Definitions

• the present invention relates to growing and testing any cell type in a multitest format.
• the multitest format utilizes a gel forming matrix for the rapid screening of clinical and environmental cultures.
• the present invention is suited for the characterization of commonly encountered microorganisms (e.g., E. coli, S. aureus, etc.), as well as commercially and industrially important organisms from various and diverse environments (e.g., the present invention is particularly suited for the growth and characterization of the actinomycetes and fungi).
• the present invention is also particularly suited for analysis of phenotypic differences between strains of organisms, including cultures that have been designated as the same genus and species.
• the present invention provides methods and compositions for the phenotypic analysis and comparison of eukaryotic (e.g., fungal and mammalian), as well as prokaryotic (e.g., eubacterial and archaebacterial) cells.
• eukaryotic e.g., fungal and mammalian
• prokaryotic e.g., eubacterial and archaebacterial
• the present invention relates to growing and testing any cell type in a multitest format.
• the multitest format utilizes a gel-forming matrix for the rapid screening of clinical and environmental cultures.
• the present invention is suited for the characterization of commonly encountered microorganisms (e.g., E. coli, S. aureus, etc.), as well as commercially and industrially important organisms from various and diverse environments.
• the present invention is particularly suited for the growth and characterization of bacteria, as well as the actinomycetes and fungi (e.g., yeasts and molds).
• the present invention provides methods for testing microorganisms comprising the steps of: providing a testing means comprising redox purple and one or more test substrates; introducing microorganisms into the testing means; and detecting the response of the microorganism to the one or more test substrates.
• the testing substrates are selected from the group consisting of carbon sources and antimicrobials.
• the testing means further comprises one or more gel- initiating agents.
• the gel-initiating agent comprises cationic salts.
• the testing means further comprises one or more gelling agents.
• the microorganisms are in an aqueous suspension.
• the aqueous suspension further comprises one or more gelling agents.
• gelling agents include, but not limited to agar, gellan gum (e.g., GelriteTM and PhytagelTM), carrageenan, and alginic acid.
• the microorganisms are bacteria, while in another embodiment, the microorganisms are fungi. It is also contemplated that the methods of the present invention will be used with members of the Order Actinomycetales.
• the testing means comprises at least one microplate (e.g., MicroPlateTM microtiter plates, available from Biolog), while in an alternative embodiment, the testing means comprises at least one miniaturized testing plates or cards (e.g., MicroCardTM test cards, available from Biolog). In yet another embodiment, the testing means comprises at least one petri plate.
• microplate e.g., MicroPlateTM microtiter plates, available from Biolog
• miniaturized testing plates or cards e.g., MicroCardTM test cards, available from Biolog
• the testing means comprises at least one petri plate.
• the present invention also provides a kit, comprising redox purple and one or more test substrates.
• the test substrates are selected from the group consisting of carbon sources and antimicrobials.
• the kit further comprises one or more gel-initiating agents.
• the gel initiating agent comprises cationic salts.
• the kit further comprises one or more gelling agents.
• the gelling agent is selected from the group consisting of agar, gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the kit further comprises a suspension of microorganisms.
• the kit further comprises a testing means. It is contemplated that various testing means formats will be used successfully in various embodiments of the kits of the present invention, including microplates (e.g., MicroPlateTM microtiter plates),
• testing plates or cards e.g., MicroCardTM miniaturized test cards
• petri plates e.g., petri plates, and any other suitable support in which the testing reaction can occur.
• the present invention provides a kit, comprising redox purple and one or more gelling agents. It is contemplated that various gelling agents will be used successfully in the various embodiments of the kits of the present invention, including but not limited to agar, gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the kit further comprises one or more gel-initiating agents.
• the gel-initiating agent comprises cationic salts.
• the kit further comprises a suspension of microorganisms.
• the kit further comprises one or more test substrates. It is contemplated that the test substrates included in the kit of the present invention be selected from the group consisting of carbon sources and antimicrobials.
• the kit further comprises a testing means. It is contemplated that various testing means formats will be used successfully in various embodiments of the kits of the present invention, including microplates (e.g., MicroPlateTM microtiter plates), miniaturized testing plates or cards (e.g., MicroCardTM miniaturized test cards), petri plates, and any other suitable support in which the testing reaction can occur.
• the present invention describes test media and methods for the growth, isolation, and presumptive identification of microbial organisms.
• the present invention contemplates compounds and formulations, as well as methods particularly suited for the detection and presumptive identification of various diverse organisms.
• the present invention combines a gel-forming suspension with microorganisms that are already in the form of a pure culture. This is in contrast to the traditional pour plate method which involves heated agar and a sample that contains a mixed culture (see e.g., J.G. Black, Microbiology: Principles and Applications. 2d ed., Prentice Hall, Englewood Cliffs, NJ, p. 153 [1993]; and American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 16th ed., APHA, Washington, D.C., pp. 864-866 [1985]).
• colloidal gel-forming substances are used at low concentrations, forming soft gels or viscous colloidal suspensions that do not need to, and in fact work best, when not completely solidified into a rigid gel.
• the present invention provides a method for introducing microorganisms into a testing device, comprising the steps of providing a testing device comprising a plurality of testing wells or compartments, wherein each compartment contains one or more gel-initiating agents; preparing a suspension comprising a pure culture of microorganisms and an aqueous solution containing a gelling agent, under conditions such that the suspension remains ungelled; and introducing the suspension into the testing device under conditions such that the suspension contacts the gel-initiating agents present in the compartments and results in the production of a gel or colloidal matrix.
• the present invention provides a method for testing microorganisms comprising the steps of providing a testing device comprising a plurality of testing compartments, wherein the compartments contain a testing substrate and one or more gel-initiating agents; preparing a suspension comprising a pure culture of microorganisms and an aqueous solution comprising a gelling agent under conditions such that the suspension remains ungelled; introducing the suspension into the compartments of the testing device under conditions such that the suspension forms a gel matrix within the compartment; and detecting the response of the microorganisms to the testing substrate.
• the testing device is a microplate (e.g., MicroPlateTM microtiter plates).
• the microorganisms tested in this method will be bacteria, including members of the Order Actinomycetales, or fungi (e.g., yeasts and molds).
• the gelling agent is selected from the group consisting of gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the gelling agent is carrageenan which contains predominantly iota-carrageenan.
• the gel-initiating agent comprises cationic salts.
• the testing substrates are selected from the group consisting of carbon sources and antimicrobials.
• the method further includes a colorimetric indicator, wherein the colorimetric indicator is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the present invention encompasses a kit for growth and identification of microorganisms comprising: a testing device comprising a plurality of testing compartments containing one or more gel-initiating agents; and an aqueous solution comprising a gelling agent.
• the testing compartments further contain testing substrates, such as carbon sources and antimicrobials.
• the gel-initiating agent comprises cationic salts.
• the testing device is a microplate (e.g., MicroPlateTM microtiter plates).
• the kit contains a gelling agent that is selected from the group consisting of gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the gelling agent is a carrageenan which predominantly contains the iota form of carrageenan.
• the gel-initiating agent comprises cationic salts.
• kit of the present invention will be used with microorganisms such as bacteria, including members of the Order Actinomycetales, as well as fungi (e.g., yeasts and molds).
• microorganisms such as bacteria, including members of the Order Actinomycetales, as well as fungi (e.g., yeasts and molds).
• kit will also include a colorimetric indicator selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the present invention comprises a kit for characterizing and identifying microorganisms comprising: a testing device containing a plurality of compartments, wherein the compartments contain one or more gel-initiating agents and one or more testing substrates, wherein the testing substrates are selected from the group consisting of antimicrobials and carbon sources and an aqueous suspension comprising a gelling agent.
• the testing device is a microplate (e.g.,
• the testing device is a miniaturized testing plate or card (e.g., MicroCardTM miniaturized testing cards).
• the kit contains a gelling agent that is selected from the group consisting of gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the gelling agent is a carrageenan which predominantly contains the iota form of carrageenan.
• the gel-initiating agent comprises cationic salts. It is contemplated that the kit of the present invention will be used with microorganisms such as bacteria, including members of the Order Actinomycetales, as well as fungi (e.g., yeasts and molds).
• kit will include a colorimetric indicator selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the present invention also provides methods for comparing the function of a gene in at least two cell preparations, comprising the steps of: providing a testing device comprising a plurality of testing wells, wherein the wells contain a testing substrate and one or more gel-initiating agents; preparing a first suspension comprising a first cell preparation, in an aqueous solution comprising a gelling agent, and a second suspension comprising a second cell preparation in an aqueous solution comprising a gelling agent, under conditions such that the first and second suspensions remain ungelled; introducing the first and second suspension into the wells of the testing device under conditions such that the first and second suspensions form a gel matrix within the wells, such that the first and second cell preparations are within the gel matrix; detecting the response of the first and second cell preparations to the testing substrate; and comparing the response of the first and second cell preparations.
• the first and second cell preparations comprise microorganisms selected from the group consisting of bacteria and fungi.
• the first and second cell preparations contain cells of the same genus and species, while in still other embodiments, the first and second cell preparations contain cells that differ in one or more genes.
• the gelling agent is selected from the group consisting of gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the testing substrates are selected from the group consisting of carbon sources, nitrogen sources, sulfur sources, phosphorus sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials.
• the gel-initiating agent comprises cationic salts.
• the methods further comprise a colorimetric indicator.
• the colorimetric indicator is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the oxidation-reduction indicator is tetrazolium violet, while in other embodiments, the oxidation-reduction indicator is redox purple.
• the testing device is at least one microplate (e.g., MicroPlateTM microtiter plates), while in other preferred embodiments, the testing device is at least one miniaturized testing plate or card (e.g., MicroCardTM testing cards).
• the response is a kinetic response.
• kits suitable for determining the phenotype of at least two organisms comprising: a testing device containing a plurality of wells, wherein the wells contain one or more gel-initiating agents and one or more testing substrates; a first aqueous suspension comprising a gelling agent; and a second aqueous suspension comprising a gelling agent.
• the testing substrates are selected from the group consisting of carbon sources, nitrogen sources, sulfur sources, phosphorus sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials. Indeed, it is not intended that the present invention be limited to any particular testing substrates, as it is contemplated that any testing substrate suitable for use with the present invention will be utilized.
• the gelling agent is selected from the group consisting of gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the gel initiating agent comprises cationic salts.
• the testing device further comprises a colorimetric indicator selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the oxidation-reduction indicator is tetrazolium violet, while in other embodiments, the oxidation-reduction indicator is redox purple.
• the present invention further provides methods and compositions for extrapolating the functions of genes or genetic sequences in various cell types.
• the present invention provides methods for extrapolating the function of genes or genetic sequences in eukaryotic cells.
• microbial genomes are examined to identify sequences that are homologous to the gene(s) or genetic sequence(s) of interest in the eukaryotic cell. Then, mutations are introduced into the homologous microbial gene. Next, the phenotypes of the wild-type and mutant microbial cells are analyzed and/or compared, as desired.
