CA1071711A - Microbial detection and enumeration method and 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.

Description

107171~.

MICROBIAL DETECTION AND ENUMERATION
~, METHOD AND APPARATUS__ .- BACKGROUND OF T~IE INVENTIOI~ . .

Field of the Invention The present invention re1ates to a method for detecting microorganisms : which may be present in or on any source desired to be tested for the pre-sence of m;croorganisms. More particularly, the present invention relates . .
to a method of detecting the presence of microorgatlisnls by depositing a .
source of microorganisms in a cell contain;ng two electrodes, one of which .
IO is protected from exposure to the microorganisms and a nutrient medium and detecting the change in potential between the measuring electrode expos d to . the growing microorganism and the reference electrode.
Description of the Prior Art Presently, several methods are known for the detection of microorganisms lS which may be present in various sources which include aqueous media such as blood, plasma, fermelltation media and the like. The methods are generally .
divided into two classes of detection in which the first is a screening ;
test to determine 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. Mo connlonly, nethr,ds which establish brth the ;dentlty and ". .

.~
. .
, .. , ,~
. ~ .... , amount of microoryanism are based upon a sequence of steps of culturing, growth and observation of the microorganism. Growth rates are observed in the culture which are derived from multiple dilutions of the same sample.
By observing the time at which the diluted samples reach observable pop-ulations, the concentration of microorganisms in the original san!ples can be estimated.
The estimation of microorganism popu1ations is most generally accomplished by one of three techniques. The first technique is a nutrient agar plat;ng technique in which a lilicroorganism is allowed to grow on~an agar nutrient substrate and the growth of the microorganislll is initially observed visually and thereafter by microscopic observation. This is the most common method ;n use clinically.
The second type of technique includes several methods which can be classified as chemical methods. One method of analysis involves supplying a nlicroorganism in a growth medium with carbon-14 labeled glucose. The m;croorganism llletabolizes the radioactive glucose and evolves C1402, 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 hazardous from the standpoint of the necessity of handling radioactive samples. Another analytical method of determing the type and amount of bacteria present in ; a sample is based UpOIl the chemiluminescentreaction 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. ~he liberated ATP
: 25 reacts with e luci~erase-luci~erin COI bination whereùy chemilunlinescent ~, "Il ' . . .
-`! 107171l 1~

¦ light is emitted which ;s detected by à photomultiplier and used to deter-¦ mine the amount of the organism present. The principal disadvantage of this¦ technique is the expense of the materials involved in the reaction. Still I another chemical method involves a measurement based upon the metabol;c ¦ conversion of nitrite ion to nitrate ion. This method, however, is only ¦ applicable to some bacteria and yeasts.
¦ The third type of technique used for the detection of microorganisms ¦ involve non-chemical methods. One method involves the evaluation of rela-¦ tively clear microorganism suspens;ons by modified particle counters. , 10 ¦ However, the nlethod is non-specific and does not provide a distinction between ¦ viable m;croorgan;sms and dead ones, or even non-biologic particulate matter.¦ A second method ;nvolves the detection of Inicroorganisms by impressing ¦ an alternating current across a pair of electrodes which have been placed ¦ ;n a microorganism containing mediuln and observing the change in impedance of the current as a function of the growth of the microorganisln. This technique however, is apparently 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 l;quid and gaseous media. The sensor ;s constructed by placing a measuring electrode such as an inert wire gauge cylinder of nickel, platinum, stainless steel or the like upon which is impregnated a colony of a microorganism or ~.
an enzyme and a reference electrode such as the standard Calomel electrode ~:
in an electrolyte. The leads from each electrode a~e attached to a volt-meter. The organism or enzyme impregnated or in close proximity to the .

measuring electrode yenerates a current within the cell by causing chemical reactions at the surface of the electrode or by promoting chemical reactions to produce materials which in turn provide depolarization reactions at the electrode. The device functions by admitting a trace element or a poison into the device which deactivates the enzyme or kills or deactivates the microorganism thus causing a change in the potential difference between the electrodes wl!ich is detected by a change in the voltmeter readings. Con-sidering the fact that the enzyme or microorganism promotes or causes a considerable chemical reaction at the measuring electrode which is detect~d lO ¦ by a standard voltmeter in the circuitry of the cell, the current generated ¦ within the cell must be substantial. The electroanalytical device of the present invention which is used to detennine the type and amount of a microorganism in a solution, on the other hand, does not require the impreg-¦ nation of a sizable colony of a microorganisl11 on the measuring electrode 15 ¦ but rather operates by detecting a microorganism in solution which yradually ¦ concentrates about the measuri1lg electrode. A further critical distinction ¦ between the method and apparatus of the present invention and the method of ¦ the reference is that the circuitry of the present system must conta;n a high inlpedance potentiometer and not the conventional voltmeter used in the ~; 20 reference's process, because the present system is dependent upon the measure-ment of an electrostatic-like potential difference between the measuring electrode and the microorganism in solution. If a standard voltmeter were -used in the circuitry of the present inventiorl, far too nluch current would be drawn by the voltmeter which would destroy the relatively delicate electrostatic-like potential difference between the measuring electrode and the microorganism concentrated about the eiectrodè's surface.

~71711 Consequently, a need continues to exist for a method of rapidly, automatically and economically determining types and amounts of various microorganism by a conceptually simple and economic technique.

The invention in one aspect comprehends a method for detecting the presence of a microorganism in a fluid sample, which comprises culturing a microorganism in a liquid growth medium which is in contact with a measuring electrode and a reference electrode, and detecting the changing potential between the electrodes. The changing potential arises by the ;~
migration and accumulation of the microorganism adjacent the surface of the measuring electrode thus forming a charge-charge interaction between the measuring electrode and accumulat-ed microorganisms and the potential change is measured with a high impedance potentiometer, having an input impedance Gf 107 to lol0 ohms.
- Another aspect pertains to a method for detecting the presence of hydrogen producing bacteria in a fluid sample, 20 which includes culturing a hydrogen producing bacterium in a growth medium which is in contact with a measuring electxode and a reference electrode which are conductively attached to a high impedance potentiometer having an input impedance of 107to 101 ohms, wherein the growing bacteria gradually concentrate about the measuring electrode thus concentrating the evolved hydrogen gas about the measuring electrode and forming a charge-charge interaction between the measuring electrode and accumulated microorganisms. The evolved hydrogen gives rise to a modified measuring electrode which has the nature of a hydrogen electrode. The method further includes achieving a stable baseline response from the potentiometer prior to reaching the minimim detectable concentration of the 71'711 bacterium and monitoring the change in potential between the modified electrQde and the reference electrode.
A still further aspect of the invention comprehends `
a method for measuring the influence of an anti-microbial agent or event on a growing microorganism, which includes culturing a mlcroorganism in a liquid growth medium which is in contact with a measuring electrode and a reference electrode which are conductively attachéd to a high impedance potent-iometer having an input impedance of 107 to 10l ohms, wherein the growing bacteria gradually accumulate about the measuring electrode thus forming a aharge-charge interaction between the measuring electrode and accumulated microorganism. The method further includes achieving a stable baseline response from the potentiometer prior to reacting the minimum detectable concentration of the microorganism, subjecting the micro-organisms to an anti-microbial agent or event and monitoring the change in potential between the electrodes as a result of the influence of the anti-microbial agent or event on the microorganism.
Another aspect of the invention pertains to an apparatus for ths electrochemical detection of living microorganisms in a fluid medium. The apparatus includes a chamber with an opening containing a growth medium suitable for the culturing of microorganisms and means are provided for sealing the open-ing of the chamber which means is self-sealing after having been punctured by a means for delivering a microorganism containing sample to the chamber. Measuring electrode means are fixedly attached to the exterior of the chamber, one end of which penetrates through the chamber into the interior thereof and is in contact with the growth medium, about which accumulatec the microorganisms thereby setting up a charge-charge inter-action at the measuring electrode means. The measuring electrod~
means is responsive to the charge-charge interaction established '''' \~O
~ ~ - 6 a -10717~1 at the measuring electrode means. ~eference electrode means are fixedly attached to the exterior of the chamber, one end of which protrudes into the interior of the chamber and which is covered with a growth medium permeable shielding means that prevents contact of the reference electrode means with the microorganisms, for establishing the potential of the cell in cooperation with the measurins electrode means. A potentiometer having an input impedance of 107 to 101 ohms is conduct-ively connected to the measuring and reference electrode means.
A further aspect of the invention pertains to an apparatu~
for the electrochemical detection of microorganisms in a fluid medium. The apparatus includes a chamber closed at one end and open at the other end thereof and containing a growth medium suitable for the culturing of microorganisms. Measuring elect-rode means are fixedly attached to the exterior of the chamber, one end of which protrudes through the chamber into the interior thereof and is in contact with the growth medium about which accumulates the microorganisms thereby setting up a charge-charge interaction at the measuring electrode means.
The measuring electrode means are responsive to the charge-charge interaction established at the measuring electrode means. Reference electrode means are fixedly attached to the exterior of the chamber, one end of which protrudes into the interior of the chamber and which is covered with a growth medium permeable shielding means that prevents contact of the reference electrode means with the microorganisms, for establishing the potential of the cell in cooperation with the measuring electrode means. Conduit means penetrate through the base of the chamber and the shielding means such that the internally protruding open end thereof is exposed to the culture medium for transport of the fluid medium into the chamber.

