Methods and compositions for controlling biofouling using fluorosurfactants

The present invention relates to a method to inhibit bacteria from adhering to a submergible surface. The method contacts the submergible surface with an effective amount of at least one fluorosurfactant to inhibit bacterial adhesion to the submergible surface. The present invention also relates to a method for controlling biofouling of an aqueous system. This method adds an effective amount of at least one fluorosurfactant to inhibit bacteria from adhering to a submerged surface within the aqueous system. This method effectively controls biofouling without substantially killing the bacteria fouling organisms. The fluorosurfactant used in the method of the invention is an anionic or nonionic fluorosurfactant selected from: R.sub.f CH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.2 CO.sub.2 Li, (R.sub.f CH.sub.2 CH.sub.2 O)P(O) (ONH.sub.4).sub.2, (R.sub.f CH.sub.2 CH.sub.2 O).sub.2 P(O) (ONH.sub.4), (R.sub.f CH.sub.2 CH.sub.2 O)P(O) (OH).sub.2, (R.sub.f CH.sub.2 CH.sub.2 O).sub.2 P(O) (OH), R.sub.f CH.sub.2 CH.sub.2 O (CH.sub.2 CH.sub.2 O).sub.x H, R.sub.f CH.sub.2 CH.sub.2 O(CH.sub.2 CH.sub.2 O).sub.y H, R.sub.f CH.sub.2 CH.sub.2 SO.sub.3 H, or mixtures thereof. The group R.sub.f is F(CF.sub.2 CF.sub.2).sub.3-8, x varies from 2-20, and y varies from 2-20. The present invention also relates to a compositions containing a fluorosurfactant and useable in the above methods. The compositions comprise at least one fluorosurfactant in an amount effective to inhibit bacteria from adhering to submergible or submerged surfaces.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention uses fluorosurfactants to inhibit bacterial adhesion to 
submergible or submerged surfaces, particularly those surfaces within an 
aqueous system. The invention also relates to methods and compositions for 
controlling biological fouling. 
2. Description of Related Art 
Microorganisms adhere to a wide variety of surfaces, particularly surfaces 
in contact with aqueous fluids which provide a suitable environment for 
microbial growth. For example, microorganisms are known to adhere to ship 
hulls, marine structures, teeth, medical implants, cooling towers, and 
heat exchangers. Adhering to such submerged or submergible surfaces, 
microorganisms may foul the surface or cause it to deteriorate. 
In mammals, (e.g., humans, livestock, pets), microorganisms adhered to a 
surface may lead to health problems. Plaque, for example, results from 
microorganisms adhering to the surfaces of teeth. Medical implants with 
unwanted microorganism adhered to their surfaces often become crusted over 
and must be replaced. 
Scientific studies have shown that the first stage of biofouling in aqueous 
systems is generally the formation of a thin biofilm on submerged or 
submergible surfaces, i.e., surfaces exposed to the aqueous system. 
Attaching to and colonizing on a submerged surface, microorganisms such as 
bacteria, are generally thought to form the biofilm and modify the surface 
to favor the development of the more complex community of organisms that 
make up the advanced biofouling of the aqueous system and its submerged 
surfaces. A general review of the mechanisms and importance of biofilm as 
the initial stage in biofouling is given by C. A. Kent in "Biological 
Fouling: Basic Science and Models" (in Melo, L. F., Bott, T. R., Bernardo, 
C. A. (eds.), Fouling Science and Technology, NATO ASI Series, Series E, 
Applied Sciences: No. 145, Kluwer Acad. Publishers, Dordrecht, The 
Netherlands, 1988). Other literature references include M. Fletcher and G. 
I. Loeb, Appl. Environ. Microbiol. 37 (1979) 67-72; M. Humphries et. al., 
FEMS Microbiology Ecology 38 (1986) 299-308; and M. Humphries et. al., 
FEMS Microbiology Letters 42 (1987) 91-101. 