• the functions of the microbial and eukaryotic genes are compared by utilizing genetic engineering methods to prepare transferable expression vectors (e.g., plasmids, phages, etc.) containing the eukaryotic gene(s) or genetic sequence(s) of interest.
• This expression vector is transferred into and expressed in a microbial host cell.
• the phenotype of the host microbial cell i.e., the cell containing the expression vector
• untransformed microbial cells i.e., control cells comprising the same microbial cell line, but not containing the expression vector
• the vector comprises eukaryotic genes that have been modified (i.e., the genes are modified such that they are not the same as the wild type gene sequences).
• the present invention also provides methods for comparing at least two cell preparations, comprising the steps of: providing a testing device comprising a plurality of testing wells, wherein the wells contain at least one test substrate selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements; preparing a first suspension comprising a first cell preparation in an aqueous solution, and a second suspension comprising a second cell preparation in an aqueous solution; introducing the first and second suspensions into the wells of the testing device; detecting the response of the first and second cell preparations to the testing substrate; and comparing the response of the first and second cell preparations.
• the first and second cell preparations comprise microorganisms selected from the group consisting of bacteria and fungi. In still other embodiments, the first and second cell preparations contain cells of the same genus and species, while in other embodiments, the first and second cell preparations contain cells that differ in one or more genes.
• the testing substrates further comprise substrates selected from the group consisting of carbon sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials.
• the method further comprises a colorimetric indicator.
• the colorimetric indicator is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the oxidation-reduction indicator is tetrazolium violet or redox purple.
• the testing device is at least one microplate (e.g., MicroPlateTM microtiter plates), while in other preferred embodiments the testing device is a miniaturized test plate or card (e.g., MicroCardTM miniaturized testing cards).
• the response is a kinetic response.
• the present invention also provides methods for comparing the function of a gene in at least two cell preparations, comprising the steps of: providing a testing device comprising a plurality of testing wells, wherein the wells contain one or more gel-initiating agents, and at least one testing substrate selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements; preparing a first suspension comprising a first cell preparation, in an aqueous solution comprising a gelling agent, and a second suspension comprising a second cell preparation in an aqueous solution comprising a gelling agent, under conditions such that the first and second suspensions remain ungelled; introducing the first and second suspensions into the wells of the testing device under conditions such that the first and second suspensions form a gel matrix within the wells, such that the first and second cell preparations are within the gel matrix; detecting the response of the first and second cell preparations to the testing substrate; and comparing the response of the first and second cell preparations.
• the first and second cell preparations comprise microorganisms selected from the group consisting of bacteria and fungi, while in other embodiments, the first and second cell preparations contain cells of the same genus and species. In still other embodiments, the first and second cell preparations contain cells that differ in one or more genes.
• the testing substrates further comprise substrates selected from the group consisting of carbon sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials.
• the gelling agent is selected from the group consisting of gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the gel-initiating agent comprises cationic salts.
• the method further comprises a colorimetric indicator.
• the colorimetric indicator is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the oxidation-reduction indicator is tetrazolium violet, while in other preferred embodiments, the oxidation-reduction indicator is redox purple.
• the testing device is at least one microplate (e.g., MicroPlateTM microtiter plates), while in other preferred embodiments the testing device is a miniaturized test plate or card (e.g., MicroCardTM miniaturized testing cards).
• the response is a kinetic response.
• kits for determining the phenotype of at least two organisms comprising: a testing device containing a plurality of wells, wherein the wells contain one or more testing substrates selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements; a first aqueous suspension; and a second aqueous suspension.
• the wells of the testing device further contain one or more gel-initiating agents, the first aqueous suspension further comprises a first gelling agent, and the second aqueous suspension further comprises a second gelling agent.
• the testing substrates further comprise substrates selected from the group consisting of carbon sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials.
• the gelling agent is selected from the group consisting of gellan gum (e.g., GelriteTM and/or PhytagelTM), carrageenan, and alginic acid.
• the gel initiating agent comprises cationic salts.
• the testing device further comprises a colorimetric indicator.
• the colorimetric indicator is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.
• the oxidation-reduction indicator is tetrazolium violet, while in other preferred embodiments, the oxidation- reduction indicator is redox purple.
• the testing device is at least one microplate (e.g., MicroPlateTM microtiter plates), while in other preferred embodiments the testing device is a miniaturized test plate or card (e.g., MicroCardTM miniaturized testing cards).
• the response is a kinetic response.
• Figure 1 is an exploded perspective view of one embodiment of the device of the present invention.
• Figure 2 is a top plan view of the device shown in Figure 1.
• Figure 3 is a cross-sectional view of the device shown in Figure 2 along the lines of 3-3.
• Figure 4 is a bottom plan view of the device shown in Figure 1.
• Figure 5 shows the synthesis pathway of redox purple.
• the present invention is based in part on the discovery that various cells (e.g., microbial strains) can be differentiated based on differential biochemical reactions. Surprisingly, it was determined during the development of the present invention that in some cases, the biochemical reactions work best when the cells are contained within a gel matrix. Thus, the present invention incorporates a multiple test format in a testing device, for presumptive and rapid microbiological screening of various clinical, veterinary, research, industrial and environmental specimens. It is also intended that the present invention will be useful for definitive identification and diagnosis. In preferred embodiments, the present invention is suitable for the comparative phenotype testing of microorganisms and other cells.
• comparative phenotypic testing will find use in functional genomics (i.e., whereby cells and/or microbial strains that differ in a defined set of genetic traits are compared). It is not intended that the invention be limited to a particular genus, species nor group of organisms. Indeed, it is also intended that the present invention will find use with cells of any type, including, but not limited to cells maintained in cell culture, cell lines, etc., including mammalian, plant, and insect cells. The compositions and methods of the present invention are particularly targeted toward some of the most economically important organisms, as well as species of clinical importance.
• the present invention contemplates an indicator plate essentially similar in structure to microtiter plates ("microplates” or “MicroPlatesTM”) which are commonly used in the art and commercially available from numerous scientific supply sources (e.g., Biolog, Fisher, etc.). It is contemplated that the present invention be used with various gelling agents, including but not limited to alginate, carrageenan, and gellan gum (e.g., GelriteTM and/or
• the present invention is a great improvement over standard microtiter plate testing methods in which liquid cultures are used. Unlike the liquid format, the gel matrix of the present invention does not spill from the microtiter plate, even if the plate is completely inverted.
• This safety consideration highlights the suitability of the present invention for use with organisms or other cells that are easily aerosolized. It is also contemplated that the present invention is highly useful in the educational setting, where safety is a primary concern.
• the present invention permits novices to work with bacteria and study their biochemical characteristics with a reduced chance of contamination, as compared to other testing systems.
• the present invention permits novices to work with infected cells (e.g., virally-infected cells harvested from cell cultures), with a reduced chance of contamination.
• the gel matrix system of the present invention also offers other important advantages. For example, over incubation periods of several hours, cells will often sink to the bottom of testing wells and/or attach or clump to other cells, resulting in a non- uniform suspension of cells within the wells. This non-uniformity can result in a non- uniform response of the cells in the well. Clumping artifacts perturb the optical detection of cellular responses.
• the present invention provides methods and compositions which trap the cells in a gel matrix within the wells, the cells are uniformly suspended, and have uniform access to nutrients and other compounds in the wells.
• the present invention serves to make this type of cell testing as reproducible and homogenous as possible.
• the gel matrix of the present invention simulates the natural state of cell growth.
• the gel matrix decreases the diffusion of oxygen to the cells and helps protect them from oxidative damage.
• the range of cell types that can be tested using the methods and compositions of the present invention includes cells that undergo complex forms of differentiation filamentation, sporulation, etc.
• organisms such as the actinomycetes are grown on an agar medium which stimulates the production of aerial conidia. This greatly facilitates the harvesting of organisms for inoculation in the present invention.
• the present invention provides methods and compositions for the testing of fungi (e.g., yeasts and molds), as well as bacteria other than actinomycetes.
• fungi e.g., yeasts and molds
• bacteria other than actinomycetes e.g., yeasts and molds
• these organisms may be grown on any primary isolation or culture medium that is suitable for their growth, although it is preferred that the primary isolation or culture medium used promotes the optimal growth of the organisms.
• the cells are grown in cell culture media (e.g., Eagle's Minimal
• a microplate e.g., a MicroPlateTM microtiter plate
• the gel-forming matrix containing suspended cells is used to inoculate the wells of a microplate or another receptacle.
• the gel-forming matrix is in liquid form, allowing for easy dispensing of the suspension into the compartments.
• These compartments contain dried biochemicals and cations.
• the suspension solidifies to form a soft gel, with the cells evenly distributed throughout. This gel is sufficiently viscous or rigid that it will not fall out of the microplate should the plate be inverted.
• a microcard format is used.
• one embodiment of the device of the present invention comprises a housing (100) with a liquid entry port through which the sample is introduced.
• the housing further contains a channel (110) providing communication to a testing region (120) so that a liquid (not shown) can flow into a plurality of wells or compartments (130).
• the channel (110) is enclosed by the surface of a hydrophobic, gas- venting membrane (140) adapted for forming one surface of the wells (130) and attached to one side of the housing (100).
• the housing (100) can be sealed on its other side by a solid base (150).
• a flexible tape (not shown) may be substituted for the solid base (150) or the solid base (150) may be molded so as to be integral with the housing (100).
• an optional non- venting material such as tape (e.g., polyester tape) (160) can be adhered to the outer surface of the gas- venting membrane (140) to seal it against evaporation of the gel matrix within the device through the gas-venting membrane.
• tape e.g., polyester tape
• the gel-forming matrix with suspended cells is in liquid form. Once the liquid comes into contact with the compounds present in the testing region, a. gel matrix is produced, trapping the suspended cells.
• the present invention is predicated in part on the discovery that various cells or cell types may be identified and differentiated based on differential biochemical reactions. In some cases this is facilitated by the use of gelled media.
• the multiple test medium of the present invention permits presumptive and rapid microbiological screening of various specimens.
• this invention in the form of a kit is suitable for the easy and rapid biochemical testing of various cells, including commonly isolated bacteria, as well as actinomycetes and fungi (i.e., yeasts and molds), in addition to mammalian, insect, and plant cells.
• the present invention provides compositions and methods for the phenotypic analysis of cells.
• Nucleic acid microarrays See e.g., DeRisi et al., Science 278:680-686 [1997]) and gene fusion arrays (See e.g., Glaser, Genet. ⁇ nginer. News, September 15, 1997, at pages 1 and 15), have been developed which can analyze the genotype and gene expression levels of cells. By determining the function of genes, the analysis can go a step further, through the ascertainment of groups of genes which are regulated similarly and which, by implication, are likely to provide related functions in the cell. Though clearly of great value, these technologies still do not indicate the function of the gene, nor do they describe the phenotypic changes that occur in the cell of interest due to the presence of different alleles of that gene.
