US3870601A - Novel diagnostic system for differentiation of enterobacteriaceae - Google Patents

Novel diagnostic system for differentiation of enterobacteriaceae Download PDF

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US3870601A
US3870601A US357318A US35731873A US3870601A US 3870601 A US3870601 A US 3870601A US 357318 A US357318 A US 357318A US 35731873 A US35731873 A US 35731873A US 3870601 A US3870601 A US 3870601A
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substrate
beta
identification
bacteria
lysine
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Bert Warren
George L Evans
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Merck Sharp and Dohme Corp
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Schering Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/848—Escherichia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/848—Escherichia
    • Y10S435/849—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/852—Klebsiella
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/873—Proteus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/879—Salmonella
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/88—Serratia

Definitions

  • ABSTRACT This invention relates to a culture medium for the rapid differentiation and identification of bacteria belonging to the family Enterobacteriaceae.
  • This invention relates to a culture medium for the rapid differentiation and identification of bacteria of the family Enterobacteriaceae. More specifically, this invention relates to single culture media, the method for their preparation and the utilization of these media for the identification of the bacteria of the family Enterobacteriaceae.
  • the Enterobacteriaceae are a ubiquitous group of bacteria consisting of frank enteric pathogens (Salmonella and Shigella) and many other opportunistic organisms capable of causing infections in every conceivable body locus. They are defined as gram negative rods that reduce nitrates, are oxidase negative and ferment glucose. Their spectrum of sensitivity to antibiotics varies considerably and in many instances therapy ought be based only upon the identity of the organism. Some members are epidemiologically significant and all require identification for specific diagnosis. Currently, they are the most frequent cause of bacterial infections and account for over million tests per year.
  • this invention relates to media wherein chromogenic B-galactoside substrates are admixed with either (a) a decarboxylase substrate, (b) a deaminase substrate, (c) a urease substrate, (d) a hydrogen sulfide detecting system, or (e) a carbohydrate fermentation system, or the chromogenic B-galactoside substrate is admixed with any combination of such systern.
  • Suitable chromogenic B-galactoside substrates are o-nitrophenyl-,B-galactopyranoside (ONPG), 5-bromo- 4-chloro-3-indolyl-,B-galactoside, and 6-bromo-2- naphthyl-B-D-galactoside, as well as any other wellknown agents.
  • Suitable deaminase substrates are such l-amino acids as phenylalanine, tryptophane, histidine, leucine, norleucine, methionine and norvaline and the like. Urea is used as the substrate for urease.
  • Suitable hydrogen sulfide detecting agents are sodium thiosulfate in the presence of an iron containing salt such as ferric ammonium citrate.
  • Suitable decarboxylase agents are lysine, ornithine and arginine, and the like.
  • Suitable fermentable carbohydrates or sugar alcohols are dextrose, mannitol, arabinose, sucrose, dulcitol, rhamnose, and the like.
  • the surprising feature of this invention is that for the first time a system has been devised wherein a chromogenic B-galactoside substrate has been combined with the abovementioned other type substrates without the color reactions of the chromogenic ,B-galactoside substrate interfering-with the efficacy of the tests. Also. as is evident from the herein described media, although it is preferred to strive for as many substrates as possible. it is of course understood that such media can be modified wherein one or more of the above-described a, b, c, d. or e components can be eliminated from the medium.
  • the preferred medium of this invention is comprised of such ingredients as bromthymol blue (used as pH indicator), yeast extract (a source of nutrient), dextrose and/or other fermentable carbohydrates, l-lysine (detection of lysine decarboxylase), ferric ammonium citrate and sodium thiosulfate (detection of hydrogen sulfide production), tryptophan, (detection of deaminase and indole), o-nitrophenyl ,B-galactopyranoside (ONPG) (detection of B-galactosidase activity), trace amounts of lactose to activate the B-galactosidase system, urea for detection of urease and agar as a supporting base, and sodium chloride (for osmotic control).
  • starch or carboxymethyl cellulose may be added to enhance gas formation and to prolong the shelf life of the medium.
  • the preferred medium is comprised and prepared as follows:
  • the medium is then dispensed in suitable quantities in sterile screw-capped tubes, a1- lowing the agar to cool while the tubes are angled to obtain a butt and slant configuration according to standard techniques.
  • the inside of the screw-cap is fitted with a p-dimethylamino-benzaldehyde-impregnated paper disc previously prepared according to standard techniques suitable for the detection of indole.
  • the media provide for possible color differentiation wherein the proteus-providence group of organisms give rise to a brown slant, hydrogen sulfide-producing organisms causing a blackening in the butt, lysine positive organisms giving a green butt, lysine negative organisms giving a yellow butt, urease positive organisms give a blue-green to blue color in the butt or at the butt/slant juction. In the case of Proteus species, this bluish color will mask the lysine reaction which is not essential for the identification of this group in the presence of urea.
  • the ONPG reacting organisms give rise to a green color in the slant while ONPG negative organisms turn the slant blue.
