• Measurement
• Second Stage Amherst Va

Books.google.com.tr - This adaptation of Bentley's Textbook of Pharmaceutics follows the same goals as those of the previous edition, albeit in a new look. The content of the old edition has been updated and expanded and several new chapters, viz. Complexations, Stability Testing as per ICH Guidelines, Parenteral Formulations. Bentley's Textbook of Pharmaceutics - E-Book.

A procedure for identification of bacteria comprises subjecting the bacteria to a combination of tests for determination of 26 bacterial enzymes, permitting rapid identification by use of tests generally adapted for determination of constitutive enzymes. The procedure generally permits more rapid identification. Patent EP0018825A1 - A process for the identification of bacteria and a kit of reagents for use in this process.

A procedure for identification of bacteria comprises subjecting the bacteria to a combination of tests for determination of 26 bacterial enzymes, permitting rapid identification by use of tests generally adapted for determination of constitutive enzymes. The procedure generally permits more rapid identification than previous bacterial identification procedures and is universally applicable to a very wide range of bacteria including most bacteria which are commonly encountered clinically. In a particular embodiment the invention includes a procedure for rapid differentation of the bacterial groups Escherichia, Klebsiella spp., Proteus and Pseudomonas spp., comprising subjecting the bacteria to a combination of tests for determination of bacterial acid phosphatase. Beta-galactosidase, glutamate decarboxylase, phenylalanine deaminase, cytochrome oxidase, diacetyl producing enzymes and urease.

Additionally the invention includes kits for carrying out the procedures of the invention. Typically comprising separate specific substrates for each of the enzymes which it is desired to determine, the kit being preferably in the form of a test card or other suitable apparatus comprising a plurality of wells or compartments which separately contain the enzyme substrates. Customary bacterial identification procedures rely-upon a series of characterisation tests on the basis of which the unknown organism is assigned to a defined group of bacteria. These tests'include tests in which bacteria are classified by their ability to metabolise various substrates, metabolism being determined by resultant changes in the substrate media, e.g. PH changes, which may be detected by use of coloured indicators.

For such metabolic tests it is necessary, however, to grow the bacteria, usually in complete growth media, and this requires considerable expenditure of time so that it is rarely possible to identify the bacteria on the same day as the specimen arrives in the laboratory. Often, if conventional bacteria identification procedures are used, definitive identification is not possible until 48 - 72 hours after receipt of the specimen.

In the meantime, the treatment prescribed by the clinician can at best be only a guess and may be initially incorrect with the consequence that the infection persists and the condition of the patient deteriorates. There is a pressing need, therefore, for increases in the speed of identification of bacteria which cause infections so that the correct treatment, e.g.

Antibiotic, may be prescribed without undesirable delay. Very recently bacterial identification procedures have been proposed which rely only to a limited extent upon bacterial growth for identification, but depend more upon determination of enzymes which are initially present in the bacteria or are induced after a relatively short period of time, e.g. A few hours, and these tests enable bacterial identification to be made earlier than previously, sometimes on the same day as receipt of the sample.

Each of these bacterial identification procedures, however, generally permit identification only of bacteria within certain limited groups and it is thus necessary to carry out a preliminary identification, usually requiring bacterial growth, to select the particular identification procedure which should be used. The procedure of the invention may be used for identification of a very wide range of bacteria, including most of those bacteria which are customarily encountered clinically. In particular, the procedure may be used to identify the commonly encountered bacterial groups: Aeromonas, Acinetobacter, Alcaligenes, Bordatella, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium, Hafnia, Klebsiella, Providencia, Proteu.s, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus and Streptococcus. For example the procedure of the invention has been used to identify the following bacterial species:. Aeromonas hydrophyla. Aeromonas formicans. Acinetobacter calcoaceticus var.