Biofouling, or biological fouling, is a persistent nuisance or problem in a 
wide varieties of aqueous systems. Biofouling, both microbiological and 
macro biological fouling, is caused by the buildup of microorganisms, 
macro organisms, extracellular substances, and dirt and debris that become 
trapped in the biomass. The organisms involved include microorganisms such 
as bacteria, fungi, yeasts, algae, diatoms, protozoa, and macro organisms 
such as macro algae, barnacles, and small mollusks like Asiatic clams or 
Zebra Mussels. 
Another objectionable biofouling phenomenon occurring in aqueous systems, 
particularly in aqueous industrial process fluids, is slime formation. 
Slime formation can occur in fresh, brackish or salt water systems. Slime 
consists of matted deposits of microorganisms, fibers and debris. It may 
be stringy, pasty, rubbery, tapioca-like, or hard, and have a 
characteristic, undesirable odor that is different from that of the 
aqueous system in which it formed. The microorganisms involved in slime 
formation are primarily different species of spore-forming and 
nonspore-forming bacteria, particularly capsulated forms of bacteria which 
secrete gelatinous substances that envelop or encase the cells. Slime 
microorganisms also include filamentous bacteria, filamentous fungi of the 
mold type, yeast, and yeast-like organisms. 
Biofouling, which often degrades an aqueous system, may manifest itself as 
a variety of problems, such as loss of viscosity, gas formation, 
objectionable odors, decreased pH, color change, and gelling. 
Additionally, degradation of an aqueous system can cause fouling of the 
related water-handling system, which may include, for example, cooling 
towers, pumps, heat exchangers, and pipelines, heating systems, scrubbing 
systems, and other similar systems. 
Biofouling can have a direct adverse economic impact when it occurs in 
industrial process waters, for example in cooling waters, metal working 
fluids, or other recirculating water systems such as those used in 
papermaking or textile manufacture. If not controlled, biological fouling 
of industrial process waters can interfere with process operations, lower 
process efficiency, wasting energy, plug the water-handling system, and 
even degrade product quality. 
For example, cooling water systems used in power plants, refineries, 
chemical plants, air-conditioning systems, and other industrial operations 
frequently encounter biofouling problems. Airborne organisms entrained 
from cooling towers as well as waterborne organisms from the system's 
water supply commonly contaminate these aqueous systems. The water in such 
systems generally provides an excellent growth medium for these organisms. 
Aerobic and heliotropic organisms flourish in the towers. Other organisms 
grow in and colonize such areas as the tower sump, pipelines, heat 
exchangers, etc. If not controlled, the resulting biofouling can plug the 
towers, block pipelines, and coat heat-transfer surfaces with layers of 
slime and other biologic mats. This prevents proper operation, reduces 
cooling efficiency and, perhaps more importantly, increases the costs of 
the overall process. 
Industrial processes subject to biofouling also include papermaking, the 
manufacture of pulp, paper, paperboard, etc. and textile manufacture, 
particularly water-laid non-woven textiles. These industrial processes 
generally recirculate large amounts of water under conditions which favor 
the growth of biofouling organisms. 
Paper machines, for example, handle very large volumes of water in 
recirculating systems called "white water systems." The furnish to a paper 
machine typically contains only about 0.5% of fibrous and non-fibrous 
papermaking solids, which means that for each ton of paper almost 200 tons 
of water pass through the headbox. Most of this water recirculates in the 
white water system. White water systems provide excellent growth media for 
biofouling microorganisms. That growth can result in the formation of 
slime and other deposits in headboxes, waterlines, and papermaking 
equipment. Such biofouling not only can interfere with water and stock 
flows, but when loose, can cause spots, holes, and bad odors in the paper 
as well as web breaks--costly disruptions in paper machine operations. 
Biofouling of recreational waters such as pools or spas or decorative 
waters such as ponds or fountains can severely detract from people's 
enjoyment of them. Biological fouling often results in objectional odors. 
