CA1088157A - Microbial detection and enumeration apparatus - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The presence of microorganisms in a sample is determined by culturing microorganisms in a growth medium which is in contact with a measuring electrode and a reference electrode and detecting the change in potential between the electrodes which arises by the migration and accumulation of said microorganisms adjacent the surface of the measuring electrode thus forming a charge-charge interaction between said measuring electrode and accumulated microorganisms, by measuring the potential change with a high impedance potentiometer. In this divisional application, the invention pertains to an apparatus for detecting micro-organisms.

Description

5~7 This~ application is a diyision of Canadian Se~ial No.

268,06a, Ei~led December 16, 1976.

MICROBIAL DETECTION AND ENUME'RATION
APPA~ATUS
. _ . . . _ .
BACKGROUND OF T~IE IN~ENTION

Field of -the Invention The present invention relates to a method for detecting microorganisms which may be present in or on any source desired to be tested for the presence of microorganisms. More partic-ularly, the present ~nvention relates to a method of detectingthe presence of microorganisms by depositing a source of micro-organisms in a cell containg two electrodes, one of which is pro-tected from exposure to the microorganisms and a nutrient medium and detecting the change in potential between the measuring electrode exposed to the growing microorganism and the reference -electrode.
Description of the Prior Art .. _ ..... __ ~
Presently, several methods are known for the detection of microorganisms which may be present in various sources which include aqueous media such as blood, plasma, fermentation media and the like. The methods are generally divided into two classes of detection in which the first is a screening test to deter-mine whether or not large numbers of microorganisms are present in a sample~ If a positive test is obtained by the first step, a second level of testing is employed to determine the type and amount of organism present. Most commonly, methods which estab-lish both the identity and amount of microorganism are based upon a sequence of steps of culturing, growth and observation of the microorganism. Growth rates are o~served in the culture which are derived from multiple dilutions of th~ same sample.

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By observing the time at which the diluted samples reach observ-able populations, the concentration o~ microo~ganisms in the original samples can be estimated.
The estimation of microorganism populations is most generally accomplished by one of three techniques. The first technique is a nutrient agar~lating technique in which a microorganism is allowed to grow on an agar nutrient substrate and the growth of the microorganism is initially observed visually and there-after by microscopic observation. This is the most common method in use clinically.
The second type of technique includes several methods which can be classified as chemical methods. One method of analysis involves supplying a microorganism in a growth medium with car-; bon-l~ labeled glucose. The microorganism metabolizes the radioactive glucose and evolves cl402, which is sampled and counted. While positive results can be obtained by this method in a relatively short period of time, the method is encumbered by various operational complexities, is expensive and is hazard-ous from the standpoint of the necessity of handling radioactive samples. ~nother analytical method of determining the type and amount of bacteria present in a sample is based upon the chemi-luminescent reaction between luciferase and luciferin in the presence of ATP. Bacteria are grown on a culture and then the cells are lyzed to free the ATP present therein. The liberated ATP reacts with the luciferase-luciferin combination whereby chemiluminescent light is emitted which is detected by a photo-multiplier and used to determine the amount of the organism present. The principal disadvantage of this technique is the expense of the materials involved in the reaction. Still another chemical method involves- a measurement based upon the metabolic ' '' . ~
' , . . ~ ` .

~~ 7 conversi~on o~ nitrite ion to nitrate ion. This method, howeyer, is only appli~cable to some bacte~i~a and yeasts.
The third type o~ technique use~ for the detection of tnicroorganisms involve non-chemical methods. One method involves the evaluati~on of relatively~ clear microorganism suspensions by modi~fied particle counters. However, the method is non-specific and does not provide a distinckion ~etween viable microorganisms and dead ones, or even non-biologic particulate matter. A second method involves the detection of microorganisms by impressing an alternating current across a pair of electrodes which have been placed in a microorganism containing medium and observing -the change in impedance of the current as a function of the growth of the microorgatiism. This technique however, is apparent-ly not as amenable to automation as has been expected for the device and method.
A bio-chemical sensor is known as disclosed by Rohrback et al U.S. Patent 3,403,081, which is used to detect trace elements and poisons in liquid and gaseous media. The sensor is construct-ed by placing a measuring electrode such as an inert wire gauge cylinder of niekel, platinum, stainless steel or the like upon whieh is impregnated a eolony of a mieroorganism or an enzyme and a reference electrode such as the standard Calomel electrode in an eleetrolyte. The leads from each electrode are attached to a voltmeter. The organism or enzyme impregnated or in close proxlmity to the measuring eleetrode generates a current within the cell by causing ehemieal reactions at the surface of the electrode or by promoting chemical reactions to produce materials whieh in turn pro~ide depolarization reactions at the eleetrode. ;
The device functions by admitting a trace element or a poison into the device which deaetivates the enzyme or kills or deacti-yates~ the~microorganlsm thu$~ causin~ a change in the potentialdifference between the electrodes, which I`S: detected b.y a chan~e i~n the voltmeter read~ngs. Cons.i~deri~ng the fact that the enzyme or microorganism promotes or causes a cons~iderable chemical .reaction at the measuring electrode which is detected by a standard voltmeter in the circuitry of the cell, the current generated within the cell must be substantial. The electroanalyt-ical device of the present invention which is used to determine the type and amount of a microorganism in a solution, on the other hand, does not require the impregnation of a sizable colony of a microorganism on the measuring electrode but rather operates by detecting a microorganism in solution which gradually concen-trates about the measuring electrode. A further critical distinc-tion between the method and apparatus of the present invention and the method of the reference is that the circuitry of the present system must contain a high impedance potentiometer and . not the conventional voltmeter used in the reference's process, because the present system is dependent upon the measurment of an electrostatlc-like potential difference between the measuring 20 electrode and the microorganism in solution. If a standard volt-meter were used in the circuitry of the present invention, far too much current would be drawn by the voltmeter which would des-troy the relatively delicate electrostatic-like potential differ- -~ence between the measuring electrode and the microorganism con-centrated about the electrode's surface.
Consequently, a need continues to exist for a method and apparatus of rapidly, automatically and economically determining types and amounts of various microorganism by a conceptually simple and economic technique.
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~ ~ IIT ~y~NTION
The parent appli~cation No. 26;8,~,60 ~ets out various as,pects of a method and apparatus ~or detecti~ng the presence of a micro-organlsm in a fluid sample. In ~his di~vi~ional application, the invention as claimed pertains to an apparatus for the electro-chemical detection of livi`ng microorganisms in a fluid medium, which apparatus includes a conduit containing a hollow bore therethrough and having a flui~d sampling end. A conductive lead is attached to and in conductive communication with the conduit so that the conduit functions as a measuring electrode which responds to the charge-charge interact;`on set up by accumulation of the microorganisms about the electrode, when the apparatus is immersed in such a flu;d medium. Insulator means are concentri-cally dispoaed within the bore of the conduit and have an inter- ,~
ior bore running therethrough. A second conductive lead is disposed throughout the length of the interior bore which trav- ~, erses the insulator means. Means are provided for attaching the end of the second conductive lead as the fluid sampling end of the conduit to the insulator, which attaching means is impervious to the microorganisms within the fluid sample such that the fluid sample does not contact the second conductive lead. The leads of the apparatus are adapted for connection to a potentio- ;~
meter having an input impedance of 107 to 101 ohms.
BRIEF DESCRIPTION OF T~E DRAWINGS

