change the sensitivity to certain antimicrobial
compounds in a positive or negative way, and/or
viscosity-increasing agents. When a buffer is present in the
medium, it may be added during the mixing of the components of the
medium or the components may be dissolved and/or suspended in the
buffer. Optionally the test medium is sterilized and usually the pH
is adjusted to the required value. Examples of substances that
change the sensitivity to certain antimicrobial compounds are
antifolates like ormethoprim, tetroxoprim and trimethoprim that
improve the sensitivity of the micro-organism towards sulfa
compounds or salts of oxalic acid or hydrofluoric acid, which
improve the sensitivity towards tetracycline. Examples of
viscosity-increasing agents are ascorbyl methylsilanol pectinate,
carbomer, carboxymethyl cellulose, cetearyl alcohol, cetyl alcohol,
cetyl esters, cocamide DEA, emulsifying wax, glucose, hydroxyethyl
cellulose, hydroxypropylmethyl cellulose, lauramide DEA,
linoleamide DEA, magnesium aluminum silicate, maltodextrins, PEG-8
distearate, polyacrylamide, polyvinyl alcohol, PVP/hexadecene
copolymer, sodium chloride, sodium sulfate, soyamidopropyl betaine,
xanthan gum and the like. Alternatively, the optional ingredients
of the test medium mentioned above may also be added exogenously.
The test medium may be contained within any type of container;
frequently used containers are tubes, microtiter plates and petri
dishes.
[0025] The term `threshold` refers to the concentration value above
which a given analyte is to be regarded as present and below which
said analyte is to be regarded as absent. Generally, a threshold
value is given for particular analytes in particular samples by
local, regional or interregional authorities but it can also be
pre-set for certain research purposes.
[0026] In a first aspect of the invention there is provided a test
system that comprises a test medium. The test medium comprises a
microorganism, a substance that provides a solid state and at least
one indicator, at least one of which has the general formula (I)
given above. Preferred examples of said indicator are Bromocresol
Green, Bromothymol Blue, Chlorocresol Green, m-Cresol Purple,
Thymol Blue and Xylenol Blue. Most preferably the indicator is
Bromothymol Blue.
[0027] Preferably, the substance providing for a solid state is a
gelling agent and/or a carrier material. The amount of gelling
agent in the test medium is between 2 and 100 g.l.sup.-1,
preferably between 5 and 50 g.l.sup.-1, more preferably between 10
and 20 g.l.sup.-1, most preferably between 12 and 15 g.l.sup.-1.
Preferably the gelling agent is agar.
[0028] In an embodiment of the first aspect of the invention, the
microorganism is a thermo stable microorganism such as a Bacillus
species, preferably Bacillus stearothermophilus, an Escherichia
coli species, or a Streptococcus species, preferably Streptococcus
thermophilus. These species may be introduced in the test as units
capable of producing colonies, or Colony Forming Units (CFU's).
Said CFU's may be spores, vegetative cells or a mixture of both.
The concentration of said CFU's is expressed as Colony Forming
Units per ml of test medium (CFU.ml.sup.-1) and is usually in the
range of 1.times.10.sup.5 to 1.times.10.sup.12 CFU.ml.sup.-1,
preferably 1.times.10.sup.6 to 1.times.10.sup.10 CFU.ml.sup.-1,
more preferably 2.times.10.sup.6 to 1.times.10.sup.9 CFU.ml.sup.-1,
most preferably 5.times.10.sup.6 to 1.times.10.sup.8 CFU.ml.sup.-1,
or still more preferably 5.times.10.sup.6 to 2.times.10.sup.7
CFU.ml.sup.-1.
