USE OF NOVEL COMPOUNDS TO RELEASE NUCLEOTIDES FROM LIVING CELLS RELATED APPLICATION This application is based on U. S. Provisional Application No. 60/495,440 titled"Use of novel compounds to release nucleotides from living cells"filed on 15 August 2003.

FIELD OF THE INVENTION The present invention relates to a method for effecting a rapid and comprehensive release of cellular contents, including nucleotides and other associated molecules of interest from living cells.

BACKGROUND OF THE INVENTION Many industries and markets have a requirement to detect the presence of contamination in their products or samples. Chief among these contaminants are microbial cells, such as bacteria, yeasts or molds. These types of cells are microscopic in size, effectively impossible to detect by visible means.

Most conventional methods to detect microbial cells rely on techniques that encourage the growth of possible contaminants until the growth can be seen by the naked eye; a process that can take many days. The delays caused by this requirement are costly and even potentially dangerous.

There is therefore a need to detect microbial contamination as rapidly as possible. One approach commonly taken to effect this is to isolate and detect specific chemical markers within contaminating microbial cells. One example is the molecule Adenosine Triphosphate ("ATP"), which is universally present in all living matter, where it serves as a source of energy for processes within cells. ATP can be quickly detected using the firefly luciferase reaction-addition of ATP to a purified preparation of the firefly enzyme luciferase and its substrate luciferin will produce an instant light emission, a phenomenon known as ATP- bioluminescence. This light is proportional to the amount of ATP present, and can be measured using a sensitive light detecting instrument such as a luminometer.

Commercial systems are available, comprising luciferase-based reagent kits and luminometer instrumentation, that enable customers to quickly test their products and samples for ATP which, if found in unusual amounts, indicates the presence of microbial contamination.

One challenge to the development of tests for microbial contamination by ATP- bioluminescence is that the ATP required for detection is'hidden'or contained within the contaminating cells. Hidden ATP is not available to trigger the luciferase/luciferin light reaction, so in this form the test will not work. To enable the test to work, ATP first has to be released from contaminating cells, in a process typically referred to as extraction.

A second challenge to the development of a successful ATP-bioluminescence test is that a majority of samples contain ATP of non-microbial origin. An example of this is bovine ATP in milk, derived ultimately from the udder cells of the cow. This non-microbial ATP may conceal or smother the microbial ATP that the test is designed to detect. To counter this, it is possible to pre-treat the sample being tested with an ATP-destroying enzyme. Such enzymes are commonly used in commercial kits, and are generally referred to as apyrase.

Treatment of a sample with apyrase is successful because the apyrase is able to remove only the ATP it finds free in solution. During this pre-treatment, ATP locked up inside microbial cells is kept safely away from the action of the apyrase; however, once release/extraction of this microbial ATP has occurred, it is vulnerable to apyrase degredation, and typically a proportion of microbial ATP is indeed lost to the action of apyrase before it can be read.

Extraction is usually accomplished by chemical means-the most common types are based around a family called Quaternary Ammonium Compounds ("QAC's"). QAC's were first described in United States Patent 4,303, 752. Specific examples include quaternary ethoxylated ammonium chlorides, benzyl trimethyl ammonium chloride, cetyl trimethyl ammonium bromide and other generic cationic detergents under various trade names.

Microbial ATP extraction poses additional challenges, in that microbial cells are typically protected by robust cell walls. A successful extractant should: Release cell components such that they are available for subsequent detection and measurement by outside systems such as ATP-bioluminescence.

Release cell components from a variety of different types of cells, with accordingly different wall compositions (See, for example, U. S. Patent No.

5,558, 986 to Lundin, which is hereby incorporated by reference).

Effect this release in as rapid manner as possible (Id.).

Effect this release in a reproducible manner.

Enable the timely, reproducible release of a maximum of cell components.

Perform the extraction in a wide potential range of sample environments- which may include, but are not limited to, milk, cream, fruit juice, oil emulsions, detergent solutions, liquid dyestuffs and combinations thereof.

Not interfere significantly with the stability of the released components (such as ATP) prior to subsequent detection and measurement.

Not interfere significantly with subsequent detection and measurement systems (such as ATP-bioluminescence).

SUMMARY OF THE INVENTION The present invention relates to a method for effecting a rapid and comprehensive release of cellular contents, including nucleotides and other associated molecules of interest from living cells, using a novel family of quaternary ammonium compounds. In addition, the invention relates to a method of using dimethyl-dialkyl-ammonium halides having varying alkyl chain lengths for selective extraction of microbial cell contents. Finally, the use of these novel compounds exhibits potential to effect the inactivation of ATP-destroying enzymes (used to deplete a sample of its non-microbial ATP).

The present invention demonstrates a superior ability to release ATP from bacteria and yeasts in dairy products. The present invention also demonstrates a superior ability to release ATP from property in case of personal care products incubated in growth media, but with the possible exception of Pseudomonas in certain cases.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 illustrates the effect of the proportion of quaternary ethoxylated amine on the reaction kinetics and level of light emulsion as a function of time after adding ionic surface active agent in bacterial sample.

Figure 2 illustrates the baselines for a prior art kit verses a kit in accordance with the present invention.

Figure 3 illustrates the spiked samples for a prior art kit verses a kit in accordance with the present invention.

Figure 4 illustrates ratios between relative light units for a prior art kit verses a kit in accordance with the present invention.

Figure 5 illustrates the average relative light units for the different organisms tested in different products for a prior art kit verses a kit in accordance with the present invention.

Figure 6 illustrates the signal/blank values for a prior art kit verses a kit in accordance with the present invention.

Figure 7 illustrates the average relative light units for the different organisms tested in different products for a prior art kit verses a kit in accordance with the present invention.

Figure 8 illustrates the signal/blank values for a prior art kit verses a kit in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION It is known that surface active agents have been used to rupture (lyze) single cells in order to eliminate ATP from somatic cells (See, for example, U. S. Patent No. 3,745, 090), but the above method has drawbacks which limit its applicability. Rupturing the cell membrane allows enzymes to be released from somatic cells and these enzymes can cause interference in the later phases of the assay of nucleotides. U. S. Patent No. 3,745, 090 suggests both non- ionic detergents (octyl phenoxy polyethoxyethanol, terpenoid saponins, steroid saponins, sulfosuccinate glycosides, and fatty acid esters of sorbitol anhydrides) and ionic detergents can be used to rupture somatic cells. Some of these, particularly the ionic surface active agents, affect the permeability of the microbial cell wall and membrane and release

nucleotides (ATP, FMN and other small molecules) from microbial cells. As a result, such surfactants cannot be used for selective rupturing of somatic cells prior to measurement of microbial ATP or other nucleotides. The present invention describes methods of applying specific surface active agents for selective release of nucleotides (purine and pyridine nucleotides, and PMN) either for somatic or microbial cells. This selective release of nucleotides is accomplished without releasing enzymes from cells and the assay of nucleotides can be made without interference from undesired enzymatic hydrolysis of assayed nucleotides.

Concentration of nucleotides is measured in metabolic studies, biochemistry, clinical chemistry and bioassays. About one third of the known 2000 enzymes use purine nucleotides (ATP, ADP, AMP GTP, GMP, ITP, etc. ) and pyridine nucleotides (NAD, NADH, NADP, NADPH, CTP, UTP, etc. ) as substrates. In conventional methods, these substrates have been extracted from cells by destroying the cells by physical or chemical means and inactivating the enzymes in the cells by freezing, heating or with chemicals. In many conventional methods proteins have to be separated from the sample before the measurement of nucleotides. Such complicated manipulations can cause errors in the assay and make the sample preparation laborious. The present invention makes the sample preparation simple, rapid and reproducible. When these methods are used in conjunction with bioluminescent assay of metabolites, the measurement is specific to ATP, FMN, NADH OR NADPH; whichever is meant to be measured. The samples need not be deproteinized nor the nucleotides separated by chromatography or liquid extraction techniques.

