CA1211349A - Composition, analytical element and method for the detection of bacteria - Google Patents

Abstract

-i-COMPOSITION, ANALYTICAL ELEMENT AND
METHOD FOR THE DETECTION OF BACTERIA
Abstract of the Disclosure A composition, an analytical element and method for the detection of bacteria in specimen samples, e.g. biological fluids, are disclosed. The composition optionally, but preferably, comprises a metabolizable substrate (e.g. glucose) and a benzin-dole dye which undergoes a detectable color change when incubated in admixture with a bacterial micro-organism. Useful dyes include particular benz[cd]-indole, benz[e]indole and benz[g]indole compounds.
The described analytical element contains this com-position, preferably, in a spreading zone. Detection of bacteria can be accomplished by bringing the com-position or element into contact with a specimen sample. This invention is particularly useful in detection of significant bacteriuria.

Description

~ ~3~9 COMPOSITION, ANALYTICAL ELEMENT AND
METHOD FOR THE DETECTION OF BACTERIA

Field of the Invention The present invention rel~tes to the detec-tion of bacteria in specimen samples with benzlndole dyes. More specific~lly, it relates to a composi-~ion~ an analytical ele~ent and a method using s~me for detection of bacteria in biological fluids, e.g.
uri.ne.
Back~round of the Invention For the rapid and effective diagnosis and treatment of infectious diseases, it ls desirable to be able to detect the bacteria causing the disease as rapidly as possible. Infections of the urinary tract are among the most common bacterial disenses, second in frequency only to infections of the res-plratory tract. In fact, in many hospitals, urinary tract infections are the most common form of noso-comial infections, often following the use of in dwelling catheters and various surgical procedureæ.
Most urinary tract infections (UTI) result from ascending infection by microorganisms introduced through the urethra and vary in severity from an unsuspected infection to a condition of severe sys-temic disease. Such infections are usually 8SSO-ciated with bacterial counts of 100,000 (105) or more organisms per mL of urine, a condition referred to as significant bacteriuria. Under normal condi-tions, urine is sterile, althou~h contamination from the external genitalia may contribute up to 1~000 ~103~ organisms pPr mL in properly collected and transported specimens.

~2~3 Significant bacteriuria may be present in a number of pathological conditions involving micro-bial invasion of any of the tissues of the urinary tract, or may result from slmple bacterial multipli-cation in the urine without tissue invasion. Theinfection may involve R Bingle site such a~ the urethra, prostate, bladder, or kidney, although fre-quently it involves more than one site. Infeetion restricted to the urine may present itself as asymp-tomatic bacteriuria, i.e., a condition which mani-fests no overt signs or symptoms of infection.
Early treatment of this condition can prevent the development of more serious conditions, e g.9 pyelo-nephritis ~inflammation of the kidney and the renal pelvis). The rapid detection of bacteris by a reli-able method would therefore facilitate an early and specific diagnosis.
Further~ in order to insure that a pre-scribed antibiotic is in fact effective in treating an infection, repeated tests during ~herapy are required. The need for simple, rapid bacteriuria tests is thus clear. Moreover, in view of the fre~
quent unsuspected asymptomatic occurrences of UTI
among children, pregnant women~ diabetics and geri-atric populations, diagnosis of which may requirecollection and testing of several specimens, bacter-iuria tests must be sufi~1ently simple and economi-cal to permit routine performance. Again, this il-lustrates the need for a rapid and inexpenslve bacteriuria detection method.
Current laboratory me~hods based on cul turing microorganisms, e.g., the calibrated loop-direct streak method, require significant ineubation periods (18~24 hours) before results can be de-termined. These laboratory methods are also time-consuming to perform and require considerable clini-cal training and faeilities.

:.

~31 3~

Known methods for the relatively rapid detection of bacteriuria include:
l. Uroscreen~ (triphenyltetrazollum chloride) is described in a brochur~ entitled "A
Simple Semi~quantitative Diagnos~ic Screening Method for PresumptivP Bacteriuria" published by Pfizer, Diagnostics Division, NY, 1974. Uroscreenl"
utilizes a dry, buffered tetrazolium reagent (colorless, soluble 2,3,5-triphenyltetrazolium chloride). Ln the presence of significant bacter-iuria3 Uroscreen~ will be reduced by the metabol--Lzing bacteria within 4 hours to a pink~red, insol-uble precipitate of triphenylformazan. This method has several disadvantages: it may not be accurate because bloody urine includes deposits resembling the pink-red precipitate of a positive test; and highly colored urine (caused by concentra~ion, bili-rubin, or drug ingestion) may obscure the results.
The accuracy of this test in detecting significant bacteriuria has been reported to be from abo~t 60 to about 90%.

2. Microstix~-3 Reagent Strips for Urinalysis are described in a brochure entltled "Test fur Nitrlte with Miniaturized Culture Tests for Total Bacterial Gram-Neg Bacterial Counts in Urine," published by Ames Co. ~Division of Miles Laboratories, Inc.), Elkhart, IN, 1976~
MicrostixNw3 is a firm, plastic strip to which ~hree separate reagen~ areas are affixed~ i.e. a chemical test area for the immediate recognition of nitrite in urine and two culture areas for semi-quantitation of bacterial population. The nitrite test, based on a modificat;on of the known Grless nitrite reaction, dPpends upon the conversion of nitrate (derived from di~tary metabolltes) to nitrite by the action of certain species of bacteriR

3~

in the urine. However, a negative result from this test does not provide proof that the urine is ster~
lle. This can be a problem, partlcularly -lf there are clinical signs or symptoms to indicate a bacter ial infection. Also, blood, Pyridium~, bilirubin, methylene blue, and other interferents in the sample may obscure the test results. This method also has several disadvantages inherent to nitrite tests: it must be performed on fir6t-morning specimens for optimum accuracy; it may give false negative results on specimens collected from patients on low-nitrate diets; it can be blocked or interfered wlth by phenzapyridine hydrochloride or any drug to which the bacteria are susceptible; it requires a large bac~erial population for positive results; and it will not detect microorganisms which do not reduce nitrate to nitrite.
3. Montgomerie, J. Z., Kalmanson, G. M., and ~uze, L. B. describe the catalase test in "The Use of the Catalase Test ~o Detect Signiflcant Bacteriuria," Am J. Med Sci., 251:94-97, 1966.
The catalase test is based on rapld gas production resulting from urea reacting with hydrogen perox-ide. This reaction is catalyzed by catalase avail-able from bacteria in the specimen sample~ Themethod has several drawbacks for detecting bacter-iuria, however: microorganisms which lack catalase, or those containing low levels of catalase may pro vide false negative results; and reduclng chemicals present in urine sapable of reacting with hydrogen peroxide may interfere with the test.
Other diagnostic agents for detecting bac-teria in biological and other fluids are described in U.S. Patents 3,496,0S6 (issued February 17, 1970 to Berger et al) and 3,621,016 (issued November 16, 1971 ~o Berger et al~. Such agents are metabollzed by bacteria present in the sample specimen to pro-duce detectable products.