Anitratus. Acinetobacter calcoaceticus var. The procedure of the invention permits very rapid identification of bacteria, the enzyme determination tests used generally requiring only a relatively short period of incubation, e.g. From about 10 minutes up to about 2 hours, usually from about 30 to about 90, minutes at about 40 C, to give sufficient product for detection e.g. By spectroscopic measurements. It is believed that generally the enzymes which are determined during the procedure of the invention are constitutive enzymes for the bacteria concerned, though in some cases the enzymes may be induced enzymes the rates of bacterial synthesis of which are very fast. Thus the tests used are generally suitably adapted for determination of constitutive enzymes.

Without prejudice to the foregoing, however, determination of tryptophanase and deoxyribonuclease appears to require bacterial growth usually requiring incubation for a period of 2 - 2t hours. The product of the enzyme interaction may be detected by spectrometric measurements including fluorimetry or colorimetry. For example, the specific enzyme substrate may comprise an umbelliferyl derivative which on interaction with the enzyme gives rise to umbelliferone which is monitored fluorimetrically, or the substrate may comprise a nitrophenyl, nitroaniline or similar type of derivative which on interaction with the enzyme gives rise to a coloured product which is monitored colorimetrically. An example of an enzyme which may be determined by spectro- metric measurement of the direct product of enzyme interaction with a substrate is cytochrome oxidase; for instance, by interaction of sample with tetraphenyl tetramethyl-p-phenylene-diamine (TMPD) an indicator which is oxidised by cytochrome oxidase to give a purple colouration.

Also acid phosphatases, beta- galactosidases and phenylalanine deaminases may be determined spectrometrically; for instance, using nitrophenyl derivatives, though it is normally necessary to treat the enzyme-substrate mixture with alkali subsequent to incubation so as to develop the nitrophenyl colouration by raising the pH above the pH optimum of the enzyme reaction. Products which require further treatment after enzyme reaction with the substrate before monitoring, may be detected spectrometrically. For example, ammonia releasing enzymes, such as leucine deaminase may be determined by reacting the ammonia produced by the enzyme interaction with a colour producing reagent such as the Nessler reagent, and monitoring the resultant colour by colorimetric measurement. The ammonia released may be measured directly in the enzyme-substrate reaction mixture or may be removed, e.g. By dialysis, from the reaction mixture before assay. The tests used for determination of the enzymes may be varied as desired, for instance to increase the organism specific selectivity of the tests.

Thus, two tests for determination of phosphatase activity are included within the procedure of the invention, one for whole cell acid phosphatase activity and one for acid phosphatase activity in the presence of agents which disrupt the bacterial cell permeability barrier. Any suitable agent may be used, though Cetrimide (Cetyl trimethyl-ammonium bromide) and lysozyme, especially in combination, are particularly preferred. The use of such an agent, for instance, has the effect of selectively decreasing the acid phosphatase activity of Proteus bacteria and increasing that of Klebsiella bacteria. The invention includes kits of reagents for use in the procedure of the invention.

Such kits typically comprise separate specific substrates for each of the enzymes which it is desired to determine in the procedure of the invention. Thus, for example, a basic kit for use in the procedure of the present invention comprises separate specific substrates, for determination of a - z listed previously. Preferably these substrates are such as to give chromogenic products on interaction with corresponding enzymes to advantageously permit colorimetric monitoring. Additionally the kits may also comprise suitable buffer solutions and other reagents, e.g. Colour developing reagents, together with the enzyme substrates. The procedure of the present invention is generally applicable to the identification of bacteria in clinical specimens including urine samples, throat swabs and sputum, wound swabs, stools and blood samples.

Bacteria may be isolated from the specimens prior to identification. For example, bacterial cultures are prepared from the specimens and colonies of the organisms to be identified are harvested from the cultures after a sufficient period of growth e.g. Normally about 18 hours, and made up into a suitable form e.g. Suspension form, for determination by the procedure of the invention. In particularly preferred embodiments, however, it is envisaged that the procedure of the invention will be carried out on samples derived directly from clinical specimens, for instance, on samples derived directly from urine samples, without need for growth of bacteria and isolation of single colonies.