~ ~ - 6b -A plunger means fits within the open end of the chamber and moves freel~ inwardly and outwardly which allows a sample containing the microorganisms to be withdrawn into or expelled from the chamber and a potentiometer having an input impedance of 107 to 101 ohms which is conductively connected to the measuring and reference electrode means.

BRIEF DESCRIPTION OF THE_DRAWIN~,S

FIGURE 1 shows a potential response curve for the growth :
~ of Pseudomonas versus time in 10 ml of Trypticase Soy Broth medium;
FIGURE 2 shows two potential versus time curves which demonstrate the growth impeding influence of bacterioPhage ~: on bacterial strains of E. coli;
FIGURE~3 is an embodiment of one form of the electroanal~tical cell of the present invention;
FIGURE 4 is another embodiment of the electroanalytical cell of the present invention wherein the cell has been adapted to function in part as a syringe;

..~

.

- 6 c -I ~07~71~
FIGURE 5 is an embodiment of the device of the present invention wherein the challlber of the device ;s equipped with means for drawing a microorganism containing fluid sample into the device under force of vacuum;
FIGURE 6 is an embo~inlent of the device of the present invention which in part is a needle suitable for the sampling of body fluidsi FIGURE 7 is a strip chart recording of potential versus time for the culturing of E. coli.;
: . FIGURE 8 is a strip chart recording of a series of cultures of E. coli wherein the bacterial concentration of the initial inocula in each sample was varied;
FIGURE 9shows a comparison plot of potential versus time for a hydrogen ¦ producing organism (E.coli) and a non-hydrogen producing organism(alkalescens), appearing with Figure 7;
FIGURE lO is a correlation diagram of potential and radioactivity at the measuring electrode as a function of cell concentratioll for radioactive alkalescens in TSB media; ,:
FIGURE ll shows two plots of potential versus time obtained from a platinum wire-ca~l~omel electrode system and a nickel-nickel electrode system in the same TSB culture mediunl of alkalescens; the nickel electrodes are positioned as in FIGURE 3.
FIGURE 12 sllows several potential versus time curves which show the growth impedillg influence of the antibiotic, ampicillin, on S. epidermidis, appearing with Figure 2; and FIGURE 13 shows several.potential versus time curves which show the growth impeding influence of cephalothin on ~_J) eumoniae.
DETAILED DESCRIPTION OF Tl-IE PREFERRED E~lBODI~lE~TS
¦ The discovery upon which the method and apparatus of the present , ."~ , : -7 `~ 1071711 . . . , . . , ~

. . . . .
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 reference electrode generates potential changes within the cell because of a difference in electrostatic charge between a live organism and the nleasuring 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 10 renders all microorgansn~s anlenable to detection by the method of the pre-; ~ sent invention. It is believed that the growing microorsanisms gradually I migrate to the exposed surface of the measuriny electrode 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 mediunl and not the .
` microorganlsms or not be sensitive to the microorganisnls's charge and that the microorganisms concentrate themselves about the measuring electrode.
For example, when a metal wire or the like is used as tlle reference elec-trode, the electrode is normally shielded from the microorganisms by a fluid permeable but organism impermeable substance. When a device such as ~1 the calomel electrode is used as the reFerence electrode, it is normally protected by the glass envelope from the microorganislns. The accumulation of microorganisms about the measuring electrode alters the potential of the measuring electrode relative to the reference electrode and consequently, a ~¦ 25 change in potential is set up between the electrodes because of the charge-~ , ,~ ` .

1~ -8- 1 ','' I . .
~ I . , .
.'' '' ' .. . .
charge interactions at the measuring electrode. The voltage change generated by this electrostatic interaction is the means by which the presence of microorganisms ;n solution can be detected.
The cell structure which confines the growing microorganisms and to ` 5 which the electrodes are attached can be manufactured fronl any convenient materials normally used in the manufacture of electroanalytical cells such as glass, plastic and the 11ke. Any material which is suitable for such use and which does not interfere with the gro~th or viability of the micro-organisms and does not affect the voltage generated within the cell can be used.
The electroanalytical cell is prov;ded with a culture medium for the growth of the microorganisnls placed within the cell. Any culture nledium which is comlnonly used for the growth of microoryanisms can be used, and therefore, the type of culture medium used is not critical. Suitable ~- 15 growth media include brain-heart infusion, trypticase soy broth (TSB), phenol red broth base + 1% glucose, trypticase soy broth + C02, milk, beer, sodium glycolate and the like. The a~ount of growth mediuln provided within the measuring cell, of course, is not critical.
The measuriny and reference electrodes attached to the cell can be made of the same conductive material or they can be of different n~terials.
Usually, the measuring electrode is fabricated of any suitable electrode material in any convenient form and is attached to the cell so that a portion .
thereof is in contact wi-th the liquid medium within the cell. Suitable ~.
materials from whicll the measuring electrode can be fabricated include the 25 nob 1 e r et such a s s i 1 ve n, pl a tl num, pa 11 adi um, go 1 d, a nd the 1 i he, ~.'' .: .
.,~,' ' . .

tungsten, molybdenulll, nickel and the like, and various nletal 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 elec-trode, which is a most unusual electrode configuration for galvanic cells.
Normally, the electrodes 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 convenient 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 ref-erence electrode in contact with the mediulll is usually shielded fron. contact with the microorganisnls in the mediuln. When the measuring and reference electrodes are of unlike metals or alloys, it is not necessary to shield the refe~ence electrode since the microorganisms will preferentially accumulate about the more positive of the two electrodes in the cell. On the other hand, when the measuring and reference electrodc 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, carrageenan ycls- acrylimide and the like. Menlb nes such as relatively larye pore ultrafiltration mclllbranes or .~ -10~ .
,:~
.
':- ` .