3~

Hence, there is a need for n simple~ reli-able and inexpensive procedure for rapid detection of bacteria, and particularly significant bacter-iuria, which procedure avoids the problems presented with known procedures.

Summary of the Inventlon The present invention provides a composi-tion, an analytical element and a method for the detection of bacteria in specimen samples, e.g.
biological fluids, by the use of certain benzindole dyes. This invention overcomes the above-described problems inherent with known detection procedures.
In particular, among the adv~ntages derived from ~he present invention are: 1) rapid detection of bacteria, i.e., generally 60 minutes or less; 2) minimized color interference due to blood or other substan~es that may be present in the sample being evaluated (the preferred dyes, the benz[cd~indoles~
absorb maximally in the 620 nm region and reduce in the 400-450 nm region); and 3) sultability for use in both solution and dry chemistry ormats.
In accordance wlth this invention, a com-position for the detection of bacteria comprises a metabolizable substrate and a benzindole dye which undergoes a detectable color change when incubated in ~dmixture with a bacterial microorganism. Such dye is a benz[cd3indole, benz[e]indole or benz[g]-indole dye.
This inve~tion also provides an element for detecting bacteria. This element comprises a sup-port and a benzindole dye which undergoes a detect-able color change when incubated in admixture with a bacterial m~croorganism. The dye is benz[cdJindole, benz[e]indole or benz[g]indole dye~

~ 2 ~ ~ 3 This invention further provides a m thod for the detection of bacteria comprising brlngin~ a specimen sample for analysis into contact with a benzindole dye which undergoes a detectable color change when incubated in admixture with a bacterifll microorganism. Such dye is a benz[cd]indole~
benz[e]indole or benz[g]indole dye. The method can be practiced in either solution or dry analytical format.
Detailed Description of the Invention The present invention relates to the detec-tion of bacteria~ and particularly significant bac-teriuria ~i.e. at least about 10 5 microorganisms per mL), in specimen samples, such as liquids.
Although any specimen sample suspected of having bacteria therein (e.g. food, tissue, ground water, cooling water, pharmaceutical products, se~age, etc.) can be assayed, the invention is particularly useful for bacterial detection in aqueous liquids, such as human and animal biological fluids (e.g.
urine, spinal fluid, blood and the like as well as stool secretions) and suspenslons of human or animal tissue. The preferred biological fluid used in practicing this invention is human urine (diluted or undiluted~.
The benzindole dyes useful in the practice of this invention are capable of undergoing a detectable color change when incubated in admixture with a bacterial mlcroorganism, e.g. Escherichia coli. Generally, such dyes are selected from the group consisting of benz[cd~indole dyes, benz[e]-indole dyes and benz[g]indole dyes. However, 1~
should be understood that not all benzindole dyes are operable for this purpose~ The determination of whether a given benzindole dye is operable and 3~

within the scope of the present inventlon can be made by a simple test comprising incubating ~he dye in admixture with an aqueous suspension of ~ partic-ular microorganism at 37~C and observing whether the dye is reduced by the microorganism ~o produce a detectable change in the visible color o~ the dye.
Generally, a visLble color ohange occurs in the 400 to 700 nm range of the electromagnetic spectrum.
More than one dye can be used in the compositions of this invention, although preferably, only one dye iæ
used.
Benzindole dyes particularly useful in the practice of this invention undergo the described color change and have a structur~l formula selected from the group consisting of:

I. ~0 il i~ CH=CH )n A

R X

25 II. ~ ~ Rl R2 ~ o ( CH=CH~ G; and R

III. /---\
~-_0~ ~-\ = / ~ N-R5 L3~

wherein:
A is selected from the groUp congi6ting of:
~Ll \ _ / N ~ ;

-- --Z~----_ ~ N Rll ; and C--~CRg=CR ~
I

_ - -Z~2 --CH ~C ~N Rll ;
-~CRg=CR ~

R, R4, R5 and Rll are indepen-dently hydrogen, alkyl, aryl, alkaryl, ar~lkyl or cycloalkyl;
Rl and R 2 are independently hydrogen or lower alkyl, or taken together complete a 5- to 6-membered carbocyclic rlng;
R 3 iS aryl;
R 6 and R 7 are independently hydro-gen, alkyl~ cycloalkyl or aryl, or taken together complete a 4- to 20-~embered heterocyclic group;
R8, Rg and Rlo are independently 3 hydrogen, halog alkyl, aryl~ alkaryl, aralkyl or cycloalkyl;
G is -ORl 2 or R6-N-R7;
R 12 iS lower alkyl;

~2~3~9 Ll and L 2 are independently hydro-gen~ or Ll represents atoms taken with R 6 to complete a 5- or 6~membered ring or L 2 repre~
sents atoms ~aken with R7 to each complete a 5- or 6-membered ring;
Ar is aryl;
m and q are independently 0 or 1, n iæ 0, 1, 2 or 3;
p is 1, 2 or 3, except when G is R6-N-R 7, p iS 2 or 3;
t is ~, 1 Or 2;
s is 0, 1 or 2, except when n is 1, s is 1 or 2;
Z represents the carbon, selenium, sulfur or nitrogen atoms needed to eomplete a heterocyclic group;
Zl and Z 2 independently represent a single bond or the carbon, selenlum, sulfur or nitrogen atoms needed to complete a heterocyelic group; and X~ is a monovalent anion.
In the above formulae I, II and III, the dyes can contain one or more other non-interfering substituents which will not advPrsely interfer2 with the dye's solubility characteristics and cap~bility to change color in the presence of bacteria (i~e.
capability of being reduced by the bacteria). ~ny of the substituents described below, i.e. R, R
R2, R3, G, A~ etc. can lik.ewise have one or more of such non~interfering substituents att~ched thereto, as is known by a worker skilled in dye chemistry.
In the above formulae I, II and III, R, R4~ Rs and Rll can be hydrogen, alkyl ~substituted or unsubstituted), aryl (substituted or unsubstituted~ 9 alkaryl, aralkyl or cycloalkyl.

