Apparatus and process for microbial detection and enumeration

An apparatus and process for detecting and enumerating 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 prior to being electrically connected via connectors 14 and 16 to strip chart recorder 11 for detecting and enumerating the microorganisms collected on filter 23.

BACKGROUND OF THE INVENTION 
The broad use of membrane filtration and electrochemical 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 numbers in large volumes and there is, 
at times, a long lag time to produce the required number of 10.sup.6 to 
10.sup.7 cells/ml for a detectable response to occur in the system. 
In the present invention an attempt is made to combine the advantageous 
features of both the membrane filtration and electrochemical detection 
systems while minimizing the disadvantages thereof. 
It is therefore an object of the present invention to provide a novel 
apparatus and process for detecting and enumerating specific 
microorganisms in a sample solution. 
It is another object of the present invention to combine the membrane 
filtration and electrochemical microbial detection systems into a simple 
system that may be readily employed by semiskilled operators to detect and 
enumerate microorganisms in a sample solution. 
Another object of the present invention is a rapid process for determining 
water contamination of small numbers in large volumes. 
Another object of the present invention is a novel apparatus for 
qualitatively and quantitatively detecting water sample impurities. 
According to the present invention the foregoing and additional objects are 
attained by filtering a sample solution through a standard membrane filter 
impervious to the microorganism tested for, placing the membrane filter 
onto an absorbent pad previously moistened with a microorganism growth 
stimulant medium, positioning a pair of platinum electrodes on the filter 
surface and covering the electrodes with a second identically treated 
absorbent pad. This assembly is then sealed within a petri dish or like 
container with one exposed end of each electrode protruding from the 
sealed container being connected to a suitable recorder for detecting and 
recording the microbial growth.

DETAILED DESCRIPTION OF THE INVENTION 
Referring now to the drawings and more particularly to FIG. 1, the combined 
membrane filtration and electrochemical microbial detection system of the 
present invention is shown and designated generally by the reference 
numeral 10. System 10 includes a strip chart 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; 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 60.times.15 mm petri dish acquired from Falcon, Division of 
Becton, Dickinson & Company, 1950 Williamsburg Drive, Oxnard, Calif. 
93030; absorbent pads 21 and 25 were 47 mm diameter and acquired from 
Millipore Corporation, Bedford, Mass. 01730; membrane filter 23 was a 0.45 
.mu.m pore size filter GN-6, obtained from the Gelman Instrument Company, 
600 South Wagner Road, Ann Arbor, Mich. 48106; and electodes 13 and 15 
were constructed from 24 gauge (0.508 mm) diameter platinum wire with a 
length ratio of electrode 15 to electrode 13 being approximately 2:1, 
i.e., 50 and 25 mm, respectively. One end of electrode 13 and both ends of 
electrode 15 were bent in hairpin fashion to provide a 90.degree. bend in 
the electrode to extend over and frictionally engage the edge surface of 
base half 19 of container 17 while maintaining a length of the individual 
electrodes adjacent the interior bottom surface of the base half 19. 
Retainer ring 27 may be formed of any suitable pliable wire and in the 
specific example illustrated was formed of 19 gauge (0.90 mm) diameter 
steel wire with the end portions thereof also bent in hairpin fashion 
90.degree. to the main wire length so as to permit engagement with the 
electrode-membrane-pad assembly when the rim portions are frictionally 
clamped over the edge of the base half 19 of container 17. As shown more 
clearly in FIG. 1 when the container 17 is assembled with its 
electrode-membrane-pad assembly in place, retainer ring 27 is disposed at 
right angles to electrodes 13 and 15 to maintain constant pressure and 
close contact between pads 21 and 25, filter 23 and electrodes 13 and 15. 
Container 17 is sealed at the edges of cover half 29 and base half 19 as 
indicated by reference numeral 20 by a conventional laboratory sealant to 
reduce moisture loss. In the specific example described herein the sealant 
material 20 was Parafilm "M", a registered tradename product of the 
American Can Company, Marathon Products Division, Neenah, Wis. 
Prior to connecting the electrodes to the strip chart recorder (Model 194, 
Honeywell Industrial Division, Fort Washington, Pa.), each channel was set 
at a zero reference point. The 25 and 50 mm electrodes were connected to 
the positive and negative terminals, respectively, of the recorder 
operated at 0.2 or 0.5 volts full scale with a chart speed of 10 min/in 
(24.5 mm). The electrodes were allowed to equilibrate for 60 to 80 minutes 
before establishing a baseline which was generally offset from the 
recorder zero reference point by 10 to 20 millivolts in the positive 
direction. Responses in the upward directon from the baseline were 
considered positive and downward negative. Millivolt measurements for peak 
height responses were made from the baseline in either the positive or 
negative direction. Detection time endpoints (lag time) were read from the 
strip chart trace and recorded as the time between challenge and the 
initial increase in voltage. Each trace was characterized as to the type 
of response curve and the maximum millivolt response was also recorded. 
In operation, the time between challenge and the initial increase in 
voltage of an electrochemical detection system is known to be a function 
of the number of cells in the inoculum. For Escherichia coli, the 
detection times, for example, for 10.sup.5 and 10.sup.1 cells/ml were four 
and eight hours, respectively. This lag time is dictated by the 
requirement for a cell population of 10.sup.6 to 10.sup.7 cells/ml at the 
time of response. In practice, when organisms are present in very low 
numbers, especially in 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 23 and the membrane placed between 
absorbent pads 21 and 25 that are moistened with a nutrient growth medium 
specific for the organism being tested. For Escherichia coli, the growth 
medium chosen was Trypticase soy broth (TBS, BBL, Division of Becton, 
Dickinson & 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 medium may be employed when employing the 
present invention to detect and enumerate other microorganisms. 
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.sup.-1 and 
10.sup.-2 dilutions of estuarine and fresh water sample and collected on 
membrane 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 colonies 
after 24 hour incubation at 35.degree. C. The procedures used to determine 
detection time endpoints with the electrochemical apparatus of the present 
invention were the same as previously described in Applied Environmental 
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 electrodes were tested in a 
broth-test tube experimental system. In the combined system of the present 
invention, 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 10.sup.8 or 10.sup.11 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 fresh 
water samples, respectively. In monitoring water quality by relating 
endpoints for 100 ml samples to the curves thus obtained, these 
preliminary results indicate that the present invention could be employed 
by semiskilled operators as an effective and rapid method for testing 
water purity by providing a readily obtainable estimate of the microbioal 
loading of different water samples. 
Although the operation of the present invention has been described in 
connection with a specific embodiment thereof, it is not so limited and 
the specific structural features described herein are to be considered as 
illustrative only and not exhaustive. 
Obviously many modifications and variations of the present invention are 
possible in the light of the above teachings. It is therefore to be 
understood that within the scope of the appended claims the invention may 
be practiced otherwise than as specifically claimed.