Prior to assay of bacterial enzymes, however, the samples containing bacteria, whether derived directly from clinical specimens or derived as single colonies after bacterial growth, may in some cases be subjected to treatment to disrupt the permeability barrier of the bacteria and release the enzymes for assay. Any suitable treatment may be used to disrupt the bacterial permeability barrier. Generally during determination of cytoplasmic and periplasmic enzymes, such as j3-galactosidases and acid phosphatases, such prior disruption of the bacterial permeability barrier may be desirable, though other enzymes, such as deaminases, which appear to be membrane associated may require the bacterial cells to be kept intact for enzyme activity to be maintained. The enzymes assay tests of the procedure of the invention may be carried out by any suitable method or means, including continuous flow and discrete sample analysis techniques, such as those which are well known in the art.

In one embodiment a discrete analyser, such as the Kem-0-Mat system., is used. In another embodiment the enzymes may be assayed by automated continuous analysis techniques. In such continuous flow analysis methods it may be desirable to include a protein determination in view of the differing protein concentrations of various bacteria, so that the absolute relative enzyme activities of the bacteria may be determined. Also a protein assay may provide a measure of the blank in spectrometric assays for the absorbance due to the concentration of the organisms. The conditions used during enzyme determinations may be varied as desired. For example, in continuous flow analysis techniques the relative organism to reagent concentration may be raised, e.g. A sample to reagent ratio in the range from about 1:3 up to about 3:1, to increase the levels of product formed on interaction of enzyme and substrate and thus permit detection of enzyme at lower bacterial suspension concentrations.

Also, preferably relatively elevated temperatures e.g. Temperatures of about 40 C or more, may be used during incubation of sample and substrate to increase the rate of interaction.

Preferably, using continuous flow techniques, it is possible to achieve bacterial identification at a very early stage, especially within about 1 hour of the sample reaching the laboratory e.g. If apparatus comprising a single channel for each enzyme test is employed. In further preferred embodiments, however, the procedure of the invention may be carried out using a test card or other suitable apparatus comprising a plurality of wells or compartments which separately contain specific enzyme substrates for each of the enzyme tests of the procedure and other reagents, as required, e.g.

Colour developing reagents. In use the sample, usually bacterial suspension, is added to each compartment and the development or absence of a detectable, e.g. Coloured, product after a relatively short incubation period e.g. From about 20 minutes up to about 2 hours in preferred embodiments, indicates the presence or absence of the corresponding enzymes in the bacterial sample. Such apparatus is included within the scope of the invention, and in particularly preferred embodiments may be adapted to handling by automated techniques including automated, preferably computerised, spectro-metric scanning techniques which identify the bacterial species directly from the responses of the enzyme tests. In a particular embodiment the invention also includes a bacterial identification procedure for rapid differentation of the commonly encountered bacterial groups Escherichia, Klebsiella spp, Proteus and Pseudomonas spp, in which a sample comprising bacteria of one of these groups is subjected to a combination of tests for determination of bacterial acid phosphatase, beta-galactosidase, glutamate decarboxylase, phenylalanine deaminase, cytochrome oxidase, diacetyl producing enzymes and urease. This limited combination of seven tests may be used to rapidly differentiate the bacterial groups Escherichia, Klebsiella spp, Proteus and Pseudomonas spp substantially as hereinbefore described with reference to the full identification procedure incorporating the 26 test (a) - (z).

In general the procedures of the invention rely upon determination of the enzyme activity profiles of the bacteria undergoing identification, and in accordance with the invention the combination of enzyme tests chosen, i.e. Tests (a) - (z) or the limited combination of 7 tests, typically gives a unique 'fingerprint' for the bacteria. The unique 'fingerprint' for each species or group of bacteria may be determined with reference to the enzyme activity profiles of previously identified bacteria; for instance, from bacteria obtained from culture collections. Enzyme activity profiles may be determined qualitatively or quantitatively. The use of data-processing techniques may be desirable to facilitate the identification by comparison of enzyme profiles of unknown bacteria with those of previously identified bacteria.