• the present invention solves these problems, by providing methods and compositions to assay the function of genes directly in cells. Unlike previous methods and compositions, the present invention permits the analysis of thousands of cell phenotypes simultaneously. This cellular approach is nicely complementary to the molecular techniques; it is contemplated that those skilled in the art will utilize the present invention in conjunction with molecular methods to characterize a wide variety of cell types.
• the present invention is intended for use with eukaryotic, as well as prokaryotic cells. Indeed, the ease of finding phenotypic changes has also been demonstrated recently in yeast. As of 1996, of the 6000 genes in the chromosome of S. cerevisiae, less than one half had been known, and 30% could not be assigned a function (Goffeau et al., Science 274:546-567 [1996]). Subsequently, Smith and coworkers developed a method that allowed the introduction of Tyl insertion mutations into 97% of the genes on chromosome V.
• the present invention provides useful, practical, efficient and cost-effective systems, including in one embodiment, an instrument which is used in conjunction with disposable testing panels, to allow the direct and simultaneous analysis of cells and cell lines for thousands of phenotypes.
• the present invention provides methods and compositions for the phenotypic analysis of prokaryotic, as well as eukaryotic cells. Indeed, the present invention is not limited to any particular organism, cell, or testing format.
• the present invention provides one or more testing panels, with each test panel including substrates for 95 phenotypic tests.
• the substrates in the test panel include various carbon sources, while in other embodiments, the test panels include nitrogen, sulfur, phosphorus, and/or other substrates.
• the present invention encompass testing panels with test substrates of any type suitable for the phenotypic testing of various cells.
• the present invention encompasses methods and compositions for the phenotypic testing of E. coli, which is an important "model" organism for many biochemical systems.
• the present invention provides methods and compositions for the testing of isogenic strains with known mutations, in order to identify and characterize unexpected and/or misleading phenotypes.
• the present invention provides methods and compositions to determine the function of genes of interest.
• the present invention provides means to analyze and compare source strains and daughter strains for their phenotypic differences.
• the gene of interest, with an unknown function in the source strain is completely or partially inactivated by creating an altered allele in an isogenic daughter strain. Then, the source strain and the daughter strains are cultured simultaneously under identical conditions and tested in the testing panels described above in order to determine the phenotypic consequences of the alteration of gene function.
• a third cell strain is created.
• This third strain is a revertant of the mutation, derived from the daughter strain. It is intended that this approach will find use in situations in which the cells contain mutations that strongly select for secondary suppressor mutations in the cell line that otherwise can easily go unnoticed. By analyzing a revertant along with the source and daughter strains, one can tell whether any and all phenotypic differences between source and daughter are due to the original mutation or to second site mutations.
• a gene of interest from another cell type is sequenced and its homolog is mutated in E. coli and/or S. cerevisiae.
• a gene of interest from another cell type is cloned and expressed at a physiologically appropriate level in E. coli and or S. cerevisiae.
• the present invention provides methods and compositions for the direct phenotypic analysis of cells which have been mutated. The present invention further contemplates knocking out expression of genes transiently with antisense RNA, and performing phenotypic analysis on cells with a transiently inactivated gene.
• one or more sets of 95 tests will be aimed toward each of the following groups of tests, which encompasses the majority of the catabolic functions of cells, as well as the majority of the biosynthetic functions of cells, and much of the macromolecular machinery of the cell including the ribosome, DNA and RNA polymerases, cellular respiration, transport and detoxification systems, cell wall, and inner and outer membranes: (1) carbon source oxidation tests (including peptide substrates), (2) carbon source fermentation tests, (3) amino and or carboxy peptidase tests, (4) nitrogen source tests, (5) phosphorus source tests, (6) sulfur source tests, (7) auxotrophic tests for all essential metabolites such as amino acids, vitamins, polyamines, fatty acids, and/or nucle
• any number of additional carbon sources of interest will be included in the present invention.
• peptides be included as carbon sources, as during the development of the present invention, it was observed that these carbon sources can provide very useful phenotypic tests.
• E. coli can use D- and L-alanine, D- and L-serine, D- and L-threonine, D- and L-aspartate, L-asparagine, L-glutamine, L-glutamate, and L-proline as carbon sources.
• various chromogenic amino and carboxypeptidase substrates be used in the present invention.
• Carbon source fermentation tests measure acid production from a variety of sugars, and therefore they can provide phenotypic information that is different from carbon source oxidation tests. These tests are performed using a chromogenic pH indicator, including, but not limited to such compounds as bromthymol blue, bromcresol purple, and neutral red.
• a chromogenic pH indicator including, but not limited to such compounds as bromthymol blue, bromcresol purple, and neutral red.
• the present invention also provides methods and compositions to observe utilization of nitrogen, phosphorus, and/or sulfur sources, using an indicator system (e.g., tetrazolium reduction) to demonstrate substrate utilization.
• Various nitrogen sources are contemplated for use in the present invention, including, but not limited to D-alanine, L- alanine, L-arginine, D-asparagine, L-asparagine, D-aspartic acid, L-aspartic acid, L- cysteine, L-cystine, D-glutamic acid, L-glutamic acid, L-glutamine, glycine, L-histidine, L- homoserine, D,L-B-hydroxy-glutamic acid, L-isoleucine, L-leucine, L-phenylalanine, L- proline, D-serine, L-serine, L-tryptophan, L-tyrosine, glutathione (as well as any peptide containing the
• phosphorous sources are contemplated for use in the present invention, including, but not limited to pyrophosphate, trimetaphosphate, 2'-mononucleotides, 3'- mononucleotides, 5'-mononucleotides, 2',3'-cyclic nucleotides, 3',5'-cyclic nucleotides, aryl-phosphates (e.g., p-nitrophenyl phosphate), phosphonates (e.g., aminoethyl phosphonate), sugar phosphates (e.g., glucose- 1 -phosphate), acid phosphates (e.g., 2- phospho-glyceric acid), aldehyde phosphates (e.g., glyceraldehyde-3 phosphate), ⁇ -glycerol phosphate, ⁇ -glycerol phosphate, inositol phosphates (e.g., phytic acid), pho
• sulfur sources are contemplated for use in the present invention, including, but not limited to sulfur, thiosulfate, thiophosphate, metabisulfite, dithionite, tetrathionate, polysufide, cysteine, cystine, cysteic acid, cysteamine, cysteine sulphinic acid, cystathionine, lanthionine, ethionine, methionine, N-acetyl-methionine, N-acetyl-cysteine, glycyl-methionine, glycyl-cysteine, glutathione, L-djenkolic acid, L-2-thiohistidine, S- methyl-cysteine, S-ethyl-cysteine, methionine sulfoxide, methionine sulfone, taurine, thiourea, and thioglycolate.
• Example 18 provides a description of experiments conducted using various sulfur sources
• amino and carboxy peptidases are contemplated for use in the present invention, including, but not limited to dipeptides containing all natural L-amino acids on the amino terminal, and all natural L-amino acids on the carboxy terminal, as well as suitable non-protein occurring amino acids, such as pyroglutamate, ornithine, ⁇ - amino butyrate, D-amino acids, etc.
• the present invention also provides methods and compositions for auxotrophic testing using a minimal medium supplemented with various single nutrients.
• the growth in the well where the organism is capable of using the nutrient results in a color change via tetrazolium reduction.
• mutations that result in auxotrophy cause the strain to fail to grow in all wells except the one containing the necessary nutrient.
• the wells contain more than one nutrient, in order to allow analysis of genes that affect more than one biosynthetic pathways (e.g., isoleucine+valine (ilv), arginine+uracil (car), and purine+pyrimidine+histidine+tryptophan+nicotinamide (prs)).
• Various compounds are contemplated for use in this embodiment of the present invention, including, but not limited to L-amino acids, D-glutamic acid, D-aspartic acid, D-alanine, vitamins, nucleosides, polyamines, and fatty acids.
• a "drop out" medium or substrate is used.
• a complex defined supplement is used and one nutrient is missing in the substrate dispensed in each well (i.e., the medium lacks one nutrient of the substrate complex).
• Example 18 provides a description of experiments conducted to determine the auxotrophic requirements of an organism.
• a minimal medium is used, while in other cases, an enriched, defined medium is preferable.
• the present invention be limited to any particular testing substrates, as it is contemplated that any testing substrate suitable for use with the present invention will be utilized.
• growth in the wells can result in a color change via tetrazolium reduction.
• the optimal concentration for use in testing for sensitivity/resistance is determined for the cell type to be tested.
• sensitivity tests are contemplated, including tests utilizing compounds including, but not limited to oxidizing agents, reducing agents, mutagens, antibiotics, amino acid analogs, sugar analogs, nucleoside and base analogs, dyes, detergents, toxic metals, and toxic organics.
• the present invention also provides methods and compositions for testing growth at extremes of pH and salt, and the compensatory effect of several compatible solutes.
• diauxic testing is performed with a limiting amount of a favored nutrient present in a well.
• the cells need to adapt from a more favored to a less favored nutrient, and the lag and growth kinetics for numerous substrates can be measured quickly and efficiently in a microplate format.
• the present invention be used with various gelling agents, including, but not limited to agar, pectin, carrageenan, alginate, alginic acid, silica, gellans and gum.
• the pectin medium of Roth U.S. Patent Nos. 4,241,186, and 4,282,317; herein incorporated by reference
• pectin is not a colorless compound itself.
• the gellan of Kang et al. U.S. Patent Nos. 4,326,052 and 4,326,053, herein incorporated by reference
• carrageenan is used as the gelling agent.
• carrageenan type II or any carrageenan which contains predominantly the iota form of carrageenan is used.
• the cells to be tested are mixed in a suspension comprising a gelling agent, and then inoculated into a well, compartment, or other receptacle, which contains the biochemical(s) to be tested, along with a gel-initiating agent such as various cations.
• a gel-initiating agent such as various cations.
• the suspension solidifies to form a viscous colloid or gel, with the cells evenly distributed throughout.
• the present invention also contemplates a multitest indicator plate that is generally useful in the phenotypic characterization, as well as identification and antimicrobial sensitivity testing of microorganisms.
• This medium and method are particularly targeted toward some of the most economically important organisms, as well as species of clinical importance. It is not intended that the invention be limited to a particular genus, species nor group of organisms. Indeed, it is contemplated that any cell type (e.g., microorganisms, as well as plant, mammalian, and insect cells) will find use in the present invention.
• the present invention contemplates a testing device that is a microplate similar in structure to commonly used microtiter plates (i.e., "microplates” or “MicroPlatesTM”) commonly used in the art and commercially available from numerous scientific supply sources (e.g., Biolog, Fisher, etc.).