  • the pdimethylaminobenzylaldehyde impregnated paper disc turns red to show the presence of indole.
  • Glucose lf lysine is not decarboxylated and glucose is fermented, the pH of the butt will drop below 6.2, resulting in yellow color.
  • ONPG lf the organism has an inducible ,B-galactosb dase, galactose is split from ONPG liberating the yellow-collored o-nitrophenol.
  • ONPG lf the organism has an inducible ,B-galactosb dase
  • galactose is split from ONPG liberating the yellow-collored o-nitrophenol.
  • the blue slant that forms in this medium if no deaminase activity is present, the combination of the yellow o-nitrophenol and blue, produce a green colored slant. Blue slants occur if the organism tested is both ONPG and deaminase negative.
  • Urea If the organism produces urease. ammonia is formed causing a rise in pH above 7.0. With strong urease producers such as Proteus species, the color of the butt will turn blue/green to blue. Due to the deaminase activity combined with urease activity with Proleus' species, the slant will turn blue/green or green/brown (olive). With weaker urease producers such as Klebsiella, a blue/green color may only be produced at the buttslant junction.
  • lndole lf indole.is formed as a result of tryptopha nase activity, the disc insert in the cap will turn red to violet. It will remain colorless if indole is not produced.
  • Group I Hydrogen Sulfide Positive H 5 Tryptophan lndole Lysine ONPG UREA Arizona Eclwardriellu Salmonella P. mirabilis P. vulgaris Cilrobacler freululir' d
  • Group ll Tryptophan Positive (excluding Group I organisms) H 5 Tryptophan lndole Lysine ONPG UREA l. nmrganii I. rt'llgz'rl 1 Provide/win Group lll: lndole Positive (excluding Groups l and ll organisms) H- S Tryptophan lndole Lysine ONPG URliA E.
  • a culture medium having a pH in the range of about 6.7-7.2 suitable for determining the identification of bacteria of the family Enterobacteriaceae which comprises a chromogenic B-galactosidase substrate in combination with a member of the group consisting of (a) a decarboxylase substrate, (b) a deaminase substrate, (c) a urease substrate, (d) a hydrogen sulfide detecting system, or (e) a carbohydrate fermentation system.
  • a culture medium of claim 1 wherein the chromogenic B-galactosidase substrate is chosen from the group consisting of o-nitrophenyl-B-galactopyranoside, 5-bromo-6-chloro-3-ind'olyl-,B-D-galactoside and 6- bromo-2-naphthyl-B-D-galactoside
  • the decarboxylase agents are lysine, ornithine and arginine
  • the urease substrate is urea and a carbohydrate fermentation system selected from the group consisting of dextrose, mannitol, arabinose, sucrose, dulcitol, rhamnose
  • the deaminase substrate is selected from the group consisting or l-aminoacids, preferably tryptophane, phenylalanine, and histidine
  • the hydrogen sulfide detection system containing an iron salt in combination with hydrogen sulfide detecting agent preferably
  • a culture medium having a pH in the range of about 6.7-7.2 suitable for determining the identification of bacteria of the Enterobacteriaceae which comprises the following ingredients, said ingredients being present in proportions indicated:
  • compositions of claim 8 which comprises admixing the bromthymol blue, yeast extract, dextrose, lysine, ferric ammonium citrate, sodium thiosulfate, agar, lactose and sodium chloride ingredients in suitable quantities of water, adjusting the pH to about 7.0, autoclaving the resulting solution followed by adding sterile o-nitrophenyl-ngalactopyranoside, urea and tryptophan, with q.s. water to form the defined concentrations.

Abstract

This invention relates to a culture medium for the rapid differentiation and identification of bacteria belonging to the family Enterobacteriaceae.

Description

United States Patent [1 1 Warren et al.
[ 1 NOVEL DIAGNOSTIC SYSTEM FOR DIFFERENTIATION OF ENTEROBACTERIACEAE [75] Inventors: Bert Warren, Tuxedo Park, N.Y.; George L. Evans, Hopatcong, NJ.
[73} Assignee: Schering Corporation, Bloomfield,
[22] Filed: May 4, 1973 [21] Appl. No.: 357,318
[52] US. Cl. 195/1035 R, 195/100 [51] Int. Cl Cl2k 1/04 {58] Field of Search 195/99-103.5 R
[' r 3,870,601 Mar. '11, 1975 Primary Examiner-Samih N. Zaharna AssistantExaminer-Robert J. Warden Attorney, Agent. or Firm-Raymond A. McDonald; Stephen B. Coan [57] ABSTRACT This invention relates to a culture medium for the rapid differentiation and identification of bacteria belonging to the family Enterobacteriaceae.
18 Claims, No Drawings NOVEL DIAGNOSTIC SYSTEM FOR DIFFERENTIATION OF ENTEROBACTERIACEAE This invention relates to a culture medium for the rapid differentiation and identification of bacteria of the family Enterobacteriaceae. More specifically, this invention relates to single culture media, the method for their preparation and the utilization of these media for the identification of the bacteria of the family Enterobacteriaceae.
