CA1112140A - Bacterial sensitivity test kit - Google Patents
Bacterial sensitivity test kitInfo
- Publication number
- CA1112140A CA1112140A CA289,457A CA289457A CA1112140A CA 1112140 A CA1112140 A CA 1112140A CA 289457 A CA289457 A CA 289457A CA 1112140 A CA1112140 A CA 1112140A
- Authority
- CA
- Canada
- Prior art keywords
- pad
- bacteria
- antibacterial agent
- redox indicator
- inoculum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
Abstract
BACTERIAL SENSITIVITY TEST KIT
Abstract of the Disclosure Disclosed herein is a method for the rapid determination of the susceptibility of pathogenic bacteria to antibacterial agents that correlates to the Kirby-Bauer method. An apparatus and test element, composed of a mixture of an antibacterial agent and a redox indicator dispersed in an inert pad, also are disclosed. The apparatus includes a tray divided into a plurality of compartments each of which contains the test element, each test element comprising the pad impregnated with a redox indicator and a different antibacterial agent. Each pad is inoculated with the bacteria in a nutrient medium and the apparatus is incubated. The redox indicator color change determines the sensitivity or resistance of the tested bacteria, and the change correlates with the accepted Kirby-Bauer method. The apparatus and method for the determination of the susceptibility of pathogenic bacteria to antibacterial agents is particularly useful since it is rapid, simple and requires inexpensive equipment and supplles.
Abstract of the Disclosure Disclosed herein is a method for the rapid determination of the susceptibility of pathogenic bacteria to antibacterial agents that correlates to the Kirby-Bauer method. An apparatus and test element, composed of a mixture of an antibacterial agent and a redox indicator dispersed in an inert pad, also are disclosed. The apparatus includes a tray divided into a plurality of compartments each of which contains the test element, each test element comprising the pad impregnated with a redox indicator and a different antibacterial agent. Each pad is inoculated with the bacteria in a nutrient medium and the apparatus is incubated. The redox indicator color change determines the sensitivity or resistance of the tested bacteria, and the change correlates with the accepted Kirby-Bauer method. The apparatus and method for the determination of the susceptibility of pathogenic bacteria to antibacterial agents is particularly useful since it is rapid, simple and requires inexpensive equipment and supplles.
Description
Backaround of the Invent on a) Fieid of the Invention This invention relates to a method, and an apparatus and ~` test elements for use therewith, for the rapid determination of the susceptibility of bacteria to antibacterial agents. The method correlates to the art accepted standards of the Kirby-Bauer method for the sensitivity testing of pathogenTc bacteria to antibacterial agents.
b) DescrlDtion of the Prior Art In the past, varlous methods have been used to determine anttblotlc sensltlvlty; for example, agar diffusion and serial dllutlon. As a result, the correlat10n of sensitivity testing results from vartous hospltals was dlfficult. In 1966, A.W. Bauer et al., Amerlcan Journal of Cltnlcal Pathology, 45, 493(1966), reported a standardlzed dlsc procedure in an attempt to allevlate this sltuation.
In 1972, the FDA publlshed 1n the Federal Register, 37, No.l91, 20527~1972), a notice that the latter standard7zed disc procedure, whtch ts hereln refered to as the Klrby-Bauer method, Is to be used as the standard method tn ~11 cllnl CD I laboratorles for the sensltivlty testlng of pathogenlc bacterla to antlb~cterlal agents. However, the Klrby-i~uer method ls not wlthout certatn dlsadvantages. Some of the dlsadvantages are: fallure to use Mueller-Hlnton agar, fallure to measure the pH of the medlum, Inadequate standardlzatlon of broth culture denslty, fallure to press surplus fluld from swab before Inoculatllng,plates, excess delay between culture standardization and plate InoculatTon, excess delay in applying the dlsc after Inoculation of plates, wrong Incubatlon temperature and tlme and fallure to measure .; .
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zones carefully. If the Kirby-Bauer method is conducted as outlined - in the Federal Register with the errors listed above being eliminated, then any new method and apparatus can be justified only after experimental data has demonstrated that the aIternate method is at least as accurate and precise as the recommended Kirby-Bauer method.
Although thTs accepted standard has been established for the determTnation of bacterial sensitivity to antibacterial agents, the prlor art does not solve a numb-r of problems. For example, the existing ; apparatus and methods for performing the KirbyC~auer test are noteasily standardlzed to glve conslstent results. In addition, a skllled technlclan Is necessary for the operatlon of the apparatus and methods.
Another difflculty encountered in the prior art is that the determination of the bacterial sensitTvTty to the antibacterial agent requires various measurements and suTtable standard bacterTal strains and the InterpretatTon Ts only qualltatTve. Thus, a method to gTve an easTly measurable and dTstTnct end potnt for determTnTng the bacterTal sensTtivity to the antlbacterTal agent Is clearly needed.
An attempt to solve thls latter problem of correlDtlng th0 bacterlal sensltlvlty to the antlbacterlal agent Is reported by Y. Kanazawa and T. Kuramata, The Journal of Antlblotlcs, Ser. A, XIX ~5), 229(1966).
In thls report they used oxTdatlon - reductlon dyes or redox indlcators to glve an Indlcatlon of bacterTal sensltTvTty to the antTbacterial agent.
However, a number of problems remalned unsolved; for example, the malntenance ; of the antTbacterTal agent Tn the dTsc and suTtable concentrations of the redox Indlcator and antTbacterTal agent to give a correlation of results wTth an accepted standard.
An obJect of the present Inventlon Ts to provTde an apparatus, test element and method for the rapid determTnatTon of bacterial .
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14~1 susceptibility to antibacterial agents, the method giving results equivalent to those of the accepted Kirby-~auer method.
Another object of the present invention is to provide an -apparatus that gives easily measurable and distinct end points for determining the susceptibility of bacterial pathogens to all useful antibacterial agents.
Still another obJect of the present invention is to provide a new and improved method that is rapid, simple and requires inexpenslve equlpment and supplies for determtning the suscepttbtItty of bacterlal pathogens to all useful anttbactertal agents.
Summ ~ entlon According to one aspect of the present invention, an apparatus is provlded for the indlcation of bacterial susceptibtlity to anttbactertal agents. The apparatus Includes a tray dlvlded tnto a plurallty of compartments, each compartment contalnlng a pad formed from inert material.
A redox Indtcator that changes c'olor In the presence of growlng bacteria and a particular antibacterial agent is tmpregnated In each pad. The tray Is provlded wlth a removable tray cover In order to protect and contaln the pads In the tray. The tray or tray cover can Include Indlcla to enable a determlnatlon of the character of the antlbactertal agent In the partlcular compartment.
Another aspect of thls Inventlon Involves a new method for obtalnlng results equlvalent to those obtalned by the Kirby-i3auer method.
General DescrlPtlon of the Inventlon The term "antlbactertal agent" as used heretn tncludes anti~
bacterlal, antlblotlcs and synthettc antibactertal agents.
The efflcacy of the anttbacterial agents ts evident by observation of the color change of the pad. A color change tndicates that the organlsm grows In the presence of the anttbacterial agent in the pad ; ~ AHP-6781 14i~
.' and is not susceptible to it, whereas if the pad does not change color the organism does not grow and is susceptible to the antibacterial agent in the pad. The pads are standardized so that the color change and the lack of color change indtcate reslstance and susceptibility of the bacterium,-rospectlvely, and correlate to the interpretatlon of the accepted Klrby-Elauer method tt.e., to the standards as publlshed in the Federa:
RegTster, clted above).
The nature of the apparatus lends Itself to almost complete standardlzatlon so that ITttle Is left for the technlclan to do but add a standard volume of the standardlzed Inoculum of bacteria under test and Incubate the Inoculated apparatus. The characteristlcs of the test ; element also provide the apparatus or kit wtth a very practical shelf-life.
The present apparatus is an tntegrated, relatively tnexpenslve package, unlquely adapted for standardlzatlon and can be discarded '5 upon completlon of the test. The apparatus thus affords a rapid and practlcal means for determlnlng the suscept7billty of bacterla to particular antlbacterlal agents. The redox Indlcator, the antlbacterlal agents and thelr ~rrangement are standardlzed and are an Integral part of the of the apparatus. All that the user needs to do Is add a standardlzed inoculum of the b~cterlum under test dlrectly to each pad contalning a glven antlbacterlal agent and examlne each pad for color change after the speclfled perlod of Incubatton.
Sultable Tnert materlals for the pad are selected from porous or flbrous materlals havlng an absorptlve property. Examples of such Inert materlals Include alpha cellulose paper (I.e. fllter paper), cellulose acetate and polyurethane foam. The Inert materlal must be clean and contaln no substances whlch will Inhiblt or enhance the actlvlty of the antlbacterTal agent absorbed thereln, nor affect the pH.