Dimethyl-dialkyl-ammonium halides represent a novel class of Quaternary Ammonium compounds that can be used for efficient and effective release of cellular contents, such as nucleotides, from microbial organisms. Variation of the alkyl chain lengths of dimethyl- dialkyl-ammonium halides may affect their ability to act as nucleotide extractants. It is believed a shorter chain length of approximately 8 to 10 carbon atoms may represent a rapid, quantitative release. It is further believed that extending the chain length beyond 10 carbon atoms may allow for selectively extracting nucleotides from different microbial sources. For example, having an alkyl chain length greater than 10 carbon atoms may allow for the release of bacterial cell contents while yeast cell contents remain largely intact.

Dimethyl-dialkyl-ammonium halides also are able to inactivate apyrase enzymes while leaving luciferase enzyme molecules untouched and fully active. It is believed that this

property can be used to improve and optomise current test methods-inactivating apyrase eliminates the loss of microbial ATP that currently occurs in the interval between extracting microbial contents and adding the ATP-sensitive luciferase enzyme.

DESCRIPTION OF A PREFERRED EMBODIMENT In the present invention, the nucleotides are released from single cells in suspension or from mono-and bilayers of cells through the cell wall and cell membrane made permeable by means of the action of surface active agents. Certain surface-active agents change the permeability of the cell membrane by affecting the integrity of the lipid and phospholipid layer in the membrane. It is possible to select certain non-ionic surface active agents which do not lyze the somatic cell but make the cell membrane permeable to small-size molecules, such as purine and pyridine nucleotides. When the cell membrane is made permeable to small molecules, nucleotides diffuse out of the cells instantaneously, but enzymes and proteins having a large molecular size will not be able to penetrate the membrane and stay inside the cells. This allows a simple and rapid extraction of nucleotides from cells without having to inactivate enzymes in the cells. Released nucleotides in extracellular solution are stable for several minutes if the solution does not have endogenous enzymes or broken cells in high enough quantities to cause a rapid, undesired enzymatic hydrolysis of nucleotides in the solution.

By selecting a non-ionic surfactant that does not lyze somatic cells but only changes the permeability of the membrane, does not affect microbial cell walls, and does not inhibit the bioluminescent reaction, it is possible to release nucleotides selectively from somatic cells for assaying by bioluminescent systems. However, microbial cells, such as bacteria, yeasts, fungi and slime molds have a cell wall that is resistant to chemical and environmental factors.

The cell wall contains substances, such as muramic acid (a peptidoglycan) in bacteria, and chitinous substances in fungi which protect the more fragile cell membrane. Therefore, it is necessary to use stronger surface active agents, that is, ionic surface active agents to make the cell wall of microbes permeable for small molecules. The ionic surfactants are selected by their specific properties, such as the length of alkyl chain, degree of ethoxylation (lipophility or hydrophility) and presence of radicals, such as quaternary salts, to affect the permeability of the cell wall of microbial cells for releasing nucleotides. Since many ionic surfactants precipitate proteins and inactivate enzymes, it is necessary to use a surfactant that does not inactivate the luciferase enzyme or other enzymes used in the bioluminescent reaction, or the

reaction conditions have to arranged to such that the interference from the surfactant can be eliminated, for example, by dilution.

The sample preparation for releasing nucleotides from a suspension of somatic or microbial cells requires only the mixing of the surfactant in a concentration from about 0.02 to about 2% by volume of the total combined volumes of the sample suspension and surface active agent. The diffusion of metabolites from the cells through the membrane is so rapid that even ATP, which has a turnover time in the metabolism within the cell of less than one second, can be quantitatively released.

Samples having primarily microbial cells, for example activated sludge and microbial cultures, can be extracted for nucleotides without prior elimination of nucleotides of non- microbial cells. Microbial cell walls have substances, such as peptidoglycans, chitin and mucoidal substances that make the wall resistant to chemicals. Therefore, it is more difficult to release nucleotides from microbial cells than from somatic cells. According to the present invention, the application of ionic surface active agents changes the permeability of the cell wall and membrane of microbes to make them permeable for small size molecules, such as nucleotides, but not for enzymes. By"ionic surface active agents"is meant anionic or cationic surface active agents to the exclusion of non-ionic surface active agents. This selective permeability is obtained by treating microbes with ionic surface active agents, the best of which are those that contain quaternary ammonium salts and a fatty group with a chain length of 12 carbon atoms; however, any chain length of carbon atoms from 8 to 18 can be used.

Examples of suitable ionic surfactants for quantitative release of nucleotides from microbial cells for bioluminescent measurement with firefly and photobacterial systems are: ethoxylated amines, ethoxylated diamines, polyethylene glycol esters of fatty acids, and ethoxylated amides having a chemical structure of :

where R is a fatty alkyl group having 8-18 carbon atoms and x, y and z are numbers ranging from 2 to 50, and quaternary ammonium salts having a formula of : where R, and R2 are an alkyl, alkyl-aryl-alkyl, ethoxyalkyl, hydroxyalkyl, or ethoxylated alkylphenol with a 4 to 22 carbon atom chain and the ethoxylated alkyls having 2 to 15 ethoxyl groups, and R3 and R4 are alkyl groups having a 1 to 15 carbon atom chain and y, for example, a halogen, sulphate, sulphite or phosphate.

Combinations of the aforesaid amines and quaternary ammonium salts are particularly advantageous, since such combinations facilitate a precise release rate for the nucleotides.

Such surface active agents also include hyamine chloride, that is, diisobutyl cresoxy ethoxy ethyl dimethyl ammonium chloride.

Ethoxylated amines release nucleotides quantitatively from microbes, but the quaternary ammonium salts of ethoxylated amines penetrate the cell wall of microbes faster and quaternary salts are affected less by buffers, pH, and other agents possibly encountered in the sample than are ethoxylated amines. A 0.02-0. 5% solution of a mixture of 1 to 19 parts of an ethoxylated amine, and 1 to 19 parts of a quaternary ammonium salt of an ethoxylated amine,

both having a carbon atom chain length from 8 to 18, provide a complete and rapid release of nucleotides from bacteria, yeasts, fungi, and slime molds as well as from certain bluegreen, green, brown and red algae. Nucleotides are also released from somatic cells with these reagents, but due to the precipitation of some proteins by the reagents, the release can be incomplete. These reagents do not inhibit firefly luciferase in the degree that would interfere with the measurement. On the contrary, the presence of a low concentration of a quaternary ethoxylated amine (0. 001-0. 03%) enhances the turnover rate of the firefly luciferase and produces up to about twice as many photons per second during the first part of the reaction as is produced by the same concentration of ATP with the same luciferin-luciferase reagents in plain buffer solution.

The rate of release of ATP and the bioluminescent reaction are affected by the proportions of the ethoxylated amine and the quaternary salt of the ethoxylated amine. The release of ATP and other nucleotides from microbial cells is slow, but still quantitative, and the reaction rate of luciferase is the same as in buffer when ethoxylated amines are used for the release. By including a quaternary salt of an ethoxylated amine with the ethoxylated amine the rate of nucleotide release and the bioluminescent reaction can be increased from moderately slow to moderately fast depending on the proportion of the quaternary ethoxylated amine in the reagent.