More importantly, particularly in recreational waters, biofouling can 
degrade the water quality to such an extent that it becomes unfit for use 
and may even pose a health risk. 
Sanitation waters, like industrial process waters and recreational waters, 
are also vulnerable to biofouling and its associated problems. Sanitation 
waters include toilet water, cistern water, septic water, and sewage 
treatment waters. Due to the nature of the waste contained in sanitation 
waters, these water systems are particularly susceptible to biofouling. 
To control biofouling, the art has traditionally treated an affected water 
system with chemicals (biocides) in concentrations sufficient to kill or 
greatly inhibit the growth of biofouling organisms. See, e.g., U.S. Pat. 
Nos. 4,293,559 and 4,295,932. For example, chlorine gas and hypochlorite 
solutions made with the gas have long been added to water systems to kill 
or inhibit the growth of bacteria, fungi, algae, and other troublesome 
organisms. However, chlorine compounds may not only damage materials used 
for the construction of aqueous systems, they may also react with organic 
materials to form undesirable substances in effluent streams, such as 
carcinogenic chloromethanes and chlorinated dioxins. Certain organic 
compounds, such as methylenebisthiocyanates, dithiocarbamates, 
haloorganics, and quaternary ammonium surfactants, have also been used. 
While many of these are quite efficient in killing microorganisms or 
inhibiting their growth, they may also be toxic or harmful to humans, 
animals, or other non-target organisms. 
One possible way to control the biofouling of aqueous systems, which 
include the associated submerged surfaces, would be to prevent or inhibit 
bacterial adhesion to submerged surfaces within the aqueous system. This 
can be done, of course, using microbicides which, however, generally 
suffer from some of the disadvantages mentioned above. As an alternative, 
the present invention provides methods and compositions useful to 
substantially inhibit bacterial adhesion to a submerged or submergible 
surface and to control biofouling of aqueous systems. The invention 
obviates the disadvantages of prior methods. Other advantages of this 
invention will become apparent from a reading of the specifications and 
appended claims. 
SUMMARY OF THE INVENTION 
The present invention relates to a method to inhibit bacteria from adhering 
to a submergible surface. The method contacts the submergible surface with 
an effective amount of at least one fluorosurfactant to inhibit bacteria 
from adhering to the submergible surface. The fluorosurfactant used in the 
method is an anionic or nonionic selected from: R.sub.f CH.sub.2 CH.sub.2 
SCH.sub.2 CH.sub.2 CO.sub.2 Li, (R.sub.f CH.sub.2 CH.sub.2 O)P(O) 
(ONH.sub.4).sub.2, (R.sub.f CH.sub.2 CH.sub.2 O).sub.2 P(O) (ONH.sub.4), 
(R.sub.f CH.sub.2 CH.sub.2 O) P (O) (OH).sub.2, (R.sub.f CH.sub.2 CH.sub.2 
O).sub.2 P (O) (OH), R.sub.f CH.sub.2 CH.sub.2 O(CH.sub.2 CH.sub.2 
O).sub.x H, R.sub.f CH.sub.2 CH.sub.2 O(CH.sub.2 CH.sub.2 O).sub.y H, 
R.sub.f CH.sub.2 CH.sub.2 SO.sub.3 H, or mixtures thereof, wherein R.sub.f 
is F(CF.sub.2 CF.sub.2).sub.3-8, x and y each vary independently from 
2-20. 
The present invention relates also to a method for controlling biofouling 
of an aqueous system. This method adds to an aqueous system an effective 
amount of at least one fluorosurfactant described above to inhibit 
bacteria from adhering to submerged surfaces within the aqueous system. 
This method effectively controls biofouling without substantially killing 
the bacteria. 
The present invention also relates to a composition for controlling 
biofouling of an aqueous system. The compositions comprises at least one 
fluorosurfactant described above in an amount effective to inhibit 
bacteria from adhering to submerged surfaces within the aqueous system. 
