WO1991018085A1

WO1991018085A1 - Multi-nutrient growth culturing system and a method of use

Info

Publication number: WO1991018085A1
Application number: PCT/US1991/003104
Authority: WO
Inventors: Joseph B. Hippensteel
Original assignee: Future Medical Technologies, Inc.
Priority date: 1990-05-11
Filing date: 1991-05-06
Publication date: 1991-11-28
Also published as: CA2082634A1

Abstract

A device (10) and method for culturing and identifying microorganisms in a liquid sample. A container (100) has a transverse wall (102), an upwardly extending peripheral skirt (104) defining an internal compartment (106) and terminating in an upper surface (108) which defines an opening into the compartment (106), and partitions (112) for sectioning the compartment. A porous material (300) including a dry nutrient is located in each section of the compartment (106). A porous filter member (200) overlies the porous materials (300) and traps microorganisms on its upper surface when the liquid sample is applied thereto, while permitting the liquid sample to move transversely along the upper surface (202) of the filter member (200) and then therethrough to activate the nutrients of the porous materials (300). A container (100) forming part of the device also is a part of the present invention.

Description

MULTI-NUTRIENT GROWTH CULTURING SYSTEM AND A METHOD OF USE

Field of the Invention This invention relates generally to microorganism . culturing and identification, and more specifically to a Multi-Nutrient Growth Culturing System and a Method of Use.

Background of the Invention

The prior art is replete with disclosures of units for the collection and growth of microorganisms from a liquid sample.

A number of units include filter members used to trap microorganisms present in the liquid sample, for subsequent exposure to a solid or liquid media contained in a single compartment. Representative patents disclosing such units are U.S. Patent Nos. 4,829,005 (Friedman et al.), 3,197,384 (Gold¬ man) , 4,777,137 (Lemmonier) , 2,879,207 and 2,923,669, (Poitras) , and 3,929,583 (Sharpe et al.).

In one embodiment of the Lemmonier '137 patent a sealing member covers the opening into a media-retaining compartment of a container positioned below the filter member, and a series of small openings about the perimeter of the container, which are not covered by the sealing member, permits the liquid of a sample to be tested to pass out of the device, while microorganisms in the sample are trapped on the filter member. Thereafter the sealing member is removed to permit a nutrient therein to "feed" the microorganisms previously deposited on the filter member.

A unit employing a pervious sheet to cultivate microorganisms for subsequent exposure to different liquid media contained in a segmented Petri dish is disclosed in U.S. Patent No. 4,775,628 (Takakura et al.). In this device the specimen (e.g., puss) containing the microorganisms to be cultivated is applied to the pervious sheet with a spiral plater; not by being flowed onto and through a filter member to rehydrate plural dehydrated media. Units also have been designed which have segmente media sections onto which a user directly applies a test sampl for growth and testing. However, these units do not employ filter membrane to collect microorganisms for exposure t multiple types of dehydrated media. Representative patent disclosing such units are U.S. Patent Nos. 3,102,082, (Brewer), 4,072,578 (Cady et al.), Design Patent No. 258,761 (Graham), 3,912,596 and 4,042,463 (Haque et al.) , 4,076,591 (Heden) , 4,912,037 (Lemmonier), and 4,668,633 (Walton).

In addition, it is known in the art to utilize dehydrated media to culture microorganisms, which may b activated upon wetting, as is disclosed in U.S. Patent Nos. 4,485,171 (Ikeda et al.) , 4,245,043 (Lund) , 4,587,21 (Malecki) , 4,250,256 (Wielinger et al.).

Moreover, the use of a filter membrane to tra microorganisms used in connection with a dried media also i disclosed in U.S. Patent Nos. 2,761,813 (Goetz) , 4,485,17

(Ikeda et al.), 2,677,646 (Lovell et al.) , and 3,741,87

(Shaufus) .

In spite of all the above teachings, however, it i believed that a need still exists for a single unit in whic microbes may be effectively collected and quickly filtered an grown without the need to handle the filter membrane upon whic the sample is deposited, which is not susceptible to leakage o dehydration of media during storage, handling and/or use, an which, for at least certain applications (e.g., the testing o urine) does not require the use of additional vacuum devices.

Objects of the Invention

Accordingly, it is a general object of this inventio to provide a device which permits the user to quickly collec and grow microorganisms in a liquid sample while minimizing th dangers of contamination from outside sources.

