Note: Descriptions are shown in the official language in which they were submitted.
e~
~lS
PUI,SED VOLI`~MMETRIC DETECTION OF B~CTERIA
The present invention relates to a method for the detection of
microorganisms. More particlllnrly, the present invention relates to a simple,
efficient and reIiable electrochernical method îor the detection of bacteria
by measllring the decrease in polarographic oxygen current passin~ throu~h
an electroanalytical cell contnining two dissimilar wire electro~les immersed
in a liquicl culture medium.
The determination of whether or not a substance is contaminated with
biolo~icaUy active agents such as bacteria is of great importance to the
medical field, the pharmaceutical inclustry, the public health Iielcl, the
cosmetic industry, the food processing industry, and in the preparation of
interplanetary space vehicles. One of the most widely used techniques for
making this determination, especia11y in medical applications, has been
nutrient agar plating. In this method a microorganism is allowed to grow
on an agar nutrient substrate, and the growth of the microorganism is
observed, at first visually and thereafter by microscopic examination. This
technique, which is most commonly used clinically, requires overni~ht
incubation of plates before results are available.
Another technique widely used for the determination of microorganisms
involves supplying a microorganism in a growth rnedium with carbon-14 labeled
~lucose or the like. See Waters IJ.S. Patent No. 3,6~6,679 and Waters U.S.
Patent No. 3,935,073. The microorg~nism metabolizes the radioactive gIucose
and evolves C1~02, which is sampled and counted. While positive results
can be obtained by this radiometric method in a relatively short period of
time, this method requires the use of comparatively expensive and complex
apparatus and involves the handling of radioactive materials.
~,~
'7~ ~
Th~ pl ior nrt also ciescribes a number o~ detection techniques basecl
on electrochemical phenomenn. (1enerally these techn;qlles employ very
clelis~ate ancl e~pensive electronic equipment nnd nre extremely di~ficuIt to
use in nn on-going detection progrnm. One of these described rnethocls
involves the measurem ent of polarographic oxygen current in an
electroanalytical cell. CeU current is a function o~ the dissolvecl oxygen
content of the electrolyte, and the metabolic activity of any oxygen-
consumillg microorganisms present will, therefore, cause the current vaIues
to fall off. ~or a general discussion of this electroanalytical t~(hnique see
Hitchmall, Measurerrlent of Dissolved O2cy~en (1~77); Fntt,
Oxygen Sensors (1~)76)~ and Norris, Methocls in Nlicrobiolo~ (1970). Modern
techniques of polarographic oxygen meas1lrement rely almost exclusively on
the so called Clark-type electrodes which employ a semi-permeable membrane
to prevent the electrocles from contncting the solution; see Clark U.S. Patent
No. 2,913,386.
The commercially available membrane polarographic oxygen de-tector
(MPOD) is presently used to determine dissolved oxygen in BOD studies,
marine ecology, wastewater treatment and the like. The MPOD is usually
constructed with an inert cathode material (gold, platinllm) and a silver-
silver chloride reference electrode, and uses a relatively concentrated (0.3
- 3.0M) potassium chloride electrolyte. 'I'he electrode areas are relatively
large (ca. 1.0cm2) and are prevented from contacting the solution to be
analyzed by a semi-permeable membrane, usually of polyethylene or
polytetrafluoroethylene. The potential applied to the electrodes is normalIy
about 0.8V, cathode negative. This potential must be applied to the MPOD
several minutes prior to any use of the detector, and must remain applied
throughout the duration of any measurements to be made. The MPOD îs
thus a "steady-statell device, in that all electrode reactions stabillze at new
equilibrium values under the influence of an applied potential constant in
time. The steady-state cell current detected under these circumstances is
a measure of the dissolved oxygen content of the solution. Because proper
electrode operation depends upon diffusion of dissolved oxygen through the
membrane to reach the cathode, the solution must be stirred or agitated
constantly to prevent the depletion of oxygen from the sample solution in
the immediate vicinity of the membrane from affecting the results. Some
-
investi~ntion of !~'00 or~elation under non-steacly state or pulsecl conditions t
hns been unclet takell; see T-litchman~ Sllpra~ Cllapter G.
ï he Clark-type polnrograpl- sensors, however, suffer from serious r
drnwbacks which malce them undesilnble for the detection of microorgranisms.
These sensors are expensive and cumbersome to use. The relatively high
cost of the electrocles precludes the use o~ a separate electrode for each t
sample. Thus in order to prevent cross contamination of samples, the
electrode Sut faces have to be sterili~ed between samples using a strong
bactericicle, and then rinsecl completely with a sterile rinse solution so as
not to kill org~nisms in or contaminate the contents o~ the next sflmple
cell tested.
The electroanalytical detection of microorganisms by measurement of
oxiclation-recluction potentinl has also been described in the prior art; see
generally Norris, suprfl, Chapter 4. Tn the redox potential method a platinum
electrocle in combination with nny commonly usecl reference electrode such
as the calomel electrode will cvidence an eguilibrium potenti~l in ~rowth
medium proportional to dissolved oxygen in the medium and to any other
oxidation-reduction (electron transport) reactions taking place in the solution.Because this is an equilibrillm measurement, a voltmeter with very high
input impedarlce must be used to measul e the potential existing between
the electrodes so as not to clisplace the equilibrium as a result of current
flow. Microorganisms ~rowing in the medium use oxygen from the solution,
and may possibly contribute to other redox reactions which cause the
measured potential, usua11y greater than +lOOmV (cathode or platinum positive
with reference to the standard calomel electrode) in sterile médium, to
shift toward more negative values. l~erobic organisms are able to reduce
the solution enougrh to yield measured potentials of -lOûmV to -200mY (vs.
SCE). This is also the range of redox potentials where the voltammetric
m ethods cease to function; the cell current due to dissolved oxygen is by
this time very small, and is usually swamped by the residual cell current
d~le to solution impurities and electrode imperfeetions. Facultative lmaerobes,
however, may reduce the solution extensively~ An exhausted culture of P.
mirabilis will have reduced the medium in a sealed container to a value of
. .
around -550mV (vs. SCE) before ceasing growth. The redox potential method
by itself is not well suited to the detection of bacteria because it is
1,
relntively slow ~n(l the response will clepend on the type of organism being
cletected.
From the foregoing it is clear thnt a need exists for a simple rapid
and reliable method of detecting the presence of microorganisms in a suspect
sample.
~ ccordingly, it is an object of the present invention to provide a
method for cletecting the presence of microorganisms which empIoys apparatus
which is relatively simple in both construction flnd operation Rnd which uses
relatively inexpensive non-radiolabeled materials.
It is nlso an object of the present invention to provide a method for
the cletection of microortrallisms which facilitates computer controlled
automation and which can incorporate disposable components.
Further objects of the invention will be apparent from a consideration
of the following description,
These and other objects of the invention are achieved by providing
an electroanalytical method for detecting the presence of oxygen-consuming
microorganisms in a sample comprising the steps of providin~ a mixture of
said sample and a fluid culture medium capable of supporting microorganism
growth in an electroanalytical cell equipped with two electrodes
which are in contact with said mixture; applying a series of voltage pulses-
of substantially constant amplitude and duration across said e]ectrodes; and
measuring the resulting current prior to the trailing edge of each of said
applied voltage pulses; the presence of oxygen-consuming microorganisms
being indicated by a decrease in cell current which is a function of the
dissolved oxygen content of said mixture.
In a preferred embodiment the present invention also cantemplates a
process for the detection of microorganisms as described above and
ad~itionally comprising measuring the open-circuit voltage potential across
said electrodes during the interval between successive applied voltage pulses.
