Culture media device and method of use

A culture media device comprised of a body member including self-supporting substrate, and coated on its upper surface with a layer of water-based adhesive composition and a layer of a cold-water-soluble powder, is provided. The culture media device can also include an optional cover sheet, covering at least a portion of the body member, and an air-permeable membrane affixed to the upper surface of the substrate to allow for the growth of aerobic microorganisms. In addition, processes of making and using the culture media device are disclosed.

FIELD OF THE INVENTION 
This invention relates to culture media devices for growing microorganisms. 
In particular, the present invention relates to culture media devices 
utilizing various agents, such as nutrient and selective agents, and a 
dry-powdered gelling agent. Application of an aqueous test sample to the 
dry-powdered gelling agent forms a reconstituted medium capable of 
containing and growing microorganisms for quantitative determination. 
BACKGROUND OF THE INVENTION 
For many years, agar-filled pour plates provided the best method of 
determining the number of microorganisms in a liquid sample, such as water 
or milk. However, the use of agar medium is particularly inconvenient and 
time-consuming. For example, agar medium must be sterilized, melted, and 
cooled prior to addition of the liquid sample. Furthermore, the sample and 
medium must be mixed, solidified, and incubated prior to counting of the 
number of microorganism colonies which grow on the plate. 
To date, the prior art has provided several devices useful for assaying 
liquid specimens for microorganisms which are easier and quicker to use 
than traditional agar pour-plate technology. For example, German patent 
application No. 2055741, published May 19, 1971, discloses a 
microbiological growth medium comprised of an inert card or strip coated 
or impregnated with a dry-gelled nutritive medium. In one embodiment, the 
inert card or strip may include optional side walls to prevent shifting of 
the medium after wetting with a liquid sample. In addition, the nutritive 
medium may include an adhesive component or be adhered to the card or 
strip by an intervening adhesive layer. Furthermore, an optional sponge 
material can be disposed between the card or strip and the nutritive 
medium, and the nutritive medium can be covered by a semi-permeable 
membrane. 
U.S. Pat. No. 4,565,783 (assigned to the Assignee of the present invention) 
provides a culture media device comprised of a dry-powdered gelling agent 
and/or nutrient composition adhered to a waterproof substrate by a layer 
of water-insoluble adhesive which is non-inhibitory to the growth of 
microorganisms. Upon application of a liquid sample to the device, the 
gelling agent(s) hydrates to form a gelatinous medium useful for growing 
microorganisms contained in the liquid sample. In addition, the device can 
also include a transparent cover sheet and/or a hydrophobic spacer element 
with side walls to maintain a pre-determined amount of a liquid sample in 
contact with the dry-powdered gelling agent(s) and/or nutrient composition 
of the culture media device. Any nutritive components and/or other agents 
are incorporated along with the gelling agent(s) into the dry-powdered 
media coating the device. Alternatively, the nutritive component and/or 
other agents can be incorporated into a substantially water-free, 
non-adhesive composition coated onto the waterproof substrate. However, 
dry-powdered gelling agents cannot be utilized to coat such an embodiment. 
Commercial embodiments of such devices include Petrifilm.TM. brand growth 
media, available from 3M, St. Paul, Minn. 
European patent application No. 0374905, published Jun. 27, 1990, also 
discloses a device for culturing microorganisms comprised of a base sheet 
composed of a lower water repellent sheet and an upper hydrophilic sheet, 
such as filter paper. A gel agent or gelatinizer is dispersed in the upper 
hydrophilic sheet and then solidified. Thereafter, a water-repellent sheet 
is applied to cover the upper surface of the hydrophilic upper sheet. 
U.S. Pat. application Ser. No. 07/354,627, allowed on Sep. 16, 1991, now 
U.S. Pat. No. 5,089,413, and assigned to the Assignee of the present 
invention, provides yet another microbiological dry culture medium device. 
The device is constructed in an analogous fashion to the culture media 
device of U.S. Pat. No. 4,565,783, described above, except that the base 
of the device is comprised of an air-permeable membrane adhered to the 
upper surface of the waterproof substrate. Utilization of the 
air-permeable membrane provides a means for growing oxygen dependent 
microorganisms, such as molds, even when an air-impermeable cover sheet is 
placed over the inoculated culture medium. 
The above-described devices have not addressed several areas that are 
important to the successful construction and use of culture media devices. 
For example, many conventional adhesives inhibit microorganism growth due 
to their strongly anionic or cationic nature, or through the deliberate 
incorporation of antimicrobial agents. Thus, culture devices that 
incorporate such an adhesive component in, or adjacent to, the nutritive 
medium may inhibit rather than facilitate the growth of microorganisms. 
Even when non-inhibitory adhesives are utilized, such as in U.S. Pat. No. 
4,565,783, the water-insoluble nature of the adhesive renders them 
essentially incapable of holding all but the smallest quantities of 
water-soluble nutrients and/or other hydrophilic agents. Thus, these 
nutrients and/or other agents must be incorporated into other 20 
water-soluble layers, such as the substantially water-free, 
cold-water-reconstitutable material of U.S. Pat. No. 4,565,783, and/or 
various other dry-powdered media. However, these forms of media typically 
may not provide for adequate control on the release rates of the nutrients 
and/or other agents. Furthermore, concentration gradients of these 
components also can occur when these dry media are hydrated. 
The lack of control of release rates and creation of concentration 
gradients is of particular concern with inhibitory agents. Rapid and high 
dosage release of inhibitory agents may in fact lead to non-selective 
inhibition of microorganism growth. In addition, creation of concentration 
gradients of such inhibitory agents may lead to growth inhibition on one 
portion of the device, but not on another. Under either scenario, the 
ability to grow and accurately quantify microorganism colony growth is 
lost. 
Finally, utilization of water-absorbing elements, such as sponges and/or 
filter paper, may not sufficiently contain colony growth, thereby limiting 
the quantitative value of such devices, and making microorganism colony 
isolation impractical. In addition, neither of these structures are 
sufficiently transparent to allow for the counting of colonies through the 
substrate, thereby also rendering the accurate counting of microorganism 
colonies nearly impossible. 
SUMMARY OF THE INVENTION 
The present invention overcomes the deficiencies of the previously 
described devices by providing a culture media device that includes a 
layer of a water-based adhesive composition coated on a self-supporting 
substrate. This adhesive composition is non-inhibitory to microorganism 
growth, can incorporate significant quantities of nutrients and/or other 
hydrophilic agents therein at substantially uniform concentrations 
throughout the layer of the adhesive composition, and releases the 
nutrients and/or other hydrophilic agents contained therein in a gradual, 
controlled manner. In addition, the use of cold-water-soluble powder, 
containing at least one gelling agent, adhered to the adhesive layer 
eliminates the need for side walls, hydrophobic spacer elements, sponges, 
or filter paper to absorb and contain an aqueous test sample. Instead, the 
cold-water-soluble powder rapidly hydrates after addition of the aqueous 
test sample into a reconstituted medium capable of growing microorganisms 
contained within the aqueous test sample. 
Specifically, the present invention provides a culture media device 
comprising: (a) a body member formed of a self-supporting, substrate with 
upper and lower surfaces; (b) a layer of water-based adhesive composition 
comprising a water-insoluble adhesive, a non-inhibitory emulsifying agent, 
and at least one hydrophilic agent selected from the group consisting of a 
hydrophilic nutrient for growing microorganisms, hydrophilic selective 
agents and combinations thereof, coated on the upper surface of the 
substrate; and (c) a layer of a cold-water-soluble powder, comprising at 
least one gelling agent, uniformly adhered to the layer of the water-based 
adhesive composition. 