_ . . . _ _ , FIGURE l shows a potential response curve for the growth of Pseudomonas versus time in 10 ml of Trypticase Soy Broth ~, medium; , FIGURE 2 shows two potenti`al versus~ time curves which demonstrate the growth, impedi~n~ i~nfluence of bacteriophage on bacterial strains of`E. coli;

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FIGURE 3 is an embodiment of one form of the electro-analytical cell of the present invention;
FIGURE 4 is another embodiment of the electroanalytieal cell of the present invention wherein the cell has been adapted to function in part as a syringe;
FIGURE 5 is an embodiment of the device of the present in-vention wherein the chamber of the device is equipped with means for drawing a microorganism containing fluid sample into the device under force of vacuum;
FIGURE 6 is an embodiment of the device of the present in-vention which in part is a needle suitable for the sampling of body fluids;
FIGURE 7 is a strip chart recording of potential versus time for the eulturing of E. coli.;
FIGURE 8 is a strip ehart recording of a series of cultures of E. coli wherein the baeterial concentration of the initial inoeula in eaeh sample was varied; :
: FIGURE 9 shows a comparison plot of potential versus time for a hydrogen produeing organism (E. eoli) and a non-hydrogen pro~
dueing organism (alkaleseens), appearing with FIGURE 7;
FIGURE 10 is a correlation diagram of potential and radio- .
activity at the measuring eleetrode as a function of cell con-centration for radioactive alkaleseens in TSB media;
FIGURE 11 shows two plots of potential versus time obtained from a platinum wire-ealomel eleetrode system and a nie~el-niekel eleetrode system in the same TSB eulture medium of alkalescens;
the nickel eleetrodes are positioned as in FIGURE 3;
FIGURE 12 shows several potential versus time curves which show the growth impeding influence of the antibiotic, ampicillin, : .
on S. epidermidis, appearing with FIGURE 2; and :

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~ U~ 13 s,how~s, s.eye"ral p,ot~nt:ial ~ers,us, ti~e cu~es which, show the ~rowth impedi~ng i~n1uence o~ cephalo-thin on K.' pneu-_ _nlae.

DETAILED DBSCRIPTION OP T~E P~E~E~ED EMBODI~lENT5 The discovery upon which. the method and apparatus of the present invention is based is that the presence of certain population levels of a given microorganism in a fluid medium confined in a cell containing a measuring electrode and a re- :
ference electrode generates potential changes within the cell because of a difference in electrostatic charge between a live ~:.
organism and the measuring electrode. The potential is measured .:
by a high impedance potentiometer which is fastened through ,', conductive leads to the electrodes of the cell. It has been .~':
found that all microorganisms exhibit a relatively negative ~ :
electrostatic charge in solution versus the electrode which is used as the measuring electrode which consequently renders all microorganisms amenable to detection by the method of the pre-sent invention. It is believed that the growing microorganisms gradually migrate to the exposed surface of the measuring elec- :
trode which is somewhat positive relative to the microorganisms. ' ~:~
In order that a potential difference exist between the measuring and reference electrodes, it is essential that the reference ~:
electrode either only contact the growth medium and not the micro- .
organisms or not be sensitive to the microorganisms's charge and that the microorganisms concentrate themselves about the measur- ' ,.
ing électrode. For example, when a metal wire or the like is '.
used as the reference electrode, the electrode is normally shield- '.
ed from the microorganisms, by a fluid permeable but organism impermeable substance. When a device such as the calomel elec-trode is used as the reference electrode, it is normally protect- -ed b~ the glass envelope from the microorganisms. The accumula-tion oE microor~ani,sm~ about the measuring electrode alters the potential of the measurin~ electrode relat~ve to the reference electrode and consequently, a change in potential is set up between the electrodes because of the charge-charge interactions at the meas-uring electrode. The voltage change generated by this electrostatic interaction is the means by which the presence of microorganisms in solution can be detected.
The cell structure which confines the growing microorganisms and to which the electrodes are attached can be manufactured from any convenient materials normally used in the manufacture of electroanalytical cells such as glass, plastic and the like.
Any material which is suitable for such use and which does not interfere with the growth or viability of the microorganisms ' and does not affect the voltage generated within the cell can be used.
The electroanalytical cell is provided with a culture medium for the growth of the microorganisms placed within the cell. Any culture medium which is commonly used for the growth of micro-,, organisms can be used, and therefore, the type of culture medium ' 20 used is not critical. Suitable growth media include brain-heart infusion, trypticase soy broth (TSB), phenol red broth base * 1%
glucose, trypticase soy broth + CO2, milk, beer, sodium glycolate ~, and the like. The amount of growth medium provided within the measuring cell, of course, is not critical.
The measuring and reference electrodes attached to the cell can be made of the same conductive material or they can be of different-~materials. Usually, the measuring electrode is fabri-cated of an~ suitable electrode material in any conven~ent form and is attached to the cell so that a portion thereof is in con-tact with the liquid medium within the cell. Suitable materials . _ g _ ' ` ' ..

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from which the measuring electrode can be fabricated include thenoble metals such as silver, platinum, palladium, gold, and the like, tungsten, molybdenum, nickel and the like, and various metal alloys such as stainless steel, nickel-chromium, and silver-palladium. The form of the electrode is not critical and can be of any suitable shape such as a wire, ribbon, coil or the like.
The reference electrode can be fabricated of any of the suitable electrode materials used for the measuring electrode.
Thus, conceivably the reference electrode can be of the same material as the measuring electrode, which is a most unusual electrode configuration for galvanic cells. Normally, the elec-trodes employed in galvanic cells are fabricated of different materials in order to have an operable cell. The reference electrode is also attached to the cell by any cDnvenient means in a manner such that a portion (such as one end of a wire or ribbon) of the electrode is in contact with the liquid medium in the cell. The portion of the reference electrode in contact with the medium is usually shielded from contact with the microorga-nisms in the medium. When the measuring and reference electrodes are o~ unlike metals or alloys, it is not necessary to shield the reference electrode since the mic~oorganisms will prefer-entially accumulate about the more positive of the two electrodes in the cell. On the other hand, when the measuring and reference electrodes are the same material, the reference electrode must -be shielded. Any means by which the reference electrode can be shielded from the growing microorganisms but at the same time allows contact with the liquid medium can be used. Thus, for example, the exposed portion of the electrode can be embedded within a gel such as an agar gel, gelatin, dextran gel, carrage nin gels- acrylimide and the like. Membranes such as relatively :