107'171~ ~
.
even large pore n~embranes, frit, ceranlic or porous filmsof plastic are all suitable if they are impermedble to the microoryanisllls. If the reference electrode and the measuring electrode are fabricated of the same material such as two sta;nless steel electrodes whicll is d preferred embodiment of the invention, 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 thermocoup1e effects. If the electrodes are fabricated of unlike metals or metal alloys, then the therlllocouple effect can exist and the potential readings obtained should compensate for that portion of voltage attributable to the thermocouple effect. Thus, it can be appreciated that the themlocouple effect wllich can arise with certain electrode combinations is not critically restr;ctive andone operating according to the present method need only be aware of this factor.
The reference electrode besides being of the type described above, can also be a standard reference electrode such as one of the standdrd 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 microorganisnls, because either the construotion of these electrodes in a glass envelope nonllally eliminates contact of the interior working portions of the elec- ~
trode with the microoryranisms in solution or they have a potential which .
is not effected by microorgdnisms.
: -..' .'~ -11 - . - ' ~ , . . .

. In order to measure the voltage generated within the electroanalyt;cdl cell containing the growing microorganism7 it is necessary to connect both electrodes of the cell to a high ;mpedance potentiometer. ~he type of potentiometer used is not critical with the only requirement being that it .
S be of the high impedance type. The potentiometer must have an illlpUt : . impedance over the range of 107 ohms to 101 ohms, preferably greater than 10~ ohnls. If a relatively low impedance potentiometer is used, too much current would be drawn through the measuring device thus upsetting the charge-charge interaction between the measuring electrode and the micro- :.
organisms. With the destruction of the electrostatic potential at the elec-trode, 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 perfornlance of a measurement according to the procedure of :: the present invention, a sa!nple of a microorgallisnls is introduced into the . growth mediulll within the cell. In.one embodinlent, a fluid sample of a microorganisll1 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 intro-. duced into the growth medium of the ceil. A basic feature of the present technique is that a stable baseline can be established by the recording ~ r instrument which is attached to the hiyh inlpedance potentiometel and which .. .
25 plots challyes in potent;al as a function of til;le before the millimulll detect-.,. . .,~,.
~ . . -12-able concentration (MDC) of the growing l~icroorganism is attaine~. In other words, the stable baselin~ is equivalent to a value of zero for the function of dE/dt. Growth of the microorganism occurs and once the population level reaches the minimum detectable concentration normally of about sxlo4 to 5X105.or~anismsll ml for all except the slower qrowing microorganisms, the microorganism can be detected by the voltage generate-l. The voltage as measured in millivolt readings always changes in a nogative direction.
; This is because the charge on the microorganisllls is more negative than that on the nleasuring electrode. For slow growing m;croorganisms suchas the baci lu~
- lO- of tuberculosis, however, the lower detectable concentration can be as low as l x lO3 organisms/l ml. In instances whele the method of the invention is used to detect slow growing microorganisms, the cell can be agitated by shaking, for instance, or by any other suitable means. Agitation of the microorganisms containing solution facilitated growth of the microorganis Isin 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 the measuring electrode in order to have the ~ necessary electrical charge-charge interactions, too vigorous stirring may : sweep the microorganisms away and eliminate the potential change. The temperature range over which the microorganisms are grown and eventually .~ detected span the range of 15 to 60C, prefera~ly 32-37C. Pressure is not a critical factor in the measurements since growth and detection of the microorganisnls can occur under partial vacuurn conditions as well as under .
¦ super-atmospheric pressures. Thus, for instance, the method of the present ¦ in,vention is amerlable to the detection of microor~allisllls which grow under ¦ pressure s ;n the manufacturing of beer.

~ -13-, I .
-' I . .
. : , . . .
..

1071711 ,~:~ ''` . , . . 1:
. The method of the present invention can be used to successfully detect any type of microorganisnl whicll can be cultured in the nutrient - medium provided within the electroanalytical cell or added to the cell in a suitable concentration. Also it is not absolutely necessary for the microorganisms to be growing or metabolizing provided they remain alive.
This is because it is their viability that g;ves them their negative charge, which they loose at déath. Thus, the method is applicable to the detection tof yeasts, fungi and bacteria. Spec;fic examples of bacteria which can be ¦-:. detected by the method of the present invention include the non-hydrogen producing bacteria, Staphylococcus aureaus, Staphylococcus epider!nidls, Streptococcus, Listeria monocytogenes, Pseudomonas aeruginosa, ~loraxella, Shigella alkalescens, Diplococcus pneumoniae, Bacillus subtilus, and Hemophilus influenzae. Suitable examples of yeasts which can be detected include Candida species such as Candida albicans and Candida rugosa, Hansenula species such as Hansenula anolllala, Pichia species such as Pichia me!llbranaefaciens, Torulopsis species and Saccharonyces cerevisiae. Suitable . examples of fungi include the various species of Aspergillus such as Aspergillus auricularis, L barbae, A. bouffardi, A. calvatus, A concentricus L falvus, A. funligatus, L ~iganteus, L glaucus, L gliocladiu!n, A. muco- , ;
roides, A. nidulans, A. niger, A. ochraceus, A. pictor, and A. rel?ens and the species of the large group of fun~i known as Fungi lmperfecti.
- A number of bacteria form a class of bacteria which are known for their .;~ ability to rPlease hydrogen during their growth. These bacteria include Escherichia coli, Enterobacter aerogenes, Serratia !nalcescens, Pro~eus ..
-. mirabilis, ~itr~ r i~t~ e~i~n, Citrobacter frrullclii, Salmonella and :~ 14 . .
- .

1f~71711 ::

and Klebsie!1a. Since t~lese bacteria n~igrate toward the measuring electrode as all microorganisms do, their release of hydrogen tends to concentrate ~ .
about the measuring electrode.
The presence of hydrogen at the measuring electrode very substantially .
amplifies tlle characteristics of the electrode such that it becomes similar to the well known hydrogen electrode. Thus, in effect a conlpletely diff-erent type of measuring electrode exists for the measurement of hydrogen producing oryanisms than for non-hydrogen producing organisms. The principle behind the operation of the hydrogen electrode is that the following equil-- 10 ibrium exists at the surface of the metal electrode, usually platinum or gold:
H2 ~ Pt ' 2H + 2e.
It is clear from this expression that an equilibrium exists between mole-cular hydrogen and hydrogen ions in solution and it is the variations within the equilibrium that detennines the potential of the electrode.
Once an equilibrium has been established at the electrode surface, the electrode is termed as a "nonpolari~able" or reference electrode. ~he nonnal hydrogen ~reference) electrode is platinum in an acid solution of pH=O with a saturated solution of molecular hydrogen at a defined temper-ature. The nonnal hydrogen electrode (NHE) is defined to have a potential -~, of O.OO volts and forms the relative basis ~or the potential scale of all -~- other electrode reactions as well as establishes the basis for the electro--, motive series of metals.
~' ~ ____ _ ___.
..
.
.. ",~

... . . . I
In a more specific aspect of the nleasurement of llydrogen producing bacteria a calon~el electrode is used as the reference electrode in combin-ation with a metal measuring electrode~ The calomel electrode has a potential .~; of about +0.23 volts with respect to NHE and since the growth medium has a pH of about neutral or 7, the measuriny hydrogen electrode has a potential ofabout-0.42 volts with respect to NHE. Thus, tlle measuring electrode for hydrogen producing bacteria has a potential of about -.65 volts; i.e. ~
. , ;~ ~ t-0.42)-~+0.23)= -0.65 in a negative direction relative to calomel. Be-I cause, in reality a pressure of one atmosphere of hydroyen is ne~er achieved at the measuring electrode because of atmospheric dilutioneffects due to C02, nitrogen and the like, a leveling-off potential of approximately -0.4 to -0.5 volts versus calomel is achieved in the measurelnents obtained ¦ for hydrogen producing bacteria.
In the measurelllent of both hydroyen and non-hydrogen producing ;.
microorganisms, a lag time is initially observed from the bme the analytical cell is inoculated with the microorganisnl until the time the microorganism reaches sufficient population levels to be detected. There-after, the potential continues to increase in a negative direction until a peak potential is attained where the potential levels off. Thereafter, with advancing time the potential drifts back to more positive potentials. For non-hydrogen producing microorganisnls, the total change in millivolt readings ranges from about 200-300 millivolts. In the case of hydrogen producing organisms, the initial negative increases in potential are attri- ;butable to the presence of the organism only. Thereafter, as the effects of 25 ~ the hydrogen build-up at the electrode become significant, the potential 16- ~