13~
-10~
Wher2 any of these groups is alkyl~ the alkyl chain can be straight or branched and preferably, but not necessarily, has from 1 ~o 12 carbon atoms (e.g.
methyl, ethyl, n propyl, n-butyl 3 n-heptyl~ n-nonyl, n-undecyl, n-dodecyl, etc. and isomer~ thereof).
More preferably, each of these groups is hydrogen or a lower alkyl having from 1 to 4 carbon atoms (2 . g .
methyl, n-propyl, isopropyl, t-butyl, etc.) Where R, R4, Rs or Rll is substi~uted, it preferably contains halo, hydroxy, (alkoxycarbonyl-alkyl)carbamoyloxy.
Where R, R4, Rs or Rll is aryl, it preferably, but not necessarily, has from 6 to 20 carbon atoms forming ring systems which are more preferably phenyl or naphthyl, and which can, if desired, h~ve one or more non-interfering substitu-ents attached thereto as described hereinabove (e.g.
halo, hydroxy, alkyl, oxyalkyl, etc.).
When R, R4, Rs or Rll is alkaryl or aralkyl, it is also pref~rred, but no~
necessary, that it have from 7 to 20 carbon atoms (e.g. benzyl, ethylenephenyl, 2-ethylenephenyl, etc.). Preferably, it is benzyl. Alsol any of these groups can be cycloalkyl ha~ing~ preferably from about 6 to about 20 carbon atoms (e.g. cyclo-hexyl, cycloheptyl, etc.). PrefPrably, the cyclo-alkyl group is cyclohexyl.
R~ and R2 in formula II abo~e are independently hydrogen or lower alkyl (preerably of from 1 to 4 carbon atoms as described for R 12 below), or taken together comp~ete a 5- to 6-membered carbocyclic rin8 (e.g. cyclopentyl, cyclo-hexyl, phenyl, etc.). It is preferred that R
and R2 be identically hydro~en or lower alkyl.
Where each is lower alkyl, it is particularly pre-ferred that each is methyl.

3~9 In formula III hereinabove, R 3 iS aryl as defined for R, R4, R5 or Rl,. Pre-ferably, R 3 iS phenyl (unsubstituted or substi-tuted as noted hereinabove).
S G in formula II hereinabove can be -OR~ 2 wherein Rl 2 iS lower alkyl of from l to 4 carbon atoms (e.g. methyl, chloromethyl, ethyl, isopropyl, t~butyl, etc.). Alternatively and pre-ferably, G is R6 N-R7 wherein R6 and R7 (here as well as in the I

group A) are independently alkyl or aryl as de-scribed hereinabove for R, R~, R 5 and Rll~ or taken together, R 6 and R7 can compl~te a 4- to 20-membered heterocycli~ group.
Preferably, R6 and R7 are independently lower alkyl of from 1 to 4 carbon atoms as described for R 12 hereiaabove. Most preferably, both - R6 and R7 are methyl.
R8, Rg and Rlo are independent-ly hydrogen, halo (fluoro, chloro, bromo and iodo), alkyl, aryl, alkaryl, aralkyl or cycloalkyl which are defined similar to R, R41 Rs and R~
hereinabove.
Ll and L 2 in formulae I and II are independently hydrogen, or Ll represents atoms taken~with R6 to complete a 5- or 6-membered ring and L 2 represents atoms taken with R 7 to each complete a 5- or 6-membered ring. Those rings could be a part of a larger ring having from 1~ to 12 membersO Preferably, each of Ll and L 2 iS hydrogen.
Ar ln formula III above is aryl preferably;
but not necessarily, of from 6 to 12 carbon atoms as described ~bove for R3. More preferably, Ar is phenyl or naphthyl.

34~31 Z in formula III pr~ferably represent6 the carbon, selenium, sulfur or nitrogen atoms needed to complete a 4 to 20-membered heterocyclic group, such as nitrobenzothiaæole, nitrobenzoxazole, nitro-benzoselenazole, nitro-3H-indole, imidazo[4,5~b]-quinoxaline, and pyrrolo[2,3-b]pyridine.
Zl and Z 2 independently represen~
either a single bond (e.g. between a nitrogen and a carbon atom), or the carbon, selenium, sulfur or nitrogen atoms needed to complete a heterocyclic group as defined for Z.
In formula I abo~e A is preferably ~ 6 and n is at least 1.
In formula II above, p is preferably 2 or 3.
X~ in all of the formulae represents a monovalent anion, which can be, for example?
p-toluenesulfonate; halide, such as iodide, chloride~ or bromide; acet~te; or perchlorate.
The benzindole rlng systems in all of the above formulae, I 9 II, and III, can be substituted with an appropriate electron withdrawing or electron-donating group, such as alkyl, aryl, alkoxy, cyano, nitro, halo or others known to those skilled in the art which will not interfere wlth the solubllity characteristics or other desirablc pro-perties of the dye.
As would be recognized by a worker skilled in the art, the dyes represented by formula III can exist in several tautomerie forms, depending upon what m and q are. The structure shown here~nabove clearly shows the dye structure when both m and q are 1. If both m and q are 0, the dye structure i8 represented by the formula:

IIIa. /-=-\

R-N~ =CH-CH - - - N Rs t X

wherein R, R3~ R5, Z and X~ are as defined hereinabove. If ~ is 1 and q is 0, the dye ~tructure is represented by the formula:

20 IIIb. =-O
R-N~ C -CH=CV- ~ N-R5 I Ar wherein R, R3, R5, Z and X~ are as defined hereinabovel If m is 0 ~nd q is 1, the dye structure i.s represented by the formula:

IIIc.

\~= ./ ~z--_~
R-N/~ CH-CH ~ ~ ~-Rs R3 ~4 2~ ~ 3 wherein R, R3, R4, Rs~ Z and X~ are as defined hereinabove. In formulae lIlb and IIIc, the dye cont~ins two positive charges and, heDce~ two balancing monovalent anions (which c~n be the same or different).
The dyes employed in the practlce o~ the present invention Are known in the art and m~ny of them are commercially avallable, for example, from Eastman Organic Chemicals, Rochester, N.Y. Some of these dyes are described in published references including, for example, U.S. Patents 3,501,312 (issued March 17, 1970 to Mee et al); and 3,505,070 (issued April 7, 1970 to Lit~erman et al~. Some of the dyPs are advantageously water soluble. However, in ~he practice of this invention as described in detail in the Examples hereinbelow, all dyes were prepared and stored as 10- 3 molar methanolic solu-tions although depending upon the extinctlon coeffi-cient of the dye, it can ke used in a concentration up to about 10 l M, and preferably in a range of from about 10- 7 to about 10- 2 M in the composi-tions of this invention. To minimize the possible effects of light a all the work reported in the Examples were performed under yellow lights and, un-less otherwise specified, all test tubes were incu-bated in the dark. Exemplary dyes useful in ~he practice of ~his invention are listed in Table I
hereinbelow. A preferred dye is:

~ \.
+N ~ - - CH= CH~ N ~C
C2Hs p-toluenesulfonate Although i~ is not necessary to include a metabolizable ~ubstrate in the elements and meth~ds of this invention, use of such substrates is prefer-red in order to stimulate bacterial metabolism and consequently, to facilitate detection. Any conven-~ional metabolizable substrate can be used ~9 i~
known to one skilled in the art Other terms for metabolizable substrate are "energy source" and "carbon source", the latter ~errn indic~ting that the compound is a source of metabolizable carbon for the bacteria. Suitable substrates are described in U.S.
Patent 4,035,~37 (issued July 12, 1977 to Masurekar et al) and include sugars, e.g. glucose, fructose, sucrose, lactose, maltose, raffinose, etc.; starch, salts of carboxylic acids, e.g. lactate, ~itrate, malate, succinate, etc.; glycols, e.g. glycerol, sorbitol, dulcitol, mannitol, etc.; and others known to a skilled worker in the microbiology art. The concentration of the metabolizable substrate gener-~lly ranges from about 0.01 to about 1 percent, byweight, of total composition weight when the compo-sition is used ln solution assay procedures.
The method and composition of this lnven-tion are adaptable to both solutlon and dry element assays. Thus, an aqueous or alcoholic solution con-taining the described ben~indole dye and, prefer-ably, one or more metabolizable substrates i~ pre-pared and bacteria are detected by contacting a sample of the specimen to be tested (e~g. a urine specimen) with a predetermined volume of the dye solution. Alternatively, the metabolizable sub-strate can be present in the test sample prior to addition of dye.

:

~Z3~3 In solution assay, generally the dye solu-tion is added to the specimen sample ~o be tested which is in a suitable container (e.g. tes~ tube, petrie dish beaker, other suita~le laboratory con-tainer). The resulting solution is gently mixed andincubated Eor a relatively short time ~i,e. less than about 60 minutes) at 37C. The color of the dye in the incubated solution is then observed (over a time up to several hours if necessary) and com-1~ pared to a control solution similarly prepared andhandled but withou~ any bacteria therein. If the incubated solution contains microorganisms, the dye therein will exhibit a visible color change, i.e. a change from one color to another, from a color to colorless, or from colorless to a color.
Alternatively, the benzindole dyes can be incorporated into a porous lamina, i.e. a a ma~rix or support of absorben~ material, such as filter paper strips 9 by impregnation or otherwise, to yield an ~n analytical element suitable for the detection of bacteria in a specimen sample deposited thereon.
In addition, ~he method is used to partic-ular advantage when carried out in an analytical element having a support and a spreading zone of the type described in U.S. Patents 3,992,158 (issued ~ovember 16, 1976 to Przybylowicz et al) or

4,258,001 (issued March 24~ 1981 to Pierce e~ al~.
The presence or absence of bacteria is then deter-mined by contactlng (e.g. spotting3 the element with 3 n the specimen sample suspected of containing bacter-ia. The specimen sample here is usually a suspen-sion of bacteria in a liquid (eOg. an aqueous liquid)~ The presence of bacteria in significant numbers will then cause a visible color change in 35 the benzindole dye located in the element.

i . ,,i~ ~

~2~3~

Ihe analytical element of this invention generally has a reagent zone containing the benzin-dole dye, and optionally, a metabollzable substrate described herein. This zone can be sel-supporting9 or alternatively, the element c~n also comprise a support. ~ere a single zone ls used, it is æome~
times called a spreading/reagent zone. The element preferably includes a support ~nd a plurality (at least a first and second) of zones. Preferably, the first zone is adjacent the suppor~. These zones are in fluid contact with each other, meaning that fluids can pass between superposed regions of adja-cent zones. Stated in another manner, fluid contact refers to the abîlity to transport components of a fluid between the zones in fluid contact. Prefer-ably, the zones are separate coated lay~rs~ although one or more zones can be in a single layer of an element. Suitable dry element formats are kn~wn in the art and described, for example, in U.S. Patent 399929158 no~ed hereinabove; as well as ln U.S.
Patents 4,042,335 (issued ~ugust 16, 1977 to Clçment~; 4,144,306 ~lssued March 13, 1979 to Figueras), 4,132,528 (issued January 2, 1979 to Eikenberry et al~; 4,050,898 (issued September 27, 1977 to Goffe et al); and ~eissue 30,267 (reissued May 6, 1930 to Bruschi).
The support for the element can be composed of any dimensionally stable material (e.g. poly-(ethylene terephthalate)) and is preerably trans parent.
~ ther materials and elements whlch are adapted for use in the practice of thls invention are ~escribed, for example, in U.S. Patentæ
3,092,4659 3,418,099, 3,~18,083, 2,893,~43, ~,8939~44~ 2,912,309l 39~0~,8799 3,~029~423 3,798,0649 3,~98,739, 3,915,6~7, 3,9179453, ~ Z ~ ~ 3 3,9939594, 3,936,357, 4,270,92~, 4,248,829, 4,255,384, 4,256,693, U.K. Patent 2~52,057 and Research Disclo~ure, Vol. 146, June 1976, Item 14638.
In a preferred embodiment of this inven-tion, the element includes a support having ~hereon and in fluid contact, reagent and spreading zones.
The benzindole dye described herein i8 preferably in the reagent zone which is a reagent layer ad~acent the support. The spreading zone is a spreading l~yer preferably adjacent the reagent layer, al-though there can be one or more intervening layers.
Preferably, this spreading layer comprises beads composed of poly(vinyltoluene-co-p~t-butylstyrene-co-methacrylic acid) and a suitable binderS if desired.
In the elements of this invention, the amount of the benzindole dye can be varie~ widely, but it is present generally in a coverage of up to about 5 g/m~ and prPferably, from about 0.05 to about 2 g/m2. Similarly, when present, the amount of the metabolizable substrate can be widely varied, but it is generally present in a coverage of from about 10- 3 to about 1 g/m 2 and preferably, from abou~ 0.01 to ebout 0.5 g/m2. The substrate can be present in any zon~ (or layer) of the element, but it is preferably in the same zone (or layer~ as the benzindole dye.
One or more of the zones of the elements of this invention can contain a variety of other desir able, but optional, components, including buffers, surfactants, and binders ~typically hydrophilic) as is known in the art.
Further details of the elements, particu-larly suitable components of the spreading zones,are given ~n U.S. Patents 3,992,158 and 4,258~001 3~3 -lg-noted hereinabove; and U.K. Patent Applicatlon 2,052,057 (published January 71, 1981). The ~pread-in~ zones, for example, can be composed of either fibrous or non-fibrous mater-lals, or both.
Exempl,ary elements are illustratQd herein-below in Examples 5 and 6.
The following examples are provided to illustrate the practice of thls invention. In these examples, Enterobacter cloacae ~ATCC 23355), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), Proteus w l~aris ~ATCC 13315), Pseudomonas aeruginosa (ATCC 27853), Serratia marcescens (ATCC 81003, Staphylococcus aureus (ATCC
25923), Staph~lococcus e~dermidis (ATCC 12228), and St~ptococcus pyrogenes (ATCC 19615) were obtained from Difco Laboratories, De~rolt, MI.
All cultures were routinely grown in 125 mL
flasks contaîning 50 mL of commercially-available brain-heart infusion medium and incubated at 37C
prior to use.
Where cell suspensions were used~ a 24-hour culture was centrifuged at 5000 xg for 10 minutes.
The resulting pellet was washed twice in 0.05 M
potassium phosphate buffer (p~ 7.0), and resuspended (1:4 aqueous dilution), to give a final optlcal density of 2.0, as measured on a Bausch and Lomb Spectronic~ 20 spectrophotometer at 620 nm.
To each 5 mL of cell suspension, 0.1 mL of 10% wlv glucose was added.
The urine samples employed in the examples were obtained from a local hospitalO To minimize microbial contamination and growth in the samples, two 5 mL urine samples rom the same source were placed in separate transport tubes supplied ln B-D
Urine Culture Kits obtained from Becton Dickinson and Company, Rutherford, NJ. These tubes contained a preservative, 0.5 mL borlc acld-glycerol-sodium orma~e, which maintains a stable bacterial popula-tion. The tubes were refrigerated and ~ll s~mples were used within 24 hours.
The urine samples were analyzed ~ccording to the following procedure: the contents of two 5 mL urine samples were combined in R 15 mL centrifuge tube and centrifuged at 10,000 xg for 10 minutes, after which the clear supernatant was discarded.
The resulting pellet was resuspended in 1.5 ~L of 0.05 M potassium phosphate buffer (p~ 7.0), and thoroughly mixed using a conventional high speed mixer. Unless otherwise specified, 25 ~L of a benzindole dye solution (10- 3 M dye iD methanol) were added to each centrifuge tube. The conten~s of each tube were ~hen gently agitated and incubated for 30 minutes at 37C in the dark. Preferably, a metabolizable subs~rate i 6 present during this incu-batioll. A scan of the vislble spectrum of each tube was then made using a Perkin-ElmPr 572 spectrophoto-meter. In most cases a a slit width of 1 nm and a scan speed of 120 nm/min. were used.
Control tubes containing 0.05 M potassium phosphate buffer (pH 7.3) and 25 ~L of benzindole dye solution were used in all studies. Where a metabolizable substrate was used, it was Qlso in-cluded in the control tubes.
Example 1: Color Change in Selective Benzindole Dyes in Pres_nce of Microor~anisms Cell suspensions of a urinary tract bacter-ia, E. coli were prepared as described above. To e~ch 5 mL of cell suspension, 0.1 mL of each dye solution conta~ning the dyes shown in Table I here-inbelow, was added. A readily utilizable metaboliz-able substrateg 0.1 mL of 10% w/v glucose, was added to each resulting mixture. Con~rol tubes without microorganisms were prepared as des~ribed above.
The contents of each tube were gently mix2d and incubated for 30 minutes at 37VC in the dark. The color of the dye was noted in each sample tube con-taining a cell suspension and ~ompared to the colorin the corresponding control tube. The r~sults, summarized in Table I, show benzindole dye6 that are useful in the practice of the present invention as well as dyes that are not as useful. All of the dyes which gave a color change (I, II, IV, V~
VII-XII, XXIII-XXVII and XXXI-XXXV) are useful in the practice of this invention, although some of them are particularly superior a notably those within formulae I, II and III described herein. DyP I is lS most preferred.

