CA2082634A1 - Multi-nutrient growth culturing system and a method of use - Google Patents

Abstract

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

V~og~ 08 2081>2~

r~U~ T~IENT G~O~T~ CUTTU~ING SYSTE~ AND A ~TH0~ ~r u 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.

Backqround of the Invention The prior art is replete with disclosures o units for the collection and growth of microorganisms from 2 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. 4,82,005 (Friedman et al.), ~,i97,38'. (Gold-man), 4,777,137 (Lemmonier), 2,879,207 and 2,923,569, (Poitras), and 3,929,583 (Sharpe et al.).
In one embodiment of the Lemmonier '1~7 patenl 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 perime_e~ 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 sheel to cultivate microorganisms for subsequent exposure to different liquid media containec in a segmented Petri dish is disclosed in U.S.
Patent No. 4,775,628 (~akakura et al.). In this device the specimen (e.g., puss) containing the microorganisms to be cultivated is applied to the pervious sheet ~ith a spiral plater; no~ by being flowed onto and through a filter member to rehydrate plural dehydrated media.

WO91/1808~ 2 0 8 2 6 ~ ~ PCT/i S9]/0~1()1 ~ nits also have heen designed which :~ave seg~,en ed media sections onto which a user directly applies a te.~ samplP
for gro;:th and testing. However, these units do not emplo~ a filter membrane to collect microorganisms fo~- e~:posure Ic multiple types of dehydrated media. Represen~al_ive paten_s disclosing such units are U.S. Patent Nos. 3,102,0i32, (~re-.er`, 4,072,573 (Cady et al.), Design Patent No. 25~3,761 (C-raham), 3,912,~96 and ~,042,463 (Haque et al.), 4,076,591 (r:eden), 4,12,037 (Lemmonier), and 4,568,633 (Walton).
In addition, it is known in the art '_o utilize a dehydrated media to culture microorganisms, whic}l ~av be activated upon wetting, as lS disclosed in ~.S. Paten_ ~os.
4,485,171 (Ikeda et al.), ~,245,043 (Lund), ~,5~7,21, (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 is disclosed in ~.S. Patent ~os. 2,761,813 (Goetz), 4,~35,171 (Ikeda et al.), 2,677,646 (Lovell et al.), and 3,7~1,877 (Shaufus).
In spite of all the above teachings, however, i', is believed that a need still exists for a single unit in which microbes may be effectively collected and quickly filtered and grown without the need to handle the filter membrane upon whic~
the sample is deposited, which is not susceptible to leakage or dehydration of media during storage, handling and/or use, and which, for at least certain applications (e.g., the testing of urine) does not require the use of additional vacuum devices.

Obiects of the Invention Accordingly, it is a general object of this invention to provide a device which permits the user to quickly collect and grow microorganisms in a liquid sample while minimizing the dangers of contamination from outside sources.
It also is an object of this invention to permi. â
user to reliably subject collected microorganisms tc a number of different dehydrated media.

~'091/18~ 2~82~ PCT/~S91/0~

It is a further object of this invention ~o effec-tively collect microorganisms to be tested from a liq~lid 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 per~it 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 sur.ace 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 WO 91/1808~ PCr/~S91/03~

2~82~3~

nutrient. The filter member has pores of a size fcr preventing the microorganisms of the liquid sample fro: passing therethrough while permitting the li~uid Oc _he liquid sample to pass therethrough. The liquid sample is applied to he upper filter surface to cause said sample to flow transverselv along the upper surface of the filter member into overly~ng relationship with the at least two sections of the compartment, before passing completely through the filter member and into the porous materials. The porous materials aid in pulling ~he liquid of the liquid sample through the pores of the filte-member by capillary action for rehydrating the nutrient, to thereby permit the rehydrated nutrient to be directed th~ough the filter member to the microorganisms.
In the preferred embodiment of the invention the porous material including the dry nutrient is in the form of a porous carrier having the dry nutrient retained therein.
Accordingly, in the preferred construction the porous structure is of a defined geometric shape.
In the preferred embodiment the upper surfaces of ~he peripheral skirt and partition means are in substantially the same plane to effectively isolate the nutrients in each of the sections of the container from each other.
In the preferred embodiment the device has a cover with a liquid receiving means therein, to permit the introduc-tion of liquid into the device. The cover preferably includes a downwardly extending peripheral skirt providing a downwardly facing surface contiguous to the upper surface of the peripheral sXirt of the container. The filter member is located between these latter two surfaces, and preferably is bonded to one of them. Preferably one or more vents extend through the partition means for the venting of the device.
Quite surprisingly it has been found that although the filter member permits transverse flow o~ the liquid sample applied to its upper surface, so that the liquid substantially covers the upper surface of the filter before being absorbed into each of the porous materials including dehydrated media, the different media, which are rehydrated by the liquid, are ~'0 91/180~ PC~ 'S')l/0~
2082~ 2~