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lar~e pore ultra,filtration ,m,e.mbranes or eyen lar~e po,re mernbranes, frit, ceramic or porous ~ilms o~ plastic are all sultable if they are impermeable to the microorc~anisIms. If the reference electrod~ and the measuring electrode are ~abricated of the same material such as two stainless steel electrodes which is a pre- -ferred embodiment of the invent;on, two platinum electrodes or the like, the potential change generated at the sensing electrode cell is relatively free of thermocouple effects. That is, if the electrodes are the same, no voltage will be generated which is attributable to thermocouple effects. If the electrodes are .
fabricated of unlike metals or metal alloys, then the thermocouple effect can exist and the potential readings obtained should com- ., pensate for that portion of voltage attribu~able to the thermo- ,' couple effect. Thus, it can be appreciated that the thermocouple ' ef~ect which can arise with certain electrode combinations is : not critically restrictive and one operating according to the present method need only be aware of this factor.
l'he reference electrode besides being of the type described above, can also be a standard reference electrode such as one of the standard calomel electrodes or the mercury-mercurous sulfate electrode, the Ag/AgCl electrode, or the quinhydrone electrode. If one of these standard types of reference electrodes is used, the portion of the electrode within the liquid medium in the cell.need not be shielded from the microorganisms, because either the construction of these electrodes in a glass envelope normally eliminates contact of the interior working portions of the electrode with the microorganisms in solution or they have a potential which iS not ef~ected by micro~rganisms.

In ordex to measure the voltage generated within the electro-analytical cell contatning the growing microor~anism, it ls ~31~
necessary to connect both electrodes of the cell to a high impedance potentiometer~ The type of potentiometer used :1~
not critical with the onl~ requirement being that it be of the high impedance type. The potentiometer must have an input impedance over the range of 107 ohms to 101 ohms, preferably greater than 108 ohms. I~ a relatively low impedance potentio-meter is used, too much current would be drawn through the mea-suring device thus upsetting the charge-charge ~nteraction be-tween the measuring electrode and the microorganisms. With the destruction of the electrostatic potential at the electrode, no potential readings can be obtained. Of course, other apparatus accessories compatible with the high impedance potentiometer such as an amplifier and a recording device can also be added to the instrumentation package.
In the performance of a measurement according to the proce-dure of the present invention, a sample of a microorganism is introduced into the-growth medium within the cell. In one em-' bodiment, a fluid sample of a microorganism can be injected into the cell through a self sealing cap which seals the cell to the .... ....
atmosphere or is drawn into the cell under force of vacuum.
~, Microorganisms can also be introduced into the cell in the form of a gaseous sample with the stipulation that the microorganisms are introduced into the growth medium of the cell. A basic fea-i, ture of the present technique is that a stable baseline can be established by the recording instrument which is attached to the high impedance potentiometer and which plots changes in potential as a functlon of time be~ore the minlmum detectable concentration ~MDC) o~ the ~rowlng m~croorganism ~s attained. In other words,~:
the stable baseline is equivalent to a value of zero for the 30 function of dE~dt. Growth of the microorganism occurs and once ... .

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, the population leyel xeaches the minimum detectable concentrati~n normally of about 5X104 to 5X105 or~anisms/l ~1 for all except the slower ~rowing microorgan;~sms, the microorganism can be detected by the voltage generated. The voltage as measured in millivolt readings always changes in a negatiye direction.
This is because the charge on the microorganisms is more negative than that on -the measuring electrode. For slow growing micro-organisms such as the bacillus of tuberculosis, however, the lower detectable concentratlon can be as low as 1 X 103 organisms/
1 ml. In instances where the method of the invention is used to detect slow growing microorganisms, the cell can be agitated by shaking, f~rlinstance, or by any other suitable means.
Agitation of the microorganisms containing solution facilitated growth of the microorganisms in some cases and may thus reduce the delay time for the detection of the microorganism. However, since the present invention requires a net accumulation of organisms at th~ measuring electrode in order to have the neces-sary electrical charge-charge interactions, too vigorous stirring may sweep the microorganisms away and eliminate the potential change. The te~perature range over which the microorganisms are grown and eventually detected span the range of 15 to 60C, p~eferebly 32-37C. Pressure is not a critical factor in the measurements since growth and detection of the microorganisms can occur under partial vacuum conditions as well as under super-atmospheric pressures. Thus, for instance, the method of the present invention is amenable to the detection of microorganisms which grow under pressure as in the manufacturing of beer.
The method of the p~esent inYention can be used to success-: full~ detect an~ type o~ microorganism which can be cultured in the nutrient medium provided within the electroanalytical cell ~ 13 -,. -~ . . . ' .

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or added to the cell in a suitalbe concentration. Al~o it i.s not absolutely necessary for the microor~an~sms to be growiny or metabolizing pro~ided they remain alive. This is because it is their viability that gives them their negative charge, which they loose at death.. Thus, the method is applicable to the detection of yeasts, fungi and bacteria. Speci~ic examples of bacteria which can be detected by the method of the present invention include the non-hydrogen producing bacteria, Staphy-lococcùs aureaus, Staphylococcus epidermidls, Streptococcus, Li'~teria monocytogenes, Pseudomonas'a'eruginosa,'Moraxella, Shigella alkalescens, Diplococcus pneumoniae, Bacillus subtilUs, --and ~emophilus influenzae. Suitable examples of yeasts which can be detected include Candida species such as Candida albicans '' "'' "
and Candida ~ , Hansenula species such as Hansenula anomala, ~' Pichia species such as Pichia membranaefaciens, Torulopsis . . . _ . . .
species and Saccharomyces cerevisiae. Suitable examples of fungi .
include the various species of Aspergillus such as Aspergillus auricularis, A. barbae, A. bouffardi, A. calvatus, A. concentri-cus, A. falvus, A. fumigatus, A. giganteus, A. glaucus, A. glio-cladium, A. mucoroides, A. nidulans, A. niger, A. ochraceus, A. pictor, and A. repens and the species of the large group of -- ' -- .
fungi known as Fun~ Imperfecti.-A number of bacteria form a class of bacteria which areknown for their ability to release hydrogen during their growth.
These bacteria include Escherichia coli, Enterobacter aerogenes, ' Serratia marcescens, Proteus mirabilis, Citrobacter intermedium, ''' C'i'trobacter'freùndil,''S`a'lmonelIa and Klebsiella. Since these ''~
bacter1a ~igrate toward the measuring electrode as all micro- ~ ' organisms do, their release of hydro~en tends to concentrate :

about the measuring electrode.