The Enterobacteriaceae are a ubiquitous group of bacteria consisting of frank enteric pathogens (Salmonella and Shigella) and many other opportunistic organisms capable of causing infections in every conceivable body locus. They are defined as gram negative rods that reduce nitrates, are oxidase negative and ferment glucose. Their spectrum of sensitivity to antibiotics varies considerably and in many instances therapy ought be based only upon the identity of the organism. Some members are epidemiologically significant and all require identification for specific diagnosis. Currently, they are the most frequent cause of bacterial infections and account for over million tests per year.
Although certain individual principles and aspects to which this invention relates have been known and used in hospital and field conditions for the determination of bacteria, such factors have generally employed the use of multiple media requiring at least three days and as many as 10-20 separate tests. Although other procedures have been produced to obviate some of the problems involved, such procedures have been complex, awkward and expensive multi-media devices. The media of this invention provide a relatively simply prepared single confluent medium which affords the diagnostician with a large number of differential tests in a single tube. Other advantages and distinguishing characteristics of this invention over the media of the prior art will also be apparent to the skilled artisan as the details of this invention are explored.
1n its broad concept this invention relates to media wherein chromogenic B-galactoside substrates are admixed with either (a) a decarboxylase substrate, (b) a deaminase substrate, (c) a urease substrate, (d) a hydrogen sulfide detecting system, or (e) a carbohydrate fermentation system, or the chromogenic B-galactoside substrate is admixed with any combination of such systern.
Suitable chromogenic B-galactoside substrates are o-nitrophenyl-,B-galactopyranoside (ONPG), 5-bromo- 4-chloro-3-indolyl-,B-galactoside, and 6-bromo-2- naphthyl-B-D-galactoside, as well as any other wellknown agents. Suitable deaminase substrates are such l-amino acids as phenylalanine, tryptophane, histidine, leucine, norleucine, methionine and norvaline and the like. Urea is used as the substrate for urease. Suitable hydrogen sulfide detecting agents are sodium thiosulfate in the presence of an iron containing salt such as ferric ammonium citrate. Suitable decarboxylase agents are lysine, ornithine and arginine, and the like. Suitable fermentable carbohydrates (or sugar alcohols) are dextrose, mannitol, arabinose, sucrose, dulcitol, rhamnose, and the like. The surprising feature of this invention, as the below described media and color formations indicate, is that for the first time a system has been devised wherein a chromogenic B-galactoside substrate has been combined with the abovementioned other type substrates without the color reactions of the chromogenic ,B-galactoside substrate interfering-with the efficacy of the tests. Also. as is evident from the herein described media, although it is preferred to strive for as many substrates as possible. it is of course understood that such media can be modified wherein one or more of the above-described a, b, c, d. or e components can be eliminated from the medium.
Essentially the preferred medium of this invention is comprised of such ingredients as bromthymol blue (used as pH indicator), yeast extract (a source of nutrient), dextrose and/or other fermentable carbohydrates, l-lysine (detection of lysine decarboxylase), ferric ammonium citrate and sodium thiosulfate (detection of hydrogen sulfide production), tryptophan, (detection of deaminase and indole), o-nitrophenyl ,B-galactopyranoside (ONPG) (detection of B-galactosidase activity), trace amounts of lactose to activate the B-galactosidase system, urea for detection of urease and agar as a supporting base, and sodium chloride (for osmotic control). Optionally, starch or carboxymethyl cellulose (or any other cellulose) may be added to enhance gas formation and to prolong the shelf life of the medium.
More specifically, the preferred medium is comprised and prepared as follows:
After weighing the components of part A of this medium, sufficient distilled water is added to bring the volume, to 700 ml. The resulting suspension is then heated and with the aid of a magnetic stirrer is brought into solution. The pH of the solution is adjusted to about 6.9, after which it is autoclaved at 15 psi. for 15 minutes. The remaining components of the medium, (B) are brought to about pH 6.9 and sterilized by filtering through a Nalgene filter. Both (A) and (B) components are then admixed under sterild conditions. The final pH is adjusted to about 7.0. The medium is then dispensed in suitable quantities in sterile screw-capped tubes, a1- lowing the agar to cool while the tubes are angled to obtain a butt and slant configuration according to standard techniques. The inside of the screw-cap is fitted with a p-dimethylamino-benzaldehyde-impregnated paper disc previously prepared according to standard techniques suitable for the detection of indole.
Although the foregoing formulation is the most preferred ingredient-concentration, quite naturally modifications may be made to achieve substantial, but varying degrees of success. Thus, it is comtemplated that the foregoing ingredlent-concentrations may be modified and still be within the spirit of this invention, as follows: Bromthymol blue (0.025-O.15 g/l) yeast extract 1 .59 g/l) dextrose (0.5-5 g/l) l-lysine (5.0-20.0 g/l) ferric ammonium citrate (0. ll .2 g/l) sodium thiosulfate (0.1-1.2 g/l) tryptophan (1.2l0.5 g/l), agar (10-20 gms) urea (0.5-1.5 g/l) with the adjustment to the pH to about 6.7-7.1.
