U.S. patent number 4,018,652 [Application Number 05/647,802] was granted by the patent office on 1977-04-19 for process and apparatus for ascertaining the concentration of microorganism in a water specimen.
This patent grant is currently assigned to McDonnell Douglas Corporation. Invention is credited to Norman L. Fadler, James T. Holen, James W. Lanham.
United States Patent |
4,018,652 |
Lanham , et al. |
April 19, 1977 |
Process and apparatus for ascertaining the concentration of
microorganism in a water specimen
Abstract
The concentration of microorganisms in a known volume of a water
specimen is ascertained by introducing the water specimen into a
plurality of wells which have known volume and contain a nutrient
medium. The mixture of water specimen and nutrient medium is
incubated and the wells are observed for a change in the appearance
thereof which indicates metabolic activity, that is, the existence
of microorganisms in the wells. If all the wells change appearance,
then it is known that the concentration exceeds a certain limit,
that is, at least one cell per specific well volume. On the other
hand, if none of the wells change, then it is most likely the
concentration is less than one cell per total volume of specimen in
the wells. A change in appearance of some but not all of the wells
indicates a concentration between the foregoing limits, and this
concentration is estimated by statistical evaluation based on
proven statistical computations.
Inventors: |
Lanham; James W. (St. Louis
County, MO), Holen; James T. (Florissant, MO), Fadler;
Norman L. (St. Peters, MO) |
Assignee: |
McDonnell Douglas Corporation
(St. Louis, MO)
|
Family
ID: |
24598323 |
Appl.
No.: |
05/647,802 |
Filed: |
January 9, 1976 |
Current U.S.
Class: |
435/36; 422/504;
435/38; 435/822; 435/852; 435/874; 435/40; 435/848; 435/885;
435/288.4 |
Current CPC
Class: |
C12M
23/12 (20130101); C12M 41/36 (20130101); G01N
2035/00148 (20130101); Y10S 435/874 (20130101); Y10S
435/822 (20130101); Y10S 435/848 (20130101); Y10S
435/852 (20130101); Y10S 435/885 (20130101) |
Current International
Class: |
C12M
1/20 (20060101); C12M 1/16 (20060101); G01N
35/00 (20060101); C12K 001/04 () |
Field of
Search: |
;195/103.5,127,139
;23/253R,259 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Martin Frobisher, Fundamentals of Microbiology, 8th Edition, W. B.
Saunders Company, 1968, pp. 46-49..
|
Primary Examiner: Jones; Raymond N.
Assistant Examiner: Warden; Robert J.
Attorney, Agent or Firm: Gravely, Lieder & Woodruff
Claims
What is claimed is:
1. An apparatus for determining the concentration of microorganisms
in a water specimen, said apparatus comprising a rigid body having
a plurality of first wells and a plurality of second wells therein,
the first wells being of equal volume and the second wells being of
equal volume with the volume of the second wells being different
from that of the first wells, the rigid body also having an inlet
which opens outwardly from the body and forms an entry into which
the water specimen may be introduced into the body, the rigid body
further having filling channels leading up to and opening into the
first and second wells, there being a separate filling channel for
each first well and for each second well with each filling channel
communicating with the inlet such that a water specimen introduced
into the inlet passes into the filling channels and then into the
wells, the upstream end of each filling channel being in
communication with the inlet independently of the other filling
channels so that the water specimen will flow into each well
without passing through another well; means closing the ends of the
wells and the sides of the filling channels for isolating the
interiors of the wells and filling channels from the surrounding
atmosphere except through the inlet, said means being transparent;
and a culture medium in the wells.
2. An apparatus according to claim 1 and further comprising means
at the inlet for isolating the channels from the surrounding
atmosphere.
3. An apparatus according to claim 2 wherein the means at the inlet
for isolating the channels comprises a septum in the inlet of the
rigid body.
4. An apparatus according to claim 1 wherein the means for closing
the ends of the wells is a light-transmitting tape adhered to those
surface areas of the rigid body out of which the wells open.
5. A process for ascertaining the concentration of microorganisms
in a water specimen, said process comprising: placing the water
specimen in communication with filling channels leading to sealed
first cavities which are of known and equal volume; placing the
water specimen in communication with additional filling channels
leading to sealed second cavities which are of known and equal
volume, the volume of the second cavities being less than the
volume of the first cavities; placing the water specimen under a
vacuum so that air is evacuated from the first and second cavities
and filling channels through the water specimen; releasing the
vacuum and subjecting the water specimen to atmospheric pressure so
that the water specimen is forced through the filling channels and
into the first and second cavities to take the place of the
evacuated air; mixing the water specimen with a nutrient medium
such that a mixture of water specimen and nutrient medium exists in
the first and second cavities; observing the water specimen in the
first and second cavities for a change in the appearance thereof to
determine the number of cavities which change appearance, and
comparing the number of cavities which do change appearance with
tabulated results derived from statistical evaluations to determine
the concentration of microorganisms.