Determination of the dissolved oxygen content of the cell is
accomplished by pulsing the electrodes briefly with a known potential (cathode
negative) and measuring the resulting current through the cell prior to the
trailing edge of the applied voltage pulse, The growth medium in the cell
is used as both the analyte and the electrolyte for the deterrnination. The
very low duty cycle of the pulse with r espect to the overall sampling
2l(~
interval obviatcs the necd for constant agil:ation or ;tlrrlng
of the sample solukion required by conventional stea~ly~Y~ e
methodology, and permits the same electrodes to be uscd to
determine the relative oxidation-reduction potential in tlle
cell through the rneasurcment of the open-circuit potent:lal
existing between the electrodes. Bact:erial dctection is bcst
accomplished by measuring -the decrease in pulsed volt.lmmetrlc
oxygen current, while inEorrnation characteristic of the ty~e
of organism present is best furnished by the relative cell
potential determination.
Times-to-detection for all organisms studied vary
wlth inoculum strength in a predictable fashion, permittillg
accurate quantification of the organism in question when results
are compared with times-to-detection obtained using known
inocula of the same organism.
The process of the present invention provides numer-
ous advantages compared to traditional manual methodology and
present automated systems. This process requires a cell of very
simple construction, provides ample opportunity or the
creation of disposables, promotes automated quality control,
prevents any chance of cross-contamination, and can be configurcd
- as an instrument very sophisticated in operation, yet extrcmcly
simple to operate.
Figure 1 is a front elevation view of an electro-
analytical cell useful in the process of the present invelltion.
Figure 2 is a sectional view of the electro-
analytical cell of Fig. 1 taken along line 3-3.
Figure 3 is a top plan view of the electroallalytical
cell of Fig. 1.
Figure 4 is a simplified schematic diagram o an
analog conditioning circuit useful in the process of ~lle
present invention.
~t3~'7~,~
Fi~ure 5 is c3raph showing the ccll currcl-lt rc~sponse
oE a Eully-c3rown E. c~oli culture at vari.ous pulse wi(lt}ls as a
function of applied cell voltage.
Figure 6 is a graph showing the cell cul-rcllt
respon.se of a ste~ile cell with constant appl.ied po~cntial as
a fullction of elapsed ti.me since pulse applicati.on.
Figure 7 is a graph showing the cell current
response as in Figure 6 with the elapsed time extendcd to
300 seconds.
Figure 8 is a graph showing the cell current
response as a function
-5a-
of applie(l potential for n sterile electroanalytical ce~l and for a cell
containing fully-grown F. coli cultut e.
~ ;gure 9 is a graph showillg voltammetric cell current response for
a sterile ceLl purgre{l ~vitll dry nitrogen; cell current is recorded as a function
of elasped time during the purge.
Figure 10 is a graph showing the cell current response for the sterile
cell of Fig. 9 purged with dry nitrogen, then purged with roorn air.
Figllre 11 is n schematic representation of the sampling and dikltion
scheme used in the preparation of electroflnfllytical cells and pOUI' plates
for the e~amples.
Figrure 12 is n graph showinog normaliYed voltammetric cell current
response as a functioll of incubation time for varying inoculum strengths of
the organism E. coli.
Figure 13 is a ~aph showing normalizecl cell potential response as a
function of incllbation time for varying inoculum strengths of the organism
E. coli.
Figure 14 is a graph showin~ normalized- voltammetric eell current
response as a function of incubation time for varying inoculum strengths oE
the organism E. cloacae.
Figure 15 is a graph showing normalized cell potential response as a
function of incubation time for varying inoculum strengths o~ the organism
E. cloacae.
. .
Figure 16 is a graph showing normalized voltammetric cell curr ent
response as a function of incubation time for varying inoculum strengths of
the organism P. mirabilis.
... .. _.
Figure 17 is a graph showing normalized cell potential response as a
function of incubation time for varying inoculum strengths of the organism
P. mirabilis.
Figure 18 is a graph showing normalizecl voltammetric cell current
response as a function of incubation time for varying inoculum strengths of
the organism P. aeru~osa.
Figure 19 is a graph showing normalized cell potential rcsponse as a
function of incllbation time for varying inoculum strengths of the organism
P. aeruFinosa.
Figure 20 is a graph showing normalized voltammetric cell current
response as a function o~ incubation time for var!~incr inoculurn strcngths of
the orgrnnism S. a~!reus.
Figure 21 is a grnph sho~vingr nol malizeù cell potential response as a
function of incubation time for varying inocull1m strengths of the organism
S, aureus.
Fiv~ure 22 is a graph showing normalized voltammetric cell current~
response as a function of incubation time for varying inoculum strengths of
the organism S. bovi~s.
Figure 23 is a graph showing norma1ized cell potential response as a
function of incubation time for vnrying inoculum strengths OI the organism
S. bovis.
.__
Figure 2~ is a graph showing normalized cell current response as a
unction of incubation time for the orgnnism E. coli with four decades of
initial inocu1um concentration.
~ igure 25 is a g,raph showing the logarithm of the initial inoculum
dilution ratio as a function of time--to-detection at a 60% detection threshold
for the data shown in Pig. 2~.
Figure 26 is a graph showing normalized voltammetric cell current
response as a function of incubation times for varying inoculum strengths
of the organism E. coli using cells fitted with golcl cathodes.
~ igure 27 is a graph showing normali~ecl voltammetric cell current
response as a function of incub~tion times for varying inoculum strengths
of the organism P. mirabilis using cells fitted with gold cathodes.
Figure 28 is a graph showing normalized voltammetric cell current
response as a function of incubation times for varying inoculum strengths
of the organism P. aeruginosa using cel1s fitted with goId cathodes.
The present invention detects the presence of bacteria in a suspect
sample primarily by measuring the decrease in voltammetric oxygen currerlt
passing through an electroanalytical cell containing the sample and a fluid
growth medium. Viable organisms capable of utilizing dissolved oxygen
during rnetabolism will cause the deteeted oxygen current to decrease with
continued incubation, signifying detection. Sterile inocula will evidence no
such current decrease. ~clditional means is provided to measure the open-
circuit voltage of the analytical cell in order ~o obtain information as to
the type of bacteria (primarily aerobic or facultative anaerobic) present in
the cell. Organisrns thflt consume little or no oxygen from the growth
- ~ -
meclium, yet which have the ability to alter the solution redox potential,
rnny be cletected by notirlg the change in the solution reclox potential with
incubation, as furnished by the open-circllit cell potential measurernent.
The methocl of the present invention ean be used to deteet the
presence of any aerobie or faeultntive organisms ~Jhieh consume oxygen frorn
a liquid meclium during metabolism. Specifie exarnples of such organisms
include bacteria such as E. coli, E. cloncae, ~. mirabilis, P. aeruginosa? S.
aurells~ S. bovis T~. peneumoniae, S. albus ~. o~ytoca, E. aerogen~ E.
? ~
ag~lomerans~ C. freullclii~ E'. morga~L P. stuartii, S. rmarcescens~ Group B.
Beta strep, C.rp. D. Strep, ~nd yeasts such as C~ albicans.
In the process of the present invention a small portion of a s~speet
sample is first introdllced into an electroflnalytical cell containing a liquid
growth medium. Tlle growth medium also serves flS the primary electrolyte
in the cell. ~ny medium which will support the g~owth of vxygen -
eonsuming mieroorganisms may be utiliæed.
Typical growth media generally contain water, a earbon souree, a
nitrogen source, caleium, magnesium, potassium, phosphate, sulfate, and trace
amounts of other minor elements. The carbon source may be a carbohydrate,
amino acid~ mono- or clicarboxylic acid or salt thereof, polyhydroxy alcohol,
hydroxy acid or other m etabolizable earbon compouncl. Usually the carbon
source will comprise at least one sUcrar such as glucose, sucrose, fructose,
xylose, maltose, lactose etc. ~mino acids such as lysine, glycine, alanine,
tryrosine, threonine, histidine, leucine, etc. also frequently comprise part of
the culture media earbon source.
The nitrogen souree may be nitrate, nitrite, ammonia, urea or any
other assimilable organic or inorganic nitrogen source. ~n amino acid might
serve as both a carbon and a nitrogen source. Sufficient nitrogen should be
present to facilitate cell growth.
A variety of calcium, potassium and magnesium salts may be empIoyed
in the growth medium inclucling chlorides, sulfates, phosphates ancl the like.
Similarly, phosphate and sulfate ions can be supplied as a variety of salts.