It also is an object of this invention to permit user to reliably subject collected microorganisms to a numbe of different dehydrated media. It is a further object of this invention to effec¬ tively collect microorganisms to be tested from a liquid sample and which is not susceptible to leakage of the growth media or the liquid sample during storage, handling and/or use.

It is a further object of this invention to permit the user, in some applications (e.g. , the testing of urine) , to quickly collect and grow microorganisms from a liquid sample without the need to use capital intensive equipment, including but not limited to expensive incubation and vacuum devices.

It is yet still a further object of this invention to provide the user with a method of collecting and culturing microorganisms which is easy to accomplish, fast and reliable.

It is still a further object of this invention to reliably permit a user to collect and grow microorganisms, for the purpose of identification, and then to save or store the cultured microorganisms for future reference and/or use.

It is still a further object of this invention to effectively collect microorganisms from both large and small liquid sample sizes, and, if necessary or desired, to permit a vacuum assist to remove excess liquid.

Summary of the Invention The above and other objects of this invention are achieved by providing a device and method for culturing and identifying microorganisms in a liquid sample. The device includes a container having a transverse wall and a peripheral skirt extending upwardly therefrom to define an internal compartment, with the peripheral skirt terminating in an upper surface to define an opening into the internal compartment. A partition means in the internal compartment divides the compartment into at least two sections, and a porous material including a dry (dehydrated) nutrient is located in each of the at least two sections. A filter member is contiguous to the upper surface of the peripheral skirt and overlies the at least two sections of the internal compartment of the container, with the lower surface of the filter member closely adjacent to, and preferably in contact with the porous material including dry nutrient. The filter member has pores of a size for preventin the microorganisms of the liquid sample from passin therethrough while permitting the liquid of the liquid sampl to pass therethrough. The liquid sample is applied to th upper filter surface to cause said sample to flow transversel along the upper surface of the filter member into overlyin relationship with the at least two sections of the compartment before passing completely through the filter member and int the porous materials. The porous materials aid in pulling th liquid of the liquid sample through the pores of the filte member by capillary action for rehydrating the nutrient, t thereby permit the rehydrated nutrient to be directed throug the filter member to the microorganisms.

In the preferred embodiment of the invention th porous material including the dry nutrient is in the form of porous carrier having the dry nutrient retained therein Accordingly, in the preferred construction the porous structur is of a defined geometric shape.

In the preferred embodiment the upper surfaces of th peripheral skirt and partition means are in substantially th same plane to effectively isolate the nutrients in each of th sections of the container from each other.

In the preferred embodiment the device has a cove with a liquid receiving means therein, to permit the introduc tion of liquid into the device. The cover preferably include a downwardly extending peripheral skirt providing a downwardl facing surface contiguous to the upper surface of th peripheral skirt of the container. The filter member i located between these latter two surfaces, and preferably i bonded to one of them. Preferably one or more vents exten through the partition means for the venting of the device.

Quite surprisingly it has been found that althoug the filter member permits transverse flow of the liquid sampl applied to its upper surface, so that the liquid substantiall covers the upper surface of the filter before being absorbe into each of the porous materials including dehydrated media the different media, which are rehydrated by the liquid, ar not subjected to this same transverse flow when transmitted through the lower surface of the filter member into contact with the microbes, even when the filter member has the same properties throughout its entire structure.

Thus, the transverse flow of the liquid sample along the filter media permits the microbes to be distributed over the filter member into overlying relationship with each of the different media. Moreover, this transverse flow of the liquid sample permits the liquid to rehydrate the media, with the assistance of capillary attraction. Although this transverse flow is extremely important to the proper operation of the device, a corresponding transverse flow of the rehydrated media, if it did occur, would cause an intermixing of the different media on the filter member; resulting in decreased efficiency of the system. Since significant transverse flow of the rehydrated media does not take place in the filter member, and is precluded by the partition means in the container from taking place within the container, the present invention has proven to be successful in exposing microbes to selective and/or differential media in discrete, selected areas of a filter member overlying each of the media, for the purposes of quickly and efficiently culturing and identifying such microbes.