6. A process according to claim 5 wherein the water specimen and
nutrient medium are mixed together in the first and second
cavities.
7. A process according to claim 5 and further comprising placing
the water specimen in communication with third cavities which are
of known and equal volume, the volume of the third cavities being
less than the volume of the second cavities, whereby when the water
specimen is placed under a vacuum, the air is evacuated from the
third cavities as well as from the first and second cavities, and
when the vacuum is thereafter released, the water specimen is
forced into the third cavities, and comparing the number of third
cavities which change appearance with tabulated results derived
from statistical evaluations to determine the concentration of
microorganisms; and wherein the step of mixing the water specimen
with the nutrient medium is such that a mixture of water specimen
and nutrient medium also exists in the third cavities.
Description
BACKGROUND OF THE INVENTION
This invention relates in general to microorganisms and more
particularly to a process and apparatus for enumerating
microorganisms in a water sample.
Contamination of public waters such as streams, rivers and lakes by
residential and industrial sewage constitutes a major environmental
problem, since such waters are the primary source of drinking water
and the water used for the preparation and processing of foods,
drugs and cosmetics. Moreover, such bodies of water represent a
principal source of recreation. While the solution to the
contamination problem of course involves eliminating harmful
microorganisms from effluents which enter such bodies of water; it
also requires continuous microbiological monitoring of the
effluents as well as the public waters themselves. Current
techniques for microbiological analysis are time-consuming,
laborious and expensive.
There are two widely practiced procedures for ascertaining the
bacterial count of water or aqueous suspensions. The first,
commonly called the Most Probable Number, or MPN procedure,
requires serial 10-fold dilutions of a sample into a nutrient
medium with each successive dilution usually being 1 ml. of the
previous dilution mixed into 9 ml. of nutrient medium. The
microorganisms are then allowed to grow and the various dilutions
are observed for turbidity, change of color, or some other
indication of growth. The reciprocal of the next to the last
dilution showing growth is taken as the concentration in cells per
milliter.
In the second procedure cells are collected on a membrane filter,
and the filter is placed either on an absorbent pad saturated with
culture media, or on solid media containing bacteriological agar.
The cells are visualized as colonies on the membrane filter after a
defined period of incubation and the number of colonies, related to
the dilution used and amount filtered, provides the number of cells
in the original sample. This procedure is commonly called the
membrane filter process.
Since the membrane filter procedure is limited to samples which
will not clog the membrane pores, and is not effective when
chlorinated samples are tested, the MPN procedure has become the
most widely accepted procedure for determining bacterial counts in
water.
In its simplest form, the MPN procedure involves a straight 10-fold
dilution to extinction using a single set of test tubes. Measured
aliquots of these dilutions are then added to tubes containing
nutrient medium. For example, the first dilution tube will contain
10 ml. of sample without any dilution (10.degree. dilution). The
second dilution tube will contain 1 ml. of sample and 9 ml. of
diluent (10.sup.-.sup.1 dilution). The third dilution tube will
contain 0.1 ml. of sample in 9.9 ml. of diluent (10.sup.-.sup.2
dilution). The fourth, fifth, etc. dilution tubes are filled with 9
ml. diluent and 1 ml. of suspension derived from their immediate
predecessors in the series. Thus, the concentration of sample in
the fourth dilution tube will be 1/1000 that of the original sample
(10.sup.-.sup.3 dilution), while the concentration in the fifth
dilution tube will be 1/10000 that of the sample (10.sup.-.sup.4
dilution).
One ml. aliquots of each dilution tube are then added to
corresponding tubes containing nutrient medium (culture tubes),
thus resulting in a series of tubes. The first culture tube of this
series will contain 1.0 ml. of the 10.degree. dilution, the second
tube 1.0 ml. of the 10.sup.-.sup.1 dilution, and so on to a final
tube containing 1.0 ml. of the 10.sup.-.sup.5 dilution. These
culture tubes are then incubated and observed for changes. If the
fifth culture tube (which received 1.0 ml. of the 10.sup.-.sup.5
dilution) is the last to exhibit growth, then at least one cell was
present in the 1 ml. derived from the 10.sup.-.sup.5 dilution of
the dilution tube and added to the fifth culture tube. It is
assumed, therefore, that each ml. in the sixth dilution tube
contained one cell also, or a concentration therein of one cell/ml.
Since this dilution constitutes 1/100,000 of the 10.degree., or
10.sup.-.sup.5, then the concentration in the 10.degree. sample is
the reciprocal of 10.sup.-.sup.5 which is 10.sup.5 cells per
ml.
A single series of tubes gives only a rough estimate. To provide
greater accuracy, usually 5 tubes of each dilution are employed.
Under normal circumstances not all of the series will dilute to
extinction at the same dilution. For example,three of the tubes of
10.sup.-.sup.4 dilution may show growth, while the two remaining do
not. Tables prepared from statistical evaluations are available to
provide the bacteria count for any combination of growth and no
growth tubes at various dilutions. Of course, the greater the
number of series, the more accurate is the count.