The use of the foregoing media presumes the primary isolation of cultures of pure cultures. When used, the butt is stabbed and the slant streaked with the test cul ture in the usual manner that is used to inoculate other tubed media having a slant and butt, and the screw cap loosely replaced onto the tube. Tne inoculated culture is permited to grow at 37C for about 24 hours and read within 24-72 hours from inoculation. Of course, it is possible to permit the inoculated culture at temperatures below 37C but in such instances the growth period is proportionately lgnger.
The media provide for possible color differentiation wherein the proteus-providence group of organisms give rise to a brown slant, hydrogen sulfide-producing organisms causing a blackening in the butt, lysine positive organisms giving a green butt, lysine negative organisms giving a yellow butt, urease positive organisms give a blue-green to blue color in the butt or at the butt/slant juction. In the case of Proteus species, this bluish color will mask the lysine reaction which is not essential for the identification of this group in the presence of urea. The ONPG reacting organisms give rise to a green color in the slant while ONPG negative organisms turn the slant blue. The pdimethylaminobenzylaldehyde impregnated paper disc turns red to show the presence of indole.
The expected reactions of the above-exemplified medium are as follows:
Glucose: lf lysine is not decarboxylated and glucose is fermented, the pH of the butt will drop below 6.2, resulting in yellow color. However, if the organism is strongly urease positive, an overriding alkaline reaction ONPG: lf the organism has an inducible ,B-galactosb dase, galactose is split from ONPG liberating the yellow-collored o-nitrophenol. lo the presence of the blue slant that forms in this medium if no deaminase activity is present, the combination of the yellow o-nitrophenol and blue, produce a green colored slant. Blue slants occur if the organism tested is both ONPG and deaminase negative.
Urea: If the organism produces urease. ammonia is formed causing a rise in pH above 7.0. With strong urease producers such as Proteus species, the color of the butt will turn blue/green to blue. Due to the deaminase activity combined with urease activity with Proleus' species, the slant will turn blue/green or green/brown (olive). With weaker urease producers such as Klebsiella, a blue/green color may only be produced at the buttslant junction.
lndole: lf indole.is formed as a result of tryptopha nase activity, the disc insert in the cap will turn red to violet. It will remain colorless if indole is not produced.
The following tables illustrate the identification of the organisms of the Enterobacteriaciae.
Table I Group I: Hydrogen Sulfide Positive H 5 Tryptophan lndole Lysine ONPG UREA Arizona Eclwardriellu Salmonella P. mirabilis P. vulgaris Cilrobacler freululir' d Group ll: Tryptophan Positive (excluding Group I organisms) H 5 Tryptophan lndole Lysine ONPG UREA l. nmrganii I. rt'llgz'rl 1 Provide/win Group lll: lndole Positive (excluding Groups l and ll organisms) H- S Tryptophan lndole Lysine ONPG URliA E. (all d Slrigella or Klebriellu or d Group IV: lndole Negative S Tryptophan lndole Lysine ONPG Urea Shigella or Salmonella I Cirrobacler d Klebsiella or d E. (loacue d E. acrogvnes 1:. liufniae or Bl E. liqm'fut'it'nx or or d Scrramu d Different biochemical types. -With exception of Shigellu .rumrci or majority positive.
or majority negative.
COLOR REACTIONS OF ENTEROBACTERIACEAE WITH EXAMPLE I MEDIUM Organisms Slant Butt lndole l) Irm'idenciu Brown Yellow (2) I. re/lgeri BrownlBlue-Olive Blue (3) P. morganii Brown/Blue-Olive Blue (4) l. miruhilis Brown/Blue-Olive Blue/Black (5) I. rulgurii Brown/Blue-Olive Blue/Black l (l l K lvhiivllu Green Blue/Yellow (7 ('umlmilvr jmlm/ii Green Black/Yellow (X Ari mm Green Black/Green (*1) Iii/wurrlsir'I/u Blue Black/Green I0) I-.'. mli Green Green or Yellow (H1 Si'rru/iu Green Green l2) l2. liqmfucicus Blue or Green or Green Yellow l3) l5. m'rqgcnes Green Green (14) If. (/Oflflll Green Yellow 15) S. smim'i Green ellow (no gas) (I6) Salmonella Blue Black l7) Sliigellu Blue Yellow or (18) If. lltlfllllll. Blue or Green Green Two color in the butt.
Numbers cross-correlate with FIGS. l and 2.
We claim:
1. A culture medium having a pH in the range of about 6.7-7.2 suitable for determining the identification of bacteria of the family Enterobacteriaceae which comprises a chromogenic B-galactosidase substrate in combination with a member of the group consisting of (a) a decarboxylase substrate, (b) a deaminase substrate, (c) a urease substrate, (d) a hydrogen sulfide detecting system, or (e) a carbohydrate fermentation system.