Description of the Drawings

Other objects and many attendant features of this invention will become readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:

Fig. 1 is a isometric view of a culturing system in accordance with this invention;

Fig. 2 is a exploded isometric view of the culturing system shown in Fig. 1;

Fig. 3 is a sectional view taken along line 3-3 of Fig. 2; and Fig. 4 is a sectional view taken along line 4-4 of Fig. 3.

Detailed Description of the Preferred Embodiments

Referring now to various figures of the drawing where like reference numerals refer to like parts there i shown at 10 in Fig. 1, a device constructed in accordance wit this invention for use in culturing and identifyin microorganisms in a liquid sample.

As shown most clearly in Fig. 2 , the device comprise a container 100, and a filter member 200 disposed above porou carriers 300, with each of said carriers containing at leas one dehydrated media therein. In the most preferre embodiment, the device 10 also comprises a cover 130 with liquid receiving aperture 140 therein.

As shown in Figs 2 and 3, the container 100 has transverse wall 102 and a peripheral skirt 104 extendin upwardly therefrom, to define an internal compartment 106. Th peripheral skirt 104 terminates in an upper surface 108 t define an opening into the internal compartment 106. Th compartment 106 has partition means 112 extending upwardly fro upper surface 120 of the transverse wall 102 for dividing th compartment 106 into separate sections. In the preferre embodiment the partition means 112 are in the form of a pair o raised dividers 114 which intersect at the central axis 116 o the container to divide the compartment 106 into four separat sections 122A to 122D. However, it is within the scope of thi invention to vary the number and geometry of the dividers 114 to thereby vary the number and geometry of the separat sections within the container 100. The dividers 112 provid the important function of preventing the migration of medi between compartments, by separating the porous carriers 30 contained therein. Additionally, the device has in its mos preferred embodiment an outside wall 124 which is concentri with peripheral skirt 104, for frictional engagement with th cover 130, as is more specifically described hereinafter. Still referring to Figs. 2 and 3, a plurality of ribs 118 is provided on the upper surface 120 of transverse wall 102 within each of the sections 122A to 122D. These ribs provide open areas 123 between the porous carriers 300 and the upper surface 120 of the transverse wall 102, when the carriers 300 are disposed within the sections 122A-122D of the container 100. These open areas 123 constitute reservoirs beneath the carriers 300 for any excess' liquid which may not be retained in the carriers during use of the device 10.

In addition, the ribs 118 maintain the porous carriers 300 in their required position relative to the filter member 200. Specifically, the height of the ribs is such that when the porous carriers 300 are placed thereon, the porous carriers are sufficiently close to the overlying filter member 200, to permit the carriers to absorb liquid through the filter member via capillary action. In the most preferred embodiment, of the invention the container 100 is molded as a unitary member with the ribs 118 and raised dividers 114 therein.

As shown most clearly in Figs. 3 and. 4, a porous carrier 300 is located in each of the sections 122A to 122D, and each carrier has an upper surface 304 closely adjacent the upper surface 108 of the peripheral skirt 104. Each carrier 300 includes a dry nutrient or media therein, which is exposed to any microorganisms present in the liquid sample to be investigated, in a manner to be described in detail hereinafter. Additionally, the carriers 300 also may hold inhibitory substances, such as antibiotics, antimicrobial agents, etc., or any other substances which may affect microorganism growth. Reference to "nutrient(s) " and/or "media" throughout this application includes these inhibitory substances, in addition to substances that promote the growth of the microorganisms.

The carriers 300 in accordance with the preferred embodiment of this invention preferably are made of any suitable porous material for retaining the dehydrated nutrient therein, and are capable of holding and retaining a volume of the liquid sample to be investigated. This volume of the liquid sample functions to activate (i.e., rehydrate) th dehydrated media. Carriers made from materials such as nitro cellulose, and available from Sartorius GmbH, Gόttingen, Fed. Rep. of Germany, are preferred. However, other materials, such as foam, may be utilized to form the carriers 300.

In accordance with the method of this invention, a described in detail hereinafter, a liquid sample, such a water, urine, blood, soft drinks, etc., to be investigated, i applied to and travels transversely over filter member 200 before being drawn completely through the filter member vi capillary action, by the underlying porous carriers 300, t thereby activate the dehydrated media in the carriers. Th sample is then incubated in the device 10 for the requisit time period, and thereafter, any resulting microorganism growt or lack thereof, is analyzed.