, , ~ AHi~6781 14~) The color of the inert materi~l can vary; however, the color selected should not interfere with the detection of the color change of the dye.
The shape, sTze and thickness of the pad are not critlcal and can be varled so long as the pad contains the required amount of redox indicator and antTbacterTal agent, and wlll absorb the standardized tnoculum. The pads can be preformed and impregnated by conventional means. A convenlent means includes the use of an inert, volitile liquid carrler for the impregnation. Alternatively, the redox indicator and antibacterlal agent are absorbed into a sheet of the tnert materTal, followed by cuttlng out the pads from the sheet. A useful and preferred Inert materlal pad Is the 0.5 Inch dlameter, 0.034 Inch thlck, hlgHly absorbent paper dlsc No.740-E, ava11able from Schelcher and Schnell Inc., Keene, New Hamshlre.
~arlous redox Indlcators can be absorbed Into the pad, ~5 for example resazurln, trlphenyltetrazollum chlorlde, 2,6-dichloro-phenollndophenol and the llke. Requlrements for selecting a redox Indlcator dye are that the redox Indlcator must be responslve to changes In the redox potentlal whlch re9ult from met~bollc processcs of llvlng bacterla and that the response, once It has taken place, is not reverslble. A sultsble range of redox potentlal Is an oxldatlon-reductlon potentlal between +0.2 and +0.05 volts. In addltlon, the redox Indicator should not Inhlblt or enhance the actlvlty of the antl-bacterlal agent absorbed theretn, act on the bacterlum under test nor act as pH Indlcator, for example In the pH range of from 6.0 to 8Ø The amount of the redox Indlcator absorbed Into the pad Is selected so that the redox Indlcator changes color from the metabollc processes of llvlng bacterla wlthln a perlod of 3 to 36 hours, preferably 6 to 24 hours, at about 37~C.
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A preferred redox Indicator satTsfylng these requirements is resazurin ~7-hydroxy-3H-phenoxazin-3-one 10-oxide). Resazurin (blue) is reduced to resorufin (pink) at an oxidatton - reduction potential between +0.2 and +0.05 volts and this phase of reductlon is not reversible. The concentration or amount of the redox indlcator present fn each pad Is dependent upon whether gr~m posi+ive organisms or gram negative organtsms are to be tested. For example, when resazurin Is used as an indicator of bacterial growth, the recazurin at a concentratlon of I to40 ~g, preferably 2 to 4 ~9, per 0.5 tnch diameter paper dlsc ~descrlbed above) glves a vlslble color change of the Indlcator ~blue ' plnk) wlthln a slx hour poriod of Incubation at 37C wlthout a subsequent change after 24 hours Incubatlon at 37C for gram posltlve bacterta. For gram negatlve organlsms, a concentration of l.Oto 100 1l9, preferably 10 to 20 ~9, per the 0.5 Inch dlameter paper dlsc Is requlred to obtaln a vlslble color change of the Indlcator ~blue plnk) wlthln a slx hour perlod of Incubatlon at 37C
wlthout any subsequent change after 24 hours incubation at 37C.
Vlrtually any of the avallable ~ntlbacterl~l agents can be Impregnated In the p~ds. Examples of these antlbacterlal agents are as follows: the penlcllllns, exempllfled by penlclllln G, penlclllln V, amplclll1n, cloxaclllln, dlcloxac~llln, methlclllln, c~rbenlclllln and the llke; the cephalosporlns, exemi~llfled by cephalosporln C, cephalothln, cephalorldlne, cephaglycln, cephalexin, cefazolln, cefamandolo, cefoxltln and the llke; the sulfa drugs exempllfled by sulfathlazole, sulfaguanldlne, sulfamethizole, sulfamethoxazole and the llke; the tetracycllnes, exempllfled by tetracycllne, chlorotetracyclln-, oxytetracycllne and the llke; the amino glucosldes, exempllfled by streptomycln, neomycln, gentamlcin and the llke; mlscellaneous antlbacterlal agents, ex-mpllfled by novoblocin, --7~
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colimycln, lincomyctn, trlmethoprim, clindomycin, erythromycin, chloramphenicol, nitrofuranto7n, furazol1done, nalidixlc acid, polymyxin ~, kanamycin and the like; combtnattons of antTbactertal agents, exempltfled by a combtnatlon of sulfamethoxazole and trtmethoprim ~i.e., i3actrlm~ and the llke; In thelr pharmaceutlcally acceptable forms. "In thelr pharmaceutically acceptable forms" refers to the form In whlch such antlbacterlal agents are admlnlstered therapeutically;
for example, alkall metal salts thereof such as cephslothtn sodium, cefazolin sodlum, potasslum penlcllltn G, and the Itke, the ammontum salt of cefamandole, Internal salts such as the zwltter tons of amplclllln, cephalogiycln and cephalextn, and the betaln of cephaloridine.
The concentratlon or amount of the antibactertal agent absorbed Into each pad must glve an antlbacterlal sensltlvlty correspondlng to that of the Klrby-Bauer method. The amount of the antlbacterlal agent 1n the pad Is determlned by preparlng a serles of pads eoch contolnlng the redox Indlcator and a dlfferent amount of the antlbacterlal agent. The Inoculum of bacterla Is added to each pad followed by Incub~tlon of thls serle~ of padfi. Eoch pad 1~ examlned for the ~en~ltlvlty of the b~cterl~ to the concentratlon of the antl-bacterlal agent, and from thls serles of concentratlons, the concentration of the antlbacterl41 agent Is selected so thot the senstttvtty of the bact~rla to thc antlbacterlal agent corresponds to the senslttvlty exhlblted by the bacterla In the Klrby-i3auer method.
~ore speclf1cally, the amount of antlbacterla! agent In each pad Is determlned by: , a) preparlng a plurallty of standard Inocula of bacteria,each Inoculum betng of a dlfferent stroln of a parttcular bactertal .
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' specles and the selectTon of straTns TncludTng those known to be sensTtTve and those known to be resTstant to the antlbacterTal agent accordTng to the Klrby-Bauer method;
b) preparTng a correspondTng plurallty of a serTes of pads, each pad Tmpregnated with the antTbacterTal agent 7n a different concentratlon and a redox Tndicator Tn a concentration selected so that the redox Indlcator changes color in response to the growth of bacterta wlthln 3 to 36 hours at about 37C
when Inoculated wlth a predetermTned amount of saTd standard Inoculum;
c) Inoculatlng eech serles of pads wlth the predetermined ~mount of a partlcular Inoculum of bactert fl;
d) Incubatlng the serles of pads at about 37C; and e) selectlng the concentrotlon of the antlbacterlal agent ; 15 thot glves results equlvalent to those obtaTned by the KTrby-Ebuer method Not worthy Is the flndlng that the concentratlon of the partlculor antlbocterlal agent, determlned In the precedlng manner, Is sultoble for determlnlng the susceptlblllty of other bacterla to that portlculor anttbocterlol agent, and con be employed as the standard concentrotlon for that partlcular antlbacterlal ogent In the tray of the opparatus The only llmltatlon to thls flndlng Ts~that dTfferent concentrotlons ore requtrsd for gram neg~tlve m d gram posTtlve bacterTa Any nutrlent broth Is sultoble as the medium for preparlng the 2S Inocull~m; for example, the broths d~scrlbed by W.R,BalIey and E G Scott Dlognostlc Mlcrobiology The C.V. Mosby Company, Salnt Louls, U S A , 1974, pp 363-388 , ... . ..
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A convenient and preferred standardizatton of the inoculum ; of bacterta makes use of the brain heart infusion broth. In this method the tops of isolated colonies, of a slmllar morphological type, are transferred with a wire loop to 0.5 ml of brain heart Infusion broth to glve a heavy suspensTon. The broth is incubated for thlrty mlnutes at 37C. The turbidity is then adjusted with brain heart Infuslon broth to a barlum sulfate standard correspondlng to a MacFarland tube No. I or by adJustlng the suspenslon to an optical denslty of 0.07 at 625 nm in a 13 mm dlameter test tube. Thls adjusted suspenslon Is then dlluted 1/100 wlth braln heart Infuslon broth. Thls dlluted suspenslon ~0.15 ml) Is odded to each of the 0.5 Inch dlameter paper dlscs (descrlbed above) contalnlng the approprlate concentratlon of Indlcator and antlbacterlal agent.
