An apparatus and process for detecting and enumerating specific microorganisms from large volume samples containing small numbers of the microorganisms is presented. The large volume samples are filtered through a membrane filter to concentrate the microorganisms. The filter is positioned between two absorbent pads and previously moistened with a growth medium for the microorganisms. A pair of electrodes are disposed against the filter and the pad electrode filter assembly is retained within a petri dish by retainer ring. The cover is positioned on base of petri dish and sealed at the edges by a parafilm seal prior to being electrically connected via connectors to a strip chart recorder for detecting and enumerating the microorganisms collected on filter

Grana, D.

Wilkins, J. R.

English

NASA Technical Reports Server

United States Patent 1191 
Wilkins et al. 
El 11 4,335,206 
E451 Jun. 15, 1982 
[54] APPARATUS AND PROCESS FOR 
MICROBIAL DETECTION AND 
ENUMERATION 
[75] Inventors: Judd R. Wilkins; David C. Grana, 
[73] Assignee: The United States of America as 
both of Hampton, Va. 
represented by the Administrator of 
the National Aeronautics and Space 
Administration, Washington, D.C. 
[21] Appl. No.: 235,796 
[22] Filed: Feb. 19,1981 
[Sl] Int. C l . 3  ............................................... C12Q 1/04 
[52] U.S. Cl. ........................................ 435/34; 435/39; 
435/291; 204/195 B 
1581 Field of Search ....................... 435/29, 30, 34, 39, 
435/287, 291, 817; 204/195 B 
~561 References Cited 
U.S. PATENT DOCUMENTS 
2,677,646 5/1954 Love11 et al. ................... 435/297 X 
3,743,581 7/1973 Cady et al. ..................... 435/291 X 
3,890,201 6/1975 Cady ................................. 435/34 X 
3,929,583 12/1975 Sharpe et al. ................... 43?/291 X 
3,741,877 6/1973 Shaufus et al. ................... 435/30 X 
4,072,578 2/1978 Cady et al. .......................... 435/291 
4,149,938 4/1979 Young et al. ....................... 435/291 
4,264,728 4/1981 Wilkins ........................... 435/291 X 
Primary Examiner-Robert J. Warden 
Attorney, Agent, or Firm-Wallace J. Nelson; John R. 
Manning; Howard J. Osborn 
ABSTRACT t571 
An apparatus and process for detecting and enumerat- 
ing specific microorganisms from large volume samples 
containing small numbers of the microorganisms 
wherein the large volume samples are filtered through a 
membrane filter 23 to concentrate the microorganisms 
and filter 23 is positioned between two absorbent pads 
21 and 25 previously moistened with a growth medium 
for the microorganisms. A pair of electrodes 13 and 15 
are disposed against filter 23 and the pad-electrode-filter 
assembly retained within a petri dish 17 by retainer ring 
27. Cover 29 is positioned on base 19 of petri dish 17 and 
sealed at the edges thereof by a parafilm seal prier to 
being electrically connected via connectors 14 and 16 to 
strip chart recorder 11 for detecting and enumerating 
the microorganisms collected on filter 23. 
9 Claims, 3 Drawing Figures 
17 
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APPARATUS AND PROCESS FOR MICROBIAL 
DETECTION AND ENUMERATION 
ORIGlN OF THE INVENTION 
The invention described herein was made by employ- 
ees of the U. S. Government and may be manufactured 
and used by or for the Government for governmental 
purposes without the payment of any royalties thereon 
or therefor. 
BACKGROUND OF THE INVENTION 
The broad use of membrane filtration and electro- 
chemical systems to detect microorganisms in various 
samples is well known. Membrane filtration normally 
requires a minimum of twenty-four hours incubation 
time for adequate growth to appear and requires the use 
of an operator to examine and count the microorganism 
colonies. Electrochemical detection systems presently 
employed have the inherent disadvantage of a high 
probability of missing cells when present in small num- 
bers in large volumes and there is, at times, a long lag 
time to produce the required number of 106 to lo7 
L 
FIG. 2 is an exploded view of the container assembly 
of the apparatus shown in FIG. 1, and 
FIG. 3 is a graphic illustration of the number of spe- 
cific microorganism cells (Escherichia coli) retained on 
5 membrane filters and detection times as obtained for a 
series of tests utilizing the present invention. 