The accompanying Figure illustrates the effect of the proportion of quaternary ethoxylated amine on the reaction kinetics and level of light emulsion as a function of time after adding ionic surface active agent in bacterial sample. Numbers refer to the percentage of ethoxylated amine and quarternary ethoxylated amine, respectively in a total concentration of 0. 1% in the sample. Both ethoxylated amine and quarternary ethoxylated amine had a chain length of 12 carbons. "A"refers to the time of adding the surface active agent and"B"to the time of adding the firefly reagent. In the tests 100 microliters of 0.2% aqueous solution of the ethoxylated amine and a quaternary ethoxylated amine were pipetted to 100 microliters volume of bacterial suspension (E. coli) and the sample was mixed by shaking for fifteen seconds before being measured in a photometer where 100 microliters of luciferin-luciferase reagent was injected to the sample in a light-tight reaction chamber just prior to the measurement. The ethoxylated amine alone releases ATP from bacteria in 15 seconds and produces the same reaction kinetics as ATP in plain buffer, that is a continuous emission of

light that decays 1 to 10% per minute depending on the proportions of the components in the luciferin-luciferase reagent.

The quaternary salt of an ethoxylated amine used alone causes too fast a reaction rate to allow easy and reproducible measurement of ATP. A mixture of the ethoxylated amine and a quaternary salt of an ethoxylated amine, having both a 12 carbon chain length, gives the possibility to select a desired rate of nucleotide release and reaction kinetics. As can be seen in the Figure, the level of light emission is increased about two times by the use quaternary salt over the ethoxylated amine alone as a releasing agent. More than one half of a quaternary salt of an ethoxylated amine in the reagent causes inactivation of the luciferase enzyme; thus the proportions have to be controlled.

While ethoxylated amines do release nucleotides reasonably rapidly from bacteria, they produce a slow release from yeasts and fungi which have an even stronger cell wall than bacteria. Therefore, it is beneficial to include a quaternary salt of an ethoxylated amine as part of the release reagent. One third of a quaternary ethoxylated amine and two thirds of ethoxylated amine give a complete release of nucleotides, such as ATP and FMN from most bacteria in a few seconds, and from Mycobacteria, yeasts and fungi in 30-60 seconds.

If the sample contains microbial cells only, or if the proportion of non-microbial cells is insignificant (for example, activated sludge, soil, sediments), the release of nucleotides from the sample is accomplished by simply adding, for example a mixture of an ethoxylated amine and a quaternary ethoxylated amine in an end-concentration of 0.05-0. 5% by volume of the sample. The reagent and sample are mixed and the reagent is allowed to remain in contact with the cells for a sufficient time to complete the release of nucleotides. After this the sample should be measured within five minutes to avoid any breakdown of nucleotides by the enzymes left inside the cells. These ionic reagents do inactivate some enzymes, among which is photobacterial luciferase. Therefore, the sample should be diluted 5 to 100 times after treatment and before measurement if FMN or pyridine nucleotides are measured in the sample with the bacterial bioluminescent system. The nucleotide release reagent for microbial cells, consisting of ethoxylated amines, can be adjusted to acidic and alkaline pH; thus the reduced and oxidized pyridine nucleotides can selectively be extracted from microbial cells.

The release reagent for microbial nucleotides requires a direct contact with the cell wall, in order to give a quantitative release. Therefore, the cells have to be in suspension. If cells form clumps or flocculates in the sample, they should preferably be dispersed prior to application of the reagent. The dispersion or homogenizing must not stress the cells because stress would affect the level of nucleotides in the cells.

Table 1, below, shows that a linear relationship is obtained between the number of bacterial cells and the concentration of released ATP when a 3: 7 mixture of an ethoxylated quaternary amine and ethoxylated amine were used to extract this purine nucleotide from E. coli suspension: Number of bacteria per milliliter Relative light units for 10 second integration 150, 000 1, 480 500, 000 4, 800 1,000, 000 9, 580 2,000, 000 18, 900 Table 1 The procedure was as follows: 100 microliters of 0.2% releasing reagent was added to 100 microliters of sample and the solution was mixed for 15 seconds. The sample was placed in the light-tight reaction chamber of the photon counter. Just prior to starting a 10-second integration, 100 microliters of firefly luciferin-luciferase reagent was added to the sample in a transparent cuvette in the reaction chamber. The bacteria were grown on a liquid nutrient medium and the number of cells was determined by standard colony counting. The different dilutions were made in physiological saline solution.

The extraction efficiency of the release reagent based on a quaternary ethoxylated amine and an ethoxylated amine mixture was also tested against the boiling tris-EDTA buffer and the perchloric acid extraction methods. The samples consisted of whole blood diluted with physiological saline 200 times, a suspension of E. coli bacteria in concentration of one million cells per milliliter, and a suspension of green algae, Chlorella sp.

Sample treatments were: Ionic surface active nucleotide releasing reagent: To a 100 microliter aliquot of sample solution, 100 microliters of a 0.2% aqueous solution of a 3: 7 part

mixture of an ethoxylated quaternary amine and ethoxylated amine was pipetted and mixed for 15 seconds (for blood and bacteria) or 60 seconds (for algae).

Perchloric acid method: 0.1 ml 1 N perchloric acid was pipetted to 1 ml of sample and mixed for one minute. Sample was neutralized by adding 0.1 ml 1 N NaOH.

Boiling tris-EDTA method: Nine milliliters of tris (0.02 molar) -EDTA (0.002 molar) at pH 7.4 was heated to boiling and 1 ml of sample solution was pipetted on the boiling buffer. Samples were boiled for three minutes and cooled on ice.

Measurement of ATP by bioluminescence: 100 microliter aliquots of extracted sample solution were pipetted into transparent glass cuvettes and these were placed into the light-tight reaction chamber of a photon counter. Just prior to the measurement, 100 microliters of firefly luciferin-luciferase reagent was injected into the sample. The light emission was integrated for 10 seconds and the results read on the digital display as relative light units. The relative light units were converted to ATP by internal standardization whereby a known quantity of ATP standard was added into an aliquot of the extracted sample and measured as above. The standard was added in 10 microliters in order not to change the total volume of the sample significantly. By subtracting the reading of the sample from that of the sample + added ATP standard a conversion factor for relative light units to ATP was calculated. The results of the extraction efficiency test are given in Table 2, below: Sample Ionic surface active Perchloric Tris-EDTA nucleotide releasing agent acid method boiling buffer Whole blood 100% 7% 80% E. Coli suspension 100% 3% 95% Chlorella sp., alga 100% 100% Table 2 In all sample types the ionic surface active reagent gave the highest extraction efficiency.

Boiling tris-EDTA buffer method extracted the same quantity of ATP from algae, but less from bacteria and blood. The low values obtained with perchloric acid method are due to co-

precipitation of ATP with the perchlorate during the neutralization step. Perchloric ion is also a strong inhibitor of the firefly bioluminescent reaction.

Examples Appendix A shows comparative results of bacterially spiked samples between an older dairy products test, using less effective releasing agents (Dessert Kit available from Celsis, Inc., 400 West Erie, Suite 300, Chicago Illinois 60610-6910 USA) compared to a new test in accordance with the present invention, using a 0.8 percent aqueous solution of didodecyl dimethyl ammonium chloride as an extractant or releasing reagent. These spiked samples were then measured. As a test sample various milk based dairy products were spiked with a number of bacterial strains. 50 microliters of milk based dairy product was incubated with 25 microliters of an apyrase enzyme solution in order to reduce non-microbial ATP, followed by an injection of 50 microliters of extractant. The samples were then allowed to stand for 5 seconds and 100 microliters of Luciferase-Luciferin was injected into the sample. The light signal was then recorded and expressed as relative light units (RLU). Figure 2 illustrates the baselines for a prior art kit verses a kit in accordance with the present invention. Figure 3 illustrates the spiked samples for a prior art kit verses a kit in accordance with the present invention. Figure 4 illustrates ratios between relative light units for a prior art kit verses a kit in accordance with the present invention.