In applylng the method of thts InventTon for dstermlning the susceptlblllty of bacterla to antlbacterlal agents equlvalent to the standards of the accepted Klrby-eauer method, the followlng steps are performed: applylng an Inoculum of bacterla to a pad, sald pad conslstlng essentlally of an Inert mDterlal Impregnated wlth ~ redox Indlcator and an antlbacterlal agent; Incub~tlng sald pad contalnlng sald applled bacterla; and detectlng Dny change In sald redox Indlcator as a mearure of the susceptlblllty of sald bacterla to sald antlbacterlal agent; sald antlbacterlal agent, sald Inoculum of bacterla and sald redox Indlcator belng so proportloned so that a color change Indlcates reslstance of sald bacterla to sald antlbacterlal agent In accordance wlth the results obtalned by the Klrby-eauer method.
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1~1214~
Other obJects and features of the Inventlon wlll become apparent from conslderatlon of the followlng descr~ptlon of the preferred embodlments taken In connectlon wlth the accompanying drawlngs.
Brl ~ e Drawlnqs Flg. I Is a perspectlve vlew of an embodlment of the apparatus accordlng to the present Inventlon;
Flg. 2 Is a cross-soctlonal vlew of the mbodlment of Flg.l taken along the llne 2-2;
Flg. 3 15 a dotalled vl-w of a Jlngle Inert pad shown Impregnat-d wlth a redcx Indtcator ~nd an antlbact-rlal agent; and Flg. 4 Is a perspectlve vlow of another embodlment of an apparatus accordlng to the present Inventlon.
pescrlDtlon of the Preferred Embodlm~nts , Referrlng now to the drawlngs and partlcularly to , ~ F!gs. I and 2 ther- Is Illustrated an apparatus or device comprlsing a rectangular tray 1~ and a complemental overlylng cover ll whlch 1~
plvotally mounted to the tray by hlnges 12 locatod at the bock of the tray. Th- cov-r ~nd tr~y ~re ach formsd of a ultable pl~stlc materlal preferably transparent and unaffocted by the materlals to be placed In tho tray.
The tr~y 10 Is provlded wlth a plurallty of compartments 13 In the form of walls whlch can b- molded or oth-rwlse Integrally formed ag part of the tray. Each compartm nt 13 contalns ~ pad 14 and each pad 14 Is Impr-gnatod wlth the redox Indloator and an antlbacterla!
agent. Tho pad 14 Is advantageously slzed snugly to flt the compartment 13 so as to be securod thereln. the pad 14 can optionally b~ attached to the bottom of the corpartment 13 by an adh~61ve.
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, ~ ;. ' ;~ ' ' ~, ~~ AHP-6781 The front of the tray 10 and cover 11 is provided with compl-mental snap fittings 15 whlch enable the cover and tray to be snap-fitted or clamped together Tn tight fltting relation in order that the pads 14 do not fall out of the compartment 13. The cover 11 or tray 10 can be provlded wlth an Informatlon bearing marker ~not shown) ad~iacent to each compartment 13 contalnlng the pad 14 for indicating the type of antibacterial agent Impregnated In the pad 14.
One of the pads 14 can serve as a control. For lnstance, a control pad can conta1n no antlbacterlal agent and the resultlng color change wlll serve as an Indlcatlon of growth of the bacterla.
A socond control can conslst of a pad that contalns no antlbacterial agent and remalns unlnoculated. It wtll show no color change.
Flg. 3 Illustrates the pad 14. Each pad 14 Is formed of an Inert materlal, and Is Impregnated wlth a redox Indlcator that 15~ changes color In the presence of growlng bacterla, and In thls Instance an antlblotlc of speelfled concentratlon.
Flg.4 Illustrates a perspectlve vlew of another embodiment of an apparatus accordlng to the present Inventlon. In thts embodlment the tray 10 Is fltted wlth a slldlng cover 16. Thc cover 16 slldes In th- guldes 17 whlch are formsd as ~ part of the tray 10.
The slldlng cov-r 16 may Include a handle 18 to provlde a handhold for slldlng the cover l6. The tray 10 Is provlded wlth two parallel row~ of comp~rtments 13, each contalnlng a pad 14 Impregnated with the redox Indlcator and antlbacterlal agent. An extra compartment 19 Is provlded In the tray 10, sald extra compartmont contalnlng ~ control pad 20 havlng the redox Indlcator but no antlbacterlal agent.
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~ AHP-6781 14a~1 In use, the apparatus constitutes an integrated package which can readtiy be used by a technlclan. The cover 11 or 16 of the apparatus of Flgure I or 4, respectively, Is opened so that the pads 14 In the various compartments 13 can be Tnoculated with an Tnoculum of bacterla.
Next, the cover 11 or 16 Is closed and the apparatus is Incubated at approxlmately 37C for 6 to 24 hours. Subsequent examination of the color of the pads In the varlous compartments will indicate the susceptlbillty or lack of susceptlbility of th0 organlsms to the varlous antlbacterlal agents. Furthermore, the results wlll corre1ate to those of the accopted Klrby-Bbuer method.
In a preferred embodlment of thls Inventlon we have selected a preferred concentratlon of 3 ~9 of resazurln per 0.5 Inch dlameter paper dlsc for gram posTtlve organlsms and 15 ~9 per 0.5 Inch diameter paper dlsc for gram-negatlve organlsms.
Tho antlbacterlal agent llsted In the tables have the followlng forms and potencles: amplclllln ttrlhydrate, 830 ~g/mg);
carbenlctllln tsodlum, 800 ~g/mg); cepholothln tsodlum, 945 ~g/mg):
chloramphenlcol (1,000 ~g/mg); cloxaclllln ~odlum, 910 ~g/mg);
erythromycln ~b~se, 910 ~g/mg); gontamlcln ~sulfate, 600 ~g/mg);
k~n~mycln ~sulfate, 805 ~g/mg); llncomycln ~hydrochlorlde monohydrate, 860 ~gtmg); neomycln (sulfato, 673 ~g/mg); penlclllln G tpotasslum, 1,580 unlts/mg), polymyxln B ~ulfate, 7,475 unlts/mg), streptomycln ; tsulfDte, 745 ~g/mg) and tetracycllne Ihydrochlorlde, 1,000 ~g/mg).
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Table I gives the results of the combination of 3 ~9 per dlsc of resazurtn and 0.0375 jug/dtsc of pentcllltn G when compared to the Ktrby-8auer method agatnst six stratns of Sta~hylococcus Dvo,qenes after 6 and 24 hours tncubation at 37C. In Table 1, and the followtng tables, the pad employed Is the above descrlbed paper dtsc No. 740-E.
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TABLE I
Test Orqanism - StaDhvlococcus pyoqenes, Strains No. 12,40,41,126,127,128. .
Inoculum- Resazurin disc - 0.15 ml of standard7zed suspension is ~dded to the dlsc. --KTrbv-Bauer - as outlined in the Federal Reglster, 37, (191), 20527, 1972.
Resylts - S - sensltive R - resistant R E S U L T S
~nKTrby-Bauer Resazurln t24 hr) 6 hr 24 hr ,Y~ ~OCCUS
PYoaenes No.12 S S S
R R R
f 127 S S S
12~ 5 S S
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:~ ~4~3 AHP-6781 Table 2 gives the results of the combination of 15 ,ug of resazurin per disc and 4.5 1l9 of ampîcillin when compared to the Kirby-Bauer method against various species of gram negative bacteria after 6 and 24 hours - incubation at 37C.
Test Oraantsms: Proteus sD. No. 384; E. coli No. 119; ~E. coli No. 219 E. coli No. 253 Citrobacter sp. No. 305 Citrobacter sp.
No. 313 Klebsiella SD. No. 89, K!ebstella SD. No. 236.
Inoculum. - Resazurln dtscs - 0.15 ml of standardized inoculum .~ Is ~dded to the dlsc.
Ktrb,~-E3auer - as outlIned In Federal Reglster, 37, (191), .; 20527, 1972.
Results: S - sensltlve R - reslstant R E S U L T S
~peeles Klrbv-8~uer Rosazurlr~
t24 hr) 6 hr 24 hr i'rote4s SD. No. 384 S S S
,~,. eoll No. 119 S ... ~ S S
- ~QLL No. 219 S S S
E. ~,L No. 253 S S S
Cltrob~cter sP. No. 305 R R R
Cltro_acter SD . No. 313 S S S
Klebslella sP. No. 89 R R R
Klebslella sP. No. 236 R R R
The results reported in Table I and Table 2 demonstrate that the apparatus and method of this invention correlates at 6 hours incubation with the Kirby-Bauer method with no subsequent change in sensitivity if read after 24 hours incubation at 37C. This allows the laboratory to obtain the results of the new sensitivity test of this Inven;lon at any time between 6 and 24 hours incubation at 37C
knowing that the results will correlate with those obtained with the Klrby-i3auer method after 24 hours Incubation.