Preferably, the self-supporting substrate is substantially water-proof. In 
addition, the culture media device according to the present invention can 
optionally include a cover sheet, such as a transparent film, which is 
releasably adhered to at least a portion of the body member. The inner 
surface (i.e., powder facing) of this cover sheet is preferably capable of 
being coated with a noninhibitory adhesive and a layer of 
cold-water-soluble powder comprising at least one gelling agent. This 
additional quantity of cold-water-soluble powder increases the capacity of 
the culture media device, such that larger volumes of aqueous test samples 
can be contained and hydrated into the reconstituted medium for growing 
microorganisms without the need to resort to spacer elements, side walls, 
sponges, or filter paper. 
In another embodiment, the present invention can provide a culture media 
device capable of growing aerobic microorganisms when an air-impermeable 
cover sheet is used to cover the reconstituted medium hydrated by an 
aqueous test sample. Specifically, the body member comprises an 
air-permeable membrane affixed to the upper surface of the substrate. A 
layer of the water-based adhesive composition is coated on the top surface 
of the membrane, and a layer of cold-water-soluble powder is adhered to 
the layer of the adhesive composition. Utilization of the air-permeable 
membrane in this culture media device allows a constant source of air to 
reach the microorganisms growing in the reconstituted medium during 
incubation of the inoculated culture media device. 
In yet another embodiment, the present invention can provide a method of 
making a culture media device comprising the steps of: (a) providing a 
body member in the form of a self-supporting substrate having upper and 
lower surfaces, and a water-based adhesive composition comprising a 
water-insoluble adhesive, a non-inhibitory emulsifying agent, and at least 
one hydrophilic agent selected from the group consisting of hydrophilic 
nutrients for growing microorganisms, and hydrophilic selective agents; 
(b) coating the water-based adhesive composition on the upper surface of 
the substrate; and (c) affixing a uniform layer of cold-water-soluble 
powder, comprising at least one gelling agent to the layer of the 
water-based adhesive composition. 
In yet a further embodiment, the present invention can provide a method of 
using a culture media device comprising the steps of: (a) providing a 
culture media device comprising a body member including a self-supporting 
substrate with upper and lower surfaces, and a layer of water-based 
adhesive composition coated on the upper surface of the substrate, wherein 
the water-based adhesive composition comprises a water-insoluble adhesive, 
a non-inhibitory emulsifying agent, and at least one hydrophilic agent 
selected from the group consisting of a nutrient for growing 
microorganisms, a selective agent, and combinations thereof, and wherein a 
uniform layer of cold-water-soluble powder comprising at least one gelling 
agent is adhered to the layer of the water-based adhesive composition; (b) 
inoculating the culture media device with a predetermined volume of an 
aqueous test sample to form a reconstituted medium; (c) incubating the 
culture media device for a predetermined period of time; and (d) counting 
the number of microorganism colonies growing on the reconstituted medium. 
These and various other advantages and features of novelty which 
characterize the invention are pointed out with particularity in the 
claims annexed hereto and forming a part hereof. However, for a better 
understanding of the invention, its advantages, and objects obtained by 
its use, reference should be had to the Drawing which forms a further part 
hereof, and to the accompanying descriptive matter, in which there is 
illustrated and described preferred embodiments of the invention. 
DEFINITIONS 
For the purposes of this invention, 
"aqueous test sample" refers to an aqueous medium, including food samples 
that are homogenized, diluted, or suspended in the aqueous medium, that 
can contain various microorganisms therein; 
"powder" refers to a particulate material (e.g., of one or more gelling 
agents) wherein the particles have an average diameter suitable for use in 
the culture media device(s) of the present invention, preferably a 
diameter of from about 400 .mu. to about 10 .mu., more preferably a 
diameter of from about 90 .mu. to about 30 .mu.; 
"cold-water-soluble powder" refers to a powder that forms a gel in room 
temperature water (e.g., from about 18.degree. C. to about 24.degree. C.) 
when combined with an aqueous test sample; 
"non-inhibitory emulsifying agent" refers to an emulsifying agent, 
preferably a nonionic emulsifying agent, that is suitable to disperse a 
water-insoluble adhesive in an aqueous medium, and which does not 
substantially inhibit the growth of the microorganisms intended to be 
grown; 
"reconstituted medium" refers to a solution or gel formed from the 
reconstitution of a cold-water-soluble powder with water or an aqueous 
test sample; 
"air-permeable" refers to a membrane that, when substantially exposed at 
its edges to air, is sufficiently permeable to air in the horizontal 
direction (i.e., parallel to its top and bottom surfaces) to provide an 
adequate supply of air to an overlying reconstituted medium in order to 
support the growth of aerobic microorganisms in the reconstituted medium; 
"water-insoluble adhesive" refers to a hydrophobic adhesive that is 
substantially insoluble in an aqueous medium, and which is preferably 
formed by aqueous emulsion polymerization techniques; 
"water-based adhesive composition" refers to an adhesive composition of a 
water-insoluble adhesive that is dispersed in an aqueous medium by a 
non-inhibitory emulsifying agent prior to coating onto a substrate; 
"substantially impermeable to microorganisms and water vapor" refers to a 
cover sheet that prevents undesired contamination and hydration of the 
underlying layers of the water-based adhesive composition and 
cold-water-soluble powder during shipping, storage, and use of the culture 
media device(s), and that avoids desiccation of the reconstituted medium, 
such that the reconstituted medium is suitable to support the growth of 
microorganisms during an incubation period; and 
"selective agent" refers to any element, compound, or composition that 
functions to inhibit the growth, and/or facilitate the identification, of 
microorganisms grown on the culture media device(s) according to the 
present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION 
Culture Media Devices 
A first embodiment will be described with reference to FIG. 1, which 
illustrates a culture media device 10 in accordance with the present 
invention. Culture media device 10 includes body member 11 comprising 
self-supporting substrate 12 having upper and lower surfaces 14 and 16, 
respectively. Substrate 12 is coated on its upper surface 14 with a layer 
of water-based adhesive composition 18. Cold-water-soluble powder, 
comprising one or more gelling agents, is adhered in a thin, relatively 
uniform layer 20 to the layer of water-based adhesive composition 18. Once 
inoculated with an aqueous test sample (not shown), the layer of 
cold-water-soluble powder 20 quickly hydrates to form a reconstituted 
medium (not shown), which in turn is capable of containing and growing 
microorganisms present in the aqueous test sample. In addition, culture 
media device 10 can optionally include cover sheet 22, to cover and 
further contain the reconstituted medium after inoculation of culture 
media device 10 with the aqueous test sample. 
In an alternative embodiment illustrated in FIG. 3, culture media device 30 
includes a body member 32 comprising self-supporting substrate 34, having 
upper and lower surfaces 36 and 38, respectively. Air-permeable membrane 
40, including top and bottom surfaces 41 and 42, respectively, is affixed 
to upper surface 36 of substrate 34 by adhesive layer 43. A layer of 
water-based adhesive composition 44 is shown coated on top surface 41 of 
air-permeable membrane 40. In a like manner to culture media device 10 
illustrated in FIG. 1, cold-water-soluble powder, comprising one or more 
gelling agents, is shown adhered in a thin, relatively uniform layer 48 to 
the layer of water-based adhesive composition 44. Furthermore, culture 
media device 30 can optionally include cover sheet 50. 
