Antimicrobial liquid compositions and methods for using them

A liquid composition for applying a non-leachable antimicrobial coating on a surface. The liquid composition consists of a solution, dispersion or suspension of a biguanide polymer reacted with a cross-linking agent to form an adduct, and an antimicrobial metal material. The resulting antimicrobial coating does not release biocidal levels of leachables into surrounding solution.

FIELD OF THE INVENTION 
The present invention relates to liquid dispensers, specifically, the 
provision of liquid dispensers capable of maintaining the sterility of 
sterile solutions during storage, during dispensing, and subsequent to 
dispensing of the solution, as well as methods of manufacture and use of 
such dispensers. 
BACKGROUND OF THE INVENTION 
Many indications require administration of sterile solutions. In general, 
such solutions, and the dispensers in which they are stored, are 
sterilized prior to closure of the dispenser. Contamination can occur, 
however, after the dispenser is opened and used. Various approaches have 
been employed in attempts to minimize this contamination problem. 
Single dose dispensers are available. Such dispensers, however, are made 
only for one time use, and then are discarded, adding considerably to 
packaging costs and waste. Moreover, more sterile solution than is 
required for a single dose usually is packaged which adds to the expense 
of the treatment. Another problem is that persons may attempt to use the 
single dose dispenser multiple times, which can result in contamination of 
the liquid being dispensed. 
Alternatively, preservatives have been added to multi-dose dispensers to 
prevent microbial contamination after the dispenser is initially used. 
Such preservatives, however, often are toxic to mammalian cells, as well 
as microbial cells. For example, many preservatives used in eye drop 
formulations are toxic to the goblet cells in the eye. Such toxicity is 
detrimental to persons requiring prolonged application of the solutions. 
Moreover, persons often develop chemical sensitivity to the preservative, 
resulting in significant allergic reactions to the preparations. Such 
allergies can appear in some persons after prolonged exposure, as well as 
in others after only a single exposure. 
Membrane filters have also been used in liquid dispensers in attempts to 
prevent microbial contamination of the stored sterile liquid. If a 
hydrophilic filter is used, however, the filter can allow the phenomenon 
known as "grow-through," in which microbial progeny on the downstream 
(non-sterile) side of the filter can pass through the filter pores because 
of their smaller size during cell division, and thereby contaminate the 
sterile solution contents in the dispenser. 
Hydrophobic filters have been employed in liquid dispensers. Hydrophobic 
surfaces are non-wetting, and therefore are significantly more difficult 
for microbes to grow on. Such filters, however, because of their 
hydrophobicity prevent the flow of sterile aqueous solutions through the 
filter. 
SUMMARY OF THE INVENTION 
It is an object of the invention to provide a multi-dose liquid dispenser 
which prevents external microbial contamination of the liquid during 
repeated use. 
According to the invention, an article of manufacture is provided--a liquid 
dispenser for dispensing a sterile liquid. The liquid dispenser comprises 
a container for storing the sterile liquid and a nozzle assembly which is 
attached to the container. The nozzle-assembly has a passageway which 
enables the sterile liquid to flow from the container through the 
passageway in order to dispense the liquid. The liquid dispenser further 
comprises an integral, non-leaching antimicrobial element for inhibiting 
microbial contamination of the solution. In one embodiment, the 
antimicrobial element comprises a filter or filters attached to the nozzle 
assembly and positioned across the passageway so that liquid and air flow 
are directed through the filter. 
The filter comprises a substrate having an antimicrobial agent attached to 
the surface and in the pores thereof. The substrate may be an organic or 
inorganic material. The antimicrobial agent can be a metallic or 
non-metallic material having anti-bacterial, anti-viral and/or anti-fungal 
properties, or a combination thereof. In one embodiment, the filter is at 
least partially coated on the downstream surface with a metallic material, 
e.g., a metal, metal oxide, metal salt, metal complex, metal alloy, or 
mixtures thereof, which are bacteriostatic or bacteriocidal. The filter 
has pores of a size which precludes passage of microorganisms through the 
filter while permitting passage of the sterile liquid from the container 
through the filter. The pores are preferably approximately 0.1 microns to 
approximately 1.2 microns, and more preferably approximately 0.22 microns 
to approximately 0.65 microns in diameter. 
In another embodiment, the upstream surface of the filter may be at least 
partially coated with the antimicrobial material. 
In another embodiment, the surfaces and plurality of pores of the filter 
are at least partially coated with an additional different bacteriostatic 
or bacteriocidal material. A variation is the liquid dispenser having a 
second filter that is serially aligned with the first metal coated filter, 
and which is at least partially coated on at least one surface and within 
a plurality of its pores with a different bacteriostatic or bacteriocidal 
material. 
In certain embodiments, the filter and a plurality of the pores can have at 
least a partial coating with a non-metallic antimicrobial compound that 
has an anti-viral, anti-fungal or anti-bacterial property. The 
non-metallic antimicrobial compound may be used in lieu of or in addition 
to the metallic materials. 
In another embodiment, the filter includes a hydrophobic portion for 
allowing air to enter the container to replace the sterile liquid that is 
dispensed from the filter dispenser. In another variation, the dispenser 
may contain a second port separate from the dispensing nozzle for allowing 
replacement air into the container after the liquid is dispensed. In order 
to ensure the sterility of the air entering this second port, the port 
opening would be spanned by a hydrophobic membrane having a pore size that 
precludes bacterial migration into the dispenser, or having an 
antimicrobial agent attached thereto or coated thereon. 
In yet another embodiment of the invention, the passageway walls in the 
nozzle assembly, at least on the downstream side of the filter, are coated 
with an antimicrobial material that is bacteriostatic or bacteriocidal. 
The antimicrobial material may be any of the metallic or non-metallic 
antimicrobial materials described herein. 
In other variants, the liquid dispenser can have a prefilter which is 
spaced upstream from the filter for providing a barrier to the passage of 
particulate matter through the prefilter and for permitting the passage of 
sterile liquid from the container through the prefilter. A support means 
can also be spaced upstream from the filter for reinforcement of the 
filter. 
In another embodiment of the present invention, the dispenser may contain 
an antimicrobial element in lieu of or in addition to the filter, the 
surface of which is at least partially coated with an antimicrobial agent. 
The element is disposed within the body of the container such that it 
remains in contact with the sterile solution at all times, e.g., during 
storage and dispensing. This is accomplished by providing a substrate 
having permanently attached thereto or coated thereon an antimicrobial 
agent. The substrate may be a bead or plurality of beads, a membrane, 
cartridge, filter, wool, cotton, baffle or fibrous bundle, for example. 
The element may be free-floating in the solution within the container or 
may be attached to or immobilized within the container. In this 
embodiment, the antimicrobial element remains in contact with the solution 
thereby insuring its sterility even after repeated doses have been 
dispensed by a user. 
In another aspect of the above embodiment, the inside wall of the container 
which is in contact with the solution is coated with or has attached 
thereto an antimicrobial material. 
Another aspect of the invention provides a membrane, element or surface 
which has an antimicrobial material coated thereon or attached thereto. In 
one embodiment, a microporous membrane having pores or other perforations 
which provide liquid conduits interconnecting the upstream and downstream 
surfaces of the membrane for liquid passage from one surface to the other 
is treated such that at least one surface and at least some of the pores 
are coated or otherwise derivatized with the antimicrobial material. The 
pores are of a size so as to preclude passage of microorganisms through 
the membrane and so as to permit passage of liquid and air through the 
membrane. Variations include all surfaces, including the pores, being at 
least partially coated with the antimicrobial material. The membrane 
surfaces and a plurality of the pores can be at least partially coated 
with an additional antimicrobial material that has an anti-viral, 
anti-fungal or anti-bacterial properties. 
The invention also includes a method in which a liquid can be dispensed by 
applying pressure to the container of the liquid dispenser of this 
invention so as to discharge the liquid from the container. The container 
preferably is formed at least in part of a resiliently deformable 
material, such as an elastic polymer, which permits manual squeezing to 
discharge a dose of medicament, and subsequent elastic recovery of the 
material to its original configuration by drawing gas from a surrounding 
atmosphere into the container while the gas is sterilized by the filter in 
passing therethrough. 
In a preferred embodiment of the invention, the liquid dispenser is used 
for eyecare in an individual, in which a sterile eyecare liquid, e.g., 
liquid artificial tears, a solution for contact lens care or a medicament, 
is dispensed from the liquid dispenser into the eye or onto an object that 
is to be placed into the eye. Preferably, the eyecare liquid is 
preservative-free. 
The invention also features methods for attaching antimicrobial agents to 
the surfaces of a substrate. This may be accomplished by a number of 
methods. For example, metallic compounds may be applied to a surface by 
metal vapor deposition, electroless plating, chemical derivatization or 
coating. Non-metallic antimicrobial materials may be applied to such 
metallic surfaces by chemical derivatization or coating, for example. 
In one embodiment, a metallic silver coating is accomplished by contacting 
the substrate with a carbonyl compound, e.g., an aldehyde such as 
glutaraldehyde, a sugar such as glucose, or an aldehyde functionality 
generating compound, drying the substrate, contacting the dried substrate 
with a metal salt, e.g., silver nitrate, or metal carboxylate salt 
solution, e.g., silver tartrate, and an amine-containing compound 
solution, e.g., ammonium hydroxide, so as to deposit the metal on the 
surface and within a plurality of the pores of the substrate. In an 
alternative embodiment, the drying step is omitted. One or more techniques 
may be combined to accomplish the desired result. For example, metal vapor 
deposition deposits metal on a surface of a membrane, but not in the 
pores. Therefore, antimicrobial material can be deposited in the pores of 
the membrane by an appropriate technique, followed by metal vapor 
deposition to coat the surface, thereby forming a membrane in which both 
the pores and the surface are coated with antimicrobial material. 
In another embodiment, the substrate is contacted with an activator, e.g., 
a tin dichloride solution, is dried, and then contacted with a metal salt 
or metal carboxylate salt solution, either with or without an 
amine-containing compound solution, so as to deposit the metal on the 
surfaces of the substrate. 
In another embodiment of the present invention, non-metallic antimicrobial 
agents are covalently attached to or coated onto a metal coated substrate 
such as a filter or an element disposed in the reservoir of the container. 
Non-metallic antimicrobial agents may include any anti-bacterial, 
anti-viral and/or anti-fungal materials which are capable of being 
immobilized on a surface and which are compatible with the sterile liquid. 
Most preferred are the class of agents which cause dissolution of the 
lipid bilayer membrane of a microorganism. For this purpose, surface 
active agents, compounds such as cationic or polycationic compounds, 
anionic or polyanionic compounds, non-ionic compounds and zwitterionic 
compounds may be used. Preferred agents include biguanide compounds or 
benzalkonium compounds. These agents may be attached to the substrate by 
covalent bonding, ionic interaction, coulombic interaction, hydrogen 
bonding, crosslinking (e.g., as crosslinked (cured) networks) or as 
interpenetrating networks, for example. 
In another embodiment of the present invention, non-metallic antimicrobial 
agents are covalently attached to or coated onto a substrate such as a 
membrane, filter, an element disposed in the reservoir of the container or 
the walls of the reservoir in contact with the solution. These 
non-metallic agents are attached or coated directly onto the surface of 
the substrate in lieu of the metal coating. Non-metallic antimicrobial 
agents useful for his purpose include any anti-bacterial, anti-viral 
and/or anti-fungal materials which are capable of being immobilized on a 
surface and which are compatible with the liquid. Most preferred are the 
class of agents which cause dissolution of the lipid bilayer membrane of a 
microorganism. For this purpose, surface active agents, compounds such as 
cationic or polycationic compounds, anionic or polyanionic compounds, 
non-ionic compounds and zwitterionic compounds may be used. Preferred 
agents include biguanide compounds or benzalkonium compounds. These agents 
may be attached to the substrate by covalent bonding, ionic interaction, 
coulombic interaction, hydrogen bonding or interpenetrating networks, for 
example. 