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The presence of h~dro~en at the measuring elect~ode very substanitally amplifies the characteristics of the electrode such that it becomes similar to the well known hydrogen electrode.
Thus, in effect a completely different type of measuring elec-trode exists for the measurement of hydrogen producing organisms tha~ for non-hydrogen producing organisms. The principle behind the operation of the hydrogen electrode is that the following equilibrium exists at the sur~ace of the metal electrode, usually platinum or gold:

H - Pt ~2 ~

2-~ - H + 2e.
It is clear from this expression that an equilibrium exists be-tween molecular hydrogen and hydrogen ions in solution and it is the variations within the equilibrium that determines the potential of the electrode. Once an equilibrium has been estab-lished at the electrode sur~ace, the electrode is termed as a "nonpolarizable" or reference electrode. The normal hydrogen (reference) electrode is platinum in an acid solution of pH=O
with a saturated solution of molecular hydrogen at a defined temperature. The normal hydrogen electrode (NHE) is defined to have a poten~ial of 0.00 volts and forms the relative basis for ; the potential scale of all other electrode reactions as well as establishes the basis for the electro-motive series of metals. -In a more speciEic aspect of the measurement of hydrogen producing bacterla a calomel electrode is used as the reference ~; electrode in combination with a metal measuring electrode. The , :
calomel electrode has a potentlal of about +0.23 volts with espect to NHE ~nd since the growth medium has a pH of about neutral or 7, the me~surlng hydrogen electrode has a potential o~ about -0.42 volts with respect to NHE. Thus, the measuring electrode for hydrogen producing bacteria has a potential of , , ~ . : - ,, , . . . , ., : . . , . ::
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about -.65 yolts; 1,e, (-0.~2~ 0,23)- -0,65 in a ne~ti~e direction relative tv calvmel. Because~ in ~eality a pressure of one atmosphere of hydrogen is ne~er achieved At the measuring electrode because of atmos~heric dilution effects due to CO2, nitrogen and the like, a leveling-off potentlal of approximately -0.4 to -0.5 ~olts versus calomel is achieved in the measurements obtained for hydrogen producing bacteria.
In the measurement of both hydrogen and non-hydrogen produc-ing microorganisms, a la~ time is initially observed from the time the analytical cell is inoculated with the microorganism until the time the microorganism reaches sufficient population levels to be detected. Thereafter, the potential continues to increase in a negative direction until a peak potential is at-tained where the potential levels off. Thereafter, with advanc-ing time the potential drifts back to more positive potentials.
For non-hydrogen producing microorganisms, the total change in millivolt readings ranges from about 200-300 millivolts. In the case of hydrogen producing organisms, the initial negative in-creases in potential are attributable to the presence of the organism only. Thereafter, as the effects of the hydrogen build-up at the electrode become significant, the potential ; sharply increases to significantly more negative potentials than ~ -are obtained for non-hydrogen producing microorganisms. The negative increase in potential attributable to the presence of ~ -hydrogen at the measuring electrode amounts to about 500 milli-volts.
The potential xeadings obtained for each measurement repre-sent the sum of the change in potential ca~sed by the presence of the microorganism in solution and the potential changes att-ributable to other solution and system factors which cause minor ., ` ' ', ' ', changes in the poten-tial. These fact~rs a~e char~cteristic of the particular liquid medium present in the cell as well as the electrodes used and should be ascertalned by a potentlal reading of the medium ~ree o~ the presence o~ detectable amounts of particular microorganism to be tested. By achievin~ a measure of this back~round potential, one there~ore can readily determine that the total potential change is caused by the presence of the microorganism. ~n interestlng embodiment of the present inven-tion is that it affords a method of detecting a mixture of micro-organisms in a sample desired to be tested. When a sample con-taining a mixture of 2 or more types or species of microorganisms is cultured within the analytical cell, the microorganim which reaches its minimum detectable limit first i5 the one which will be detected by the system. With this knowledge, it is therefore apparent that the skilled artisan can alter the growth conditions such as temperature and the -type of culture medium to favor the growth of one type of microorganism to the detriment of other microorganisms and thus preferential:Ly detect one microorganism over another. For example, in order to detect fecal coliform ~0 in a mixture of microorganisms, the temperature o~ the growth medium can be ralsed to preferentially kill the other micro-organisms so that only the coliform remains.
The volume of the liquid medium within the electroanalytical cell is not critical. Of course, the liquid medium must be in ionic contact with both electrodes. The volume of the liquid growth medium can be any reasohable size which the skilled arti-san can readily determine. Of course, as the volume of the liq-uid ~s increased, the greater the dilution of the mlcroorganism, and the lower the resulting response obtaIned. ~t will be appreciated that the sealed electroanalytical cell can contain ' :::
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-a growth medium therein prior to introduckion of the microoryan-ism or the growth medium and sa~ple o~ microoryanisrQ can be introduced into the cell at the same time. The quantity of microorganisms introduced into the cell is not critical and need not be within any set limits.
A further understanding of the method of -the present in~en-tion can be achieved by reference to FIGURB 1 which shows two simultaneous potential versus time recordings of a Pseudomonas ; aeroginosa, initial inocula 10~ cells/ml sample culture in a trypticase soy broth mediu~ at 37 C over a period of several hours, in two separate electroanalytical cells. Each curve shows the potential response of the organism as a function of time. For the first hour or two the chart shows no significant response.
Thereafter, the organism begins to reach minimum detectable con-centrations as evidenced ~y the gradual increase in the potential reading in both cells. The curve then begins to recede gradually after reaching its peak value due to the eventual death of some of the cell population.
The invention thus far has been described in terms of directly ~
measuring the presence of living microorganisms in an electro- ~ ~-chemical cell. In another embodiment of the invention, substances or events can be monitored which change or destroy microbial via-bility such as exposing the microorganisms in the cell to an anti-biotic, serum antibodies, chemical toxins, viruses, e.g~ bacterio-'~ phage, or any other anti-microbial or anticellular event. Thus, `~ the effect of an anti-microbial agent or event can be monitored ` ~ by two methods:
by adding antimicrobial agent to standard dilutions of -i cultures at some concentration prior to reaching the MDC: and ~:~ 3n 2) by add;ng the anti-mlcrobial agent at or above the MDC.