When using either of culture media devices 10 or 30 illustrated in FIGS. 1 
or 3, an accurate count of the colonies of microorganisms present is often 
desirable. As illustrated in FIG. 2, the counting of colonies of 
microorganisms, such as bacteria colonies, can be facilitated by 
imprinting square grid pattern 60, either on substrate 12 or 34, or on 
air-permeable membrane 40 of culture media device(s) 10 or 30. In 
addition, it will also be appreciated that square grid pattern 60 could be 
imprinted on cover sheets 22 and 50 to aid in the counting of 
microorganism colonies. 
Body Member 
In both of culture media devices 10 and 30 illustrated, respectively, in 
FIGS. 1 and 3, body members 11 and 32 include self-supporting substrates 
12 and 34. Substrates 12 and 34 preferably comprise a relatively stiff 
film of a polymeric material, including without limitation, polyolefins 
such as polypropylene and polyethylene, polyesters, polystyrenes, or 
mixtures thereof. Preferably, the self-supporting substrates 12 and 34 are 
substantially water-proof, such that they will not substantially absorb or 
otherwise be affected by water. Polyester films approximately 100.mu. to 
180.mu. thick, polypropylene films approximately 100 .mu. to 200.mu. 
thick, and polystyrene films approximately 300.mu. to 380.mu. thick have 
been found to work well with the present invention. Other suitable 
substrates include paper with a polyethylene or other substantially 
water-proof coating, such as "Schoeller Type MIL" photoprint paper 
(Schoeller, Inc., of Pulaski, N.Y.). In addition, substrate 12 and 34 can 
be transparent, translucent, or opaque, depending on whether one wishes to 
view and count microorganism colonies through substrate 12 and 34. 
In addition to self-supporting substrate 34, body member 32 of culture 
media device 30, illustrated in FIG. 3, includes air-permeable membrane 40 
affixed to upper surface 36 of substrate 34. In addition to facilitating 
the growth of aerobic organisms, air-permeable membrane 40 is also useful 
in instances where the microorganisms require air for reasons in addition 
to, or other than for growth, for example, to oxidize a dye that renders 
the microorganism colonies more easily visible, as discussed more fully 
below. 
Horizontal passage of air for a particular membrane is most conveniently 
estimated by evaluating the vertical air permeability of the membrane 
(i.e., permeability in a direction normal to top and bottom surfaces 41 
and 42 of membrane 40). Vertical air permeability can be determined by any 
suitable means. For purposes of the instant specification and claims, 
vertical air permeability is determined by ASTM-D-726-58, Method A, using 
a Gurley.TM. densitometer to measure the time in seconds needed to pass 50 
ml of air through air-permeable membrane 40 (i.e., generally air-permeable 
membrane 40 itself, absent any layer of water-based adhesive composition 
44, cold-water-soluble powder 48, substrate 32, etc.). This permeability 
is referred to herein as "Gurley Porosity". In this regard, it is 
preferred that air-permeable membrane 40 have a Gurley Porosity value of 
less than about 100 seconds, more preferably less than about 50 seconds, 
and most preferably less than about 25 seconds. 
Those skilled in the art will recognize that the optimum thickness of 
air-permeable membrane 40 will depend in part upon the air and water 
permeability of membrane 40. In general, a uniform thickness between about 
10.mu. and about 500.mu. is suitable, a uniform thickness between about 
20.mu. and about 100.mu. is preferred, and a uniform thickness between 
about 40.mu. and about 80.mu. is particularly preferred. 
Suitable materials useful for air-permeable membrane 40 include, but are 
not limited to, microporous films and microporous non-woven webs of 
synthetic or natural materials. Such materials are readily available, and 
methods of preparing them are well known to those skilled in the art. 
Preferred materials for use in a device of the invention include 
microporous membranes such as those prepared according to Example 23 of 
U.S. Pat. No. 4,539,256, the disclosure of which is incorporated herein by 
reference. These preferred materials can be made of any polymer suitable 
for use in the method of preparation described in the '256 patent. 
Particularly preferred are air-permeable membranes 40 made of 
polypropylene, polyethylene, polyethylene terephthalate, polybutylene 
terephthalate, nylon, polyvinylidine fluoride, or copolymers or blends 
thereof. Examples of preferred air-permeable membranes include Exxaire.TM. 
breathable polyolefin film (50 thick; Gurley Porosity about 50 seconds; 
product number 10-B04; Exxon Chemical Co., Polymers Group); Exxaire.TM. 
breathable polyolefin film (50.mu. thick; Gurley Porosity about 100 
seconds; product number 7-B03; Exxon Chemical Co., Polymers Group); 
microporous polyethylene film (20 thick; Gurley Porosity about 25 
seconds); and 3M Micropore.TM. tape, which has a non-woven rayon web as 
backing, and as an adhesive tape, is 125.mu. thick and has a Gurley 
Porosity about 0.1 seconds (product number 1530; 3M Company, St. Paul, 
Minn.). 
Water-Based Adhesive Composition 
Preferably, the layer of water-based adhesive composition 18 and 44 is 
sufficiently transparent when wetted by an aqueous test sample to enable 
the viewing of the colonies of microorganisms through body member 11 and 
32 and/or cover sheet 22 and 50 of culture media devices 10 and 30. 
Water-based adhesive composition layers 18 and 44 which turn milky upon 
exposure to water are less preferred, but may be used in conjunction with 
a non-transparent substrate 12 and 34 and/or air-permeable membrane 40, or 
where colony visualization is not required. 
It is preferred that the water-insoluble adhesive of the water-based 
adhesive composition be a pressure-sensitive adhesive. More preferably, 
the water-insoluble adhesive is a pressure-sensitive adhesive comprising a 
copolymer of an alkyl acrylate monomer and an alkyl amide monomer. 
Preferably the weight ratio of alkyl acrylate monomer to alkyl amide 
monomer in these copolymers is from about 90:10 to 99:1, more preferably 
95:5 to 98:2. 
In a preferred embodiment, the alkyl acrylate monomer comprises a lower 
alkyl (C.sub.2 to C.sub.10) monomer of acrylic acid, including, without 
limitation, isooctyl acrylate (IOA), 2-ethylhexyl acrylate, butyl 
acrylate, ethyl acrylate, isoamyl acrylate, and mixtures thereof, while 
the alkyl amide monomer can comprise, without limitation, acrylamide 
(ACM), methacrylamide, N-vinylpyrrolidone (NVP), N-vinylcaprolactam 
(NVCL), N-vinyl-2-piperidine, N-(mono- or di-lower alkyl (C.sub.2 to 
C.sub.5))(meth)acrylamides, N-methyl (meth)acrylamide, 
N,N-dimethyl(meth)acrylamides, or mixtures thereof. Particularly preferred 
water-insoluble adhesive copolymers in accordance with the present 
invention include a copolymer of IOA and ACM, or a copolymer of IOA and 
NVP, both formed in a weight ratio of about 98:2. 
The water-insoluble adhesive component of the water-based adhesive 
composition is preferably formed by aqueous emulsion polymerization. In 
preparing the water-insoluble adhesive via emulsion polymerization, the 
above-described alkyl acrylate and alkyl amide monomers and a 
polymerization initiator are combined according to the preferred weight 
ratios in an aqueous medium that includes a noninhibitory emulsifier. (See 
e.g., pending patent application, U.S. Ser. No. 07/804,296, filed Dec. 9, 
1991, M. Crandall et al., assigned to the Assignee of the present 
invention, the disclosure of which is herein incorporated by reference.) 
A typical process for producing the emulsified water-based adhesive 
composition according to the present invention involves first preparing an 
aqueous solution of a nonionic emulsifier and water. A previously-prepared 
mixture of the alkyl acrylate and alkyl amide monomers in the desired 
weight ratios, and a nonionic oleophilic polymerization initiator, is then 
mixed and dispersed in the aqueous solution via the nonionic emulsifier. 