Articles made in accordance with these methods are also included in this 
invention. 
A multidose dispenser containing a hydrophilic filter having immobilized 
thereon an antimicrobial agent that prevents bacterial grow-through while 
maintaining high flow rates of aqueous solutions was unknown in the art 
prior to Applicants' invention. 
The present invention is unique in the following respects: 
i) A multi-dose dispenser that incorporates a hydrophilic membrane which is 
surface modified (including pores) with a bound antimicrobial agent. 
ii) The ability of the filter to prevent microbial grow through in long 
term contact applications, while maintaining high flow rates of aqueous 
solutions. 
iii) The unique nature of the antimicrobial agent that utilizes a 
synergistic effect of it's components. This results in surface high 
biocidal activity, while maintaining almost no significant leachables into 
solutions it is in contact with 
iv) The mechanism of action being essentially a surface mediated one, 
whereby organisms succumb only upon contact with the filter due to the non 
leaching property associated with it. 
v) The ability of such surfaces to remain viable over multiple organism 
challenges with no decrease in their bioactivity. 
vi) The utilization of such biocidal coatings on the dispensing tip of the 
device, thereby eliminating the possibility of microbial colonization in 
the dead volume of the tip downstream to the filter. 
vii) User friendliness and cost effectiveness of the device for all types 
of applications. 
viii) Adaptability to existing manufacturing technology, thereby enabling 
large scale manufacture without added cost. 
ix) Applicability to a variety of ophthalmic formulations over a wide range 
of solution viscosity including artificial tears, saline, anti-glaucoma 
and ocular hypertensension drugs, and contact lens cleaning solutions. 
x) Readily adaptable for varied flow requirements (single drop or stream). 
xi) Readily adaptable for the delivery of other types of medicaments or 
solutions where preservatives have been used such as ear and nasal drug 
formulations. 
The above and other objects, features and advantages of the present 
invention will be better understood from the following specification when 
read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION 
One embodiment of the present invention, as shown in FIGS. 1-3, provides a 
liquid dispenser 1 for dispensing a sterile liquid 2. The liquid dispenser 
1 has a container 4 for storing sterile liquid 2 and a nozzle assembly 3 
which is mounted on top of container 4. Nozzle assembly 3 has a passageway 
5 which enables sterile liquid 2 to flow from container 4 through 
passageway 5 when sterile liquid 2 is dispensed. 
Container 4 is designed to permit manual squeezing so as to force sterile 
liquid 2 from container 4 through filter 6 out of orifice 7 of nozzle 
assembly 3. In normal operation, liquid dispenser 1 is inverted and 
container 4 is squeezed. 
Container 4, as shown in FIG. 1, has a circular cross-section extending 
along a vertical axis 8, with a flat bottom 9 and an upper end 10. 
Sidewall thickness is preferably in the range of 0.01 to 0.25 inch. 
However, various sizes and configurations can be used. The shape of the 
container can be round, elliptical, polygonal, irregular, or the like, and 
in some cases may be in tube form. The particular sidewall thickness can 
vary greatly, as can the volume of the chamber within container 4 that 
holds the medicament or other liquid to be dispensed. Thus, various sizes 
ranging from cubic millimeters to cubic centimeters or more can be used 
for the container chamber. 
In a preferred embodiment, the nozzle assembly 3 is an ovoid form. The 
nozzle assembly has a cross-section formed of a plastic material which is 
self-supporting and defines a generally ovoid configuration having an 
inverted lip portion 11 mating with and sealed to the top of container 4 
at upper end 10. Nozzle assembly 3 includes within it a ring-shaped lower 
spacer 12 of a solid material having a central passage connecting the 
chamber of container 4 with the filter or filters and an upper spacer 13 
which acts to hold the filter or filters in place. The lower spacer 12 
comprises a supporting screen 30. A disc 14 carries channel means. As 
shown in FIG. 3, disc 14 carries a plurality of concentric channels 22 
which are interconnected by radial channels 23 to a central passageway 5 
so that liquid coming from container 4 will pass through the filter or 
filters and be distributed on the surface of disc 14 so as to cause 
dispensed sterile liquid 2 to coalesce into a single drop or a stream of 
liquid when expelled from container 4. Depending on factors including, 
e.g., the applied pressure, the viscosity of the expelled liquid and the 
surface area of disc 14, either a single drop or a stream of liquid will 
be dispensed. Disc 14 is held in place by being adhesively secured, e.g., 
by ultrasonic welding or by a mechanical force, to the upper spacer 13. 
Support 16, prefilter 15, filter 6 and second filter 17 can be suspended 
by spacers 12 and 13. In some embodiments, only filter 6 need be used and 
one or more of the support, prefilter or second filter, can be eliminated. 
Various combinations of these elements can be used in different 
embodiments as desired. 
While the prefilter and filter, as well as the second filter, are shown as 
planar members, various configurations can be used. These members can be 
in the form of cones, polygonal or other shaped devices as may be 
desirable for specific applications. Planar sheet-type materials as shown 
are most preferred. 
While passageway 5 is preferably axially extending with a circular 
cross-section, it can have any configuration as desired for specific 
applications. 
Container 4 can be formed from a flexible material; e.g., an elastically 
deformable polymer which may be a thermosetting or thermoplastic polymeric 
material, including, for example, polypropylene, polyethylene, 
polyvinylchloride, polyethylene terephthalate, polytetrafluoroethylene, 
polysulfone and polyethersulfone polymers or copolymers. In some cases the 
container can be a deformable metallic or plastic medicament container, 
such as a toothpaste tube, where the container may remain deformed after 
each dose is dispensed. 
Nozzle assembly 3 can be formed from the same or a more rigid type of 
material than container 4. In one embodiment, nozzle assembly 3 is 
permanently attached to container 4 with a liquid-tight connection so as 
to aid in maintaining the sterility of sterile liquid 2 in container 4. 
Such a connection can be formed by standard techniques, e.g., ultrasonic 
welding, heat press sealing, adhesive sealing or mechanical sealing. 
Filter 6 is sealingly attached to nozzle assembly 3 so that filter 6 
extends across the entire expanse of passageway 5 to direct liquid and air 
flow out of and into containers through filter 6. Filter 6 can be attached 
to nozzle assembly 3 by any method which results in such a seal, 
including, e.g., ultrasonic sealing, heat press sealing and adhesive 
sealing. 
By filter is meant any material which can function as a microbial filter. 
Microporous membranes are preferred filter materials. As used herein, the 
term "microporous" means having pores of an average diameter of 5 m or 
less. Membranes used in the filter of this invention may be formed from 
organic or inorganic materials. Organic materials include polymeric 
materials which can be used for the preparation of membranes or filter 
papers. Examples of organic polymeric materials include polysulfone, 
polyethersulfone, polyamide (e.g., nylon), polycarbonate, polyacrylate, 
polyvinylidene fluoride, polyethylene, polypropylene, cellulosics (e.g., 
cellulose), and Teflon.RTM.. The hydrophobic materials, e.g., 
polypropylene or Teflon.RTM., may require prior surface activation with 
techniques such as plasma, chemical oxidation or metallic sensitization, 
e.g., a primer. Inorganic filters include glass fiber filter paper, 
ceramic membranes (e.g., alumina or silica), and metal filters. Sintered 
glass and sintered ceramic blocks also can be used. The filters can be 
either hydrophilic or hydrophobic. If a hydrophobic filter is used, the 
metal coating, described below, converts it to a filter with hydrophilic 
properties. 
Filter 6 has pores which form interconnecting liquid conduits extending 
from an upstream surface of the filter to a downstream surface. The pore 
size for filter 6 is chosen so that the pores permit passage of sterile 
liquid 2 from container 4 through filter 6, but preclude passage of 
microorganisms through filter 6, thereby maintaining the sterility of 
sterile liquid 2 in container 4. By microorganism is meant bacteria, 
blue-green algae, fungi, yeast, mycoplasmids, protozoa and algae. The pore 
size can range from approximately 0.1 microns to approximately 1.2 
microns. Preferably, the pore size is approximately 0.22 microns to 
approximately 0.65 microns. Most preferably, the pore size is about 0.65 
microns. Whereas 0.22 microns is the pore size used in most bacterial 
filtration systems, this invention can produce a sterile filtrate with 
larger pore sizes, e.g., 0.45 and 0.65 microns, thus permitting a device 
which gives a faster flow rate for the dispensed liquid. 
A major problem in multi-dose liquid dispensers is that residual liquid may 
accumulate downstream of the filter subsequent to dispensing liquid from 
the container. By downstream of the filter is meant the side of the filter 
that liquid from the container which has passed through the filter would 
be on, e.g., the surface of the filter exposed to the outside atmosphere. 
By upstream of the filter is meant the side of the filter facing the 
liquid in the container which has not yet passed through the filter. 
Microorganisms can multiply in this accumulated downstream liquid and 
contaminate liquid which is subsequently dispensed. Moreover, certain 
microorganisms in this accumulated liquid can, because of their smaller 
size during cell division, pass through the pores of the filter, a 
phenomenon known as "grow-through," and contaminate the sterile liquid in 
the container. 
This invention addresses this problem by providing that filter 6 be at 
least partially coated with or has attached thereto on the downstream 
surface and within a plurality of the pores with an antimicrobial material 
that is bacteriostatic or bacteriocidal. By bacteriocidal is meant the 
killing of microorganisms. By bacteriostatic is meant inhibiting the 
growth of microorganisms, which can be reversible under certain 
conditions. The antimicrobial agent may be a metal or metal compound, a 
non-metallic compound, or a combination of both. 
In a preferred embodiment, the antimicrobial agent is a metal, metal oxide, 
metal salt, metal complex, metal alloy or mixture thereof which is 
bacteriocidal or bacteriostatic. By a metallic material that is 
bacteriostatic or bacteriocidal is meant a metallic material that is 
bacteriostatic to a microorganism, or that is bacteriocidal to a 
microorganism, or that is bacteriocidal to certain microorganisms and 
bacteriostatic to other microorganisms. In certain embodiments, filter 6 
is also at least partially coated on the upstream surface with a metallic 
material, e.g., a metal, metal oxide, metal salt, metal complex or metal 
alloy or mixtures thereof. Any metal which is bacteriostatic or 
bacteriocidal can be used. Examples of such metals include, e.g., silver, 
zinc, cadmium, mercury, antimony, gold, aluminum, copper, platinum and 
palladium. The appropriate metal coating is chosen based upon the use to 
which the sterile liquid passing over the metal coated filter is to be 
put. Preferably, metals which are not toxic are attached to filters which 
are to be used for filtering solutions that are to be applied to humans 
and other organisms. The currently preferred metal is silver. 
In another embodiment of the present invention, filter 6 is first coated 
with an antimicrobial metal, metal salt or metal complex material, and a 
non-metallic antimicrobial agent is covalently attached to or coated onto 
the metal coated substrate. Non-metallic antimicrobial agents useful for 
this purpose include any anti-bacterial, anti-viral and/or anti-fungal 
materials which are capable of being immobilized on a surface and which 
are compatible with the sterile liquid. Most preferred are the class of 
agents which cause dissolution of the lipid bilayer membrane of a 
microorganism. For this purpose, surface active agents, compounds such as 
cationic or polycationic compounds, anionic or polyanionic compounds, 
non-ionic compounds and zwitterionic compounds may be used. Preferred 
agents include biguanide compounds or benzalkonium compounds. These agents 
may be attached to the substrate by covalent bonding, ionic interaction, 
coulombic interaction, hydrogen bonding, crosslinking (e.g., as 
crosslinked (cured) networks) or as interpenetrating networks, for 
example. 