DETAILED DESCRIPTION OF THE INVENTION 
In one embodiment, this invention relates to a method to inhibit bacteria 
from adhering to a submergible surface. A submergible surface is one which 
may at least partially be covered, overflowed, or wetted with a liquid 
such as water or another aqueous fluid or liquid. The surface may be 
intermittently or continually in contact with the liquid. As discussed 
above, examples of submergible surfaces include, but are not limited to 
ship or boat hulls, marine structures, teeth, medical implants, surfaces 
within an aqueous system such as the inside of a pump, pipe, cooling 
tower, or heat exchanger. A submergible surface may be composed of 
hydrophobic, hydrophilic, or metallic materials. Advantageously, using an 
anionic or nonionic fluorosurfactant according to invention can 
effectively inhibit bacteria from adhering to hydrophobic, hydrophilic, or 
metallic submergible or submerged surfaces. 
To inhibit the adhesion of a bacteria to a submergible surface, the method 
contacts the submergible surface with a fluorosurfactant. The surface is 
contacted with an effective amount of a fluorosurfactant, or mixture of 
fluorosurfactants, to inhibit bacterial adhesion to the surface. The 
fluorosurfactant may be applied to the submergible surface using means 
known in the art. For example as discussed below, the fluorosurfactant may 
be applied by spraying, coating or dipping the surface with a liquid 
formulation containing the fluorosurfactant. Alternatively, the 
fluorosurfactant may be formulated in a paste which is then spread or 
brushed on the submergible surface. Advantageously, the fluorosurfactant 
may be a component of a composition or formulation commonly used with a 
particular submergible surface. 
"Inhibiting bacteria from adhering" to a submergible surface means to allow 
a scant or insignificant amount of bacterial adhesion for a desired period 
of time. Preferably, essentially no bacterial adhesion occurs and more 
preferably, it is prevented. 
The amount of fluorosurfactant employed should allow only scant or 
insignificant bacterial adhesion and may be determined by routine testing. 
Preferably, the amount of fluorosurfactant used is sufficient to apply at 
least a monomolecular film of fluorosurfactant to the submergible surface. 
Such a film preferably covers the entire submergible surface. 
Contacting a submergible surface with a fluorosurfactant according to this 
method allows the surface to be pretreated against bacterial adhesion. 
Accordingly, the surface may be contacted with a fluorosurfactant then 
submerged in the aqueous system. 
The present invention relates also to a method for controlling biofouling 
of an aqueous system. An aqueous system comprises not only the aqueous 
fluid or liquid flowing through the system but also the submerged surfaces 
associated with the system. Submerged surfaces are those surfaces in 
contact with the aqueous fluid or liquid. Like the submergible surfaces 
discussed above, submerged surfaces include, but are not limited to, the 
inside surfaces of pipes or pumps, the walls of a cooling tower or 
headbox, heat exchangers, screens, etc. In short, surfaces in contact with 
the aqueous fluid or liquid are submerged surfaces and are considered part 
of the aqueous system. 
The method of the invention adds at least one fluorosurfactant to the 
aqueous system in an amount which effectively inhibits bacteria from 
adhering to a submerged surface within the aqueous system. At the 
concentration used, this method effectively controls biofouling of the 
aqueous system without substantially killing the bacteria. 
"Controlling biofouling" of the aqueous system means to control the amount 
or extent of biofouling at or below a desired level and for a desired 
period of time for the particular system. This can eliminate biofouling 
from the aqueous system, reduce the biofouling to a desired level, or 
prevent biofouling entirely or above a desired level. 
According to the present invention, "inhibiting bacteria from adhering" to 
a submerged surface within the aqueous system means to allow a scant or 
insignificant amount of bacterial adhesion for a desired period of time 
for the particular system. Preferably, essentially no bacterial adhesion 
occurs and more preferably, bacterial adhesion is prevented. Using a 
fluorosurfactant according to the invention can, in many cases, break up 
or reduce other existing attached microorganisms to undetectable limits 
and maintain that level for a significant period of time. 