For the selection of antibacterial agents concentrations to be added to the pad in combination wlth the approprTate concentration of resazurtn, the bacterla are dlvlded Into two groups, e.g., gram posltlve and gram negatlve. In cllnlcal laboratorles perform7ng the Klrby-Bauer sensltivity test, the choice of antlbacterial agents to be us-d In the test Is based on whether the bacteria are gram positive or gram negatlve. For the present Inventlon, the concentratlon of antlbacterlal agents for the gram posltlve organisms 7s 17sted 7n Table 3 and the concentrat70ns for the gram negat7ve bacter7a are llsted In Table 4. Further examples of the correlatlon wlth the Klrby-Ebuer m~thod for the grDm posltlve bacterla Is shown In Table 5 and for the gram negatlve bacterla, Table 6.
, :
~ _17_ . .
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. AHP-6781 ~' 14~) Preferred concentration of antibacterial agents (~g/disc) in combination with 3 ~g/disc of resazurin for gram positive bacteria in general and Enterococci in part1cular J 5 Anttbacterial Agent Antibacterial Agent ; concentration (~g/dtsc) for concentration AnttbacterTal Gram Postttve ~g/dtsc) for Aaent _ 8acterla _ Entorococct , . . -_ _ __ , Penicllltn G 0.0375 same Cloxaclllln 1.5 same Cephalothln 0.3 same Chloramphenlcol 1.5 7.5 Erythromyctn 0.3 same . Lincomycln 0.15 same :~ Tetracycllne 1.5 same Neomycln 1.5 same Streptomycln 4.5 same Amplclllln -* 1.5 * Note that sensltlvlty to penlclllln G Indlcates sensltlvlty to amplclllln.
. -18--~~ AHP-6781 4~
Preferred concentration of antibacterial agents (~g/disc) in combination with 15 ~g/dtsc of resazurin for gram negative bacteria . Antlbacterial Agent Antlbacterlal concentration Agent ~g/disc) for Gram Ne~ative ~acteria Ampiclll1n 4~5 Cephalothln lO
Chloramphenlcol 7.5 10 Carbenlclllln 24 Gentamlcln 3 Kanamycln 4.5 Polymyxln B lOO
TetracyclIne 1.5 Neomycln 7.5 :' , :: ' :.
, ~, . :
AHP~
$~14~) .: Percent correlatlon of the resazurin-antibacterial agent disc method ~results read at 6 hours) to the Kirby-Bauer method against 79 clTntcal isolates of StaDhy~occus pyoqenes.
Antlbacterial Aaent ~ Correlation Penlcillln G 98.7 Cloxaclllln 10O
Cephalothin 100 Chloramphenlcol 100 *Erythromycln 100 Lincomycln 100 Tetracycllne 98.7 *After 18 hours Incubatlon.
-20- .
~~ AHP-6781 .
TAE~LE 6 Percent correlation of the resazurin-antibacterial agent disc method (results read at 6 hours) to the Ktrby-Bauer method against gram negatlve bacterla , .
~ecle~_~nd ~ mfbers E. coll - 20 Ente~obacter sp. - 15 Proteus ~ 6 Proteus vulqacl~ - 8 .Prote~la mlr3bl I ts - 10 ~lebslella SD.- 12 Cltrobacter SD.- I
SeLcg~l~ SD-- 1O
Pseud,omQna~ aerualnos3 - 17 Total - 99 Gram negatlve bacterla.
: An ~ Aqent % Corre!atlon Amplcllltn 92 *Cephalothln 95 *Chloramphenlcol 98 Gentamicln 99 ; KDnamycln 99 *Polymyxln B 98 *Tetracycllne 99 , 25 f*18 hours Incubatlon necessary for: Cephalothln - E- co!l, E~tC9k~E~
Pro~ mlra,blll~si Chloramphenlcol - Proteus SD-, Klebsiella s~.;
Polymyxln B - ~err~tL~ SD-, ~S~y~ vu!~arls; Tetracycline - Serratia SD.
; ~ .
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.
b) DescrlDtion of the Prior Art In the past, varlous methods have been used to determine anttblotlc sensltlvlty; for example, agar diffusion and serial dllutlon. As a result, the correlat10n of sensitivity testing results from vartous hospltals was dlfficult. In 1966, A.W. Bauer et al., Amerlcan Journal of Cltnlcal Pathology, 45, 493(1966), reported a standardlzed dlsc procedure in an attempt to allevlate this sltuation.
In 1972, the FDA publlshed 1n the Federal Register, 37, No.l91, 20527~1972), a notice that the latter standard7zed disc procedure, whtch ts hereln refered to as the Klrby-Bauer method, Is to be used as the standard method tn ~11 cllnl CD I laboratorles for the sensltivlty testlng of pathogenlc bacterla to antlb~cterlal agents. However, the Klrby-i~uer method ls not wlthout certatn dlsadvantages. Some of the dlsadvantages are: fallure to use Mueller-Hlnton agar, fallure to measure the pH of the medlum, Inadequate standardlzatlon of broth culture denslty, fallure to press surplus fluld from swab before Inoculatllng,plates, excess delay between culture standardization and plate InoculatTon, excess delay in applying the dlsc after Inoculation of plates, wrong Incubatlon temperature and tlme and fallure to measure .; .
, ' AHP~
zones carefully. If the Kirby-Bauer method is conducted as outlined - in the Federal Register with the errors listed above being eliminated, then any new method and apparatus can be justified only after experimental data has demonstrated that the aIternate method is at least as accurate and precise as the recommended Kirby-Bauer method.
Although thTs accepted standard has been established for the determTnation of bacterial sensitivity to antibacterial agents, the prlor art does not solve a numb-r of problems. For example, the existing ; apparatus and methods for performing the KirbyC~auer test are noteasily standardlzed to glve conslstent results. In addition, a skllled technlclan Is necessary for the operatlon of the apparatus and methods.
Another difflculty encountered in the prior art is that the determination of the bacterial sensitTvTty to the antibacterial agent requires various measurements and suTtable standard bacterTal strains and the InterpretatTon Ts only qualltatTve. Thus, a method to gTve an easTly measurable and dTstTnct end potnt for determTnTng the bacterTal sensTtivity to the antlbacterTal agent Is clearly needed.
An attempt to solve thls latter problem of correlDtlng th0 bacterlal sensltlvlty to the antlbacterlal agent Is reported by Y. Kanazawa and T. Kuramata, The Journal of Antlblotlcs, Ser. A, XIX ~5), 229(1966).
In thls report they used oxTdatlon - reductlon dyes or redox indlcators to glve an Indlcatlon of bacterTal sensltTvTty to the antTbacterial agent.
However, a number of problems remalned unsolved; for example, the malntenance ; of the antTbacterTal agent Tn the dTsc and suTtable concentrations of the redox Indlcator and antTbacterTal agent to give a correlation of results wTth an accepted standard.
An obJect of the present Inventlon Ts to provTde an apparatus, test element and method for the rapid determTnatTon of bacterial .
~r1r~
14~1 susceptibility to antibacterial agents, the method giving results equivalent to those of the accepted Kirby-~auer method.
Another object of the present invention is to provide an -apparatus that gives easily measurable and distinct end points for determining the susceptibility of bacterial pathogens to all useful antibacterial agents.
Still another obJect of the present invention is to provide a new and improved method that is rapid, simple and requires inexpenslve equlpment and supplies for determtning the suscepttbtItty of bacterlal pathogens to all useful anttbactertal agents.
Summ ~ entlon According to one aspect of the present invention, an apparatus is provlded for the indlcation of bacterial susceptibtlity to anttbactertal agents. The apparatus Includes a tray dlvlded tnto a plurallty of compartments, each compartment contalnlng a pad formed from inert material.
A redox Indtcator that changes c'olor In the presence of growlng bacteria and a particular antibacterial agent is tmpregnated In each pad. The tray Is provlded wlth a removable tray cover In order to protect and contaln the pads In the tray. The tray or tray cover can Include Indlcla to enable a determlnatlon of the character of the antlbactertal agent In the partlcular compartment.
Another aspect of thls Inventlon Involves a new method for obtalnlng results equlvalent to those obtalned by the Kirby-i3auer method.
General DescrlPtlon of the Inventlon The term "antlbactertal agent" as used heretn tncludes anti~
bacterlal, antlblotlcs and synthettc antibactertal agents.