Appendix B is similar to Appendix A and gives a comparison between a current, commercially available test (Biotrace kit available from Biotrace International Plc., The Science Park, Bridgend, Wales CF31 3NA United Kingdom) and a new test in accordance with the present invention, which uses halide containing quaternary ammonium compounds as releasing agents as disclosed in this application.

Appendix C also gives a comparison between an older test (Rapid Screen Kit available from Celsis, Inc. ) and a new test in accordance with the present invention, which uses halide containing quaternary ammonium compounds as releasing agents. Specifically, this Appendix shows test results for non-dairy types of samples incubated over 16 hours in a nutrient solution in order to allow microbes in the samples to multiply. The results are expressed as ratio of the light signal divided by the background noise signal-this ratio is used regularly to show differences in detection sensitivity. Figure 5 illustrates the average relative light units for the different organisms tested in different products for a prior art kit

verses a kit in accordance with the present invention. Figure 6 illustrates the signal/blank values for a prior art kit verses a kit in accordance with the present invention. Figure 7 illustrates the average relative light units for the different organisms tested in different products for a prior art kit verses a kit in accordance with the present invention. Figure 8 illustrates the signal/blank values for a prior art kit verses a kit in accordance with the present invention. This incubation technique is common for products such as shampoos and tooth pastes, which have lower numbers of microbes than other products. Incubation of such samples is a multiplying technique which allows a low number of microbes to be increased and thereby made more easily detectable. The new kit allows for much better and faster detection of bacterial strains which grow slowly such as Burkholderia and Candida.

Appendix D is similar to Appendix C except that the results are expressed as relative light units instead of signal to blank ratio as in Appendix C.

Appendix E describes the use of didodecyl dimethyl ammonium bromide as a selective releasing reagent for Gram negative bacteria in presence of yeast cells.

While the invention has been described with specific embodiments, other alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it will be intended to include all such alternatives, modifications and variations set forth within the spirit and scope of the appended claims.