Table I
Microbially Catalyzed Color Changes with Benzindole Dyes Dye Visible Color Change I. ~.\

25ll/ ~i +~--CH=CH~ Blue to yPllow C 2H 5 p-toluenesulfonate II.
3~

-CH=CH--~ N/ Blue to yellow .~ N \. ./
C2Hs p-toluenesulfonate ~22-III. .
No ch~nge /+ ~ cH=cH-cH= ~

C2H5 CzH5 IV.
Il i Blue / \./ ~. to green -CH=CH-C}l'CH-CH=~

C2Hs C2H5 V.
o~ \c li I Blue-green ! 1l / '.' ~. to green 2~ ll i N+~ ~H C~-CH=CH-CH-CH-CH=~

C2H5 C2Hs VI.

~ 2 No change +~'-c~=cH-CH=, !l !

C2Hs C2H5 p-toluenesulfonate 3~

VII.

CH
o CH2 .~ \. . I
~ . Blue to green /+~-cH~cH-CH= - 1 11 !

C2Hs CH2 CH

p-toluenesulfonate VIII.

ll l-N~ Pink to clear C2H5 .~ \.
~0/ ClO 4 IX.

~ !! \O~ . 0~ Pink to clear \-~ N

3QC2~s .~
l~ ~I ClO 4 ` 24-X.
~o ! i! '.=.' ~. .~ Purple to yellow i1 ~1 +~--CH=CH~

C2Hs ~ \~
i~ !1 p-toluenesulfonate XI.
0~ \. .~ \.

15 i\ ~--N~ . Purple to yellow ./-~.

p-toluenesulfonate XII.

.~ \0 !~ /!1\ /cl Pin~ to 2 s / ~ /+~ \ colorles s !i !
ClO 4 XIII.
-~ CH3 CHg O~ \, CH3 CH3/-\ /~ No change \;/ ~i/+~-CHzCH~CH~

C2Hs C2Hs ~a3 XIV .
.~ CH3 CH3 .~ ~-li I/ - -\CH 3CH 3 / ~ NG Change \;/ ~ *~-CH=CH-CH

XV. CH3 CH3 \CH 3 CH 37 \O / ~. NO Change i! +~--CH=CH-CH=D~,N !l !

!l ~ lGH 3 1 ~ I

XVI .

\~ ~CH=CH~-~ / N~ NO Change Br 25 XVII.
.~ CH3 O_ .
+~-~CH=CH~-~ ~ N/ NO Change Br -~6-XVIII .

+\s-~CH=CH~ N/ No change Br XIX.

. / ~ . 1 3 li I +f--CH=CH~ N/~ No change C2~5 Cl04 ~X.
O~ CH3 /+f--CH-CH~f ~ / No change C2Hs XXI.