For example, processing of results obtained by discriminant function analysis, e.g. Using the SPSS package (Statistical Package for Social Sciences) has been found to be particularly useful. A stream of organism suspension 1 is mixed with an air segmented buffer stream 2 in the first single mixing coil (SMC) 3, and is then mixed with the substrate stream 4 in the second SMC 5 and incubated for 18 minutes in a glass coil maintained in an oil bath 6 at 40 oC. The reaction is stopped by addition of a strong alkaline solution 7 (1.9M NH 4OH, 0.68M, NaOH, Triton-X-100 0.3 g/1) which also acts as a colour developer for the released p-nitrophenyl molecule.

The stream is then de-bubbled and passed through a 20mm flow-cell in a photometer 8 and the absorbance measured at 405nm for beta-galactosidase and acid phosphatase and at 480 nm for phenylalanine deaminase. The absorbance readings obtained are registered on a recorder 10.

The figures given in the enclosed area 11 in Figure 1 are the flow rates used for the various reagent and reactant streams. With reference to Figure 2 the method used for assay of ammonia-releasing enzymes, i.e. Leucine deaminase and urease, is based on that of Bascomb and Grantham (1975 'Some Methods for Microbiological Assay' ed. Board & Lovelock pp 20-54, Academic Press, N.Y.) but using different sized tubes. Ammonia released from the substrates is assayed by adding the Nessler reagent to ammonia which has been collected by dialysis into a 0.005 MHC1 recipient stream. Attempts to measure the ammonia directly in the organism/substrate stream are not satisfactory due to troublesome base-line drift and non-reproducibility of the standards. Urease activity is determined in the absence of tris maleate or phosphate buffer as their presence caused a noisy base-line, the enzyme being measured in the presence of distilled water only (pH approximately 4.7-5.0).

NH 4Cl solutions are used as standards in both assays. The substrates used are 5mM 1- leucine in 0.5M phosphate borate, pH 8.0 and 100mM urea in fresh distilled water. The Nessler reagent is prepared as described by Bascomb and Grantham in the abovementioned publication and diluted 1:10 in fresh glass distilled water daily. A 20mm flow cell is used in the photometer and absorbance is measured at 420nm. The Voges-Proskauer reaction for the presence of acetoin/ diacetyl is used, in the manifold illustrated in Figure 3, for determination of diacetyl-producing enzymes. The method used is developed from that described by Kamoun et al (Clin.

1972, 18, 355-357). The substrate solution contains 0.3M sodium pyruvate, 0.1M acetate buffer (pH 4.5), 0.1mM thiamine pyrophosphate and creatine 2g/1. The 1-naphthol colour-developing reagent is dissolved (25g/1) in 2M sodium hydroxide. Diacetyl solutions are used as standards. Absorbance is measured at 420nm using a 20mm flow cell.

Glutamate decarboxylase. Glutamate decarboxylase is determined, with reference to Figure 4, by interaction with substrate comprising glutamate to produce C0 2 which is dialysed from the reaction mixture into a buffered cresol red solution where its presence causes a colour change in the indicator. The method used is based on those described by Leclerc (Annls. Pasteur, Paris (1967) 112, 713-731), Moran and Witler (1976 J.

41, 165-167) and Technicon Methodology AA 11-08. Technicon sodium carbonate standards are used. The substrate used comprises 0.1M acetate buffer (pH 3.8), 0.05M sodium glutamate and pyridoxal phosphate 20 mg/l. After incubation of the sample and substrate 0.5M sulphuric acid diluent containing Brij-35 (30% Technicon) 1ml/l is added to raise the pH and assist removal of CO 2 produced by dialysis through a hydrophobic dialysis membrane. The colour reagent used contains 0.4M Tris, ammonia solution 28pl/l, Brij-35 20µl/l and cresol red 20µg/l.