From the foregoing, it is quite apparent that the MPN method with
all its dilutions of dilutions, is quite laborious and
time-consuming and requires a considerable amount of culture
medium.
SUMMARY OF THE INVENTION
One of the principal objects of the present invention is to provide
an apparatus and method for enumerating microorganisms within a
sample in a relatively short time and with a minimum amount of
effort. Another object is to provide an apparatus and method of the
type stated which does not rely on or require a large number of
serial dilutions for enumeration, nor does it require the user to
prepare media. A further object is to provide a process of the type
stated which is highly aseptic so that it is not easily subjected
to contamination or rendering false positives from such
contamination. An additional object is to provide an apparatus and
process of the type stated which may be used to enumerate specific
groups of microorganisms such as fecal streptococci, coliforms,
aerobes and anaerobes, or even individual genera such as
Esherichia, Klebsiella, Enterobacter, Pseudomonas and Proteus.
Still another object is to provide a process and apparatus of the
type stated which is ideally suited for preforming bacterial counts
of water derived from lakes, rivers, streams and the like or from
the effluents of sewage treatment plants which discharge into such
bodies. These and other objects and advantages will become apparent
hereinafter.
The present invention is embodied in a process including obtaining
a liquid specimen, filling a plurality of cavities of known volume
with the specimen, mixing the specimen in the cavities with a
nutrient medium, and observing the cavities for a change in the
appearance thereof to determine the number of cavities which change
appearance. The invention also resides in an apparatus for
performing the foregoing process, that apparatus having a plurality
of wells of equal volume. The invention also consists in the parts
and in the arrangements and combinations of parts hereinafter
described and claimed.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plan view showing the top of a cassette used in the
process of the present invention;
FIG. 2 is a plan view showing the bottom of the cassette;
FIG. 3 is a sectional view taken along line 3--3 of FIG. 1;
FIG. 4 is a fragmentary sectional view taken along line 4--4 of
FIG. 2 and showing one of the viewing wells in the cassette and its
associated overflow chamber and filling channel;
FIG. 5 is a perspective view showing the cassette of FIGS. 1-4
being loaded with a liquid specimen in a loading device;
FIG. 6 is a top plan view of a modified cassette containing 28
wells;
FIG. 7 is a bottom plan view of the modified cassette of FIG.
6;
FIG. 8 is a sectional view taken along line 8--8 of FIG. 7;
FIG. 9 is a plan view of still another modified cassette containing
55 wells; and
FIG. 10 is a sectional view taken along line 10--10 of FIG. 9.
DETAILED DESCRIPTION
Referring now to the drawings (FIGS. 1 and 2), C designates a
cassette used in the process of the present invention for
enumerating microorganisms in a water sample such as might be
derived from a lake, river, stream or other body of water or from
the effluent of a sewage treatment or industrial plant. The
cassette C is rectangular in shape, measuring about 2.5 inches by
3.5 inches and is about 0.125 inches thick.
The cassette C includes (FIGS. 1-4) a rigid transparent plate 10,
which is preferably formed from a suitable plastic such as
polycarbonate and is the same size and shape as the cassette C. The
plate 10 has 20 viewing wells 12 arranged in four transverse rows
of five each. Each viewing well 12 extends completely through the
plate 10 (FIGS. 3 and 4), that is, from one major surface area to
the other, and is of known volume which may be virtually any
desired volume, depending on the size of the cassette C. Volumes
from 0.005 ml. to approximately 0.10 ml. are representative. All of
the wells 12 are of equal volume. Located adjacent to each well 12
is an overflow chamber 14 (FIGS. 2 and 4), and these chambers 14
extend only partially through the plate 10 and hence open out of
only one of the two major surface areas. Each overflow chamber 14
is connected to its adjacent viewing well 12 through shallow
connecting channels 16 which likewise opens out of the same surface
area as the overflow chambers 14. The overflow chambers 14 assure
complete filling of the wells 12.
The plate 10 further has separate filling channels 18 (FIGS. 1 and
3), leading to each of the viewing wells 12 from a longitudinally
extending feeder channel 20 which in turn is located close to one
of the longitudinal edges of the plate 10. The filling and feeder
channels 18 and 20 are quite shallow and open out of the opposite
major surface of the plate 10, that is, the surface opposite from
which the overflow chambers 14 and connecting channels 16 open.
Each filling channel 18 is of a dog leg configuration.
Approximately, midway between its ends the feeder channel 20 opens
into a short inlet channel 22 (FIGS. 1 and 3) which likewise opens
out of the same surface area on the plate 10 as does the feeder
channel 20, but the inlet channel 22 is of substantially greater
depth, extending almost all the way through the plate 12. The inlet
channel 22 projects laterally from the feeder channel 20 and aligns
with a bore 24 which opens out of the adjacent longitudinal edge of
the plate 10. The bore 24 contains a tightly fitted septum 26
formed from a suitable elastomeric material and is connected with
the inlet channel 22 through a short connecting bore 28 of reduced
diameter.