Appendix A (Sheet 1 of 4) Chicano Lab Study 3-Concell'Unilait'kit vs current Concell 'Dessert' kit - further baselines and spiked samples Sheet 1 - All data, Plate Layout Sheet 2-Sorted data - Baselines Sheet 3-Sorted data-Spiked Samples Sheet 4-Protocol Author-A. Hearn Date-8 June 03 Raw data Controls unilait clrls dessert ctds Inst sulk 4 S Inst blk 4 7 ATP Ve 23S603 173312 ATP +ve 225945 162699 Plate Layout 1 2 3 4 5 6 7 8 9 10 11 12 A Choc FLCe Milk Choc Rlce Milk Choc So milk Choc So milk UHT whole UHT whole Irlsh Creme Iriah Creme French Vanllla French Vanilla HezeInut Hamlnut Butter Pecan Buter Pecsn Canee Canel9 Ameretlo Amaretlo Venllle HaYnul Venille HeYnut TaAee Coramel To% ee Carame e9 wM vee ee whl ue C eav whl cre av wlvl crea Hall d Helf Helf 8 Hall No vam la No sam le No aem le No sam le No nem la No sem le No nem le No sem le Choc Rice Milk Choc Rice Milk Choc Rice Mllk Choc Rica MiIk Choc Rlce Mllk Choc Wco Mllk Choc filce Mllk Choc Rice Milk Choc Wce Mllk Choc Rica Milk CAOC Rice Ulk Choc Rice Mllk a E. coll E. coli E. coll P. aeru P. aeru P. aeru S, aureus S. eureua S. eurws A. cereus B. cereus A. ceraua Choc Soy mllk Ghoc Soy mllk CAOC Soy milk Chx Soy milk Coc 5oy mllk Choc Soy milk Cx Soy milk Coc Soy mllk Choc Soy milk Coc Soy mllk Choc Soy milk Coc Soy mllk E v E. mli E. coll, E. coli P. eeru P. aerv p, eeru S, eureus s S. euraua 9. eureus B. ceraua B. cereus B. cereua UHT whole UHT whole UHT whole UHT whole UHT whole UHT whole UHT vmola UHT whole UHT whole UHT whole UHT whole UHT whole F E. mi E. coll E. rnll P. emu P. eeru P. earu 9. aureua S, emeus S. eureue B. ceraus 8. ureus B. cereus Vanilla Haz'nut Vamlle Hez'nut Venllle HezhW Vanilla Hazhut Vanllle HeYnut Venlllo Hezhut Vanlila HeYnul Venilla HaYnut Vanilla HeYnut Vanills HaYnut Vanllle HoYnut Venllle HaYnut G a E coll E. coli E. coll P. eeru . aeru P. eeru . aU eUa + e. aureu S. eureua B. cereus + B. cereua B. cereus Helf 8 Helt Half 8 Helf Hell 8 Hall Hell 8 Helf Hall 8 HBIf Hell 6 Helf Hell 8 Holf HdlB Hdl Hell d HBII Hnlt 8 HNI Helf 8 Nelf NBI (8 Hell H. E COll + E COll + E COII + P XrUP + P eEnUP + P e « ; rUg + S AUreU-+ S eUraUX + S eUreUX + B CZr UI + B C + B CerEUS PlaWb 1-UnilAlt 5s 1 8 +Eml 155464322 9 11 17 14 5 7 10 10 8 7 341 296 71 75 16 14 2 1 2 1 2 2 1 1 27864 27183 34333 50105 50576 48572 74260 90455 72605 213784 237511 234508 82974 76708 74174 64175 64869 68114 92060 96096 96012 5 119246 90674 243555 224857 248390 402434 306369 318080 71576 84569 87522 152128 139454 141273 77 78 75 11 11 12 422 430 408 12 7 44 189899 199880 187738 275010 305506 285873 48821 49803 53788 119250 125970 124947 Plate 2-unllalt tOs 1 9 12 4 369445543 9 6 22 19 7 6 12 13 8 7 219 220 61 91 19 17 2 2 1 1 2 1 1 1 16073 25757 26120 39302 35181 41031 59905 59145 48816 242592 244894 249230 50475 57805 56143 74971 82833 57502 76797 96570 79788 85074 86216 96926 124891 170198 177573 300038 305244 267718 96679 104557 93703 99148 95940 86743 48 64 81 16 14 15 334 325 329 17 10 10 140709 141311 155070 277482 272485 263110 88216 63843 70248 114336 113329 102955 Plate 3-Dessert 1 3 85 3 174776757 13 15 35 41 11 12 21 23 19 17 41 25 28 45 13 9 2 1 1 1 1 1 1 1 2006 2863 3186 18332 17818 13458 17213 15953 15721 568 87 66 22896 24865 32900 47288 46056 53096 29823 32479 29255 59803 63648 54504 32198 48288 41306 81859 67620 83837 MM 26738 27753 27209 27618 23523 28 44 38 21 24 24 111 91 108 24 23 22 32872 34954 28307 55577 57980 54142 14871 16284 11984 33003 29012 30061 Plate 4-Dessert 2 4 4 5998799 29 7 17 16 36 35 15 15 24 20 17 18 114 20 21 29 12 10 2 1 2 4 2 2 7 2 1208 1852 2061 12488 13320 12200 14482 10841 11108 1828 46 98 17922 18301 25603 31546 40334 45315 18868 27378 22889 42320 42393 39069 24890 31962 36349 68846 67641 66497 23351 22654 28430 32276 19833 20619 31 38 38 28 24 28 79 101 91 23 29 28 22489 28341 24788 63516 70775 47996 12307 8953 13112 34922 24530 21888 Plate 5-unlialt 10s 2 6 6 2 258456336 9 10 22 21 6 11 13 13 9 8 159 56 28 75 14 15 1 1 1 1 2 1 1 1 5227 11622 12577 19824 15864 14757 30680 37676 32381 196582 184029 191661 20914 23220 31910 37339 39141 44375 60383 69317 59755 125404 108192 105461 61412 73490 93481 196356 224458 222569 76987 75483 81172 91718 90092 80497 29 40 34 18 19 17 263 233 298 13 11 13 67357 86275 78391 265851 271971 296780 53085 57011 18888 85318 &55J3 77808 Plate 6-unifait Ss 2 7 7 3 4 3 9 6 6 5 5 5 3 is 10 25 24 8 9 16 16 9 9 41 42 49 110 16 14 2 1 1 1 1 1 1 1 5278 11330 12197 17594 15351 16668 34604 43370 37804 225054 200048 241477 26232 31682 43720 42823 46920 45127 57707 78513 80811 147592 123265 127190 bob052 87858 99912 247019 223983 217455 69919 75679 76789 111793 109440 101701 25 39 39 17 21 19 272 224 280 27 11 14 75304 119059 118713 328278 321280 324569 51905 49360 57456 103591 96487 91668 Appendix A (Sheet 2 of 4)<BR> Sample Baselines (sporadic 'flyers' highlighted in red; not used in average/SD/CV calculation UNILAIT KIT 5SEC EXTRACTION Choc Rice Milk Choc So milk UHT whole trish Creme French Vanilla Hazelnut Butter Pecan Canela Amaretto Vaniila Haz'nut Toffee Caramel Hea whi cream. Heavy whip cream Hbtf & Half 8 4 5 4 4 2 9 17 5 10 8 341 71 16 Plate 1 9 1 5 8 3 2 11 14 7 10 7 296 75 14 Plate 1 7 3 3 6 5 5 15 25 8 16 9 41 49 16 Plate 6 7 4 9 6 5 3 10 24 9 15 9 42 110 14 Plate6 8 3 6 6 4 3 11 20 7 13 8 180 76 5 Average 1 1 3 2 1 1 3 5 2 3 1 161 25 1 S 12 47 46 27 23 47 23 27 24 25 12 89 33 a CV UNILAIT KIT 10SEC EXTRACTION Choc Rice Miik Choc So milk UHT whale rish Gme French Vanilla Hazelnut Butter Pecan Canela Amaretto Vanilla Haz'nut Toffee Caramel Hea wh'cream Hea whi ceam Flalf 8 Half 9 4 6 4 5 4 9 22 7 12 8 219 61 19 Plate 2 12 3 9 4 5 3 8 19 6 13 7 220 91 17 Plate 2 6 2 5 4 6 3 9 22 6 13 9 159 28 14 Plate 5 8 2 8 5 3 6 10 21 11 13 8 56 75 15 Plate 5 9 3 7 4 5 4 9 21 8 13 8 164 64 16 Average 3 1 2 1 1 1 1 1 2 1 1 77 27 2 SD 29 35 26 12 26 35 9 7 32 4 10 47 42 14 CV CONCELL DESSERT KIT Choc Rice Milk Choc So milk UHT whole Irish GAme French Vanilla Hazelnut Burier Pecan Canela Amaretto Vanilta Haz'nut Toffee Caramel Hea whi aeam Hea whi am Half 8 Halt 3 3 7 7 6 5 13 35 