;l I

., . . .
sharply incre~ses to significantly more negative potentials than are obtained for non-hydrogen producing microorganisms. The negative increase in potentlal attributable to the presence of hydrogen at the measuring electrode amounts to about 500 millivolts.
The potential readings obtained for each medsurenlent represent the sum of the change in potential caused by the presence of the microorganisn in solution and the potential changes attributable to other solution and system factors which cause m;nor changes in the potential. These factors are characteristic of tlle particular liquid medium present in the cell as, well as the electrodes used and should be ascertained by a potential reading of the medium free of the presence of detectable amounts of particular microorganisnl to be tested. By achieving a measure of this background potential, one therefore can readily determine that the total potential change is caused by the presence of the microorgallislll. An interesting embodilnent of the present invention is that it affords a method of detecting a mixture of microorganisllls in a sample desired to be tested. When a sample containing a mixture of 2 or more types or species of microorganisms is c~ltured within the analytical cell, the microorganisnl which reaches its minimum detectable limit first is the one which will be detected by the system. With this knowledge, it is therefore apparellt that the skilled artisan can alter the growth conditions such as temperature and the type of culture mediunl to favor the growth of one type of microorganism to the detriment of othernlicrool^ganisnls and thus preferentially detect one micro-organism over another. For example, in order to detect fecal coliform in a mix~ure of I ~roorgariSIns, the temperature o~ the ~rowth nled;ulll can be ..

1071 ~1 ;

raised to preferentially kill the other microorganisms so that only the ~-coliform ren~ains.
The volume of the liquid medium within the electroanalytic cell is not critical. Of course, the liquid mediuln must be in ionic contact with both electrodes. The voluIne o~ the liquid growth mediuIll can be any reason-able size which the skilled artisancan readily determiIle. Of course, as the volume of the liquid is increased, the greater the dilution of the microorganism, and the lo~Jer the resulting response obtained. It will be - appreciated that the sealed electronanalytical cell can contain a growth medium therein prior to introduction of the microorganisn1 or the growth . nledium and sample of microorgaIlism 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.
I A further understanding of the method of the present invention can 15 ¦ be achieved by reference to FIGURE 1 which shows two simultalleous potential ; ¦ versus time recordings of a Pseudomonas aeroginosa, initial inocula 104 ¦ cells/mI sample cultured in a trypticase soy broth medium at 37~ 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 significallt response. There-after, the organism begins to reach mininlunl detectable concentrations as evidenced by 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 popula~ion.

~ 18- .

10717~1 1 The invention thus far has been described in terms of directly measuring the presence of living microorganisn1s in an electrocl1eltlical cell. In another embodiment of the invention, substances or events can be monitored which change or destroy microbial viability such as exposing the microorganis s in the cell to an antibiotic, serum antibodies, chemical toxins, viruses, e.g. bacteriophage, or any other anti-microbial or anticellular event.
. ¦ Thus, the effect of an anti-microbial agent or event can be monitoled by ¦ two methods:
. ¦ 1) by adding antimicrobial agent to standard dilutions of cultures at :~ 10 ¦ some concentration prior to reaching the MDC: and 1 2) by adding the anti-l11icrobial agent dt or above the MDC.
i . I The method of the present invention is therefore useful in antimicrobial susceptibility testing for the investigation of the activity of new anti-biotics, or in the surveillance of developments in an organism such as : 15 the development of antibiotic res;stance by the organism. Most importantly, in Vitl'O evidence of bacterial-antibiotic interactions endbles a clinician .~ ~ to predict the in vivo efficacy of a particular drug. The present method is .. useful in antimicrobial suscpetibility testiny when the causative oryanism doesn't respond predictably to antibiotics as shown in Table 1 below, and al when the organisnl is invariably susceptible to a particular drug as ,` shown i a-1e 2 bel~w.

., ,~
. ' '' . .
:~

1~ 10,1711 . TAULE 1 MICROORGANIS~S WITH UNPREDICTABLE ANTIMICROBIAL RESPONSE
I . .
¦ Orgdnism A
gent to which or~allism llldy be sensiti Staphylococcus aureus Penicillin G., methic;llin, 1, 1 , cephalothin, Vd nconwcin , ¦ Escherichia coli ~nll)icillin, cephalothin, tetracycline, kanamycin .
Klebsiella pneumonide Cephalothin, tetracycline, . kanamycin, chloranlpllenicol , .. ~.
Pseudomonds aerugillosa Gentamycill, tobramycin, ~:
carbenicillin, poly~lixin B
. Proteus mirdbilis Penicillill (moderately resistant~
:, ampicillin, kanamycin Proteus vulgaris Tetracycline, kanamycin, . chlorampllenicol , .,", Enterobacter aerogene,s Tetracyclines, chlorampllenicol .. ' kanalnycin . Sal!nonella Ampicillin, tetracycline, ~, .~` cephal,othin, kanamycin ,.
~;, 20 Shigella Same as above (Salmonelld) . List ria nlollocytogenes Penicillin G, erytllromycin, ch10rdlD~ en col ~ I ., . ~
.~ , ..
I _ ~ t1 ' -20, F
'.
. . `.

``1 107~711 ¦ TABLE 2 ¦ MICROORGANISMS ~IITH PREDICTABLE ANTIMICROBIAL 17ESPONSE

i ¦ ORGANISM AGENT TO ~11IIC11 ORGANISr1 IS UNIFOR~1LY SENSITI~
I ¦ Neisseria gonorr?loeae . Penicillin G, erythromycin ¦ Diplococcus pneumoniae All penicillins, cephalothin erythromycin, vancon1ycin . . ¦ St1-eetococcus p~enes Same as Diplococcus : ¦ Clostridium perfringens All penic;llins, cephalothin, . I tetracycline ¦ Hae1nophilus influenzae Penicillin G, ampicillin, . tetracycline, chloramphenicol ¦ Haen1ophilus pertussis Same as !~. influenzae ¦ Brucella Tetracycline ¦ Corynebacteri Ulll diph theria Erythronwcin, penicillin G
~ 15 ¦ Neisseria meningitidis Penicillin G, ampicillin ,~ , I .
.,,/,"~, . . . . ~
`:'i :' . , ~ ~
. ~ -21-: ' . .

~'~ ' . . , ' ' ' .
,~
, ~'.
.~ ;
.