In the preferred method of the invention the volum of the liquid sample introduced into the device 10 is matche to the liquid retaining volume of the porous carriers, suc that the liquid passing through the filter member 20 preferably will be completely absorbed within the porou carriers 300, without any excess (overflow) of the liqui sample being present in the device 10. As an alternative, th particular carriers utilized may be chosen to absorb or retai a desired liquid sample size. This permits the user to var the liquid holding capacity of the carriers for applications o use where larger or smaller liquid sample sizes ar advantageous, while still permitting the rehydrated nutrient(s to be directed to the filter member and the microorganism thereon.

For many medical applications, such as in the testin of urine, only small liquid samples are required for use in th device 10 of this invention. In fact, as will be discussed i greater detail hereinafter, applicant has obtained excellen results in the testing of urine with liquid samples of approxi mately only 3 is.

However, in the testing of beverages, and for othe non-medical applications, it may be necessary to use large liquid volumes, on the order of 100 is. When such larger volumes are utilized it may be necessary to increase the size of the individual carriers 300, so that they can retain more of the liquid sample. Moreover, as will be explained in detail hereinafter, in accordance with one aspect of this invention a vacuum assist can be employed to remove excessive quantities of liquid from the device.

As shown in Fig. 3, the filter member 200 has an upper surface 202 for initially receiving the liquid sample thereon, and a lower surface 204 which is contiguous to the upper surface 108 of the peripheral skirt 104. The filter member 200 is positioned to overly the various sections 122A- 122D of the internal compartment 106 of the container 100, with the lower surface 204 of the filter member 200 being closely adjacent to, and preferably in engagement with the upper surface 304 of each of the porous carriers 300. The filter member 200 may rest on or be retained unsecured to the device 10, or alternatively may be bonded in place, as will be described in detail below.

The filter member 200 has pores of a size for preventing microorganisms of the liquid sample from passing therethrough, while permitting the liquid of the liquid sample to pass therethrough into the underlying porous carriers 300. The type of filter member used in the device may vary depending upon the particular applications of use, and such filter members are well known to people skilled in the art. For example, in order to trap microorganisms suspected of being present in a human urine sample, it is preferable to use a cellulose nitrate membrane having a pore size of .45 μm, available from Sartorius GmbH, Gottingen, Fed. Rep. of Germany.

In the preferred embodiment of the invention the filter member 200 causes the liquid sample, when applied to the upper surface 202, to flow transversely along the filter member into overlying relationship with the various sections 122A-122D of the compartment 106, before completely passing through the filter member into the porous carriers 300. As stated earlier, the porous carriers 300 assist in pulling the liquid sample through the pores of the filter member 200 by capillary attrac¬ tion to rehydrate the media in each of the carriers. In the most preferred embodiment of the invention the upper surface 304 of each of the carriers 300 is in contact with the filter member lower surface 204, which enhances the capillary attraction for the liquid and also permits the rehydrated media to be directed to the microorganisms which may be trapped on the upper surface of the filter member 200, after the liquid sample has been directed into the device 10.

In order to fully rehydrate the nutrient(s) in the carriers 300, while preventing or minimizing the likelihood of fluid leakage, it is preferable that the volume of the liquid sample be approximately the same as, or only slightly less than the liquid holding capacity of the carriers 300. In an exemplary embodiment of the invention, in which the liquid sample is urine, approximately 3.0 illiliters (mis) of the urine is directed into the device 10 having four carriers 300, and each of said carriers has the capability of retaining, without leakage, approximately 0.75 mis.

The use of a filter member 200, in conjunction with carriers 300 which absorb the liquid of the sample to be investigated is extremely advantageous. One advantage in utilizing a filter member to trap and isolate microorganisms on its upper surface, and then exposing the filter member to a media, is that the filter member is able to collect a larger number of microorganisms (almost 100% of those contained in the liquid sample) in a small, defined surface area. Upon incubation, these microorganisms are much easier to identify as colonies, due to their higher initial concentration on the filter, as opposed to the concentration achieved when the microorganisms are spread over a wide surface area, such as when being inoculated on a conventional agar plate with a wire loop. Due to this higher initial concentration of microorganisms on the filter member 200, it takes much less time for the microorganisms to grow to detectable colonies. Another factor which enhances the growth rate of the colonies, at least in some applications, is that growth inhibitors initially present in the liquid sample also are directed through the filter member 200, to separate the inhibitors from the microbes.