In another embodiment of the present invention, filter 6 is treated with 
non-metallic antimicrobial agents which are covalently attached to or 
coated onto the surfaces and/or pores of the filter. These non-metallic 
agents are attached or coated directly onto the surface and/or pores of 
the substrate in lieu of the metal coating. Non-metallic antimicrobial 
agents useful for his purpose include any anti-bacterial, anti-viral 
and/or anti-fungal materials which are capable of being immobilized on a 
surface and which are compatible with the liquid. Most preferred are the 
class of agents which cause dissolution of the lipid bilayer membrane of a 
microorganism. For this purpose, surface active agents, compounds such as 
cationic or polycationic compounds, anionic or polyanionic compounds, 
non-ionic compounds and zwitterionic compounds may be used. Preferred 
agents include biguanide compounds or benzalkonium compounds. These agents 
may be attached to the substrate by covalent bonding, ionic interaction, 
coulombic interaction, hydrogen bonding, crossinking (e.g, as crosslinked 
(cured) networks) or as interpenetrating networks, for example. 
In another embodiment of the present invention, filter 6 is treated with 
non-metallic antimicrobial agents which are covalently attached to or 
coated onto the surfaces and/or pores of the filter. These non-metallic 
agents are attached or coated directly onto the surface and/or pores of 
the substrate. Non-metallic antimicrobial agents useful for this purpose 
include any anti-bacterial, anti-viral and/or anti-fungal materials which 
are capable of being immobilized on a surface and which are compatible 
with the liquid. Most preferred are the class of agents which cause 
dissolution of the lipid bilayer membrane of a microorganism. For this 
purpose, surface active agents, compounds such as cationic or polycationic 
compounds, anionic or polyanionic compounds, non-ionic compounds and 
zwitterionic compounds may be used. Preferred agents include biguanide 
compounds or benzalkonium compounds. These agents may be attached to the 
substrate by covalent bonding, ionic interaction, coulombic interaction, 
hydrogen bonding, crosslinking (e.g. as crosslinked (cured) networks) or 
as interpenetrating networks, for example. An antimicrobial metal, metal 
salt or metal complex material is introduced into the non-metallic 
antimicrobial coating either prior to or after coating the surface in the 
form of either as particles or as a homogeneous solution. 
In one embodiment, filter 6 is a membrane having both hydrophilic and 
hydrophobic regions. For example, a hydrophobic filter which has only been 
coated with a metal or metal oxide or metal salt on a portion of the 
filter, will be hydrophilic for the coated portion and hydrophobic for the 
uncoated portion. In another example, a hydrophilic or hydrophobic filter 
is coated with a metal, metal oxide or metal salt, so as to make the 
filter hydrophilic, and then a portion of this metallic surface is 
rendered hydrophobic by incorporation of a hydrophobic coating via 
formation of a spontaneously self-assembled monolayer that is covalently 
attached to the metallic surface, e.g., formation of an alkyl thiolate 
monolayer on a silver coated surface by treatment with a solution of an 
alkyl thiol, such as dodecane thiol. In another example, a portion of a 
hydrophilic filter may be rendered hydrophobic by treatment with a 
polymeric siloxane, a perfluoro polymer, or silyl monomer(s) or perfluoro 
monomer(s) that may be polymerized thermally, photolytically or 
chemically. Such a dual purpose filter is preferred when multiple doses of 
liquid are dispensed in quick succession to each other, in order to more 
quickly replace the liquid which has been dispensed from the container 
with air from outside the container, so as to equalize the pressure. The 
dispensed liquid can pass through the hydrophilic portion of the filter, 
and the replacement air can pass through the hydrophobic portion without 
being hampered by the presence of liquid on the hydrophilic portion. 
In the embodiment of FIG. 2, an air port or vent (not shown) can be 
provided through upper spacer 13 positioned directly above the hydrophobic 
portion of the filter so as to allow air passage to container 4 as the 
liquid is dispensed from container 4. The air port or vent provides for 
compensation of the air pressure as liquid is dispensed from the container 
so as to avoid formation of a vacuum. The device will work with or without 
the air port or vent, however, if a constant and sustained flow is 
desired, better flow rates may be obtained with the use of an air port or 
vent. In those cases where a hydrophobic/hydrophilic membrane is used, the 
air port or vent described may be particularly desirable to equalize 
pressure as liquids leave the container. In such a case, it is preferred 
that the air port or vent be positioned above the hydrophobic portion of 
the filter. 
The invention also provides for a filter in which the downstream surface 
and a plurality of the pores are at least partially coated with an 
additional second antimicrobial material. In one embodiment, the upstream 
surface is also at least partially coated with a second metal, metal 
oxide, metal salt, metal complex or metal alloy, or mixture thereof. 
Examples of metals that can be used are discussed above in relation to the 
single metal coating. The use of two different metals can enhance the 
antimicrobial properties of the filter. Different types of microorganisms 
can exhibit different degrees of sensitivity to different metals. In 
addition, the use of two different metals can significantly reduce the 
problem of selection for microorganisms having resistance to the metal in 
the metal coating that can occur when only one metal is used. 
Another variation of the invention is a liquid dispenser which has a second 
filter 17 with pores of a size that permits passage of sterile liquid 2 
from container 4, that is serially aligned with filter 6. Second filter 17 
is at least partially coated on at least one surface and within a 
plurality of its pores with a different antimicrobial material, e.g., a 
metal, metal oxide, metal salt, metal complex or metal alloy or mixtures 
thereof, that is bacteriostatic or bacteriocidal, than is used for the 
coating on filter 6. The presence of different antimicrobial materials on 
different filters in the liquid dispenser is advantageous for the same 
reasons as discussed above regarding the embodiment where two different 
antimicrobial materials are applied to a single filter. In other 
embodiments, more than two different antimicrobial filters are present. 
In another embodiment of this invention, the surfaces of the filter (or 
other membrane) and a plurality of pores of the filter or membrane is 
coated with or has attached thereto, a non-metallic compound that has 
antimicrobial properties, e.g., antiviral, antibacterial and/or antifungal 
properties. By anti-viral is meant capable of killing, or suppressing the 
replication of, viruses. By anti-bacterial is meant bacteriostatic or 
bacteriocidal. By antifungal is meant capable of killing or suppressing 
replication of fungi. This non-metallic antimicrobial material may be used 
in lieu of or in addition to the metallic coating. Use of these materials 
in conjunction with the metallic agent as an additional coating can allow 
for more effective anti-bacterial liquid dispensers, in that different 
anti-bacterial compounds can exhibit different degrees of effectiveness 
for different types of microorganisms. Multiple anti-bacterial compounds 
also significantly reduce the problem of selection for microorganisms 
having resistance to the metal in the metal coating, as discussed above. 
Moreover, a combination of antimicrobial materials can allow for joint 
anti-bacterial/anti-viral/anti-fungal liquid dispensers. Preferably, this 
compound is bound to at least a portion of the first antimicrobial coating 
on the filter. Any compound which has anti-bacterial, anti-fungal or 
anti-viral activity can be used. Examples of such compounds include 
cationic or polycationic compounds, anionic or polyanionic compounds, 
non-ionic compounds and zwitterionic compounds. Preferred compounds 
include benzalkoniumchloride derivatives (see, for example, Example 9), 
a-4-1-tris(2-hydroxyethyl) 
ammonium-2-butenyl!poly1-dimethylammonium-2-butenyl!-.omega.-tris(2-hydro 
xyethyl)ammonium chloride, and biguanides of the general formula: 
##STR1## 
or their water soluble salts, where X is any aliphatic, cycloaliphatic, 
aromatic, substituted aliphatic, substituted aromatic, heteroaliphatic, 
heterocyclic, or heteroaromatic compound, or a mixture of any of these, 
and Y.sub.1 and Y.sub.2 are any aliphatic, cycloaliphatic, aromatic, 
substituted aliphatic, substituted aromatic, heteroaliphatic, 
heterocyclic, or heteroaromatic compound, or a mixture of any of these, 
and where n is an integer equal to or greater than 1. Preferred compounds 
include, e.g., chlorhexidine or polyhexamethylene biguanide (both 
available from Zeneca of Wilmington, Del.). These compounds may be 
modified to include a thiol group in their structure so as to allow for 
the bonding of the compound to the metallic surface of the filter. 
Alternatively, these compounds may be derivatized with other functional 
groups to permit direct immobilization on a non-metallic surface. For 
example, the above-mentioned antimicrobials may be suitably functionalized 
to incorporate groups such as hydroxy, amine, halogen, epoxy, alkyl or 
alkoxy silyl functionalities to enable direct immobilization to the 
surface in lieu of a metal. 
Antimicrobial elements having the various antimicrobial compounds coated or 
attached thereon described above also are included in this invention. 
The invention also includes an embodiment in which the liquid dispenser has 
a prefilter 15 which is spaced upstream from filter 6 and provides a 
barrier to the passage of particulate matter through prefilter 15, while 
permitting passage of sterile liquid 2 from container 4 through prefilter 
15. In this manner, particulate matter that may be present in sterile 
liquid 2 in container 4 does not need to be filtered by filter 6, and thus 
prevents clogging of filter 6, thereby aiding in preserving the capacity 
of, and flow rate through, filter 6. Preferably, the pore size of 
prefilter 15 is approximately 1 micron to approximately 50 microns. The 
prefilter material includes, e.g., glass fibers, synthetic polymer fibers, 
e.g., hydrophilic polypropylene fibers, nylon and cellulosic fibers. 
Preferably, prefilter 15 is attached to filter 6 in embodiments where 
there is only one filter, or attached to the most upstream filter where 
there is more than one filter, and is also attached to nozzle assembly 3. 
Preferably, the attachments are by welding. 
In another embodiment, the liquid dispenser has a support 16 which is 
spaced upstream from the filter to act as a reinforcement for the filter. 
Preferably, support 16 is perforated. Support 16 can be made from any 
material that the container or nozzle assembly is made from. 
In other embodiments of the invention, the internal walls 18 of nozzle 
assembly 3 are at least partially coated, with an antimicrobial agent As 
described herein above, the agent may be a metallic material, e.g., a 
metal, metal oxide, metal salt, metal complex, metal alloy or mixtures 
thereof, or may be a non-metallic organic material that is bacteriostatic 
or bacteriocidal or a combination of the two. After the liquid dispenser 
of the invention has initially been used to dispense liquid from container 
4, some residual liquid may remain on internal walls 18 of nozzle assembly 
3 downstream from filter 6. Microorganisms can grow in this residual 
liquid and contaminate any future drops of liquid which are dispensed. By 
coating these walls with an anti-bacterial material, this contamination is 
reduced. Examples of metals and non-metallic materials which can be used 
to coat the nozzle assembly walls were discussed above in relation to 
coating filter 6. Example 12 describes a method for depositing silver onto 
the walls of the nozzle assembly. 
In another preferred embodiment, shown in FIGS. 4-5, the container 4 has 
disposed therein an antimicrobial element in contact with sterile liquid 
2. In this embodiment, the antimicrobial element may be present in lieu of 
or in addition to the filter. The element comprises an organic or 
inorganic substrate having an antimicrobial agent attached thereto as 
coated thereon. The substrate may have any shape or configuration and may 
be free-floating within the container or may be attached to or be an 
integral part of the container. In the embodiment shown in FIG. 4, the 
substrate comprises beads 32. In the embodiment shown in FIG. 5, the 
substrate comprises a cotton ball 34. Other substrate configurations 
including cartridges, fibrous bundles, membranes or baffles may be used. 