107 ~711 FIGURE 2 shows another example of an agent which disrupts the growth of a microorganism. Curves A and B'show the potential-time responses for the yrowth of a strain of E. coli in a growth mediunl wherein different concentrations of bacteriophage 0x i~e. 104 phage/ml and 105 phage/n11 respectively were added to the gro~ing microorganism after the ~IDC was reached. The time required for the potential to reach its peak after the ;;
virus ;s added is inversely related to the logaritlloll of tl-e virus concen-tration at the time of addition i.e.
log C virus C~ Ipeak ~ ~ MDC
By this technique the analysis of viral concentration in less than one hour can be achieved.
The following drawings represent various embodimellts of the electro-analytical cell of the present invention:
FIGURE.3 shows an embodil11ent of an electroanalytical cell in ~hich chamberll is sealed with a resilient cap 2 which is self sealing. ~1easuring electrode 3 is attached and secured to the chamber through an opening (not shown) in the chal1lber wall. Portion 4 of electrode 3 extends through the chamber wall so that its surface is exposed to culture nledium 5 in the . chamber through an opening (not shown) in the base thereof. The portion 7 of the reference electrode within the chamber is shielded fron1 any micro-organisnl which is present within the culture mediull1 by shielding means 8.
$hielding meat)s ~ measuring electrode 3 and reference electrode 6 can be formed from the materials described above. In the use of the above described device an appropriate quantity of a microorganism containing j~
fluid sample is injected into the chamber through the cap. ~he exterior j terminals of the measuring and reference electrodes are tllen attaclled by ; leads to the appropriate terminals of a hitJh impedance potentio1l1eter and the medium is monitored as ~;e microorganism grows. ^ -.~. ~ 1071711 A~other.e~nbodilllent of the electroanalytical cell of the present 1 I invention is shown in FIGURE 4. In this device, the cell is essentially : . . I a syringe of challlber 11 and plunger 12. The base of the chamber is provided with a suitable hollow conduit 13 which can be a hollow needle, rubber l . S I tube, or the like, one end 14 of which protrudes throuyh the chanlber and ;
~, microorganism shielding means lS into the bulk of.the culture medium 16 within the chanlber. The device is equipped with measuring electrode 17 ..
which is attached to the challlber through an opening (not shown). Portion 18 of the.electrode is in contact with the culture medium. Reference ~.
. ~ : . 10 electrode 19 is attached and secured to the base of the chamber through , :. an opening (not shown). That portion of the electrode within the c~lamber 20 terminates within sheilding means 15.
The device of FIGURE 4 is adapted so that the same device can be used to directly withdraw or expel fluids from a living body into a culture 15 mediuIn and be imnetliately attaclled to a potentialleter so that the growth -~
of any microorganislll present in the body fluid can be monitored.
Yet another embodiment of the cell of the present invention is shown . ¦ in FIGURE 5. Ttle device in this case is adapted so that d fluid containing ~ ~ ¦ a microorganisnl to be analyzed can be witlldrawIl into challlber 30 through entry 1 20 I line 31 via a vacuum applied to vacuum line 32. Both lines are shown as protruding throuyh resilient seal.ing 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 1 the vacuwll, it falls upon and ;s mixed within culture nletIiulil 34. Measuring ..
25 ¦ electrode 35 is attached to the device througtl an opening (not shown) so ¦ that.the nleasuriIl9 end of the electrode (36) protrudes into the culture . ~ -23-t,.
.~ , , . . ~
.. . .. ..

¦~ medi m. Reference elec~rode 37 is attached to the device 1n a sin1ilar ¦~ :
¦ n7anner at the base of the chamber so that the end 38 w;thin the chamber ¦ projects into a shielding means 39 which shields the end of the electrode ¦ from Illicroorganisllls in the culture medium. The device can be used in the ; S ¦ 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 ~IGURE 6, the functioning parts are present within a needle.
¦ Thus, the device is fabricated of a conduit 40 whicll can be a hollow ¦ needle inside of ~Ihich is concentrically disposed electrically insulating ¦ means ~l. Lead 42 ;s attached to the exterior surface of the needle so ¦ that it functions as the measuring electrode. Wire 43 is disposed with;n ¦ the hollow inner core 44 of the insulating mea1ls and attached at the base ¦ of the insulating core 4~ by a shielding means 46. The needle apparatus is 1 then attached to an evacuated, sealed chanlber (not shown) which can contain a growth medium. When used, the needle is injected into a subject and ¦ fluids containing the microorganisln desired to be measured are withdrawn.
The leads attached to the needle can then be attached to d hi9h illlpedallCe potentiometer to nleasure the microorganisllls in the fluid.
¦ In another embodi1llent of the device oF the present invention (not 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 challlber of the device ¦ is provided with a sealed hollow needle whose attached end projects through the base and shielding means into the culture mediun). The device can be `'!

, I : ._ , 1. 1071711 used to withdraw a fluid containing a microorganism into the chamber by simply injecting the free end of the needle into the body of an animal r for instance, whereby the force of the injection and/or the fluid pressure breaks the seal within the needle and the fluid is withdrawn into the device by the force of the vacuum.
-~ The present invention enjoys a wide variety of fields of application, because the present invention is applicable wherever 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 bacteria in a biological or non-biological fluid mediunl. The method of the invention can be used as an antibiotic 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 inf1uence of the antibiotic on the potent;al readings obtained. Biological applications include the detection of microorganisms in such body fluids as urine, blood, cerebro-spinal fluids, sputum, amniotic fluid, synovial fluid, plasma and artifical kidney dialysate. The present technique is also -amenable to throat tissue cultures, vaginal and cervical tissue cultures and tissue biopsies. The present technique flnds further application for the detect;on of microorganisms in alcohol producing media such as from wood, grain, molasses, sulfite and waste liquors; from the production of w;ne and beers and from the production of glycerol. Still another area of ;
application irt il~ the fernlelltation of organic acids such as lactic acid, citric ac;d, fulllaric acid and acetic acid. Ye~ ?nolher general area of appl;cab;l;ty IS the food processing ;ndustry, particularly in thc ; -25~

`~, 1071.11 ~,`

; packaging of and the production of beverayes and canned goods.
Still further areas of applicdbility include rural, city and reyional water supplies, water holding tanks and septic tanks.
Having generally described the invention, a further understan~ing can be obtained by reference to certain specific examples which are provided herein for purposes of illus-tration only and are not intended to be limiting unless otherwise specified.
EXAMPLE 1 .
For the specific experimelltal procedure described below, the following hydrogen producing bacteria were obtained from the American Type Culture Collection (Rockville, Maryland) U.S.A.
E. coli (12014), E. aero~enes (13046), S. marcescens (138~0), C. intermediuln (6750), C. freundii (~090), and P. mirabilis (12453).
; ~Cultures were maintained at 5C on trypticase soy agar slants (TSA, BBL) and transferred monthly.
Inoculum p~eparation, viable counts, and !~!edia.
Inocula for the hydrogen measulelnents 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 appropriate ~ilutions to 27 ml of phenol 20 ¦ red broth base with l.0~ glucose (Difco) prewarmed to 35C. In a limited ¦. number of tests, members of the coliform group were tested in lauryl tryptose¦ broth (Difco). Viable counts were made by spreading appropriate dilutions ;
.from the 10-fold series on TSA and counting colonies after 35 h of incubdtion at 35C. Viable counts were also made on eacll organislll at the time of hydro~n ev ion and at the end of 24 h of in~ubatioll lQ71711 .' . ' . ..
~Iydrogen Illeas~relllellts The experimental apparatus for n~easuring hydroyen evolution by the test organisms consisted of a test tube (25 by 90 mm) containing two elec-trodes plus broth and organisms and positioned in a 35C water bath. Leads from the electrodes were connected to a dc buffer amplifier (type 122, Neff, Inc., Duarte, Calif.) which in turn was connected to a strip chart recorder ~-(model 194, Honeywell Industrial Div., Fort Washington, Pa.). The dc buffer amplifier served to match the high impedence of the electrode test system with the strip-chart recorder. Hydrogen evolution was measured by ,~n increase in voltaye in the negative (cathodic) direction and was recorded on the strip-chart recorder.
The electrodes employèd in the apparatus were a standard calomel (SCE-Beckman Instruments Inc., Fullerton, Cal;f.) as the reference electrode cemented to a plastic cap and a platinum electrode was fornled by shaping a strip of platinum to f;t the circumference of the test tube; a section of the platinum was positioned outside of a test tube for attachment to the anlplifier lead. During operation, the platinum electrode and test tube were steam-sterilized by conventional autoclave procedures. The reference electrode (SCE) attached to the plastic cap, was sterilized by exposure for 30 minutes to two ultraviolet lamps (15T~, General Electrio lieights, Ohio) housed in a clear plastic box. A number of tests demonstrated this technique to be effective in ster;lizing the reference electrodes. j~
The strip chart recording of the millivolt response curve for 1.9 x 10 `.j cells of E. coli per milliliter is shown in FIGURE ~. Characteristically, for hydrogen producing o~ganisms, the recording shows a lag period during which the m;croorganislll is growing but is present in insufficient popu-lations to give a response. Once the microorganism population has reached a sufficient level, a response is noted which is attributable to the microorganism. As the amount of hydrogen evolved becomes sufficlent, the electrode response characteristics rapidly change as noted by the sudden increase in potential. The period of decline after the nlaxinlum potential was reached (400 to 500 millivolts) occurred over a 3 to 4 hour period.
. The relationshp between inoculum size and length of the lag period for various inocula of E. coli is shown in FIGURE 8. Lag times ranged from 1 hour for lo6 cells/ml-to 7 hours for 10 cells/ml which indicated 1) that ea h10-fold increment of cells reduced the lag tin~e by 60 to 70 minutes and