As such materials are conventional in fermentation media, the selection of
specific materials as well as their proportions is within the skill of the art.
The so called minor elements whieh are present in trace amounts are
com monly understood to include manganese, iron, zinc, cobalt and possibly
others. Due to the ~act that most biologically active species cannot function
.' , . ~
'C~~ ~ ~ ~
- ~ -
in s~rongly acidic or strong1y alkaline medi~, sui~able buf~ers such ~s
potassiurn or ammoniurn phosp}lates rnay be employed, if desired, to maint~in
thc p~-3 oî the gro~th medillm neal ncutrality.
Exarnples of well known gro-vth media which may be used in the
present invention are peplone broth, tryptic soy broth, nutrient broth or
thioglycolate broth. I`ryptic soy broth-based rnedium (6Es Medium, Johnston
Laboratories Inc., Cockeysville, Md.) has been found to work welI. The
amollnt oî growth mediurn provided in the electroanalytical cell is not overly
critical. 5.0cc of 6B medium has proven very e~fective.
The analytical cell useful in the process~of the present invention may
be of any convient si~e and shape. The cell can ~ie formed from any
materials norrn~lly used in the rnanufncture of e1ectroanalytical cells such
as ~lnss, plastic and the llke. ~ny material ~hich does not affect the
growth the microorganlsms or the measurement o~ electrochemical
phenome)-on in the cell can be employed. In the preferred form, the
electronalytical cell useful in the process of the present invention comprise
a plastic container of the general configuration shown in ~igs. 1-3. Cell
volume may vary accordingr to the cell design and is not critical. A ce~I
of the type shown in Figs. 1-3 has been effectively used at a capacity of
about 10-15 mls. In the preferred manner of operation a number of these
cells can be utilized in the form of an array to permit testin~ of multiple
samples.
The electroanalytic~l cell is a~so equipped with two
electrodes in electrical contact with the growth medium. The ~vorkin~
electrode (cathode) is normally a noble metal, for example, gold,
silver or platinum. When only voltametric measurements are to be
taken, gold ,platinum or silver are preferred ~or the cathode.
When potentiometric (redox) measurements are also taken gold should
not be used as the cathode material. The reference e~ectrode is
preferably pure silver ~99 95% or better) electrolyzed in place
using a basic electrolyte to deposit Ag20 on the silver. Silver
chloride may be electrolytically deposited from HCl solution to
form the alternative Ag/AgCl reIerence electrode, but this electrode
has been found less stable in this application. In actual practice
clean, unprepared silver wire will quickly become covered with a mixture
OI Ag20 and AgCl due to Cl`ion in the medium and to the very high pH
around the anode when pulsed . Al though pure silver is known to be
- ]O -
bactericidal, no evidence of such toxicity has been noted using oxidized
silver r cference clectrodes.
The electl odes may be used in any convenient form. Preferred are
wires o~ the above materials al~hough other forms such as printed circuit
traces can be used. Most preferred are l~-shaped staples inserted through
the bottom of the cell as best seen in Fig. 3. The elec~rodes, however,
can be of other conventional ~orms, including spaced apart vertically
disposed hair pin shaped eLectrodes. The electrode wire diameter l
is not critical. ~Yires as small as 0.010" rnay be used, provided their ¦
frangibility nnd low sensitivity can ~e tolerated. Wires approaching 0.040"
probably represent a practical upper limit since these materials are quite
expensive. Preferre(l electrode diameters nre in the range of from about
.015 to .050", with about 0.020" to 0.040" being most preferred. Electrode
lengths are lil<ewise noncritical. In practice, lengths of from about 0.5cm
to 2.0cm and preferably about l.0 to 1.5 cm are suitable. The wires may
be separated by about 0.5 to 2.5cm and preferably about l.0 to 2.0 cm.
It wil} be apparent to those skilled in the art that solid precious metal
wires can be replaced with less expensive wire electroplated with the precious
metals of choice.
The electrode pair may be covered with a porous gel, preferably a
nutrient gel such as tryptic soy agar (TSA). Other gel materials which
may be used include gelatin, dextran gel, carrageenan gel and the like.
Best results are obtained when the gel just covers the electrodes. The
main benefit of the gel is to reduce measurernent baseline drift sometimes
caused by the introduction of biological samples (urine, etc.), presumably by
preventing the migration of large charged molecules to the electrodes. The
quantity of gel is r,ot critical; l.0cc of TSA has served to just cover the
electrodes in the type of ce71 shown in Figs. l-3. It may be appreciated
that some ionic conduction is necessary in the gel; hence its equivalent
conductance when saturated with growth meclium/electrolyte should approach
that of the medium alone.
Ihe electrode pair may also be isolated from the effects of large
charged molecules by positioning a layer of porous material such as ordinary
filter paper over the electrodes. ~hile this will not prevent contact of
the electrodes with tne analyte or even with the microorganisms in the
sample, it ~.lill limit migration of lflrge charged molecules to the electrode
region.
-
In nnother embodiment of the present invention the re!felence electrode
can be isvlate(1 Erorn tlle analyte mi.Ytul e by the use of a salt briclge or
othel conventionnl means. In this rnnnner it i5 possible to employ a single
reference electroc3e in conjunction with a plurality of worlcing electrodes in
scparate analytical half cells.
~ fter the cell has been inoculated with a sample to be tested a
series of voltage pulses of substanti~lly constant amplitucle àncl cluration areappliecl ncross the electrodes. The voltnge amplitude of the pulses can vary
from about -0.35 v. to about -0.90v. Preferred is an applied voltage pulse
of abollt -0.70v. Tlle pulse cluration should be at least abollt 600 milliseconds.
The llpper limit of the pulse duratioll is not critieal. ~\s a practical matter,times mllch over nbout 3 seconds reslllt in a redllced current signal but
may be used. Preferably the pulse cluration can be from about ~00 to
2000ms. with about 1200 ms. being most preferred. The pulse intervaI is
not critical ancl should be short enougll to follow the biolo~,ical changes but
long enol1gh to allow the cell to app~oach equilibrium conditions for redox
potential measurements in the pulse intervals. Times OI from about 5 min.
to 20 min. are suit~ble. A pulse interval OI about 10 minutes is preferred.
During the testing period the analytical cell and its contents should
be held at a constant temperature, preferably 37C ~0.2~C. It is understood,
however, that not all biologically active agents exhibit maximurr1 growth
within the cited temperature range. I~ it is of interest to determine whether
or not a specific microorganism which grows better at some other
temperature is present, then the temperature at which the organism in
question exhibits maximum growth should be employed.
Current readings from the cell are taken prior to the trailing edge
of each of the applied voltage pulses. The pulsed, periodic nature of the
measurements involved obviates the need for constant agitation or stirring
of the sample as would be required for conventional steady-state polarographic
oxygen determination. The requirement of extremely hi~h input impedance
for the potential measuring circuitry is similarly relaxed, since the analyticalcell is connected to the external electronics only long enough for appropriate
potential and current readings to be taken. The simplicity of the analytical
cell coupled with the complexities of sample selectio1l and interrogation
suggest that the technique of the present invention be practiced in a fully
J ~i
-- 12 --
nl1tolnate(l fashion. A microcoi-nputel sy~st~m t an be use(l to control all
aspects of expel ilnental measllrelrlent an(l to nnaly~e~ tabulate, plot, and
store on floppy clislc the information gatllerecl during each experiment.