The substrate may be formed from any material compatible with the sterile 
liquid, for example, plastic, metal or cellulosic material. The substrate 
preferably is inert and non-degradable in the sterile liquid. The 
currently preferred materials for use as a substrate include glass or 
polymeric beads or pellets, fibers and non-woven materials such as cotton 
or cellulose, or metal foils. The preferred substrate will be 
antimicrobial, either naturally or will have attached thereto or coated 
thereon an antimicrobial agent. Examples of naturally antimicrobial 
materials include certain metals, metal oxides, metal salts, metal 
complexes and metal alloys. For example, a metal foil such as silver foil 
may be used. Substrates which are not anti-microbial have immobilized 
thereon an antimicrobial agent. Antimicrobial agents which may be used 
include the metallic and non-metallic agents discussed in detail herein. 
The amount and/or type of the antimicrobial agent which is used in a 
particular application will vary depending on several factors, including 
the amount of liquid which must be maintained in a sterile environment, 
the type and amount of contamination which is likely to occur, and the 
size of the antimicrobial surface present in the dispenser or other 
container. For example, certain metals, such as silver, are highly 
effective against most bacteria, but less effective against yeast (such as 
Candida albicans). However, non-metallic agents such as biguanide 
compounds, are toxic to yeast. Therefore, if the sterile liquid is likely 
to be exposed to contamination by both bacteria and yeast, a combination 
of silver and a biguanide compound can be used as the antimicrobial. The 
amount of antimicrobial used will be a minimum amount necessary to 
maintain the sterility of the liquid. As stated above, this amount will 
vary depending upon various considerations. 
In a preferred embodiment, glass beads coated with metallic silver or a 
silver salt are added to the sterile liquid. The silver compound is 
attached to the beads such that the silver is substantially non-leachable. 
As used herein, the term "substantially "non-leachable" means that none or 
very small amounts (e.g., below a certain threshold) of the non-leachable 
material dissolves into the sterile liquid. It is understood that minute 
amounts of silver or other anti-microbial agent may dissolve into the 
solution, but these amounts are minute. The amount of dissolved silver in 
the sterile liquid may be substantially reduced by passivating the silver 
coated surface by reaction with compounds which form silver salts or salt 
complexes. For example, reaction with halogens, e.g., chlorine, iodine, 
bromine or mixtures thereof may be performed, thereby generating a layer 
of silver halide on the metallic silver coating. Other embodiments 
include, for example, pellets or beads formed from an inert polymeric 
material, such as polyethersulfone, having an an organic, inorganic or a 
combination of the two types of antimicrobial agents attached or coated on 
the surface. Preferred inorganic antimicrobial agents include elemental 
silver or silver compounds. Preferred organic antimicrobial agents include 
cationic or polycationic compounds, anionic or polyanionic compounds, 
non-ionic compounds and zwitterionic compounds. Preferred compounds 
include benzalkonium chloride derivatives and biguanide compounds, all of 
which are discussed in detail hereinabove. 
In methods of the invention the surfaces and pores of a filter or other 
substrate are coated with a metallic or non-metallic antimicrobial 
compound, or a combination of the two types. In one embodiment, a filter 
having pores is provided, the filter is contacted with a carbonyl 
compound, the filter is dried, and the dried filter is contacted with a 
metal salt solution or metal carboxylate salt solution and an 
amine-containing compound solution so as to deposit the metal on the 
surface and within a plurality of the pores. In one embodiment, this 
filter is then washed and dried. Other elements such as glass or polymeric 
beads, glass wool, glass or polymeric fibers, membranes, cotton or other 
fibrous material or cartridges etc. can be treated in like manner to coat 
or attach an antimicrobial agent. 
The carbonyl compound includes, e.g., aldehydes, sugars, and aldehyde 
functionality generating compounds. Aldehydes include compounds with the 
formula R(CHO).sub.n, where R is any aliphatic, aromatic or heteroaromatic 
group and n is an integer greater or equal to 1. Examples of water soluble 
aldehydes are glutaraldehyde, formaldehyde, acetaldehyde, butyraldehyde, 
glyceraldehyde, glyoxal, glyoxal disodium bisulfite, paraldehyde and 
cyclic trioxanes. Examples of water insoluble aldehydes are cinnamaldehyde 
and benzaldehyde. By sugar is meant a reducing sugar. Sugars include, 
e.g., fructose, glucose, lactose, maltose and galactose. By an aldehyde 
functionality generating compound it is meant a compound capable of 
generating aldehyde group(s). Examples of such compounds include acetals 
and hemiacetals. Polymeric acetals, e.g., paraformaldehyde and polyacetal, 
can also be used in this invention. The carbonyl compound acts as a 
reducing agent, so that the metal ion is reduced to the metal, e.g., 
silver ion is reduced to metallic silver. This electroless redox reaction 
occurs in situ in solution or in the solid state. The carbonyl compound 
has affinity for aqueous and non-aqueous phases and therefore can be used 
in the process of coating either hydrophilic or hydrophobic filters. If 
hydrophobic filters are used, the resulting metal coating confers 
hydrophilic properties on the coated filter. 
After treatment with the carbonyl compound, the filter or other substrate 
is either immersed directly into the metal salt solution or metal 
carboxylate salt solution, or is dried first and then immersed in this 
solution. Preferably, the filter is first dried. The drying step increases 
the metal coating within the pores of the filter and produces a more 
uniform and adhesive metal coating thickness on the surface and within the 
pores of the filter. Coating within the pores enhances the bacteriostatic 
or bacteriocidal properties of the filters. 
Any metal which has bacteriostatic or bacteriocidal properties, as 
described above, can be used in this invention to coat the substrate. In a 
preferred embodiment, the metal is silver. The silver salts that can be 
used in the metal coating process are salts that are capable of 
solubilizing, even to a limited degree, in aqueous media, to produce 
silver ions. Such salts include, e.g., silver nitrate, silver benzoate, 
silver tartrate and silver acetate, silver citrate or any silver 
carboxylate. 
Metal carboxylate salts include compounds with the formula R(COO.sup.- 
M.sup.+).sub.n, where R is any aliphatic, aromatic or heteroaromatic group 
and n is an integer greater or equal to 1. Examples of metal carboxylate 
salts include, e.g., silver, zinc, cadmium, mercury, antimony, gold, 
aluminum, copper, platinum and palladium salts of acetic, propanoic, 
lactic or benzoic acid; and mono- or di- sodium or potassium salts of 
diacids, e.g., oxalic, malonic, glutaric or tartaric acids. The term metal 
carboxylate salts is also meant to include carboxylic acids which are 
capable of forming carboxylate salts in situ under conditions including 
the presence of a base and a metal ion, and compounds which are capable of 
forming carboxylic or carboxylate groups in situ, including, e.g., esters, 
lactones, anhydrides and amides. 
By amine-containing compound it is meant a compound capable of producing a 
metal-amine complex when metal salts react with amines under basic 
conditions. Examples of amine-containing compounds include ammonium 
hydroxide, ammonia, and aliphatic, aromatic and heteroaromatic amines. 
In another embodiment, a filter having pores is provided, the filter is 
contacted with an activator, the filter is dried, and the dried filter is 
contacted with a metal salt or metal carboxylate salt solution so as to 
deposit the metal on the surface and within a plurality of the pores of 
the filter. The activator is a salt of a metal including, e.g., tin, 
titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, 
zinc, germanium, selenium, zirconium, niobium, molybdenum, technetium, 
ruthenium, rhodium, palladium, antimony, tellurium and lead. A preferred 
activator is tin dichloride. An alternative embodiment is to contact the 
dried filter with an amine containing compound in addition to the metal 
salt or metal carboxylate salt solution. 
Additionally, many types of metals can be plated onto the surface of 
suitably primed polymeric materials using standard well known 
electroplating techniques or by electroless methods. It is necessary to 
prime the polymer surface to allow for the electroplating process to occur 
because most polymers are electrically insulating and do not carry an 
electrical current. Priming deposits a very small amount of metal onto the 
surface of the polymer allowing for the subsequent electrolytic deposition 
of a metal from solution. 
The metal coating on the filters derived from any of the methods discussed 
above, can be further treated to produce a metal oxide coating, as 
described in Example 8, or a metal halide coating, as described in Example 
16. 
The invention further provides methods for attaching or coating 
non-metallic antimicrobial agents to a surface. In this embodiment 
antimicrobial agents including the anti-bacterial, anti-viral and/or 
anti-fungal agents described herein are non-leachably immobilized on the 
surface of a filter or other element. These non-metallic agents may be 
used in lieu of or in addition to the metallic antimicrobial agents. The 
non-metallic agents may be immobilized by any suitable method, including 
covalent bonding, ionic attraction, coulombic interaction, hydrogen 
bonding and interpenetrating networks, for example. Methods for attaching 
organic antimicrobial agents to a metal surface are described in Examples 
16 and 17. 
The invention further provides methods for attaching or coating a 
combination of a metallic and non-metallic antimicrobial agents to a 
surface. In this embodiment antimicrobial agents including the 
anti-bacterial, anti-viral and/or anti-fungal agents described herein are 
non-leachably immobilized on the surface of a filter or other element. The 
non-metallic agents may be immobilized by any suitable method, including 
covalent bonding, ionic attraction, coulombic interaction, hydrogen 
bonding, cross-linking (curing) and interpenetrating networks, for 
example. The metallic antimicrobial may be introduced in the non metallic 
antimicrobial coating either prior to or after applying the coating to the 
surface. The metallic antimicrobial may consist of a metal, metal salt or 
metal complex may be introduced into the non-metallic antimicrobial either 
as particles or as a homogeneous solution. Methods for attaching organic 
antimicrobial agents to a metal surface are described in Examples 16 and 
17. 
The substrate may be pretreated, if necessary, to activate the surface. In 
one embodiment, the surface is silylated to render it more receptive to 
binding antimicrobial agents. Silylation can be carried out by 
art-recognized techniques including direct coupling reactions, grafting 
reactions and dendrimer-type reactions. The antimicrobial agent then is 
reacted with the resulting silyl functionality, or to a group attached to 
the silyl functionality. Methods for immobilizing antimicrobial compounds 
on silylated surfaces are described in Example 17. 
In another embodiment, metallic and non-metallic antimicrobial agents may 
be attached to non-silylated surfaces. In this embodiment, the surface is 
treated to obtain carboxylic or amine functionalities as described above, 
and the antimicrobial agent is attached by reaction with these 
functionalities. Methods for immobilizing antimicrobial materials on a 
non-silylated surface are described in Example 18. 
This invention also includes the products made in accordance with these 
methods. 
This invention also provides a method for dispensing sterile liquid by 
applying pressure to the container of the liquid dispenser of this 
invention so as to discharge the sterile liquid from the container. In one 
embodiment, the container has an elastically deformable wall, pressure is 
applied to deform the wall and force the sterile liquid from the container 
through the filter, and the wall is allowed to recover so as to draw gas 
from the surrounding atmosphere into the container, the gas being 
sterilized as it passes through the filter. 
The liquid dispenser can be used for any purpose which requires dispensing 
a sterile solution from a container. Such uses include, e.g., medical 
related purposes, e.g., dispensing sterile liquids onto any part of the 
body of an organism or onto an object that is to be placed into the body 
of an organism, e.g, for use in eye, ear, or nose care. For example, this 
invention provides a method for using the liquid dispenser of this 
invention for eyecare in an organism in which a sterile eyecare liquid is 
dispensed into an eye of the organism or onto an object that is to be 
placed into the eye of the organism. Preferably, the sterile eyecare 
liquid is preservative-free. The sterile eyecare liquid includes, e.g., 
liquid artificial tears, a solution for contact lens care or a medicament. 