2. A culture medium of claim 1 wherein the chromogenic B-galactosidase substrate is chosen from the group consisting of o-nitrophenyl-B-galactopyranoside, 5-bromo-6-chloro-3-ind'olyl-,B-D-galactoside and 6- bromo-2-naphthyl-B-D-galactoside, the decarboxylase agents are lysine, ornithine and arginine, the urease substrate is urea and a carbohydrate fermentation system selected from the group consisting of dextrose, mannitol, arabinose, sucrose, dulcitol, rhamnose, the deaminase substrate is selected from the group consisting or l-aminoacids, preferably tryptophane, phenylalanine, and histidine, and the hydrogen sulfide detection system containing an iron salt in combination with hydrogen sulfide detecting agent preferably in the form of a thiosulfate.
3. A culture medium of claim lwherein the chromogenic B-galactasidase substrate is in combination with a decarboxylase substrate.
4. A culture medium of claim 1 wherein the chromogenic B-galactosidase substrate is in combination with a deaminase substrate.
5. A culture medium of claim 1 wherein the chromo- 0 genie B-galactosidase substrate is in combination with a hydrogen sulfide detecting system.
6. A culture medium of claim 1 wherein the chromo- 8. A culture medium having a pH in the range of about 6.7-7.2 suitable for determining the identification of bacteria of the Enterobacteriaceae which comprises the following ingredients, said ingredients being present in proportions indicated:
9. A process for preparing compositions of claim 8 which comprises admixing the bromthymol blue, yeast extract, dextrose, lysine, ferric ammonium citrate, sodium thiosulfate, agar, lactose and sodium chloride ingredients in suitable quantities of water, adjusting the pH to about 7.0, autoclaving the resulting solution followed by adding sterile o-nitrophenyl-ngalactopyranoside, urea and tryptophan, with q.s. water to form the defined concentrations.
10. The process for the identification of bacteria of the Enterobacteriaceae which comprises inoculating the media defined by claim 8 allowing the inoculated media to grow for at least about 24 hours at 37C, followed by the identification of the bacteria according to the herein described changes for lysine, hydrogen sulfide, tryptophan, ONPG, urea and indole.
11. The process for the identification of bacteria of the Enterobacteriaceae which comprises inoculating the media defined by claim 8 allowing the inoculated media to grow for at least about 24 hours at 37C, followed b the idenification of the bacteria according to the herein described color changed for glucose, lysine, hydrogen sulfide, tryptophan, o-nitrophenyl ,B-galactopyranoside urea and indole.
12. A sterile culure medium having a pH of about Table-Continued Group l: Hydrogen Sulfide Positive 6.7-7.2 comprising: H 5 Tryptophan lndole Lysine N G Urea l v P Salmonella lngredient Grams/liter Citrgbacfe v d Klebsl'ella or d Bromthymol Blue 0.05 E. cloacae d Yeast Extract 3.0 E. aeragenes Dextrose 0.9 E. hufniae or L-lysine 12.0
Ferric Ammonium Citrate 0.4 E. liquefaciens or d Sodium Thiosulfate 1.5 Agar 12.0 Serratl'a d Lactose 0.02 Sodium Chloride 0.75 Different biochemical typesv O-nltrophenyl-B-galactopyranosrde 0.75 with exception of Slii ellu sonei. Tryptophan 3.75 or majority positive. Urea 1.0 -l-- or majority negative. Water q.s. to make I000 cc.
16. The process of claim 14 wherein the color change r 13- A process for preparing composltlon of clam 12, results are ead according to the following chart which comprises admixing the bromthymo] blue, yeast extract, dextrose, lysine, ferric ammonium citrate, 50- COLOR REACTIONS OF ENTERUBACHiRMCEA/i WITH EXAMPLE rum thlosulfate, agar, lactose and sodium chloride ingredients in suitable quantities of water, adjusting the I pH to about 7.0, autoclaving the resulting solution followed by adding sterile o-nitrophenyl-B- ORGANISMS SLANT BUTr gt galactopyranoside urea and tryptophan, with q.s. water to form the defined concentrations. (1) Providenc 'a Brown Yellow 14. The process for the identification of bacteria of (2) g an Blue the Enterobacteriaceae which comprises inoculating 3 P, morganl'i do, Blue the media defined by claim 12, allowing the inoculated (4) 'l' Blue/Black O (5) P. vulgarls do. Blue/Black media to grow for at least about 24 hours at 37 C, fol- (6) Klebsieua Green Blue/Green lowed by the identification of the bacteria according to z qc r G Bl k/Y H the herein described changes for lysine, hydrogen sul- (8) igi: gg f f tide, tryptophan, ONPG, urea and lndole. (9) Edwardsiella Blue Black/Green 15. The process of claim 14 wherein the color change (10 Green results are read according to the following table: (11) s l reen Green (12) E. liquefacl'ens Blue or Green or Table Green Yellow 4O (l3) E. aerogenes