Each viewing well 12 contains a culture medium which is preferably
in a dehydrated condition. The medium may be a universal medium in
the sense that it sustains and responds to practically all
microorganisms. On the other hand, it may be a selective medium
which will both sustain and respond to only a selected species or
group of microorganisms. The response should be such that the
mixture of culture medium and water will exhibit a change in
appearance or light transmitting characteristics when a
microorganism capable of triggering the culture medium is present.
Thus, in the case of a universal medium practically all
microorganisms will effect a change in appearance. On the other
hand, where a selective medium is present, only the microorganism
to which the medium is specific will cause a change in appearance.
Suitable selective media are disclosed in U.S. patent application
Ser. No. 461,249 of Clifton Aldridge, Jr. et al which was filed
Apr. 16, 1974 and is entitled PROCESS AND APPARATUS FOR ANALYZING
SPECIMENS FOR THE PRESENCE OF MICROORGANISMS THEREIN, now U.S. Pat.
No. 3,963,355 .
The two major surface areas of the plate 10 are covered with
transparent tapes 30 (FIG. 3) which are wide enough to cover the
wells 12. Thus, the two tapes 30 close the ends of the wells 12 and
retain the culture medium in them. The one tape 30 further covers
the overflow chambers 14 and connecting channels 16, while the
other tape 30 further covers the inlet channel 22, the feeder
channel 20, and the filler channels 18. Hence, the tapes 30
together with the septum 28 completely isolate the interior of the
cassette C, that is, the inlet channel 22, the feeder channel 20,
the filling channels 18, the wells 12, the connecting channels 16,
and the overflow chambers 14 from the surrounding atmosphere, and
since the culture medium is in the wells 12, it is likewise
isolated and protected from contamination. The tape 30 is slightly
permeable in the sense that it will admit air to the wells 12, but
the permeability is such that neither water nor microorganisms can
escape from the wells. Furthermore, the tapes 30 admit air to the
wells 12 so slowly that they permit the interior of the plate 10 to
be placed under a vacuum of at least 28 inches Hg and held at that
condition for at least 3 minutes. FEP 5430 tape marketed by the 3M
Company is suitable for the tapes 30.
The cassette C is loaded with a water specimen in a loading device
L (FIG. 5) including a flat base 32, a tube 34 projecting upwardly
from the base 32, and a pair of parallel guide webs 36 interposed
between the base 32 and the tube 34. The spacing between the webs
36 is slightly greater than the thickness of the cassette C so that
the cassette C may be fitted between them. The tube has a hollow
needle 38 projected radially from it into the space between the
webs 36 and the distance between this needle 38 and the base equals
the distance between one of the transverse edges on the cassette C
and the bore 24 containing the septum 26. Hence, when the cassette
C is inserted between the webs 36 with its transverse edge resting
on the base 32, the needle 38 will align with the septum 26. To
couple the cassette C with the loading device L, the cassette C is
advanced toward the tube 34 until the needle 38 is projected
through the septum 26. This provides communication between the
interior of the tube 34 and the interior of the cassette C. The
upper end of the tube 34 is open and is fitted with a flexible
stack 40 which serves to connect the tube to a vacuum line (not
shown), and that vacuum line is evacuated by means of a vacuum
pump.
To estimate the bacterial count in a known volume of specimen using
the cassette C, the cassette C is first coupled with the loading
device L such that the needle 38 on the tube 34 of the loading
device L is projected through the septum 26 of the cassette C. This
places the interior of the cassette C, that is, the inlet channel
22, the feeder channel 20, the filling channels 18, the viewing
wells 12, the connecting channels 16 and the overflow chambers 14,
in communication with the interior of the tube 34. Next, the tube
34 is filled with the water specimen. The tube 34 is large enough
to receive the total volume of specimen.
Once the tube 34 is filled with the specimen, the flexible stack 40
thereon is connected with a vacuum line and a vacuum of at least 28
inches Hg is created. As a result, the air formerly trapped in the
interior of the cassette C passes out of the cassette C through the
needle 38 and bubbles through the water specimen in the tube 34,
thus, establishing the vacuum in the interior of the cassette C
also. In this regard, it will be recalled that the permeability of
the tapes 30 is such that the vacuum is maintained for at least the
time required to develop it.
After the desired vacuum is established in the tube 12 and cassette
C connected thereto, that vacuum is released by venting the upper
end of the tube 34 to the surrounding atmosphere. The resulting
force developed on the water specimen within the tube 34 by the
atmosphere forces the water specimen into the cassette C. In
particular, the water specimen flows in order through the inlet
channel 22, the feeder channel 20, the filling channels 18, the
viewing wells 12, the connecting channels 16 and the overflow
chambers 14. In other words, the water specimen replaces the air
evacuated from the cassette C. Any air which is not evacuated
accumulates in the overflow chambers 14. The water upon entering
the viewing wells 12 rehydrates the culture medium therein.
Bacteria in the water sample are thus randomly separated into the
wells 12.