[0029] In a second aspect of the invention, there is provided a
method for the determination of an antibiotic in a fluid comprising
the steps of contacting a sample of said fluid with a test medium
according to the first aspect of the present invention in the
presence of nutrients. Advantageously, the method provides for
conditions that there is no growth of microorganism prior to the
addition of fluid sample, by keeping the test medium at conditions
that prevent growth, such as a relatively low temperature and/or in
the absence of nutrients essential for growth. After addition of
the fluid sample, growth of the microorganism is allowed to take
place during a period sufficiently long for the microorganisms to
grow in case no antibiotics are present, by adding nutrients,
optionally before the contacting of said fluid sample, and/or
raising the temperature, and/or providing for a pH-value at which
the microorganism is able to grow; and detecting growth of the
microorganism by observing the presence or absence of a change of
an indicator. The method of the present invention also includes
mixing samples (e.g. with other samples, but also with salts,
buffering compounds, nutrients, stabilizers, isotope-labeled
compounds, fluorescence-labeled compounds and the like),
concentrating and/or diluting (e.g. with diluting liquids such as
water, milk or liquids derived from milk, blood or liquids derived
from blood, urine and/or solvents) samples prior to addition to the
test medium.
[0030] In one embodiment of the second aspect of the present
invention, the antibiotic is a .beta.-lactam antibiotic such as a
cephalosporin or a penicillin derivative. Examples of such
derivatives are amoxicillin, ampicillin, cefadroxil, cefradine,
ceftiofur, cephalexin, penicillin G, penicillin V and ticarcillin,
but of course many other similar .beta.-lactam derivatives are
known and applicable in the method of the present invention. In
another embodiment the antibiotic is an aminoglycoside such as, for
instance, neomycin.
[0031] Advantageously, it was established that the method of the
present invention displays selectivity with regard to antibiotics,
in particular with regard to .beta.-lactam antibiotics and
aminoglycosides.
[0032] In another embodiment of the second aspect of the invention,
the growth of the microorganism is to take place during a
predetermined period, preferably within a time span of 0.5 to 4
hours, more preferably between 1 to 3.5 hours, most preferably
between 2.0 to 3.25 hours. Preferably the growth of the
microorganism is conducted at a predetermined temperature,
preferably the optimal growth temperature of the micro-organism.
When, for example, thermo stable microorganisms are used, said
temperature preferably is between 40 and 70.degree. C., more
preferably between 50 and 65.degree. C., most preferably between 60
and 64.degree. C. Optionally said reaction can be carried out with
the aid of a thermostatic device. Alternatively, the time required
for growth of the micro-organism is equal to the time that is
required for a calibration sample without any analyte to induce a
change in the indicator.
[0033] In still another embodiment of the second aspect of the
invention, nutrients are added as a separate source, e.g. as a
tablet, disc or a paper filter. Also other compounds such as the
indicator(s), microorganism, stabilizers and/or antifolates may be
added as a separate source, optionally incorporated in the nutrient
medium.
[0034] In yet another embodiment of the second aspect of the
invention, there is provided a method for determining the presence
or absence of an antibiotic in a fluid sample whereby the ratio of
the fluid sample to test medium exceeds 2:3 (0.68:1) (v/v).
Preferably, said ratio is at least 20:27 (0.74:1) (v/v), more
preferably said ratio is at least 25:27 (0.93:1) (v/v); most
preferably said ratio is at least 2:1 (v/v). It has been found that
there is no technical reason for an upper limit to the amount of
fluid sample. In practice this volume should not exceed the maximum
content of the container that holds the test medium. For example,
in a 2 ml container having 0.2 ml test medium, no more than 1.8 ml
of fluid sample should be added. In practice, containers for
performing the method of the present invention have a volume that
rarely exceeds 50 ml and hence the amount of fluid sample to be
added shall not exceed 50 ml, preferably 10 ml, more preferably 5
ml, still more preferably 2 ml, most preferably 1 ml. Thus, in
general, the upper limit of the ratio of the volume of fluid sample
to the volume of test medium is 250:1 (v/v), preferably 50:1 (v/v),
more preferably 25:1 (v/v), still more preferably 10:1 (v/v), most
preferably 5:1 (v/v). Preferably, the volume of fluid sample is
greater than the volume of test medium.
[0035] The result of the method of the present invention is
determined by the observation of the presence or absence of a
change of the indicator or indicators used. When, for example such
a change is a color change, said color change may be observed
visually. However in one embodiment of the invention said color
change is determined using an arrangement that generates digital
image data or an arrangement that generates analog image data and
converts said analog image data into digital image data followed by
interpretation of said digital image data by a computer processor.