• microtiter plates i.e., "microplates” or “MicroPlatesTM”
• standard 96-well microtiter plates or “microplates”
• microtiter plates with more wells are used (e.g., 384 well and 1536 well microtiter plates or microplates).
• the microtiter plate (or microplate) format is suited for methods for kinetic analysis of substrate utilization by cells.
• a test panel for detailed phenotypic testing of E. coli and S. typhimurium called the ⁇ S MicroPlateTM (Biolog) was used.
• This panel contains 95 carbon sources, which can be utilized by most strains of these species.
• identical cell suspensions of isogenic parental and mutant strains are prepared and pipetted into the 96 wells of a microtiter plate (e.g., a MicroPlateTM). The cells are incubated for approximately 16-24 hours and if a substrate oxidation occurs in a given well, a violet purple color is produced due to coupled reduction of a tetrazolium dye.
• the MicroPlatesTM can be read at frequent time intervals to determine the kinetics of color formation (i.e., carbon source oxidation rates) in each of the 96 wells. For a typical strain, perhaps 80 to 85 wells provide positive reactions and useful data.
• An alternate embodiment of the invention generally relates to a "microcard” (i.e., such as the MicroCardTM developed by Biolog) device for the multiparameter testing of chemical, biochemical, immunological, biomedical, or microbiological samples in liquid or liquid suspension form in a small, closed, easy-to-fill device, and is particular suitable for multiparameter testing and identification of microorganisms. It is not intended that the present invention be limited to a particular sized device. Rather, this definition is intended to encompass any device smaller than the commonly used, 96-well microtiter plates.
• the miniaturized cards e.g., MicroCardTM
• the miniaturized cards is approximately 75 mm in width and 75 mm in length, and approximately 3 mm in depth.
• the present invention contemplates a device comprising: a) a housing; b) a testing region contained within the housing; c) a liquid receiving means on an external surface of the housing; d) a liquid flow-directing means providing liquid communication between the testing region and the liquid receiving means; and e) a gas-venting, liquid barrier in fluidic communication with the testing region.
• a non-venting, sealing tape can be applied to the device to cover the gas- venting, liquid barrier to reduce the evaporation of the liquid from the device.
• the tape can permit the molecular diffusion of oxygen and/or carbon dioxide into or out of the device to maintain the desired chemical or biochemical environment within the device for successful performance of the test.
• the liquid receiving means comprises liquid entry ports
• a similar closing tape can be applied to close the port or ports to prevent spilling and evaporation of the liquid therefrom.
• the visual result that is detected by eye or by instrument can be any optically perceptible change such as a change in turbidity, a change in color, a change in fluorescence, or the emission of light, such as by chemiluminescence, bioluminescence, or by Stokes shift.
• Color indicators may be, but are not limited to, redox indicators (e.g., tetrazolium, resazurin, and/or redox purple), pH indicators, or various dyes and the like.
• redox indicators e.g., tetrazolium, resazurin, and/or redox purple
• Bochner Nature 339:157 (1989); and B.R. Bochner, ASM News 55:536 (1990).
• a generalized indicator useful for practice of the present invention is also described by Bochner and Savageau. See B. Bochner and M. Savageau, Appl. Environ. Microbiol.,
• a cell suspension is prepared and introduced into the testing compartments of the device. Each compartment is prefilled with a different substrate.
• all wells are prefilled with test formula comprising a basal medium that provides nutrients for the cells, a color-change indicator, as well as testing substrate(s) in sufficient concentration to trigger a color response when the testing substrate is utilized by the cell suspension upon inoculation into the wells for testing (i.e., each well contains either the same or a different testing substrate).
• test formula comprising a basal medium that provides nutrients for the cells, a color-change indicator, as well as testing substrate(s) in sufficient concentration to trigger a color response when the testing substrate is utilized by the cell suspension upon inoculation into the wells for testing (i.e., each well contains either the same or a different testing substrate).
• redox purple is used as a redox indicator in the present invention.
• the present invention contemplates microbiological testing based on the redox technology discussed above wherein a sample of a pure culture of microorganism is removed from a culture medium on which it has been grown and suspended at a desired density in saline, water, gel, gelling agent, buffer, or solution (e.g., PPS) . This suspension is then introduced into the compartments of the testing device which have been prefilled with basal medium, indicator, and substrate chemicals.
• PPS solution
• the present invention involves the use of instruments such as the Biolog MicroStationTM, an instrument system that allows the reading of testing panels inoculated with cells, and analyzes the data obtained from the testing panels. This allows the rapid analysis of multiple phenotypic characteristics for many cell types (e.g., microbial strains) in a short time.
• instruments such as the Biolog MicroStationTM, an instrument system that allows the reading of testing panels inoculated with cells, and analyzes the data obtained from the testing panels. This allows the rapid analysis of multiple phenotypic characteristics for many cell types (e.g., microbial strains) in a short time.
• sample and “specimen” in the present specification and claims are used in their broadest sense. On the one hand, they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, as well as solid tissue. These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms.
• body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, as well as solid tissue.
• CSF cerebrospinal fluid
• saliva as well as solid tissue.
• Biological samples may be animal, including human, fluid or tissue, food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste.
• Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable, and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention. Whether biological or environmental, a sample suspected of containing microorganisms may (or may not) first be subjected to an enrichment means to create a "pure culture" of microorganisms.
• enrichment means or “enrichment treatment”
• the present invention contemplates (i) conventional techniques for isolating a particular microorganism of interest away from other microorganisms by means of liquid, solid, semi-solid or any other culture medium and/or technique, and (ii) novel techniques for isolating particular microorganisms away from other microorganisms. It is not intended that the present invention be limited only to one enrichment step or type of enrichment means. For example, it is within the scope of the present invention, following subjecting a sample to a conventional enrichment means, to subject the resultant preparation to further purification such that a pure culture of a strain of a species of interest is produced. This pure culture may then be analyzed by the medium and method of the present invention.
• culture refers to any sample or specimen which is suspected of containing one or more microorganisms or cells.
• the term is used in reference to bacteria and fungi.
• Purure cultures are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to “mixed cultures,” which are cultures in which more than one genus and/or species of microorganism are present.
• organism is used to refer to any species or type of microorganism, including but not limited to bacteria, yeasts and other fungi.
• fungi is used in reference to eukaryotic organisms such as the molds and yeasts, including dimorphic fungi.
• spore refers to any form of reproductive elements produced asexually (e.g., conidia) or sexually by such organisms as bacteria, fungi, algae, protozoa, etc. It is also used in reference to structures within microorganisms such as members of the genus Bacillus, which provide advantages to the individual cells in terms of survival under harsh environmental conditions. It is not intended that the term be limited to any particular type or location of spores, such as “endospores” or “exospores.” Rather, the term is used in the very broadest sense.
• the terms “microbiological media” and “microbiological culture media,” and “media” refer to any substrate for the growth and reproduction of microorganisms. "Media” may be used in reference to solid plated media which support the growth of microorganisms. Also included within this definition are semi-solid and liquid microbial growth systems including those that incorporate living host organisms, as well as any type of media.
• culture media refers to media that are suitable to support the growth of cells in vitro (i.e., cell cultures). It is not intended that the term be limited to any particular cell culture medium. For example, it is intended that the definition encompass outgrowth as well as maintenance media. Indeed, it is intended that the term encompass any culture medium suitable for the growth of the cell cultures of interest.
• basic medium refers to a medium which provides nutrients for the microorganisms or cells, but does not contain sufficient concentrations of carbon compounds to trigger a color response from the indicator.
• cell type refers to any cell, regardless of its source or characteristics.
• cell line refers to cells that are cultured in vitro, including primary cell lines, finite cell lines, continuous cell lines, and transformed cell lines.
• primary cell culture refers to cell cultures that have been directly obtained from animal, plant or insect tissue. These cultures may be derived from adults as well as fetal tissue.
• primary culture refers to cell cultures that are capable of a limited number of population doublings prior to senescence.
• continuous cell lines refer to cell cultures that have undergone a "crisis” phase during which a population of cells in a primary or finite cell line apparently ceases to grow, but yet a population of cells emerges with the general characteristics of a reduced cell size, higher growth rate, higher cloning efficiency, increased tumorigenicity, and a variable chromosomal complement. These cells often result from spontaneous transformation in vitro. These cells have an indefinite lifespan.
• transformed cell lines refers to cell cultures that have been transformed into continuous cell lines with the characteristics as described above.
• Transformed cell lines can be derived directly from tumor tissue and also by in vitro transformation of cells with whole virus (e.g., SV40 or EBV), or DNA fragments derived from a transforming virus using vector systems.
• whole virus e.g., SV40 or EBV
• hybridomas refers to cells produced by fusing two cell types together. Commonly used hybridomas include those created by the fusion of antibody-secreting B cells from an immunized animal, with a malignant myeloma cell line capable of indefinite growth in vitro. These cells are commonly cloned and used to prepare monoclonal antibodies.
• the term "mixed cell culture,” refers to a mixture of two types of cells.
• the cells are cell lines that are not genetically engineered, while in other preferred embodiments the cells are genetically engineered cell lines.
• monolayer As used herein, the terms “monolayer,” “monolayer culture,” and “monolayer cell culture,” refer to cells that have adhered to a substrate and grow in as a layer that is one cell in thickness. Monolayers may be grown in any format, including but not limited to flasks, tubes, coverslips (e.g., shell vials), roller bottles, etc. Cells may also be grown attached to microcarriers, including but not limited to beads.
• Suspension refers to cells that survive and proliferate without being attached to a substrate. Suspension cultures are typically produced using hematopoietic cells, transformed cell lines, and cells from malignant tumors.
• carbon source is used in reference to any compound which may be utilized as a source of carbon for bacterial growth and/or metabolism. Carbon sources may be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, and peptides.
• nitrogen source is used in reference to any compound which may be utilized as a source of nitrogen for bacterial growth and/or metabolism.
• nitrogen sources may be in various forms, such as free nitrogen, as well as compounds which contain nitrogen, including but not limited to amino acids, peptones, vitamins, and nitrogenous salts.
• sulfur source is used in reference to any compound which may be utilized as a source of sulfur for bacterial growth and/or metabolism.
• sulfur sources may be in various forms, such as free sulfur, as well as compounds which contain sulfur.
• phosphorus source is used in reference to any compound which may be utilized as a source of phosphorus for bacterial growth and/or metabolism.
• carbon, nitrogen, and sulfur sources phosphorus sources may be in various forms, such as free phosphorus, as well as compounds which contain phosphorus.
• auxotroph is used in reference to an organism that can be grown only in the presence of nutritional supplements (e.g., growth factors).
• auxotrophs will only grow in the presence of the supplement(s) that is/are necessary for their growth, and will not grow in media that lack the necessary supplement(s).