11 21 19 41 28 13 Plate 3 a5 1 4 7 7 7 15 41 12 23 17 25 45 9 Plate 3 4 3 9 8 9 29 17 36 15 24 17 114 21 12 Pbte 3 | 65 | 1 | 4 | 7 | 7 | 7 | 15 | 41 | 12 | a | 17 | 25 | 45 | 9 Fbte3 | 4 | 3 | 9 | 8 | 9 | 29 | 17 | 36 | 15 j 24 | 17 | 114 | 21 j 12 Plate 4 4 5 9 7 9 7 16 35 15 20 182029 10 Plate 4 4 3 7 7 8 6 15 37 13 22 18 29 31 11 Average 1 221212322 1 11 10 2 SD 16 54 33 7 19 18 11 8 16 8 5 38 33 17 CV Choc Rice Milk Choc Soy milk UHT whole Irish GBme French Vanilla Hazelnut Butter Pecan Caneta Amaretto Vanilla Haz'nut Toffee Caramel Heavy whip aeam Heavy whip aeam Half & Half Choc Rice Milk Choc Soy milk UHT whole Insh CT#me Fnanch Vanilla Hazelnut Butter Pecan Caneta Amaretto Vanilb Haz'nut Toflee Car3met Heavv whip aeam Heavv whip aeam Hbtf & Hbif 8 3 6 6 4 3 11 20 7 13 8 180 76 15 UNILAJT Krr SSEC EXTRACTION 9 3 7 4 5 4 9 21 8 13 8 164 64 16 UNliAtTKIT10SECEXTRACTION 4 3 7 7 8 6 15 37 13 22 18 29 31 11 CONCELL DESSERT KIT Appendix A (Sheet 3 of 4)<BR> Spiked Sample Data UNILAII KII SStC E tXI RA ( : IIVN E. tri P. aeru inosa S. aureus Bacillus cereus Gnc RiceMN'k Cbc SoymiA UHT wlvle VaNle HeYM Half 8 Half Choc Rica Mlk CAOC mnlc UFfT wMb Vmvla HuhN Hal ! & Hall Ghoc Ro Mlk Chx So mi UiIT Wnb Van7le HaYV Half 8 HM Gnc Rice Mk Choc Sa m VM Mnole VeMe HazTM Ha1 8 Half 27964 82974 243555 77 89599 50105 64175 402434 1) 275010 74260 92060 7) 576 422 46621 213784 105936 152128 12 119260 Plab I 271&3 78708 224857 76 199680 50576 66E69 3063 1 305505 90455 96096 BC569 430 49803 Z17511 119246 139454 7 125970 PI. W 31333 74174 248390 75 87739 46572 68114 316060 12 265673 72605 96012 87522 408 53708 234508 9067A 147273 91 124947 Plata 3 5276 26232 68052 25 5304 7594 42623 247019 17 328278 34804 57707 69919 272 51905 225054 147592 111793 27 103591 P) a) e 6 11330 31662 87856 39 19059 5351 46920 223983 21 321280 43370 78513 75679 224 49360 200048 123265 109440 11 96487 PIaL6 12197 G3720 99912 39 118713 16668 <5f27 217455 19 32d569 37804 6081 75789 280 51456 241477 127190 f0171 14 14969 Rate6 1969 ! 5621E 163f01 55 1167l9 731N SE677 2lSEt7 75 306T79 58l81 60700--P509 779 Si513 125797 11l967 115965 19 110721 A-.'115"26M 25139 23 ii 19213 16436 70301 4 =115 23179 17a2 7060 91 na 15911 I 797E7 70l07 I 1t I 119l9 I SD 9 15 S1 I 31 55 7D 15 39 1 79 _ _ 73 9 27 T 7 16 17 T7 1 cl UNILAIT KIT 5SEC EXTRACTION E. coli P. aeru inosa S. aureus Baud ! tus cereus Gnc Rice A4k Chx So mi& Vlff whob Vmmn HetM Helf 8 Half CAx Fi » Milk Ctnc mio UHT MW Vmila HeYrW Haf 8 HaG Gnc Rim Afi Gvc So milk UHT vAOb Vmvlle HM Helf 8 Half Goc Woe Afk Gx So mM UM Ma VaNa HailNt Ha118HHNf 16073 $D475 124891 <B 1<0709 39302 74971 300038 16 277482 59905 76797 96679 334 68216 242592 65074 99146 17 114336 Patata 2 25757 57805 170196 64 t4l311 35161 62833 305244 14 272465 59145 96570 104557 325 63643 244694 66216 95940 10 113329 Rab 2 26120 56113 177573 61 ISW70 41031 57502 287716 15 263110 48816 79708 93703 329 702d8 24923 96926 86743 10 102955 Pbb2 5227 20(1914 61412 29 67357 19821 3739 196356 16 Zfi5A5i 30680 60383 ? 6987 263 53085 1965A2 125404 91)118 13 85316 PbbS It6Zl 23320 73C90 40 84275 15864 39141 22N558 9 27a911 37676 69317 75483 233 57011 1A4029 108192 90092 71 85533 runes 12577 31910 934BI 34 79391 14757 GC375 2225 17 296780 3Zlet 755 7172 298 46666 191861 105461 su97 13 77608 PIabS 16229 1007L 716041 16 111352 27660 526%4 5606t 16 275103 44767 7376E Mpyl7 297 60113 27619 ! 101312 90W0 13 96517 Awoe MSB 1672S 491M t4 MM9 21<t 1490S 46990 2 119M 1M72 t3a61 11667 41 6576 90329 1S203 66M 3 15761 Sn 51 42 z m u u4 e e n a : s is u u m u s a s cl CONCELL DESSERT KIT PLATE 3 E. co) P. aeruqinosa S. aureus Bacillus cereus Goc Rca A9c Gcc So mio UIiT wMb Vada HaYru Ha &Half Onc RiosChac GxymAt UHT vAOb VaHa HuhN He118 Half Choc Rice Ac Gnc So mdk UMTwfcb V WlH HaYM Natf & Ne Cdoc Rioe Mk Gx So mik UH1 v. lnb VaJla HazhN HNI b Haif 2006 22896 32198 28 37872 16332 47288 81859 21 55577 77213 29823 26942 11t 14871 568 59803 27209 24 33003 PIalo3 2863 24B65 4B268 44 34954 7616 48056 67620 24 57980 15953 32479 26738 91 162A4 87 63648 2761B D 29012 Pub3 3186 32900 41306 38 2A307 1345A 53096 83837 24 54142 5m 29255 27753 109 1964 66 54504 23523 22 30061 Plia 3 1208 17922 24890 31 22109 12488 31546 68846 28 67516 14482 78868 D351 79 12307 1828 42320 32276 23 34922 Rab14 152 18301 31962 38 26341 1S0 40334 67641 24 70T75 10841 2737 22654 701 8953 46 42393 19833 29 24530 He 4 2061 25603 36349 36 24788 2200 45315 66497 28 47996 1108 22689 26430 91 3112 98 39069 20619 26 2là66 PIOb 77% 1371E 75E29 36 26292 74269 1I27J 72717 35 58931 f4220 26749 25976 97 . zss4u 50290 251 xs xeass Aveny) f7 551E 615E 6 17% 127t 7N11 7M 3 7922 260 5051 22 12 2536 TO 10373 4745 3 4961 9o it 17 73 16 17 16 17 11 71 f1 79 19 9 1 30 157 21 19 11 17 CV E. cri P. aeruginosa S-aureus Badllus cereus dnc Rirn Mik ChocSovm UFIT wMb VmmarlleYrul Ha 8 Halt Cx Ree N5A Choe Sov mik tIFR wMb Vadt HaznW FWf B Ha ! ChC Riw hS Gac Sovmt UHT wtnle Vm 1e Hax71 Ha118 ilaC Chx Ris AA Che Sov m UHT. wlab Vmie HaYnd Had 8 Heli 19698 562B 16210A 55 148349 33144 58671 2&5BB7 15 306719 5888 0200 77509 339 51522. 16667 225397 118967. 1667 125964. 633 19. 16666667 110320. 5 UMWTI (fr 6016 21600 56762 26 51055 11811 22056 979J5 0 t0AB85 20521 2B423 26637 121 18028 75419 43122 43576 11 38433 UNRAFT Kn 2196 t3718 358m 36 26292 14269 44273 72717 25 56331 14220 26749 2597B 97 12910. 5 4488333333 502895 251796667 245 2wus9 CONCEAL DESSERT KILT CONCEAL DESSERT KIT E-coli P. aeruginosa S. aureus Badllus cereus doc Rioe AMk Clx SoY mIc UM whde Vmda FIaYM Half 8 Nelf CMc Rice A Chx Soy mik UHT wfcb Vada Haxhul Ha18 Hao Gac itios Ak CMc SoV UM Mvb Verde Haz'M Hall 6 llall Qoc Ro h9u Goc Sov m t1lif wtnk VaMa HaYnM Hd18 Hell CDMO ! HDNNI #DNNI NWIIII 7IDNNI #DIV/01 RONN ! ltONNt 710NNI riDIVIO ! tmIVA01 #ONNI tNn01 60IVNI 7lDIVrVI 7tOM01 #DNNI AOIVn91 oON. O ! CON01 RaW L"@ 10, OD 41 #DrVdOl ODNM ! #MM ! OMM ! mi #DIVM'*DrV/O-. 