In order to properly identify microorganisms grown on a conventional agar plate, the inoculated plate must usually be incubated for approximately 24 to 48 hours and more. In contrast, the current device and method permits rapid isolation and identification of colonies in as little as 4 hours. For example, using the device and method of the above invention with a test sample of approximately 3.0 milliliters of urine, when incubated with as little as 300 to 3,000 E.coli cells/ml, colonies were visible at 4 hours, countable by 8 hours and were confluent (complete lawn of bacteria) on the filter member 200 in 10 hours. A presumptive diagnosis generally can be made in 4-10 hours when the device 10 is used with a different dehydrated media in each of the sections 122A-122D. A significant advantage of this invention is that it permits a rapid diagnosis of a bacterial infection to be made, to thereby permit a physician to begin treatment to control and/or eradicate the infecting agent much more quickly than with the use of prior art culturing systems. For example, in order for a typical diagnosis to be made utilizing some of the prior art devices and methods, an inoculum typically requires at least 100,000 bacteria cells/ml and requires 24 to 48 hours for detection and analysis.

As shown in Figs. 2 and 3, in the preferred embodiment the device 10 additionally comprises a cover 130 having a top wall 132, a downwardly extending peripheral skirt 134, and a liquid receiving aperture 140 extending through the top wall for permitting the introduction of liquid sample into the device. The peripheral skirt 134 has a transversely extending intermediate section 135 which provides a downwardly facing, annular surface 136 contiguous to the upper surface 108 of the container 100. In addition the skirt 134 has a downwardly extending lower section 137 for frictional engagement with the outside wall 124 of an outer peripheral skirt of the container 100. The cover 130 preferably is made of clear plastic, and, if desired, may also additionally comprise a clear magnifying section (not shown) , which permits the user to easily view smaller colonies.

In the preferred embodiment the filter member 200 is bonded to the downwardly facing surface 136 of the cover. However, it is within the scope of this invention to merely clamp the filter member in proper position within the device, between the surfaces 136, 108. In either event the filter member 200, with the microbes cultured thereon, can be removed from the device 10 and retained as part of a patient's medical record, or otherwise stored. This is an extremely beneficial feature of the present invention.

Referring to Figs. 2 and 3, the liquid receiving aperture 140 preferably includes a removable plug 142 therein to provide a sealed, breathable environment, and the plug may be removed (see Fig. 2) to permit the sample to be introduced into the device 10. As can be seen in the drawings, the inner surface defining the aperture 140 is provided with a pair of diametrically opposed, axially extending recesses 143 therein to provide small air passages through which vapors can escape from within the device, when a lower sealing section 145 of the plug is within said aperture. This minimizes undesired condensation of vapors on the cover 130.

In an alternative embodiment the plug 142 is permanently secured to the aperture 140, and is comprised of a material, such as rubber, or latex, which permits the entry and removal of a syringe or other dispensing instrument (not shown) to deliver the liquid sample to the filter member 200 for testing, without requiring removal of the cover 130. The same arrangement of ribs 145 can be provided in this embodiment, for the same purpose of minimizing undesired condensation of vapors on the cover 130.

As shown in Figs 2 and 4, the device 10 additionally comprises one or more vents 150 which permits venting of the device, for a number of purposes. First, the vent(s) permit removal of any air present in the device when the liquid sample is introduced. Second, these vents 150 also permit the exchange of gases such as oxygen, etc. which may be necessary for the growth of aerobic organisms, while minimizing the possibility of contaminating the device. Third, these vents 150 can be used .with a vacuum assist to remove excess liquid from the device 10, when needed or desired.

As can be seen best in Figs. 2-4, in the preferred embodiment the vents 150 comprise channels or passages which extend vertically through the dividers 114 of the container 100. In the illustrated embodiment one vent 50 is provided at the central axis 116 of said container, and two other vents are equally spaced-apart along each of the four "spokes" of the dividers 114. The number and orientation of the vents 50 may be varied. However, when it is anticipated that a vacuum assist will be required or desired, for removing excess liquid from the device, it is preferred that a plurality of vents be provided along the dividers 114, as is shown in Figs. 2 and 4. This arrangement permits a substantially uniform suction effect to be created along the lower surface of the filter member 200, to thereby remove any pools of excess liquid from both above the filter member 200 and from between the filter member and carriers 300.