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 th oughout its entire structure.
lhus, the transverse flow of the liquid sampie along the filter media permits the microbes to be distributed ove~
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 ta~ing 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 ~uickly and efficiently culturing and identifying such microbes.

Descri~tion of the Drawinqs 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 sho-~n in Fig. l;
Fig. 3 is a sectional view taken along line 3-3 of Fig. 2; and 'VO g]/]8(!8~ PC!/I`S~l/0~

6 2~82~3~
~ ig. 4 is a sectional view taken alor.c line 4-~. of Fig. 3.

_tailed Descri~tion of the Preferred Embodiments Referring now to various figures of ~he drawings where like reference numerals refer to like parts there is shown at 10 in Fig. 1, a device constructed in accordance with this invention for use in culturing and identifying microorganisms in a liquid sample.
As shown most clearly in Fig. 2, the device comp-ises a container 100, and a filter member 200 disposed above porous carriers 300, with each of said carriers containing at least one dehydrated media therein. In the most preferred embodiment, the device 10 also comprises a cover 130 with a liquid receiving aperture 140 therein.
As shown in Figs 2 and 3, the container 100 has a transverse wall 102 and a peripheral skirt 104 extending upwardly therefrom, to define an internal compartment 106. The peripheral skirt 104 terminates in an upper surface 108 to define an opening into the internal compartment 106. The compartment 106 has partition means 112 extending upwardly from upper surface 120 of the transverse wall 102 for dividing the compartment 106 into separate sections. In the preferred embodimen~ the partition means 112 are in the form of a pair of raised dividers 114 which intersect at the central axis 116 of the container to divide the compartment 106 into four separate sections 122A to 122D. However, it is within the scope of this invention to vary the number and geometry of the dividers 114, to thereby vary the number and geometry of the separate sections within the container 100. The dividers 112 provide the important function of preventing the migration of media between compartments, by separating the porous carriers 300 contained therein. Additionally, the device has in its most preferred embodiment an outside wall 124 which is concentric with peripheral skirt 104, for frictional engagemen_ with the cover 130, as is more specifically descri~ed hereinafter.

`' ') 9 1 / 2 ~S08 ' l'CI'/ ~
7 2082~3~
Still referring to Figs. 2 and 3, a plu-a'it~ or -ibs 118 is provided on the upper surface 120 o~ transverse ~:all 102 within each of the sections 122A to 122D. These ri~s provide open areas 123 between the porous carriers 300 and the upper surface 120 of the transverse wall 102, when ~he c2~r iers 300 are disposed within the sections 122A-122D of the container 100. These open areas 123 constitute reservoirs bereath 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 fi'ter 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 there~n.
As shown most clearly in Figs. 3 and ~, a porous carrier 300 is located in each of the sections 122A to 122C, and each carrier has an upper surface 304 closely adjacen' 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 li~uid 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. ~his volume of the ~091/1808~ ~CT/~'591/0~