The mixing is carried out under homogenization conditions for about one 
minute in order to prepare an oil-in-water emulsion. 
Preferably, the monomers comprise from about 20 to 60 percent by weight, 
and more preferably about 30 to about 50 percent by weight, of the total 
weight of the monomers, emulsifier, polymerization initiator, and water 
combined. In addition, the reaction mixture can optionally contain other 
additives, including neutral nonionic cross-linking agents, such as 
4-acryloyloxy benzophenone or 1,6-hexanediol diacrylate (HDDA), at a level 
of from about 0.01% to about 0.5%, preferably about 0.02% to about 0.1%, 
and most preferably about 0.03% to about 0.08% by weight based on the 
total weight of the monomers present. 
The resulting oil-in-water emulsion is heated to induction temperature and 
stirred under nitrogen until polymerization occurs, as signaled by a 
reaction exotherm. Stirring is continued, at an elevated temperature 
(e.g., from about 50.degree. C. to about 90.degree. C.), for about two 
hours, after which the reaction vessel is cooled to room temperature and 
the polymeric product is recovered by filtration. If the resulting 
composition is to be coated directly, any additives such as nutrients and 
hydrophilic selective agents, are added with stirring. Water is added or 
removed to reach an appropriate coating viscosity, and the mixture is 
coated onto an appropriate substrate. Typically, the adhesive particle 
diameter ranges from about 0.1.mu. to about 0.9.mu., and the filtered 
reaction mixture has a Brookfield viscosity of about 5 to about 15 cps. In 
addition, appropriate adjustments to the pH of the adhesive composition 
are made, as needed, to insure that the water-based adhesive composition 
is non-inhibitory to the growth of microorganisms. Typically, the pH of 
the water-based adhesive composition should be maintained at a pH of about 
5 to about 9, more preferably at a pH of about 6 to about 8. 
The non-inhibitory emulsifying agent utilized in the formation of the 
water-insoluble adhesive, and resulting water-based adhesive composition, 
is preferably a nonionic emulsifying agent. Typical nonionic emulsifying 
agents capable of being used in the present invention are formed by the 
reaction of ethylene oxide with active hydrogen compounds such as phenols, 
alcohols, carboxylic acids, amines, and amides. Furthermore, these 
nonionic emulsifying agents also typically exhibit a 
hydrophilic-lipophilic balance (HLB) of from about 10 to about 20, 
preferably from about 12 to about 18. 
Suitable nonionic emulsifiers according to the present invention include, 
without limitation, polyethers, e.g., ethylene oxide and propylene oxide 
condensates in general, which include straight- and branched C.sub.2 and 
C.sub.18 alkyl, alkylaryl and alkenyl alcohol based copolymers of ethylene 
oxide and propylene oxide such as the Tergitol.TM. X series of emulsifiers 
(Union Carbide Co.), block copolymers of ethylene oxide and propylene 
oxide such as Pluronic.TM. and Tetronic.TM. emulsifiers (BASF Co.), and 
Tweens.TM. and Spans.TM. nonionic emulsifiers (ICI, Inc.), which denote 
polyoxyalkylene derivatives of sorbitan and fatty acid esters. Specific 
examples of nonionic emulsifiers include, but are not limited to, 
ethoxylated fatty alcohols, ethoxylated alkylphenols, ethoxylated fatty 
acids, ethoxylated fatty acids, sorbitan derivatives, sucrose esters and 
derivatives, ethylene oxide-propylene oxide block copolymers, fluorinated 
alkyl polyoxyethylene ethanols, and mixtures thereof. 
A preferred nonionic emulsifying agent according to the present invention 
is an octyl phenoxy poly(ethylene oxide) ethanol (e.g., IGE.TM. CA-897; 
Rhone Poulenc of Princeton, N.J.). Preferably, the nonionic emulsifier is 
used at a level of about 2% to about 10%, more preferably about 3% to 
about 5%, and most preferably about 4% by weight, based on the total 
weight of the monomers, emulsifier and polymerization initiator combined. 
Preferably, the polymerization initiator used in the formation of the 
water-based adhesive composition comprises a nonionic oil-soluble 
initiator. Non-limiting examples of suitable polymerization initiators 
include peroxides such as benzoyl peroxide or lauroyl peroxide, as well as 
azo initiators, such as 2-(carbamoylazo)- isobutyronitrile (e.g., "V-30 
initiator"; Wako Chemicals, Dallas, Tex.) or azobisisobutyronitrile ("AIBN 
initiator"; DuPont Co., Wilmington, Del.). Particularly preferred among 
these is lauroyl peroxide, used at level of about 0.02% to about 0.3%, 
more preferably about 0.05% to about 0.25%, and most preferably about 
0.07% to about 0.2% by weight, based on the total weight of the monomers. 
As noted above, the water-based adhesive composition incorporates one or 
more hydrophilic agents, including nutrients, selective agents, or 
combinations thereof. The specific nutrients and/or selective agents used 
in the water-based adhesive composition will be apparent to those skilled 
in the art in view of the present specification depending upon the 
particular organisms to be grown and/or to be selectively dyed or 
inhibited. After incorporation of the hydrophilic agents, and prior to 
coating, the pH of the water-based adhesive composition is normalized to 
about pH 6.5 to about pH 7.5, preferably about pH 7, to help ensure that 
the water-based adhesive composition does not inhibit the growth of 
desired microorganisms. 
Non-limiting examples of suitable nutrients include meat peptone, casein 
peptone, beef extract, lactose, glucose, galactose, as well as fats, 
minerals and vitamins. Specific examples of nutrient formulations suitable 
for use in the present invention include, without limitation, Violet Red 
Bile, Standard Methods, and Baird-Parker nutrient formulations (Acumedic, 
Inc., Baltimore, Md.) (See e.g., Tables 3 and 4 herein). 
The hydrophilic selective agents that can be incorporated into the 
water-based adhesive composition provide a means for selectively 
inhibiting or identifying microorganisms transferred to culture media 
devices 10 and 30 from the aqueous test sample. Suitable selective agents 
can include antibiotics, such as colistin methane sulfonate or nalidixic 
acid, for inhibition of unwanted organisms. Other suitable inhibitory 
selective agents include inhibitory salts, such as bile salts which, for 
example, can be used to selectively grow gram-negative microorganisms 
(i.e., inhibit the growth of gram-positive microorganisms). 
Another useful class of hydrophilic selective agents include dyes that are 
metabolized by, or otherwise react with, growing microorganisms, and in so 
doing cause the microbial colonies to be colored or fluoresce for ease of 
visualization and quantification. Non-limiting examples of such dyes 
include triphenyl tetrazolium chloride, p-tolyl tetrazolium red, 
tetrazolium violet, veratryl tetrazolium blue, crystal violet, neutral 
red, and 5-bromo-4-chloro-3-indolyl phosphate disodium salt. Particularly 
preferred dyes in accordance with the present invention include crystal 
violet, neutral red and 5-bromo-4-chloro-3-indolyl phosphate disodium 
salt. However, it will be appreciated that other suitable dyes can be used 
depending on the particular organism(s) to be identified. 
After formation, the water-based adhesive composition is coated 
(preferably, knife-coated) onto body member 11 and 32 at a thickness that 
is preferably less than the diameter of the particles of the 
cold-water-soluble powder to be adhered to adhesive layer 18 and 44. When 
coating the water-based adhesive composition, the object is to apply 
enough adhesive composition to facilitate adherence of the 
cold-water-soluble powder to upper surface 14 of substrate 12, or upper 
surface 41 of air-permeable membrane 40, but not so much that the 
particles comprising the cold-water-soluble powder become completely 
embedded in the layer of water-based adhesive composition 18 and 44. 