Once the cassette C is filled, it is withdrawn from the loading
device L and the septum 26 seals, isolating the interior of the
cassette C from the surrounding atmosphere. The cassette C is then
incubated at the desired incubation temperature for about 12 to 18
hours. At the end of the time the viewing wells 12 are observed to
determine how many exhibit a change in light transmitting
characteristics which is a manifestation of growth or more
accurately metabolic activity. Of course, if the medium is a
universal medium any microorganism will trigger the change. On the
other hand, if it is a selective medium, only the species or group
of microorganisms to which the medium is specific will cause the
change in light transmitting characteristics.
Since the volume of each viewing well 12 is known, the number of
microorganisms in the specimen may be ascertained as falling beyond
or within certain limits. Assuming that each of the 20 viewing
wells has a volume of 0.1 ml., then if all of the wells 12 exhibit
growth it is most likely that the concentration of microorganisms
in the specimen is greater than one cell or microorganism per 0.1
ml. or greater than 10 cells per ml. On the other hand, if none of
the wells 12 show growth, then it is most likely that there is less
than one cell or microorganism in every 2.0 ml. (the total volume
of the 20 wells 12) or 0.5 cells per ml. Thus, the cassette C and
its associated process is suitable for use by one seeking to
determine if the bacteria count is less than or in excess of
certain specified limits, those limits being 10 cells per ml. and
0.5 cells per ml. in the foregoing example. The example assumes
perfect random distribution throughout the cassette C. Actually,
the numerical limits are only arithmetic approximations. More
accurate estimations may be established statistically. When
considered statistically, the lower limit is about the same at 0.5
cells per ml., but the upper limit rises to 30 cells per ml. (see
Table A).
When some but not all of the wells 12 exhibit growth it is
reasonable to assume that the bacteria count falls somewhere
between the statistical limits established for the cassette C,
which in the foregoing example is somewhere between 0.5 cells per
ml. and 30 cells per ml. Statistical evaluations similar to Most
Probable Number charts may be used to estimate the number of cells
in the range with reasonable accuracy. With reference to the
previously discussed example, statistical evaluations in the form
of a table (see Table A) show that when 13 of the 20 wells 12
exhibit growth, the bacterial count is about 10.5 cells per ml.
Still more latitude is acquired by running several tests each in
separate cassettes C and with different dilutions of the specimens.
In the example given above an initial 1/10 dilution of the specimen
would adjust the statistical upper limits of the range to 300 cells
per ml., while a 1/100 dilution would adjust the upper limit to
3000 cells per ml. The lower limit would remain the same at 0.5
cells per ml. Thus, the cassette C makes it a relatively simple
matter to determine if the bacterial count exceeds a specified
limit.
Greater latitude may also be obtained by using separate cassettes C
provided with wells 12 of different volume. For example, the one
cassette C may have 0.1 ml. wells 12, and the next might have 0.01
ml. wells 12.
EXAMPLE NO. 1
Each of the three cassettes C contained 20 wells 12 and each well
12 had a volume of 0.1 ml. Moreover, the culture medium in each
well 12 was specific to the fecal coliform group of organisms. That
group is one of the most significant insofar as public waters are
concerned, since its concentration provides a good indication of
the suitability of such water for drinking, recreational, and other
purposes. The first cassette Cl was loaded with a water specimen
derived from a cooling fountain; the second cassette C2 was loaded
with a specimen derived from a creek; and the third cassette C3 was
loaded with a specimen acquired from a sewage treatment lagoon.
After incubating the cassettes C for 12 to 18 hours, none of the
wells 12 of the cassette C1 exhibited a change in the light
transmitting characteristics thereof, thereby indicating that the
fecal coliform count in the cooling fountain water was less than
0.5 cells per ml. The cassette C2 containing the creek water had 14
wells 12 which showed growth, while the remaining did not. From the
statistical table below it was determined that the fecal coliform
count approximated 12 cells per ml. The cassette C3 had growth in
all wells 12, indicating that the fecal coliform count exceeded 30
cells per ml.
______________________________________ Most Probable Number Count
Wells Positive (cells/ml.) ______________________________________ 0
<0.5 1 0.51 2 1.05 3 1.60 4 2.20 5 2.90 6 3.55 7 4.30 8 5.10 9
6.00 10 6.95 11 8.00 12 9.15 13 10.50 14 12.00 15 13.85 16 16.00 17
19.00 18 23.00 19 30.00 20 >30.00
______________________________________
EXAMPLE NO. 2
Three cassettes C of the same type and volume as described in
Example 1 were employed. However, the first cassette C1 was loaded
with a specimen acquired from a sewage treatment lagoon. The second
cassette C2 was loaded with a 10-fold dilution of the same sample,
while the third cassette C3 was loaded with a 100-fold dilution of
the same sample. After a 12.18 hour incubation at 35.degree. C. the
following results were obtained. The first cassette C1 showed 20
positive wells 12, indicating a concentration of greater than 30
cells per ml. The second cassette C2 also showed 20 positive wells
12 indicating a concentration in the original sample of greater
than 300 cells per ml., since only a 10-fold dilution was loaded
into that cassette. The cassette C3 showed 9 positive wells 12.