Green Green (14) E. cloacae Green Yellow Group I: Hydrogen Sullidc Positive (15) S. .t'lfllllll Green cllow H25 Tryptophan lndole Lysine Urea (no as) (l6) Salmonella Blue Black (l7) Shi ella Blue Yellow or Arizona m+ (18) E. lafniae Blue or Green Edwards-fella Green .S'ulnlmwllu I. ltll'rullilis Two colors in the butt. P. i'ulgarls Numbers cross-correlate with FIGS. l and 2. Cilrubajler d reuli ii f I 17. The process for the identification of bacteria of Group TWPIOPhimPOsitlve (excludmg Group I Orgamsms) the Enterobacteriaceae which comprises inoculating H25 Tryptophan lndole Lysine Urea the media defined by claim 12, allowing the inoculated media to grow for at least about 24 hours at 37 C, followed b the identification of the bacteria accordin to P IIIOF'UIIH g the herein described color changes for glucose, lysine, Pftll'lllt'llllll hydrogen sulfide, tryptophan, o-nitrophenyl B-galactopyranoside urea and indole. Grou lll: lndole Positive (excludln Groups I and ll or anlsms) p g g 18. The process ofclaim 17 wherein the color change "2 yp p lndflle Lysine Urea results are read according to the following chart:
E. (Oli shigellaz or COLOR REACTIONS OF ENTEROBAC I'ERIACEAE WITH Klehsialla or d EXAMPLE I Group IV: lndole Negative MEDIUM H 8 Tryptophan lndole Lysine Urea Organisms Slant Butt lndole S/ligrlln or -F' J l Pm VllIl'IKll! Brown Yellow Continued I COLOR REACTIONS OF ENTEROBACTERIACEAE WITH EXAMPLE 1 Organisms Slant Bun lndole (2) P. retlgeri Blue-brown to Olive Blue (3) P. morganii do. Blue (4) P. mirabilis do., Blue/Black (5) P. vulgaris do. Blue/Black (6) Klebsiella Green Blue/Green i (7) Cilrobncler freuna'ii Green Black/Yellow (8) Arizona Green Black/Green (9) Edwardsiella Blue Black/Green

Claims (18)

1. A CULTURE MEDIUM HAVING A PH IN THE RANGE OF ABOUT 6.7-7.2 SUITABLE FOR DETERMINING THE IDENTIFICATION OF BACTERIA OF THE FAMILY ENTEROBACTERIACEAE WHICH COMPRISES A CHROMOGENIC B-GALACTOSIDASE SUBSTRATE IN COMBINATION WITH A MEMBER OF THE GROUP CONSISTING OF (A) A DECARBOXYLASE SUBSTRATE, (B) A DEAMINASE SUBSTRATE, (C) A UREASE SUBSTRATE, (D) A HYDROGEN SULFIDE DETECTING SYSTEM, OR (E) A CARBOHYDRATE FERMENTATION SYSTEM.
1. A culture medium having a pH in the range of about 6.7-7.2 suitable for determining the identification of bacteria of the family Enterobacteriaceae which comprises a chromogenic Beta -galactosidase substrate in combination with a member of the group consisting of (a) a decarboxylase substrate, (b) a deaminase substrate, (c) a urease substrate, (d) a hydrogen sulfide detecting system, or (e) a carbohydrate fermentation system.
2. A culture medium of claim 1 wherein the chromogenic Beta -galactosidase substrate is chosen from the group consisting of o-nitrophenyl- Beta -galactopyranoside, 5-bromo-6-chloro-3-indolyl-Beta -D-galactoside and 6-bromo-2-naphthyl- Beta -D-galactoside, the decarboxylase agents are lysine, ornithine and arginine, the urease substrate is urea and a carbohydrate fermentation system selected from the group consisting of dextrose, mannitol, arabinose, sucrose, dulcitol, rhamnose, the deaminase substrate is selected from the group consisting or 1-aminoacids, preferably tryptophane, phenylalanine, and histidine, and the hydrogen sulfide detection system containing an iron salt in combination with hydrogen sulfide detecting agent preferably in the form of a thiosulfate.
3. A culture medium of claim 1 wherein the chromogenic Beta -galactasidase substrate is in combination with a decarboxylase substrate.
4. A culture medium of claim 1 wherein the chromogenic Beta -galactosidase substrate is in combination with a deaminase substrate.
5. A culture medium of claim 1 wherein the chromogenic Beta -galactosidase substrate is in combination with a hydrogen sulfide detecting system.
6. A culture medium of claim 1 wherein the chromogenic Beta -galactosidase substrate is in combination with a urease substrate.
7. A culture medium of claim 1 wherein the chromogenic Beta -galactosidase substrate is in combination with a carbohydrate fermentation system.
8. A culture medium having a pH in the range of about 6.7-7.2 suitable for determining the identification of bacteria of the Enterobacteriaceae which comprises the following ingredients, said ingredients being present in proportions indicated:
9. A process for preparing compositions of claim 8 which comprises admixing the bromthymol blue, yeast extract, dextrose, lysine, ferric ammonium citrate, sodium thiosulfate, agar, lactose and sodium chloride ingredients in suitable quantities of water, adjusting the pH to about 7.0, autoclaving the resulting solution followed by adding sterile o-nitrophenyl- Beta -galactopyranoside, urea and tryptophan, with q.s. water to form the defined concentrations.