While some fluorosurfactants may exhibit biocidal activity at 
concentrations above certain threshold levels, fluorosurfactants 
effectively inhibit bacterial adhesion at concentrations generally well 
below such threshold levels. According to the invention, the 
fluorosurfactant inhibits bacterial adhesion without substantially killing 
the bacteria. Thus, the effective amount of a fluorosurfactant used 
according to the invention is well below its toxic threshold, if the 
fluorosurfactant also has biocidal properties. For example, the 
concentration of the fluorosurfactant may be ten or more times below its 
toxic threshold. Preferably, the fluorosurfactant should also not harm 
non-target organisms which may be present in the aqueous system. 
A fluorosurfactant, or a mixture of fluorosurfactants, may be used to 
control biofouling in a wide variety of aqueous systems such as those 
discussed above. These aqueous systems include, but are not limited to, 
industrial aqueous systems, sanitation aqueous systems, and recreational 
aqueous systems. As discussed above, examples of industrial aqueous 
systems are metal working fluids, cooling waters (e.g., intake cooling 
water, effluent cooling water, and recirculating cooling water), and other 
recirculating water systems such as those used in papermaking or textile 
manufacture. Sanitation aqueous systems include waste water systems (e.g., 
industrial, private, and municipal waste water systems), toilets, and 
water treatment systems, (e.g., sewage treatment systems). Swimming pools, 
fountains, decorative or ornamental pools, ponds or streams, etc., provide 
examples of recreational water systems. 
The effective amount of a fluorosurfactant to inhibit bacteria from 
adhering to a submerged surface in a particular system will vary somewhat 
depending on the aqueous system to be protected, the conditions for 
microbial growth, the extent of any existing biofouling, and the degree of 
biofouling control desired. For a particular application, the amount of 
choice may be determined by routine testing of various amounts prior to 
treatment of the entire affected system. In general, an effective amount 
used in an aqueous system may range from about 1 to about 500 parts per 
million and more preferably from about 20 to about 100 parts per million 
of the aqueous system. 
The fluorosurfactant is an anionic or nonionic fluorsurfactant selected 
from R.sub.2 CH.sub.2 CH.sub.2 SCH.sub.2 CH.sub.2 CO.sub.2 Li, (R.sub.f 
CH.sub.2 CH.sub.2 O) P (O) (ONH.sub.4).sub.2, (R.sub.f CH.sub.2 CH.sub.2 
O).sub.2 P(O) (ONH.sub.4), (R.sub.f CH.sub.2 CH.sub.2 O)P(O) (OH).sub.2, 
(R.sub.f CH.sub.2 CH.sub.2 O).sub.2 P(O) (OH), R.sub.f CH.sub.2 CH.sub.2 O 
(CH.sub.2 CH.sub.2 O).sub.x H, R.sub.f CH.sub.2 CH.sub.2 O (CH.sub.2 
CH.sub.2 O).sub.y H, R.sub.f CH.sub.2 CH.sub.2 SO.sub.3 H, or mixtures 
thereof. In these fluorosurfactants, R.sub.f is F(CF.sub.2 
CF.sub.2).sub.3-8, x and y each vary independently from 2 to 20 and more 
preferably from 5 to 12. The fluorosurfactants useful in the present 
invention are available from DuPont Chemical Company and are sold as 
ZONYL.RTM. fluorosurfactants. 