(2) that the mean cell concentration at time of rapid buildup in hydrogen was 1 x 106 cellstml. Because initial studies showed no differences in the response curves for washed or unwashed cells, these studies were con-ducted ~ith unwashed cells. In addition, no differences in response curves or lag tilnes were noted from coliforms, E. coli, E. aero~enes, and C. inter-mediunl, when tested in lauryl tryptose broth or phenol red broth supplemented - with glucose. Gas chronlatogral)hic analysis of headspace gas showed that - for the cultures used in this study, the level of H2 for 24 h cultures wasbetween 4 and 10% by volume. The only exception was S. marcescens in which H2 was estimated as a trace (~ 1%). Limited studies indicated that pH did not change markedly before or during the time H2 was detected. .

FIGURE 10 shows a comparison of the potential characteristics of a hydrogen producing bacterium tE. coli) and a nonllydrogen producillg , . , .

.' 1~

¦ bacterium (~lkalscens). Each bacterium was cultivated in a broth lllediulll of lduryl tryptose glucose by inoculating each nledium with 1 ml of sample containiny 100 cells/ml. The response characteristics were measured by a ¦ platinum-calomel electrode couple. The recordings show the much greater S ¦ response characteristics in the system containing the hydrogen producing -¦ bacteria compared to the response characteristics of the non-hydrogen ¦ producing bacteria. Note that the same lag time is observed when the ¦ initial inoculum and growth rates are the same.
; EXAMPLE 3 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 con-centration. A 1 ml sample of radioactive alkalescens (initial innoculum size 100 cells/ml) was cultured in a 10 ml Tryptlcase soy broth mediunl 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 tlle electrode and that the potential response is caused by this build-up of cells about the electrode.
EXA~IPLE 4 A 1 ml sample of alkalescens was cultured in 10 ml of Trypticase soy broth medium for 7 hours. The growth of the bacterium was monitored at , the same tinle by two different electrode systems i.e. a platinum wire- i calomel systenl and a nic~el-nickel system in whicll one nickel electrode WdS
~5 sh elded rrm organisllls by a glass r~it. As can be ascertain ~ b~ reference ~ ;~ 1071711 to FIGUR 11, botII curves obtainea are essentially tlle same excep~ that , ~, the nickel-nickel electrode system does notappear to be quite as , ~ sensitive as the platinum-caloIllel system.
: ~. EXAMPLE S
A 1 ml sanIple of PseudomoIlas aerogeIlosa was cultured in trypticase soy broth in a cell described in FIGURE 3 in whicIl both the measurement and reference electrodes werè type 3 or 4 stainless steel. The shielding material in, this example was peptone agar with 10 mg/Illl NaCl. llhen the ' cell population of 9 x 104 cells/ml was reached, the potential change was detected as shown in FIGURE 1.

The same procedure described in Example 5 was followed with the exception that the organism cultured was E. coli in a mediuIll of Trypticase soy broth. A potential reading was observed at a population level of 1.2 x ' ~' 15 105 cells/ml .

, The same procedure of Examples 5 and 6 was followed with the exception ,that tl7e growth mediunl was sodium glycolate mediuIll and the organism WdS
- Shigella alkalescens. A potential reading was observed at a population 2 0 level of 8.6 x 104 cells/ml. ' ' The same procedure of Examples 5-7 was followed except that the growth nledium has sodiulll glycolate + 0.5 ml of human blood and the organism was , lIeIllophilus influenzae. A potential reading was observed at a population level of 2.4 x 105 cells/ml. ' :~" . , ,,., . -~~ . .
,, .
' , . .

~ I 1071711 The same procedure oF Examples 5-~ was followed except tllat the organism was Bacillus subtilus in l~ nutrient broth with 0.5X NaCl at .
30C. A potential reading was observed at a population level of 2.3 x 103 cells/ml.

.
The sanle procedure of Examples 5-9 was followed except that the organism was Candida tropicanas and the growth medium was 10% nutrient broth with 30% dextrose at 30C. Detection was at 3.l x 105 cells/ml.

The cell of FIGURE 4 was used to culture the fungus Aspergillus ni~er I in Trypticase soy broth at 25~C. A potential reading was observed at a population level of 104 cells/ml.

A culture of Pseudomonas aero~ll a_ was washed in non-nutrient buffer (Phosphate buffer solution or PBS) and "starved" at 15C for 48 hours. Cells mai1ltained in this condition are not dividing or metabolizing but are technically alive. Therefore they still have a net negative charge. These cells were then added to a cell described in FIGURE 4 with sta;nless steel electrodes but the nutr;ent broth was replaced with PBS.
At a population of Z x 10 viable cells/ml a signal was observed.

Two samples of bovine milk were added to cells of the type described ¦ in FIGURE 3. One sanlple was Pasteurized and one was "raw" nlilk. The nlilk25 ¦ served both as a growth mediunl and inoculum in both cases. The electrodes I

.. .. .. :.... ... .

' ! , : ' j ~.. I
': ` .`,, I . ' .

were stainless steel and the me~nbrane was saline agar. The experiment was conducted at a temperature of 37C. Each sample produced a signal ; at around 105 cells/ml but the "raw" milk developed the signal in 2 and 1/2 hours whereby the Pasteurized nlilk reached 105 cells and gave a signa1 at 5 ~i 4 and 1/2 hours.

Antimicroblal Suscepti~ y Tests Rea~ents and Media Identical lots of lnedia and antimicrobial agents were used in the lO ~; ~ susceptibility tests performed. The media used were trypticase soy agar (TSA) and TSB froln Baltinlore Biological Laboratories (BBL), Cockey-sville, Maryland. The 12 antin~icrobial agents used in the experime~ts, which were chosen on the basis of their clinical usefulness, were labor-atory reference standards including the following antibiotics: penicillin `~ G potassiuln, caphalothin sodium, tobranwcin, vancomycin hydrochloride and streptonlycin sulfate obtained frall Eli Lilly and Co.; tetracycline and carbenicillin obtained froln Pfizer Laboratories; ampicillin and sodium naf,cillin from Wyeth Laboratories; kanamycin and methicillin from Bristol Laboratories; gentamicin froln Schering.
0~ The action of an antibacterial drug against a susceptible bacterium is either bacteriostatic or bactericidal. A bacteriostatic agent merely inhibits bacterial growth, an effect which is reversible u~on renloval of ` the antimicrobial ayent. Bactericidal agents produce an irreversible, - ) .~ killing effect on the microorganislll susceptible to the drug. Of the 12 ~l antibiotics, all are bactericidal with the exception of tetracyeline which is bacteriostatic. Penicillin G, ampicil1in, nafci11in, Inethici11in, and carbenicillin al1 belong to the penici11in fallli1y and as such their mode of action involves interference with the synthesis of ce11 walls of ~ .
.~,, .