.f ~ 1 9 i I +~--CH=CH~-/ ~--N/ 3 No change CH2CH~OH
Br 34~

XXII.
./ ~ CH3 \i/ ~i/+~-~CH=CH-~ -N ~ No change ~CH2)30H
Br XXIII.
./ ~ CH3 ~ /+~--CH-CH-~ OCH3 ~16 hrs-) CH2CH20H Br XXIV.
2~ /~ CH3 \ _ / \CH3 co(l61hss `CH2CH20CONHCH2COOC2H 5 X~V .

o/ ~o \J/ ~ /+~--CH=CH-CH=CH~ N~ Blue to L3~

XXVI .
.~ CH3 il ~t +~--cH=cH-cH=cH~ N/ 3 Blue to H3 (16 hrs.) CH zCH 20H
Br 10 XXVII.
-~ C~13 il ~t +~--CH=CH-CH-CH~ --N/ 3 Blue to N ~= . H3 (16 hrs . ) (CH2) 30H
Br XXVIII .

s~ ~. /+~ - C~= CH~ N~ No s::hangP

XXIX .

~1 1 -CH3 .\- ~ t~ \i1 N~ ~hange \ / ~i~+~~CH=CH- \.=~

I

L3~L9 X~.

0 ~ n H 3C N/; \-=CH-CH=CH~ -CH 3 ./ No change i~
~ N
Br XXXI .
.= .
./' ._. ~_./ .
H3C N/~ \-=~-CH=CH~ CH3 0 Yellow to \ I colorless . (16 hrs~) ~i ~./ \. 1 1.1 l~ / ClO 4 XXXII .

N ~

H 3C-N/; \-=C-CH=CH- ~/ ~-CH3 ./ Green to \ l colorless \. (16 hrs/) ll ~. / ! ll - Br ~ 3 XXXIII

./ \._ 0~
\.=./ ~ \ /-~ ~ O2 H3C-N ~ /-=CH-CH=- il i ./ ~ / \.~ Yellow to I I colorless i~ l.l CH3(16 hrs.) p-toluenesulfonate ~XXIV
./ \._. C2Hs H3C-N ~ /-=CH-CH=~
./ ~ / ~ ~ \.~ Or~nge to 1 I colorless ~O~ C2Hs(16 hrs.

XXXV
.~'\.
.~. I !
~ O
H3C N/~ ;/-=CH-CH=~
1~ I Red to ~ / pink XXXVI .

CH2CH2N(C2~5) 2 CH3 .~ \.
C~ ~-\ /N\ CH~-\ ,!~ ,! No change i!~ CH=CH-CH- ~ i! !

C2Hs CH3 Example 2: Color Changes Induced in a Preferred -Dye Solution Using Urine Samples_with and without a Si&nificant Number of E.
-c_ 1~ The effects of urine samples, with and without a significant number of a common urinary tract bacteria (E. coli), on the visible spectrum of a preferred dye, were compared using the following procedure: Two urine samples 9 (A) containing at least 10 5 E. colilmL~ as determined by a localhospital and ~B) normal having little bacteria, were collected and prepared a~ dPscribed ~bove. Twenty-five microliters of a solution of benz~cd~indol~ dye I of Table I, prepared as above, was add~d to each tube. The contents o each tube were gently mixed and incubated for about 30 minutes at 37~C~ in the dark, after which the visible spectrum of each tube was scanned from 300 nm to 700 nm. A control tube ~C) prepared as described above was also ficanned.
large decrPase in the dye peak ~600-620 nm) and a concomitant increase in the ultraviolet reg~on of the spectrum was observed in the positive sample (A) as compared to the spectra of the normal urine sample (B~ and the control dye solution ~C).

. . ..

3~

Example 3: Color Changes Induced in Dye Solution6 Incubated with Pure Cultures o~ Micro-organisms Often Encountered ln Urinary Tract Infections The procedure of Example 1 was repea~ed with eight bacterial microorganisms often encoun~
tered in urinary tract in~ectlons (UTI~ using dye I
of Table I. The microorganisms tested were: (a~ ~.
coli, (b) S. epidermidis, (c) E. cloacae, (d) S.
a reus, (e) S. marcescens, (f) K. pneumoniae, (g) P.
aeruginosa, and (h) P. w lgaris. After about 30 minutes of incubation at 37C~ a visual color change was noted in each tube containing a cell suspen-sion. 5 Example 4: Comparison of the Rapid Scre~ &
Met~od of the Present Inve~tion with Standard Plate Culture Procedures The method of the present invention, util ized as a rapid screening prosedure for detecting significant bacteriuria, was compared to results determined by a conventional screening procedure, i.e., the calibrated loop direct s reak method.
Urine samples, obtained from local hospi tals, were evaluated using both the loop method and the method of this invention. Of 96 urine samples tested9 18 w~re determined to be positive ~i.e., contained 1007000 or more microorganisms~mL) based on the conventional calibrated loop method. Slxteen of these "po~ltive" samples were consldered to be positive by ~he method of the pre~ent invention.
The two samples determined as "positive" by the calibrated loop method and "negative" by the method of this invention were obtained from patients re~
ceiving long~term antibiotic treatment. It is believed ~hat the presence of an~ibiotics in the ~2~L~3~

urine for an extended period of time may affcct ~he physiological state of the bacterial cell and inhl-bit its reactlon with the benzindole dye useful in the practice of this inventlon.
In addition to the 16 "positivel' samples noted above, 7 additional samples showed "positive"
results using the method of the present lnvention.
These 7 samples 7 however, gave "negative" results using the calibrated loop method, although 5 of the 7 samples showed some bacterial growth on the cali-brated loop streak plates, as shown in Table II
hereinbelow. The "false positives" of the cali-brated loop method may be attributable to additional bacterial growth or changes in the physiological state of the cell between tests or to contamination durlng transport. On the other hand, they could also be attributable to errors inherent in the standard calibrated loop procedure.
The overall results, however, indicate good correlation between the two methods and substantiate the use of the method of the presen~ invention as a sui~able rapid screening procedure.

Table II
Plate Count Results of "False Positives"
Microorganism Number Proteus mirabilis 80J000 -E. coli 60,000 Mixed flora 50,000 E. coli 30,000 Pseudomonas species 10,000 Two samples had no significant bacter~al growth.

~L21~3~

Example 5: Multilayered Element for the Detection of Bacteri~l Activity Utilizin~ Benz-indole Dyes An analytical element was prepared ~ccord-ing to the following procedure:
A polyethylene film support was coated toprovide a spreading/reagent layer comprised of:
poly~vinyltoluene-co-p-t~butylstyrene-co~me~h~crylic acid~ (61:37:2 wt. ratio) beads tl7.2 g/m2)~ poly-(n-butylacrylate-co-styrene-co-2-acryl-amido-2-methylpropane, sulfonic acid) (50:40:10 wt. ratio~
binder (4 wt~%, based on bead weight) and a drop of Tween~ 80 surfactant (av~ilable from Altas Chemi-cal located in Wilmington, Delaware). Prior to coating, the beads had been soaked in an excess of a m~thanolic solution of benz[cd]indole dye I of Table I (10-3 M) 9 centrifuged and ~ashed several times with phosphate buffer to remove nonadsorbed dye.
Cell suspensions of E. coli were prepared as described above.
Bacterial evaluation was made by placing a 2 cm2 element sample into each of two test tubes, one tube containing 5 mL of the E. coli suspension ~nd the other tube containing only 5 mL of phosphate buffer (control). Both tubes were incubated at 37~Co for 35 minutes. The element samples were then removed from the tubes, allowed to dry, and reflec-tance measurements were determined on each sample with a Zeiss DMC 2~ spectrophotometer.
A higher reflectance density, measured between 600 and 650 nm, wa~ obtained from the element sample exposed to the microorganism than from the control sample.