The constr~ tion of a typicul analytical cell useflll in the process oî
the present invention is shown in ~igs. 1-3. ~ semi-flexible plastic container
(1) receives the t-vo wire electrodes (2,3) in the form of U-shaped "staples"
inserted through the hottom of the container. The worlcing electrode (2)
is analytical-grflde platinurn, Q.035" diameter. Re~ere~nce ele~trode (3) is
0.0~0" d;ameter ~\g/AgO. The elect~ocle pnir is covered with a nutrient
gel (4) such as tryptic soy ngar (rSA). ~ quantity oî liquid growth rnedium
(5) is nlso present in the ceLl to serve ns the primary electrolyte and to
facilitnte the growth of rnicroorganisms.
l`he per~ormance of a single experimental clata-gathering cycle may
be understood by considering 1 single two-electt ode cell as part of the
signfll c onversion CirCIIitry, as presented in simplifiecl form in ~igure ~. A
clflta-gathering cycle begins with the selection oE this particular cell. After
a short delay to allow the cell selection relays to settle, a potential reading
is taken with relay 11 open via operational arnpli~iers OAl and Q~2~ Because
the cell redox potentinls on platinum are bipolar with respect to the reference
electrode (-~150mv to -550mv) witll incubation of n facultative anaerobe,
provision is rnade to offset the potential r eacling using OA2 to present a
unipolar, positive sigrnal o~ adjustable gain to the computer A/l~ converter.
Immediately following the potential reading, relay 11 closes, and a positive-
going pulse derived from the computer D/A converter is applied to OA3.
The inverted, unity-gain output of 0~3 causes current to flow through the
selected cell in proportion to the dissolved oxygen content. The cell current
is sensed by OA4, connected as a current-to-voltage converter. The output
of OA4 is sampled by a second input of the A/D converter card prior to
the termination of the voltage pulse from OA3. ~11 relays are switched
off and allowed to settle prior to the selection of the next cell to be
tested, at which point the process begins again for the newly selected cell.
After all cells have been tested, all relays are deselected while the
programmed time interval between readings, usually ten minutesJ is allowed
to elapse.
The voltage signal input resistor (Rinp, OAl in ~igure 4) has the value
- 13 --
of 2~2Megollrns. Although this is by no means an electrornetric input
resistnnce ns is normnlly employecl to menslll e electroallalytical cell
potentinls, it must be remembered tlult the cell is loaded with this resistance
for only a few h~lndred milliseconds before the voltage reading is stored,
and that the foLlowing voltage pulse clrives the cell far ~rom equilibrium in
any cflse. The 2.2M resistor provicles a goocl compromise between ceII
loadin~ and noise pickup in the ceII environment.
The eell current and potent~al values measured respectively durino;
ancl between the successive applied pulses can be cornparecl to the initial
values to determine when the thresholcl of detection has been reached. This
process is facilitated by normalizntioll of the conected values as clescribed
more fully in Example 2. ~Vhen the current level has fallen to a
predetermined percenta~e of the initial value, e.~., 60-80%, detection is
found to hnve occurred. It is also possible to malce deterrninat;ons by
comparing the collectecl datfl to that obtalined in a separate reference well.
The method of the present invention can be used in any application
where the detection of microorganisms in a sample is desired. This method
finds particular utility in the detection of bacteria in biological fluids such
as blood, urine and the lilce.
~ s will be reaclily appreciated by one skillecl in th~ art, bacterial
testing can include screening, identi~ication, and antibiotic susceptibility
testing. Other ureas of utility include the detection of microorganism
contamination in food, pharmaceutical and cosmetic products.
The following examples are included for illustrative purposes only and
are not intended to limit the scope of the invention.
E~ PI.E 1
- This example demonstrates the selection of optimum values of the
voltage pulse amplitude and duration.
In order to separate the effect of pulse width from that of pulse
amplitude in a semi-quantitative fashion, a test was conducted to determine
the cell current at various pulse widths for applied voltages ranging from
-O.OOV to -1.00V in steps of 0.05V in a cell eontaining a spent culture. As
in all examples, the platinum electrode received the pulse~ while the reference
electrocle ~as helcl at virtl1al grollnd. r~ e clltlent-to-voltlge conversion gain
~vas held constant.
A sing~le sample cell o~ an at rny consisting of 8 cells as shown in
FigLIrc I ~vas prepared using l.0cc of molten TSA (~ûC) transferred to the t
cell Vicl sterile syringe. 5.0cc o~ a fully-grown E. coli cul~ure in 6B medium
(oxygen depleted~ was similarly transferred to the cell a~ter the agar had
solidi~ied. The cell m ray was plnced in a wat m-air incubator at 37 C and
allowe(l to equilibr~te Ior 40 minutes, at which time readings were begun
under computer control. ~\ voltage scan Oe the cell WflS obtaiTled and
plotted for each chosen pulse width. The interval between pulses was
nppro~imntely 10 seconcls, depending somewhnt upon th~ selected pulse width.
C~ll culrent tesponses for selectecl pulse widths are presented in
Figure 5. It may be noted tha t the desired cell response is achieved with
pulse widths greater thnn about fi4(1 milliseconds. ln practice, pulses of
about 1200ms. duration 11nve beed used with goocl success. Because cell
current begins to fall rapidly from the instant the pulse is appliecl, there
is little to be gained ~rom the use oE a pulse width in excess OI that
necessary to produce tile desired response. This effect may be noted
graphicaUy in Figures 6 and ~ which depict the results obtained when a
single cell containing 1.0cc TSA and 5.0cc 6B medium is subjected to a
continuous applied voltage oE -0.70V, with current readings obtained every
û.5 seconds beginning 0.5 seconds after vol-tage application (Fi~. 6). In
Figure 8, one point is plotted for each 20 collected, stored and tabulated.
The cell current has decreased to less than half its value at 0.5 seconds
(217) after only 4.0 seconds (106); after 5 minutes, cell current has fallen
to 16.6% of the initial measurement value (36), yet steady-state conditions
have not been achieved. Once the desired cell response of low residual
~urrent when containing microbiologically reduced med;um has been achieved7
further increases in pulse width merely decrease the desired current signal
and displace the cell further from thermodynamic equilibrium (Fig 5).
Conversely, pulses shorter than about 600ms provide unsatisfactory results.
Figure g illustrates a voltage scan overlay for two cells, one containing
l.Occ of a fully-grown culture oE E. coli in 6B, the other containing sterile
6B medium, both ineubated at 37~ C for 4 hours prior to measurement. A
pulse width of about 1300ms was employed. The cell responses are observed
.'
r
- 15 -
to scparate nt abollt -0~35V~ r~acll ma~;i1nl1m c1ivergence near ~0~75V~ and
converge ngnin nhollt -O.~)OV. Tlle cletection of microorgnnisms, tl1en, requires
a pulse potential near -0.7SY for best efficiency under t11ese conditions.
Consicleration of the results presented in Figures 5 and 6, together with
similar c1ata obtained during the early phases of experimentation Ied to the
selection of -0.70V as the pulse amplitude, with ~ wic1th o~ about 1200ms
for use in the remainder of the examples~
It is also observed that several minutes are required for the cell to
return to conditions approaching equi1ibril1m fo1lowing the application of a
voltage pu1se. Ideally, the time bet~veen pulses shoulc1 be very large compared
to the applied pulse wic1th. Ten min~1tes was selected as the sampling
interval, since it is a short period of time microbiologically speaking, yet
it provides for a pulse duty cycle of only 0.2~,~ while at the same time
permitting the acquisition of semi-continuous data for rapidly growing
orgcmisms. Even at this low duty cycle, measured ceU potential values are
depressed somewhat from their equilibrium values. The measured potentials
do, however, adequately represent relative reclox potentiRl changes in the
grow th m edium .
Finally, in order to insure that dissolvecl oxygen content is in fact
the major parameter determined by the pulse polarographic technique under
the chosen experimental conditionsJ a single cell o~ the 8-cell array was
prepareà as usual, containing l.Occ TSl~ and lO.Occ 6E~ medium. The cell
was incubated at 37 C for 30 minutes~ then read every 60 seconds for lO
minutes. Dry nitrogen gas (Matheson, High-Purity) was then bubbled through
the cell by means of a disposable glass capillary pipette. ~e~dings were
continued until the cell current dropped to a reasonably constant value. In
a separate experiment conducted in the same cell, nitrogen was again bubbled
through the cell, and readings begun just prior to reaching the purge plateau.
The gas supply tubing was quickly transferred to a small aquarium pump
~ith flow constrictor, Imd readings continued for twenty additional minutes.