The carriers 300 in the sections 122A-122D may hold one or a number of different dehydrated media, depending upon the desired use of the device 10. For example, in attempting to determine the presence and identity of microorganisms in urine, four different types of media have been employed to selectively cultivate microorganisms commonly found in urine. After cultivation, the types of microorganisms present in the liquid sample were identifiable, based on the different growth characteristics exhibited on the sections of the filter member 200 overlying the various selective and/or differential media, in comparison with conventional standardized growth data.

Dehydrated media are extremely advantageous in the above application because their use permits the device and media to be completely sealed and stored for years until use, without refrigeration or any special handling. Moreover, the possibility that the media will break down or become contaminated is significantly reduced when the device 10 is properly aseptically sealed. The device 10 also is more easily transported when dehydrated media are used because the chances of spilling and/or contamination are also significantly reduced. These advantages permit users such as field testers, technicians, etc. to transport the device to the location where a sample is to be taken, if desired, and permits the sample to be brought back in the device for growth and analysis.

Various types of dehydrated media which may be used in conjunction with human urine testing include, but are not limited to, ENDO, CASO, TERGITOL TTC, TEEPOL, CHAPMAN, M-FC, SABAURAUD and CETRIMIDE available from Sartorius GmbH, Gόttingen, Fed. Rep. of Germany. Additionally, the same media, but in different concentrations may be used in the sections 122A-122D.

The media commonly known as ENDO, is used to isolate gram negative organisms such as Escherichia coli and other coliforms. These organisms generally form colonies which appear dark red with a greenish metallic sheen when grown in the presence of ENDO. ENDO is slightly selective based on the fermentation of lactose in the presence of basic fuchsin which suppresses the growth of gram positive organisms. ENDO conforms to the American Public Health Association Standards, American Public Health Association, Washington, DC (14th Ed. 1975) .

CASO is a selective media for the detection of staphylococci _f such as staphy1ococcus aureus, which yields yellow colonies when used with approximately 7.5% sodium chloride (NaCl) , by volume, as a wetting agent, or with the addition of phenylethyl alcohol as in Trip Soy Agar with sheep blood. CASO may also be used for the growth of fastidious pathogenic organisms more likely found in blood than in urine by adding approximately 10% serum, by volume. CASO also inhibits the spread of proteus species. Staphylococci may alternatively be grown and isolated in the presence of phenyl¬ ethyl agar or CNA media (Columbia Co.). TERGITOL TTC is a selective and differential media for the isolation of coliforms, inhibiting gram positive behavior and the spread of the proteus species. Escherichia coli colonies and aerobacter can reduce TTC and hence appear orange to yellow when grown in the presence of TERGITOL TTC, while coliforms appear red in color. TERGITOL TTC also resembles Simmons Citrate media conventionally used to differential coliforms. G.H. Chapman, Superior Culture Media for the Enumeration and Differentiation of Coliforms, Journal Bacteria, 53:504 (1947).

TEEPOL media is similar to TERGITOL TTC and is used for the isolation of E.coli and fecal coliforms. When grown in the presence of TEEPOL, E.coli colonies appear yellow, while colonies of non-lactose fermenting organisms appear dark red in color.

CHAPMAN media is a mannitol-sodium chloride-phenol red medium for use in detecting pathogenic staphylococci in foods and other materials. Staphylococcus aureus develops golden yellow to orange-colored colonies with a yellow zone (mannitol-positive) . Staphyloccoccus epidermidis forms whitish colonies without changing color.

M-FC media is employed for the detection of E.coli and fecal coliform bacteria. E.coli and fecal coliform forms blue colonies with diameters of 1-2 mm.

SABOURAUD media is employed for culturing yeasts, molds, acid-tolerant and acidophilic bacteria, and also for detecting yeasts and molds in beverages, such as fruit juices and for sterility testing of pharmaceuticals and for isolating dermatophathogenic yeasts and fungi. Yeasts usually develop smooth white or colored colonies. Molds generally form velvety or fluffy, cotton-like colonies in the early growth phase, and may take on various colors after conidiospore production.