~ 3 Example 6: EffQct of Anaerobic_Incubation Conditions on Microorgansim/Dye Reactions~ Usin~ _ Multilayered lement A polyethylene terephthalate support WRS
coated with a reagent layer compri6ing th~ benz~cd~-indole dye I of Table I (17~2 ~/m2 of a 10 3 M
methanolic solution)~ poly(acrylamide-co-2-~ceto acetoxyethyl methacrylate) (90:10 wt. ratio) and Zonyl~ FSN surfactant (available from DuPont located in Wilmington, Delaware3 and a spreading layer like that describ~d in Example 5 exc~pt without the dye.
A 1 cm2 sample of the element was put into each of 4 rubber-stoppered vials. Nitrogen gas~ saturated in water, was bubbled into the vials for 10 minutes by means of a 20 gauge canula. A
16 gauge needle served as the gas outlet.
Ten microliters of a suspension of E~ coli cells was injected into 2 of the vi81s and the same amount of potassium phosphate buffer was in~ected into the remaining 2 vials designated to serve as controls. All vials were continuously purged with N2 gas for 30 minutes. The element samples were then removed from their vials, air dried~ and the reflectance densi~y of each was measured at ~20 nm~
The two elements expos~d to the E. coli exhibited higher reflectance densities compared to the con-trols, shown ~s follows:
Vi~l ~
E. coli 1.85 -E. coli 1.83 Control 1~50 Control 1.52 3~

Exam~e 7: Com~arison of V~rious Benzindole Dyes This is a comparative example showing the absence of color change using selected ben~ndole dyes outside the scope of this inventiorl. Thes S dyes (~XXVII-XLVII) are listed ln Table III below and are described itl U.S. Patent 4,232,121 ~issued November 4, 1980 to Gilman, Jr. et al). These dyes are compared to benz[cd]indole dye I of Table I
which is within the scope of this invention.
The tests were performed by adding 5 mL of cell suspension containing E. coli to a test tube.
After the optical density was adjusted ~o 1 at 620 nm, 100 ~L of a 10- 3 M methanolic solutio of each dye and 100 ~L of 10% w/v gluco~e were added to the cell suspension. Control ~ubes without microorganisms were also prepared as described above. The conten~s of each tube were gently mixed and incubated for 4 hours at 37C. Observations for any color change w~re made at 30 minutes, 1 h~ur 2Q after the beginning and at ~.he end, of the incuba-tion p~riod. The results are listed in Table III
below.

Table III
Dye Visible Color Chan~e I.
a~ `\-~ /CH3 Blue to yellow i +~--CH=CH~ N ~
C2H 5 p toluenesulfonate 3~9 XXXVII .
/\./ ~, No change il ~ +~- ~ CH=CH-CH - ~
C2Hs C2Hs 10 XXXVIII.
~o //C)\ / ~ No change / ~i +~--CH=CH-CH= oi1 I

C2Hs C2H5 XXXIX .
No change 20~ Cl CH ~N, ~
C2Hs C2Hs Br 25 XL.

Insoluble;
CH~ pr~cipitated C2Hs C2Rs , ~, L3~L9 XLI .

CH=CH-CH= ~ i\C No ~hange XLII .

CH_CH_CH=~ i I i No change C2Hs C2Hs p-toluenesulfonate XLIII.
2Q li ~ CH=CH-CH= ~ prec~pitated C2Hs C2Hs p-toluenesulfonate XLIV, C 2H 5 alkyl o~0 change 30 ~I E ~ CH=CH-CH=~

C2H5 alkyl perchlorate 3~

X~V .

Il i ,/
\-~

~ H CH CH

;1 i i1 \.~ `.~
p-toluenesulfonate XLVI.

-N=N~CH=~ No change G2Hs C2H5 XLVII.
02N\ / ~ 2 Ij ~ c~=cH-cH-~ i No change C2~5 - ~2~1s The invention has been described in detail with particular reference ~o preferred embodiments thereof, but it will be understood th~t variations and modifications can be effected within the spirit and scope of the invention.

~;

: .

Claims (25)

We claim:

1. In a composition for the detection of bacteria, said composition comprising a metaboliz-able substrate and A benzindole dye which undergoes a detectable color change when incubated in ad-mixture with a bacterial microorganism, the improve-ment wherein said dye is selected from the group consisting of benz[cd]indole dyes, benz[e]indole dyes and benz[g]indole dyes.

2. The composition of claim 1 wherein said benzindole dye has a structural formula selected from the group consisting of:

I. II. ; and III. wherein:
A is selected from the group consisting of:

;

; and ;
R, R4, R5 and R11 are indepen-dently hydrogen, alkyl, aryl, alkaryl, aralkyl or cycloalkyl;
R1 and R2 are independently hydrogen or lower alkyl, or taken together complete a 5- to 6-membered carbocyclic ring;
R3 is aryl;
R6 and R7 are independently hydrogen alkyl, cycloalkyl or aryl, or taken together complete a 4- to 20-membered heterocyclic group;
R8, R9 and R10 are independently hydrogen, halo, alkyl, aryl, alkaryl, aralkyl or cycloalkyl;
G is -OR12 or R6-?-R7;

R12 is lower alkyl;
L1 and L2 are independently hydro-gen, or L1 represents atoms taken with R6 to complete a 5- or 6-membered ring or L2 repre-sents atoms taken with R7 to complete a 5- or 6-membered ring;
Ar is aryl;
m and q are independently 0 or 1 n is 0, 1, 2 or 3;
p is 1, 2 or 3, except when G is R6-?-R7, p is 2 or 3;
t is 0, 1 or 2;
s is 0, 1 or 2, except when n is 1, s is 1 or 2;
Z represents the carbon, selenium, sulfur or nitrogen atoms needed to complete a heterocyclic group;
Z1 and Z2 independently represent a single bond or the carbon, selenium, sulfur or nitrogen atoms needed to complete a heterocyclic group; and X- is a monovalent anion.

3. The composition of claim 2 wherein:

A is ;

R is hydrogen or lower alkyl;
G is R6-?-R7, R1 and R2 are independently hydrogen or lower alkyl;

R4 and R5 are independently hydrogen or alkyl;
n is at least 1; and p is 2 or 3.