Such an arrangement, which may for instance be a sample-reading
device such as a scanner coupled to a personal computer, is
described in International Patent Application WO 03/033728,
incorporated by reference, and briefly summarized below.
[0036] The arrangement can be suitably used for instance for
detecting residues of antibiotics in milk. With this arrangement it
is possible to scan the bottom side of each of the samples in a
test plate. The color and the brightness of the reflected light are
registered in three variables, each describing one color component,
for instance the so-called L*a*b* model. In the L*a*b* model, the
color spectrum is divided in a two-dimensional matrix. The position
of a color in this matrix is registered by means of the two
variables "a" and "b". The variable L indicates the intensity (for
instance, from light blue to dark-blue). It is possible to make a
criterion comprising the a-value, b-value and L-value to make a
composite function as follows: Z=w.sub.LL+w.sub.aa+w.sub.bb
[0037] where w.sub.L, w.sub.a and w.sub.b are weighting factors for
the L-value, a-value and b-value, respectively. The values of these
weighting factors can be calculated by means of "discriminent
analysis", such that the group means show a maximum distance in
relation to the spreading. By combining two or more of the color
components in the L*a*b* model in a predetermined manner that
depends on the type of residue and the sample, an accurate
detection is possible. In practice, a certain value of Z at which a
test should switch between positive and negative result is
experimentally predetermined.
[0038] In a third aspect of the invention there is provided a kit
for carrying out the method of the second aspect of the present
invention. Such a kit comprises one or more containers filled with
test medium as described in the first aspect of the invention and
optionally a sampling device. The containers may be test tubes of
any shape and size and from any material available, provided that
observation of indicator changes is possible. Also, the containers
may be wells such as those incorporated in micro-titer plates.
[0039] Said sampling device is a device with the aid of which fluid
can be added to said test medium. Preferably, such a device is a
container, optionally with volume markings. More preferably, such a
device is a syringe, a pipette or an automated pipetting system.
Such a syringe or pipette may be designed in such a fashion that
with only one mode of operation a predetermined volume can be
withdrawn from the fluid to be analyzed. Optionally, systems known
in the art with which more than one syringe or pipette can be
operated with one single handling may be applied. It is the object
of the second aspect of the present invention to provide a kit that
allows for simple addition of the amounts of fluid to be added
according the first aspect of the invention. Optionally, said kit
comprises means for sealing of said containers filled with test
medium during incubation and/or an insert with instructions for use
and/or a means for setting the time needed for incubation.
[0040] In one embodiment of the third aspect of the invention, said
kit comprises nutrients. Preferably said nutrients are contained
within a medium such as a tablet, disc or a paper filter. The
advantages of providing nutrients contained within a medium are
that the user can easily add them to the test medium and that the
amounts can be predetermined so as to avoid errors in dosing the
required amounts. Also other compounds such as the indicator(s),
stabilizers and/or antifolates may be added as a separate source,
optionally incorporated in the nutrient medium.
[0041] In another embodiment of the third aspect of the present
invention, said kit comprises a thermostatic device, With the aid
of which test samples can be kept at a pre-set temperature, such as
the temperature at which the microorganism shows sufficient growth.
Preferably, said thermostatic device is designed in such a fashion
that it can hold said containers filled with test medium.
Optionally the thermostatic device is coupled to a means for
setting the time needed for incubation such that heating and/or
cooling is stopped after lapse of a pre-set period.
[0042] In yet another embodiment of the third aspect of the
invention, said kit comprises a data carrier loaded with a computer
program suitable for instructing a computer to analyze digital data
obtained from a sample-reading device. Said data carrier may be any
carrier suitable for storing digital information such as a CD-ROM,
a diskette, a DVD, a memory stick, a magnetic tape or the like.
Advantageously, said data carrier loaded with a computer program
provides for easy access to the latest available computer programs
suitable for use in the method of the present invention.
[0043] In a fourth aspect of the present invention there is
provided the use of a compound having the general formula (I) to
improve the sensitivity for an antibiotic in a microbial inhibition
test.