From Table A 9 positive wells correspond to 6.0 cells per ml. But
since a 100-fold dilution was loaded into the cassette C3, the
Figure must be multiplied by 100 to give the cell concentration in
the original sample, which would be 600 cells per ml.
MODIFICATION
A modified cassette D (FIGS. 6-8) differs from the cassette C in
that it provides its own 10-fold dilutions. The cassette D
possesses substantially the same external configuration as the
cassette C and likewise has a rigid plastic plate 50 covered on
both of its major surface areas by transparent tapes 52 (FIG.
8).
The plate 60 contains 14 large wells 54 and 14 small wells 56, with
the latter being 1/10 the volume of the former. The large wells 54
are elongated in the direction of the longitudinal axis of the
plate 50 and are arranged in two rows at the end of the plate 50.
The small wells 56 are likewise arranged in two rows, those rows
being between the rows of large wells 54. Both the large wells 54
and the small wells 56 extend completely through the plate 50,
their ends being closed by the tapes 52. The large wells 54 may
have a volume of 0.180 ml. and the small wells a volume of 0.0180
ml.
Extended along the ends of the wells 54 and 56 in each row
transverse feeder channels 58 (FIG. 6) which are connected to the
individual wells 54 and 56 through separate filling channels 60.
All but one of the transverse feeder channels 58 connect with a
longitudinal feeder channel 62 which intermediate to its ends opens
into an inlet cavity 63. The one feeder channel 58 which does not
connect with the channel 62, opens directly into the inlet cavity
63. The cavity 63 in turn communicates with a bore 64 having a
septum 66 fitted therein. The channels 58, 60 and 64 open out of
the plate 50 through one major surface area thereof, and these
outwardly opening sides of the channels 58, 60 and 64 are covered
with one of the tapes 52. All of the viewing wells 54 and 56
contain a dehydrated nutrient medium which may be universal or
selective.
The cassette D is coupled with the loading device L and filled with
a water specimen in the same manner as the cassette C. Once filled,
the cassette D is incubated at 35.degree. C. for 12 to 18 hours.
Then it is observed to determine the number of large wells 54 and
the number of small wells 56 which exhibit growth.
Assuming the purposes of illustration that the large wells 54 are
0.180 ml. and the small wells 56 are 0.0180 ml., and that all of
the wells 54 and 56 exhibited growth, then it is certain that the
concentration of microorganisms is greater than one cell per 0.0180
ml. or 55.5 cells per ml. when considered arithmetically and 146
cells per ml. when considered statistically (see Table B). On the
other hand, if none of the wells 54 and 56 show growth, then most
likely there is less than one cell per 2.772 ml. (the total volume
of all the wells 54 and 56) or 0.36 cells per ml. For varying
combinations of growth and no growth in both the large wells 54 and
the small wells 56, tables based on proven statistical computations
are referred to (see Table B).
The presence of the two sets of wells 54 and 56 having different
volumes considerably extends the range of concentration over which
the cassette D is functional. Without dilution the range extends
from 0.36 to 146 cells per ml. With a 1/10 dilution the range
extends from 0.36 to 1460 cells per ml.
EXAMPLE NO. 3
Three specimens of water were obtained, one from a cooling
fountain, another from a creek, and still another from a sewage
treatment lagoon. The specimens from the fountain and creek were
loaded into cassettes D1 and D2 directly, while the specimen from
the lagoon was diluted 10 fold and loaded into the cassette D3. The
wells 54 and 56 of these cassettes D1, D2 and D3 contained a
universal nutrient medium.
After incubation at 35.degree. C. for 12 to 18 hours, all of the
wells 54 and 56 of the cassette D3 (lagoon) exhibited growth
indicating that one cell existed for each 0.0180 ml. or that the
concentration was in excess of 146 cells per ml. of diluted
specimen when considered statistically. Since the specimen was
originally diluted to 10 times its original volume, the actual
concentration was in excess of 1460 cells per ml. of undiluted
specimen.
In the cassette D2 containing the creek specimen, all of the large
wells 54 exhibit growth but only 7 of the small wells 56 did so.
From the following Table B prepared from statistical evaluations,
it was determined that the actual cell count was 39.01 cells per
ml. In the cassette D1 containing the fountain specimen, only three
of the large wells 56 exhibited growth, while none of the small
wells 54 did so. From Table B it was determined that the
concentration of microorganisms in the specimen was 1.2 cells per
ml.