4. The composition of claim 2 wherein said benz-indole dye is selected from the group consisting of:

;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ;
X- ; and X- ;
X-wherein X- is a monovalent anion.

5. The composition of claim 3 wherein said benzindole dye is p-toluenesulfonate

6. The composition of claim 2 wherein X-is p-toluenesulfonate, halide, acetate or perchlorate.

7. The composition of claim 1 wherein said benzindole dye is present in a concentration of up to about 10-1 M.

8. The composition of claim 1 wherein said metabolizable substrate is a sugar, starch, salt of a carboxylic acid or glycol.

9. The composition of claim 8 wherein said metabolizable substrate is glucose.

10. An analytical element for detecting bacteria, said element comprising a reagent zone containing a benzindole dye which undergoes a detectable color change when incubated in admixture with a bacterial microorganism, said dye selected from the group consisting of benz[cd]indole dyes, benz[e]indole dyes and benz[g]indole dyes.

11. The element of claim 10 whereia said benzindole dye has a structural formula selected from the group consisting of:

I. ;
X-II. ; and X-III, X-where in:
A is selected from the group consisting of:

;

; and ;

R, R4, R5 and R11 are indepen-dently hydrogen, alkyl, aryl, alkaryl, aralkyl or cycloalkyl;
R1 and R2 are independently hydrogen or lower alkyl) or taken together complete a 5- to 6-membered carbocyclic ring;
R3 is aryl;
R6 and R7 are independently hydro-gen, alkyl, cycloalkyl or aryl, or taken together complete a 4- to 20-membered heterocyclic group;
R8, R9 and R10 are independently hydrogen, halo, alkyl, aryl, alkaryl, aralkyl or cycloalkyl;
G is -OR12 or R6-?-R7;
R12 is lower alkyl;
L1 and L2 are independently hydro-gen, or L1 represents atoms taken with R6 to complete a 5- or 6-membered ring or L2 repre-sents atoms taken with R7 to complete a 5- or 6-membered ring;
Ar is aryl;
m and q are independently 0 or l;
n is 0, 1, 2 or 3;
p is 1, 2 or 3, except when G is R6-?-R7, p is 2 or 3;
t is 0, 1 or 2;
s is 0, 1 or 2, except when n is 1, s is 1 or 2;

Z represents the carbon, selenium, sulfur or nitrogen atoms needed to complete a heterocyclic group;
Z1 and Z2 independently represent a single bond or the carbon, selenium, sulfur or nitrogen atoms needed to complete a heterocyclic group; and X- is a monovalent anion.

12. The element of claim 10 comprising a spreading/reagent zone containing said benzindole dye.

13. The element of claim 12 wherein said spreading/reagent zone comprises polymeric beads of poly(vinyltoluene-co-p-t-butylstyrene-co-methacrylic acid).

14. The element of claim 10 wherein said benzindole dye is present at a coverage of from about 10-8 to about 1 g/m2.

15. The element of claim 10 comprising a metabolizable substrate.

16. An analytical element for detecting significant bacteriuria, said element including a support having thereon and, in fluid contact, rea-gent and spreading zones, said reagent zone contain-ing a benzindole dye which undergoes a detectable color change when incubated in admixture with a bac-terial microorganism, said benzindole dye selected from the group consisting of benz[cd]indole dyes, benz[e]indole dyes and benz[g]indole dyes.

17. The element of claim 16 wherein said reagent zone is a layer adjacent said support, and said spreading zone is a layer comprising polymeric beads of poly(vinyltoluene-co-p-t-butylstyrene-co-methacrylic acid).

18. A method for the detection of bacteria comprising bringing a specimen sample for analysis into contact with a benzindole dye which undergoes a detectable color change when incubated in admixture with a bacterial microorganism, said dye selected from the group consisting of benz[cd]indole dyes, benz[e]indole dyes and benz[g]indole dyes.

19. The method of claim 18 wherein said benzindole dye has a structural formula selected from the group consisting of:

I. ;
X-II. ; and X-III. X-wherein:
A is selected from the group consisting of:
;

; and ;

R, R4, R5 and R11 are indepen-dently hydrogen, alkyl, aryl, alkaryl, aralkyl or cycloalkyl;
R1 and R2 are independently hydrogen or lower alkyl, or taken together complete a 5- to 6-membered carbocyclic ring;
R3 is aryl;
R6 and R7 are independently hydro-gen, alkyl, cycloalkyl or aryl, or taken together complete a 4- to 20-membered heterocyclic group;

R8, R9 and R10 are independently hydrogen, halo, alkyl, aryl, alkaryl, aralkyl or cycloalkyl;
G is -OR12 or R6-?-R7;
R12 iS lower alkyl;
L1 and L2 are independently hydro-gen, or L1 represents atoms taken with R6 to complete a 5- to 6-membered ring or L2 repre-sents atoms taken with R7 to each complete a 5- or 6-membered ring;
Ar is aryl;
m and q are independently 0 or 1;
n is 0, 1, 2 or 3;
p is 1, 2 or 3 except when G is R6-?-R7, p is 2 or 3;
t is 0, 1 or 2;
s is 0, 1 or 2, except when n is 1, s is 1 or 2;
Z represents the carbon, selenium, sulfur or nitrogen atoms needed to complete a heterocyclic group;
Z1 and Z2 independently represent a single bond or the carbon) selenium, sulfur or nitrogen atoms needed to complete a heterocyclic group; and X- is a monovalent anion.

20. The method of clalm 18 wherein:

A is ;

R is hydrogen or lower alkyl;
G is R6-?-R7;
R1 and R 2 are independently hydrogen or lower alkyl;
R4 and R5 are independently hydrogen or alkyl;
p is 2 or 3;
n is at least 1; and X- is p-toluenesulfonate, halide, acetate or perchlorate.

21. The method of claim 18 wherein said incubation occurs in the presence of a metabolizable substrate.

22. The method of claim 21 wherein said metabolizable substrate is glucose.

23. A method for the detection of signifi-cant bacteriuria, said method comprising contacting a urine sample with a benzindole dye which undergoes a detectable color change when incubated in admixture with a bacterial microorganism, said dye selected from the group consisting of benz[cd]indole dyes, benz[e]indole dyes and benz[g]indole dyes.

24. A method for the detection of bacteria in a specimen sample, said method comprising bringing said sample into contact with an analytical element including a support and a spreading/reagent zone con-taining a benzindole dye which undergoes a detectable color change when incubated in admixture with a bac-terial microorganism, said dye selected from the group consisting of ben[cd]indole dyes, benz[e]-indole dyes and benz[g]indole dyes.

25. The method of claim 24 wherein said specimen sample is spotted onto said spreading/reagent zone.