A
.~ ., ' .
t', ' ` .. ,, ~ ;, ' '~ ,, . ''.

~;` ~
l ::
:: ~ ~
~ 1071711 , bacteria. This is also true of cephalothin, a cephalosporin, and vancomycin, a glycopeptide. Tne aminoglycoside antibiotics kanamycin, streptomycin, and gentamicin cause specific misreadings of the genetic code at the ~, ribosomal level, thereby interfering with the protein syntllesis of the . bacterium. This also results from the use of tetracycline, which specifical prevents attachn~ent of amino acid activated transfer RNA to the ribosomes.
~ ,~ Cultures `
; ~ `The following cultures were obtained from Hoffnlann-La~oche, lnc.
and were derived from the American Type Culture Collection (ATCC) organisms 10~ as indicated: Escherichia coli, "Seattle Strain" ATCC 2592Z, Klebsiella , pneumoniae ATCC Z7736; Pseudonlonas aeruginosa, ATCC 9721~ These bacteria are qram negative bacteria. The following bacteria are gram positive:
Staphylococcus aureus, "Seattle Strain" ATCC 25923; Staphylococcus epiderdi!n s ATCC 14990; and Streptococcus pyogenes, nTcc 10389. In addition, clinical 15~r~` 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 epidemlidis), were chosen because they are some of the most frequently encountered organisllls in clinical isolates.
Inoculum Preparation and Viable Counts Inocula were prepared by making a 100-fold di1ution x 2 of an over-night TSB culture of the organism to be tested in sterile TSB prewarmed to 35C. Thus, the total dilution of overnight stock was 10 4. From the~' 10 4 series, 0.3 m1 was transferred to a test tube (Falcon, disposable #2057) .~25 ` containing 2.7 ml oF TSI3 if the growth con~rol, or 2.6 ml af TSB plus : 1~ 0.1 ml o particular concentration of an appropr;ate antibiotic to be tested. he antibiotic was weighed on an aD~Iytical 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/lnl wh;ch Yas then frozen in small aliquots. Further dilutions as necessary for susceptibility testing were also achieved through the addition of sterile distilled water to this stock concentration. Viable counts were made by plat;ng appropriate dilutions From the 10 ~ series on TSA and counting colonies after 24 hours incubation at 35C. Viable counts were also made on each microorganisnl at the time of MDC and again at the end of the 24 hour test period to confirm the minilnuln inhibitory concentration (MIC)., 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 100 mm, each containing a sterilized (by boiling) Pt-SGE combination electrode (Sargent-l~elch, S-30101-15), plus TSB, microorganisms, and antibiotic where appropriate, ~ositioned in a 35C heating l~lock. Leads ~rom the electrode were connected to a high impedance (greater than 10~ ohms) potentiometer, or voltage following device, whicll in turn was connected to a strip chart recorder (Hewlett- .
Packard type 680M). Microbial growth was measured by an increase in voltage in the negative direction with time as recorded Oll the strip chart recorder.
FIGURE 12 is a stripchart recording of the antimicrobial response curve of a four channeled expriment involving a clinical specimen of the non-hydrogen producing, Gram-positive bacteria Staphylococcus epidernlidis treated with 0.012, 0.5 andl.0 mcg/ml of the antibiotic alopicillin as shown in curves B, C and D respectively. Curve A shows the respollse curve of a control sample of S. epiderl!lidis which has not been treated -34, ~ ' ~',. .
,'- .' .
.. , . ,1 ` ~ l 1071~
.. :
with ampicillin. FIGURE 13 is astrip chart recording of the antimicrobial response curve of a six channeled experiment involving an ATCC culture sample of the pathogenic hydrogen producing, Gram-negative bacteria ~ Klebsiella pneumonlae treated with 0.3, 0.4, 0.5 and l.0 and 2.0 mcg/ml ¦ ~ ~ of cephalothin as shown in curves B', C', D', E' and F' respectively.
Curve A' shows the response curve of a control sample of K. pneumoniae wh1ch has not been treated with cephalothin. Both figures show that the growth of the respective bacteria is increasingly inhibited by the parti- .
~- ~ cular antibiotic material used. The minimum inhibitory concentration10 ~ (MlC) is defined as that amount of antimicrobial agent which totally inhi-;~. bits the growth of the microorganislll being tested. Tables 3 and4 below show the MIC values of various antibiotics for a number of microorganisms ¦ as detemlfned by the method of the present invention.

;~; . ~ I
~

. . . .
~ ' . .
.' ~;; , . - ~ , ~ . .
.
". . ' . , ; . . . i.
"; ''`'``,''''' " . . . ., ' ' ~ .

''`~ . ' .
~,'' ' .
,~`.

`' ~07171~
-,:~.,' " ' ` ' i~^. . ,~ r~, ~ ~n ~s~ _ _ , , ~ , ,~ , _ , ~ ~J ,n _ ,n -.r> ~ o , ~ ~ *, ~i ~ l I .

. E <~ ¦_ ~ r-- ~_ ~1 ~D r-- O _ _ E ~1 ~7 O ~ O ~ ~D E v r-- r-- _ r~ J l ¦

~, OL O O =) i~o _ _ . ~r ~ _ _ O O _ _ i , ~ n J; ,~ ,~ ,- aJ ,r~ r~ r~ irh. l l i

3~r'.,a' Ic _ _ _ __ _ . 1~ __ _ _ o o _ _ Il)C Oi=~ O O O i1_ IJ r-- r h ' D L~i i . ~ ~ l ~D ~ i ~,) i~ ~n ,n W¦ Cli~Ji_~DO3') r~ m ~I m o ~ ~D _ n ,, _ _ .
m ~ r- L O O`30 ~J r~J ~ ~ ~J.~ O? r-- O r ri_) _ nr-- r ID i~O i i ~r- Cl r--_ r-- ~ _ _ J, . ~ i O O ioO ~0 O ~ Ln Ir) O
: .c ~ -.? _ c E . . ~J l ~ i ~J ,~ ~ ~ o c l r--~ ~ ~ ~ ~ ~t l . . -o ,,.,,,,j,, . Z . .~ .

~1 . r-- r~ i 'C
-- ~_ i_) r- (_J r- O . r~ i_ ~ ~r~ ~ r--_, 1 rr r0i~ r~ ~S r-- cJ ~_ r~ _ ~_ i~_~ ~ r-'~ i! ~ V ' r- a) aJ ,n ,u v ,-- r-- r Ir ?-- r J ~ r~ ~r- .~ O O _ _ r- ~ iJl L~7 2 E r- U E E r- 7~ z ~E~ v ~ E E aJ a) i ~ r-r-- _i~J W L t ~ ~r- L L . _ r- O O
C O O ~ ~ ';IJ '~U i~. ~ ~ ~ O. 0 ~) ~1 '~ J L V i,;~ i t i i n~ 'C) i~_~ L . ~ IIJ aJ ~ ¦
LIO LlJ Li :~C ~ i~ i' ~J O 'J) I~ iJ~ if) i.~ ~1 ~ r't'-~ _ ~ . ~b O ,n ~

~.. t, ,~ ' 3 6 ~ I
~ ~ 1071711 :
' . .

. Having now fully described this invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can .
t~ be made thereto without departing from the spirit or scope of the inven- :~
, tion as set forth herein ' . . . .