CETRIMIDE is a media which permits the isolation and presumptive identification of pseudomonas aeruqinosa, whose colonies appear as a greenish color when grown in its presence. This type of media inhibits the growth of almost all other organisms. It is preferable that the device 10 be pre-packaged to include the carriers 300 with the different types of dehydrated media to be used in the investigation, to minimize potential contamination. However, it should be apparent that the carriers with dehydrated media therein may be inserted before the liquid sample is applied to the device 10, by removing the cover 130 and the filter member 200 (which may be secured to the cover) and inserting the desired dehydrated media containing carriers 300 into sections 122A-122D of the container 100.

In an exemplary embodiment of this invention a liquid sample of human urine, having an approximate volume of 3.0 mis, is directed into the container 100, on the upper surface 202 of filter member 200. As the liquid is applied to the upper surface of the filter member it travels transversely across the filter member 200, substantially uniformly in all directions, to substantially cover the upper surface of the filter member, before any significant quantity of the liquid passes through the filter member. Therefore, by first flowing transversely along the filter member 200, substantially equal volumes of urine are position over each of the carriers 300. Thereafter, the liquid is directed downwardly through the filter member 200 towards and into each of the underlying dehydrated porous carriers 300 in the sections 122A-122D. The downward movement of the liquid sample through the filter member is enhanced by capillary action provided by the carriers 300. As the liquid (e.g., urine) enters the carriers 300 it activates the various dehydrated media, and the media is thereafter directed through the filter membrane to any microorganism trapped on the upper surface thereof. Moreover, each of the carriers has a liquid holding capacity of approximately 0.75 mis., and therefore the four carriers 300 are capable of retaining virtually all of the 3.0 mis. of the liquid sample directed through the filter member 200. This substantially eliminates the presence of free liquid in the device 10, which, if present, could leak from the device 10 during handling. Thereafter, the inoculated device is incubated at 37°C for approximately 4-10 hours. Analysis of any resultant microorganism growth may then readily be undertaken by comparing the microorganism colonies grown in a specific media with conventional standardized data.

As stated earlier, when larger liquid samples are employed with the device 10, e.g., on the order of 100 mis. for the testing of beverages, it may be necessary or desirable to utilize a vacuum assist to remove excess quantities of liquid from the device. In such a case the container 100 can be placed on a suitable vacuum device (not shown) , and a suction applied through the various vents 150 extending through the dividers 114. This vacuum assist will act to direct excess liquid located above the filter and/or in the region between the carriers 300 and the lower surface of the filter 200, out of the device 10, to minimize the possibility of undesired fluid leakage from the device.

Without further elaboration the foregoing will so fully illustrate my invention that others may, by applying current or future knowledge, readily adapt the same for use under various conditions of service.

Claims

What is claimed as the invention is:

1. A device for use in culturing and identifying microorganisms in a liquid sample, said device including: a container having a transverse wall and a peripheral skirt extending upwardly therefrom to define an internal compartment, said peripheral skirt terminating in- an upper surface to define an opening into said internal compart¬ ment, and partition means in said internal compartment for dividing said compartment into at least two sections; a porous material including a dry nutrient located in each of said at least two sections; a filter member having upper and lower surfaces, said filter member being contiguous to the upper surface of the peripheral skirt and overlying said at least two sections of the internal compartment of the container with the lower surface of said filter member closely adjacent said porous materials, said filter member having pores of a size for preventing the microorganisms of said liquid sample from passing therethrough while permitting the liquid of said liquid sample to both pass through the filter members and also to flow transversely along the upper surface of said filter member into overlying relationship with said at least two sections of the compartment before passing completely through the filter member into said porous materials, said porous materials being suffi¬ ciently closely adjacent to the lower surface of the filter member for aiding in pulling the liquid of said liquid sample through the pores of the filter member by capillary action for rehydrating the nutrient and for permitting said rehydrated nutrient to be directed to said microorganisms on the filter member.

2. The device of Claim 1 additionally comprising vent means extending through said partition means.

3. The device of Claim 2 wherein said vent means includes a plurality of channels, each of said channels extending through said partition means.

4. The device of Claim 1 wherein each porous material including a dry nutrient is a porous carrier with dry nutrient therein.

5. The device of Claim 4 wherein said partition means terminates in an upper surface, said upper surface of the partition means and the upper surface of the peripheral skirt being substantially in the same plane.

6. The device of Claim 4 wherein the liquid sample is of a volume which is substantially completely absorbed by said porous carriers and said filter member, without an excess of said liquid sample being present in said device.