• antimicrobial is used in reference to any compound which inhibits the growth of, or kills microorganisms. It is intended that the term be used in its broadest sense, and includes, but is not limited to compounds such as antibiotics which are produced naturally or synthetically. It is also intended that the term includes compounds and elements that are useful for inhibiting the growth of, or killing microorganisms.
• testing substrate is used in reference to any nutrient source (e.g., carbon, nitrogen, sulfur, phosphorus sources) that may be utilized to differentiate bacteria based on biochemical characteristics. For example, one bacterial species may utilize one testing substrate that is not utilized by another species. This utilization may then be used to differentiate between these two species. It is contemplated that numerous testing substrates be utilized in combination. Testing substrates may be tested individually (e.g., one substrate per testing well or compartment, or testing area) or in combination (e.g., multiple testing substrates mixed together and provided as a "cocktail"). Following exposure to a testing substrate such as a carbon or nitrogen source (or any other nutrient source), or an antimicrobial, the response of an organism may be detected.
• a testing substrate such as a carbon or nitrogen source (or any other nutrient source), or an antimicrobial
• This detection may be visual (i.e., by eye) or accomplished with the assistance of machine(s) (e.g., the Biolog MicroStation ReaderTM).
• machine(s) e.g., the Biolog MicroStation ReaderTM
• the response of organisms to carbon sources may be detected as turbidity in the suspension due to the utilization of the testing substrate by the organisms.
• growth can be used as an indicator that an organism is not inhibited by certain antimicrobials.
• color is used to indicate the presence or absence of organism growth/metabolism.
• the terms "chromogenic compound” and "chromogenic substrate,” refer to any compound useful in detection systems by their light absorption or emission characteristics.
• chromogenic all enzymatic substrates which produce an end product which is detectable as a color change. This includes, but is not limited to any color, as used in the traditional sense of "colors,” such as indigo, blue, red, yellow, green, orange, brown, etc., as well as fluorochromic or fluorogenic compounds, which produce colors detectable with fluorescence (e.g. , the yellow-green of fluorescein, the red of rhodamine, etc.). It is intended that such other indicators as dyes
• luminogenic compounds be encompassed within this definition.
• pH indicator encompasses all compounds commonly used for detection of pH changes, including, but not limited to phenol red, neutral red, bromthymol blue, bromcresol purple, bromcresol green, bromchlorophenol blue, m-cresol purple, thymol blue, bromcresol purple, xylenol blue, methyl red, methyl orange, and cresol red.
• redox indicator and "oxidation-reduction indicator” encompass all compounds commonly used for detection of oxidation/reduction potentials (i.e., "eH”) including, but not limited to various types or forms of tetrazolium, resazurin, methylene blue, and quinone-imide redox dyes including the compounds known as "methyl purple” and derivatives of methyl purple.
• the quinone- imide redox dye known as methyl purple is referred to herein as "redox purple.”
• redox purple comprises the compound with the chemical structure shown in Figure 5, VI.
• analogous derivatives of the reagent e.g., alkali salts, alkyl O-esters
• modified properties e.g., solubility, cell permeability, toxicity, and/or modified color(s)/absorption wavelengths
• redox purple e.g., salts, etc.
• testing means and “testing device” are used in reference to testing systems in which at least one organism is tested for at least one characteristic, such as utilization of a particular carbon source, nitrogen source, or chromogenic substrate, and/or susceptibility to an antimicrobial agent.
• This definition is intended to encompass any suitable means to contain a reaction mixture, suspension, or test. It is intended that the term encompass microplates, petri plates, microcard devices, or any other supporting structure that is suitable for use.
• a microplate having at least one gel- initiating agent included in each of a plurality of wells or compartments comprises a testing means.
• Other examples of testing means include microplates without gel-initiating means included in the well.
• the definition encompasses the MicroPlateTM microtiter plates for characterization of microorganisms (available from Biolog).
• the definition is also intended to encompass a "microcard” or miniaturized plates or cards which are similar in function, but much smaller than standard microtiter plates (for example, many testing devices can be conveniently held in a user's hand).
• the microcards are the MicroCardTM device described in U.S. Patent Nos. 5,589,350, and 5,800,785, both of which are herein incorporated by reference (available from Biolog). It is not intended that the present invention be limited to a particular size or configuration of testing device or testing means.
• microtiter plates including but not limited to MicroPlatesTM
• miniaturized testing plates e.g., MicroCardTM miniaturized testing cards
• petri plates petri plates with internal dividers used to separate different media placed within the plate, test tubes, as well as many other formats.
• gelling agent is used in a broad generic sense, and includes compounds that are obtained from natural sources, as well as those that are prepared synthetically. As used herein, the term refers to any substance which becomes at least partially solidified when certain conditions are met.
• GelriteTM a gellan which forms a gel upon exposure to divalent cations (e.g., Mg 2+ or Ca 2+ );
• GelriteTM is a gellan gum, produced by deacetylating a natural polysaccharide produced by Pseudomonas elodea, and is described by Kang et al. (U.S. Patent Nos.
• gelling agents obtained from natural sources, including protein-based as well as carbohydrate-based gelling agents.
• bacteriological agar a polysaccharide complex extracted from kelp.
• gelatins e.g., water-soluble mixtures of high molecular weight proteins obtained from collagen
• pectin e.g., polysaccharides obtained from plants
• carrageenans and alginic acids e.g., polysaccharides obtained from seaweed
• gums e.g., mucilaginous excretions from some plants and bacteria.
• gelling agents used in the present invention may be obtained commercially from a supply company, such as Difco, BBL, Oxoid, Marcor, Sigma, or any other source.
• gelling agent be limited to compounds which result in the formation of a hard gel substance.
• a spectrum is contemplated, ranging from merely a more thickened or viscous colloidal suspension to one that is a firm gel. It is also not intended that the present invention be limited to the time it takes for the suspension to gel.
• the present invention provides a gelling agent suitable for production of a matrix in which organisms may grow (i.e., a "gel matrix").
• the gel matrix of the present invention is a colloidal-type suspension of organisms produced when organisms are mixed with an aqueous solution containing a gelling agent, and this suspension is exposed to a gel-initiating agent. It is intended that this colloidal- type gel suspension be a continuous matrix medium throughout which organisms may be evenly dispersed without settling out of the matrix due to the influence of gravity.
• the gel matrix must support the growth of organisms within, under, and on top of the gel suspension.
• gel-initiating agent refers to any compound or element which results in the formation of a gel matrix, following exposure of a gelling agent to certain conditions or reagents. It is intended that "gel-initiating agent” encompass such reagents as cations (e.g., Ca 2+ , Mg 2+ , and K + ). Until the gelling agent contacts at least one gel-initiating agent, any suspension containing the gelling agent remains "ungelled” (i.e., there is no thickening, increased viscosity, nor hardening of the suspension). After contact, the suspension will become more viscous and may or may not form a rigid gel (i.e., contact will produce "gelling").
• cations e.g., Ca 2+ , Mg 2+ , and K +
• inoculating suspension or “inoculant” is used in reference to a suspension which may be inoculated with organisms to be tested. It is not intended that the term “inoculating suspension” be limited to a particular fluid or liquid substance.
• inoculating suspensions may be comprised of water, saline, or an aqueous solution which includes at least one gelling agent.
• an inoculating suspension may include a component to which water, saline or any aqueous material is added. It is contemplated in one embodiment, that the component comprises at least one component useful for the intended microorganism. It is not intended that the present invention be limited to a particular component.
• kit is used in reference to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as carbon sources, nitrogen sources, chromogenic substrates, antimicrobials, diluents and other aqueous solutions, as well as specialized microplates (e.g., GN, GP, ES, YT, SF-N, SF-P, and other MicroPlatesTM, obtained from Biolog), inoculants, miniaturized testing cards
• the present invention contemplates other reagents useful for the growth, identification and/or determination of the antimicrobial susceptibility of microorganisms.
• the kit may include reagents for detecting the growth of microorganisms following inoculation of kit components (e.g., tetrazolium or resazurin included in some embodiments of the present invention). It is not intended that the term "kit” be limited to a particular combination of reagents and/or other materials.
• the present invention involves inoculation of a testing plate in which the organisms are suspended within a gel-forming matrix.
• primary isolation refers to the process of culturing organisms directly from a sample.
• primary isolation involves such processes as inoculating an agar plate from a culture swab, urine sample, environmental sample, etc.
• Primary isolation may be accomplished using solid or semi-solid agar media, or in liquid.
• isolation refers to any cultivation of organisms, whether it be primary isolation or any subsequent cultivation, including “passage” or “transfer” of stock cultures of organisms for maintenance and/or use.
• Presumptive diagnosis refers to a preliminary diagnosis which gives some guidance to the treating physician as to the etiologic organism involved in the patient's disease. Presumptive diagnoses are often based on “presumptive identifications,” which as used herein refer to the preliminary identification of a microorganism based on observation such as colony characteristics, growth on primary isolation media, gram stain results, etc.
• the term “definitive diagnosis” is used to refer to a final diagnosis in which the etiologic agent of the patient's disease has been identified.
• the term “definitive, identification” is used in reference to the final identification of an organism to the genus and/or species level.
• KS Oxoid
• Basingstoke, England BBL (Becton Dickinson Microbiology Systems, Cockeysville, MD); DIFCO (Difco Laboratories, Detroit, MI, now part of Becton-Dickinson); Acumedia (Acumedia, Baltimore, MD); U.S. Biochemical (U.S. Biochemical Corp., Cleveland, OH); Fisher (Fisher Scientific, Pittsburgh, PA); Sigma (Sigma Chemical Co., St. Louis, MO.); Biolog (Biolog, Inc., Hayward, CA); ATCC
• Tables list the principal bacterial strains used in the following Examples, with Table 2 listing the various actinomycetes, and Table 3 listing other species of microorganisms.
• Sporulation Agar also known as m-Sporulation Agar
• m-Sporulation Agar comprises agar (15 g/1), glucose (10 g/1), tryptose (2 g/1), yeast extract (1 g/1), beef extract (1 g/1), and FeSO 4 • 7H 2 O (1 ⁇ g/1), pH 7.2 ⁇ 0.2 at 25°C.
• These ingredients are added to 1 liter of distilled/deionized water, and mixed thoroughly with heat to boiling. After the mixture has dissolved, it is autoclaved at 15 psi (121 °C) for 15 minutes, and dispensed into plates.
• YEMEWG Agar comprises Bacto yeast extract (4 g/1; Difco), and Bacto-malt extract (10 g/1; Difco). These ingredients are added to 1 liter of distilled/deionized water and mixed thoroughly. The pH is adjusted to 7.3, and agar (20 g/1) is added to the mixture. The mixture is then autoclaved at 121°C for 15-20 minutes, and dispensed into
• YEMEWG was used because preliminary studies indicated that, while glucose-containing YEME agar was adequate for growth of the Streptomyces species, genera such as Nocardiopsis and Actinoplanes grew better when glucose was omitted from the medium recipe. Because of the interest in obtaining spores, media that encourage sporulation were tried. For example, YEMEWG was found to be particularly useful, as this medium gave satisfactory growth and sporulation of most strains tested within 2-4 days of incubation at 26°C.