The methods according to the invention may be part of an overall water 
treatment regimen. The fluorosurfactant may be used with other water 
treatment chemicals, particularly with biocides (e.g., algicides, 
fungicides, bactericides, molluscicides, oxidizers, etc.), stain removers, 
clarifiers, flocculants, coagulants, or other chemicals commonly used in 
water treatment. For example, submergible surfaces may be contacted with a 
fluorosurfactant as a pretreatment to inhibit bacterial adhesion and 
placed in aqueous system using a microbicide to control the growth of 
microorganisms. Or, an aqueous system experiencing heavy biological 
fouling may first be treated with an appropriate biocide to overcome the 
existing fouling. A fluorosurfactant may then be employed to maintain the 
aqueous system. Alternatively, a fluorosurfactant may be used in 
combination with a biocide to inhibit bacteria from adhering to submerged 
surfaces within the aqueous system while the biocide acts to control the 
growth of microorganisms in the aqueous system. Such a combination 
generally allows less microbicide to be used. 
"Controlling the growth of the microorganisms" in an aqueous system means 
control to, at, or below a desired level and for a desired period of time 
for the particular system. This can be eliminating the microorganisms or 
preventing their growth in the aqueous systems. 
The fluorosurfactant may be used in the methods of the invention as a solid 
or liquid formulation. Accordingly, the present invention also relates to 
a composition containing a fluorosurfactant. The composition comprises at 
least one fluorosurfactant in an amount effective to inhibit bacteria from 
adhering to a submergible surface or a submerged surface within an aqueous 
system. When used in combination with another water treatment chemical 
such as a biocide, the composition may also contain that chemical. If 
formulated together, the fluorosurfactant and water treatment chemical 
should not undergo adverse interactions that would reduce or eliminate 
their efficacy in the aqueous system. Separate formulations are preferred 
where adverse interactions may occur. 
Depending on its use, a composition according to the present invention may 
be prepared in various forms known in the art. For example, the 
composition may be prepared in liquid form as a solution, dispersion, 
emulsion, suspension, or paste; a dispersion, suspension, or paste in a 
non-solvent; or as a solution by dissolving the fluorosurfactant in a 
solvent or combination of solvents. Suitable solvents include, but are not 
limited to, acetone, glycols, alcohols (such as isopropanol), ethers, or 
other water-dispersible solvents. 
Aqueous formulations are preferred. The composition may be prepared as a 
liquid concentrate for dilution prior to its intended use. Common 
additives such as surfactants, emulsifiers, dispersants, and the like may 
be used as known in the art to increase the solubility of the 
fluorosurfactant or other components in a liquid composition or system, 
such as an aqueous composition or system. In many cases, the composition 
of the invention may be solubilized by simple agitation. Dyes or 
fragrances may also be added for appropriate applications such as toilet 
waters. 
A composition of the present invention may also be prepared in solid form. 
For example, the fluorosurfactant may be formulated as a powder or tablet 
using means known in the art. The tablets may contain a variety of 
excipient known in the tableting art such as dyes or other coloring 
agents, and perfumes or fragrances. Other components known in the art such 
as fillers, binders, glidants, lubricants, or antiadherents may also be 
included. These latter components may be included to improve tablet 
properties and/or the tableting process. 
The following illustrative examples are given to disclose the nature of the 
invention more clearly. It is to be understood, however, that the 
invention is not limited to the specific conditions or details set forth 
in those examples.

EXAMPLES 
Test Method: The following method effectively defines the ability of a 
chemical compound to inhibit bacterial adhesion, or attack the formation 
of existing attached bacteria on various types of surfaces. As an 
overview, bioreactors were constructed in which approximately 1 
in..times.3 in. slides (glass or polystyrene) were fixed to the edge of 
the bioreactor. The lower ends (approx. 2 in.) of the slides dipped into a 
bacterial growth medium pH 7 within the bioreactor which contained a known 
concentration of the test chemical. Following inoculation with known 
bacterial species, the test solutions were stirred continuously for 3 
days. Unless otherwise indicated in the results below, the medium within 
the bioreactor was turbid by the end of three days. This turbidity 
indicated that the bacteria proliferated in the medium despite the 
presence of the chemical tested. This also shows that the chemical, at the 
concentration tested, showed substantially no biocide (bactericidal) 
activity. A staining procedure was then used on the slides in order to 
determine the amount of bacteria attached to the surfaces of the slides. 