0m, VDIVM ! #DIVIO-#Wlo ! ODIVMI #DCVIO ! % ONNI CDNNI #DIW91 l3DNn01/fDIVNI Reto UedM Ss . Oesxn EXHIBIT A (Sheet 4 of 4) Unilait kit 1 Reagents ATX Lot No. : 14010503 Ref. No. : 321613000 Apyrase reagent Dilutor ED Lot No 05200301. Ref.no. 030501235 Apyrase buffer Cellsotver PD Lot. No. : 05200302 : Ref. No.: 090502175 cellsolvin reagent Sensilux DE200 tests Cat. No. : 321714000 Lot. No. : 11051002 light reagent DilutorPM Lot. No.: 05210303 Ref no: 090503575 light reagent buffer Instrument and settings sample volume 50 µL Instrument M 596 Instrument serial no. 801545 RLU coefficient 0, 24 Protocol name Unilait Protocol type well by well Vol ins 1 2S Vol inj 2 50 Vol inj 3 100 Timing scheme 1st/2nd ini Incub Incubation time 10 minutes Delay 2nd/3rd inj 5 seconds Delay 3rd inj/meas 0. 5 Measuring time 2 seconds Shaking duration (sec.) 15 Shaking interval (sec.) 30 Shaking speed (steps/s) 5000 injector speed 3500 total volume : 22SpL room temperature 23°C Concell Dessert 2 Reagents ATX Lot No.: 14061002 Ref. No. : 321613000 Apyrase reagent Dilutor ED Lot No. 15031002, Ref. no. 321726235 Apyrase buffer CellsolverPD. Ref. 321736175. lot. no. 13021102 cellsolvin reagent SensiluxDE 200 tests Cat. No.: 321714000 Lot. No : 11051002 light reagent DitutorPM Lot. No.: 12010203 Ref. no: 321723575 light reagent buffer Instrument and settings sa le volume 50 L Instrument M 596 Instrument serial no. 801545 RLU coefficient 24 Protocol name dessert1 Protocol type well by well Vol inj 1 25 Vol inj 2 50 Vol inj 3 100 Timing scheme 1st/2nd inj Incub Incubation time 10 minutes Delay 2nd/3rd inj 7 seconds Delay 3rd inj/meas 0.5 Measuring time 2 sec. Shaking duration (sec.) 15 Shaking interval (sec.) 30 Shaking speed (steps/s 5000 injector speed 3500 total volume : 225µL room temperature 23'C CONFIDENTIAL EXHIBIT B (1 sheet) European dairy Products Sample New Kit New Kit Competitor Kit 2 sec extr. Milk, 3.5% fat M596 M596 M596 Blank 10 11 11 Pseudomonas Inosa 24 208096 27920 Ratiu 2 18918 2538 : 1 5 91225 10210 Ratio 2 8293 928 -2 16 4709 537 Ratio 2 428 49 -3 32 368 63 Ratio 3 33 6 Staphylococcus aureus 67144 713264 32783 Ratio 6714 64842 2980 -1 11215 53894 5964 Ratio 1122 4899 542 -2 1151 3357 557 Ratio 115 305 51 114 242 78 Ratio 11 22 7 Bacillus stearothermophilus 57512 401707 56518 Ratio 5751 36519 5138 -1 3421 18170 1878 Ratio 342 1652 171 -2 401 755 251 Ratio 40 69 23 3 64 124 56 Ratio 6 11 5 Escherichla coll 75719 156671 44983 Ratio 7572 14243 4089 -1 19454 94886 17439 Ratio 1945 8626 1585 -2 1860 7377 1401 Ratio 188 671 127 -3 181 603 145 Ratio 18 55 13 Madibic, Volume extra Blank 14 41 30 Pseudomonas aerugino-ia 103468 267670 62198 Ratio 7392 6529 2073 -1 14597 15988 4351 Ratio 1043 390 145 -2 1530 1021 317 Ratio 109 25 11 -3 149 266 123 Ratio 11 6 4 Staphyiococcusaureus 77104 86614 16722 Ratio 5507 2113 557 -1 4517 4260 932 Ratio 323 104 31 -2 437 416 171 Ratio 31 10 6 -3 101 214 84 Ratio 7 5 3 Bacillus stearothermophilus 256900 65258 13445 Ratio 18350 1592 448 -1 24042 3097 782 Ratio 1717 76 26 -2 4119 335 156 Ratio 294 8 5 -3 347 138 67 Ratio 25 3 2 Escherlchla coll 29010 146836 43660 Ratio 2072 3581 1455 13785 17897 3853 Ratio 985 437 128 -2 1758 1159 361 Ratio 126 28 12 . 3 222 189 92 Ratio 16 5 3 Appendix C (Sheet 1 of 2) RapiScreen Kit New Kit, containing novel extractant strains Sample signal blank Aspergillus niger A. n. Eucerin 2 2 Candida albicans C. albi. Eucerin 1 637 4 265 Pseudomonas aeruginosa Ps ae. Eucerin 26 330 10 952 Staphylococcus aureus St. au. Eucerin 26 330 46 468 Aspergillus niger A. n. Toothpaste 1 1 Candida albicans C. albi. Toothpaste 17 99 Pseudomonas aeruginosa Ps. ae. Toothpaste 9 234 10461 Staphylococcus aureus St. au. Toothpaste 74 136 Aspergillus niger A. n. Listerine 1 3 Candida albicans C. albi. Listerine 989 2 879 Pseudomonas aeruginosa Ps. ae. Listerine 28 090 13 205 Staphylococcus aureus St. au. Listerine 28 090 57 937 Aspergillus niger A. n. Nyquil 2 2 Candida albicans C. albi. Nyquil 1 096 2 839 Pseudomonas aeruginosa Ps. ae. N uil 28 885 12 767 Staphylococcus aureus St. au. Nyquil 28 885 54 407 Candida albicans C. albi. Cleansin lotion 733 2 693 Pseudomonas aeruginosa Ps. ae. Cleansing lotion 21 645 11 774 Staphylococcus aureus St. au. Cleansing lotion 21 645 48 544 Burkholderia cepacia B. cep. Cleansing Lotion 8 2212 Aspergillus niger A. n. Eucerin 18 1504 Burkholderia cepacia B. cep. Eucerin 77 79 Burkholderia cepacia B. cep. Listerine 10 1114 Aspergillus niger A. n. Toothpaste 18 1721 Burkholderia cepacia B. cep. Toothpaste 87 62 Burkholderia cepacia B. cep. Nyquil 149 1526 Rapid Screen Kit Rapid Screen Kit III strains Sample Average RLU TAT Control* 2 386 2 084 Eucerin Control 3 798 2 152 Aspergillus niger A. n. Eucerin 6 948 4 725 Candida albicans C. albi. Eucerin 6 218 673 9 178 709 Pseudomonas aeruginosa Ps. ae. Eucerin 99 999 999 23 568 014 Staphylococcus aureus St. au. Eucerin 99 999 999 99 999 999 Toothpaste Control 1 661 765 Aspergillus niger A. n. Toothpaste 1 325 608 Candida albicans C. albi. Toothpaste 28 23375494 Pseudomonas aeruginosa Ps. ae. Toothpaste 153371868002780 Staphylococcus aureus St. au. Toothpaste 123 165 104 164 TAT Control 3 713 1 949 Listerine Control 3 560 1 726 Aspergillus niger A. n. Listerine 4 849 4 333 Candida albicans C. albi. Listerine 3 521 922 4 969 957 Pseudomonas aeruginosa Ps. ae. Listerine 99 999 999 22 791 965 Staphylococcus aureus St. au. Listerine 99 999 999 99 999 999 TAT Control 3 359 1 594 Nyquil Control 3462 1838 Aspergillus niger A. n. Nyguil 6 304 3 864 Candida albicans C. albi. Nyquil 3 794 861 5 217 875 Pseudomonas aeruginose Ps. ae. Nyquil 99 999 999 23 465 081 Staphylococcus aureus St. au. Nyquil 99 999 999 99 999 999 Cleansing Lotion Control** 4 620 2 060 Candida albicans C. albi. Cleansing lotion 3 387 935 5 548 598 Pseudomonas aeruginosa Ps. ae. Cleansing lotion 99 999 999 24 254 605 Staphylococcus aureus St. au. Cleansing lotion 99 999 999 99 999 999 Appendix C (Sheet 2 of 2) Rapid Screen Kit | Rapid Screen Kit III RS 3 vs. RS 1 strains Sample signal I blank AspN Candida Psdm Staph Aspergillus niger A. n. Eucerin 2 2 1. 2 1. 2 Candida albicans C. albi. Eucerin 1 637 4 265 2. 6 2. 6 Pseudomonas aeruginosa Ps. ae. Eucerin 26 330 10 952 0. 4 0. 4 Staphylococcus-aureus St. au. Eucerin26 33046468 1. 