The efflcacy of the anttbacterial agents ts evident by observation of the color change of the pad. A color change tndicates that the organlsm grows In the presence of the anttbacterial agent in the pad ; ~ AHP-6781 14i~
.' and is not susceptible to it, whereas if the pad does not change color the organism does not grow and is susceptible to the antibacterial agent in the pad. The pads are standardized so that the color change and the lack of color change indtcate reslstance and susceptibility of the bacterium,-rospectlvely, and correlate to the interpretatlon of the accepted Klrby-Elauer method tt.e., to the standards as publlshed in the Federa:
RegTster, clted above).
The nature of the apparatus lends Itself to almost complete standardlzatlon so that ITttle Is left for the technlclan to do but add a standard volume of the standardlzed Inoculum of bacteria under test and Incubate the Inoculated apparatus. The characteristlcs of the test ; element also provide the apparatus or kit wtth a very practical shelf-life.
The present apparatus is an tntegrated, relatively tnexpenslve package, unlquely adapted for standardlzatlon and can be discarded '5 upon completlon of the test. The apparatus thus affords a rapid and practlcal means for determlnlng the suscept7billty of bacterla to particular antlbacterlal agents. The redox Indlcator, the antlbacterlal agents and thelr ~rrangement are standardlzed and are an Integral part of the of the apparatus. All that the user needs to do Is add a standardlzed inoculum of the b~cterlum under test dlrectly to each pad contalning a glven antlbacterlal agent and examlne each pad for color change after the speclfled perlod of Incubatton.
Sultable Tnert materlals for the pad are selected from porous or flbrous materlals havlng an absorptlve property. Examples of such Inert materlals Include alpha cellulose paper (I.e. fllter paper), cellulose acetate and polyurethane foam. The Inert materlal must be clean and contaln no substances whlch will Inhiblt or enhance the actlvlty of the antlbacterTal agent absorbed thereln, nor affect the pH.
, , ~ AHi~6781 14~) The color of the inert materi~l can vary; however, the color selected should not interfere with the detection of the color change of the dye.
The shape, sTze and thickness of the pad are not critlcal and can be varled so long as the pad contains the required amount of redox indicator and antTbacterTal agent, and wlll absorb the standardized tnoculum. The pads can be preformed and impregnated by conventional means. A convenlent means includes the use of an inert, volitile liquid carrler for the impregnation. Alternatively, the redox indicator and antibacterlal agent are absorbed into a sheet of the tnert materTal, followed by cuttlng out the pads from the sheet. A useful and preferred Inert materlal pad Is the 0.5 Inch dlameter, 0.034 Inch thlck, hlgHly absorbent paper dlsc No.740-E, ava11able from Schelcher and Schnell Inc., Keene, New Hamshlre.
~arlous redox Indlcators can be absorbed Into the pad, ~5 for example resazurln, trlphenyltetrazollum chlorlde, 2,6-dichloro-phenollndophenol and the llke. Requlrements for selecting a redox Indlcator dye are that the redox Indlcator must be responslve to changes In the redox potentlal whlch re9ult from met~bollc processcs of llvlng bacterla and that the response, once It has taken place, is not reverslble. A sultsble range of redox potentlal Is an oxldatlon-reductlon potentlal between +0.2 and +0.05 volts. In addltlon, the redox Indicator should not Inhlblt or enhance the actlvlty of the antl-bacterlal agent absorbed theretn, act on the bacterlum under test nor act as pH Indlcator, for example In the pH range of from 6.0 to 8Ø The amount of the redox Indlcator absorbed Into the pad Is selected so that the redox Indlcator changes color from the metabollc processes of llvlng bacterla wlthln a perlod of 3 to 36 hours, preferably 6 to 24 hours, at about 37~C.
;
, . , . ., . . . , --;214~
A preferred redox Indicator satTsfylng these requirements is resazurin ~7-hydroxy-3H-phenoxazin-3-one 10-oxide). Resazurin (blue) is reduced to resorufin (pink) at an oxidatton - reduction potential between +0.2 and +0.05 volts and this phase of reductlon is not reversible. The concentration or amount of the redox indlcator present fn each pad Is dependent upon whether gr~m posi+ive organisms or gram negative organtsms are to be tested. For example, when resazurin Is used as an indicator of bacterial growth, the recazurin at a concentratlon of I to40 ~g, preferably 2 to 4 ~9, per 0.5 tnch diameter paper dlsc ~descrlbed above) glves a vlslble color change of the Indlcator ~blue ' plnk) wlthln a slx hour poriod of Incubation at 37C wlthout a subsequent change after 24 hours Incubatlon at 37C for gram posltlve bacterta. For gram negatlve organlsms, a concentration of l.Oto 100 1l9, preferably 10 to 20 ~9, per the 0.5 Inch dlameter paper dlsc Is requlred to obtaln a vlslble color change of the Indlcator ~blue plnk) wlthln a slx hour perlod of Incubatlon at 37C
wlthout any subsequent change after 24 hours incubation at 37C.
Vlrtually any of the avallable ~ntlbacterl~l agents can be Impregnated In the p~ds. Examples of these antlbacterlal agents are as follows: the penlcllllns, exempllfled by penlclllln G, penlclllln V, amplclll1n, cloxaclllln, dlcloxac~llln, methlclllln, c~rbenlclllln and the llke; the cephalosporlns, exemi~llfled by cephalosporln C, cephalothln, cephalorldlne, cephaglycln, cephalexin, cefazolln, cefamandolo, cefoxltln and the llke; the sulfa drugs exempllfled by sulfathlazole, sulfaguanldlne, sulfamethizole, sulfamethoxazole and the llke; the tetracycllnes, exempllfled by tetracycllne, chlorotetracyclln-, oxytetracycllne and the llke; the amino glucosldes, exempllfled by streptomycln, neomycln, gentamlcin and the llke; mlscellaneous antlbacterlal agents, ex-mpllfled by novoblocin, --7~
. , ' . ,. , , . : :
, . . ~ .
colimycln, lincomyctn, trlmethoprim, clindomycin, erythromycin, chloramphenicol, nitrofuranto7n, furazol1done, nalidixlc acid, polymyxin ~, kanamycin and the like; combtnattons of antTbactertal agents, exempltfled by a combtnatlon of sulfamethoxazole and trtmethoprim ~i.e., i3actrlm~ and the llke; In thelr pharmaceutlcally acceptable forms. "In thelr pharmaceutically acceptable forms" refers to the form In whlch such antlbacterlal agents are admlnlstered therapeutically;
for example, alkall metal salts thereof such as cephslothtn sodium, cefazolin sodlum, potasslum penlcllltn G, and the Itke, the ammontum salt of cefamandole, Internal salts such as the zwltter tons of amplclllln, cephalogiycln and cephalextn, and the betaln of cephaloridine.
The concentratlon or amount of the antibactertal agent absorbed Into each pad must glve an antlbacterlal sensltlvlty correspondlng to that of the Klrby-Bauer method. The amount of the antlbacterlal agent 1n the pad Is determlned by preparlng a serles of pads eoch contolnlng the redox Indlcator and a dlfferent amount of the antlbacterlal agent. The Inoculum of bacterla Is added to each pad followed by Incub~tlon of thls serle~ of padfi. Eoch pad 1~ examlned for the ~en~ltlvlty of the b~cterl~ to the concentratlon of the antl-bacterlal agent, and from thls serles of concentratlons, the concentration of the antlbacterl41 agent Is selected so thot the senstttvtty of the bact~rla to thc antlbacterlal agent corresponds to the senslttvlty exhlblted by the bacterla In the Klrby-i3auer method.
~ore speclf1cally, the amount of antlbacterla! agent In each pad Is determlned by: , a) preparlng a plurallty of standard Inocula of bacteria,each Inoculum betng of a dlfferent stroln of a parttcular bactertal .
.
~ -8-.
' ,r~ AHP-67BI
' specles and the selectTon of straTns TncludTng those known to be sensTtTve and those known to be resTstant to the antlbacterTal agent accordTng to the Klrby-Bauer method;
b) preparTng a correspondTng plurallty of a serTes of pads, each pad Tmpregnated with the antTbacterTal agent 7n a different concentratlon and a redox Tndicator Tn a concentration selected so that the redox Indlcator changes color in response to the growth of bacterta wlthln 3 to 36 hours at about 37C
when Inoculated wlth a predetermTned amount of saTd standard Inoculum;
c) Inoculatlng eech serles of pads wlth the predetermined ~mount of a partlcular Inoculum of bactert fl;
d) Incubatlng the serles of pads at about 37C; and e) selectlng the concentrotlon of the antlbacterlal agent ; 15 thot glves results equlvalent to those obtaTned by the KTrby-Ebuer method Not worthy Is the flndlng that the concentratlon of the partlculor antlbocterlal agent, determlned In the precedlng manner, Is sultoble for determlnlng the susceptlblllty of other bacterla to that portlculor anttbocterlol agent, and con be employed as the standard concentrotlon for that partlcular antlbacterlal ogent In the tray of the opparatus The only llmltatlon to thls flndlng Ts~that dTfferent concentrotlons ore requtrsd for gram neg~tlve m d gram posTtlve bacterTa Any nutrlent broth Is sultoble as the medium for preparlng the 2S Inocull~m; for example, the broths d~scrlbed by W.R,BalIey and E G Scott Dlognostlc Mlcrobiology The C.V. Mosby Company, Salnt Louls, U S A , 1974, pp 363-388 , ... . ..