Generally, a water-based adhesive composition level of from about 0.20 to 
about 0.001 g/cm.sup.2, more preferably from about 0.12 to about 0.006 
g/cm.sup.2, and most preferably from about 0.08 to about 0.008 g/cm.sup.2 
is suitable. The layer of water-based adhesive composition 18 and 44 is 
then preferably dried to remove excess remaining water before coating with 
the layer of cold-water-soluble powder 20 and 48. 
Cold-Water-Soluble Powder 
Suitable gelling agents for inclusion in the cold-water-soluble powder 
include both natural and synthetic gelling agents that form solutions in 
water at room temperature. Standard gelling agents, such as hydroxyethyl 
cellulose, carboxymethyl cellulose, polyacrylamide, locust bean gum, guar 
gum, and algin, as well as super-absorbent materials, including glycol 
modified polysaccharides, such as Ucargel.TM. super absorbent agents 
(Union Carbide, Boundbrook, N.J.), and starch-graft-poly(sodium 
acrylate-co-acrylamides), such as Water Lock.TM. super absorbent agents 
(Grain processing Corp., Muscatine, Iowa), form solutions in water at room 
temperature, and are suitable gelling agents for providing powders which 
are "cold-water-soluble." 
Preferably, the cold-water-soluble powder is comprised of a mixture of 
super-absorbent materials exhibiting water absorbency of from about 50 
ml/g to about 200 ml/g, more preferably 100 ml/g to about 180 ml/g, and 
standard gelling agents with water absorbency of from about 1 ml/g to 
about 20 ml/g, more preferably about 5 ml/g to about 10 ml/g. Use of a 
mixture of super-absorbent materials and standard gelling agents in the 
cold-water-soluble powder of the present invention provides a powder 
coating that can rapidly hydrate to contain a relatively large sample 
volume (e.g., about 5 ml) on a substrate surface area of a size which is 
easily handled and stored (e.g., about 75 cm.sup.2), while using a 
relatively small amount of cold-water-soluble power (e.g., only a single 
layer of powder). In this regard, the cold-water-soluble powder of the 
present invention preferably comprises Ucargel.TM. powder and/or Water 
Lock.TM. A-100 powder in combination with standard gelling agents, such as 
locust bean gum and/or xanthum gum. In a particularly preferred 
embodiment, Ucargel.TM. powder, Water Lock.TM. A-100 powder, locust bean 
gum, and xanthum gum are combined in a 1:1:1:1 weight ratio to provide the 
cold-water-soluble powder of the present invention. 
The gelling agent is included in the cold-water-soluble powder in a 
sufficient amount so that a predetermined quantity of an aqueous test 
sample can be applied and maintained on body member 11 and 32 without 
having any of the aqueous test sample run off the edge of body member 11 
and 32. Preferably, sufficient gelling agent is provided so that from 
about 1 ml to about 5 ml of an aqueous test sample, placed on 
powder-coated body member 11 and 32, will form a gelatinous reconstituted 
medium. In this regard, it is particularly preferred that the combination 
of the cold-water-soluble powder and aqueous test sample form from about a 
5% to about a 15% solution, more preferably from about a 7% to about a 12% 
solution of the mixture. Gels such as these will allow convenient handling 
and stacking, and provide distinct colony identification. In most cases 
2.5 mg to 5 mg of cold-water-soluble powder on a surface area of 1 
cm.sup.2 will provide a sufficiently viscous gel when hydrated with 1 ml 
to 5 ml of an aqueous test sample. No mixing is required, and there is no 
need for a user to heat the medium or otherwise treat it to obtain the 
gelled reconstituted medium. 
Furthermore, the size of the cold-water-soluble powder particles can be 
used to control the coating weight per unit area. For example, 
approximately 100 mesh powder coats to a weight of about 50 mg/5 cm 
diameter disc and a 400 mesh powder coats to a weight of about 25 
mg/.sup.5 cm diameter disc. If additional amounts of gelling agent are 
required, optional cover sheet 22 and 50 of culture media devices 10 and 
30 can also be coated with cold-water-soluble powder. 
In some embodiments, it will also be desirable to incorporate nutrients 
into the cold-water-soluble powder, along with the gelling agent(s). 
Inclusion of the nutrients is particularly useful to help facilitate the 
initial growth of microorganisms transferred to culture media device 10 
and 30 through the aqueous test sample. Further, a dye or other reagent 
can also be included in the cold-water-soluble powder to further enhance 
the visualization of microorganism colonies. 
Cover Sheet 
In a preferred embodiment, cover sheet 22 and 50 is affixed to one edge of 
body member 11 and 32. Cover sheet 22 and 50 is preferably transparent to 
facilitate counting of the microorganism colonies, and is substantially 
impermeable to bacteria and water vapor. Generally, cover sheet 22 and 50 
will have the same properties, such as transparency and preferred water 
impermeability, as substrate 12 and 34, but need not be as stiff. 
Furthermore, cover sheet 22 and 50 can have patterns imprinted thereon, 
such as square grid pattern 60, or a mask-edge (not shown) to aid in the 
counting of microorganisms in colonies, to provide a target for placement 
of the aqueous test sample, and/or for aesthetic reasons. 
Cover sheet 22 and 50 can be selected to provide the amount of oxygen 
transmission necessary for the type of microorganism desired to be grown. 
For example, polyester films have a low oxygen permeability (less than 
0.78 g/100 cm.sup.2 /24 hours per 25.mu. of thickness), and would be 
suitable for growing anaerobic bacteria, or aerobic bacteria when utilized 
with air-permeable membrane 40 as a component of body member 32 of culture 
media device 30. On the other hand, some forms of polyethylene have a 
relatively high oxygen permeability (approximately 78 g/100 cm.sup.2 /24 
hours per 25.mu. of thickness), and would be suitable for the growth of 
aerobic organisms, with or without the use of an air-permeable membrane 
40. The presently preferred material for cover sheet 22 and 50 is a 1.6 
mil biaxially-oriented polypropylene film. In addition, cover sheet 22 and 
50, can also be coated with optional layers of noninhibitory adhesive and 
cold-water-soluble powder (not shown). It is understood that cover sheet 
22 and 50 can alternatively be affixed to body member 11 and 32, and that 
it can be free of any coating, or may be coated only with a layer of 
noninhibitory, pressure-sensitive adhesive. 
Although both of the embodiments illustrated in FIGS. 1 and 3 have cover 
sheet 22 and 50 attached to culture media device 10 and 30, it is also 
contemplated within the scope of the invention that culture media devices 
10 and 30 can be uncovered, and simply placed in a sterile environment 
during storage and incubation. 
The noninhibitory adhesive optionally used on cover sheet 22 and 50 can 
comprise the preferred water-based adhesive composition of the present 
invention, or any other suitable, noninhibitory adhesive, including the 
adhesives disclosed in U.S. Pat. No. 4,565,783, the disclosure of which is 
herein incorporated by reference. Suitable gelling agents for inclusion in 
the cold-water-soluble powder coating of cover sheet 22 and 50 (if such 
are contained in the coating) include the above-described gelling agents, 
which form a gelatinous reconstituted medium in water at room 
temperatures. 
