MICROBIAL CELL VIABILITY ASSAY FOR DETECTION OF OR DETERMINING SLURRY CONTAMINATION

Abstract

A method is described for detecting or determining an amount of microbial contamination in an aqueous preparation. Also described, is a use of a luciferin/luciferase reagent containing at least one lytic agent for detecting or determining an amount of microbial contamination in an aqueous preparation including at least one particulate material. In addition, a use of such a method is described for detecting or determining an amount of microbial contamination in an aqueous preparation.

Claims

1. A method for detection of or determining an amount of microbial contamination in an aqueous preparation, the method comprising the steps of a) providing an aqueous preparation comprising at least one particulate material; b) providing at least one lytic agent; c) contacting the aqueous preparation of step a) with the at least one lytic agent of step b) at a concentration and for a time sufficient to lyse a microbial contamination present in the aqueous preparation such that ATP which is set free from the microbial contamination is dissolved in the aqueous preparation; and d) determining an amount of ATP content present in the aqueous preparation of step c), wherein the presence of microbial contamination is detected or the amount of microbial contamination is determined when the amount of ATP content is >0.

2. The method according to claim 1, wherein the at least one particulate material is a white mineral having a whiteness of R.sub.y of at least 65% measured according to DIN 53163.

3. The method according to claim 1, wherein the at least one particulate material of step a) is selected from the group consisting of natural ground calcium carbonate, natural and/or synthetic precipitated calcium carbonate, dolomite, surface-modified calcium carbonate, hydromagnesite, hydroxyapatite, kaolin, talcum, barite, aluminium hydroxide, aluminium silicate, titanium dioxide and mixtures thereof.

4. The method according to claim 1, wherein the aqueous preparation of step a) has (i) a pH value of from 2 to 12, and/or (ii) a solids content of up to 85.0 wt.-%, based on the total weight of the aqueous preparation.

5. The method according to claim 1, wherein the aqueous preparation of step a) is a paper making formulation, a paper coating formulation, fibre formulation, food formulation, pharmaceutical formulation, cosmetic formulation, plastic formulation and/or a paint formulation.

6. The method according to claim 1, wherein the at least one particulate material is not separated from the aqueous phase of the aqueous preparation between method steps a) to d) and/or the aqueous preparation of step a) is not diluted beyond a factor of 100.

7. The method according to claim 1, wherein the ATP is originated from a microbial contamination comprising gram-negative bacteria, gram-positive bacteria, fungi, molds, yeasts, algae or mixtures thereof.

8. The method according to claim 1, wherein the at least one lytic agent of step b) is selected from the group consisting of trichloroacetic acid (TCA), perchloric acid (PCA), citric acid, cetyl trimethylammonium bromide (CTAB), dodecyl trimethyl ammonium bromide (DTAB), chlorhexidine (CHEX), ultrasonication, other nonionic, anionic and cationic detergents, bacterial specific lytic enzymes, proteinase, antibiotics, alkylglucoside or alkylthioglucoside, betane detergents, quaternary ammonium salts, protamines, amines, and cationic, antibacterial, pore forming, membrane-active, and/or cell wall-active polymers, bacteriophage, surfactants, and mixtures thereof.

9. The method according to claim 1, wherein the at least one lytic agent of step b) yields sufficient lysis without inhibition by the presence of calcium and/or magnesium ions.

10. The method according to claim 1, wherein the at least one lytic agent of step b) is provided together with a sufficient amount of at least one chelating agent, preferably EDTA.

11. The method according to claim 1, wherein the amount of ATP content is determined by using a luciferin/luciferase reagent.

12. The method according to claim 1, wherein steps c) and d) are carried out simultaneously, or separately in the given order.

13. The method according to claim 1, wherein steps c) and d) are carried out simultaneously by using a luciferin/luciferase reagent comprising at least one lytic agent.

14. The method according to claim 1, wherein the presence of microbial contamination is detected or the amount of microbial contamination is determined in step d) by monitoring a luminescence signal.

15. The method according to claim 1, wherein the presence of microbial contamination is detected or the amount of microbial contamination is determined in step d) by using a luminometer.