~ AHP-6781 4~
A convenient and preferred standardizatton of the inoculum ; of bacterta makes use of the brain heart infusion broth. In this method the tops of isolated colonies, of a slmllar morphological type, are transferred with a wire loop to 0.5 ml of brain heart Infusion broth to glve a heavy suspensTon. The broth is incubated for thlrty mlnutes at 37C. The turbidity is then adjusted with brain heart Infuslon broth to a barlum sulfate standard correspondlng to a MacFarland tube No. I or by adJustlng the suspenslon to an optical denslty of 0.07 at 625 nm in a 13 mm dlameter test tube. Thls adjusted suspenslon Is then dlluted 1/100 wlth braln heart Infuslon broth. Thls dlluted suspenslon ~0.15 ml) Is odded to each of the 0.5 Inch dlameter paper dlscs (descrlbed above) contalnlng the approprlate concentratlon of Indlcator and antlbacterlal agent.
In applylng the method of thts InventTon for dstermlning the susceptlblllty of bacterla to antlbacterlal agents equlvalent to the standards of the accepted Klrby-eauer method, the followlng steps are performed: applylng an Inoculum of bacterla to a pad, sald pad conslstlng essentlally of an Inert mDterlal Impregnated wlth ~ redox Indlcator and an antlbacterlal agent; Incub~tlng sald pad contalnlng sald applled bacterla; and detectlng Dny change In sald redox Indlcator as a mearure of the susceptlblllty of sald bacterla to sald antlbacterlal agent; sald antlbacterlal agent, sald Inoculum of bacterla and sald redox Indlcator belng so proportloned so that a color change Indlcates reslstance of sald bacterla to sald antlbacterlal agent In accordance wlth the results obtalned by the Klrby-eauer method.
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--î 0-- . . , ' - : .
1~1214~
Other obJects and features of the Inventlon wlll become apparent from conslderatlon of the followlng descr~ptlon of the preferred embodlments taken In connectlon wlth the accompanying drawlngs.
Brl ~ e Drawlnqs Flg. I Is a perspectlve vlew of an embodlment of the apparatus accordlng to the present Inventlon;
Flg. 2 Is a cross-soctlonal vlew of the mbodlment of Flg.l taken along the llne 2-2;
Flg. 3 15 a dotalled vl-w of a Jlngle Inert pad shown Impregnat-d wlth a redcx Indtcator ~nd an antlbact-rlal agent; and Flg. 4 Is a perspectlve vlow of another embodlment of an apparatus accordlng to the present Inventlon.
pescrlDtlon of the Preferred Embodlm~nts , Referrlng now to the drawlngs and partlcularly to , ~ F!gs. I and 2 ther- Is Illustrated an apparatus or device comprlsing a rectangular tray 1~ and a complemental overlylng cover ll whlch 1~
plvotally mounted to the tray by hlnges 12 locatod at the bock of the tray. Th- cov-r ~nd tr~y ~re ach formsd of a ultable pl~stlc materlal preferably transparent and unaffocted by the materlals to be placed In tho tray.
The tr~y 10 Is provlded wlth a plurallty of compartments 13 In the form of walls whlch can b- molded or oth-rwlse Integrally formed ag part of the tray. Each compartm nt 13 contalns ~ pad 14 and each pad 14 Is Impr-gnatod wlth the redox Indloator and an antlbacterla!
agent. Tho pad 14 Is advantageously slzed snugly to flt the compartment 13 so as to be securod thereln. the pad 14 can optionally b~ attached to the bottom of the corpartment 13 by an adh~61ve.
, ' , .. . . ..
, ~ ;. ' ;~ ' ' ~, ~~ AHP-6781 The front of the tray 10 and cover 11 is provided with compl-mental snap fittings 15 whlch enable the cover and tray to be snap-fitted or clamped together Tn tight fltting relation in order that the pads 14 do not fall out of the compartment 13. The cover 11 or tray 10 can be provlded wlth an Informatlon bearing marker ~not shown) ad~iacent to each compartment 13 contalnlng the pad 14 for indicating the type of antibacterial agent Impregnated In the pad 14.
One of the pads 14 can serve as a control. For lnstance, a control pad can conta1n no antlbacterlal agent and the resultlng color change wlll serve as an Indlcatlon of growth of the bacterla.
A socond control can conslst of a pad that contalns no antlbacterial agent and remalns unlnoculated. It wtll show no color change.
Flg. 3 Illustrates the pad 14. Each pad 14 Is formed of an Inert materlal, and Is Impregnated wlth a redox Indlcator that 15~ changes color In the presence of growlng bacterla, and In thls Instance an antlblotlc of speelfled concentratlon.
Flg.4 Illustrates a perspectlve vlew of another embodiment of an apparatus accordlng to the present Inventlon. In thts embodlment the tray 10 Is fltted wlth a slldlng cover 16. Thc cover 16 slldes In th- guldes 17 whlch are formsd as ~ part of the tray 10.
The slldlng cov-r 16 may Include a handle 18 to provlde a handhold for slldlng the cover l6. The tray 10 Is provlded wlth two parallel row~ of comp~rtments 13, each contalnlng a pad 14 Impregnated with the redox Indlcator and antlbacterlal agent. An extra compartment 19 Is provlded In the tray 10, sald extra compartmont contalnlng ~ control pad 20 havlng the redox Indlcator but no antlbacterlal agent.
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... , ~.. ,.. ".. ~,~........ .
~ AHP-6781 14a~1 In use, the apparatus constitutes an integrated package which can readtiy be used by a technlclan. The cover 11 or 16 of the apparatus of Flgure I or 4, respectively, Is opened so that the pads 14 In the various compartments 13 can be Tnoculated with an Tnoculum of bacterla.
Next, the cover 11 or 16 Is closed and the apparatus is Incubated at approxlmately 37C for 6 to 24 hours. Subsequent examination of the color of the pads In the varlous compartments will indicate the susceptlbillty or lack of susceptlbility of th0 organlsms to the varlous antlbacterlal agents. Furthermore, the results wlll corre1ate to those of the accopted Klrby-Bbuer method.
In a preferred embodlment of thls Inventlon we have selected a preferred concentratlon of 3 ~9 of resazurln per 0.5 Inch dlameter paper dlsc for gram posTtlve organlsms and 15 ~9 per 0.5 Inch diameter paper dlsc for gram-negatlve organlsms.
Tho antlbacterlal agent llsted In the tables have the followlng forms and potencles: amplclllln ttrlhydrate, 830 ~g/mg);
carbenlctllln tsodlum, 800 ~g/mg); cepholothln tsodlum, 945 ~g/mg):
chloramphenlcol (1,000 ~g/mg); cloxaclllln ~odlum, 910 ~g/mg);
erythromycln ~b~se, 910 ~g/mg); gontamlcln ~sulfate, 600 ~g/mg);
k~n~mycln ~sulfate, 805 ~g/mg); llncomycln ~hydrochlorlde monohydrate, 860 ~gtmg); neomycln (sulfato, 673 ~g/mg); penlclllln G tpotasslum, 1,580 unlts/mg), polymyxln B ~ulfate, 7,475 unlts/mg), streptomycln ; tsulfDte, 745 ~g/mg) and tetracycllne Ihydrochlorlde, 1,000 ~g/mg).
~ ' .' ' , , ~ .. . . ~ , , , ~ .. , . . ~
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Table I gives the results of the combination of 3 ~9 per dlsc of resazurtn and 0.0375 jug/dtsc of pentcllltn G when compared to the Ktrby-8auer method agatnst six stratns of Sta~hylococcus Dvo,qenes after 6 and 24 hours tncubation at 37C. In Table 1, and the followtng tables, the pad employed Is the above descrlbed paper dtsc No. 740-E.
~ AHP-6781 4~
TABLE I
Test Orqanism - StaDhvlococcus pyoqenes, Strains No. 12,40,41,126,127,128. .