Examples of medicaments are antibiotics, decongestants, 
anti-inflammatories, anti-glaucoma agents, anti-bacterial agents, 
anti-viral agents, anesthetics, mydriatics, anti-cholingerics and miotics. 
An object that is to be placed into the eye includes, e.g., a contact 
lens. Other uses include process filters for sterilization of all types of 
solutions, e.g., drug solutions and instillation solutions; intravenous 
catheters, where a membrane unit is employed for the admittance of air but 
prevents back flow of blood or other liquids; process filters for food 
products where sterility is required; dispensation of items such as baby 
formula where the presence of a preservative would be undesirable; and 
membrane filter units, e.g., for campers and hikers where the generation 
of microbial free water is desired without the possibility of future 
contamination. 
EXAMPLES 
Example 1 
Metal Vapor Deposition (MVD) of Silver Onto a Polyethersulfone Membrane 
This example illustrates a method for depositing silver onto a surface, but 
not within the pores, of a membrane filter. A precut polyethersulfone 
membrane (Supor 450, pore size 0.45 4M, hydrophilic, obtained from Gelman 
of Ann Arbor, Mich.) was mounted on a plate such that the surface to be 
coated faced the heating source of a metal evaporator. An approximately 
4-6 inch long silver wire (obtained from Johnson Matthey of Wardhill, 
Mass.) was rolled into a coil and placed on the metal bridge in the 
evaporator. The evaporator was pumped down to 10.sup.-5 Torr and a current 
of approximately 60-70 amperes was applied to melt the silver. A uniform 
silver coating of the membrane surface resulted in about 15-30 secs. The 
current was turned off and the evaporator chamber was allowed to return to 
atmospheric pressure. The membrane was turned over and the procedure 
repeated. The resulting membrane had a uniform coating of silver on both 
surfaces, but not within the pores, as determined by scanning electron 
microscopy (SEM) and energy dispersive X-ray analysis (EDAX). 
Example 2 
Electroless Coating of Silver Onto a Polyethersulfone Membrane (Method 1) 
This example illustrates a method for depositing metallic silver onto a 
surface, and within the pores, of a membrane filter. A polyethersulfone 
membrane Gelman Supor 450, pore size 0.45 mM, hydrophilic) was precut into 
a 47 mm disk. This membrane was immersed in 5 ml of glutaraldehyde (25% 
solution, obtained from Aldrich of Milwaukee, Wis.) for 1 min. at 
22.degree. C., removed from the aldehyde solution and air dried 
thoroughly. The treated membrane was then immersed in 5 ml of the silver 
coating solution described in Example 7A, at pH approximately 12 (the pH 
can range from approximately 8-14), at 35.degree. C. for 15 secs. The 
plated membrane was thoroughly rinsed with distilled water and dried in a 
vacuum oven at 20.degree. C. for 2 hrs. SEM coupled with EDAX showed 
uniform silver coating on the membrane surface and within the pores. 
(Ag:S=0.4-0.5:1) 
Example 3 
Electroless Coating of Silver Onto a Polyethersulfone Membrane (Method 2) 
This example illustrates a method for depositing metallic silver onto a 
surface and within the pores of a membrane filter. A polyethersulfone 
membrane (Millipore, pore size 0.45 mM, hydrophobic, obtained from 
Millipore Corp. of Bedford, Mass.), was precut into a 47 mm disk. This 
membrane was immersed in 5 ml of 0.1M a-D-glucose in an aqueous solution 
containing 10% ethanol for 5 mins. at 22.degree. C., removed from the 
sugar solution and air dried thoroughly. The treated membrane was then 
immersed in 5 ml of the silver coating solution described in Example 7A, 
at pH approximately 12 at 35.degree. C. for 2 mins. The plated membrane 
was thoroughly rinsed with distilled water and dried in a vacuum oven at 
20.degree. C. for 2 hrs. SEM coupled with EDAX showed uniform silver 
coating on the membrane surface and within the pores. (Ag:S=0.8:1) 
Example 4 
Electroless Coating of Silver Onto a Polyethersulfone Membrane (Method 3) 
A polyethersulfone membrane (Gelman, pore size 0.45 mM, hydrophilic) was 
precut in the form of a 47 mm disk. This was immersed in 5 ml of a 0.1M 
a-D-glucose in an aqueous solution containing 10% ethanol for 5 minutes at 
22 deg. C. It was then removed from the sugar solution and air dried 
thoroughly. The treated membrane was then immersed in 5 ml of a silver 
plating solution 1 (pH .about.12) at 35 deg. C. for 2 minutes. A rapid 
deposition of metallic silver on the membrane surface ensued. The plated 
membrane was thoroughly rinsed with distilled water and dried in a vacuum 
oven at 20 deg. C. for 12 hours. SEM coupled with EDAX showed uniform 
silver coating on the membrane surface and within the pores. (Ag:S=0.3:1) 
Example 5 
Electroless Coating of Silver Onto a Polyethersulfone Membrane (Method 4) 
This example illustrates a method for depositing metallic silver onto a 
surface and within the pores of a membrane filter. A polyethersulfone 
membrane (Gelman Supor 450, pore size 0.45 mM, hydrophilic) was precut 
into a 47 mm disk. This membrane was immersed in 5 ml of the silver 
coating solution described in Example 7B, which was then heated to 
55.degree. C. and maintained at this temperature for 5 mins. Rapid 
deposition of metallic silver onto the membrane ensued. The plated 
membrane was thoroughly rinsed with distilled water and dried in a vacuum 
oven at 20.degree. C. for 12 hrs. SEM coupled with EDAX showed uniform 
silver coating on the membrane surface and within the pores. (Ag:S=0.05:1) 
Example 6 
Electroless Coating of Silver Onto a Polyethersulfone membrane: (Method 5) 
This example illustrates a method for depositing metallic silver onto a 
surface and within the pores of a membrane filter. A polyethersulfone 
membrane (Gelman Supor 450, pore size 0.45 mM, hydrophilic) was precut 
into a 47 mm disk. This membrane was immersed in 5 ml of a solution 
containing 1 g tin (II) chloride dihydrate (Aldrich), 1 ml concentrated 
HCl and 9 ml distilled water, at room temperature for 5 mins. The membrane 
was dried and immersed in the silver coating solution, described in 
Example 7B, which was then heated to 55.degree. C. and maintained at this 
temperature for 3 mins. Rapid deposition of metallic silver on the 
membrane surface ensued. The plated membrane was thoroughly rinsed with 
distilled water and dried in a vacuum oven at 20.degree. C. SEM coupled 
with EDAX showed uniform silver coating on the membrane surface and within 
the pores. (Ag:S=0.3:1) 
Salts of other metals including titanium, vanadium, chromium, manganese, 
iron, cobalt, nickel, copper, zinc, germanium, selenium, zirconium, 
niobium, molybdenum, technetium, ruthenium, rhodium, palladium, antimony, 
tellurium and lead may be used as activators in place of tin dichloride 
prior to silver plating. 
Example 7 
Preparation of Silver Coating Solutions 
(A) This example illustrates the preparation of the silver coating solution 
that is used in Examples 2, 3, 4 and 10. 3 ml of a silver nitrate solution 
(10 g silver nitrate dissolved in 10 ml distilled water) was added to 3 ml 
of a sodium hydroxide solution (10 g sodium hydroxide dissolved in 10 ml 
distilled water) at 22.degree. C. A brown precipitate of silver oxide 
formed rapidly. Concentrated ammonium hydroxide (28% ammonia, obtained 
from EM Science of Gibbstown, N.J.) was added dropwise to the solution 
until the silver oxide dissolved completely to give a clear solution of a 
soluble silver amine complex with a pH of 12. 
(B) This example illustrates the preparation of the silver coating solution 
that is used in Examples 5 and 6. Three (3) ml of a sodium tartrate 
solution (tartaric acid disodium salt dehydrate, obtained from Aldrich) 
dissolved in 20 ml distilled water) was added to 3 ml of a silver nitrate 
solution (silver nitrate dissolved in 10 ml distilled water) at 22.degree. 
C. A white precipitate of silver tartrate formed rapidly. Concentrated 
ammonium hydroxide, 28% ammonia (obtained from EM Science), was added 
dropwise to the solution until the silver tartrate dissolved completely to 
give a clear solution of ammoniacal silver tartarate with a pH of 
approximately 12. 
Example 8 
Oxygen Plasma Treatment of Silver Coated Membrane Filters 
This example illustrates a method for treating a silver coated membrane 
filter with oxygen plasma so as to produce a silver oxide coating. A 
silver coated polyethersulfone membrane, obtained from either Example 1 or 
2, was mounted on a glass holder and placed inside the reaction chamber of 
a plasma reactor so that both surfaces of the filter were exposed to the 
plasma. The reaction chamber was purged with oxygen three times. The 
pressure of the chamber was adjusted to 300 mTorr, the power maintained at 
100 watts, and the membrane subjected to oxygen plasma for 2 mins. 
Example 9 
Surface Modification of Silver Coated Membrane Filters 
This example illustrates a method for treating a silver coated membrane 
filter with a second compound that has anti-bacterial or anti-viral 
properties, 3.44 mg (20 mmol) of benzalkoniumchloride thiol (BAC-S), 
obtained as described in Example 9, was dissolved in 5 ml of absolute 
ethanol that was degassed for 1 hr under dry nitrogen. A freshly silver 
coated polyethersulfone membrane obtained from Example 1 or 2, was 
immersed in this solution at 22.degree. C. for 16 hrs, rinsed in absolute 
ethanol, and dried under a stream of nitrogen. 
Example 10 
Synthesis of Benzalkoniumchloride Thiol (BAC-S) 
This example illustrates a method for the synthesis of BAC-S from 
10-chlorodecanethiol which in turn is synthesized from 
w-chlorodecane-thioacetate. 
(a) .omega.-Chlorodecanethioacetate 
Triphenylphosphine (6.53 g, 25 mmol) (obtained from Aldrich) was dissolved 
in 95 ml of dry, distilled tetrahydrofuran and the solution was cooled to 
0.degree. C. under dry nitrogen. 4.9 ml (25 mmol) of 
di-isopropylazodicarbonate (obtained from Aldrich) was added to the 
solution. The reaction mixture was stirred for 30 min. at 0.degree. C. 
during which time a white precipitate formed. A 1M solution of 
10-chloro-1-decanol (4.82 g in 25 ml THF (tetrahydrofuran) (obtained from 
VWR Scientific of Boston, Mass.) was added. 2.3 g of thiolacetic acid 
(obtained from Aldrich) in 20 ml THF was subsequently added. The resulting 
clear solution was stirred at 0.degree. C. for 15 mins and warmed to room 
temperature. Two drops of water were added. The solvent was removed under 
reduced pressure, the residue was dissolved with ethyl ether and crystals 
of triphenylphosphine oxide were removed by filtration. Evaporation of the 
ether resulted in the crude product, .omega.-chlorodecanethioacetate, as a 
yellow oil. This product was distilled under reduced pressure 
(92.degree.-95.degree. C./10 mM) to give the pure compound as a pale 
yellow oil (3.23 g, 57%). IR neat, KBr plates cM-1: 1728,s, (O.dbd.C--S), 
TLC, silica (hexane:dichloromethane, 60:40 eluent) Rf=0.7. 