Data from these tests is plotted in I; igs. 9 ancl 10. Cell response is shown
to be clearly related to the oxygen content of the solution; response also
appears to be totally reversible in the case of this sterile cell In actual
use with bacteria, however, cell reversibiIity becomes a strong function of
the amount of time the cell is exposed to a fully-grown culture.
I
EX~MPT.E 2
In nll tile following e:calTIples, electrochemical measurements~ unless
otherwise notecl~ were carriecl ont in an S-ce11 arrny using platinum (0.035"~
ancl silver/silver oxicIe (0.040") wire electrocle~; of l.O~m length separated hy
l.Ocrn. Prior to each run, the cel1s were carefully rinsed with deionized
water nnd vigorously shaIcen to clislodge the larger droplets. I.Occ TS~
(Trypic Soy ~gar~ ~O.Og/17 ~BL, Cockeysville, MD) at about 90 C was then
transferre(I to each cell usin~ a sterile 3.0cc syringe with 18ga. needle.
The arrRy ~,vas then covered, and the agar ~11owed to solidiey. 5.0cc sterile
6B medium (Johnston LaboIatories, Inc., Cockeysville, MD) was then added
to ~ach cell, together with O.lcc of a ~.5g/20ml stcrile glucose solution
and/or O.lcc of a 1.5g/20ml sterile glycine solutioIl flS desired. The prepared
ce11s were covered ~md set aside while the inoculum dilutions and pOUl- plate
dilutions were preplL~ed.
~ resh cultures of the orgaIlisms to be studied were prepared in 6B
medium the clay before each test, ancl al1owed to incubate at room
temperature until neecIec]. Previously preparecl sterile 20cc vials fitted with
rubber septa and aluminum closures containin~ 9.0cc TSB (27.5~/l,BBL) were
used for all inoculum dilutions. Similar lOOcc vials containing 99~9cc 1/2-
strengrth TSB were used to prepare pour plate dilutions. The test was
initiated by preparing n sterile l.Occ syrin~e containing about 0.5cc of the
overnight culture of the desired organisrn. This culture was adcled dropwise
to a 9.0cc dilution vial with agitation until visual turbidity was achieved.
After complete mixing, l.Occ was removed from this vial and used to
inoculate a second (X O.l) vial containing 9.0cc, achieving 1:10 dilution. l.Occfrom this vial was used to inoculate a third (X 0.01) vial, whieh was in
turn used to inoculate a fourth tX 0.001). ~dditionally, O.lcc was removed
from the X l.O vial and used to inoculate one 99.9cc vial to obtain l:1000
dilution for pour plate preparation. Similarly, O.lcc was withdrawn ~rom
the X 0.01 vial and used to inoculate a second 99.9cc vial to obtain 1:10(),000
dilution. A fifth vial containing 9.0cc TSB was used as the source of
sterile, control inocllla.
l.Occ of the X 1.0 vial was then transferred to cell (1) of the array.
Cells (2) and (3) received l.Occ each from the X 0.1 dilution vial; cells (4)
.1,~ g
-- 17 --
and (5) were inoculated with l.Occ flom the X o~nl vial, while cel1s (6) and
(7) cach receivecl an inocul~lrn from the ~C 0.001 vinl. Cell (8) was inoculatedWitll l.Occ sterile l`SB to serve ns the control.
l`he inoculated cells were placed in a warm-air incubator held at
37 C ~ 0.2, connected to the analog conditioning electronics, and the
measu1ements begurl. No preinoculation incubation of the array was
employecl. Cell current and cell potential readings were obta;ned at 10-
minute intervals using a pulse nmplitude of -0,70V ancl a pulse duration of
1200 rnilliseconcls.
Dllplicate pour plates were prepared containing 10-15ml TSA t40g/1)
usin~ l.Vcc and O,lcc from each of the ~9.~cc clilution vials to yielcl pairs
of plates at 1:103, 1:10~, 1:105, ancl l:lOfi dilutions. The plates were allowedto harden at room temperature prior to 24-hour incubation, Details of the
sampling and clilution scheme are set out in Figure 11.
All values of current and potential recorded by comp~lter in these
examples range from O to 255 as a c~-nsequence of unipolar 8-bit conversion
of the input signals. These raw data values are storecl in the appropriate
memory array during the experiment. All data manipulation is performed
after the experiment is terminated. I~aw data and experimental specifics
are storecl on a floppy disk for future retrieval.
Cell current l(T,N) as a function of time (T) and sample number (N)
is normalized at a chosen time interval ll for sample N by dividing cell
currents observed at all times T for sample N by the cell current value
observed at time T=Tl, and then rmultiplying by lOn.O, e.g.: -
X(T N) = I(T7N) x 100 0
I(Tl,N)
'rhe same normalization time (Tl) is used for all samples. The normalized
current values X(T,N), now ranging nominaUy from O to lOO, are then scaled
for plotting $hrough division by a scale factor, herein 2.0, so that aU data
together with the machine-generated coordinate time axis will fit on the
80-character CRT/printer line~ Cell currents for each sample are thus easily
presented as ~ percentage of the normalized current value, usually taken
as the current value observed after 30 minutes experiment time.
- lR -
Ccll potclltial res-llts V( r,N) are normali~ecl by simple ~-axis
translation alld ulliforrn scnlin~. ~ constant is first derived from the voltageobserved at the normalir~ing time intcr val Tl:
,
C = V(Tl,N) - 2n
whiclr is in turn llsecl to translate all observed voltage vaIues for a given
sample N:
Z(T,N) = V(T,N) - C
The translatecl values Z(TtN) are then scale~l to page width by dividing by
the scale factor, herein 3.5? and then printed. Potential norrnalization is
usually performed a-t 60-100 minutes after the experiment hns begun. Cell
potential readings require about 60 minutes longer, on average, to stabilize
than do the current readings.
For the purposes of the fo~lowing examples, detection of the organism
is said to occur when the pulse voltammetric cell current hns fallen to 80%
of its value at the normalization time interval. Potential measurements
are considered positive when R relative normalized value of 20 is attained.
All values obtained for cell (1) were used as high-inoculum marlcers only,
and do not appear in the results.
This example demonstrates the detection and quantifieation of E. coli~
A fresh overnight culture of E. coli in 6B medium was used as the inoculum
souree. The sampling and dilution scheme of Figure 11 was employed to
prepare sample cells and pour plates. The sample cell medium was enriehed
with O.Icc (oî the~ 4.5g/20ml glucose stock solution. Ineubation of alI vials,
sample cells and plates was at 37~ C. Cell readings were continued for 8
hours.
Pulsed voltammetric current responses for three deeade dilutions of
the organism plus control are shown in Figure 12. The related cell potential
results are presented in Figure 13. The Y-axis indicates potential increasing
in the negative direetion. The short plateau evidenced at relative potential
values Oe 30-40 probably indicates a ehange in the metabolism of the organism
triggered by the redueed oxygen tcnsion in the medium. Both eell parameters
_ 19 _
give times-to-detection which vary in a predictable manner with inoculum
strength. Silver cathodes have also been used without rendering
the process inoperative . Pour plate results indlcate that 1. O x lO
cfu/ml E, coli were present in the freshly inoculated X O, 1 cell .
Times-to-detection Eor the duplicate cell current and potential
measurements are presented in Table 1.
Initial Inoculum Cell Current Cell Potential
in Cell _ B A B
1.0 x 105cfu/ml 120 120 160 180
1.0 x 1o4 " 150 160 220 230
1.0 x 10 " 200 200 260 27U
Table 1
l`imes-to-I)etection in Minutes for
Cell Current and Cell Potential
for the Organism E. coli
EX~MPLE 3
This example demonstrates the detection and quantification of E.
cloacae. A fresh culture of E. cloacae was incubated overnight at room
temperature to serve as the inoculum source. The sampling and dilution
scheme presented in Figure 11 was used to prepare duplicate sample cells
and pour plates. The cell medium was enriched with 0.1cc glucose stock
solution. All incubations were performed at 37~ C. The inoculated ce'll
array ~as covered with clean aluminum foil~ placed in the incubator, and
connected to the analog conditioning electronics moments before the start
of the test. The test was continued for 520 minutes (8-2/3 hours).