8 1. 8 Aspergillus niger A. n. Toothpaste 1 1 1. 0 1. 0 Candida albicans C. albi. Toothpaste 17 99 5. 8 5. 8 Pseudomonas aeruginosa Ps. ae. Tooth aste 9 234 10 461 1. 1 1. 1 Staphylococcus aureus St. au. Toothpaste 74 136 1. 8 1. 8 Asperillus niger A. n. Listerine 1 3 1. 8 1. 8 Candida albicans C. albi. Listerine 989 2 879 2. 9 2. 9 Pseudomonas aeruginosa Ps. ae. Listerine 28 090 13 20S 0 5 0. 5 Staphylococcus aureus St. au. Listerine 28 090 57 937 2. 1 2. 1 Aspergillus niger A. n. Nyquil 2 2 1. 2 1. 2 Candida albicans C. albi Nvquil 1 096 2 839 2 6 2. 6 Pseudomonas aeruginosa Ps. ae. Nyquil 28885 12767 0. 4 0. 4 Staphylococcus aureus St. au. N uil 28 885 54 407 1. 9 1. 9 Candida albicans C. albi. Cleansin lotion 733 2 693 3. 7 3. 7 Pseudomonas aeruginosa Ps. ae. Cleansing lotion 21 645 11 774 0. 5 0. 5 Staphylococcus aureus St. au. Cleansing lotion 21 645 48 544 2. 2 2. 2 Average 1. 3 3. 5 0. 6 2. 0 B. ce acia and 48 Hour Mold Results P Rapid Screen Kit Rapid Screen Kit III strains Sample Average RLU TAT Control 2 180 1 474 Cleansing Lotion Control 2601 1 092 Burkholderia cepacia B. cep. Cleansing Lotion 20408 2415979 Eucerin Control 3 538 1 558 Aspergillus niger A. n. Eucerin 64 059 2 343 051 Burkholdena cepacia B cep. Eucerin 273 429 122 759 Listemine Control 2988 1 335 Burkholderia cepacia B. cep. Lisierine 30 414 i 486 930 Toothpaste Control 580 254 Aspergillus niger A. n. Toothpaste 10 712 437 025 Burkholderia cepacia 8. ce. Toothpaste 50 397 15 771 Nyquii Control 2 861 i 124 Burkholderia cepacia B. cep. Nyquil 427 695 1 715 364 B. cepacia and 48 Hour Mold Results Rapid Screen Kit Rapid Screen Kit III RS 3 vs. RS 1 strains Sample signal/blank Burkh AspN 282 282 Burkholderia cepacia B cep Cleansing Lotion 8 | 2212 83 83 Aspergillus niger A. n. Eucedn 18 1504 1 1 Burkholderia cepacia B. cep. Eucenn 77 79 109 109 Burkholderia cepacia 8. ce. Listerine 10 1114 93 93 Aspergillus niger A. n. Toothpaste 18 1721 1 Burkholderia cepacia B. cep. Toothpaste 87 62 10 10 Burkholdena cepacia B. cep. Nyquil 149 1526 Average 81 88 Appendix D (1 shoot) Prapmation of oraontams 1. streins were token from siate and diluted in peptone water for CFU detormination with McFmtand stondord or microacope Burkholderte cepacia (ATCC 25416) inoculation with 500 CFU Pseudomones aeruginose (ATCC 10 145) Inoculation with 620 CFU Pseudomones seruginose (ATCC 9027) Inoculation with 640 CFU Candida albion (ATCC 10231) inoculation with 200 CFU Aspergillus niger inoculation with 200 Ascospores 2. Inocutated into modie with the PCP product (1%:use of TAT Broth with Tween:) grown after inoculation 48 h 3. Asseyod poreformed on Calsis Advonce Luminomater 4. during the whole exporiment the prepored cuttures were starnd in the retrgetator in or det to prevent further <BR> <BR> <BR> <BR> <BR> <BR> growth<BR> Kits exondned 1) Rapid Screen Kit II from Celsis no.: 1230834 (Rapid Screen) with LuminHance z) New Kit 200 pL New releasing reagent No.: 9020505 100 uL Luciferin-Luclferase Reagent : LuminATE (rHS), No.: 11020103 dissolved in LuminATE buffer for rHS : No. : 9120008 d vte prAed u ed RapiScreen II New Kit 0 0 .. in 0 0 ca, vd 0 0 vrdma nene : PCP 5 PCP I stmpitvdumphiSL 50 50 Vd 1 nj) 50 Vau 2 200 200 Vd kq 3 100 100 mmuroq 4ms 0 0 ^v « 3StepT. cel. 0 0 dSay 900sec. 0 dtiay 2 0 0 d « ay3 0 0 beigmundmet ; ulogubw O O lemptrsule O O ynturcmvd 0 0 RLU-mftwt 0. 586 0. 586 avera e RLU massurad p,. dut-. : I$ T-Mp-t. u. hyd. ; 7. d. fwr-. h : P.-4-Ph-l-,- (u saenrm diution Kittested RapisCnden 11 New Klt 10° 2503 153 10544611 BuikIaICMe cspaNe (ATCC26ei6) » 9i8B 13776 to'3847 5 190 to, 770943 740642 Ta cef 10'10 706 10 568 10 1 963 1 961 producl m. : 18 Mtlavptk MauNwmh'. mge0entr Thymd O. O6A % : Eucdyptol 0. 082'A. melyl salkytaW 0. % : d : 0. On1 % : wua. van n. md dil tion Ktt testeA RapiSceen il New Kit 10°795 841 4490095 t ATCC 2b16 t 10 10 312 16 581 10 S 3dS 7 745 (ATCC 25A76) to, 5345 7745 to, 50699963 52525 182 Mparyau, Npoi 7 p v 155254 1B464 10"4964 6090 p, d. 1-. : 20 C. 1d m. dl. i. ; lng, knl. : d,., . id FD & bi no 1 ; FD & C, W a 40 ; ft- : high (-I-... n w sUemned dilulion Kil tested Rap) Screen)) New Kit 1912992 7205929 au7hddwrsg 13511--1. 20. (ATCC p5116) to, 7523 9407 to, 1400108 1645284 to, 25930 29062 to'6386 6942 product m. : xt NmW Clww : aawe : punMa wr> o m : C I Cmrc 1 sHen ; Cav alcwlery wroMedecene ; rFn- dilution Kit tested Ra IScreen II New Kit 10° 651344 3324474 ß h7drSa evelf tO 5 066 b 940 (ATCC 2511 ) 10 <50666940 to, 99999999 12296746 AapsrpnuaNper pv 31015 12119 10'S 708 8 258 . oam,. : u Moi. wai mm, r. aw, u. : . r ; m. r w aeo. yrw. xea o. aa m ; i. a,.. m. , e : sodwm . nme : u. es mdW nea Cllution K'rttestad Ra iScreen II New K ! t 10° 490 2334294 RapiScmen 11 New Kh (A7CC 35418 tu 1102922569 10° 2379615 99999999 p., to"40 D61 51 453 to'6 184 10448 Appendix E RLU % vs DTAB Explanation Dodecyl trimethyl ammonium bromide 0. 50 Bacteria Dodecyl trimethyl 1. 35% 985963 100% to-1. 35% aqueous solution (DTAB) Dodecyl trimethyl 0. 50% 918394 93% Trioctyl 0. 05 % 929203 94% Trioctyl 0. 20 % 872688 89% Trioctyl methyl ammonium chloride 0. 05 to Didodecyl 0. 05 % 847920 86% 0. 20 % aqueous solution Didodecyt 0. 20 % 888448 90% Dodecyl trimethyl 1. 35% 1189402 100% Didodecyl dimethyl ammonium bromide 0. 05 Dodecyl trimethyi 0. 50% 949932 80% to 0. 20 % Trioctyl 0. 05 % 950118 80% Trioctyl 0. 20 % 896398 75% D ! dodecy) 0. 05% 1046059 88% Extraction time 3 sec Measurement with Sensilux 25 2sec All aqueous soln contained 1. 5 mM EDTA Dodecyl trimethyl 1. 35% 226997 100% Dodecyl trimethyl 0. 50% 204146 90% Trioctyl 0. 05 % 191689 84% Trioctyl 0. 20 % 174756 77% Didodecyl 0. 05 % 152311 67% Didodecyl 0. 20 % 148147 65% Yeast'S Dodecyl trimethyl 1. 35% 1336856 100% Dodecyl trimethyl 0. 50% 1247739 93% Trioctyl 0. 05 % 1148033 86% Trioctyl 0. 20 % 1263582 95% Didodecyl 0. 05 % 5845 0% Didodecyl 0. 20 % 19186 1% Dodecyi trimethyl 1. 35% 232206 100% Dodecyl trimethyl 0. 50% 217253 94% Trioctyl 0. 05 % 173872 75% Trioctyl 0. 20 % 191694 83% Didodecyl 0. 05 % 0 0% Didodecyl 0. 20 % 0 0%