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Publication numberUS3870601 A
Publication typeGrant
Publication dateMar 11, 1975
Filing dateMay 4, 1973
Priority dateMay 4, 1973
Publication numberUS 3870601 A, US 3870601A, US-A-3870601, US3870601 A, US3870601A
InventorsBert Warren, George L Evans
Original AssigneeSchering Corp
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Novel diagnostic system for differentiation of enterobacteriaceae
US 3870601 A
Abstract
This invention relates to a culture medium for the rapid differentiation and identification of bacteria belonging to the family Enterobacteriaceae.
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Description  (OCR text may contain errors)

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

Non-Patent Citations
Reference
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Classifications
U.S. Classification435/12, 435/880, 435/34, 435/822, 435/848, 435/38, 435/879, 435/873, 435/849, 435/852, 435/14, 435/253.6
International ClassificationC12Q1/10
Cooperative ClassificationC12Q1/10, Y10S435/852, Y10S435/849, Y10S435/879, Y10S435/873, Y10S435/822, Y10S435/88, Y10S435/848
European ClassificationC12Q1/10