Advantages of the Invention 
Culture media devices 10 and 30 according to the present invention provide 
several advantages over previously known culture media devices. For 
example, the water-based adhesive composition of the present invention 
comprises an aqueous emulsion that allows for the incorporation of 
significantly greater amounts of hydrophilic agents into the layer of 
water-based adhesive composition 18 and 44 of culture media devices 10 and 
30 than was possible with previously-used solvent-based adhesive 
compositions. In particular, the hydrophilic agents can comprise from 
about 2:3, to about 1:10, more preferably from about 1:2 to about 1:4, and 
most preferably about 1:3 parts by weight of a hydrophilic agent to total 
parts of the water-based adhesive composition. In contrast, typical 
solvent-based adhesive compositions used in known culture media plates can 
incorporate no more than a weight ratio of about one part-by-weight 
hydrophilic agent to 8000 parts of the solvent-based adhesive composition. 
For example, in a forty percent (40%) solution of the water-based adhesive 
composition of the present invention, up to about fifty percent (50%), 
more preferably from about twenty percent (20%) to about forty percent 
(40%) by weight, based on the weight of the water-insoluble adhesive, 
non-inhibitory emulsifying agent, and hydrophilic agent(s) combined, can 
comprise a standard nutrient composition, such as a Baird-Parker or Violet 
Red Bile nutrient formulation, whereas virtually none of the same nutrient 
composition can be incorporated into a solvent-based adhesive composition, 
such as disclosed in U.S. Pat. No. 4,565,783. 
In addition to having increased solubility, the incorporated hydrophilic 
agents are dispersed in a relatively uniform manner throughout the layer 
of water-based adhesive composition 18 and 44. Accordingly, these agents 
will diffuse from this layer at a relatively even rate and at a relatively 
constant gradient across the surface of the layer of water-based adhesive 
composition 18 and 44. The control of release rates that is provided by 
the even dispersion of hydrophilic agents throughout the layer of 
water-based adhesive composition 18 and 44 can be particularly critical 
when using selective inhibitory agents. By applying a uniform rate of 
inhibition throughout the reconstituted medium, a more accurate 
quantitative measure of the microbial colonies growing in the medium can 
be obtained. In addition, the slower release of inhibitory agents will 
help prevent the nonselective toxic effects of a large dosage of 
inhibitory agent on the microorganisms transferred to the reconstituted 
medium of the device via the aqueous test sample. 
The ability to incorporate substantial quantities of hydrophilic agents 
into the layer of water-based adhesive composition 18 and 44 also provides 
advantages in the construction and usage of culture media devices 10 and 
30 according to the present invention. For example, many desirable 
selective agents typically could not be used with previous culture media 
devices. In particular, heavy-metal salts and antibiotic selective agents 
should not be incorporated into culture media devices or otherwise handled 
without the use of appropriate safety equipment, such as respirators, 
gloves, or other protective equipment. This is especially true when such 
agents comprise a component of powders or other dry particulates used in a 
culture media device. However, such concerns are substantially eliminated 
or reduced by incorporating these potentially hazardous selective agents 
into the layer of water-based adhesive composition 18 and 44 of culture 
media devices 10 and 30 according to the present invention. In particular, 
incorporating such agents into the layer of water-based adhesive 
composition 18 and 44 should substantially prevent these agents from 
dispersing in the air, thereby preventing a contamination risk to the 
users of culture media devices 10 and 30. 
Furthermore, incorporation of the hydrophilic agents into the layer of 
water-based adhesive composition 18 and 44 allows additional quantities of 
cold-water-soluble powder to be applied as relatively uniform layer 20 and 
48 to the layer of water-based adhesive composition 18 and 44. In 
particular, use of the preferred mixture of super absorbent materials and 
standard gelling agents to form the cold-water-soluble powder provides a 
powder which hydrates rapidly, with a high absorbency potential, to form 
the reconstituted medium. This in turn provides culture media devices 10 
and 30 which can accept larger volumes of aqueous test sample to form the 
reconstituted medium without the need to resort to spacer elements, side 
walls, sponges, filter paper, and the like. 
Usefulness of the Invention 
Use of culture media devices 10 and 30 of the present invention will be 
discussed with specific reference to the device of FIG. 1, although the 
same method of use would apply equally well to the device of FIG. 3. To 
use the device of FIG. 1 as a substitute for a standard, liquid 
media-filled pour plate, cover sheet 22 is pulled back and a predetermined 
quantity (e.g., 1 ml to 5 ml) of an aqueous test sample is placed on the 
layer of cold-water-soluble powder 20 coated on body member 11. The 
gelling agent quickly hydrates to form a reconstituted medium capable of 
supporting microorganism growth. Cover sheet 22 is then replaced over body 
member 11, and a weighted plate (not shown) is placed on top to completely 
spread the aqueous test sample and reconstituted medium. The device is 
then incubated for a predetermined period of time. Any colonies of 
microorganisms which grow in the medium can then be counted through 
transparent cover sheet 22, and/or body member 11. 
Device 10 can also be conveniently used to test the surfaces of various 
objects to determine the extent of microbial contamination (i.e., "Rodac 
testing"). Specifically, cover sheet 22, coated only with a 
pressure-sensitive adhesive, is pulled back and touched to the surface 
being tested, thereby picking up any microorganisms present on the surface 
being tested. The cold-water-soluble powder of culture media device 10 is 
then hydrated to form the reconstituted medium, cover sheet 22 is 
replaced, and device 10 is incubated. 
A further test for Staphylococcus bacteria can also be performed when using 
culture media devices 10 and 30 of the present invention. To test for 
Staphylococcus bacteria, the water-based adhesive composition incorporates 
a phosphatase-indicating dye, such as 5-bromoy-4-chloro-3-indolyl 
phosphate disodium salt. Upon inoculation of culture media device 10 with 
an aqueous test sample, any Staphylococcus bacteria present in the sample 
produce phosphatase, which then breaks down the selective dye to form 
blue-colored Staphylococcus colonies. However, some Streptococcus Group D 
bacteria also produce phosphatase. Therefore, an esculin-impregnated disc 
at a concentration of 5 mg of esculin per disc is placed onto the 
reconstituted medium after an initial incubation period of 48 hours at 
37.degree. C. The esculin (Sigma Chemical, Inc., St. Louis, Mo.) in the 
disc results in a brown precipitate forming around any Streptococcus Group 
D colonies growing on culture media device 10. Accordingly, Staphylococcus 
colonies can be identified and quantified on device 10, as the only 
blue-colored, non-precipitate-containing, microorganism colonies growing 
on the reconstituted medium of culture media device 10. 
The invention will be further illustrated by reference to the following 
non-limiting Examples. All parts and percentages are expressed as parts by 
weight unless otherwise indicated. 
COMATIVE EXAMPLES 1-6 
EXAMPLES 7-12 
Separate mixtures of 68.8 g lGE.TM. CA-897 (nonionic surfactant) and 32 
g N-vinylpyrrolidone (NVP) in 2400 g deionized water, and 2.4 g lauroyl 
peroxide in 1568 g isooctyl acrylate (IOA) were prepared, then mixed 
together in a Waring blender, and homogenized for one minute. The 
resulting homogenate was added to a nitrogen-purged 5-liter reaction flask 
equipped with a paddle stirrer, and heated with stirring to 60.degree. C. 
The start of the polymerization reaction was signaled by heat liberation, 
which was allowed to progress to a peak temperature of about 90.degree. C. 
The reaction was then allowed to cool to 70.degree. C., and was held at 
that temperature, with stirring, for two hours. After cooling to room 
temperature, the reaction mixture was filtered through cheesecloth, 
Baird-Parker Nutrient formulation added, and the filtrate coated onto a 
suitable substrate. The resulting mixture comprised about 40% adhesive 
solids, with the remainder being water and displayed a particle size of 
about 0.3.mu. to about 0.8.mu., a pH range of about 6 to about 8, a 
Brookfield viscosity of about 5 to about 15 cps. In addition, another 
water-based adhesive composition according to the present invention was 
made using the same procedure as described above, except acrylamide (ACM) 
monomer was substituted for the N-vinylpyrrolidone (NVP) monomer. 