• Example 2 a method more optimal for preparation of a homogeneous inoculum was determined. For example, it was found that an easy and reproducible method was to grow the organisms as described in Example 1 on YEMEWG- prepared with 25 g/1 agar, or other suitable agar medium. A low density inoculum (i.e., 0.01 to 0.1 OD 590 ) was then prepared by moistening a cotton swab and rubbing it across the top of the colonies to harvest mycelia and spores. It was determined that sterilized water and 0.85% sterile saline worked reasonably well as a suspension medium for all strains. However, some strains exhibited a preference for one or the other.
• a low density inoculum i.e. 0.01 to 0.1 OD 590
• Streptomyces coeruleoribidus, S. hygroscopicus, and S. ⁇ lbidofl ⁇ vus produced an average of ten additional positive reactions when water was used as the suspension medium, whereas thirteen additional positive reactions were observed for S. l ⁇ vendul ⁇ e when saline was used as the suspension medium. The majority of the Actinomycetes performed better when water was used. Therefore, water was used routinely to prepare the suspensions.
• Example 2 The inocula prepared as described in Example 2 were used to inoculate various Biolog MicroPlatesTM , including the commercially available GN, GP, and YT MicroPlatesTM . A few strains worked well upon inoculation into the GN or GP
• MicroPlatesTM e.g., S. lavendulae.
• strains e.g., A. ferruginea, and N dessertville ⁇
• positive reactions were observed in all of the test wells for some organisms (e.g., S. hirsuta), indicating that there was a problem with false positive results.
• Much improved results were obtained when the wells located in the bottom five rows of the YT MicroPlateTM were used. It was thought that this observation was due to the absence of tetrazolium in these wells, as the tetrazolium present in the other wells appeared to inhibit the growth of the organisms.
• MicroPlateTM without tetrazolium were then tested. These plates were inoculated with water or saline suspensions of various actinomycetes, and incubated at 26°C for 1 -4 days. Increased turbidity (i.e., growth of the organisms) was readable visually, or with a microplate reader (e.g., a Biolog MicroStation ReaderTM, commercially available from
• Example 3 Although growth was observable in the multi-test system described in Example 3, the results were still not completely satisfactory, due to the unique growth characteristics of the actinomycetes. Many of these strains adhered to the plastic walls of the microplate wells, thereby making detection of increased turbidity less than optimal.
• the inoculating suspension is a liquid, turbidity often was concentrated along the outer circumference of the wells, rather than producing a uniform dispersion of turbidity throughout the wells.
• a gelling agent was added to the suspension to prevent individual cells from migrating to the well walls.
• GelriteTM for example, preparations of GelriteTM (commercially available from Sigma, under this name, as well as “Phytagel”) were found to be highly satisfactory.
• GelriteTM does not form a gel matrix until it is exposed to gel- initiating agents, in particular, positively charged ions such as divalent cations (e.g., Mg 2+ and Ca 2+ ).
• gel- initiating agents in particular, positively charged ions such as divalent cations (e.g., Mg 2+ and Ca 2+ ).
• the GelriteTM comes into contact with the salts present in the bottom of the microplate wells, the gelling reaction begins and results in the formation of a gel matrix within a few seconds.
• the entire procedure for growth and testing of the actinomycetes required a total of 3-7 days, including primary inoculation on YEMEWG medium and other suitable media to determination and analysis of the final results. Importantly, a minimum amount of personnel time was required (i.e., just the few minutes necessary to inoculate the primary growth medium and then prepare the suspension for biochemical testing).
• the present invention provides a much improved means for the rapid and reliable identification of actinomycetes.
• EXAMPLE 5 Comparison of Water and GelriteTM
• the eleven actinomycetes listed in Table 2 were tested in both water and gel suspensions.
• a water suspension of organisms with an optical transmittance of 70% was diluted 1:10 in either water or 0.4% GelriteTM.
• two samples of each organism were produced, one sample being a water suspension and one sample being a suspension which included GelriteTM.
• SF-P MicroPlatesTM GP MicroPlatesTM without tetrazolium; commercially available from Biolog.
• the MicroPlatesTM were incubated at 27°C for 48 hours, and observed for growth. As shown in the table below, the number of positive reactions increased dramatically for the organisms suspended in GelriteTM, as compared to water.
• gelling agents were tested in this Example.
• alginic acid, carrageenan type I, carrageenan type II, and pectin were tested for their suitability in the present invention. All of these compounds are commercially available from Sigma.
• pectin was found to be unsuitable when tested by adding 1% pectin to SF-P MicroPlatesTM .
• Pectin has a yellowish cast to it, and is therefore not a colorless or clear compound.
• gelling was dependent upon the presence of sugars in the microplate wells. Because many of the substrates tested in this multitest format do not contain sugars, gelling did not occur uniformly in all wells.
• Carrageenan type II was slightly better than type I and it was also comparable to or better than GelriteTM. Surprisingly, the carrageenan type II functions as effectively as the GelriteTM, although the carrageenan does not form a rigid gel. This indicates that it is not necessary that a rigid gel be formed in order for the beneficial effects of these colloidal gelling agents to be observed. EXAMPLE 8 Testing of Other Bacterial Species
• the present invention is also suitable for the rapid characterization of numerous and diverse organisms, such as those listed in Table 3.
• the gram-negative bacteria tested covered a range of genera and tribes, including Pseudomonas cepacia, Providencia stuartii, Neisseria lactamica, Xanthomonas maltophilia, Vibrio metschnikovii, Cedecea neteri, and Escherichia coli.
• Various gram-positive bacteria were also tested, including Rhodococcus equi and Staphylococcus aureus.
• MicroPlatesTM used to test the gram-positive organisms.
• ES MicroPlatesTM Biolog were also tested with some of the gram-negative species. Inoculation in 0.4% GelriteTM was compared to inoculation in 0.85% saline. The inoculation densities used were those normally recommended for these MicroPlateTM test kits (55% transmittance for the gram-negative organisms, and 40% for the gram-positive organisms). Following inoculation of the MicroPlatesTM with 150 ⁇ l suspensions of organisms in either saline or GelriteTM per well, the MicroPlatesTM were incubated at 35°C for 16-24 hours.
• This experiment was designed to determine the suitability of the present invention for use in identification of eukaryotic microorganisms, such as yeasts.
• two types of reactions were observed to establish a metabolic pattern: a) assimilation reaction tests which are based on turbidity increases due to carbon utilization by the organisms; and b) oxidation tests, which also test for carbon utilization, but which detect utilization via a redox color change of the organism suspension.
• yeasts were first grown on BUY Agar (Biolog) a solid agar medium, and harvested from the agar surface as described in Example 2 above.
• the organisms included in this example are listed in Table 3 (D. ovetensis, C. laurentii, C. terreus, K. marxianus, S. cerevisiae, and W. saturnus).
• Biolog YT MicroPlatesTM available commercially from Biolog
• Each well of the YT MicroPlateTM was inoculated with 100 ⁇ l of either the water or 0.4% GelriteTM suspension of organisms.
• the inoculated MicroPlatesTM were incubated at 27°C, and the results observed at 24, 48, and 72 hours of incubation.
• yeast identification systems such as the Minitek (BBL), API 20C (API), expanded Uni-Yeast-Tek System (Flow), and Vitek (Biomerieux) were overcome or avoided in the present example (see e.g., G.A. Land (ed.), "Mycology,” in H.D. Isenberg (ed.), Clinical Microbiology Procedures Handbook. American Society for Microbiology, in particular "Commercial Yeast Identification Systems,” pp. 6.10.1 through 6.10.5, [1994]).
• the Vitek system heavily encapsulated yeasts and isolates with extensive mycelial growth are sometimes difficult to suspend.
• this limitation is avoided by the present invention, allowing for reliable and reproducible testing procedures and systems.
• the GelriteTM was shown to be clearly superior to water for the rapid identification of eukaryotic microorganisms.
• This experiment was designed to determine the suitability of the present invention for use in identification of eukaryotic microorganisms, such as molds.
• the molds were first grown on modified Sabouraud-Dextrose agar (commercially available from various sources, including Difco).
• This medium is prepared by thoroughly mixing dextrose (20 g/1), agar (20 g/1), and neopeptone (1 g/1) in 1 liter of distilled/deionized water. Heat is applied, until the mixture boils. The medium is autoclaved for 15 minutes at 15 psi (121°C). After cooling, the medium is distributed into petri plates.
• the organisms included in this example are listed in Table 3 (P. notatum, P. chrysogenum, R. pusillus, A. niger and T. mentagrophytes).
• Example 1 After they were grown on Sabouraud-Glucose agar, an inoculum was prepared as described in Example 1. YT and SP-F MicroPlatesTM (Biolog) were then inoculated with a 1:10 dilution of a starting inoculum having an optical transmittance of 70%, in water, 0.2% carrageenan type II, or 0.4% GelriteTM.
• Each well of the SF-P MicroPlatesTM was inoculated with 100 ⁇ l of organisms suspended in either water, 0.2% carrageenan type II, or 0.4% GelriteTM.
• 100 ⁇ l of organisms suspended in either water, or 0.4% GelriteTM were used to inoculate the wells.
• the inoculated MicroPlatesTM were incubated at 25°C, and the results observed by eye and using a MicroStation ReaderTM (Biolog) at 24 hour increments for a total of 4 days of incubation.
• MicroPlatesTM without tetrazolium were tested (all of these plates were obtained from Biolog).
• E. coli was inoculated into the SF-N MicroPlatesTM, and S. aureus was inoculated into the SF-P MicroPlatesTM.
• 25 mg/1 of resazurin was added as a color indicator as an alternative to tetrazolium.
• 12.5 ⁇ l of 10% glucose solution and 15 ⁇ l of each antimicrobial dilution were added to each well, as described in the paragraph above.
• the results in the GelriteTM agreed with the results obtained with saline as an inoculant within one two-fold dilution. This is considered satisfactory according to the National Committee on Clinical Laboratory Standards (NCCLS) guidelines (see e.g., J. Hindler (ed.), "Antimicrobial Susceptibility Testing," in H.D. Isenberg (ed.), Clinical Microbiology Procedures Handbook. American Society for Microbiology, pp. 5.0.1 through 5.25.1, [1994]).
• NCLS National Committee on Clinical Laboratory Standards
• the MIC was slightly lower in saline as compared to GelriteTM.
• the MIC's were slightly lower in GelriteTM, than in saline.
• the present invention provides a novel and useful alternative method for determination of antimicrobial sensitivities of microorganisms. Another advantage of this invention is that the test may be conducted in a format that cannot be accidentally spilled.
• Redox Purple the redox indicator referred to as "Redox Purple” was synthesized for use in the present invention.