. .
`'', ' . ' . ~ ' , .

y~ ~
~ ; I 37 :~ " , , . "

~ '" . . ' ..,",,.
. . .
!, , , .. ':. . . ' ' ' `

Claims (31)

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

1. A method for detecting the presence of a microorganism in a fluid sample, which comprises:
culturing a microorganism in a liquid growth medium which is in contact with a measuring electrode and a reference electrode; and detecting the changing potential between said electrodes, which arises by the migration and accumulation of said microorganism adjacent the surface of said measuring electrode thus forming a charge-charge interaction between said measuring electrode and accumulated microorganisms, by measuring said potential change with a high impedance, potentiometer, having an input impedance of 107 to 1010 ohms.

2. The method of Claim 1, wherein the portion of said reference electrode exposed to said growth medium is shielded from said microorganism by a growth medium permeable but organism impermeable shielding means.

3. The method of Claim 2, wherein said shielding means is a gel.

4. The method of Claim 3, wherein said gel is agar gel.

5. The method of Claim 2, wherein said measuring electrode and said reference electrode are made of the same metal.

6. The method of Claim 5, wherein said electrodes are fabricated of nickel, gold, platinum, silver or stainless steel.

7. The method of Claim 2, wherein said electrodes are fabricated from dissimilar metals.

8. The method of Claim 1, wherein said measuring electrode is a metal electrode and said reference electrode is one of the standard calomel electrodes or the mercury-mercurous sulfate electrode.

9. The method of Claim 1, wherein said growth medium is selected from the group consisting of brain-heart infusion, trypticase soy broth, phenol red broth base + 1%
glucose, trypticase soy broth + CO2, milk, beer and sodium glycolate.

10. The method of Claim 1, wherein said microorganism is a bacterium, fungi or yeast.

11. The method of Claim 10, wherein said bacterium is a non-hydrogen producing organism selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus, Listeria monocytogenes, Pseudomonas aeruginosa, Moraxella, Shigella alkalescens, Diplococcus pneumoniae, Bacillus subtilus and Hemophilus influenzae.

12. The method of Claim 9, wherein said yeast is a species of Candida, Hansenula, Pichia, Toruloysis or Saccharomyces.

13. The method of Claim 1, wherein said microorganism is cultured at a temperature of 15 to 60° C.

14. The method of Claim 1, wherein said microorganism is a mixture of at least two types of microorganisms.

15. A method for detecting the presence of hydrogen producing bacteria in a fluid sample, which comprises:
culturing a hydrogen producing bacterium in a growth medium which is in contact with a measuring electrode and a reference electrode which are conductively attached to a high impedance potentiometer having an input impedance of 107 to 1010 ohms, wherein the growing bacteria gradually concentrate about said measuring electrode thus concentrating the evolved hydrogen gas about said measuring electrode and forming a charge-charge interaction between said measuring electrode and accumulated microorganisms, whereby the evolved hydrogen gives rise to a modified measuring electrode which has the nature of a hydrogen electrode;
achieving a stable baseline response from said potentiometer prior to reaching the minimum detectable concentration of said bacterium; and monitoring the change in potential between said modified electrode and said reference electrode.

16. The method of Claim 15, wherein said growth medium is selected from the group consisting of brain-heart infusion, trypticase soy broth, phenol red broth base + 1%
glucose, trypticase soy broth + CO2, milk, beer and sodium glycolate.

17. The method of Claim 10 or 15, wherein said bacterium is a hydrogen producing organism selected from the group consisting of Escherichia coli, Enterobacter aerogenes, Serratia marcescens, Proteus mirabilis, Citrobacter intermedium, Citrobacter freundii, Salmonella and Klebsiella.

18. A method for measuring the influence of an anti-microbial agent or event on a growing microorganism, which comprises:
culturing a microorganism in a liquid growth medium which is in contact with a measuring electrode and a reference electrode which are conductively attached to a high impedance potentiometer having an input impedance of 107 to 1010 ohms, wherein the growing bacteria gradually accumulate about said measuring electrode thus forming a charge-charge interaction between said measuring electrode and accumulated microorganism;
achieving a stable baseline response from said potentio-meter prior to reacting the minimum detectable concentration of said microorganism;
subjecting said microorganisms to an anti-microbial agent or event; and monitoring the change in potential between said electrodes as a result of the influence of said anti-microbial agent or event on said microorganism.

19. The method of Claim 18, wherein said microorganism is subjected to said anti-microbial agent or event before said microorganism attains the minimum detectable concentration in said medium.

20. The method of Claim 18, wherein said microorganism is subjected to said anti-microbial agent or event after said microorganism attains the minimum detectable concentration in said medium.

21. The method of Claim 18 wherein said microorganism is a bacterium, fungi or yeast.

22. The method of Claim 18, wherein said anti-microbial agent is an antibiotic, serum antibodies, a chemical toxin or virus.

23. An apparatus for the electrochemical detection of living microorganisms in a fluid medium, which comprises:
a chamber with an opening containing a growth medium suitable for the culturing of microorganisms;
means for sealing said opening of said chamber which means is self-sealing after having been punctured by a means for delivering a microorganism containing sample to said chamber measuring electrode means fixedly attached to the exter-ior of said chamber, one end of which penetrates through said chamber into the interior thereof and is in contact with said growth medium, about which accumulates said microorganisms thereby setting up a charge-charge interaction at said measuring electrode means, said measuring electrode means being responsive to said charge-charge interaction established at the measuring electrode means;
reference electrode means fixedly attached to the exterior of said chamber, one end of which protrudes into the interior of said chamber and which is covered with a growth medium permeable shielding means that prevents contact of said reference electrode means with said microorganisms, for establishing the potential of said cell in cooperation with said measuring electrode means; and a potentiometer having an input impedance of 107 to 1010 ohms which is conductively connected to said measuring and reference electrode means.

24. The apparatus of Claim 23, wherein said measuring electrode means and said reference electrode means are fabricated of the same metal.

25. The apparatus of Claim 23, wherein said electrode means are fabricated from stainless steel.

26. The apparatus of Claim 23, wherein said shielding means is a gel.

27. The apparatus of Claim 26, wherein said gel is agar gel.

28. The apparatus of Claim 23, wherein an inlet means for introducing a fluid sample of a microorganism into said cell and a means for drawing a vacuum within said sealed cell are attached and secured to said cell.

29. The apparatus of Claim 28, wherein said inlet means and vacuum means are attached and secured to said chamber.

30. The apparatus of Claim 28, wherein said inlet means and vacuum means are attached and secured to said sealing means.

31. An apparatus for the electrochemical detection of microorganisms in a fluid medium, which comprises:
a chamber closed at one end and open at the other end thereof and containing a growth medium suitable for the culturing of microorganisms;
measuring electrode means fixedly attached to the exterior of said chamber, one end of which protrudes through said chamber into the interior thereof and is in contact with said growth medium about which accumulates said microorganisms thereby setting up a charge-charge interaction at said measuring electrode means, said measuring electrode means being responsive to said charge-charge interaction established at the measuring electrode means;
reference electrode means fixedly attached to the exterior of said chamber, one end of which protrudes into the interior of said chamber and which is covered with a growth medium permeable shielding means that prevents contact of said reference electrode means with said microorganisms, for establishing the potential of said cell in cooperation with said measuring electrode means;

Claim 31 - cont'd ...

conduit means penetrating through the base of said chamber and said shielding means such that the internally protruding open end thereof is exposed to said culture medium for transport of said fluid medium into said chamber;
plunger means fitting within said open end of said chamber which moves freely inwardly and outwardly which allows a sample containing said microorganisms to be withdrawn into or expelled from said chamber; and a potentiometer having an input impedance of 107 to 1010 ohms which is conductively connected to said measuring and reference electrode means.