16. The method according to claim 1, wherein the amount of microbial contamination is determined in step d) by (i) determining the amount of ATP content present in the aqueous preparation of step c), (ii) treating the aqueous preparation of step a) such that the ATP content present in the aqueous preparation is essentially completely hydrolyzed and/or the microbial contamination present in the aqueous preparation is essentially completely killed, and processing the aqueous preparation through method steps a) to d), and (iii) subtracting the amount of ATP content determined after step (ii) from the amount of ATP content determined in step (i).

17. A method of detecting or determining an amount of microbial contamination in an aqueous preparation, the method comprising detecting or determining the amount using a luciferin/luciferase reagent comprising at least one lytic agent, wherein the aqueous preparation comprises at least one particulate material as defined in claim 1.

18. (canceled)

19. The method according to claim 1, wherein the lower limit of detection (LoD) or limit of quantification is 110.sup.4 cfu/mL.

20. The method according to claim 3, wherein the at least one particulate material comprises natural ground calcium carbonate and/or synthetic precipitated calcium carbonate.

21. The method according to claim 4, wherein the pH value is from 6 to 12.

22. The method according to claim 4, wherein the pH value of from 7 to 10.5.

23. The method according to claim 4, wherein the solids content is from 10.0 wt.-% to 82.0 wt.-%.

24. The method according to claim 4, wherein the solids content is from 20.0 wt.-% to 80.0 wt.-%.

25. The method according to claim 6, wherein the aqueous preparation of step a) is not diluted beyond factor of 10.

26. The method according to claim 6, wherein the aqueous preparation of step a) is not diluted beyond factor of 2.

27. The method according to claim 6, wherein the aqueous preparation is not diluted between method steps a) to d).

28. The method according to claim 8, wherein for the at least one lytic agent at least one of the following applies: the bacterial specific lytic enzyme is lysostaphin or lysozyme; the proteinase is proteinase K; and the surfactant is triton X.

29. The method according to claim 9, wherein sufficient lysis without inhibition occurs by the presence of bivalent cations.

30. The method according to claim 9, wherein sufficient lysis without inhibition occurs by the presence of at least one particulate material of the aqueous preparation provided in step a).

31. The method according to claim 10, wherein the at least one chelating agent is EDTA.

32. The method according to claim 19, wherein the LoD or limit of quantification is 110.sup.3 cfu/m L.

33. The method according to claim 19, wherein the LoD or limit of quantification is 110.sup.2 cfu/m L.

Description

EXAMPLES

1. Materials

[0185] Calcium carbonate slurries A, B and C have been used. The characteristics of these calcium carbonate slurries are outlined in the following table 1:

TABLE-US-00001 TABLE 1 Characteristics of the calcium carbonate slurries Slurry A Slurry B Slurry C Residue > 45 m 10 ppm 50 ppm Residue > 75 m 10 ppm Top cut (d.sub.98) 4 m 30 m Median particle size by 0.7 m 5.7 m Sedigraph 5120: weight at constant density 1.6-2.2 m (Sedigraph), or by volume Sympatec HELOS: (HELOS) (d.sub.50) 2.2-3.0 m Particle fraction by weight 90% 24% Sedigraph 5120: at constant density 50-65% (Sedigraph), or by volume Sympatec HELOS: (HELOS) < 2 m 30-45% Tappi-whiteness [%] 94 95-97.5 Whiteness CIE L*, a*, b* 98/0.3/0.9 Ry (C/2) [%] 96% Solid content [wt.-%] 76.50 70 48-53 Density [kg m.sup.3] 1 929 1 790 pH 10 9.2 8-10 Viscosity [mPa * s] 350 1 000 600

2. Measurement Methods

[0186] The following measurement methods are used to evaluate the parameters given in the examples and claims.

[0187] Slurry pH Measurement

[0188] The pH of a slurry (or suspension) is measured at 20 to 25 C. using a Mettler Toledo Seven Easy pH meter and a Mettler Toledo InLab InLab413 SG pH electrode. A three point calibration (according to the segment method) of the instrument is first made using commercially available buffer solutions (e.g. Hamilton Duracal buffer solutions) having pH values of 4.01, 7.0 and 9.2 at 21.7 C. (from Aldrich). The reported pH values are the endpoint values detected by the instrument (the endpoint is when the measured signal differs by less than 0.1 mV from the average over the last 6 seconds).