(b) 10-Chlorodecanethiol 
.omega.-Chlorodecanethioacetate (3.2 g, 1.2 mmol) was hydrolyzed in 30 ml 
of methanol, that was degassed under dry nitrogen for 4 hrs, containing 
1.6 g (1.2 mmol) anhydrous potassium carbonate (obtained from Aldrich) at 
22.degree. C. The suspension was stirred for 1 hr, and then quenched with 
0.75 ml glacial acetic acid (obtained from VWR Scientific). The potassium 
carbonate was filtered and the solvent removed under reduced pressure to 
yield 10-chlorodecanethiol as a pale yellow oil, 2.4 g, 1.1 mmol). TLC, 
silica (hexane:dichloromethane, 60:40 eluent) Rf.dbd.O 
(c) Benzalkoniumchloride Thiol (BAC-S) 
2.4 g (11 mmol) of 10-chlorodecanethiol was reacted with 1.8 g (14 mmol) of 
N,N-dimethylbenzylamine (obtained from Aldrich) in 50 ml of dry THF. The 
reaction mixture was refluxed for 20 hrs and cooled. White crystals of 
BAC-S separated out. These crystals were filtered, washed with THF and 
dried, yielding 0.85 g of product. 
Example 11 
Synthesis of Alkane Thiol Derivative of Polyhexamethylene Biguanide 
(PHMB-S) and Chain Extended Polyhexamethylene Biguanide (PHMBCE-S) 
This example illustrates a method of the synthesis of alkane thiol 
derivative of polyhexamethylene biguanide (PHMB-S) and chain extended 
polyhexamethylene biguanide (PHMBCE-S): 
(a) Neutralization of Polyhexamethylene biguanide-bis hydrochloride 
Polyhexamethylene biguanide-bis-hydrochloride (Zeneca, Wilmington, Del.), 1 
g, was neutralized by addition of 2 ml. of a 10% NaOH solution. The 
solvent was evaporated and the residual solid was washed rapidly with 
water to minimize dissolution and dried to give polyhexamethylene 
biguanide (PHMB). 
(b) Synthesis of Chain extension of chain polyhexamethylene biguanide 
(PHMBCE) 
Polyhexamethylene biguanide (PHMB) was reacted with 
ethyleneglycol-bis-glycidylether (Aldrich) in a 1:0.9 mole ratio 
respectively in anhydrous DMSO at 50 deg.C. for 12 hours. The solvent was 
evaporated under reduced pressure and the solid residue was washed with 
ether and dried. 
(c) Synthesis of alkanethiol derivative of polyhexamethylene biguanide 
(PHMB-S) and chain extended polyhexamethylene biguanide (PHMBCE-S) 
Polyhexamethylene biguanide (PHMB-S) and chain extended polyhexamethylene 
biguanide (PHMBCE-S) respectively 1 mole equivalent) were reacted with 
10-chlorodecanethiol (0.5 mole equivalent) described in Example 10b in 
anhydrous DMSO at 50 deg.C for 12 hours. The solvent was evaporated under 
reduced pressure and the solid residue was washed with ether and dried to 
yield the title compounds. 
Example 12 
Electroless Coating of Silver Onto Tubing For Use in Nozzle Assembly 
This example illustrates a method for depositing silver onto the inner 
surface of tubing which can be used for the passageway walls in the nozzle 
assembly of the liquid dispenser. Polyethylene tubing (2 inches long, 800 
mM ID) (obtained from Putnam Plastics Corp. of Dayville, Conn.) was 
immersed in a 25% aq. glutaraldehyde solution and ultrasonicated at 
20.degree. C. for 2 mins. The tubing was then dried thoroughly and silver 
coating solution, as described in Example 7A, was drawn into the tubing 
with a pipette. The plating solution was allowed to soak inside the tubing 
for 3 mins at 20.degree. C., the excess solution was then expelled and the 
inside of the tubing was flushed with distilled water. A uniform silver 
coating resulted on the inside surface of the tubing. 
The tubing was tested for bacteriocidal activity. Control or silver treated 
plastic tubing was inoculated with 10 mls of a suspension of Pseudomonas 
dimunata containing 5.times.10.sup.7 organisms. The tubes containing 
bacterial suspension were incubated 15 hours at 37.degree. C., at which 
time the tubes were placed in thioglycollate bacterial culture medium (1 
cc) and vortexed. Aliquots of this solution were removed and serially 
diluted and 100 mls of these dilutions were plated onto NZY agar plates. 
The plates were incubated overnight at 37.degree. C. and the bacterial 
concentrations were determined by counting bacterial colonies. 
Example 13 
Surface Modification of Silver Plated Membrane to Give 
Hydrophobic/Hydrophilic Surface 
Method A 
This example illustrates a method for producing a silver coated membrane 
filter that is partially hydrophobic and partially hydrophilic. 20.2 mg 
(20 mmol) of 1-dodecanethiol (obtained from Sigma Chemical Co., St. Louis, 
Mo.) was dissolved in 5 ml of absolute ethanol that was degassed for 1 hr 
under dry nitrogen. A freshly silver coated (by MVD or electroless 
process) polyethersulfone membrane (Gelman Supor 400, pore size 0.45 mM, 
hydrophilic) was partially immersed in this solution at 22.degree. C. for 
16 hours. The membrane was then rinsed in absolute ethanol and dried under 
a stream of nitrogen. The resulting surface treated membrane was 
hydrophobic (non-wetting) in the area treated with the alkyl thiol while 
remaining hydrophilic in the non-treated area. 
Method B 
This example illustrates a method for producing a partially hydrophobic 
area in a hydrophilic membrane (with or without a silver coating). 
The hydrophilic membrane was immersed in a 2% n-hexane solution containing 
a 1:1:1 mixture (by weight) of methyltriacetoxy silane, 
ethyltriacetoxysilane and silanol terminated polytrimethylsiloxane 
(obtained from Huls America, Piscataway, N.J.). The membrane was air dried 
at room temperature for 30 min., after which it was heated at 120.degree. 
C. for 30 min. to render the treated area totally hydrophobic. 
Example 14 
Silver Coating of Polymethylmethacrylate (PMMA) Sheets 
This example illustrates methods for depositing metallic silver onto the 
surface of PMMA sheets. 
Method A 
A commercially obtained PMMA sheet was cut in the form of a slide and the 
surface was cleaned by ultrasonication in absolute ethanol for 1 minute at 
room temperature. The cleaned slide was then immersed in 30 ml of an 
activator solution consisting of 10% tin dichloride dihydrate (SnCl.sub.2 
2H.sub.2 O, obtained from Allied Chemicals of New York, N.Y.), 45% 
absolute ethanol and 45% distilled water and ultrasonicated at 45.degree. 
C. for 15 minutes. The slide was rinsed several times with distilled 
water. It was then immersed in a 10% aqueous solution of silver nitrate 
for 15 minutes at room temperature. During this time the slide acquired a 
brown tinge due to the deposition of silver. The coated slide was then 
rinsed with distilled water and dried. The silver coating was adherent to 
the polymer and did not cause loss of transparency of the slide. 
Method B 
A PMMA slide was treated with plasma as described in Example 8, followed by 
immersion into a 2% aqueous solution of polyethyleneimine (PEI) (Aldrich 
Chemical Co., Milwaukee, Wis.) for 15 min. The slide was rinsed with 
water, after which it was first immersed in a 5% aqueous glutaraldehyde 
solution (Aldrich) for 5 min., followed by a 10% silver nitrate solution 
for 5 min. The surface was then subjected to electroless silver plating in 
a silver tartarate solution as described in Example 7B to give a uniform, 
adhesive silver mirror. 
Example 15 
Silver Coating of Polypropylene (PP) 
This example illustrates a method for depositing a metallic silver coating 
onto the surface of polypropylene slides (obtained from Eastman Chemical 
Product, Inc. Kingsport, Tenn.). The slides were immersed in 30 ml of a 5% 
solution of chlorosulfonic acid (obtained from Aldrich of Milwaukee, Wis.) 
in chloroform at 50.degree. C. for 5 mins. They were allowed to dry for 15 
mins. then immersed in a 1M aqueous solution of sodium hydroxide after 
which they were rinsed thoroughly with distilled water. The surface 
treated slides were subjected to the silver coating procedure described in 
Example 4 using the plating solution described in Example 7B. This 
resulted in a uniform silver coating that is adherent to the polypropylene 
surface. 
Example 16 
Modification of Metallic Surfaces with Antimicrobial Compounds Generation 
of Adherent Silver Halide Surfaces 
Method A 
Freshly silver plated membranes (by MVD or electroless method) were exposed 
to either vapors of chlorine gas (generated in situ by reaction of sodium 
hypochlorite with concentrated HCl) or to bromine vapors for 0.5 to 1 min. 
at room temperature. The metallic surface was rapidly coated with a layer 
of silver chloride and silver bromide respectively. The membranes were 
then washed with water and dried. 
Method B 
Freshly silver plated membranes (by MVD or electroless method) were 
immersed in (i) an aqueous solution of 0.9% NaCl, or (ii) a 2% aqueous 
solution of bromine, or (iii) a 2% aqueous solution consisting of 
equimolar amounts of bromine, iodine and potassium iodide at room 
temperature for 15 minutes. A rapid coating of silver halide (or halides) 
resulted on the surface of the metallic coating. The membranes were then 
washed with water and dried. 
Method C 
Freshly silver plated membranes (by MVD or electroless method) were 
immersed in a 2% aqueous solution consisting of equimolar amounts of 
iodine and potassium iodide at room temperature for 15 minutes. A rapid 
coating of silver iodide resulted on the surface of the metallic coating. 
The membranes were then washed with ethanol followed by water and dried. 
Modification of silver plated membrane with antimicrobial 
(a) with benzalkoniumchloridethiol (BAC-S): 3.44 mg (10.sup.-3 mmol) of 
benzalkoniumchloridethiol (BAC-S) was dissolved in 5 ml of absolute 
ethanol that was degassed for 1 hr under dry nitrogen. A freshly silver 
coated (by MVD or electroless process) polyethersulfone membrane (Gelman 
Supor 450, pore size 0.45 mM, hydrophilic) was immersed in the resulting 
solution at 22 deg. C. for 16 hours. The membrane was then rinsed in 
absolute ethanol and dried under a stream of nitrogen. 
(b) with alkanethiol derivative of polyhexamethylene biguanide (PHMBCE-S): 
3 mg of polyhexamethylenebiguanide (PHMB-S) and chain extended 
polyhexamethylenebiguanide (PHMBCE-S) respectively were dissolved in 5 ml 
of degassed water. A freshly silver coated (by MVD or electroless process) 
polyethersulfone membrane (Gelman Supor 450, pore size 0.45 uM, 
hydrophilic) was immersed in the resulting solution at 22 deg. C. for 16 
hours. The membrane was then rinsed with water, 1% HCl solution followed 
by water and dried. 
Modification of gold foil with antimicrobial 
Commercially obtained gold foil was treated with oxygen plasma for 3 
minutes to clean the surface and improve wetability. The foil was then 
immersed in the resulting solution at 22 deg. for 16 hours. The membrane 
was then rinsed in absolute ethanol and dried under a stream nitrogen. 
Modification of silver and gold surfaces with biguanides 
Freshly silver plated membranes and gold foil (cleaned as described above) 
were immersed in aqueous solutions of diamine terminated 
polyhexamethylenebiguanide (ICI Zeneca) its chain extended analog 
(synthesized) that were suitably modified to incorporate a thiol group at 
22 deg. C. for 16 hours. The membrane was then rinsed with water followed 
by a 1% HCl solution. After rinsing thoroughly with water they were dried. 