3 ~0~2~
liquid sample functions to activate (i.e., rehydrate) the dehydrated media. Carriers made from materials such as nitro-cellulose, and available from Sartorius GmbH, Gottingen, Fed.
Rep. of Germany, are preferred. However, other materials, such as foam, may be utilized to form the carriers 300.
In accordance wi~h the method of this invention, as described in detail hereinafter, a liquid sample, such as water, urine, blood, soft drinks, etc., to be investigated, is applied to and travels transversely over filter member 200, before being drawn completely through the filter member via capillary action, by the underlying porous carriers 300, to thereby ac~ivate the dehydrated media in the carriers. The sample is then incubated in the device lO for the requisite time period, and thereafter, any resulting microorganism growth or lac~. thereof, is analyzed.
In the preferred method of the invention the volume of the liquid sample introduced into the device lO is matched to the liquid retaining volume of the porous carriers, such that the liquid passing through the filter member 200 preferably will be completely absorbed within the porous carriers 300, without any excess (overflow) of the liquid sample being present in the device lO. As an alternative, the particular carriers utilized may be chosen to absorb or retain a desired liquid sample size. This permits the user to vary the liquid holding capacity of the carriers for applications of use where larger or smaller liquid sample sizes are advantageous, while still permitting the rehydrated nutrient(s) to be directed to the filter member and the microorganisms thereon.
For many medical applications, such as in the testing of urine, only small liquid samples are required for use in the device lO of this invention. In fact, as will be discussed in greater detail hereinafter, applicant has obtained e~cellent results in the testing of urine with liquid samples of appro~i-mately only 3 mls.
However, in the testing of beverages, and for other non-medical applications, it may be necessary to use larger ~ T'CT/lS91/0~]()~

9 ~2~
liquid volumes, on the o~der of 100 mls. ~.her. 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 detai' hereinafter, in accordance with one aspect OI 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 sec_ions 122~-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 pcrous carriers 300.
The type of filter member used in the device may vary depending upon the particular applications of use, and suc~ filter members are well known to people skilled in the art. Fo 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 ~m, 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 membe-into overlying relationship with the various sections 122A-122D
of the compartment 106, before completely passing throug~ the filter member into the porous carriers 300. As stated earlier, `~'091/1~08~ Pcr/ ~ ss l /0~1 (\ 1 20~2~
the porous carriers 300 assist in pullins the llqui sample through the pores of the filter membe- 200 by capillary a-tlrac-tion to rehydrate the media in each of the carriers. In the most preferred embodiment of the invention the upper surface 30~. of each of the carriers 300 is in contact witn the filter member lower surface 204, which enhances the capillary attraction for the liquid and also permits the rehydrated mediz to be directed to the microorgan sms which may be trapped on the upper surface of the filter member 200, afte~ 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 li~elihood 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 embodimen~ of the inven-ion, in which the liquid sample is urine, approximately 3.0 milliliters (mls) of the urine is directed into the device 10 having four carriers 300, and each of said carriers has the capability cf retaining, without lea~age, approximately 0.75 mls.
The use of a filter member 200, in conjunction "ith 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 numbe- 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 ta~es much less time for the microorganisms to grow to detectable colonies.
Anothe- factor which enhances the growth rate of the colonies, '- 9~/1808' PC~t~ l/0:~1() ' 2~2~3/~

at least in some applications, is that gro;.th inhibito~s initially present in the liquid sample also are ~irecte~
through the filter member 200, to separate the inhibitors from the microbes.
In order to properly identify microorganisms grown on a conven~ional 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 ~. hours. For example, using the device and method of the above invention with a test sample of approximately 3.0 milliliters o~ urine, when incubated with as little as 300 to 3,000 E.coli cells/ml, colonies were visible at 4 hours, countable bv 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 -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 .ith 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 10~
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 ~o s~ ns~ Pcr/~,ssl/Q~ln-~

12 2~2~
skirt of the container 100. The cover 130 preferably is made of clear plastic, and, if desired, may also additiona ly comprise a clear magnifying section (not shown), whlch 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 cove-.
However, it is within the scope of this invention to merely clamp the filter member in proper posi~ion 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 extremelv beneficial feature of the present invention.
Referring to Figs. 2 and 3, the liquid receiving aperture 140 preferably includes a removable plug 1~2 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 1~0 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 1~5 of the plug is within said aperture. This mini~,izes 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 re~uiring removal of the cover 130. The same arrangement of ribs 145 can be provided in this embodiment, fo-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 ` ~91/1~08~ r/~.~s~