DETAILED DESCRIPTION OF THE 
INVENTION 
Referring now to the drawings and more particularly 
to FIG. 1, the combined membrane filtration and elec- 
trochemical microbial detection system of the present 
invention is shown and designated generally by the 
reference numeral 10. System 10 includes a strip chart 
15 recorder 11 and a petri dish assembly 17. A pair of 
electrical leads 14 and 16 serve to connect recorder 11 
to platinum electrodes 13 and 15 of dish assembly 17. 
Referring now to FIG. 2, petri dish 17 includes base 
half 19; a bottom absorbent pad 21; membrane filter 23; 
20 electrodes 13 and 15; a top absorbent pad 25; a retainer 
ring 27; and, a cover half 29. 
In a specific example, container 17, consisting of base 
half 19 and cover half 29, was a 6 0 ~  15 mm petri dish 
acquired from Falcon, Division of Becton, Dickinson & 
10 
cells/ml for a detectableresponse to occur in the sys- 25 Company, 1950 Williamsburg Drive, Oxnard, Calif. 
tem. 93030; absorbent pads 21 and 25 were 41 mm diameter 
In the present invention an attempt is made to com- and acquired from Millipore Corporation, Bedford, 
bine the advantageous features of both the membrane Mass. 01730; membrane filter 23 was a 0.45 pm pore size 
filtration and electrochemical detection systems while GN-6, obtained from the Gelman Instrument 
minimizing the disadvantages thereof. 30 Company, 600 South Wagner Road, Ann Arbor, Mich. 
It is therefore an object of the present invention to 48106; and electodes 13 and 15 were constructed from 
provide a novel apparatus and process for detecting and 24 gauge (0.508 mm) diameter Platinum wire with a 
enumerating specific microorganisms in a sample solu- length ratio of electrode 15 to electrode 13 bekg ap- 
tion. proximately 2:1, Le., 50 and 25 mm, respectively. One 
35 end of electrode 13 and both ends of electrode 15 were is another object of the present invention to 
bine the membrane filtration and electrochemical mi- 
crobial detection systems into a simple system that may 
bent in 
electrode to extend Over and 
fashion to provide a bend in the 
engage the 
be readily employed by semiskilled operators to detect 
and enumerate microorganisms in a sample solution. 
process for determining water contamination of small 
numbers in large volumes. 
Another object of the present invention is a novel 
edge surface of base half 19 of container 17 while main- 
taining a length of the individual electrodes adjacent the 
40 interior bottom surface of the base half 19. Retainer ring 
specific example illustrated was formed of 19 gauge 
(0.90 mm) diameter steel wire with the end portions 
thereof also bent in hairpin fashion to the main wire 
water sample impurities. membrane-pad assembly when the rim portions are 
additional objects are attained by filtering a sample container 17. As shown clearly in FIG. when 
solution through a standard membrane filter impervious the container 17 is assembled with its electrode-mem- 
to the microorganism for, placing the E%3Ilbrane 50 brane-pad assembly in place, retainer ring 27 is disposed 
filter onto an absorbent pad previously moistened with at right angles to electrodes 13 and 15 to maintain con- 
a microorganism growth stimulant medium, Positioning stant pressure and close contact between pads 21 and 
a pair of platinum electrodes on the filter surface and 25, filter 23 and electrodes 13 and 15. Container 17 is 
covering the ekctrodes with a second identically sealed at the edges of cover half 29 and base half 19 as 
treated absorbent pad. This assembly is then sealed 55 indicated by reference numeral 20 by a conventional 
within a petri dish or like container with one exposed laboratory sealant to reduce moisture loss. In the spe- 
end of each electrode protruding from the sealed con- cific example described herein the sealant material 20 
tainer being connected to a suitable recorder for detect- was Parafilm “M”, a registered tradename product of 
ing and recording the microbial growth. the American Can Company, Marathon Products Divi- 
BRIEF DESCRIPTION OF THE DRAWINGS Prior to connecting the electrodes to the strip chart 
A more complete appreciation of the invention and recorder (Model 194, Honeywell Industrial Division, 
many of the attendant advantages thereof will be Fort Washington, Pa.), each channel was set at a zero 
readily apparent as the same becomes better understood reference point. The 25 and 50 mm electrodes were 
by reference to the accompanying drawings wherein: 65 connected to the positive and negative terminals, re- 
FIG. 1 is a part schematic view of a typical combined spectively, of the recorder operated at 0.2 or 0.5 volts 
membrane-electrochemical microbial detection appara- full scale with a chart speed of 10 midin (24.5 mm). 