Absorbance is measured at 420nm in a 10mm flow cell. Cytochrome Oxidase and Protein Assays. Voice activated commands keygen for mac. The protein assay used is based on that of Lowry et al (1951, J.

Chem., 193, 265-275) and as described by Bascomb and Grantham in the abovementioned publication, though using smaller tubes. The reagents used are alkaline copper solution prepared daily by mixing two ml sodium potassium tartrate (10.g/1) with 2 ml CuS0 4. 5H 20 (5 g/l) and 46 ml 0.2M NaOH containing 0.37M NaCO 3 (anhydrous), in the order described. The alkaline copper reagent is introduced prior to the first single mixing coil. Folin-ciocalto reagent (BDH) is diluted 1:8 in distilled water daily and introduced prior to the second single mixing coil. The incubation period used is 17 minutes at room temperature and absorption is measured at 660nm in a 10mm flow cell.

Bovine serum albumin solutions are used as standards. A few drops of chloroform are added to the sodium tartrate, carbonate and protein standard solutions to prevent microbial contamination. The enzyme assays described above are carried out in a combined continuous flow analysis system, the flow chart of which is illustrated in Figure 6, comprising three channels which are run simultaneously: one channel (A) for assaying enzymes utilising nitrophenyl derivatives, the second channel (B) for assaying protein and cytochrome oxidase, and the third channel (C) for assaying diacetyl producing, ammonia-producing and CO - producing (glutamate decarboxylase) enzymes. The NH 3 and C0 2 products are dialysed from the reaction mixtures and the Nessler and 1-naphtol colour-developing reagents added where appropriate between the incubation coil and the third SMC. The stream from the single sample probe is divided into three, providing bacterial suspensions to each of the channels A, B and C. The bacterial suspensions are maintained in cups on a revolving sample plate D, while substrates and buffer solutions are provided in continuous streams via the tubes.

All bacterial suspensions are first tested for activity of three enzymes, one in each of the channels A', B and C. When the cycle is complete, the substrate, buffer and sample lines are transferred manually to reagents for the next batch of three tests and the sampling of bacteria is restarted. This process is repeated four times including control runs with distilled water in the substrate lines to obtain the absorbance values of bacterial suspensions in the acid phosphatase, cytochrome oxidase, leucine deaminase and urease assays. Bacterial suspensions each comprising ten colonies in 5 ml saline are prepared in a separate location and are brought to the automated system to ensure that automated identification is carried out without any knowledge of the colonial appearance of the samples. A 0.1 ml aliquot is removed from each suspension and added to 5 ml nutrient broth which was then incubated at 37°C for 2 hours on a Matburn rotary mixer.

These suspensions are used for inoculation of a chosen set of conventional test media and a nutrient agar slope to be kept for further testing. The remaining saline suspension is tested directly by the automated procedure.

Cetrimide and lysozyme are included in the buffer solutions for acid phosphatase and β-galactosidase assays and mixed with the saline organism suspensions for 2i minutes at room temperature prior to addition of the substrate to effect disruption of the bacterial permeability barrier. This treatment halved the absorbance at 340nm of all gram-negative bacteria tested. A total of 96 suspensions of different organisms is tested by the automated method described above in Example 1 and the preceding description except in this case each suspension comprises a single colony of organisms in 1 ml of saline. The automated test procedure adopted is the same as in Example 1 though with fewer control runs and similarly conventional tests are carried out on aliquots of each suspension for the sake of comparison. The results obtained, in terms of the success rate, are given below in Table 3 which also includes results for the success rate of the automated tests of Example 1. As can be seen the agreement rate achieved with the automated system in Example 1 is 98%. The three strains that are not identified include a strain of Klebsiella spp.