7. The device of Claim 4 wherein said porous carriers and said filter member have a fluid retaining capacity sufficient to retain a predetermined volume of the liquid sample without leakage therefrom.

8. The device of Claim 4 additionally comprising a cover, said cover having a top wall, a downwardly extending peripheral skirt and a liquid receiving means to permit the introduction of liquid into said device.

9 .^' The device of Claim 8 wherein said downwardly extending peripheral skirt provides a downwardly facing surface contiguous to said upper surface of the peripheral skirt of said container, said filter member being located between said downwardly facing surface and said upper surface of said peripheral skirt.

10. The device of Claim 9 wherein said filter member is bonded to said downwardly facing surface.

11. The device of Claim 9 additionally comprising vent means extending through said partition means.

12. In a method of culturing and identifying micro¬ organisms in a liquid sample comprising the steps of: positioning at least two porous materials including a dry nutrient closely adjacent a lower surface of a filter member, said filter member having pores of a size for preventing the microorganisms of said liquid sample from passing therethrough while permitting the liquid of said liquid sample to pass therethrough; directing the liquid sample onto an upper surface of the filter member and causing said sample to flow along said upper surface into regions of said filter member overlying said at least two porous materials prior to said liquid of said sample completely passing through said filter member; and directing the liquid of the liquid sample through the filter member and into each of said at least two porous materials by capillary action of said porous materials, to thereby cause said liquid to rehydrate the dry nutrient and cause the rehydrated nutrient to be exposed to microorganisms located substantially only in regions of said upper surface of the filter member overlying said each of said materials.

13. The method of Claim 12, wherein said porous materials are in the form of porous carriers with dry nutrient therein, said carriers being capable of retaining a predetermined volume of liquid therein, including the step of directing a volume of the liquid sample onto the upper surface of the filter member which is no greater than the predetermined volume of liquid which the carriers are capable of retaining.

14. The method of Claim 12, including the step of directing substantially the same volume of the liquid into each of the materials.

15. The method of Claim 12, including the step of positioning the filter member in engagement with each of the materials.

16. The method of Claim 12, including the step of physically separating said at least two materials with a barrier to prevent the nutrients of said at least two materials from intermixing with each other when said nutrients have been rehydrated.

17. In a method of culturing and identifying micro¬ organisms in a liquid sample comprising the steps of: providing a container with dividers therein to form at least two separate compartments; positioning a porous carrier with a dry nutrient therein in each of said compartments, with upper surfaces of said carriers substantially in the same plane as upper surfaces of said compartments; positioning a filter member in overlying relationship with said compartments, with a lower surface of said filter member closely adjacent an upper surface of each of said carriers; directing the liquid sample onto an upper surface of the filter member to cause the sample to flow along said upper surface into overlying relationship with each of said carriers prior to the liquid of said sample completely passing through said filter; directing the liquid of the liquid sample through the filter member into each of said carriers by capillary action, to rehydrate the dry nutrient in each of said carriers and causing said rehydrated nutrients to move through the overlying filter member into contact with the microorganisms captured on said filter member.

18. The method of Claim 17, wherein said carriers are capable of retaining a predetermined volume of liquid therein, including the step of directing a volume of the liquid sample onto the upper surface of the filter member which is no greater than the predetermined volume of liquid which the carriers are capable of retaining.

19. The method of Claim 17, including the step of directing substantially the same volume of the liquid into each of the carriers.

20. The method of Claim 17, including the step of positioning the filter member in engagement with each of the carriers.

21. The method of Claim 17, including the step of storing the filter member, after culturing and identifying microorganisms thereon, for future use and/or reference.

22. The method of Claim 17 wherein vent means extend through the dividers of the container, including the additional step of applying a vacuum through the vent means for removing access liquid from regions above the filter member and/or between the filter member and porous carriers.

23. A container employed in a device for use in culturing and identifying microorganisms in a liquid sample, said container including a transverse wall and a peripheral skirt extending upwardly therefrom to define an internal compartment, said peripheral skirt terminating in an upper surface to define an opening into said internal compartment, and partition means in said internal compartment for dividing said compartment into at least two sections, said at least two sections being adapted to receive nutrient for culturing microorganisms, and vent means extending through the partition means for venting the container.

24. The container of Claim 23, wherein said vent means includes a plurality of elongate passages extending through the partition means.