tus of the present invention; The electrodes were allowed to equilibrate for 60 to 80 
Another Object Of the present invention is a rapid 27 may be formed of any suitable pliable wire and in the 
apparatus for qualitatively and quantitatively detecting 45 length so as to permit engagement with the electrode- 
According to the present invention the foregoing and frictionally clamped Over the edge ofthe base half 19 of 
Neenahy wis* 
4,3 3 5,206 
3 4 
minutes before establishing a baseline which was gener- fresh water samples, respectively. In monitoring water 
ally offset from the recorder zero reference point by 10 quality by relating endpoints for 100 ml samples to the 
to 20 millivolts in the positive direction. Responses in curves thus obtained, these preliminary results indicate 
the upward directon from the baseline were considered that the present invention could be employed by semi- 
positive and downward negative. Millivolt measure- 5 skilled operators as an effective and rapid method for 
ments for peak height responses were made from the testing water purity by providing a readily obtainable 
baseline in either the positive or negative direction. estimate of the microbioal loading of different water 
Detection time endpoints (lag time) were read from the samples. 
strip chart trace and recorded as the time between chal- Although the operation of the present invention has 
lenge and the initial increase in voltage. Each trace was 10 been described in connection with a specific embodi- 
characterized as to the type of response curve and the ment thereof, it is not so limited and the specific struc- 
maximum millivolt response was also recorded. tural features described herein are to be considered as 
In operation, the time between challenge and the illustrative only and not exhaustive. 
initial increase in voltage of an electrochemical detec- Obviously many modifications and variations of the 
tion system is known to be a function of the number of 15 present invention are in the light of the above 
cells in the inoculum. For Escherichia coli, the detection 
times, for example, for lo5 and lo1 cells/ml were four the scope of the appended claims the invention may be 
and eight hours, respectively. This lag time is dictated 
cells/ml at the time of response. In practice, when or- 20 Letters Patent of the United States is: 
large volumes, in addition to extended lag times there is 
an increased probability of missing cells when samples 
are limited to 1, 10 or even 50 ml. To circumvent this 
problem, the sample is filtered through membrane filter 25 
23 and the membrane placed between absorbent pads 21 
and 25 that are moistened with a nutrient growth me- 
dium specific for the organism being tested. For Escher- 
ichia coli, the growth medium chosen was Trypticase 
soy broth (TBS, BBL, Division of Becton, 
Company, Cockeysville, Md. 21030). Absorbent pads 
21 and 25 were each moistened with 2.0 ml of this broth 
for the tests described herein. A different nutrient me- 
teachings. It is therefore to be understood that 
practiced otherwise than as specifically claimed. 
by the requirement for a cell population Of lo6 to lo' 
ganisms are present in very low numbers? in 
What is claimed as new and desired to be secured by 
1. An e]ectrochemical detection method for deter- 
mining the presence and quantity of specific microor- 
ganisms in a sample solution comprising: 
filtering the sample solution through a membrane 
filter impervious to the organism to be detected 
and quantified; 
assembling a pair of electrodes in spaced adjacency 
relative to each other and each electrode being 
positioned in abutting relationship with the mem- 
brane filter; 
positioning the membrane filter and electrodes be- 
tween two absorbent pads adapted to fit within an 
3o 
dium may be employed when employing the present 
invention to detect and enumerate other microorgan- 35 
isms. 