Example 17 
Modification of Silver halide surfaces with biguanides 
Membranes coated with silver halides were immersed in either one of the 
coating solutions (Examples 18a or 18b) for 1 minute, excess solution 
removed from the membrane surface, after which they were allowed to dry at 
ambient temperature for 0.5 hour. The membrane was then cured at 
130.degree. C. for 30 minutes. It was then extracted with a 50% aqueous 
ethanol solution for 30 minutes at 80.degree. C., followed by 3 times with 
water (1 hour each, 80.degree. C.) after which they were air dried for 0.5 
hour at 80.degree. C. The coated membranes were then immersed in a 
solution containing 0.5% of silver iodide and 12.5% (wt/vol.) potassium 
iodide in 100 mL of 50% aqueous ethanol at ambient temperature for 5 
minutes. The membranes were rinsed with 50% aqueous ethanol followed by 
water extraction (3.times.100 mL, 1 hour each at 80.degree. C. They were 
then air dried. 
Example 18 
Preparation of biguanide coating solutions 
Polyhexamethylenebiguanide (PHMB) was precipitated from a 20% aqueous 
solution of polyhexamethylene biguanidedihydrochloride ((Zeneca Biocides, 
Wilmington, Del.) by addition of two volume equivalents of 5% aqueous 
NaOH. The resulting precipitate was separated from the alkaline solution, 
dissolved in anhydrous DMF and reprecipitated with acetonitrile. The 
precipitate was filtered and dried under vacuum at 50.degree. C. for 6 
hours. 
(a) 2 mL of a 5% (weight/volume) PHMB solution in anhydrous ethanol was 
added to 2 mL of 5% (weight/volume) solution of 
4,4'-methylene-bis(N,N-diglycidylaniline) (Aldrich Chemical Company, 
Milwaukee, Wis.) dissolved in a 4:1 (vol/vol) ethanol/acetonitrile 
mixture. The solution was stirred at room temperature for 20 minutes and 
diluted with 76 mL of anhydrous ethanol to a solution containing 0.25% 
solids. 
(b) 2 mL of a 5% (weight/volume) PHMB solution in anhydrous ethanol was 
added to 2 mL of 5% (weight/volume) solution of 
4,4'-methylene-bis(N,N-diglycidylaniline) (Aldrich Chemical Company, 
Milwaukee, Wis.) dissolved in a 4:1 (vol/vol) ethanol/acetonitrile 
mixture. The solution was refluxed at 90.degree.-95.degree. C. for 15 
minutes. The solution was cooled and diluted with 76 mL of anhydrous 
ethanol to a solution containing 0.25% solids. 
(c) 2 mL of a 5% (weight/volume) PHMB solution in anhydrous ethanol was 
added to 2 mL of 5% (weight/volume) solution of 
4,4'-methylene-bis(N,N-diglycidylaniline) (Aldrich Chemical Company, 
Milwaukee, Wis.) dissolved in a 4:1 (vol/vol) ethanol/acetonitrile 
mixture. The solution was refluxed at 90.degree.-95.degree. C. for 15 
minutes. The solution was cooled diluted with 16 mL of anhydrous DMF. 50 
mg of finely ground silver iodide was dispersed in the diluted solution 
and the resulting suspension was heated to 80.degree. C. with stirring to 
produce a clear solution. This was cooled and filtered to give a clear, 
homogeneous solution 
(d) 2 mL of a 5% (weight/volume) PHMB solution in anhydrous ethanol was 
added to 2 mL of 5% (weight/volume) solution of 
4,4'-methylene-bis(N,N-diglycidylaniline) (Aldrich Chemical Company, 
Milwaukee, Wis.) dissolved in a 4:1 (vol/vol) ethanol/acetonitrile 
mixture. The solution was refluxed at 90.degree.-95.degree. C. for 15 
minutes. The solution was cooled diluted with 16 mL of anhydrous DMF. 50 
mg of finely ground silver iodide was dispersed in the diluted solution to 
give a well dispersed suspension. 
Example 19 
Introduction of Antimicrobial Compounds on Silylated Surfaces 
The following reaction schemes demonstrate methods for preparing a 
silylated surface and reacting an antimicrobial agent to the silyl 
functionality. 
##STR2## 
Example 20 
Introduction of Antimicrobial Compounds on Non-Silylated Surfaces 
Incorporation of silver salts on PMMA surfaces 
I) Polyacrylic acid was introduced on the surface of PMMA by graft 
polymerization of acrylic acid to obtain surface functionalization. Base 
treatment followed by an aqueous silver nitrate solution resulted in the 
formation of silver salt of polyacrylic acid 
II) The polyacrylic acid coating obtained on PMMA by the above method was 
further modified by coupling the carboxylic groups of the acrylic acid 
with cysteine using DCC. The membrane was then treated with base followed 
by an aqueous silver nitrate solution to give a mixture of silver 
carboxylate and silver thiolate. 
Attachment of antimicrobials to coulombic multilayers on 
activated-polymeric surfaces 
Polymeric surfaces activated by (i) oxidative chemical procedures, (ii) 
plasma or (iii) e-beam are then treated sequentially with 
polyethyleneimine (PEI) and polyacrylic acid. This process may be repeated 
over several cycles to obtain the desired amplification. Antimicrobial 
compounds (or suitably modified derivatives thereof) are then covalently 
attached to the amine functionalities in PEI. 
##STR3## 
Coating of antimicrobials to activated polymeric surfaces 
Polymeric surfaces are activated by (i) oxidative chemical methods or (ii) 
plasma. Solutions of antimicrobial compounds (or suitably modified 
derivatives thereof) are then applied on the surfaces and cured 
(thermally, photochemically or chemically) to result in stable, non 
leachable coatings 
Example 21 
Antimicrobial Coating of Polymeric Surface 
(a) Polypropylene coupons (10.times.10 cm) were surface oxidized by known 
methods (chemical or plasma). The coupons were immersed in the coating 
solution described in Example 18a or 18b for 1 minute, excess solution 
removed from the membrane surface, after which it was allowed to dry at 
ambient temperature for 0.5 hour. The coupons were then cured at 
130.degree. C. for 30 minutes. They were then extracted with a 50% aqueous 
ethanol solution for 30 minutes at 80.degree. C., followed by 3 times with 
water (1 hour each, 80.degree. C.) after which they were air dried for 0.5 
hr. at 80.degree. C. The coated membranes were then immersed in a solution 
containing 0.5% of silver iodide and 12.5% (wt/vol.) potassium iodide in 
100 mL of 50% aqueous ethanol at ambient temperature for 5 minutes. The 
membranes were rinsed with 50% aqueous ethanol followed by water 
extraction (3.times.100 mL, 1 hour each at 80.degree. C. They were then 
air dried. 
(b) Polypropylene coupons (10.times.10 cm) were surface oxidized by known 
methods (chemical or plasma). The coupons were immersed in the coating 
solution described in Examples 18c or 18d for 1 minute, excess solution 
removed from the surface, after which it was allowed to dry at ambient 
temperature for 0.5 hour. The coupons were then cured at 130.degree. C. 
for 30 minutes. They were then extracted with a 50% aqueous ethanol 
solution for 30 minutes at 80.degree. C., followed by 3 times with water 
(1 hour each, 80.degree. C.) after which they were air dried for 0.5 hr. 
at 80.degree. C. 
Example 22 
Preparation of surface coating solutions 
(a) 2 mL of a 5% (weight/volume) PHMB solution in anhydrous ethanol was 
added to 2 mL of 5% (weight/volume) solution of 
4,4'-methylene-bis(N,N-diglycidylaniline) (Aldrich Chemical Company, 
Milwaukee, Wis.) dissolved in a 4:1 (vol/vol) ethanol/acetonitrile 
mixture. The solution was refluxed at 90.degree.-95.degree. C. for 15 
minutes. The solution was cooled diluted with 16 mL of anhydrous DMF. 50 
mg of finely ground silver iodide was dispersed in the diluted solution 
and the resulting suspension was heated to 80.degree. C. with stirring to 
produce a clear solution. This was cooled and filtered to give a clear, 
homogeneous solution. 
(b) 2 mL of a 5% (weight/volume) PHMB solution in anhydrous ethanol was 
added to 2 mL of 5% (weight/volume) solution of 
4,4'-methylene-bis(N,N-diglycidylaniline) (Aldrich Chemical Company, 
Milwaukee, Wis.) dissolved in a 4:1 (vol/vol) ethanol/acetonitrile 
mixture. The solution was refluxed at 90.degree.-95.degree. C. for 15 
minutes. The solution was cooled diluted with 16 mL of anhydrous DMF. 50 
mg of finely ground silver iodide was dispersed in the diluted solution to 
give a well dispersed suspension. 
Example 23 
Anti-bacterial Properties of Differently Treated Membrane Filters 
This example illustrates a method for testing different types of membrane 
filters with different pore sizes, which have been coated with different 
compounds by different procedures, for their bacteriocidal/bacteriostatic 
properties. 
Filters, either control or treated, were placed into Gelman plastic filter 
holders and the entire unit was autoclaved 30 minutes at 121RC. Using 
asceptic techniques under a laminar flow hood, sterile bacterial media, 
sterile saline solution or a preservative free artificial tear solution 
("Tears Plus," obtained from Johnson and Johnson) was introduced to 
eliminate air pockets and to ensure proper flow through the assembled 
filter apparatus. The challenge organism, either Pseudomonas dimunata, 
Candida albicans or a "cocktail" consisting of (i) Pseudomonas dimunata, 
Bacillus subtilis, and Escherichia coli, or (ii) Staphylococcus aureus and 
Pseudomonas aeruginosa at a concentration of 10.sup.7 organisms per ml, 
was purged through the filter using a 3 cc syringe and manual pressure. 
Approximately 1 mL of liquid was expelled first and was checked for 
sterility so to ensure that the membrane was properly sealed. Three drops 
of eluate were collected and tested for sterility. The outlet tip was 
maintained in a sterile environment using a clean sterile cover and the 
entire unit was stored at 37.degree. C. for the course of the experiment. 
The sterility of the eluate was tested daily by collecting three drops 
(approximately 150 ml) of bacteria/media inoculum into sterile 
thioglycollate medium which was then placed in a 37.degree. C. shaker 
overnight and assessed the next day for sterility. As the medium/bacteria 
inoculum level lowered over the course of the experiment, the input 
syringe was removed and fresh sterile media was added and reattached to 
the filter holder unit. A filter was considered to have "failed" when the 
sterility check failed on two consecutive tests, the failed filter 
apparatus still held air pressure under water, and the failed sterility 
check demonstrated by gram stain the expected morphology of the test 
organism. 
Table I summarizes the results. "GS" is Gelman Supor 400 membranes; "GH" is 
Gelman HT650 membranes; "M" is Millipore membranes. The type of material 
that the membranes are composed of is indicated by "PES" for 
polyethersulfone and "PVDF" for polyvinylidene fluoride. "mM" indicates 
the micron pore size of the membrane. "-" indicates that the membrane was 
untreated. "MVDAG" indicates the membrane was treated to produce a silver 
coating by the metal vapor deposition method as described in Example 1. 
"TAg" indicates the membrane was treated to produce a silver coating by 
the electroless method of this invention as described in Example 2, Method 
1. "+P" indicates the silver coated membrane was treated with oxygen 
plasma to produce a silver oxide coating as described Example 7. "+B" 
indicates that the silver coated membrane was treated with BAC-S 
(benzalkoniumchloride thiol) to produce a layer of BAC-S over the silver 
coating as described in Example 8. "+BG" indicates that the silver coated 
membrane was treated with PHMB-S or PHMBCE-S. "+Cl" indicates that the 
membrane was treated with chlorine or NaCl. "+Br" indicates that the 
membrane was treated with bromine. "+Br+I" indicates that the membrane was 
treated with a mixture of bromine and iodine. "+I" indicates that the 
membrane was treated with iodine. "+I+PHMB" indicates that the silver 
iodide coated membrane additionally with a PHMB coating. "+PHMB" indicates 
that the membrane was coated with PHMB. "+PHMB+I" indicates that the 
membrane was coated with PHMB followed by silver iodide introduction. 