The cell current response for each of the three decade dilutions of
E. cloacae plus control is presented in Figure 14. Cell currents are seen
to rise relatively rflpidly from their attained minimum values probably as a
consequence of electrode-active metabolic products synthesized by the
organism in its latter stages of growth under reduced conditions. The
related cell potential data is shown in Figure 15. A short plateau in redox
potential values is again noted at relative normali7,ed values between 30
~ "~
- 2n-
and ~0, ag~in attriblltecl to an or~mism metabolic pathway change. Duplicate
pour plate counts were used to determine the initial inoculum level in the
X 0.1 cells to be 3.7 x 10 cfu/ml. The incubation times required to detect
the or~anism are listed in Table 2.
Initial Inoculum Cell Current Cell Potential
in Cell ~ B ~ B
.
3.7 x 105c~u/ml 150 150 230 200
3.7 ~c 10~ " 210 210 260 2~0
3.7 x 103 " 270 270 300 310
Table_2
Times-to-Detection in Minutes for Cell
Current and Cell Potential
for the Organism E. cloacae
EX~MPLE ~
This example demonstrates the detection and quantification of P.
mirabilis. ~ fresh overnight culture of P. mirabilis in 6~ medium w~s used
as the inoculum source. Sample cells and pour plates were prepared as
per the sampling and dilution scheme presented in Figure 11. 5.0cc 6B
medium was enriched with 0.1cc sterile glucose stock solution in each of
the cells. All incubations were performed at 37 C. The electroanalytical
measurement was begun immediately following cell inoculations without pre-
inoculation incubation, and was continued for 540 minutes (9 hrs).
Pulsed voltammetric cell current responses for the three decade
dilutions of the organism are presented in l~igure 16. Cell responses appear
normal as oxygen is consumed and cell current falls to the residual level,
then rapid vertical transitions appear lasting only 20-30 mim~tes. Cell
current values seem to stabilize following these events, but do not return
to pre-transition levels.-
Cell potential responses are shown in l?ig-lre 17. ~gain, a .slight
plateau is observed at normalized relative potentials between 30 and 41)
-- 21 --
urlits. Instcn(l o~ the rapiclly incrensill~ negat;ve potentials noted for E.
coli nnd 1~. cloacae, P. mirnbilis potelltials fall sharply after a sli~ht increase
following the plateau. The time intervals noted for this potential decrease
correlate well with the observed vertical transitions in cell current previouslynoted. Since P. mirabilis is a facultative org~mism known to efficiently
.
reduce growth media, and has been used as a standard organism for medium
reduction measurements, these flnomalous results in the long-incubation
regime are best explained by the formation of electrode-active nnetabolic
by-products, most probably sulfide-containing molecules (H2S, CH3$CH3~
CI13SGH2C~I3 etc.) which certainly would perturb the clectrocle system. The
silver/silver oxide electrocle is noticenbly blackened by ea~posure to P
mirabilis for extendecl periods. The electrodes clo not seem to be permanently
dnm~ged by such exposure, and may be returned to their initial conàition
by careful washing nnd wiping of the electrocle surfaces. The discoloration
can be removed only by rnechanical polishing. The X0.1 cells (2) and ~3)
contained 2.0 x 105 cfu/ml of r. n!irabilis at inoculation as determined by
duplicate pour plate counts. Times-to-detection for cell current and cell
potential are listed in Table 3.
Initial Inoculùm Cell Current Cell Potential
in Cell A B A B
_
2.2 x 105cfu/ml 190 170 250 230
2.0 x 10a~ " 220 230 270 280
2.0 x 103 " 290 300 3A~0 33
Table 3
Times-to-Detection in Minutes for
Cell Current and Cell Potential
for the Organism P. mirabllis
EXAMPLE 5
This example demonstrates the detection and guantification of P.
aeruginosa.
A freshly inoculated vial of 6B medium was incubated overnight at
~ i5~r7;~
- ~2 --
roorn ternperature for use ~s the soul ce of inocula. 'I'h~! samplirlg and
dillltioll scheme illustrated in Figure ll wa.s emp~oyecl to prepnre sample cells
and pour plates. The sampl~ cell medium was enriched with 0.1cc sterile
1.5g/20rnl glycine stock solutiorl in each cell~ All cells and plates were
incubated at 37 C. Electroanalytical cell readings were b~gun immediately
a~ter inoculation and were continued for a~90 minutes (8 1/6 hours).
Cell current response of the organism with decade dilution and of
the control cell containin~ sterile medium nre shown in Figure 18. Normal
cell current behaviol is observed. The related cell potential responses are
presentecl in ~:igure l9. Becnllse P. aerutrinos_ is a relatively slow growing
obligflte aerobe, cell potential response at en~!h inoculum level changes more
slowly ancl reaches a limitin~ valtle oî considerably less amplitude than do
the fncultative anaerobes. Pour plate COUlltS in d~lplicate were used to
determine the initi~l inoculum level in tlle X0.1 cells as 7.7 x 10~ cfu/ml.
Times-to-detection for the detection methods are presented in Table ~.
Initial Inoculum Cell Current Cell Potential
in Cell A B A B
7.7 x 10~cfu/ml 190 160 220 170
7.7 x 103It 220 250 2a~0 310
7.7 x 102.~ 300 310 390 390
Table 4
Times-to-Detection in Minutes for
Cell Current and Cell Potential
for the Organism P. aeru~inosa
EXAMPLE 6
,
This example demonstrates the detection and quarltification of S.
aureus. A freshly inoculated vial of 6B medium was incubated overni~ht
at roorn temperature to serve as the source of all inocula. The sampling
and dilution scheme of Figure 11 was again employed to prepare sample cells
snd pour plates used in the test. The growth mediunn in eacll cell wrs
- 23 -
enriclle(l with ().lcc sterile ~1ucose stocl; solution. AlL incubations were
carried Otlt at 37 C in a wnrm-air incubator. Cell current and potential
readings were reco~clecl every 10 minutes uncler cornputer control. The
experiment was continued for ~8~ millutes (8 hours). Cell current response
for the organism nt decade inocu1um levels is shown in Figure 20. Norrnal
cell current behavior is obtainecl. The related ceLI potential variations are
presented in Figure 21. Note thnt very little potential change occurs with
continued growtll o~ S. aureus; thresho1d cletection is barely achievecl.
Dup1icate 2~-hour pour plate COUIltS were used to determine the inoculllm
level in the X0.1 cc11s to be n.2 ~ lO~ cflltml. Times-to~detection for cell
current and cell potenti~l methocls are given in Table 5.
Initial Inoculllrn Cell Currellt Cell Potential
in Cell ~ B ~ B
8.2 x 105 c~u/ml l50 130 290 200
8.2 x l0~ " 180 1~0 250 260
8.2 x 1û3 " 250 260 320 3a~0
Table 5
Times-to-Detection in Minutes for
Cell Current and Cell Potential
for the Organism S. aureus
EX~MPLE 7
This example demonstrates the detection and quantifiction of S. bovis.
A fresh overnight culture of S. bovis in 6B medium was used as the inoculum
source. Sample cells and pour plates were prepared with reference to ~igure
11. Eflch cell also received 0.1cc glucose stoclc solution as enrichment. All
incubations were carried out at 37~ C. The measurements were continued
for ~5~ minutes (7 112 hours). Cell current measurements are presented in
Fi~lre 22~ Normal current response is observed prior to the current minimum
at each inoculum level. Values recorded after each rninimum r ise more
rapidly then usual. Cell potential responses are shown in Figure 23r S~
`
- 2~ -
bovis causes little change in the potcntial observed as growth progresses,
save for the srnaII singularity usually observed in conjunction with the cllrrent
minima in Figure 22. Duplicate 2~1-hour pour plate counts indicated 6.5 x
I05 cfu/ml to be present in the ~o.i cells initially. Times-to-detection for
the detection methods are presented in Table 6.