Ten milliliter volumes from a ten-fold series were 
filtered in dose-response studies for both Escherichia coli 
and Staphylococcus aureus. Bacteria were also taken 
from 100 ml of undiluted and 10-1 and IO-2dilutions of 40 
estuarine and fresh water sample and collected on mem- 
brane filters. Viable counts for both the dose-response 
studies and the water samples were conventionally 
made by spreading appropriate dilutions from a ten-fold 
series on Trypticase soy agar (BBL) and counting colo- 45 
nies after 24 hour incubation at 35" C. The procedures 
used to determine detection time enduoints with the 
open container; 
positioning the absorbent pad-filter-electrode assem- 
bly within the container so as to leave at least one 
exposed end of each electrode extending from the 
container; 
providing a retaining clip extending over and con- 
tacting the edges of the container and having por- 
tions thereof abutting and retaining the absorbent 
pad-filter-electrode assembly within the container; 
sealing a cover over the container so as to leave the 
exposed end of each electrode extending from the 
container; and, 
attaching a strip chart recorder to the exposed ends of 
the electrodes to detect and record the electrical 
electrochemical apparatus of the present invention were 
the same as previously described in Applied Environmen- 
tal Microbiology, Volume 36, pages 683-687, 1978. 
The dose-response curve fore Escherichia coli is 
shown in FIG. 3 and the linear regression parameters 
were; slope 0.0184; intercept 9.6590; and correlation 
coefficient 0.9508. These results compare favorably 
with previously published data in which platinum elec- 
trodes were tested in a broth-test tube experimental 
system. In the combined system of the present inven- 
tion, no response, i.e., no increase in voltage was noted 
when Staphylococcus aureus was tested at the normal 
strip chart input resistence of 1 megohm. Responses 
were observed when the input resistance was increased 
to either IO8 or 1011 ohms but the endpoints were erratic 
and not reproducible. 
Fifty estuarine and forth-six fresh water samples were 
tested with the present invenion and the plotted results 
obtained for the estuarine and fresh water samples were 
quite similar although slight differences were noted in 
the slopes, viz., 0.0101 and 0.0092 for the estuarine and 
output of the electrodes as a function of the pres- 
ence and quantity of the specific microorganism 
obtained on the filter from the sample solution. 
2. The method of claim 1 wherein the container is a 
petri dish having a base half and a cover half, and the 
exposed ends of the electrodes are bent in hairpin fash- 
ion so as to extend from the interior base of the petri 
55 dish over the rim edge thereof and frictionally engage 
the rim edge to assist in retaining the electrodes in abut- 
ting relationship with the membrane filter. 
3. The method of claim 2 wherein both electrodes are 
formed from platinum wire having a 2:l relative length 
60 ratio and one electrode has both end portions bent in 
hairpin fashion to frictionally engage the rim edge of 
the petri dish. 
4. The method of claim 1 wherein the microorganism 
being tested is Escherchia coli and wherein the absorbent 
65 pads are moistened with a growth medium specific 
therefor prior to being positioned about the membrane. 
5. Apparatus for rapidly determining trace microor- 
ganism impurities in a water sample comprising: 
50 
5 
4,33 5,206 
6 
filter means for isolating trace quantities of microor- 6. n e  apparatus of claim 5 wherein said electrodes 
are formed of platinum wire and said means electrically 
connected to said electrodes is a strip chart recorder. ganisms from a water sample; 
7. The apparatus of claim 6 wherein said container is 
approximately 2:1 and constructed from the Same 5 a petri dish and said electrodes are provided with por- 
tions thereof frictionally engaging said petri dish to 
assist in maintaining said electrodes in a fixed position material; 
within said petri dish. 
8. The apparatus Of claim 7 including a retainer clip 
IQ having ends thereof formed to frictionally engage the 
petri dish and having an intermediate elongated portion 
abutting the pad-electrode-filter sandwich assembly to 
assist in retaining the sandwich assembly within said 
petri dish, said retainer clip being disposed essentially 
15 90" relative to said electrodes, and means sealing said 
petri dish so as to portions of said electrodes 
extending from said dish. 
pads include a nutrient medium for stimulating specific 
a pair of electrodes having a relative length ratio of 
said pair of electrodes being disposed in spaced ad- 
jacencey to each other and in contact with said 
filter means; 
a Pair of absorbent Pads in contact with and forming 
a sandwich configuration with said electrodes and 
said filter means; 
container means for housing the absorbent pad-filter 
means-electrode sandwich configuration; and 
detect and enumerate microorganism isolated on 20 microorganism growth on said filter means. 
said filter means. * * * * *  
means e1ectricalIy connected to said ele&odes to 9' The apparatus Of wherein said absorbent 
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Apparatus and process for microbial detection and enumeration

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