Construction of Bioreactors: The bioreactors comprised a 400 ml glass 
beaker over which a lid (cover from a standard 9 cm diameter glass petri 
dish) was placed. With the lid removed, slides of the material of choice 
were taped at one end with masking tape and suspended inside the 
bioreactor from the top edge of the beaker. This allows the slides to be 
submerged within the test medium. Typically, four slides (replicates) were 
uniformly spaced around the bioreactor. The score presented below are the 
average of the four replicates. A magnetic stirring bar was placed in the 
bottom of the unit, the lid positioned, and the bioreactor autoclaved. 
Glass slides were used as examples of hydrophillic surfaces and 
polystyrene (polystyr.) as examples of hydrophobic surfaces. Bacterial 
Growth Medium: The liquid medium utilized in the bioreactors was described 
previously by Delaquis, et al., "Detachment Of Pseudomonas fluorescens 
From Biofilms On Glass Surfaces In Response To Nutrient Stress", Microbial 
Ecology 18:199-210, 1989. The composition of the medium was: 
______________________________________ 
Glucose 1.0 g 
K.sub.2 HPO.sub.4 5.2 g 
KH.sub.2 PO.sub.4 2.7 g 
NaCl 2.0 g 
NH.sub.4 Cl 1.0 g 
MgSO.sub.4.7H.sub.2 O 0.12 g 
Trace Element 1.0 ml 
Deionized H.sub.2 O 1.0 L 
Trace Element Solution: 
CaCl.sub.2 1.5 g 
FeSO.sub.4.7H.sub.2 O 1.0 g 
MnSO.sub.4.2H.sub.2 O 0.35 g 
NaMoO.sub.4 0.5 g 
Deionized H.sub.2 O 1.0 L 
______________________________________ 
The medium was autoclaved and then allowed to cool. If a sediment formed in 
the autoclaved medium, the medium was resuspended by shaking before use. 
Preparation of Bacterial Inocula: Bacteria of the genera Bacillus, 
Flavobacterium, and Pseudomonas were isolated from a paper mill slime 
deposit and maintained in continuous culture. The test organisms were 
separately streaked onto plate count agar and incubated at 30.degree. C. 
for 24 hours. With a sterile cotton swab, portions of the colonies were 
removed and suspended in sterile water. The suspensions were mixed very 
well and were adjusted to an optical density of 0.858 (Bacillus), 0.625 
(Flavobacterium), and 0.775 (Pseudomonas) at 686 nm. 
Biofilm Production/Chemical Testing: To four separate bioreactors was added 
200 ml of the sterile medium prepared above. Chemicals to be evaluated 
were first prepared as a stock solution (40 mg/2 ml) using either water or 
a 9:1 acetone: methanol mixture (acet./MeOH) as a solvent. A 1.0 ml 
aliquot of the stock solution was added to the bioreactor using moderate, 
continuous magnetic stirring. This provided an initial concentration of 
100 ppm for the test compound. One bioreactor (Control) contains no test 
compound. Aliquots (0.5 ml) from each of the three bacterial suspensions 
were then introduced into each bioreactor. The bioreactors were then 
provided with continuous stirring for three days to allow for 
proliferation of the bacterial population and deposition of cells onto the 
surfaces of the slides. 
Evaluation of Results: ZONYL.RTM. FSA fluorosurfactant and ZONYL.RTM. FSN 
fluorosurfactant were evaluated using the above procedure. After the test 
was complete, the slides were removed from the bioreactors and were 
positioned vertically to permit air drying. The degree of adhesion of 
bacteria to the test surface was then estimated using a staining 
procedure. The slides were briefly flamed in order to fix the cells to the 
surface, and then transferred for two minutes to a container of Gram 
Crystal Violet (DIFCO Laboratories, Detroit, Mich.). The slides were 
gently rinsed under running tap water, and then carefully blotted. The 
degree of bacterial adhesion was then determined by visual examination and 
subjective scoring of each slide. The intensity of the stain is directly 
proportional to the amount of bacterial adhesion. 