Initial Inoculum Cell Current Cell Potential
in Cell A B A B
6~5 x 105 cfu/ml200 180 280 2~0
6.5 x 104 " 220 220 290 280
6.5 x 103 " 280 290 330 340
Table 6
Times-to-Detection in Minutes for
Cell Current and Cell Potential
for the Organism S. bovis
EXAMPLE_8
This example demonstrates the extended quantification of E. coli using pulsed
voltammetric detection. A fresh overnight culture of E. coli in 6B medium
was used as the inoculum souree. Dilution vials and pour plates were
prepared with reference to Figure 11, except that dilution vials were prepared
out to a dilution ratio of 1:106. The growth medium in each of the cells
was enriched with 0.1cc sterile glucose stock solution. The first seven cells
in the 8-cell array eaeh received 1.0ce from the appropriate dilution vial
as inoeulum. Cell (8) reeeived 1.0ec sterile ISB inoculum as the eontrol.
Beeause the cells had previously been meehanically po3ished and reeonditioned,
the current sensitivity for this experiment was reduced somewhat to insure
that all recorded values would remain within the dynamie range of the A/D
converter. Incubation and testing were carried out at 37 G. No pre-
inoeulation incubation period was used.
The normalized cell current responses of cells (3) through (8) are
shown in Figure 24. Cell (1) was used as a high-inoculum marker only,
- 25 -
since the cell assembl~v rcc3uires nt least 30 minutes to nttnin temperature
cquilibriurn. Cell (2) reslllts were nllomnlolls Witil respect to cletection time,
prohnhly due to sligilt contaminatiorl of tlle c ell walls or clectrode surfMcesduring reconditionin~, nnd are not shown. Over tlle foul decades of inoculu~n
level considered, t;m~to-cletection is seen to vary linearly with the logarithm
of inoculum strength. Note that the detection threshold has been reduced
to 6û,~ o~ the value observed at tlle time of normali~ation; this provides
more clepenclable tirme-to-detectioll vallles in prolonged tests where the slight
downwarcl baseline clri~t with time can gellerate cletection times slightly
shorter than the correct vnllles. Note that the 60% current level occurs
near the point of ma:~imum slope of the ~owth curves. Experience with
the system has shown thnt best qllanti~ication when usin~ this technique is
obtained when the detection time is taken to be the time at which ma~cimum
slope of the ~rowth curve is eviclenced. ~igure 2S illustrates the good
quantification achieved for inocula of 1.5 x 105 cfu to 1.8 x 101 cfu in the
present exarnple. Repeated trials with the system has shown that inocula
greater than about 5 x 105 cfu require sligh-tly longer to detect than
predicted. This is pai tly due to the lag induced by the time required for
the cell array to reach temperature equilibrium. The remainder of the
problem is most likely caused by the finite time required for the orgranisrn
in question to acl~pt to the new environmellt imposecl by dilution and
sampling. The short time interval between recorded data points (10 minutes3
makes cven slight devia-tions from expected behavior noticeable.
, .
EX~MPI.E 9
The results for the organisms studies in Examples 2-7 using the û-cell array
(platinum cathodes) are summarized in Table 7. Data from parallel
radiometric tests (BACTEC) are included for comparison. Cell current
duplicate resl~ts are shown to differ by a maximum of 30 minutes at any
inoculum level for all organisms. Cell potential detection is less reliable
for quantification, differing in duplicates by as much as 90 minutes in one
case (S. aureus).
.
Pulsed voltammetric detection of the test organisms compares well
with detection based upon the B~CTEC system; E coli and E. cloacae are
- 2fi -
.
ietcct~cl with nppro~cimat~ly ~!qual ~acility by both m~thods. P. mirubilis
~ncl most notably, P. aerllginosa are detec!ted si~ni~icantly faster usintr the
cell culrent measurement. ~etection of S. aureus by the cell current
method is abo~lt 40 minutes faster then BACTE:C, while S. bovis cletec-tion
is accomplished about 1 hour sooner by the B~CTEC system.
Cell potential detection of organism growth compared to either the
cell current cletermination or to the B~CTEC system leaves much to be
desired. Results can be quite unreliable for organisms such as S. aureus
(Fig. 21) and S. bovis (Fig. 23) which produce little chflnge in solution redox
potential with ~owth. Thresholds for detection are approached slowly and
barely exceeded by such organisms ns compared to the Enterobacteriacae,
thlls promoting a test of widely varying sensitivity as a function of the
organism being deteeted. The widely àifferin~ reclox potential patterns do,
however, provide good clues as to the type of organisrns simultaneously
detectecl by other means.
Table 7
_
TIMES-TO-DETECTION, MINUTES
Tested Inoculum Level BACT~C Cell Current CeU Potential
Or~anism in Cell or Vial ~ B A
E. coli 1.0 x 105 cfu/ml 120120 120160 180
1.0 x 103 cfu/ml 180150 160220 230
1.0 x 10 cfu/ml 240200 200260 270
F.. _acae 3.7 x 10,~cfu/ml 120 150 150 230 200
- 3.7 x 103 cfu/ml 180210 210260 240
3.7 x 10 cfu/ml 240270 270300 310
P. mirabilis 2.0 x 104 cfu/ml 240I90 170250 230
2.0 x 10 cfu/ml 300220 230270 280
2.0 x 103 cfuiml 420290 300340 330
P. aerll~osa 7.7 x 104 cfu/ml ~80190 160220 170
7.7 x 12 cfu/ml 600220 250240 310
7.7 x 10 cu/ml 660300 310390 390
~ 27 ~
,1'
Table 7 (Contld) r
.
TIMES-TO-DETECT~C)N, MINUTES
Tested Inoculurrl Level BACTEC Cell Current Cell Potential
Orgnnism in Cell or Vinl A B A B
S. aureus 8.2 x 105 cfu/ml 180 150 130 290 200
8.2 x 103 cEu/ml 240 180 180 250 260
8.2 x 10 c~u/ml 300 250 260 320 3~0
S. bovis 6.5 x 105 cfu/ml 120 200 1~0 280 2~0
6.5 x 10~ cfu/ml 180 220 ~20 290 280
6.5 x 10 cîu/ml 2~10 280 290 330 340
.
Summary OI Results
Times-to-Detection for all Organisms
by All Methods
E~PLE 10
.
This example demonstrntes the use of electrodes of other materials and
sizes then those used in the fore~oing examples. A 6-ccll array constructed
with 0.020" gold wire as cathode and 0.020" silver wire as anode in each
cell was used to test the response of the system toward pulse polaro~raphic
detection only. The gold cathode prevented collection of potential data,
but gold is somewhat less expensive then platinum to use in cases where
detection alone will suffice. The gold and silver wires, arranged in parallel
fashion in the bottom of each cell, were each 1.4cm long and were separated
by 1.6cm. The observed cell currents were appreciably lower than those
observed when using the 8-cell array with larger diameter wires and platinum
cathode. No attempt was made to measure true current sensitivity. The
cells were prepared with either l.5.cc or 2.0cc TSA and 5.0cc enriched 6B
medium, and were tested with various organisms under the same conditions
as for the previously noted experiments. No electrode pretreatment was
employed. The pulse arnplitude (-D.70V) and the pulse duration (1200ms)
were the same as in the previous examples. Duplicate pOUI plates were
also prepared for these experiments. Cell current data normfllization was
~:~
-- 2~ --
cnrried ollt as previollsly describetl. I~resh overni~rht cultul es of ~L P-
mirabilis and P. fleru~inostl itl 6B med;um were useci as inoculum sources.
All snmple flnCI pour plate preparations were preformed with reference to
Figure 1l. Cell current readin, s were obtained at 10-minute intervals. No
pre-inocnlation incubation was employecl.