__________________________________________________________________________
Number Positive Number Positive MPN MPN 0.18 ml 0.018 ml (cells/ml)
0.18 ml 0.018 ml (cells/ml)
__________________________________________________________________________
0 0 <.36 8 0 4.07 1 0 0.37 8 7 8.26 1 7 3.06 8 14 13.26 1 14
5.90 9 0 4.87 2 0 0.77 9 7 9.54 2 7 3.58 9 14 15.36 2 14 6.58 10 0
5.82 3 0 1.20 10 7 11.15 3 7 4.15 10 14 18.21 3 14 7.34 11 0 6.95 4
0 1.67 11 7 13.28 4 7 4.70 11 14 22.49 4 14 8.19 12 0 8.39 5 0 2.18
12 7 16.39 5 7 5.49 12 14 30.10 5 14 9.16 13 0 10.32 6 0 2.74 13 7
21.94 6 7 6.28 13 14 48.70 6 14 10.28 14 0 13.32 7 0 3.36 14 7
39.01 7 7 7.19 14 13 146.61 7 14 11.62 14 14 >146.61
__________________________________________________________________________
The foregoing Table contains only selected portions of the entire
Table, which includes all combinations possible from 0-0 to
14--14.
EXAMPLE NO. 4
Three cassettes D1, D2 and D3 each having 14 wells 54 of 0.180 ml.
and 14 wells 56 of 0.018 ml. volume were loaded with a lagoon
sample as follows: cassette D1 was loaded with undiluted sample,
cassette D2 was loaded with a 10-fold dilution of the sample, and
cassette D3 was loaded with a 100-fold dilution of the sample.
After incubation at 35.degree. C. for 18 hours the following
results are obtained:
Cassette D1 contained 14 positive large wells 54 and 14 positive
small wells 56, indicating the concentration to be greater than 146
cells per ml. Cassette D2 showed 12 large wells 54 positive and 7
small wells 56 positive. From Table B this combination was found to
equate to 16.39 cells per ml. Since a 10-fold dilution was used,
the count in the original sample was 163.9 cells per ml. The third
cassette D3 showed 4 positive large wells 54 and no positive small
wells 56. From Table B this combination equated to 1.67 cells per
ml. But since a 100-fold dilution was used, the actual count in the
original sample is 100 .times. 1.67 or 167 cells per ml.
FURTHER MODIFICATION
A modified cassette E provides a three fold dilution, thus
increasing still further the range of concentration over which the
cassette E remains functional. The cassette E includes a plate 70
which on one of its major surface areas is stepped so that plate
contains a thick segment 72 and a thin segment 74.
The thick segment 72 contains five large wells 76 which are
arranged in a row and extend completely through the thick segment
72. The large wells 76 are of equal volume, which may be 0.5 ml.
All of the large wells 76 are tied together through a major feeder
channel 78 having lateral branch channels 80 leading therefrom into
the large wells 76. The major feeder channel 78 near its
mid-portion communicates with a bore 82 which opens out of the side
edge extended along the thick segment 72 of the plate 70. The bore
82 contains a septum 84 which is fitted tightly therein.
The thin segment 74 contains a plurality of intermediate wells 86
which are arranged in two rows therein parallel to the row of large
wells 76. Successive intermediate wells 86 are located in different
rows. Each intermediate well extends completely through the thin
segment 74 and may possess a volume of 0.05 ml.
The intermediate wells 86 are arranged in groups of five, and each
group of five is connected with a single large well 76 through a
short discharge channel 88, a secondary feeder channel 90, and
individual supply channels 92 which branch off of the secondary
feeder channel 90. The channels 88, 90 and 92 open out of the
uninterrupted surface of the plate 70, that is the surface located
opposite from the stepped surface out of which the channels 78 and
80 open.
The thin segment 74 further contains small wells 94 which likewise
extend entirely through the thin segment 74, but are considerably
smaller in diameter than the intermediate wells 86. Indeed, the
small wells 94 may have a volume of only 0.005 ml. Each small well
94 is connected with a different intermediate well 86 through a
filler channel 96 which opens out of the stepped surface of the
plate 70.
Located beyond each small well 94 is an overflow well 98, and each
overflow well 98 is connected with its small well 94 through a
short connecting channel 100 which opens out of the uninterrupted
surface of the plate 70.
Both are uninterrupted surface and the step surface of the plate 70
are covered with tapes 102 which close the ends of the wells 76, 86
and 94 as well as the sides of the channels 78, 80, 88, 90, 92, 96
and 100.
In use, the cassette E is filled with a mixture of speciment and
liquid culture medium by utilizing a loading device quite similar
to the loading device L. In other words, the cassette E is loaded
by evacuating air from the interior thereof and then replacing the
evacuated air with a mixture of specimen and liquid nutrient
medium. The nutrient medium is not contained within the wells 76,
86 and 94 themselves, but constitutes a broth into which the
specimen is diluted. This procedure is utilized inasmuch as it is
difficult to contain the proper amount of dried medium in the large
wells 76 since there is a tendency for such medium to wash through
into the intermediate wells 86 and the small wells 94. This would
provide too great of concentration of culture medium in the
intermediate and small wells 86 and 94 and too small a
concentration in the large wells 76. However, by locating each well
76, 86 and 94 at the end of its own filling channel as in the
cassettes C and D, it is possible to use a dehydrated culture
medium in each well 76, 86 and 94 of the cassette E.