,. -' 5q The method of the present invention is th.erefore use~ul in anti-microbial susceptibility testing for the investiya,tion of the activity of new antibiotics, or in the sur~eillance of develop-ments in an organism such as the development of antibiotic resistance by the organism~ Most importantly, n vitro evidence of bacterial-antibiotic interactions enables a clinician to predict the in vivo efficacy of a particular drug. The present method is useful in antimicrobial susceptibility testing when the causative organism doesn't respond predictably to antibiotics as shown is Table 1 below, and also when the organism is invariably susceptlble to a part cular drug as shown in Table 2 below.

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, 19 ' , , MICROORG~NISMS WITH UNPREDICTABLE ANTIMICROBIAL RESPONSE

Organism Agent to which organism may be_sensitive . .
. Staphylococcus aureus Penicillin G., methicillin, cephalothin, vancomycin Escherichla coli Amptcillin, cephalothin, tetracycline, kanamycin Klebsiella pneumoniae Cephalothin, tetracycline, : -kanamycin, chloramphenicol Pseudomonas aeruginosa Gentamycin, tobramycin, carbenicillin, polymixin B
Proteus mirabilis Penicillin (moderately resistant) ampicillin, kanamycin Proteus vulgaris Tetracycline, kanamycin, chloramphenicol Enterobacter aerogenes Tetracyclines, chloramphenicol :
kanamycin Salmonella Amplcillin, Tetracycline, cephalothin, kanamycin Shigella Same as above (Salmonella~
Listeria monocytogenes Penicillin G, erythromycin, chloramphenicol -- 20 - . .

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MICROORGANSIMS WITH PREDICTABLE ANTIMICROBIAL RESPONSE

AGENT TO WHICH ORGANISM
ORGANISM IS ~NIFORMLY SENSITIVE
Neisseria gonorrhoeae Penicillin G. erythromycin Diplococcus p_ eumoniae All penicillins, cephalothin erythromycin, vancomycin Streptococcus py-o--g-enes Same as Diplococcus Clostridium p_rfringens All penicillins, cephalothin, ~.
tetracycline Haemophilus influenzae Penicillin G, ampicillin, tetracycline, chloramphenicol Haemophilus pertussis Same as H. influenzae . . . _ . _ _ , Brucella Tetracycline _ _ , Corynebacterium dip heria Erythromycin, penicillin G
~ Penicillin G, ampicillin '"~ '.

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FIGURE 2 shows another exa~ple o,~ an a~ent which disrupts the growth of a microorganism. Curves ~" and B" show the potential-time responses for the yrowth of a strain of E. coli in a yrowth medium wherein d~f~erent concentrations of bacteriophage ~x~
i.e. 104 phage/ml and 105 phage~ml respectively were added to the growing microorganism after the MDC was reached. The time required for the potential to reach its peak after the virus is added is inversely related to the logarithon of -the virus concentration at the time of addition, i.e.

log C virus ~
J peak -~ MDC
By this technique the analysis of viral concentration in less than :
one hour can be achieved.
The followiny drawinys represent various embodiments of the electroanalytical cell of the present invention:
FIGURE 3 shows an embodiment of an electroanalytical cell in which chamber 1 is sealed with a resilient cap 2 which is self sealing~ Measuring electrode 3 is attached and secured to the chamber throuyh an opening (not shown) in the chamber wall.
Portion 4 of electrode 3 extends through the chamber wall so that its surface is exposed to culture medium 5 in the chamber through an opening (not shown~ in the base thereof. The portion 7 of the reference electrode ~ithin the chamber is shielded from any micro-organism which is present within the culture medium by shielding means 8. Shielding means 8, measuring electrode 3 and reference electrode 6 can be formed from the materials described abo~e. In the use of the above described de~ice, an appropriate quantity of a microorgamism containing fluid sample is injected into the chamber through the cap. The exterior terminals of the measuring and reference electrodes are then attached ~y leads to the appropriate terminals of a high impedance potentiometer and the medium is moni-tored as the microorganism grows.
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Another embodiment of the electroanalytical cell of the present invention is shown in FIGURE 4. In this device, the cell is essentially a syringe of chamber 11 and plunyer 12.
The base of the chamber is provided with a suitable hollow con-duit 13 which can be a hollow needle, rubber tube, or the like, one end 14 of which protrudes through the chamber and micro-organism shielding means 15 into the bulk of the culture medium 16 within the chamber. The device is equipped with measuring electrode 17 which is attached to the chamber through an opening (not shown)O Portion 18 of the electrode is in contact with the culture medium. Reference electrode 19 is attached and secured to the base of the chamber through an opening (not shown). That portion of the electrode within the chamber 20 terminates within shielding means 15.
The device of FIGU~E 4 is adapted so that the same device can be used to directly withdraw or expel fluids from a living body into a culture medium and be immediately attached to a potentiometer so that the growth of any microorganism present in the body fluid can be monitored.
Yet another embodiment of the cell of the present invention is shown in FIGURE 5. The device in this case in adapted so that a fluid containing a microorganism to be analyzed can be withdrawn into chamber 30 through entry line 31 via a vacuum .
applied to vacuum line 32. Both lines are shown as protruding ~:
through resilient sealing cap 33, however, it can be appreciated that both lines could be attached to the device through the walls ; of chamber 30 if desired. As the fluid sample is drawn into the device by the force of the vacuum, it falls upon and is mixed within culture medium 34. Measuring electrode 35 is at-tached to the device through an opening (not shown) so that the measuring end of the electrode (36) protrudes into the culture '~