• the method of Graan et al. T. Graan, et al. , "Methyl Purple, an Exceptionally Sensitive Monitor of Chloroplast Photosystem I
• the benzoquinone-4-chloroimide ( Figure 5, II) was produced by dissolving 5 g 4-aminophenol (Figure 5, I) in 1 N aqueous HCl (75 mL) (0°C), followed by the addition of 200 mL sodium hypochlorite (NaCIO, 5% w/v) to produce a chloroimide derivative shown in Figure 5, Panel A.
• NaCIO sodium hypochlorite
• the solution was continuously stirred and the temperature maintained below 4°C during addition of the sodium hypochlorite. After stirring at room temperature for 12 hours, the yellow to orange colored product was isolated by filtration, washed with cold distilled water and dried in air and in vacuo.
• the product was vacuum filtered using a Buchner funnel, washed with a minimal amount of ice-cold water (approximately 30 ml) in the funnel, dried in air for approximately 24 hours, and dried overnight in a vacuum desiccator.
• the ring closure was accompanied by a change in the solution color to a dark purple.
• the reaction mixture was filtered and the precipitate washed with minimal cold water as described above.
• the filtrate was saturated with an excess of solid sodium chloride (approximately 100 g), the solution was decanted off the excess salt on the bottom of the container, and the solution extracted with diethylether (5 X 100 mL) until no more orange-colored material was removed from the aqueous phase. Vigorous shaking of the ether and aqueous phases was avoided, as this was found in some experiments to result in formation of an intractable emulsion.
• the combined ether layers were back- extracted with 70 mM aqueous sodium carbonate solution (25 mL), the pH of the sodium carbonate solution reduced to 4.5 with glacial acetic acid, and the resulting mixture refrigerated overnight at 4°C.
• the redox purple precipitated as the free acid. Additional redox purple was obtained by acidifying the original aqueous phases with glacial acetic acid (pH 4.5) and repeating the above purification. The total yield obtained by this synthesis method was approximately 25%.
• the purity of the redox purple synthesized according to this method was 95-98%, as determined by thin-layer chromatography, a method that is well know in the art (A. Braithwaite and F.J.
• analogous derivatives of the reagent e.g., alkali salts, alkyl O-esters
• modified properties e.g., solubility, cell permeability, toxicity, and/or modified color(s)/absorption wavelengths
• redox purple e.g., salts, etc.
• redox purple was used as the redox indicator in the test system.
• E. coli 287 (ATCC #11775) was cultured overnight at 35°C, on TSA medium supplemented with 5% sheep blood.
• a sterile, moistened, cotton swab was used to harvest colonies from the agar plate and prepare six identical suspensions of organisms in glass tubes containing 18 ml of 0.85% NaCl, or 0.2% carrageenan type II. The cell density was determined to be 53-59% transmittance.
• One saline and one carrageenan suspension were used to inoculate Biolog GN MicroplatesTM, with 150 ⁇ l aliquots placed into each well.
• the wells of this plate contain tetrazolium violet as the redox indicator.
• Two ml of a 2 mM solution of redox purple (sodium salt)(prepared as described in Example 12), or two ml of a 2 mM solution of resazurin (sodium salt) were added to the other tubes, to produce a final dye concentration of 200 ⁇ M.
• These suspensions were used to inoculate Biolog SF-N MicroplatesTM. As with the GN MicroplatesTM, aliquots of 150 ⁇ l were added to each well in the plates.
• the SF-N MicroplatesTM are identical to the GN
• MicroPlatesTM with the exception being the omission of tetrazolium violet from the wells of the SF-N plates.
• the inoculated plates were incubated at 35°C for approximately 16 hours. The plates were then observed and the colors of the well contents recorded.
• redox indicators i.e., redox purple, tetrazolium violet, and resazurin
• wells containing the following carbon sources ⁇ -cyclodextrin, adonitol, D-arabitol, cellobiose, i-erythritol, xylitol, citric acid, D-glucosaminic acid, ⁇ -hydroxybutyric acid, ⁇ - hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, ⁇ -ketovaleric acid, malonic acid, quinic acid, sebacic acid, L-histidine, hydroxy L-proline, L-leucine, and D,L-carnitine.
• the negative control wells containing water, instead of a carbon source were also negative for all three redox indicators.
• the oxidized form of redox purple spectrally matches the reduced form of tetrazolium violet (i.e., with a maximum absorbance at 590 nm). This may provide an advantage, as detection methods such as spectrophotometry settings may be used interchangeably with tetrazolium violet and redox purple.
• T. harzianum DAOM 190830 was cultured for seven days at 26°C on malt extract agar (Difco). A sterile, moistened cotton swab was used to harvest conidia from the culture and prepare a suspension in 16 ml of 0.25% GelriteTM. The cell density was determined to be 75% transmittance. A 2 ml aliquot of a 2 mM solution of redox purple was added to the suspension, along with 2 ml of 1 M triethanolamine-SO 4 , pH 7.3. The final concentration of redox purple was 200 ⁇ M, and the final concentration of triethanolamine-SO 4 was 100 mM.
• the final suspension was mixed well and used to inoculate the wells of a Biolog SF-N MicroplateTM.
• 100 ⁇ l of the suspension was added to each well.
• the inoculated SF-N MicroplateTM was incubated at 30°C for approximately 24 hours, and observed.
• MicroCardTM testing inocula were prepared in IF1 at a density of 35%T (as measured in the Biolog turbidimeter) in 12x75 tubes. The inocula were dispensed into MicroPlatesTM (150 ⁇ l/well) or MicroCardsTM, as appropriate, and incubated at 35°C, for 24 hours. While results were obtained using both the MicroPlatesTM and MicroCardsTM, the results were more consistent with MicroPlatesTM. Some wells in the MicroCardTM trapped air bubbles and gave false negative results.
• the MicroPlateTM results are indicated in Table 14, below, as well as described further in the text following the Table. In Table 14, "+" indicates that the organism tested was capable of utilizing the carbon source listed, while “-” indicates that the organism tested was not capable of utilizing the carbon source listed, and "w” indicates weak positive reactions.
• Strain 14444 has been reported to be a xylA (i.e., xylose-negative) mutant of strain 14443. The results of this experiment indicated that while strain 14443 is xylose-positive (i.e., capable of utilizing xylose), strain 14444 is xylose-negative (i.e., incapable of utilizing xylose) However, strain 1444 was found to be negative also for maltose, maltotriose, L-proline, and L-threonine. While the results observed with L-proline and L- threonine may not be significant as these traits have been observed to be inconsistent between strains, the results obtained with maltose and maltotriose are significant, as discussed below.
• Strain 14445 has been reported to be an himA mutant of strain 14443. Prior to this experiment, it was unknown what phenotypic changes due to the himA allele, would be observed in 14445, as compared with strain 14443. Differences between 14443 and 14445 were observed in eight tests. Strain 14445 was negative for utilization of maltose, maltotriose, ⁇ -ketobutyric acid, ⁇ -hydroxybutyric acid, propionic acid, glycolic acid, L- glutamic acid, and L-threonine.
• strains are supposed to be the same strain, and both were obtained from Dr. Barbara Bachmann, at the E. coli Genetic Stock Center. Prior to testing in this experiment, strain 14443 was maintained by Dr. Blattner's laboratory, while strain 6321 was stored at Biolog. As indicated in Table 14, these two strains were shown to have differences, some of which may be insignificant, but some of which may have resulted from improper storage and maintenance, which caused the culture to change over time.
• Strain 6322 is the originating strain of the genetically important E. coli K12 culture. Strains 6321 and 6320 were reported as being derived from 6322 via genetic manipulations that eliminated the lambda phage and F+ episome. Strain 6321 was created using careful genetic manipulations, and as indicated in Table 14, its pattern of carbon utilization observed in this experiment was very similar to that of strain 6322. However, strain 6320 was created through harsh treatment (exposure to X-rays), and it differs from strain 6322 in many traits.
• strains 11547, 13671, 1367, and 13675 are all of the 0157 serological line, and are considered to be human pathogens. These strains are similar to each other, but are rather different from the K- 12 strains. It is well known that most 0157 strains are sorbitol negative, and this was observed for these four strains. However, it was also found that these strains have other special traits. For example, all four of these strains were also negative for D-saccharic acid, and D-galactonic acid-g-lactone. In addition, three of the four strains were positive for sucrose. The negative result observed for D-galactonic acid-g-lactone is particularly interesting.
• the genes involved in metabolism of D-galactonic acid-g-lactone (dgo) map at 82 minutes on the E. coli genome.
• Recent genome sequencing data have indicated that in at least one 0157 strain, a large "pathogenicity island" has been inserted in the E. coli genome at 82 minutes. It is possible that the insertion of this pathogenicity island may have resulted in the inactivation of the dgo genes.
• MicroPlateTM S. cerevisiae strains are grown on suitable media (e.g., as described in Example 9), and inoculated into the wells of the YT MicroPlateTM as described in Example 9. The ability of the strains to utilize different carbon sources (e.g., D-galactose) is then observed and compared, in order to assess the phenotypic differences between the strains. As indicated in Example 9, water or GelriteTM may be used as the inoculation suspension medium, as well as 0.85% NaCl or PPS (e.g., as described in Example 15), above with 100 ⁇ l inoculated per well, rather than the 150 ⁇ l used with bacteria.
• suitable media e.g., as described in Example 9
• PPS e.g., as described in Example 15
• two E. coli strains constructed so as to be isogenic with the exception of a single allele are compared for their ability to utilize 95 different carbon sources in the Biolog ES MicroPlateTM.
• the strains are cultured under identical conditions by growing them at room temperature on blood agar plates (TSA with 5% sheep blood). Suspensions are prepared in PPS, as described in Example 15, above. Then, 150 ⁇ l of the suspensions are used to inoculate all of the wells of two ES MicroPlatesTM (i.e., one MicroPlateTM for each strain).
• the metabolic response i.e., purple color formation
• MicroStationTM for a 24-hour period, and recorded, using SOFTmax®PRO software (Molecular Devices).
• Kinetic measurements are made using one of two methods. In the first method, each of the two MicroPlatesTM are placed inside a kinetic microplate reader and read at 15 minute intervals over a 24-hour period. In the second method, each of the two MicroPlatesTM are cycled in and out of a microplate reader using a ROBOmax® in- feed stacking device (Molecular Devices). The MicroPlatesTM are read at 15 minute intervals over a 24-hour period. The kinetic readings are then converted into 24-hour kinetic response patterns. The two patterns obtained are compared, in order to identify differences in the organisms' responses to each of the 95 carbon sources tested.