The following biofilm scores are given: 
______________________________________ 
0 = essentially none 3 = moderate 
1 = scant 4 = heavy 
2 = slight 
______________________________________ 
Chemical treatments were evaluated relative to the Control which typically 
receive an average score for the four bioreactor slides in the 3-4 range. 
Compounds which receive an average score in the 0-2 range were considered 
effective to prevent the adhesion of bacteria to the submerged slides. The 
results are shown in the following Table. 
______________________________________ 
Compound Solvent Conc. ppm MIC.sup.1 
Slides Score 
______________________________________ 
ZONYL .RTM. FSA 
acetone 100 &gt;500 glass 1 
ZONYL .RTM. FSA 
acetone 100 polystyr. 
0 
ZONYL .RTM. FSN 
acetone 100 &gt;500 glass 1 
ZONYL .RTM. FSN 
acetone 100 polystyr. 
0 
______________________________________ 
.sup.1 Minimum Inhibitory Concentration (MIC) for each compound against 
the bacteria E. Aerogenes using an 18 hour Basal Salts test both at pH 6 
and at pH 8 unless otherwise noted. 
ZONYL .RTM. FSA fluorosurfactant is a product of DuPont Chemicals, 
Wilmington Delaware. ZONYL .RTM. FSA fluorosurfactant contains 23-25 wt % 
lithium 3(1H,1H,2H,2Hflouroalkyl)thiopropionate (CAS No. 6553069-0); 0-2 
wt % telomer B 2carboxyethyl sulfide (CAS No. 6553083-8); 35-40 wt % 
isopropyl alcohol (CAS No. 6763-0); and 35-40 wt % water. ZONYL .RTM. FSN 
fluorosurfactant is a product available from DuPont Chemicals, Wilmington 
Delaware. The ZONYL .RTM. FSN product contains 40 wt % R.sub.f CH.sub.2 
CH.sub.2 O(CH.sub.2 CH.sub.2 O).sub.12 H, where R.sub.f is F(CF.sub.2 
CF.sub.2).sub.3-8, (CAS No. 6554580-4); 30 wt % isopropyl alcohol (CAS No 
6763-0); 30 wt % water; and less than 0.1 wt % 1,4dioxane (CAS No. 
12391-1). 
Minimum Inhibitory Concentration (MIC) for each compound against the 
bacteria E. Aerogenes using an 18 hour Basal Salts test both at pH 6 and 
at pH 8 unless otherwise noted. 
ZONYL.RTM. FSA fluorosurfactant is a product of DuPont Chemicals, 
Wilmington, Del. ZONYL.RTM. FSA fluorosurfactant contains 23-25 wt 5 
lithium 3-(1H,1H,2H,2H-flouroalkyl)thiopropionate (CAS No. 65530-69-0); 
2-2 wt. % telomer B 2-carboxyethyl sulfide (CAS No. 65530-83-8); 35-40 wt 
% isopropyl alcohol (CAS No. 67-63-0); and 35-40 wt % water. ZONYL.RTM. 
FSN fluorosurfactant is a product available m DuPont Chemicals, 
Wilmington, Del. The ZONYL.RTM. FSN duct contains 40 wt % R.sub.f CH.sub.2 
CH.sub.2 O(CH.sub.2 CH.sub.2 O).sub.12 H, where R.sub.f is F(CF.sub.2 
CF.sub.2).sub.3-8, (CAS No. 65545-80-4); 30 wt % isopropyl alcohol (CAS 
No. 67-63-0); 30 wt % water; and less than 0.1 wt % 1,4-dioxane (CAS No. 
123-91-1). 
While particular embodiments of the invention have been described, it will 
be understood, of course, that the invention is limited to those 
embodiments. Other modifications may be made. The appended claims are 
intended to cover any such modifications as fall within the true spirit 
and scope of the invention.