Table I below compares growth figures for several species of Staphylococcus 
bacteria on standard self-supporting substrates (with and without an 
air-permeable membrane) coated only with Baird-Parker Nutrient formulation 
(Acumedia, Inc., Baltimore, Md.), vs. substrates coated with a 
conventional water-based adhesives of isooctyl acrylate (IOA) and acrylic 
acid (AA) in a weight ratio of 95:5, prepared using an ionic emulsifier 
and an ionic initiator. The conventional IOA/AA adhesive incorporated 
Baird-Parker Nutrient formulation in a 1:1 (column 3, Table 1) and a 1:2 
(column 4, Table 1) ratios by weight of adhesive to nutrient. As can be 
seen in Table I, the standard water-based adhesives completely suppressed 
the growth of the various strains and species of Staphylococcus bacteria. 
In contrast, the substrates coated only with nutrient, and no adhesive, 
show normal Staphylococcus colony growth patterns. 
Table II below gives growth figures (i.e., the number of colonies counted) 
for the same species/strain of Staphylococcus bacteria as used in 
Comparative Examples 1-6 on a Control pour-plate filled with agar media 
and Standard Methods Nutrient formulation (Acumedia, Inc., Baltimore, Md.) 
(column 1); in the presence IGE.TM. CA897 nonionIc emulsifier (2% 
solution) incorporated into the control pour-plate (column 2); and for the 
two formulations IOA and ACM, and IOA and NVP, at 98:2 weight ratios) of 
water-based adhesive compositions of the present invention coated onto 
self-supporting substrates (Columns 3 and 4). The growth results disclosed 
in Table II indicate that the water-based adhesive compositions of the 
present invention provide growth for Staphylococcus bacteria which is 
equivalent to, if not better than, the Control and IGE.TM. pour-plates. 
TABLE 1 
______________________________________ 
Number of colonies counted of various species/strains 
of Staphylococcus bacteria grown on nutrient-coated 
substrates (with and without an air-permeable membrane) 
versus substrates coated with conventional water-based 
adhesive compositions incorporating nutrient therein. 
Conven- 
Conven- 
Nutri. Nutri. tional tional 
Comp. Staph. coated coated IOA/AA IOA/AA 
Ex. Species/ Subst. Subst. Adhesive 
Adhesive 
No. Strain (mem) (no mem) 
(1:1) (1:2) 
______________________________________ 
1 S. intermedius 
255 342 0 0 
2 S. aureus/ 418 479 0@ 0@ 
Z288 
3 S. aureus/ 131 14 0 0 
6538 
4 S. aureus/ 381 342 0 0 
fish 
5 S. aureus/ 140 110 0 0 
F265 
6 S. aureus/ 269 186 0 0 
Fla. Halo 
______________________________________ 
@Approximately 400 microorganism colonies counted, but no blue colonies, 
indicating the presence of Staphylococcus aureus, Strain Z288, were 
recorded. 
TABLE 2 
__________________________________________________________________________ 
Number of colonies counted of various species/strains of 
Staphylococcus bacteria grown on a control pour plate, the 
Control pour plate with IGE .TM. nonionic emulsifying 
agent therein, and two formulations of the water-based 
adhesive compositions of the present invention. 
New New 
Pour Adhesive 
Adhesive 
Staph. Control 
Plate Comp. Comp. 
Ex. Species/ 
Pour IGE .TM. 
(IOA/ACM) 
(IOA/NVP) 
No. Strain Plate 
(2%) (98:2) (98:2) 
__________________________________________________________________________ 
7 S. intermedius 
450 430 650 560 
8 S. aureus/ 
450 480 940 810 
Z288 
9 S. aureus/ 
120 90 195 195 
6538 
10 S. aureus/ 
850 850 1300 1300 
fish 
11 S. aureus/ 
540 500 650 600 
F265 
12 S. aureus/ 
270 270 300 300 
Fla. Halo 
__________________________________________________________________________ 
EXAMPLE 13 
One side of 0.13 mm thick polyethylene-coated paper (Schoeller Paper Inc., 
of Pulaski, N.Y.) was knife-coated with a water-based adhesive composition 
at a level (measured when dried) of 6.2 mg/cm.sup.2, and dried. The 
water-based adhesive composition was formed by dissolving 300 g of Violet 
Red Bile nutrient formulation (Acumedia Inc., Baltimore, Md.) (Table 3 
below) and 2.5 g of guar gum (Hi-Tek Polymers Inc. of Louisville, Ky.), 
with stirring, in 1 liter of an emulsion suspension of a water-insoluble 
adhesive copolymer of isooctyl acrylate (IOA) and N-vinylpyrrolidone (NVP) 
at a 98:2 weight ratio (IOA:NVP). The components of the water-based 
adhesive composition included 1568 g of IOA (38.5 parts by weight), 32 g 
of NVP (0.8 parts), 2400 g of deionized water (59 parts), 68.8 g of 
IGE.TM. CA897 nonionic surfactant (1.7 parts), and 24 g of lauroyl 
peroxide (0.06 parts by weight). Next, 0.01 g of crystal violet dye and 
0.48 g of neutral red dye (Sigma Chemical, St. Louis, Mo.) were dissolved 
in 100 ml of methanol, and this solution was added with stirring to above 
solution. The combined solutions were allowed to stand overnight at a 
temperature of 4.degree.-8.degree. C., coated onto the substrate, and 
dried in an air oven at 93.degree. C., to yield a sticky layer of the 
water-based adhesive composition on the surface of the substrate. 
A mixture of cold-water-soluble powders, formed of equal proportions by 
weight of xanthan gum (Keltrol.TM., Kelco Inc., San Diego, Calif.), locust 
bean gum (Myprodyne.TM.; Hi-Tek Polymers, Louisville, Ky.), Ucargel 
XLG-100.TM. (Union Carbide, Boundbrook, N.J.), and Water Lock.TM. A-100 
(Grain Processing Corp., Muscatine, Iowa), was dusted over the surface of 
the water-based adhesive layer. Any excess powder was shaken loose. This 
adhesive-coated and powder-coated paper was used to form the bottom 
portion of the culture media device. 
A cover sheet was made from a sheet of 0.04 mm thick, transparent, 
biaxially-oriented, corona-treated polypropylene film, coated with a 
noninhibitory adhesive copolymer of isooctyl acrylate (IOA) and acrylamide 
(ACM) in a 98:2 weight ratio (IOA:ACM), at a level (measured when dry) of 
0.93 mg/cm.sup.2, and dried. The adhesive was then dusted uniformly with a 
mixture of xanthan gum (Keltrol.TM.), Locust bean gum (Myprodyne.TM.), 
Ucargel XLG-lOO.TM. and Water Lock.TM. A-100 in a 1:1:1:1 weight ratio. 
The excess powder was shaken loose. 
Both the adhesive-coated and powder-coated bottom portion and cover sheet 
were cut into 10 cm .times. 10 cm pieces, placed together with the 
powdered sides facing each other, and heat-sealed together along one edge. 
The completed culture media device was enclosed in a foil package and 
sterilized with gamma radiation. 