• MG1655 Prior to inoculating MicroPlateTM microtiter plates, MG1655 was pre-grown overnight on the limited nutrient medium, R2A (Acumedia). MG1655 cells were streaked onto the R2A agar, and grown overnight at 35°C. Individual colonies were picked from the agar surface, using a sterile cotton swab. The cells were suspended in GN/GP-IF inoculating fluid (Biolog), at a density corresponding to 50% transmittance in a turbidimeter (Biolog), using a 20 mM diameter tube. The suspension was then diluted 8- fold, and inoculated onto the MicroPlateTM microtiter plates.
• R2A Acumedia
• R2A was chosen after careful examination of a number of pre-growth media, including Luria-Bertani (LB), TSA, TSA with 5% sheep blood, BUGTM (Biolog), and BUGTM with blood.
• Organisms pre- cultured on R2A were the only cultures that exhibited no growth and therefore, no purple color in the negative control wells (i.e., wells that did not contain either a nitrogen source
• the complete minimal medium used in the MicroPlateTM microtiter plates contained 100 mM NaCl, 30 mM triethanolamine-HCl (pH 7.1), 25 mM sodium pyruvate, 5.0 mM NH 4 CI, 2.0 mM NaH 2 PO 4 , 0.25 mM Na 2 SO 4 , 0.05 mM MgCl 2 , 1.0 mM KC1, 1.0 ⁇ M ferric chloride, and 0.01% tetrazolium violet.
• the ability of MG1655 to grow on the defined medium served as a positive control in each experiment.
• this medium was supplemented with various nutrients and/or growth factors, with vitamins and Tweens provided at 0.25 ⁇ M, nucleotides/nucleosides at 100 ⁇ M, amino acids at 10 ⁇ M, N- ⁇ -acetyl-L-ornithine, L-ornithine, L-citrulline, putrescine, spermidine, and spermine at 50 ⁇ M; and 4-amino-imidazole-4(5)-carboxamide at 1 mM.
• the NH 4 C1 in the medium was replaced with 3.0 mM of the nitrogen source being examined.
• the NaH 2 PO 4 or Na 2 SO 4 in the medium were replaced with 1.0 mM or 100 ⁇ M respectively, of the various phosphorus and sulfur sources tested.
• the pH of the stock solutions containing the various test chemicals was tested, and if necessary, adjusted to approximately pH 7 with either NaOH or HCl, prior to dispensing the chemicals in the appropriate test panel(s). All of the chemicals tested were obtained from Sigma.
• Nitrogen-free, sulfur-free, and phosphorous-free media were used in the negative control wells of the EN and EPS panels, and consisted of the defined minimal medium described above, with the omission of NH 4 C1, NaH 2 PO 4 , or Na 2 SO 4 . Lack of growth/purple color in the negative control wells indicated the absence of significant quantities of nitrogen, phosphorous and sulfur-containing contaminants that might have been present due to transfer of these elements when the organisms were inoculated in the wells of the MicroPlateTM microtiter plates from the R2A medium.
• the nitrogen sources tested included ammonium chloride, sodium nitrite, potassium nitrate, urea, glutathione (reduced form), alloxan, L-citrulline, putrescine, L-ornithine, agmatine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,
• L-glutamine glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- phenylalanine, L-proline, L-serine, L-tyrosine, L-threonine, L-valine, D-alanine, D- asparagine, D-aspartic acid, D-glutamic acid, D-lysine, D-serine, D-valine, N-acetyl- glycine, L-pyroglutamic acid, L-homoserine, met-ala, n-amylamine, n-butylamine, ethylamine, ethanolamine, ethylene diamine, histamine, (R)-(+)- ⁇ -phenylethylamine, ⁇ - phenylethylamine, tyramine, acetamide, formamide, glucuronamide, lactamide, D(+)
• the phosphorus sources tested included phosphate, pyrophosphate, trimetaphosphate, tripolyphosphate, hypophosphite, thiophosphate, adenosine 2 '-monophosphate, adenosine 3 '-monophosphate, adenosine 5 '-monophosphate, adenosine 2':3'-cyclic monophosphate, adenosine 3 ':5 '-cyclic monophosphate, dithiophosphate, DL- ⁇ -glycero-phosphate, ⁇ -glycero-phosphate, phosphatidyl glycerol, phosphoenol pyruvate, phosphocreatine, 2' deoxy glucose 6-phosphate, guanosine 2' -monophosphate, guanosine 3 '-monophosphate, guanosine 5 '-monophosphate, guanosine 2':3'-cyclic monophosphate,
• the sulfur sources tested included sulfate, thiosulfate, tetrathionate, thiophosphate, dithiophosphate, L-cysteine, cys-gly, L-cysteic acid, cysteamine, L-cysteine-sulphinic acid, cystathionine, lanthionine, DL-ethionine, glutathione (reduced form), L-methionine, glycyl- DL-methionine, S-methyl-L-cysteine, L-methionine sulfoxide, L-methionine sulfone, taurine, N-acetyl-DL-methionine, N-acetyl cysteine, isethionate, thiourea, thiodiglycol, thioglycolic acid, thiodiglycolic acid, 1-dodecane-sulfonic acid, taurocholic acid, tetramethylene
• the auxotrophic supplements tested included L-alanine, L-arginine, L-asparagine, L-aspartic acid, adenine, adenosine, 2'-deoxyadenosine, adenosine 3 ':5' -cyclic monophosphate, adenosine 3 '-monophosphate, adenosine 5 '-monophosphate, L-cysteine, L- glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, guanine, guanosine, 2'- deoxyguanosine, guanosine 3 ':5 '-cyclic monophosphate, guanosine 3 '-monophosphate, guanosine 5 '-monophosphate, L-leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine,
• the inoculated test panels were observed.
• the contents of the wells in which E. coli was able to grow i.e., the well contained a nitrogen, phosphorus, or sulfur source suitable for the organism
• the well contained a nitrogen, phosphorus, or sulfur source suitable for the organism turned purple.
• phenotypes that were stimulated by histidine or various pyrimidine compounds produced a purple color in the wells where Salmonella was growth was stimulated.
• the following compounds resulted in a weak positive test result: D(+)- galactosamine, D-mannosamine, and ⁇ -amino-n-butyric acid.
• the following compounds were not suitable nitrogen sources (i.e., there was no MG1655 growth in wells containing these compounds: negative control (medium without any nitrogen source), sodium nitrite, potassium nitrate, urea, glutathione (reduced form), alloxan, L-citrulline, putrescine, L- ornithine, agmatine, L-alanine, L-cysteine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-tyrosine, L-threonine, L-valine, D-asparagine, D-aspartic acid, D-glutamic acid, D-lysine, D-
• EPS phosphorus and sulfur test panel
• positive phosphate control (medium with phosphate), positive sulfur control (medium with sulfate), trimetaphosphate, thiophosphate, hypophosphite, adenosine-2' -monophosphate, adenosine 3-monophosphate, dithiophosphate, DL- ⁇ -glycerophosphate, ⁇ -glycerophosphate, phosphoenol pyruvate, phosphocreatine, 2'-deoxyglucose 6-phosphate, guanosine 2'- monophosphate, guanosine 3 '-monophosphate, guanosine 5 '-monophosphate, guanosine 2':3'-cyclic monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 1- phosphate, fructose 6-phosphate, mannose 1-phosphate
• the following compounds resulted in a weak positive test result: 2-aminoethyl phosphonate, S-methyl-L-cysteine.
• the following compounds were not suitable phosphorous or sulfur sources (i.e., there was no MG1655 growth in wells containing these compounds: negative control (medium without any phosphorus or sulfur source), pyrophosphate, tripolyphosphate, adenosine 5 '-monophosphate, adenosine 2':3'- cyclic monophosphate, adenosine 3 ':5 '-cyclic monophosphate, phosphatidyl glycerol, guanosine 3':5'-cyclic monophosphate, cytidine 2 '-monophosphate, cytidine 3' :5' -cyclic monophosphate, uridine 3':5'-cyclic monophosphate, inositol hexaphosphate, nitrophenyl phosphate,
• MG1655 is not auxotrophic for any nutrients or growth factors, this strain was capable of growing in all wells of the EA panel. Instead, two S. typhimurium auxotrophs were used in the EA experiments. With one strain, hisF645, only the well containing L-histidine turned purple, while with the other strain, pyrC ⁇ 73, wells containing a pyrimidine (i.e., uracil, cytosine, uridine, cytidine, 2-deoxyuridine, 2- deoxycytidine, uridine 3 '-monophosphate, uridine 5 '-monophosphate, cytidine 2'- monophosphate, cytidine 3 '-monophosphate, and cytidine 5 '-monophosphate) turned purple and wells containing a purine (i.e., adenosine 2 '-monophosphate, adenosine 3'- monophosphate, adeno
• the present invention represents an unexpected and much improved system for the broad-based, rapid biochemical testing and/or phenotypic testing of microorganisms and/or other cell types, in many uses and formats (or configurations).
• the present invention provides a major advance in the testing of actinomycetales, fungi, and other spore-forming microorganisms. The results are highly surprising in view of the obligate aerobic nature of most of these organisms. Using the novel approach of embedding the organisms in a gel matrix, the biochemical test reactions are dispersed uniformly throughout the testing well, providing an easy to read indicator of organism growth and metabolism.
• both automated and manual systems with fixed time point or kinetic reading may be used in conjunction with the present invention.
• the results may be observed visually (i.e., by eye) by the person conducting the test, without assistance from a machine.
• the results may be obtained with the use of equipment (e.g., a microtiter plate reader) that measures transmittance, absorbance, or reflectance through, in, or from each well of a multitest device such as a microtiter testing plate (e.g., MicroPlateTM) or a miniaturized testing card (e.g., MicroCardTM).
• Kinetic readings may be obtained by taking readings at frequent time intervals or reading the test results continuously over time.
• a device particularly suited for incubating and conducting the methods of the present invention includes the device described in co-pending U.S. Patent Application Serial No. 09/277,353.
• the present invention provides methods and compositions for easily performing comparative testing of numerous phenotypes, thereby providing means to determine the functions of various genes.
• the embodiments of the present multitest gel-matrix invention provide numerous advances and advantages over the prior art, including: (1) much greater safety, as there is no spillage, nor aerosolization of cells, mycelia, nor spores, while performing or inoculating test wells; (2) faster biochemical reactions are produced, giving final results hours or days earlier than commonly used methods; (3) more positive biochemical reactions are obtained, giving a truer picture of the microorganisms' metabolic characteristics; (4) darker, more clear-cut biochemical reactions and color changes are obtained; (5) more uniform color and/or turbidity are obtained, as the cells, mycelia, and/or spores do not settle and clump together at the bottom of the wells, nor do they adhere to the sides of the wells; (6) the reactions are much easier to observe visually or with optical instruments (e.g., the Biolog MicroStation ReaderTM); and (7) the overall process for performing multiple tests is extremely simple and efficient, requiring very little labor on the part of the microbiologist. All of these advantages enhance

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 1999
  • 1999-06-16 EP EP99928683A patent/EP1088097A4/de not_active Withdrawn

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