10. The process for the identification of bacteria of the Enterobacteriaceae which comprises inoculating the media defined by claim 8 allowing the inoculated media to grow for at least about 24 hours at 37*C, followed by the identification of the bacteria according to the herein described changes for lysine, hydrogen sulfide, tryptophan, ONPG, urea and indole.
11. The process for the identification of bacteria of the Enterobacteriaceae which comprises inoculating the media defined by claim 8 allowing the inoculated media to grow for at least about 24 hours at 37*C, followed b the idenification of the bacteria according to the herein described color changed for glucose, lysine, hydrogen sulfide, tryptophan, o-nitrophenyl Beta -galactopyranoside urea and indole.
12. A sterile culure medium having a pH of about 6.7-7.2 comprising:
13. A process for preparing composition of claim 12, which comprises admixing the bromthymol blue, yeast extract, dextrose, lysine, ferric ammonium citrate, sodium thiosulfate, agar, lactose and sodium chloride ingredients in suitable quantities of water, adjusting the pH to about 7.0, autoclaving the resulting solution followed by adding sterile o-nitrophenyl- Beta -galactopyranoside urea and tryptophan, with q.s. water to form the defined concentrations.
14. The process for the identification of bacteria of the Enterobacteriaceae which comprises inoculating the media defined by claim 12, allowing the inoculated media to grow for at least about 24 hours at 37*C, followed by the identification of the bacteria according to the herein described changes for lysine, hydrogen sulfide, tryptophan, ONPG, urea and indole.
15. The process of claim 14 whErein the color change results are read according to the following table:
16. The process of claim 14 wherein the color change results are read according to the following chart:
17. The process for the identification of bacteria of the Enterobacteriaceae which comprises inoculating the media defined by claim 12, allowing the inoculated media to grow for at least about 24 hours at 37*C, followed by the identification of the bacteria according to the herein described color changes for glucose, lysine, hydrogen sulfide, tryptophan, o-nitrophenyl Beta -galactopyranoside urea and indole.
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US3957584A (en) * 1974-09-30 1976-05-18 Warner-Lambert Company Detection of beta-galactosidase producing micro-organisms
US4070247A (en) * 1977-03-30 1978-01-24 Indiana University Foundation Diagnostic media
FR2443062A1 (en) * 1978-11-29 1980-06-27 Bioresearch Inc FAST DETECTION OF BACTERIA
FR2461000A1 (en) * 1979-06-29 1981-01-30 Minnesota Mining & Mfg DEVICE FOR IDENTIFYING MICROORGANISMS AND CORRESPONDING IDENTIFICATION METHOD
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US4591554A (en) * 1979-10-31 1986-05-27 Ajinomoto Co., Inc. Rapid method for detecting microorganisms
US4603108A (en) * 1979-05-02 1986-07-29 National Research Development Corp. Method for identification of bacterial species
EP0254771A2 (en) * 1986-06-30 1988-02-03 Stephen C. Edberg Detection of microbes in a sample
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US5527667A (en) * 1993-05-25 1996-06-18 Virginia Polytechnic Institute And State University Water sample viral contamination detection system
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US20070020719A1 (en) * 2003-01-10 2007-01-25 Duran Vila Anabel Selective culture medium for the isolation and/or detection of species in the streptococcus genus

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Cited By (45)

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US3957584A (en) * 1974-09-30 1976-05-18 Warner-Lambert Company Detection of beta-galactosidase producing micro-organisms
US4070247A (en) * 1977-03-30 1978-01-24 Indiana University Foundation Diagnostic media
FR2443062A1 (en) * 1978-11-29 1980-06-27 Bioresearch Inc FAST DETECTION OF BACTERIA
US4242447A (en) * 1978-11-29 1980-12-30 Bioresearch Rapid detection of bacteria
US4603108A (en) * 1979-05-02 1986-07-29 National Research Development Corp. Method for identification of bacterial species
FR2461000A1 (en) * 1979-06-29 1981-01-30 Minnesota Mining & Mfg DEVICE FOR IDENTIFYING MICROORGANISMS AND CORRESPONDING IDENTIFICATION METHOD
EP0030964A1 (en) * 1979-06-29 1981-07-01 Flow Lab Negative control media for microbiologic biochemical tests.
EP0030964A4 (en) * 1979-06-29 1981-11-11 Flow Lab Negative control media for microbiologic biochemical tests.