Inoculum- Resazurin disc - 0.15 ml of standard7zed suspension is ~dded to the dlsc. --KTrbv-Bauer - as outlined in the Federal Reglster, 37, (191), 20527, 1972.
Resylts - S - sensltive R - resistant R E S U L T S
~nKTrby-Bauer Resazurln t24 hr) 6 hr 24 hr ,Y~ ~OCCUS
PYoaenes No.12 S S S
R R R
f 127 S S S
12~ 5 S S
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:~ ~4~3 AHP-6781 Table 2 gives the results of the combination of 15 ,ug of resazurin per disc and 4.5 1l9 of ampîcillin when compared to the Kirby-Bauer method against various species of gram negative bacteria after 6 and 24 hours - incubation at 37C.
Test Oraantsms: Proteus sD. No. 384; E. coli No. 119; ~E. coli No. 219 E. coli No. 253 Citrobacter sp. No. 305 Citrobacter sp.
No. 313 Klebsiella SD. No. 89, K!ebstella SD. No. 236.
Inoculum. - Resazurln dtscs - 0.15 ml of standardized inoculum .~ Is ~dded to the dlsc.
Ktrb,~-E3auer - as outlIned In Federal Reglster, 37, (191), .; 20527, 1972.
Results: S - sensltlve R - reslstant R E S U L T S
~peeles Klrbv-8~uer Rosazurlr~
t24 hr) 6 hr 24 hr i'rote4s SD. No. 384 S S S
,~,. eoll No. 119 S ... ~ S S
- ~QLL No. 219 S S S
E. ~,L No. 253 S S S
Cltrob~cter sP. No. 305 R R R
Cltro_acter SD . No. 313 S S S
Klebslella sP. No. 89 R R R
Klebslella sP. No. 236 R R R
The results reported in Table I and Table 2 demonstrate that the apparatus and method of this invention correlates at 6 hours incubation with the Kirby-Bauer method with no subsequent change in sensitivity if read after 24 hours incubation at 37C. This allows the laboratory to obtain the results of the new sensitivity test of this Inven;lon at any time between 6 and 24 hours incubation at 37C
knowing that the results will correlate with those obtained with the Klrby-i3auer method after 24 hours Incubation.
For the selection of antibacterial agents concentrations to be added to the pad in combination wlth the approprTate concentration of resazurtn, the bacterla are dlvlded Into two groups, e.g., gram posltlve and gram negatlve. In cllnlcal laboratorles perform7ng the Klrby-Bauer sensltivity test, the choice of antlbacterial agents to be us-d In the test Is based on whether the bacteria are gram positive or gram negatlve. For the present Inventlon, the concentratlon of antlbacterlal agents for the gram posltlve organisms 7s 17sted 7n Table 3 and the concentrat70ns for the gram negat7ve bacter7a are llsted In Table 4. Further examples of the correlatlon wlth the Klrby-Ebuer m~thod for the grDm posltlve bacterla Is shown In Table 5 and for the gram negatlve bacterla, Table 6.
, :
~ _17_ . .
' , :'' , , :. . , ~:
. AHP-6781 ~' 14~) Preferred concentration of antibacterial agents (~g/disc) in combination with 3 ~g/disc of resazurin for gram positive bacteria in general and Enterococci in part1cular J 5 Anttbacterial Agent Antibacterial Agent ; concentration (~g/dtsc) for concentration AnttbacterTal Gram Postttve ~g/dtsc) for Aaent _ 8acterla _ Entorococct , . . -_ _ __ , Penicllltn G 0.0375 same Cloxaclllln 1.5 same Cephalothln 0.3 same Chloramphenlcol 1.5 7.5 Erythromyctn 0.3 same . Lincomycln 0.15 same :~ Tetracycllne 1.5 same Neomycln 1.5 same Streptomycln 4.5 same Amplclllln -* 1.5 * Note that sensltlvlty to penlclllln G Indlcates sensltlvlty to amplclllln.
. -18--~~ AHP-6781 4~
Preferred concentration of antibacterial agents (~g/disc) in combination with 15 ~g/dtsc of resazurin for gram negative bacteria . Antlbacterial Agent Antlbacterlal concentration Agent ~g/disc) for Gram Ne~ative ~acteria Ampiclll1n 4~5 Cephalothln lO
Chloramphenlcol 7.5 10 Carbenlclllln 24 Gentamlcln 3 Kanamycln 4.5 Polymyxln B lOO
TetracyclIne 1.5 Neomycln 7.5 :' , :: ' :.
, ~, . :
AHP~
$~14~) .: Percent correlatlon of the resazurin-antibacterial agent disc method ~results read at 6 hours) to the Kirby-Bauer method against 79 clTntcal isolates of StaDhy~occus pyoqenes.
Antlbacterial Aaent ~ Correlation Penlcillln G 98.7 Cloxaclllln 10O
Cephalothin 100 Chloramphenlcol 100 *Erythromycln 100 Lincomycln 100 Tetracycllne 98.7 *After 18 hours Incubatlon.
-20- .
~~ AHP-6781 .
TAE~LE 6 Percent correlation of the resazurin-antibacterial agent disc method (results read at 6 hours) to the Ktrby-Bauer method against gram negatlve bacterla , .
~ecle~_~nd ~ mfbers E. coll - 20 Ente~obacter sp. - 15 Proteus ~ 6 Proteus vulqacl~ - 8 .Prote~la mlr3bl I ts - 10 ~lebslella SD.- 12 Cltrobacter SD.- I
SeLcg~l~ SD-- 1O
Pseud,omQna~ aerualnos3 - 17 Total - 99 Gram negatlve bacterla.
: An ~ Aqent % Corre!atlon Amplcllltn 92 *Cephalothln 95 *Chloramphenlcol 98 Gentamicln 99 ; KDnamycln 99 *Polymyxln B 98 *Tetracycllne 99 , 25 f*18 hours Incubatlon necessary for: Cephalothln - E- co!l, E~tC9k~E~
Pro~ mlra,blll~si Chloramphenlcol - Proteus SD-, Klebsiella s~.;
Polymyxln B - ~err~tL~ SD-, ~S~y~ vu!~arls; Tetracycline - Serratia SD.
; ~ .
.
.
Claims (11)
1. An apparatus for determining the susceptibility of bacteria to antibacterial agents according to the standards of the accepted Kirby-Bauer method, said apparatus comprising:
a tray having a plurality of compartments; and a pad in each of said compartments, said pad consisting essentially of an inert material impregnated with a redox indicator and an antibacterial agent whereby said redox indicator gives a detectable indication of the susceptibility to said antibacterial agent of an inoculum of bacteria applied to said pad, each said pad being free from nutrient media.
a tray having a plurality of compartments; and a pad in each of said compartments, said pad consisting essentially of an inert material impregnated with a redox indicator and an antibacterial agent whereby said redox indicator gives a detectable indication of the susceptibility to said antibacterial agent of an inoculum of bacteria applied to said pad, each said pad being free from nutrient media.
2. An apparatus as claimed in claim 1, wherein said anti-bacterial agent is different for different one of said pads.
3. An apparatus as claimed in claim 1, wherein the concentration of said antibacterial agent is different for different ones of said pad.
4. An apparatus as claimed in claim 1, wherein said redox indicator is 7-hydroxy-3H-phenoxazin-3-one 10-oxide.
5. An apparatus as claimed in claim 1, wherein said inert material is absorbent paper.
6. A method for determining the susceptibility of bacteria to antibacterial agents equivalent to the standards of the accepted Kirby-Bauer method, said method comprising:
applying an inoculum of bacteria to a pad initially free from nutrient media, said pad consisting essentially of an inert material impregnated with a redox indicator and an antibacterial agent;
incubating said pad containing said applied bacteria; and detecting any change in said redox indicator as a measure of the suscepti-bility of said bacteria to said antibacterial agent;
said antibacterial agent, said inoculum of bacteria and said redox indicator being proportioned so that a color change indicates resistance of said bacteria to said antibacterial agent in accordance with the results obtained by the Kirby-Bauer method.
applying an inoculum of bacteria to a pad initially free from nutrient media, said pad consisting essentially of an inert material impregnated with a redox indicator and an antibacterial agent;
incubating said pad containing said applied bacteria; and detecting any change in said redox indicator as a measure of the suscepti-bility of said bacteria to said antibacterial agent;
said antibacterial agent, said inoculum of bacteria and said redox indicator being proportioned so that a color change indicates resistance of said bacteria to said antibacterial agent in accordance with the results obtained by the Kirby-Bauer method.