~ - 23 -medium. Reference electrocle 37 is attached to the device in a similar manner at the base of the chamber so tha-t the end 38 within the chamber projects into a shieldiny means 3g which shields the end of the electrode from microorganisms in the culture medium. The device can be used in the same manner as the device under a force of vacuum rather than by injection through the sealing cap.
In still another embodiment of the device of the present invention, as shown in FIGURE 6, -the functioning parts are present within a needle. Thus, the device is fabricated of a conduit 40 which can be a hollow needle ins~ide oE which is concentrically disposed electrically insulating means 41. Lead 42 is attached to the exterior surface of the needle so that it functions as the measuring electrode. Wire 43 is disposed within the hollow inner core 44 of the insulating means and attached at the base of the insulating core 45 by a shielding means 46.
The needle-like device can be used by simply immersing the detecting end of the device into a microorganism containing fluid sample and after connectiny the leads of the device to a high impedance potentiometer, detecting the presence of - microorganisms in the sample. Alternatively, the needle-like device can be inserted into a subject, whereby the presence of microorganisms in the bodily fluids of a host subject can be detected in the manner described above. After simple removal of both insulator 41 and wire 43, or in the alternative simply wire 43, fluids can then be withdrawn from the subject in a conventional manner by the use of a syringe or equivalent device.
In another embodiment of the device of the present inven-tion (no t shown), the device of FIGURE 3 can be sealed or packed under a vacuum to provide a vacuum within the device. The base of the chamber of the device is provided with a sealed hollow needle whose attached end projects through the base and 35 shielding means into the culture medium. The device can be ~, ' , .
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used to withdraw a fluid contai~ning a mi~c~oorganism into the chamber by simply injectin~ th~e fEee end of the needle into the b-ody of an ani~al for instance, whereby -the force of the injec-tion and/or the ~lui~d pressure breaks the seal wi~thi~n the needle and the flui~d i`s wi~thd~a~n i~nto t~e devi~ce by the fo~ce of the vacuum.
The present i~nventi~on enjoys a wide vari~ety of fields of applicati~on, because the present i~nventi~on is applicable where-ever it is desired to detect the presence and quantity of microorganisms in a sample. Thus, the present invention can be used to detect the presence of ~acteria in a ~iological or non-biological fluid medium~ The method of the invention can be used as an antib;otic screening technique to determine the effectiveness of an antibiotic. For example, a microorganism can be cultured in a cell and an antibiotic added thereto.
The medium would then be observed to detect the influence of the antibiotic on the potential readings obtainedO Biological applications include thé detection of microorganisms in such body fluids as urine, blood, cerebro-spinal fluids, sputum, amniotic fluid, synoYial fluid, plasma and artificial kidney dialysate. The present technique is also amenable to throat tissue cultures, vaginal and cervieal tissue cultures and tissue biopsies. The present technique finds further application for . .
the detection of microorganisms in alcohol producing media such - -as from wood, grain, molasses, sulfite and waste liquors; from the production of wine and beers and from the production of glyc-erol. Still anot~er area of appl~cation is in the fermentation of organi`c acid$ such a~ lacti~c aci~d, ci`tric acid, fumaric acid and acetic aci~d. Yet another ~eneEal a~ea of appli~ca~i~lity i`s ~` 30 the food processi~ng i`ndustry~, parti~cularly i~n the packaging of ~ :~ ' ' .

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and the production of beYeEages and canned ~oods. Still further areas of appli~ca~ ty i~nclude Eu~al, ci~ty and ~egi~onal water suppli~es, water holdi\ng tan~s and septic tan~s.
Havi~ng generally descri~bed the i`n~ention, a further under-standing can be obtai~ned by~refe~ence to certain specific ex-amples whIch are provided ~erei~n for purposes of i~llustration only and are not intended to be li~iting unless otherwise speci-fied.
EXAMæLE 1 For t~e specific exper~mental procedure described below, the following hydrogen produci~ng bacterla were obta~ned from the American Type Culture Collection (Rockvi1le, Maryland~ U.S.A.
E. coll (12014), E. aerogenes ~13046), S~ marcescens ~13880), C._ _ termed`ium (6750), C. freundii (8090), and P. mirabilis (12453~. Cultures were maintai~ned at 5C on trypticase soy agar slants (TSA, BBL) and transferred monthly.
Inoculum preparation, viable counts, and media.
Inocula f~or the hydrogen measurements were prepared by making 10-fold dilutions of a 24-h Trypticase soy broth culture (BBL) in sterile 0.05% peptone broth and adding 3 ml of appropri-ate dilutions to 27 ml of phenol red broth base with 1.0% glucose (Difco~ prewarmed to 35C. In a limited number of tests, members --of the coliform group were tested in lauryl tryptose broth (Difco). Vlable counts were made by spreading appropriate dilutions from the 10-fold series on TSA and counting colonies after 35`hof incubation at 35~C. Viable counts were also made on each org`anism at the time of hydrogen e~olution and at t he end of 24 h of incubation.

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331 5t7 EIydrogen me:asurements~
The. experimental appa~atus: ~or mea5~uring ~y~ro~en eYolu-ti~on by the test or~ani~s~s cons~i~sted of a test tu~e ~25 by 90. mm.) contai~ni~ng two electrodes plus bPoth and organisms and posi~tioned i~n a 35C water bath. Leads from t~e electrodes were connected to a dc buffer ampli~fi~er (type 122, Neff, Inc., Duarte, Calif.l whi`ch in turn was connected to a strip chart recorder (model 194, Honeywell Indus-trial Div., ~ort Washington, Pa.). The dc buffer ampl~fier served to match the h~g.h i~pedence of the electrode test system with the str;p-chart recorder. Hydrogen evolution ~as measured by an increase in voltage in the negative ~cathodic) direction and ~as recorded on the strip-chart recorder.
The electrodes employed in the apparatus were a standard calomel (SCE-Beckman Instruments Inc., Fullerton, Calif.) as the reference electrode.cemented to a plastlc cap and a platinum :~ :
electrode was formed by shaping a str~p of platinum to fit the .:
circumference of the test tube; a section of the plati.num was positioned outside of a test tube fox attachment to the amplifier lead. During operation, the platinum electrode and test tube 20 were steam-sterilized by conventional autoclave procedures. The reference electrode (SCEl attached to the plastic cap, was.
~sterilized by exposure for 30 minutes to two ultraviolet lamps .
115T8~ General Electric Heights, Ohiol housed in a clear plastic ~ ~.
box. A number of tests demonstrated this technique to be . ~ :
effectlve in sterilizing the reference electrodes.
The strip chart recording of the millivolt response curve for l.q x 10 cells. of E. coli per millillter is s.hown in FIGURE
8. Characteristically~, for h~drogen produci`ng organisms, the record;`n~ shows~ a lag peri`od during which the mi~croorganism is grow~ng but i~s- pEesent in insu~ficient populations to give a r2sponse. Once the mi~croo-rgani~sm population has reached a . ..

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sufficient level, a response i5 noted whi~ch is attrihutable to the mi~croorganism. ~s the a~ount o~ hydrogen evolyed becomes suffi~cient, the electrode ~esponse characteristics rapidly change as noted by the sudden increase in potential. The period of decline after the maximum potential was reached (400 to 500 millivolts) occurred over a 3 to 4 hour period.
The relationship between inoculum si~ze and length of the lag period for various inocula of E. coli is shown in FIGURE 8 .
Lag times ranged from 1 hour for 106 cells~ml to 7 hours for 10 cells/ml which indicated 1) that each 10-fold increment of cells reduced the lag time by 60 to 70 minutes and 2) that the mean cell concentration at time of rapid buildup in hydrogen was l x 106 cells/ml. Because initial studies showed no diff-erences in the responsecurves for washed or unwashed cells, these studies were conducted with unwashed cells. In addition, no differences in response curves or lag times were noted from coliforms, E. coli, E._a rogenes, and C. intermedium, when tested ~` in lauryl tryptose broth or phenol red broth supplemented with glucose. Gas chromatographic analysis o~ headspace gas showed that for the cultures used in this study, the level of H2 for 24 h cultures was between 4 and 10% by volume. The only except-ion~was S. marcescens in which H2 was estimated as a trace (< 1%). Limited studies indicated that pH did not change marked-ly before or during the time H2 was detected.