In use, the device was placed on a level surface, and the top cover sheet 
folded back, exposing the powder-coated surface of the bottom section of 
the device. A 5 ml aqueous test sample containing coliform bacteria was 
carefully placed in the center of the bottom section of the device, and 
the cover sheet replaced, powder-coated side down. A weighted spreader was 
applied to evenly spread the aqueous test sample over the powder-coated 
surfaces of the culture media device. The inoculated device was placed in 
an incubator and incubated in the normal manner. After incubation, the 
device was read just as with a standard pour-plate. The crystal violet and 
neutral red dye selective agents included in the nutrient formulation 
acted as selective agents, and were metabolized by the coliform bacteria, 
which were thereby dyed a red color for ease of quantification. In 
addition, the bile salts of the nutrient formulation also served as 
selective agents which inhibited the growth of gram-positive bacteria. 
TABLE 3 
______________________________________ 
Components of Violet Red Bile nutrient formulation as 
measured in grams/liter for culture media device of 
Example 13. 
Component Weight g/l 
______________________________________ 
yeast extract 18 
pancreatic digest of gelatin 
39 
bile salts 3 
lactose 40 
sodium chloride 10 
meat peptone 3 
______________________________________ 
Example 13 shows that a culture media device according to the present 
invention can be constructed and used to contain and grow gram negative 
bacteria using a 5 ml test sample. Previously, samples of this volume 
could not be contained without additional structures, such as side-walls, 
spacer elements, or absorbent elements. Furthermore, incorporation of 
selective agents, such as dyes and inhibitory salts, can be utilized to 
selectively grow desired bacteria species and/or help to visualize 
colonies of those species. 
EXAMPLE 14 
Culture media devices were made in the same manner as that described in 
Example 13, except that an Advent.TM. microporous membrane (3M, St. Paul, 
Minn.) was laminated to the upper surface of the polyethylene-coated paper 
substrate by a layer of an IOA:ACM adhesive copolymer (98:2 weight ratio) 
at a level (when measured dry) of 0.93 mg/cm.sup.2. The total thickness of 
the two-layered body member of the microporous membrane and 
polyethylene-coated paper was 0.5 mm. 
The top surface of the membrane was knife-coated with a water-based 
adhesive composition, and powder-coated as in Example 13, except that the 
nutrient composition included in the water-based adhesive composition 
included a Baird-Parker Broth formulation (Acumedia, Baltimore, Md.), the 
specific composition of which is given below in Table 4. In addition, 10.0 
g/l of lithium chloride, 0.017 g/l of colistin methane sulfonate, and 
0.026 g/l of nalidixic acid (available from Sigma Chemical, St. Louis, 
Mo.) as selective agents were added to the solution. In particular, each 
of these selective agents served to selectively inhibit the growth of 
non-Staphylococcus bacteria. Furthermore, 0.1 g/l of 
5-bromo-4-chloro-3-indolyl phosphate dye (B-C-I phosphate) as a disodium 
salt was added to the composition to facilitate the visualization and 
counting of Staphylococcus colonies. 
The cover sheet was fabricated according to the procedure of Example 13, 
and was cut into rectangular pieces of about 7.5 cm .times. 10 cm, along 
with the bottom portions of the culture media device, and both were then 
heat-sealed together along one edge, sterilized, and packaged to form the 
completed culture media devices. 
In use, the culture media devices were inoculated by the procedure 
described in Example 13. However, Staphylococcus bacteria, instead of 
coliform bacteria, were used as the microbiological organism in the 
aqueous test sample. A total of 1 ml of test sample was placed on the 
powder-coated surface of the bottom portion of the devices. After 
inoculation, spreading, and incubation, the Staphylococcus bacteria were 
identified according to the usual procedure utilized with standard 
pour-plates, including the use of an esculin-impregnated disc to 
distinguish certain strains of Streptococcus Group D bacteria from the 
desired Staphylococcus bacteria. 
TABLE 4 
______________________________________ 
Components of Baird-Parker Broth nutrient formulation as 
measure in grams/liter for Example 14. 
Component Weight g/l 
______________________________________ 
beef extract 5 
casein peptone 10 
yeast extract 1 
glycine 12 
sodium pyruvate 10 
mannitol 5 
B-C-I phosphate 0.1 
______________________________________ 
Example 14 shows that the culture media device of the present invention can 
be made to contain and grow a 1 ml sample of Staphylococcus bacteria 
without resorting to side-walls, spacer elements, or absorbent elements, 
such as sponges or filter paper. 
EXAMPLES 15-20 
A culture media device in accordance with the present invention was 
constructed with the materials and according to the procedures disclosed 
in Example 13. The effectiveness of this device was compared with a 
standard Petrifilm.TM. Coliform Count Plate (3M, St. Paul, Minn.). The 
Example culture media device was inoculated with a 5 ml sample aqueous 
suspension of different species of coliform bacteria, while a 1 ml sample 
was used with the Petrifilm.TM. device. Each were incubated in accordance 
with standard procedures. The colonies were then counted. The comparative 
count results, shown below in Table 5 are expressed in terms of Colony 
Forming Units (CFU) per ml of inoculum. 
TABLE 5 
______________________________________ 
Comparative colony counts (CFU) of different species 
of coliform bacteria grown on a culture media device 
of the present invention and a prior art 
Petrifilm .TM. device. 
Species of Device of Petrifilm .TM. 
Example Coliform Ex. 13 Device 
______________________________________ 
15 Enterobacter cloace 
160 145 
16 Klebsiella oxytoca 
250 150 
17 Enterobacter cloace 
350 250 
18 coliform sp. 36 43 
19 coliform sp. 160 190 
20 Escherichia coli 
150 160 
______________________________________ 
The comparative results from Examples 15 through 20 illustrate that the 5 
ml culture media device in accordance with the present invention performs 
at least as well, and often better, for the enumeration of coliform 
bacteria than does the present 1 ml sample-size Petrifilm.TM. device. 
EXAMPLES 21-30 
A culture media device in accordance with the present invention was 
constructed with the materials and according to the procedures given in 
Example 14. The effectiveness of this device was compared with a standard 
Petri dish pour-plate using Baird-Parker agar medium (See Table 4). Both 
the Inventive culture media device and the comparative pour-plate were 
inoculated with 1 ml aqueous suspension of different strains of 
Staphylococcus aureus bacteria, and incubated in accordance with standard 
procedures. The colonies were then counted. The comparative count results, 
shown below in Table 6, are expressed in terms of Colony Forming Units 
(CFU) per ml of inoculum. 
TABLE 6 
______________________________________ 
Comparative colony counts (CFU) of different strains 
of Staphylococcus aureus bacteria grown on a culture 
media device of the present invention and a Petri dish. 
Strain Device of 
Example Number Example 14 
Petri Dish 
______________________________________ 
21 1043 336 275 
22 1060 365 315 
23 1068 275 300 
24 1072 215 250 
25 1078 135 120 
26 1081 145 135 
27 1112 290 345 
28 1117 260 300 
29 1119 330 390 
30 1168 250 200 
______________________________________ 
The comparative results from Examples 21-30 illustrate that the 1 ml 
culture media device in accordance with the present invention performs at 
least as well, and often better, for the enumeration of Staphylococcus 
aureus bacteria than do the more cumbersome Petri dish pour-plates. 
While in accordance with the patent statutes, description of the preferred 
weight fractions, processing conditions, and product usages have been 
provided, the scope of the invention is not to be limited thereto or 
thereby. Various modifications and alterations of the present invention 
will be apparent to those skilled in the art without departing from the 
scope and spirit of the present invention. The Examples described in this 
application are illustrative of the possibilities of varying the size of 
the culture media devices, and the amounts and types of water-based 
adhesive compositions and nutrient formulations to achieve properties for 
specific purposes. 
Consequently, for an understanding of the scope of the present invention, 
reference is made to the following claims.