[0189] The pH range of the calcium carbonate slurries tested is between [0190] 8.5-10 (100 g/L [20 C.]) for calcium carbonate slurry A [0191] 8.5-10.7 (100 g/L [20 C.]) for calcium carbonate slurry B [0192] 8.5-11.5 (100 g/L [20 C.]) for calcium carbonate slurry C

[0193] Solids Content of an Aqueous Slurry

[0194] The slurry solids content (also known as dry weight) is determined using a Moisture Analyser HR73 commercially available from Mettler-Toledo, Switzerland, with the following settings: temperature of 120 C., automatic switch off 3, standard drying of 5-20 g of slurry.

[0195] Particle Size Distribution (Mass % Particles with a Diameter <X) and Weight Median Diameter (d.sub.50) of a Particulate Calcium Carbonate-Containing Material

[0196] Weight median grain diameter and grain diameter mass distribution of a particulate calcium carbonate-containing material were determined via the sedimentation method, i.e. an analysis of sedimentation behaviour in a gravimetric field. The measurement was made with a Sedigraph 5120. Alternatively, the measurement can be made via laser diffraction, i.e. the particle size is determined in respect to particle volume by measuring the intensity of light scattered as a laser beam passes through a dispersed particulate sample assuming the particles approximate to a sphere. The measurement was made with a HELOS particle-size-analyser of Sympatec, Germany, and may be considered equivalent to weight distribution assuming a constant density throughout the particle size distribution.

[0197] The method and the instruments are known to the skilled person and are commonly used to determine grain size of fillers and pigments. The measurement is carried out in an aqueous solution of 0.1 wt.-% Na.sub.4P.sub.2O.sub.7. The samples are dispersed using a high speed stirrer and supersonics.

[0198] Density

[0199] Density measurements were made in accordance with DIN EN 323.

[0200] Viscosity

[0201] For the purpose of the present invention, the term viscosity or Brookfield viscosity refers to Brookfield viscosity. The Brookfield viscosity is for this purpose measured by a Brookfield (Type RVT) viscometer at 25 C.1 C. at 100 rpm using an appropriate spindle of the Brookfield RV-spindle set and is specified in mPa.Math.s. Based on his technical knowledge, the skilled person will select a spindle from the Brookfield RV-spindle set which is suitable for the viscosity range to be measured. For example, for a viscosity range between 200 and 800 mPa.Math.s the spindle number 3 may be used, for a viscosity range between 400 and 1 600 mPa.Math.s the spindle number 4 may be used, and for a viscosity range between 800 and 3 200 mPa.Math.s the spindle number 5 may be used.

[0202] Residues

[0203] The residues were determined in accordance with ISO 787/7.

[0204] Tappi-Whiteness

[0205] Pigment whiteness R457 was measured using an ELREPHO 3000 from the company Datacolor according to ISO 2469:1994 (DIN 53145-2:2000 and DIN 53146:2000).

[0206] Whiteness CIE L*, a*, b*

[0207] The CIELAB L*, a*, b* coordinates were measured using an ELREPHO 3000 from the company Datacolor according to DIN 6174 and barium sulphate as standard.

[0208] R.sub.y (C/2)

[0209] R.sub.y (C/2) was determined in accordance with DIN 53163.

3. Examples

Example 1: CaCO.SUB.3 .Slurry a with 75% Solid Content

[0210] Three samples of calcium carbonate slurry A were autoclaved. One autoclaved slurry was inoculated with a defined bacterial inoculum in order to receive a sample with 10.sup.3 cfu/mL and one with 10.sup.4 cfu/mL. The three different slurry samples (autoclaved, 10.sup.3 cfu/mL, 10.sup.4 cfu/mL) were measured 2-5 times with the Glomax 20/20 Luminometer (available from Promega) and the BacTiter Glo Reagent (available from Promega) and were plated in parallel on TSA plates in order to quantify the bacteria.

[0211] The results for the calcium carbonate slurries A with 75% solid content, tested without further dilution (overall dilution by addition of test reagents was 2) are shown in FIG. 1. Assay sensitivity was about 1E3 cfu/mL as defined by all measured samples above background level (here about 1 100 RLU). RLU was measured with Glomax 20/20 Luminometer (available from Promega) versus cfu/mL evaluated on TSA. Datapoints represent the mean of triplicates with standard deviation.