FIGURE 10 shows a comparison of the potential characteristics of a hydrogen producing bacterium ~E. c~li) and a nonhydrogen producing bacterium (~lkalscen$~. Each bacterium was cultivated n a broth medium of laury~l tryptose glucose ~y Inoculating each medium with 1 ml o~ sample contai`ni~ng 100 cells/ml. The response . .
~ . .

characteri~s,tics, were meas,ure~ b~ a platinu~-calo~el electrode couple. The recordi~ngs,~ s~ow the much greater Ee~pons~ chaPacter-i`sti~cs i~n the sy~stem contai~ni~ng t~e hydrogen produci~ng bacteria compared to the response characteristi`cs of the non-hydrogen prodùcing bacteri~a. Note that the same la~ time i~s obser~ed when the i~nitial inoculum and gro~th rates are the same.

The data in FIGURE 10 provide a correlation which show that the change in potential of a culture medium of alkalescens as a function of cell concentration. A 1 ml s-ample of radioactive alkalescens ~initial innoculum size 100 cells/ml) was cultured ~, in a 10 ml Trypticase soy broth medium at 37C. Each time a potential reading was made, a sample of the electrode was taken and measured for radioactivity-. The plots clearly indicate corresponding increases in potential and radioactivity as a function of cell concentration. The data clearly indicate that the cells increasingly concentrate about the surface of the ' electrode and that the potent;~al res~ponse is caused by this --~
build-up of cells about the electrode.

.
A 1 ml sample of alkalescens was cultured in 10 ml of Tryp- ;
ticase soy broth medium for 7 hours. The growth of the bacterium was monitored at the same time by two different electrode systems, , i.e., a platinum wire-calomel system and a nickel-nickel system ~' ' in which one nickel electrode was shielded from organisms by a ~ ' '' glass frit. As can be ascertained by reference to FIGURE 11, both curves obtained are essentially the same except that the ni`ckel-nickel electrode system does not appear to ~e-quite as sensitive as the platinum--calomel s~ystem.

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A l ml sample of Pseu omonas aerogenosa was cultured in trypticase soy broth in a cell ~escri~bed ~n FIGURE 3 in which both the measurement and reference electrodes were type 3 or 4 stainles-s steel. The shi~eldi~ng material in this example was peptone agar with lO mg/ml NaCl. When the cell population of 9 x 104 cells/ml was reached, the potential change was detected às shown in FIGURE l.

. _ The same procedure described in Example 5 was followed with the exception that the organism cultured was E. coli in a medium f Trypticase soy broth. A potential reading was observed at a population level of 1.2 x 105 ce~ls/ml.

i The same procedure of Examples 5 and 6 was followed with the exception that the growth medium was sodium glycolate medium and the organism was _igella alkalescens. A potential reading was observed at a population level of 8.6 x 104 cells/ml.

The same procedure of Examples 5-7 was followed except that the growth medium was sodium glycolate ~ 0.5 ml of human blood and the organism was Hemophilus influenzae. A potential reading was observed at a population level o 2.4 x 105 cells/ml.

The same procedure of Examples 5-8 was followed except that the organism was Bacillus eubtilus in l~ nutrient broth with 0.5~ NaC1 at 30C. ~ potential readi~ng was o~served at a pop-ulati~on level o~ 2.3 x 103 cell~/ml.
EXAMP`LE-lO
---.. _ _ ~ 30 The same procedure of Examples 5-9 was followed except that ;

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the or~anism was Candida tro~i~canas and the growth ~edi~m was 10% nutxient broth ~i~th~ 30~ ~extEose at 3UC. Detection was at 3.1 x lU5 cells/ml.

.
The cell of FI`GURE 4 was~used to culture the fungus Asper-gillus niger i~n trypti~case soy broth at 25 C. A potential readi~ng was observed at a populati~on level of 104 cells/ml.

A culture of P'se'udomon'as aer'oy'i`nosa was washed in non-nutrient buffer (Phosphate ~uffeP solution or PBS)i and "starved"
at 15C for 48 hours. Cells maintained in this condition are not dividing or metabolizing bu~ are technically alive. There-fore they still have a net negative charge. These cells were then added to a cell described in FIGURE 4 with stainless steel electrodes but the nutrient ~roth was replaced with PBS. At a population of 2 x 10 viable cells/ml a signal was observed.