13 208~ 4 is introduced. Second, these vents 150 also permit the e~change of gases such as oxygen, etc. which ma~ be necessary for the growth of aerobic organisms, while r,inimizing the possibility of contaminating the device. Third, Ihese vent, 150 can be used with a vacuum assist to remove excess liquid from the device lO, when needed or desired.
As can be seen best in Figs. 2-4, in ~he preferred embodiment the vents 150 comprise channels or passages which extend vertically through the dividers 114 of the container lO0. In the illustrated embodiment one vent 5C 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 plu-ality of vents be provided along the dividers 114, as is shown in Figs. 2 and 4.
This arrangement permits a substantially uniform suction erfect 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 lO. 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 commonlv 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 'he fil'er 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. Moreove~, the `~'091/1X0~ J~

14 2082~
possibility that ~he media ~"ill break do;;n c. becor,e contamina-ted is signlficantly reduced when the devic~ l0 ~ 5 properly aseptically sealed. The device l0 also is more easily transported when dehydrated media are used because the chances of spilling and/or contamination are also signi icantly reduced. These advantages permit use s such as fie'd testers, technicians, etc. to transport the device to ~he locatior. where a sample is to be taken, if desired, and permits the sample 'o 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, CHAPI~.A1~ FC, SABAURA7JD and CETRIMIDE available from Sartorius GmbH, Gottingen, Fed. Rep. of Germany. Additionally, the same media, but in different concentrations may be used in the sections l22A-l22D.
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 me_allic sheen when qrown 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. EN~o conforms .o the American Public Health Association Standards, American Public Health Association, h7ashington, DC (l~th Ed.
1975)-CASO is a selective media for the detection ofsta~h~lococci, such as staphYlococcus aureus, which yields yellow colonies when used with appro~imately 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 7?roteus species. _aphYlococci may alternatively be grown and isolated in the presence of phenyl-ethyl agar or CNA media (Columbia Co.).

~9l/180~
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TERGITOL TTC is a selective and differen ial 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 TE~GI~OL TI~C, 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 o' Coliforms, Journal Bacteria, 53:50~ (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 yellcw, while colonies of non-lactose fermenting organisms appear dar}; 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 vellow 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 aeruqirosa, whose colonies appear as a greenish color when grown in its presence.
This type of media inhibits the growth of almost all other organisms.

g l / ~ ~308 ' PCr/ ~
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It is preferable that the device 10 be pre-pacXaged to include the carriers 300 with the differenl~ types of dehydrated media to be used in the investigation, to Imini~i7e potential contamination. However, it should be apparen. tna~
the carriers with dehydrated media therein may be inserted before the liquid sample is applied to the device '0, by removing the cover 130 and the filter mem~er 200 (which may be secured to the cover) and inserting the desired dehydrated media cor.'aining carriers 300 into sections 122A-i22D of the container 100.
In an exemplary embodiment of this invention a liquid sample of human urine, having an approximate volume of 3.0 mls, is directed into the container 100, on the upper surface 202 or filter member 200. As the liquid is applied to the upper surface of the filter member it travels transversely ac-oss the filter member 200, substantially uniformly in all directions, to substantially cover the upper surface of the filter member, before any significan_ 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 p- -~ over each of the carriers 300. Thereafter, the liauid is Glrected downwardly through the filte~ mem~er 20G
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 li3ui d (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 appro~imately 0.75 mls., and therefore the four carriers 300 are capable of retaining virtually all of the 3.0 mls. of the liquid sample directed through the filter member 200. This substantially eliminates the presence of free liqlid in the device 10, which, if present, could leaX from the device 10 during handling.

~ 91/1808' PCT/~91/031()~

17 ~2~34 Thereafter, the inoculate~ device is incubated at 37DC for approximately 4-lO hours. Analysis of any resultan~
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 lO, e.g., on the order of lO0 mls. 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 lO0 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 lO, to minlmize 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 (24)

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.

?91/18085 PCT/US91/03104

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;

?91/18085 PCT/US91/03104 directing the liquid sample onto an upper surface of the filter member and causing said sample to flow along said upper surface into regions o 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 ?91/18085 PCT/US91/03104 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.