Example 2: CaCO.SUB.3 .Slurry B 60 with 70% Solid Content

[0212] A sample of the contaminated calcium carbonate slurry B was autoclaved. For different points of the curve, a 10-fold serial dilution of the contaminated slurry in the autoclaved slurry was prepared as follows: [0213] 1:10 dilution: 1 mL contaminated slurry (original contaminated calcium carbonate slurry B)+9 mL autoclaved calcium carbonate slurry B [0214] 1:100 dilution: 1 mL of the 1:10 diluted slurry+9 mL autoclaved calcium carbonate slurry B [0215] 1:1 000 dilution: 1 mL of the 1:100 diluted slurry+9 mL autoclaved calcium carbonate slurry B [0216] 1:10 000 dilution: 1 mL of the 1:1 000 diluted slurry+9 mL autoclaved calcium carbonate slurry B

[0217] Then the different diluted slurries were tested with the Glomax 20/20 Luminometer (available from Promega; 3 measurements of each diluted slurry) and the BacTiter Glo Reagent (available from Promega) and were plated on TSA in 10-fold dilutions in order to quantify the bacteria on the TSA plates.

[0218] The results are as follows: Autoclaved calcium carbonate slurry B for background level: [0219] <100 cfu/mL on TSA, 1 862 RLU with Luminometer

[0220] Contaminated calcium carbonate slurry B: [0221] 200 cfu/mL on TSA, 3 729 RLU with Luminometer [0222] 2.36E+03 cfu/mL on TSA, 15 090 RLU with Luminometer [0223] 5.18E+04 cfu/mL on TSA, 133 948 RLU with Luminometer [0224] 1.98E+05 cfu/mL on TSA, 825 758 RLU with Luminometer [0225] 3.60E+06 cfu/mL on TSA, 1 417 575 RLU with Luminometer

[0226] The results for the CaCO.sub.3 slurry calcium carbonate slurry B with 70% solid content, tested without further dilution (overall dilution by addition of test reagents was 2) are also shown in FIG. 2. Assay sensitivity was about 1E3 cfu/mL as defined by all measured samples above background level (here about 5E3 RLU). RLU was measured with Glomax 20/20 Luminometer (available from Promega) versus cfu/mL evaluated on TSA. Datapoints represent the mean of triplicates with standard deviation.

Example 3: CaCO.SUB.3 .Slurry C with 50% Solid Content

[0227] A sample of the contaminated calcium carbonate slurry C was autoclaved. For different points of the curve, a 10-fold serial dilution of the contaminated slurry in the autoclaved slurry was prepared: [0228] 1:10 dilution: 1 mL contaminated slurry (original contaminated calcium carbonate slurry C)+9 mL autoclaved calcium carbonate slurry C [0229] 1:100 dilution: 1 mL of the 1:10 diluted slurry+9 mL autoclaved calcium carbonate slurry C [0230] 1:1 000 dilution: 1 mL of the 1:100 diluted slurry+9 mL autoclaved calcium carbonate slurry C

[0231] Then the different diluted slurry's were tested with the Glomax 20/20 Luminometer (available from Promega; 3 measurements of each diluted slurry) and the BacTiter Glo Reagent (available from Promega) and were plated on TSA in 10-fold dilutions in order to quantify the bacteria on the TSA plates.

[0232] The results are as follows:

[0233] Autoclaved calcium carbonate slurry C for background level: [0234] <100 cfu/mL on TSA, 12 464 RLU

[0235] Contaminated calcium carbonate slurry C: [0236] 100 cfu/mL on TSA, 15 356 RLU with Luminometer [0237] 800 cfu/mL on TSA, 19 397 RLU with Luminometer [0238] 5.36E+03 cfu/mL on TSA, 68 358 RLU with Luminometer [0239] 5.25E+04 cfu/mL on TSA, 476 893 RLU with Luminometer

[0240] The results for the calcium carbonate slurry C with 50% solid content, tested without further dilution (overall dilution by addition of test reagents was 2) are also shown in FIG. 3. Assay sensitivity was about 1E2 cfu/mL as defined by all measured samples above background level (here about 20 000 RLU). RLU was measured with Glomax 20/20 Luminometer (available from Promega) versus cfu/mL evaluated on TSA. Datapoints represent the mean of triplicates with standard deviation.