7. A method as claimed in claim 6, wherein said redox indicator is 7-hydroxy-3H-phenoxazin-3-one 10-oxide.
8. A method as claimed in claim 6, wherein said inert material is absorbent paper.
9. The method of claim 8, wherein the inoculum is 0.15 ml, prepared by diluting 100 times a brain heart infusion broth suspension of the bacteria, said suspension having an optical density of 0.07 at 625 nm in a 13 mm diameter test tube; the absorbent paper is a 0.5 inch diameter disc; the redox indicator is resazurin present in the amount of 1 to 40 µg per pad for gram positive bacteria; and in the amount of 1.0 - 100 µg per pad for gram negative bacteria.
10. A method for determining the concentration of an anti-bacterial agent in the pad of claim 1, said pad having a concentration of redox indicator so that the redox indicator changes color in response to the growth of bacteria within 3 to 36 hours at about 37°C, comprising:a) preparing a plurality of standard inocula of bacteria, a) preparing a plurality of standard inocula of bacteria, each inoculum being of a different strain of a particular bacterial species and the selection of strains including those known to be sensitive and those known to be resistant to the antibacterial agent according to the Kirby-Bauer method;
b) preparing a corresponding plurality of a series of pads, each pad impregnated with the antibacterial agent in a different concentration and a redox indicator in a concentration selected so that the redox indicator changes color in response to the growth of bacteria within 3 to 36 hours at about 37°C when inoculated with a predetermined amount of said standard inoculum;
c) inoculating each series of pads with the predetermined amount of a particular inoculum of bacteria;
d) incubating the series of pads at about 37°C; and e) selecting the concentration of the antibacterial agent that gives results equivalent to those obtained by the Kirby Bauer method.
b) preparing a corresponding plurality of a series of pads, each pad impregnated with the antibacterial agent in a different concentration and a redox indicator in a concentration selected so that the redox indicator changes color in response to the growth of bacteria within 3 to 36 hours at about 37°C when inoculated with a predetermined amount of said standard inoculum;
c) inoculating each series of pads with the predetermined amount of a particular inoculum of bacteria;
d) incubating the series of pads at about 37°C; and e) selecting the concentration of the antibacterial agent that gives results equivalent to those obtained by the Kirby Bauer method.
11. An apparatus for determining the susceptibility of bacteria to antibacterial agents according to the standards of the accepted Kirby-Bauer method, said apparatus comprising:
a tray having a plurality of compartments; and a pad in each of said compartments, said pad consisting essentially of an inert material impregnated with a redox indicator and an antibacterial agent whereby said redox indicator gives a detectable indication of the susceptibility to said antibacterial agent of an inoculum of bacteria applied to said pad, said pad having a concentration of redox indicator so that the redox indicator changes color in response to the growth of bacteria within 3 to 36 hours at about 37°C, and the concentration of said antibacterial agent in said pad being determined by:
a) preparing a plurality of standard inocula of bacteria, each inoculum being of a different strain of a particular bacterial species, the strains including those known to be sensitive and those known to be resistant to the antibacterial agent according to the Kirby-Bauer method;
d) preparing a corresponding plurality of a series of pads, each series of pads being impregnated with a different antibacterial agent and each pad within a series being impregnated with the antibacterial agent in a different concentration and a redox indicator in a concentration selected so that the redox indicator changes color in response to the growth of bacteria within 3 to 36 hours at about 37°C when inoculated with a predetermined amount of said standard inoculum;
c) inoculating each series of pads with the predetermined amount of a particular inoculum of bacteria;
d) incubating each series of pads at about 37°C; and e) selecting the concentration of the antibacterial agent for each series of pads that gives results equivalent to those obtained by the Kirby-Bauer method.
a tray having a plurality of compartments; and a pad in each of said compartments, said pad consisting essentially of an inert material impregnated with a redox indicator and an antibacterial agent whereby said redox indicator gives a detectable indication of the susceptibility to said antibacterial agent of an inoculum of bacteria applied to said pad, said pad having a concentration of redox indicator so that the redox indicator changes color in response to the growth of bacteria within 3 to 36 hours at about 37°C, and the concentration of said antibacterial agent in said pad being determined by:
a) preparing a plurality of standard inocula of bacteria, each inoculum being of a different strain of a particular bacterial species, the strains including those known to be sensitive and those known to be resistant to the antibacterial agent according to the Kirby-Bauer method;
d) preparing a corresponding plurality of a series of pads, each series of pads being impregnated with a different antibacterial agent and each pad within a series being impregnated with the antibacterial agent in a different concentration and a redox indicator in a concentration selected so that the redox indicator changes color in response to the growth of bacteria within 3 to 36 hours at about 37°C when inoculated with a predetermined amount of said standard inoculum;
c) inoculating each series of pads with the predetermined amount of a particular inoculum of bacteria;
d) incubating each series of pads at about 37°C; and e) selecting the concentration of the antibacterial agent for each series of pads that gives results equivalent to those obtained by the Kirby-Bauer method.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US73524776A | 1976-10-26 | 1976-10-26 | |
| US735,247 | 1976-10-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1112140A true CA1112140A (en) | 1981-11-10 |
Family
ID=24954961
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA289,457A Expired CA1112140A (en) | 1976-10-26 | 1977-10-25 | Bacterial sensitivity test kit |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA1112140A (en) |
| GB (1) | GB1535643A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5501959A (en) * | 1989-01-17 | 1996-03-26 | Alamar Biosciences Laboratory, Inc. | Antibiotic and cytotoxic drug susceptibility assays using resazurin and poising agents |
| US6982152B2 (en) | 2002-04-17 | 2006-01-03 | Promega Corporation | Cytotoxicity assay |
| WO2012051437A1 (en) | 2010-10-13 | 2012-04-19 | Life Technologies Corporation | Compositions and assays for determining cell viability |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4518565A (en) * | 1983-06-06 | 1985-05-21 | Miles Laboratories, Inc. | Reagent test device holder |
| US4728607A (en) * | 1984-03-22 | 1988-03-01 | J. K. And Susie L. Wadley Research Institute And Blood Bank | Miniaturized yeast identification system |
| US4847128A (en) * | 1984-03-22 | 1989-07-11 | Wadley Technologies, Inc. | Miniaturized yeast identification system |
| US5081033A (en) * | 1984-03-22 | 1992-01-14 | Wadley Technologies, Inc. | Miniaturized yeast identification system |
| GB2177508A (en) * | 1985-07-01 | 1987-01-21 | Dudley Roger Lester | Testing of soil |
| DD253570A1 (en) * | 1986-09-09 | 1988-01-27 | Medizin Labortechnik Veb K | COVERING FOR HOSE SYSTEMS |
| DD252543A1 (en) * | 1986-09-09 | 1987-12-23 | Medizin Labortechnik Veb K | PACKAGING FOR URIN AND SECRET |
| GB8915938D0 (en) * | 1989-07-12 | 1989-08-31 | Proteus Biotech Ltd | Apparatus for microbiological testing |
| US5206151A (en) * | 1990-06-11 | 1993-04-27 | Nalco Chemical Company | Rapid selection of biocide using a reduction oxidation indicator system |
| FR2676122B1 (en) * | 1991-05-02 | 1994-08-05 | Rsim | REACTIVE SUBSTRATE AND IDENTIFICATION PLATE FOR BODIES AND / OR ORGANISMS PRESENT IN A MEDIUM AND APPLICATION IN A BIOLOGICAL MEDIUM. |
| US6284526B1 (en) * | 1998-03-07 | 2001-09-04 | Wardlaw Partners Lp | Method and apparatus for determining the sensitivity of a microorganism to a growth altering agent |
| DE102013225037B4 (en) * | 2013-12-05 | 2016-08-25 | Asklepios Kliniken Verwaltungsgesellschaft mbH | Method for detecting resistant germs and device for carrying out the same |
-
1977
- 1977-10-24 GB GB4410877A patent/GB1535643A/en not_active Expired
- 1977-10-25 CA CA289,457A patent/CA1112140A/en not_active Expired
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5501959A (en) * | 1989-01-17 | 1996-03-26 | Alamar Biosciences Laboratory, Inc. | Antibiotic and cytotoxic drug susceptibility assays using resazurin and poising agents |
| US6982152B2 (en) | 2002-04-17 | 2006-01-03 | Promega Corporation | Cytotoxicity assay |
| US7282348B2 (en) | 2002-04-17 | 2007-10-16 | Promega Corporation | Kit for measuring cytotoxicity of a test agent |
| WO2012051437A1 (en) | 2010-10-13 | 2012-04-19 | Life Technologies Corporation | Compositions and assays for determining cell viability |
Also Published As
| Publication number | Publication date |
|---|---|
| GB1535643A (en) | 1978-12-13 |
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