The cassette E, once it is loaded, is incubated and then observed
in the manner of the cassettes C and D. Since the cassette E in
effect provides a three fold dilution, the range of concentration
through which the cassette E is functional is quite broad.
EXAMPLE NO. 5
Three cassettes E1, E2 and E3 have large wells 76, intermediate
wells 86 and small wells 94 of 0.5 ml., 0.05 ml., and 0.005 ml.
respectively. From a statistical standpoint, this gives the
cassettes an effective range of 0.258 to 643.77 cells per ml. (see
Table C).
Into the cassette E1 was loaded a sample derived by mixing 10 ml.
of drinking fountain water with 10 ml. of double strength universal
medium. Another mixture was prepared by mixing 1 ml. of a lagoon
sample with 100 ml. of single strength medium and that mixture was
vacuum loaded into the cassette E2. Likewise, 1 ml. of the sewage
effluent is mixed with 100 ml. of single strength universal medium
and that mixture is loaded into the cassette E3. All three
cassettes E1, E2 and E3 were incubated.
The cassette E1 showed one positive large well 76, but no positive
intermediate wells 86 or small wells 94. From the following Table
C, it was determined that the concentration in the mixture within
the cassette E1 was 0.27 cells per ml. but since drinking sample
was diluted in an equal volume of diluent, the concentration in the
actual water sample was 2 .times. 0.27 or 0.54 cells per ml.
The cassette E2 had two positive large wells 76, 8 positive
intermediate wells 86, and 8 positive small wells 94. From the
Table C it was determined that the concentration of the mixture in
the cassette E2 amounted to 6.16 cells per ml., but since the
sample was diluted 100 times, the concentration of the sample was
616 cells per ml.
The cassette E3 containing the sewage effluent had five positive
large wells 76, 16 positive intermediate wells 86, and no positive
small wells 94. This amounted to a concentration of 17.44 cells per
ml. within the cassette E3 itself, and since the sample was diluted
100 times by the culture medium, the actual concentration in the
sample amounted to 1,744 cells per ml.
TABLE C
For a 55 well cassette containing 5 wells of 0.5 ml., 25 wells of
0.05 ml. and 25 wells of 0.005 ml. Only selected portions of entire
table are presented.
__________________________________________________________________________
Number Positive MPN Number Positive MPN 0.5 ml 0.05 ml 0.005 ml
(cells/ml) 0.5 ml 0.05 ml 0.005 ml (cells/ml)
__________________________________________________________________________
0 0 0 <0.25 3 4 4 3.95 1 0 0 .27 3 8 8 7.59 1 1 0 .55 3 16 0
8.31 1 1 1 .83 3 16 25 22.30 1 2 0 .84 4 0 0 1.45 1 4 0 1.43 4 1 0
1.85 1 4 4 2.60 4 1 1 2.25 1 8 8 5.15 4 2 0 2.27 1 16 0 5.47 4 4 0
3.17 1 16 25 14.31 4 4 4 5.04 2 0 0 0.59 4 8 8 9.85 2 1 0 0.90 4 16
0 11.18 2 1 1 1.21 4 16 25 31.28 2 2 0 1.22 5 0 0 2.07 2 4 0 1.87 5
1 0 2.56 2 4 4 3.19 5 1 1 3.05 2 8 8 6.16 5 2 0 3.09 2 16 0 6.60 5
4 0 4.27 2 16 25 17.40 5 4 4 6.81 3 0 0 .97 5 8 0 7.29 3 1 0 1.32 5
16 0 17.44 3 1 1 1.67 5 16 25 54.95 3 2 0 1.68 5 25 24 643.77 3 4 0
2.43 5 25 25 >643.77
__________________________________________________________________________
The foregoing Table includes only a selected number of examples
from the actual table, which includes all possible combinations
from 0-0-0 to 5-25-25.
FURTHER CONSIDERATIONS
The cassettes C, D and E may be observed during incubation by the
naked eye, in which case turbidity, color change or some other
change in the light transmitting characteristics of the wells is
clearly visible. On the other hand, the cassettes C and D may be
monitored electro-optically during the incubation period for
changes in the optical density of the wells 54 and 56. An
electro-optical detector which projects light through the wells,
measures the intensity of the light leaving the wells, and records
that intensity in graphic form as a function of time, is disclosed
in U.S. Pat. No. 3,963,355, previously referred to herein.
Where wells of different volume are employed such as in the
cassettes D and E, the volumes need not be related by factors of
10. For example, in the cassette D the small cells 56 may be 1/20
the volume of the large cells 54. This is easily achieved in the
cassette D by extending the small cells 56 only half way through
the plate 50 instead of entirely through it.
Tables A, B and C were derived from statistical computations
prepared much in the same manner as MPN Tables are formulated.
This invention is intended to cover all changes and modifications
of the example of the invention herein chosen for purposes of the
disclosure which do not constitute departures from the spirit and
scope of the invention.
* * * * *