US4591554A (en) * 1979-10-31 1986-05-27 Ajinomoto Co., Inc. Rapid method for detecting microorganisms
US4492761A (en) * 1982-04-05 1985-01-08 Duke University Complement assay method
US6783950B2 (en) 1986-06-30 2004-08-31 Stephen C. Edberg Method of detecting a microbe in a liquid water sample
US6730496B2 (en) 1986-06-30 2004-05-04 Stephen C. Edberg Method of detecting first generation environmental-sourced microbes in an environmentally-derived sample
US5780259A (en) * 1986-06-30 1998-07-14 Edberg; Stephen C. Medium and method for detecting a target microbe
EP0254771A3 (en) * 1986-06-30 1988-08-24 Stephen C. Edberg Detection of microbes in a sample
EP0254771A2 (en) * 1986-06-30 1988-02-03 Stephen C. Edberg Detection of microbes in a sample
US5292644A (en) * 1987-11-05 1994-03-08 Berg James D Rapid process for detection coliform bacteria
EP0332752A1 (en) * 1988-01-05 1989-09-20 Giuseppe Giammanco Enzymatic confirmation of coliform bacteria
US5210022A (en) * 1990-04-20 1993-05-11 Rcr Scientific, Inc. Method test media and chromogenic compounds for identifying and differentiating general coliforms and Escherichia coli bacteria
US5393662A (en) * 1990-04-20 1995-02-28 Rcr Scientific, Inc. Test media for identifying and differentiating general coliforms and Escherichia coli bacteria
US6699685B1 (en) 1990-04-20 2004-03-02 Rcr Scientific, Inc. Method, test media and chromogenic compounds for identifying and differentiating general coliforms and escherichia coli bacteria
US5358854A (en) * 1990-04-20 1994-10-25 Research Organics, Inc. Method, test media and chromogenic compounds for identifying and differentiating general coliforms and Escherichia coli bacteria
US5633144A (en) * 1990-05-03 1997-05-27 University Of Florida Research Foundation, Inc. Assay pad and method for determination of the presence of total coliforms
WO1991018111A1 (en) * 1990-05-14 1991-11-28 The Regents Of The University Of California Novel and improved method for determination of e. coli in water
US5643743A (en) * 1990-05-14 1997-07-01 The Regents Of The University Of California Method for detecting coliform and E. coli bacteria
US5364767A (en) * 1993-02-11 1994-11-15 Research Organics, In. Chromogenic compounds and methods of using same
US5527667A (en) * 1993-05-25 1996-06-18 Virginia Polytechnic Institute And State University Water sample viral contamination detection system
WO1995003424A1 (en) * 1993-07-21 1995-02-02 Merck Patent Gmbh Culture medium for the simultaneous detection of coliform bacteria and escherichia coli
US5650290A (en) * 1994-04-01 1997-07-22 Hach Company Method & Medium for use in detecting E. coli and total coliforms
US5849515A (en) * 1994-04-01 1998-12-15 Hach Company Method and medium for use in detecting E. coli and total coliforms
US5541082A (en) * 1994-04-29 1996-07-30 Biolog, Inc. Microbiological medium
US5464755A (en) * 1994-04-29 1995-11-07 Biolog, Inc. Microbiological medium and method of assay
ES2079319A1 (en) * 1994-05-16 1996-01-01 Aguayo Jose Maria Garcia Bacteriological culture medium and process for its preparation
FR2728587A1 (en) * 1994-12-22 1996-06-28 Pasteur Sanofi Diagnostics Non-selective gel culture medium
US6136554A (en) * 1995-06-07 2000-10-24 Biolog, Inc. Microbiological media for isolation and indentification of enteric pathogens such as E. coli and salmonella
WO1996040861A1 (en) * 1995-06-07 1996-12-19 Biolog, Inc. Microbiological media for isolation and identification of enteric pathogens such as e. coli and salmonella
US6287797B1 (en) 1995-11-14 2001-09-11 Biocontrol Systems, Inc. Method for quantification of biological material in a sample
US6509168B2 (en) 1995-11-14 2003-01-21 Biocontrol Systems, Inc. Method for quantification of biological material in a sample
US20040018585A1 (en) * 1995-11-14 2004-01-29 Crouteau Andrew J. Method for quantification of biological material in a sample
US5985594A (en) * 1995-11-14 1999-11-16 Idexx Laboratories, Inc. Method for quantification of biological material in a sample
US5700655A (en) * 1995-11-14 1997-12-23 Idexx Laboratories, Inc. Method for quantification of biological material in a sample
US7122338B2 (en) 1995-11-14 2006-10-17 Biocontrol Systems, Inc. Method for quantification of biological material in a sample
US5888760A (en) * 1997-04-10 1999-03-30 Dade Microscan Inc. Universal test systems and methods of use thereof for identifying multiple families of microorganisms
US6548021B1 (en) 1997-10-10 2003-04-15 President And Fellows Of Harvard College Surface-bound, double-stranded DNA protein arrays
US6764832B2 (en) * 2001-08-22 2004-07-20 Lawrence Restaino Plating media for the presumptive identification of the genus Shigella and the species Shigella sonnei and shigella boydii
US20070020719A1 (en) * 2003-01-10 2007-01-25 Duran Vila Anabel Selective culture medium for the isolation and/or detection of species in the streptococcus genus

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