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Two samples of bovine milk were added to cells of the type described in FIGURE 3. One sample was Pasteurized and one 2Q was "raw" milk. The milk served both as a growth medium and inoculum in both cases. The electrodes were stainless steel ~"` ''' and the memb~ane was saline agar. The experiment was conducted at a temperature of 37~C. Each sample produced a signal at i around 10'--,cells/ml but the "raw" milk developed the signal in ,~ 2 and 1/2 hours whereby the Pasteurized milk reached 105 cells and gave a signal at 4 and 1/2 hours.
EXAMPLE' 14 Antimicro~a'l'Su's'cept'i~i`li'ty~ Tests Re'ag'en't~s,_an'd Medi~a Identical lots of media and anti~i~crobial agents were used i . ' .
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in the susceptibility tests performed. The media used were trypticase soy agar (TSA) and TSB from Baltimore sioloyical Laboratories (BBL), Cockeysville, Maryland. The 12 antimicrobial agents used in the experiments, which were chosen on the basis of their clinical usefulness, were laboratory reference standards including the following antibiotics: penicillin G potassium, caphalothin sodium, tobramycin, vancomycin hydrochloride and streptomycin sulfate obtained from Eli Lilly and Co.; tetracycline and carbenicillin obtained from Pfizer Laboratories; ampicillin and sodium nafcillin from Wyeth Laboratories; kanamycin and meth-icillin from Bristol Laboratories; gentamicin from Schering.
The action of an antibacterial drug against a susceptible bacterium is either bacteriostatic or bactericidal. A bacterio-static agent merely inhibits bacterial growth, an effect which is reversible upon removal of the antimicrobial agent. Bacteri-cidal agents produce an irreversible, killing effect on the ` microorganism susceptible to the drug. Of the 12 antibiotics, all are bactericidal with the exception of tetracycline which is bacteriostatic. Penicillin G, ampicillin, nafcillin, meth-icillin, and carbenicillin all belong to the penicillin family and as such their mode of action involves interference with the synthesis of cell walls of bacteria. This is also true of cephalothin, a cephalosporin, and vancomycin, a glycopeptide.
The aminoglycoside antibiotics kanamycin, streptomycin, and gentamicin cause specific misreadings of the genetic code at the ribosomal level, thereby interfering with the protein synthesis of the bacterium. This also results from the use of tetracycline, which specifically prevents attachment of amino acid activated transfer RNA to the ribosomes.
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CU1 tures, The followin~ cultures were obtai~ned from ~lof~an-LaRoche, Inc. and were deri~ed from t~e kmePlcan Type Culture Collection (ATCC~ organisms as indicatea: E's'che`r'i~chia coli, "Seattle Strain"
ATCC 25~22, Klebsiell'a pneumoniae ATCC 27736, Pseud`omon'as aeru-. . , ginosa, ATCC 9721. These bacter;~a are gram negative bacteria.
The following bacteria are gram positive: Staphylococcus aureus, "Seattle Strain" ATCC 25923; Staphu`l'o'c'oc'cus ep`i'd'ermidis ATCC
149~0; and Streptococcus pyogenes, ATCC 10389. In addition, clinical specimens of these same six organisms were obtained from sensitivity isolates from the University of Virginia Hospital Department of Clinical Pathology Bacteriology Section. These organisms, both pathogenic and non-pathogenic (Staphylococcus epidermidis), were chosen because they are some of the most frequently encountered organisms in clinical isolates.
Inoculum Preparation and Viable Counts ~ .
Inocula were prepared by making a 100-fold dilution x 2 of an overnight TSB culture of the or~anism to be tested in sterile TSB prewarmed to 35C. Thus, the total dilution of overnight stock was 10 . From the 10 series, 0.3 ml was transferred to a test tube (Falcon, disposable ~2057) containing 2.7 ml of TSB if the growth control, or 2.6 ml of TSB plus 0.1 ml of a partlcular concentration of an appropriate antibiotic to be tested. The antibiotic was weighed on an analytical balance, according to each agent's activity standard, and diluted with sterile distilled water in'a volumetric flask to achieve a stock solution of 1000 mcg/ml which was then frozen in small aliquots.
Further d;`lutions as necessa~y for susceptibility testi`ng were '' also achieYed throu~h the a~di~ti~on of sterilè dis~ti~lled water to this stock concentration~ Viable counts were made by plating -`
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appropri~ate dilutiorls from the ].0 4 seri~es. on TSA and counting colonies~ after 24 hours i.ncubati~on at 35~C. Via~,le counts were also made on each mIcroo~ganism at tKe time of ~DC ~nd again at the end of the 24 hour test period to confirm the minimum inhibitory concentration (~IC~,.
The experimental apparatus used in this study to monitor the inhibition of growth due to the addition of antibiotics consisted of test tubes, up to and including 8, size 17 x lO0 mm, each containing a sterilized (by Boiling) Pt-SOE combination . 10 electrode (Sargent-Welch, S-30101-15), plus TSB, microorganisms, -and antibiotic where appropriate, positioned in a 35~C heating block. Leads f~om the electrode were connected to a high imped-ance (.greater than 108 ohms~ potentiometer, or voltage following device, which in turn was connected to a strip chart recorder (~Iewlett-Packard type 680M). Microbial growth was measured by an increase in voltage in the.negative direction with time as recorded on the strip chart recorder.
FIGURE 12 is a strip chart recording of the antimicrobial response~:c:urve of a four channeled experiment involving a clinical specimen of the non-hydrogen producing, Gram-positive bacteria ;~ Staph-l~c.-c~ rmidis treated with 0.012, 0.5 and 1.0 mcg/ml .~ of the antibiotic ampicillin as shown in curves B, C and D ~.:
, respectively. Curve A shows the response curve of a control -sample of S. epiderm_dis which has not been treated,with-ampicill'in~
FIGURE 13 is a strip chart recording of the antimicrobial response ~ -~ curve.of a six channeled experiment involving an ATCC culture : sample o~ the pathogenic hydrogen producing, Gram-negative --~
bacteria'K'leb'si'e`l'la_~neumoni'ae treated with Q.3, 0.4, 0.5 and l.0 and 2.0 mcg/ml of cephalothin as shown is curves B', C', ,: 30 D', E' and F' respectively. Curve A' shows the response curve ~ 34 -.

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of a control sample of K. pneu~oniae which has not been t~eated with cephalotnin. soth ~ ures show that the growth of the respective bacteria I5 InCPeaS~ingly inhibited ~y~ the particular antibIotic material used. The minimum inhibitory concentration (MIC~ is defined as that amount of antimicrobial agent which totally inhibits the grow-th of the microorganism being tested.
Tables 3 and 4 below-show the MIC values of various antibiotics for a num~er of microorganisms as determined by the method of the present invention. ~

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Hayi~ng now ~ully des,c~i~hed thi~s i~nyention, it will he apparent to one o~ ordi~nary~skill in the art that many chan~es, and modificatIons can be made thereto without departi~n~ from the spirit or scope of the in~ention as s-et forth herein.

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Claims (3)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An apparatus for the electrochemical detection of living microorganisms in a fluid medium, the apparatus comprising:
a conduit containing a hollow bore therethrough and having a fluid sampling end;
a conductive lead attached to and in conductive communication with said conduit so that said conduit functions as a measuring electrode which responds to the charge-charge interaction set up by accumulation of microorganisms about said electrode, when in operative association with such a fluid medium;
insulator means concentrically disposed within the bore of said conduit and having an interior bore running therethrough;
a second conductive lead disposed throughout the length of said interior bore which traverses said insulator means;
means for attaching the end of said second conductive lead as said fluid sampling end of said conduit to said insulator which attaching means, while being perviuos to the fluid medium is impervious to the microorganisms within such a fluid medium; and the leads of said apparatus being adapted for connection to a potentiometer having an input impedance of 107 to 1010 ohms.

2. An apparatus for the electrochemical detection of living microorganisms in a fluid medium which comprises:
a conduit containing a hollow bore therethrough and having a fluid sampling end;
a conductive lead attached to and in conductive communication with said conduit so that said conduit functions as a measuring electrode which responds to the charge-charge interaction set up by accumulation of said microorganism about said electrode;
insulator means concentrically disposed within the bore of said conduit and having an interior bore running therethrough;
a second conductive lead disposed throughout the length of said interior bore which traverses said insulator means;
means for attaching the end of said second conductive lead as said fluid sampling end of said conduit to said insulator which attaching means, while being pervious to said fluid medium into which said apparatus is immersed, is impervious to the microorganisms within said fluid medium; and a potentiometer having an input impedance of 107 to 1010 ohms which is conductively connected to said measuring electrode and to said second conductive lead.

3. The apparatus of claim 1 or 2, wherein said attaching means is a plug of gel, pervious to said fluid medium and impervious to the microorganisms within said fluid medium.