Showing only acid phosphatase activity, a strain of E. Coli showing only beta-galactosidase activity, and a strain of Pseudomonas spp. For which the cytochrome oxidase activity results are not available. All three organisms are originally classified as unidentified and on repeat testing are identified correctly.

Measurement

Thus with the choice of 8 enzyme activity tests used together with a protein assay test it is possible to correctly identify all four bacterial groups, namely Escherichia, Klebsiella spp., Proteus spp., Pseudomonas spp., by the automated testing procedure. Culture collection strains were from the National Collection of Type Cultures, the Computer Trial Laboratory at Colindale and the Bacteriology Department, St. Mary's Hospital Medical School; all had been charcterised and identified by conventional methods. Fresh strains were isolated from routine urine specimens and identified using API strip 20E. All strains were cultured overnight at 37°C on MacConkey agar plates, without added sodium chloride (Difco Laboratory and Tissue Culture Services). Bacterial suspensions of 8 colonies in 4ml sterile saline were used for all enzyme assays. The discrete analyser, Kem-0-Mat, (Coulter Electronics) was used to perform the tests listed in Table 5.

The bacterial suspensions were placed in the sample cups, buffers were dispensed by the diluent syringe, substrates and reagents (for developing the colour of the reactant end-products) were dispensed by the reagent syringe. To increase sample throughput and lengthen incubation periods cuvettes containing bacterial suspensions, buffer and substrate were incubated outside the Kem-0-Mat. The cuvette changer was used for simultaneous insertion of all 32 cuvettes and for their removal. Two program cartridges were used to perform each test. Program cartridge 1 was used for distribution of bacterial suspensions, buffer, cofactors and substrate to each cuvette and for reading the initial absorbance of each cuvette. The cuvettes were then removed and placed in a 40 0C incubator.

The diluent and reagent syringes and probes were rinsed and charged with the reagents for the next enzyme test. After incubation the cuvettes of each enzyme test were returned to the analyser. Program cartridge 2 was used for adding the second reagent and for reading the final absorbance. The details of reagent composition are given in Table 5. For all tests 350µl of diluent and 70µl of reagent 1 were dispensed into each cuvette. Bacterial suspensions were dispensed in 50 91 aliquots for the DNAase test and in 25µ 1 aliquots for all other tests.

Minimum incubation periods for tryptophanase and DNAase were 2h, for VP and PNPA 1½h, and for the remaining tests one hour. At the end of the incubation period reagent 2 was added to the cuvettes containing substrate and bacterial suspension in 50µl aliquots for the VP, PNPA, PNPP, PNPP+C+L and PNPG+C+L tests and in 350µl aliquots for the INDOLE test. Final absorbance was read 2 min after addition of the second reagent, except for VP and INDOLE tests which were read after 20 min. Absorbance measurements from the 3 different analytical systems were fed into a computer for calculation of enzyme activity and specific enzyme activity of each organism suspension in each enzyme test. Three hundred and four cultures falling into 35 species were tested by both Kem-0-Mat and continuous flow systems on a total of 26 tests and a protein assay.

Thirty one strains were excluded from the calculations because of missing values, mixed cultures and doubtful identity by conventional testing. The remaining 273 cultures were divided into a known set (Training Set) comprising of between 210 and 221 cultures of mainly culture collection strains; and an unknown set (Test Set) of 52-73 cultures, mainly of strains freshly isolated from cultured urine specimens and tested the day after the urine specimen was received.

Non-Patent Citations Reference 1. CHEMICAL ABSTRACTS Vol. 5, January 30, 1978, ref. 34280y, page 230 Columbus Ohio (US) & EDRO SARAP Res. 1976, 1, Paper 9-73, 29pp E.H. PETERSON: '.

Abstract. 2. CHEMICAL ABSTRACTS, Vol. 17, October 27, 1969, ref.

78067n, page 83 Columbus Ohio (US) & Appl. 1969, 18(2), 156-8 A.L.