EXAMPLES
Example 1
Comparison of Bromothymol Blue and Bromocresol Purple in Penicillin
G Determination
[0044] Commercially, available Delvotest.RTM. MCS, prepared
according to the methods described in EP 0005891, was adapted by
the replacement of the indicator Bromocresol Purple by Bromothymol
Blue (160 mg.l.sup.-1). The sensitivity for penicillin G was
determined by investigating samples containing different
concentrations of penicillin G in different test systems. The
results are summarized in the Table below (sensitivity values in
ppb). TABLE-US-00001 Bromocresol Purple Bromothymol Blue Penicillin
G.sup.1 4 2 Penicillin G.sup.2 3 2 .sup.1Penicillin G
concentrations of 0, 2, 4 and 6 ppb were investigated
.sup.2Penicillin G concentrations of 0, 1, 2, 3 and 4 ppb were
investigated
Example 2
[0045] Comparison of Bromothymol Blue and Bromocresol Purple in
Determination of Various Antibiotics
[0046] Commercially, available Delvotest.RTM. MCS, prepared
according to the methods described in EP 0005891, was adapted by
the replacement of the indicator Bromocresol Purple by Bromothymol
Blue ('160 mg.l.sup.-1). The sensitivity for different antibiotics
was determined by investigating two sets of six experiments using
either a plate test or a tube test system. The sensitivity was
determined by reading the test at the moment at which an
antibiotic-free control changed color. From the results as
summarized in the Table below (sensitivity values in ppb), it can
be seen that Bromothymol Blue gives superior sensitivities in
comparison with Bromocresol purple (BP) for all antibiotics
investigated with the exception of sulfadiazine in which case the
results for the two indicators are identical. TABLE-US-00002
Bromothymol Blue Type of test BP.sup.1 Plate test Tube test
Experiment # 0 1 2 3 4 5 6 7 8 9 10 11 12 Penicillin G 3 1 2 1 1
1-2 1 2 2 2 2 2 2 Ampicillin 8 2 2 2 2 2 2 2 2 2 2 2 2 Amoxicillin
8 4 2 2 2 2-4 2 4 4 4 4 4 4 Cephapirin 8 2 2 4 6 2 4-6 4 4 4 4 4 4
Cloxacillin 40 10 10 10 10 10 10 20 20 20 20 20 20 Neomycin 600 100
100 100 100 100 100 200 200 200 200 200 200 Sulfadiazine 50 50 50
50 50 50 50 50 50 50 50 50 50 .sup.1BP: Bromocresol Purple
Example 3
Concentration Effect in the Comparison of Bromothymol Blue and
Bromocresol Purple
[0047] Two test systems were prepared. The first system differs
only from a commercially available Delvotest.RTM. MCS in that the
concentration of Bromocresol Purple is 160 mg.l.sup.-1, and the
second series is as the first with Bromocresol Purple replaced with
Bromothymol Blue (160 mg.l.sup.-1). The sensitivity for penicillin
G was determined reading the test at the moment at which an
antibiotic-free control changed color. From the results as shown in
the Table below (sensitivity values in ppb) it can be seen that the
sensitivity of Bromothymol Blue is better than that of Bromocresol
Purple at the same concentrations of indicator. TABLE-US-00003
Bromocresol Purple Bromothymol Blue (160 mg l.sup.-1) (160 mg
l.sup.-1) Penicillin G 3 2
Example 4
Comparison of Bromothymol Blue and Bromocresol Purple in Production
Scale Microbiological Inhibition Test
[0048] Two series of test systems were prepared on production
scale. The first series is a commercially available Delvotest.RTM.
MCS and the second series is as the first with Bromocresol Purple
replaced with Bromothymol Blue (160 mg.l.sup.-1). The sensitivity
for different antibiotics was determined reading the test at the
moment at which an antibiotic-free control changed color. The
results are in the Table below (sensitivity values in ppb).
TABLE-US-00004 Bromocresol Purple Bromothymol Blue Penicillin
G.sup.1 3 2 Penicillin G.sup.2 3 2 Ampicillin.sup.2 6 2
Amoxicillin.sup.2 8 4 Cephapirin.sup.2 8 4 Cloxacillin.sup.2 40 20
Neomycin.sup.2 400 200 .sup.1Batch #1, immediately after production
.sup.2Batch #1, three weeks after production
* * * * *