Results obtained for separate tests us;ng E. coli, P. mirabilis and P.
nerutrinosa are shown in Fi~ures 26, 27 nncl 28, respec~ively. The organism
inoclllllm level for the most concentrated cell in each experiment is listed
below:
E. coli 1.5 x 105 cfu/ml
P. mirabilis 1.2 x 10 cfu/ml
P. aeruginosa 1.~3 x 105 cfu/ml `
The other tracings in each Eigure are for decade dilutions and a
sterile control cell. Tn all cases, detection of the organism was readily
accomplished, as indicated by a drop in recorded cell current to 80% of
the normalization value. Times-to-cletection for all dilutions in each of the
figures are seen to depend upon the inoculum level in a predictable manner,
varying essentially as the logarithm o~ inoculum conce~ntration. The slight
scatter noted in som e of the data is due to stray pickup by the analog
conditioning electronics, a capacitor was addecl across the voltage-to-current
conversion operational amplifier to alleviate this problem.` Downward baseline
drift noted for the control samples is most likely due to the lack of any
preconditioning of the cells. Any increase in times-to detection noted at
a given inoculum level of a specific organism when using the 6-cell array
is probably a function of the thickness of TSA over the electrodes, particularlyin the case where 2.0cc was used. Once the electrodes are covered,
detection times increase as the level of TS~ in the cell increases as a
consequence of the increased diffusion path. 2.ûcc TSA was used in the
experiment run with P. aeruginosa. The E. coli and P. mirabilis experiments
both used 1.5cc TSI~.
OJ`,.~'~'t.~
..
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~ 9 ~
EXAM PI,E 11
This exa1nple clemonstrates a clinical trial of the pulsed voltammetric
detection techniqlle of the present inventor for the detection of si~nificant
bacteriuria. The test was conc~lcted in conjunction with Sinai ~Iospital of
Baltimore. Over the 33-dfly period o~ the study, 389 urine samples were
collected from Sinfli nnd testecl at Johnston Laboratories using the pulsed
voltammetric technique in parallel with the BACTEC radiometric system.
TSA pour plates were employed to checlc actual organisrn counts.
The test was carried out as follows. Urine specimens sampled foLIowingr
Sinni eollection ancl plnnting were pielced up from the hospital at
approx;mately U:OOAM each clay. Prior to sample arrival at JLI, the 16~
eell assembly to be used with the P~llsed Voltammetric instrllmentation was
filled with scaldin~ hot water and aUowed to stand for at least ten minutes.
The assembly was then rinsed twice with sterile deionized water, then shaken
vigorously to dislod~e any large water droplets. I.Occ sterile Tryptic Soy
Agar (~O.Og/l~ BBL or l)IFCO~ at about 95 C was then added by sterile
syringe to each cavity of the assembly. The assembly was then covered
with a double thicl~ness of aluminum îoil sterilized with isopropanol, and
the agar allowed to solidify. 5.0cc of sterile TSB (27.5g/1; BBL or I~IFCC))
containing De~trose (2.5g/1; M~\~LINCKRODl` or J.T. BAKER) was then adcled
to each cavity via syringe, and the cover replaced.
Upon arrival at JLI, urine samples were cataloged as to JLI daily
and conseeutive sequence numbers, Sinai reference number, and gross physical
characteristics. l.Oec of eaeh urine specimen was inoculated via syringe
into one cell of the assembly. Similarly, l.Occ was used to inoculate a
septum-fitted vial of nominal 50cc capacity containing 5vOec ~ JLI ~A
Urine Sereening Medium (JLI B/N 0379ûlU-1.5uCi/vial) for use in the para}lel
BACTEC study. O.lcc of each speeimen was inoculated into previously
refrigerated, septum-fitted vials containing 99.9ce 1/2-strength TSB to obtain
1:1000 sample dilution for the pour plate studies. Plates were prepared at
1:103 and 1:104 dilutions for each sample using 10-15 ml TSA ~40.ûg/1) and
l.Occ and Olcc from each dilution vial, respectively.
The inoculated cell assembly was placed in a 37C warm air incubator
without agitation and pulsed voltammetric testing begun under compu~er
~s~
- 30 -
cont~ ol. TC!St vnllles were r ecor~le~l for all samples at 10-m;n~lte intervals.
Dntn nol mali~ution ~vas bnsed uporl sample dnta v~llues recorclecl after the
first 10 minutes for tabulation; all datR values recorded for each sample
were ultimately expressed as a percentage of the 10-minute value. A sample
was considered to be positive when the normalizecl data value for that
sample at any given time interv~l a~ter normalizfltion ~eU below 70 or rose
above 140. The latter criterion was employed to permit detection of some
highly positive tca. 10~ cfu/ml) samples which produced data minima slightly
greater than 70, yet whieh inter~ered with normal operntion suf~iciently to
procklce data maxima over 1~0.
Each ~lay of testing concll~cled with the generation of a computer
printout which inclllded plots of relative cell potential readincrs~ and a tableof normalized cell current readings for each sample, all as n function of
incubation time. The normali~ed, tabulated results were used to determine
sample result classifications.
Of the 389 tested samples, 45 were omitted from the s-tudy usually
for experimental reasons (Incubator Pailure, 12; CeU Reconditioning Failure,
14; Contaminated Petri Dishes, 12; JLI/Hospital Datfl Discrepancy, 7).
Contaminated samples numbered 30, and were similarly omitted from further
consideration. Of the remainin~ 31~L samples, 8a~ were considered significant
clinically. Table 8 lists the organisms identiEied by Sinai Hospital found to
be present in the sigrnificant samples. The number of samples containing
each organism is also noted, as is the percentage of the total containing
that organism. Non-integer sample numbers are due to samples containing
more than one organism.
Or~anism No. of Samples Percentage
E. coli 3a~ 5 41.07
r. aeru~inosa 11).5 12.50
K. pneumoniae ~.0 10~71
.
P. mirabilis 6.0 7.14
~east, unspecified 3.5 d~.17
S. aureus 3.0 3.57
C. albic ns 2.5 2.98
Grp. B, Beta Strep 2.5 2.98
~ ~~~
7,4~
-- 31 -
~:~n No. of S~lmE~ Percenta~e
Grp. 1) Strep 2.0 2.38
S. alblls 2.0 ~.38
... .
K. o~;ytoca 1.5 I.79
E. aero~enes 1.0 1.19
E. n~g~lon~erans1.0 1.19
E. cloacne 1.0 1.19
C. freunclii 1.0 1.19
.
P. morf~anii 1.0 1.19
Grnm-Negative Rod1.0 1.19
P. stunrtii 0.5 0.60
. . . _ _ _ _
S. marcescens 0.5 0.60
8~.0 100.01
T~BI.E 8
Organisms Contributing to Significant ~amples
Results of the test for the 314 samples consiclered for data analysis
are set out in l'able 9.
True Positives 80 (25.48%)
True ~egatives 215 (68.47%3
Palse Positives 15 ( 4.78,o)
False Negatives 4 ( 1~27%)
314 100.û%
T~BLE 9
OI the 84 samples considered clinically significant, the pulsed
voltammetric method detected 80 (95.24%3. Of the four samples missed by
the pulsed voltammetric technique, two were missed by BACTEC as l,vell.
ZiJI
One of these is known to be from a patient receiving anti-
biotic therapy; the other contained S. Aureus. The remaining
two false negative samples contained P. Aeruginosa and an
unspecified yeast. The BACTEC system detected 76 ~90.48%) of
the 84 samples considered clinically significant.
The pulsed voltammetric detection technique properly
identified 95.24~ of urines considered significant in the
study, yielding a total sample false negative rate of 1.27%,
with a false positive rate of 4.78%. The BACTEC system detected
90.48~ of significant urines, with a total sample false negative
rate of 2.55~ and a false positive rate of 1.27~. BACTEC
required 3 hours to achieve the reported level of performance;
the pulsed voltammetric technique required 4 hours.
The technique of the present invention is thus shown
to provide competent detection of significant bacteriuria, with
clinically acceptable levels of false negative and false posi- !
tive results. The rapidity and sensitivity of the method compare
favourably with parallel results obtained using the BACTEC
system.
While certain specific embodiments of the invention
have been described with particulars herein, it should be
recognized that various modifications thereof will occur to
those skilled in the art. Therefore, the scope of the invention
is to be limited solely by the scope of the claims appended
hereto.
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