The numbers in the "Days" column indicate the average number of days until 
the filter failed, as determined by the criteria discussed above. A "&gt;#" 
indicates that no failure was detected for the duration of the test, i.e., 
the number of days indicated. 
The various growth media used are indicated by "B" for sterile bacterial 
media, "S" for sterile saline solution, and "T" for "Hypo Tears." a 
preservative free artificial tear solution by Johnson & Johnson. 
Sterilization of the membranes was achieved either by autoclaving at 121RC 
for 30 minutes ("A"), or placement in absolute ethanol for 20 minutes 
("Et"). The "No." indicates the number of samples of membranes that were 
tested 
TABLE 1 
______________________________________ 
Bacterial Challenge Experiments on Silver Coated Membranes 
Growth 
Steril- 
Mfgr Type mm Treatment 
Days Medium 
zation 
No. 
______________________________________ 
GS PES 0.45 -- 7 B A 3 
GS PES 0.45 MVDAG + P 
10 B A 3 
GS PES 0.45 TAg 22 B A 3 
GS PES 0.45 TAG + P 16 B A 3 
GS PES 0.45 MVDAG + B 
14 B A 3 
GS PES 0.45 TAG + B &gt;77 B A 3 
GS PES 0.45 7 B A 9 
GS PES 0.45 TAg &gt;25 B A 6 
GS PES 0.45 10 T A 9 
GS PES 0.45 TAg &gt;175 T A 9 
GS PES 0.45 3 S A 6 
GS PES 0.45 TAg &gt;57 S A 6 
GH PES 0.65 4 T A 3 
GH PES 0.65 TAg &gt;82 T A 3 
GS PES 0.65 1 S A 5 
GS PES 0.65 TAg &gt;145 S A 5 
m PVDF 0.22 5 B Et 1 
m PVDF 0.22 MVDAG &gt;14 B Et 1 
m PVDF 0.22 MVDAG + P 
&gt;14 B Et 1 
m PVDF 0.22 TAg &gt;14 B Et 1 
m PVDF 0.22 TAg + p &gt;14 B Et 1 
m PVDF 0.22 TAG + B &gt;14 B Et 1 
m PVDF 0.22 15 B A 3 
m PVDF 0.22 TAg &gt;170 B A 3 
GS PBS 0.45 TAg + B &gt;71 S A 3 
GS PES 0.45 THE + I + 
&gt;97 S Et 3 
PHMB 
GS PES 0.65 TAG + B &gt;71 S A 3 
GS PES 0.65 TAG + BG &gt;71 S A 3 
GS PES 0.65 TAG + Cl &gt;62 S A 3 
GS PES 0.65 TAG + Br &gt;36 S A 2 
5 
GS PES 0.65 TAG + Br + I 
&gt;20 S A 1 
5 
m PVDF 0.45 2 B Et 1 
m PVDF 0.45 MVDAG 5 B Et 1 
m PVDF 0.45 MVDAG + P 
&gt;11 B Et 1 
m PVDF 0.45 TAg &gt;11 B Et 1 
m PVDF 0.45 7 B A 3 
m PVDF 0.45 TAg &gt;70 B A 3 
______________________________________ 
Example 24 
Anti-Microbial Properties of Differently Treated Substances 
This example illustrates the anti-microbial effect of various substrates 
having immobilized thereon anti-microbial agents. The substrates include 
glass beads, polyethersulfone (PES) pellets, gold foil and PES membranes. 
The test was carried out as described in Example 20. The results are shown 
in Table 2. 
TABLE 2 
__________________________________________________________________________ 
TYPE MEDIUM 
ORGANISM 
COATING COUNT 1(24 hrs) 
NUMBER 
__________________________________________________________________________ 
1 Tear Soln (P)* 
PD 0 
2 Tear Soln (PF)* 
PD 6.2 .times. 10.sup.3 
3 Glass Beads 
A PD Uncoated 1.0 .times. 10.sup.4 
7 
4 Glass Beads 
A PD Silver coated 
63 7 
5 Glass Beads 
A PD Uncoated 2.3 .times. 10.sup.3 
2 
6 Glass Beads 
A PD Silver coated 
33 2 
7 PES Pellets 
A PD Uncoated 4.0 .times. 10.sup.3 
25 
8 PES Pellets 
A PD Silver coated 
0 25 
9 PES Pellets 
A PD Uncoated 2.0 .times. 10.sup.3 
5 
10 PES Pellets 
A PD Silver coated 
2 5 
11 Tear Soln (P)* 
A BC1 0 
12 Tear Soln (PF)* 
A BC1 3.5 .times. 10.sup.6 
13 PES Pellets 
A BC1 Uncoated 3.5 .times. 10.sup.6 
25 
14 PES Pellets 
A BC1 Silver coated 
0 25 
15 PES Pellets 
A BC1 Silver + Add. Coat..sup.1 
0 25 
16 PES Pellets 
B BC1 Uncoated 8.0 .times. 10.sup.6 
25 (New beads) 
17 PES Pellets 
B BC1 Silver coated 
0 25 (New beads) 
18 PES Pellets 
B BC1 Uncoated 8.0 .times. 10.sup.6 
25 (Used beads**) 
19 PES Pellets 
B BC1 Silver + Add. Coat..sup.1 
0 25 (Used beads**) 
20 PES Pellets 
B BC1 Silver + Add. Coat..sup.2 
0 25 (Used beads**) 
21 Tear Soln (P)* 
A CA 0 
22 Tear Soln (PF)* 
A CA 2.5 .times. 10.sup.4 
23 PBS Control 
B CA 8.4 .times. 10.sup.4 
24 PES Pellets 
B CA Uncoated 1.5 .times. 10.sup.5 
25 
25 PES Pellets 
B CA Silver + Add. Coat..sup.2 
10 25 
26 PES Pellets 
B CA Silver + Add. Coat..sup.3 
3.5 .times. 10.sup.2 
25 
27 Gold Foil 
B BC1 Uncoated 9.6 .times. 10.sup.5 
2.5 .times. 1.25.sup.+ 
28 Gold Foil 
B BC1 Silver + Add. Coat..sup.3 
0 2.5 .times. 1.25.sup.+ 
29 Gold Foil 
B BC1 Silver + Add. Coat..sup.3 
0 2.5 .times. 1.25.sup.+ 
30 Gold Foil 
B CA Uncoated 7.4 .times. 10.sup.3 
2.5 .times. 1.25.sup.+ 
31 Gold Foil 
B CA Silver + Add. Coat..sup.2 
1.6 .times. 10.sup.3 
2.5 .times. 1.25.sup.+ 
32 Gold Foil 
B CA Silver + Add. Coat..sup.3 
0 2.5 .times. 1.25.sup.+ 
33 Polypropylene (PP) 
B BC1 1 .times. 10.sup.6 
2.5 .times. 1.25.sup.+ 
34 Polypropylene (PP) 
B BC1 Silver salt + Add. Coat..sup.4 
0 2.5 .times. 1.25.sup.+ 
35 Polypropylene (PP) 
B BC2 Silver salt + Add. Coat..sup.4 
0 2.5 .times. 1.25.sup.+ 
36 Polypropylene (PP) 
B PSA Silver salt + Add. Coat..sup.4 
0 2.5 .times. 1.25.sup.+ 
37 Polypropylene (PP) 
B BC1 Silver salt + Add. Coat..sup.5 
0 2.5 .times. 1.25.sup.+ 
38 Polypropylene (PP) 
B BC2 Silver salt + Add. Coat..sup.5 
0 2.5 .times. 1.25.sup.+ 
39 Polypropylene (PP) 
B BC1 Silver salt + Add. Coat..sup.6 
0 2.5 .times. 1.25.sup.+ 
40 Polypropylene (PP) 
B BC2 Silver salt + Add. Coat..sup.6 
0 2.5 .times. 1.25.sup.+ 
41 Polypropylene (PP) 
B PSA Silver salt + Add. Coat..sup.6 
0 2.5 .times. 1.25.sup.+ 
Bacterial Challenge on 0.65 uM (Gelman PES, MT 650) membranes 
42 PBS Control BC2 2.5 .times. 10.sup.6 
43 0.65 uM PES 
B BC2 silver 0 13 mm.sup.++ 
44 0.65 uM PES 
B BC2 Silver salt 
0 13 mm.sup.++ 
1/30 Bacterial Challenge on 0.45 uM (Gelman PES, Super 450) membranes 
45 0.45 uM PES 
B BC1 1 .times. 10.sup.6 
13 mm.sup.++ 
46 0.45 uM PES 
B BC2 1 .times. 10.sup.6 
13 mm.sup.++ 
47 0.45 uM PES 
B BC1 Silver salt + Add. Coat.sup.4 
0 13 mm.sup.++ 
48 0.45 uM PES 
B BC2 Silver salt + Add. Coat.sup.4 
0 13 mm.sup.++ 
49 0.45 uM PES 
B PSA Silver salt + Add. Coat.sup.4 
0 13 mm.sup.++ 
50 0.45 uM PES 
B BC1 Silver salt + Add. Coat.sup.5 
0 13 mm.sup.++ 
51 0.45 uM PES 
B BC2 Silver salt + Add. Coat.sup.5 
0 13 mm.sup.++ 
52 0.45 uM PES 
B PSA Silver salt + Add. Coat.sup.5 
0 13 mm.sup.++ 
53 0.45 uM PES 
B BC1 Silver salt + Add. Coat.sup.6 
0 13 mm.sup.++ 
54 0.45 uM PES 
B BC2 Silver salt + Add. Coat.sup.6 
0 13 mm.sup.++ 
55 0.45 uM PES 
B PSA Silver salt + Add. Coat.sup.6 
0 13 mm.sup.++ 
__________________________________________________________________________ 
A: *Johnson & Johnson HypoTears Tear Solution 
B: B: Phosphate Buffered Saline 
(P) Preservative containing 
(PF) Preservative free 
Uncoated: No coating, control 
Silver coated: Coated with metallic silver 
Silver + Additional coating: 
.sup.1 Benzalkonium chloride thiol 
.sup.2 poly(hexamethylenebiguanide) thiol 
.sup.3 chainextended poly(hexamethylene biguanide) thiol 
.sup.4 Silver halide + poly(hexamethylenebiguanide) overcoat 
.sup.5 Silver iodide + poly(hexamethylenebiguanide) coating solution 
.sup.6 Poly(hexamethylenebiguanide) coating followed by AgI/KI 
introduction 
Silver salt: Inorganic salt of silver halide salts such as chloride or 
bromide or mixed salts of chloride, bromide and iodide 
Avg. surface area of each bead: 0.07 cm.sup.2 
Avg. surface area of each pellet: 0.02 cm.sup.2 
BC1: Cocktail contains p. dimunuta, B. subtlis, and E. Coli 
BC2: Cocktail contains S. aureus and p. aeruginosa 
PD: Pseudomonmas dimunate 
PSA: Pseudomonas aeruginosa 
CA Candida albicans 
**Stored for one month 
.sup.+ Dimension of foil (cm) 
.sup.++ Membrane diameter 
Equivalents 
Those skilled in the art will be able to ascertain, using no more than 
routine experimentation, many equivalents of the specific embodiments of 
the invention described herein. These and all other equivalents are 
intended to be encompassed by the following claims.