BARRY et al.: '. Abstract. 3. CHEMICAL ABSTRACTS, Vol. 17, April 29, 1974, ref. 93098c, page 182 Columbus Ohio (US) & Clin.

1973, 19(11), 1248-9 P.L. WOLF et al.: '. Abstract. 4. CHEMICAL ABSTRACTS, Vol. 3, July 18, 1977, ref. 18619q, page 274 Columbus Ohio (US) & Lab.

Delo 1977, (5), 291-2 N.I. GRIDNEVA et al.: '. Abstract. 5.

CHEMICAL ABSTRACTS, Vol. 23, June 5, 1978, ref. 166658n, page 302 Columbus Ohio (US) & Ann. 1978, 8(2), 111-16 P.M.

TIERNO et al.: '. Abstract. 6. CHEMICAL ABSTRACTS, Vol. 9, August 28, 1978, ref.

Second Stage Amherst Va

72775s, page 206 Columbus Ohio (US) & J. 1978, 105(2), 275-85 J.L. GRAMOLI et al.: '.

Abstract. 7. CHEMICAL ABSTRACTS, Vol. 3, January 15, 1979, ref.

18831q, page 289 Columbus Ohio (US) & J. 1978, 11(3), 225-31 N.J. LEGAKIS et al.: '. Abstract. Referenced by Citing Patent Filing date Publication date Applicant Title. Mar 7, 1984 Oct 17, 1984 E-Y Laboratories, Inc. Colorimetric assay for enzymes, diagnostic article therefor and a method for forming such article.

Jul 17, 1984 Mar 27, 1985 MERCK PATENT GmbH Method for sensitivity testing bacteria. Jul 17, 1984 Mar 26, 1986 Merck Patent Gesellschaft Mit Beschrankter Haftung Method and means for sensitivity testing bacteria. Nov 2, 1984 Jun 19, 1985 Syntex (U.S.A.) Inc. Assay pretreatment method, compositon therefor and agglutination assay kit containing it. Apr 12, 1985 Dec 11, 1985 TERUMO KABUSHIKI KAISHA trading as TERUMO CORPORATION Medium for beta-galactosidase test. Jun 18, 1985 Dec 27, 1985 The Board Of Regents Of The University Of Michigan Diagnostic assay method for the detection of oral pathogenic bacteria mixtures.

May 8, 1986 Dec 10, 1986 Barry James Marshall, M.D. Compositions and methods for the detection of urease for the diagnosis of campylobacter pyloridis infection. May 8, 1986 May 27, 1987 Barry James Marshall, M.D. Compositions and methods for the detection of urease for the diagnosis of campylobacter pyloridis infection.

Mar 16, 1998 Mar 19, 2003 Dade Microsan Inc. Universal test system and methods of use thereof for indentifying multiple families of microorganisms. Mar 16, 1998 Jan 19, 2005 Dade Behring Inc. (a Delaware corporation) Universal test system and methods of use thereof for indentifying multiple families of microorganisms.

Jun 13, 1985 May 31, 1988 Marshall Barry J Compositions and methods for the diagnosis of gastrointestinal disorders involving urease. Apr 21, 1993 Apr 4, 1995 Biocontrol Systems, Inc. Precipitate test for microorganisms. Apr 10, 1997 Mar 30, 1999 Dade Microscan Inc. Universal test systems and methods of use thereof for identifying multiple families of microorganisms May 28, 1998 Feb 11, 2003 Barry J. Marshall Process for preparing a reactive pharmaceutical product for the detection of gastrointestinal disorder caused by bacteria in the gastrointestinal superior tract Oct 15, 2001 Feb 14, 2006 Donald J. McMichael Method for detecting Helicobacter pylori.

May 21, 1986 Dec 4, 1986 University Of Wales College Of Medicine Bacterial cell extractant. Mar 16, 1998 Oct 15, 1998 Dade Microscan Inc.

Universal test systems and methods of use thereof for identifying multiple families of microorganisms.